Publish canine-aging-corpus

.gitattributes

@@ -58,3 +58,4 @@ saved_model/**/* filter=lfs diff=lfs merge=lfs -text
 # Video files - compressed
 *.mp4 filter=lfs diff=lfs merge=lfs -text
 *.webm filter=lfs diff=lfs merge=lfs -text
+records.jsonl filter=lfs diff=lfs merge=lfs -text

LICENSE

@@ -0,0 +1,10 @@
+Mixed licences; see README.md.
+
+- records.jsonl and manifest.parquet: bibliographic metadata and abstracts retrieved from the
+  Europe PMC REST API (https://europepmc.org/RestfulWebService), redistributed under Europe
+  PMC's terms (https://europepmc.org/Copyright). Abstract copyright remains with the
+  respective publishers and authors.
+- fulltext.parquet and fulltext/md/: article full text, only for articles whose Europe PMC
+  licence field is CC BY (any version) or CC0. Each row/file carries the article's licence
+  and identifiers; attribute the original authors and journal.
+- The compilation itself (selection, conversion, manifest): CC BY 4.0.

README.md

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+---
+pretty_name: Canine Aging Corpus
+language:
+- en
+license: other
+license_name: europe-pmc-and-per-article-licences
+license_link: https://europepmc.org/Copyright
+tags:
+- biology
+- veterinary
+- dogs
+- aging
+- longevity
+- geroscience
+- europe-pmc
+- literature
+task_categories:
+- text-retrieval
+size_categories:
+- 1K<n<10K
+configs:
+- config_name: records
+  data_files: records.jsonl
+  default: true
+- config_name: fulltext
+  data_files: fulltext.parquet
+- config_name: manifest
+  data_files: manifest.parquet
+---
+
+# Canine Aging Corpus
+
+A versioned, reproducible corpus of the canine aging and longevity literature, built from
+the [Europe PMC](https://europepmc.org/RestfulWebService) REST API by the
+[`canine-aging-corpus`](https://github.com/w0lph/k9/tree/main/corpus) pipeline. It exists so
+that agents and retrieval systems can work over a fixed, citable snapshot of what has been
+published about dogs as a model of aging, instead of live search results that change daily.
+
+| | |
+|---|---|
+| Records | 3,565 works (`records`), of which 2,185 match the strict `core` query |
+| Full text | Markdown for the open-access articles whose licence allows redistribution: CC BY or CC0 (`fulltext`, also as `fulltext/md/PMC*.md`) |
+| Manifest | one row per record with identifiers, year, journal, licence, and which artefacts exist (`manifest`) |
+| Snapshot | `corpus_version.json`: query definitions, counts by tier / source / year, SHA-256 of `records.jsonl`, build time (2026-09-29) |
+
+## Query tiers
+
+Every record carries the list of tiers it matched, so consumers can filter.
+
+- **core**: `(dog OR dogs OR canine OR "Canis familiaris") AND (aging OR ageing OR lifespan OR longevity OR geroscience)` in title or abstract. High precision.
+- **extended**: adds geriatric, senior, age-related, healthspan, frailty, senescence, cognitive dysfunction, rapamycin, life expectancy. Higher recall, more clinical noise.
+
+## Fields (`records`)
+
+`key` (Europe PMC `source:id`, e.g. `MED:38263575`), `source`, `id`, `pmid`, `pmcid`, `doi`,
+`title`, `abstract`, `authors`, `author_string`, `journal`, `year`, `first_publication_date`,
+`pub_types`, `keywords`, `mesh` (descriptor, major flag, qualifiers), `grants`,
+`is_open_access`, `license`, `in_pmc`, `in_epmc`, `has_pdf`, `fulltext_urls`, `cited_by_count`,
+`language`, `tiers`, `retrieved_at`, `raw_page` (which raw search page the record came from).
+
+`fulltext` rows: `pmcid`, `pmid`, `doi`, `title`, `journal`, `year`, `license`, `source`
+(which service served the JATS XML: `ncbi` or `europepmc`), `article_type`, `chars`,
+`markdown` (front matter, title, abstract, body sections with heading levels, table and
+figure captions; references dropped).
+
+## How it was built
+
+```bash
+cd corpus && uv sync
+uv run cac fetch-metadata --tier core && uv run cac fetch-metadata --tier extended
+uv run cac fetch-fulltext --source ncbi      # batched NCBI E-utilities efetch; Europe PMC is the fallback
+uv run cac convert && uv run cac manifest
+```
+
+JATS XML is converted deterministically (no model involved). Raw API pages are kept by the
+pipeline for provenance and `cac rebuild-records` reconstructs `records.jsonl` from them.
+
+## Limitations
+
+- Full text covers 1,140 of the 1,347 records with a PMCID locally; 207 are restricted by
+  their publisher from XML distribution. Only the CC BY / CC0 subset is redistributed here;
+  articles under CC BY-NC variants or without a licence statement are excluded from this
+  repository (`fulltext_manifest.json` has the counts) and can be fetched by the pipeline.
+- Europe PMC's `license` field is only populated for part of the open-access set; records
+  with an empty licence are treated as non-redistributable.
+- Query-based corpora have recall limits: some classic papers that do not use the query
+  vocabulary in their title or abstract are absent.
+
+## Licence
+
+Metadata and abstracts: retrieved from Europe PMC and redistributed under
+[Europe PMC's terms](https://europepmc.org/Copyright); abstract copyright stays with the
+publishers and authors. Full text: each article's own licence (CC BY or CC0), stated per
+row and in each Markdown file's front matter. The compilation: CC BY 4.0. See `LICENSE`.
+
+## Related
+
+- [`w0lph/canine-geroscience-questions`](https://huggingface.co/datasets/w0lph/canine-geroscience-questions): a grounded question set over this corpus.
+- [`w0lph/dog-geroscience-mcp-data`](https://huggingface.co/datasets/w0lph/dog-geroscience-mcp-data): the SQLite database that indexes this corpus for the `dog-geroscience-mcp` server.
+- Pipeline and documentation: https://github.com/w0lph/k9/tree/main/corpus

corpus_version.json

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+{
+  "corpus_version": "2026.09.29",
+  "tool_version": "0.1.0",
+  "built_at": "2026-09-29T02:05:29+00:00",
+  "queries": {
+    "core": "(TITLE_ABS:(dog OR dogs OR canine OR \"Canis familiaris\") AND TITLE_ABS:(aging OR ageing OR lifespan OR longevity OR geroscience))",
+    "extended": "(TITLE_ABS:(dog OR dogs OR canine OR \"Canis familiaris\") AND TITLE_ABS:(aging OR ageing OR lifespan OR longevity OR geroscience OR geriatric OR senior OR \"age-related\" OR healthspan OR frailty OR senescence OR \"cognitive dysfunction\" OR rapamycin OR \"life expectancy\"))"
+  },
+  "counts": {
+    "records": 3565,
+    "by_tier": {
+      "core": 2185,
+      "extended": 3565
+    },
+    "core_only": 0,
+    "with_pmcid": 1347,
+    "open_access": 1051,
+    "fulltext_xml": 1140,
+    "fulltext_md": 1140,
+    "fulltext_md_over_3k_chars": 1109,
+    "fulltext_md_chars_total": 42150014,
+    "by_source": {
+      "MED": 3286,
+      "PPR": 145,
+      "PMC": 58,
+      "PAT": 42,
+      "AGR": 21,
+      "ETH": 12,
+      "CBA": 1
+    },
+    "by_year": {
+      "1918": 1,
+      "1922": 1,
+      "1947": 2,
+      "1952": 2,
+      "1953": 1,
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+      "1960": 2,
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+      "1969": 4,
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+      "1971": 1,
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+      "1973": 6,
+      "1974": 3,
+      "1975": 13,
+      "1976": 7,
+      "1977": 14,
+      "1978": 14,
+      "1979": 26,
+      "1980": 13,
+      "1981": 17,
+      "1982": 18,
+      "1983": 25,
+      "1984": 24,
+      "1985": 13,
+      "1986": 24,
+      "1987": 23,
+      "1988": 30,
+      "1989": 34,
+      "1990": 22,
+      "1991": 22,
+      "1992": 32,
+      "1993": 30,
+      "1994": 32,
+      "1995": 26,
+      "1996": 39,
+      "1997": 37,
+      "1998": 38,
+      "1999": 26,
+      "2000": 55,
+      "2001": 52,
+      "2002": 54,
+      "2003": 51,
+      "2004": 55,
+      "2005": 71,
+      "2006": 60,
+      "2007": 60,
+      "2008": 55,
+      "2009": 69,
+      "2010": 64,
+      "2011": 71,
+      "2012": 102,
+      "2013": 80,
+      "2014": 72,
+      "2015": 84,
+      "2016": 98,
+      "2017": 135,
+      "2018": 115,
+      "2019": 140,
+      "2020": 135,
+      "2021": 153,
+      "2022": 161,
+      "2023": 210,
+      "2024": 197,
+      "2025": 281,
+      "2026": 245
+    }
+  },
+  "records_sha256": "f15d39ad3f91379d2a558079cf5ea6af043e5ef1306f29c3f5c2e48b90748467"
+}

fulltext.parquet

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fulltext/md/PMC10006577.md

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+---
+pmcid: "PMC10006577"
+pmid: "36915903"
+doi: "10.1093/geroni/igac080"
+title: "Pet Ownership and Maintenance of Physical Function in Older Adults-Evidence From the Baltimore Longitudinal Study of Aging (BLSA)."
+journal: "Innovation in aging"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Pet Ownership and Maintenance of Physical Function in Older Adults—Evidence From the Baltimore Longitudinal Study of Aging (BLSA)
+
+## Abstract
+
+### Background and Objectives
+
+Pet ownership or human–animal interaction has been associated with better health outcomes in individuals with disease or disability. We hypothesized that pet ownership, as well as dog ownership and cat ownership separately, are associated with maintaining physical function, and leisure time physical activity and that among dog owners, dog walking is associated with maintaining these outcomes for generally healthy community-dwelling older adults participating in the Baltimore Longitudinal Study of Aging.
+
+### Research Design and Methods
+
+A total of 637 men (44.1%) and women aged 50–100 years (*M* = 68.3, standard deviation [*SD*] = 9.6) completed a comprehensive pet ownership questionnaire that ascertained pet ownership history 10–13 years and had serial assessments of physical function every 1–4 years prior. Linear or generalized linear mixed models with time varying pet ownership were used to examine change in physical function over a mean of 7.5 years (range 1–13, *SD* = 3.6) according to pet ownership.
+
+### Results
+
+Pet owners (*n* = 185) were significantly younger (*p* < .001) and had fewer comorbidities (*p* = .03) than nonowners; thus, age and comorbidities were included as covariates in the longitudinal analyses. Physical function and leisure time physical activity declined with aging across all outcomes (*p* < .001); the decline was slower among pet owners in overall physical performance (*p* < .001), rapid gait speed (*p* = .03), usual gait speed (*p* = .032), cardiorespiratory fitness (*p* < .001), and physical well-being (*p* = .002) controlling for age and comorbidities. Changes in leisure time physical activities with aging did not differ between pet owners and nonowners. Dog walking was not independently related to the maintenance of physical function or leisure time physical activity with aging.
+
+### Discussion and Implications
+
+This study provides the first longitudinal evidence that pet ownership is associated with maintained physical function among community-dwelling generally healthy older adults.
+
+**Translational Significance:** Prior research indicates that human–animal interaction promotes physical function of older adults residing in care communities. There is a dearth of data relating pet ownership to longitudinal changes in physical function for generally healthy older adults residing in community settings. Older adult pet owners experienced less decline in physical function as they aged, after considering both their preexisting health and age. Pet ownership was not related to changes in leisure time physical activity. Policy makers can use these outcomes to consider possible contributions of pet ownership in the design and assessment of strategies for maintaining physical function in this population.
+
+Physical function typically decreases as adults age, with greater decreases occurring in the later years of life (Metti et al., 2018). Declines in function and increases in disease and disability frequently lead to poorer quality of life during the final years. As the proportion of the population in the older age groups increases over the next several decades, addressing older adults’ health and care needs will become a more pressing problem (Vespa, 2018). Promoting healthy aging is intended to reduce the current 5–7 year gap between high life quality and total life expectancy and enable people to live with the best function possible for as long as possible (Beltrán-Sánchez et al., 2015). Healthy aging depends on using many strategies to help minimize the declines in physical and cognitive function, in the development and progression of disease and disability, and in active engagement with life.
+
+Human–animal interaction in several forms is suggested to promote physical, psychological, and social health in an aging population (Friedmann, 2019; Friedmann & Gee, 2018; Gee, 2021; Gee & Galik, 2019; Gee & Mueller, 2019; Gee et al., 2021). Pet ownership is common in community-dwelling older adults with estimates ranging upwards from 50% among individuals over the age of 50 (Mueller et al., 2018). Evidence supports the contribution of pet ownership to some aspects of healthy aging (Friedmann, 2019; Friedmann & Gee, 2018), including improved cardiovascular health and decreased mortality (Gee & Mueller, 2019; Levine et al., 2013).
+
+The biopsychosocial model provides potential mechanisms for the impact of human–animal interaction on human health. In this conceptualization, biological, psychological, and social variables affect each other and integrate into health. Human–animal interaction can be conceptualized as a source of enhancement in each variable group (Friedmann, 2019; Gee et al., 2021). The psychological and biological effects of interacting with an animal are often closely interwoven; human–animal interaction leads to reductions in a variety of biological stress indicators such as cortisol, blood pressure, and heart rate. Likewise, the psychological and social variable groups are tightly interwoven; human–animal interaction may provide a source of social support or attachment, which in turn may contribute to lower stress and better psychological health (Friedmann, 2019).
+
+Pet ownership is theorized to support physical health and thereby promote healthy aging (Friedmann & Gee, 2018; Gee & Mueller, 2019). Research links pet ownership with better health outcomes in older adults, particularly those with chronic health conditions, with the strongest evidence from studies of individuals with heart disease (Gee & Mueller, 2019; Levine et al., 2013). The first evidence of the association of pet ownership with physical health was from a study of patients hospitalized with coronary heart disease (Friedmann et al., 1980). That and follow-up studies confirmed that the association of pet ownership with survival was independent of disease severity and social support (Friedmann & Thomas, 1995; Friedmann et al., 2011). The evidence-base for the link between pet ownership and cardiac health in older adults is not consistently positive. In one study examining patients hospitalized for acute cardiac symptoms, pet ownership, specifically cat ownership, was related to the composite outcome of mortality or readmission (Parker et al., 2010). One of the ways pet ownership is suggested as a mechanism for improved cardiovascular health is to encourage physical exercise, and this function is expected to be limited to dog owners, specifically those who walk their dogs (Levine et al., 2013). The American Heart Association validated the idea that pet ownership supported physical activity when they conducted and published a review of the existing evidence, carefully weighing all the results, and issued a statement that pet ownership, particularly dog ownership, likely plays a causal role in reducing the risk of cardiovascular disease. Much of that benefit was attributed to physical activity with the dog (Levine et al., 2013). Another frequently suggested mechanism is that pets decrease stress and stress responses, which decrease cardiovascular-related risk. A repeated measures study using ecological momentary assessment of blood pressure during their daily lives individuals with pre- to mild-hypertension found that stress was lower when they were in the presence of their pets in their homes compared with when their pets were not present (Friedmann et al., 2013).
+
+Cross-sectional studies of the general older adult population also link any current pet ownership to positive health outcomes. Community-residing older adults who owned dogs or cats in the past, but not current owners, experienced reduced odds of becoming frail over 2 years than those who never owned a dog (Taniguchi et al., 2019). Several cross-sectional studies of older adults suggest that dog walking is associated with overall greater physical activity (Coleman et al., 2008; Dall et al., 2017; Mein & Grant, 2018; Thorpe et al., 2006). For example, comparing community-dwelling older adult dog owners with matched nonowners, dog-owners averaged 22 min more of walking time, walked 2,760 more steps, and had fewer prolonged sedentary events per day than nonowners (Dall et al., 2017). Dog walkers on average walk their dogs 160 min per week divided into an average of four walks (Christian et al., 2013). Overall community-dwelling older adult dog owners were 12% more active than nonowners after controlling for potential confounders (Feng et al., 2014). Other large population-based studies of older adults indicated that individuals with a history of pet ownership had greater motor fitness and completed more walking activities than those with no history of pet ownership (Taniguchi et al., 2018). A small prospective observational study of older adults revealed that heart rate variability, a stress indicator, was better (higher) when walking with a dog than when walking alone (Motooka et al., 2006). Older adults who walk their dogs may also experience other benefits, such as greater community engagement (Toohey et al., 2013; Wood et al., 2005), positive feelings about their neighborhoods (Mein & Grant, 2018), and social benefits (Antonacopoulos & Pychyl, 2014; McNicholas & Collis, 2000; Taniguchi et al., 2018; Wells, 2004).
+
+Longitudinal studies have an advantage over cross-sectional studies in establishing a temporal relationship between exposure and outcomes. In this study, we use longitudinal data to examine the associations of pet ownership with maintaining physical function and leisure time physical activity in the generally healthy community-dwelling older adult participants in the Baltimore Longitudinal Study of Aging (BLSA).
+
+This study provides an opportunity to examine this speculation. We expected deterioration in physical function and leisure time physical activity as participants aged. We hypothesized that pet ownership, as well as dog ownership, and cat ownership separately, are associated with maintaining physical function and leisure time physical activity and that among dog owners, dog walking is associated with maintaining physical function and leisure time physical activity for generally healthy community-dwelling older adults participating in the BLSA.
+
+## Design
+
+The study used a cohort design with health data obtained in the BLSA, an ongoing National Institute on Aging (NIA) Intramural Research Program-funded cohort study. The BLSA is America’s longest-running scientific study of human aging. Started in 1958, BLSA is a longitudinal observational study that addresses critical questions about normal and pathological age-related change. BLSA uses a standardized battery of surveys and tests to measure cognitive and physical changes associated with aging in real-time. Each BLSA visit consists of three consecutive days of testing. Participants who are 20–60 years old complete BLSA visits every 4 years; those 60–79 years old complete them every 2 years; and those 80 years and older complete them annually. Following IRB approval by the National Institutes of Health Intramural Research Program Institutional Review Board, a group of pet ownership-related questions, including a 10-year pet ownership history, were added to the battery of surveys completed during visits starting in March 2017. The BLSA visit that included the individual’s first assessment of human–animal interaction is termed the survey visit. Individuals aged 50 and older at the survey visit and with two or more BLSA visits were included in the study.
+
+Participants included in this study had been completing visits to the BLSA for varying times prior to the survey visit. They also continued to attend their regularly scheduled BLSA visits for up to 3 years after the survey visit. Thus, the BLSA participants in this study attended BLSA visits and provided functional data at specified BLSA intervals over a period of 1–13 years, with a mean follow-up of 7.5 years (standard deviation [*SD*] = 3.6). Pet ownership data from the pet ownership history component of the human–animal interaction assessment were associated with each BLSA visit for the 10 years prior to the survey visit and for any visits during the subsequent 3 years of the study. The first BLSA visit associated with pet ownership data is termed the index visit (see Figure 1).
+
+*Figure: Figure 1. Human–animal interaction (HAI) assessment module and pet ownership history assessment in the context of the Baltimore Longitudinal Study of Aging (BLSA). The survey visit is the first BLSA visit with a human–animal interaction assessment (March 2017 to March 2020). The index visit is the first BLSA visit in the 10 years prior to the survey visit.*
+
+## Participants
+
+A total of 637 participants in the BLSA were aged 50–100 years (*M* = 75.1, *SD* = 10.15). The sample was predominantly white (66.98%) with smaller proportions of Black (28.12%), Asian (1.26%), and Hawaiian/Pacific Islanders (0.32%) represented. For analyses, Asians and Hawaiian/Pacific Islanders were grouped with Whites. The sample was relatively equally distributed between male (44.11%) and female (55.89%). A large majority of the sample held postgraduate degrees (67.03%). The sample was also largely married or partnered (62.20%) and living with at least one other person (70.96%), and in a single-family home (78.74%). Only 16.61% of the sample had an income less than $50,000 per year. Less than half of the sample currently work (32.60%), while 54.17% volunteered.
+
+Among the 637 participants who completed the survey visit, 185 (29.0%) owned pets, 112 (17.58%) owned cats and 95 (14.91%) owned dogs; 22 (7.33%) owned either other species of pets only or a combination of multiple species of pets including cats or dogs. Among dog owners 58 (69.05%) indicated they walked their dogs. There were notable differences in race and housing type between pet owners and nonpet owners, summarized in Table 1. Pet ownership was significantly more common among whites than blacks and among those who resided in single-family homes than those who did not (*p*’s < .001).
+
+**Table 1. Demographic and Pet Ownership Characteristics of Respondents at the Baltimore Longitudinal Study of Aging Study Survey Visit When Participants First Completed the Human–Animal Interaction Questionnaire**
+
+| Characteristic | Overall (*n* = 637) | Nonpet owner (*n* = 452) | Pet owner (*n* = 185) | Test statistic | *p* |
+| --- | --- | --- | --- | --- | --- |
+| *N* | % | *M* | *SD* | *N* | % | *M* | *SD* | *N* | % | *M* | *SD* |
+| Dog owner | 112 | 17.58 |  |  | 0 | n/a |  |  | 112 | 60.54 |  |  |  |  |
+| Cat owner | 95 | 14.91 |  |  | 0 | n/a |  |  | 95 | 51.35 |  |  |  |  |
+| Age in years^a^ | 75.09 | 10.15 |  |  | 76.76 | 9.67 |  |  | 71.01 | 10.15 |  |  | 6.71 | **<.001** |
+| Black^b^ | 178 | 27.94 |  |  | 146 | 32.30 |  |  | 32 | 17.30 |  |  | 14.68 | **<.001** |
+| Female | 356 | 55.89 |  |  | 257 | 56.86 |  |  | 99 | 53.51 |  |  | 0.60 | .440 |
+| <College Graduate^b^ | 80 | 12.62 |  |  | 65 | 14.44 |  |  | 15 | 8.15 |  |  | 5.03 | .081 |
+| Married or partnered^b^ | 395 | 62.20 |  |  | 263 | 58.31 |  |  | 132 | 71.74 |  |  | 10.02 | **.002** |
+| Live alone^b^ | 176 | 29.04 |  |  | 143 | 33.41 |  |  | 33 | 18.54 |  |  | 13.49 | **<.001** |
+| Live in single family house^c^ | 500 | 78.74 |  |  | 328 | 72.73 |  |  | 172 | 93.48 |  |  | 33.62 | **<.001** |
+| Family income exceeds $50,000^b^ | 527 | 83.39 |  |  | 359 | 83.10 |  |  | 168 | 92.31 |  |  | 8.92 | **.003** |
+| Currently works^b^ | 207 | 32.60 |  |  | 131 | 29.11 |  |  | 76 | 41.30 |  |  | 8.83 | **.003** |
+| Currently volunteers^b^ | 344 | 54.17 |  |  | 241 | 53.56 |  |  | 103 | 55.98 |  |  | 0.31 | .578 |
+| Body mass index^a^ |  |  | 26.98 | 4.81 |  |  | 27.10 | 4.87 |  |  | 26.54 | 4.66 | 0.96 | .339 |
+| Obese^b^ | 148 | 23.23 |  |  | 107 | 23.67 |  |  | 41 | 22.16 |  |  | 0.17 | .682 |
+| Comorbidities^a^ |  |  | 1.66 | 1.48 |  |  | 1.73 | 1.49 |  |  | 1.50 | 1.46 | 1.84 | .066 |
+| Follow-up years^a^ |  |  | 6.84 | 3.57 |  |  | 7.01 | 3.34 |  |  | 6.43 | 3.73 | 1.91 | .057 |
+| 400-m Walk time (wln)^d^ |  |  | 289.38 | 68.17 |  |  | 297.7 | 70.01 |  |  | 270 | 59.44 |  | **<.001** |
+| Usual speed^a^ |  |  | 1.12 | 0.24 |  |  | 1.09 | 0.24 |  |  | 1.20 | 0.24 | −5.06 | **<.001** |
+| Rapid speed^a^ |  |  | 1.66 | 0.38 |  |  | 1.61 | 0.37 |  |  | 1.78 | 0.38 | −4.97 | **<.001** |
+| Physical performance^a^ |  |  | 2.53 | 0.62 |  |  | 2.45 | 0.63 |  |  | 2.72 | 0.57 | −4.89 | **<.001** |
+| Physical well-being(rln)^d^^,^^e^ |  |  | 51.67 | 7.59 |  |  | 51.26 | 7.74 |  |  | 52.65 | 7.15 |  | **.005** |
+| Leisure activity^d^ |  |  | 3,867.68 | 3,788.39 |  |  | 3,450.30 | 3,394.00 |  |  | 4,868.01 | 4,451.70 |  | **<.001** |
+| Leisure walking (ln)^a^ |  |  | 3.31 | 3.56 |  |  | 3.04 | 3.66 |  |  | 3.95 | 3.21 | −2.88 | **.004** |
+| Brisk exercise (ln)^a^ |  |  | −0.49 | 4.60 |  |  | −0.75 | 4.55 |  |  | 0.14 | 4.65 | −2.18 | **.030** |
+| Weekly exercise (ln)^a^ |  |  | 1.17 | 4.54 |  |  | 1.005 | 4.55 |  |  | 1.56 | 4.48 | −1.37 | .171 |
+| Recommended exercise^b^ | 149 | 24.55 |  |  | 101 | 23.60 |  |  | 48 | 26.82 |  |  | 0.71 | .401 |
+
+Annual pet ownership data were obtained retrospectively during the survey visit for the preceding 10 years. These pet ownership data were mapped onto the BLSA physical assessment visits over that same period. The earliest occasion when pet ownership data could be associated with a BLSA assessment was used to produce the index visit data. Pet ownership data were associated with each BLSA physical assessment within the last 10 years as well as the 3 years of the study after the survey visit.
+
+The characteristics of the participants at the index visit are included in Supplementary Table 1. These data are baseline data for all further analyses. Average age at index visit was 68.25 years (*SD* = 9.64). The participants were generally in good health; participants’ mean number of comorbidities was 0.95 (*SD* = 1.17). Only 20.88% of the participants were obese.
+
+At the index visit, 188 (29.51%) of the 637 participants owned pets; 67 (10.52%) owned cats, and 84 (13.19%) owned dogs. Other pet owners did not indicate the types of pets they owned. Pet owners were significantly younger (*p* < .001) and had fewer comorbidities (*p* = .030) than nonowners. There were notable differences in race and housing type between pet owners and nonpet owners, like those reported for the survey visit.
+
+There were no significant differences in the demographic characteristics of cat owners and dog owners at the index visit. Dog owners were not significantly younger and did not have a better initial physical function, although they did have significantly fewer comorbidities (*p* = .039) than cat owners. At the index visit, individuals who owned dogs but not cats (dog owners exclusively) and individuals who owned cats but not dogs (cat owners exclusively) shared similar demographic characteristics (Table 2). Dog owners exclusively (*n* = 79) were 65.52 years old on average, while cat owners exclusively (*n* = 62) were 66.60 years old at the time of initial assessment. Most cat owners exclusively and dog owners exclusively were white (83.87% and 78.67%, respectively) and predominantly female (*n* = 77, 54.61%). Both cat owners exclusively (77.42%) and dog owners exclusively (70.51%) had high levels of postgraduate education. Cat owners exclusively had significantly more comorbidities (*M* = 1.05, *SD* = 1.25) than dog owners exclusively (*M* = 0.75, *SD* = 1.15, *p* = .041). Cat owners exclusively were followed longer in the study (*M* = 7.71, *SD* = 3.38) than dog owners exclusively (*M* = 6.16, *SD* = 4.29, *t* = −2.33, *p* = .02). None of the physical function variables differed significantly between cat owners exclusively and dog owners exclusively.
+
+**Table 2. Demographic and Pet Ownership Characteristics of Pet Owners Comparing Those That Own Cats Exclusively (Without Owning Dogs) to Those Who Own Dogs Exclusively (Without Owning Cats) Index Visit**
+
+| Characteristic | Overall (*n* = 141) | Cat owners (*n* = 62) | Dog owner(*n* = 79) | Test Statistic | *p* |
+| --- | --- | --- | --- | --- | --- |
+| *N* | % | *M* | *SD* | *N* | % | *M* | *SD* | *N* | % | *M* | *SD* |
+| Age in years^a^ |  |  | 65.99 | 9.22 |  |  | 66.60 | 8.58 |  |  | 65.52 | 9.72 | −0.69 | .489 |
+| Black^b^ | 26 | 18.44 |  |  | 10 | 16.13 |  |  | 16 | 20.25 |  |  | 0.39 | .531 |
+| Female^b^ | 77 | 54.61 |  |  | 37 | 59.68 |  |  | 40 | 50.63 |  |  | 1.15 | .284 |
+| <College graduate^c^ | 8 | 5.71 |  |  | 4 | 6.45 |  |  | 4 | 5.13 |  |  |  | .441 |
+| Married or partnered^b^ | 108 | 77.14 |  |  | 49 | 79.03 |  |  | 59 | 75.64 |  |  | 0.23 | .635 |
+| Lives alone^b^ | 25 | 17.86 |  |  | 9 | 14.52 |  |  | 16 | 20.51 |  |  | 4.41 | .221 |
+| Single family housing^c^ | 132 | 94.96 |  |  | 60 | 98.36 |  |  | 72 | 92.31 |  |  |  | .135 |
+| Family income exceeds $50,000^b^ | 120 | 86.96 |  |  | 53 | 86.89 |  |  | 67 | 87.01 |  |  | 0.001 | .982 |
+| Currently works^b^ | 84 | 60.00 |  |  | 35 | 56.45 |  |  | 49 | 62.82 |  |  | 0.58 | .445 |
+| Currently volunteers^b^ | 79 | 57.25 |  |  | 39 | 63.93 |  |  | 40 | 51.95 |  |  | 2.00 | .158 |
+| Body mass index^a^ |  |  | 26.49 | 4.58 |  |  | 25.81 | 4.28 |  |  | 27.03 | 4.76 | 1.57 | .118 |
+| Obese^b^ | 24 | 17.02 |  |  | 6 | 9.68 |  |  | 18 | 22.78 |  |  | 4.23 | **.040** |
+| Comorbidities^d^ |  |  | 0.80 | 1.20 |  |  | 1.05 | 1.25 |  |  | 0.75 | 1.15 |  | **.041** |
+| Follow-up years^a^ |  |  | 6.84 | 3.98 |  |  | 7.71 | 3.38 |  |  | 6.16 | 4.29 | −2.33 | **.021** |
+| 400-m Walk time (wln)^d^ |  |  | 251.94 | 48.53 |  |  | 256.9 | 55.77 |  |  | 247.79 | 41.52 |  | .376 |
+| Usual speed^a^ |  |  | 1.23 | 0.22 |  |  | 1.22 | 0.20 |  |  | 1.23 | 0.23 | 0.23 | .818 |
+| Rapid speed^a^ |  |  | 1.88 | 0.35 |  |  | 1.87 | 0.34 |  |  | 1.89 | 0.36 | 0.36 | .722 |
+| Physical performance^d^ |  |  | 2.74 | 0.47 |  |  | 2.71 | 0.38 |  |  | 2.77 | 0.53 |  | .240 |
+| Physical well-being (rln)^d^^,^^e^ |  |  | 53.61 | 6.02 |  |  | 53.17 | 6.52 |  |  | 53.96 | 5.60 |  | .657 |
+| Leisure activity^a^ |  |  | 6,433.07 | 4,322.20 |  |  | 5,023.85 | 4,027.53 |  |  | 6,713.54 | 4,954.53 | 1.97 | .051 |
+| Leisure walking (ln)^a^ |  |  | 3.88 | 3.12 |  |  | 4.38 | 2.96 |  |  | 3.26 | 3.23 | 2.12 | **.036** |
+| Brisk exercise (ln)^a^ |  |  | 0.67 | 4.53 |  |  | 1.05 | 4.59 |  |  | 0.19 | 4.45 | 1.12 | .26 |
+| Weekly exercise (ln)^a^ |  |  | 1.82 | 4.33 |  |  | 2.22 | 4.28 |  |  | 1.33 | 4.37 | 1.2 | .231 |
+| Recommended exercise^b^ | 41 | 29.29 |  |  | 26 | 33.33 |  |  | 15 | 24.19 |  |  | 1.39 | .238 |
+
+Among the 73 dog owners who indicated whether they walked their dogs at the survey visit, 58 (79.5%) indicated they walked their dogs. Index visit characteristics of dog owners who walked and did not walk their dogs are included in Table 3. The demographics of the two groups did not differ, but health status did. Dog walkers had significantly lower impairment in activities of daily living and lower triglycerides. Comorbidities did not differ significantly between dog walkers and nonwalkers.
+
+**Table 3. Demographic and Pet Ownership Characteristics of Dog Owners (*n* = 73) Who Walk and Do Not Walk Their Dogs**
+
+| Characteristic | Overall (*n* = 73) | Dog walkers (*n* = 58) | Nondog walkers (*n* = 15) | Test statistic | *p* |
+| --- | --- | --- | --- | --- | --- |
+| *N* | % | *M* | *SD* | *N* | % | *M* | *SD* | *N* | % | *M* | *SD* |
+| Age in years^a^ |  |  | 64.48 | 9.89 |  |  | 64.11 | 9.36 |  |  | 65.92 | 12.00 | 0.63 | .532 |
+| Black^b^ | 12 | 16.44 |  |  | 10 | 17.24 |  |  | 2 | 13.33 |  |  |  | 1.000 |
+| Female^b^ | 38 | 52.05 |  |  | 27 | 46.55 |  |  | 11 | 73.33 |  |  | 3.43 | .064 |
+| <College graduate^c^ | 3 | 4.17 |  |  | 1 | 1.75 |  |  | 2 | 13.33 |  |  |  | .541 |
+| Married or partnered^b^ | 55 | 76.39 |  |  | 41 | 71.93 |  |  | 14 | 93.33 |  |  |  | .100 |
+| Lives alone^b^ | 14 | 19.72 |  |  | 10 | 17.86 |  |  | 4 | 26.67 |  |  |  | .475 |
+| Single family housing^c^ | 66 | 91.67 |  |  | 53 | 92.98 |  |  | 13 | 86.67 |  |  |  | .598 |
+| Family income exceeds $50,000^b^ | 62 | 87.32 |  |  | 48 | 84.21 |  |  | 14 | 100 |  |  |  | .189 |
+| Currently works^b^ | 47 | 65.28 |  |  | 38 | 66.67 |  |  | 9 | 60.00 |  |  | 0.23 | .629 |
+| Currently volunteers^b^ | 33 | 46.48 |  |  | 26 | 46.43 |  |  | 7 | 46.67 |  |  | 0.001 | .987 |
+| Body mass index^a^ |  |  | 26.72 | 4.51 |  |  | 26.51 | 3.79 |  |  | 27.54 | 6.72 | 0.57 | .575 |
+| Obese^b^ | 15 | 20.55 |  |  | 11 | 18.97 |  |  | 4 | 26.67 |  |  | 0.43 | .511 |
+| Comorbidities^d^ |  |  | 0.68 | 1.17 |  |  | 0.50 | 0.80 |  |  | 1.40 | 1.92 |  | .175 |
+| Follow-up years^a^ |  |  | 5.46 | 4.31 |  |  | 5.44 | 4.41 |  |  | 5.51 | 4.06 | 0.06 | .955 |
+| Attachment^a^ |  |  | 65.70 | 11.57 |  |  | 66.39 | 11.34 |  |  | 63.21 | 12.48 | −0.94 | .349 |
+| 400-m Walk time (wln)^d^ |  |  | 242.92 | 38.85 |  |  | 241.41 | 35.62 |  |  | 250.45 | 53.68 |  | .973 |
+| Usual speed^a^ |  |  | 1.25 | 0.23 |  |  | 1.24 | 0.19 |  |  | 1.28 | 0.34 | 0.39 | .700 |
+| Rapid speed^a^ |  |  | 1.91 | 0.36 |  |  | 1.91 | 0.30 |  |  | 1.94 | 0.53 | 0.26 | .795 |
+| Physical performance^d^ |  |  | 2.84 | 0.54 |  |  | 2.84 | 0.42 |  |  | 2.82 | 0.90 |  | .615 |
+| Physical well-being (rln)^d^^,^^e^ |  |  | 54.16 | 5.19 |  |  | 55.05 | 3.29 |  |  | 50.81 | 8.82 |  | .0518 |
+| Leisure activity^a^ |  |  | 6,602.87 | 4,895.25 |  |  | 6,489.20 | 4,193.20 |  |  | 5,518.70 | 4,210.60 | −0.78 | .441 |
+| Leisure walking (ln)^a^ |  |  | 4.45 | 2.90 |  |  | 2.47 | 4.74 |  |  | 4.93 | 2.02 | −3.01 | **.004** |
+| Brisk exercise (ln)^a^ |  |  | 0.86 | 4.61 |  |  | -0.09 | 5.07 |  |  | 1.11 | 4.50 | −0.89 | .376 |
+| Weekly exercise (ln)^a^ |  |  | 2.11 | 4.32 |  |  | 0.63 | 5.12 |  |  | 2.50 | 4.04 | −1.51 | .136 |
+| Recommended exercise^b^ | 23 | 31.94 |  |  | 4 | 26.67 |  |  | 19 | 33.33 |  |  | 0.24 | .622 |
+
+## Measures
+
+### Pet Ownership Measures
+
+Pet ownership-related variables were assessed using multiple sources, including a 10-year pet ownership history questionnaire and the NIA Health and Retirement Study (HRS) pet ownership interaction module (National Center for Health Statistics, 2020; NIA, 2012). The HRS pet ownership module queried individuals who owned dogs about dog walking behavior, including whether they walked their dog, how frequently they walked their dog, and the average duration of walks (Friedmann et al., 2020).
+
+### Physical Function Measures
+
+The BLSA physical function measures include measures of cardiorespiratory fitness (400-m walk time), lower body strength (usual and rapid gait speed), overall physical performance (physical performance), participants’ perceptions of their physical function (physical well-being), and leisure time physical activities (leisure activity, leisure walking, brisk exercise, and recommended exercise).
+
+#### Cardiorespiratory fitness
+
+Cardiorespiratory fitness was assessed using 400-m walk time Participants walked 10 laps around a 40-m course on a carpeted interior corridor while accompanied by a study member counting the laps. They were instructed to do so as quickly as possible. Each lap time was recorded. Participant scores were calculated based on the use of assistive devices and their ability to complete the distance either with or without a walking aid (Simonsick et al., 2001a). Participants received standard encouragement and were told the number of laps remaining (Martinez-Amezcua et al., 2021). Time to complete the 400-m walk has previously been associated with both physical activity level and cardiovascular disease, with lower walk time predicting increased physical activity level and decreased cardiovascular disease (Trombetti et al., 2016; Winger et al., 2020). Four hundred meter walk time is validated as a measure of physical fitness by a negative correlation (−0.79) with peak VO2 (Simonsick et al., 2006). Lower time indicates better function.
+
+#### Lower body strength
+
+Lower body strength was assessed over a 6-m indoor carpeted course with participants walking at their usual walking speed (usual gait speed) for 6 m twice and then at their maximum walking speed (rapid gait speed) for 6 m twice. Time for each walk was measured with a stopwatch to the hundredth of a second. The time for the fastest of the two maximum speed walks was divided into six and provided the rapid gait speed (m/s; Schrack et al., 2012). This measure is validated in older adults by treadmill validation of an association with maximum oxygen consumption (Simonsick et al., 2006). Higher scores reflect faster gait in meters per second. The ideal usual gait speed is ≥1.0 m/s, however, certain daily activities may necessitate faster speeds (Sprague et al., 2021).
+
+#### Overall physical performance
+
+The Healthy Aging and Body Composition Study physical performance battery was used to assess the overall physical function of each participant. The score was derived as a composite of the four components from the Short Physical Performance Battery: usual gait speed as described earlier, time to stand-up from and sit back down on an armless chair five times, ability to hold three progressively more challenging balance-related stances (semitandem, full-tandem and single-leg for up to 30 s) each, and ability and time to walk a narrow (20 cm wide) 6-m course. For the narrow walk, three attempts were completed with the fastest successful walk time used as the score; having three failures was scored as zero. Each test was scored on a ratio scale with maximal performance as the denominator and actual performance as the numerator. Maximal performance for usual gait speed and the narrow walk was 2.0 m/s, for five chair stands was 5 s, and for the standing balance test was 90 s. For the narrow walk, three attempts were permitted to walk without stepping on or outside the limits of the course more than twice. Any performance exceeding the defined maximum was assigned a score of 1.0 for that test. The physical performance score constitutes the sum of these four ratio scores for a maximum total of 4.0 (Simonsick et al., 2016; Simonsick et al., 2001b). Higher values on this well-validated measure (Simonsick et al., 2006) indicate better physical performance.
+
+#### Physical well-being
+
+The Medical Outcomes Study Short Form-12 (SF-12) was used to provide individuals’ assessments of their own well-being. It consists of 12 self-rated items that capture eight domains of self-rated health that generate physical component summary (physical well-being) and mental component summary (mental well-being) scores calibrated to population norms (Ware, 2005; Ware et al., 1996). According to the Centre for Health Service Development (Marosszeky & Sansoni, 2005), a meta-analysis instrument review revealed SF-12 to be a psychometrically sound tool with test–retest reliability for the physical well-being score of 0.89; higher scores indicate greater physical well-being (Frantzen et al., 2021; Marosszeky & Sansoni, 2005)
+
+### Leisure Time Physical Activity Measures
+
+Five measures of weekly leisure time physical activity were calculated: total leisure time physical activity (leisure activity), time spent walking (leisure walking), minutes spent in “brisk” or exercise-related walking (brisk exercise), minutes spent exercising (weekly exercise), and a dichotomous variable (recommended exercise) indicating whether the participant got the recommended 150 min of weekly moderate-intensity exercise (Brach et al., 2004). Leisure time physical activity was determined using a standardized questionnaire designed specifically for the Health ABC study, modeled from commonly used physical activity assessments, including the leisure time physical activity questionnaire. Participants were asked if they participated in specific activities at least 10 times in the past 12 months; if so, whether they had done them in the past 7 days; and if so, the amount of time spent doing each. They were also asked to name other physical activities they participated in and answer similar questions about them. Physical activity amounts were calculated based on activities in the past 7 days and metabolic equivalent values of the tasks (Brach et al., 2004). Higher values indicate greater leisure time physical activity.
+
+### Covariates
+
+Both increasing age and comorbidities are generally associated with diminished physical function. These variables have the potential to confound the relationship of pet ownership with physical function because pet owners were younger and had fewer comorbidities than nonowners. Therefore, age and comorbidities were added as predictors in analyses of changes in physical health with aging. Age and comorbidities at the first time for which both pet ownership and BLSA functional data are available were used as covariates where indicated. A summary of physical comorbidities at the same time was also used as a covariate where indicated. Comorbidities scores were calculated as the sum of the number of eight conditions that the respondents reported (Friedmann et al., 2020). Conditions assessed were heart disease (including angina, myocardial infarction, heart failure, angioplasty, and coronary artery bypass graft), diabetes, pulmonary disease, cerebral vascular disease, lower extremity arthritis, lower extremity pain, minor functional difficulty, and exertional pain while walking.
+
+## Statistics
+
+Descriptive statistics were used to portray the participants and their index and survey visit characteristics. Characteristics of pet owners and nonowners at both index and survey visits were compared using *t*-tests for normally distributed continuous variables, Wilcoxon rank sum test for nonnormally distributed continuous variables, chi-square tests for categorical variables, and Fisher’s exact tests for categorical variables with expected cell frequencies lower than five. Differences in index visit physical function between cat and dog owners, and in dog owners who walked their dogs at survey visit and those who did not were examined in a similar manner. Prior to multivariable analysis, data were cleaned and examined for outliers and normality. Four-hundred-meter walk time, leisure walking, brisk exercise, and weekly exercise were log-transformed and physical well-being was reflected, and natural log-transformed. Unconditional intraclass correlations indicated considerable dependence ranging from 0.38 (400-m walk time) to 0.75 (physical performance; Supplementary Table 2).
+
+A linear mixed models (LMM) approach was used to examine changes in physical function and leisure time physical activities and compare changes by pet ownership status, except a generalized logistic model was used for recommended exercise, which had a dichotomous outcome. Pet ownership associated with each BLSA assessment was used as a time-varying covariate, unless noted otherwise. Thus, pet ownership at each data collection point was considered in all analyses except when pet ownership at index visit is specifically stated. Linear, or generalized linear, mixed models were used to examine changes in outcomes over up to 13 years (*M* = 7.5, *SD* = 3.6). Separate models were used to examine the association of pet ownership, dog ownership, and cat ownership to change in each outcome over time. Subsequently, these analyses were rerun to include age and comorbidities as covariates. In addition, LMMs that simultaneously included cat ownership, dog ownership, and the covariates were used to examine the independent associations of cat and dog ownership to longitudinal changes in each physical function and leisure time physical activity. We then ran LMM adjusting for covariates that tested the associations of cat ownership versus dog ownership to longitudinal changes in physical function and leisure time physical activity. A similar LMM approach was used to examine differences in both overall physical function/leisure time physical activities and changes in physical function/leisure time physical activity between dog owners who walked their dogs at the survey visit and those who did not. Dog walking analyses modeled the effect of this first dog walking status (time-invariant). Power was adequate (>80%) with a medium effect size for the changes in outcomes according to pet ownership, dog ownership, and cat ownership, but not for the smaller samples comparing dog owners who walked and did not walk their dogs. We calculated Cohen’s *d* for the significant interactions of aging and pet ownership status. We calculated the difference in changes in outcomes over time between pet owners and nonowners and divided this by the raw baseline outcome standard deviation.
+
+## Results
+
+At the index human–animal interaction assessment, 185 (29.04%) participants owned pets; 95 (14.91%) owned cats, and 112 (17.58%) owned dogs. Fewer than 5% of the participants owned a pet other than a cat or dog (Friedmann et al., 2020). Index visit physical function of pet owners were compared with those of nonowners (Supplementary Table 1). At the index visit, pet owners took significantly less time to walk 400 m, indicating better cardiorespiratory fitness, had faster usual and rapid gait speed, better overall physical performance, and reported more total leisure time physical activity but no other measures of leisure time physical activity, than nonowners. There were no significant differences in physical well-being between pet owners and nonowners.
+
+### Changes in Outcomes With Aging
+
+As hypothesized, all physical function outcomes declined over time, *p*’s < .001 (Supplementary Table 3).
+
+### Association of Pet Ownership With Changes in Outcomes With Aging
+
+Pet ownership was associated with less decline in most physical functional domains, but not in leisure time physical activities, after controlling for age and comorbidities (Table 4). Pet owners experienced significantly smaller increases in time to walk 400 m (Figure 2A, effect size [ES] = −0.40), decreases in physical performance score (Figure 2B, ES = 0.35), usual and rapid gait speed (Figures 2C and D, ES = 0.15, 0.13), and physical well-being than nonowners (Figure 2E, ES = −0.25).
+
+**Table 4. Changes in Physical Function Variables With Aging According to Pet Ownership, Dog Ownership, and Cat Ownership, Adjusted for Age and Comorbidity (*n* = 637)**
+
+| Outcome | Interaction | With | Follow-up | Interaction | With | Follow-up | Interaction | With | Follow-up |
+| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
+| PO est | PO *se* | PO *p* | DO est | DO *se* | DO *p* | CO est | CO *se* | CO *p* |
+| 400-m Walk time (wln) | −0.006 | 0.001 | **<.001** | −0.002 | 0.001 | .113 | −0.005 | 0.001 | **<.001** |
+| Physical performance | 0.015 | 0.003 | **<.001** | 0.009 | 0.004 | **.046** | 0.015 | 0.004 | **<.001** |
+| Usual speed | 0.003 | 0.002 | **.032** | 0.003 | 0.002 | .244 | 0.002 | 0.002 | .346 |
+| Rapid speed | 0.005 | 0.002 | **.030** | 0.008 | 0.003 | **.015** | 0.002 | 0.003 | .507 |
+| Physical well-being (rln)^a^ | −0.008 | 0.003 | **.002** | −0.002 | 0.004 | .501 | −0.009 | 0.003 | **.009** |
+| Leisure activity | −21.594 | 25.549 | .398 | −36.534 | 36.648 | .319 | −3.633 | 35.162 | .918 |
+| Leisure walking (ln) | 0.040 | 0.031 | .195 | 0.056 | 0.044 | .202 | −0.010 | 0.042 | .820 |
+| Brisk exercise (ln) | −0.015 | 0.041 | .717 | −0.057 | 0.058 | .331 | 0.052 | 0.057 | .361 |
+| Weekly exercise (ln) | 0.019 | 0.039 | .630 | −0.042 | 0.056 | .447 | 0.092 | 0.054 | .092 |
+| Recommended exercise | −0.005 | 0.033 | .888 | −0.036 | 0.044 | .821 | −0.035 | 0.047 | .455 |
+
+*Figure: Figure 2. Changes in five measures of physical function according to pet ownership status with 10 years of aging, estimates based on linear mixed models that include age and comorbidities as covariates.*
+
+### Associations of Dog Ownership and Cat Ownership With Changes in Outcomes With Aging
+
+In separate analyses, both dog ownership and cat ownership were associated with less decline in some physical function measures (Table 4). Dog owners experienced significantly less deterioration in overall physical performance (*p* = .046, ES = 0.20) and rapid gait speed (*p* = .015, ES = 0.22) than people who did not own dogs after controlling for age and comorbidities. Cat owners exhibited significantly less deterioration in 400-m walk speed (*p* < .001, ES = −0.36), physical function (*p* < .001, ES = 0.35), and physical well-being (*p* = .009, ES = −0.29) than people who did not own cats after controlling for age and comorbidities. Dog and cat ownership were not associated with changes in leisure time physical activity measures.
+
+Additional analyses examined the independent associations of dog ownership and cat ownership to physical function outcomes by entering both dog ownership and cat ownership into the models simultaneously. They provided similar results (Supplementary Table 4). Sensitivity analysis (Supplementary Table 5) using index visit pet ownership characteristics instead of time-varying pet ownership characteristics at the time of each physical function assessment as predictors produced similar results as those reported in Table 4, with two exceptions. Pet ownership at the index visit was not related to changes in rapid gait speed, and dog ownership at the index visit was not related to changes in overall physical performance.
+
+Analyses comparing changes in the physical function of dog owners exclusively and cat owners exclusively with aging (Table 5) demonstrated no significant difference between the groups. Changes with aging in 400-m walk time, physical performance, usual gait speed, rapid gait speed, physical well-being, and leisure time physical activity did not differ significantly between dog owners exclusively and cat owners exclusively.
+
+**Table 5. Estimates for Interaction Parameters From Linear Mixed Models Examining the Associations of Dog Ownership Exclusively (Without Cat Ownership) Versus Cat Ownership Exclusively (Without Dog Ownership) and the Association of Dog Walking to Changes in Physical Function Variables With Aging, Adjusted for Age and Comorbidity (*n* = 141, 73, Respectively)**
+
+| Outcome | Interaction | With | Follow-up | Interaction | With | Follow-up |
+| --- | --- | --- | --- | --- | --- | --- |
+| DvC est | DvC *se* | DvC *p* | DW est | DW *se* | DW *p* |
+| 400-m Walk time(wln) | 0.003 | 0.002 | .110 | −0.001 | 0.003 | .828 |
+| Physical performance | −0.011 | 0.006 | .068 | −0.0004 | 0.010 | .968 |
+| Usual speed | −0.002 | 0.003 | .543 | −0.002 | 0.005 | .660 |
+| Rapid speed | 0.002 | 0.005 | .645 | 0.011 | 0.009 | .239 |
+| Physical well-being (rln)^a^ | 0.004 | 0.005 | .481 | 0.005 | 0.009 | .593 |
+| Leisure activity | −15.557 | 52.786 | .768 | −16.318 | 99.130 | .869 |
+| Leisure walking (ln) | 0.102 | 0.056 | .069 | −0.265 | 0.081 | .001 |
+| Brisk exercise (ln) | −0.069 | 0.084 | .413 | −0.071 | 0.144 | .624 |
+| Weekly exercise (ln) | −0.111 | 0.081 | .171 | −0.045 | 0.140 | .751 |
+| Recommended exercise | 0.094 | 0.067 | .163 | 0.094 | 0.067 | .163 |
+
+### Dog Walking and Change in Outcomes With Aging
+
+There were no significant differences in changes in physical function or and most leisure time physical activity variables with aging between dog owners who walked their dogs and those who did not (Table 5). However, time spent in leisure walking changed differently for dog owners who walked and did not walk their dogs. Leisure time walking was greater for dog owners who walked their dogs at index visit. Dog-walkers’ leisure time walking decreased over time, while nondog-walkers’ leisure time walking increased (*p* = .001, es = −0.74). At 10 years, dog walkers’ and nonwalkers’ leisure time walking did not differ significantly (point estimate = 4.374, 95% confidence interval [CI] 3.770 to 4.979, point estimate = 4.496, 95% CI 3.319 to 5.619, *p* = .886).
+
+## Discussion
+
+The results of this study indicate that pet ownership is associated with better-preserved physical function in community-dwelling older adults. This work provides an important extension of previous cross-sectional evidence discussed in the introduction by providing a longitudinal perspective on pet ownership and physical function. It establishes clear evidence that pet ownership is associated with slower deterioration over time in physical functioning among older adults. Of note, the study includes longitudinal objective measures of physical function as well as the more typical questionnaire-based assessments (Koohsari et al., 2020). In the current study, pet ownership was related to less deterioration in longitudinal objective measures of physical function, but not to changes in leisure time physical activity assessed with questionnaires.
+
+The study did not produce evidence that dog ownership or cat ownership was superior in relationship to maintenance of physical function in older adults. This suggests that aspects other than the physical activity associated with dog ownership are associated with the maintenance of physical function. We can speculate that the need to move about and care for any animal could be a helpful impetus for physical activity that promotes function. Similarly, dog walking was not associated with different trajectories of change in objective measures of physical function. A possible explanation is that dog walking might not add to total walking; the same people who walk with the dog might also perform equal activities to maintain physical function with or without it. This conjecture is supported by the finding that by the end of the study, when fewer individuals owned pets, total walking did not differ significantly between those who walked and did not walk their dogs. Of note, the comparison of dog walkers with dog owners who do not walk their dogs lacks power due to small sample sizes and requires further replication.
+
+In the current study, we controlled for age to address the staggered entry of individuals into the study because older adults’ physical function does decline over time, and it declines faster with age. It is important that the results of this study not be generalized to a younger population, where the physical function may not deteriorate, and the relationship of pet ownership to health may be different. In a population-based study of Australian adolescents, neither pet ownership nor time spent caring for the pet were related to health or well-being (Mathers et al., 2010). It also is possible that time spent with a pet is a factor in how pet ownership is related to health. In the aforementioned study, despite having high rates of pet ownership, adolescents spent little time with their pets (Mathers et al., 2010). Older adults may spend more time with their pets than younger adults or adolescents who spend more time in other leisure time physical activities.
+
+In the current study, we also controlled for comorbidities. The comorbidity index included minor difficulties in physical function. Including this variable may over-adjust and thus obscure some pet ownership or dog walking-related differences in changes in physical function over time.
+
+The findings in this study confirm the importance of using a longitudinal approach to examine of the relationship of exposure and outcomes as well as controlling for confounders. In bivariate cross-sectional analysis, all outcomes except recommended physical activity and high-intensity exercise minutes were significantly related to pet ownership. In contrast, in the longitudinal analyses that controlled for age and comorbidities, changes in all physical function variables were related to pet ownership, but changes in leisure time physical activity were not. This finding suggests that leisure time physical activity would not change whether people kept pets or did not.
+
+In the current study, dog ownership and cat ownership both made independent associations to longitudinal changes in physical function in the same direction but not to leisure time physical activity. The results of this study indicate that dog ownership and cat ownership are related to less deterioration in physical functioning as older adults age. The finding that dog ownership was not related to higher intensity leisure time physical activity is consistent with evidence that that owning a dog was not associated with medium-to-high-intensity physical activities (Koohsari et al., 2020). This implies that the type of person who owns a dog would do more physical activity even if they did not have a pet. No previous studies of which we are aware evaluate the relationship of cat ownership to leisure time physical activity.
+
+Any benefit of dog ownership for older adults would be particularly important for individuals who are socially isolated (Carr et al., 2021). A recent population-based cross-sectional study of 9,856 community-dwelling older adults in Japan found that socially isolated older adults who never owned a dog were more likely to report lower psychological health than current or past socially isolated dog owners (Ikeuchi et al., 2021). Dog ownership may increase opportunities for socially isolated older adults to engage in physical as well as social activities, potentially improving both function and psychosocial health in this population. It is important to recognize that individuals with more impaired ADLs who live alone will have difficulty caring for a pet, especially a dog, without outside assistance. The current study addressed a healthy population, and thus these findings cannot be generalized to a more impaired group.
+
+Of course, it is also possible that individuals who experienced decreases in physical function rehomed their pets, and this led to the apparent relationship of not owning a pet to deterioration in physical function. The data are less supportive of this idea because the association of pet ownership with the maintenance of physical function was present for both cat and dog owners and was not attributable to dog walking.
+
+Several previous cross-sectional studies associated dog ownership, and dog walking in particular, with more physical activity or better physical function (Christian et al., 2013; Coleman et al., 2008; Dall et al., 2017; Taniguchi et al., 2019; Thorpe et al., 2006). They differ from the current study that focuses on the trajectory of change, rather than the relative amount of, physical activity. In fact, the graphs suggest that there are differences in some measures of physical activity at specific time points. For example, Figure 3 demonstrates that at index visit, dog walkers spent more time walking than did dog owners who did not walk their dogs. However, this difference disappeared as the participants aged.
+
+*Figure: Figure 3. Changes in dog owners’ weekly time spent walking according to dog walking status with 10 years of aging, estimates based on linear mixed models that include age and comorbidities as covariates (*n* = 73).*
+
+Use of linear mixed models with random intercepts enhances the interpretation of differences in changes in outcomes according to pet ownership-related characteristics. We treat pet ownership as a time-variant covariate. Participants only count as pet owners during the assessment occasions when they had a pet. This minimizes the impact of participants rehoming their pets on the results of the analysis. They are considered as nonowners in the next assessment. Furthermore, the random intercept models used for analysis, provide a unique baseline for each person which is used to examine trajectories of change independent of the starting point. This minimizes the effects of participants’ overall activity levels on changes in physical function.
+
+### Limitations
+
+Using the BLSA participants for this study had the advantage of a large well-described group of older adults with an extensive longitudinal objective evaluation of physical function. However, this sample is more affluent, White, and educated that the general aging population. The findings in this sample should be replicated in a more representative sample. This study examines dog relationships and dog related activities at one point in time, the survey visit. Data about dog walking may not accurately reflect the entire period over which physical function was assessed. Furthermore, attachment to the pet and the amount of time spent dog walking were not included in the analyses. These variables may have an impact on the magnitude of the physical benefits from pet ownership. The BLSA data are collected over time, allowing for longitudinal examination of change in physical function. Additional longitudinal assessment of attachment to pets and dog walking will permit future evaluation of their relationships with changes in physical function. Self-selection bias is a factor in this study because individuals are not randomly assigned to pet ownership groups. This limits causal inferences.
+
+## Conclusion
+
+This study provided longitudinal evidence suggesting that pet ownership moderates age-related declines in physical functional status later in life. Pet ownership was associated with less deterioration with aging over a mean of 7.5 years in five of the six measures of physical function, including the Health ABC Physical Function Battery, in community-dwelling older adults. There was no evidence that the association of dog and cat ownership with physical function differed or that dog walking was related to changes in physical function with aging. There also no evidence that pet ownership moderated age-related declines in leisure time physical activity in this population.

fulltext/md/PMC10015816.md

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+---
+pmcid: "PMC10015816"
+pmid: "36922800"
+doi: "10.1186/s12917-023-03575-7"
+title: "Canine distemper virus N protein induces autophagy to facilitate viral replication."
+journal: "BMC veterinary research"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Canine distemper virus N protein induces autophagy to facilitate viral replication
+
+## Abstract
+
+### Background
+
+Canine distemper virus (CDV) is one of the most contagious and lethal viruses known to the *Canidae*, with a very broad and expanding host range. Autophagy serves as a fundamental stabilizing response against pathogens, but some viruses have been able to evade or exploit it for their replication. However, the effect of autophagy mechanisms on CDV infection is still unclear.
+
+### Results
+
+In the present study, autophagy was induced in CDV-infected Vero cells as demonstrated by elevated LC3-II levels and aggregation of green fluorescent protein (GFP)-LC3 spots. Furthermore, CDV promoted the complete autophagic process, which could be determined by the degradation of p62, co-localization of LC3 with lysosomes, GFP degradation, and accumulation of LC3-II and p62 due to the lysosomal protease inhibitor E64d. In addition, the use of Rapamycin to promote autophagy promoted CDV replication, and the inhibition of autophagy by Wortmannin, Chloroquine and siRNA-ATG5 inhibited CDV replication, revealing that CDV-induced autophagy facilitated virus replication. We also found that UV-inactivated CDV still induced autophagy, and that nucleocapsid (N) protein was able to induce complete autophagy in an mTOR-dependent manner.
+
+### Conclusions
+
+This study for the first time revealed that CDV N protein induced complete autophagy to facilitate viral replication.
+
+### Supplementary Information
+
+The online version contains supplementary material available at 10.1186/s12917-023-03575-7.
+
+## Background
+
+Canine distemper virus (CDV) can infect *Canidae* causing canine distemper (CD), one of the most contagious diseases in the *Canidae*, which can result in high morbidity and mortality in unimmunized host [1]. CDV has multicellular tropism (epithelial cells, lymphocytes, and nerve cells) and can cause systemic infections. [2]. The clinical symptoms of CD characterized by fever, increased ocular and nasal discharge, conjunctivitis, diarrhea, vomiting, rash, lymphopenia, and neurologic symptoms such as encephalitis, with severe immunosuppression, but also lifelong immunity in surviving hosts [3–6]. CDV has an expanding host range, mainly comprising mammals from at least 6 orders and more than 20 families [4, 7], especially all families of *Carnivora* [8, 9]. It poses a great threat to the pet industry, fur industry, and conservation of endangered species worldwide [10], especially the recent large outbreaks of CD in primate populations of Macaca mulatta and Macaca fascicularis, which have raised concerns about possible zoonotic diseases and public health concerns after measles eradication [11, 12]. CDV is a single-stranded, negative-stranded, non-segmented RNA virus, which belongs to the *Morbillivirus* genus of the *Paramyxoviridae* family [13, 14]. The viral particles have a double-layered envelope with dense spikes. The full-length genome is 15.69 kb and contains 6 transcription units (N-P-M-F–H-L), and 8 proteins are encoded, of which the P gene encoding two non-structural proteins C and V [15].
+
+Autophagy is an evolutionarily conserved catabolic process that begins with the isolation of "cargo" (unwanted proteins, damaged organelles, invading microorganisms, etc.) to be degraded into autophagosome with a double membrane, which then fuse with lysosome to form autophagolysosome, in which hydrolytic enzymes degrade the luminal contents, and metabolic byproducts can be reused to maintain the dynamic balance of the intracellular environment [16, 17]. Autophagy (programmed cell death type II) also occurs at basal levels in normal cells as a cytoprotective mechanism [18, 19]. Autophagy can also be up- or down-regulated in response to various stress stimuli both inside and outside the cell, such as nutrient deficiency, oxidative stress, endoplasmic reticulum stress, pathogen infection, and pathogen-associated molecular patterns [20, 21]. In a process known as “xenophagy”, the isolation of viral particles within the autophagosome may lead to their degradation and destruction and/or delivery to compartments involved in pathogen recognition and antigen presentation to activate immune responses against the virus [22]. On the other hand, viruses have evolved strategies to evade or hijack autophagy to facilitate their own propagation, and viruses can use autophagosomal membranes as replication platforms or interact with autophagy-related-gene (ATG) proteins to aid in the assembly, maturation, and encapsulation of viral particles [23–25]. Measles virus (MeV) [26], peste des petits ruminants virus (PPRV) [27], newcastle disease virus (NDV), hepatitis C virus (HCV) [28], foot-and-mouth disease virus (FMDV) [29, 30], and Influenza A virus [31] use the autophagic process for replication.
+
+The mammalian target of rapamycin (mTOR) signaling pathway is a key pathway regulating cell growth, proliferation and survival [32]. mTOR is a serine/threonine kinase belonging to the phosphatidylinositol 3-kinase (PI3K)-related protein kinase family, and the PI3K/protein kinase B (PKB, generally known as AKT) pathway is the main upstream regulator of mTORC1, whose activation inhibits autophagy [32, 33]. The relationship between other members of the *Paramyxoviridae* family and autophagy has been studied. Previous study found that the number of stained cells and the intensity of the immunoreaction for autophagy-associated microtubule-associated protein 1 light chain 3 (LC3) were significantly elevated in the cerebellums of CDV-infected dogs, which indicated that autophagy increased in virus-infected cells [34]. Another study showed that CDV induced virus-cell or cell–cell fusion triggering autophagy which in turn enhanced cell-to-cell transmission of the virus [35]. Both studies suggest that CDV infection induces the onset of autophagy, but the impact of the autophagy machinery on CDV infection remains unclear. In this study, the relationship between CDV and autophagy was systematically investigated for the first time. The data provided the first evidence that CDV induced complete autophagic flux to promote viral replication. For the first time, it was revealed that nucleocapsid protein induced complete autophagy in an mTOR-dependent manner and played an important role in CDV-induced autophagy to facilitate viral replication.
+
+## Results
+
+### Infection of CDV triggers autophagy in vero cells
+
+The CDV3-CL strain can infect Vero cells and passage on Vero cells, resulting in syncytial lesions (Fig. 1A). The microtubule-associated protein 1 light chain 3β (MAP1LC3B/LC3B) was known to bind to mature autophagosome membranes and was a marker of autophagosome formation [36, 37]. The results showed that the amount of LC3-II increased with the progression of CDV infection (Fig. 1B, C), and the level of LC3-II were found to increase starting at 20 hpi, consistent with the level of N protein (Fig. 1C, E). Because the amount of LC3-II was closely related to the number of autophagosomes, the ratio of LC3-II to β-tubulin in cells was now considered to be an accurate indicator of autophagic activity [38]. The gray scale ratio of LC3-II to β-tubulin bands was higher in CDV-infected Vero cells than that in mock-infected cells from 20 hpi onwards (Fig. 1D). Furthermore, we next performed immunofluorescence assays. Consistent with the results with immunoblotting, a large amount of punctate GFP-LC3 protein was observed in or near the syncytium induced by viral infection, and similarly, in Rapamycin-treated positive control cells. In contrast, mock-infected Vero cells exhibited a weak diffuse staining pattern with almost no LC3 punctate accumulation (Fig. 1F). These results suggested that CDV infection induced the onset of autophagy in Vero cells.Fig. 1CDV infection triggers autophagy in vero cells. **A** The cytopathic effect (CPE) in CDV-infected Vero cell at 28 hpi (magnification, × 4). **B** and **C** Vero cells were mock-infected or infected with CDV (MOI = 1) for 2, 4, 8, 12, 16, 20, 24, 28, 32, and 36 h. The expression levels of LC3B, CDV-N, and β-tubulin (loading control) were analyzed by western blot with specific antibodies. **D** The LC3-II levels relative to the β-tubulin levels were determined by densitometry. **E** The CDV-N levels relative to the β-tubulin levels were determined by densitometry. **F** Vero cells were transfected with GFP-LC3 for 28 h. The cells were then fixed and processed for indirect immunofluorescence using antibodies against CDV-N protein, followed by the corresponding secondary antibodies. The fluorescence signals were visualized by confocal immunofluorescence microscopy. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ****P* < 0.001
+
+### CDV infection enhances autophagic flux
+
+To investigate whether CDV-induced autophagy is a complete process, we determined the degradation of polyubiquitin-binding protein p62/SQSTM1 (sequestosome 1), a marker of autophagy-mediated protein degradation pathway, which is considered as a marker of autophagic flux [37]. As shown in the Fig. 2A, the level of p62 protein did not change significantly during the mid to late phase of infection (from 24 to 32 hpi) when compared to mock cells, but decreased significantly at late stage of the infection (from 36 to 40 hpi), and similarly, the gray scale ratio of p62 protein to β-tubulin bands in CDV-infected Vero cells was much lower than that in negative control cells (Fig. 2B). E64d is a membrane-permeable inhibitor of histones B, H and L. The upregulation of LC3-II and p62 in the presence of the lysosomal protease inhibitor represents an increase in autophagic flux. Both LC3-II and p62 degradation were significantly inhibited in CDV-infected cells treated E64d. The same results were also obtained from E64d and Rapamycin-treated Vero cells (Fig. 2C-F).Fig. 2CDV infection enhances autophagic flux. **A** Vero cells were mock-infected or infected with CDV (MOI = 1) for 24, 28, 32, 36 h and 40 h. The expression levels of p62/SQSTM1, CDV-N, and β-tubulin (loading control) were analyzed by western blot with specific antibodies. **B** The p62 levels relative to the β-tubulin levels were determined by densitometry. **C** Vero cells pre-treated with rapamycin for 4 h and then mock-infected with DMEM, and cells infected with CDV (MOI = 1) were further cultured in the absence and presence of 10 mg/mL E64d for 24 and 28 h. The cell samples were then analyzed by western blot with anti-CDV-N, anti-LC3B, anti-p62, and anti-β-tubulin (loading control) antibodies. **D**, **E** and **F** The CDV-N, LC3B and p62 levels relative to the β-tubulin levels were determined by densitometry in mock-infected, rapamycin-pre-treated, and CDV-infected Vero cells in the absence and presence of E64d. **G** Cells were transfected with pmCherry-GFP-LC3B for 24 h, followed by CDV infection (MOI = 1) and treatment with E64d. The fluorescence signals were visualized by confocal immunofluorescence microscopy. **H** Vero cells were mock-infected or infected with CDV (MOI = 1) for 28 h. The cells were fixed and processed for indirect immunofluorescence using antibodies against LC3 and LAMP1 protein. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; # *P* > 0.05; ***P* < 0.01; ****P* < 0.001
+
+To further confirm the above results, Vero cells were transfected with the dual fluorescent-tagged plasmid mCherry-GFP-LC3B. The green signaling protein (GFP) is degraded and quenched under low pH conditions in the lumen of the lysosome, whereas the red signaling protein (mCherry) exhibits more stable fluorescence under acidic conditions [37, 39]. As shown in Figs. 2G and S1A, almost all of the green and red fluorescent puncta colocalized in the CDV-infected Vero cells at 24 hpi. In contrast, the number and intensity of the red fluorescent dots are greater than the green fluorescent point at 32 hpi (Figs. 2G, S1B). Subsequently, E64d treatment dramatically recovered green fluorescent puncta and increased yellow puncta in CDV-infected Vero cells (Figs. 2G and S1C). These results showed that CDV infection could promote GFP to be degraded by autolysosome. Lysosome-associated membrane protein 1 (LAMP1), a lysosomal marker, co-localizes with LC3 during autophagosome maturation [40]. There was a punctiform distribution of LC3 with LAMP1 in CDV-infected cells. More importantly, partial overlap of LC3 with LAMP1 was observed, and the same phenomenon was observed in Rapamycin-treated positive control cells. In contrast, both LC3 and LAMP1 exhibited weaker diffuse staining in negative control cells (Fig. 2H). In summary, CDV infection increased autophagosome formation, promoted the fusion of autophagosome with lysosome to form autophagosome, enhanced autophagic degradation, and enhanced autophagic flux.
+
+### Induction of autophagy with rapamycin promotes virus replication
+
+Treatment of cells with the autophagy inducer rapamycin did induce autophagy, which not only upregulated the expression of LC3-II in CDV-infected Vero cells (Fig. 3A, B), but also decreased the expression of p62 (Fig. 3A, C). Importantly, rapamycin treatment increased the level of CDV N protein in a non-dose dependent manner (Fig. 3A, D). In addition, the effect of rapamycin treatment was also found to significantly increase viral RNA copy numbers and viral titer (Fig. 3E, F). These findings suggested that the autophagy contributed to CDV replication.Fig. 3Induction of autophagy with rapamycin promotes virus replication. **A** Vero cells were pre-treated with rapamycin for 4 h and then infected with CDV (MOI = 1) for 26 and 30 h. The cell samples were then analyzed by western blot with anti-CDV-N, anti-LC3B, anti-p62, and anti-β-tubulin (loading control) antibodies. **B**, **C** and **D** The CDV-N, LC3B and p62 levels relative to the β-tubulin levels were determined by densitometry. **E** and **F** Vero cells were pre-treated and infected as described in (**A**). At 26 and 30 hpi, copy numbers of CDV were measured by qRT-PCR; virus titers were measured by TCID~50~. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; # *P* > 0.05; **P* < 0.05; ***P* < 0.01; ****P* < 0.001
+
+### Inhibition of autophagy reduces CDV replication
+
+To further confirm the effect of autophagy on CDV replication, we treated Vero cells with chloroquine (CQ) and wortmannin (WM) and examined CDV replication. CQ can inhibit endosomal acidification, causing accumulation of autophagosomes and autophagolysosomal contents, and inhibits autophagy at a late stage [41]. Wortmannin, a specific inhibitor of the PI3K pathway, blocks autophagy at an early stage [42]. In CDV-infected Vero cells, chloroquine treatment increased the accumulation of LC3-II and p62 (Fig. 4A, B), and Wortmannin treatment decreased LC3-II expression and p62 degradation (Fig. 4E, F). Importantly, The CDV-N expression levels were reduced after treatment of cells with CQ and WM (Fig. 4A, B, E, F). Similarly, Chloroquine and Wortmannin treatment resulted in a significant reduction in viral RNA levels and titers (Fig. 4C, D, G, H). In addition, E64d treatment inhibited autophagic degradation, resulting in reduction of CDV N protein expression levels (Fig. 2C, D). These data suggested that pharmacological inhibition of autophagy negatively regulated CDV replication.Fig. 4Autophagy inhibition reduces CDV replication. **A** and **E** Vero cells were pre-treated with Chloroquine and Wortmannin for 4 h and then infected with CDV (MOI = 1) for 26 and 30 h. The cell samples were then analyzed by western blot with anti-CDV-N, anti-LC3B, anti-p62, and anti-β-tubulin (loading control) antibodies. **B** and **F** Vero cells were pre-treated and infected as described in (**A** and **E**). At 26 and 30 hpi, the target protein levels relative to the β-tubulin levels were determined by densitometry in control, chloroquine- and wortmannin-pre-treated cells. **C-D** and **G-H** Vero cells were pre-treated and infected as described in (**A** and **E**). At 26 and 30 hpi, copy numbers of CDV were measured by qRT-PCR; virus titers were measured by TCID~50~. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; # *P* > 0.05; **P* < 0.05; ***P* < 0.01; ****P* < 0.001
+
+We next examined the effect of target-specific RNA interference with reduced expression of the endogenous autophagy-associated protein ATG5 on CDV replication. The ATG5 protein, with ATG12 and ATG16L1, forms a conjugation system that is essential for the phagophore elongation process. Knockdown of ATG5 can block the autophagy process [43]. As shown in the Fig. 5A, C and D, Vero cells transfected with ATG5-specific small interfering RNA (si-ATG5) exhibited significantly lower levels of endogenous ATG5 and LC3-II protein compared to cells transfected with scrambled siRNA, indicating that the expression of the target protein was successfully suppressed in Vero cells. There was no significant difference in the effect of syncytial lesions caused by CDV infection of the two cell types (Fig. 5B). Importantly, ATG5 knockdown significantly inhibited CDV-induced autophagy (Fig. 5C, E). These results suggested that CDV infection can induce autophagy in Vero cells through an ATG5-dependent pathway. More importantly, inhibition of autophagy strongly reduced CDV N protein expression (Fig. 5C, E), viral RNA copy number and viral titer (Fig. 5F, G). These data further revealed that inhibition of autophagy at the genetic level is detrimental to CDV replication.Fig. 5Inhibition of autophagy with specific siRNA targeting ATG5 reduces CDV replication. **A** and **C** Vero cells were transfected with siRNA-ATG5 or scrambled siRNA for 48 h; then, the cells were infected with CDV (MOI = 1). The cell samples were then analyzed by western blot with anti-ATG5, anti-CDV-N, anti-LC3B, and anti-β-tubulin (loading control) antibodies. **B** Cell lesions in CDV-infected Vero and siRNA-ATG5-Vero cell at 32 hpi (magnification, × 4). **D**-**E** Vero cells were pre-treated and infected as described in (**C**). At 24, 28 and 32 hpi, the target protein levels relative to the β-tubulin levels were determined by densitometry. **F**-**G** Vero cells were pre-treated and infected as described in (**C**). At 24, 28 and 32 hpi, copy numbers of CDV were measured by qRT-PCR; virus titers were measured by TCID~50~. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; **P* < 0.05; ***P* < 0.01; ****P* < 0.001
+
+### CDV N proteins induce autophagy in an mTOR-dependent manner
+
+We inactivated CDV by ultraviolet (UV) irradiation [28]. Infectious CDV could infect cells producing significant syncytial lesions and viral titers, whereas the cells treated with UV-inactivated CDV after 72 hpi produced none (Fig. 6A, B). These data suggested that UV-inactivated CDV had lost its ability to infect and replicate. More importantly, both infectious CDV and UV-inactivated CDV inoculated Vero cells increased the expression level of LC3-II compared to mock-infected cells (Fig. 6C, D). This indicated that UV-inactivated CDV can also induce the onset of autophagy. Therefore, structural proteins of CDV may play an important role in the process of virus-induced autophagy.Fig. 6N Proteins Induce Autophagy in an mTOR-Dependent Manner. **A** The CPE in mock-, UV-CDV-, CDV-infected Vero cell at 72 hpi (magnification, × 4). **B** Vero cells were infected as described in (**A**). virus titers were measured by TCID~50~. **C** Vero cells were infected as described in (**A**). The cell samples were then analyzed by western blot with anti-CDV-N, anti-LC3B, and anti-β-tubulin (loading control) antibodies. **D** The LC3B levels relative to the β-tubulin levels were determined by densitometry. **E** Vero cells were transfected with p3 × Flag-CMV and p3 × Flag-CMV-N plasmids expressing the fusion protein for 48 h. The cell samples were then analyzed by western blot with anti-Flag antibody. **F** Vero cells were infected as described in (**E**). The cell samples were then analyzed by western blot with anti-p-mTOR, anti-mTOR, anti-p-AKT, anti-AKT, anti-p62, anti-LC3B and anti-β-tubulin (loading control) antibodies. **G** Vero cells were infected as described in (**E**). The target protein levels relative to the β-tubulin levels were determined by densitometry. The blots were cropped. The samples derived from the same experiment and that blots were processed in parallel. The data represent the mean ± SD of three independent experiments. Two-way ANOVA; ***P* < 0.01; ****P* < 0.001
+
+To analyze the effect of different viral proteins of CDV on autophagy induction. We constructed expression vectors with flag-tagged proteins and transfected them with Vero cells. Western blot assays revealed that Flag-N, Flag-P, Flag-V, Flag-C proteins were expressed in Vero cells (Figs. 6E, S2A). When we transfected the same concentration of plasmids, Flag-N caused the LC3-II level to increase the most significantly (Fig. S2B). Additionally, Flag-N promoted the degradation of p62 protein (Fig. 6F, G). This suggested that CDV N protein induces complete autophagic flux. The mammalian target of rapamycin (mTOR) is an important signaling molecule that negatively regulates autophagy and is also a downstream target of phosphatidylinositol 3 kinase (PI3K) and AKT pathways [44]. The expression of phosphorylated AKT (Ser 473) and phosphorylated mTOR (Ser 2448) was reduced in Vero cells expressing Flag-N protein compared to controls, but there was no significant change in total AKT or mTOR expression (Fig. 6F, G). These data suggested that CDV N protein induced autophagy by inhibiting the AKT-mTOR pathway, indicating that N protein play an important role in CDV-induced autophagy.
+
+## Discussion
+
+Autophagy relies on lysosomal degradation and plays an important role in maintaining intracellular homeostasis by recycling long-lived proteins and obsolete or damaged organelles during nutrient deficiency, energy recycling, and cell survival. Subsequently, it has been shown that autophagy can also play an important role in defense against pathogens as an immune mechanism after pathogen invasion of cells [45, 46]. Although autophagy can transport viruses to the lysosomal compartment for degradation [47], it has been shown that sindbis virus, tobacco mosaic virus and vesicular stomatitis virus infection-induced autophagy exerts a protective function by limiting intracellular virus replication [48–50]. However, an increasing number of pathogens have evolved strategies to avoid or hijack autophagy for their own benefit. Members of the *Paramyxoviridae* family have been shown to induce autophagy in target cells, including MeV [26], PPRV [27], NDV [51], sendai virus (SeV) [52], human parainfluenza virus type 3 (HPIV3) [53], human respiratory syncytial virus (HRSV) [54], and simian parainfluenza virus 5 (SV5) [55]. In this study, the date suggested that CDV infection of Vero cells led to increased autophagosome formation and induced autophagy. Consistent with the results of our study, the number of GFP-LC3 spots increased and autophagosome accumulation increased after infection of GFP-LC3-transfected VerodogSLAMtag cells by CDV wild-type strain 5804P and MeV vaccine strain MeVvac [35]. Application of immunohistochemistry to detect the expression of microtubule-associated protein 1 light chain 3 (LC3) protein showed increased autophagy in CDV-infected canine cerebellums [34].
+
+Autophagy is a dynamic process and the accumulation of LC3 dot may be the result of increased autophagosome formation or decreased autophagosome degradation [36]. Many date have recently shown that viruses can induce the early stages of autophagy but prevent the fusion of autophagosomes with lysosomes, such as coxsackievirus B3 (CVB3) [56], herpes simplex virus [57], influenza A virus [58], porcine reproductive and respiratory syndrome virus [59] and hepatitis C virus [60]. MeV [26], PPRV [27], NDV [51], SeV [51], and RSV [54], members of the *Paramyxoviridae* family, induce increased autophagic flux, whereas HPIV3 induces incomplete autophagy by blocking autophagosome-lysosome fusion [53]. In this study, we found that CDV infection induced complete autophagy and increased autophagic flux. The previous study showed that CDV or MeV infection of VerodogSLAMtag cells induced the formation of autophagosome, but prevented their subsequent fusion with lysosomes [35]. Another study showed no change in the intensity of LC3 expression in cerebellar tissue during the chronic phase of CDV infection as in the acute phase, suggesting that the virus delayed or prevented the degradation of autophagosome [34].
+
+Several members of the *Paramyxoviridae* family, including MeV [26], HRSV [54], PPRV [26], NDV [51], and HPIV3 [53], can promote replication using an autophagic mechanism. Kabak et al. suggested that CDV does not use autophagy for replication and supported the suggestion by Delpeut et al. that CDV induces autophagy through virus-cell and cell–cell fusion and that autophagy enhances cell-to-cell transmission of morbilliviruses rather than replication [34, 35]. Our data showed that both pharmacological and genetic promotion and inhibition of autophagy were important for CDV replication in Vero cells. The reason for the difference between the results of the above study and our study may be the difference in the CDV strains used and the infected cells. This also suggests that the autophagic response is virus and cell type specific and even related to cellular pathogen receptor. It has been shown then that infection of different cells with the same rabies virus strain induces different autophagic flux responses [61].
+
+mTOR signaling is essential for many cellular processes such as growth, survival, and proliferation [32]. As a major autophagy regulator, mTOR negatively regulates autophagy when activated by signals from nutrients, growth factors, and stress stimuli [62]. Among them, the PI3K/Akt/mTOR signaling pathway plays a key role in autophagy [33]. PPRV [27], CVB3 [63], FMDV [29], Avian influenza H5N1 virus and avian reovirus trigger autophagy by inhibiting AKT-mTOR signaling [64, 65]. Our data suggested that CDV N protein induced autophagy by inhibiting the AKT-mTOR pathway, suggesting that N protein plays an important role in CDV-induced autophagy for viral replication. The nucleoprotein of MeV and the cytoplasmic nucleocapsid of CDV interact with 70 K heat shock proteins (HSP70K) [66, 67]. PPRV-N interacts with HSPA1A to play an important role in PPRV-induced second autophagic wave [27]. Which key nodal proteins interact with CDV proteins to affect autophagy deserve further investigation.
+
+## Conclusions
+
+This study is the first systematic investigation of the relationship between autophagy and CDV replication. For the first time, it was demonstrated that CDV induces complete autophagic flux in Vero cells to promote viral replication. It was revealed for the first time that nucleocapsid protein induces complete autophagy in an mTOR-dependent manner, which plays an important role in CDV-induced autophagy to facilitate viral replication. Given that autophagy facilitates CDV replication, our study reveals a novel approach to improve the efficiency of live attenuated CDV vaccine production that may enhance its immune effect in host cells by targeting the autophagic pathway. Our study also provides novel mechanisms of CDV-cell interactions and may provide theoretical references for the development of strategies against CDV infection and CDV-host cell interactions.
+
+## Materials and methods
+
+### Antibodies, reagents and plasmids
+
+The primary antibodies used in this study included antibodies against rabbit LC3B (Abcam, UK); SQSTM1/p62, Phospho-mTOR (Ser2448), Phospho-Akt (Ser473), AKT (all from Cell Signaling Technology, USA); LAMP1 (Proteintech, USA), ATG5 (Novus Biologicals, USA); mouse mTOR (Proteintech, USA); β-Tubulin (Affinity, USA); CDV-NP (VMRD, USA). Secondary antibodies were goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP antibody (Bioworld, USA); Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, USA); Alexa Fluor 594 goat anti-rabbit IgG (Abmart, China). Pharmaceuticals were Rapamycin, Wortmannin, E64d (Selleck, USA); Chloroquine (Sigma-Aldrich, USA). Plasmids pEGFP-LC3B (Cat.: P0199) and pmCherry-EGFP-LC3B (Cat.: P0446) was obtained from miaolingbio (Wuhan, China). Most of the primary antibodies used in the study are against human, and the similarities between the target proteins from the two species (human and monkey) are high.
+
+### Cell culture and virus propagation
+
+African green monkey kidney epithelial cell line (Vero) was obtained from ATCC. Cells were grown in DMEM medium (JSBio, China) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin (BI, ISR) at 37 °C with 5% CO~2~ in a humidified atmosphere. The CDV3-CL vaccine strain was obtained from the Institute of Special Animal and Plant Sciences, Chinese Academy of Agricultural Sciences (Changchun, China). The CDV3-CL strain were passaged in Vero cells. The viral titer was determined by the Reed-Muench method in 96-well plates and expressed as a 50% tissue culture infectious dose (TCID~50~) [68].
+
+### Virus infection and pharmaceutical treatment
+
+Vero cells were not treated or treated with 200 nM, 500 nM, 1000 nM Rapamycin, 5 μM Chloroquine, 200 nM (final concentration) Wortmannin for 4 h, followed by CDV infection (multiplicity of infection, MOI = 1). Cells were covered with DMEM medium containing 2% FBS and certain concentration of the above inducer or inhibitor and cultured at 37 °C with 5% CO2.
+
+### Plasmid construction
+
+CDV N gene was amplified from the CDV3-CL genomic cDNA used in this study and cloned into the p3 × Flag-CMV vectors according to the manufacturer's instructions of the ClonExpress MultiS Cloning Kit (Vazyme, China).
+
+### Plasmid transfection
+
+Vero cells were cultured in Petri dishes until 70% fusion. 2 μg of plasmid was diluted in 200 μL of JetPRIME buffer, then 4 μL of JetPRIME transfection reagent was added, mixed well and incubated at room temperature for 10 min. The mixture was added to each dish and mixed well. If transfection is performed in laser confocal-specific Petri dishes, the plasmids and reagents are halved.
+
+### Transfection and gene silencing with siRNAs
+
+siRNAs targeting the Vero cell ATG5 were designed using the online design tool Block-iTRNAi Designer (Invitrogen, USA). siRNAs were synthesized by GENEWIZ Biotechnology (Suzhou, China). Vero cells were transfected with 100 pmol siRNA using JetPRIME transfection reagent (Ployplus, FR) at a 35 mm dish according to the manufacturer's instructions. Scrambled siRNA was used as a negative control. Silencing efficiency was measured by Western blot analysis. The sequence of the siRNA targeting ATG5 used in this study: GATTCATGGAATTGAGCCA (5’-3’).
+
+### Immunoblotting
+
+Cells were collected and lysed with RIPA strong lysis buffer (Beyotime) containing the protease inhibitor PMSF (Beyotime, China), and 5 × loading buffer (BOSTER, China) was added, and the mixture was sonicated on ice for 15 s. Denaturation was performed at 100 °C for 10 min. Proteins were separated on an electrophoretic sodium dodecyl sulfate–polyacrylamide gel and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked for 1 h or more with 5% skim milk (BBI, China) and cut according to the size of the target protein before hybridization with the antibody. The membranes were incubated with primary antibody overnight at 4 °C, then incubated with peroxidase-coupled secondary antibodies. Protein bands were detected using chemiluminescence image analysis system (Amersham Imager 600, GE, USA), with an ECL kit (Advansta, USA). The intensity of the target protein blots was analyzed using ImageJ software (NIH, USA). The relative protein level was normalized to β-tubulin. The dilution ratios of antibodies were shown in Table 1.Table 1The dilution of all the antibodies used for the immunoblottingAntibodiesDilutionLC3B1∶2000p621∶1000p-mTOR1∶1000mTOR1∶2000p-AKT1∶1000AKT1∶1000ATG51∶2000CDV-N1∶5000β-Tubulin1∶5000goat anti-mouse IgG-HRP1∶8000goat anti-rabbit IgG-HRP1∶8000
+
+### Immunofluorescence and confocal microscopy
+
+Cells were grown in glass-bottom culture dishes (NEST, China). The CDV-infected at 1 MOI or plasmids-transfected or pharmaceutical-treated cells fixed with 4% paraformaldehyde (Beyotime, China) for 15 min. and then treated with 0.2% Triton X-100 (Solarbio, China) for 15 min and blocked with 5% bovine serum albumin (MP, USA) for 1 h. Cells are then incubated with primary antibody overnight at 4 °C or 2 h at 37 °C, followed by incubation with the appropriate fluorescent dye-conjugated secondary antibody for 1 h at 37 °C under dark conditions. Cells were then stained with DAPI (Beyotime, China) for 5 min. Samples were then imaged using a confocal fluorescence microscope (Objective magnification, × 60) (Leica SP8, Germany). The dilution ratios of antibodies were shown in Table 2.Table 2The dilution of all the antibodies used for immunofluorescenceAntibodiesDilutionLC3B1∶300CDV-N1∶500LAMP11∶300Alexa Fluor 488 goat anti-rabbit IgG1∶500Alexa Fluor 594 goat anti-mouse IgG1∶500Alexa Fluor 594 goat anti-rabbit IgG1∶300
+
+### Quantitative real-time PCR
+
+Vero cells were treated with pharmaceuticals or siRNA, and infected with CDV (MOI = 1). Total RNA was extracted using the Viral RNA/DNA Extraction Kit (TaKaRa, China) according to the manufacturer's protocol. cDNA was synthesized by reverse transcription using PrimeScript RT Master Mix reagent (TaKaRa, China). Luna Universal qPCR Master Mix (New England Biolabs, USA) was used to perform quantitative real-time PCR. For CDV-specific detection, primer pairs targeting the region corresponding to the N gene were used, and a recombinant plasmid containing the CDV N gene was used to construct a standard curve for calculating the viral RNA copy number in different samples. qPCR was performed with the primers shown below:CDV N Forward: 5′-GAGAATTAACAACTATTGAATC-3′CDV N Reverse: 5′-CATAGCATAACTCCAGAG-3′
+
+### Statistical analysis
+
+The Data are expressed as mean ± standard deviation (SD). The significance of the variability between treatment groups was analyzed by two-way analysis of variance (ANOVA) test by GraphPad Prism software (version 8.3, USA). Differences were considered statistically significant at *P* < 0.05.
+
+## Supplementary Information
+
+**Additional file 1:**
+**Figure S1.** Fluorescence intensity and co-localization analysis of Cherry and GFP. **Figure S2.** N protein significantly increased the level of LC3-II.**Additional file 2.** The original blots for the figures.

fulltext/md/PMC10019356.md

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+---
+pmcid: "PMC10019356"
+pmid: "36937006"
+doi: "10.3389/fvets.2023.1125618"
+title: "Potential revival of cholinesterase inhibitors as drugs in veterinary medicine."
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "review-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Potential revival of cholinesterase inhibitors as drugs in veterinary medicine
+
+## Abstract
+
+The cholinergic system is involved in the regulation of all organ systems and has acetylcholine (ACh) as almost its only neurotransmitter. Any substance is called cholinergic if it can alter the action of acetylcholine. Cholinesterases (ChEs) are enzymes that enable the hydrolysis of acetylcholine and in this way ensure homeostasis in cholinergic synapses. Cholinesterase inhibitors (ChEi) are a group of indirect-acting cholinergic agonists that influence the activity of the cholinergic system. Several compounds that can inhibit cholinesterases are of importance to veterinary medicine from pharmacological and toxicological perspective. The frequency of their use in veterinary medicine has fluctuated over the years and is now reduced to a minimum. They are mainly used in agriculture as pesticides, and some are rarely used as parasiticides for companion animals and livestock. In recent years, interest in the use of new cholinesterase inhibitors has increased since canine cognitive dysfunction (CCD) became a recognized and extensively studied disease. Similar to Alzheimer's disease (AD) in humans, CCD can be treated with cholinesterase inhibitors that cross the blood–brain barrier. In this review, the mammalian cholinergic system and the drugs that interact with cholinesterases are introduced. Cholinesterase inhibitors that can be used for the treatment of CCD are described in detail.
+
+## Introduction
+
+A variety of substances are known to have inhibitory effects on cholinesterases (ChEs). Organochlorine, organophosphorus, carbamate, and other substances are known to interact with ChEs and temporarily or permanently inactivate these enzymes. Other compounds, such as divalent ions, e.g., magnesium, can also inhibit ChEs (1). These compounds were commonly used in agriculture as pesticides or insecticides in the past, so they must be recognized as a possible cause of poisoning from a clinical perspective. Unintentionally, pets, livestock, and humans can easily come into contact with them. However, their use has changed over the years. Currently, their use is limited, although the horizon is opening for some of them as medicines for canine cognitive dysfunction (CCD).
+
+Organochlorine compounds have been used extensively for decades as very effective insecticides. They have been used in households, in agriculture, and as parasiticides in veterinary medicine, but because of their toxicity, their use for these purposes has been banned in Europe and the United States since the early 1970s, but they continue to be used in some Asian countries (2). Currently, animals or humans do not usually encounter organochlorine preparations, but because of their long half-life, organochlorines used decades ago are still present in the environment and living organisms may be exposed to low concentrations through food and drinking water. The concentration of these persistent organic pollutants is too low to affect the cholinergic system in the organism. However, prolonged exposure to low levels of organochlorine compounds has been confirmed to cause endocrine disruption (3, 4).
+
+Organophosphorus compounds are esters of phosphoric acid. They are used in agriculture as pesticides to control insect and arthropod pests in intensive crop production. As in mammals, they inhibit acetylcholinesterase (AChE) in pests and cause their death. Although approved organophosphorus pesticides have a much shorter half-life than organochlorine compounds, animals and humans can be accidentally exposed to higher concentrations that lead to poisoning. The debate over poisoning and its treatment is outside the scope of this report and can be found elsewhere (5). On the other hand, some organophosphorus compounds are approved as parasiticides and can be used to treat pets, livestock, and bees. Organophosphorus compounds, however, include agents that act as chemical nerve agents.
+
+Several well-described drugs (physostigmine, neostigmine, pyridostigmine, etc.) act as reversible AChE inhibitors and are carbamates. Carbamates are derived from carbamic acid. As drugs, they are used in human rather than veterinary medicine, although they can be used in non-food-producing animals. In addition, some carbamates are also used as pesticides to control insect and slug invasion and as parasiticides in veterinary medicine.
+
+In recent years, interest in the use of new cholinesterase inhibitors (ChEi) has increased since canine cognitive dysfunction (CCD) became a recognized and extensively studied disease. Similar to Alzheimer's disease (AD) in humans, CCD can be treated with ChE inhibitors that cross the blood–brain barrier. In addition, intensive work is underway to develop agents that inhibit ChEs while having adrenergic properties, and their pharmacological properties are being investigated. It is expected that these types of agents will allow better treatment of the symptoms of AD and CCD in the future.
+
+## Cholinergic system
+
+The cholinergic system was named after acetylcholine (ACh), which plays the role of a neurotransmitter in the central and peripheral nervous systems. The action of ACh is expressed by binding to cholinergic (muscarinic and nicotinic) receptors in postsynaptic neurons or effector cells, while the homeostasis of cholinergic transmission is regulated by ChEs, mainly by the enzyme acetylcholinesterase (AChE) and less importantly by butyrylcholinesterase (BChE). Thus, cholinergic neurons, ACh as a neurotransmitter, cholinergic receptors, and ChEs constitute the cholinergic system (6).
+
+### Acetylcholine (ACh)
+
+Although ACh had previously been synthesized in the laboratory, it was first extracted from biological material by Sir Henry Hallet Dale in 1914 (7). A few years later, Otto Loewi found that ACh was involved in the action of the vagus on heart rate and neurotransmission (8). Acetylcholine is a neurotransmitter in the cholinergic system. It provides neurotransmission in parasympathetic neurons and preganglionic sympathetic neurons at the neuromuscular junction and is widely distributed in the CNS. Thus, ACh is involved in regulating the activity of all organ systems, blood vessels, skeletal muscles, the immune system, and the brain. Acetylcholine is mainly synthesized in presynaptic neurons from choline and acetyl coenzyme A, which requires the action of choline acetyltransferase (ChAT), and is stored in intracellular vesicles. After depolarization of the presynaptic neuron, it is released into the synaptic cleft by exocytosis and provides neurotransmission (Figure 1). Acetylcholine action ends with its cleavage into choline and acetate, which occurs by AChE in the synaptic cleft. The reuptake of choline enables the synthesis of new ACh molecules in presynaptic neurons (9, 10). As shown in Table 1, ACh, as a neurotransmitter in the parasympathetic nervous system, regulates vascular tension; increases gastrointestinal motility, uterine contraction, and bronchial dilation; and decreases heart rate and blood pressure. In the sympathetic nervous system, ACh is released from preganglionic neurons and modulates synaptic transmission to postganglionic neurons, acting as a fight–flight response through noradrenaline release from axon terminals. In the CNS, ACh acts as the major excitatory neurotransmitter and regulates many cognitive functions, such as attention, learning, and memory (6).
+
+*Figure: Figure 1 Schematic representation of a cholinergic neuron. Shown are the cell membrane of the pre- and postsynaptic neuron with synaptic cleft and ongoing processes. Synthesis of acetylcholine (ACh) by choline acetyltransferase (ChAT) from choline and acetyl-CoA, storage, and release occur in the presynaptic neuron, whereas hydrolysis of ACh by acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) occurs in the synaptic cleft. The two types of cholinergic receptors in the postsynaptic membrane are muscarinic (M1–M5) and nicotinic (nAChR) receptors (Created with BioRender.com).*
+
+**Table 1 Effects on various organs of the stimulated cholinergic system.**
+
+| **Effector** | **Response** | **Acetylcholine receptor** | **Reference** |
+| --- | --- | --- | --- |
+| CNS | Cognitive functions | mAChs | (21) |
+|  | Neuromodulation | nAChRs |  |
+| Heart | Negative chronotropic, bathmotropic, and dromotropic | M2, M4 | (24) |
+| Blood vessels | Dilatation | M1, M2, M3 | (24) |
+|  | Decrease of blood pressure |  |  |
+| Gastrointestinal tract | Increased motility | M2, M3 | (25) |
+|  | Secretion of glands |  |  |
+|  | Sphincter relaxation |  |  |
+| Bronchioles | Smooth muscle contraction | M2 | (26) |
+|  | Secretion of glands |  |  |
+| Urinary bladder | Contraction of fundus | M2, M3 | (27) |
+|  | Sphincter relaxation |  |  |
+| Eye | Miosis, decrease of intraocular pressure | M1, M2, M3 | (28) |
+| Reproductive system | Erection |  | (29) |
+|  | Spermatogenesis |  |  |
+|  | Uterine contractions |  |  |
+|  | Gland excretion |  |  |
+| Sweat glands | Excretion | M3 | (30) |
+| Skeletal muscles | Contractions | nAChR | (20) |
+|  | Activity |  |  |
+
+### Choline acetyltransferase (ChAT)
+
+Choline acetyltransferase is the only enzyme involved in the biosynthesis of ACh; it transfers the acetyl group from acetyl-CoA to choline to form ACh. There are at least two isoforms (one common and one peripheral); alternative splicing has been studied in humans and to a lesser extent in mice and rats. Its activity is higher in motor fascicles than in sensory fascicles and is expressed in different proportions in different cortical regions and the retina. In addition to ChAT activity, the need to transport acetyl-CoA from mitochondria to the cytoplasm and intracellular choline concentrations may be rate-limiting for ACh synthesis (11).
+
+Choline acetyltransferase activity is thought to play a central role in basic brain processes such as learning, memory, consciousness, and sleep (12). Mutations of the enzyme are the cause of congenital myasthenic syndromes (13). The amount of ChAT protein and ChAT isoforms was found to correlate with the progression of AD (14). In addition, the activities of the enzyme in plasma and/or CSF and as a marker of disease severity have been suggested but have not (yet) been tested as drug targets (15). Choline acetyltransferase is also a potential target for pharmacotherapy in several other diseases, including blood pressure disorders and cholinergic overstimulation by organophosphorus nerve agents, but a lack of useful inhibitors has limited preclinical research to date (16).
+
+### Cholinergic receptors
+
+The action of ACh as a neurotransmitter is mediated by binding to two types of cholinergic receptors: muscarinic (mAChR) and nicotinic (nAChR) receptors. Both families of receptors are widely distributed throughout the organism but differ in their mode of action; mAChRs are metabotropic receptors, while nAChRs are ionotropic receptors. Muscarinic receptors M1, M3, and M5 are coupled to protein Gq and activate phospholipase C, leading to the formation of diacylglycerol and inositol trisphosphate as second messengers (17). M2 and M4 are coupled to the protein Gi/o, leading to inhibition of adenylate cyclase (18). M4, as an autoreceptor, inhibits the release of ACh in the CNS (19). nAChRs are ligand-gated ionotropic receptors that regulate the transport of positively charged ions (Na^+^, K^+^, and Ca2+) through membrane channels. Structurally, they are pentamers composed of five transmembrane subunits organized in a cylindrical structure with a central pore (20, 21). To date, 17 different subunits (the muscular α1-, β1-, δ-, γ-, and ε-subunits, as well as the neuronal α2–10 and β2–4 subunits) have been identified (21). nAChR subtypes occur in homomeric or heteromeric form and generally consist of both α- and β-subunits. For example, the adult muscle receptor has an α2β1ε1δ1 composition, whereas the major ganglionic subtype is α2β3, with the exception of the homomeric (α7)5 subtype, which is mainly found in the brain (22, 23). Given these different combinations of subunits, modulation of neurotransmitter release is subtype-specific, and each subtype may mediate a different physiological function; moreover, the subunits contribute to pharmacological specificity within each receptor subtype (22, 23). As described by Picciotto et al. m- and nAChRs are located pre- and postsynaptically in the CNS. Presynaptic muscarinic receptors (M2 and M4) exert inhibitory effects as autoreceptors that control further ACh release (21). Presynaptic nicotinic receptors are involved in the release of various excitatory and inhibitory neurotransmitters, such as dopamine, noradrenaline, ACh, glutamate, and GABA. Postsynaptic M2 and M4 have an inhibitory effect, whereas M1, M3, and M5 are excitatory. Postsynaptic nAChRs provide postsynaptic neuron depolarization and neurotransmission. At the neuromuscular junction, nAChRs act as ligand-gated channels to provide sodium ion transport into muscle cells, depolarization, and skeletal muscle contraction (20). In addition, ACh alters the response of neurons to the stimulation (or inhibition) of neurotransmitters and thus acts as a neuromodulator, which is particularly important for the regulation of cognitive functions (21). The effects of the cholinergic system on various organs are listed in Table 1.
+
+### Cholinesterases
+
+As mentioned earlier, there are two enzymes with cholinesterase activity, AChE and BChE, which hydrolyze acetylcholine into choline and acetate (31). Acetylcholinesterase and butyrylcholinesterase have a similar structure, and it is estimated that they are more than 50% identical (1, 9). However, both enzymes have their own structural features and therefore differ in their physiological roles, which will be discussed later. Each tissue contains both BChE and AChE, but in very different proportions. In the brain, BChE is expressed at a much lower level than AChE, and the change in the ratio is an important factor indicating neurodegenerative processes (31, 32).
+
+#### Acetylcholinesterase (AChE)
+
+Acetylcholinesterase, named after its endogenous substrate, the neurotransmitter ACh, is produced mainly in neurons but also in muscle and hematopoietic cells. Acetylcholinesterase is one of the most efficient enzymes due to its high catalytic rate. It is involved in the homeostasis of the cholinergic system in the brain, autonomic nervous system, and neuromuscular junction. Homeostasis is enabled by the hydrolysis of ACh and the termination of its action. It is also located in the membranes of red blood cells, where its role is to metabolize plasma ACh (1). In the cholinergic system, AChE is present mainly in the tetramer form, although some water-soluble dimers are also present.
+
+As is shown in Figure 2 the active site of AChE is located at the bottom of the cavity called the aromatic gorge, covered by aromatic amino acids (1, 9, 33). The active site consists of two parts. The anionic site (α anionic site) is composed of amino acids (Trp, Tyr, Phe) and interacts with the positively charged nitrogen (ammonium) in ACh, placing the ACh molecule in the correct position to bind to the nearby esteratic site of AChE (1, 9, 33). The esteratic site is another part of the active site. It contains the catalytic triad, the site where the substrate is metabolized. The catalytic triad is formed from three amino acids (Ser, His, Glu). Once ACh encounters the catalytic triad, the bond between choline and acetate is transmitted to serine, and choline is released. In addition, acetate reacts with a water molecule and releases the catalytic triad (9). On the surface of AChE, there is another important active part called the peripheral anionic site (PAS), which is also a β-anionic site. It is formed mainly by Tyr, Asp, and Trp residues. It interacts with the substrate and is located to allow the substrate to enter the gorge (34). It is believed that PAS interacts with amyloid and therefore plays an important role in the pathogenesis of AD in humans (1).
+
+*Figure: Figure 2 Schematic representation of the acetylcholinesterase (AChE) **(left)** and (butyrylcholinesterase) BChE **(right)**. AChE and BChE differ in the size of the binding pocket, with BChE having an approximate gorge volume of 1,500 Å3 and AChE of 1,300 Å3. Peripheral aromatic site (PAS) is located near the entrance and plays an important role in substrate entry. PAS of AChE contains serine, tyrosine, aspartic acid, and tryptophan, in BChE it is asparagine, aspartic acid, glutamine, serine, and tyrosine. The residues in BChE are more aromatic, resulting in a more negatively charged site. The acyl and choline binding sites are close to the esteratic site and consist of tryptophan, tyrosine, and phenylalanine in human AChE and are replaced by leucocin, alanine, and valine in BChE. The main role of the anionic site is to position the ligand for catalysis. Substitution with more aromatic residues leads to conformational changes that result in a deeper gorge in BChE. This allows BChE to interact with and potentially hydrolyze a much broader range of substrates and inhibitors than AChE. The catalytic triad of the esteratic site is located deep down in the gorge and consists of serine, glutamic acid, and histidine. The oxyanine hole (OAH) is a two- or three-pronged site in BChE. The Ω (omega) loop (red in this Figure) helps the enzyme “breathe”—a process that can help substrates move from the surface to deeper regions. It consists of about 30 amino acid residues (33) (Created with BioRender.com).*
+
+In addition, some toxins and drugs (fasciculin, berberine, donepezil, huperzine A) react with the PAS and inhibit AChE activity (1, 35, 36). However, because the catalytic rate of AChE is one of the most active in the organism, it could be inhibited by an excess of substrate (1). Despite the primary function of AChE, researchers have found that AChE also plays a role in neuronal development. Embryologically, it is involved in nervous system development and is expressed by developing neurons and during periods of axonal growth (31).
+
+#### Butyrylcholinesterase (BChE)
+
+Butyrylcholinesterase (BChE), formerly called “pseudocholinesterase,” is produced mainly by the liver (33). The richest sources of BChE enzymes in the body are plasma and liver, as well as leg muscles and skin (37). Although BChE can metabolize ACh, it has a higher affinity for butyrylcholine (BCh), a synthetic compound that does not occur naturally in the body and is a choline-based ester with neurotransmitter activity. It is used as a clinical laboratory tool to distinguish between enzymes (33). The main structural difference between BChE and AChE is that 6 of the 14 aromatic amino acids lining the active site gorge of AChE are replaced by aliphatic residues in BChE (33). These conformational changes result in a larger gorge volume in BChE, which allows BChE to interact with and potentially hydrolyze a much broader range of substrates than AChE. Butyrylcholinesterase is therefore referred to as the promiscuous or non-specific “bigger brother” of the smaller and much more specific AChE (33, 38).
+
+Although ChEs share some of these functions, the biological role of BChE has a much more obscure purpose. The higher concentration of BChE in plasma led to the theory that BChE evolved from AChE to be a general detoxifying agent while still retaining some function in the process of neurotransmission (33, 37). However, it is known that it has no function that could not be compensated for by other enzymes (37). On the other hand, BChE is known to play an important role in drug detoxification (examples: physostigmine, cocaine, aspirin, succinylcholine, mivacurium, heroin) (31, 33, 39). Moreover, BChE restores cholinergic transmission when AChE activity is impaired or absent, e.g., in the brains of AD patients (9). This was suggested in studies using the drugs donepezil and huperzine A in BChE knockout mice (39). Similar changes in the physiological functions of both enzymes have been observed in humans, where AChE concentration decreases and BChE concentration increases with the progression of AD, increasing the ratio of BChE to AChE from 0.2 to up to 11. In addition, BChE may be involved in lipid metabolism and the development of inflammation (37).
+
+### Drugs that inhibit ChE activity
+
+Several compounds can bind to ChEs and inhibit their activity, and only a few compounds are used as drugs affecting the cholinergic system of mammals. To achieve the desired pharmacological effect, these drugs must inhibit ChEs for a short period of time so that they are spontaneously hydrolyzed after binding to active sites in ChEs. In addition to drugs that interact with animal ChEs, drugs that interact with the cholinergic system and are used as parasiticides are relatively common in veterinary medicine. Since there is a possibility of interaction with the host AChE, these drugs are also important from a toxicological point of view.
+
+### Carbamates
+
+The effects of these classic drugs are well-described in the literature. Carbamates are reversible inhibitors of ChEs and block the catalytic site of AChE, resulting in decreased activity of AChE and thus increased concentration of ACh in the synapses and neuromuscular junction and prolonged cholinergic action. Therefore, they are indirect-acting cholinergic agonists. The effects of carbamates can be predicted in terms of the anatomic location of muscarinic and nicotinic receptors and pharmacokinetic properties. However, treatment with neostigmine and physostigmine may lead to adverse effects because these compounds stimulate preganglionic sympathetic nerves and adrenal glands and may produce unexpected effects by releasing catecholamines (40).
+
+#### Edrophonium
+
+Edrophonium is a small molecule containing a positively charged nitrogen ion that forms an electrostatic bond with the anionic site of ChEs. Since the anionic site is blocked by edrophonium, the natural substrate cannot reach the esteratic (catalytic) site of the enzyme. This inhibits the activity of the ChEs for a short time until the electrostatic force between the ions is removed when the ChEs are reactivated (40). Unlike other carbamates, edrophonium is not hydrolyzed, but the anionic site is spontaneously released from the drug. Edrophonium is used for diagnostic procedures in myasthenia gravis-like disorders primarily in dogs and cats (41). Short-term administration of edrophonium improves skeletal muscle weakness in myasthenia or myasthenia-like disorders (42).
+
+#### Physostigmine
+
+Physostigmine is a tertiary amine that reversibly blocks ChEs. Transient binding between ChEs and physostigmine occurs at the esteratic site of the enzyme. It is then hydrolyzed, and the ChEs are reactivated. It has few indications and is rarely used in veterinary medicine. It has been reported that physostigmine can be used to treat glaucoma in dogs and cats (43), but safer drugs such as carbonate anhydrase inhibitors or some beta-blockers (timolol) are more commonly used for this purpose. Physostigmine crosses the blood–brain barrier and can be used to treat cognitive dysfunction and intoxication with atropine, scopolamine, or belladonna. It has been reported that physostigmine can be used for symptomatic treatment of ivermectin toxicosis in collies (44). Theoretically, rumen or intestinal atony can be treated with physostigmine in ruminants, but the drug is not approved for use in food-producing animals. Physostigmine can also be used to prevent organophosphate (or chemical weapon) poisoning. Administration of sublethal doses of physostigmine (via the skin or eyes) protects against lethal doses of organophosphorus compounds such as soman and sarin, which are used as chemical weapons (45, 46).
+
+#### Neostigmine
+
+Administration of neostigmine acts more intensely on the neuromuscular junction than on the cholinergic transmission that activates muscarinic receptors in the digestive system. Because it is a quaternary nitrogen compound, it does not cross the blood–brain barrier and does not exhibit central effects. However, it has been described to treat rumen atony or caecal dislocation without torsion in ruminants (47). It can also be used as an antidote in curare-like poisoning (48). Neostigmine preparations are not approved for veterinary use, which limits their use in livestock. After general anesthesia, it is used in dogs to rapidly interrupt neuromuscular blockade caused by non-depolarizing muscle relaxants (49).
+
+#### Pyridostigmine
+
+Pyridostigmine is a carbamate containing quaternary ammonium. It binds to the catalytic site of ChEs and temporarily blocks their activity. Its action is in the peripheral nervous system, as the positively charged ammonium ion can cross the blood–brain barrier (50). Pyridostigmine is a drug of choice for the prevention of the nerve agent intoxication and treatment of myasthenia gravis-like disease in dogs and cats (51, 52). Because it is not approved for veterinary use, human preparations, usually in syrup and tablet form, are used. The recommended dose is given in Table 2, but the dose can be individualized depending on the stage of the disease.
+
+**Table 2 Use of drugs that act as AChE inhibitors in animals.**
+
+| **Drug** | **Animal species** | **Dose (mg/kg) Route of administration** | **Indication** | **Reference** |
+| --- | --- | --- | --- | --- |
+| Edrophonium chloride | Dogs and cats | 0.1–0.2 IV | Diagnosis of myasthenia-like diseases | (42) |
+| Physostigmine | All species | Individual regimen | Atropine intoxication | (50) |
+| Physostigmine | Dogs | 2.5 mcg/kg/h | Prevention of nerve agent intoxication | (45, 46, 51) |
+| Physostigmine | Dogs and cats | Eye drops 2% | Glaucoma | (43) |
+| Physostigmine | Cattle | 0.04–0.08 IV | Curare-like intoxications (e.g., *Delphinium barbeyi*) | (48) |
+| Pyridostigmine | Dogs and cats | 0.5 Orally | Myasthenia gravis | (52) |
+| Neostigmine | Cattle | 0.02 SC | Rumen atony | (47) |
+| Neostigmine | Sheep | 0.01–0.02 SC | Rumen atony | (47) |
+|  |  |  | Caecal dislocation |  |
+| Neostigmine | Cattle | 87.5 mg in 10 L of sodium-glucose infusion: 2 drops/s | Caecal dislocation | (47) |
+| Neostigmine | Cattle | To the effect | Curare and curare-like intoxications | (48) |
+| Neostigmine | Dogs | 0.02–0.07 IV | To terminate myorelaxation during anesthesia | (49) |
+
+### Organophosphates and carbamates as parasiticides
+
+Although the main era of using organophosphates and carbamates as parasiticides has already passed, some of them are still approved in some countries for use in veterinary medicine. Approved organophosphates are used as parasiticides for the control of ecto- and endoparasites. Trichlorfon is a pro drug that is metabolized to dichlorvos after administration. Both of them were used to control nematodes and ectoparasites, mainly in dogs (53). Additionally, coumaphos is used for the treatment of bee colonies against infestation with Varroa destructor. Since it accumulates in beeswax after each use, it should be used sparingly (54). Additionally, two carbamate drugs, carbaril and propoxur, are used for the control of ectoparasites in pets and livestock. It is expected that these organophosphate and carbamate drugs interact with the cholinergic system of the parasites and do not affect the host; thus, their characteristics are not a topic of this review. However, it must be mentioned that in addition to organophosphates and carbamates as veterinary drugs, they are also used as pesticides in agriculture, and accidental contact of animals with these compounds can cause severe intoxication due to AChE blockage (5, 55).
+
+### Existing acetylcholinesterase inhibitors for the treatment of cognitive impairment
+
+Rivastigmine, galantamine, and donepezil are AChE inhibitors approved for the treatment of Alzheimer's disease (AD). Their use is considered suboptimal therapy because they do not halt disease progression but only improve symptoms that lead to better cognitive function (56). Based on research and literature, there are limited data on the clinical use of these drugs in animals, particularly in dogs with cognitive impairment. Matsunami et al. reported that “canine cognitive dysfunction syndrome criteria” improved after a 2-week study with donepezil hydrochloride (57). In the study by Araujo et al. the role of cholinergic hypofunction in the canine model was investigated (58). In two tasks, a delayed non-matching-to-position (DNMP) task to assess working memory and an oddity-discrimination task to assess complex learning, they examined the effect of the cholinesterase inhibitors phenserine and donepezil on performance in older dogs. Phenserine (0.5 mg/kg; orally) and donepezil (1.5 mg/kg; orally) significantly improved performance (58). Phenserine is an AChE inhibitor whose research was abandoned after clinical phase III due to some methodological problems (59). Out of curiosity, we searched the commercial website for the clinical use of AChE inhibitors in dogs. One website recommends and sells donepezil 23 and 10 mg tablets under the brand name Aricept for dogs with CCD. This drug is otherwise approved for human use in the United States (60). We could not find any clear information or reports from the FDA or EMA on its use in dogs.
+
+## Use of ChE inhibitors for the treatment of canine cognitive dysfunction
+
+Acetylcholine is also responsible for learning and memory formation (9, 61). The pathological processes in Alzheimer disease (AD) lead to a deficiency of ACh and consequent loss of cholinergic neurons. Neurotransmission is therefore impaired, leading to cognitive deficits (56). This so-called cholinergic hypothesis is the basis for treating AD with drugs that increase ACh levels. Cholinergic hypofunction is also present in dogs with CCD (62, 63). Cognitive dysfunction in dogs is defined as a chronic progressive neurodegenerative disease that affects 14–60% of old dogs but is diagnosed in less than 2% of all affected individuals (64). It most commonly occurs in dogs older than 8 years and results in changes in behavior and cognitive abilities (65). Dogs also show age-related cognitive impairments that parallel the behavioral symptoms of AD. In addition to behavioral symptoms, the neuropathology of both diseases is similar in several aspects (63). For this reason, dogs with CCD represent one of the potentially most important animal models for studying the treatment of AD due to their functional and neuroanatomical similarity to humans (66, 67).
+
+As mentioned earlier, phenserine and donepezil have been successfully tested in dogs (58). In another study by Studzinski et al. the efficacy of two AChE inhibitors—CP-118,954 and phenserine—was tested in a modified delayed non-matching to position task (68). Minimal cognitive enhancing effects were seen after treatment with CP-118,954. In contrast, phenserine was able to improve learning and memory in dogs and showed positive effects on memory. In humans, CP-118,954 was discontinued after reaching clinical phase II, as was the case with phenserine (59, 68).
+
+More important, however, is focusing on the area that most of the ChE research has focused on in the last two decades: the inhibition of one or both enzymes, AChE and BChE, by different compounds from different chemical groups. The reason is the almost desperate search for a drug that could improve the symptoms of AD and CCD. Recently, a new series of selective nanomolar BChE inhibitors have been developed (61, 69). The rationale for the development of selective BChE inhibitors is that during the progression of AD and CCD, cholinergic transmission, and AChE activity decrease. To compensate, BChE takes over and terminates up to 90% of cholinergic signaling in the brain. In addition, these agents have no peripheral parasympathomimetic side effects, which are quite common with AChE inhibitors due to the selective inhibition of BChE (61). In the last clinical trial, dogs with CCD were treated with newly developed BChE inhibitors (69, 70). Their cognitive abilities were assessed using a questionnaire (CADES score) and two cognitive performance tests: Food search test and Problem Solving Test. The dogs participated in therapy for at least 6 months, and the dogs with moderate cognitive impairment showed improvement in clinical signs and performance-related tests compared to the untreated group (*p* < 0.001). In addition, owners reported improved quality of life for the treated dogs and for themselves. This argues for the use of cholinergic medications in older dogs and allows for future screening of therapeutics for AD (70).
+
+The lack of information on clinical trials of CCD with ChEi is the main reason why we cannot make general conclusions or suggestions about the efficacy and safety of these drugs. Several studies on this problem are expected to be conducted in the next few years, and the issue of CCD treatment will become clearer.
+
+## Drug interactions
+
+It is well-known that some drugs may potentiate or inhibit the action of cholinesterase inhibitors. Thus, atropine counteracts the muscarinic actions of physostigmine, neostigmine, and pyridostigmine; therefore, atropine decreases the action of carbamates (pyridostigmine), and conversely, carbamates block the action of atropine (50). The interaction of carbamates and depolarizing myorelaxants (succinylcholine) results in enhanced and prolonged myorelaxation. The effect of non-depolarizing myorelaxants is shortened or reversed by administration of neostigmine and other carbamates. This combination therapy is used for faster recovery after the use of myorelaxants such as pancuronium or rocuronium for myorelaxation during anesthesia (71). In addition, some antibiotics, e.g., aminoglycosides, polymyxins, tetracyclines, and bacitracin, can prolong neuromuscular blockade when used with non-depolarizing myorelaxants (50, 72). Adrenergic beta inhibitors may attenuate the effect of pyridostigmine in the treatment of myasthenia gravis. As mentioned earlier, pretreatment with carbamates can prevent poisoning and severe effects of chemical weapons such as sarin, soman, etc. (45, 46, 51).
+
+## Safety and adverse effects of cholinesterase inhibitors (ChEi)
+
+One of the main disadvantages of approved ChEi is its frequent side effects. This is due to an increased total amount of ACh caused by inhibition of ChEs not only in the brain but also in the peripheral nervous system, parasympathetic autonomic nervous system, and basal ganglia. In humans, gastrointestinal side effects, such as diarrhea, vomiting, and nausea, are the most common signs of an overstimulated parasympathetic nervous system (69, 73). In addition to gastrointestinal issues, other common side effects cited include dizziness, headache, and weight loss (73). With regard to donepezil, sleep disturbances have been reported with its use, and there are case reports of cardiac side effects. However, studies to date have not shown serious arrhythmogenic, hypotensive, or negative chronotropic effects (73). Such symptoms may also occur in patients with mild AD but may increase in severity and frequency as the disease progresses. Their frequency and intensity may depend on the dose and route of administration (73). Nevertheless, the three marketed cholinesterase inhibitors are considered safe and remain the first choice in AD therapy (74). However, little is known about the safety and proper dosing of these drugs in animals, particularly dogs (62). In Araujo's study, side effects such as tremors, vomiting, and hypersalivation occurred in dogs receiving 6 mg/kg donepezil. Side effects occurred less frequently in the placebo and 1.5 mg/kg groups (58). Dogs treated with medium and high doses of CP-118,954 showed slightly impaired performance in behavioral tests, probably due to side effects (68). At the same time, no side effects have been reported with the use of phenserine, although it is possible that they may occur with higher doses and prolonged administration (58, 68).
+
+In a clinical trial with a newly developed BChE inhibitor, Košak et al. and Zakošek Pipan et al. reported that the timing of treatment may be critical (69, 70). In their study, vomiting and nausea occurred in all dogs with severe cognitive impairment after the beginning of treatment. In any case, no side effects were observed when using the new BChEi to treat dogs exhibiting moderate cognitive impairment. Therefore, the new inhibitor is unlikely to have muscarinic effects in the early stages of the disease. The same observation was made in a preclinical study in mice, in which the newly developed BChEi had no negative effects on the motor coordination of the mice and did not cause motor deficits. Moreover, no acute cholinergic side effects were observed even at a high dose (100 mg/kg) in mice (69, 70).
+
+Finally, the differential effects of dose and timing indicate the need for comprehensive pharmacokinetic and pharmacodynamic profiling in target animals before veterinary use can be considered (58).
+
+## Discussion
+
+The time when some cholinesterase inhibitors such as organochlorines were used as veterinary drugs is certainly over. Since other effective drugs are currently available, organophosphorus and carbamate parasiticides are not used as often as they were. However, organophosphates and carbamates are still used in agriculture as pesticides, so there is a possibility of accidental poisoning if animals come into contact with them. Carbamate drugs are used primarily for diagnostic purposes and are more widely used in human medicine. Nevertheless, edrophonium can be used for diagnostic purposes in myasthenia gravis-like disorders, especially in dogs and cats (41), and physostigmine can be used to treat glaucoma in dogs and cats (43) and for antimuscarinic toxicity. Pyridostigmine and neostigmine are the most used AChE inhibitors for the treatment of myasthenia gravis, but pyridostigmine is preferred because of its longer duration of action and lower potential for adverse gastrointestinal effects (52). However, some ChE inhibitors are gaining importance because their mechanism of action plays a role in neurodegenerative diseases.
+
+Alzheimer' disease is one of the most common causes of dementia in the elderly and a major public health concern in the world. In veterinary medicine, CCD is also a problem in older dogs and is becoming more well-known. Researchers are trying to develop new therapeutics to alleviate the symptoms of this neurodegenerative disorder. In addition, old dogs (and, more recently, cats) have been recognized as animal models for AD (75). The main advantage over non-transgenic aged rodent species is that dogs are more similar to humans from a neuroanatomical and physiological perspective (63, 67), which means that they spontaneously develop neurodegenerative diseases, unlike rodents. In addition, the use of multiple animal models could also be crucial for safety reasons. This was evident in the AN-1792 clinical trials that were initiated in humans based on positive findings from the transgenic mouse model. The trials were stopped after 18 patients developed meningoencephalitis, some of whom died. The preclinical studies had not predicted this adverse event, probably because the Aβ vaccine in the transgenic mouse targeted a foreign peptide, whereas in humans, it targeted an endogenous peptide and triggered an autoimmune response. Perhaps this result could have been avoided if the preclinical data had also been obtained from primates or dogs that naturally deposit amyloids (68, 76). As a result, there is increasing interest in the use of new ChE inhibitors. It has been reported that approved AChE inhibitors for AD have been tested in dogs (58, 68), but these studies are not supported by new trials, and little is reported on their clinical use. In most cases, other drugs are used for symptomatic treatment. However, some BChE inhibitors have been tested recently (61, 69, 70, 77). It is recommended to focus only on BChE inhibition as a target for symptomatic treatment because it offsets the diminishing effect of AChE in nervous tissue and fewer side effects have been reported compared with AChE inhibitors. Owners have reported great satisfaction with the tested drug, as it has improved their dogs' lives and their own (70). In view of the above findings, the new BChEi shown in our study may be a good alternative to the currently available AD and CCD treatments (70).
+
+## Future prospects
+
+The way to approve drugs for the treatment of CCD is still long, but there is hope as the research and development of new chemical mediators progresses. Recently, a new series of BChE inhibitors have been optimized (77). Additionally, the development of new compounds, the so-called multitarget direct ligands, is very interesting and encouraging. They target multiple signaling pathways in the neurodegenerative process present in AD and CCD and could not only alleviate symptoms but also counteract cellular processes associated with neurodegeneration (74, 78).
+
+The treatment of canine cognitive dysfunction with ChEi in veterinary medicine is, in fact, the only new application we have been able to glean from recent publications. Considering the increasing awareness of a better quality of life of geriatric pets, we can assume that the number of drugs considered for this purpose will increase and that other age-related treatable pathologies will probably be recognized, which prompted us to write the current review. Although a very similar neurodegenerative syndrome has also been recognized in feline patients, no (successful) attempt has been made to treat them with ChE inhibitors (75, 79, 80).
+
+Finally, we must not forget that CCD is still an underdiagnosed disease. The reason for this is the inability of owners to recognize the disease in its early stages, the inexperience of veterinarians, and the lack of diagnostic tools, which too often leads to symptoms being mistaken for the normal aging process. However, early detection of cognitive aging disorders in older pets is of paramount importance. The fact that dogs are good models for AD opens up the possibility of testing new drugs in dogs and treating their cognitive issues. Another thing to consider is that pets are exposed to similar environmental risk factors as humans, and one could even say that we lead the same lifestyle. This could bring many interesting questions and answers in the future.
+
+## Author contributions
+
+NŽ: data selection, preparation of the figures, writing, and editing. MŠ and NG: review and editing. TS: design of the manuscript, data selection, writing, review, and editing. All authors contributed to the article and approved the submitted version.

fulltext/md/PMC10019679.md

@@ -0,0 +1,122 @@
+---
+pmcid: "PMC10019679"
+pmid: "36928870"
+doi: "10.1371/journal.pone.0282859"
+title: "Using cultured canine cardiac slices to model the autophagic flux with doxorubicin."
+journal: "PloS one"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Using cultured canine cardiac slices to model the autophagic flux with doxorubicin
+
+## Abstract
+
+Chemotherapy-induced impairment of autophagy is implicated in cardiac toxicity induced by anti-cancer drugs. Imperfect translation from rodent models and lack of *in vitro* models of toxicity has limited investigation of autophagic flux dysregulation, preventing design of novel cardioprotective strategies based on autophagy control. Development of an adult heart tissue culture technique from a translational model will improve investigation of cardiac toxicity. We aimed to optimize a canine cardiac slice culture system for exploration of cancer therapy impact on intact cardiac tissue, creating a translatable model that maintains autophagy in culture and is amenable to autophagy modulation. Canine cardiac tissue slices (350 μm) were generated from left ventricular free wall collected from euthanized client-owned dogs (n = 7) free of cardiovascular disease at the Foster Hospital for Small Animals at Tufts University. Cell viability and apoptosis were quantified with MTT assay and TUNEL staining. Cardiac slices were challenged with doxorubicin and an autophagy activator (rapamycin) or inhibitor (chloroquine). Autophagic flux components (LC3, p62) were quantified by western blot. Cardiac slices retained high cell viability for >7 days in culture and basal levels of autophagic markers remained unchanged. Doxorubicin treatment resulted in perturbation of the autophagic flux and cell death, while rapamycin co-treatment restored normal autophagic flux and maintained cell survival. We developed an adult canine cardiac slice culture system appropriate for studying the effects of autophagic flux that may be applicable to drug toxicity evaluations.
+
+## Introduction
+
+With profound advances in cancer survivorship, cardiovascular disease has become the leading cause of non-cancer-related morbidity and mortality in cancer patients following treatment [1–3]. Traditional chemotherapies such as anthracyclines (*e*.*g*., doxorubicin), as well as many novel therapies such as small molecule kinase inhibitors, increase the risk of heart failure in cancer patients. In a pooled analysis in the metastatic cancer setting, reductions in left ventricular ejection fraction or clinical heart failure occurred in a dose-dependent manner from 6% of those treated with 250 mg/m^2^ up to 76% of patients at a cumulative doxorubicin dose of 600 mg/m^2^ [4]. Even at lower cumulative doses of doxorubicin commonly used in breast cancer (240 mg/m^2^), 10–15% of patients develop cardiomyopathy and >5% develop heart failure [5, 6]. Newer targeted agents and combination therapies are generally considered safer, but evidence now suggests that many of these have potential for cardiotoxicity through mechanisms not yet clearly defined [7]. Furthermore, there are currently no routinely used cardioprotective strategies to prevent toxicity, in part due to gaps in knowledge regarding mechanistic drivers [1, 8]. Like humans, dogs develop cancer spontaneously with over 4 million dogs diagnosed annually [9]. Importantly, dogs develop similar cardiotoxicity from treatment of their cancer. For example, doxorubicin-induced cardiotoxicity can occur in dogs who have received a total cumulative dose as low as 90 mg/m^2^ [10]. As with humans, the lack of cardioprotective strategies has hampered treatment advances.
+
+One potential cause of chemotherapy-induced cardiotoxicity is dysregulated autophagy, resulting in cardiomyocyte cell death [11, 12]. Autophagy is activated in response to a myriad of environmental and cellular stresses, and is manifested by sequestration of parts of the cytoplasm to form double-membraned vesicles (autophagosomes) that are subsequently trafficked to the lysosomes for degradation. The degraded cellular material is subsequently recycled, used as nutrients, or exocytosed. Disruption of this multi-step process can lead to dysregulation of the autophagic flux, a dynamic measure of the degradative capacity of autophagy, followed by cell death rather than cell repair. Individual steps in autophagic flux can be influenced by both intrinsic regulators (for example: mTOR signaling, reactive oxygen species) and extrinsic regulators (drugs). The integrity of the autophagic pathway can be assessed by quantifying autophagic markers including lipidated Light Chain 3 (LC3-II) and autophagic cargo adaptor p62 [13, 14]. Specifically, LC3-II formation from LC3-I indicates the successful formation of autophagosomes, and p62 marks the trafficking of intracellular contents to the autophagosomes [15]. Previous preclinical *in vitro* and *in vivo* (murine) studies linked impaired autophagy to doxorubicin-induced cardiotoxicity and further demonstrated that restoration of autophagic flux can be achieved by pharmacological, physiological, or genetic means to improve cardiomyocyte salvage and overall cardiac function [16–19].
+
+Successful clinical translation of agents that restore autophagic flux has been challenging, in part due to a lack of credentialed large animal model systems that accurately recapitulate the process of autophagy following cardiac insult. While such processes can be evaluated *in vitro*, there are no readily available stable culture systems for mature cardiac tissues that faithfully model human cardiac responses. Rodent-derived cardiomyocytes are most often generated from neonates and lack the typical ion channel and myofilament composition found in human cardiomyocytes [20]. Human iPSCs-derived cardiomyocytes can be used in place of those from rodents, but the resultant cardiomyocytes often lack adult cell phenotypes [21, 22], and these immature cells have different ion handling behavior [23]. Furthermore, studies employing individual cardiomyocytes, either through enzymatic digestion or differentiation from iPSCs ignore critical interactions with other cells in the heart including endothelial cells, fibroblasts, and immune cells. Given that many of the newer anti-cancer drugs may induce cardiotoxicity by targeting key support cells within cardiac tissues, it is critical that an *in vitro* model system include these elements. Consequently, we sought to credential a novel platform using cardiac slices, a format in which the natural cardiac microstructure is preserved, to study how cancer treatments influence autophagic flux [20].
+
+For the following studies, we chose to focus on heart tissue derived from dogs. Canine cardiomyocytes are more similar in ion channel behavior to human cardiomyocytes than those of rodents [24, 25]. Furthermore, dogs develop spontaneous cancers and similar to humans, receive a variety of chemotherapy and small molecule treatments [26]. Indeed, canine cancer patients treated with cumulative doxorubicin doses of >180–240 mg/mm^2^ develop similar signs of cardiotoxicity including development of life threatening heart failure [10]. Finally, precision-cut tissue slices derived from canine cancer patients can be readily obtained and used in *ex vivo* studies. As such, the purpose of this study was to demonstrate that cardiac tissue derived from adult canines can be successfully cultured as intact slices and that this novel platform can be readily employed to evaluate changes in autophagic flux following exposure to anti-cancer agents.
+
+## Methods
+
+### Canine cardiac slice culture
+
+Adult canine hearts were collected from client-owned dogs whose owners elected euthanasia at the Tufts University Foster Hospital for Small Animals with owner consented through the tissue donation program at Tufts University Cummings School of Veterinary Medicine. Tissue collected from the client-owned patient donation program has received Tufts Institutional Animal Care and Use Committee (IACUC) exemption and does not require IACUC review and approval. Hearts were collected from dogs without known cardiac disease and who have not received any cancer treatment. The demographic information of these dogs is shown in Table 1. Humane euthanasia was performed by the attending veterinarian according to standard protocols in veterinary hospitals, and each heart was harvested within 10 minutes of euthanasia and kept in cold (4°C) sterile cardioplegic solution (5.5 mM glucose, 0.5 mM MgSO~4~, 24 mM KCl, 15 mM NaHCO~3~, 109 mM NaCl, 0.9 mM NaH~2~PO~4~, 1.8 mM CaCl~2~, adjusted to pH of 7.4) for 24 hours. Cardiac slicing protocol was modified from previous report [27]. Briefly, trans-mural sections of the LV free wall measuring 1 cm × 1 cm were excised. These tissue blocks were then affixed to the cutting block of a vibrating microtome (Campden Model 5100mz) using cyanoacrylate glue with the epicardial surface facing down, and then submerged in cold oxygenated slicing solution (30 mM 2,3-butanedione monoxime, 1 mM glucose, 10 mM HEPES, 6 mM KCl, 140 mM NaCl, 1 mM MgCl~2~, 1.8 mM CaCl~2~, adjusted to pH of 7.4). The top ¼ section of the tissue at the endocardial surface was first sliced and removed to create a flat cutting surface. Then, slices of 350-μm thickness were cut with a frequency of 75 Hz and amplitude of 1.5 mm, advancing at 0.1 mm/s. Resulting slices were washed once in culture media (M199 with 0.1% BSA, 1× ITS, 10 mM 2,3-butanedione monoxime, 1× chemically defined lipid, 100 U/ml penicillin/streptomycin, 1× HEPES) before being placed on a 0.3 μm pore size transwell surface of a 6-well transwell culture plate at the air-media interface at 37°C with 5% CO~2~. Slices were subsequently treated with doxorubicin (Cayman Chemical, 15007), rapamycin (Cayman Chemical, 13346), or chloroquine (Cayman Chemical, 14194).
+
+**Table 1 Demographic information of dogs used in this study.**
+
+| Breed | Age (Years) | Sex | Weight (kg) |
+| --- | --- | --- | --- |
+| West highland terrier | 14 | Castrated male | 15 |
+| English Springer Spaniel | 7 | Spayed female | 17 |
+| Boxer | 10 | Female | 26 |
+| Dachshund | 6 | Castrated male | 10 |
+| Mixed breed | 13 | Spayed female | 25 |
+| German wirehaired pointer | 9 | Castrated male | 25 |
+| Yorkshire terrier | 12 | Spayed female | 4 |
+
+### Mouse cardiac slice culture
+
+The study complied with all institutional and national requirements for the care and use of laboratory animals (approved by Tufts IACUC, protocol number G2022-122). Adult B6 (Jackson Laboratory) male mice, a strain of mice with normal hearts, were euthanized using CO~2~, and each heart was harvested immediately after euthanasia and kept in cold (4°C) sterile slicing solution (as described above) for transport and immediate slicing. The base of the heart was transected to remove the great vessels and atria and create a flat surface perpendicular to the long axis of the heart. This surface was then affixed to the cutting block of a vibrating microtome using cyanoacrylate glue and submerged in cold oxygenated slicing solution. 300-μm slices were cut with a frequency of 75 Hz and amplitude of 1.5 mm, advancing at 0.1 mm/s. Resulting slices were washed once in Claycomb media with 100 μM norepinephrine, 10% fetal bovine serum, and 4 mM L-glutamine, before being placed on a 0.3 μm pore size transwell in the same Claycomb media (or the canine media above) at the air-media interface at 37°C with 2% CO~2~. A flow chart summarizes the experiments performed with the cardiac slices is shown in S1 Fig.
+
+### Cardiac slice MTT assay
+
+Cell viability within the slices was assessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Invitrogen, M6494). First, the slices were incubated in 1 ml of 0.5 mg/ml MTT solution at 37°C with 5% CO~2~ (canine) or 2% CO~2~ (mice) for 1 hour. The slices were then placed in 1 ml of PBS for 5 minutes at 37°C with 5% CO~2~ (canine) or 2% CO~2~ (mice), and subsequently incubated in DMSO for 30 minutes at 37°C with 5% CO~2~ (canine) or 2% CO~2~ (mice). The absorbance of the resulting supernatant was assessed by Optical Density measured at 555 nm and normalized by the weight.
+
+### Cardiac slice histology
+
+Slices were fixed in 10% formaldehyde in PBS for 24 hours, then in 70% ethanol prior to sectioning. Hematoxylin and eosin (H&E) staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) (Abcam, ab206386) were then performed. Quantification of the number of TUNEL+ nuclei was assessed by examining at least five light microscopy images from independent biological samples by treatment-blinded investigators. Since we were unable to validate any antibodies to caspase 3 for accurate quantification of canine protein, TUNEL staining was used to quantify apoptosis for the canine cardiac slices.
+
+We used the Bellingham grading system to describe severity of pathologic changes in heart tissue secondary to doxorubicin treatment as it was developed to characterize tissue damage specifically in the context of doxorubicin cardiotoxicity [28]. Grade 0 indicates no change from normal myofibrillar structure, grade 1 indicates early myofibrillar loss, grade 2 indicates marked myofibrillar loss with cytoplasmic vacuolization, and grade 3 indicates diffuse cell damage with marked changes.
+
+### Tissue fluorescence imaging
+
+Direct imaging of the inherent doxorubicin fluorescence was carried using an IVIS spectrum (Perkin Elmer). After exposure to 0–50 μM of doxorubicin for 24 hours, the slices were washed with PBS overnight before imaging. Imaging was performed with the filter setting 500 nm excitation/540 nm emission, 20 second exposure, with 135 μm in-plane resolution. Doxorubicin fluorescence signal was quantified in ImageJ (NIH) by calculating the signal-to-noise ratio (mean fluorescence intensity of heart/standard deviation of air).
+
+### Protein immunoblotting
+
+Cardiac slices were flash-frozen in liquid nitrogen, sonicated, and homogenized on ice in radio-immunoprecipitation assay buffer supplemented with protease inhibitors (Boston BioProducts) per manufacturer’s instructions. Proteins were extracted by constant agitation for 30 minutes before centrifugation. Protein concentration was measured by Bradford assay (BioRad), and 20–40 μg of total protein was loaded for electrophoresis and transferred to the polyvinylidene difluoride membrane. The membrane was processed for immunoblotting against LC3 (CST 3868, rabbit monoclonal, clone: D11; 1:1000, antibody ID: AB_2797680), p62 (CST 5114, rabbit polyclonal, 1:1000, antibody ID: AB_10624872), and actin (Sigma A5316, mouse monoclonal, clone: AC-74; 1:5000, antibody ID: AB_476743) and imaged with a ChemiDoc XRS molecular imager (BioRad). Immunoblot images are analyzed using ImageJ by measuring the LC3-I, LC3-II, p62, and actin signal intensity and calculating the LC3-II to LC3-I, LC3-II to actin, and p62 to actin ratios.
+
+### Immunostaining and confocal microscopy
+
+Cardiac slices were fixed, sectioned, permeabilized, and stained with the LC3 antibody (MBL M152-3, mouse monoclonal, 1:100, antibody ID: AB_1279144) followed by fluorescent anti-mouse secondary antibodies (Jackson ImmunoResearch Lab 715-585-151, donkey polyclonal, 1:200, antibody ID: AB_2340855). After nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI), the sections were visualized on a Falcon SP8 system (Leica) with images acquired at 1024×1024 resolution. The images were analyzed in ImageJ.
+
+### Statistical analysis
+
+Normality of data distribution was assessed using the D’Agostino-Pearson normality test. For normally distributed data, statistical analysis was performed using the one-way ANOVA test with Tukey post-test for comparison of three or more groups or Dunnett’s test for multiple comparison test against a single baseline. All statistical analysis and plots of mean ± SEM were performed using Prism (GraphPad). *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.
+
+## Results
+
+### Canine cardiac slices maintain viability in culture longer than mouse cardiac slices
+
+The duration of cell viability within canine and mouse cardiac slices was first quantified in culture by MTT. Cultured canine slices maintained consistently high viability for 7 days, and by day 14, cardiac tissue remained on average 82.4% viable compared to the day of collection (n = 7, p = 0.99 day 14 vs. day 1) (Fig 1A). Histological staining throughout the first 7 days of culture showed minimal myofibrillar damage based on the Billingham pathologic grade [29], and no change in TUNEL staining as a measure of cell death (Fig 1B and 1C). By comparison, the mouse slices were not viable immediately after slicing when cultured in the same media. They were, however, able to maintain viability in the Claycomb media in a 2% CO~2~ environment. Despite no significant change in tissue viability by MTT assay in the Claycomb media through the first 7 days of culture (n = 4, p = 0.54 day 7 vs. day 1), a significant increase in TUNEL+ staining was noted after 3 days of culture, suggesting increase in apoptotic cell death (p = 0.006, day 3 vs. day 1) (Fig 1D–1F). These results also suggest that TUNEL staining analysis is more sensitive to changes in cellular viability than MTT assay. Based on these data, the canine slice model was deemed more suitable for long term culture than mouse slices, and hence all subsequent studies were performed using canine cardiac slices to study autophagic flux.
+
+*Figure: Fig 1 Canine cardiac slices are viable for >7 days in culture.A. No significant reduction in adult canine cardiac slice viability through 14 days of culturing (arrow) by MTT staining was noted. N = 7 hearts. B. H&E staining demonstrated minimal myofibrillar damage during Day 1 (Billingham grade 0), 3 (Billingham grade 0), 5 (Billingham grade 1), and 7 (Billingham grade 1) of culture, and TUNEL staining showed minimal TUNEL+ apoptotic cells. Red arrows: TUNEL+ nuclei. C. Quantification shows a low baseline of TUNEL+ nuclei which did not increase during 7 days in culture (n = 3 per condition). D. Adult mouse cardiac slices had much lower viability when cultured in the same canine media (CM) compared to the Claycomb media (MTT purple staining indicates viable tissue), E. in which tissues showed no significant decrease in viability within the 7 days of culture by MTT staining. F. However, TUNEL staining showed significant increase in apoptotic cells by day 3 of culture. N = 4, ANOVA with Dunnett’s post-test. *p<0.05, **p<0.01, ns = no significant change.*
+
+### Characterization of basal autophagy in canine cardiac slices
+
+Autophagic flux was quantified by measuring the protein levels of autophagy markers LC3-II and p62 in the cardiac slices over the course of culture or post treatment with well-established autophagy modulators (S2 Fig) [14, 30]. Expression levels of these proteins did not significantly change during the 7 days of culture (Fig 2A–2D). Rapamycin inhibits mTOR complex, triggering downstream signaling cascades that upregulate autophagic flux, evidenced by increased LC3 lipidation to form LC3-II (p<0.0001) without impacting the level of p62 (Fig 2E–2H), consistent with autophagy induction that had reached equilibrium [13, 14]. Conversely, autophagy inhibition with chloroquine, which disrupts lysosomal degradation of autophagosomes, led to an accumulation of both LC3-II (p<0.0001) and p62 (p = 0.0004), consistent with autophagic flux blockade. These data support the notion that canine cardiac slices maintain autophagic flux during culture and that the impact of known activator/inhibitor of autophagy can be accurately quantified in these tissue slices.
+
+*Figure: Fig 2 Basal autophagy is preserved in cultured canine cardiac slices.A-D. Representative immunoblots for cardiac slices cultured for 1–7 days. Quantification showed no change in expression of autophagy proteins LC3-II and p62. E-H. Autophagy was induced by 0.1 μM rapamycin (Rap) or blocked by 20 μM chloroquine (Cq) for 24 hours. Rapamycin significantly increased LC3-II expression but not p62 compared to vehicle (Veh) control. Chloroquine led to the accumulation of LC3-II and p62. Protein quantifications were normalized to Actin. N = 3, ***p<0.001, ****p<0.0001, ns = no significant change, ANOVA with Tukey post-test.*
+
+### Doxorubicin induces apoptotic cell death and impairs autophagy in canine cardiac slices
+
+Next, we evaluated whether cardiac tissue damage and changes in autophagic flux secondary to doxorubicin treatment could be modeled using canine cardiac slices. We tested increasing doses of doxorubicin between 10–50 μM and quantified the impact on apoptotic cell death and disruption in autophagic flux. Higher doses of doxorubicin were tested to definitively perturbed the autophagic flux. When incubated with doxorubicin for 24 hours, a dose-dependent increase in tissue damage was observed in the H&E and TUNEL stained sections, as evidenced by mild cardiomyocyte disorganization at 10 μM (Bellingham grade 2) and moderate disorganization with 30 and 50 μM (Bellingham grade 3) (Fig 3A). This correlated with the dose-dependent retention of doxorubicin in the cardiac tissue measured by the inherent fluorescence signature of doxorubicin (Fig 3B). To further quantify apoptosis, slices exposed to 10 μM doxorubicin exhibited an average 141.7% increase in TUNEL+ nuclei compared to untreated slices, which increased significantly to 504.7% (p = 0.0005) at 30 μM, and 430.7% (p = 0.0032) at 50 μM (Fig 3C). Concomitant with the rise in TUNEL+ nuclei, doxorubicin treatment also caused autophagy blockade in the cardiac slices. Specifically, there was a dose-dependent accumulation of LC3-II and p62 (Fig 3D–3G and S3 Fig). Consistent with the blockade of autophagic flux, immunostaining showed increased LC3 accumulation in doxorubicin-treated slices compared to untreated slices (Fig 3H and S4 Fig).
+
+*Figure: Fig 3 Doxorubicin induces cardiac tissue damage, apoptotic cell death, and impairs autophagy.Canine cardiac slices were exposed to increasing concentrations of doxorubicin (Dox, 10–50 μM) for 24 hours. A. Representative H&E and TUNEL stained sections exposed to 0–50 μM of doxorubicin. 0 μM: Bellingham grade 0; 10 μM: Bellingham grade 2; 30 and 50 μM: Bellingham grade 3. Blue arrows: disorganized myocyte fibers. Red arrows: TUNEL+ nuclei. B. Quantification of doxorubicin retention in cardiac slices by direct fluorescence tissue imaging. SNR = signal-to-noise ratio. N = 4. C. Quantification of TUNEL+ nuclei in cardiac slices exposed to 0, 10, 30, or 50 μM of doxorubicin for 24 hours (n = 4). D-G. Representative western blots and quantification of autophagy proteins (LC3-II, p62) in slices treated with increasing concentrations of doxorubicin for 24 hours. N = 3. H. Immunostaining of LC3 demonstrated robustly increased LC3 accumulation (green = LC3, blue = DAPI) in cardiomyocytes of slices exposed to doxorubicin. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 compared to untreated controls unless otherwise noted, ANOVA with Tukey post-test.*
+
+### Rapamycin rescued cardiomyocyte cell death by restoring autophagic flux
+
+To test whether the canine slice model could be used to screen for potential cardioprotective strategies that reverse the detrimental impact of doxorubicin, we investigated the effects of rapamycin co-treatment with doxorubicin on autophagic flux and apoptosis. Rapamycin significantly reduced the accumulation of LC3-II (p = 0.0040) and p62 levels (p = 0.0016) due to doxorubicin treatment (Fig 4A–4D). This was associated with a significant reduction in apoptotic cell death in rapamycin treated cardiac slices compared to treatment with 50 μM doxorubicin alone (p = 0.0045) (Fig 4E and S5 Fig). Effects on the autophagic flux and the impact on apoptotic cell death are summarized in Fig 4F.
+
+*Figure: Fig 4 Autophagy induction by rapamycin significantly attenuated cardiomyocyte apoptotic cell death.Cardiac slices were exposed to 50 μM doxorubicin with or without rapamycin (Rap) co-treatment. A-D. Representative immunoblots and quantification of LC3-II and p62. E. Expression increases of LC3-II and p62 with doxorubicin were mitigated with rapamycin co-treatment for 24 hours, leading to cardiomyocyte apoptosis reduction. N = 3. F. The effects of doxorubicin, rapamycin, and chloroquine treatment on LC3-II and p62 expression, and the overall effect on autophagic flux and apoptotic cell death are summarized. **p<0.01, ***p<0.001, ****p<0.0001, ANOVA with Tukey post-test.*
+
+## Discussion
+
+We report here the use of adult canine hearts from organ donors to generate a LV cardiac tissue culture model relevant for dissecting the mechanisms and identifying protective strategies secondary to toxin-induced cardiac damage. Specifically, we demonstrate that canine cardiac slices: 1) remain viable in culture for at least a week without induction of apoptosis; 2) maintain autophagic flux for at least 7 days in culture and respond as expected to autophagic flux modulators; 3) develop loss of structural integrity, impaired autophagic flux, and increased apoptosis in response to the cardiotoxic doxorubicin; 4) and can be rescued from doxorubicin-induced toxicity by co-treatment with rapamycin, an autophagy activator. This improved cardiac tissue culture technique allows for the study of the autophagic flux in intact adult cardiac tissue without subjecting the tissue to enzymatic digestion. Importantly, this translational model has the potential to generate critical insights into acute toxic responses to drugs and their associate cellular and molecular effects with the cardiac cells in the context of their native structure, as well as to evaluate the impact of novel treatment strategies designed to prevent cardiac toxicities.
+
+In contrast to the canine cardiac slices, mouse cardiac slices could not be maintained in culture under the same conditions. Despite improved tissue viability with modifications of the culture parameters including the use of Claycomb media with 2% CO~2~, increased apoptotic cell death was still noted after just 3 days. Not only do canine cardiac tissues remain viable in culture for longer periods of time, canine cardiomyocytes possess cellular and molecular features that are more similar to human cardiomyocytes than those from mice. Thus, data generated from canine tissue slices are more likely to have translational relevance for human disease. Furthermore, given the shorter life expectancy of canines compared to humans, especially after cancer diagnosis, longitudinal studies involving terminal tissue harvest can be accomplished within 1–2 years, as opposed to decades required for similar human studies. Consequently, the impact of cardiotoxic treatments can be readily assessed in the context of treating canine patients with spontaneous cancer, permitting interactions among co-morbidities, cancer specific factors and drugs to be studied in this model system [26, 31–34].
+
+Our data further demonstrated that the canine cardiac slice culture system can be used to study the effects of cancer treatment on the autophagic process. The slices showed stable basal levels of autophagy and responded appropriately to rapamycin, an activator of autophagic flux, and chloroquine, an inhibitor that blocks autophagosome and lysosome fusion. Furthermore, we noted that autophagic flux blockade by doxorubicin led to cardiomyocyte cell death, similar to previous reports using isolated murine and human cardiomyocytes and murine hearts *in vivo* [16, 18]. It is worth noting that the canine cardiac slices exhibited LC3-II accumulation and increased p62 expression, indicative of acute autophagy blockade [13], in contrast to the autophagy precursor-limited flux blockade previously reported to occur in human embryonic stem cell-induced cardiomyocytes where p62 was noted to decrease in conjunction with an increase in LC3 [18]. This acute impairment in autophagic flux in the canine cardiac slices can be restored by rapamycin co-treatment, mitigating cardiac cell death induced by doxorubicin.
+
+While boosting autophagy prior to doxorubicin administration can be cardioprotective, excessive initiation of autophagy after doxorubicin exposure may be detrimental [35]. Doxorubicin exposure for 24 hours induced cell death in canine cardiac slices and is associated with autophagic flux blockade, evident by the accumulation of autophagy proteins (LC3-II, p62) and the increase in apoptosis. This is consistent with previous reports in neonatal rat cardiomyocytes and mouse hearts, supporting the pathophysiological role of cardiac autophagy in doxorubicin-induced toxicity, where blockade of autophagic flux and accumulation of autophagosomes exacerbates cell death [18, 36, 37]. Our results confirm that the doxorubicin-induced autophagic blockage phenomenon also occurs in canine adult intact heart tissues and in a species regularly treated with doxorubicin for cancer therapy. As such, the canine cardiac culture system provides a platform to investigate effects of autophagic blockage in heart tissue acquired from actual canine patients treated with doxorubicin.
+
+Current clinical options to prevent chemotherapy-induced cardiotoxicity are limited and not routinely used [38]. Autophagy modulation achievable by a myriad of FDA-approved drugs represents a viable cardioprotective strategy, but approaches exploiting this biology have not yet been translated into the clinical setting [39, 40]. While the cardiotoxic effects of doxorubicin are attributed predominantly to direct cardiomyocyte injury, newer targeted therapies such as tyrosine kinase inhibitors may act through vascular cell damage and impact cardiomyocyte autophagy, both of which may lead to systolic dysfunction or heart failure [41–43]. The cardiac slice culture addresses such limitations by encompassing the various cell types and tissue architecture, which will allow visualization of protein important in characterizing autophagic flux, such as LC3 and p62, by immunostaining as well as quantification by protein blot in slices while preserving structural and electrophysiological interactions between these cell types [20, 24, 25, 44, 45]. Consequently, the canine cardiac slice model represents a viable platform for screening cancer treatments for cardiotoxicity while also providing a mechanism to screen novel strategies designed to mitigate these toxicities.
+
+### Limitations
+
+Several limitations should be acknowledged. Our *ex vivo* system does not include electrical stimulation of the cardiac tissue, which would replicate even more closely the native state of the cardiomyocytes. Despite this, we were still able to demonstrate stable basal levels of autophagy with normal flow of the autophagic process. Comparison of results with human cardiac slices would also help determine any cross-species differences. However, healthy human donor hearts are difficult to obtain to perform such comparisons. This highlights the advantage of using canine hearts that can be easily obtained in a humane fashion from patients. Because we are utilizing heart tissues from client-owned dogs of various breeds, age group, and may have other systemic diseases not known at the time of tissue collection; for example, many of them were obese, these factors may affect cardiac function. Given our sample size, we cannot make any conclusions regarding how these factors may influence the tissue responses to doxorubicin or rapamycin. Future work will be needed to better understand the effects of these factors on autophagy. Nevertheless, a potential advantage is that these are similar co-morbidities experienced by human patients, allowing such studies to reflect more clinically relevant conditions.
+
+## Conclusion
+
+We report here the validation of adult canine cardiac slice culture as a novel platform relevant for use in drug-induced cardiotoxicity studies, including those designed to elucidate the mechanistic underpinnings of autophagy and its disruption in cancer drug-induced injury. Our data further support testing of autophagy modulation as a protective therapy during cardiotoxic cancer treatment. This *ex vivo* system will enable future studies that incorporate the influence of cancer intrinsic factors on cardiotoxicity with the use of hearts acquired from canine oncology patients at the time of euthanasia.

fulltext/md/PMC10061196.md

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+---
+pmcid: "PMC10061196"
+pmid: "36852498"
+doi: "10.1111/jvim.16644"
+title: "Thyroidectomy in dogs with thyroid tumors: Survival analysis in 144 cases (1994-2018)."
+journal: "Journal of veterinary internal medicine"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Thyroidectomy in dogs with thyroid tumors: Survival analysis in 144 cases (1994‐2018)
+
+## Abstract
+
+### Background
+
+Few studies have assessed predictors of outcome in dogs with thyroid tumors undergoing thyroidectomy.
+
+### Objective
+
+To estimate the survival and identify prognostic factors in dogs with thyroid tumors treated by thyroidectomy.
+
+### Animals
+
+A total of 144 client‐owned dogs with thyroid neoplasia that underwent thyroidectomy.
+
+### Methods
+
+Retrospective study. Data for analysis included hospital attended and year of surgery, signalment, thyroxine concentration, thyroid tumor features (lobe involvement, size, invasiveness, histopathological type), thrombosis, metastasis, additional surgery and therapy, administration of adjuvant chemotherapy. The association of predictors with survival (time from surgery to death) were assessed by calculating cause‐specific hazard ratios (HR~cs~) and 95% confidence intervals (CI). Causes of death were classified as thyroid‐related or because of other cause.
+
+### Results
+
+Overall median survival time was 802 days (CI95% = 723‐1015 days); 89 dogs (77.4%) survived >500 days. Metastases were identified at admission in 12 (8.3%) dogs and were associated with higher thyroid cancer‐related fatality (HR = 5.83, CI95% = 1.56‐21.78; *P* = .009). Thrombosis occurred in 40 dogs and was associated with increased risk of death because of other cause (HR = 2.73, CI95% = 1.18‐6.35; *P* = .019). Nonfollicular carcinoma (HR = 4.17, CI95% = 1.27‐13.69; *P* = .018) and administration of chemotherapy (HR = 3.45, CI95% = 1.35‐8.82; *P* = .01) were associated with higher risk of thyroid cancer‐related death.
+
+### Conclusions and Clinical Importance
+
+Dogs with thyroid tumors undergoing thyroidectomy have a long life expectancy. Despite the rare presence of nonfollicular carcinoma and metastases, thyroidectomy should still be considered in some of these dogs.
+
+AbbreviationsCIconfidence intervalCIFcumulative incidence functionHRhazard ratioHR~OC~hazard ratio for other cause of deathHR~TC~hazard ratio for thyroid cancerMSTmedian survival timeORodd ratio
+
+1
+
+## INTRODUCTION
+
+Thyroid tumors in dogs have a prevalence of 1‐4% among all neoplasms, and represent the most common tumor of the neuroendocrine system.^1^, ^2^, ^3^, ^4^, ^5^ Older dogs are affected more frequently,^6^, ^7^ and the Beagle, Boxer and Golden Retriever breeds are overrepresented.^2^, ^3^, ^6^, ^8^ Unilateral thyroid lobe involvement is more common than bilateral,^6^, ^9^, ^10^ and approximately 50‐60% of affected dogs are euthyroid, 30‐40% are hypothyroid, and 10‐20% are hyperthyroid.^5^
+
+Carcinomas are the most frequent thyroid tumors in dogs and, among them, 70% arise from follicular cells (ie, follicular thyroid carcinomas) and 30% from parafollicular cells (ie, medullary thyroid carcinomas), both are invasive. Adenomas, on the other hand, are rare in dogs.^3^, ^4^, ^5^, ^11^, ^12^ In dogs, follicular thyroid carcinomas are histologically classified as well‐differentiated (ie, follicular, compact, follicular‐compact and papillary), poorly differentiated, undifferentiated, and carcinosarcoma (malignant mixed thyroid tumor).^12^
+
+At diagnosis, 16‐38% of dogs with thyroid tumors have distant metastasis.^4^, ^13^ Thyroidectomy represents the treatment of choice for freely movable tumors and sometimes could be indicated also for nonresectable ones, along with external beam radiation or radionuclide therapy, chemotherapy or a combination of these therapies.^10^, ^14^, ^15^
+
+There are many purported factors potentially associated with less favorable outcome. Undifferentiated carcinomas have a less favorable outcome,^5^, ^16^, ^17^ however there are conflicting reports in the literature regarding local invasiveness and outcome.^7^, ^18^, ^19^ Tumor size was a negative prognostic factor that appeared also related to the presence of metastases, in an investigation with specimens obtained at necropsy,^20^ but not in other reports with measurements performed at the time of thyroidectomy.^14^, ^16^, ^18^ Thrombosis of the tumor and cervical vessels negatively affects the outcome.^7^ Higher WHO staging and metastatic disease (local or distant) might not result in a poor outcome.^14^, ^21^
+
+A median survival time (MST) of 500‐1100 days is reported after thyroidectomy.^7^, ^18^, ^19^, ^22^, ^23^ In particular, MST >960 days is documented both in dogs undergoing unilateral and bilateral thyroidectomy,^18^, ^23^ and 630 days in dogs with gross vascular invasion treated with unilateral or bilateral thyroidectomy.^19^ Bilateral thyroidectomy negatively affects outcome and the MST of these dogs is 365 days.^14^ Of dogs with functional tumors undergoing thyroidectomy the MST is 2190 days.^24^
+
+No benefit of adjuvant chemotherapy on MST is observed in dogs treated with thyroidectomy alone vs thyroidectomy combined with chemotherapy, irrespective of metastasis.^14^ Administration of toceranib phosphate on macroscopic disease to affected dogs, for a minimum of 10 weeks is beneficial in 75% of cases (ie, complete response to stable disease).^25^ Conversely, no benefit on MST is observed in dogs treated with thyroidectomy alone vs thyroidectomy combined with chemotherapy, irrespective of metastasis.^14^
+
+The aims of the present study were to retrospectively assess survival in dogs with thyroid tumors treated by thyroidectomy, and to identify prognostic factors using competing risk analysis.
+
+2
+
+## MATERIALS AND METHODS
+
+2.1
+
+### Experimental design
+
+This is a multi‐institutional retrospective case series study.
+
+2.2
+
+### Case selection
+
+Medical records of 4 of the authors' institutions (EMM, FM, GR, SN) were retrospectively reviewed in order to identify dogs diagnosed with thyroid tumors that had undergone thyroidectomy between July 18, 1994 and June 20, 2018. Thyroidectomies were performed by both boarded (EMM, FM, GR) and specialized surgeons (SN). Dogs were included if the thyroidectomy had been performed by 1 of the above authors and if follow‐up was available.
+
+For each dog included in the study, variables retrieved from medical records were collected and used to determine their influence on MST, defined as the time from thyroidectomy to death or administrative censoring (last visit). These variables were: the institution where thyroidectomy was performed and the year of surgery, the age (in years, at the time of surgery), gender, neutering status (ie, intact, spayed and castrated), breed, dog size (ie, small, <10 kg; medium, 10‐25 kg; large, >25 kg), presurgical serum thyroxine (T4) concentration (ie, increased, normal or decreased), thyroid lobe involvement (ie, unilateral or bilateral), type of thyroidectomy (ie, unilateral or bilateral), additional surgeries performed (ie, cervical lymphadenectomy and muscular, internal jugular vein resection and parathyroid gland autotransplantation^26^), tumor major axis size, histopathological tumor type, local invasiveness (determined by palpation characteristics of the ventral cervical area combined with computed tomography examination of the same area in some dogs and histopathological examination in others, or histopathological examination in others), thrombosis, metastatic disease at the time of surgery (determined by computer tomography and histopathological examination), administration of adjuvant chemotherapy, radiation therapy after thyroidectomy, levothyroxine and calcitriol supplementation following surgery. In dogs diagnosed with bilateral thyroid lobes tumor involvement, who undergone bilateral thyroidectomy, at least 1 of the parathyroid glands was transplanted. The parathyroid gland was freed from his thyroid capsule, inserted in a muscular pocket, which allowed revascularization and regaining functionality after few weeks. The effect of each aforementioned variable on survival was studied in relation to the cause of death which was eithers thyroid cancer or other cause.
+
+2.3
+
+### Statistical analysis
+
+Competing risk analysis was used in this study. This was because the classical Kaplan‐Meier analysis, which censors the observed survival time when a competing event intervenes, violates the assumption of independence between time to event and censoring, yielding an underestimate of the survival probability^27^ and overestimating the probability of death. Two competing events were considered, that is, death because of thyroid cancer or death because of another cause. The (cause specific) cumulative incidence function (CIF) estimates the marginal probability for each competing event (ie, the probability of experiencing the event of interest before time t and before the occurrence of a different type of event) as a function of time and was used to estimate the likelihood of the occurrence of each of the 2 causes of death. Gray's test was used to compare cause‐specific CIFs in dogs classified according to each variable gathered at surgery.^28^ The extended Cox proportional hazards model was used to estimate the cause‐specific hazard ratio (HR) for each variable.^29^, ^30^ The model accounted for the multicentric nature of the study by allowing different baseline hazards for the 4 clinics. The proportional hazards assumption was evaluated by studying the Schoenfeld residuals.^31^ Linearity was assumed based on the analysis of the martingale residuals. After an initial assessment, the effect of neutering was not considered.
+
+In order to preserve the sample size, the random survival forest algorithm for imputing missing data was used.^32^ Bivariate associations between variables were investigated by Fisher's exact test and *t*‐test. Logistic regression was used to model the probability of concurrent metastasis and thrombosis at diagnosis, and odds ratios (ORs) were calculated. All analyses were performed using R with *mstate*, *cmprsk*, *randomForestSRC* and *survival* packages.^31^, ^33^, ^34^, ^35^
+
+3
+
+## RESULTS
+
+3.1
+
+### Dogs
+
+Medical records of 144 dogs with thyroid tumors that had undergone thyroidectomy fulfilled the inclusion criteria and were considered for analysis. Dogs were treated in the 4 authors' institutions, named A, B, C and D; of which, 78 (54.2%) were treated in A, 24 (16.7%) in B, 34 (22.9%) in C and 9 (6.3%) in D. Seventy‐four dogs (51.4%) were female and 66 (45.8%) were male; gender was not available in 4 cases (2.8%). Among females, 39 (52.7%) were intact and 35 (47.3%) were spayed, and among males 16 (24.2%) were intact and 50 (75.8%) were neutered. Median age at the time of surgery was 10 years (range: 4‐15). Forty‐two (29.2%) dogs were crossbreed and 102 (70.8%) were purebred. Beagles (n = 11) and Boxers (n = 11) were most represented, followed by Golden Retrievers (n = 10), English Setters (n = 10) and Siberian Huskies (n = 7). Of the 144 dogs, 13 (9%) were small (<10 kg), 17 (11.8%) medium (10‐25 kg), and 73 (50.7%) large (>25 kg); size was unknown in 41 cases (28.5%).
+
+The affected thyroid lobe was recorded in 135 (93.8%) dogs; 122 (91.1%) cases had unilateral and 13 (9%) had bilateral involvement. Presurgical serum T4 concentration was measured in 103 (71.5%) dogs; 9 (8.7%) cases had abnormally high T4, all with unilateral involvement, 7 (6.8%) cases had abnormally low T4, including 6 cases with unilateral and 1 with bilateral involvement, and 87 (84.5%) cases were euthyroid. Of the euthyroid cases, thyroidal lobe involvement was recorded in 52 (59.8%), including 45 (86.5%) with unilateral, and 7 (13.5%) with bilateral involvement.
+
+The year of surgery was available for all dogs; surgery was performed from July 18, 1994 to June 20, 2018 (Table 1). Thyroidectomy alone was performed in 109 of the 144 (75%) dogs, while in 35 (24.3%) additional surgery was required, including cervical lymphadenectomy, muscular and internal jugular vein resection, and parathyroid gland autotransplantation.^26^ Often, even with marginal resection it was possible to obtain a complete removal of the thyroid tumors, which was considered curative. Whenever possible, the surgical procedures performed have led to complete removal of the thyroid tumors, even with small margins. In those cases where local invasion of the blood vessels or nerves was noted, resection of the venous structures was performed, while the arterial and nervous structures were subjected to debulking with safeguarding of the latter. This approach has still allowed an excellent local control of the thyroid tumors. At surgery, the mean major axis of the excised lobe was measured in 142 (98.6%) dogs which was 4.6 cm (range, 4‐16). Of 141 (97.9%) dogs with histopathologic assessment of the thyroid sample, 128 (90.8%) had differentiated and 13 (9.2%) had undifferentiated thyroid tumors. One (0.8%) of the 129 dogs with a differentiated thyroid tumor had an adenoma, while the remaining 128 (99.2%) had a carcinoma. Computed tomography of the skull, ventral cervical area, thorax and abdomen was performed in 91 (62.8%) dogs; tumor and jugular vein thrombosis was identified in 40 of 144 (27.8%) cases, and lymphadenopathy and suspected metastases in 12 (8.3%). Other dogs included in the study had similar staging tests (ie, thoracic x‐rays, abdominal ultrasound scan). In case of regional lymphadenopathy (eg, prescapular, mandibular, retropharyngeal), a fine needle aspiration was performed in order to evaluate the presence of metastases, while the lung nodules were suspected of being metastases by means of computer tomography images.
+
+**TABLE 1 Descriptive statistics of dogs with thyroid tumors treated by thyroidectomy.**
+
+|  | Surviving (N = 55) | Death by thyroid cancer (N = 29) | Death by other cause (N = 60) | Overall (N = 144) |
+| --- | --- | --- | --- | --- |
+| Sex |  |  |  |  |
+| Female | 27 (49.1%) | 18 (62.1%) | 29 (48.3%) | 74 (51.4%) |
+| Male | 26 (47.3%) | 10 (34.5%) | 30 (50%) | 66 (45.8%) |
+| Not available | 2 (3.6%) | 1 (3.4%) | 1 (1.7%) | 4 (2.8%) |
+| Age (years) |  |  |  |  |
+| Mean (SD) | 9.97 (2.33) | 9.11 (2.47) | 9.81 (2.43) | 9.73 (2.41) |
+| Median [Min, Max] | 11 [4, 15] | 9 [5, 13] | 10 [5, 14] | 10 [4, 15] |
+| Not available | 2 (3.6%) | 1 (3.4%) | 1 (1.7%) | 4 (2.8%) |
+| Breed |  |  |  |  |
+| Crossbreed | 13 (23.6%) | 13 (44.8%) | 16 (26.7%) | 42 (29.2%) |
+| Purebred | 42 (76.4%) | 16 (55.2%) | 44 (73.3%) | 102 (70.8%) |
+| Size |  |  |  |  |
+| Small | 8 (14.5%) | 2 (6.9%) | 3 (5%) | 13 (9%) |
+| Medium | 8 (14.5%) | 1 (3.4%) | 8 (13.3%) | 17 (11.8%) |
+| Large | 26 (47.3%) | 14 (48.3%) | 33 (55%) | 73 (50.7%) |
+| Not available | 13 (23.6%) | 12 (41.4%) | 16 (26.7%) | 41 (28.5%) |
+| T4 levels |  |  |  |  |
+| Normal | 30 (54.5%) | 17 (58.6%) | 40 (66.7%) | 87 (60.4%) |
+| Increased | 5 (9.1%) | 2 (6.9%) | 2 (3.3%) | 9 (6.3%) |
+| Decreased | 2 (3.6%) | 1 (3.4%) | 4 (6.7%) | 7 (4.9%) |
+| Not available | 18 (32.7%) | 9 (31%) | 14 (23.3%) | 41 (28.5%) |
+| Year of surgery |  |  |  |  |
+| 1994 | 0 (0%) | 2 (6.9%) | 2 (3.3%) | 4 (2.8%) |
+| 1997 | 1 (1.8%) | 0 (0%) | 2 (3.3%) | 3 (2.1%) |
+| 1998 | 1 (1.8%) | 1 (3.4%) | 1 (1.7%) | 3 (2.1%) |
+| 1999 | 2 (3.6%) | 0 (0%) | 1 (1.7%) | 3 (2.1%) |
+| 2000 | 0 (0%) | 0 (0%) | 2 (3.3%) | 2 (1.4%) |
+| 2001 | 2 (3.6%) | 2 (6.9%) | 1 (1.7%) | 5 (3.5%) |
+| 2002 | 1 (1.8%) | 0 (0%) | 4 (6.7%) | 5 (3.5%) |
+| 2003 | 1 (1.8%) | 1 (3.4%) | 3 (5%) | 5 (3.5%) |
+| 2004 | 0 (0%) | 0 (0%) | 2 (3.3%) | 2 (1.4%) |
+| 2005 | 0 (0%) | 2 (6.9%) | 5 (8.3%) | 7 (4.9%) |
+| 2006 | 2 (3.6%) | 3 (10.3%) | 1 (1.7%) | 6 (4.2%) |
+| 2007 | 0 (0%) | 0 (0%) | 4 (6.7%) | 4 (2.8%) |
+| 2008 | 1 (1.8%) | 1 (3.4%) | 7 (11.7%) | 9 (6.3%) |
+| 2009 | 2 (3.6%) | 5 (17.2%) | 4 (6.7%) | 11 (7.6%) |
+| 2010 | 1 (1.8%) | 2 (6.9%) | 5 (8.3%) | 8 (5.6%) |
+| 2011 | 3 (5.5%) | 2 (6.9%) | 3 (5%) | 8 (5.6%) |
+| 2012 | 3 (5.5%) | 0 (0%) | 3 (5%) | 6 (4.2%) |
+| 2013 | 3 (5.5%) | 2 (6.9%) | 3 (5%) | 8 (5.6%) |
+| 2014 | 6 (10.9%) | 2 (6.9%) | 3 (5%) | 11 (7.6%) |
+| 2015 | 6 (10.9%) | 1 (3.4%) | 3 (5%) | 10 (6.9%) |
+| 2016 | 4 (7.3%) | 3 (10.3%) | 0 (0%) | 7 (4.9%) |
+| 2017 | 10 (18.2%) | 0 (0%) | 0 (0%) | 10 (6.9%) |
+| 2018 | 6 (10.9%) | 0 (0%) | 1 (1.7%) | 7 (4.9%) |
+| Major axis (cm) |  |  |  |  |
+| Mean (SD) | 4.65 (2.8) | 6.26 (2.88) | 5.33 (2.18) | 5.25 (2.63) |
+| Median [min, max] | 4 [1, 16] | 6 [2.5, 13] | 5 [2, 12] | 4.75 [1, 16] |
+| Not available | 0 (0%) | 0 (0%) | 2 (3.3%) | 2 (1.4%) |
+| Histology |  |  |  |  |
+| Undifferentiated | 3 (5.5%) | 7 (24.1%) | 3 (5%) | 13 (9%) |
+| Follicular | 50 (90.9%) | 22 (75.9%) | 56 (93.3%) | 128 (88.9%) |
+| Not available | 2 (3.6%) | 0 (0%) | 1 (1.7%) | 3 (2.1%) |
+| Lobar involvement |  |  |  |  |
+| Unilateral | 46 (83.6%) | 29 (100%) | 47 (78.3%) | 122 (84.7%) |
+| Bilateral | 4 (7.3%) | 0 (0%) | 9 (15%) | 13 (9%) |
+| Not available | 5 (9.1%) | 0 (0%) | 4 (6.7%) | 9 (6.3%) |
+| Metastasis |  |  |  |  |
+| No | 52 (94.5%) | 21 (72.4%) | 59 (98.3%) | 132 (91.7%) |
+| Yes | 3 (5.5%) | 8 (27.6%) | 1 (1.7%) | 12 (8.3%) |
+| Thrombosis |  |  |  |  |
+| No | 43 (78.2%) | 17 (58.6%) | 44 (73.3%) | 104 (72.2%) |
+| Yes | 12 (21.8%) | 12 (41.4%) | 16 (26.7%) | 40 (27.8%) |
+| Additional surgery |  |  |  |  |
+| No | 45 (81.8%) | 17 (58.6%) | 47 (78.3%) | 109 (75.7%) |
+| Yes | 10 (18.2%) | 12 (41.4%) | 13 (21.7%) | 35 (24.3%) |
+| Chemotherapy |  |  |  |  |
+| No | 43 (78.2%) | 11 (37.9%) | 43 (71.7%) | 97 (67.4%) |
+| Yes | 11 (20%) | 16 (55.2%) | 13 (21.7%) | 40 (27.8%) |
+| Not available | 1 (1.8%) | 2 (6.9%) | 4 (6.7%) | 7 (4.9%) |
+| Levothyroxine |  |  |  |  |
+| No | 46 (83.6%) | 28 (96.6%) | 50 (83.3%) | 124 (86.1%) |
+| Yes | 8 (14.5%) | 1 (3.4%) | 9 (15%) | 18 (12.5%) |
+| Not available | 1 (1.8%) | 0 (0%) | 1 (1.7%) | 2 (1.4%) |
+| Calcium or vitamin D |  |  |  |  |
+| No | 49 (89.1%) | 28 (96.6%) | 51 (85%) | 128 (88.9%) |
+| Yes | 5 (9.1%) | 1 (3.4%) | 8 (13.3%) | 14 (9.7%) |
+| Not available | 1 (1.8%) | 0 (0%) | 1 (1.7%) | 2 (1.4%) |
+| Center |  |  |  |  |
+| A | 27 (49.1%) | 14 (48.3%) | 37 (61.7%) | 78 (54.2%) |
+| B | 15 (27.3%) | 6 (20.7%) | 3 (5%) | 24 (16.7%) |
+| C | 8 (14.5%) | 8 (27.6%) | 17 (28.3%) | 33 (22.9%) |
+| D | 5 (9.1%) | 1 (3.4%) | 3 (5%) | 9 (6.3%) |
+
+Among all dogs, 40 (27.6%) received adjuvant chemotherapy (Table 1); in 7 out of 144 (4.8%), the information regarding the use of adjuvant chemotherapy was missing. Radiotherapy (once, unknown dose) and doxorubicin (30 mg/m^2^, q3w, 3 times) was administered in 1 dog, cyclophosphamide (15 mg/m^2^, q24h, metronomic) and doxorubicin (1 mg/kg, q3w) in 1 dog, toceranib (2.8 mg/kg, 3 times/week, 120 times) in 1 dog, cyclophosphamide (10 mg/m^2^, q24h, metronomic) in 1 dog, carboplatin (300 mg/m^2^, q3w, 4 times) in 8 dogs, doxorubicin (1 mg/kg, q3w, 2 times) in 2 dogs, doxorubicin (1 mg/kg, q3w, 3 times) in 4 dogs, doxorubicin (1 mg/kg, q3w, 5 times) in 6 dogs, while the remaining dogs have received doxorubicin (1 mg/kg, q3w, 4 times).
+
+Eighteen (12.4%) dogs received levothyroxine supplementation and 14 (9.7%) calcitriol and calcium following surgery, or calcium following surgery. Of the latter, 12 (85.7%) cases had bilateral thyroid lobes involvement and benefited also from autotransplantation of 1 of the parathyroid glands. Twelve (66.7%) of these 18 dogs were euthyroid before thyroidectomy, 2 (11.1%) had decreased T4, 2 (11.1%) had increased T4, and in the remaining 2 (11.1%) the thyroid status was not recorded before surgery.
+
+3.2
+
+### Risk of death during study period
+
+The observational period ranged between 7 and 3240 days (median 610 days), with the accrual of 280 dog‐years. During this interval, 29 (20.1%) dogs died because of thyroid cancer and 60 (41.7%) died because of other causes (laryngeal paralysis in 1 dog, dog fight in 2 dogs, pulmonary thromboembolism in 1 dog, hepatocellular carcinoma with metastasis in 1 dog, and euthanasia because of unrelated thyroid cancer diseases, hypothesized based on clinical records in the remaining dogs); necropsies were not performed, and the causes of death were inferred from the clinical records. The remaining 55 (38.2%) dogs were still alive at the time of data collection or were lost to follow‐up and their survival time was right censored at the time of the last examination. The median time to censoring was 581 (minimum 30, maximum 1453). Cumulative mortality functions for both causes of death (because of thyroid cancer or other cause) and overall (ie, for all the causes) survival of dogs are reported in Figure 1. The MST for the 144 dogs was 802 days (CI95% = 723‐1045); of which, 89 (74.04%) survived >500 days. The MST and cumulative mortality function for both causes of death is detailed in Table 2. The probability of dying because of thyroid cancer was estimated to be 10.4% after 1 year, whereas the probability of dying from other cause was 10.8%. Two years after surgery, the probability of dying because of thyroid cancer was 18.6%, which after 5 years increased to approximately 26%, leveling off to 28.6% after 6 years. The risk of death for other causes continued to increase over time.
+
+*Figure: FIGURE 1 Kaplan‐Meier estimate of overall survival (ie, regardless of the death cause) and cumulative incidence (mortality) functions for each of the 2 outcomes (death by thyroid cancer and death by other cause).*
+
+**TABLE 2 Cumulative incidence (mortality) and Kaplan‐Meier survival at various time points in dogs with thyroid tumor treated by thyroidectomy.**
+
+|  | Cumulative mortality (%) |
+| --- | --- |
+|  | Thyroid cancer | Other cause | Overall survival |
+| One month | 0.3 | 1.4 | 98.3 |
+| Two months | 1.9 | 2.1 | 96.0 |
+| Three months | 3.5 | 2.1 | 94.4 |
+| Six months | 7.2 | 7.3 | 85.5 |
+| One year | 10.4 | 10.5 | 79.2 |
+| Two years | 18.6 | 25.9 | 55.5 |
+| Three years | 21.8 | 42.3 | 35.9 |
+| Four years | 21.8 | 49.4 | 28.8 |
+| Five years | 26.3 | 61.3 | 12.4 |
+| Six years | 28.6 | 67.2 | 4.2 |
+| Seven years | 28.6 | 68.2 | 3.2 |
+| Eight years | 28.6 | 69.2 | 2.2 |
+
+3.3
+
+### Cause‐specific CIF
+
+The estimated effect of metastasis on cause‐specific CIFs is shown in Figure 2 and Table 3. A higher risk of death because of thyroid cancer was observed in dogs with metastasis (CIF = 0.00 at 30 days, CIF = 0.35 at 365 days; *P* < .001), while the presence of metastasis did not affect risk of death because of other causes of death. No dog with metastasis survived more than 2 years and a half.
+
+*Figure: FIGURE 2 CIFs for each of the 2 outcomes for subjects with or without metastasis at the time of surgery.*
+
+**TABLE 3 Cause‐specific cumulative mortality by presence or absence of metastasis, at various time points after surgery, [CIF].**
+
+|  | Death by other cause | Death by thyroid cancer |
+| --- | --- | --- |
+| Years | No metastasis | Metastasis | No metastasis | Metastasis |
+| 1 | 0.11 | 0.00 | 0.08 | 0.35 |
+| 2 | 0.27 | 0.11 | 0.14 | 0.68 |
+| 3 | 0.45 | 0.11 | 0.17 | 0.78 |
+| 4 | 0.52 |  | 0.17 |  |
+| 5 | 0.64 |  | 0.21 |  |
+| 6 | 0.71 |  | 0.24 |  |
+| 7 | 0.71 |  | 0.24 |  |
+| 8 | 0.74 |  | 0.24 |  |
+
+Regarding histological type, prognosis in dogs with thyroid carcinoma with undifferentiated type (CIF = 0.00 at 30 days, CIF = 0.46 at 365 days) was worse than in those with follicular type (CIF = 0.00 at 30 days, CIF = 0.09 at 365 days; *P* < .001). The histological type did not affect risk of death because of other cause (Figure 3, Table 4).
+
+*Figure: FIGURE 3 CIFs for each of the 2 outcomes for subjects with follicular or nonfollicular thyroid cancer.*
+
+**TABLE 4 Cause‐specific cumulative mortality by histological type, at various time points after surgery.**
+
+|  | Death by other cause | Death by thyroid cancer |
+| --- | --- | --- |
+| Years | Follicular | Other types | Follicular | Other types |
+| 1 | 0.11 | 0.09 | 0.07 | 0.46 |
+| 2 | 0.27 | 0.09 | 0.15 | 0.55 |
+| 3 | 0.42 |  | 0.19 |  |
+| 4 | 0.5 |  | 0.19 |  |
+| 5 | 0.62 |  | 0.23 |  |
+| 6 | 0.69 |  | 0.26 |  |
+| 7 | 0.69 |  | 0.26 |  |
+| 8 | 0.72 |  | 0.26 |  |
+
+The presence of thrombosis was associated with a higher risk of death because of other causes (CIF = 0.03 at 30 days, CIF = 0.62 at 2000 days; *P* = .044), but did not affect risk of death because of thyroid tumor (Figure 4, Table 5).
+
+*Figure: FIGURE 4 CIFs for each of the 2 outcomes for subjects with or without thrombosis at the time of surgery.*
+
+**TABLE 5 Cause‐specific cumulative mortality by presence or absence of thrombosis, at various time points after surgery.**
+
+|  | Death by other cause | Death by thyroid cancer |
+| --- | --- | --- |
+| Years | No thrombosis | Thrombosis | No thrombosis | Thrombosis |
+| 1 | 0.08 | 0.17 | 0.08 | 0.17 |
+| 2 | 0.25 | 0.27 | 0.14 | 0.3 |
+| 3 | 0.42 | 0.43 | 0.16 | 0.38 |
+| 4 | 0.51 | 0.43 | 0.16 | 0.38 |
+| 5 | 0.61 | 0.56 | 0.22 | 0.38 |
+| 6 | 0.71 | 0.56 | 0.26 | 0.38 |
+| 7 | 0.71 | 0.56 | 0.26 | 0.38 |
+| 8 | 0.71 | 0.62 | 0.26 | 0.38 |
+
+Administration of adjuvant chemotherapy was associated with an increase in risk of death because of thyroid cancer (CIF = 0.00 at 30 days, CIF = 0.16 at 365 days; *P* < .001) but not because of other causes (Figure 5, Table 6).
+
+*Figure: FIGURE 5 CIFs for each of the 2 outcomes for subjects with or without postoperative chemotherapy.*
+
+**TABLE 6 Cause‐specific cumulative mortality by administration of postoperative chemotherapy, at various time points after surgery.**
+
+|  | Death by other cause | Death by thyroid cancer |
+| --- | --- | --- |
+| Years | No chemotherapy | Chemotherapy | No chemotherapy | Chemotherapy |
+| 1 | 0.12 | 0.05 | 0.07 | 0.16 |
+| 2 | 0.27 | 0.18 | 0.11 | 0.34 |
+| 3 | 0.44 | 0.32 | 0.13 | 0.41 |
+| 4 | 0.52 | 0.38 | 0.13 | 0.41 |
+| 5 | 0.64 | 0.48 | 0.16 | 0.47 |
+| 6 | 0.76 |  | 0.16 |  |
+| 7 | 0.76 |  | 0.16 |  |
+| 8 | 0.8 |  | 0.16 |  |
+
+3.4
+
+### Cause‐specific hazard ratios
+
+The cause‐specific hazard ratios for thyroid cancer (HR~TC~) and for other cause of death (HR~OC~) are shown in Table 7 and represent the adjusted effect of the covariates on the hazard of the specific event. As expected, older age was associated with death because of other cause (HR~OC~ for an increase of 1 year of age = 1.39, CI95% = 1.19‐1.61; *P* < .001); older age was not associated with death because of thyroid cancer. Neither sex nor breed were associated with the risk of death because of thyroid cancer and other cause. In addition, the risk of death because of other cause was higher for large sizes compared to small size dogs (HR~OC~ = 4.3, CI95% = 1.05‐18; *P* = .043); no association was found with thyroid cancer‐related death. Serum T4 concentration, tumor size, additional surgery, administration of levothyroxine, calcium and calcitriol were not associated with any causes of death.
+
+**TABLE 7 Cause‐specific hazard ratios and 95% confidence interval for each covariate based on the extended Cox Proportional Hazard model calculated for the 2 competing causes of death.**
+
+|  | Thyroid cancer | Other cause |
+| --- | --- | --- |
+|  | HR | HR 95% lower limit | HR 95% upper limit | *P* value | HR | HR 95% lower limit | HR 95% upper limit | *P* value |
+| Age | 0.944 | 0.767 | 1.162 | .587 | 1.383 | 1.188 | 1.611 | <.001 |
+| Sex (male vs female) | 0.573 | 0.23 | 1.427 | .231 | 0.926 | 0.495 | 1.729 | .808 |
+| Breed (pure vs mixed) | 1.04 | 0.383 | 2.826 | .939 | 1.59 | 0.782 | 3.235 | .2 |
+| Size (medium vs small) | 0.341 | 0.027 | 4.333 | .407 | 4.483 | 0.864 | 23.259 | .074 |
+| Size (large vs small) | 0.262 | 0.04 | 1.713 | .162 | 4.34 | 1.047 | 17.992 | .043 |
+| Histology (undifferentiated vs follicular) | 4.175 | 1.273 | 13.689 | .018 | 1.964 | 0.496 | 7.775 | .336 |
+| T4 increased (vs normal) | 0.401 | 0.072 | 2.231 | .297 | 0.574 | 0.112 | 2.949 | .507 |
+| T4 decreased (vs normal) | 1.353 | 0.141 | 12.987 | .794 | 0.888 | 0.213 | 3.701 | .871 |
+| Major axis (cm) | 1.148 | 0.964 | 1.366 | .122 | 1.036 | 0.906 | 1.185 | .605 |
+| Thrombosis (yes vs no) | 2.144 | 0.735 | 6.25 | .163 | 2.732 | 1.178 | 6.336 | .019 |
+| Metastasis (yes vs no) | 5.831 | 1.561 | 21.778 | .009 | 0.507 | 0.055 | 4.653 | .548 |
+| Levothyroxine (yes vs no) | 0.347 | 0.038 | 3.179 | .349 | 1.366 | 0.533 | 3.504 | .516 |
+| Additional surgery (yes vs no) | 0.593 | 0.19 | 1.857 | .37 | 0.868 | 0.308 | 2.447 | .79 |
+| Chemotherapy (yes vs no) | 3.448 | 1.347 | 8.821 | .01 | 0.383 | 0.145 | 1.015 | .053 |
+| Year of surgery (years) | 0.952 | 0.855 | 1.059 | .363 | 0.917 | 0.857 | 0.981 | .012 |
+
+Histological tumor type was associated with the probability of dying because of thyroid cancer but not to other cause. Notably, dogs with nonfollicular tumors had an approximate 4‐fold higher risk of death because of thyroid cancer than those with the follicular type (HR~TC~ = 4.18, CI95% = 1.27‐13.69; *P* = .018).
+
+Thrombosis was associated with a higher risk of death because of other causes: pulmonary thromboembolism in 2 dogs, acute kidney injury in 1 dog, cardiac tamponade in 1 dog, congestive heart failure in 1 dog, frontal sinus carcinoma in 1 dog, mediastinal tumor in 1 dog, splenic hemangiosarcoma in 1 dog, gastric dilatation and volvulus in 1 dog, cardiac base tumor in 1 dog, lung disease not caused by neoplasia in 1 dog, transitional cell carcinoma in 1 dog, euthanasia because of aggressiveness in 1 dog; the remaining dogs were euthanized because of diseases unrelated to thyroid carcinoma (HR~OC~ = 2.73, CI95% = 1.18‐6.33; *P* = .019) but not to thyroid cancer.
+
+Administration of adjuvant chemotherapy was associated with a higher risk of death because of thyroid cancer (HR~TC~ = 3.45, CI95% = 1.35‐8.82; *P* = .01) but not to other cause; overall MST of dogs treated with adjuvant chemotherapy was shorter than the overall MST of dogs that did not receive adjuvant chemotherapy (710 days, CI95% = 550‐1015; 910 days, CI95% =760‐1465; but the difference was not significant at the logrank test *P* = .064).
+
+The year of surgery was associated with better survival because of other cause (HR~OC~ = 0.92 per year, CI95% = 0.86‐0.98; *P* = .012) highlighting a better outcome for the most recent cases.
+
+Metastasis at surgery was associated with death because of thyroid cancer (HR~TC~ = 5.83, CI95% = 1.56‐21.78; *P* = .009) but not with other cause, with a 6‐fold increase in the risk of death by thyroid cancer in dogs compared to those without tumor spread; overall MST of dogs with metastasis was 540 days, and 818 days for dogs without metastasis, 5 out of 12 (41.7%) dogs with tumor spread survived longer than 500 days. Regarding metastasis, associations with other variables were also studied in a multivariate logistic regression model that considered tumor size, histological tumor type, presence of thrombosis and the number of thyroid lobes involved; histological tumor type (OR = 8.1, CI95% = 1.8‐35.2, *P* = .005) was associated with tumor spread.
+
+The presence of metastasis was also associated with that of tumor thrombosis (OR = 3.44, CI95% = 1.07‐16.1; *P* = .045), but not with the other variables.
+
+Tumor thrombosis was observed in 4 cases of the 13 undifferentiated thyroid tumor type and in 8 cases of the 120 follicular thyroid tumor type (*P* = .015).
+
+When modeling the probability of tumor thrombosis in a multivariate logistic regression model, conjointly considering histological type (all other types vs follicular), sex, age, body size, major axis, bilateral thyroid lobe involvement, and metastasis, associations were found with tumor size (OR for 1 cm increase = 1.26, CI95% = 1.09‐1.49; *P* = .003), bilateral thyroid lobe involvement (OR = 4.67, CI95% = 1.4‐17.06; *P* = .014) and presence of metastasis (OR = 3.85, CI95% = 1.02‐15.76; *P* = .048).
+
+4
+
+## DISCUSSION
+
+We investigated survival in dogs affected by thyroid tumors treated with thyroidectomy. We also examined the outcome predictors by classifying death as thyroid related or because of other cause, using competing risk analysis. The overall (ie, for all causes of death) MST of affected dogs was longer than 2 years. Undifferentiated carcinoma, metastasis and administration of adjuvant chemotherapy were associated with a higher hazard of death because of thyroid tumor. Increasing age, larger body size, presence of thrombosis and less recent surgery were associated with shorter survival time because of other cause.
+
+Regarding the survival of affected dogs, the results of this series are similar to previous investigations that indicated an MST ranging between 500 and 1100 days.^11^, ^19^, ^22^, ^23^, ^36^ Based on the cumulative mortality assessment, in the present study the risk of death because of thyroid cancer during the first year after thyroidectomy was approximately 10%, and at 5 years approximately 25%. Not unexpectedly, the latter was significantly lower than the risk of death because of other cause, which was approximately 60%. Thus, most dogs diagnosed with thyroid tumors that survived the thyroidectomy died because of causes unrelated to thyroid cancer.
+
+With regard to outcome predictors, the present study confirms previous observations that undifferentiated thyroid carcinomas are more aggressive, and have a poorer prognosis.^4^, ^17^, ^36^ In fact, in our study, dogs diagnosed with a nonfollicular carcinoma had a 4‐fold higher risk of death because of thyroid cancer compared to follicular carcinoma. The reported local aggressive behavior of nonfollicular thyroid tumors likely explains the guarded outcome.^7^ Conversely, in some studies the histopathological tumor type did not influence survival in dogs, possibly because of the more limited number of cases included compared to the present investigation.^22^, ^23^, ^37^
+
+In this case series, metastasis at diagnosis was a major risk factor of death in dogs with thyroid cancer, and survival was 35% lower in those with tumor spread. However, although the presence of metastasis at diagnosis was associated with a poor outcome, 6 dogs with metastasis lived longer than 500 days. Hence, thyroidectomy could be taken into consideration in certain cases with metastatic disease, despite the pros and cons that need to be clearly discussed with the owner.
+
+Notably, in a study the presence of metastases in 15 dogs undergoing thyroidectomy did not negatively affect the outcome.^14^ Recently, in an investigation into the prognosis of canine thyroid carcinoma based on computed tomography assisted staging, the presence of metastatic disease had no influence on MST, although a trend toward significance was observed.^21^ The latter study included 58 dogs and given that the whole abdomen was not routinely imaged, the rate of metastatic disease might have been underestimated. Although, in another study it was found that from 15 dogs diagnosed with thyroid carcinoma, 9 dogs had pulmonary metastases, while 1 dog had abdominal metastases spread to the liver and spleen.^38^
+
+In our study, the administration of adjuvant chemotherapy was negatively associated with survival, irrespective of metastasis at surgery, with a hazard of dying of about 3.5 times greater in those that received chemotherapy. It cannot be excluded that this finding was caused by a “confounding by indication” phenomenon, because of the retrospective nature of the study. Although unproven, it is possible that adjuvant chemotherapy was recommended more often and administered to dogs presenting other negative prognostic factors, hence the outcome was poor regardless of chemotherapy. This is supported by the literature, which suggests chemotherapy in dogs presenting with negative prognostic factors, such as metastatic disease, thrombosis, and invasive tumor. In addition, adjuvant chemotherapy could have played a permissive role in some complications, however medical records prevented further clarification. Caution is however required in inferring a causal relationship since chemotherapy was not administered at random but upon clinical indication.
+
+Similar to the present study, adjuvant chemotherapy did not improve survival compared to surgery‐only treated cases.^14^ However, it has also been reported that 30‐50% of dogs treated with doxorubicin or cisplatin had a more than 50% reduction in the thyroid tumor volume,^39^, ^40^ indicating some benefits of chemotherapy on gross disease. Moreover, in dogs with thyroid cancer that had received adjuvant 9‐cis retinoic acid after thyroidectomy, the outcome was better than surgery alone or combined with doxorubicin,^17^ despite the limited number of cases included. In humans diagnosed with differentiated thyroid cancer, systemic cytostatic chemotherapy does not seem to be beneficial for survival.^41^
+
+As expected, in the present series higher age^3^ and body size at the time of surgery were associated with a higher risk of death because of other cause. This is in line with the literature, showing that medium and large‐breed dogs are at increased risk to develop thyroid tumors.^3^, ^6^, ^16^, ^20^ The reasons for this is unclear, but an explanation could be related to the fact that the risk of mutation associated with normal processes of cells replication are enhanced in large breed dogs.^42^
+
+Thrombosis was also identified as a cause of death unrelated to thyroid cancer. In humans with thyroid carcinoma tumor, thrombosis is associated with an increased chance of death because of thyroid cancer but not to all other causes.^43^ The underlying explanation in dogs remains unclear.
+
+In our study, an association between histological tumor type and metastasis was observed. In turn, metastasis was associated with thrombosis, thyroid tumor size, and bilateral tumor lobe involvement. Thus, not unexpectedly, tumors that spread to other sites are frequently those that have the ability to grow larger and invade the vessels.^20^ In addition, similarly to a previous study where larger thyroid tumors were more locally invasive in dogs,^7^ we found that tumor thrombosis was associated with bilateral lobe involvement and a longer tumor major axis. Therefore, it could be suspected that in the pathophysiology of thyroid tumors in dogs, metastasis, thrombosis, and size could be connected, but further investigation is needed in order to verify if this association is consistent.
+
+With regard to the year of surgery, earlier years were associated with an increased risk of death because of other cause. This might be because of improved veterinary care in the last decade, possibly increasing the overall life expectancy of the most recent cases. Moreover, the use of the latest generation surgical instruments allows the closure of the thyroid vascularization in a much safer way in humans. LigaSure, seems to generate less thermal damage to the treated tissues.^44^ In veterinary medicine, their use is associated with a reduction in surgery time but does not seem to influence the complication rate and postsurgical hospitalization time.^45^
+
+None of the remaining risk factors assessed in this investigation, including sex, neutering, breed, presurgical T4 concentration, thyroid lobe involvement, type of surgery, additional surgery performed, thyroid tumor size, local invasiveness, postsurgical levothyroxine calcitriol and calcium supplementation, and institution where surgery was performed, were associated with survival.
+
+Unlike previous findings in dogs and humans,^7^, ^20^, ^46^, ^47^, ^48^ thyroid tumor size, bilateral involvement and local invasiveness did not influence outcome in our study. However, other investigations in affected dogs have highlighted that tumor diameter and volume, as well as bilateral thyroidectomy, had no effect on survival.^7^, ^18^, ^23^ Regarding local invasiveness, it is possible that the increasing attention of clinicians helped to achieve an earlier diagnosis and prevented disease progression in the affected dogs. Similarly to this series, dogs diagnosed with thyroid carcinoma undergoing thyroidectomy have an excellent outcome despite gross vascular invasion, with an MST of 621 days.^19^
+
+The present investigation has some limitations. Because of its retrospective nature, some data were incomplete. There was also no standardized adjuvant chemotherapy protocol used to treat the affected dogs. The histopathological evaluation of the excised thyroid tumors was performed by different pathologists and samples could not be collected to review the diagnosis and add further testing.
+
+In conclusion, dogs with thyroid tumors undergoing thyroidectomy have a long life expectancy. Undifferentiated carcinomas and metastases are major risk factors for thyroid cancer‐related death. Although metastases have a guarded outcome, extended survival is observed in certain dogs. Despite the rare presence of nonfollicular carcinoma and metastases, thyroidectomy should still be considered in some of these dogs. Similar to prior investigations, adjuvant chemotherapy did not prove to be beneficial in affected dogs. Unlike some studies, but confirming others, thyroid tumor size, invasiveness and bilateral thyroidectomy did not affect outcomes. The underlying reasons for thrombosis being a risk factor for death unrelated to thyroid cancer are unclear.
+
+## CONFLICT OF INTEREST DECLARATION
+
+Eric Zini serves as Associate Editor for the Journal of Veterinary Internal Medicine. He was not involved in review of this manuscript. No other authors have a conflict of interest.
+
+## OFF‐LABEL ANTIMICROBIAL DECLARATION
+
+Authors declare no off‐label use of antimicrobials.
+
+## INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) OR OTHER APPROVAL DECLARATION
+
+Authors declare no IACUC or other approval was needed.
+
+## HUMAN ETHICS APPROVAL DECLARATION
+
+Authors declare human ethics approval was not needed for this study.

fulltext/md/PMC10097543.md

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+---
+pmcid: "PMC10097543"
+pmid: "37064063"
+doi: "10.1093/jscr/rjad170"
+title: "Successful replantation of total scalp amputation due to dog bites in geriatric patient with prolonged tissue ischemia and comorbidities: a case report."
+journal: "Journal of surgical case reports"
+year: "2023"
+license: "cc by"
+article_type: "case-report"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Successful replantation of total scalp amputation due to dog bites in geriatric patient with prolonged tissue ischemia and comorbidities: a case report
+
+## Abstract
+
+Traumatic scalp amputation is a rare and life-threatening case causing massive blood loss and serious tissue damage. This report aims to record a successful scalp replantation on a 69-year-old woman with total scalp amputation due to dog bites. The challenges of this case were age, prolonged tissue ischemia and several comorbidities such as high glucose index (uncontrolled diabetes mellitus), hypertension and obesity. After 2 hours of admission, 9 hours of stabilization and 7 hours of revascularization, left superficial temporal artery and vein end-to-end anastomosis was successful, adding to 18 hours total of warm tissue ischemia time. The patient was discharged on Day 8 without signs of reperfusion injury or infection. Elderly patients with comorbidities and long-term tissue ischemia may be at risk for reperfusion injury and infection. However, sufficient vascular anastomosis, good tissue quality and optimal evaluation can achieve a good result.
+
+## INTRODUCTION
+
+Replantation is a method of reconnecting tissue or limb followed by the application of microsurgical technique. The first successful scalp replantation was conducted by Miller in 1976 [1]. Reportedly, only a few cases of partial or total scalp amputation can be found in the trauma unit [2, 3].
+
+The most common incidents that led to this trauma are rotating machine accident, animal bite and assault [4, 5]. Scalp amputation can lead to severe injury, permanent hair loss and massive blood loss. Partial defects can be managed by local flaps or skin grafts, while complete detachment may require free flap or replantation, with consideration of ischemia time and blood supplies for scalp survival [1, 6, 7].
+
+Elderly and prolonged ischemia time, followed by a combination of comorbidities, are very susceptible to reperfusion injury or ischemia-induced necrosis. When not properly treated, people who receive suboptimal care will lead to poor clinical outcomes [8].
+
+## CASE REPORT
+
+A 69-year-old woman was referred to Mohammad Hoesin Hospital in Palembang, South Sumatra, with total scalp amputation due to dog bites 2 hours before admission. The scalp tissue was 30 × 26 cm in full thickness, with multi-level laceration, irregular edges and exposed skull stump (Fig. 1). The patient also has comorbidities of high glucose index (uncontrolled diabetes mellitus), hypertension and obesity (body mass index > 32), and there are no signs of concussion or airway obstruction.
+
+*Figure: Figure 1 69-year-old woman with traumatic scalp amputation; (**a**) a total circular scalp avulsion with multi-level lacerated wounds; (**b**) an intact skull stump; (**c**) total detachment of the scalp exposing the skull stump; (**d**) scalp attachment with sutures to support the head shape and (**e**) an end-to-end anastomosis of the right superficial temporal artery and vein.*
+
+Hemorrhage control was performed, as well as blood loss resuscitation with fluid and blood transfusion. After obtaining good vital signs, comorbidities were treated preoperatively; this took 9 hours of tissue ischemia time after the incident. Although replantation success may be small due to prolonged tissue ischemia and comorbidities’ time-consuming management, replantation on the scalp was still being carried out.
+
+After removing damaged cells and visible dirt, the scalp was washed several times with normal saline and disinfected with iodine solution. The patient was supine during the operation and under general anesthesia. Because attempts to identify the supratrochlear artery were futile, vessels in the temporal lobe were selected for the anastomosis. Prolene 9.0 microsurgery under a dissecting microscope was used to perform end-to-end anastomosis of the right superficial temporal artery and vein. After 7 hours of anastomosis, the scalp was sutured to the periosteum with prolene 4.0–5.0, and heparin lavage was immediately performed. Nerve anastomosis cannot be performed due to patient morbidity from long hours surgery. The amputated scalp had undergone a total of 18 hours of warm ischemia time.
+
+Post-operatively (Fig. 2), the scalp showed a positive pin-prick test, restored capillary refill time and adequate blood flow as measured by doppler ultrasonography. Every 12 hours, antibiotics and 10 000 IU of heparin were given intravenously. The patient was then admitted to the ICU in an intubated state with close monitoring and was able to be discharged in a stable condition 8 days after surgery.
+
+*Figure: Figure 2 Post-operative.*
+
+After 17 days of surgery, several areas along the line of scalp avulsion and multi-laceration wounds were found to be moderately necrotic. The patient was referred for debridement, and honey dressing was applied. Fresh granulation tissue appeared a month later, indicating a successful wound healing.
+
+The wound healed well after 2 months, and hair began to grow (Fig. 3). The patient did not require any additional revisions.
+
+*Figure: Figure 3 2-month follow-up.*
+
+During 9 months follow-up (Fig. 4), the patient regained consequent hair growth, and small alopecia area can be covered through hair styling.
+
+*Figure: Figure 4 9-month follow-up.*
+
+After 2 years (Fig. 5), the patient has no complaints except for paresthesia in several areas of the scalp. However, the patient was very pleased with the outcome.
+
+*Figure: Figure 5 2-year follow-up.*
+
+## DISCUSSION
+
+This replantation of total scalp amputation on an elderly patient has a high risk of failure due to 18 hours of warm ischemia time, patient’s comorbidities and reperfusion injury. Skin and subcutaneous tissue, which are relatively resistant to ischemia, can optimally survive up to 6 hours or more [6]. However, this case of prolonged tissue ischemia has shown an effective result of scalp survival from replantation even for the hair that is least ischemia resistant.
+
+In cases of prolonged ischemia time, predicting whether the scalp can survive is still a debate among reconstructive surgeons. Therefore, scalp survival can be predicted from the quality of the scalp and severity of scalp inner vasculature injury. After surgery, scalp necrosis might occur caused by arterial injury, hence good comprehensive management should be ensured [6].
+
+The presence of comorbidities must be addressed both before and after surgery, as they may be at risk of complications and mortality [8]. Antibiotics are also important for contaminated wounds, particularly for the bacteria most commonly found in dog mouths (*Pasteurella multocida*) [4, 6, 9].
+
+Replantation surgery in the elderly requires an adapted and individualized approach as well as a combination of microsurgery advances. Apart from replantation, other options for total scalp amputation are the Antero-lateral Thigh (ALT) Free flap, which may require up to two thighs due to the large defect, as well as Split-thickness Skin Graft (STSG) after previously chipping the skull and awaiting granulation, which is time-consuming and carries a risk of osteomyelitis.
+
+This case concludes that replantation is the best option for total scalp amputation requiring successful anastomosis, good management of comorbidities and long-term evaluation in order to achieve optimal reconstruction and good esthetic outcome.

fulltext/md/PMC10099574.md

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+---
+pmcid: "PMC10099574"
+pmid: "36336823"
+doi: "10.1111/jsap.13567"
+title: "Use of urinalysis during baseline diagnostics in dogs and cats: an open survey."
+journal: "The Journal of small animal practice"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Use of urinalysis during baseline diagnostics in dogs and cats: an open survey
+
+## Abstract
+
+### Objectives
+
+To describe how veterinarians utilise and perform urinalyses for dogs and cats.
+
+### Materials and Methods
+
+A survey, developed and distributed through the Veterinary Information Network, enlisted veterinarians who perform urinalyses for dogs and cats. Participants were directed to question banks based on whether urinalyses were performed in‐house, by an outside diagnostic laboratory, or using an in‐house automated instrument. Participants using multiple methods were directed to questions that related to the chosen methods.
+
+### Results
+
+A total of 1059 predominantly first‐opinion clinicians from the USA and Canada completed the survey. Participants performed urinalyses much less frequently than blood work during a routine examination. The most common factors preventing participants from performing a urinalysis with blood work included clients' financial constraints, difficulty obtaining urine and lack of perceived diagnostic need. The most common reasons for submission to a diagnostic laboratory included efficiency, more trusted results and convenience. Speed of obtaining results was the most common reason for performing urinalyses in‐house. Of the participants who performed in‐house urinalyses, fewer always performed a manual sediment examination (79%) as compared with urine‐specific gravity (99%) and manual dipstick (87%).
+
+### Clinical Significance
+
+This survey documents that urinalysis is often not used in senior patients as recommended by recent clinical guidelines for dogs and cats which can result in decreased diagnosis and impaired management of subclinical disease. There is significant variability in urinalysis methods despite veterinary guidelines promoting standardisation, and this could lead to inaccurate results.
+
+## INTRODUCTION
+
+Veterinarians commonly perform urinalyses during the evaluation of their patients. It constitutes the third arm of the minimum database, along with a complete blood count (CBC) and chemistry panel. Routine urinalysis allows veterinarians to gain information about renal glomerular and tubular function, lower urinary system health and other body systems (Fry 2011, Callens & Bartges 2015). Urine‐specific gravity (USG) and serum biomarkers of glomerular filtration rate help evaluate renal function (Barsanti 2012). Urinalyses and chemistry panels synergistically provide a better understanding of patients' health. Some of the most common diseases in dogs and cats require urinalysis for diagnosis. For example, Banfield's State of Pet Health report found that cystitis and chronic kidney disease were two of the most common diagnoses in cats (Banfield Pet Hospital 2016). Cystitis affected over 2% of their cat population ranging from young adult to senior, and chronic kidney disease affected over 10% of their geriatric cat population. These conditions require urinalyses for complete and correct diagnosis.
+
+As perceptions of the roles of companion animals in society change, clients and veterinarians alike increasingly prioritise prevention and early diagnosis of diseases. Recently, canine life stages guidelines have been established by the American Animal Hospital Association (AAHA) for primary care diagnostics and treatments to help veterinarians optimise medical practices (Creevy *et al*. 2019). Five life stages are established: puppy, young adult, mature adult, senior and end‐of‐life. The guidelines for baseline blood work and urinalysis are determined by age group. In three of these life stages (young adults, mature adults and senior animals), urinalysis constitutes a component of the recommendations to allow veterinarians to determine a dog's baseline values and follow trends overtime. Following trends allows veterinarians to diagnose occult and early stages of specific diseases, such as chronic kidney disease, which often occur without clinical signs early in the disease course, but require an early diagnosis to implement measures that might slow disease progression (Jacob *et al*. 2002, Jacob *et al*. 2005, Grauer *et al*. 2008, Littman *et al*. 2013, Cortadellas *et al*. 2014). Similarly, AAHA and the American Association of Feline Practitioners (AAFP) have developed guidelines for evaluating laboratory work during different life stages. In cats 7 years of age and older, urinalyses are recommended as part of wellness examinations to allow for early detection of disease and evaluate for trends in laboratory parameters that may be concerning. In young adults (1 to 6 years), urinalyses should be considered based on the individual patient (Quimby *et al*. 2021). These guidelines highlight the need to have a better understanding of how, why and what factors determine when veterinarians perform urinalyses.
+
+Urinalyses are composed of four parts – physical characteristics, USG, chemical analysis using multi‐test semiquantitative dipsticks and sediment examination (Duncan & Prasse 1976). Dipstick analyses identify urinary protein, glucose, ketones, pH, bilirubin and heme, whereas sediment exams identify cells (*i.e*. leukocytes, erythrocytes, epithelial cells), casts, crystals and infectious organisms. Recently, automated urine dipstick readers and urine sediment analysers have been developed to perform urinalyses in‐house more efficiently and consistently (Ferreira *et al*. 2018, Hernandez *et al*. 2019). These new analysers have expanded the methods used for performing urinalyses in‐house.
+
+Although the utility of urinalyses is well‐established, the methodology and use of urinalyses in small animal veterinary practice have not been documented. The purpose of this study was to establish the factors that determine when, why and how veterinarians perform urinalyses.
+
+## METHODS
+
+We created a survey to explore factors that contribute to how, why and when veterinarians perform urinalyses for dogs and cats. An e‐mail with a link to the survey was sent to member veterinarians of the Veterinary Information Network (Appendix S1). No incentives were provided for veterinarians to complete the survey.
+
+The survey had two sets of questions with different focuses. The first set of questions focused on demographics, including practice type, geographic area, species seen in the practice, practice size and graduation year from veterinary school. The second set of questions focused on general urinalysis practices, such as how urine is obtained for urinalysis, urinalysis methodology and how often urinalyses are performed compared to CBCs and chemistry panels. Four categories about how urinalyses are performed were available: in‐house manual examination (in‐house exam), sent to an outside diagnostic laboratory (outside lab), in‐house using an automated dipstick reader (dipstick reader), and/or in‐house using an automated sediment analyser (sediment analyser). Based on this question, the participants were given a select question bank that applied to each of the methodologies they selected. For example, if a participant only performed urinalyses using outside labs, then they were directed to an applicable question bank. However, if a participant used both outside labs and in‐house exams, then the participant answered two question banks that included questions about both.
+
+Participants that performed in‐house exams were asked several questions regarding how urinalyses are performed, by whom, their reasons for performing urinalyses in‐house rather than sending samples to outside labs, and whether they have considered using an in‐house automated analyser. Questions for participants that sent urinalyses to outside labs included how they store the urine, factors that determine why they send urinalyses to laboratories rather than performing them in‐house, what component of the urinalysis is the main limiting factor for performing urinalyses in‐house, and if they would consider using an automated in‐house instrument, if available. Disease entities that may have driven the selection of measured urine components were not queried in the study. Veterinarians who reported using an in‐house dipstick reader and/or sediment analyser were asked which type of analyser they use along with questions regarding sample preparation and storage. Participants using sediment analysers were asked questions focused on if and when they send samples to outside labs or perform in‐house exams instead of, or in addition to, the sediment analyser. Additionally, participants in this group were asked how often they review images generated by the analyser and what prompts them to review images or send them to a clinical pathologist for review.
+
+Descriptive statistics were performed in this study to evaluate the different facets of the data generated from the survey. The participants were divided into categories depending on the method of urinalyses performed. The data were reported as percentages and medians. Figures were generated using Microsoft Excel.
+
+### Ethics statement
+
+This study was performed as a collaboration between Texas A&M University and Veterinary Information Network. The study was deemed not to be human research and was therefore exempted from IRB approval.
+
+## RESULTS
+
+### Demographics
+
+A total of 1182 participants filled out the online survey between September and October 2019. Thirty participants who did not complete the survey were excluded. Seven participants who completed the survey were excluded because they reported they do not see patients directly and/or do not perform urinalyses for dogs or cats. Eighty‐six participants (7.5%) were from countries other than the USA or Canada or did not state their country and were excluded. Of the remaining 1059 participants, 918 were from the USA (87%) and 141 were from Canada (13%) (Table 1). The median year that the participants graduated from veterinary school was 2000 (range: 1961 to 2019; 1059 responses). Most participants described their practice as first opinion with exclusive care for dogs and cats (Table 1).
+
+**Table 1 Demographics of participants from the USA and Canada who completed a survey regarding canine and feline urinalyses**
+
+|  | Number of respondents |
+| --- | --- |
+| Practice type |  |
+| First opinion | 963 |
+| Emergency | 60 |
+| Shelter | 19 |
+| Referral | 11 |
+| Academia | 6 |
+| Total | 1059 |
+| Practice locale |  |
+| Rural | 173 |
+| Suburban | 595 |
+| Urban | 281 |
+| Other | 7 |
+| No response | 3 |
+| Total | 1059 |
+| Species |  |
+| Dog and cat only | 746 |
+| Mixed | 87 |
+| Mixed with exotics | 226 |
+| Total | 1059 |
+| Number of full‐time veterinarians |  |
+| 1 | 279 |
+| 2 to 5 | 629 |
+| >5 | 146 |
+| No response | 5 |
+| Total | 1059 |
+
+### Performing the minimum database
+
+Two‐thirds of all participants (68%; 725 of 1059) reported that they do not perform a urinalysis with every/almost every CBC or chemistry profile, most commonly because of financial constraints (n=452), because they do not think the urinalysis is warranted (n=386), and/or difficulty obtaining urine (n=372). This contrasts to 89% of respondents (945 of 1059) who stated that they would perform a urinalysis when the patient has indications for urine testing. The most common reasons for not testing in this situation are difficulty obtaining urine (n=93) and financial constraints (n=60).
+
+The number of participants that often or always perform urinalyses with CBC and chemistry panels varied widely based on the age of the patients (Table 2). For patients less than 7 years old, sevenfold more participants perform CBCs and chemistry panels compared to urinalyses. However, for patients over 7 years of age, only twice as many participants performed CBCs and chemistry panels compared to urinalyses.
+
+**Table 2 Relative frequency with which participants who completed a survey regarding canine and feline urinalyses perform a complete blood count (CBC), chemistry panel or urinalysis on patients as part of pre‐anaesthetic or annual testing**
+
+|  | Patients <7 years old, n (%) | Patients ≥7 years old, n (%) |
+| --- | --- | --- |
+| CBC | Chemistry | Urinalysis | CBC | Chemistry | Urinalysis |
+| Never | 36 (3.4) | 22 (2.1) | 137 (12.9) | 8 (0.8) | 3 (0.3) | 40 (3.8) |
+| Rarely | 142 (13.4) | 80 (7.6) | 463 (43.7) | 32 (3.0) | 13 (1.2) | 167 (15.8) |
+| Sometimes | 278 (26.3) | 265 (25.0) | 371 (35.0) | 108 (10.2) | 75 (7.1) | 354 (33.4) |
+| Often | 310 (29.3) | 336 (31.7) | 72 (6.8) | 367 (34.7) | 354 (33.4) | 356 (33.6) |
+| Always | 293 (27.7) | 356 (23.6) | 16 (1.5) | 544 (51.4) | 614 (58.0) | 142 (13.4) |
+| Total | 1059 | 1059 | 1059 | 1059 | 1059 | 1059 |
+
+### Overview of methods used for urinalyses and urine collection
+
+The majority of participants indicated that they perform urinalyses by multiple methods with most performing in‐house exams, followed by using outside labs (Table S1). The urinalysis method did not impact the median number of estimated urinalyses performed weekly (Table S1). For urine collection, most participants stated that they use cystocentesis (98.6%; 1044 of 1059) and free catch urine (owner‐obtained free catch: n=759, mid‐stream free catch: n=698, any free catch: n=609). The median percentage of time each method is used to collect urine was 50% for cystocentesis, 20% for mid‐stream free catch, 15% for any free catch, 10% for owner‐obtained free catch and 5% each for catheterization, owner‐obtained litter box, and “other.”
+
+### In‐house manual examinations
+
+Of the participants that use both in‐house exams and outside labs, the median percentage of time that they perform in‐house exams was 50% (range: 1 to 99%). For all participants that perform in‐house exams (n=728), common reasons for performing urinalyses in‐house (*versus* outside labs) were the ability to obtain rapid results (n=680), having the required equipment (n=305), availability of trained staff (n=323) and economics (n=263). Of 141 participants who chose “other” reasons, 75 explained that they believe the results are more accurate when done in‐house due to time delays causing artefacts with submission to outside labs, with crystal formation being a common concern.
+
+Of participants that indicated they perform in‐house exams, 99% (720 of 728) always measure the USG when doing a urinalysis. Approximately 95% (694 of 728) of participants use a refractometer for USG measurement, whereas 0.1% (1 of 728) use urine dipstick alone and 4.4% (32 of 728) use both the urine dipstick and refractometer (Table S2), despite the inaccuracy of the urine dipstick for USG. In contrast, 87% of participants (495 of 570) who perform manual dipstick evaluation without dipstick readers stated they always perform a dipstick evaluation when doing in‐house exams, and 79% of participants that perform manual sediment examinations without sediment analysers always perform manual sediment examinations with their urinalyses (Fig. 1). Circumstances selected that would prompt a sediment examination for the participants who do not always perform these exams include lower urinary tract signs (80%; 94 of 117), abnormal urine appearance (71%; 83 of 117), white or red blood cells identified on the urine dipstick (45 and 42%; 53 and 49 of 117, respectively), and protein identified on the dipstick (44%; 51 of 117). Multiple participants commented that evaluation for crystals prompts sediment evaluation (n=10).
+
+*Figure: FIG 1 Relative frequency that participants without an automated sediment analyser perform urine sediment examinations as part of in‐house manual urinalyses in dogs and cats*
+
+Of the time categories provided, a plurality of participants indicated that manual sediment examination takes between 6 and 10 minutes to prepare and perform (Fig. 2). Practices most commonly use a combination of veterinarians and either licensed/certified veterinary technicians, or other trained staff, or both, to perform manual sediment examinations (43%; 256 of 595). Fewer use only veterinarians (30%; 178 of 595), only technicians (19%; 114 of 595) or only other trained staff (2%; 14 of 595), with the remainder using a combination of technicians and other trained staff. At the practices that use a combination of veterinarians and other employees (technicians and/or trained staff), veterinarians perform sediment examinations an average of 31 to 48% of the time.
+
+*Figure: FIG 2 Time required to prepare and to perform manual urine sediment examinations in veterinary practices*
+
+Most participants that perform in‐house exams without sediment analysers complete both the dipstick and sediment evaluations within 30 minutes of collection (Fig S1). Few participants indicated that they refrigerate urine before in‐house analysis (n=51 for dipstick and sediment examination). Of those, 57% (29 of 51) and 37% (19 of 51) indicated that they perform the dipstick analysis or sediment examination, respectively, immediately or within 10 minutes of removal from the refrigerator. For manual dipstick analysis, 61% (346 of 570) of participants use unspun urine and 24% (139 of 570) sometimes use unspun urine and sometimes supernatant.
+
+For participants that perform in‐house exams with or without sediment analysers, 84% of respondents centrifuge <3 mL or 3 to 6 mL of urine (345 and 267 of 728 responses, respectively). Additional information regarding how participants perform urinalyses are presented in Table S2.
+
+Of the participants who only perform in‐house exams, 35% (86 of 246) have considered obtaining a sediment analyser. Reasons for not obtaining a sediment analyser are described in Fig. 3.
+
+*Figure: FIG 3 Number of participants who perform in‐house manual urine sediment examinations in dogs and cats grouped by reasons they do not use an in‐house automated urine sediment analyser*
+
+### Outside diagnostic laboratories
+
+Eighty‐nine percent (108 of 121) of participants that use outside labs store the urine in the refrigerator before collection by the courier. For these participants, the most common factors that contribute to sending urinalyses to outside labs are efficiency (105 of 121), trusted/reliable results (81 of 121), lack of expertise in urinalysis and/or sediment examination (79 of 121), and convenience (78 of 121). When asked which component of the urinalysis is the main limiting factor for performing urinalyses in‐house, 54 participants chose the sediment examination, two participants chose urine dipstick testing, and none chose USG. The time to analysis for outside diagnostic laboratories was not included in the survey.
+
+Of participants who exclusively send urinalyses to outside labs, 28% (34 of 121) stated they would use a sediment analyser if one was available and 32% (39 of 121) stated they would not; 36% (44 of 121) of participants stated that they would consider using a sediment analyser. Participants indicated cost, accuracy and personnel time were the major deciding factors.
+
+### In‐house automated instruments
+
+Approximately one‐third of participants indicated that they use either dipstick readers, sediment analysers or both. However, only 7% of participants selected that they perform urinalyses exclusively using these methods (Table S1). Of the participants that reported using dipstick readers, 85% (275 of 325) use the IDEXX™ VetLab™ UA™ Analyzer and 13% (43 of 325) use the Abaxis™ VetScan™ Urinalysis Analyzer. One participant uses the Bayer™ Clinitek™. When asked the time between sample collection and performing the dipstick using a dipstick reader, 81% (264 of 325) of participants reported performing the analysis within 30 minutes.
+
+Of the participants who use sediment analysers, 89% (285 of 319) use the IDEXX™ SediVue���, whereas 8% (27 of 319) use the Abaxis™ VetScan™ Sediment Analyser, with the remaining participants being unaware of the brand they use. Of the participants who use sediment analysers, approximately two‐thirds also submit urine to outside labs. The most common factors leading to submission of urine to outside labs despite having a sediment analyser include the need for additional expertise for interpretation and unexpected/uncertain/confusing results from the automated analyser (Fig. 4). Another reason that the participants wrote‐in as an “other” response was that the urinalysis is part of package pricing with blood work or additional urine testing (*e.g*. culture or urine protein‐to‐creatinine ratio).
+
+*Figure: FIG 4 Number of participants with an automated urine sediment analyser grouped by reasons for performing a urine sediment exam manually in‐house (in‐house exam) or through an outside laboratory for dogs and cats*
+
+Eighty‐four percent (269 of 319) of participants who use sediment analysers indicated that they perform an automated sediment analysis on 90 to 100% of urine samples that undergo urine dipstick testing. Eighty percent (256 of 319) of participants stated that they perform the automated sediment evaluation within 30 minutes of urine collection. Twenty‐six participants indicated that they sometimes refrigerate samples before using the sediment analyser, and 18 (69%) run the urine sediment immediately or within 10 minutes of removal from the refrigerator. Of the participants who use sediment analysers, 79% (251 of 319) perform a manual sediment examination as a follow‐up or substitute for the sediment analysers, although 76% (191 of 251) of these respondents indicated that they rarely or sometimes do *versus* 24% (59 of 251) reporting that they often or always do this. The most common reasons for performing in‐house exams with a sediment analyser are described in Fig. 4. Additional reasons provided included exotic species patients, crowded samples or search for abnormal epithelial cells.
+
+Of the participants who use sediment analysers, approximately 74% (236 of 319) reported often or always reviewing images selected by the analyser. The factors that prompt participants to review images included suspected bacteria (n=100); an unclassified crystal (n=68), cell (n=63) or cast (n=47); unknown material (n=58); confirmation of cells (n=52), casts (n=42) or the report in general (n=52); and/or the opportunity to learn (n=50). Most participants reported never (247 of 319) or rarely (n=62) sending images to a clinical pathologist for review.
+
+## DISCUSSION
+
+Our survey of first‐opinion small animal clinicians revealed that urinalyses are commonly excluded as part of the minimum database, even for older patients. Furthermore, participants who perform in‐house urinalyses omit examination of the urine sediment more often than other components of the urinalysis. Our survey suggests that clinicians in first‐opinion practice underutilize urinalyses in older patients. The results of our study also demonstrate the variability and lack of standardisation of urinalysis techniques in veterinary clinics despite published clinical guidelines (Gunn‐Christie *et al*. 2012, Arnold *et al*. 2019).
+
+Two‐thirds of all participants reported that they do not perform a urinalysis with every or almost every CBC and/or chemistry profile. Even for patients 7 years or older, urinalyses are performed half as often as blood work. Despite AAHA canine life stages guidelines that recommend routine urinalyses for senior or geriatric dogs (Creevy *et al*. 2019), many participants omit urinalyses because they do not believe urinalyses are warranted. However, urinalyses are necessary to detect certain common diseases associated with ageing, such as glomerular disease (Macdougall *et al*. 1986). Studies have found that 13 to 19% of apparently healthy elderly dogs showed evidence of persistent proteinuria (Willems *et al*. 2016, Marynissen *et al*. 2017). Willems *et al*. (2016) reported that approximately 77% of dogs with proteinuria had evidence of hypertension with systolic blood pressure being 160 mmHg or above. Missing proteinuria and related hypertension can lead to progressive injury to multiple organ systems, including the kidneys and cardiovascular system, which are commonly associated with morbidity and mortality in older dogs and cats (Lees *et al*. 2005, O'Neill *et al*. 2013, Acierno *et al*. 2018). Therefore, clinicians who fail to routinely perform urinalyses likely fail to detect certain common diseases in dogs and cats at the frequency with which they occur.
+
+The present study found that approximately 20% of participants who perform in‐house urinalyses do not always perform sediment examinations as part of the urinalysis. Of these participants, the most common factors contributing to performing a sediment examination were protein, white blood cells, or red blood cells identified on dipstick exam in addition to lower urinary tract signs. This is in line with the fact that an active urine sediment examination can negate the ability to interpret the protein present in the urine with regard to kidney disease (Bagley *et al*. 1991). Conversely, leukocyte identification by urine dipstick has little or no diagnostic value in dogs and cats (Callens & Bartges 2015). Therefore, limiting urine sediment examinations to only cases that yield a positive dipstick test result for white blood cells will likely fail to identify urogenital inflammatory conditions in some affected dogs or cats. In addition, without sediment examination, crystals and casts cannot be recognised, leading to missed indicators of disease.
+
+Over 60% of veterinarians use multiple methods for performing urinalyses. Different strengths and weaknesses of each modality impacted veterinarians' decisions about which method to use. Strengths of in‐house manual examinations include faster and low‐cost results with fewer artefacts of time‐delayed samples, whereas weaknesses include the time it takes to perform this test and potential inaccuracies. In 30% of practices, veterinarians are the only personnel performing the urine sediment examinations. Even when other technical staff are utilised, veterinarians perform urine sediment examinations an average of one of three of the time. Given the average time of 6 to 10 minutes to prepare and perform urine sediment examinations, procedure time might limit veterinarians and technicians from performing urinalyses in‐house, or it might lead to incomplete analyses, with omission of the most time‐consuming component (*i.e*. the sediment examination). The strengths and weaknesses of sending urinalyses to outside labs are largely opposite of performing urinalyses in‐house. Some strengths of sending to outside labs include time efficiency and more trusted results due to more expertise with urine sediment examinations, whereas weaknesses of this method include increased costs and delayed analysis and results, which can affect clinical application.
+
+Given that time constraint was one of the factors cited for why urinalyses are not done in‐house, increased efficiency afforded by using an automated urine sediment analyser could be helpful for increasing the consistency of performing a sediment examination with every urinalysis. For example, in our survey, 84% of the participants who use a sediment analyser perform sediment exams most of the time *versus* 79% of those who perform in‐house exams. Users of sediment analysers also reported performing the sediment evaluation within 30 minutes of urine collection more frequently (80%) than participants using in‐house exams (69%). The American Society of Veterinary Clinical Pathologists (ASVCP) quality assurance guidelines state that urinalyses should ideally be performed within 30 minutes of collection (Arnold *et al*. 2019); increasing the proportion of urinalyses within this window will help with consistency and accuracy of results. Short‐term refrigeration is acceptable; for example, sediment elements (*e.g*. leukocytes, erythrocytes) are stable for a maximum of 4 hours in the refrigerator (Arnold *et al*. 2019). However, many of those who refrigerate the sample perform the urinalysis within 10 minutes of removal from the refrigerator, in contrast to the recommendation to warm the sample to room temperature first.
+
+Manual dipstick examinations were not considered the main limiting component for most participants. Moreover, the use of dipstick readers did not appear to increase the percentage of dipstick analyses performed within 30 minutes when compared to manual dipstick analysis (81 *versus* 79%, respectively). In addition, the present study found that 67% of participants that send urinalyses to outside labs did so because of the lack of expertise in performing urinalyses and/or sediment examination. Automated sediment analysers might allow participants to gain familiarity and experience with sediment examinations by reviewing the images provided, as supported by the participants who indicated that reviewing the images provides them with an opportunity to learn. Over time, this could allow veterinarians to feel more confident and educated about performing urine sediment examinations. However, there was some concern among participants about the accuracy of these analysers, and more studies are needed to determine the role of these new analysers in veterinary medicine.
+
+Our survey highlights the variability in the protocol used for performing urinalyses and in particular, sediment examinations, in veterinary practices. In human medicine, there is less variability, largely due to more strict regulations regarding who can perform diagnostic testing. A urine sediment examination is considered a non‐waived test as part of the Clinical Laboratory Improvement Amendments (CLIA) (United States Food and Drug Administration 2020). The CLIA regulations are federal standards that are required to test human specimens for health assessment that must meet specific regulations. After these regulations were implemented, urinalyses were less commonly performed in doctor offices and more likely to be sent to outside diagnostic laboratories (Binns *et al*. 1998).
+
+The ASVCP quality assurance guidelines for urinalyses provide an overview of preanalytical, analytical and postanalytical factors that are important in performing urinalyses (Gunn‐Christie *et al*. 2012, Arnold *et al*. 2019). These guidelines address many factors beyond time to analysis, including urine storage, handling and transport, sediment examination preparation, instrumentation and personnel knowledge. While our study examined several of these factors, others were out of the scope of the study (*e.g*. instrument quality control testing). Based on our survey, some of the more variable practices involved urine sediment preparation. Almost half of the participants use less than 3 mL of urine to prepare the urine sediment despite the recommendation that a standard volume of at least 3 to 5 mL be centrifuged (Barsanti 2012). Similarly, 63% of participants use less than the recommended 0.5 mL of supernatant to resuspend the sediment (Arnold *et al*. 2019). These reduced volumes can lead to errors in evaluating or interpreting the urine sediment as well as prevent comparisons from different samples. The ASCVP guidelines also recommend examination of a stained sediment smear or cytocentrifuged preparation for confirmation of bacteria as this results in higher specificity and sensitivity for detection of bacteriuria than wet‐mount alone (Arnold *et al*. 2019). However, only 19% of participants who perform in‐house exams state that they often or always use stain to confirm bacteria. Therefore, many participants could be misidentifying or missing bacteria as part of their sediment examinations.
+
+We neither examine why deviations from recommended practice occur in veterinary clinics, nor why they occur so frequently; however, one possibility is a lack of awareness of such guidelines by most first‐opinion clinicians. The ASVCP guidelines were published in a specialty journal targeting mostly clinical pathologists (Gunn‐Christie *et al*. 2012, Arnold *et al*. 2019). Therefore, most first‐opinion clinicians would probably not encounter the guidelines in their reading. Further investigation is necessary to determine the reasons for the lack of standardisation and adherence to the guidelines in veterinary practices, since decreasing the variability in performing urinalyses may allow for increased accuracy and better interpretation of results.
+
+The present study has some potential limitations, inherent with being a survey‐based study. Although a large number of participants were recruited for this study, the recruitment process involved veterinarians who are members of Veterinary Information Network, and this might introduce selection bias. Another potential limitation is that manual dipstick and sediment evaluations were included in the same category, which could have affected responses for manual sediment examinations, such as how often a sediment examination is performed. Other factors affecting the frequency of sediment examination could also have influenced participant responses if participants completed the survey considering patients in which a particular situation warranted a focused urine evaluation (*e.g*. urine glucose in a diabetic patient). Time to analysis after shipment to outside laboratories was also not queried but can significantly change the quality of the result. In addition, during data analysis and review of responses, some participants appeared to confuse automated urinalysis instruments and manual urinalysis examinations. When identified, these participants were excluded from analysis for the questions related to methodology. Because of the automated nature of the survey, computerised code was used to direct participants to specific question banks, and that is a potential source for error. There were eight participants who were funnelled to the wrong category as an error of the online survey protocol; these participants were excluded for the related questions. No other obvious discrepancies were identified on visual inspection of the data. Furthermore, given the large number of respondents, a minor number of participants being misdirected would likely have little or no impact on our findings.
+
+In conclusion, the present study described the factors and methodologies of urinalyses in first‐opinion veterinary clinics. These results should be helpful for understanding the limitations for performing urinalyses, the variability that exists in practices, as well as opportunities for targeting methodologies to increase the efficiency and consistency of urinalyses.
+
+## Author contributions
+
+**Nicole Gibbs:** Conceptualization (supporting); data curation (equal); formal analysis (lead); methodology (equal); writing – original draft (lead); writing – review and editing (equal). **Johanna Heseltine:** Conceptualization (lead); data curation (supporting); formal analysis (equal); methodology (equal); project administration (equal); supervision (equal); writing – original draft (supporting); writing – review and editing (equal). **Mark Rishniw:** Data curation (lead); formal analysis (equal); methodology (lead); project administration (supporting); resources (lead); software (lead); writing – review and editing (equal). **Mary Nabity:** Conceptualization (equal); data curation (supporting); formal analysis (equal); methodology (equal); project administration (equal); supervision (equal); writing – original draft (supporting); writing – review and editing (equal).

fulltext/md/PMC10099803.md

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+---
+pmcid: "PMC10099803"
+pmid: "36336849"
+doi: "10.1111/jsap.13561"
+title: "Infective endocarditis in dogs in the UK: 77 cases (2009-2019)."
+journal: "The Journal of small animal practice"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Infective endocarditis in dogs in the UK: 77 cases (2009‐2019)
+
+## Abstract
+
+### Objectives
+
+To determine the causative organisms, clinical features and outcome of canine infective endocarditis in the UK.
+
+### Materials and Methods
+
+Medical records of three veterinary referral hospitals were searched for dogs with infective endocarditis between December 2009 and December 2019. Signalment, clinical signs, causative organism, valve affected, treatment and survival data were recorded.
+
+### Results
+
+Seventy‐seven cases with possible or definite infective endocarditis (according to the modified Duke criteria) were included. The majority were large breed (40/77 – 51.9%). There were 47 of 77 (61%) male dogs and the mean age was 7.3 ±3 years. A causative organism was identified in 26 of 77 (33.8%) cases. The most common organisms were *Escherichia coli* (7/27 – 25.9%), *Pasteurella* spp. (5/27 – 18.5%), *Staphylococcus* spp. (4/27 – 14.8%) and *Corynebacterium* spp. (4/27 – 14.8%). *Bartonella* spp. were not detected in any patients. The mitral valve was most commonly affected (48/77 – 62.3%). Clinical features were non‐specific, with lethargy being the most common clinical sign observed (53/77 – 68.8%). Fifty‐three dogs (68.8%) survived to discharge. The median survival time post discharge was 425 days (2 to 3650 days). The development of congestive heart failure was associated with a poorer outcome. Cardiac troponin concentration, antithrombotic use and the development of thromboembolism or arrhythmias were not significantly associated with outcome.
+
+### Clinical Significance
+
+Some dogs with infective endocarditis that survive to discharge can have a long lifespan. The inability to detect an underlying organism is common and *Bartonella* spp. may be a less prevalent cause of canine infective endocarditis in the UK than in the USA.
+
+## INTRODUCTION
+
+Infective endocarditis (IE) is a life‐threatening disease that is difficult to diagnose and manage in veterinary patients (Miller *et al*. 2004). It is caused by bacterial infection of the valvular endothelium and results in proliferative or erosive lesions leading to valvular insufficiency (Häggström 2010). The prevalence of IE varies between publications but is considered low in canine patients. An incidence of <1% has been reported in one veterinary hospital (MacDonald *et al*. 2004). Males appear to be at a greater risk of IE than females (Sisson & Thomas 1984, Miller *et al*. 2004). Previous studies have contradictory findings regarding breed predilections. Some suggest small breed dogs are more predisposed due to their predisposition to congenital heart defects, while others suggest medium to large breed dogs are over‐represented (Sisson & Thomas 1984, Miller *et al*. 2004, Romero‐Fernandez & Palermo 2019). Valvular endocardiosis appears to be a predisposing factor for IE in humans; however, this does not appear to be the case in dogs (Kiefer & Bashore 2012, Romero‐Fernandez & Palermo 2019).
+
+IE can be difficult to diagnose ante‐mortem due to the non‐specific and variable clinical signs and limited diagnostic capabilities in general practice (Häggström 2010). However, a modified version of the human Duke criteria used for diagnosis of IE in dogs has been described (Sykes *et al*. 2006a). Although not part of the Duke criteria, cardiac troponin‐I (cTnI) is another supportive test for IE (Kilkenny *et al*. 2021). The most common bacteria identified in previous studies of dogs with IE were *Streptococcus* spp. and *Bartonella* spp. according to two case series in the USA (Sykes *et al*. 2006a, Reagan *et al*. 2022). Other commonly implicated organisms include *Staphylococcus* spp., *Escherichia coli*, *Pseudomonas* spp., *Erysipelothrix rhusiopathiae*, *Pasteurella* spp. and *Corynebacterium* spp. (Peddle & Sleeper 2007, Reagan *et al*. 2022). Previous studies indicate that *Streptococcus* spp. most commonly infects the mitral valve, *Bartonella* spp. tend to affect the aortic valve while *Staphylococcus* spp. display no valve predilection (MacDonald *et al*. 2004, Sykes *et al*. 2006a). Other Gram‐negative bacteria showed a predilection to infect the mitral valve (Sykes *et al*. 2006a). A bacteraemia is required for the development of IE; however, many cases have no clinically detectable source of infection, possibly because many dogs are already receiving antibiotic therapy before the start of a diagnostic work‐up (Romero‐Fernandez & Palermo 2019). The use of antithrombotics has been shown to increase survival time in IE patients (Reagan *et al*. 2022).
+
+The prognosis for IE and its sequelae in dogs is guarded; in one retrospective case series, a survival rate of 50% was reported (Reagan *et al*. 2022). This appears to be dependent on the valve affected, however, with a shorter median survival time of just 3 days in dogs with aortic valve IE and of 476 days in dogs with mitral IE according to a previous study (MacDonald *et al*. 2004). The shorter survival time of aortic valve infections is thought to be due to its predisposition to *Bartonella* spp. colonisation (Sykes *et al*. 2006b). This can lead to aortic regurgitation which is less well tolerated than mitral regurgitation as it is associated with high afterload and possibly myocardial failure. A recent study has found a longer survival time of 71 days in dogs with aortic valve IE due to *Bartonella* spp. than previous studies (Reagan *et al*. 2022). Complications associated with IE include congestive heart failure (CHF), immune‐complex disease and thromboembolic disease (TED) which can manifest in many organs including the kidneys (Reagan *et al*. 2022). The development of CHF, TED and acute kidney injury (AKI) has been shown to be negatively correlated with survival (Reagan *et al*. 2022).
+
+The scientific literature on canine IE is limited and focused on veterinary hospitals in the USA. The aim of this study was to address the gap in the literature on canine IE cases in the UK, specifically to describe signalment, presenting clinical signs, valve affected, causative bacterial species and outcome in these patients.
+
+## MATERIALS AND METHODS
+
+### Study design and inclusion criteria
+
+This was a retrospective study and the medical records of three UK veterinary referral hospitals were searched for dogs diagnosed with IE between January 2009 and December 2019. Cases were classified as definite or possible IE based on the modified Duke criteria described by Keene (2002), Sykes *et al*. (2006a) and Ljungvall & Häggström (2017) (Table 1) or definite when the diagnosis was confirmed by *post‐mortem* examination. Cases were excluded if they had had previous cardiac surgery associated with the mitral valve repair programmes at two of the veterinary hospitals. Positive findings for IE on echocardiogram as described in Table 1 involved documenting changes in the normal heart anatomy such as thickening of the valves, vegetative lesions, which are often irregularly outlined and oscillatory (*i.e*. move independently from the valve) and associated valvular insufficiencies and/or elevated valve velocities (Ljungvall & Häggström 2017). The mitral valves were viewed from several angles to help distinguish between myxomatous nodular lesions (if degenerative valvular disease was present) and vegetations (Ljungvall & Häggström 2017). The echocardiograms were carried out by residents or board‐certified veterinary cardiologists at the veterinary hospitals. A simultaneously acquired single‐lead electrocardiogram (ECG) was reviewed during echocardiography, with a six or 12 lead ECG recorded according to clinical indication or clinician's preference. The presence (or absence) of arrhythmias was noted.
+
+**Table 1 The modified Duke criteria for the diagnosis of infective endocarditis in dogs**
+
+| Major criteria | Minor criteria |
+| --- | --- |
+| Positive findings for endocarditis on echocardiogram, *e.g*. vegetative or erosive lesion, abscess | Pyrexia (≥39.3°C) |
+| New valvular insufficiency: moderate to severe aortic insufficiency without subaortic stenosis (SAS) | New or worsening heart murmur |
+| At least two separate microbial culture of blood (MCB) positive for a typical organism or three if common skin contaminant | Single positive MCB or serologic evidence of infection by indirect fluorescent antibody assay and/or by polymerase chain reaction |
+|  | Detection of vascular or embolic event, *e.g*. thromboembolism |
+|  | Immunologic event, *e.g*. immune‐mediated thrombocytopenia, glomerulonephritis, etc |
+|  | Prolonged intravenous (iv) catheterisation or infected iv catheterisation |
+|  | SAS |
+|  | Medium to large dog (>15 kg) |
+|  | History of steroid use with any of the above conditions |
+
+### Medical record search
+
+Electronic medical records from each referral hospitals were searched for dogs diagnosed with endocarditis between 2009 and 2019. The software used included VetCompass, Tristan and Rx‐Works. Medical records were searched using the keywords “endocarditis” in three centres and “new heart murmur,” “pyrexia” and “lethargy” in one centre. The medical records were searched by two operators in one centre and one operator each in both other centres. The dates the medical records were searched were May and July 2021.
+
+### Data extracted from records
+
+Clinical features were recorded including patient signalment, presenting clinical signs, Duke criteria fulfilment, microbial culture of blood (MCB) and *Bartonella* spp. polymerase chain reaction (PCR), valves involved, circulating cTnI concentrations, antibiotic therapy (before and post IE diagnosis), other therapy started post IE diagnosis, any comorbidities, hospitalisation length, complications (development of CHF, TED, arrhythmias and renal complications) and patient outcome. To evaluate outcome for dogs that survived hospital discharge, the primary veterinary practices of patients were contacted to determine whether they were known to be alive, or if they had died or were euthanased. The date of their euthanasia or natural death was gathered to the nearest month. If a dog died and a *post‐mortem* consent was provided, *post‐mortem* examination was carried out. Bacterial culture samples were taken aseptically from cardiac tissue. Ethical approval was gained to contact the veterinary practices from all institutions (ethical review reference number SR2020‐0120; VREC1092). The ethical approval SR2020‐0120 covers two centres. The development of complications was recorded as follows; CHF was diagnosed either on *post‐mortem* by board‐certified clinical pathologists or by findings of cardiogenic pulmonary oedema (*e.g*. enlarged cardiac silhouette, enlarged pulmonary vasculature and infiltrative pulmonary patterns) by thoracic radiographs identified by board‐certified veterinary radiologists. Thromboembolic events were defined as visualisation of infarcts or thrombus either at *post‐mortem* examination or by abdominal ultrasound or computed‐tomography by board‐certified clinical pathologists or radiologists respectively. Renal complications were defined as cases with serum creatinine concentrations above the normal reference range in animals with concurrent isosthenuria or hyposthenuria and no history of chronic kidney disease.
+
+### Bartonella detection
+
+Detection of *Bartonella* spp. for this study was carried out by DNA extraction and qPCR for centre A, PCR alone for centres B and C.
+
+### Collection of blood cultures
+
+In centre A, aseptic collection of three, 3 to 10 ml aliquots were collected from three different veins following sterile preparation, all taken at the same time. The whole blood was then subcultured onto blood agar and MacConkey agar for aerobic and anaerobic cultures and incubated at 37°C for 7 days. In centre B, aseptic collection of three, 5 ml aliquots were collected from three different veins following sterile preparation in a time frame of 60 minutes. The whole blood was then subcultured onto blood agar for aerobic and anaerobic cultures and incubated at 37°C for 7 days. In centre C, aseptic collection of three, 2 to 5 ml aliquots were collected from three different veins following sterile preparation, 30 minutes apart. The whole blood was then subcultured onto Signal Blood Culture System (ThermoFisher) for aerobic and anaerobic cultures and incubated at 38°C for 4 to 7 days.
+
+### Statistical analysis
+
+Continuous variables were assessed for normality using the Shapiro–Wilk test. Normally distributed data were reported as mean ±sd and non‐normally distributed data as median (minimum to maximum range). Data were analysed using the statistical analysis programme GraphPad Prism Version 9.0 (GraphPad Software). Definite and possible cases of endocarditis were analysed together. Kaplan–Meier survival curves were constructed and the log‐rank test was used to compare the following populations: dogs with different valve infections, use of antithrombotics, development of CHF, TED and arrhythmias. Survivors were censored on the last day of follow‐up. Cases lost to follow‐up before 1 month after discharge were excluded from the patient outcome analysis. Values of P < 0.05 were set as significant.
+
+## RESULTS
+
+The medical record search identified 287 patients at referral centre A, 49 records at centre B and 42 records at centre C that were eligible for assessment. In total, 77 cases were eligible for the study, the rest were excluded as the final diagnosis was not IE and they did not fulfil enough criteria to be defined as possible or definite cases of IE.
+
+### Signalment
+
+A total of 77 cases were included in this study. There were 37 cases from centre A, nine cases from centre B and 31 cases from centre C. There were more male (neutered: n=33/77, 43%; entire: n=14/77, 18%) than female (neutered: n=21/77, 27%; entire: n=9/77, 12%) dogs. The mean age of all dogs was 7.3 ±3 years. The most common breeds were Labrador retrievers and their crosses (n=15/77, 19%), followed by Border collies (n=9/77, 12%) and boxers and their crosses (n=8/77, 10%). There were more large breed dogs (>25 kg) (n=40/77, 52%) than medium breed dogs (10 to 25 kg) (n=28/77, 36%) and small breed dogs (<10 kg) (n=9/77, 12%).
+
+### Common clinical signs
+
+The median duration of illness before admission was 7 days (0 to 334 days). The most common clinical signs on admission are summarised in Table 2. Other clinical signs recorded were blindness, ptyalism and epistaxis in one dog each. Seventy‐one (92%) dogs presented with multiple clinical signs.
+
+**Table 2 Summary of the main clinical signs observed in this study**
+
+| Clinical signs | Number of cases |
+| --- | --- |
+| Lethargy/weakness | 53 |
+| Pyrexia (≥39.3°C) | 47 |
+| Locomotor problems, *e.g*. shifting, acute or chronic lameness, joint pain or effusions | 34 |
+| Neurological signs, *e.g*. obtundation, head tilt, nystagmus | 16 |
+| Collapse | 13 |
+| Diarrhoea | 11 |
+| Weight loss | 8 |
+| Vomiting | 7 |
+| Polydipsic/polyuric | 7 |
+| Cough | 5 |
+| Oculonasal discharge | 4 |
+| Abdominal pain | 2 |
+
+### Comorbidities
+
+Thirty‐five (45%) of 77 patients had no co‐morbidities reported before the development of IE. Of the 42 remaining cases, the most common comorbidities were osteoarthritis (n=8, 19%), skin disorders (aural infection, cellulitis, grass seed foreign body and associated infection, aural haematoma and wounds) (n=7, 17%), dental disease (n=6, 14%), urinary tract infections (n=5, 12%), discospondylitis (n=3, 7%), prostatitis (n=2, 5%), gastroenteritis (n=2, 5%), septic peritonitis (n=2, 5%), closed pyometra (n=1, 2%) and bronchopneumonia (n=1, 2%). Other comorbidities included neoplasia (n=4, 10%), myxomatous mitral valve disease (MMVD) (n=2, 5%), keratoconjunctivitis sicca (n=1, 2%), epilepsy (n=1, 2%), conjunctivitis (n=1, 2%) and meningitis of unknown aetiology (n=1, 2%). Some dogs had multiple comorbidities (n=4).
+
+### Duke criteria fulfilment
+
+Out of 77, 67 (87%) dogs were classified as definite endocarditis, and 10 (13%) as possible according to the modified Duke criteria. Exclusion of possible cases did not impact the results or statistical analyses. Figure 1 summarises how many cases fulfilled each of the modified Duke's criteria.
+
+*Figure: Fig 1 Histogram showing the number of cases which fulfilled each of the modified Duke's criteria. MCB Microbial culture of blood, iv Intravenous, IMTP Immune‐mediated thrombocytopaenia, TED Thromboembolism, SAS Sub‐aortic stenosis*
+
+Three dogs had prolonged intravenous (iv) catheterisation sites and one had an infected iv catheter site. One of the dogs with the prolonged catheterisation sites had a vascular access port placed. This dog subsequently developed a tricuspid IE. The dog with the infected catheter site developed aortic IE and the other two dogs developed mitral IE.
+
+Ten dogs were submitted for *post‐mortem* at which point a definite diagnosis of endocarditis was confirmed by bacterial culture of cardiac tissue and characteristic valve pathology. Microbiology laboratory reports of these samples did not interpret any of the cultures to be potentially contaminated.
+
+Eight of the 10 dogs were already diagnosed as definite endocarditis cases before *post‐mortem* examination. However, the remaining two cases were initially classed as “possible” endocarditis cases before the *post‐mortem* examination. One of these dogs did not have an MCB submitted ante‐mortem and another had a negative MCB result. Following confirmation of the *post‐mortem* results, these two dogs were then classified as “definite” endocarditis. In four cases, the records did not state how many MCBs were collected; these were classed as a single positive MCB.
+
+### Infecting organism and valve involvement
+
+A causative organism was identified in 26 of the 77 cases (34%). Seven dogs had multiple organisms grown on MCB (in four cases this was detected on *post‐mortem* examination). Figure 2 shows the distribution of organisms confirmed by blood culture and which valve they infected. There was a negative blood culture or blood cultures in 36 (59%) of 77 dogs, with this being more common in referral centre B (n=7/8, 87.5%) compared to referral centre A (n=13/32, 37.5%) and referral centre C (n=16/22, 72.7%). One dog had no blood culture taken but IE was confirmed by *post‐mortem* examination. No blood culture or *post‐mortem* examination was performed in 14 (18.2%) of the 77 cases. Six of these cases died or were euthanased within 3 days of admission (range 1 to 5 days). Two of these 14 dogs were classified as possible endocarditis, the other 12 were classified as definite endocarditis. Table 3 shows how the 12 cases that were classified as definite endocarditis fulfilled this classification despite not having a blood culture or *post‐mortem* carried out. A PCR test for *Bartonella* spp. was performed for 13 dogs, the results were negative for all 13.
+
+*Figure: Fig 2 Histogram showing the infecting organisms and cardiac valves involved in the 26 cases with positive blood culture*
+
+**Table 3 Summarising which criteria were fulfilled in the 12 cases with no blood cultures or no *post‐mortem* examinations performed**
+
+| Minor criteria |
+| --- |
+| Case | Positive findings for endocarditis on echocardiogram | Over 15 kg | Pyrexia | New onset heart murmur | Infected iv site | Detection of TE | Detection of IMTP | Subaortic stenosis |
+| 1 | Yes | Yes | Yes | Yes | Yes |  |  |  |
+| 2 | Yes |  | Yes | Yes |  | Yes | Yes |  |
+| 3 | Yes | Yes | Yes |  |  |  |  |  |
+| 4 | Yes |  | Yes | Yes |  |  |  |  |
+| 5 | Yes | Yes |  | Yes |  |  |  |  |
+| 6 | Yes | Yes |  | Yes |  |  |  | Yes |
+| 7 | Yes | Yes |  | Yes |  |  |  |  |
+| 8 | Yes | Yes | Yes |  |  |  |  |  |
+| 9 | Yes | Yes | Yes |  |  |  |  |  |
+| 10 | Yes | Yes | Yes | Yes |  |  |  |  |
+| 11 | Yes | Yes | Yes |  |  |  |  |  |
+| 12 | Yes |  | Yes | Yes |  |  |  |  |
+
+Out of the 77 cases, the mitral valve was infected in 48 cases (62.3%), the aortic in 18 cases (23.7%) and the tricuspid in two cases (2.6%). The aortic and mitral were both infected in six cases (7.9%), while the aortic and tricuspid, and the aortic, tricuspid and pulmonic were infected in one case each (1.3%). One dog did not have an echocardiogram done. This dog was classified as possible endocarditis as it did not fulfil any of the major criteria and had four minor criteria. Six dogs had mural lesions in addition to a valve lesion (8%): two dogs with aortic IE had a lesion on the right interatrial septum, one dog with aortic IE had a lesion extending into the right atrium, one dog with aortic and tricuspid IE had a lesion extending into the myocardium of the atrioventricular region, one dog with mitral IE had an lesion extending into the myocardium of the left ventricle and one dog with mitral IE had a lesion in the ventricular apical lumen and another lesion extending into the left ventricular outflow tract.
+
+### Hospitalisation and patient outcome
+
+Out of the 77 patients, 19 dogs were euthanased and five died spontaneously at the hospital (30%). Of the 19 that were euthanased, 13 (68%) had mitral IE, four (21%) had aortic IE, one (5%) had tricuspid IE and one had mitral and aortic valve IE (5%). Of the five that died, three (60%) had mitral IE and two (40%) had aortic IE. The median hospitalisation length of cases that died or were euthanased was 2 days (0 to 10 days). Of the 77 cases, 53 (69%) survived to discharge with a mean length of hospitalisation of 7.1 days ±3.9 days. Dogs that were discharged from the hospitals with aortic valve endocarditis lived a median of 480 days (range 22 to 3650 days) while dogs with mitral valve endocarditis lived a median of 440 days (range 2 to 2769 days) (Fig 3); survival times were not significantly different for the site of endocarditis. Fifteen dogs (28%) were lost to follow‐up and excluded from this analysis. The dog with tricuspid valve endocarditis lived 152 days. Dogs with both aortic and mitral valve endocarditis lived a median of 121 days (2 to 1065 days) and the dog with aortic, tricuspid and pulmonic valve endocarditis lived for 1825 days.
+
+*Figure: Fig 3 Kaplan–Meier survival curves of 31 dogs that were discharged from each referral centre with previously diagnosed aortic and mitral valve infective endocarditis*
+
+The median hospitalisation time of dogs with mitral valve IE was 5 days (n=48, 0 to 15 days), for aortic valve IE it was 4.5 days (n=18, 1 to 11 days), and for tricuspid valve IE it was 4.5 days (n=2, 0 to 9 days). The mean hospitalisation length of dogs with mitral and aortic valve IE was 7 days (n=5, ±4.5 days). The hospitalisation time of the dog with aortic and tricuspid valve IE was 8 days and the dog with aortic, tricuspid and pulmonic valve IE was 5 days. The mean hospitalisation time of dogs with single valve IE was 5.7 days (n=68, ±3.8 days) and that of multiple valve IE was 6.9 days (n=7, ±3.8 days).
+
+### Antimicrobial therapy
+
+Of the 77 cases, 52 (68%) had received either injectable or oral antimicrobial therapy when they presented at the referral hospitals, as summarised in Table 4. The median time antimicrobial therapy was prescribed by the referring veterinary practice was 7 days before referral to the hospitals (1 to 56 days). Topical antibiotics were excluded from this analysis.
+
+**Table 4 Summary of systemic antibiotic therapy received before admission at the referral hospitals**
+
+| Antibiotic | Number of cases |
+| --- | --- |
+| Amoxicillin–clavulanic acid | 18 |
+| Amoxicillin–clavulanic acid, fluoroquinolone (enrofloxacin, marbofloxacin) | 6 |
+| Amoxicillin–clavulanic acid, metronidazole, fluoroquinolone (enrofloxacin, marbofloxacin) | 5 |
+| Fluoroquinolones (enrofloxacin, marbofloxacin, pradofloxacin) | 4 |
+| Metronidazole | 3 |
+| Amoxicillin–clavulanic acid, metronidazole | 3 |
+| Penicillins (amoxicillin, amoxicillin–clavulanic acid), clindamycin | 2 |
+| Tetracyclines (doxycycline, oxytetracycline) | 2 |
+| Cefalexin, marbofloxacin | 1 |
+| Cefalexin, metronidazole | 1 |
+| Enrofloxacin, metronidazole | 1 |
+
+Of the 52 dogs that received antimicrobial therapy before referral, 24 subsequently showed negative MCB at the referral hospitals (46%). Of the remaining 25 dogs that had not received antibiotics before referral, 12 had negative MCB (48%).
+
+The most common antibiotic therapies prescribed at the referral centres were amoxicillin–clavulanic acid (Synulox; Zoetis) (Augmentin; GSK) (Co‐amoxiclav; Sandoz limited) and a fluoroquinolone (Baytril; Elanco) (Marbocyl; Vetoquinol) (Marfloquin; Virbac) (n=15, 22%) amoxicillin–clavulanic acid and a fluoroquinolone with metronidazole (Metrobactin; Dechra) (Metronidazole; Braun) (n=10, 15%), fluoroquinolone, cephalosporin (Zinacef; GSK) (Therios; Ceva), (Convenia; Zoetis) (Rilexine; Virbac) and metronidazole (n=4, 6%). These were administered either iv, subcutaneously or by mouth. Nine (12%) of the 77 dogs did not receive antibiotic treatment at the hospital as they either died or were euthanased before therapy was started.
+
+Of the 53 dogs that survived to discharge, the most common antibiotic protocol prescribed once discharged was 2 to 12 weeks of amoxicillin–clavulanic acid and a fluoroquinolone (n=23, 43%) by mouth. Four dogs had markedly prolonged therapy for periods of 5 to 22 months. Amoxicillin–clavulanic acid, enrofloxacin and metronidazole was used in nine dogs (17%).
+
+### Other therapies
+
+A list of other therapies initiated on the diagnosis of IE is summarised in Table 5. Other therapies used included metoclopramide (Emeprid; CEVA), mexiletine (Mexiletine HCl; Summit) and amiodarone (Amiodrone; Covetrus) in one case each. Therapies intended solely for analgesia were excluded from this analysis, *e.g*. non‐steroidal anti‐inflammatory drugs, opioids etc. Antithrombotic medication (clopidogrel and/or aspirin) was used in 18 (23%) of 77 dogs, four of which developed TED. Figure 4 summarises the survival curves between dogs that received antithrombotic medication and those that did not. Sixteen dogs were excluded from this graph as they were lost to follow‐up. The log‐rank test showed no significant difference in the survival time between these groups.
+
+**Table 5 Summary of medication other than antimicrobials administered to patients diagnosed with infective endocarditis at the referral hospitals**
+
+| Medication | Number of cases |
+| --- | --- |
+| Clopidogrel | 14 |
+| Maropitant | 10 |
+| Omeprazole | 9 |
+| Furosemide | 8 |
+| Pimobendan | 8 |
+| Benazepril | 7 |
+| Aspirin | 6 |
+| Ranitidine | 5 |
+| Sucralfate | 4 |
+| Beta blocker (sotalol, atenolol) | 4 |
+| Prednisolone | 3 |
+| Amlodipine | 3 |
+| Anticoagulant (dalteparin) | 3 |
+| Mirtazapine | 2 |
+
+*Figure: Fig 4 Kaplan–Meier survival curves comparing dogs that received antithrombotic medications and those that did not*
+
+### Complications of IE
+
+Eleven (14%) out of 77 dogs developed CHF. These included nine (82%) dogs that developed left‐sided CHF, five (56%) of which were mitral valve IE, two (22%) had mitral and aortic valve IE and two (22%) had aortic valve IE. One (9%) dog developed biventricular CHF with an aortic valve IE and one (9%) dog developed mitral valve IE but the *post‐mortem* analysis did not specify what side CHF the dog developed. The median survival time of dogs that developed CHF with IE was 5 days (range 0 to 908 days). Figure 5 summarises the survival curves between dogs that developed CHF and those that did not. Fifteen dogs were excluded from this analysis as they were lost to follow‐up. The log‐rank test showed a significant difference between the survival time of these two groups (P=0.0440).
+
+*Figure: Fig 5 Kaplan–Meier survival curves comparing dogs that developed congestive heart failure and those that did not*
+
+Sixteen (21%) out of 77 dogs developed TED. Eight (50%) dogs had renal TED, seven (43%) dogs had splenic TED, four (25%) dogs had TED in their musculature, two (13%) dogs had liver TED and one (6%) dog had an aortic TED. Some dogs developed TED in multiple locations. Figure 6 summarises the survival curves of dogs that developed TED and those that did not. Fifteen dogs were excluded from this analysis as they were lost to follow‐up. The log‐rank test showed no significant difference between the survival time of dogs that developed TED and those that did not.
+
+*Figure: Fig 6 Kaplan–Meier survival curves comparing dogs that developed thromboembolic events and those that did not*
+
+Twenty‐seven (35%) out of 77 dogs developed arrhythmias. Ventricular arrhythmias recorded included ventricular premature complexes (n=14, 52%), accelerated idioventricular rhythm (n=12, 44%), ventricular tachycardia (n=4, 15%) and ventricular bigeminy or trigeminy (n=2, 7%). Atrial arrhythmias recorded included supraventricular tachycardia (n=4, 15%) and supraventricular premature complexes (n=2, 7%). Four dogs had atrioventricular block, which was characterised as first degree in three dogs, second degree in one dog and third degree in one dog. Some dogs showed multiple types of arrhythmias. Figure 7 summarises the survival curves of dogs that developed arrhythmias and those that did not. Fifteen dogs were excluded from this analysis as they were lost to follow‐up. The log‐rank test showed no significant difference between the survival times of dogs that developed arrhythmias and those that did not.
+
+*Figure: Fig 7 Kaplan–Meier survival curves comparing dogs that developed arrhythmias and those that did not*
+
+AKI was observed in four (5%) of the 77 cases and the median survival time of these cases was 2��days (range 0 to 908).
+
+One dog was diagnosed with a tract connecting the left ventricle and right atrium (Gerbode defect), presumed as a complication of IE. This was diagnosed on echocardiographic examination and confirmed at *post‐mortem*. This dog had aortic valve IE and had been diagnosed with congenital SAS. The dog subsequently developed third degree AV block and was euthanased on the second day of hospitalisation due to clinical worsening.
+
+### Cardiac troponin level measurement
+
+Table 6 summarises the cTnI concentrations in the 30 dogs in which measurements were taken.
+
+**Table 6 Summary of cTnI levels**
+
+|  | Median cTnI level (ng/ml) (range) (reference, <0.23 ng/ml) |
+| --- | --- |
+| All dogs (n=30) | 2.9 (0.104 to 180) |
+| Dogs that survived until discharge (n=25) | 2.9 (0.104 to 180) |
+| Dogs that died or were euthanased (n=5) | 6.3 (1.05 to 27.5) |
+
+## DISCUSSION
+
+This multi‐centre study represents the first review of canine IE in a referral population of dogs in the UK and the second largest case series of IE to date. Large and medium breed dogs appear to be more predisposed to developing IE than small breed dogs, as has been described in previous veterinary studies (Sisson & Thomas 1984, Peddle & Sleeper 2007, Kilkenny *et al*. 2021, Reagan *et al*. 2022). However, reasons for this remain unclear. The mean age at which dogs were infected with IE in this study was similar in males and in females. A higher proportion of middle‐aged to older dogs were reported with IE in this study as noted in previous studies (Sisson & Thomas 1984, Sykes *et al*. 2006b, Kilkenny *et al*. 2021, Reagan *et al*. 2022). This may be due to age‐related senescence of the immune system, which has been shown to increase the incidence of infection in older pets (Day 2010). Similar to previous studies, we show that male dogs have a greater predisposition to developing IE than female dogs (Sisson & Thomas 1984, MacDonald *et al*. 2004, Reagan *et al*. 2022). Studies have shown sex differences in immune components with female dogs displaying stronger cell‐mediated and humoral responses, greater numbers of CD8 T‐cells and higher immunoglobulin levels than males which may account for this difference (Blount *et al*. 2005, Sundburg *et al*. 2016).
+
+The most common organisms that were cultured in this study were *E. coli*, *Staphylococcus* spp. and *Pasteurella* spp., which have all been reported in previous USA studies (MacDonald *et al*. 2004, Sykes *et al*. 2006a, Reagan *et al*. 2022). The mitral valve was most commonly infected in this study as shown in recent studies (Kilkenny *et al*. 2021; Reagan *et al*. 2022). This differs from previous studies however where both the aortic and mitral valve were frequently affected (Sykes *et al*. 2006a). One of the reasons for this is likely linked to the lack of *Bartonella* spp. IE cases, which appear to preferentially affect the aortic valve (MacDonald *et al*. 2004). However, it is also possible that *Bartonella* infections were missed due to a low level of PCR testing (in only 17% of cases), particularly given the high level of cases where no causative organism was detected (66%). A recent study found a 3% seroprevalence of *Bartonella* spp. in UK dogs (Alvarez‐Fernandez *et al*. 2018). A similar seroprevalence was found in USA dogs at 3.6%; however, this increased to 36 and 52% when dogs were co‐exposed to *Ehrlichia canis* or *Babesia canis*, respectively (Alvarez‐Fernandez *et al*. 2018). Neither *E. canis* or *B. canis* are thought to be endemic in the UK, which may explain why *Bartonella* spp. were not detected in our patients (Bird 2016, Wright 2018). Research shows that PCR testing is no more sensitive at detecting *Bartonella* spp. than blood cultures (Meurs *et al*. 2011, Roura *et al*. 2018), however, this depends on what samples were used to run the PCRs and how the blood samples were cultured. Studies have shown that using only valve tissue samples rather than blood samples and a pre‐enrichment culture before PCR testing may increase *Bartonella* positive results (MacDonald *et al*. 2004, Davis *et al*. 2020). A recent study utilised serology, PCR and blood cultures to aid their identification of *Bartonella* spp. as a cause of IE (Reagan *et al*. 2022). These techniques were not utilised in this study. Thus, performing both MCB, serology and PCR simultaneously may improve the detection of *Bartonella* spp. in IE patients, and may be required to prove that *Bartonella* spp. is not a major cause of IE in the UK (Meurs *et al*. 2011).
+
+Nearly, half the MCBs in this study were negative and this was not related to antimicrobial therapy before referral. This was shown by the lack of differences in the number of MCBs between groups that did and did not receive antimicrobials before referral. This may be due to the ability of some bacteria to invade macrophages and reside in cells as quiescent intracellular reservoirs, which may help protect it against the immune system and antimicrobial therapy (Croxen & Finlay 2009). Other common reasons for obtaining negative MCBs include infections by non‐bacterial organisms such as *Aspergillus* spp. or fastidious organisms such as *Chlamydia* spp. or *Mycoplasma* spp. which have been shown to cause endocarditis in humans (Sykes *et al*. 2006a, Habib *et al*. 2010). *Aspergillus* spp. were cultured in two dogs in this study; unfortunately, its diagnosis can be missed as it is a slow‐growing organism and therefore takes longer to isolate from MCBs (Pasha *et al*. 2016). Fungal endocarditis lesions have been shown to embolize easily in humans and should therefore be suspected in patients with negative MCBs and signs of embolic disease.
+
+Successful treatment of IE is based on early diagnosis and immediate, aggressive treatment to minimise secondary complications. Selection of the appropriate treatment is based on culture and sensitivity testing; however, while the culture results are pending, empirical treatment with a broad‐spectrum antibiotic such as an aminoglycoside, beta‐lactam or fluoroquinolone is recommended (Häggström 2010). The most common antibiotic therapy protocol used in this study (amoxicillin–clavulanate and enrofloxacin in 43% of patients) was similar to that proposed in previous literature (MacDonald 2010). Although current expert opinion suggests 4 to 6 weeks of antibiotic therapy (Häggström 2010), some patients in this study received much longer courses. Such long courses need to be carefully considered and patients monitored closely to determine if antibiotic therapy is still required as poor antimicrobial stewardship increases the risk of antimicrobial resistance (Schuts *et al*. 2016). Current guidelines in human cases of IE also suggest 4 to 6 weeks of antibiotic therapy, and longer courses are only indicated in cases of prosthetic valve IE (Baddour *et al*. 2015).
+
+The comorbidities in dogs with endocarditis noted in this study are similar to those previously described (Sykes *et al*. 2006a, MacDonald 2010). The most common comorbidities were a history of osteoarthritis, skin infections and periodontal disease.
+
+Although osteoarthritis is unlikely to be related to the development of IE in dogs, it can be a precursor to immune‐mediated polyarthritis or septic arthritis when combined with a generalised infection and any lameness or joint effusions should be investigated (MacDonald 2010). Skin abscesses and wounds have also been shown as portals of entry in a previous study (Sykes *et al*. 2006a). A link between endocarditis and periodontal disease has been shown in dogs (Pereira dos Santos *et al*. 2019). One study suggests that chronic inflammation of the oral cavity in the presence of bacterial flora may lead to endocarditis due to the development of high bacteraemia particularly in dogs with stage 3 periodontal disease (Glickman *et al*. 2009). However, other studies challenge this association (Sykes *et al*. 2006a, Peddle *et al*. 2009). Unfortunately, the stage of dental disease was not recorded in the patients in this study. The canine oral microbiome has been shown to be highly diverse and up to 38.2% of species are unculturable, thus these may also account for some of our negative MCB (Riggio *et al*. 2011). Although only six of our patients presented with a history of periodontal disease, up to 64.5% of dogs are affected with the disease in the general population (Robinson *et al*. 2016) and so it is likely that this was under‐reported in the patient records. In addition, the incidence and severity of periodontal disease increases with age which correlates with the higher number of middle age to older dogs affected by endocarditis as seen in this study (Wallis *et al*. 2019). From our data, endocarditis seems a rare sequela of periodontal disease.
+
+Congenital and acquired cardiac diseases were previously shown to predispose dogs to endocarditis (Romero‐Fernandez & Palermo 2019). Only two dogs had acquired cardiac disease (MMVD), thus this was not considered to be a major predisposition to IE in this referral population. Six dogs had underlying congenital heart disease (SAS) which has been previously suggested to predispose dogs to IE due to creating turbulent blood flow and damage to the aortic cusps (MacDonald 2010). In addition, SAS is one of the most common congenital heart conditions in large breed dogs which may account for their predisposition to IE (Ontiveros & Stern 2021). Male dogs have also been shown to be predisposed to SAS which may also partly explain their higher prevalence (Schrope 2015). A recent study did not diagnose any congenital SAS in their IE cases thus further studies are indicated to investigate this link (Reagan *et al*. 2022). One dog showed a Gerbode type defect which is thought to be secondary to destruction of the interventricular septum by bacterial IE (Peddle *et al*. 2008).
+
+Contrary to previous studies (Sykes *et al*. 2006b, Reagan *et al*. 2022), the development of TED and the use of antithrombotics were not shown to have a significant effect on survival. However, it appears logical that the use of antithrombotics would be beneficial in helping to decrease the size of vegetative lesions as research has shown that lesions may shelter bacteria from the immune system (Liesenborghs *et al*. 2020). To confirm these results, studies with larger sample size are necessary.
+
+The development of arrhythmias was not shown to have an effect on survival in this study, as previously shown (Sykes *et al*. 2006b). It is possible that in the majority of cases that developed arrhythmias they were not severe or prolonged enough to affect survival. Previous studies indicated that the development of AKI was associated with mortality; however, too few dogs developed AKI in this study to allow analysis (Sykes *et al*. 2006b; Reagan *et al*. 2022). In fact, in agreement with Reagan *et al*., CHF was the only complication of IE that was found to have a significant effect on survival in this study (Reagan *et al*. 2022).
+
+The most common clinical signs were non‐specific and similar to those described in the literature (Peddle & Sleeper 2007). Interestingly, a new or worsening heart murmur was only diagnosed in 47 of the 77 patients. In some cases, dogs had a pre‐existing heart murmur and therefore did not meet this criterion. As this is a minor criterion in the modified Duke criteria, it is essential that the lack of a new or worsening heart murmur on initial examination does not rule out endocarditis as a differential diagnosis in a septic patient. Although only 30 of our patients had serum cTnI levels measured, it was not shown to be helpful as a prognostic indicator. A recent study has shown that serum cTnI concentrations above >0.625 ng/ml are supportive of a diagnosis of IE (Kilkenny *et al*. 2021). This cut‐off could be useful as an additional minor criterion in the Duke's modified criteria; however, it has a high specificity and a low sensitivity, therefore, it must be used within the context of the overall clinical picture.
+
+The survival to discharge of dogs with IE in this study was found to be better than in older USA studies, 68% compared to 22 and 56% previously reported (MacDonald *et al*. 2004, Sykes *et al*. 2006b). This correlates with a more recent USA study on IE which also found a higher survival to discharge (70%) (Reagan *et al*. 2022). Interestingly, there was no significant difference between the survival times of dogs with mitral and aortic IE compared to previous USA studies which reported mitral valve infections to have the longest survival time and aortic valve infections to have the shortest survival time (MacDonald *et al*. 2004). These differences may be linked to the lack of *Bartonella* spp. detected in our patients as infection with this bacterium has been shown to be negatively correlated with survival and preferentially infects the aortic valve (Sykes *et al*. 2006a). Further studies with larger sample numbers may help validate these findings in both the UK and the USA. Interestingly, dogs with both mitral and aortic valve IE had the shortest survival times as in a previous study and likely represent advanced disease leading to degenerative structural changes in the heart and therefore a worsened prognosis (Reagan *et al*. 2022). Of the dogs that developed a tricuspid and/or pulmonic valve IE, only one tricuspid valve IE case had a history of having a jugular vascular access port placed. This likely would have been the portal of entry of the infection. Tricuspid and pulmonic valves are rarely affected by IE due to the higher pressures sustained on the left‐sided valves which predisposes the mitral and aortic valve to endothelial damage (Frontera & Gradon 2000). It is thought the relatively higher oxygen concentration of the left‐sided circulation is also more supportive of bacterial growth (Frontera & Gradon 2000).
+
+There are a number of limitations in this study, many common to retrospective studies relying on data retrieval. One limitation is that there were multiple different operators who carried out the echocardiographic scans, which may have led to a different interpretation of echocardiographic images (*i.e*. a small endocarditis lesion may have been picked up by one cardiologist but not another and vice‐versa). In addition, some rhythm abnormalities may have been missed depending on how long the ECG was run for. Furthermore, although the handling and analysis of the aseptic blood cultures were largely similar between each centre, a standardised protocol was not used which could cause some variation in the results. Another limitation was the lack of blood cultures that were positive and the small number of *Bartonella* spp. PCR assays performed. Unfortunately in this study, there were not enough data to allow the analysis of survival between different microorganism causing IE infections. Furthermore, data on routine complete blood work (haematology, biochemistry) were not analysed as part of this study but may have provided useful information to readers. In some cases, the cause of euthanasia may have been due to clients' financial concerns which may not reflect the actual outcome of IE. Unfortunately, this was unlikely to have been written in the clinical notes and must be considered when studying the outcome of the disease.
+
+The results of this study have shown that the bacteria causing this disease are largely similar to those in USA studies, apart from the lack of *Bartonella* spp. and the higher prevalence of mitral compared to aortic valve endocarditis. The number of cases in this study highlights the low frequency of IE out of the total referral population. This study has shown that the mitral valve and large breed dogs appear predisposed to IE which can be caused by a variety of bacteria. Although the prognosis for the disease remains poor, once patients survive to discharge, they can survive for prolonged periods.
+
+### Author contributions
+
+**Margot Berrezaie:** Conceptualization (supporting); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); visualization (lead); writing – original draft (lead). **David Connolly:** Writing – review and editing (equal). **Judith Cruzado:** Investigation (supporting). **Elzbieta Mederska:** Investigation (supporting). **Joanna Dukes‐McEwan:** Writing – review and editing (equal). **Karen Humm:** Conceptualization (lead); formal analysis (supporting); methodology (supporting); writing – review and editing (equal).
+
+### Conflict of interest
+
+None of the authors of this article has a financial or personal relationship with other people or organisations that could inappropriately influence or bias the content of the paper.

fulltext/md/PMC10135127.md

@@ -0,0 +1,96 @@
+---
+pmcid: "PMC10135127"
+pmid: "37106953"
+doi: "10.3390/ani13081390"
+title: "Integrated miRNA Changes in Canine Testis and Epididymis According to Age and Presence of Cryptorchidism."
+journal: "Animals : an open access journal from MDPI"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Integrated miRNA Changes in Canine Testis and Epididymis According to Age and Presence of Cryptorchidism
+
+## Abstract
+
+### Simple Summary
+
+MicroRNAs have been actively studied for their diagnostic, prognostic, and therapeutic purposes for various diseases in animals. In this study, we analyzed the changes in miRNA expression under different conditions in the testis and epididymis of male dogs. We identified differentially expressed miRNAs in the testis and epididymis depending on age and the presence of cryptorchidism in male dogs, which suggests that they could help better understand aging and diseases of the reproductive system in male dogs.
+
+### Abstract
+
+In the present study, we aimed to investigate age-, cryptorchidism-, and testicular tumor-related changes in miRNAs in the testis and epididymis of dogs. Twelve healthy male dogs were divided into two groups: young (<1 year, n = 8) and old (>3 years, n = 4). Five dogs with unilateral cryptorchidism, one with a Sertoli cell tumor, and one with seminoma were referred to a veterinary hospital. After surgery, the testes and epididymis tails were collected. A high-throughput miRNA array analysis was performed to identify miRNAs affected by age, cryptorchidism, and testicular tumors. The expression of only cfa-miR-503 was downregulated in the epididymis of younger dogs, whereas the expression of 64 miRNAs was upregulated. Among them, the top five miRNAs were cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a. The expression of cfa-miR-148a and cfa-miR-497 was considerably lower in cryptorchid testis than in healthy dog testis. In the epididymis, the cfa-miR-1841 level was significantly decreased. We observed a significant difference in the expression of 26 cfa-miRNAs between testicular tumors and normal tissues. This study demonstrated that aging and cryptorchidism have a causal relationship with miRNA expression. The identified miRNAs may be candidate genes for male reproductive traits and could be applied in molecular breeding programs.
+
+## 1. Introduction
+
+Male reproductive function declines with age in both humans and dogs. It is well-known that testis volume, germ cell and Sertoli cell number, serum testosterone levels, and sperm viability decrease with age in humans [1]. Reproductive aging is associated with a reduction in the number of Leydig cells and seminiferous tubules, as well as oxidative stress [2,3]. In addition, changes in the expression of genes involved in the regulation of apoptosis and DNA repair have been observed in aging testis [4]. A negative correlation has been reported between age and normal ejaculated sperm characteristics in dogs [5]. A previous study has shown that epididymal sperm quality and fertility decrease with age in male dogs [6]. Furthermore, fresh and thawed semen from old male dogs display decreased sperm motility and mitochondrial function [7]. Therefore, age is one of the most important factors influencing infertility in male animals, including dogs.
+
+Cryptorchidism is the most common pathological condition in dogs, in which the testis fails to descend to the base of the scrotum, with an informed morbidity rate of 0.8–10% [8]. Hereditary defects caused by sex-limited autosomal recessive genes are considered an etiology of cryptorchidism in male dogs. Although cryptorchidism is often considered a mild malformation, it can seriously affect the health of dogs, as a very high-risk factor for sterility and testicular tumor. As cryptorchid testes generally have considerably higher temperatures than normally descended testes, unilateral cryptorchids reduce semen quality or cannot produce normal sperm [9]. Cryptorchidism is associated with testicular tumors in dogs [10,11]. The incidence of testicular tumors in undescended testes is approximately 13 times greater than that in normally descended testes [8]. Although testicular tumors are typically malignant, the excess production of endogenous estrogen by the tumor cells can lead to a condition known as feminization syndrome and bone marrow suppression, which can be fatal [12].
+
+MicroRNAs (miRNAs) are a group of short non-coding RNA molecules and are approximately 20–24 nucleotides long; they function by epigenetically downregulating gene expression. miRNAs play a crucial role in controlling both mRNA stability and protein synthesis, thereby influencing the processes of spermatogenesis [13] and sperm maturation during sperm passage through the epididymis [14]. Although the differentially expressed miRNAs in the testicular tissue in cryptorchidism have been investigated in humans and various animal species, including horses, rats, and mice [15], direct correlation studies between miRNAs and cryptorchidism in dogs are limited. miRNAs can be utilized as biomarkers to elucidate changes in tissues affected by several disorders. In our previous study [16], we identified several miRNAs associated with uteropathies and age markers in female dogs. To the best of our knowledge, there are no reports of alterations in miRNA expression within the testes and epididymis of canines of different ages or with cryptorchidism and testicular tumors. The purpose of this study was to (i) assess the relationship between aging and disease status and the expression of miRNAs in male dog testis and epididymis and (ii) determine the potential of miRNAs as biomarkers and therapeutic targets for infertility and various diseases in male dogs.
+
+## 2. Materials and Methods
+
+### 2.1. Collection of Tissue Sample
+
+Male reproductive organ tissues were collected from two local animal hospitals and the veterinary teaching hospital of Seoul National University, Korea, at the request of the dog owners when general neutralization surgery or castration was required according to clinical findings, such as cryptorchidism, Sertoli cell tumor, and seminoma, with owners’ informed consent. Testicular tumor diagnosis was based on histological examination (IDEXX, Seoul, Republic of Korea) of the surgically removed tissues. Furthermore, testicular tumor tissue collection was approved only by the Seoul National University Institutional Animal Care and Use Committee (approval number: SNU-200217-3-2). The dogs with testicular tumors were not in the cryptorchidism state. The testes were obtained from 19 male dogs and epididymis cauda tissues (tail part of the epididymis) were collected from 17 male dogs, excluding two dogs with testicular tumors, during castration for the prevention and treatment of disease at the request of the owners, with informed consent obtained from the owners. In dogs, the final diagnosis of cryptorchidism can only be made after 6 months of age [17]. All cryptorchid testes were palpable in the right inguinal area and the left testis was present within the scrotum. Before anesthesia for castration, all dogs underwent appropriate clinical evaluations such as blood chemistry tests for total protein, glucose, blood urea nitrogen (BUN), creatinine, alkaline phosphatase (ALKP), and alanine aminotransferase (ALT) to identify any potential pathological conditions and determine their suitability for systemic anesthesia. The serum chemistry results for the dogs used in this clinical examination are shown in Table 1. This study involved eight healthy male puppies under the age of 1 year, four healthy male dogs over the age of 3 years, five dogs with cryptorchidism under the age of 1 year, one dog with Sertoli cell tumor, and one dog with seminoma over the age of 10 years. The average results and reference ranges of each parameter of the serum chemistry are presented in Table 1. Before the tissues dried or were damaged, each testis and tail of the epididymis were promptly separated after surgery and collected. As the testis and epididymis are surrounded by the tunica vaginalis, removal of the tunica vaginalis was performed after scrotal incision. The immature spermatozoa within the testes gain the ability to move and become capacitated as they pass through the epididymis. Therefore, we collected the testes and tail parts of the epididymis cauda to compare miRNA expression in all tested dogs. To collect the epididymis cauda tissue, the surrounding tissues such as the ligament of the epididymis were excised and separated from the testis. Some of the cryptorchid and descended tissues of the testis and epididymis were stored in neutral buffered formalin for histological examination. In addition, the remaining testis and epididymal cauda tissues from each male dog were collected in RNAlater (Thermo Fisher, Waltham, MA, USA) and stored at −80 °C for miRNA array analysis.
+
+### 2.2. Hematoxylina and Eosin Staining
+
+Both cryptorchid and non-cryptorchid sides of the testes and epididymis of dogs with unilateral cryptorchidism were collected for hematoxylin and eosin (H&E) staining and analysis. The testis and epididymis cauda were fixed in neutral buffered formalin, dehydrated with a gradient series of alcohol from 60% to 90%, and embedded in paraffin. According to the standard protocol, H&E-stained 4-µm sections (Leica Microsystems GmbH, Wetzlar, Germany) placed on silane-coated slides were observed under a microscope (BX53; Olympus, Tokyo, Japan).
+
+### 2.3. RNA Isolation and Quality Check, and cDNA Synthesis
+
+The total RNA was extracted following the protocol provided by the manufacturer. To extract the total RNA from each tissue sample, weighing a minimum of 50 mg, an easy-spin™ Total RNA Extraction Kit (Intron Biotechnology, Seoul, Republic of Korea) was used to homogenize the samples with 1 mL of RNA lysis solution. To perform gene microarray hybridization, the Agilent RNA 6000 nano kit and 2100 Bioanalyzer (G2939BA; Agilent, Santa Clara, CA, USA) were used to assess both the quantity and quality of the RNA. Only samples that fulfilled the following criteria were selected for microRNA analysis: A260/A280 and A260/A280 > 1.0, concentration > 50 ng/µL, volume > 10 µL, total amount > 0.7 µg, rRNA ratio > 1.0, and RIN > 7.0 with visible small RNA peaks. Before cDNA synthesis, the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, MA, USA) was used to assess the quantity and quality of the RNA. The Maxime RT-PCR premix kit (Intron Biotechnology) was used to reverse transcribe the RNA samples (500 ng) to cDNA, with a reaction mixture of a total volume of 20 µL.
+
+### 2.4. MicoRNA Hybridization, Scanning, and Data Processing
+
+All procedures were performed in accordance with the guidelines in the Affymetrix Expression Analysis Technical Manual (Affymetrix Inc., Santa Clara, CA, USA). Total RNAs were labeled using the FlashTag Biotin HSR RNA Labeling Kit (Thermo Fisher). The GeneChip^®^ Affymetrix miRNA microarray (Affymetrix Inc.) was used to hybridize the biotin-labeled samples. The Affymetrix GeneChip^®^ scanner was used to examine all arrays, and raw data analysis was carried out using the Affymetrix GeneChip^®^ Command Console^®^ Software (AGCC) (Affymetrix Inc., Santa Clara, California, USA). The measured intensity of each array probe was obtained from CEL files, which contained the raw data images developed by the scanner. A *p*-value of <0.05 and fold change of >2 served as cut-off criteria to restrict a broad range of differentially expressed microRNAs.
+
+### 2.5. Statistical Analysis
+
+The clinical data of the dogs are presented as mean ± standard deviation. An unpaired *t*-test was used to compare the groups. All statistical analyses were conducted with SPSS software (version 25.0; SPSS Inc., Chicago, IL, USA). The level of statistical significance was defined as *p* < 0.05.
+
+## 3. Results
+
+### 3.1. H&E Staining of Cryptorchid Testis and Epididymis
+
+The cryptorchid side testis showed severe diffuse tubular atrophy of the seminiferous tubules and only Sertoli cells with germ cell depletion (Figure 1A). However, the other (non-cryptorchid) side testis showed tubular atrophy, which was less severe than that of the cryptorchid-side testis (Figure 1B). Degeneration of germ cells and depletion of elongating spermatids were observed. In the cryptorchid-side epididymis, no spermatozoa showed epithelial tubule degeneration (Figure 1C). Cell debris and germ cell exfoliates were observed; however, no spermatozoa were observed in the lumen of the non-cryptorchid side epididymis (Figure 1D).
+
+### 3.2. MiRNA Expression in the Testis and Epididymis Cauda in the Dogs according to Age
+
+The testis and epididymis cauda miRNA volume plots for the comparison of immature male dogs younger than 1 year of age versus mature dogs older than 3 years of age are presented in Figure 2. According to the fold change cut-off of >2 (upregulation or downregulation) and *p* value of <0.05, there were no significantly differentially expressed miRNAs in the testes between immature and mature dogs. In the epididymis, the top five miRNAs with noticeable differential expression were confirmed by considering the fold change, *p*-value, and volume value. Sixty-five significant miRNAs that were differentially expressed were identified. Among them, the top five meaningful miRNAs, considering fold change and volume values, were cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a. The only significant miRNA that was less expressed in mature dogs compared with that in immature dogs was miR-503.
+
+### 3.3. MiRNA Expression of Testis and Epididymis Cauda in Cryptorchid Dogs
+
+As shown in Figure 3A, two significantly downregulated miRNA genes, cfa-miR-148a and cfa-miR-497, were found in the testes of cryptorchid dogs compared with age-matched normal dogs. As shown in Figure 3B, cfa-miR-1841 was significantly downregulated in the epididymis of cryptorchid dogs.
+
+### 3.4. MiRNA Expression in the Testis of Dogs with Sertoli Cell Tumor and Seminoma
+
+One hundred and seven miRNAs, with significant differences in expression between Sertoli cell tumors and normal testes, were identified. Sixteen miRNAs were downregulated, and most of the remaining ninety-one miRNAs were upregulated in one case of Sertoli cell tumor compared with those in the normal testis. The top five miRNAs with a fold change cut-off of >2 (upregulation or downregulation) between Sertoli cell tumor and normal testis were cfa-miR-27b, cfa-miR-20a, cfa-miR-93, cfa-miR-502, and cfa-miR-500 (Supplementary Figure S1A). Cfa-miR-27b, cfa-miR-20a, and cfa-miR-93 were highly expressed in Sertoli cell tumors, and cfa-miR-502 and cfa-miR-500 were less expressed. In seminoma, 86 miRNAs, different from those in normal testes, were identified. It was confirmed that 21 miRNAs were downregulated and 65 were highly expressed. The top five miRNAs with a fold change cut-off of >2 (upregulation or downregulation) were cfa-miR-378, cfa-miR-29a, cfa-miR-27b, cfa-miR-16, and cfa-miR-106a (Supplementary Figure S1B). The overlapping and non-overlapping differentially expressed miRNAs between the Sertoli cell tumor and seminoma groups are presented in Supplementary Figure S2 as Venn diagrams. The miRNAs indicated in red were differentially upregulated and those in blue were downregulated. The miRNAs indicated in green were upregulated in one comparison, which showed contradictory results for each comparator group, but downregulated in the other comparison. While there was an overlap between 55 significant miRNAs in “Sertoli cell tumor vs. normal testis” and “Seminoma vs. normal testis,” the expression of 11 miRNAs indicated in green were interestingly discordant in each comparison, indicating that a significant portion of the “tumor type” could affect the miRNA expression pattern. Twenty-nine miRNAs were commonly identified in the comparison between Sertoli cell tumor and normal testis and between seminoma and normal testis. Next, both Sertoli cell tumors and seminomas, regarded as testicular tumors, were grouped and compared to age-matched normal testes. Significant changes in the expression of 28 miRNAs were observed in testicular tumors compared with that in the normal testes. All 28 miRNAs (100%) overlapped, with the expression of the miRNAs frequently changing compared with that in the comparison of Sertoli cell tumors versus normal testes. Two miRNAs, miR-216b and miR-449, were commonly downregulated in the comparisons of testicular tumors versus normal testes and Sertoli cell tumors versus normal testes. The overlapping parts of the three circles represent 26 miRNAs—23 upregulated and 3 downregulated.
+
+## 4. Discussion
+
+The results of this study provide conclusive evidence regarding the effect of age and cryptorchidism on miRNA expression in the male reproductive system. The miRNA expression in the epididymis observed in this study was affected by age, with increased expression of cfa-let-7 members, cfa-miR-26a, and cfa-miR-200c in dogs older than 3 years of age. We focused on miRNA expression in dogs with cryptorchidism and testicular tumors as (1) cryptorchidism is the most common hereditary disorder in male dogs and (2) it may be a risk factor for infertility and testicular cancer. Our results showed that in cryptorchid dogs, the expression of cfa-miR-148a and cfa-miR-497 was downregulated in the testes and that of cfa-miR-1841 was downregulated in the epididymis compared with those in age-matched dogs. In addition, testicular tumors showed different miRNA expression levels compared with those in age-matched dogs.
+
+In addition to advanced maternal age, paternal age tends to be significantly associated with a decline in undesirable embryonic development and poor pregnancy outcomes because semen volume, sperm motility, and normal morphology can significantly decline with age in humans [18]. Similarly, in various animals, including dogs, ferrets, and cats, age negatively correlates with male reproductive capacity [5,6,19,20]. To the best of our knowledge, there is no report on the miRNA expression patterns in the reproductive organs of dogs across different age groups. In this study, the dogs were categorized into two distinct age groups (<1 year old and >3 years old) for the purpose of evaluating miRNA expression in the testes and cauda epididymis. Interestingly, the miRNA levels were not significant in the testis but were significant in the epididymis, with higher expression of cfa-miR26a, miR-200c, and let 7 family members (let 7a, let7b, and let 7c). These results suggest that the testis and epididymis are different in terms of miRNA expression and processing.
+
+The epididymis serves as the location for the post-maturation of testicular sperm, resulting in the acquisition of sperm motility and the capacity to recognize and fertilize oocytes. In this study, epididymal tissue was harvested from the cauda epididymis, where mature sperm are stored until ejaculation. To minimize the influence of prostatic fluid and immature sperm, the cauda epididymis may be a more appropriate tissue to examine the effects of age and cryptorchidism on miRNA expression in dogs. Furthermore, as the incidence of canine prostatic disorders markedly increases with age, the miRNA expression results may have been affected by a mixture of prostatic fluid.
+
+miR-26a is a functional miRNA that regulates sperm metabolism and apoptosis. It has been reported that miR-26a can have a major effect on the quality of semen in Holstein bulls as it plays a negative regulatory role in the expression of phosphoenolpyruvate carboxykinase-1 (PCK1) [21]. Additionally, it is suggested that miR-26a is involved in the regulation of bull sperm motility [22]. In boars, miR-26a is associated with decreased sperm motility [23]. A previous study demonstrated that there is a considerable increase in the expression of miR-26a in highly motile frozen-thawed sperm compared with that in low-motile frozen-thawed sperm [24]. Furthermore, the sperm transcript level of miR-26a-5p is lower in men with unexplained infertility than in fertile control, and high phosphatase and tensin homolog (PTEN) expression is associated with ejaculated spermatozoa [25]. PTEN signaling is a major negative regulator of PI3K signaling and is involved in the maintenance of spermatogonial stem cells in mice [26]. Interestingly, our study showed that cfa-miR-26a was highly expressed in the epididymis of mature dogs compared with that in immature dogs. These findings offer compelling evidence supporting the hypothesis that miR-26a may play a role in the regulation of epididymal aging and, consequently, have an effect on sperm metabolism or motility.
+
+Let-7 (lethal-7) is among the earliest identified miRNAs. As the expression of let-7 family members gradually increases during development, it is not surprising that high levels were observed in the cauda epididymis of mature dogs compared with those of younger dogs. However, in a previous study, female germ cells did not show changes in let-7 miRNA expression, but male germ cells showed increased expression during development [27]. Boars with low sperm motility and a high percentage of abnormal sperm showed higher levels of let-7a, let-7d, and let-7e miRNAs in their spermatozoa [28], indicating that the let-7 family members may be markers for infertility. Interestingly, although the expression of miR-26a and let-7 family members was increased in the ovaries of dogs with uteropathies in our previous study, significant differences in the expression of these miRNAs were observed in the epididymis of mature dogs in this study. Therefore, miR-26a and the let-7 family members may be related to female reproductive organ disease in dogs as well as the aging of male reproductive organs. In Yorkshire boars, endogenous miR-26a and let-7 have anti-apoptotic and pro-survival functions in sperm cells by targeting *PTEN* and *PMAIP1* [23]. The most abundant miRNAs in the epididymis of bovine species are let-7 family members and miR-200a/b tumor suppressors [29]. miRNA-mediated inactivation of cellular oncogene products could play a role in maintaining the stability of the epididymis, which is an organ with a unique ability to evade tumorigenicity. Consistent with these findings, the let-7 family members and miR-200a/b tumor suppressors, whose expression was confirmed in the epididymis of younger dogs, were not identified in the testicular tumors in the present study.
+
+The miR-200 family, which includes miR-141, miR-200a, miR-200b, miR-200c, and miR-429, is the most prevalent family in the miRNA system, and all members are highly conserved. It has been reported that the miR-200 family members regulate epithelial–mesenchymal transition, which is an important step for breast cancer infiltration and metastasis [30]. Furthermore, miR-200 has a functional role in the regulation of cell invasion and migration by targeting PTEN [31]. The expression of miR-200a, miR-200c, and miR-141 in both male and female mouse germ cells is downregulated gradually during development [27]. In a human study, miR-200a-3p and miR-200c-3p were identified as potential biomarkers for male subfertility [32]. As miR-200b and miR-200c accumulate in spermatozoa during passage through the epididymis [33,34], the present study results indicate that sperm maturation in the epididymis cauda may be differentially regulated according to dog age.
+
+Cryptorchidism is a congenital defect commonly found in dogs, where one or both testicles fail to descend normally into the scrotum. The retained testicle(s) may remain in the abdomen or become lodged in the inguinal canal, causing potential health issues such as infertility, testicular tumors, and torsion. In recent years, several studies have focused on miRNA expression in the testes of various animals with cryptorchidism, including horses [35], rats [36], and mice [37]. miR-148a was identified as differentially expressed in the seminal plasma extracellular microvesicles of men with oligoasthenozoospermia subfertility compared with that in men with normozoospermic fertility, demonstrating that it may be a marker for male infertility [38]. Similarly, the present study suggests that cryptorchid-side testes and epididymis do not have spermatozoa and cryptorchid-side testes display differential expression of miR-148a. In agreement with the fact that miR-497-5p has been identified only in the testicular tissues of spermatozoa and seminal plasma in humans [39], cfa-miR-497 was found to be expressed at low levels in the testes of cryptorchid dogs.
+
+In testicular tumors, miR27b was highly expressed in both Sertoli cell tumors and seminomas; it has been found to be differentially expressed in the mature spermatozoa of infertile men. The high expression of cysteine-rich secretory protein 2 (CRISP2), which is predominantly expressed in the testis, is correlated with the expression of miR-27 in humans [40]. miR-27b expression is downregulated during sheep fetal testis development from D42 to 75 and plays an important role in regulating cellular differentiation [41].
+
+A major limitation of this study is that the miRNA expression was analyzed in a limited number of dogs, especially in one case each of Sertoli cell tumor and seminoma. The present analysis of miRNA expression in testicular tumor tissues lays a foundation for more extensive and larger-scale studies. The results of this study should be interpreted with caution. Furthermore, age and breed predisposition may have affected the miRNA expression results of this study. In order to reduce this expected bias, we classified the dogs into two age categories, those under 1 year old and those over 3 years old; evaluated tissue samples obtained only from small dog breeds; and analyzed age-matched dogs as a control group for the comparison of cryptorchidism testes and testicular tumors.
+
+## 5. Conclusions
+
+In conclusion, miRNA expression in the male reproductive tissue of dogs with cryptorchidism and testicular tumors was comprehensively analyzed and the effect of age on miRNA expression in male reproductive organs was examined. The present study demonstrated that epididymal cfa-miR-26a, cfa-miR-200c, cfa-let-7c, cfa-let-7b, and cfa-let-7a expression regulated via PTEN may be involved in aging in dogs. Furthermore, cfa-miR-148a and -497 expression were consistently lower in the testes and cfa-miR-1841 expression was lower in the epididymis of dogs with cryptorchidism, suggesting that these miRNAs may be useful biomarkers for cryptorchidism and male infertility. This study provides insights into the development and causes of cryptorchidism and testicular tumors. We hope that our study will help develop new diagnostic methods and preventive medication for spontaneous cryptorchidism and testicular tumors and improve male fertility with aging in dogs.

fulltext/md/PMC10140049.md

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+---
+pmcid: "PMC10140049"
+pmid: "37105983"
+doi: "10.1038/s41597-023-02139-6"
+title: "Generating Detailed Spectral Libraries for Canine Proteomes Obtained from Serum and Urine."
+journal: "Scientific data"
+year: "2023"
+license: "cc by"
+article_type: "data-paper"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Generating Detailed Spectral Libraries for Canine Proteomes Obtained from Serum and Urine
+
+## Abstract
+
+Domestic dogs (*Canis lupus familiaris*) are popular companion animals. Increase in medical expenses associated with them and demand for extending their lifespan in a healthy manner has created the need to develop new diagnostic technology. Companion dogs also serve as important animal models for non-clinical research as they can provide various biological phenotypes. Proteomics have been increasingly used on dogs and humans to identify novel biomarkers of various diseases. Despite the growing applications of proteomics in liquid biopsy in veterinary medicine, no publicly available spectral assay libraries have been created for the proteome of canine serum and urine. In this study, we generated spectral assay libraries for the two-representative liquid-biopsy samples using mid-pH fractionation that allows in-depth understanding of proteome coverage. The resultant canine serum and urine spectral assay libraries include 1,132 and 4,749 protein groups and 5,483 and 25,228 peptides, respectively. We built these complimentary accessible resources for proteomic biomarker discovery studies through ProteomeXchange with the identifier PXD034770.
+
+## Background & Summary
+
+Disease diagnosis is important for the welfare of companion dogs, including enabling proper treatment and reducing medical expenses that may arise in the future^1–3^. Biomarker-driven research and development improves both the sensitivity and specificity of diagnosis in various dog diseases, which can contribute towards extending the lifespan of dogs^4–7^. In addition, short-lived dogs living in the human-like circumstances form an animal model suitable for understanding human diseases and support translational discoveries^8^. Longitudinal studies that use multiomics for analyzing different types of samples collected throughout the life cycle from various breeds of dogs are recently being conducted^8–10^. Collection of such large veterinary medical data sets provide an opportunity to find more information about health and diseases in dogs^11^.
+
+The proteomics approach derived from mass spectrometry (MS) has been useful in the discovery of novel protein biomarkers by aiding in acquisition of quantitative protein information from complex biological samples;^12–16^ it enables biomarker-based disease diagnosis, prognosis, and therapeutic monitoring. Deep proteomic spectral libraries created using fractionated samples are built through data-dependent acquisition (DDA) mass spectrometry and are mainly employed to analyze data-independent acquisition (DIA)-mass spectrometry of unfractionated samples^17^. Comprehensive spectral libraries are required for robust quantification of maximum number of proteins in complex samples.
+
+Shotgun proteomics has been used in searching for protein biomarkers using canine serum and urine samples. Several studies on dilated cardiomyopathy^18^, dyssynchrony heart failure^19^, mitral valve disease^20^, chronic valve disease^21^, osteoarthritis^22^, babesiosis^23–25^, ehrlichiosis^26^, leishmaniasis^27–29^, hemangiosarcoma^30^, high-grade multicentric lymphoma^31^, osteosarcoma^32^, and mammary tumors have utilized serum samples^33^, whereas studies on X-linked hereditary nephropathy^34^, chronic kidney disease^35,36^, dirofilariasis^37^, transitional cell carcinoma^38^, and venomous bite from a European viper have utilized urine samples^39^.
+
+Publicly available spectral libraries for canine serum and urine proteome are necessary for conducting studies to discover the uses of new disease biomarkers. Serum contains certain high-concentration proteins (i.e., albumin) that make it difficult to measure low-concentration proteins using liquid chromatography-MS (LC-MS), which remains the primary reason that more than 1,000 canine proteins have not yet been identified in urine^40^. Therefore, individual spectral libraries for serum and urine are needed and could help in identifying more proteins.
+
+Comprehensive serum and urine spectral libraries were built from 24 fractionated serum and urine proteome using a high-resolution Orbitrap mass spectrometer. The serum protein library includes 5,483 peptides mapped to 1,132 serum proteins (Supplementary Table 1). The urine protein library has 25,228 peptides mapped to 4,749 urinary proteins (Supplementary Table 2). We analyzed the characteristics of the two libraries using DIALib-QC^41^. Spectral libraries can be used to interpret DIA data collected on other instruments and for qualitative and quantitative analysis of peptides and proteins through spectral matching with DDA data. We deposited raw MS data from the instrument and spectral libraries with ProteomeXchange^42^ Consortium (http://proteomecentral.proteomexchange.org) through the Proteomics Identification Database (PRIDE)^43^ and allied repository with identifier PXD034770.
+
+## Methods
+
+### Materials and reagents
+
+The materials and reagents used in this study were purchased from various sources: HPLC grade acetonitrile and water from Avantor (Radnor, PA, USA); LC-MS grade formic acid (FA) and bicinchoninic acid (BCA) assay kit from Thermo Fisher Scientific (Pittsburgh, PA, USA); MS-grade trypsin/lys-C Mix from Promega (Madison, WI, USA); suspension-trapping (S-trap) digestion kit from Protifi (Huntington, NY, USA); and triethyammonium bicarbonate (TEAB) from Sigma-Aldrich (Burlington, MA, USA).
+
+### Canine serum and urine collection
+
+The features of the dogs sampled in this study are summarized in Table 1. Data was collected from 82 dogs belonging to 19 breeds consisting of 44 neutered males, 28 spayed females, 5 females, and 5 males. Serum and urine samples were collected from 12 dogs with acute pancreatitis, 12 dogs with chronic pancreatitis, 18 dogs with lymphoma, and 40 healthy dogs. Blood was drawn from the cephalic vein, and sera were separated by centrifuging for 10 min. Urine samples were collected by free catch. The sample procedures followed the guidelines of the Institutional Animal Care and Use Committee (IACUC) of Seoul National University (Approval number, SNU-200701-6-2; Approval date, 2020.09.28)^44^.Table 1Study population of 19 breeds of 82 dogs. Average age, sex, and disease status were represented.BreedNAverage age (years)SexDisease statusMNFSFMHealthyAcute pancreatitisChronic pancreatitisLymphomaBichon Frise12.0——1—1———Chihuahua86.616—1512—Cocker Spaniel39.73——————3Coton de Tulear111.01—————1—Dachshunds35.32—1—1——2English Cocker Spaniel13.01———1———French Bulldog10.51———1———Golden Retriever10.61———1———Labrador Retriever21.2———21——1Maltese217.21271114313Maltipoo21.72———2———Pomeranian46.8121—12—1Poodle107.055——1522Poongsan dog10.9—1——1———Shetland Sheepdog13.0—1——1———Shih Tzu88.353——4—13Toy Poodle11.01——1———Welsh Corgi22.31——12———Yorkshire Terrier119.6821—2153
+
+### Serum protein sample preparation
+
+Serum samples (40 μL) were passed through a MARS14 column (100 × 4.6 mm; Agilent Technology, Palo Alto, CA, USA) on a binary HPLC system (20 A Prominence; Shimadzu, Tokyo, Japan) to reduce 14 high serum proteins; the unbound part was quantified by BCA assay and only 100 μg of it was freeze-dried with a cold trap (CentriVap Cold Traps; Labconco, Kansas City, MO, USA).
+
+### Urine sample preparation
+
+One milliliter of urine was lyophilized using a cold trap. Dried urine samples were resuspended in 100 μL of 5% SDS in 50 mM TEAB (pH 8.5) and 100 μL of protein was lyophilized after BCA quantification.
+
+### Peptide sample preparation
+
+One hundred microgram of dried serum or urine sample was reconstituted with 400 μL of 5% SDS in 50 mM TEAB (pH 8.5). Dithiothreitol was added to a final concentration of 20 mM denatured sample, and the reaction was carried out at 95 °C for 10 min. The reduced sample was then placed in iodoacetamide at a final concentration of 40 mM and incubated for 30 min at 25 °C in the dark. Using 1.2% phosphoric acid, samples were attached to S-Trap mini columns (ProtiFi, Farmingdale, NY, USA, Cat. No: CO2-mini-80). Following the manufacturer’s protocol, we performed S-trap proteolysis by adding 10 μg Lys-C/trypsin mixture and incubating at 37 °C for 16 h^45^. The digested peptide mixture was freeze-dried with a cold trap and stored at −80 °C.
+
+### Mid-pH reverse-phase fractionation
+
+Pooled digested serum and urine peptide mixtures were prepared from the 25 μg of 82 individual canine digested serum and urine peptides, the concentrations of which were measured with a UV/Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific, Waltham, MA, USA) at a wavelength of 280 nm, with the sample type option set to “1 Abs = 1 mg/mL”. Their separation was performed on an XBridge C-18 column (4.6 mm i.d. × 250 mm length; pore size, 130 Å; and particle size, 5 μm; Waters Corporation, USA), using a Shimadzu Prominence HPLC instrument (Shimadzu) at a flow rate of 0.5 mL/min for 120 min. We used 10 mM TEAB (pH 8.5) in HPLC water as mobile phase A and 10 mM TEAB (pH 8.5) in 90% acetonitrile and 10% HPLC water as mobile phase B. The sample was dissolved in 80 μL of mobile phase A and then injected into a 100 μL sample loop. A gradient of 5-5% B for 8 min, 5–40% B for 57 min, 40–44% B for 5 min, 44–60% B for 4 min, 60-60% B for 14 min, 60-5% B for 2 min and 5-5% B for 30 min were applied. From 8 to 94.4 min, the eluents were collected in increments of 0.45 mL in a 96-well plate on an FRC-10A of the Shimadzu Prominence (Shimadzu). The resulting 96 fractions were mixed in a concatenate manner to form 24 fractions (Table 2). Each fraction was dried and stored at –80 °C.Table 2Sample overview.Sample typeFractionsLibrary nameSerum24DogSerum_LibraryUrine24DogUrine_Library
+
+### Nano LC-MS/MS
+
+Peptide mixtures were separated by using the Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific, Waltham, MA, USA). The dried sample was resuspended in 0.1% formic acid to a concentration of 1 μg/μL, and 5 μL of which was loaded on a C18 Pepmap trap column (20 mm × 100 μm i.d., 5 μm, 100 Å; Thermo Fisher Scientific) and separated with an Acclaim™ Pepmap 100 C18 column (500 mm × 75 μm i.d., 3 μm, 100 Å; Thermo Fisher Scientific) over 200 min (250 nL/min) using a 0–48% acetonitrile gradient in 0.1% formic acid and 5% DMSO for 150 min at 50 °C. The LC was connected with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific) with an EASY-Spray nano-ESI source. In a data-dependent mode, mass spectra were obtained with an automatic switch between a full scan with top 20 data-dependent MS/MS scans. Resolution was set to 60,000 at m/z 200, and target value of 3,000,000 for MS scan type was selected. The ion target value for MS/MS was set at 100,000 with a resolution of 15,000 at m/z 200. The maximum ion injection time was set to 100 ms for the full scan and 50 ms for MS2 scan. Isolation width was 1.7 m/z, and normalized collision energy was set at 27. Dynamic exclusion for measurements of repeated peptides was set for 40 s.
+
+### Data processing
+
+Software Spectronaut v14 (Biosynosis, Switzerland) was used for data processing with default settings to build spectral libraries with the UniProt SwissProt and TrEMBL integrated database (*Canis lupus familiaris* (Taxon ID 9615); downloaded on 05/04/2022; 113,977 entries including both reviewed (838) and unreviewed (113,139) entries). N-terminal acetylation and methionine oxidation were set as variable modifications, and cysteine carbamidomethylation was set as a fixed modification. We set the false discovery rate (FDR) <1% at the peptide-spectrum match (PSM), peptide, and protein levels, respectively. Spectral libraries for canine serum and urine proteomes were created using 24-DDA raw mass spectrometry data.
+
+## Data Records
+
+The two spectral libraries (.xls and .kit) produced from raw mass spectrometry data (.raw) and DIALib-QC evaluation reports for both were saved to the ProteomeXchange Consortium through PRIDE with the dataset identifier PXD034770^46^. The mass spectrometry files were named “Dog(name of the sample type)-(fraction number).raw”. The spectral libraries were named “Dog(name of the sample type)_Library.kit” and imported to Spectronaut software. Individual DIALib-QC evaluation reports for the two spectral libraries were included in this process.
+
+## Technical Validation
+
+High-level standard assay libraries are needed for accurate quantification of peptides and proteins. To get as many peptides as possible, a library was constructed with DDA-MS datasets obtained by reducing sample complexity through mid-pH separation. The search engine performed FDR calculation through target-decoy method using Biognosys’s Pulsar, and the levels of PSM, peptide, and protein group were controlled to 1% or less. To build the spectral library for the serum and urine proteome, library-wide FDR controls were applied to <1% at the three levels.
+
+The canine serum spectral library contained 97,888 transitions, identifying 6,159 peptide precursors and representing 5,483 stripped peptides and 1,132 protein groups. The canine urinary spectral library included 347,749 transitions identifying 27,922 peptide precursors interpreting 25,228 stripped peptides and 4,749 protein entries. Comparison between the spectral library for each dog specimen showed that 589 protein groups were common. In addition, there were 543 and 4,160 protein groups that were uniquely identified in canine serum and urine, respectively (Fig. 1a). A total of 4,055 peptides were found to be common between the spectral library for each dog specimen. In addition, there were 1,428 and 21,173 peptides that were uniquely identified in canine serum and urine, respectively (Fig. 1b).Fig. 1Number of constituent peptides and proteins in the canine serum and urine proteome spectral libraries. (**a**) Venn diagram of the number of peptides in two spectral libraries. (**b**) Venn diagram of the number of proteins in two spectral libraries.
+
+We evaluated the quality and features of the canine serum proteome library by DIALib-QC. When contrasting the retention times (RTs) between the 2+ and 3+ ion charge states of precursors with the identical peptide, the two RTs with an R^2^ value of 1 showed almost identical values, proving the high level of chromatography (Fig. 2a). The normalized RT used here was calculated by the standard peptides in the spectral library. A higher proportion of y than of b ions (72.4 vs. 27.6%), that is previously known as higher-energy collisional dissociation fragmentation in Orbitrap^47^ (Fig. 2b). In addition, >99.4% of peptide fragment ions in the library have 1+ or 2+ charge states (Fig. 2c). Precursor ions containing five or fewer fragment ions do not account for 5.7% of the library (Fig. 2d). In the library, 98% of the precursors lie between 400 and 1,200 m/z (Figs. 2e), and 98.5% of the precursor states of charge range from +2 to +4 (Fig. 2f). More than 99.6% of all peptides in the library have 30 or fewer sequences (Fig. 2g). Proteins with two or more peptides possess 63.3% of the total amount, of which proteins with five or more peptides constitute 26.9% of the proteins in the library (Fig. 2h).Fig. 2Characteristics of the canine serum proteome spectral assay library. (**a**) Scatter plot of the retention times (RTs) of the 2+ and 3+ charge ion states of the corresponding peptide. (**b**) Number of detected fragment ion type (b- and y-ion). (**c**) Histogram of the charge states of the detected fragment ions. (**d**) Frequency of precursors as a function of the number of fragment ions per precursor. (**e**) Histogram of precursors by precursor m/z values over the entire range of mass-to-charge ratio detected. (**f**) Histogram of the charge states of the detected precursor ions. (**g**) Frequency of peptides along sequence length. (**h**) Frequency of proteins as a function of number of peptides per protein.
+
+We also applied DIALib-QC to appraise the canine urine proteome library. The RTs between the 2+ and 3+ ion charge states of the identical peptide show high positive correlation (R^2^ = 1, Fig. 3a). The proportion of b and y fragment ions are 75.1% and 24.9% (Fig. 3b). In the library, more than 99.5% of the fragment ions in charge states of 1+ or 2+ are included (Fig. 3c). As shown in Fig. 3d, peptides containing more than six fragment ions per the corresponding precursor accounted for beyond 89.6% of the total. In the library, 98.2% of the precursors belong to 400–1,200 m/z (Fig. 3e), and the charge state of 99.3% precursors range from +2 to +4 (Fig. 3f). More than 99.2% of all peptides in the library are in the sequence length range of 7–52 and have less than 30 mer in total (Fig. 3g). Proteins containing two or more peptides make up 72.3% of the total, of which proteins containing five or more peptides make up 35.5% of the proteins in the library (Fig. 3h).Fig. 3Characteristics of the canine urine proteome spectral assay library. (**a**) Scatter plot of the retention times (RTs) of the 2+ and 3+ charge ion states of the corresponding peptide. (**b**) Number of detected fragment ion type (b- and y-ion). (**c**) Histogram of the charge states of the detected fragment ions. (**d**) Frequency of precursors as a function of the number of fragment ions per precursor. (**e**) Histogram of precursors by precursor m/z values over the entire range of mass-to-charge ratio detected. (**f**) Histogram of the charge states of the detected precursor ions. (**g**) Frequency of peptides along sequence length. (**h**) Frequency of proteins as a function of number of peptides per protein.
+
+## Supplementary information
+
+Supplementary Table 1
+Supplementary Table 2

fulltext/md/PMC10142166.md

@@ -0,0 +1,129 @@
+---
+pmcid: "PMC10142166"
+pmid: "37106394"
+doi: "10.1186/s13071-023-05682-8"
+title: "Treatment of dogs with Bravecto<sup>®</sup> (fluralaner) reduces mosquito survival and fecundity."
+journal: "Parasites & vectors"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Treatment of dogs with Bravecto^®^ (fluralaner) reduces mosquito survival and fecundity
+
+## Abstract
+
+### Background
+
+Mosquitoes serve as the vector of canine heartworm (*Dirofilaria immitis*), which represents a significant and persistent threat to canine health. A reduction in the longevity and/or reproductive success of mosquitoes that take a blood meal from fluralaner-treated dogs may consequently reduce the local transmission of heartworm and prevent new infections. A novel secondary effect of an oral formulation of the ectoparasiticide fluralaner (Bravecto^®^) against a laboratory strain of the mosquito *Aedes aegypti*, a potential major vector of canine heartworm, was investigated in this study.
+
+### Methods
+
+Six dogs were administered a single dose of fluralaner orally in the form of Bravecto^®^ Chews (at the labeled fluralaner dose of 25 mg/kg body weight), while six control dogs received no treatment. Mosquitoes were fed on blood that was collected from each dog prior to treatment and weekly for 15 weeks post-treatment to assess the continued effects of fluralaner as its serum level decreased. Mosquito fitness was assessed by three parameters: rate of successful blood-feeding, survival, and egg laying.
+
+### Results
+
+Successful blood-feeding rate was similar between control and treatment groups. In the fluralaner treatment, mosquito survival was significantly reduced within the first 24 h after blood-feeding, for the first 12 weeks post-treatment of the dogs (efficacy range = 33.2–73.3%). Survival of mosquitoes up until a potentially heartworm-infective timepoint (14 days post-blood-feeding) was significantly reduced in the fluralaner-treated group at several timepoints (1, 2, 5, 11, 12, 13, 14, and 15 weeks post-treatment; efficacy range = 49.4–91.4%), but was less consistently reduced at the other timepoints. Egg laying by mosquitoes was almost completely suppressed for the first 13 weeks following treatment of the dogs with fluralaner (treatment efficacy ≥ 99.8%).
+
+### Conclusions
+
+Mosquitoes fed blood from fluralaner-treated dogs experienced a significant reduction in survival and fecundity. These findings support the potential for a reduction in heartworm transmission directly by lethal effects on the vector and indirectly through a reduction of the local vector population when mosquitoes are exposed to animals treated with fluralaner.
+
+### Supplementary Information
+
+The online version contains supplementary material available at 10.1186/s13071-023-05682-8.
+
+## Background
+
+While mosquitoes serve as vectors for several viral and bacterial pathogens of dogs, canine heartworm (*Dirofilaria immitis*) stands out as one of the most significant infectious diseases in companion animal health that is transmitted by mosquitoes. This filarial parasite has a broad distribution from tropical to temperate regions around the world and, due to its zoonotic potential, is also of concern for human health [1–3]. Furthermore, the expansion of its vector range and the increasing mobility of pets through travel and migration appear to be responsible for the introduction of heartworm to areas where it was once rare or absent [2, 4–6]. Several species of mosquito serve as potential vectors of heartworm, including *Aedes aegypti*, *Aedes albopictus*, and *Culex quinquefasciatus* [1]. In the United States, a national heartworm prevalence of 1.3% has been reported, with regional rates reaching 14.6–48.8% [7, 8]. Transmission is most intense during the warmer months, when mosquitoes are most active; the southern United States experiences especially high levels of transmission due to its climate, with prevalence rates increasing in this and several other regions over recent years [9, 10]. Infection is managed by the use of preventives in the macrocyclic lactone drug class, which, when administered regularly, kill larval worms before their development into adults.
+
+Other arthropods affecting dogs (e.g., fleas and ticks) may be routinely managed with ectoparasiticides, which effectively prevent infestation and the transmission of many of the pathogens that they vector. One such compound is fluralaner, a member of the isoxazoline class, which acts as an antagonist of γ-aminobutyric acid receptors and glutamate-gated chloride channels, inducing neurotoxicity that results in paralysis and death of the affected arthropods. This toxicity is significantly more selective for arthropod than mammalian neurons [11, 12]. Fluralaner has also been shown to exhibit toxicity against mosquitoes, with lethal effects on the larval and adult stages of *Ae. aegypti* demonstrated via multiple routes [13]. More recently, the target of fluralaner in *Ae. aegypti* was identified as the resistance to dieldrin subunit of the ionotropic γ-aminobutyric acid receptor [14]. These lethal effects of fluralaner against *Ae. aegypti* indicate its potential efficacy as a novel compound for the interruption and/or prevention of transmission of mosquito-borne pathogens.
+
+In dogs, fluralaner has a long elimination half-life and residence time in plasma, which enables 12 weeks of persistent efficacy against fleas and ticks after treatment with a single oral dose [15–17]. This efficacy relies on the attachment of the arthropod to the host and its ingestion of the active compound from the host’s circulatory system. Exposure to fluralaner rapidly kills ticks after attachment, which affects the transmission potential for *Borrelia burgdorferi* and *Babesia canis*, and may also reduce the transmission potential for *Anaplasma phagocytophilum*, *Ehrlichia canis*, and *Babesi microti* [18–21]. The efficacy of fluralaner against *Lutzomyia longipalpis*, the main vector of *Leishmania infantum*, has also been demonstrated through vector mortality [22]. For heartworm, as the duration of the blood meal of the mosquito vector is relatively brief, at least two feeding events are required for the parasite to complete its life cycle: one for the uptake of microfilariae by the mosquito vector from the peripheral blood of an infected host, and another at least 10 days later for the transmission of the infective third-stage larvae to a new host [23]. For compounds like fluralaner with a prolonged presence in the circulatory system of dogs, these feeding events offer opportunities to interrupt the heartworm life cycle when fluralaner is present in the host at concentrations lethal to the mosquito vector.
+
+The aim of the present study was to evaluate the efficacy of fluralaner against *Ae. aegypti* blood-feeding, survival, and reproduction following the treatment of dogs with a single oral dose. To this end, mosquitoes were fed blood that was drawn from treated dogs or untreated controls once prior to treatment and weekly for 15 weeks thereafter.
+
+## Methods
+
+### Study design
+
+The study protocol was approved by the Institutional Animal Care and Use Committee of the University of Georgia (protocol A2020 10-009). A randomized block design was utilized in this controlled efficacy study. Purpose-bred dogs 15 months of age were used. Mosquitoes were fed on blood drawn from each study dog (*n* = 12) prior to its assignment to any treatment group, and the percentage blood-feeding rate was determined for blood from each dog. None of the dogs had received any endectocide or heartworm prevention in the 60 days prior to the study. The dogs were sorted by mosquito blood-feeding rate and blocked into pairs. For each block, the treatment group was randomly assigned. No adverse events related to treatment were observed during the course of the study.
+
+On study day 0, dogs in the treatment group (*n* = 6) were administered fluralaner in the form of Bravecto^®^ Chews (13.64% weight/weight fluralaner chewable tablets for dogs) in accordance with their weight and the label directions. As all the dogs fell within the same body weight range (4.9–7.1 kg), they were each administered 250 mg fluralaner. Dogs in the control group (*n* = 6) were handled similarly to the treated dogs, but received no fluralaner treatment. Immediately prior to treatment on day 0 and then weekly for the following 15 weeks (study weeks), a maximum volume of 25 ml blood was collected from each dog. Approximately 200–300 µl plasma was separated from this volume of blood and stored at − 80 °C for pharmacokinetic analysis, and 15 ml of the whole blood was used for the blood-feeding of mosquitoes. Blood for the feeding of mosquitoes was collected in heparin tubes, while that from which plasma was isolated for the pharmacokinetic analysis was collected in K2-EDTA-treated tubes.
+
+### Mosquito blood-feeding
+
+The blood collected as described in the previous section was used to feed adult *Ae. aegypti* mosquitoes (black-eyed Liverpool strain) [24] that had hatched 12 days earlier. The mosquitoes were maintained in an environmental chamber at 27 °C and a relative humidity of 75%, in accordance with standard procedures [25]. Water-jacketed membrane feeders were used for the feeding of adult mosquitoes on heparinized blood collected from the study animals. Adult mosquitoes were kept in three containers per study animal, each of which housed approximately 100 adult female mosquitoes. The adult mosquitoes were fed on 5 ml heparinized blood per container, and the membrane feeders were removed after 2 h. Mosquitoes in one of the three containers were used to assess blood-feeding rate, while those in the other two containers were maintained for a further 14 days to monitor survival and egg laying.
+
+### Mosquito feeding rate
+
+The mosquitoes were processed within 1 h of completion of blood-feeding to determine their feeding rate. One container per study animal was chilled at − 20 °C to immobilize the mosquitoes within, and each female mosquito was examined for visible engorgement of the midgut; the presence of any blood in the midgut was recorded as successful blood-feeding. Feeding rate was defined as the percentage of successfully blood-fed mosquitoes out of the total female mosquitoes per container (approximately 100).
+
+### Mosquito survival
+
+Two containers of adult, blood-fed mosquitoes per study animal were maintained for 14 days following blood feeding, a timeframe known to allow the development of infective third-stage *D. immitis* larvae from microfilaremia ingested during a blood meal under the conditions utilized in this study [26]. Mosquitoes were provided with cotton pads soaked with reverse-osmosis water and sugar cubes, which were replaced as needed. Dead mosquitoes were removed from each container daily for the first 4 days post-feeding and every other day thereafter. Dead mosquitoes were counted and sexed. On day 14 post-feeding, all of the remaining mosquitoes were counted and sexed to calculate a final survival rate. Adult mosquito survival was defined as the percentage of adult female mosquitoes surviving per container at each timepoint examined (1, 2, 3, 4, 6, 8, 10, 12, and 14 days post-blood-feeding) out of the total female mosquitoes per container (approximately 100).
+
+### Mosquito egg laying
+
+The adult mosquitoes in each container were provided with a 70-mm filter paper disc (Fisherbrand^™^ Qualitative P2; Fisher Scientific, Pittsburgh, PA) on which they could lay their eggs. The disc was pressed flush against the top screen of the container and kept damp with a pad of reverse-osmosis water-soaked cotton. The disc was removed and replaced with a fresh disc 3, 6, and 14 days following blood-feeding. Eggs were counted by an automated image capture and analysis process; Python script utilizing the OpenCV module and predefined parameters for the size and shape of *Ae. aegypti* eggs provided an estimated total number of eggs per disc. Egg laying per container was defined as the sum of egg counts for all three discs collected, and egg laying per mosquito was calculated from the number of live female adults remaining on the last day of collection.
+
+### Plasma fluralaner concentration
+
+Aliquots (100 µl) of dog plasma were extracted with acetonitrile containing a fixed concentration ofD4-fluralaner (racemic). After shaking or mixing in a vortex for 10 min and centrifugation for 10 min, 400-µl aliquots of the supernatant were transferred into separate new 96-well plates and analyzed directly for*R*- and*S*-fluralaner. Fluralaner occurs as a racemic mixture of*S* and*R* enantiomers, the former being the active component of the drug and the latter the inactive one; analysis of both components is used to show the persistence of the compound in the circulatory system. The final extracts were analyzed by liquid chromatography–tandem mass spectrometry for*R*- and*S*-fluralaner using a triple quadrupole mass spectrometer in positive ion mode. For each analyte, any observed sample concentration below the limit of quantification (2.5 ng/ml) was reported as zero.
+
+### Data analysis
+
+Treatment efficacy was calculated using arithmetic means for blood-feeding rate and survival, and geometric means for egg laying, all with Abbot’s formula:\documentclass[12pt]{minimal}
+ \usepackage{amsmath}
+ \usepackage{wasysym} 
+ \usepackage{amsfonts} 
+ \usepackage{amssymb} 
+ \usepackage{amsbsy}
+ \usepackage{mathrsfs}
+ \usepackage{upgreek}
+ \setlength{\oddsidemargin}{-69pt}
+ \begin{document}$$ {\text{Efficacy}}\,{\text{(\% )}}\,{ = }\,{100 } \times \,{{\left( {{\text{Mean}}_{{{\text{Control}}}} - {\text{Mean}}_{{{\text{treatment}}}} } \right)} \mathord{\left/ {\vphantom {{\left( {{\text{Mean}}_{{{\text{Control}}}} - {\text{Mean}}_{{{\text{treatment}}}} } \right)} {{\text{Mean}}_{{{\text{Control}}}} }}} \right. \kern-0pt} {{\text{Mean}}_{{{\text{Control}}}} }} $$\end{document}Efficacy(\% )=100×MeanControl-Meantreatment/MeanControl
+
+For all comparisons, significant differences between the values of control and fluralaner-treated groups were assessed using a linear mixed model with treatment group as a fixed effect. Comparisons of adult mosquito survival were made following blood-feeding for each study week, while all other comparisons were made for blood-feeding across all of the study weeks. Comparisons for mosquito egg laying were made using log-transformed egg counts. The two-sided level of significance was set at *P* ≤ 0.05 and the Holm-Šídák multiple comparisons test was selected as the post hoc test where significant differences were observed. All analyses were performed in Graphpad Prism 8.0 (GraphPad Software, La Jolla, CA).
+
+## Results
+
+### Mosquito feeding rate
+
+No significant differences in mean blood-feeding rate were observed between mosquitoes fed on blood from control animals and fluralaner-treated animals (*P* > 0.05; Fig. 1; Additional file 1: Table S1). The rate of blood-feeding in each replicate ranged from 4.8 to 100% in the fluralaner treatment group and 3.1–98.8% in the control group, with a mean across all timepoints of 73.7% and 71.6%, respectively. Treatment efficacy against blood-feeding ranged from − 25.5 to 16.8%. An abnormally low rate of blood-feeding was observed for study week 3 (mean < 25%) due to a technical issue with the blood-feeding apparatus, and thus the data for this study week were excluded from analysis.Fig. 1Feeding rate of adult female mosquitoes on blood collected on day 0 and then weekly from fluralaner-treated dogs (single oral dose) and untreated control dogs over a 15-week period. Data points represent the mean and SD for mosquitoes fed blood from six dogs per group (approximately 100 female mosquitoes per replicate). Data for study week 3 were excluded from analysis due to technical issues with the blood-feeding procedure. No significant differences were observed between the groups at any timepoint
+
+### Mosquito survival
+
+Feeding on blood from the fluralaner-treated dogs resulted in mortality of the adult mosquitoes, with significant differences in adult female mosquito survival between control and fluralaner treatment groups for each study week examined, at a minimum of one timepoint post-feeding (Additional file2: Table S2). For the majority of study weeks, significant differences in mosquito survival were observed between control and fluralaner treatment groups at earlier timepoints post-feeding; there were fewer significant differences at later timepoints post-feeding because mortality was then similar between the control and the treatment groups. For blood collected for the first 12 weeks post-treatment of the dogs, rapid lethal effects of fluralaner were evidenced by a significant reduction in mosquito survival at 24 h post-feeding, with an efficacy range of 33.2–73.3% (*P* ≤ 0.041; Fig. 2; Additional file 2: Table S2; Additional file 5: Fig. S1). No differences in the survival of mosquitoes were observed when they fed on blood taken prior to treatment of the dogs with fluralaner (week 0; *p* > 0.05). Treatment efficacy 14 days after blood-feeding ranged from 14.2 to 91.4%, with significant differences observed for study weeks 1, 2, 5, 11, 12, 13, 14, and 15 (*P* ≤ 0.04).Fig. 2Rate of adult female mosquito survival 24 h post-feeding on blood collected on day 0 and then weekly from fluralaner-treated dogs (single oral dose) and untreated control dogs over a 15-week period. Data points represent the mean and SD for mosquitoes fed blood from six dogs per group (two replicates per dog; approximately 100 female mosquitoes per replicate). Asterisks indicate significant differences (*P* ≤ 0.05) between treatment groups at each timepoint
+
+### Mosquito egg laying
+
+A significant reduction in mean number of eggs laid by mosquitoes was observed for the fluralaner treatment group at timepoints up to and including 13 weeks post-treatment (*P* ≤ 0.0016; Fig. 3; Additional file 3: Table S3). Treatment efficacy remained above 99.8% during this period, but dropped to 95% by study week 15 (Additional file5: Fig. S1). Egg counts in the fluralaner treatment group never exceeded 762 per mosquito for any replicate for any individual study week, while control egg counts ranged from three to 2814 per mosquito. No differences in egg production between the mosquitoes were observed when they fed on blood collected from the dogs prior to treatment (*P* > 0.05).Fig. 3Number of eggs laid per mosquito after feeding on blood collected on day 0 and then weekly from fluralaner-treated dogs and untreated control dogs over a 15-week period; six dogs were treated with a single oral dose of fluralaner and six dogs comprised the control (two replicates per dog). Data points represent the geometric mean and SD calculated from the estimated number of mosquito eggs laid. Asterisks indicate significant differences between treatment groups at each timepoint (*P* ≤ 0.05)
+
+### Fluralaner plasma concentration
+
+Fluralaner was detected in the plasma of all treated dogs at all timepoints during the 15-week study. The mean peak concentration of 4720 ng/ml, observed in study week 1, decreased over the course of the study to 47.7 ng/ml at the final timepoint (Fig. 4; Additional file 4: Table S4). The*R* enantiomer (inactive enantiomer) of fluralaner became undetectable in two of the treated dogs at week 10, and was undetectable in five out of the six treated dogs at week 15. The elimination half-life of total fluralaner was calculated to be 14.9 days. No fluralaner was detected in any of the untreated dogs at any timepoint, nor was fluralaner detected in the plasma of any dog prior to treatment.Fig. 4Plasma fluralaner concentration in dogs over a 15-week period following a single oral administration. Data points represent the mean and SD of the*R* and*S*enantiomers and total fluralaner concentration of six dogs
+
+## Discussion
+
+The results of this study demonstrated effects of orally administered fluralaner (Bravecto^®^) in mosquitoes fed blood from treated dogs. The survival and fecundity of *Ae. aegypti* mosquitoes were reduced when they fed on blood collected weekly over a period of 12 weeks from dogs administered a single dose of fluralaner. During this period, a significant reduction in adult female mosquito survival was observed at 24 h post-blood-feeding, while egg laying was almost completely suppressed. These findings demonstrate a novel secondary effect of the ectoparasiticide fluralaner, which is currently labeled for use against fleas and ticks.
+
+Concentrations of fluralaner that were capable of significantly reducing mosquito survival persisted for approximately six elimination half-lives in the circulatory system of the treated dogs. The relative lethal concentration 50% (LC50) of fluralaner at 24 h post-feeding was determined to be 58 ng/ml, which is in agreement with a mean LC50 of 32–100 ng/ml reported from blood-feeding studies conducted using *Anopheles* spp. [27]. A far greater LC50 for fluralaner at 24-h post-feeding (LC50 = 49 mg/ml) was reported for *Ae. aegypti* [13], but a sucrose solution was utilized in these feeding assays rather than blood, and the LC50 of the surface contact assays (LC50 = 13 ng/ml) agrees better with the result of the present study. Apart from the noted exception, the lethal effects of fluralaner in the circulatory system of the treated dogs appear to corroborate findings from in vitro systems.
+
+A possible consequence of the lethal effect of fluralaner on blood-fed mosquitoes is that dogs receiving this treatment will serve as poorer reservoirs for heartworm transmission. Furthermore, the same formulation of fluralaner that remains effective against fleas and ticks for 12 weeks [15–17] conferred a significant mosquito-killing effect over the same timeframe in the present study. Thus, regular administration of fluralaner is likely to maintain this efficacy for as long as treatment with this ectoparasiticide is continued. It should be noted that, ideally, in an assessment of heartworm transmission potential the survival of adult female mosquitoes should be evaluated up to a plausibly infectious timepoint (i.e., 14 days after blood-feeding under the environmental conditions used in this study), at which point third-stage larvae, which are infectious to the mammalian host, are present in the mouthparts. While we monitored mosquito survival up to this timepoint, waning survival in the control groups over the 14-day period precluded meaningful comparisons. However, the rapid death of mosquitoes at earlier timepoints after their blood meal strongly indicated that the significant reduction in their survival was specifically related to their uptake of fluralaner.
+
+Fluralaner does not, conversely, appear likely to prevent heartworm transmission to treated dogs, as treatment did not influence the rate of successful blood-feeding in mosquitoes at any point during the study. As third-stage heartworm larvae emerge from the mosquito mouthparts and rapidly enter the host during the infection process [28, 29], unlike pathogens vectored by ticks, which often require more than 24 h for transmission [18], an extremely rapid killing or deterrent effect due to fluralaner would be required to prevent the heartworm infection process.
+
+Even more pronounced than the effect of fluralaner on the survival of blood-fed adults was its effect on their reproduction. Egg laying was almost completely suppressed in mosquitoes fed on blood collected from the dogs for up to 13 weeks after they were treated with a single dose of fluralaner. This suggests that mosquitoes feeding on dogs regularly administered fluralaner as an ectoparasiticide (i.e., every 12 weeks) will largely fail to reproduce and maintain their local population size. Fecundity is a key determinant of mosquito population modeling [30], and a significant reduction in egg laying is expected to likewise reduce the vector equilibrium population on a local level. This, in turn, should translate into a potential reduction in not just heartworm transmission, but also the transmission of other mosquito-borne pathogens. Owing to the relatively short dispersal ranges of some of the most significant vectors of *D. immitis*, which may not exceed a few hundred meters [31–33], it is not unreasonable to expect some degree of localized reduction in the population size and transmission potential of mosquitoes provided that they are sufficiently exposed to fluralaner-treated animals.
+
+Routine fluralaner treatment may also fill gaps where conventional heartworm preventives fall short. Animals may benefit from possible reduced heartworm transmission via the passive mosquito-killing effect conferred by fluralaner (i.e., through blood-feeding), and while this is unlikely to directly prevent heartworm infection in dogs not receiving preventives, it may reduce their role as reservoirs for transmission. Additionally, the macrocyclic lactone-resistant isolates of *D. immitis* that have been identified over the past decade highlight an ongoing need for alternative control strategies [34–36]. Again, while fluralaner may not prevent infection with heartworm, it may assist in reducing its transmission from dogs in which resistant isolates are suspected while treatment is administered. Because this supplemental therapy targets the vector and not the parasite, its effect is not expected to depend upon macrocyclic lactone susceptibility, nor is it expected to exert selective pressure for resistance. Thus, the conventional use of fluralaner as an ectoparasiticide can have beneficial secondary effects, i.e. control of the mosquito vectors of heartworm and its transmission.
+
+## Conclusions
+
+*Aedes aegypti* mosquitoes exhibited significantly reduced survival and almost completely suppressed egg laying compared to untreated controls when fed blood collected from dogs over a period of 12 weeks following their treatment with a single oral dose of fluralaner. While blood-feeding was not affected by the fluralaner treatment, the mosquito-killing effects of fluralaner may contribute directly to a reduction in heartworm transmission potential in dogs receiving this ectoparasiticide, which may thus serve as a useful supplement to conventional preventives and therapies. Additionally, the pronounced inhibition of mosquito egg laying may contribute indirectly to a reduction in heartworm transmission potential in dogs, through a reduction in the local vector population.
+
+## Supplementary Information
+
+**Additional file 1****:****Table S1.** Mean rate of mosquito blood-feeding, range, and percentage efficacy prior to and 15 weeks following a single oral administration of fluralaner to dogs.**Additional file 2: Table S2.** Mean survival rate of adult female mosquitoes, range, and percentage efficacy prior to and 15 weeks following a single oral administration of fluralaner to dogs.**Additional file 3: Table S3.** Mean number of mosquito eggs laid, range, and percentage efficacy prior to and 15 weeks following a single oral administration of fluralaner to dogs.**Additional file 4****:****Table S4.** Mean and SD of plasma fluralaner enantiomer concentrations over 15 weeks after a single oral administration to dogs.**Additional file 5****:****Fig. S1.** Percent efficacy of fluralaner treatment compared to untreated controls prior to and 15 weeks following a single oral administration of fluralaner to dogs. Asterisks indicate significant differences between treatment groups at each timepoint for both survival and egg laying (*P* ≤ 0.05). Dagger represents a significant difference only in egg laying (*P* ≤ 0.05).

fulltext/md/PMC10142813.md

@@ -0,0 +1,162 @@
+---
+pmcid: "PMC10142813"
+pmid: "37104429"
+doi: "10.3390/vetsci10040274"
+title: "Gadolinium Neutron Capture Therapy for Cats and Dogs with Spontaneous Tumors Using Gd-DTPA."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Gadolinium Neutron Capture Therapy for Cats and Dogs with Spontaneous Tumors Using Gd-DTPA
+
+## Abstract
+
+### Simple Summary
+
+Neutron capture therapy, or NCT, is a unique irradiation-based method that is used to treat invasive advanced tumors as it minimizes the impact on healthy cells and tissues. Modern neutron sources for NCT include accelerators that can be installed in treatment facilities. One of the main conditions for the successful application of NCT is sufficient accumulation of the drug in tumor cells which allows it to capture neutrons, be activated, and release energy that destroys cancer cells. Among the elements capable of capturing neutrons, boron, gadolinium, and lithium are considered to be the most suitable for NCT. In our study, we showed the results of neutron capture therapy with gadolinium in a group of dogs and cats with spontaneous tumors to clarify the technical aspects of the method while adjusting the treatment conditions as close as possible to those of clinical trials.
+
+### Abstract
+
+We conducted a clinical veterinary study on neutron capture therapy (NCT) at a neutron-producing accelerator with seven incurable pets with spontaneous tumors and gadolinium as a neutron capture agent (gadolinium neutron capture therapy, or GdNCT). Gadolinium-containing dimeglumine gadopentetate, or Gd-DTPA (Magnevist^®^, 0.6 mL/kg b.w.), was used. We observed mild and reversible toxicity related to the treatment. However, no significant tumor regression in response to the treatment was observed. In most cases, there was continued tumor growth. Overall clinical improvement after treatment was only temporary. The use of Gd-DTPA for NCT had no significant effects on the life expectancy and quality of life of animals with spontaneous tumors. Further experiments using more advanced gadolinium compounds are needed to improve the effect of GdNCT so that it can become an alternative to boron neutron capture therapy. Such studies are also necessary for further NCT implementation in clinical practice as well as in veterinary medicine.
+
+## 1. Introduction
+
+Certain malignancies are extremely difficult to treat and can still remain lethal following treatment due to both their invasive nature and the inability of all modern treatment modalities to influence single tumor cells that migrate into healthy tissues and lead to the development of new tumor masses at a distance from the initial tumor that seemed to be cured. This may happen in the case of malignant melanoma with distant metastases, malignant glioma with fast local recurrence, or another type of invasive cancer that leaves no chance of survival despite the best efforts of modern medicine. Therefore, the search for more effective cancer therapies, including more advanced irradiation methods capable of targeting single tumor cells, is still ongoing.
+
+The principle of low-energy neutron capture by a drug followed by a local release of energy that destroys tumor cells has been employed in neutron capture therapy (NCT), which is a unique and sometimes the only applicable treatment for certain types of malignancies. The main advantage of NCT is its selectivity such that only tumor cells are affected and healthy tissues are preserved due to the precise intracellular accumulation of a compound that captures neutrons, decays, and produces energy that disrupts tumor cell DNA integrity [1,2,3]. Several elements have been considered as neutron capture agents. boron-10 (^10^B)-based compounds have already been tested clinically, with one of them, boronophenyl alanine (BPA), finally approved for clinical application in Japan [4,5]. The gadolinium-152 isotope (^157^Gd) possesses the best ability to capture neutrons and releases gamma rays that ionize matter in tumor cells [6]. Experiments on lithium-6 (^6^Li) used as a neutron capture compound for NCT have been recently initiated, and additional details related to its application are yet to be discovered [7].
+
+In the case of ^10^B, a concentration greater than 20 µg/g in tumor tissue is needed to achieve a therapeutic effect [1,2]. The optimal Gd load for successful GdNCT was estimated to be 50–200 µg/g in wet tumor tissue [6]. According to the recommendations, these two elements’ tumor/normal tissue concentration ratios should be ~3 or higher during irradiation [1]. ^10^B has a smaller neutron capture cross-section and the energy released due to its interaction with a neutron is limited by cell size, which plays a key role in treatment selectivity. Clinical trials on NCT at nuclear reactors using ^10^B (boron neutron capture therapy, or BNCT) have demonstrated effectiveness in treating different malignancies [8,9,10,11,12,13,14]. For example, Fukuda et al. showed the effectiveness and limitations of BNCT for malignant melanoma [8]; Kato et al. [9] and Kankaanranta et al. [12] reported on initial clinical trials on BNCT for recurrent head and neck cancer; Japanese research groups, including Yamamoto et al. [11] and Miyatake et al. [13], demonstrated the efficacy of the method in patients with malignant brain tumors; and Wittig et al. showed the ability of ^10^B compounds to accumulate in liver metastases of colorectal cancer [10].
+
+However, the efficacy limitations of previously and currently used boron compounds, the complexity of the boron enrichment process required for their production, and their high cost restrict the use of ^10^B-containing compounds in research and clinical practice. Therefore, Gd-based drugs, which are already registered in many countries and used clinically for purposes other than NCT, could be alternatives to ^10^B compounds.
+
+Thus, beginning in 1985, dimeglumine gadopentetate (Gd-DTPA), or Magnevist^®^, became one of the first contrast agents to be used in clinical trials and was originally approved by the FDA for clinical use in 1988 [15,16,17]. In magnetic resonance imaging (MRI), contrast agents are used for tumor tissue visualization improvement due to their accumulation in tumors, leading to an increased image signal. The increase in tumor tissue signal persists for about 1–2 h depending on the type of tumor, its blood supply, the contrast agent used, and its dosage. The pharmacokinetics, toxicity, and biodistribution of different gadolinium-based compounds used for contrast enhancement in MRI, including Gd-DTPA, have been extensively studied [18,19,20]. It is estimated that gadolinium concentrations as high as 300 µg ^157^Gd/g can be achieved in brain tumors with an MRI contrast agent such as Gd-DTPA when injected intravenously at 0.5 mmol/kg body weight [21]. The effectiveness of Gd compounds in NCT has been predicted in numerous studies examining their accumulation and biodistribution using tumor cell lines and in tumor-bearing laboratory animals [22,23,24,25,26,27,28,29].
+
+Regarding the irradiation facility for NCT, a specialized nuclear reactor channel or a charged particle accelerator is needed for neutron generation [30,31,32,33]. For various reasons, nuclear reactor-based NCT has ceased to exist in many countries except for clinical and preclinical trials in Taiwan, Argentina, and China and new compound development and other research purposes in Japan [14,34,35,36,37,38]. Thus, improving NCT relies on the results of state-of-the-art accelerator research being conducted or planned in Japan, China, South Korea, Finland, Argentina, and Russia [32,33,39,40,41,42,43]. Among the most advanced projects, we can mention the BNCT System NeuCure^®^ by Sumitomo Heavy Industries, Ltd. (Tokyo, Japan), based on a cyclotron accelerator operated at an energy level of 30 MeV with a current of 2 mA and a beryllium target, which is already installed in clinical treatment facilities in Japan [3,5,32]. A tandem accelerator with vacuum insulation and a solid lithium target has been developed at the Budker Institute of Nuclear Physics in Novosibirsk, Russian Federation and later, together with TAE Life Sciences (Foothill Ranch, CA, USA), modified and installed in Xiamen Humanity Hospital (Xiamen, Fujian, China) [3]. The University of Tsukuba, together with Mitsubishi Heavy Industry Co., Ltd. (Tokyo, Japan) and several other organizations, have developed a linear accelerator with a beryllium target that has been tested preclinically, with plans to initiate clinical trials in 2023 [3,33]. A BNCT center in Helsinki, Finland, with an Hyperion™ electrostatic accelerator with a rotating lithium target developed by Neutron Therapeutics Inc. (Danvers, MA, USA) and operated at 2.6 MeV and 30 mA was commissioned in 2019 and should have met the conditions required for clinical trials by the present time [3,40].
+
+In Japan in 2020, accelerator-based BNCT using the drug Borofalan^®^ containing boron phenylalanine (BPA) was approved for head and neck cancer treatment in adults, with national insurance coverage [4,5,44]. This has led to even greater public interest in the BNCT technology and a more thorough search for agents that can surpass BPA in efficacy [38,44,45,46].
+
+In many countries, preclinical radiobiological studies are required prior to treating human malignancies with accelerator-based NCT. In this case, studies using cells and animal tumor models play a key role [45,47]. However, cell and tumor models in laboratory animals cannot fully repeat the complex morphology of spontaneous tumors. In addition, body weight, metabolic rate, and radiation tolerance in animals are different from those in humans.
+
+In this case, enrollment of large animals of the mammal family, particularly domestic animals, such as cats and dogs, is the most suitable for estimating the radiobiological effects of NCT. Such animals have a large body mass, which allows for the selection of dosimetry parameters similar to those in humans. In addition, malignant tumors in large animals are easy to examine morphologically. The localization and nature of spontaneous tumors in domestic animals are similar to human tumors in many ways. Accordingly, the applied surgical treatment, chemotherapy, and radiation therapy methods show similar results.
+
+Since malignant tumors are a common cause of death in pets [48] and standard therapies do not always show satisfactory results, interest in using alternative therapies has increased. It has been shown previously that treating animals with spontaneous tumors using BNCT can be quite successful and justified [36,49]. Thus, conducting preclinical studies in large animals with spontaneous tumors is an important step to confirm the effectiveness of the method and potentially introduce it as a new treatment option in clinical practice.
+
+NCT is designed to be applied mainly in head and neck cancer, intracerebral tumors, melanoma, and metastatic lesions, which are very common in pets. We are the first to conduct a preclinical GdNCT effectiveness study in large mammals with malignant tumors using a registered gadolinium-containing MRI contrast agent, Magnevist^®^ (dimeglumine gadopentetate), and a neutron beam obtained at the accelerator developed at the Institute of Nuclear Physics in Novosibirsk, Russian Federation [49] with irradiation conditions similar to those in our previous experiments on BNCT [50].
+
+Thus, in this study, we aim to investigate the effect of GdNCT to increase the knowledge of NCT and optimize its therapeutic effectiveness.
+
+## 2. Materials and Methods
+
+This study on gadolinium neutron capture therapy was performed based on ethical standards and approved by the Ethics Committee of the Institute of Cytology and Genetics, Novosibirsk, Russian Federation. In addition, the informed consent of the animal owners for this type of therapy was obtained in each case. For this study, we selected animals with spontaneous tumors in satisfactory somatic condition with the possibility of anesthesia and clinical observation for more than three months or those with tumors that were difficult or impossible to remove surgically, taking into account complications. We also included animals with tumors that had relapsed after surgical treatment or previous chemotherapy. A total of seven animals (five cats and two dogs) received GdNCT—the data are presented in Table 1.
+
+Before treatment, a computed tomography (CT) scan was performed to visualize tumor location, size, and possible metastases. The imaging data were used for treatment planning. Before the GdNCT session, the neutron spectrum and the absorbed dose depth distribution in the simulated tumor, skin, and surrounding tissues were calculated. To calculate the minimum effective dose with a median of 30 Gy-Eq in the tumor, a tumor/surrounding tissues ratio of 3:1 was used, with an assumed average gadolinium concentration of 5.5 mg/g in the tumor at the time of irradiation and 1.8 mg/g in the surrounding tissues. The tumor-to-blood ratio was assumed to be 1:1. The upper dose limit for the skin was 18 Gy-Eq and 12 Gy-Eq for the mucosa [8]. Tumors were irradiated from both sides sequentially, with a change of position in the middle of the irradiation session, to ensure uniform exposure to the neutron flux.
+
+The irradiation settings included a proton energy level of 2.05 MeV and an integral current between 2.2 and 4.4 mAh, depending on the presence of previous radiation therapy, tumor volume, and localization. The average irradiation session lasted for 2 h.
+
+Dimeglumine gadopentetate (Magnevist^®^), manufactured by Bayer AG (Leverkusen, Germany), was administered fractionally at the maximum dose for humans recommended by the manufacturer (0.6 mL/kg). This was accomplished via an intravenous injection immediately before the radiation (0.3 mL/kg) and another injection (0.3 mL/kg) in the middle (at the half-time point) of the radiation session. Animals received intravenous anesthesia using a standard dose of Dexdomitor, dexmedetomidine plus Zoletil 100, or tiletamine plus zolazepam.
+
+After irradiation, 1 mL of peripheral vein blood was collected in a test tube for subsequent determination of the gadolinium concentration. Blood samples were analyzed using inductively coupled plasma atomic emission spectrometry (ICP-OES). The average blood concentration of gadolinium was 182 ± 217 ppm. Since this study was also performed to infer how much gadolinium accumulates within tumors, the amount of gadolinium in the tumors was predicted using an assumed tumor-to-blood ratio of 1:1 and a tumor-to-surrounding tissues ratio of 3:1.
+
+Animal positioning during therapy, collection of blood samples for analysis, and post-treatment monitoring of the animals for three months were performed according to the methodology of the previous study [50].
+
+## 3. Results
+
+Animals (cats and dogs) are described in the sequence of their examination and treatment with GdNCT.
+
+### 3.1. Female Cat “Ayuta”
+
+- Age: 11 years.
+- Symptoms: worsening of nasal breathing and nasal discharge.
+- Tumor localization: right nasopharyngeal area. The nasopharyngeal tumor was visualized using CT.
+- Treatment other than GdNCT (surgery, radiation, or chemotherapy): none.
+- Final pathological diagnosis: highly differentiated adenocarcinoma of the nasal mucosa.
+- The course of the disease after GdNCT: during follow-up, the overall condition was satisfactory, and nasal breathing improved. At the examination, the visible part of the tumor decreased in size. A CT scan performed 3 months after GdNCT revealed no significant changes compared to the pretreatment visualization (Figure 1).
+
+### 3.2. Male Dog “John”
+
+- Age: 14 years.
+- Symptoms: impaired nasal breathing and nasal discharge.
+- Localization and size of the tumor: nasal area; according to CT, the tumor size was 32 × 39 × 47 mm.
+- Treatment other than GdNCT: surgical removal of the nasal tumor, twice.
+- Final pathological diagnosis: chondrosarcoma.
+- The course of the disease after GdNCT: after one month of follow-up observation, the overall condition was satisfactory. However, starting from the second month, a gradual deterioration of the condition, nasal bleeding, difficulty in breathing, and decreased appetite were noted (Figure 2).
+
+### 3.3. Male Cat “Sausage”
+
+- Age: 6 years.
+- Symptoms: impaired nasal breathing and nasal discharge.
+- Tumor localization and size: left nasal area with bone destruction and regional lymphadenopathy. According to the CT scan, the tumor size was 33 × 26 × 26 mm.
+- Treatment other than GdNCT: standard X-ray radiotherapy with doses of 35.4 Gy for the tumor (6 sessions) and 30 Gy for the lymph nodes (6 sessions). The outcome of that treatment was tumor recurrence.
+- Pathological diagnosis: squamous cell carcinoma.
+- The course of the disease after GdNCT: after irradiation, the animal was lethargic and had facial edema and a necrotic area (circled in the photo) with exudate. Two months after GdNCT, there were signs of tumor recurrence in the back of the nose, upper jaw, and corner of the eye (Figure 3).
+
+### 3.4. Male Cat “Lev”
+
+- Age: 11 years.
+- Symptoms: gingival swelling and ulceration, bleeding, decreased appetite, regional lymphadenopathy, and lacrimation from the right eye.
+- Tumor localization and size: oral cavity, upper jaw on the right side, with metastasis to the submandibular lymph node. Based on a head CT, the tumor size was 25 × 40 × 15 mm.
+- Treatment other than GdNCT: none.
+- Pathological diagnosis: highly differentiated squamous cell carcinoma.
+- The course of the disease after GdNCT: the animal’s condition and appetite after GdNCT were normal, and there was a visual reduction in tumor size during the early observation period. There was no discharge. After irradiation, the eye continued to tear. Twenty-three days after GdNCT, the condition worsened. The tumor began to disintegrate, the gum was covered with bloody ulcers, and holes appeared in the cavity; the cat also experienced spittle vomiting and black liquid stools, a decrease in appetite, and would rub the tumor site. After 42 days of treatment, the tumor had not become visually smaller, the gum was bleeding, the animal’s appetite was not quite stable, and constant use of analgesics was necessary. Three months after GdNCT, active tumor progression was found at the examination (Figure 4).
+
+### 3.5. Female Dog “Lily”
+
+- Age: 13 years.
+- Tumor localization and size: basal neoplasm of the middle lobe of the right lung with involvement of the cranial and caudal lobes and single metastases to the lungs and regional lymph nodes. According to the CT scan, the tumor size was 33 × 35 × 42 mm.
+- Treatment other than GdNCT: none.
+- Pathological examination was not done.
+- The course of the disease after GdNCT: general improvement, breathing recovery, and right lung excursions one month after treatment. Activity and appetite improved. According to the CT scan, 2 months after GDNCT, the mass decreased in size to 32 × 38 × 36 mm (Figure 5).
+
+### 3.6. Female Cat “Marusya”
+
+- Age: 10 years.
+- Symptoms, localization, and size of the tumor: soft tissue mass in the upper jaw on the left side with invasion into the nasal cavity and left orbital space. Destruction of the upper jaw bone. According to the CT scan, the tumor size was 16 × 31 mm.
+- Treatment other than GdNCT: none.
+- Pathological diagnosis: squamous cell carcinoma.
+- The course of the disease after GdNCT: the cat’s appetite improved, their eye was open a little, and their activity was unchanged. Sneezing disappeared after irradiation but then resumed at a lower frequency than before irradiation; grunting sounds (which were present before irradiation) also disappeared. The overall condition improved. One month after GdNCT, the condition worsened, with the tumor size increasing to 38 × 59 mm, according to CT. The condition further deteriorated, and subsequently, the animal died (Figure 6).
+
+### 3.7. Male Cat “Semyon”
+
+- Age: 6 years.
+- Localization and size of the tumor: right femur and iliac bone; according to a CT scan, the tumor was 34 × 32 × 37 mm in size.
+- Treatment except for GdNCT: four courses of chemotherapy.
+- Pathological diagnosis: osteogenic sarcoma.
+- The course of the disease after GdNCT: on the tenth day after NCT, the animal was eating, drinking, and feeling well. A CT scan three months after irradiation revealed an increase in the tumor size to 58 × 38 × 46 mm (Figure 7).
+
+## 4. Discussion
+
+Previously, our research group conducted studies on BNCT in laboratory animal tumor models and spontaneous tumors in pets (cats and dogs) using BSH [45,50,51].
+
+BSH and BPA are standard compounds typically used in BNCT. They are expensive and difficult to obtain in some countries. For example, a single clinical NCT session might require a volume of the drug that costs about 30,000 USD in equivalent. Since gadolinium compounds are currently used in clinical practice for contrast enhancement during MRI, such agents represent perfect theranostic drugs capable of combining NCT and MRI. Although this is an advantage of GdNCT, the use of ^157^Gd in NCT has been limited to cell experiments and small animal studies so far. Despite the advantages of gadolinium, such as a high gradient of accumulation ratio in tumors relative to healthy tissues, the large cross-section for thermal neutron absorption, and the possibility of using highly efficient Auger electrons from gadolinium to destroy tumor cells, there are several drawbacks associated with its use. During neutron capture by gadolinium, the most important issue is the effect of the generated gamma radiation on the healthy tissues surrounding the tumor. This radiation possesses high energy (the total energy of 7.9 MeV) and travels a considerable distance through biological tissues, which might cause irradiation of healthy cells and an insufficient irradiation dose to the tumor. Electrons and X-rays account for a small fraction of the energy released in a Gd(n, y) reaction, which is 63 keV. However, these electrons and X-rays can play a significant role in affecting the tumor, with their specific effects depending on the tumor’s size and the gadolinium location in relation to the tumor cell nuclei. Initially, many studies considered only the dose distribution from gamma radiation from Gd(n, y) reactions. However, Martin et al. (1989) and Laster et al. (1996) found that in their experiments with cells, the effect of gadolinium-related neutron capture reactions was more significant than identified in previous studies where it was associated only with gamma radiation [52,53]. In these studies, it was concluded that the additional effect was caused by the internal conversion of electrons and Auger electrons. However, for an exact explanation of this phenomenon, data on the dose distribution from all types of radiation arising in the reaction with gadolinium were needed. In subsequent studies, Cerullo et al. (2009) showed that according to dosimetry and microdosimetry of absorbed doses in both tumor and healthy tissues, gadolinium NCT appears to be a feasible treatment for malignant tumors [54].
+
+Karpovich et al. proposed a method of therapy planning for GdNCT that relies on quantitative assessment of the content of Gd (III) complexes in the tumor and surrounding tissues based on a multicompartment phenomenological pharmacokinetic model adapted to real patient study data, with the possibility of visual assessment by MRI [55]. In patients receiving Magnevist^®^, there is a retention of gadolinium in the glial tumor, which is already clearly visible during the visual analysis of MR images, and at the same time, there are no changes in the intensity of the surrounding white matter. The ratios between the transport coefficients of Magnevist^®^ in the directions from blood to tissue and from tissue to blood were calculated. For glial tumor tissue, this ratio was 9.5–10, i.e., the reverse transfer was slower by one order of magnitude. Obviously, this situation will ensure sufficient retention time of Magnevist^®^ molecules in the tumor tissue with negligible accumulation in normal tissues, which is the key to successful NCT with selective tumor cell targeting [55]. These results were in good agreement with previously published quantitative autoradiography data [56].
+
+Despite promising dosimetry data, the efficacy of Gd-DTPA and Gd-DOTA as therapeutic agents for GdNCT was initially predicted to be low due to a potentially insufficient accumulation of gadolinium in tumor cell nuclei. However, De Stasio et al. showed that Gd-DTPA penetrates cancer cell nuclei in vitro, which led to a reconsideration of the GdNCT approach using that compound [23]. In vivo studies have also suggested that multiple injections could lead to the penetration of Gd into more tumor cell nuclei [24].
+
+To increase the effectiveness of Gd-DTPA in GdNCT, continuous intra-arterial infusions have been proposed [22]. In a study by Akine et al. (1993), Gd-DTPA was injected into the femoral artery on the side of a VX-2 tumor growing in the hind limbs of New Zealand white rabbits, which created a concentration of gadolinium 5–6 times higher than in a contralateral tumor on the opposite limb. At the site of injection, tumor growth was significantly suppressed between days 16 and 23 after Gd-NCT compared to control tumors [22]. The therapeutic effect of Gd-DTPA in vivo was also reported in rats with Jensen’s sarcoma [57].
+
+A proof of principle for cell death by Gd neutron capture in U87 glioblastoma multiforme (GBM) cells preloaded with Magnevist^®^ has been presented [26]. Later, the authors described autophagy progression after neutron capture by gadolinium in U87 GBM cells [28].
+
+Gadolinium NCT agents that are potentially more effective than Magnevist^®^ have also been studied. Brugger et al. (1989) published one of the first articles reporting on Gd chelates used as NCT agents [6]. The possibility of using Gd-DTPA and Gd-DOTA to treat brain tumors was investigated. It was demonstrated that these Gd derivatives could reach therapeutic concentrations in brain tumors. In addition, a good ratio of Gd concentration in tumor tissue compared to normal tissues was observed [6]. Matsumura et al. (2003) demonstrated the efficacy of the drug Gd-BOPTA (Multihance^®^) as an agent for neutron capture therapy [58]. Gd-BOPTA showed twice as long accumulation in tumors compared to other drugs used for MR imaging.
+
+Different researchers suggested using liposomal compositions or chitosan to increase the concentration and long-term deposition of gadolinium in tumors [25,27,29]. Le et al. studied the biodistribution and accumulation of Gd-DTPA-encapsulated liposomes in tumor-bearing mice [25]. The amount of Gd accumulated in the tumors in this study not only exceeded the calculated Gd concentration required for successful NCT but also reached an average value of 158.9 ± 48.7 µg/g 12 h after intravenous injection [25].
+
+Fujimoto T. et al. (2009) used gadopentecic acid (Gd-DTPA) with chitosan to develop gadolinium-loaded chitosan nanoparticles (Gd-nanoCPs) and studied their accumulation and MR imaging visualization enhancement effect compared to Gd-DTPA (Magnevist^®^) in human malignant fibrosis histiocytoma (MFH) cells [27]. The authors showed more efficient Gd accumulation after using Gd-nanoCPs; however, the longitudinal relaxation time (T1) reduction was more prominent in the case of Gd-DTPA [27]. In a later study by the same research group, Ichikawa et al. (2014) used Gd-nanoCPs for controlled Gd delivery in NCT [29]. In vitro experiments were performed using a melanoma cell line; mice with a subcutaneous tumor xenograft were also used. In vitro, smaller Gd nanoparticles accumulated in cells in higher concentrations and were thus more suitable for NCT. Nanoparticles incorporating 1.2 mg of natural Gd were injected once or twice intratumorally into subcutaneous B16F10 melanoma-bearing mice. Eight hours after the last injection, the tumor areas of the mice were irradiated with thermal neutrons. The Gd-NCT group showed significant tumor growth suppression, although nanoparticle effectiveness was found to depend on their micrometric properties [29]. Other authors have proposed using hybrid complexes with MR imaging capabilities, including gadolinium chelates, to treat tumors with NCT [59].
+
+Regarding studies on neutron capture therapy in large animals using gadolinium compounds, a group led by Professor Mitin treated dogs aged between 9 and 15 years with histologically confirmed diagnoses of oral mucosal melanoma [60]. Dipentast^®^ and BPA were used. In the GdNCT group, 70% of cases achieved complete regression of the tumor within 30–45 days; in 14% of the animals, recurrence occurred after one month, and life expectancy was between four and six months. In the BNCT group, complete tumor regression was achieved in 95% of cases; in 20% of cases, recurrence occurred after three to four months, and the life expectancy was approximately eight months. In the gamma therapy group, 90% of the dogs showed tumor recurrence one month after treatment, metastases after 2–3 months, and a life expectancy of approximately three months.
+
+In our study, according to the Magnevist^®^ drug instructions, we used the maximum recommended dosage of 0.6 mL per kg of body weight or 0.3 mmol per kg of body weight, which in a proportional ratio should provide up to 180 µg of ^157^Gd/g in tumor tissue. We injected the drug twice at half the dose calculated per body weight to ensure a homogeneous concentration of the drug in the tumor for the entire duration of the GdNCT session.
+
+Despite meeting all the conditions for maximum accumulation of the drug in the tumor tissue, which suggests a maximum NCT effect, we observed negative changes that manifested as increases in tumor volume and clinical deterioration during the follow-up period one to three months after GdNCT in most of the animals. We observed a short-term absence of disease progression only in some cases; there were also no adverse reactions of the skin or fur at the irradiated sites.
+
+Thus, we could not show a significant positive effect of NCT for spontaneous malignant tumors in domestic animals. Computed tomography performed three months after GdNCT showed no changes or increases in tumor volumes, indicating an insufficient tumor response in all cases. Survival with poor quality of life was observed up to three months after irradiation, and the survival rate and quality of life did not improve over time. Typically, within two weeks (14 days) after GdNCT, the initial positive treatment response was replaced by a deterioration of the overall condition and quality of life. No change in tumor size after therapy and continued tumor growth could be related to the spread, depth, and infiltrative nature of spontaneous tumors as well as an insufficient concentration and rapid washout of Gd-DTPA from tumor tissues despite two injections during irradiation sessions, the extracellular localization of Gd-DTPA, and the presence of Gd-DTPA mainly in the tumor periphery. As a result, large tumors received insufficient radiation doses in the central and deeper parts. Thus, the small number of animals, different morphological characteristics of the tumors, and the lack of computed tomography data in some cases may represent the current study’s limitations.
+
+A solution to improve the therapeutic outcome at this stage would be using gadolinium drugs that can accumulate inside tumor cells, performing several GdNCT sessions, and combining GdNCT with other treatment modalities. Therefore, it is obvious that new gadolinium-containing agents capable of selective accumulation in tumors are required for GdNCT. Based on recent publications, one of the most promising theranostic agents is AGuIX, a radiosensitizer and MRI contrast agent with a high gadolinium concentration in its structure [61]. It has been employed in phase II clinical trials in patients with brain metastases and in phase I trials in patients with glioblastoma, lung, cervical, and pancreatic cancer [61]. Other newly synthesized gadolinium nanoparticles have recently been introduced, and further development of GdNCT is in progress [62,63].
+
+## 5. Conclusions
+
+Neutron capture therapy often remains the last option for the treatment of spontaneous tumors in animals, while other methods prove to be ineffective. Studies on large animals allow for treatment conditions to be adjusted as close as possible to those of clinical trials and help clarify the technical aspects of NCT. Using a unique accelerator source for neutron generation, as done in these studies, represents a pioneering application of the method worldwide. The combination of gadolinium administration and accelerator-based neutron irradiation provides additional data on the current conditions of GdNCT. These data can then be compared with those obtained by scientific groups conducting BNCT and other methods of radiation therapy for malignant tumors. At this stage, it is evident that successful GdNCT requires new gadolinium-containing drugs capable of sufficient tumor accumulation. Furthermore, the results of preclinical studies in large animals will allow us to prepare for the initiation of appropriate clinical trials.

fulltext/md/PMC10143758.md

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+---
+pmcid: "PMC10143758"
+pmid: "37104458"
+doi: "10.3390/vetsci10040303"
+title: "Can Chemotherapy Negatively Affect the Specific Antibody Response toward Core Vaccines in Canine Cancer Patients?"
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Can Chemotherapy Negatively Affect the Specific Antibody Response toward Core Vaccines in Canine Cancer Patients?
+
+## Abstract
+
+### Simple Summary
+
+The life expectancy of dogs has doubled in past decades. However, this positive effect has been accompanied by a concomitant increase in neoplasms. The aim of this study was to investigate the impact of antiblastic chemotherapy on the specific antibody response toward core vaccines in cancer-bearing dogs. Twenty-one patients with different types of malignancies were sampled before, during, and after different chemotherapy protocols to determine their actual levels of seroprotection against CPV-2, CDV, and CadV-1. No statistically significant changes in antibody titration emerged for any of the chemotherapy protocols used, suggesting that chemotherapy does not have an evident immunosuppressive effect on the post-vaccine antibody response.
+
+### Abstract
+
+The life expectancy of our pets has been getting longer in recent years due to new therapeutic opportunities, better nutrition, and better diagnostic approaches. This positive effect, however, has been accompanied by a concomitant increase in neoplasms, particularly in canine patients. Therefore, veterinarians inevitably face new issues related to these diseases, poorly or never investigated in the past, such as the possible side effects resulting from chemotherapy. The aim of this study was to investigate whether and how chemotherapy influences the antibody response against CPV-2, CDV, and CAdV-1 in dogs vaccinated before starting chemotherapy. Twenty-one canine patients with different types of malignancies were sampled before, during, and after different chemotherapy protocols to determine their actual levels of seroprotection against CPV-2, CDV, and CadV-1 by using the in-practice test VacciCheck. Differences related to sex, breed size, type of tumor, and chemotherapy protocol were evaluated. No statistically significant changes in antibody protection emerged for any of the chemotherapy protocol used, suggesting that, contrary to expectation, chemotherapy does not have a marked immunosuppressive effect on the post-vaccine antibody response. These results, although preliminary, may be useful in improving the clinical approach to the canine cancer patient, helping veterinarians fully manage their patients, and enabling owners to feel more confident about their pets’ quality of life.
+
+## 1. Introduction
+
+The life expectancy of pets has doubled in the past 40 years thanks to different life prolonging-factors, such as quality nurturing, regular veterinarian check-ups, and new therapeutic, diagnostic, and prophylactic approaches. This longer survival, however, has been accompanied by a higher frequency of diagnosis of various morbid conditions, among which neoplasms are the most common [1,2]. Veterinarians are therefore facing new issues related to these diseases, poorly investigated to date, such as the side effects of antineoplastic chemotherapy on the immune system [1,3,4]. The most common tumor types affecting canine patients are well known (e.g., mast cell tumors, lymphomas, hemangiosarcomas, transitional cell bladder carcinomas, soft tissue sarcomas), as are the chemotherapy protocols applied for their treatment (monochemotherapy or polychemotherapy) [5,6]. Notwithstanding, there are yet many aspects to be clarified in the field of immuno-oncology, particularly the impact of different tumor types and their therapies on the immune system [6,7,8]. In human medicine, it has long been known that cancer and chemotherapy increase susceptibility to opportunistic infections in patients, reactivate vaccine-preventable agents, and decrease the immune response after vaccination as a result of immune function impairment [7,9,10]. However, in canine oncology very little is known about these issues. One of the least understood aspects to date concerns the success of vaccination in canine cancer patients undergoing chemotherapy. Vaccines are the primary preventive tools for widespread and dangerous diseases both in human and veterinary practice. All international guidelines on good vaccination practices [11,12,13,14,15] classify pet vaccines as core and non-core. Those in the first category protect against some contagious, hazardous, and fatal diseases, and therefore are intended for all subjects, while those in the second category are suggested only for animals at risk of contracting specific diseases based on their lifestyle and geographical distribution, and therefore they are optional. The core vaccines for dogs are strongly recommended since they protect against three highly contagious, widespread, and often lethal diseases, i.e., canine parvovirus infection (caused by canine parvovirus type 2, CPV-2) [16,17,18,19], canine distemper (caused by canine distemper virus, CDV) [20,21,22], and infectious canine hepatitis (caused by canine mastadenovirus A, CAdV-1) [23,24].
+
+In human medicine, chemotherapy and radiotherapy still represent fundamental curative treatments for patients affected by malignancies, and their increasing use in recent years has led to significant improvements in the survival of cancer patients. Nevertheless, these successful results may be associated with drawbacks that must be taken into account in the management of oncologic patients. One of the possible inconveniences of chemotherapy in human patients (and in children above all) is hematologic toxicity, which results in transient immunodeficiency involving mainly B lymphocytes. As already mentioned, antibody titers induced by vaccination could then be seriously compromised. However, the consequences for the immune system and vaccination appear to be highly variable as these are also influenced by factors other than chemotherapy [9,10].
+
+The aim of this study was to investigate whether and how antiblastic chemotherapy impacts the antibody response against CPV-2, CDV, and CAdV-1 in cancer-bearing dogs vaccinated before starting chemotherapy.
+
+## 2. Materials and Methods
+
+### 2.1. Study Population and Study Protocol
+
+The plasma samples used for this study were initially collected over a period of about one year (January 2021–March 2022) from 54 dogs with diagnoses of malignant neoplasm. The inclusion criteria are reported in Table 1. Eligibility for inclusion required mainly that the dogs were vaccinated with the core vaccines (CPV-2, CDV, and CAdV-1) and were undergoing antiblastic chemotherapy treatment in agreement with their cancer cytotype or histotype based on evidence from the literature given their cancer prognosis. Twenty-one dogs met the inclusion criteria and were therefore included in the study. All the other dogs were excluded from the analysis since they did not meet the inclusion criteria.
+
+All patients included in the study underwent chemotherapy treatment in the dedicated room of the Veterinary Teaching Hospital (Department of Veterinary Medicine and Animal Sciences, University of Milan, Lodi, Italy). For each dog, key data were detailed: (1) sex and reproductive status: intact or neutered male or female; (2) age: adults, seniors, and geriatrics based on canine size according to the rule that small dogs live longer than large ones and vice versa [25,26]; (3) breed (purebred or crossbred) and animal size: small (<10 kg), medium (≥10–<25 kg), large (≥25–<45 kg), and giant (≥45 kg); (4) histologic/cytologic diagnoses of tumor; (5) type of dose-intensive chemotherapy (monochemotherapy or polychemotherapy and relative drugs) or metronomic chemotherapy; (6) number of antiblastic chemotherapy administrations and relative blood samples; (7) vaccination history, considering time elapsed since last vaccination: <1 year, ≥1 year–<2 years, or ≥2 years.
+
+An extra group of 50 dogs in similar situations (vaccinated oncologic patients sampled before receiving any chemotherapy protocol) was used as a control group.
+
+### 2.2. Sample Collection
+
+All blood samples were collected in K~3~EDTA for the blood analyses required by the oncologic control, and residual aliquots were used for the antibody titrations specific for the core vaccines. According to the decision of the Ethical Committee of the University of Milan, residual aliquots of samples collected with the informed consent of the owners can be used for research purposes without any additional formal request for authorization (EC decision 29 October 2012, renewed with the protocol n. 02-2016). For each patient, one blood sample was collected before the beginning of the chemotherapy, and then again before each other chemotherapy administration. A final sample was collected at the end of the chemotherapy protocol. Whole blood samples were centrifuged for 15 min at 1500× *g*, and plasma was collected, recorded, and then stored at −20 °C until use.
+
+Once the sampling was completed, three samples were tested for each dog: (1) the initial sample collected before the beginning of chemotherapy (T0); (2) a sample collected exactly in the middle of the patient’s chemotherapy protocol (T1); (3) the final sample collected at the time of the last clinical examination one month after the end of chemotherapy (T2). In this way, 63 samples were tested in order to detect eventual changes in the antibody titers specific for core vaccines due to chemotherapy treatment.
+
+### 2.3. Detection of Specific Antibodies by VacciCheck
+
+Each plasma sample was analyzed using the in-clinic test Canine VacciCheck (Biogal, Kibbutz Galed, Israel, supplied in Italy by Agrolabo, Scarmagno, Italy), following the manufacturer’s instructions. The kit is a dot-ELISA-based rapid semi-quantitative system approved to measure the specific antibody titers (IgGs) against CDV, CPV-2, and CAdV-1. VacciCheck has high specificity and sensitivity for each microorganism and is approved to be used for both research and diagnostic purposes [11,15,27,28,29,30,31,32,33]. In this rapid test, the antibody concentration is defined by the color intensity of the resulting spots compared with a scale from 1 to 6. The S0 value was standardized by the manufacturer as being equivalent to an antibody titer of <1:8 for CDV, <1:20 for CPV-2, and <1:4 for CAdV-1, while an S value of 3 (S3) was defined as being equivalent to 1:32 for CDV, 1:80 for CPV-2, and 1:16 for CAdV-1. A value equal or higher than S3 indicates that the individual is protected against each of these three diseases (Table S1).
+
+### 2.4. Statistical Analysis
+
+A statistical analysis was performed using the Graph Pad Prism 9 program (GraphPad Software, La Jolla, CA, USA). Antibody titer data were transformed with Log~2~. The normality of the data was tested (D’Agostino–Pearson normality test, Shapiro–Wilk test, and Kolmogorov–Smirnov test), and the following non-parametric tests were used: a Friedman test (with a Dunn’s multiple comparison test), a Mann–Whitney test, and a Kruskal–Wallis test. Values at *p* < 0.05 were considered statistically significant. Tendency was considered at *p*-value < 0.1.
+
+## 3. Results
+
+At the end of the study (March 2022), a total of 188 plasma samples from 21 canine patients with different malignancies, and who had been treated with different chemotherapy protocols, were collected. Of these, 14 (66.7%) were females (1 sexually intact and 13 neutered) and 7 (33.3%) were males (5 sexually intact and 2 neutered). Collectively, 6 dogs (28.6%) were intact while 15 (71.4%) were neutered. Their ages ranged from 2 to 13 years, with 4 being adult (19.0%), 11 senior (52.4%), and 6 geriatric (28.6%) patients. Regarding their breeds, 16 dogs were of pure breed (76.2%) while 5 were crossbred (23.8%); there were no breeds more represented than others (two Border Collies and only one Doberman Pinscher, one Newfoundland Dog, one Pitbull, one Cocker Spaniel, one Beagle, one American Staffordshire Terrier, one Bull Terrier, one Bernese Mountain Dog, one Greyhound, one Bobtail, one Golden Retriever, one Rottweiler, one Belgian Shepherd, and one French Bouledogue). Regarding their size, 2 were small (9.5%), 10 were medium (47.6%), 6 were large (28.6%), and 3 were giant in size (14.2%). Of the 21 oncologic patients, 10 (47.6%) suffered from a lymphoma, and of these 6 (28.6%) from a multicentric B cell lymphoma, 3 (14.3%) from a multicentric T cell lymphoma, and 1 (4.7%) from a cutaneous lymphoma; the other 11 dogs suffered from a variety of neoplasms: 3 (14.3%) from mast cell tumors, 2 (9.5%) from cutaneous soft tissues sarcoma, 2 (9.5%) from transitional carcinoma of the urinary bladder, and the others (1 per type, 4.8%) from undifferentiated testicular neoplasm, splenic hemangiosarcoma, pulmonary histiocytic sarcoma, and undifferentiated renal neoplasm. These oncologic patients were all treated with a chemotherapy protocol: 12 out of the 21 (57.1%) were treated with monochemotherapy, 5 (23.8%) using lomustine, 3 (14.3%) using vinblastine, 2 (9.5%) using doxorubicin, and 2 (9.5%) using carboplatin, while the remaining 9 (42.9%) were treated with polychemotherapy, 6 (28.6%) using a cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based protocol, 2 (9.5%) using a combination of thalidomide/cyclophosphamide, and 1 (4.8%) using LOPP. The number of chemotherapy administrations and the corresponding blood samples ranged from 3 to 18 (1 dog each, 4.8%), with a variety of other possibilities: 8 for 4 dogs (38.1%), 6, 7, and 9 for 3 dogs (14.3%), 10 for 2 dogs (9.5%), and 4, 13, 14, and 17 for 1 dog each (4.8%). Finally, regarding the time elapsed since their last core vaccination, 14 (66.7%) had been vaccinated <1 year before sampling, 6 (28.6%) received their last vaccination ≥1–<2 years earlier, and only 1 (4.8%) had been vaccinated more than 2 years earlier. The main characteristics of the 21 canine patients of this study are reported in Table 2.
+
+### 3.1. Antibody Titers and Kinetics of Protection
+
+For each of the 21 dogs in this study, 3 samples were tested (at the beginning, in the middle, and 1 month after the end of the chemotherapy protocol) in order to analyze the kinetics of the impact of the chemotherapy on measurable antibody levels over time. The results of these analyses are reported in Table 3.
+
+Almost all the dogs maintained excellent protection against the three diseases over the time of the chemotherapy treatment, i.e., 20 out of 21 (95.2%) for distemper, 17 out of 21 (81%) for infectious canine hepatitis, and 16 out of 21 (76.2%) for parvovirus infection. A statistical analysis did not show any significant difference among the different timepoints for any of the three diseases, and consequently the measurable antibody levels did not seem to be negatively affected by the chemotherapy protocols. Distemper antibody titers were found to be even higher at T2 than at T0 and T1, with a slight decrease at T1 and a subsequent increase at T2 (Figure 1a). Most of the samples had values for distemper fluctuating between 1:32 and 1:64, and they never decreased below the protective threshold.
+
+Different statistical analyses were performed taking into consideration different biological variables (i.e., type of treatment, dog size, sex, and age), and the obtained significant results are presented below. The antibody levels of the 21 individuals of this study were also compared with the antibody levels of the control group (vaccinated oncologic patients sampled before any chemotherapy protocol, *n* = 50), and no statistical differences were found.
+
+### 3.2. Evaluation of Influence of the Type of Chemotherapy
+
+The peculiar increase in antibody titers for distemper virus at T2 was particularly evident in the subjects treated with vinca alkaloids (vinblastine and vincristine, alone or in combination with other drugs in the CHOP and LOPP protocols), and the difference between T1 and T2 was statistically significant (*p*-value = 0.0476), probably due to the higher antibody titers at T2 after an initial decrease at T1 (Figure 1b). For the other two viruses (CPV-2 and CAdV-1), no statistically significant differences were observed.
+
+The 10 patients treated with vinca alkaloids were then compared with the other 11 dogs treated with different molecules, considering their values at T1, and statistically significant differences (*p*-value = 0.0237) were identified between them (Figure 1c).
+
+Even if the antibody titers against distemper virus in the patients treated with vinca alkaloids were significantly lower at T1 than those of the patients treated with other drugs, both would remain in the same protective range (1:32–1:64). This statistically significant difference suggests that dogs treated with drugs other than vinca alkaloids have higher antibody levels against distemper than dogs treated with vinca alkaloids, at least halfway through their chemotherapy treatment, while at the end of chemotherapy (T2) the distemper antibody titers increase to the extent that they became highly protective.
+
+### 3.3. Evaluation of Influence of the Type of Tumor
+
+No statistically significant differences in antibody titers were observed in the groups of dogs affected by lymphoma (*n* = 10) and by tumors other than lymphoma (*n* = 11) for any of the diseases, even if a tendency toward a statistical significance was observed between T1 and T2 when parvovirus infection was considered (Figure 2).
+
+An increase in antibody titers between T1 and T2 was noted in both groups (lymphoma and non-lymphoma), with a tendency toward significance (*p*-value = 0.0837) in the lymphoma group. For a more precise analysis, antibody titers were analyzed according to the type of immunophenotype of the multicentric lymphoma (B-cell or T-cell), but no statistically significant differences related to the type of lymphoma were found.
+
+### 3.4. Evaluation of Influence of the Dog Size
+
+Patients were also analyzed according to their size and divided into two groups: small/medium dogs (<25 kg, *n* = 12) and large/giant dogs (≥25 kg, *n* = 9).
+
+A statistically significant difference in antibody titers was detected only for CAdV-1. Specifically, large/giant dogs had statistically higher antibody titers than small/medium dogs at T0 (*p*-value = 0.0321) and T2 (*p*-value = 0.0105), while no statistically significant differences were found at T1 (Figure 3). In any case, all the values were equal or higher than the threshold values, and all the dogs were found to be protected, regardless of their size.
+
+## 4. Discussion
+
+Studies investigating the possible negative effects of chemotherapy on specific immune responses in veterinary oncologic patients are rare, but all seem to confirm the results of this study, suggesting that chemotherapy does not have a strong impact on the specific immune response, or at least that the impact is not as severe as might have been expected [6,8].
+
+In a study by Henry et al. (2001) [8], 21 dogs with different malignancies and 16 dogs with lymphoma, all treated with chemotherapy, were analyzed in order to highlight any changes in antibody response against parvovirus infection, distemper, and rabies infections. The lack of significant changes detected in the antibody titers following chemotherapy allowed the conclusion that an established immunity due to previous vaccinations is not significantly compromised by the chemotherapy applied to treat oncologic dogs. Some years later, Walter et al. (2006) [6] conducted a study to evaluate the effects of two different chemotherapy protocols (doxorubicin and a multi-drug chemotherapy) on humoral immunity to de novo vaccination in 12 dogs with lymphoma and 9 dogs with osteosarcoma. Antibody titers after vaccination were not significantly different between the control group and the dogs treated with chemotherapy, suggesting once again that chemotherapy may have less of an impact on the immune response than might be expected. Finally, in 2014, Elias et al. [7] evaluated whether the same immune system impairment described in human medicine could be observed in small animals undergoing chemotherapy. The study was conducted on eight dogs with lymphoma treated with a CHOP-based protocol and correctly vaccinated against CPV, and on another eight naturally CPV-infected, symptomatic dogs. Blood was collected from the lymphoma group prior to the CHOP chemotherapy and during the protocol (weeks 3, 6, and 9), and at the same timepoints samples were collected from the control group. The authors concluded that there was no evidence of a decreased immune response even after two chemotherapy cycles, indicating that the previously established immunity against CPV was not significantly impaired.
+
+In this study, the dogs that underwent chemotherapy with vinca alkaloid showed an unexpected increase in antibody titers against distemper at the end of the therapeutic protocol (T2), following a transient decrease in T1. Although there is a lack of studies in the veterinary literature on the positive effects of chemotherapy with vinca alkaloids on the immune system, in human medicine some studies report such an effect. A study by Gameiro et al. (2011) [34] has reported that following the administration of cisplatin and vinorelbine (the latter is a vinca alkaloid) to mice with non-small-cell lung cancer (NSCLC), there was an immediate (around 1–2 days) immunosuppressive effect on the T lymphocytes, but this was followed (3–4 days later) by a strong increase in the lymphocyte population, and particularly in the Treg subpopulation. It is known that T helper lymphocytes act as regulators and can promote the maturation of plasma cells and consequently boost antibody synthesis. Further investigations are needed to determine whether the increase in the Treg subpopulation also occurs in dogs, and whether it accounts for the increase in distemper antibody titers observed at T2 in dogs treated with a similar vinca alkaloid. Moreover, this could indicate a recovery of the immune system once chemotherapy is completed, helped by the “stimulating” effect of the alkaloids used in the chemotherapy protocol. It is not clear why only distemper antibodies show such an increase, and not other antibodies directed toward other viruses.
+
+The splitting of the oncologic dogs into two groups (lymphoma and tumors other than lymphoma) was based on the study by Elias et al. [7], in which the authors suggested that the variations in antibody titers observed in cancer patients mostly depended on the nature of the tumor itself rather than on other variables. The increase in antibody titers toward CPV noted at T2 in both the lymphoma and non-lymphoma groups could also be explained by the environmental persistence of the parvovirus, which exhibits strong resistance. It is possible that the patients had somehow come into contact with the pathogen in the environment, resulting in the stimulation of a specific antibody response. The tendency toward significance observed in the lymphoma group could be related to the fact that this group included most of the dogs that had reached the final stage of sampling, which was performed about a month after the end of the chemotherapy protocol. It is therefore very likely that by that time the immune system was recovering itself. A very interesting feature is that the patient who showed the highest T2 antibody increase was particularly young (2 years old), and thus of a very different age compared with the other patients in the group (all senior or geriatric), and this could have helped in the immune recovery. Even in absence of statistically significant differences related to the type of lymphoma (B- or T-cell), a B-cell neoplasm was generally linked to a major decrease in antibody titers (one or two dilution factors), while the T-cell lymphoma showed a milder decrease. The most plausible explanation for this finding is that chemotherapy is more aggressive toward B lymphocytes, which temporarily cease to perform their main function (antibody production) until a new stimulation by T helper lymphocytes comes.
+
+The statistically significant difference found in dogs of different sizes, with larger dogs having higher antibody titers against CAdV-1 than smaller ones, needs further investigation. Generally, small dogs have a better response after vaccination or natural stimulation than larger ones, and for this reason they are often referred to as high-responders; by contrast, many large or giant breeds often include a high number of non-responder dogs [5,11,15,33,35]. Since this difference was already present before treatment, and therefore was not induced by chemotherapy, it is possible that the immune response was further enhanced by the contact with CAdV-2. This virus is spread from dog to dog through coughing and, like CAdV-1, survives in the environment for a long time. It is therefore possible that after coming into contact with this virus (attending dog parks, boarding facilities, or grooming salons), the dogs may have developed a good cross-immunity [24,32].
+
+Killey et al. [33] observe that still little is known about the effects of different diseases on vaccine-induced protection. However the antibody titration results of the individuals in our study are very similar to those obtained using the same in-clinic test in a previous study with healthy dogs [32].
+
+Due to the small sample size enrolled in this study, however, these preliminary results need to be confirmed with further investigations on a larger cohort of patients with different neoplasms (above all, of the lymphoid and myeloid lines), and treated with other chemotherapy protocols and/or with a more uniform sampling for histotype/citotype neoplasms and chemotherapy protocols. It would also be interesting to investigate other features of the immune system (i.e., cell-mediated and innate immunity) in order to better understand the real effects of chemotherapy on the immune system, since an individual may have a very low antibody titration against a specific pathogen while still maintaining good protection based on the cell-mediated immunity, or even the innate immunity [11,33]. Moreover, VacciCheck is a laboratory test with good sensitivity (optimal for CDV, 100%) and specificity (optimal for CPV-2, 100%), and it is officially recognized for its reliability, and is the test most recommended by different international authorities and researchers from among all the in-clinic tests specific for dogs available on the market [36].
+
+Finally, it would be interesting and useful to investigate whether chemotherapy treatment produces any adverse effects on a vaccine intervention performed during antiblastic therapy. Nowadays, performing vaccine prophylaxis during antiblastic treatments is generally not recommended by oncologists, and the testing of a patient’s antibody titers specific for core vaccines is not currently a common practice, especially in the early stages of chemotherapy protocols, when the adverse effects an individual patient may suffer as a result of the antiblastic protocol are unknown (e.g., severe febrile neutropenia or gastrointestinal alterations). This study demonstrates that protection against known pathogens is not dramatically altered by chemotherapy as immune memory seems to work reasonably well. However, since every oncologic patient has his or her own clinical and therapeutical history and his or her own individual immune response in terms of efficiency and durability, an antibody titer evaluation prior to beginning chemotherapy could be a major advantage, helping veterinarians choose whether to vaccinate in the short term or to postpone vaccination boosters without taking any risk.
+
+## 5. Conclusions
+
+This study represents the first in Italy aimed at assessing the effects of chemotherapy on the immune response specific for core vaccines in oncologic canine patients. The results of this study confirm those of the other few analogue veterinary studies and allow us to state that antibody titers against canine parvovirus infections, canine distemper, and infectious canine hepatitis are not negatively influenced by chemotherapy, at least in the medium to short term, and with the protocols and for the neoplasms analyzed.
+
+These preliminary results represent a valuable starting point for future investigations. They are very useful for veterinarians wishing to comprehensively manage their oncologic patients, and we suggest avoiding booster vaccinations for dogs undergoing chemotherapy. In addition, these results could allow owners to be reassured about their pet’s quality of life during treatment, ruling out the risk of incurring dangerous and sometimes fatal viral diseases.

fulltext/md/PMC10147816.md

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+---
+pmcid: "PMC10147816"
+pmid: "36995432"
+doi: "10.1007/s00429-023-02625-y"
+title: "Central nodes of canine functional brain networks are concentrated in the cingulate gyrus."
+journal: "Brain structure & function"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Central nodes of canine functional brain networks are concentrated in the cingulate gyrus
+
+## Abstract
+
+Compared to the field of human fMRI, knowledge about functional networks in dogs is scarce. In this paper, we present the first anatomically-defined ROI (region of interest) based functional network map of the companion dog brain. We scanned 33 awake dogs in a “task-free condition”. Our trained subjects, similarly to humans, remain willingly motionless during scanning. Our goal is to provide a reference map with a current best estimate for the organisation of the cerebral cortex as measured by functional connectivity. The findings extend a previous spatial ICA (independent component analysis) study (Szabo et al. in Sci Rep 9(1):1.25. 10.1038/s41598-019-51752-2, 2019), with the current study including (1) more subjects and (2) improved scanning protocol to avoid asymmetric lateral distortions. In dogs, similarly to humans (Sacca et al. in J Neurosci Methods. 10.1016/j.jneumeth.2021.109084, 2021), ageing resulted in increasing framewise displacement (i.e. head motion) in the scanner. Despite the inherently different approaches between model-free ICA and model-based ROI, the resulting functional networks show a remarkable similarity. However, in the present study, we did not detect a designated auditory network. Instead, we identified two highly connected, lateralised multi-region networks extending to non-homotropic regions (Sylvian L, Sylvian R), including the respective auditory regions, together with the associative and sensorimotor cortices and the insular cortex. The attention and control networks were not split into two fully separated, dedicated networks. Overall, in dogs, fronto-parietal networks and hubs were less dominant than in humans, with the cingulate gyrus playing a central role. The current manuscript provides the first attempt to map whole-brain functional networks in dogs via a model-based approach.
+
+### Supplementary Information
+
+The online version contains supplementary material available at 10.1007/s00429-023-02625-y.
+
+## Introduction
+
+To map similarities and uniqueness of the mammalian brains’ functional connectivity, it is vital to cover a wide array of species from various taxa (Thiebaut de Schotten et al. 2019; Striedter 2002). In this endeavour, species outside of the Primate order are of particular interest, as during Primate evolution, the prefrontal region’s relative size compared to the rest of the cortex increased considerably, which accompanied the differentiation of cortical areas (Wise 2008; Carlén 2017).
+
+Our model, the family dog, a distant taxon, with a population not bred and reared under laboratory conditions but living in its natural social environment, provides a good fit and useful addition to the rapidly increasing field of awake rs-fMRI studies.
+
+The dog brain is currently the focus of several projects, including, among others, EEG (Iotchev et al. 2020), brain banking efforts (Urfer et al. 2021; Sandor et al. 2021, 2022), with fMRI being the most proliferate field in recent years (Aulet et al. 2019; Karl et al. 2021; Prichard et al. 2021). Recent dog fMRI studies are venturing into a wide array of topics, from epilepsy (Beckmann et al. 2021) to comparative face and voice processing (Bunford et al. 2017; Boros et al. 2021; Bunford 2020).
+
+While the default mode network (DMN) is among the most studied human rs-networks, the organisation of the dog DMN is currently disputed in the literature. Although our previous paper described a putative DMN network showing anterior and posterior connectedness, two other research groups reported it to be dissociated to an anterior and posterior hub (Kyathanahally et al. 2015; Beckmann et al. 2020).
+
+In other mammalian species investigated so far (rat Liang et al. 2015, voles Ortiz et al. 2018, ferret Zhou et al. 2016, marmoset Belcher et al. 2013 and macaques Hutchison et al. 2011), the putative DMN contained both the posterior cingulate cortex and frontal cortical areas.
+
+Our main goal was to create a map of functional brain organisation for a non-primate species with well-studied, complex cognition and social behaviour (Miklosi and Kubinyi 2016) in similar detail and approach to that suggested by recent papers (Thiebaut de Schotten et al. 2019; Keifer and Summers 2016; van den Heuvel et al. 2016).
+
+Comparative connectomics (van den Heuvel et al. 2016) aims to understand the common architectures and species-specific differences of brain topology. To achieve this, we need to reconstruct connectomes of different species suitable for such comparisons.
+
+In our previous paper (Szabo et al. 2019) we described a functional connectivity-based parcellation of the dog brain. The main reason we had to rely on a model-free approach was the lack of published dog brain atlases with voxel-level anatomical labelling. Since then, several new stereotaxic atlases have been published with better spatial resolution and more detailed parcellation (Czeibert et al. 2019; Johnson et al. 2020), including probability tissue masks for white and grey matter (Liu et al. 2020).
+
+While our previous approach allowed us to describe the spatial characteristics of the networks without the selection of a priori seed regions, it was limited by the lack of anatomically bound, standardised ROIs, which makes comparisons across studies and species difficult. The ICA approach is less suited to provide information about the functional organisation beyond certain voxels belonging to the same, statistically independent source signal (Beckmann 2012). A model-based approach can reveal the network of regions with the strongest functional connections to the respective ROI (Van Den Heuvel and Pol 2010) and inform us about the strength of individual intra- and inter-network connections via semi-partial correlations. Semi-partial correlations indicate a unique relationship between an independent and the dependent variable (ROI time series), where the variance in a dependent variable is explained only by that independent variable, excluding the variance explained by other independent variables.
+
+In our current study, we utilise ROI based approach and analyse the resulting undirected graph to group ROIs into networks to describe their characteristics and determine the most central nodes of the resulting graph. We also aimed to compare the outcome of the two approaches to understand their impact.
+
+## Materials and methods
+
+### Subjects
+
+We measured 33 family dogs *(Canis familiaris)* (age 6.70 ± 3.62 years (mean \documentclass[12pt]{minimal}
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+ \begin{document}$$\pm$$\end{document}± SD), range 1–14 years, 17 females and 16 males, 9 border collies, 7 golden retrievers, 5 mongrels, 2 Australian shepherd, 1 English cocker spaniels, 1 Labrador retriever, 1 labradoodle, 1 mudi, 1 Swiss shepherd dog, 1 Tervueren, 1 springer spaniel, 1 Chinese crested dog, 1 Cairn terrier, 1 Hungarian vizsla). The training procedure has been described in detail in a previous study (Andics et al. 2014) and was based on individual and social learning using positive reinforcement.
+
+### Procedure
+
+The study consisted of a 2-minute-long habituation phase to familiarise the dogs with the semi-continuous scanning procedure, followed by two 6-minute-long data collection runs. To provide sound protection, the dogs were wearing ear muffs. During scanning, dogs were lying with their eyes open, without presenting a fixation cross, with their handler visible, but avoiding eye contact with the subject. Figure 1a shows the dogs’ position in the scanner. The strap over the head was used to fixate the circular coil on the top of the dog’s head, not to restrain dog motion (Fig. 1b). Dogs were not fixed (could leave their position at any time) or sedated in any way. The motion threshold for successful runs was set to a maximum of 3 mm (for each translation direction) and 2 degrees (for each rotation direction) during the whole run.Fig. 1Dogs in scanner positioning
+
+### Ethics statement
+
+All procedures were approved by the Ethical Committee of Eötvös Loránd University (KA-1719/PEI/001/1490-4/2015) and by the Government Office of Pest County Directorate of Food Chain Safety and Animal Health (XIV-I-001/520-4/2012) and conducted in accordance with relevant guidelines and regulations. Owners provided informed consent and were able to stop the procedure at any time.
+
+### Image acquisition
+
+Functional MRI acquisitions were performed on a Siemens Prisma 3T scanner Siemens Healthcare, Erlangen, Germany) using a Gradient Echo Echo Planar Imaging (GRE-EPI) sequence with TR = 2640 ms, including a 500-ms delay at the end of each volume, and TE = 31 ms. We decided to introduce the delay for welfare reasons. During piloting, we realised that our dogs seemed to find continuous scanning less comfortable than protocols with short gaps between subsequent scans. The 500 ms delay was selected empirically during piloting to include the shortest gaps that the dogs are still comfortable with. The protocol had an in-plane Field-of-View 128 mm \documentclass[12pt]{minimal}
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+ \begin{document}$$86^{\circ }$$\end{document}86∘. Phase-encoding direction was set to foot \documentclass[12pt]{minimal}
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+ \begin{document}$$\hbox {d}=11$$\end{document}d=11 cm) was used for signal detection, fixed to the table of the scanner. These straps kept the coil in position on the head of the dog. The dogs were able to pull out their heads from these straps. In this case, the headphones would have remained still on their head as that was fastened with a separate layer. This event occurred only during the training sessions.
+
+In each run, 137 volumes were acquired, with the first five discarded before processing, resulting in a total functional scanning time of 362 s/run. A T1-weighted anatomical scan was carried out separately as part of another study on each awake dog for spatial registration on a 3T Philips Ingenia scanner (Philips Medical Systems, Best, The Netherlands), using a 3D Turbo Field Echo (TFE) sequence, with TR = 9.85 ms, TE = 4.6 ms, and an isotropic resolution of 1 mm.
+
+### Data analysis
+
+FMRI preprocessing relied on SPM12 (http://www.fil.ion.ucl.ac.uk/spm/) and CONN (Alfonso Nieto-Castanon 2020). The process included the following steps: affine realignment (6 parameters, least square approach), reslicing, manual coregistration of the mean image to the individuals’ own structural T1 image in Amira 6.0 (Thermo Fisher Scientific), normalisation of the functional images to this transformed mean functional image via SPM’s standard nonlinear warping function with 16 iterations.
+
+The following steps were carried out in CONN software: 1. functional outlier detection (ART-based identification of outliers scans for scrubbing), 2. segmentation of CSF, WM, GM (Liu et al. 2020), 3. regressing out confounding effects, 4. functional smoothing (spatial convolution with 4 mm Gaussian kernel), 5. denoising with bandwidth filtering (0.001 Hz–0.1 Hz), 6. linear detrending.
+
+CONN’s default denoising pipeline implements an anatomical component-based noise correction procedure (aCompCor), which estimates and removes confounding effects separately for each voxel/run/subject using Ordinary Least Squares (OLS) regression to project each BOLD signal time series to the sub-space orthogonal to all potential confounding effects. The pipeline covers noise components from WM and CSF, estimated subject-motion parameters, identified outlier scans or scrubbing, constant and first-order linear session effects (Alfonso Nieto-Castanon 2020).
+
+The analysis was carried out in the BBT stereotaxic template space (Liu et al. 2020). We relied on the ROI segmentation of (Liu et al. 2020), apart from the cingulate gyrus, where we used the more detailed segmentation provided by Johnson et al. (2020). We renormalised these ROIs to the BBT. This approach assigns hubs/nodes of functional interests based on myeloarchitectonic-based histology atlases. The analysis space was set via an explicit brain mask from a liberal brain template and was relying on percent signal change instead of the bold signal raw value.
+
+From the available 111 ROIs, we analysed only the 97 cortical and sub-cortical ROIs. We decided to exclude ROIs belonging to the brainstem and olfactory regions to focus our analysis on the cortical functional brain organisation. The list of the excluded ROIs can be found in the appendix.
+
+### Statistical analysis
+
+To evaluate the similarities and differences between the resting state network organisation described in our current and previous paper (Szabo et al. 2019), we calculated ROI-based Jaccard similarity.
+
+To obtain an ICA network to ROI mapping, we relied on the ICA mask files containing the results of our previous paper (Szabo et al. 2019), normalised it into the current atlas space and projected it over the ROI atlas. For each network, we extracted the number of overlapping voxels with each ROI. Due to the nature of the ICA approach, several networks involved the same ROIs, therefore, we allocated each ROI to the network with the largest overlapping volume/majority vote. In the case of bilateral ROIs, we averaged the number of overlapping voxels for the two ROIs for each ICA network before allocation to ensure we allocate bilateral ROIs to the same network.
+
+In the current study, we analysed seed-based connectivity (“Does the average connectivity between ROIs differ from zero?”). For first-level functional connectivity maps, semi-partial correlation and weighted GLMs were calculated. For generating the reported 2nd level connectivity map results, we relied on applied parametric multivariate statistics with the following post hoc thresholds (Benjamini and Hochberg 1995): cluster threshold p-FDR corrected\documentclass[12pt]{minimal}
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+ \begin{document}$$<0.05$$\end{document}<0.05 (MVPA omnibus test) and connection threshold p uncorrected\documentclass[12pt]{minimal}
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+
+This approach addresses the multiple comparison problems inherent to this type of analysis and is suited to handle the high rates of false positives. It calculates weighted graphs representing the level of effective or direct connectivity between an individual ROI and every other analysed ROI in the brain after discounting effects that may be mediated or accounted for by other ROIs.
+
+We transformed the ROI matrix into a network and analysed it by using Python and the NetworkX (Hagberg et al. 2008) library. In this weighted undirected network, every node corresponds to a brain region, while the strength of their connection is proportional to their ROI values.
+
+We captured the so-called small-worldness property, which quantifies how well the neighbouring node’s neighbours are connected—how easily different nodes can reach other nodes within the network (Watts and Strogatz 1998). We did so by computing the classical small-world scalar (\documentclass[12pt]{minimal}
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+ \begin{document}$$\Sigma$$\end{document}Σ) (Humphries et al. 2006), normalised clustering coefficient (\documentclass[12pt]{minimal}
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+
+Next, we extracted network communities—network subgraphs whose’ nodes are densely interconnected compared to the rest of the network—by following widely-used methodologies (Lambiotte et al. 2008).
+
+Finally, we visualised and explored the network communities by using Gephi 0.9.2 (Bastian et al. 2009) with two layers. Firstly, we plotted all analysed ROIs (except cerebellum) with various modularity thresholds to find the optimal value empirically. This gave us the initial structure with 10 modules (with a modularity threshold of 0.6). Then we ran 10 repeats to produce a consensus voting for this structure. For the second layer, we took the most populated two lateralised modules and plotted these two in a separate round, which resulted in the final, combined 12 (8+4) module structure.
+
+## Results and short discussion
+
+### ROI to ROI analysis
+
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+ \begin{document}$$(mean \pm SD)$$\end{document}(mean±SD). There was a moderate positive correlation between the subject’s age and mean framewise displacement (FD) value (\documentclass[12pt]{minimal}
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+
+Due to the significant correlation between age and FD, we decided not to look for any relationship between age and connectivity, as our sample size was not suited to tackle the significant correlation (even though all subjects did pass the motion threshold determined for a successful run and were analysed). Recent research shows that normal ageing produces a significant increase in head motion parameters, therefore, this relationship is not unexpected (Sacca et al. 2021).
+
+While cerebellar regions were included in the data, no connection survived post hoc corrections, hence they are not reported and listed in the visuals. In our previous publication, the cerebellar networks also showed up as separate, isolated network (Szabo et al. 2019). Another possible reason for the lack of such connections can be the exclusion of brainstem ROIs from the current analysis.
+
+An overview of the resulting parcellation can be found in Fig. 3Fig. 3Selected views of the functional brain networks overlaid on 3D stereotaxic brain atlas. Different colours represent different functional networks. **A** Left, **B** Ventral, **C** Extracted cingulate gyrus region, **D** Right, **E** Dorsal
+
+### Similarities to previous dog ICA rs-networks
+
+Despite the inherently different approach of ICA and ROI-based analysis, the resulting networks showed a remarkable similarity (Table 1), with Jaccard similarity values between 0.64–0.80. This means that the majority of the ROIs were part of the corresponding networks in our previous study (Szabo et al. 2019) as well.
+
+However, unlike in our previous study, we could not detect a clear, distinct auditory network candidate. Instead, we had two lateralised networks (Sylvian L, Sylvian R), including the respective Sylvian (auditory) regions among other regions (Fig. 3). Possible explanations include insufficient parcellation (current stereotaxic atlases segment this gyrus into three subregions, but these subjectively drawn regional boundaries lack clearly defined and constant anatomical margins) next to individual variation in gyrification and functional boundaries. For the reliable detection of auditory regions, individual, voxel-based localisation is currently a better approach in dogs, as they are not following anatomic boundaries, and are located in a small region containing several functions, without sub-millimetre resolution being feasible in awake dog fMRI. Future work is needed to further decrease the uncertainty regarding functional boundaries in dog auditory cortex, to functionally delineate A1 and subsidiary functional auditory regions with a large sample size, accompanied with cytoarchitecture work and tracing to establish a clearly anatomically defined extent of auditory regions and their anatomical connectivity in the dog. The two, extensive, lateralised functional networks appear in both studies despite the different phase encoding directions and analytical approaches.
+
+While we reported the anterior and posterior dog default mode network (DMN) to show functional connectedness in the previous study, the connection between them in the current seed-based analysis is less convincing. Although we found connections between the splenial gyrus and anterior cingulate regions (XMII, GI), the splenial gyrus was listed as part of the Visual network, not as part of the Ventral posterior cingulate network. A more detailed segmentation of this region in future atlases may reveal that this area plays a role in multiple networks. Other recent publications found no sign of antero-posterior connectedness of the DMN in the dog brain via rs-fMRI. The insufficient resolution, susceptibility artefact, cross-subject variations (Bijsterbosch et al. 2018), or possible breed/skull shape-related differences are all possible alternative explanations which need to be excluded before we can determine whether the dog DMN is indeed dissociated. Previous dog tractography studies aimed specifically to describe the structural basis of specifically the dog DMN (Robinson et al. 2016) and whole dog brain (Jacqmot et al. 2013). Robinson et al. (2016) reported that the antero-posterior tract between putative aDMN and pDMN regions was found only in 9 out of 23 dogs which, unlike in humans, points toward a significant individual variability in dogs. Further structural connectome studies obtained with tracing and optical imaging are necessary to uncover the underlying reasons responsible for this divergence.Table 1Comparing the resulting resting state network organisation across the two approachesICA based network (Szabo et al. 2019)Taxonomy (Uddin et al. 2019)ROI based networkJaccard indexAM-FPNHippocampal0.66Ventral posterior cingulateBM-FPNMedial prefrontal0.64CD-FPN/L-FPNDorsal/lateral prefrontalDM-CINAmygdala0.71Partial Sylvian LEM-CINPartial Sylvian RFPNPartial Sylvian R (Sensorimotor)IPNPartial Sylvian L & partial Sylvian R (Auditory)JM-CINMid-cingulateKPNSensoritomotor (peri-cruciate)0.74M-CINAnterior cingulateHM-FPNDorsal posterior cingulate0.80LONVisual0.75MON
+
+### Network characteristics
+
+#### Small world
+
+The analysed network consisted of 97 nodes and 560 edges, containing 427 triangles (Fig. 4). The connection density of the resulting network was 12%. This is within the reported range of fMRI connectivity studies in other species (5% to 30%) (Bassett and Bullmore 2017), reflecting that the applied strict threshold was adequate to account for the high level of noise inherent in rs-fMRI data (Tables 2, 3).Fig. 4The organisation of the functional network nodes in the dog brain visualised by Gephi using ForceAtlas layout. The ForceAtlas layout simulates a physical system consisting of particles linked by different attractive and repulsive forces that organize the network into an organic topology reflecting the general connectivity patterns. Node and edge colouring correspond to community membership, while the edge widths measure the semi-partial correlation values. The list of numbered nodes can be found in the supplementary material, while the main brain regions are labelled with appropriate coloursTable 2Overall network properties of the dog brainVariableSymbolNormalised valueRaw valueNormalised clustering coefficientGamma (\documentclass[12pt]{minimal}
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+ \begin{document}$$\Lambda$$\end{document}Λ)1.022.15Classical small-world scalarSigma (\documentclass[12pt]{minimal}
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+These network characteristic values are also similar to those reported in Bassett and Bullmore (2017) for macaque and mouse brain networks. Small-world networks have normalised clustering coefficient \documentclass[12pt]{minimal}
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+ \begin{document}$$> 1$$\end{document}>1Humphries et al. (2006), meaning the dog brain network satisfies the requirements of a small-world network.
+
+### Modularity
+
+For bilateral homologous regions, both node degree (\documentclass[12pt]{minimal}
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+
+We looked at modularity structure on two levels. First, we included all analysed ROIs, which resulted in 10 modules.
+
+Out of these, the two largest communities were lateralized, consisted of 15–15 ROIs and covered most of the temporal and parietal surface of the brain. In these two entities, insular, sylvian, ectosylvian, rostral composite regions got split based on laterality (Fig. 5).Fig. 5Colorwheel visualisation of positive (red) and negative (blue) ROI to ROI connections. Width and opacity of edges proportional to the strength of the connection
+
+Further lowering modularity resolution did not result in breaking up these two large groups. Therefore, we decided to carry out a second analysis restricted to the nodes of these two entities to understand their functional organisation in more detail. This resulted in dividing each into two further communities, therefore the final segmentation contained 12 modules in total (Fig. 3).Fig. 6Thresholded semipartial correlation matrix of the ROI to ROI connections. Only significant connections are displayed. P values p-FDR corrected\documentclass[12pt]{minimal}
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+ \begin{document}$$<0.01$$\end{document}<0.01Table 3Main regions involved and taxonomy of the brain network types based on Uddin et al. (2019)NameNetwork typeMain regions/gyri involvedVisualONSplenialis, ectomarginalis, marginalis, occipitalis and suprasylvius caud.Dorsal/Lateral prefrontalD-FPN/L-FPNGenualis, olfactorius lateralis, frontalis and proreusMedial prefrontalM-FPMArea subcallosa, paraterminalis, rectus, diagonalis, area septalis, nucleus caudatusHippocampalM-FPMHippocampus, parahippocampalisVentral posterior cingulateM-FPMLPVI, LPVIIDorsal posterior cingulateM-FPMFSSA, LPDI, LPDII, LPL, presplenialisMid-cingulateM-CINFPSL, GII, LAL, LAV, LMAnterior cingulateM-CINGI, XMII, remaining cinguliAmygdalaM-CINAmygdala, lobus piriformisSensoriomotorPNPre- and postcruciatus, suprasylvius rostrSylvian LPN(Ventral post anchored) comp. caud., L regionsSylvian RPN(Dorsal anchored) ectosylvius rost., suprasylvius medius, R regions
+
+#### Density within communities
+
+All resulting network communities (subgraphs) had a significantly higher edge density than the whole network (Table 4), meaning that in a network community, any pairs of nodes are more likely to be connected to each other than to any other node not part of that community. The densest network communities only contained ROIs from the cingulate gyrus (ventral posterior cingulate, dorsal posterior cingulate, anterior cingulate, middle cingulate), showing the core role of this region within the dog brain (Fig. 6).Table 4Number of nodes and density values within network communities for the respective functional networksNetworkNo. of nodesDensityWhole970.12Visual80.47Hippocampal40.5Ventral posterior cingulate40.75Dorsal/lateral prefrontal80.34Dorsal posterior cingulate100.62Medial prefrontal120.46Anterior cingulate60.55Mid-cingulate100.52Amygdala50.4Sensoriomotor70.49Sylvian L100.34Sylvian R100.3
+
+### Most central nodes and edges
+
+We present the top nodes listed by weighted degree, degree and betweenness centrality values. In the case of bilateral regions, we calculated the average of the two nodes.
+
+Ten of these ROIs were ranked within the top 20 for all three categories (Table 5): area subcallosa, prorean gyrus, splenial gyrus, posterior cingulate gyrus (LPDI, LPVI, LPVII), anterior cingulate gyrus (GI, XMII, G cinguli) and mid-cingulate gyrus (LM).Table 5Most central nodes by weighted degree (WD), degree (D) and betweenness centrality (BC) values. Top 20 bilateral nodes listed in each ranking. Bilateral values were calculated by averaging the two nodes prior to rankingROIWDROIDROIBCLPDI101.15LPDI19LM121.19LPL92.37LM19g. cinguli107.07LPDII76.38G. cinguli18g. proreus90.8LPVI76.38LPVI16.5LPDI83.6g. cinguli71.93XMII16LPVI82.55LAV65.39LPL14.5Area subcallosa82.3XMII64.17LPDII14.5G. suprasylvius rostralis81.6LM62.2FSSA14.5lobus piriformis78.17G. paraterminalis60.25LPVII14.5LPVII76.82LAL56.44LAV14.5XMII75.36FSSA56.27Area subcallosa13.5G. precruciatus75.19GII44.35GII13G. ectomarginalis74.88LPVII53.55G. suprasylvius rostr13G. suprasylvius caud72.04Area septalis50.99G. proreus13G. compositus rostr71.92Area subcallosa46.4GI12.5G. splenialis69.83G. splenialis45.93LAL12.5GI69.3l piriformis44.74G. splenialis12.5G. ectosylvius caud63.44GI44.34G. precruciatus12.5G. compositus caudalis62.82G. proreus41.59G. ectomarginalis12.5G. sylvius rostr59.41G. marginalis40.98G. compositus rostr12G. rectus58.32
+
+7 of the 10 most central nodes are part of the cingulate gyrus, while all 7 nodes belong to separate networks.
+
+There were two networks without inter-network connections (with a direct link stronger than our threshold), the amygdala network and the medial prefrontal network (Fig. 5). We found no direct links between the anterior cingulate and dorsal posterior cingulate gyrus, nor between the ventral posterior cingulate and mid-cingulate areas, which would have been our expectation for a direct aDMN and pDMN connection. This does not mean that these networks are functionally isolated from other parts of the brain. The lack of direct links surviving the current threshold could be the result of mediation by other regions, because the utilised semi-partial correlation only shows the unique relationship between two ROIs, excluding variance shared with other independent variables.
+
+The two lateralised Sylvian networks were highly connected. Next to covering associative and sensorimotor cortices, these networks also included the auditory regions and the insular cortex. The dorsal/lateral prefrontal network was only connected to the anterior cingulate network. The anterior cingulate network also had links to the ventral posterior cingulate, mid-cingulate, dorsal/lateral prefrontal and the visual network.
+
+## Conclusions
+
+We scanned awake dogs in a “task-free condition”, a cognitive state hard to define objectively (Gorges et al. 2017), especially in the case of animals where scanner fixation is widely used (e.g. rabbits Weiss et al. 2018). Our subjects, similarly to humans, remain willingly motionless during scanning. Recently, a condition called “naturalistic viewing” (no fixation-cross, high in social content, evoking variable gaze positions) was found to be outperforming the standard resting condition in humans both in regard to functional connectivity-based prediction of behaviour (Finn and Bandettini 2021) and test-retest reliability (Wang et al. 2017). This supports our claim that the free-viewing and fixation-free condition we measured the dogs in provides relevant data.
+
+We found that even in the case of successful sessions (defined by session-level total motion thresholds), there is a significant correlation between the age of the subject and framewise displacement. This means that in dogs, similarly to humans (Sacca et al. 2021), normal ageing results in increasing head motion. Differences between the age groups’ motion levels have to be considered and adequately addressed if the goal is to interpret differences in functional connectivity networks across such groups. Future studies (apart from collecting data from more subjects) may try to utilise special, extra preprocessing steps which address this in humans, for example, via taking into account framewise displacement and DVARS during scrubbing as proposed by Power et al. (2012). Due to the currently available sample size, we were unable to investigate the putative effects of age or breed differences on the organisation of functional networks. Future multi-site studies with standardised scanning protocols are more likely to achieve the required sample size for such endeavours.
+
+Due to the susceptibility artefact around the sinuses, we decided to exclude olfactory regions from our analysis. The level of noise and signal loss makes data from this area inaccessible/unreliable with the current hardware and coil. This prohibited the exploration of a possible olfactory network and could contribute to the list of reasons why we did not detect a clear antero-posterior DMN connectivity (although we found connections between the splenial gyrus and anterior cingulate regions), as in our previous study, we did not exclude these regions from the analysis.
+
+The existence of the antero-posterior DMN connectivity in dogs is debated, with several studies not finding supporting evidence for it Kyathanahally et al. (2015), Robinson et al. (2016), Beckmann et al. (2020). Given that the current result is also inconclusive in this regard (strong individual ROI to ROI connections between the respective networks were present, but no connection on the network level surviving post hoc corrections), we do not feel we can make a binary conclusion regarding the definitive presence or absence of this connection based on the currently available information.
+
+The currently described network organisation closely resembles our previous findings, despite the different approaches (model-free vs. model-based) between the two studies.
+
+The human lateral prefrontal cortex (LPFC) is disproportionately expanded even when compared to non-human primates (Vendetti and Bunge 2014). In dogs, on the other hand, it is a small area, including less than 10% of the cortical regions. It consists of mostly the frontal and prorean gyrus, which have a combined total volume of c.a 400 voxels/3200 cm^3^. This area also suffers from susceptibility artefact/signal loss due to the extensive sinuses.
+
+The human LPFC, thought to be related to relational reasoning (Vendetti and Bunge 2014) plays a part in three functional network groups (D-FPN, L-FPN, M-CIN) (Uddin et al. 2019). In contrast, the whole dog LPFC belongs to a single resting state network (Dorsal/lateral prefrontal). The lack of separate attention and control networks in dogs could be a byproduct of the limits of the currently available image resolution, which can be further improved by developing coils specifically tailored to measure dogs (Guran et al. 2022). Alternatively, in dogs, the cingulate cortex may be handling some integrative functions which were taken over by frontoparietal regions during primate brain evolution.
+
+The cingulate cortex has been consistently identified as a hub with a high level of centrality, as well as dense and widespread connectivity in comparative studies across humans, chimpanzees, macaques, and cats (van den Heuvel and Sporns 2013). The human posterior cingulate gyrus shows high node degree and betweenness centrality, suggesting it is essential for integrating and passing information across networks. In macaques, the posterior cingulate gyrus showed the highest node degree and betweenness centrality values (Miranda-Dominguez et al. 2014). Our results regarding the centrality of the cingulate cortex in dogs are in line with these findings. Areas across the anterior (GI, XMII), medial (LM) and posterior (LPDI, LPVI, LPVII) cingulate gyrus are all found among the most central nodes, next to the prorean gyrus and splenial gyrus. Functional dog fMRI studies also report the involvement of the cingulate gyrus across several tasks, such as word detection and odour discrimination (Prichard et al. 2018, 2020)
+
+The cingulate cortex plays a central role in various functions related to behavioural flexibility, ranging from autonomic regulation to action selection and performance monitoring. It is a processing hub for the regulation of autonomic responses, assessing emotional and motivational aspects of internal and external information (seeing emotional faces or listening to emotionally charged voices), cognitive control, such as response selection, attentional processing, monitoring conflict, detecting errors, and reward-based decision-making (van Heukelum et al. 2020).
+
+Dogs’ cingulate cortex is relatively larger than in humans when compared to the total cortex volume. Functional studies testing whether and where in the dog cingulate cortex all the above-mentioned processes are localised, or determining the level of frontoparietal integration in these tasks in dogs are yet to be carried out. Future studies should also include validated cytoarchitecture work and extensive tracing to allow us to establish a clearly anatomically defined extent of cingulate and its anatomical connectivity in the dog. This is a prerequisite for a more detailed parcellation of the splenial and cingulate gyrus, which in turn is necessary to determine whether e.g., the cingulate gyrus is handling integrative functions that were taken over by frontoparietal regions in the case of the human brain.
+
+To overcome some disadvantages of the model-blind approach utilised in our previous paper, the current research set out to describe the functional organisation via a model-driven analysis. This allowed us to look at both inter- and intra-network connections, describe the most central nodes, and evaluate the whole brain as a single large network while providing more straightforward interpretability,
+
+Both approaches have advantages and disadvantages, focusing on a different aspect of brain networks, yielding similar, but not identical network structures. Neither is able to provide definitive answers, or certainty about the functional organisation of the dog brain, as both are mere models and approximations. By applying the model-based approach, we adhere to how the first layers of knowledge were built up at the early stage of human functional brain mapping, as early human rs-fMRI studies typically characterised functional connectivity via a small number of large-scale spatial maps as well Smith et al. (2013).
+
+We relied on the universal resting state network taxonomy proposed by Uddin et al. (2019) to categorise dog networks. Currently, compared to the field of human fMRI, we have very few studies describing the functions of respective brain areas in dogs. Therefore, we had to resort to anatomical and evolutionary parallels where the functional parallels are yet untested. We found examples for most major network types and that anatomical modules correspond to groups/communities of functional networks in the dog brain.
+
+Stimulus-based fMRI studies are needed to assign functions to the networks described in this paper. The current reference map allows researchers to carry out future connectivity fingerprint matching studies (Thiebaut de Schotten et al. 2019) across even more species, now including the dog as well.
+
+## Supplementary Information
+
+Below is the link to the electronic supplementary material.Supplementary file 1 (PDF 97KB)

fulltext/md/PMC10155720.md

@@ -0,0 +1,58 @@
+---
+pmcid: "PMC10155720"
+pmid: "37152338"
+doi: "10.24908/pocus.v8i1.16145"
+title: "'Teaching old dogs new tricks' - POCUS Education for Senior Faculty."
+journal: "POCUS journal"
+year: "2023"
+license: "cc by"
+article_type: "other"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# “Teaching old dogs new tricks” – POCUS Education for Senior Faculty
+
+## Abstract
+
+Point of Care Ultrasound (POCUS) is a growing diagnostic modality across a variety of specialties and is increasingly being taught in undergraduate medical education. Uptake within internal medicine has been slow but is becoming more commonplace. Training of extant hospital medicine faculty, including senior members, in POCUS is an unmet need in graduate medical education with significant pedagogical and patient safety implications. With this in mind, we created a training program for the core teaching faculty at our academic internal medicine residency program. The experiential, hands-on curriculum explored the reasoning behind concepts and emphasized psychological safety for senior faculty learners and was successful and well-received. In our piece, we aim to explore the existing literature around training this unique population in POCUS and report on our single-center experience. We also provide a framework for how our program succeeded, collate tips derived from the expert ultrasound teachers and list pearls learned while teaching these experienced educators. Although this worthwhile effort requires planning and support, it was appreciated even by senior faculty.
+
+## Introduction
+
+Over the past 25 years, the practice of point of care ultrasound (POCUS) has vastly expanded and is now utilized across a variety of specialties ranging from emergency medicine to rheumatology. POCUS differs from consultative or comprehensive ultrasound imaging in that it is performed by the patient’s clinician at the bedside to answer a focused set of questions that aid in diagnosis 1. Though initially slow to uptake the skill compared to other specialties, internists have begun to utilize POCUS to care for both inpatients and outpatients 2. Simultaneously, incorporation of POCUS education in undergraduate medical education curricula has grown, with now almost 73% of medical schools including POCUS in their preclinical courses as well as clinical education 3. This poses a unique challenge within academic internal medicine, as many incoming first-year medical residents will arrive each year trained in skills that their teachers lack. Modern medical residents are eager to use POCUS to care for patients, however at least one study has shown significant amount of diagnostic inaccuracy in residents with cursory training 4. It is possible and indeed likelier every day, that a resident will start to make diagnostic and therapeutic decisions using POCUS. Thus, training of extant faculty in POCUS represents a growing need amongst internal medicine residency training programs nationwide, one that has important patient safety implications as well. In this perspective piece, we seek to denote the challenges in teaching senior learners a dynamic and multifaceted skill such as POCUS and to report on our experience in training such a group at one institution.
+
+## Challenges in teaching senior faculty
+
+Mid-to-late career faculty, herein defined as faculty with at least 5 years of practice since completing training, represent a unique population from a pedagogical standpoint and one for which there is sparse educational guidance in the literature. The data available for POCUS education in this population within the internal and emergency medicine literature would suggest that these learners are able to learn POCUS at similar rates to trainees5 but may ultimately not incorporate it into their clinical practice 6. In many ways, they are the standard adult learner: self-motivated and ready to learn if the subject matter is relevant to their goals. Employing the principles of learning science is likely to be high-yield, and thus the curriculum should be experiential, problem-focused and presented in a way that both emphasizes the “why” and allows for discovery rather than emphasizing memorization 7. However, many individuals in this career stage are likely to be seasoned or expert clinicians who have not sat in the novice’s seat for some time and thus may feel vulnerable in the process of acquiring new skills. There must be an emphasis on creating psychological safety in the teaching of a new skill, especially if taught in a group setting amongst peers. Additionally, there are likely to be kinesthetic and spatial learning considerations with regards to POCUS image acquisition and interpretation. With initial training, the emphasis for mid-career or senior faculty should be on comfort over competence. Achieving competence in this complex clinical skill can be overwhelming and the goal at the end of an introductory course should be that individuals should be comfortable enough with the technology and interpretation to begin practicing on their own between scheduled trainings. Techniques such as task deconstruction for the specific transducer movements can go a long way to establishing a strong foundation in the fundamental skills of POCUS. Implementing measures such as task deconstruction for a kinesthetic skill such as POCUS is especially relevant to those who practice in a predominantly cognitive specialty like internal medicine, and for those who likely have not performed bedside procedures since their residency training. The opportunity for repeated, hands-on practice either on a POCUS model or a live volunteer with consistent, calm, and non-judgmental instruction is key. The goal as instructors is for the faculty to feel comfortable at the end of the introductory course and achieve competency at the end of longitudinal training.
+
+## The MGH Experience
+
+Acknowledging the need for POCUS-trained faculty in our Internal Medicine Residency Program, we set out to begin training our Core Educator Faculty in POCUS during the fall of 2019. Our goal was to pursue a “train-the-trainer” approach wherein a cadre of dedicated POCUS teachers could more easily train the much larger population of residents in POCUS. The Core Educator Faculty (CEF) for the Massachusetts General Hospital Internal Medicine Residency Training Program is a 16-member group of hospitalist clinician-educators at varying career stages who are responsible for most of the residency’s clinical teaching on the inpatient medical services. All members of the CEF had completed residency training more than five years prior, and none had any formal education regarding diagnostic POCUS. Many in the group had been in practice for more than 20 years and had never held an ultrasound probe or interpreted ultrasound images before.
+
+We designed a so-called “bolus/drip” curriculum wherein the dissemination of content was accomplished in an intensive and hands-on single-day retreat – representing the “bolus.” Following this, a select group of 3-4 faculty “super-users” would engage in longitudinal competency acquisition over the subsequent months – the “drip.” Once these select faculty members completed the longitudinal training, a new set of “super-users” would be selected for intensive training.
+
+The “bolus” was taught by a group of 3 hospitalists with extensive POCUS education experience as well as an educational sonographer. This “bolus” training consisted of 6 total POCUS applications: heart, lung, inferior vena cava (IVC)/jugular venous pressure, abdominal fluid evaluation (Focused Assessment of Free Fluid – or FAFF) 8, renal/bladder and soft tissue POCUS. Both handheld and cart-based ultrasound machines were used for teaching purposes in order to expose learners to different device types. The day-long session was designed to employ didactic modules for content dissemination followed immediately by hands-on practice sessions on live human models. The learning content covered the anatomy for each POCUS application, scanning technique, the normal sonographic findings, and a brief overview of select pathologic states. This hybrid didactic/hands-on model was intentional and served to punctuate the large amount of didactic material being presented with breaks in which direct application of the content could be performed. We designed the session to have a low faculty-to-learner ratio with only three or four learners per station with one faculty member.
+
+The “drip” was largely comprised of scheduled and ad hoc scanning sessions taught by a designated ultrasound-fellowship-trained hospitalist. Following the “bolus”, CEF members interested in more intensive training were asked to write a short paragraph to be selected as one of three “super-users” and were considered based on level of interest, bandwidth and overall clinical footprint on the teaching services. These super-users met with the trainer to scan live inpatients who either volunteered for an educational scan or for whom a scan was clinically warranted. These scanning sessions occurred up to three times per month and lasted around two hours. In addition, all faculty were offered roughly monthly virtual sessions for image review, as a content refresher. To complete their training, each “super user” was required to complete a total of 150 scans (25 scan per application), including both supervised and unsupervised scans. The latter were reviewed by the trainer, and asynchronous feedback was given. After finishing their scanning portfolio, the “super user” was required to complete an objective structured clinical examination (OSCE) with the trainer. These minimum standards were derived from the American College of Emergency Physicians’ training guidelines9 for POCUS and were finalized in collaboration with our Department of Radiology, Department of Emergency Medicine, and the MGH Credentialing Office. Upon completion of training and credentialing, the faculty members were given hospital privileges to perform diagnostic POCUS and bill on their scans. Subsequent clinical scans were reviewed for quality assurance by the trainer. In general, most super-users took 12-18 months to complete their training. Notably, this method inherently creates a large gap in POCUS exposure for faculty members not selected as super-users while the initial cadre are trained, during which time content knowledge and skills are likely to dissipate. To remediate this, all faculty are encouraged to attend the virtual refresher sessions noted above and the subsequent batch of super-users undergo a quick refresher upon selection.
+
+## Reflections, Tips and Challenges
+
+Our experience in teaching a cadre of mid-career to senior faculty was an extraordinarily positive one. Upon beginning the intensive “bolus”, it became clear that the faculty were simultaneously ecstatic and apprehensive about learning a new skill. Our plan at the outset was to maximize the benefit of frequent breakouts and time with hands on a probe. Often, we found most benefit in leveraging the physical examination knowledge that the faculty possessed – correlations of the external examination of the jugular venous pulse to that of the sonographic internal jugular assessment or the IVC were the subject of joy and intense discussion. A feature that was surprising, but not unexpected, was the degree with which these experienced teachers asked “why” when presented with new material. It was noted several times during the training program that these educators would ask their instructors to delve into details that more junior learners such as residents seldom investigate - whether due to comfort with interrupting a teacher due to inherent seniority or because their teaching had for so long consisted of deconstructing mechanisms or processes is unclear. Lastly, we found great success with spaced learning and weaving prior material into the curriculum – for example, using callbacks to similar anatomy shared in different scanning applications such as that seen when imaging the right flank during the sonographic assessment of free abdominal fluid or when searching for a pleural effusion. The training program was very well-received amongst the Core Educator Faculty and they showed an infectious enthusiasm for acquiring new skills, even amongst those not selected as a super-user later. Multiple faculty members, some decades into their careers, remarked on the joys of learning a new skill, one which allowed them to see into the human body at the bedside like they never had before. These faculty members frequently spontaneously voiced these sentiments as having the potential to mitigate burnout. Additionally, through our continuing educational review sessions, non-super users have acquired skills in image interpretation despite not recalling how to acquire them – which is in keeping with what others have found in training similar populations using asynchronous cognitive-focused curricula 10. The faculty who completed their super-user training continues to scan actively and use POCUS in their clinical decision making on a near daily basis and are actively engaged in our image review sessions. We have currently completed training for five faculty members and are actively training four additional ones. Many of the strategies utilized and phenomena witnessed share a great deal in common with principles of learning science and are universally applicable in most spheres of medical education. We have outlined the tips and tricks we felt most useful in Table 1.
+
+**Table 1 Tips and tricks in the teaching of diagnostic point of care ultrasound (POCUS) for senior faculty learners**
+
+| **Tip** | **Description** |
+| --- | --- |
+| Develop a hook | As with most learners, engagement is made much more likely if senior faculty have vested interest in learning the material. Utilizing elements of lifelong learning, patient safety and the rising numbers of POCUS-savvy house officers are powerful motivators. |
+| Use callbacks | Reference prior images, scanning zones or probe handling techniques through spaced learning and delayed recall. If possible, attempt to have the learners recall similarities or homologous concepts from other scanned areas. This will allow learners to revisit information and create cognitive connections that strengthen learning. |
+| Teach the “why” | As opposed to junior learners, senior faculty are more likely to expect their instructors to know why something is the way it is. This may result in longer time to teach concepts and require instructors to have a strong grasp of abstract topics (e.g. mirror image artifact) though it may possibly lead to stronger associations and better learning. |
+| Leverage existing knowledge | Whenever possible compare the new skill (POCUS) to skills which the senior learner has already mastered such as aspects of the physical exam with direct relevance such as the jugular venous pressure, S3s, fluid waves or egophony and sonographic findings of JVD, IVC plethora, left ventricular dysfunction and ascites. |
+| Maximize hands-on practice | Senior learners are likely to grasp concepts quickly however may have the most issue with kinesthetic skills and learning of a procedural task. Additionally, the inherent spatial considerations of scanning zones and POCUS images may be difficult for some senior learners. Spend the most time possible during introductory stages with hands on a probe. |
+
+## Conclusion
+
+The popularization of POCUS amongst hospitalists, coupled with the rising rates of ultrasound education at the undergraduate medical education level will likely lead to a large need in training of sizeable numbers of faculty to keep up with the new skills of their learners. We have found that training academic hospitalists in POCUS is feasible and well-received even by senior faculty, although this worthwhile effort requires planning and support.
+
+## Conflicts of Interest
+
+RKD served on the board of governors of the American Institute of Ultrasound in Medicine until 7/2020. She has been the Chief Medical Officer at Vave Health since 7/2020, however the bolus training and her involvement in this research had concluded by then. CMS received consulting fees from the Global Ultrasound Institute. DR and TFH have no conflicts of interest to declare.

fulltext/md/PMC10168294.md

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+---
+pmcid: "PMC10168294"
+pmid: "37180067"
+doi: "10.3389/fvets.2023.1176772"
+title: "Induced pluripotent stem cells in companion animals: how can we move the field forward?"
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "review-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Induced pluripotent stem cells in companion animals: how can we move the field forward?
+
+## Abstract
+
+Following a one medicine approach, the development of regenerative therapies for human patients leads to innovative treatments for animals, while pre-clinical studies on animals provide knowledge to advance human medicine. Among many different biological products under investigation, stem cells are among the most prominent. Mesenchymal stromal cells (MSCs) are extensively investigated, but they present challenges such as senescence and limited differentiation ability. Embryonic stem cells (ESCs) are pluripotent cells with a virtually unlimited capacity for self-renewal and differentiation, but the use of embryos carries ethical concerns. Induced pluripotent stem cells (iPSCs) can overcome all of these limitations, as they closely resemble ESCs but are derived from adult cells by reprogramming in the laboratory using pluripotency-associated transcription factors. iPSCs hold great potential for applications in therapy, disease modeling, drug screening, and even species preservation strategies. However, iPSC technology is less developed in veterinary species compared to human. This review attempts to address the specific challenges associated with generating and applying iPSCs from companion animals. Firstly, we discuss strategies for the preparation of iPSCs in veterinary species and secondly, we address the potential for different applications of iPSCs in companion animals. Our aim is to provide an overview on the state of the art of iPSCs in companion animals, focusing on equine, canine, and feline species, as well as to identify which aspects need further optimization and, where possible, to provide guidance on future advancements. Following a “step-by-step” approach, we cover the generation of iPSCs in companion animals from the selection of somatic cells and the reprogramming strategies, to the expansion and characterization of iPSCs. Subsequently, we revise the current applications of iPSCs in companion animals, identify the main hurdles, and propose future paths to move the field forward. Transferring the knowledge gained from human iPSCs can increase our understanding in the biology of pluripotent cells in animals, but it is critical to further investigate the differences among species to develop specific approaches for animal iPSCs. This is key for significantly advancing iPSC application in veterinary medicine, which at the same time will also allow gaining pre-clinical knowledge transferable to human medicine.
+
+1.
+
+## Introduction
+
+Veterinary regenerative medicine is a multidisciplinary field with a focus on developing innovative treatments for animal patients. Structural and functional healing of injured tissues and organs can be achieved by using either cells alone, cells combined with tissue engineered constructs or by delivery of the secretome without cells. These strategies involve different therapeutic mechanisms of action with the ultimate objective of enhanced treatment for diseases of veterinary interest (1–5). The advancement of the field is strongly influenced by the One Health—One Medicine approach. By adopting a panoramic view of the challenges, and exploiting the emerging positive outcomes in human and veterinary regenerative medicine, both may advance synergistically. Simply put, valuable insights can be translated in both directions to accelerate translation (6). For example, clinical experience with veterinary species can provide pre-clinical knowledge on safety and efficacy for human application, potentially reducing or eliminating the need for laboratory animals (7, 8). Therefore, a robust and inclusive One Health approach has exceptional value in human and veterinary medicine.
+
+Among different cell types currently being explored for regenerative purposes, mesenchymal stromal cells (MSCs) isolated from multiple species and tissue sources are extensively studied for a wide array of veterinary applications, mostly due to their cell regulatory abilities (9–11). The relative ease in tissue collection, processing and *in vitro* culture has made MSCs an attractive therapeutic option. However, *in vitro* expansion of these cells is limited and extensive passaging may lead to cell senescence. In addition, the differentiation potential of MSCs is limited to mesodermal cell lineages (12–14). Embryonic stem cells (ESCs) can overcome these obstacles as they are pluripotent cells with a virtually unlimited capacity for self-renewal, and can provide a constant source of cells in terms of number and types. However, obtaining ESCs requires the use of embryos, with associated ethical concerns (15). In 2006, Takahashi and Yamanaka reported for the first time an alternative type of stem cell that could overcome the limitations of both adult and embryonic stem cells: the induced pluripotent stem cells (iPSCs). These cells are not naturally occurring but produced in the laboratory: adult somatic cells, such as dermal fibroblasts, can be reprogrammed into pluripotent cells by inducing the expression of four pluripotency-associated transcription factors (Oct4, Sox2, c-Myc, and Klf4, also known as Yamanaka factors). Theoretically, any cells of the body can be transformed into pluripotent cells without the disadvantages associated with ESCs (16). Therefore, the discovery of iPSCs has revolutionized regenerative medicine, not only because of their therapeutic potential but also because of their usefulness for disease modeling, drug screening, and even species preservation strategies (17).
+
+Reprogramming somatic cells into iPSCs involves a global reset of the mature epigenome of the somatic cell, in order to go from its differentiated state back to a pluripotent one. To do that, the endogenous pluripotency-associated genes have to be re-activated while the somatic genes, associated with the specialized function of the cell, need to be repressed. These changes are initiated by inducing the ectopic expression of transcription factors Oct4, Sox2, c-Myc, and Klf4, which are proteins able to interact with the DNA to control gene expression (16, 18). These factors can be delivered to the cell by different methods that may involve or not viral vectors, and may result or not in the integration of transgenes in the genome of the cells (19). Even though the process of delivering the Yamanaka factors may seem relatively simple, complex epigenomic remodeling needs to take place (18). In order to succeed in this process, it is important to consider all the stages of reprogramming, starting from the selection of the somatic cells (20), the choice of the factors and the method to deliver them (19, 21), as well as the signaling pathways that can be regulated by adding growth factors or small molecules to the media (22, 23). Once the cells are reprogrammed, culture conditions need to be optimized to expand iPSCs while maintaining a pluripotent state. Of note, the complexity of the process may result in a mixture of cells at different stages of the reprogramming, thus making it critical a throughout characterization to confirm their identity as iPSCs (24). Therefore, there are many factors that can impact the resulting iPSCs and their subsequent applications. Therapeutic application of iPSCs is a major goal to pursue, however, the complexity of these cells and the genetic changes that they undergo during reprogramming have raised concerns that need to be addressed to move this application forward. Tumorigenicity and immunogenicity are among the main concerns, for which a number of strategies are being developed, from differentiation of iPSCs into specialized cells to the creation of haplobanks (25, 26). While the development of therapeutic applications moves forward, a number of other applications for iPSCs have emerged, being disease modeling one of the most relevant. Generating patient-specific iPSCs allows the subsequent derivation of specialized cells with specific genetic signatures or alterations that otherwise would be extremely complex to obtain primarily from tissues (27). *In vitro* disease modeling can help reducing the need of *in vivo* models and allows involving the species of interest since the pre-clinical phase of drug development. Basic research to unveil mechanisms of disease and physiology, as well as to conduct toxicological studies and drug screening can also be greatly facilitated by iPSCs (28).
+
+Although iPSC technology is still a young field needing intensive work, nonetheless important advancement has been made in the human side (29). However, the veterinary iPSC field is significantly less developed and with much fewer publications. In fact, the first reports on canine iPSCs emerged in 2010 (30), equine in 2011 (31), and felids in 2012 (32). The majority of studies in veterinary species have focused on the generation of iPSCs and/or on their *in vitro* use, mostly to derive cell types relevant for clinical or disease modeling applications, while only very few works have pursued an *in vivo* application. The knowledge on human iPSCs can greatly contribute to advance the veterinary side, as most of the interests and challenges are shared between human and veterinary medicine. However, we also need to increase our understanding on the differences among species (33). Pluripotency networks, epigenomic landscape and identity of the iPSCs, and their derivatives need to be addressed from a comparative perspective rather than directly extrapolating from the human side. Advancing the field of veterinary iPSCs is important to improve the standard point-of-care of companion animals as patients, but also because of their potential as translational models. For instance, dogs suffer several spontaneous diseases with similar pathophysiology and incidence than in humans, like diabetes, epilepsy, or various types of cancers (34). Furthermore, dogs and humans also share the genetic basis of some diseases affecting the cardiovascular, neuromuscular, or immunological systems, thus creating a unique landscape for iPSC-based research (35). The domestic cat can serve as a natural animal model of Alzheimer’s disease (36) or hypertrophic cardiomyopathy (37), and also suffers genetic diseases that affect people too, like retinal blindness or polycystic kidney disease (38). Horses have long been considered as one of the most suitable animal models to study musculoskeletal pathologies like tendon injuries or joint pathologies (39) and, more recently, they are also acknowledged as models for immune-mediated diseases and to study immune responses (40–42). Therefore, joining efforts would revert in mutual benefit for human and veterinary patients, but additionally, there are some animal-specific applications like species conservation for which iPSCs can be of great importance.
+
+The aim of this review is to provide an assessment of the current state of the art of iPSCs in companion animals, focusing on the equine, canine, and feline species because of their relevance as veterinary patients and their potential as animal models. Following a “step-by-step” approach, we firstly discuss the different stages in the generation of iPSCs from veterinary species (Figure 1) and secondly the development of different applications of iPSCs in companion animals (Figure 2). Our intention is to identify those technical aspects that need further optimization and to provide helpful guidance on future advancements.
+
+*Figure: Figure 1 Overview of the process for generating induced pluripotent stem cells (iPSCs) from companion animals. A “step-by-step” flow is presented identifying the stages of the process, along with the key considerations in each step and suggested potential strategies to address each point. OSKM: Oct-4, Sox-2, Klf-4, c-MYC; i.e., Yamanaka factors. LIF, leukemia inhibitory factor; bFGF, basic fibroblast growth factor. Created with Biorender.com.*
+
+*Figure: Figure 2 Applications of induced pluripotent stem cells (iPSCs) in companion animals for disease modeling and therapy. After reprogramming, iPSCs can be differentiated into specialized cells to recreate a “disease in a dish.” *In vitro* disease modeling can be used to better understand mechanisms of disease, physiology of the cells and to conduct drug screening, resulting in a more customized approach for therapy. Therapeutic application of iPSCs requires their differentiation into specialized cells or into intermediate progenitors like mesenchymal stromal cells (iMSCs). Gene editing can be used at this stage to obtain healthy cells from patients carrying genetic disorders. The iPSCs, iMSCs, and/or their derivatives can be manufactured and banked for therapeutic use. Created with Biorender.com.*
+
+2.
+
+## “Step-by-step” approach for generating iPSCs in companion animals
+
+2.1.
+
+### Selecting the tissue source
+
+To obtain iPSCs, the first step is to consider the type of somatic cells to be reprogrammed. Even though theoretically any cell can be induced into a pluripotent state, there is evidence that some cells are more easily reprogrammed than others. In addition, the invasiveness of the cell harvest procedure and the ease of culture should be taken in to account. Human iPSCs have been established from a wide range of tissue sources including dermal fibroblasts (43), peripheral blood mononuclear cells (PBMCs) (44), bone marrow derived MSCs (BM-MSCs) (45), and adipose derived stromal cells (ADSCs) (46). PBMCs are easily accessible while the other cell types require more invasive intervention. Indeed, waste or discarded tissues, such as foreskin fibroblasts (47), periodontal tissue (48), or renal cells from urine (49) have also been taken used. Umbilical cord blood banks are also a useful resource for iPSC reprogramming (50, 51).
+
+The differentiation status of the cell can influence the reprogramming efficiency, as the epigenomic state needs to be reset to a pluripotent state. For instance, hematopoietic stem and progenitor cells can be reprogrammed into iPSCs much more efficiently than terminally differentiated lymphocytes (52). In addition, the developmental age of the cells can affect their capacity to revert to an earlier state of pluripotency, as shown by the fact that embryonic and fetal tissues may be reprogrammed more efficiently than adult tissues (53). Furthermore, iPSCs are reported to retain epigenetic memory of the parent cell type (54, 55), which may influence the differentiation potential of iPSCs toward a desired lineage. For example, iPSCs derived from human cardiac-derived mesenchymal progenitor cells and pancreatic islet beta cells demonstrated enhanced differentiation toward the parent lineages compared to cells from other tissues (56, 57).
+
+In companion animals, adult and fetal fibroblasts have been the most commonly used to obtain iPSCs, but other cell types have been explored such as keratinocytes (58), MSCs from different tissue sources (adipose tissue, bone marrow, umbilical cord tissue, and peripheral blood) (59–61), myogenic mesoangioblasts (MAB) (60), tenocytes (62, 63), and PBMCs (64). Very few studies have directly compared the generation of iPSCs from different cell types in these species.
+
+In horses, Pessôa et al. (59) reported that the tissue of origin of the cell may significantly influence the capacity for reprogramming. Adult fibroblasts, umbilical cord tissue (UC)-MSCs, and adipose tissue (AT)-MSCs were successfully reprogrammed, with AT-MSCs showing the highest colony formation potential, whereas BM-MSCs did not produce iPSCs. In the same study, authors observed differential miRNA expression profile among iPSC lines, which may be the result of different responses to reprogramming. Direct comparison of iPSC generation from cells of adult and fetal origin has not been performed in horses. However, when comparing fibroblasts from young and old individuals, it was suggested that the derivation of equine iPSCs is not impaired by aging (65).
+
+Regarding canine iPSCs, it seems that adult cells are more “resistant” to reprogramming compared to fetal cells. Questa et al. (66) hypothesized that chromatin remodeling and accessibility were behind this resistance to reprogram since chromatin remodeling is required for the inactivation of somatic loci and activation of pluripotent ones. Therefore, these authors explored the implications of chromatin accessibility for canine somatic cell reprogramming by comparing different embryonic and adult cell types. The transduction efficiency was similar between adult and embryonic cells, however, only iPSCs from embryonic origin met pluripotent criteria whereas adult reprogrammed cells did not form stable colonies. Authors identified global patterns of chromatin openness, finding that iPSCs and embryonic fibroblasts shared substantially more features than iPSCs with adult cells. Actually, adult canine cells showed a region of closed chromatin that was open in embryonic cells and in which pluripotency associated genes are located. Findings were aligned with that expected during reprogramming and may explain why adult cells are more ‘resistant’ to reprogramming, which may help enhancing the process by targeting reprogramming barriers.
+
+The influence of epigenetic memory in the differentiation potential of iPSCs has not been deeply studied in companion animals, but it has been suggested that equine iPSCs retain some lineage commitment since those originated from MAB formed a higher quantity of muscle patches in teratomas, while iPSCs from PB-MSCs produced larger chondrogenic patches (60). The same group similarly showed that canine iPSCs derived from MAB had enhanced propensity to differentiate into skeletal muscle lineage compared to iPSCs derived from fibroblasts, which could be attributed to the DNA methylation pattern of MAB (67). Furthermore, authors also differentiated canine iPSCs from MAB and from fibroblasts into mesodermal progenitors (MiPS) and administered them in dystrophic mice, showing that the engraftment in the skeletal muscle was higher when MAB-MiPS were delivered compared to fibroblast-MiPS.
+
+Evidence in companion animals is still limited to suggest superior cell sources for iPSC reprogramming, but collectively with human evidence points at carefully considering this choice. Not all cell sources are equally suitable for reprogramming and iPSC properties can be impacted by their origin, so identifying the most suitable source for a particular application is of utmost importance. To do that, it is also important to unveil and understand the differences among distinct cell types in each species, particularly at the epigenomic level.
+
+2.2.
+
+### Reprogramming
+
+2.2.1.
+
+#### Inducing the expression of pluripotent factors
+
+Once the tissue source is selected, the choice of the pluripotent transcription factors and the method to deliver them to the cells should be carefully considered. Studies on companion animal iPSCs have mostly used human or murine factors for reprogramming, typically the four Yamanaka factors (OSKM) (16, 43). Of course, the mRNA and protein sequence homology of these transcription factors should be as high as possible. For example, in the horse, the homology is higher with human sequences than with mice (68) and, even though some works have generated equine iPSCs using murine factors (31, 69, 70), the studies that have directly compared both of them reported success only with human factors (59, 68). Studies comparing human and murine factors to generate canine iPSCs found that both were able to reprogram canine cells, but only human transgenes were silenced (71). To the best of our knowledge, only one early canine iPSC study was carried out using species-specific factors (30). Later studies used factors of human or murine origin. Based on what we understand to date, it is unclear whether species-specificity of reprograming factors is important for iPSC generation in companion animals (72). Interestingly, while the four OSKM factors are sufficient to reprogram equine and canine cells, it seems that the addition of NANOG is key in felids (32), including the domestic cat (73). Based on these considerations, it seems especially prudent to pay attention to the species origin of the factors, as well as the specific combination of factors used.
+
+Methods used to induce expression of the selected pluripotent factors may be classified as integrative/non-integrative and viral/non-viral. Human and murine iPSCs were originally generated using integrating retrovirus and lentivirus vectors to introduce the OSKM reprogramming factors (16, 43, 74). Genome-integrating methods may result in heterogeneous iPSC lines that are not suitable for clinical applications because the transgenes may become reactivated in iPSC derived cells, leading to a risk of tumor formation (21, 75). This is a limitation that will be further discussed in the Application section. Transgene free, non-integrating methods have been utilized to overcome these safety concerns, including Sendai-virus (76), episomal vectors (77, 78), and RNA based methods (79, 80). Furthermore, clinical-grade human iPSCs have been developed using modified mRNAs and non-integrating episomal vectors (45, 81). However, while significant advancement has been accomplished in the generation of human iPSCs by non-integrative methods, these strategies have been rarely applied in companion animals, with the majority of applications involving integrative viral methods (35, 82).
+
+In the generation of equine iPSCs, retroviral vectors were mainly used in early efforts and lentiviral vectors more recently. Specifically, the use of a STEMCCA cassette can increase the efficiency of reprogramming by delivering the four factors together and thus also reducing the number of integrations in the genome (83). Only a few studies in equine iPSCs report the use of non-viral methods. The Piggy-back transposon technique was indeed used in the first report on equine iPSCs (31), and the lines obtained were also used in later studies (84, 85). However, Moro et al. (86) compared the lentiviral and transposon systems and found that only the former was efficient at generating equine iPSCs from fetal fibroblasts. Transposon reprogramming allows more control of transgene expression by using excisable or inducible systems, but it is still an integrative method. Transgenes can also be excised if delivered by lentivirus, as done for example by Chauveau et al. to generate canine iPSCs (87). A relatively simpler way of controlling the expression of transgenes is by using an inducible promoter, however the transgene remains integrated (31, 70, 88). In general terms, these strategies can improve the safety profile of integrative methodologies, but integration still takes place with the potential risk associated with activation of unwanted genes such as those related to tumorigenicity.
+
+Exploration of transgene-free strategies has been more extensive in canine iPSCs, however direct extrapolation of the conditions used for human iPSCs does not seem to be straightforward. Baird et al. (61) used both retroviral and Sendai-virus based delivery in the same cells, but iPSC colonies appeared only with the former (61). Chow et al. also used the Sendai-virus system and reported that only a single colony was viable upon further passaging after colony picking (89). However, this was sufficient to establish a line that was used in this and subsequent studies (90). Similarly, Tobias et al. could not maintain stable canine iPSCs for longer than 26–30 days after reprogramming when using Sendai-virus (91). Tsukamoto et al. generated canine iPSCs with Sendai-virus that could be maintained for multiple passages, but only one line was obtained which failed to produce all three germ layers in teratoma assays in mice, suggesting that it might be a heterogeneous population (64). Later efforts by the same group showed that canine iPSC generation with Sendai-virus is possible but requires very specific conditions, including supplementation with a cocktail of small molecules. With this improved protocol, these authors were able to culture the generated canine iPSCs over 40 passages (92). Other non-integrative virus strategies have been tested for canine iPSC generation, such as the use of a vector based on the Venezuelan equine encephalitis RNA virus, which overall was not successful (93).
+
+Non-viral and non-integrative methods for pluripotent factors delivery have also been explored for canine iPSCs. Yoshimatsu et al. (94) reported on the use of episomal vectors delivered by electroporation. While colonies could be obtained with this method it was only when highly defined media was used, and still the reprogramming efficiency was very low (94). Chandrasekaran et al. also used episomal reprogramming with electroporation, but even though morphological changes were observed in the cells, complete reprograming was not achieved and lentivirus was used subsequently, resulting in generation of canine iPSCs from the same somatic cells (95).
+
+Although iPSCs generated with integrative methods can be effectively used for *in vitro* applications (basic research, disease modeling, drug screening, etc.) (96), the optimization of non-integrative methods is needed in the veterinary field to develop safer therapeutic applications. In spite of the efforts of several groups, we currently do not have a robust and widely used tool for transgene-free obtainment of iPSCs in companion animals. One possible reason may be that in companion animals iPSCs, the continuous expression of the exogenous transgenes may be required to maintain the pluripotency, as the endogenous networks might not be fully activated (33) thus significantly dampening the derivation of stable transgene-free lines. Therefore it is key to better understand such pluripotency networks in animals to provide the required conditions for iPSC generation.
+
+2.2.2.
+
+#### Culture conditions during reprogramming
+
+Following delivery of the pluripotent factors, appropriate culture conditions are needed to facilitate the changes in gene expression that allow the cell to alter its fate from somatic to pluripotent. This process can be facilitated by inhibitors of certain protein kinases, like glycogen synthase kinase 3 (GSK3), mitogen-activated protein kinase (MAPK), MAPK/extracellular signal-regulated kinase (MEK), Rho-associated kinase (ROCK), or activin-like kinase (22). Furthermore, histone acetylation facilitates the binding of transcription factors to DNA, so chemical inhibitors of histone deacetylase (HDAC) such as valproic acid, sodium butyrate, or ascorbic acid can increase chromatin accessibility and thus potentially improve reprogramming efficiency (23).
+
+Only a few equine iPSC studies have reported the use of kinase inhibitors (31, 97) or HDAC inhibitors (65) during reprogramming. However, these studies have not compared different combinations and neither have explored in detail the specific changes elicited by the inhibitors. In feline iPSCs, in spite of the limited number of studies published, the use of inhibitors has been reported (98). Optimization of iPSC generation has been further pursued in dogs. For instance, Moshref et al. (99) hypothesized that HDAC inhibitors would increase chromatin accessibility and facilitate reprogramming of adult canine cells. These authors found that neither valproic acid nor sodium butyrate effectively inhibited canine HDAC. On the other hand, panobinostat, another HDAC inhibitor, significantly increased histone acetylation and improved chromatin accessibility but without evidence of increased efficiency of generating iPSCs (99). Furthermore, Kimura et al. found that a cocktail of small molecules including some of the inhibitors mentioned (ROCK inhibitor, MEK inhibitor, GSK3b inhibitor, TGFβ antagonist, forskolin, and ascorbic acid) contributed to efficient generation of canine iPSCs (92).
+
+These findings certainly appear to indicate that each species has a unique epigenomic landscape that requires specific approaches, and this might help to explain the relatively limited success in obtaining iPSCs in companion animals. While human studies can provide a basis of knowledge, directly extrapolating the same protocols into other species would not be an optimal strategy. Thus, more studies in this direction are needed to understand the conditions required for reprogramming in each species.
+
+2.3.
+
+### Expansion of iPSCs in companion animals
+
+Once the cells are reprogrammed, the next stage is to maintain them in a pluripotent state and this generally requires the use of specific media containing selected growth factors and chemical components, as well as layers of feeder cells or matrix proteins (100, 101). Media composition for expansion of veterinary iPSCs has been reviewed elsewhere (35, 82), so this review will only focus on two aspects directly related to species-specific aspects and transferability.
+
+One of these is the use of either serum-containing or serum-free media, the latter being more suitable for therapeutic applications as it avoids potential xeno-contaminants and/or infectious diseases, as well as reduces batch-to-batch variation. Nevertheless, it should be noted that some serum-free media may still contain components of human or animal origin that are potential xeno-contaminants for veterinary species. Furthermore, it has been shown for animal MSCs that serum-free media developed for human cells may not work as well in veterinary species [reviewed by (102)]. For iPSCs, it seems that serum-free conditions may work better in equine species. Some papers have reported success in using fetal bovine serum (FBS), but studies directly comparing FBS vs. knockout serum replacement (KOSR) reported better results with the latter (69). In canine iPSCs, a majority of works have also used serum-free media [reviewed by (35)], however other studies suggest that media containing FBS result in higher colony formation compared to KOSR during reprogramming (91). On the other hand, reports in felid iPSC suggest that FBS-containing media are more advantageous (32, 73, 98, 103).
+
+A second important aspect for veterinary iPSC media composition relates to growth factor requirements. The dependence of iPSCs on either basic fibroblast growth factor (bFGF) or leukemia inhibitory factor (LIF) is related to the stage of the embryonic development that is mimicked by pluripotency induction. ESCs from the inner cell mass present a more naïve phenotype, with mounded colonies that are dependent on LIF. When ESCs are derived from the epiblast, a structure formed later during the development, these cells seem to be already primed, possibly representing a more restricted state of pluripotency, and are dependent on bFGF with colonies presenting a flat morphology (33). The majority of human iPSC lines resemble the primed phenotype (104), however there are mixed reports on the naïve/primed nature of iPSCs from companion animals (24). The scarce literature in felid iPSCs points at LIF-dependency (32, 73, 98, 103) while only bFGF-dependent (59, 60, 68, 71) and only LIF-dependent (70, 97) iPSC lines have been reported in both equine and canine species; however, the evidence so far points at a co-dependency on both factors in these two species (69, 105, 106). The reason for this co-dependency is not well understood. A study in canine iPSCs found that bFGF would act by inhibiting spontaneous differentiation toward ectoderm and mesoderm, while LIF activated the JAK-STAT3 pathway involved in pluripotency maintenance, but in a different manner than described in mouse ESCs (107). These studies collectively show that iPSCs from different species may present unique mechanisms for maintenance of pluripotency. Understanding such mechanisms is critical to provide the optimal conditions for expansion of iPSCs for different applications in the veterinary field. Moreover, LIF and bFGF used in animal studies are usually from human or murine origin. The use of species-specific factors has been suggested (108) but scarcely reported. Interestingly, only feline LIF, but not murine LIF, can maintain the pluripotent features of iPSCs in the domestic cat (73). Species-specific reagents usually present more limited availability (102), but may represent an important strategy to enhance pluripotency maintenance in these species.
+
+In addition to specific media composition, iPSCs require to grow onto layers of feeder cells, for which inactivated mouse embryonic fibroblasts (iMEF) are commonly used. This possess another concern when the application of interest is therapeutic: the presence of xeno-contaminants in the cell products, or even the risk of disease-transmission. A possible strategy to avoid xeno-contamination at this point is the use of feeder cells from the same species. For example, human iPSCs can be cultured onto neonatal foreskin fibroblasts with good results (109). Similarly, Nagy et al. used 1:1 iMEF and equine fetal fibroblasts (31), and Zhou et al. used cat fetal fibroblasts (98) to prepare feeder layers. As discussed above, fetal cells can be more easily reprogrammed, and the same cells could be used as feeders after inactivation. This strategy would be more time-consuming and less standardized than the purchase of batch-tested, ready-to-use iMEF, but it might be interesting to explore whether using species-specific feeder cells could better support iPSCs, in addition to avoid xeno-contamination.
+
+For the development of clinical grade hiPSCs, standardized and quality-controlled xeno-free and feeder-free culture products are commercially available including media such as Essential E8 (Gibco), mTeSR plus (Stemcell Technologies), StemFit (Ajinomoto) and NutriStem (Sartorius) (110–113), and matrix substrates that are more defined with less batch-to-batch variability, such as vitronectin (114), laminin-521 (115) or-511 (116), CellStart (117), and synthetic materials (118). The use of such systems is much more rapidly evolving for human iPSCs (100, 101), while most reports in companion animals rely on feeder cells and mostly on iMEF (24, 35, 82). Some attempts have been done to adapt the use of commercially available iPSC media and feeder-free substrates formulated for human iPSCs into companion animal cells. Such systems present the advantage of having a defined composition, being more stable and homogeneous, and providing serum-free, cell-free, or even xeno-free conditions. In horses, there are only brief mentions to the use of the StemFlex system (Thermofisher) to maintain equine iPSCs once the lines were established (62, 63). In cats, StemFlex media was used for reprogramming and expansion of feline iPSCs but supplementation with LIF and protein kinase inhibitors was needed (98). In dogs, Kimura et al. compared the suitability of different commercial media and feeder-free substrates and found that StemFit media (Ajinomoto) and iMatrix-511 (Nippi) provided the most suitable conditions for canine iPSC maintenance and large-scale expansion, and even LIF could be removed (108).
+
+In summary, similarly to that discussed for previous steps, there are not standardized culture systems for iPSCs from companion animals, being of great importance to develop serum-free and feeder-free options for clinical application. This would require fine-tuning of the conditions and a more in-depth understanding of animal iPSC requirements, along with increasing availability of species-specific reagents to truly avoid xeno-contamination.
+
+2.4.
+
+### iPSC characterization in companion animals
+
+Once iPSC putative lines are established, it is critical to confirm that these really are pluripotent cells. iPSCs are characterized at three levels: cellular (morphologically), molecular and functional. The cells should have a large nucleus and form compact colonies. They should endogenously express pluripotency markers at both gene and protein levels, and have the potential to spontaneously differentiate into the three embryonic germ layers [either *in vitro* via embryoid body (EB) formation assays, and/or *in vivo* via teratoma assay in immunocompromised mice]. Finally, iPSCs must have a stable karyotype as they can acquire chromosomal abnormalities after genetic reprogramming and long-term culture (43, 119, 120). Reports on the characteristics of iPSCs from companion animals at these three levels have been reviewed and compared by other authors (24, 35, 82). A detailed description is out of the scope of this review, but instead we are highlighting the main aspects to consider at each level of characterization.
+
+First, and as discussed in the previous section, at the cellular level it is not clear which type of colony morphology features each species of companion animals. Both naïve and primed-like morphology have been described, which also relates to the dependency of these cells on different growth factors. Thus, so far we do not have a strict criteria at this level to consider the cells as iPSCs in each species. This also adds complexity to the selection of colonies when these start emerging from reprogrammed cells. Colonies are picked individually mostly based on their morphology and ideally should be monoclonal, i.e., starting from a single reprogrammed cell. This process is challenging and labor intensive, and can lead to a heterogeneous selection of lines that are apparently similar but hold subtle phenotypic differences. Such differences may be very difficult to appreciate at the morphological level but could eventually result in different characterization profiles and varying differentiation capacity (58).
+
+Second, at the molecular level, different works have reported the expression at the gene and/or protein level of several pluripotent factors. It is important to note two points in this regard: the lack of a standardized panel of markers, and the relevance of ensuring the specificity of antibodies and primers used for characterization. Human and murine pluripotent cells have shown differential expression of certain markers (121), and this variability is also expected across veterinary species. To determine which pluripotent markers are expected in each species we could look at the expression pattern in ESCs. However, intra-species variability has also been noted for ESCs. For instance, in horses and dogs, the same marker has been reported as both positive and negative in ESCs of the same species [reviewed by (24)]. Furthermore, the limited number of ESC lines derived from companion animals makes it difficult to elucidate whether ESCs and iPSCs are truly equivalent and what developmental stage reflect in these species (24). An additional obstacle for the analysis of pluripotent markers in veterinary species is the complexity of finding antibodies which are either species-specific or presenting good cross-reactivity with the species of interest. Whereas the availability of suitable antibodies for veterinary species has substantially improved in the last years, it still can be difficult to find reliable antibodies for some specific markers. Moreover, the use of different clones for the same marker among different studies might contribute to the heterogeneity observed intra-species (24). The analysis of gene expression is also an important tool and designing primers specific for the species of interest is easier than developing antibodies. However, because of the high homology in the mRNA sequences between the human exogenous factors used for reprogramming and the endogenous genes activated in the cell (68), it is critical to ensure that the primers are only amplifying the target of interest.
+
+Finally, variable outcomes have been reported in the different species when it comes to the functional pluripotency of iPSCs, i.e., their ability to differentiate into cells of the three germ layers. The *in vitro* EB formation assay has provided more consistent results, which have shown successful differentiation of iPSCs. However, *in vivo* formation of teratomas has not been observed in all the reports in companion animals, or only partial differentiation has been recorded (24, 82). A potential explanation for this would be an incomplete reprogramming of the cells into the pluripotent state, even though the other criteria are met.
+
+As aforementioned, different studies have used different methodologies and conditions for reprogramming and culture, and this lack of standardization could probably influence differences observed in the characterization of iPSCs in companion animals (24). Thus, it is imperative to advance in determining the pluripotent features representative of each species and in developing suitable methods to analyze them with confidence. Standardization of iPSC characterization in companion animals is key to develop robust applications, and is tightly influenced by a previous proper establishment of reprogramming strategies and maintenance conditions.
+
+3.
+
+## Applications of iPSCs in companion animals
+
+The use of iPSCs finds multiple applications allowing development of novel treatments in human and veterinary medicine. These applications range through various biomedical disciplines, including development of cell therapies, disease modeling and drug testing, and clinical application for untreatable diseases in both people and companion animals (35, 122, 123). In addition, as a characteristic application in animal species and important for the maintenance of biodiversity, iPSCs have been generated from critically endangered mammalian (32, 103, 124, 125) and avian species (126). These are important for the study of developmental and physiological species-specific processes, and as wildlife preservation efforts they are important for the conservation of genetic resources and maintenance of a healthy eco-system.
+
+When it comes to the applicability of iPSCs in companion animals, reports are scarcer than for their generation and mainly focus on therapeutics or disease modeling. Therapeutic use of iPSCs has been mainly proposed for musculoskeletal conditions in horses (60–63, 88, 127–129) and for neurological and cardiovascular conditions in dogs (67, 95, 130–133). In terms of disease modeling, equine iPSCs can also be used for neurological conditions (58, 134). In dogs and humans, there is an interest in modeling genetic diseases by deriving patient-specific iPSCs with genetic abnormalities, which poses a remarkable scenario for translational medicine (35). In cats, to the best of our knowledge, there are no reports on therapeutic or disease modeling approaches, being the bibliography in this species the most limited. However, interestingly, iPSCs have been proposed in wild feline species as strategy to preserve biodiversity (32, 103).
+
+The different applications of veterinary iPSCs clearly remain less developed than in human research, but hold a similarly high potential yet to be explored. Nevertheless, the challenges to accomplish safe therapeutic application are essentially the same in all species. Some of these limitations arise during the generation process, as has been exposed in the first part of this review, or are inherent to the nature of these cells. In this section, we will review the current state of the art of iPSC applications in companion animals and their main limitations (the present), and we will discuss potential strategies to address such challenges and to keep moving the field forward (the future).
+
+3.1.
+
+### Where are we: present of iPSC applications in companion animals
+
+3.1.1.
+
+#### iPSC-based cell therapy in companion animals
+
+The current clinical investigation of iPSC-derived cell therapies in companion animals is still limited as the field is in its initial stage. The rationale behind the interest on these cells is that they could act as direct replacement of diseased cells with healthy and functional cells able to re-establish the tissue homeostasis, which results in an approach closer to actual tissue regeneration than with adult stem cells. The published studies in veterinary species so far offer insights on proof of concepts and initial clinical evidence in low number of clinical cases. In equines, based on the interest in treating recurrent sport injuries, the majority of studies focused on generating iPSC-derived cell types clinically relevant for musculoskeletal and wound healing injuries. In this regard, equine iPSCs have been differentiated into several cell types including osteoblasts (129), chondrocytes (60), tenocytes (62, 63, 127), myocytes (128), and keratinocytes (85). In spite of the *in vitro* evidence of iPSC differentiation potential, the functionality of the obtained cells has only been demonstrated to certain extents and would need to be further tested in the clinical setting. Similarly, the transplantation of functional neuronal cells derived from iPSCs would be of great benefit in the specific context of the nervous system, characterized by very limited regenerative abilities and accompanied by the incidence of neuropathies and traumatic spinal cord injuries in canine and equine patients. Based on this, equine functional motor neurons have been generated (58) and canine iPSC-derived neuronal progenitors have been tested for the treatment of traumatic spinal cord injury in three canine patients. While adverse effects were not noted, neither clinical improvement nor tissue remodeling were observed up to a 1-year follow-up (131).
+
+Thus, besides *in vitro* evidence of iPSC differentiation, optimal clinical use of iPSC-derived cells requires demonstration of cell engraftment and functionality *in vivo*. A proof of concept study using autologous canine iPSCs showed successful myocardial delivery in healthy dogs as monitored by non-invasive imaging techniques. Additionally, these canine iPSCs were differentiated into endothelial cells and administered *in vivo* into two murine models: hind limb ischemia and myocardial injury, in both of which these cells suggested efficient functionality (130). Similarly, canine iPSC-derived mesodermal progenitors showed engraftment and functional improvement in a murine model of cardiac and skeletal muscle, with no off-target tissue formation (67). A murine model of muscle injury was also used to test equine iPSC-derived myofibers, which showed engraftment and histological improvement, but the regeneration was not complete (70).
+
+An alternative option for delivering therapeutic cells is by generating MSCs from iPSCs (iMSCs) as an intermediate cell type that can be further used for cell therapy development with a major interest in musculoskeletal applications in companion animals. Canine iMSCs have been successfully differentiated into osteogenic and chondrogeneic lineage (90, 135). Furthermore, from a functional perspective, canine iMSCs show an immunomodulatory capacity similar to primary MSCs derived from adipose tissue and bone marrow, with similar gene expression profiles, effects on the proliferation of T cells, maturation of dendritic cells and response to priming with pro-inflammatory cytokines (89, 136). Furthermore, canine iMSCs IV injected in three healthy dogs did not produce adverse events in the short term, neither tumor formation was observed up to 15 months of monitoring (89). Equine iMSCs have also been tested *in vivo* for the treatment of a variety of naturally-occurring musculoskeletal injuries in equine patients, showing overall positive effects with absence of serious side effects (88). The heterogeneity of conditions included in this study and the lack of a control group hamper extracting definitive conclusions, but the results are valuable as a proof of concept for the therapeutic potential of iMSCs in horses.
+
+3.1.2.
+
+#### iPSC-based disease modeling and drug screening in companion animals
+
+Efficient prevention and treatment of diseases requires advanced knowledge of the altered genes and pathways responsible for the diseased phenotype of interest. To better understand the molecular background and establish the appropriate treatment, iPSCs can be exploited as *in vitro* models of disease. Furthermore, the defect of interest can be induced and subsequently multiple compounds of interest can be tested to identify a candidate drug with higher therapeutic efficiency (24, 27). In addition, when it comes to genetic disorders, genome editing tools like CRISPR-Cas9 technology can be used to correct the mutation and generate isogenic iPSC lines as controls. This allows accounting for the influence of the genetic background, since the isogenic line only differs from the original one in the disease-causing mutation (137).
+
+Compared to current advances in the use of human iPSCs in disease modeling and drug screening, there is scarce published literature in companion animals and is mostly limited to neurological disorders. In canines, an iPSCs line was generated from a West Highland White Terrier affected by mild cognitive impairment, showing an important proof of concept on successfully generating iPSCs from a geriatric patient (95), while on the equine side iPSC-based *in vitro* models have been generated to study the process of neurotropic viral infections (134). Although limited, these studies constitute relevant milestones for generating efficient *in vitro* modeling systems that will not depend on limitations of adult somatic cells and can eventually lead to a personalized/customized medicine approach where the most efficient treatment will be made available in a patient-centered approach.
+
+3.1.3.
+
+#### Limitations for iPSC applications in companion animals
+
+The iPSC field comes with as many promises as challenges, the latter being even more present in veterinary medicine. Potential applications of iPSCs are almost endless for therapy and research, however unleashing all of this potential requires overcoming several limitations, owed to the complexity and particularities of these cells. The process of generating iPSCs in companion animals faces several challenges itself, as detailed in the first part of this review. Furthermore, once iPSCs are generated, their posterior use for *in vitro* or *in vivo* applications does not come without limitations. Some of these limitations directly arise from the generation stage, such as transgene expression or xenogeneic contamination already discussed, while other handicaps derive from the inherent characteristics of these cells.
+
+One of the key challenges, particularly for *in vivo* application, is the pluripotent nature of these cells. As aforementioned as part of the functional iPSC characterization, these cells have the potential to form benign tumors composed of multiple cell types, known as teratomas, if they are administered undifferentiated in immune-compromised recipients (138). This can pose significant health risks for the recipient and limit the use of iPSCs in medical or veterinary applications. Because of this, and as it will be discussed later, *in vivo* applications aim at using differentiated cells derived from iPSCs that have lost their pluripotency. Furthermore, iPSCs also possess a risk for malignant tumorigenesis. This risk is particularly concerning if integrative methods are used for reprogramming, which are so far the most commonly reported in veterinary species. The random integration of the transgenes into the genome of the cell can activate tumorigenic genes and, even if transgene expression is silenced, they are still present and can reactivate even after differentiation. This is particularly concerning for *in vivo* applications, but can also impact the outcome of *in vitro* research and applications if iPSCs become tumorigenic (25).
+
+Another consideration concerning the therapeutic use of the iPSCs is their immunogenicity. As it will be discussed later in the Banking section, the use of allogeneic cells presents several advantages, particularly in the case of iPSCs which obtainment is highly demanding. However, the immune system of the recipient may recognize and target allogeneic cells, thus affecting the effectiveness and safety of the therapy (139). Importantly, even if the iPSCs are autologous, i.e., derived from the own patient, they can still be rejected by the immune system. This autologous rejection may be related to different factors. iPSC-derived cells are often immature and thus express low levels of the major histocompatibility complex (MHC), which make them targets of natural killer cells. In addition, the genomic and epigenomic changes that the cell undergoes during reprogramming and subsequent *in vitro* expansion and differentiation may result in immunogenic triggers (26).
+
+Another hurdle for the therapeutic application of iPSC derivatives is the lack of proper function, where differentiated iPSCs may not function properly *in vivo*, especially if they are not fully matured or are not adequately integrated into the recipient tissue (140). This issue will be covered in the next section along with strategies to enhance applicability of iPSCs in companion animals. Finally, the use of iPSCs in medical applications is also highly regulated and requires regulatory approval, which can significantly slow the development and commercialization of iPSC-based therapies. Furthermore, the development and production of iPSCs is still a relatively new, complex and expensive field, limiting the accessibility of iPSC-based therapies for many patients (96, 141, 142).
+
+3.2.
+
+### Where are we going: future of iPSC applications in companion animals
+
+3.2.1.
+
+#### Differentiation of iPSCs into specific cell types
+
+Most iPSC applications require their differentiation into the desired cell type, either if they are used for therapy, for disease modeling or for drug screening. Differentiating the iPSCs substantially reduces the potential risks associated with teratoma formation and facilitates the regulatory approval process. Additionally, these differentiated cells are more mature and functional, which can increase their effectiveness and reduce their immunogenicity in therapeutic applications. Directed differentiation methods have made significant progress in recent years in the human side, allowing for the efficient and specific differentiation of iPSCs into a variety of cell types, including neurons (143), cardiomyocytes (144), and hematopoietic cells (145), among others. Therefore, direct differentiation of iPSCs represents a promising alternative for the development of new therapies and *in vitro* applications, holding the potential to significantly impact the field of regenerative medicine. However, directed iPSC differentiation presents several obstacles. To begin with, the process of inducing iPSCs to differentiate into specific cell types can be inefficient, with high variability, low specificity and poor reproducibility, as well as constituting a time-consuming and costly process (146). Furthermore, the differentiated cells may be heterogeneous and result in a population with varying degrees of differentiation and functional activity, and the presence of residual undifferentiated iPSCs can compromise the purity of the differentiated cell population (147). Overall, the lack of control over the differentiation process and the variability of iPSC lines pose significant challenges to their practical use.
+
+To overcome the challenges in inducing differentiation of iPSCs, researchers are actively pursuing several strategies to improve the efficiency, specificity, and reproducibility of the process. The optimization of differentiation protocols is a crucial aspect of this research, as it involves refining the methods and conditions used to induce iPSC differentiation. This can involve adjusting the presence of specific growth factors that can influence the differentiation process or applying engineering-derived approaches to promote iPSC differentiation by mimicking the extracellular matrix (148). Directed differentiation is a strategy that involves directing iPSCs toward specific cell types using signaling pathways and small molecule inhibitors. This can help to increase the specificity of the differentiation process and reduce the formation of unwanted cell types, but requires deep knowledge on the embryonic development of the target cells to mimic the corresponding pathways (149), which is often complex and particularly in veterinary species. Reporter lines and cell sorting methods to identify and purify the cell population of interest is frequently used in human iPSC differentiation (150, 151). Including this approach into the strategies for veterinary iPSC differentiation would require further characterization of animal markers and species-specific antibodies to correctly identify the cells of interest. Another strategy is choosing iPSC lines that have a high propensity for differentiation into specific cell types, as has been described in the first part of the review (55). Finally, quality control measures are also essential for ensuring the purity of differentiated cell populations and minimizing contamination with residual undifferentiated iPSCs. This can be accomplished using molecular markers and other techniques that can help to distinguish between different cell types (119). Importantly, checking the identity of the obtained cells possesses its own challenges, as not all cell types exhibit a well-defined and stable pattern of markers, so several tests may be needed possibly including functional ones. Therefore, combining different strategies in a multi-faceted approach can help to address the challenges in the iPSC differentiation process from multiple angles, each one constituting a unique opportunity for research and development.
+
+3.2.1.1.
+
+##### iPSC-derived cells in companion animals
+
+As commented above, different cell types have been derived from iPSCs in the equine and canine species, but not all of them have shown functional properties *in vitro* or *in vivo*. For instance, equine iPSCs have been differentiated *in vitro* into neurons, keratinocytes, myocytes, tenocytes, osteoblasts and chondrocytes. However, only neurons and myocytes have shown functional properties such as depolarization and contraction (128, 134). Equine tenocytes are apparently challenging to obtain (127) but mechanical loading can improve differentiation (63), and osteoblasts derivation can be promoted in 3D scaffolds that would also facilitate clinical application (129). Obtainment of chondrocytes from equine iPSCs has been limited and non-conclusive. Equine chondrocytes were obtained during spontaneous differentiation of iPSCs (60), but its derivation using an intermediate MSC stage has shown mixed results (60, 88, 152). More progress has been reported in the derivation of functional specific cells from canine iPSCs, including mature megakaryocytes able to release functional platelets (153).
+
+Regarding all the considerations that have been discussed for iPSC differentiation, in companion animals it should be emphasized the need of further research into their embryological development to fine-tune differentiation protocols adapted to the particularities of each species, as well as on characterization of the obtained cells thus requiring species-specific reagents. Furthermore, the approach to generate iPSCs can later influence their differentiation potential, not only because of the epigenetic memory of the cell, but also because the permanent expression of transgenes may interfere with the differentiation process, which needs pluripotency silencing (154).
+
+3.2.1.2.
+
+##### Derivation of MSCs from iPSCs
+
+Provided the complexity of deriving specific types of cells from iPSCs and the limitations of directly using undifferentiated iPSCs, an intermediate approach has been proposed: the derivation of MSCs from iPSCs, known as iMSCs. While this might look as a considerable round about, the use of iMSCs has several notable advantages over primary MSCs. In contrast to primary MSCs that are commonly obtained from more invasive sources like bone marrow or adipose tissue and require large quantities of tissue for isolation, iPSCs can be generated from small numbers of cells obtained from less invasive sources such as skin or blood. Furthermore, while primary MSCs are subjected to considerable variability among tissue sources, iMSCs can be derived from iPSC lines coming from single cell colonies, thus substantially increasing the homogeneity of the cell population and facilitating standardization of the cell product. In addition, primary MSCs do not possess a limitless self-renewal potential and enter senescence after some time expanding *ex vivo*. This requires collecting tissue again for MSC isolation and results in a new population of cells, even if the same donor is used. On the other hand, iMSCs can be derived from the same iPSC clonal line multiple times, and both iPSCs and iMSCs can be easily cryobanked for later use. In summary, iMSCs could be used for large scale production of homogeneous population of cells leading to phenotypical, molecular and biological stability that ultimately is needed for an ideal off-the-shelf product for therapeutic use (155, 156).
+
+Despite of the advantages, the development of efficient and scalable methods for generating high-quality iMSCs remains a challenge in need of further investigation. Of note, MSCs found in adult tissues do not have all the same embryological origin. Most MSCs derive from the mesoderm, but some of them come from the paraxial mesoderm while others generate in the lateral plate mesoderm, or even in the extraembryonic mesoderm (157). Furthermore, the origin of some MSC populations has been traced back to neural crest cells generated in the ectoderm (158). These observations are important to understand the natural heterogeneity of MSCs, as well as to define strategies to derive them *in vitro* from iPSCs. General approaches for iMSC generation have been reviewed elsewhere (159) and are beyond the scope of this review, however it is worth highlighting that the derivation methodology can influence the properties of the resulting iMSCs. While main characterization features seem to be preserved, the functional properties of iMSCs may differ from their natural counterparts and among derivation strategies (160). This could constitute a limitation, but also an opportunity to direct iMSCs toward specific characteristics depending on the intended use.
+
+Mesenchymal stromal cells from iPSCs have been generated in horses and dogs to obtain multipotent progenitor cells readily available for therapeutic use. Lepage et al. (152) used equine fetal fibroblasts to generate equine iPSCs lines that were subsequently differentiated into iMSCs. These displayed a typical fibroblast morphology, testing positive for CD29, CD44, and CD90 surface markers, and when tested for tri-lineage differentiation were able to differentiate into osteogenic and adipogenic lineage while failing to achieve chondrogenesis in 3D pellet culture (152). Similarly, Chung et al. (88) generated equine iMSCs by serial passaging of iPSCs in MSC-defined media which were characterized as CD29 and CD44 positive and were able to differentiate successfully into the chondrogeneic, osteogenic, and myogenic lineages (88). On the canine side, iMSCs have been generated by inhibiting the TGF-β/Activin pathway in MSC-defined media following serial passaging. Once generated, the canine iMSCs expressed CD73, CD105, STRO1+ and CD24 (135), with variable expression of CD90 and CD44, and did not express the pluripotency marker Oct3/4 as well as the negative surface markers CD45 and CD34 (89). All the canine iMSC lines generated in these studies showed tri-lineage differentiation *in vitro* and, interestingly, when canine iMSCs were compared with BM-MSCs, there was evidence of different time and concentration-dependent effect of dexamethasone and BMP-2 on the onset of osteogenesis, which needs to be taken into consideration for the generation of clinically relevant cells (90). The risk of uncontrolled *in vivo* differentiation would not be an issue since injected iMSCs locally in immunocompromised mice and systemically in healthy Beagle dogs did not form any teratomas or abnormal tissues showing potential for therapeutic safety (89). Although limited, the number of published studies shows the feasibility to generate successfully iMSCs in companion animals.
+
+In addition to the interest on iMSCs for therapy, in human medicine patient-specific iMSCs have been exploited significantly as platforms for drug screening and toxicity for multiple conditions affecting mesenchymal lineages such as osteogenesis imperfecta (161), Fanconi anemia (162), fibrodysplasia ossificans progressiva (163), and Hutchinson-Gilford progeria syndrome (164). Based on this, future studies are needed in companion animals to assess efficiently the quality and optimize the large-scale production of iMSCs-based therapeutics and research platforms.
+
+3.2.2.
+
+#### Master banks of iPSCs for veterinary applications
+
+Genetic mutations that cause the disease can be present in the starting cells, which could be transmitted to the newly generated iPSCs. While this is valuable for disease modeling applications by generating patient-specific iPSCs, it turns out to be a barrier for therapeutic use of autologous iPSCs. Furthermore, the quality of somatic cells to generate autologous iPSCs can be diminished by such genetic diseases or by aging, leading to a reduced yield of functional iPSCs and increasing the risk of rejection. This, in turn, can prolong treatment timelines and negatively affect therapeutic outcomes (165, 166). Moreover, the complexity and cost of generating iPSCs make it currently unpractical to produce these cells from the own patient, added to the prolonged time required to obtain, expand and characterize the iPSCs significantly delaying the treatment (167).
+
+To overcome these limitations, allogeneic therapy has been proposed as a more feasible alternative. iPSCs can be generated from a healthy donor and then used to treat multiple patients. This eliminates the need for individualized cell sourcing, reducing the time and costs associated with autologous therapies. Additionally, allogeneic iPSCs can be characterized in detail prior to banking them to ensure their identity and quality, which otherwise would add significant further delay to the autologous treatment (167). Stem cell banks increase the availability and ensures the quality of the cell products, while reducing the time to administer the therapy (168). Not only iPSCs can be banked, but also their derivatives including iMSCs (169) and some types of differentiated cells (170), as well as their secretomes (171). However, it is important to note that allogeneic therapy also has its own limitations, highlighting the risk of immunological rejection (167). Even though the immune responses generated against allogeneic iPSCs and their derivatives requires further investigation, various strategies are being developed to overcome this potential hurdle. The use of immunosuppressive drugs and the genetic engineering of cells to reduce their immunogenic potential have been suggested. These approaches may be effective but also raise several concerns, such as drug side effects or further manipulation of the cell’s genome (172). Therefore, the focus could be put on the selection of donors.
+
+Haplobanks have been proposed as a solution to provide a more widely available source of allogeneic iPSCs. The underlying idea is to select donors who are homozygous for the most common haplotypes for the MHC. While the genetic diversity of MHC haplotypes is high, some haplotypes are more common within a population. Identifying which haplotypes are more prevalent and banking iPSCs from healthy donors carrying such MHC types can allow providing MHC-matched cell products to a considerable part of the population (173, 174). This way, by using a limited number of selected donors, haplobanks can reduce the genetic diversity of the iPSCs and limit the risk of immunological rejection. Furthermore, the iPSCs stored in haplobanks can be differentiated into various cell types, which can then be used for transplantation or *in vitro* disease modeling (175). For instance, a clinical-grade iPSC haplobank in Japan has been established from seven donors and can provide HLA-matched iPSCs for approximately 40% of the Japanese population. This haplobank was released in 2015 and since then has provided iPSCs for over 10 clinical trials (176). This strategy could be transferred the veterinary field owed the growing knowledge on MHC haplotype diversity in different breeds of companion animals (177–180). To the best of our knowledge, iPSC haplobanks for companion animals are not yet a reality, but there are initiatives to create haplobanks for veterinary MSCs. The impact of MHC matching in MSC therapy in veterinary patients is being increasingly acknowledged (181–184), and the most common MHC haplotypes have been defined in several equine populations (177, 178). Following this path, the same concept could be implemented for animal iPSCs in the coming years.
+
+3.2.3.
+
+#### iPSC-based cell-free therapy
+
+Another alternative to the limitations posed by iPSCs for their clinical application is the utilization of extracellular vesicles (EVs) or the entire secretome obtained from iPSCs, or from their derivative cells such as iMSCs or other cell types (185). It has been proposed that cells mainly communicate through their secretome, which consists of either packed or free components. The packed secretome, also known as EVs, are nano-sized sacs produced by a wide range of cell types, including different stem cells with therapeutic potential. EV-based therapy has gained substantial attention in recent years due to their benefits over traditional cell therapy, as it allows a cell-free modality that overcomes concerns related to immunogenicity and cell survival, and increases product standardization. The EVs comprise a diverse range of biologically active substances, including proteins, lipids, and nucleic acids, that can be delivered directly to target cells, leading to a desired therapeutic outcome (186). As intermediaries in cell therapy, EVs transmit information similar to their cells of origin. In comparison to cell therapy, where cells need to survive, migrate and differentiate for a therapeutic effect to occur, EVs can be more easily delivered to the site of injury because of their smaller size, either through direct injection or intravenous administration, without being recognized by the host immune cells. In the case of iPSC-based therapy, using their secretome also prevents additional concerns related to this specific type of cells, like their tumorigenicity (187).
+
+Induced pluripotent stem cell-derived EVs offer the possibility of personalized medicine by producing patient-specific EVs and can be engineered to contain specific drugs or components. Despite this is a relatively new field needing further work, iPSC-derived EVs have been investigated in human regenerative medicine for various diseases, including osteoarthritis, skin and auto-immune disorders (188–190), however their application in companion animals has yet to be fully explored. To date, only one study has been conducted in dogs, serving as canine model for using human iPSC-derived cell-free secretome to enhance post-pneumonectomy compensatory response. Human iPSC-derived secretome showed improved angiogenesis and alveolar remodeling leading to enhanced gas exchange after the pneumonectomy (191). This highlights the therapeutic potential of iPSC-derived secretome and EVs in human and veterinary patients, particularly due to their ease of administration. Nevertheless, further research is necessary to fully realize their potential and to scale-up and standardize the production.
+
+3.2.4.
+
+#### Manufacturing of iPSCs for veterinary applications
+
+The clinical adoption of robust and high quality iPSCs and their cell-derivatives requires a standardized and reliable cell Good Manufacturing Process (cGMP). Due to their ability to proliferate indefinitely, these cells can represent a true off-the-shelf product that can be manufactured in high number of identical doses from one cell line (192). To achieve this, the intrinsic challenges elaborated in details above for the generation of iPSCs need to be addressed in a large-scale-up context needed for cGMP. These aspects have not been assessed and published for companion animals, however, they have been implemented for human cGMP manufacturing and important considerations can be translated (112). For example, careful choice of the starting tissue source is fundamental and for large-scale manufacturing it would be ideal to use one less prone to chromosomal aberrations and epigenetic memory such as umbilical cord blood or peripheral blood due to sampling accessibility. The variability in cell reprogramming strategies represents a bottleneck and ideally, a process ensuring genomic and phenotypic stability would need to be implemented using non-integrating vectors or peptide-based delivery of transcription factors that can be easier to standardize for regulatory approvals. The laborious manual selection of iPSC colonies based on morphology would need to be automated in a high accuracy and robust process based potentially on micro-devices able to perform immunoselection for clinical grade sorting, and the *in vivo* teratoma assay could be replaced by a rapid qPCR throughput testing (193). The costs and standardization of cGMP manufacturing in general represent a limiting factor in veterinary medicine as also species-specific differences are a critical factor that needs to be taken into consideration. The differentiation toward an intermediate cell type such as iMSCs could be a beneficial step since guidelines for MSCs manufacturing in veterinary medicine have been published (102) and these can be easily translated into the process.
+
+4.
+
+## Discussion and conclusion
+
+The application of iPSCs in the veterinary field is clearly less developed than in the human side. While this is not surprising because this is a complex and relatively new field, it seems that iPSC advancement in companion animals substantially faces bigger challenges. When thinking about the whole process of developing an application using iPSCs for companion animals, one can realize that there are additional hurdles since the very beginning. The generation of iPSCs in dogs, horses or cats has proved to be less effective than in humans. While transgene-free methodologies for reprogramming are already customarily used to obtain human iPSCs, in the veterinary side the integrating virus methods remain the most common. Furthermore, even for this approach, there are no standardized protocols yet and different methodologies with variable outcomes are reported, plus the same approach does not always work depending on the cell type (59). Even when putative iPSCs are generated, their characterization can show mixed results suggesting that some lines may not be fully reprogrammed, as it is also pointed out by the permanent transgene expression detected in some works (24). Hurdles continue after iPSC lines are established and ready for application. The pluripotent nature of these cells carries higher risks of tumorigenesis than the use of adult stem cells, and immunogenicity can be a concern not only in the allogeneic scenario but also in autologous application (26), which may compromise the safety of the therapy. Furthermore, xeno-free systems need to be further developed in veterinary iPSCs to reduce the risk of xeno-antigens.
+
+Some of these limitations can be overcome by using cells already differentiated, derived from iPSCs. While theoretically iPSCs can be differentiated into any cell of the body, highly specific protocols are needed to provide the exactly required conditions, being the development of such protocols another field of great importance (194). A relatively easier alternative can be the derivation of iMSCs, which can greatly increase the availability, standardization and homogeneity of these cells compared to primary sources (160). Furthermore, manufacturing and cell banking strategies could be transferred from primary MSCs to iMSCs, or even to iPSCs and/or their derivatives like EVs (187). Creation of cell banks would facilitate the availability of different cells and cell products for several applications while reducing the time for treatment by using allogeneic cells. In this regard, the creation of haplo-banks to match donors and patients by their MHC has been proposed in human iPSCs (173) and is gaining consideration for veterinary MSCs, so this could also be transferred to veterinary iPSCs.
+
+Considering the gaps in the field of iPSCs in companion animals, we propose five main areas in which focus needs to be placed:
+
+First, we need to acknowledge and understand the differences between animal species and human. While extrapolating methodologies from the human side is common in veterinary research and certainly useful at initial stages, it is important to unveil the differences and work toward them. Better understanding on companion animal embryology is of utmost importance both to understand the pluripotency networks of ESCs and to direct the differentiation of iPSCs toward the desired cell lineage. Therefore, we need more basic research on this area and more work to transfer that knowledge into application in the iPSC generation, characterization and differentiation.
+
+Second, more basic research is also needed to characterize clinically relevant cell types in companion animals. Most iPSC applications are based on deriving cells, either for therapy or for *in vitro* research like disease modeling. We need not only to understand how to derive these cells, but also to develop the tools to ensure their identity and, importantly, their functionality.
+
+Third, even though it is crucial to firstly laying the foundations, it is equally important to start developing tools to keep building the field in the future. In this sense, parallel efforts are needed to establish approaches allowing implementation of iPSC applications in companion animals. Optimization and standardization of protocols for veterinary iPSC generation and characterization are very much needed, but in a later stage we will also need the tools to scale up the production of these cells and their derivatives in xeno-free conditions, using cGMP manufacturing and banking.
+
+Fourth and closely related to all of the above, another area in need of improvement is the production and validation of species-specific reagents, like growth factors, antibodies, or other molecules needed during the processes of generation, characterization, expansion, and differentiation of iPSCs in companion animals. While there are some commercially available products suitable for veterinary species, the increasing specialization requires further development of custom solutions. This constitutes an interesting opportunity of collaboration with industry, and is an integral part of the cell therapy field in veterinary medicine.
+
+Last, but not the least, promoting collaboration among researchers working in the veterinary iPSC field is key. Provided the many challenges we face, the best way to advance is to do it together by sharing expertise and resources, as well as experiences and failures. Some actions in this direction could include establishing a network of researchers and creating task forces, as well as considering the creation of bio-resources like cell banks, while seeking funding to support these actions. As in the fourth point above, the involvement of industry could also bring interesting opportunities to develop such networking and collaborations by taking advantage of industrial management skills and resources.
+
+In conclusion, we are in front of a field of great promise that can significantly contribute to developing new therapies for veterinary patients, but also to providing critical information for human medicine. The veterinary field can greatly benefit from the advancements in the human side, but we also need to appreciate the differences and conduct basic research, while getting ready for the application.
+
+## Author contributions
+
+LB and FB contributed to the conception and structure of the manuscript. LB, TE, AO’B, and AI contributed to the literature revision and manuscript writing. TE designed the figures. LB, TE, AO’B, AI, and FB critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.
+
+## Funding
+
+This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement no. 101026825.
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10171673.md

@@ -0,0 +1,193 @@
+---
+pmcid: "PMC10171673"
+pmid: "37163464"
+doi: "10.1371/journal.pone.0284570"
+title: "Metabolomics during canine pregnancy and lactation."
+journal: "PloS one"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Metabolomics during canine pregnancy and lactation
+
+## Abstract
+
+During pregnancy and parturition, female dogs have to cope with various challenges such as providing nutrients for the growth of the fetuses, hormonal changes, whelping, nursing, milk production, and uterine involution. Metabolomic research has been used to characterize the influence of several factors on metabolism such as inter- and intra-individual factors, feeding, aging, inter-breed differences, drug action, behavior, exercise, genetic factors, neuter status, and pathologic processes. Aim of this study was to identify metabolites showing specific changes in blood serum at the different phases of pregnancy and lactation. In total, 27 privately owned female dogs of 21 different breeds were sampled at six time points: during heat, in early, mid and late pregnancy, at the suspected peak of lactation and after weaning. A validated and highly automated canine-specific NMR metabolomics technology was utilized to quantitate 123 measurands. It was evaluated which metabolite concentrations showed significant changes between the different time points. Metabolites were then grouped into five clusters based on concentration patterns and biochemical relationships between the metabolites: high in mid-pregnancy, low in mid-pregnancy, high in late pregnancy, high in lactation, and low in lactation. Several metabolites such as albumin, glycoprotein acetyls, fatty acids, lipoproteins, glucose, and some amino acids show similar patterns during pregnancy and lactation as shown in humans. The patterns of some other parameters such as branched-chain amino acids, alanine and histidine seem to differ between these species. For most metabolites, it is yet unstudied whether the observed changes arise from modified resorption from the intestines, modified production, or metabolism in the maternal or fetal tissues. Hence, further species-specific metabolomic research may support a broader understanding of the physiological changes caused by pregnancy that are likely to be key for the normal fetal growth and development. Our findings provide a baseline of normal metabolic changes during healthy canine pregnancy and parturition. Combined with future metabolomics findings, they may help monitor vital functions of pre-, intra-, and post-partum bitches and may allow early detection of illness.
+
+## Introduction
+
+Pregnancy and parturition are challenging for mammals because they are characterized by considerable hormonal and metabolic changes [1, 2] to support fetal demands [3, 4]. During pregnancy the maternal body needs to supply appropriate nutrients for the conceptuses via the placentas and later the newborn puppies rely on lactation for several weeks [5]. These processes are governed by complex hormonal and neurological interactions and cause major alterations in the mothers metabolism [5], resulting in modified nutritional demands for both micro- and macronutrients [6, 7]. It has been claimed that even small deviations of the metabolism from the norm during pregnancy could have serious consequences for the mother and the offspring [8].
+
+In dogs, pregnancy-related changes in metabolic parameters might be even more intensive than in many other mammalian species because fetal development is rapid as pregnancy duration is, on average, only 63 days, and a bitch might carry a high number of conceptuses. Parturition usually lasts several hours, is laborious, and often exhausting [9]. Other parameters may change during pregnancy and parturition because of hormonal changes, uterine enlargement, pain, anxiety and uterine contractions [10]. In addition, the canine endotheliochorial placentation type leads to a longer and more intensive uterine involution process than in other species [11]. Finally, also tasks such as nursing the puppies, including milk production and change of diet, are challenging for the female dog [1].
+
+Understanding the physiology, endocrinology, and metabolic features during pregnancy, parturition, and lactation are required to understand and diagnose normal and abnormal conditions and provide optimal supportive management [1, 5]. In that regard, it is important to realize that some parameters change physiologically during pregnancy, parturition, and lactation [10]. However, it has been shown that inadequate supply of key metabolites can lead to abnormal fetal growth and impaired development of organs such as the heart [12] or nerval tissue [13].
+
+Metabolomics is an omics-based approach that generates comprehensive information about metabolites in blood serum, enabling an extensive view of the individual’s current state of systemic metabolism [14, 15]. Several metabolomics studies have been conducted in humans to address the physiological and pathophysiological metabolic changes during pregnancy and lactation, but no previous studies have been conducted in dogs. This study aimed to determine the metabolic changes occurring in dogs during pregnancy and lactation, to discuss their similarities or differences to the changes occurring in humans.
+
+## Materials and methods
+
+### Animals
+
+In total, 27 privately owned bitches of 21 different breeds were sampled during this study (Table 1). Female dogs were enrolled from March 2018 until December 2019. To be included, bitches had to be generally healthy within the past 6 months before mating and not receive medication, except deworming. In addition, dogs had to become pregnant and be presented for sampling at six time-points. Previous gynecological illnesses were inquired about, but none were present in any of the bitches at the time of sampling. Informed written consent and approval from the owners was obtained. The dogs were fed individual diets chosen by the owners. The blood sample collection was approved by the committee on animal welfare of the federal state Berlin (LAGeSo Berlin O 0095/18).
+
+**Table 1 Attributes of female dogs included in a study on metabolomics during canine pregnancy and lactation.**
+
+| Dog No | Breed | Bodyweight (kg) | Parity* | Age | Number of puppies born | Thereof stillborn puppies |
+| --- | --- | --- | --- | --- | --- | --- |
+| **1** | Dogue de Bordeaux | 51.3 | 4 | 7.0 | 2 | 0 |
+| **2** | Miniature Bull Terrier | 16.8 | 1 | 2.9 | 6** | 3 |
+| **3** | Rhodesian Ridgeback | 38.0 | 2 | 4.9 | 12 | 1 |
+| **4** | Saarloos Wolfhound | 28.8 | 2 | 6.2 | 5 | 0 |
+| **5** | Collie | 24.0 | 2 | 6.3 | 5 | 0 |
+| **6** | Borzoi | 36.4 | 1 | 8.1 | 8** | 0 |
+| **7** | Labrador Retriever | 22.4 | 1 | 2.6 | 9 | 0 |
+| **8** | Staffordshire Bull Terrier | 14.2 | 1 | 2.6 | 1 | 0 |
+| **9** | French Bulldog | 11.8 | 2 | 2.6 | 3 | 1 |
+| **10** | Schnauzer | 18.2 | 2 | 7.8 | 6 | 0 |
+| **11** | Olde English Bulldogge | 32.2 | 1 | 2.0 | 12 | 1 |
+| **12** | Miniature Bull Terrier | 19.0 | 2 | 4.7 | 7** | 0 |
+| **13** | Wirehaired Dachshund | 9.0 | 2 | 4.9 | 7 | 0 |
+| **14** | Boxer | 27.8 | 2 | 6.8 | 4 | 0 |
+| **15** | Hovawart | 41.6 | 2 | 6.7 | 9 | 1 |
+| **16** | Irish Wolfhound | 56.4 | 1 | 2.9 | 13 | 1 |
+| **17** | Golden Retriever | 22.8 | 1 | 4.4 | 5 | 0 |
+| **18** | Entlebucher Mountain Dog | 23.6 | 1 | 2.6 | 7 | 0 |
+| **19** | Golden Retriever | 33.0 | 1 | 4.6 | 6 | 0 |
+| **20** | Miniature Bull Terrier | 16.0 | 2 | 5.4 | 6 | 1 |
+| **21** | Eurasier | 20.8 | 2 | 3.8 | 5 | 0 |
+| **22** | Wirehaired Dachshund | 9.2 | 1 | 3.4 | 6 | 0 |
+| **23** | Kromfohrlaender | 10.4 | 1 | 2.9 | 7 | 1 |
+| **24** | English Cocker Spaniel | 11.2 | 2 | 2.9 | 8 | 0 |
+| **25** | Labrador Retriever | 28.2 | 2 | 5.3 | 6 | 0 |
+| **26** | English Cocker Spaniel | 11.2 | 2 | 2.9 | 6 | 0 |
+| **27** | Dobermann | 38.8 | 1 | 3.4 | 13 | 0 |
+|  | **Total (Average)** | **24.9** | **1.6** | **4.5** | **6.8** | **0.37** |
+
+Bitches were initially presented for ovulation timing. After they met the inclusion criteria, an initial clinical and gynecological examination was performed, including vaginoscopy and vaginal cytology.
+
+### Blood sampling and sample processing
+
+For this study, three stages of canine pregnancy were defined according to Hinderer et al. [16]. A total of six appointments for drawing blood samples were scheduled. The first sample was taken at a visit for ovulation timing in estrus (E). Three samplings during pregnancy (P) were appointed, i. e., between day 11 and 19 (P1) after mating, between day 23 and day 32 (P2), and between day 52 and 60 (P3). The P2 examination included an ultrasonic examination to assert whether the bitch was pregnant. This examination was not scheduled in the middle of the second third to respect the owners’ wishes of having a pregnancy diagnosis rather sooner than later while completing the study examination within the same appointment. A fifth sampling was performed 18 to 24 days after whelping. The last clinical examination (A) was performed 18 to 24 days after the weaning of the litter.
+
+All blood samples were collected in the morning or early afternoon.
+
+After parturition, data on the delivery (natural or cesarean section), the number and weight of puppies (alive and stillborn), and survival after three weeks were documented.
+
+Blood samples were collected by venipuncture from either the cephalic or the saphenous vein into plastic tubes (Sarstedt Tube 4.5 mL, Clotting Activator/Serum, 75 × 13 mm, Sarstedt AG & Co KG, Nümbrecht, Germany). Samples were left at room temperature for 30 to 60 min for clotting and then centrifuged at 5000 rpm or 2884 g for 5 min (Hettich Centrifuge EBA 20, Hettich, Tuttlingen, Germany). Serum was transferred into a serum tube (Simport Cryovial sterile with lip seal design, external threads, 2 mL Tubes, Boloeil QC, Canada), which was placed in a refrigerator at– 80°C until shipment. The storage temperature was checked daily, and variations of the measured temperature remained between -80°C and– 83°C. Storage duration at– 80°C of the samples ranged from 11 to 30 months.
+
+Only complete sample sets with sufficient serum and no haemolytic appearance were submitted for Nuclear magnetic resonance (NMR) analyses.
+
+For shipment preparation, the tubes were thawed for one to three hours, and then an amount of 0.3ml was transferred into a second serum tube (Simport Cryovial sterile with lip seal design, external threads, 2 mL tubes, Boloeil QC, Canada), both aliquots were again immediately frozen at– 80°C. Overnight shipment to PetBiomics, Finland, was performed in a Styrofoam box with plenty of dry ice.
+
+### Nuclear magnetic resonance (NMR) metabolomics analyses
+
+A validated and highly automated canine-specific NMR metabolomics technology was utilized for metabolomics analyses of the collected serum samples [14]. The method quantitates 123 measurands from various molecular groups, including a comprehensive lipoprotein analysis, fatty acids, triglycerides, cholesterols, amino acids, glycolysis- and fluid balance-related metabolites, and a novel inflammatory marker glycoprotein acetyls (GlycA) [14]. A similar method has been largely utilized in human metabolomics studies [17–19], including a study on the metabolic effects of human pregnancy [2]. Technical details of the method are provided elsewhere [17–19].
+
+### Statistical analysis
+
+All statistical analyses were performed using R version 4.1.3 (R Foundation for Statistical Computing, Vienna). First, the data were checked for missing observations. The data did not include any metabolite values missing at random. Values below the detection limit were treated as zero values in the following statistical analyses.
+
+For metabolite selection, a Friedman-Test (a non-parametric alternative to a repeated-measures ANOVA) was used to determine which metabolite concentrations showed significant changes between the different time points and thus are affected by the course of the pregnancy. The Friedman-Test allows taking into account the repeated measures of each animal (6 time points per animal), the corresponding null hypothesis is that there are no changes in metabolite levels within each animal over the course of the pregnancy. Based on the observed data, the normality assumption could not be reliably checked for every of the 123 parameters. Therefore, a non-parametric approach was chosen for its higher robustness. All p-values were corrected for multiple comparisons using the Benjamini-Hochberg method, and the p-value cutoff for adjusted p-values was set at p<0.05.
+
+Since p-values have limited interpretation for ‘omics data, where the number of observations is much smaller than the number of variables (27 animals << 123 metabolites), we used *k*-means Clustering for dimensionality reduction [19]. *k*-means Clustering was done using the z-scaled values of each metabolite. The optimal number of clusters was determined using the “gap” statistic (R package “cluster” version 2.1.3, [20]). Cluster were afterward modified based on the biochemical relationships between the metabolites (super/subcategories, absolute/relative values of the same metabolite), effectively creating 5 clusters from 123 variables. We assume that similar changes in the metabolites, defined by the clusters, are due to shared or related biological processes. To visualize the effect of the time points on the metabolites, “locally estimated scatterplot smoothing” (LOESS) was used. The reference intervals of the analysis [9] were added to the plots to provide reference on the typical concentrations of the metabolites and to highlight the magnitude of the change.
+
+## Results
+
+The 27 dogs enrolled in the study (Table 1) belonged to 21 different breeds and had a median body weight of 22.8 kg (Min: 9.0, Q1: 15.1, Q3: 32.6, Max: 56.4kg). The median age was 4.35 years (Min 1.98, Q1: 2.9, Q3: 5.77, Max 8.1 years).
+
+Fig 1 shows the metabolic parameters which were high in mid-pregnancy (P2). Metabolites which showed different patterns, i. e. low in mid-pregnancy, high in late pregnancy, high in lactation, and low in lactation are presented in Fig 2 (cluster 2, 3,4,5).
+
+*Figure: Fig 1 Metabolic parameters high in mid-pregnancy (Cluster 1).Change in metabolic parameters over the course of canine pregnancy (E = estrus, P1-3 = early/mid/late pregnancy, L = lactation, A = after weaning). Points show the individual measurements. The solid line shows the change in average metabolite levels (LOESS). Dashed lines show the upper and lower limits of the metabolite reference intervals [9]. The p-values were calculated using a Friedman-Test with Benjamini-Hochberg correction. The p-value cutoff for adjusted p-values was p< 0.05.*
+
+*Figure: Fig 2 Metabolic parameters low in mid-pregnancy, high in late pregnancy, high in lactation, and low in lactation (Cluster 2,3,4,5).Change in metabolic parameters over the course of canine pregnancy (E = estrus, P1-3 = early/mid/late pregnancy, L = lactation, A = after weaning). Points show the individual measurements. The solid line shows the change in average metabolite levels (LOESS). Dashed lines show the upper and lower limits of the metabolite reference intervals [9]. The p-values were calculated using a Friedman-Test with Benjamini-Hochberg correction. The p-value cutoff for adjusted p-values was p< 0.05.*
+
+## Discussion
+
+Metabolism during pregnancy and lactation is a dynamic and precisely programmed process [8], as this stage of life is defined by rapid fetal or neonatal growth and development [2, 21]. Metabolite adaptations, such as increased hepatic glucose output, decreased peripheral insulin sensitivity, unchanged hepatic insulin sensitivity and lower peripheral insulin levels, allow the dam to maintain euglycemia during pregnancy [7, 22] and to support fetal growth despite the increasing nutritional demands [7]. Nevertheless, nutrition should be adapted to the respective pregnancy phase to ensure the health of the dam and the puppies [6]. It has been shown that protein deposition in maternal and fetal tissues increases throughout pregnancy, most occurring during the third trimester [21]. Furthermore, leptin increased proportionally with increased food intake during pregnancy, although an apparent body weight gain was observed only at day 60 [23]. The concentrations of metabolites in the blood serum of the bitch affect the corresponding concentrations and the composition of amniotic and allantoic fluid [7]. These fluids are essential during pregnancy, providing metabolites, protection, and the environment for normal fetal development [24].
+
+Five distinct clusters were observed in the analysis of the patterns of the assessed parameters. The identified clusters were: parameters high (Cluster 1/Fig 1)/low (Cluster 2/Fig 2) in mid-pregnancy, parameters high at late pregnancy (Cluster 3/Fig 2), and parameters high (Cluster 4/Fig 2)/low (Cluster 5/Fig 2) in lactation. The clustering was based on similar patterns in metabolite concentrations with elevations and decreases in metabolite concentration occurring during similar time points within each cluster.
+
+### Cluster 1: Parameters high in mid-pregnancy
+
+**Total fatty acids** tended to be higher during pregnancy than at other time points, albeit the differences were not significant. However, an increase in the molar concentration of individual fatty acids was also observed, which may explain changes in the total fatty acids. In addition, elevated total fatty acids have also been observed in another study on dogs [5] and in human pregnancies because a high concentration of fatty acids is necessary to meet the demands of fetuses [3]. Fatty acids play an important role in the development of the fetus and may also be involved in modulatory effects on the mother’s immune system [25]. It is well known that omega 3 fatty acids have anti-inflammatory effects [26] and it has been shown that some that fatty acids such as DHA can increase so called resolvins, which act inflammation resolving and anti-inflammatory [27]. Another strong link between metabolism and the immune system is the fatty acid composition of membranes of immune cells affects phagocytosis capabilities, T cell signaling and antigen presentation capability [28].
+
+The concentration of **free cholesterol (significant)** was highest in mid-pregnancy. The same pattern was mirrored in **total cholesterol** and **esterified cholesterol** (both not significant). These findings are not following another study assessing 12 dogs assigned to four different diet groups in which plasma total cholesterol concentrations were depressed in early gestation and then increased in the later stages independently of the diet [29]. These differences refer mostly to a sample taken on day 42. At this time point, we did not sample the dogs. In another study no changes were found [5]. However, samples were taken at various and not exactly specified time points in pregnancy. It is well known that cholesterol also increases during human pregnancies [30].
+
+Lipoproteins are divided into four major classes: chylomicrons, very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL), with HDL being the most abundant lipoprotein in dogs [31]. Chylomicrons and VLDL are the main carriers of serum triglycerides, while HDL and LDL contain mainly cholesterol [32].
+
+Our results show that the lipoprotein parameters show different patterns in the course of pregnancy and lactation in the dog. While the concentration of HDL particles (HDL-P) and VLDL particles (VLDL-P) show no changes, many others, such as **HDL size**, **total lipids in very large HDL (XL-HDL total lipids)**, the **concentration of LDL particles (LDL particles), LDL cholesterol, total lipids in LDL (LDL total lipids)** peak in mid-pregnancy and decline after that. Other lipoprotein parameters peak in late pregnancy (see cluster 3). An increase of lipoprotein concentrations during canine pregnancy has also been shown in other studies [29]. It is well known that dogs have a unique lipid metabolism and produce substantial, cholesterol-rich HDL particles in hypercholesterolemia. Other than in dogs, in humans, these cholesterols are usually transformed into the more atherogenic LDL particles, which is believed to increase the risk of atherosclerosis in humans [32].
+
+The percentage of polyunsaturated fatty acids (**PUFA %**) (significant) and **PUFA** (not significant) were high in mid-pregnancy. The same phenomenon has been shown in humans [3, 25]. In humans, polyunsaturated fatty acids (PUFA) accumulate in maternal fat depots in early pregnancy and become available for placental transfer during late pregnancy when the fetal growth rate is maximal and fetal requirements for PUFAs are greatly enhanced [33]. A high demand for PUFAs has also been shown in dogs [13].
+
+**Omega-3** unsaturated fatty acids and **omega-3%** were high in mid-pregnancy (both significant), as well as **omega-6** (not significant) and **omega-6%** (significant). Omega-3 and Omega-6 PUFA are needed to ensure the physiologic development of nervous tissue, retinal tissue, kidney, liver, and skin functions [29].
+
+**Docosahexaenoic acid (DHA**) belongs to Omega-3 fatty acids and was significantly elevated in mid-pregnancy, as well as **DHA%**. It plays a significant role during pregnancy and lactation because it contributes to brain development, accounting for over 10% of brain fatty acids [34]. DHA increases during gestation in humans, but its proportion of total lipids decreases [35]. The authors suggest that this indicates a preferential transfer of DHA across the placenta to the fetus [35].
+
+Arachidonic acid (AA), an Omega-6 fatty acid, and AA% were significantly elevated in mid-pregnancy. At the same time, **linoleic acid (LA**), also an Omega-3 fatty acid and precursor for DHA and AA, was only slightly elevated in dogs. Concentrations of LA and AA also increase in pregnant women [35].
+
+**Total saturated fatty acids** (**SFA**), including **palmitic acid (PalA)** and **stearic acid (SteA)**, were slightly elevated during pregnancy in dogs. In humans, the increase of SFA and PalA is markedly higher until the third trimester [25].
+
+**Glycoprotein acetyls (GlycA)** is a novel spectroscopic marker of systemic inflammation combining the *N*-acetyl methyl group proton signals of several acute-phase proteins. It peaks significantly in mid-pregnancy in dogs, which is in accordance with other recent findings [5]. In humans, the intra-individual biological variation of this marker is low, and the main acute-phase protein contributors to its signal are α1-acid glycoprotein, haptoglobin, α1-antitrypsin, and α1-antichymotrypsin [36]. It has been shown that GlycA is higher in pregnant women than in non-pregnant women [3]. The placentation process might explain the rise of this parameter and peak during mid-pregnancy. It is well known that acute phase proteins such as fibrinogen and c-reactive protein increase in the second half of pregnancy [37, 38]. A rise of acute phase proteins during pregnancy has also been shown in 12 pregnant bitches [39].
+
+These findings follow the research on eight pregnant Beagle dogs, which has shown that Alpha-1-acid glycoprotein, which contributes to the GlycA signal, rises during dog pregnancy and peaks at around 45 days of gestation” [40]. In humans, Alpha-1-antitrypsin, another contributor to the GlycA signal, increases in pregnancy [41].
+
+The only amino acid which shows a marked peak during mid-pregnancy is **phenylalanine**. In humans, it has been demonstrated that phenylalanine, alanine, and histidine were increased in pregnant women [3]. In dogs, alanine and histidine seem not to increase during pregnancy. Phenylalanine is required for protein synthesis and is intracellularly converted to tyrosine, which, in turn, is either used for protein synthesis, oxidized, or converted to the important neurotransmitters epinephrine, norepinephrine, and dopamine [42]. It has been shown that excessive feeding of phenylalanine to rats impairs proliferation and hypertrophy in the brain of their fetuses [43], but the dietary requirement for phenylalanine in healthy human pregnancies or canine pregnancies has not been determined yet. Dietary phenylalanine requirements seem not to vary between adult and non-pregnant dog breeds [44].
+
+### Cluster 2: Parameters low in mid-pregnancy
+
+**Oleic acid** is a monounsaturated omega-9 fatty acid. Compared to other unsaturated fatty acids (see Cluster 1), its concentration slightly declines in mid-pregnancy. In humans, Oleic acid increases during pregnancy [25].
+
+**Free cholesterol** and **phospholipids** in large VLDL (L-VLDL) and **total lipids in extensive VLDL (XL-VLDL total lipids)** decline significantly during mid-pregnancy and increase towards the end of pregnancy. An increase in all parameters during pregnancy has been observed in humans [45].
+
+### Cluster 3: Parameters high in late pregnancy
+
+Through various mechanisms, pregnancy causes insulin resistance, suppressing the intracellular transport of glucose and increasing blood glucose concentrations in dogs [4, 46, 47]. Glucose concentrations significantly increase during late pregnancy and remain high until the time after weaning. Despite the pronounced insulin resistance in pregnant compared with non-pregnant diestrous bitches [38], gestational diabetes mellitus in dogs is rare [48]. The same mechanisms of insulin resistance have been described in humans [49, 50], albeit several groups have found lower glucose concentrations in pregnant women compared with non-pregnant ones [3]. The mechanism causing this decline is not well understood. Potential contributing factors include dilutional effects, an increased utilization by the fetoplacental unit or increased maternal uptake secondary to increased β-cell function, and/or inadequate hepatic production [50].
+
+**Total triglycerides, LDL Triglycerides, and VLDL triglycerides significantly** peak in late pregnancy. This is in accordance with findings in normal human pregnancies. Shen et al. [51] found that the levels of lipids, including TG, TC, and LDL cholesterol, increased gradually during gestation and peaked before delivery; meanwhile, HDL cholesterol amounts increased from the 1st to 2nd trimester with a slight decrease in the 3rd trimester. Furthermore, in human pregnancies, an abnormal increase in triglycerides during pregnancy is associated with gestational diabetes mellitus [51]. The possible disease correlations of abnormally high triglycerides during canine pregnancy require further study. Similar to gestational diabetes mellitus, hypertensive disorders are not common complications of canine pregnancy. Interestingly, unlike humans, dogs’ HDL triglycerides are not affected by pregnancy or lactation.
+
+### Cluster 4: Parameters high in lactation
+
+It has been shown that lactate and pyruvate are more elevated in pregnant women than in non-pregnant women [3]. The **lactate, pyruvate**, and **acetate** concentrations significantly rise towards late pregnancy and lactation, presumably reflecting the higher demands of energy metabolism, even if their medians remain within the reference intervals at all time points. Whether lactate can be used as an indicator for sepsis or other pregnancy-related disorders in dogs as it is used in humans [52] requires further study since all bitches included in our study had undisturbed pregnancies.
+
+The total concentration of **branched-chain amino acids (BCAA)**, which includes leucine, isoleucine, and valine, shows a marked increase during lactation. Interestingly, isoleucine and valine show the same pattern, whereas leucine shows low concentrations during lactation (see cluster 5). The same is true for the amino acid **glycine**. Glutamine, glycine, valine, and tyrosine were lower in pregnant women compared with non-pregnant women; no differences were found for isoleucine and leucine between pregnant and non-pregnant women [3].
+
+**Creatinine** shows a significant decrease in mid-pregnancy, which is in accordance with other findings in dogs [5] and in pregnant women. It has been stated that a physiologic increase in the glomerular filtration rate during pregnancy results in a decrease in the concentration of serum creatinine [53]. In addition, it has been shown that creatinine was lower in pregnant women compared to non-pregnant women [3].
+
+### Cluster 5: Parameters low in lactation
+
+The observed significant decline of **albumin** during late pregnancy and lactation has also been well documented in dogs [5] and humans during and after normal pregnancies [3, 54]. It is believed that this decline is caused by physiologic hemodilution but may lead to physiological edema and even may associate with conditions such as eclampsia [54]. In addition, it has been shown that albumin concentrations increase in the allantoic fluid in late canine pregnancy [24].
+
+**Docosapentaenoic acid (DPA)** is also significantly elevated during pregnancy but shows a significant decline during lactation, which is also visible using the parameter **DPA%**.
+
+The concentration of the amino acid **histidine** remains stable at all time points, except for a significant decrease during lactation. Histidine likely plays a role in milk production, as it has been shown for cows, who have an increased energy-corrected milk yield when supplemented with histidine [55].
+
+The amino acid **leucine** showed a significant increase in its concentration during early pregnancy and a decline during mid and late pregnancy and lactation. This contrasts with other branched-chain amino acids (see cluster 4). This decline is likely connected with milk production since leucine plays a vital role in milk production, as shown in cows [56].
+
+### Limitations of the study
+
+The metabolome is affected by several internal factors such as gene and protein activity and external factors, including diet and environmental factors [15]. In our study, privately owned dogs were used, which were housed and fed under different conditions. Furthermore, 21 breeds and dogs aged between 2 and 8 years were included. To what extent these heterogeneous conditions may have affected the results remains open. However, the included dogs may better reflect the real-life heterogeneity seen in pregnant dogs than laboratory animals. Experienced breeders kept most dogs so that appropriate housing and feeding under consideration of the reproductive status of the female dog is likely.
+
+A further limitation is that dogs did not fast before sampling because this would not have been acceptable for pregnant and lactating dogs. The timing of collection of the blood samples also showed a mild variation of a few hours; therefore, the impact of diurnal influence cannot be excluded. However, as they were usually sampled in the morning, diurnal variance would be expected to be small. Another potential limitation is the small number of dogs originating from one geographical area.
+
+Some comparisons of our findings with findings from human pregnancies need to be interpreted with caution. Pregnancy of dogs is around 63 days, significantly shorter, and the offspring usually is much larger. Puppies are born physiologically immature and dependent on their mother´s care and milk supply [57]. In the study, the dogs delivered between 1 and 12 puppies.
+
+In addition, research on pregnant dogs has led to several contradictory findings, which makes interpretation of actual metabolic and hematologic findings difficult [1]. For example, it was described more than 40 years ago that some bitches develop progressive, normochromic, and normocytic anemia beginning in mid-pregnancy [9]. While some authors consider this normal, other groups suggest that haematocrits should remain in the normal reference range and that bitches with anaemia should be examined for other concurrent diseases [58].
+
+The samples for this study were taken throughout the year. However, previous research using the same method has shown that only minimal seasonal variation exists in the vast majority of metabolites [59]. Therefore, the season was not taken into account during the data analysis.
+
+In our current study, samples were stored at −80°C for up to 30 months before analysis. In a previous study validating the NMR platform, all studied metabolites were stable at −80°C for at least 12 months [14]. However, the impact of more extended storage on canine samples is lacking.
+
+In many species, it can be challenging to determine whether changes detected during pregnancy are related to the presence of a conceptus or the increased progesterone concentrations [39].
+
+## Conclusions
+
+Several metabolites such as albumin, GlycA, fatty acids, lipoproteins, glucose, and some amino acids show similar patterns during canine pregnancy and lactation as shown in humans. The patterns of some other parameters, such as branched-chain amino acids, alanine and histidine, seem to differ.
+
+To date, it is unclear for most metabolites if observed changes arise from modified resorption from the intestines, modified production or metabolism in the maternal tissues, or their migration through the placenta. In addition, it has hardly been investigated so far to what degree fetal fluid concentrations of specific metabolites are influenced by maternal concentrations [7]. Hence, further species-specific research is needed to shed more light on these questions.
+
+Understanding the molecular changes during pregnancy and the relation to discrete biological events such as embryo development and placentation may support understanding of these processes, learn more about causes of infertility or diseases, and aid in identifying treatments for these conditions [59]. It is essential to understand physiological changes during pregnancy and lactation so that these changes are not misinterpreted as indicators of disease or malnutrition. Furthermore, knowledge of the physiological changes is important for designing studies on metabolites to adjust inclusion criteria, i. e. if or not to include also pregnant or lactating bitches, and for interpreting the results.
+
+Further research should provide molecular reference ranges in normal pregnancies in relation to studies of adverse pregnancy outcomes [3]. In that regard, metabolomics findings may help monitor vital functions of pre-, intra-, and post-partum bitches and early detection of illness [1].

fulltext/md/PMC10175583.md

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+---
+pmcid: "PMC10175583"
+pmid: "37187924"
+doi: "10.3389/fvets.2023.1151266"
+title: "Sleep and cognition in aging dogs. A polysomnographic study."
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Sleep and cognition in aging dogs. A polysomnographic study
+
+## Abstract
+
+### Introduction
+
+Sleep is fundamental for cognitive homeostasis, especially in senior populations since clearance of amyloid beta (key in the pathophysiology of Alzheimer's disease) occurs during sleep. Some electroencephalographic characteristics of sleep and wakefulness have been considered a hallmark of dementia. Owners of dogs with canine cognitive dysfunction syndrome (a canine analog to Alzheimer's disease) report that their dogs suffer from difficulty sleeping. The aim of this study was to quantify age-related changes in the sleep-wakefulness cycle macrostructure and electroencephalographic features in senior dogs and to correlate them with their cognitive performance.
+
+### Methods
+
+We performed polysomnographic recordings in 28 senior dogs during a 2 h afternoon nap. Percentage of time spent in wakefulness, drowsiness, NREM, and REM sleep, as well as latency to the three sleep states were calculated. Spectral power, coherence, and Lempel Ziv Complexity of the brain oscillations were estimated. Finally, cognitive performance was evaluated by means of the Canine Dementia Scale Questionnaire and a battery of cognitive tests. Correlations between age, cognitive performance and sleep-wakefulness cycle macrostructure and electroencephalographic features were calculated.
+
+### Results
+
+Dogs with higher dementia scores and with worse performance in a problem-solving task spent less time in NREM and REM sleep. Additionally, quantitative electroencephalographic analyses showed differences in dogs associated with age or cognitive performance, some of them reflecting shallower sleep in more affected dogs.
+
+### Discussion
+
+Polysomnographic recordings in dogs can detect sleep-wakefulness cycle changes associated with dementia. Further studies should evaluate polysomnography's potential clinical use to monitor the progression of canine cognitive dysfunction syndrome.
+
+## 1. Introduction
+
+Sleep is fundamental for cognitive homeostasis as some of its most relevant functions are memory consolidation and learning processing (1–3). Dogs, like humans and other mammals, experience age-related changes in their sleep-wakefulness cycle (4–8). In fact, owners of aging dogs report that sleeplessness is an age-related behavioral change with great impact on the owner-pet relationship (9). While these variations can be attributed to normal aging, in some cases they may possibly be due to underlying neurodegenerative processes (10, 11).
+
+Humans with Alzheimer's disease (AD), for example, experience sleep disruptions, such as insomnia and sleep fragmentation. These signs can occur early in the course of the disease because brain regions that regulate sleep and circadian rhythms, such as the suprachiasmatic nuclei, are some of the first to be affected (10, 12, 13). Older dogs are prone to develop canine cognitive dysfunction syndrome (CCDS), a disease similar to AD (14–16). This syndrome is characterized by disorientation, memory impairment and difficulty learning, changes in social interactions and house soiling behaviors, increased anxiety, and alteration of the sleep-wakefulness cycle (11, 17–19). Specifically, owners of dogs with CCDS report that their dogs suffer from difficulty sleeping, increased pacing and vocalizations at nighttime, and/or increased sleeping at daytime (11, 20).
+
+While sleep disturbance might be a consequence of these neurodegenerative processes, they can also contribute to the pathophysiology of the disease and to memory impairment resulting in a vicious cycle (13, 21). Clearance of amyloid beta (Aβ) occurs in the brain through the glymphatic system, and this system is primarily active during slow wave sleep (NREM sleep) (22). Therefore, sleep deprivation can increase Aβ deposition in the brain (13, 22–25). While several studies have shown that sleep is disrupted in dogs with CCDS (11, 26), this has been based on owner reports, and no study has looked at the macrostructure or at the electroencephalographic changes of sleep in these dogs. Polysomnography is the gold standard technique to objectively evaluate sleep (27) and a non-invasive technique has been developed in dogs (28). It consists of the simultaneous recording of the EEG, electrical activity of the muscles (EMG) and eye movements (EOG) and can also include the recording of the electrocardiogram (ECG) and respiratory movements.
+
+In addition to the importance of an adequate amount of time dedicated to sleep, the electroencephalographic characteristics of sleep are also associated with Aβ clearance; for example, the reduction of slow oscillations (delta) power is strongly correlated with Aβ deposition in the brain (25). In fact, studies in humans using quantitative electroencephalographic analyses (qEEG) (27) have shown electroencephalographic signatures of dementia during wakefulness and sleep states (29–33). One of the most widely employed qEEG analyses is spectral power, which provides the relative contribution of the frequency components of a signal and reflects the local degree of synchronization of the extracellular potential (34). Spectral power is calculated by means of the fast Fourier transform, and the details of its calculation can be found elsewhere (27). Usually, the frequency spectrum is divided into discrete ranges (bands); in dogs, these are delta (1–4 Hz), theta (4–8 Hz), alpha (8–12 Hz), sigma (12–16 Hz), beta (16–30 Hz), and gamma (30–45 Hz) (21). Each band is associated with different behavioral or consciousness states, for example, wakefulness is characterized by higher power in faster oscillations (alpha, beta, and gamma), while NREM sleep or anesthetic states are characterized by higher power in delta oscillations (27, 35, 36). In humans with AD, an increase in delta power and a reduction in the frequency of the peak alpha power has been demonstrated during resting state (37).
+
+Another qEEG analysis frequently used is the spectral coherence, which measures the degree of functional coupling between two cortical areas. Two brain oscillations are completely coherent at a specific frequency if their phase difference and the relationship between the amplitudes at that frequency are constant (27, 38). Coherence can be calculated between two areas within the same brain hemisphere (intrahemispheric) or between areas on the right and left hemispheres (interhemispheric) We have previously shown that, comparable to people with AD (39, 40), during wakefulness, dogs with presumptive diagnosis of CCDS show a reduction of interhemispheric coherence in the high frequency bands (41).
+
+Both power and coherence are measures of linear dynamics, but the complex EEG signal cannot be explained only by them. Non-linear analyses are a more modern method to study the complexity of the system (42). One of the most used in qEEG research is Lempel Ziv Complexity, which evaluates the randomness of finite sequences based on the number of distinct patterns in a signal and the symbolic encoding (43). The LZC analysis is usually performed on the whole signal of a wider range of frequencies, without analyzing different frequency bands. This complexity has been used in the analysis of different neurophysiological signals, for example, to study the EEG signatures of anesthesia, sleep, and altered states of consciousness, as well as brain disorders such as AD or epilepsy (44–47). During wakefulness, people with AD show a reduction in Lempel Ziv Complexity (45), but we have recently found that the opposite is true for dogs with presumptive diagnosis of CCDS (41).
+
+The aim of this study was to describe and quantify age-related changes in the sleep-wakefulness cycle macrostructure and EEG features in senior dogs and to correlate them with their cognitive performance. We predicted that cognitively impaired dogs would sleep less and would have distinct EEG features from dogs without cognitive impairment.
+
+## 2. Methods
+
+### 2.1. Study population
+
+All procedures were approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee, protocol number: 21-303. Client-owned dogs participating in the longitudinal study of neuro-aging at the North Carolina State University (NCSU), College of Veterinary Medicine were used in this study [described in our previous research (18, 48, 49)]. All owners reviewed and signed an informed consent form prior to participation. To be included, dogs had to be older than the 75% of their expected lifespan, which was calculated using the formula proposed by Greer et al. (50) that takes into consideration the dog's height and weight and can thus be applied in case of mixed-breed dogs as well. Additionally, they had to be free of comorbidities that could impede their ability to perform cognitive tests such as inability to walk or blindness.
+
+Demographic information was collected, and physical, orthopedic, and neurological examinations were conducted for every dog. Height to the dorsal aspect of the scapula (withers) and body weight were measured in order to calculate dogs' expected lifespan. Urinalysis, biochemistry panel and complete blood cell count were also performed to rule out potential comorbidities.
+
+### 2.2. Polysomnographic studies
+
+Dogs underwent polysomnographic recordings with simultaneous recording of EEG, EOG, EMG, and ECG with a slight modification of the protocol described by Reicher et al. (51). As schematized in Figure 1A, we employed four active EEG electrodes, F3, F4, Fz (left, right, and midline frontal, respectively), and Cz (at the level of the vertex). The electrode location was chosen to evaluate the frontal and parietal cortex, the frontal cortex being particularly important in cognitive decline (52). These electrodes were referenced to Oz, an electrode placed over the external occipital protuberance. Bipolar EOG signal was recorded by placing two electrodes on the left and right zygomatic arch next to the lateral canthus of each eye (F7 and F8, respectively). A ground electrode was placed over the left temporal muscles. For EMG, bipolar signal was obtained by placing two electrodes over the dorsal neck muscles on the left and right side. Finally, one electrode was placed over the fifth intercostal space and referenced to the Cz electrode to record the ECG. We used gold-coated electrodes (Genuine Grass 10 mm Gold Cup, Natus Medical Inc) which were attached to the skin with SAC2 electrode cream (Cadwell Laboratories) after applying a skin preparation and electrode solution (Signa Spray, Parker Laboratories). Recordings were performed with the software Cadwell Easy II software (Cadwell Laboratories). Electrode impedance was checked to be under 20 kΩ before starting the recording. A frequency sample of 400 Hz was used. All signals were bandpass filtered between 0.53 and 70 Hz. A notch filter was also applied during acquisition to remove the 60 Hz power-line noise.
+
+*Figure: Figure 1 Polysomnographic recordings in dogs. **(A)** Electrode locations. Four active EEG electrodes were placed over the midline, left and right frontal areas (Fz, F3, and F4, respectively), and over the vertex (Cz). Those electrodes are represented with a black dot. A reference electrode was placed over the external occipital protuberance (Oz) and it is represented with a red dot. Bipolar EOG signal was recorded by placing two electrodes on the left and right zygomatic arch next to the lateral canthus of each eye (blue dots). A ground electrode (G) was placed over the left temporal muscles (green dot). EMG and ECG were also recorded but it is not shown in the figure. **(B)** Picture of a dog undergoing a polysomnographic recording using their own bed. **(C)** Representative ECG, EOG, EMG, and EEG signals during wakefulness, drowsiness, NREM, and REM sleep. Contamination of the ECG can be observed in the EMG signal.*
+
+Recordings were performed in a very quiet room, with dim light and white noise reproduced through a laptop computer. Room temperature was maintained at 20°C. Owners were asked to bring their dogs for polysomnography recordings on 2 different days, an adaptation day [to avoid the “first-night-effect” (51)], and a recording day and were instructed to bring their dogs' usual bed or blankets to both recording days, so dogs felt more comfortable to sleep. Figure 1B shows an example of one dog during a polysomnography recording. During the adaptation day we performed a 30 min recording that served to acclimate the dog to the recording room and setup. On the actual testing day, the dogs had a 2-h polysomnography recording that started between 12:30 and 1:30 PM. This period of time was selected because it has been shown that dogs usually have naps around noon (5, 53, 54). For dogs who became anxious and attempted to leave the room or removed all the electrodes, we stopped the recordings even if the 2 h were not complete. Time between the adaptation and the actual recording day was never more than 2 weeks, and cognitive testing happened in between these days.
+
+States of wakefulness, drowsiness, NREM and REM sleep were manually scored in 3 s epochs in the Spike 2 software (Cambridge Electronic Design) using criteria described previously (28). As shown in Figure 1C, wakefulness was characterized by fast frequency EEG activity, high activity in the EMG, and frequent high-amplitude eye movements. Drowsiness was defined by EEG activity similar to wakefulness, reduced but observable muscle activity in the EMG and decreased amplitude and frequency of eye movements. NREM sleep was characterized by low-frequency and high-amplitude neural oscillations, mainly within the delta frequency band. Muscle activity during NREM sleep was significantly reduced and there were no or sporadic low-amplitude eye movements. Finally, REM sleep was recognized by high frequency activity in the EEG, absence of muscle activity and frequent rapid eye movements that can also be observed as artifacts in the EEG (28). Latency to enter drowsiness, NREM and REM sleep as well as the percentage of the total recording time spent in each behavioral state were calculated. Additionally, sleep efficiency was calculated by adding the time spent in NREM and REM sleep and dividing it by the total time recorded.
+
+### 2.3. qEEG analyses
+
+Similar to our previous study (41), raw EEG signals from the four EEG electrodes were exported from Spike 2 to MATLAB (version 2022b; The MathWorks Inc). All artifact-free, non-transition epochs were selected for quantitative EEG analysis. Power spectrum was calculated for Fz and Cz electrode locations with a 0.25 Hz resolution between 0.5 and 50 Hz by means of *pwelch* function in MATLAB using the following parameters: 1 s sliding windows with 50% overlap. Power at F3 and F4 were not calculated since we were interested in changes in frontal and parietal cortices and analyzing three different electrodes of the same area was redundant. Total power was computed by summing the power of each 0.25 Hz bin and relative power was calculated by dividing power over total power. Interhemispheric (between F3 and F4) and intrahemispheric (between Fz and Cz) coherence were calculated using the MATLAB *mscohere* function utilizing the same parameters used for power spectrum analysis. Coherence values were then normalized using Fisher's z-transform.
+
+We also evaluated the complexity of the EEG signal by means of Lempel-Ziv complexity for Fz and Cz. To perform the analysis, the signal was pre-processed by applying an order 5 butterworth band-pass filter between the frequencies 1 and 50 Hz. We then used the *lziv_complexity* function from the *AntroPy* package from Python (55).
+
+### 2.4. Cognitive evaluation
+
+We evaluated cognitive status in these dogs by using an owner-based questionnaire and a battery of cognitive tests. As in our previous studies, the questionnaire utilized was the Canine Dementia Scale (CADES), a validated clinical metrology instrument to capture behavioral changes associated with CCDS. In this questionnaire owners need to specify the frequency with which their dogs experience specific behaviors in four different domains: spatial disorientation, social interactions, sleep-wakefulness cycle and house soiling (11). CADES scores range from 0 (normal) to 95 (severely affected) and allow classification of dogs into four different categories: normal (0–7), mild cognitive impairment (MiCI, 8–23), moderate cognitive impairment (MoCI, 24–44), and severe cognitive impairment (SCI, 45–95).
+
+Cognitive testing was also performed to objectively evaluate different domains of cognition: attention (sustained gaze test), working memory and executive control [inhibitory control and detour (cylinder) tasks]. The detailed procedure for each task has been described by our group elsewhere (18, 56). In brief, the sustained gaze task measures how long the dog holds the gaze with an experimenter who is holding a treat near their face. To evaluate working memory, two red Solo^®^ cups were placed on a mat and a small food treat was hidden under one of them (while the dog is allowed to see where it was placed). The delay (up to 2 min) between placing the treat and allowing the dog to choose one of the cups to retrieve the treat was progressively increased. The upper threshold of time that dogs were able to correctly identify (at least four out of six times) where the treat was hidden was then calculated. In the inhibitory control task, dogs were asked to retrieve a treat from a transparent plastic cylinder that is open on both ends, without touching the outside walls of cylinder. The detour task is similar but adds an additional difficulty—the dog's preferred side for retrieving the treat is now covered, and they need to enter the cylinder from the opposite side (detour) in order to obtain the treat. The outcome measure for these two tests is the percentage of correct trials (retrieving the treat without touching the outer walls of the cylinder or the detour).
+
+### 2.5. Statistical analysis
+
+We calculated the correlation between the sleep architecture variables (and Lempel Ziv Complexity) and age, CADES score, and performance in each of the cognitive tests by means of Spearman correlation analyses. While fractional lifespan (instead of chronological age) was employed for inclusion criteria, we used chronological age in our analyses because previous studies that have looked at sleep and qEEG analysis in dogs have reported chronological age (28, 51, 57). Statistical significance was adjusted using the Benjamini, Krieger and Yekutieli method (58) with a false discovery rate of 0.05. Adjusted *p*-value is expressed with a “q.” To evaluate the correlation between age and cognitive performance with power and coherence we did a bin to bin spearman correlation analysis and we used the procedure known as Rüger's areas (59) previously employed in polysomnographic studies in dogs (28, 57) to address the issue of multiple comparisons. These areas can be defined as a cluster of conventionally significant (*p* < 0.05) results, which are considered significant (or not) as a whole. A Rüger's area is the cluster of all the adjacent, consecutive frequency bins which contain a significant result surrounded by bins containing non-significant results. After defining these areas of significance, the number of frequency bins within the area was counted, and it was inspected to see if at least half of the *p*-values were lower than 1/2 of the conventional *p* = 0.05 significance level (i.e., below 0.025) and at least 1/3 of them were lower than 1/3 of the conventional *p* = 0.05 (i.e., 0.0167). If both requirements were satisfied, the area as a whole was considered significant.
+
+## 3. Results
+
+### 3.1. Demographics
+
+Twenty-eight dogs [13.25 ± 1.57 years old (Range: 10.4–16.2)] were enrolled in this study. Their mean fractional lifespan was 1.06 ± 0.12 (Range 0.81–1.22). Seventeen were spayed females and 11 were castrated males. Seventeen were purebred dogs, and 11 were mix breed. The most represented breed was pit bull terrier with four dogs, followed by Labrador retriever with two dogs. There were also 1 dog of each of the following breeds: Australian shepherd, Basset hound, beagle, Border collie, Brittany spaniel, dachshund, German shorthaired pointer, golden retriever, German shepherd, Irish setter, and Pomeranian.
+
+### 3.2. Polysomnography recordings
+
+Of the 28 dogs, 17 completed 2 h of recording. Of the remaining 9, five completed at least 1.5 h of recording and the minimum recording time was 1.01 h. Twenty-six dogs entered drowsiness, 24 into NREM sleep and 15 into REM sleep. Percentage of the total recorded time spent in each behavioral state, sleep efficiency and latency to drowsiness, NREM sleep, and REM sleep are shown in Figure 2.
+
+*Figure: Figure 2 Latency to and percentage of time spent in each behavioral state during polysomnography recordings. **(A)** Box plots showing latency in minutes to drowsiness, NREM and REM sleep. Only data from dogs that entered each specific behavioral state was employed (drowsiness *n* = 26, NREM sleep *n* = 24, and REM sleep *n* = 15). **(B)** Box plots showing percentage of total time spent in each behavioral state and the sleep efficiency (sum of time in NREM and REM sleep over total time). All dogs (*n* = 28) were used. Box plots represent the median (central line of the box), the 25 and 75th percentile (lower and upper line of the box). The whiskers represent 1.5 times the interquartile range (1.5^*^IQR) and outliers are shown with a circle. W, wakefulness; Drow, drowsiness; NREM + REM, sleep efficiency.*
+
+### 3.3. Cognitive evaluation
+
+Median score in the CADES questionnaire was 18 (Range 0–70). According to their score, eight dogs (28.5%) were classified as normal, eight dogs (28.5%) with mild, four dogs (14.3%) with moderate, and eight dogs (28.5%) with severe cognitive impairment. Every dog completed the sustained gaze, and inhibitory control tasks. Memory task was not performed in three of the dogs because they failed to pass warm-ups (required as a criterion for learning; they did not remember where the treat was even immediately after showing them the location, therefore, these dogs scored 0 for memory) and one dog did not complete the detour task because they lost interest and would no longer engage with the task. For this dog, detour task data were excluded from the analysis. The median sustained gaze time was 17.0 s (range 1.3–60), median memory threshold was 40 s (range 0–120), median percentage of correct trials in inhibitory control was 100 (range 13–100%) and in detour 50 (range 0–100%).
+
+### 3.4. Correlation between sleep macrostructure and age and cognitive status
+
+As shown in Table 1, older dogs tended to spend more time awake and less time in NREM sleep. However, these correlations did not reach statistical significance after correcting for multiple comparisons. There was also a relationship between cognitive performance and sleep parameters: the CADES score was negatively correlated with time spent in NREM and REM sleep. Dogs with higher CADES scores, also tended to have a higher latency to enter into NREM sleep. Figure 3 shows the hypnogram of two representative dogs, one with a low CADES score (classified as with mild cognitive impairment) and one with a high CADES score (classified as with severe cognitive impairment). This figure clearly illustrates how dogs with higher CADES scores spend less time sleeping. Additionally, performance in the detour task was negatively correlated with time spent in wakefulness and positively correlated with time spent in NREM and REM sleep. Dogs with worse performance also tended to have longer NREM sleep latency. We did not find any association between sleep macrostructure and performance in any of the other cognitive tests.
+
+**Table 1 Correlation between sleep macrostructure and age and cognitive function.**
+
+| **Variable** | **By variable** | **Spearman ρ** | **Raw *p*-value** | ***q*-value** |
+| --- | --- | --- | --- | --- |
+| Age | Latency to Drow. | 0.229 | 0.260 | 0.439 |
+| Age | Latency to NREM | 0.440 | 0.031 | 0.116 |
+| Age | Latency to REM | 0.091 | 0.747 | 0.783 |
+| Age | Time in W (%) | 0.408 | 0.020^*^ | 0.116 |
+| Age | Time in Drow. (%) | −0.373 | 0.051 | 0.164 |
+| Age | Time in NREM (%) | −0.441 | 0.019^*^ | 0.092 |
+| Age | Time in REM (%) | −0.305 | 0.114 | 0.295 |
+| CADES | Latency to Drow. | 0.210 | 0.304 | 0.472 |
+| CADES | Latency to NREM | 0.436 | 0.033^*^ | 0.116 |
+| CADES | Latency to REM | 0.011 | 0.969 | 0.897 |
+| CADES | Time in W (%) | 0.454 | 0.015^*^ | 0.085 |
+| CADES | Time in Drow. (%) | −0.267 | 0.169 | 0.331 |
+| CADES | Time in NREM (%) | −0.554 | <0.001^*^ | **0.020**
+^*****^ |
+| CADES | Time in REM (%) | −0.553 | 0.002^*^ | **0.020**
+^*****^ |
+| Memory | Latency to Drow. | −0.197 | 0.357 | 0.496 |
+| Memory | Latency to NREM | −0.319 | 0.147 | 0.331 |
+| Memory | Latency to REM | −0.258 | 0.395 | 0.529 |
+| Memory | Time in W (%) | −0.361 | 0.077 | 0.229 |
+| Memory | Time in Drow. (%) | 0.272 | 0.189 | 0.349 |
+| Memory | Time in NREM (%) | 0.327 | 0.110 | 0.295 |
+| Memory | Time in REM (%) | 0.283 | 0.170 | 0.331 |
+| Inhibitory control | Latency to Drow. | −0.212 | 0.298 | 0.472 |
+| Inhibitory control | Latency to NREM | −0.148 | 0.489 | 0.613 |
+| Inhibitory control | Latency to REM | 0.123 | 0.661 | 0.755 |
+| Inhibitory control | Time in W (%) | 0.014 | 0.942 | 0.893 |
+| Inhibitory control | Time in Drow. (%) | −0.055 | 0.781 | 0.798 |
+| Inhibitory control | Time in NREM (%) | 0.079 | 0.691 | 0.767 |
+| Inhibitory control | Time in REM (%) | 0.111 | 0.572 | 0.674 |
+| Detour | Latency to Drow. | −0.193 | 0.355 | 0.496 |
+| Detour | Latency to NREM | −0.525 | 0.010^*^ | 0.065 |
+| Detour | Latency to REM | −0.184 | 0.529 | 0.642 |
+| Detour | Time in W (%) | −0.556 | 0.003^*^ | **0.020**
+^*****^ |
+| Detour | Time in Drow. (%) | 0.149 | 0.457 | 0.592 |
+| Detour | Time in NREM (%) | 0.683 | <0.001^*^ | **0.019**
+^*****^ |
+| Detour | Time in REM (%) | 0.596 | 0.001^*^ | **0.019**
+^*****^ |
+| Eye gaze | Latency to Drow. | −0.019 | 0.926 | 0.893 |
+| Eye gaze | Latency to NREM | −0.305 | 0.147 | 0.331 |
+| Eye gaze | Latency to REM | −0.343 | 0.211 | 0.372 |
+| Eye gaze | Time in W (%) | 0.193 | 0.324 | 0.484 |
+| Eye gaze | Time in Drow. (%) | 0.041 | 0.837 | 0.833 |
+| Eye gaze | Time in NREM (%) | −0.071 | 0.718 | 0.774 |
+| Eye gaze | Time in REM (%) | −0.273 | 0.160 | 0.331 |
+
+*Figure: Figure 3 Representative hypnograms showing the difference in sleep architecture between a dog with mild **(left)** and severe **(right)** cognitive impairment based on CADES score. W, wakefulness; Drow, Drowsiness.*
+
+### 3.5. Associations between electroencephalographic features, age, and cognitive status
+
+We calculated power, coherence, and Lempel Ziv complexity for every dog, except for one in which Fz, F3 and F4 electrodes were not analyzed because the dog removed those electrodes during the recording. Therefore, in this dog, power and complexity were analyzed only for Cz, and we could not perform coherence analysis. The power spectrum analysis is summarized in Figure 4. There were significant relationships with age, performance on the detour task and memory. For Fz, during drowsiness the power of slow oscillations (1–1.75 Hz) was negatively correlated with age and positively correlated with performance in the detour task. At each of the correlated frequencies, the strength of the correlation was higher for performance at detour task than for age. In addition to this, memory performance was correlated with the power of some frequency bands during REM sleep at Cz: dogs with higher memory scores had higher power of slow oscillations from 1 to 1.75 Hz, and lower power between 15.75 and 19 Hz (within beta frequency band). Also, at Fz, dogs with higher memory scores had lower power of higher oscillations, between 48 and 50 Hz (within gamma frequency band). Interhemispheric (F3–F4) coherence showed some correlations with age and cognitive function. As shown in Figure 5, during wakefulness, older dogs showed higher interhemispheric coherence for frequencies 6.5–13 Hz (within theta and alpha frequency bands). Dogs with higher CADES scores also had higher interhemispheric coherence at those frequencies, but the correlation was only significant between 10.5 and 12 Hz. Similarly, dogs with worse performance at the detour task showed higher interhemispheric coherence between 10.25 and 12 Hz. The strength of these correlations was higher for age than for CADES scores or performance at the detour task. We also found a negative correlation between performance in the sustained gaze task and interhemispheric coherence for slow oscillations up to 3 Hz (within delta frequency band). Furthermore, during REM sleep, older dogs had higher interhemispheric coherence for frequencies between 36.25 and 38 Hz.
+
+*Figure: Figure 4 Correlations between wakefulness, EEG power and age, CADES score, or performance in cognitive tests. Correlation coefficients for the EEG channels Fz (blue open circle) and Cz (red solid circle) are shown for each frequency bin. Frequencies with significant correlations are indicated with a blue (for Fz) or red (for Cz) shadow box. The analysis was performed only for the dogs that entered each specific behavioral state (wakefulness *n* = 28, drowsiness *n* = 26, and NREM sleep *n* =24).*
+
+*Figure: Figure 5 Correlations between EEG interhemispheric (F3–F4) coherence and age, CADES score, or performance in cognitive tests. Spearman correlation coefficients are shown for each frequency bin. Frequencies with significant correlations are indicated with a purple shadow box. The analysis included only the dogs that entered each specific behavioral state (wakefulness *n* = 28, drowsiness *n* = 26, and NREM sleep *n* =24).*
+
+Intrahemispheric (Fz-Cz) coherence was correlated with cognitive function, but not with age (Figure 6). During wakefulness, dogs who performed better at the inhibitory control task had lower Fz-Cz coherence for the frequencies between 8 and 14.25 Hz. Similarly, during drowsiness, frequencies between 0.25–6.5 and 7–12.5 Hz were lower for dogs with better performance in the same task. During NREM sleep, there was also a negative correlation between coherence and inhibitory control scores, but for higher frequencies, between 7.75–20.75 and 30.25–44.25 Hz. During REM sleep, an association was found with performance on the detour task, dogs with better performance had higher coherence for frequencies between 10.75 and 16.5 Hz.
+
+*Figure: Figure 6 Correlations between EEG intrahemispheric (Fz-Cz) coherence and age, CADES score, or performance in cognitive tests. Spearman correlation coefficients are shown for each frequency bin. Frequencies with significant correlations are indicated with a purple shadow box. The analysis included only the dogs that entered each specific behavioral state (wakefulness *n* = 28, drowsiness *n* = 26, and NREM sleep *n* =24).*
+
+We evaluated the complexity of the EEG signal for the electrodes Fz and Cz and evaluated its correlation with age, CADES score, and performance in cognitive tests and we found a negative correlation between memory performance and complexity during REM sleep at Fz (Table 2). Age showed a positive correlation and performance in the detour task showed a negative correlation with complexity during REM sleep, but the significance was lost after multiple comparisons correction. Similarly, we observed that age tended to be positively correlated, while CADES, memory, and performance at the detour task tended to be negatively correlated with complexity during REM sleep at Cz. However, none of these remained significant after multiple comparisons correction (Table 3).
+
+**Table 2 Correlation between Lempel Ziv Complexity at Fz and age and cognitive function.**
+
+| **Variable** | **By variable** | **Spearman (ρ)** | **Raw *p*-value** | ***q*-value** |
+| --- | --- | --- | --- | --- |
+| Age | LZC W | 0.172 | 0.391 | 0.708 |
+| Age | LZC Drow. | 0.291 | 0.158 | 0.398 |
+| Age | LZC NREM | 0.268 | 0.215 | 0.433 |
+| Age | LZC REM | 0.631 | 0.015^*^ | 0.124 |
+| CADES | LZC W | 0.141 | 0.483 | 0.730 |
+| CADES | LZC Drow. | 0.286 | 0.165 | 0.398 |
+| CADES | LZC NREM | 0.269 | 0.215 | 0.433 |
+| CADES | LZC REM | 0.438 | 0.117 | 0.398 |
+| Memory | LZC W | −0.036 | 0.866 | 0.950 |
+| Memory | LZC Drow. | 0.025 | 0.908 | 0.954 |
+| Memory | LZC NREM | −0.178 | 0.440 | 0.708 |
+| Memory | LZC REM | −0.813 | 0.001^*^ | **0.031**
+^*****^ |
+| Inhibitory control | LZC W | −0.078 | 0.698 | 0.887 |
+| Inhibitory control | LZC Drow. | −0.119 | 0.572 | 0.812 |
+| Inhibitory control | LZC NREM | 0.002 | 0.992 | 0.998 |
+| Inhibitory control | LZC REM | −0.228 | 0.432 | 0.708 |
+| Detour | LZC W | −0.326 | 0.104 | 0.398 |
+| Detour | LZC Drow. | −0.343 | 0.100 | 0.398 |
+| Detour | LZC NREM | −0.342 | 0.119 | 0.398 |
+| Detour | LZC REM | −0.711 | 0.006^*^ | 0.078 |
+| Eye gaze | LZC W | 0.049 | 0.806 | 0.946 |
+| Eye gaze | LZC Drow. | −0.087 | 0.679 | 0.887 |
+| Eye gaze | LZC NREM | −0.049 | 0.823 | 0.946 |
+| Eye gaze | LZC REM | −0.398 | 0.159 | 0.398 |
+
+**Table 3 Correlation between Lempel Ziv Complexity at Cz and age and cognitive function.**
+
+| **Variable** | **By variable** | **Spearman ρ** | **Raw *p*-value** | ***q*-value** |
+| --- | --- | --- | --- | --- |
+| Age | LZC W | 0.281 | 0.147 | 0.362 |
+| Age | LZC Drow. | 0.337 | 0.092 | 0.362 |
+| Age | LZC NREM | 0.304 | 0.148 | 0.362 |
+| Age | LZC REM | 0.625 | 0.017^*^ | 0.362 |
+| CADES | LZC W | 0.124 | 0.531 | 0.704 |
+| CADES | LZC Drow. | 0.312 | 0.120 | 0.362 |
+| CADES | LZC NREM | 0.275 | 0.193 | 0.362 |
+| CADES | LZC REM | 0.572 | 0.033^*^ | 0.362 |
+| Memory | LZC W | −0.136 | 0.518 | 0.704 |
+| Memory | LZC Drow. | −0.056 | 0.1794 | 0.934 |
+| Memory | LZC NREM | 0.010 | 0.966 | 1.000 |
+| Memory | LZC REM | −0.583 | 0.047^*^ | 0.362 |
+| Inhibitory control | LZC W | 0.002 | 0.992 | 1.000 |
+| Inhibitory control | LZC Drow. | 0.048 | 0.816 | 0.934 |
+| Inhibitory control | LZC NREM | 0.055 | 0.798 | 0.934 |
+| Inhibitory control | LZC REM | −0.385 | 0.174 | 0.362 |
+| Detour | LZC W | −0.232 | 0.244 | 0.362 |
+| Detour | LZC Drow. | −0.343 | 0.093 | 0.362 |
+| Detour | LZC NREM | −0.315 | 0.143 | 0.362 |
+| Detour | LZC REM | −0.376 | 0.205 | 0.362 |
+| Eye gaze | LZC W | −0.242 | 0.214 | 0.362 |
+| Eye gaze | LZC Drow. | −0.244 | 0.230 | 0.362 |
+| Eye gaze | LZC NREM | −0.257 | 0.225 | 0.362 |
+| Eye gaze | LZC REM | −0.424 | 0.131 | 0.362 |
+
+## 4. Discussion
+
+In this study we evaluated changes in sleep architecture objectively using polysomnography, the gold standard technique used to describe and quantify sleep (27, 28). We were able to obtain artifact-free EEG data using surface (cup) electrodes in elderly dogs while sleeping in the hospital setting. We demonstrated that dogs with higher dementia scores spend less time sleeping in both NREM and REM sleep in the afternoon than dogs with lower scores. This supports owner observations that disruptions of the sleep-wakefulness cycle characterize CCDS (11, 17). Similarly, humans with AD suffer from insomnia and lower sleep quality (60, 61), and a bidirectional relationship between sleep and disease has been proposed (13) in which amyloid burden in the brain disturbs sleep (62–64) while poor sleep promotes amyloid β deposition and impairs memory consolidation (65, 66).
+
+We found that sleeping time was positively associated with the performance in a problem-solving detour task. Studies in humans have demonstrated that sleep plays a critical role in enhancing the skills needed to find novel strategies to solve problems (67, 68). One study has shown sleep dependent improvement in problem solving tasks, particularly those with higher difficulty (68). In dogs, Kis et al. have shown that sleep is associated with social learning skills (21). In the current study, longer sleep time was associated only with performance at the detour task and not with the other cognitive tasks. Similarly, a previous study on senior dogs (ages 7–14 years) testing the relationship between sleep spindles and cognition found that correlations were specific to, in that case, reversal learning only (69). Some human studies have shown that sleep deprivation can affect particular components of cognition differently (70–72). For example, Tucker et al. observed a larger effect of deprivation induced sleep loss in humans on non-executive rather than executive components of cognition, with working memory being less vulnerable than other domains (71). The main difference between human studies and our study is that they artificially deprive subjects to evaluate the effect of sleep deprivation, and in our study, reduction in sleep occurs naturally, likely due to aging and neurodegeneration. In addition, interindividual differences in the effects of sleep loss on cognition have been shown; some individuals can be more susceptible to attention impairment while others show working memory reduction (73). In dogs, the specific domains of cognition that are impaired by sleep loss are vastly understudied, with a sole paper suggesting that disturbance of NREM sleep affects dogs' socio-cognitive processing (74), but without reporting on the potential interindividual differences. The more challenging nature of the detour task we used, encompassing the interaction of multiple cognitive domains (18) may make this test more susceptible to the effect of sleep loss than other tests. This task requires cognitive stability (the ability to focus) and cognitive flexibility (the ability to adapt behavioral actions due to a change in demands) (75). We have previously shown that the detour task, together with inhibitory control and sustained gaze, is better able to discriminate between dogs with and without cognitive dysfunction than other tests such as the working memory task (18).
+
+### 4.1. Power and age
+
+Quantification of the EEG signal using power spectrum, coherence and complexity in different behavioral states revealed associations with age and cognitive function (25). As previously mentioned, power spectrum calculates the relative contribution of the frequency components of a signal, and it is a measure of local synchronization. During drowsiness, power of slow oscillations was lower for older dogs and for dogs with worse performance at the detour task. These oscillations are within the delta range and are characteristic of states of diminished levels of consciousness such as NREM sleep and anesthesia (76, 77). Our findings are consistent with the observation in humans (78), rats (79), cats (80), and dogs (28), that delta power declines with aging during NREM sleep, and this is considered a hallmark of reduction in sleep depth (81–83). In our study, the reduction in delta power with age was observed during both drowsiness and NREM sleep, however it only reached statistical significance for drowsiness. Of note, these associations were only observed in the Fz EEG channel, not in the Cz. In humans, it has been observed that frontal brain areas are more sensitive to aging and to response to sleep loss, and while in young subjects there is a predominance of delta waves in the frontal cortex, this predominance is lost with aging (84).
+
+### 4.2. Power and memory
+
+We identified associations between power and memory scores but not age during REM sleep, the period in which memory consolidation occurs. By contrast, Kis et al. (28) found an age-associated reduction in delta and an increase in alpha and beta power in dogs. However, they studied dogs from 1 to 8 years of age, without including senior or geriatric dogs. Performance at the memory task was positively correlated with delta power and negatively correlated with power at higher frequencies (beta and gamma). The biological relevance of this finding should be further evaluated in future studies and include dogs across a wider range of ages together in one study. While REM sleep is usually treated as a homogenous state, two different phases have been recognized, tonic and phasic REM sleep. These phases have different roles in information processing and memory consolidation and differ in the power of different oscillations, with higher power of alpha and beta bands during the tonic phase and higher gamma power on the phasic phase (85–87). Studies in dogs are needed to characterize the electroencephalographic features of each REM phase and whether they are associated with cognition and cognitive function.
+
+### 4.3. Interhemispheric coherence and age
+
+Coherence analyses are a measure of functional coupling between two distant cortical areas. Higher interhemispheric coherence within theta and alpha frequency bands was found in older dogs during wakefulness. This result was unexpected since in people, coherence in theta and alpha band decreases with aging (88, 89). However, those human studies included a wider range of ages, including young, middle age and senior adults, while in our study, we evaluated only senior and geriatric dogs. Our current findings concur with a previous study we performed in a separate cohort of dogs in which we found that dogs at risk of developing CCDS had higher interhemispheric coherence values than normal dogs or dogs with CCDS (41). It is important to note that those dogs at risk were also older than the normal dogs, and differences between groups could also be age-related.
+
+During REM sleep we observed a positive correlation between gamma interhemispheric coherence and age. In the normal individual, absence of gamma coherence characterizes REM sleep (90, 91), and gamma coherence shows its peak during wakefulness. Therefore, higher gamma coherence during REM sleep could be caused by shallower sleep states and frequent arousals and could suggest an age-related reduction in quality sleep in these dogs.
+
+### 4.4. Interhemispheric coherence and cognition
+
+There was a positive correlation between interhemispheric coherence and CADES score and a negative correlation with performance at the detour task during wakefulness. This was true for frequencies between 10.5 and 12 Hz, some of the same frequencies for which coherence was positively correlated with age. For these frequencies, the strength of the correlations was higher for age than for CADES or performance at the detour task. We have previously demonstrated that cognitive performance decreases with age (18), and therefore, it is possible that age is the main factor driving these coherence differences.
+
+During wakefulness, delta interhemispheric coherence (1–3 Hz) showed a negative correlation with performance at the sustained gaze task, a test that measures sustained attention. Dogs with shorter attention during the task had higher coherence. This mirrors studies in people with dementia that have shown an increase in delta coherence in comparison with healthy age-matched controls (40, 92). In addition to this, patients with attention deficit-hyperactivity disorders have elevated delta coherence in the frontal area of the brain (93).
+
+In our previous study we found that dogs with higher dementia scores had lower interhemispheric coherence in the gamma frequency band during wakefulness, but those results were not replicated in this study. One of the main reasons could be that in our previous study we used needle electrodes that have a much smaller recording surface than the cup electrodes. In fact, Musteata et al. found that values of interhemispheric coherence in dogs are dependent on the type of electrode used (94). A limitation of coherence analyses is that coherence values can be overestimated due to volume conduction in which the conductive properties of the brain, cerebrospinal fluid, skull, and scalp (and the conductive gel used during the recording), cause the neural source information to diffuse before reaching the electrodes (95, 96). Therefore, coherence measurements with cup electrodes might not be completely reliable in dogs, and future studies should evaluate more advances measures of functional connectivity that are able to overcome the volume conduction problem such as the phase lag index (95, 97).
+
+### 4.5. Intrahemispheric coherence and cognition
+
+Intrahemispheric coherence showed associations with performance in cognitive tasks but not with age or CADES scores. Higher coherence in different frequency bands during wakefulness, drowsiness and NREM sleep were associated with worse performance at the inhibitory control task. In humans with AD, Sankari et al. found an increase in delta, theta, and alpha intrahemispheric coherence during wakefulness (98). Finally, during REM sleep, dogs with better performance at the detour task showed higher intrahemispheric coherence within the alpha and sigma frequency bands. REM sleep in humans is characterized by the occurrence of alpha bursts with high occipito-frontal connectivity (99). It has been proposed that these alpha bursts may contribute to higher information processing during this state, and to the incorporation of external stimuli to dreams (99, 100).
+
+### 4.6. Complexity and memory
+
+Lempel Ziv Complexity evaluates the randomness of the EEG signal. We did not find any effect of aging on EEG complexity in any of the behavioral states evaluated. Regarding its association with cognitive status, dogs with better performance in memory tasks had a lower EEG complexity during REM sleep. Similarly, dogs with better performance in the detour task had also lower REM sleep complexity, but this result did not achieve statistical significance after multiple comparisons correction. It has been shown that complexity has its maximum value during wakefulness, is reduced during NREM sleep and increases to intermediate values during REM sleep (101, 102). Therefore, higher levels of complexity during REM in dogs with worse cognitive performance can be an indication of increased arousal, or shallower REM sleep.
+
+Together these EEG findings demonstrate that dogs with cognitive impairment spend less time sleeping and show EEG features of shallower sleep than dogs without cognitive impairment. Additionally, changes in power and coherence of specific frequency bands are associated with age or performance at some cognitive tests in aging dogs.
+
+### 4.7. Limitations
+
+Polysomnographic recordings in this study were completed during afternoon sleep and thus might not reflect the sleep architecture of dogs at nighttime. While there is evidence that at nighttime, dogs sleep more and spend less time in drowsiness and awake after first drowsiness (103), there are no data showing whether day vs. night sleep patterns are differentially affected by age (which would be a limitation to the present study). Thus, we do not know if and to what extent the present results would change if replicated during night-time recordings. Recordings during nighttime require considerably more effort from both the clinical staff and the participating owners, thus afternoon recordings are much more feasible for veterinary settings. Wireless polysomnographic equipment with bandages, Elizabethan collars and or special EEG caps designed to assure that electrodes stay in place (104) were not available for this study. In addition to this, while it has been demonstrated that dogs usually take naps around noon (5, 53), there is individual variability, and for some dogs the time chosen for recordings might not be their preferred nap time. However, the results observed in this study are in line with the sleep difficulties experienced by dogs with CCDS at night, since, as expected (9, 11), dogs with higher CADES scores spent less time sleeping. Future studies could compare these afternoon polysomnographic recordings with easier evaluations of activity at night time such as activity monitors (53) or sleep questionnaires (105). In addition to this, we have already demonstrated that cognitive function decreases with aging (18), and therefore, it is challenging to determine the relative contribution of age and cognitive impairment on changes in the sleep-wakefulness cycle and the qEEG features.
+
+## 5. Conclusion
+
+In conclusion, this study evaluates for the first time the correlation between sleep architecture and cognitive performance in aging dogs using polysomnography, the gold standard technique for sleep evaluation. We have demonstrated that dogs with higher CADES scores spent less time in both NREM and REM sleep. Additionally, when evaluating different domains of cognition, problem solving abilities showed a positive correlation with time spent in NREM and REM sleep and a negative correlation with time awake. Finally, we showed that quantitative electroencephalographic analyses during different behavioral states can demonstrate differences in dogs associated with age or cognitive performance which should be further evaluated for its potential clinical use to monitor the progression of CCDS.
+
+## Data availability statement
+
+The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
+
+## Ethics statement
+
+The animal study was reviewed and approved by Institutional Animal Care and Use Committee NCSU. Written informed consent was obtained from the owners for the participation of their animals in this study.
+
+## Author contributions
+
+AM and NJO were responsible for the design, analysis, and primary writing of the manuscript for this study. AK and MEG participated in the design. AM, CL, and MK participated in the data acquisition. DM and MC participated in the analysis of the data. All authors participated in editing and review of the manuscript. All authors contributed to the article and approved the submitted version.

fulltext/md/PMC10191315.md

@@ -0,0 +1,215 @@
+---
+pmcid: "PMC10191315"
+pmid: "37196042"
+doi: "10.1371/journal.pone.0285930"
+title: "A novel kinetic energy harvesting system for lifetime deployments of wildlife trackers."
+journal: "PloS one"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# A novel kinetic energy harvesting system for lifetime deployments of wildlife trackers
+
+## Abstract
+
+Wildlife tracking devices are key in obtaining detailed insights on movement, animal migration, natal dispersal, home-ranges, resource use and group dynamics of free-roaming animals. Despite a wide use of such devices, tracking for entire lifetimes is still a considerable challenge for most animals, mainly due to technological limitations. Deploying battery powered wildlife tags on smaller animals is limited by the mass of the devices. Micro-sized devices with solar panels sometimes solve this challenge, however, nocturnal species or animals living under low light conditions render solar cells all but useless. For larger animals, where battery weight can be higher, battery longevity becomes the main challenge. Several studies have proposed solutions to these limitations, including harvesting thermal and kinetic energy on animals. However, these concepts are limited by size and weight. In this study, we used a small, lightweight kinetic energy harvesting unit as the power source for a custom wildlife tracking device to investigate its suitability for lifetime animal tracking. We integrated a Kinetron MSG32 microgenerator and a state-of-the-art lithium-ion capacitor (LIC) into a custom GPS-enabled tracking device that is capable of remotely transmitting data via the Sigfox ‘Internet of Things’ network. Prototypes were tested on domestic dog (n = 4), wild-roaming Exmoor pony (n = 1) and wisent (n = 1). One of the domestic dogs generated up to 10.04 joules of energy in a day, while the Exmoor pony and wisent generated on average 0.69 joules and 2.38 joules per day, respectively. Our results show a significant difference in energy generation between animal species and mounting method, but also highlight the potential for this technology to be a meaningful advancement in ecological research requiring lifetime tracking of animals. The design of the Kinefox is provided open source.
+
+## Introduction
+
+Tracking wild animals has revealed fascinating insights into migration routes and movement patterns and has become an essential tool for conservationists in protecting everything from individual animals to sites of species level importance [1, 2]. The first attempt at wildlife tracking for scientific purposes can be traced back to the bird-ringing practices of Hans Christian Cornelius Mortensen in the early 1900’s, an innovation which paved the way for the revolutionary introduction of VHF wildlife trackers in the 1960s [3].
+
+Continuous technological advancements in energy storage, sensors, and wireless communications technology have since helped to decrease power consumption drastically [4]. However, devices capable of lasting more than a few years, and thus enabling lifetime tracking are still unavailable for most mammalian species. This is mainly due to challenges with the energy supply of mobile tracking devices on free-roaming animals. Today, most wildlife trackers are powered by non-rechargeable lithium-based batteries. The main challenge with batteries is their energy density which, when tracking for multiple years, will add significant weight that makes it impossible to track many smaller species. Arguably, the general consensus currently suggests that wildlife trackers for mammals should not exceed 3–5% of the animal’s body weight [5], or it might harm the animals’ fitness and movement capabilities [6–10]. Consequently, permits for tracking animals often come with a requirement that devices do not exceed 3–5% of the weight of the tracked animal [11]. Nonetheless, even if tags meet the mass requirements, the possibility of tracking smaller animals for multiple years is limited by battery longevity. Non-rechargeable lithium-based battery technology is advertised to be able to last for up to several decades (e.g., [12]), but studies suggest that wildlife tracking collar malfunctions in many cases are caused by early battery drainage [13], an issue probably caused by a combination of environmental effects (extreme temperatures) and inaccurate battery life estimation (etc. unpredictable energy consumption of GPS).
+
+The challenges regarding weight and longevity of batteries are most often solved with solar cells in combination with rechargeable batteries (e.g., lithium-polymer-based) or supercapacitors. As tracking technology evolved to have a much lower power demand, solar cells gained traction, especially with bird trackers and ear-tags where low weight is critical [14]. Solar cells have become an easy and affordable way to extend the lifetime of a tracking device, but they have their limitations. Few animals live constantly under direct sun, and many animals are nocturnal or live in habitats with a small amount of sun exposure, such as dense rainforests, caves, or merely in low-sunlight habitats at high or low latitudes. Solar cells also suffer from robustness issues, because they require a thin and transparent coating to ensure optimal energy generation [15–17]. To solve some of the challenges related to batteries and solar cells, scientists have attempted to harvest energy using alternative methods. These include a 286 g thermal energy harvesting solution on sheep [18], a 200 g and 20 cm long kinetic energy harvesting solution on reindeer [19] and a 40 g, 6 cm in diameter vibration energy harvester not including casing and collar material [20]. All results are promising but still limited by mass and size.
+
+Humans, on the other hand, have spent the last centuries perfecting energy harvesting in body-worn devices. Automatic watches, also known as self-winding watches, have existed since the late 18th century, and are still widely used. Automatic watches are equipped with a kinetic energy harvester that transforms the wrist’s movement into energy used to wind up the watch [21]. These wrist-worn kinetic energy harvesters are lightweight and small enough to fit into a wildlife tracker. These types of harvesters have not previously been used to harvest kinetic energy on animals to power wildlife trackers. In this study we have developed and explored the potential of using a kinetic energy harvesting unit, a 18g, 32mm diameter micro-generator from a smartwatch (Fig 1A) to create a kinetically powered wildlife tracker (the ‘Kinefox’) for long-term and eventually lifetime tracking of wild animals.
+
+*Figure: Fig 1 The Kinefox wildlife tracker concept.We tested if a lightweight kinetic energy harvester (A), could power an alternative to batteries (B) in the form of a Lithium-Ion Capacitor (LIC). The energy from the LIC was used for low-power sensors (C), including a GPS-module and an accelerometer. Data was then compressed and sent via the low-power wide-area network Sigfox (D). Finally, the data was used to estimate how much energy the harvester generated when mounted on an animals (E).*
+
+The energy generated from the harvester was stored in a lithium-ion capacitor LIC (Fig 1B). As many wildlife tracking studies are based on collecting and evaluating data from GPS and accelerometers [22], both sensors were integrated into the Kinefox (Fig 1C). The wireless communication ‘Internet of Things’ technology Sigfox was integrated for remote data retrieval (Fig 1D). The data were transmitted to an online database for real-time data analysis and energy generation calculations (Fig 1E). Harvesting kinetic energy from animals is only dependent upon the movement of an animal, a behavior that most animals exhibit–at least intermittently—until they die. While lifetime tracking of birds is now a reality due to many decades of development in durable solar tags [23], for many mammal species lifetime tracking is still only a distant reality [24, 25]. The development of a durable kinetically powered light-weight wildlife tracking device would enable lifetime deployments and tracking of mammals. It could, for example, revolutionize studies on mammalian dispersal, a critical life history phase in many mammals when individuals often experience strong natural (mortality) selection for reasons that largely illude scientists. Constant and long-term tracking of individuals will also be a very useful tool in animal-human conflict zones, as well as in conservation and population management in general. The overall goal of this study is to investigate whether the energy generated by the Kinefox prototype, when mounted on animals, is sufficient to daily transmit essential information about an individual via low-power Internet of Things communication technologies.
+
+## Methods
+
+### Ethics statement
+
+All equipment testing on animals were given with the owners’ verbal approval and witness by multiple co-authors some of whom where the owners of the dogs used for testing the Kinefox prototypes.
+
+### Electronics
+
+The Kinefox tracker is based on the 18 g, 32 mm diameter micro-generators (MSG32, Kinetron BV, Tilburg, Netherlands) used in the commercially available Sequent Supercharger smartwatch (Fig 2A).
+
+*Figure: Fig 2 Hardware overview of the Kinefox.The MSG32 micro-generator (A) generates an AC current, which is rectified into a DC current by the voltage doubling circuit (B). The current is then used to charge a LIC (C). A Tag-Connector connector (D) is used to charge the LIC as well as program the microcontroller (E). The energy is also used to power a Sigfox module (F), transmitting via a flexible printed circuit board (FPC) antenna (G). The Kinefox V2 also contains an accelerometer (H) and a GPS module (I) which is switched on and off with a load switch (J). The GPS module uses a patch antenna (K).*
+
+The MSG32 micro-generator is a pendulum-based automatic watch movement, where the pendulum is glued to a ferromagnetic ring. The pendulum with the ferromagnetic ring is placed around a coil of copper wire. When the pendulum swings back and forth, the ferromagnetic ring induces an alternating current in the coil. We integrated a voltage doubler rectifier to transform the alternating current into direct current (Fig 2B). The voltage created by magnetic induction depends on the speed of the magnet and thus the direct current output from the rectifier fluctuates based on the speed of the pendulum. Kinetron BV states that the generator is designed to directly charge a LiPo battery, which assumes a maximum voltage output of 4.2 V, when mounted on a human wrist. For energy storage it was critical to find an alternative to LiPo batteries, due to their poor longevity. A 30 F LIC (VPC, Vinatech) was chosen as this special hybrid supercapacitor is suitable for storing power from rapid high-voltage input, have low self-discharge/leakage and vastly better charge/discharge capabilities [26] than LiPo batteries while still maintaining an acceptable energy density (Fig 2C). Most LiPo batteries are advertised to withstand between 300–500 charge cycles. The selected hybrid supercapacitor advertises 20.000 charge cycles, vastly outperforming LiPo batteries and thus increasing the potential longevity of the Kinefox device. The LIC also has the advantage of a linear relationship between state-of-charge and voltage [27], which makes it possible to estimate energy generation based on voltage change.
+
+The LIC can be charged via a TC2030 Tag-Connect connector (Fig 2D), which is also used to program the microcontroller (ATtiny2627, Microchip technology) (Fig 2E). For remote data transmission we chose Sigfox as the wireless data transmission platform due to its low energy consumption [28, 29], easy integration, and coverage in Denmark and Germany where case studies were conducted. A Sigfox module from ON Semiconductors (AX-SIP-SFEU-1-01-TX30, ON Semiconductors) was selected due to its small footprint (Fig 2F). Furthermore, Sigfox has an integrated location service called Sigfox Atlas Native, which gives a rough estimate of geolocation without requiring additional energy (Triangulation-based). Whip-antennas are traditionally used in wildlife trackers, but they are known to be fragile [30]. Since the goal of the Kinefox is to achieve lifetime operation, internal antennas were explored to maximize longevity. An FPC antenna (8/9-FPC 868, Linx Technologies) (Fig 2G) and a homemade bent-wire monopole antenna made from 1mm cobber wire (S1 Fig) were used for Sigfox. Two hardware versions of the Kinefox were tested in this study. The Kinefox V2 is the one illustrated in the figures throughout this paper. V2 contains the same components as V1, but additionally integrates an accelerometer (MC3635, MEMSIC Inc.) (Fig 2H) and a low-power GPS module (L70B-M39, Quectel) (Quectel) (Fig 2I). Furthermore, the eRIC Sigfox module (eRIC-Sigfox, LPRS) was used in V1 instead of the module from ON-Semi. This is a result of supply-chain issues (performance is almost identical). To keep the power consumption as low as possible, the power to the GPS is turned on and off with a low-power load switch (SIP32431DNP3, Vishay Semiconductors) (Fig 2J). The Adafruit 2461 patch antenna (2461, Adafruit) was used for GPS (Fig 2K). For both Kinefox versions, all the components were placed on a 2-layer printed circuit board (PCB). All design files for both versions of the Kinefox as well as a list of specific components used are open-source available on GitHub (https://github.com/TroelsG/Kinefox).
+
+### Embedded software
+
+Embedded software for the Kinefox was created in C/C++ in Microchip studio and programmed via an Arduino Nano flashed with the jtag2updi firmware [31]. Three firmware versions were used in the experiments. Version 1 and 2.1had the main purpose of sampling and transmitting the voltage of the LIC, used to estimate energy generation, based on the method presented in the Measuring energy generation section. Firmware 2.2 had the main purpose of testing the GPS module and accelerometer, as well as long-term durability and function. All three firmware versions are illustrated as a flowchart in S2 Fig. All firmware versions are based on a loop that can be divided into the following steps: sleep, wakeup, data sampling and transmission. Throughout this paper, these steps will be referred to as a “transmission cycle”.
+
+Firmware 1. This firmware was used in the experiments involving all tests with Kinefox V1. In this firmware, the device was coded to wake up and sample the voltage of the LIC. If the voltage was below 2.9V the device would go to sleep for 4h and continue this cycle until the voltage was above 2.9V. This was to avoid getting below the 2.5V cut-off voltage of the LIC. Four hours was selected as the sleep period, because it was assumed that none of the test animals could generate enough power to overcharge the LIC within this period. If the voltage on the other hand was above 2.9V and below 3.55 V, the firmware code would compress the sampled voltage into 2 bytes in HEX format, transmit via the Sigfox network, go to sleep for 4 hours, and then repeat the transmission cycle. If the voltage was above 3.55 V, the device would go to sleep for only 60 seconds and then repeat the cycle. This was done to quickly lower voltage to avoid overcharging of the LIC. If voltage was above 2.9 V and below 3.55 V every time it woke up, a transmission cycle would be 4 hours long.
+
+Firmware 2.1. This firmware was used for the experiments involving energy generation with Kinefox V2. This firmware is identical to Firmware 1, except that after every transmission cycle, the device goes to sleep 6 times, resulting in a transmission cycle length of 24 hours if voltage was between 2.9 V and 3.55 V at wakeup. After every 4 h sleep period, the device sampled voltage and if was above 3.55 V, the sleep period was lowered to 20 minutes and a transmission cycle would be executed at every wakeup to lower voltage, until the voltage was again above 2.9 V. Furthermore, in this firmware, the 2 byte payload was saved on EEPROM. This way, the data could be downloaded from the device in case of failed transmissions.
+
+Firmware 2.2. The purpose of this firmware version was to test the functionality of the GPS-module and accelerometer. This firmware is identical to Firmware 2.1, only with the addition of what data to transmit. Version 2.1 was coded to sample and transmit 4 seconds of 54 Hz VeDBA sum burst acceleration data (2 bytes), voltage of the LIC (2 bytes), TTF of the GPS fix (1 byte) and a GPS fix (7 bytes), thus transmitting a 12 bytes message.
+
+In addition to the data transmitted by the Kinefox via Sigfox, the timestamp of when the message was received by the Sigfox backend, as well as a sequence number, are part of the metadata of each message. For Firmware 2.1 and 2.2, the Sigfox Atlas positioning data is also included in the metadata.
+
+### Measuring energy generation
+
+The objective of the Kinefox prototypes is to determine how much energy can be produced by the MSG32 micro-generator. To accomplish this, we created a method for measuring the energy generated by the MSG32. This method exploits the approximately linear relationship between the state-of-charge and voltage in LICs. To determine energy generation based on this relationship, we first had to determine a baseline for how much the voltage would drop due to the energy consumption of one transmission cycle without any generated energy.
+
+Two baseline experiments were conducted with the two Kinefox versions with Firmware 1 and 2.1, respectively, which were placed on an office desk until the LIC voltage reached 2.9 V. This resulted in two datasets containing the voltage of the LIC over time, which was fitted with a simple linear regression model with the y-intercept at the origin. The resulting baseline datasets, along with their linear regression lines, are depicted in Figs 4 and 5. The slope coefficient (mV) of these two baseline datasets represents the voltage drop caused by one transmission cycle.
+
+The same experimental setup was employed in the case studies, where we also fitted the data with simple linear regression models. By calculating the difference between the slope coefficient of the linear regression model from the case studies with that of the baseline experiments, we were able to determine the voltage increase caused by the generated energy of the MSG32. Dividing this by the slope coefficient of the baseline experiment allowed us to calculate the number of potential transmission cycles that could be executed with the generated energy.
+
+```
+Generatedenergy(Numberofpotentialtransmisioncycles)=−Slopecoefficient(Baseline)−Slopecoefficient(Casestudy)Slopecoefficient(Casestudy)
+```
+
+The number of potential transmission cycles was converted into joules by multiplying it with the energy consumption of one transmission cycle.
+
+```
+Generatedenergy(Joule)=−Slopecoefficient(Baseline)−Slopecoefficient(Casestudy)Slopecoefficient(Casestudy)⋅Energyconsumptionforonetransmissioncycle
+```
+
+### Enclosure
+
+The enclosure for the Kinefox V1 was 3D-printed in PETG plastic (Ultimaker B.V, Utrecht Holland), on a FDM printer (Ultimaker B.V, Utrecht, Holland). In the Kinefox V1, the MSG32 harvester was soldered to the PCB and the enclosure was externally sealed with epoxy. The Kinefox V1 weighed approximately 75 grams.
+
+The enclosure for the Kinefox V2 was printed in CPE+ (Ultimaker B.V, Utrecht Holland). In the Kinefox V2, the PCB (Fig 3A and 3B) had the MSG32 (Fig 3C) mounted with pogo-pins and both parts were then mounted to the enclosure (Fig 3D) with a M3 screw and a heat-set threaded insert.
+
+*Figure: Fig 3 The Kinefox V2.A) Top view of the PCB. B) Back view of the PCB. C) The MSG32 micro-generator. D) Top view of the assembled Kinefox placed within the CPE+ printed casing. E) Assembled Kinefox. F) Kinefox mounted in collar made in Biothane. Kinefox and collar is wrapped in heat-shrink for increased waterproofing.*
+
+The enclosure for Kinefox V2 was sealed with a 3D-printed lid and waterproofed with a silicone greased O-ring (Fig 3E). For testing on free-ranging animals, the housing for V2 was additionally sealed with 50 mm diameter heat-shrink and CA-glued tight to the collar (Fig 3F). The total weight of the Kinefox V2 is approximately 150g, varying according to collar size. It should be noted that weight has not been optimized in this project. Instead, robustness was prioritized. If for example the enclosures were to be injection molded, similar toughness could be achieved with significantly less weight.
+
+### Fittings
+
+In order to investigate the real-world energy generation potential of the Kinetron MSG32, it is important that the collar attachment on the animals resembles “industry standard” as much as possible, as the mounting method can affect the movement of the tracker and thus the energy generation. Collars used for wildlife trackers must last for years and thus be made of a durable material. Collars also need a tight fit against the neck to avoid entanglement with vegetation, rubbing abrasions or falling off. In order to test the Kinefox V1 as early as possible, the devices were mounted in the existing collars and harnesses of the test animals. Based on the learnings from V1, we developed a custom collar (Fig 3F) for the Kinefox V2 based on Biothane (Biothane USA, USA), a nylon-webbing overmolded with PVC plastic. SmartParks use this material for their collar design [32], and we thus knew that it was field-tested to withstand the rigors of wild animals and weather. The Kinefox V2 was bolted to the collar and closed with the same flatheaded bolt and nut system as most conventional wildlife tracking collars [33].
+
+### Case-studies
+
+The Kinefox V1 was tested on two domestic dogs (*Canis lupus familiaris*) (Dog 1 and Dog 2) and a wisent (*Bison bonasus*). For Dog 1, the Kinefox was mounted in its everyday collar (Experiment 1). For Dog 2, two devices were mounted. They were mounted in a harness that the dog was used to, one on the back of the harness (Experiment 2) and one on the chest (Experiment 3). The dog was used to the harness. For the experiments with the dogs, the goal was 6 days of deployment. The Kinefox on the wisent, was mounted to the side of a Vectronics collar (Experiment 4) with the goal of being deployed until device failure. All experiments with the Kinefox V1 was flashed with Firmware 1.
+
+Based on these preliminary results we constructed the complete collar version (Kinefox V2). This prototype was fitted on two domestic dogs (Dog 3 and Dog 4) (Dog 2 and 4 is the same animal) that had their daily walks measured by the owners over a 14-day period to gain insight into how much energy was generated per hour of walking (Experiment 5 and 6). The Kinefox V2 was then mounted on a free-roaming Exmoor pony (*Equus ferus caballus*) for 12 days (Experiment 7). In the experiments with the Exmoor pony, a version with a bend wire antenna for Sigfox communication were used (S1 Fig). In experiment 5–7, the devices were flashed with Firmware 2.1.
+
+No readily available data exist on the TTF of modern GPS modules in wildlife trackers. TTF listed in datasheets of GPS-modules are often bias towards optimal conditions, and do not reflect the conditions of being mounted on a wild animal in their natural habitat. To obtain a rough estimate of TTF on wild animals, the collar fitted to the Exmoor pony was programmed to change to Firmware V2.2 after 12 days (Experiment 8). The goal was to give a rough idea about expected energy consumption by GPS on wild animals, in order to evaluate potential to use the Kinefox for GPS positioning. In addition to GPS-fixes, 4 seconds of VeDBA burst sum acceleration data was sampled at 54 Hz [34], and transmitted within the Sigfox message (12 bytes in total). One of the goals with this experiment was also to test the longevity of the device, so deployment time was set to be until the device stopped functioning. An overview of all the experiments and how they were configured can be seen in Table 1.
+
+**Table 1 Experiments overview.The table contains all information for each experiment including: firmware version, duration, what animal it was tested on and hardware. In experiments 1–7, the voltage remained between 2.9 V and 3.55 V for the entire duration of the experiment, resulting in regular transmission cycle rates. Experiment 8 generated less energy than it took to execute one transmission cycle per day and thus fell below 2.9 V. After this, the device would only sample and transmit when the voltage surpassed 2.9 V. This resulted in message intervals between 4 days and 24 days, depending on energy generation as well energy consumption by the GPS module as a result of varying TTF.**
+
+| Experiment no. | Firmware version | Kinefox Hardware version | Duration | Animal | Data sampled and transmitted | Transmission cycles per day | Fittings | Sigfox antenna | GPS antenna |
+| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
+| 1 | 1 | V1 | 6 days | Dog 1 | Voltage of LIC (2 bytes) | 6 | The device was mounted in the everyday collar of the dog. | Linx FPC 8/9 868 | None |
+| 2 | 1 | V1 | 6 days | Dog 2 | Voltage of LIC (2 bytes) | 6 | The device was mounted on the chest in the everyday harness of the dog. | Linx FPC 8/9 868 | None |
+| 3 | 1 | V1 | 5 days | Dog 2 | Voltage of LIC (2 bytes) | 6 | The device was mounted on the back in the everyday harness of the dog | Linx FPC 8/9 868 | None |
+| 4 | 1 | V1 | 17 days | Wisent | Voltage of LIC (2 bytes) | 1 | The device was mounted on the side of a Vectronics collar. | Linx FPC 8/9 868 | None |
+| 5 | 2.1 | V2 | 14 days | Dog 3 | Voltage of LIC (2 bytes) | 1 | The device was mounted in a custom collar | Homemade wire antenna | Bentoni (Not used) |
+| 6 | 2.1 | V2 | 14 days | Dog 4 | Voltage of LIC (2 bytes) | 1 | The device was mounted in a custom collar | Linx FPC 8/9 868 | Bentoni (Not used) |
+| 7 | 2.1 | V2 | 12 days | Exmoor pony | Voltage of LIC (2 bytes) | 1 | The device was mounted in a custom collar | Linx FPC 8/9 868 | Bentoni (Not used) |
+| 8 | 2.2 | V2 | 146 days | Exmoor pony | Voltage of LIC, VeDBA sum burst, GPS, TTF (12 bytes) | <1 | The device was mounted in a custom collar. | Homemade wire antenna | Adafruit patch |
+
+## Results
+
+### Power consumption
+
+We measured the average power consumption of the Kinefox V1 and V2 (Table 2) using an Otii Arc (Qoitech AB, Lund, Sweden) power analyzer at 3.3V. The recording of the power consumption can be found in the project GitHub.
+
+**Table 2 Energy consumption overview of Kinefox.Power consumption recordings were made with the Otii arc power analyzer at 3.3 V. Total energy consumption represent the energy it takes to execute one transmission cycle.**
+
+| **Kinefox V1 (Firmware 1)** |  |  |  |
+| --- | --- | --- | --- |
+| Function | Time | Current | Energy consumption (J) |
+| Sigfox transmission (2 bytes) | 8.11 s | 31.2 mA | 0.84 |
+| Wakeup and LIC voltage sampling | 40 ms | 89.2 uA | 1.18 ·10^−5^ |
+| Sleep | 4 h | 2 uA | 0.095 |
+| Total energy consumption |  |  | 0.94 |
+| **Kinefox V2 (Firmware 2.1)** |  |  |  |
+| Sigfox transmission (2 bytes) | 8.17 s | 27.8 mA | 0.75 |
+| Wakeup and LIC voltage sampling | 40 ms | 175 uA | 2.31 · 10^−5^ |
+| Sleep | 24 h | 0.95 uA | 0.27 |
+| Total energy consumption |  |  | 1.02 |
+| **Kinefox V2 (Firmware 2.2)** |  |  |  |
+| Sigfox transmission (12 bytes) | 10.2 s | 31.3 mA | 1.05 |
+| Wakeup and LIC voltage sampling | 40 ms | 175 uA | 2.31 · 10^−5^ |
+| Sleep | 24 h | 0.95 uA | 0.27 |
+| GPS | 59.56s* | 16.6 mA | 3.26 |
+| Acc | 4.16 s | 265 uA | 0.0036 |
+| Total energy consumption |  |  | 4.6 |
+
+The total power consumption per transmission cycle for the Kinefox V1 with Firmware 1 was 0.94 joules. The total power consumption per transmission cycle for the Kinefox V2 with Firmware 2.1 was 1.02 joules. The total power consumption per transmission cycle for the Kinefox V2 with Firmware 2.2 was 4.6 joules (Table 2).
+
+### Case-studies results
+
+The results from experiment 1 showed that Dog 1 with the Kinefox attached to a standard dog collar generated an average of 2.26 joules per day (Fig 4 and Table 3).
+
+*Figure: Fig 4 Chart illustrating energy generation from experiments 1–4.Experiments were conducted with four different devices on three different animals. Baseline value (grey) shows the decrease in voltage in the LIC over a 9-day period, with no energy generation from the MSG32 and transmitting a 2-byte Sigfox message every 4 hours. Gaps in chart indicate failed Sigfox transmission. Simple regression models have been fitted to all datasets and the slope coefficient is shown in the legend.*
+
+**Table 3 Experimental results of energy generation from experiments 1–4.**
+
+|  | Dog 1 (Exp. 1) | Dog 2 (Exp. 2) | Dog 2 (Exp. 3) | Wisent (Exp. 4) | Baseline |
+| --- | --- | --- | --- | --- | --- |
+| Slope coefficient (mV)* | -3.21 | 4.17 | -2.31 | -3.08 | -5.35 |
+| Generated energy (Number of potential transmission cycles)** | 2.40 | 10.68 | 3.41 | 2.55 |  |
+| Generated energy (Joule)*** | 2.26 | 10.04 | 3.20 | 2.39 |  |
+
+The Kinefox mounted on the chest of Dog 2 in experiment 2 generated an average of 10.04 joules per day, while the Kinefox mounted on the back of Dog 2 in experiment 3 generated an average of 3.20 joules per day (Fig 4 and Table 3). The Kinefox was mounted on all three dogs for 6 days, but on the last day of testing, the MSG32 broke in experiment 3 (Ferromagnetic ring broke, could hear it rattling), resulting in only 5 days. The Kinefox on the wisent in experiment 4 generated an average of 2.39 joules per day and survived 17 days (Fig 4), but data is only showed for 8 days. This was due to the fact that the wisent was kept in a small enclosure for the first week and was not representative of normal behavior. Summarized in Table 3, experiments 1–4 generated on average between 2.26 and 10.04 joules per day, which translates into enough energy to execute between 2.4–10.68 transmission cycles. The Kinefox V2 on Dog 3 in experiment 5 generated an average of 1.10 joules per day, when walked an average of 68 min/day (Fig 5 and Table 4).
+
+*Figure: Fig 5 Chart illustrating energy generation from experiments 5–7.Experiments were conducted with three different devices on three different animals. Baseline value (grey) shows the decrease in voltage in the LIC over a 14-day period, with no energy generation from the MSG32 and transmitting a 2-byte Sigfox message every 24 hours. Gaps in chart indicate failed Sigfox transmission. Regression models have been fitted to all datasets and the slope coefficient is shown in the legend.*
+
+**Table 4 Experimental results of energy generation from experiments 5–7.**
+
+|  | Dog 3 (Exp. 5) | Dog 4 (Exp. 6) | Exmoor pony (Exp. 7) | Baseline |
+| --- | --- | --- | --- | --- |
+| Slope coefficient (mV)* | 0.58 | 8.82 | -2.38 | -7.32 |
+| Generated energy (number of potential transmission cycles)** | 1.08 | 2.20 | 0.67 |  |
+| Generated energy (joule)*** | 1.10 | 2.25 | 0.69 |  |
+
+The Kinefox V2 on Dog 4 in experiment 6 generated and average of 2.25 joules per day, when walked an average of 88 min/day (Fig 5 and Table 4). Finally, the Kinefox V2 on the free-roaming Exmoor pony in experiment 7 generated an average of 0.69 joules per day. Overall, the Kinefox V2 generated on average between 0.69–2.25 joules per day, with the Exmoor pony generating the least energy, yet still enough energy to execute on average of 0.67 transmission cycles per day, while Dog 4 generated the most at 2.20 transmission cycles messages per day. The Kinefox was mounted on both dogs for 14 days.
+
+After 12 days on the Exmoor pony, experiment 8 started and the Kinefox V2 started to sample GPS fixes and accelerometer data once a day. At the submission of this paper, a total of 27 GPS fixes were obtained and transmitted, but only 16 received (due to poor antenna performance, not optimized in this study; see Fig 6 for map with GPS points).
+
+*Figure: Fig 6 Map of GPS locations from experiment 8.Red diamonds indicate GPS-fixes. All GPS-fixes are within the fenced area (dotted magenta line) that the Exmoor pony roams.*
+
+The average TTF of these 16 fixes were 59.56 seconds. The power consumption of the GPS module is not constant as it attempts to obtain a GPS fix, but an average was measured to be ~16.6 mA (Table 2). By assuming an average current of 16.6 mA for 59.56 seconds, it required an average of 3.26 joules (at 3.3 V) to obtain a GPS-fix for the Kinefox V2 on the Exmoor pony. The Kinefox V2 on the Exmoor pony reached 2.9V and went to sleep on 2022-12-19. The Kinefox has since woken up and sampled and transmitted 14 GPS fixes. The subsequent intervals between attempts to obtain GPS-fixes and transmit them via Sigfox has been between 4 and 24 days (due to variation in energy generation and GPS TTF).
+
+The accelerometer data confirmed that the Exmoor pony had various levels of activity when it was sampled (S3 Fig) and confirmed that the collar was still fitted on a living animal. The Kinefox on the Exmoor is still ongoing and last transmitted 2023-04-17, 147 days after being deployed.
+
+## Discussion
+
+In this study we demonstrate the potential of harvesting kinetic energy of animals to power a wildlife tracker continuously. Using the Kinetron MSG32 micro-generator with state-of-the-art energy storage and low-power electronics, it is demonstrated that it is possible to harvest from 0.69 J on an Exmoor pony to 10.04 J on a domestic dog. The Kinefox V1 on the chest of Dog 2 showed significantly higher values than all in all other experiments. Observations of Dog 2 (Exp. 2) in motion showed that the chest-mounted tracker swung back and forth due to the softness of the harness material. For this reason, a rigid, tighter collar, similar to what is used in conventional wildlife tracking collars, was developed for the Kinefox V2. Consequently, the results from Dog 2 (Exp. 2) are not regarded as representative of the potential of the Kinefox.
+
+The aim of this study was to evaluate if kinetic energy could be used to power a wildlife tracker for obtaining lifetime tracks of mammals [24]. In lifetime tracking the least understood phase is often juvenile dispersal, yet it is the most critical for understanding survival and how movement syndromes evolve in mammals [25]. The Kinefox V2 prototype weighs 150 g including collar material, making the Kinefox light enough to be mounted on animals as small as 3 kg when applying the 5% body mass rule [5]. It should be noted that the design of the Kinefox was not primarily optimized for weight at this point, but for robustness instead, and there is great potential for decreasing the weight for use on smaller animals or potentially in an ear-tag. In fact, the weight of the MSG32 harvester itself should be compared to the weight of conventional batteries. In the Kinefox, the MSG32 harvester and a 30 F LIC replaces a conventional battery. These two components weigh a total of 19.9 g. This is roughly equivalent to two non-rechargeable batteries (8.8 g SAFT batteries [35]) that each have a capacity of 1.2Ah (14.255 J at 3.3 V). For a wildlife tracker that uses these two batteries instead of the MSG32 and consumes 2.25 joules per day (2.25 joules is the energy generated on a domestic dog in experiment 6), a quick estimate (2 · 14255 J/ 2.25 J)/365 days) means it will last for 35 years, disregarding self-discharge of the battery. To make the case that the MSG32 is a suitable alternative to these batteries, one or more of the following assumptions must be made.
+
+- That the MSG32 and the LIC can outlast the 35 years of the batteries.
+- That batteries will discharge significantly due to age or environmental conditions before 35 years have passed.
+- That the MSG32 can generate significantly more energy on wild animals than on domestic animals.
+
+Looking at the first assumption, even if the MSG32 could survive 35 years, it is unplausible that the housing and collar of any wildlife tracker could last this long.
+
+With regards to the second assumption, it is well known that early battery drainage is a huge issue in wildlife tracking [13]. Early battery drainage is most often caused by extreme temperatures, a phenomenon very common in nature. Low temperatures increase resistance in the battery, resulting in lower voltage and thus lower efficiency and higher energy consumption. Overall capacity will also drop due to low temperatures. Oppositely, high temperatures result in increased self-discharge. The battery used in the above example is advertised to have a self-discharge of less than 1% per year [35]. There is however no documentation for exactly how much temperature will affect self-discharge and it is therefore very difficult to verify, since little to no research have been done on these types of batteries (Li-SOCI2). Regardless, without testing, this makes batteries an unreliable energy source for long-term studies.
+
+Considering the third assumption, there is amble evidence that wild animals move more than domestic. The domestic dogs from experiment 6 was walked 1.48 hour per day but were otherwise confined to be indoors. A study by Theuerkauf, Rouys [36] on 11 wolves found that they spend on average 8.6 hours a day moving. If we assume that movement characteristics are similar to the those of the domestic dog in experiment 6, the wolves could generate up to 13.07 joules per day (2.25 J *(8.6 h/1.48 h)). Assuming this, a wildlife tracker powered by batteries would only last for 6 years (under the assumption that the battery tracker powered was also set to consume 13.07 joules per day). This would change the advantage in using MSG32 drastically, assuming the Kinefox and the MSG32 can last for longer than 6 years. For the MSG32 to be superior to the two SAFT batteries, the MSG32 will have to outlive the batteries. This emphasizes not only the importance in further testing of batteries, but importance of testing the Kinefox prototype in long-term studies on multiple species of wild animals to evaluate the longevity of the MSG32 in real-world scenarios. The MSG32 consists of moving parts and all moving parts experience wear over time. In experiment 3, the ferromagnetic ring broke, a matter that also needs further investigation. It is also worth pointing out that the MSG32 is not specialized for harvesting energy from animals, but from human wrists. Many parameters can be changed to optimize for harvesting on wild animals. The weight of the pendulum, the strength of the magnet and the turns of the copper coil can all be modified.
+
+Maybe, at some point it will be possible to engineer a 5-g harvester that can be used in low-weight ear-tags, as well as on body attachment in birds, fish and small mammals.
+
+Other than optimizing the MSG32 platform itself, there is also potential in exploring placement. In this study, the MSG32 was placed horizontally within the collar (the tracker on the wisent was placed vertically). These were perhaps not the ideal directions to mount the harvester and we suggest that the ideal mounting position varies from species to species and should be optimized. As animal species differ vastly in size and shape as well as movement, it is currently difficult to predict why some species generate more energy than others, but knowing more about which specific movement patterns generate the most energy would make it easier to estimate which animals might be suited for kinetically powered trackers. It would also be interesting to explore the potential in other kinetic harvesting concepts like the spherical harvester developed by Hall and Rashidi [37], which can convert movement to energy from multiple directions.
+
+Nevertheless, even in its current form, the Kinefox might be able to close the gap in wildlife tracking of animals where lifetime data is needed for an ecological study. In order to exemplify the applications of the Kinefox, we present two conceptualized use-cases (Fig 7).
+
+*Figure: Fig 7 Potential use-cases of the Kinefox wildlife tracker inspired by real world scenarios.A) Long distance juvenile dispersal of a wolf from Germany to Denmark. From our results on domestic dogs, it is possible that a wolf moving substantially more would generate enough energy to obtain a daily GPS fix and transmit it via Sigfox. The Sigfox Native Atlas function could serve as an alternative mean of positioning the wolf, though with a larger accuracy error than GPS. B) Monitoring health of animals using accelerometry data. Using VeDBA burst sum, as in experiment 7, it would be possible to study the activity patterns on animals as an indicator for their health and mortality. A sharp change in activity could be an indication that the animal needs attention by caretakes and would serve as a useful monitoring in e.g., a rewilding program.*
+
+### Long distance dispersal
+
+When conducting ecological studies on animals that disperse hundreds or thousands of kilometers, precision GPS might not be necessary. Examples of long-distance dispersers in Europe are wolves (*Canis lupus*) that have been found to disperse in the excess of 3000 km (Fig 7) [38, 39] and red foxes (*Vulpes vulpes*) that disperse for more than a 1000 km [40]. Knowing that the domestic dogs in this study generated enough energy to transmit 1–2 Sigfox messages a day, it is reasonable to assume that a wild wolf or fox can generate at the least the same amount of energy. And as mentioned previously, it can be estimated that wolves can generate up to 13.07 joules per day, enough to take several GPS-fixes a day (Table 2) [36]. Even if wolves or foxes only generate the same amount of energy as domestic dogs, there is potential for daily positioning. Using the Sigfox Atlas Native location service (requires no additional energy), a rough position with an accuracy between 800 m to 20 km can be achieved. Even though the accuracy of the Sigfox Atlas Native location service is not in the realm of modern GPS locations it would be sufficient to create valuable data about dispersal and migration behavior over not only long distances, but for longer than any existing solutions [29, 41]. Accelerometer data could be used to indicate mortality. Thus, the Kinefox would enable studies on animals that migrate or disperse throughout their entire lives.
+
+### Health monitoring in rewilding projects
+
+Rewilding projects are increasing in numbers around the world, and especially in Denmark these projects are used to bring back nature. One of the challenges in Danish rewilding projects is that they are regulated by the same laws as domestic animals. The legislation concerns the welfare of animals and to prevent die-offs due to starvation as it happened at the Dutch rewilding project at Oostvaardersplassen. In the winter of 2017/2018 winter, 3.380 deer, horses and cattle died either by natural death or due to starvation because the carrying capacity of the area was surpassed [42]. For managers, this means that the animals must be monitored frequently, a task that can be challenging to do in person. This task could be solved by the Kinefox. Accelerometer data could be sampled several times a day, summarized, and then transmitted once a day via Sigfox (Fig 7). Accelerometry data from rewilding animals fitted with the Kinefox could provide information on how the activity patterns of the animal as an indicator of the health status, as it has been proven useful for in the studies on pigs and cattle by Chapa, Maschat [43] and Riaboff, Shalloo [44]. Four seconds of VeDBA sum burst only requires 0.0036 joules and is sufficient to inform about mortality, thus it would be possible to sample multiple times a day, even with as little energy generation as generated by the Exmoor pony. If for any reason management must attend an animal, an uplink message can be sent to the Kinefox, making it sample and transmit a GPS fix. This might only be necessary to do once a month or less. Even with only 0.69 J generated per day on the Exmoor pony, a GPS fix once a month is possible (as shown in experiment 8). The reason why the Kinefox has an advantage over battery-powered solutions in this case, is that these animals are wild and not handled. Avoiding routinely sedating the animals to replace trackers on the animals is a major advantage.
+
+These examples are based on the results achieved from the tests in this study, but different animals generate different amounts of energy and only the MSG32 micro generator have been tested. To verify the potential of kinetically powered wildlife trackers, continued development and exploration of the concept should be performed. This is true both in terms of weight, efficiency, and longevity, but also the performance on various species over longer periods of time.
+
+With the Kinefox we have shown that the combination of state-of-the-art electronics and a lightweight kinetic energy harvester can generate enough energy to make a self-powering wildlife-tracker that potentially could last for the entire lifetime of a wild animal.

fulltext/md/PMC10219152.md

@@ -0,0 +1,96 @@
+---
+pmcid: "PMC10219152"
+pmid: "37233184"
+doi: "10.3390/jcdd10050217"
+title: "Spatial and Temporal Non-Uniform Changes in Left Ventricular Myocardial Strain in Dogs with Duchenne Muscular Dystrophy."
+journal: "Journal of cardiovascular development and disease"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Spatial and Temporal Non-Uniform Changes in Left Ventricular Myocardial Strain in Dogs with Duchenne Muscular Dystrophy
+
+## Abstract
+
+Background: Understanding and effectively treating dystrophin-deficient cardiomyopathy is of high importance for Duchenne muscular dystrophy (DMD) patients due to their prolonged lifespan. We used two-dimensional speckle tracking echocardiography to analyze more deeply the non-uniformity of myocardial strain within the left ventricle during the progression of cardiomyopathy in golden retriever muscular dystrophy (GRMD) dogs. Methods: The circumferential strain (CS) and longitudinal strain (LS) of left ventricular (LV) endocardial, middle and epicardial layers were analyzed from three parasternal short-axis views and three apical views, respectively, in GRMD (n = 22) and healthy control dogs (n = 7) from 2 to 24 months of age. Results: In GRMD dogs, despite normal global systolic function (normal LV fractional shortening and ejection fraction), a reduction in systolic CS was detected in the three layers of the LV apex but not in the LV middle-chamber and base at 2 months of age. This spatial heterogeneity in CS progressed with age, whereas a decrease in systolic LS could be detected early at 2 months of age in the three layers of the LV wall from three apical views. Conclusions: Analyzing the evolution of myocardial CS and LS in GRMD dogs reveals spatial and temporal non-uniform alterations of LV myocardial strain, providing new insights into the progression of dystrophin-deficient cardiomyopathy in this relevant model of DMD.
+
+## 1. Introduction
+
+Duchenne muscular dystrophy (DMD) is an X-linked hereditary disease affecting approximately 1 in 5000 male births worldwide and is caused by mutations in the gene that encodes the 427-kDa cytoskeletal protein dystrophin. Dystrophin forms a dystrophin protein complex with dystrophin-associated proteins including dystroglycans, sarcoglycans, integrins and caveolin in the sarcolemma, linking the internal cytoskeleton to the extracellular matrix in striated muscle and playing an important role in stabilizing the cell membrane and signal transduction during muscle contraction [1]. The vast majority of DMD patients lack the dystrophin protein, making the membrane susceptible to damage by mechanical stress which can lead to muscle degeneration. DMD is characterized by progressive muscle degeneration and weakness. Beginning as early as 2 to 3 years of age, the principal symptom of DMD is muscle weakness, affecting the proximal muscles first, then the distal limb muscles and usually the lower external muscles before the upper external muscles. Generally, DMD patients are wheelchair-bound by 12 years of age and die of respiratory failure in their late teens or early twenties. Although there is currently no effective treatment for this disease, patients with DMD live significantly longer thanks to improved respiratory ventilation and other supportive care. As a result, deaths from heart failure rather than respiratory failure have increased [2]. Therefore, understanding the development of dystrophin-deficient cardiomyopathy and effectively treating it has become very important for DMD patients.
+
+Similar to DMD, golden retriever muscular dystrophy (GRMD) is also caused by the dystrophin gene mutation, a single nucleotide change that leads to exon skipping and an out-of-frame DMD transcript [3]. GRMD dogs develop symptoms and signs like DMD patients and are used to explore pathological changes and test novel therapeutic strategies for DMD [4,5,6,7,8,9,10,11].
+
+Using image-processing algorithms for two-dimensional digital echocardiographic images, two-dimensional speckle tracking echocardiography (2D-STE) identifies small stable myocardial footprints called speckles generated by ultrasound-myocardial tissue interactions within a defined region of interest and tracks speckles frame-to-frame over the cardiac cycle. Distances between speckles or their spatiotemporal displacement provide information about global and segmental myocardial deformation. Despite some technical factors influencing strain value [12], 2D-STE has been developed into a reliable method to objectively quantify left ventricular (LV) regional and global myocardial function [12,13] since strain information is independent of the Doppler angle of incidence [13]. Studies have shown that 2D-STE can be used to reveal early pathological changes in the heart of DMD patients [14,15,16] and GRMD dogs and cardiac responses in GRMD therapeutic trials [8,9,10]. Interestingly, recent technological developments have enabled 2D-STE to analyze LV wall strain in more detail, allowing, for example, the analysis of endocardial, middle and epicardial strains in different regions of the LV wall. Therefore, the aim of this study was to evaluate the changes in myocardial circumferential strain (CS) and longitudinal strain (LS) of LV endocardial, middle and epicardial layers in GRMD dogs aged from 2 to 24 months. These GRMD dogs were compared to healthy control dogs to reveal alterations in LV myocardial strain during the pathological development of dystrophin-deficient cardiomyopathy. Our results showed that spatial and temporal non-uniform alterations of myocardial strain occur within the left ventricle during the progression of the disease. This finding may provide insight into the progression of cardiomyopathy due to dystrophin deficiency in this highly relevant canine model of DMD.
+
+## 2. Materials and Methods
+
+### 2.1. Animal Model
+
+Twenty-two GRMD and seven healthy control dogs were provided by the Centre d’Elevage du Domaine des Souches (Mezilles, France) and were cared for by veterinarians throughout the study. GRMD was diagnosed by DNA analysis at 1 month of age. The experimental protocol was approved by the animal ethics committee (ComEth ANSES-ENVA-UPEC agreement #20/12/12–18) and the experimental procedures were performed in accordance with the European Union Directive 2010/63/EU.
+
+### 2.2. Echocardiographic Image Acquisition and Analysis
+
+Echocardiographic image acquisition and analysis were performed by the same investigator in GRMD dogs and their healthy littermates at 2, 6, 9, 12, 18 and 24 months of age. Using a Vivid 7 ultrasound (General Electric Medical System, Horten, Norway), echocardiographic images were acquired from conscious (i.e., without the use of sedatives or anesthetics) 2-month-old puppies in a standing position using a 7S cardiac sector array probe and from dogs of other ages in a standing position in a sling using a 5S or an M4S cardiac sector array probe after an adaptive period of 20–30 min. The standing position allows for the acquisition of images from all commonly used LV views. An ECG was recorded during the echocardiographic examination to calculate heart rate.
+
+M-mode parasternal short-axis (PSAX) images at the mid-chamber (papillary muscles) level were recorded to measure LV end-diastolic (EDD) and end-systolic (ESD) diameters, end-diastolic and end-systolic posterior and interventricular septal wall dimensions and fractional shortening (FS).
+
+High-frame-rate videos (ranging from 60 to 119 frames/s) were recorded in apical 2-chamber, 3-chamber and 4-chamber views. In some cases, the apical 5-chamber view was used when an adequate 3-chamber image could not be obtained. The PSAX-base view was acquired at the level of mitral valve leaflets. The PSAX-mid-chamber view was recorded at the level of papillary muscles. The PSAX-apex view was recorded when the LV cavity was as small as possible without visible papillary muscles. For each view, a cine loop of at least three consecutive cardiac cycles was stored digitally for offline analysis. To ensure the image quality for strain analysis, the images of young dogs were generally acquired with the highest frame rate possible.
+
+Echocardiographic images were analyzed as recommended [17]. FS (%) was calculated as [(LV EDD − LV ESD)/LV EDD × 100%]. LV end-diastolic volume (EDV, mL), end-systolic volume (ESV, mL) and ejection fraction (EF, %) were calculated by the biplane Simpson’s method using the apical 4-chamber and 2-chamber images.
+
+Using dedicated software, 2D-STE was performed offline (EchoPac version 201, GE Healthcare, Horten, Norway). CS and LS were analyzed in endocardial (inner), middle and epicardial (outer) layers of GRMD and healthy control dogs of various ages using EchoPac version 201. The cardiac cycle length was measured from the beginning of systole. After delineating an area of interest within the LV wall and performing manual adjustments to include the entire LV wall, the software automatically divided the LV wall into 6 LV wall segments for each view. Each segment was divided into endocardial and epicardial parts and averaged to obtain endocardial, middle and epicardial strains. After viewing the validation results automatically generated by the software and adequately curve fitting the area of interest within the LV wall, the adequate tracking results were considered for analysis. It is often necessary to manually adjust the area of interest several times to obtain an adequate fitting. CS was analyzed in PSAX-apex, PSAX-mid-chamber and PSAX-base views. LS was analyzed in apical 3-chamber (or 5-chamber), 4-chamber and 2-chamber views.
+
+### 2.3. Statistical Analysis
+
+Continuous variables were expressed as mean ± SEM. Statistical analysis was performed with StatView (Version 5.0, Abacus Concepts Inc., Berkeley, CA, USA). The one-way ANOVA was used to test within-group differences over time (age). The ANOVA for repeated measurements over time was performed on echocardiographic data from 8 GRMD dogs and 4 healthy control dogs aged 2 to 24 months to test between-group differences. The two-tailed unpaired two-sample *t*-test was performed on data obtained in all dogs at each time point to determine the differences between GRMD and healthy control dogs. A difference was considered statistically significant at *p* < 0.05.
+
+The assessment of inter-observer variability was not carried out in the study, because the acquisition and analysis of the echocardiographic images were carried out by a single investigator. To examine intra-observer variability, a set of images used for the calculation of EF, CS in the PSAX-apex view and LS in the 4-chamber view were taken from 6 randomly selected dogs used in the study and analyzed twice by the same sonographer in an interval of 24 h or more (3 heart beats were analyzed for each image). The interclass correlation coefficient (ICC) for these parameters was calculated using the Real Statistics Resource Pack. The coefficient of variation (CoV) for these parameters was calculated by the following equation: CoV (%) = standard deviation/mean × 100. The two measurements were used for the calculation of the ICC and the CoV for EF (0.908 and 3.1 ± 2.7%, respectively), endocardial CS (0.955 and −3.4 ± 2.9%), middle CS (0.941 and −3.8 ± 2.5%), epicardial CS (0.738 and −7.9 ± 5.7%), endocardial LS (0.958 and −3.1 ± 2.4%), middle LS (0.950 and −3.3 ± 2.7%) and epicardial LS (0.913 and −4.3 ± 4.2%).
+
+## 3. Results
+
+Of the 22 GRMD dogs included in the study, three were euthanized around the age of 6 months due to loss of ambulation; four between 9 and 12 months due to severely reduced mobility (n = 2), poor general conditions (n = 1) and severe bronchopneumonia (n = 1); three between 12 and 18 months of age due to reduced mobility (n = 2) and respiratory failure (n = 1); and two died of heart failure. No death occurred in healthy dogs throughout the study, and three of the seven healthy control dogs were rehomed after the 6-month examination.
+
+GRMD dogs had significantly lower body weights than healthy control dogs at different ages (Table 1), notably reflecting a loss in muscle mass. In GRMD and healthy control dogs, the heart rate decreased with age, and the majority of GRMD dogs had slightly higher heart rates than healthy control dogs at the same age (Table 1).
+
+### 3.1. Changes in LV Dimensional and Functional Parameters Measured by Echocardiography in GRMD and Healthy Control Dogs at Different Ages
+
+As shown in Table 1, the LV EDD of healthy control dogs increased with age until 9 months old. The LV EDD of GRMD dogs increased with age but was smaller than that of healthy control dogs until 9 months of age. Thereafter, LV EDD was similar in both groups. LV ESD increased with age in healthy control and GRMD dogs. As a result, these changes in LV EDD and ESD of GRMD dogs resulted in a significant reduction in FS at 12, 18 and 24 months (*p* < 0.05 or *p* < 0.01). The decrease in FS in some GRMD dogs reached a pathological value (i.e., less than 28%) at 18 and 24 months, whereas healthy control dogs had a stable FS throughout all ages examined. In healthy control dogs, interventricular septal wall end-diastolic thickness (IVSWEDT) and LV posterior wall end-diastolic thickness (PWEDT) increased with age and reached a stable thickness at 12 months. In contrast, IVSWEDT and PWEDT of GRMD dogs were smaller than those of healthy control dogs starting from 6 and 9 months of age, respectively. However, interventricular septal wall systolic thickening (% IVSWTh) was not significantly changed over time in healthy control dogs and decreased, without reaching significance, in GRMD dogs. Similarly, LV posterior wall systolic thickening (% PWTh) did not significantly change throughout the protocol in healthy control dogs, whereas it significantly decreased over time in GRMD dogs.
+
+LV end-diastolic volume (LVEDV) increased with age in both healthy control and GRMD dogs, but GRMD dogs had smaller LVEDV at each age examined, which is certainly due to the marked smaller size and body weight of GRMD dogs. LV end-systolic volume (LVESV) increased with age in healthy control dogs whereas GRMD dogs had a smaller LVESV at 2 and 6 months of age than healthy control dogs but a similar LVESV from 9 months of age. Consequently, GRMD dogs had smaller EF and, contrasted with healthy control dogs, EF decreased progressively with age, starting at 9 months whereas healthy control dogs had stable EF at all ages examined (Table 1). The decrease in EF in some GRMD dogs reached a pathological value (i.e., less than 50%) at 18 and 24 months old.
+
+These data obtained by conventional echocardiography indicated that significant alterations in LV global function (FS and EF) can be detected rather tardily (12–18 months of age) in GRMD dogs.
+
+### 3.2. Changes in CS in the Three LV Wall Layers Analyzed by 2D-STE from the Three Short-Axis Views in GRMD and Healthy Control Dogs through Time
+
+Figure 1 shows two typical examples of LV endocardial, middle and epicardial CS from a 2-month-old healthy control dog (panel A) and a 2-month-old GRMD dog (panel B) analyzed by 2D-STE from the PSAX-apex view. As shown in Table 2, in the LV apical region, the peak CS of endocardial, middle and epicardial layers remained rather stable with age in healthy control dogs. In contrast, GRMD dogs showed significant reduction in peak CS in the three examined layers of the LV apex (values being less negative) at 2 months, which were mainly caused by the significantly smaller peak CS of LV anterior and antero-lateral regions than those of healthy control dogs. It is worth noting that this smaller peak CS of epicardial and middle layers in the LV apical region of GRMD dogs could be seen at the different ages examined.
+
+At the PSAX-mid-chamber level, GRMD had similar peak CS in the endocardial layer as healthy control dogs except at 2 and 24 months of age and had smaller peak CS in epicardial and middle layers at 18 and 24 months of age. In the endocardial layer, this parameter significantly decreased with age in both groups. However, in the middle and epicardial layer, peak CS significantly decreased with age in GRMD dogs, whereas no significant change was detected in healthy control dogs. At the base of the left ventricle, the peak CS of endocardial, middle and epicardial layers was basically similar for GRMD and healthy control dogs except for the epicardial layer at 9, 18 and 24 months and the middle and endocardial layers at 24 months, where GRMD dogs had smaller peak CS than healthy control dogs. In the epicardial layer, basal peak CS decreased significantly with age in GRMD dogs, whereas this parameter remained stable over time in healthy control dogs.
+
+These results indicated that GRMD dogs exhibited a non-uniform spatial alteration over time in CS, characterized by an early and persistent decrease in peak CS in the epicardial and middle layers of the LV apical region, mainly due to decreased peak CS in the apical anterior and antero-lateral regions. At the middle and basal levels of the left ventricle, decreased peak CS in GRMD dogs occurred later (at 18–24 months of age). In these regions of the left ventricle, the decrease in peak CS occurred earlier in epicardial and middle layers than in the endocardial layer.
+
+### 3.3. Changes in LS in the Three LV Wall Layers Analyzed by 2D-STE from the Three Apical Views in GRMD and Healthy Control Dogs at Different Ages
+
+Figure 2 shows two typical examples of LV endocardial, middle and epicardial LS from a 2-month-old healthy control dog (panel A) and a 2-month-old GRMD dog (panel B) analyzed by 2D-STE from the apical four-chamber view. As shown in Table 3, at the apical three-chamber view, GRMD dogs had significantly smaller peak LS than healthy control dogs at 2, 6, 9 and 12 months of age in the three layers of the LV wall. In the apical four-chamber view, peak LS of the three layers of LV wall were slightly but not significantly decreased with age in healthy control dogs. Contrastingly, GRMD dogs displayed a reduction with age and significantly smaller peak LS in the three layers of the LV wall at 2, 6, 9 and 24 months of age (Figure 2 and Table 3). In the apical two-chamber view, GRMD had significantly smaller peak LS in the endocardial layer than healthy control dogs at 2, 6, 9 and 12 months and significantly smaller peak LS in epicardial and middle layer at 2, 6 and 12 months (Table 3).
+
+Thus, from a young age, a significantly smaller peak LS could be detected in the three layers of the LV wall of GRMD dogs from three apical views. The difference in peak LS between GRMD and healthy control dogs became less evident at 18 and 24 months of age.
+
+## 4. Discussion
+
+In this longitudinal study, significant alteration in global LV systolic function (i.e., decreased FS and EF) was revealed late by conventional echocardiography in GRMD dogs, as previously reported [6,8]. The decline in LV systolic function with age reflects the natural progression of cardiomyopathy in GRMD dogs in the absence of specific treatment targeting cardiomyopathy.
+
+Myocardial regional mechanics can be assessed by echocardiography in terms of four principal types of strain or deformation: circumferential, longitudinal, radial and rotational. A combined analysis of these strains can give a complete description of LV myocardial strain. At present, simultaneously analyzing myocardial strains of LV endocardial, middle and epicardial layers by echocardiography is technically available for CS and LS. This study analyzed myocardial strain in terms of the CS and LS of different LV wall layers of different regions, with the hope that assessing LV myocardial strain from two aspects gives a more detailed description of the changes in LV myocardial regional strain during the development of cardiomyopathy in GRMD dogs. In fact, more detailed analysis of LV myocardial CS and LS revealed different change trends in LV myocardial strain in both the circumferential and longitudinal directions, showing earlier alterations consistent with previous findings [6,8]. More importantly, these alterations in LV function were not uniform within the LV wall, showing differential progression of cardiomyopathy from the apex to the base and from the epicardium to the endocardium.
+
+Clinical studies using cardiac magnetic resonance image [18], tissue Doppler imaging (TDI) [19] and 2D-STE [16] showed that DMD boys (less than 10 years old) had a decreased peak CS which worsened with age. Interestingly, a study in DMD boys (aged 14.8 ± 3.1 years old) showed that peak CS in the posterior wall analyzed by TDI exhibited smaller values, more frequently, in the outer layer than in the inner layer [20]. In the present study, an analysis of endocardial, middle and epicardial CS by 2D-STE from PSAX-apex, PSAX-mid-chamber and PSAX-base views demonstrated a non-uniform change in peak CS in GRMD dogs, which occurred early (2 months of age) and manifested as a significant and persistently smaller peak CS in the epicardial and middle layers of the LV apical anterior and antero-lateral wall (Figure 2 and Table 2). Meanwhile, at the mid-chamber and basal levels of the left ventricle, a smaller CS was detected relatively later in GRMD dogs (Table 2). These results suggest that analysis of CS in the LV apical region may be a useful tool to reveal early manifestations of cardiomyopathy in GRMD dogs, paralleling the pattern described in DMD patients. This non-uniform distribution of functional alteration between the apical and basal areas was complemented by differential timing of reduced contractile function from the epicardial to the endocardial layer.
+
+Previous studies have shown that a reduction in global systolic LS can be detected in young DMD boys [14,16,19,21] and GRMD dogs [8,10]. Going further, this study analyzed LS more deeply in the three layers of the LV wall from three apical views and showed that systolic LS was lower in the three LV wall layers in GRMD dogs than in healthy control dogs from a young age, which is consistent with previous studies showing a worse global systolic LS in GRMD dogs [8,10]. These results suggest that analysis of LS remains a sensitive tool to detect early alterations in LV systolic function in GRMD dogs.
+
+A preferential alteration in subendocardial contractile function has been demonstrated in many pathological conditions such as exercise under acute increase in afterload [22], LV hypertrophy [23], coronary heart disease [24], and heart failure [25]. Previous studies have shown an early alteration of subendocardial systolic velocity analyzed by TDI in GRMD dogs [4,8]. This non-uniform change in subendocardial-subepicardial function has been considered to contribute to the progression of the disease. Interestingly, the present study showed that the left ventricle of GRMD dogs exhibits spatial non-uniform alteration in myocardial strain that occurs particularly in the apical region for CS. Because this non-uniform alteration in myocardial strain occurs early, it is reasonable to speculate that it is an important factor contributing to the progression of LV global contractile dysfunction.
+
+The study may have some limitations. (1). Due to the obvious physical differences between GRMD and healthy control dogs, it is impossible for the investigator to be blind to the study group, which may lead to study bias. (2). This study characterized the temporal changes of LV myocardial strain in GRMD dogs but did not provide cellular and molecular mechanisms to explain this early change in myocardial strain. Thus, further studies of cellular and molecular mechanisms are needed. (3). The LV myocardial wall is a three-dimensional object and has strain that can occur along three planes. During contraction, the heart is not only shortening along its long and short axis, but also rotating, tilting and displacing inside the chest. Thus, in addition to technical factors that may influence the analysis of strain [12], this may also affect linear strain analyzed by 2D-STE, which is based on a simplistic sense of lengthening, shortening, thickening or rotating. In addition, the small thickness of the myocardial wall of young dogs (to be further divided into different layers by the software) and the high heart rate may increase the difficulty for accurate analysis of myocardial strain and possibly affect the accuracy of results. Therefore, caution should be taken when interpreting these results. However, the results obtained in young and adult dogs regardless of their conditions (healthy or GRMD) provide consistent myocardial strain information which is consistent with classic notions about LV contractile function. For example, both CS and LS data showed that the myocardial strain of the subendocardial layer is stronger than that in the subepicardial layer, which is consistent with the classic concept that the subendocardial layer contracts more strongly than the subepicardial layer. Further development of three-dimensional STE may give a more physiological description of LV myocardial strain reflecting the complexity of the heart’s three-dimensionality, and a recent study showed early impairment of LV 3-dimensional global LS in young patients with DMD [26].
+
+## 5. Conclusions
+
+In young dogs with DMD who have a normal global systolic function, a non-uniform reduction in systolic CS can be detected, especially in the LV apex, while decreased systolic LS can be detected in the three LV wall layers from three apical views. Our results suggest that analysis of myocardial strain by multi-layer 2D-STE may be a useful tool for monitoring the spatial evolution of alterations in LV systolic function and their progression with age in GRMD dogs. Given the similarity in pathogenesis, pathological changes, and clinical signs and symptoms between GRMD and DMD, our results suggest that assessing LV myocardial strain mechanics may be useful in detecting the early alteration of LV systolic function in early childhood of DMD patients and monitor its progression. The significance of these findings may warrant further investigation in DMD patients.

fulltext/md/PMC10229623.md

@@ -0,0 +1,25 @@
+---
+pmcid: "PMC10229623"
+pmid: "37253931"
+doi: "10.1038/s41598-023-34875-5"
+title: "Author Correction: Evaluation of cognitive function in the Dog Aging Project: associations with baseline canine characteristics."
+journal: "Scientific reports"
+year: "2023"
+license: "cc by"
+article_type: "correction"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Author Correction: Evaluation of cognitive function in the Dog Aging Project: associations with baseline canine characteristics
+
+Correction to: *Scientific Reports*
+https://doi.org/10.1038/s41598-022-15837-9, published online 25 August 2022
+
+The original version of this Article contained errors in the Dog Aging Project Consortium list, where the following authors were omitted:
+
+Joshua M. Akey, Brooke Benton, Elhanan Borenstein, Marta G. Castelhano, Amanda E. Coleman, Kyle Crowder, Matthew D. Dunbar, Virginia R. Fajt, Annette L. Fitzpatrick, Unity Jeffrey, Erica C. Jonlin, Elinor K. Karlsson, Kathleen F. Kerr, Jonathan M. Levine, Jing Ma, Robyn L. McClelland, Stephen M. Schwartz, Sandi Shrager, Noah Snyder-Mackler, M. Katherine Tolbert, Silvan R. Urfer, Benjamin S. Wilfond.
+
+Consequently, the Supplementary Information file listing the Consortium members has been removed.
+
+The original Article has been corrected.

fulltext/md/PMC10233048.md

@@ -0,0 +1,312 @@
+---
+pmcid: "PMC10233048"
+pmid: "37275618"
+doi: "10.3389/fvets.2023.1168711"
+title: "A masked, placebo-controlled, randomized clinical trial evaluating safety and the effect on cardiac function of low-dose rapamycin in 17 healthy client-owned dogs."
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# A masked, placebo-controlled, randomized clinical trial evaluating safety and the effect on cardiac function of low-dose rapamycin in 17 healthy client-owned dogs
+
+## Abstract
+
+### Introduction:
+
+Geroscience studies of low-dose rapamycin in laboratory species have identified numerous benefits, including reversing age-related cardiac dysfunction. Cardiovascular benefits have been observed in dogs with 10 weeks of treatment, raising questions about possible benefits and adverse effects of long-term use of low-dose rapamycin. The objectives of this study were to assess the impact of 6 months of low-dose rapamycin on echocardiographic indices of cardiac function in healthy dogs and to document the occurrence of adverse events.
+
+### Methods:
+
+Seventeen client-owned dogs aged 6–10 years, weighing 18–36 kg, and without significant systemic disease were included in a prospective, randomized, placebo-controlled, masked clinical trial. Low-dose rapamycin (0.025 mg/kg) or placebo was administered three times per week for 6 months. Baseline, 6-month, and 12-month evaluation included physical examination, cardiology examination, and clinicopathology. Three-month evaluation included physical examination and clinicopathology. Owners completed online questionnaires every 2 weeks.
+
+### Results:
+
+There were no statistically significant differences in echocardiographic parameters between rapamycin and placebo groups at 6 or 12 months. No clinically significant adverse events occurred. In 26.8% of the bi-weekly surveys owners whose dogs received rapamycin reported perceived positive changes in behavior or health, compared to 8.1% in the placebo group (*p* = 0.04).
+
+### Discussion:
+
+While no clinically significant change in cardiac function was observed in dogs treated with low-dose rapamycin, the drug was well-tolerated with no significant adverse events.
+
+1.
+
+## Introduction
+
+Normal cardiac aging results in multiple structural and functional changes over time as well as increased incidence of age-related cardiovascular diseases. In humans, dilation of the left atrium, increased left ventricular wall thickness, and diastolic dysfunction are well-recognized age-related changes (1–4). In dogs, echocardiographic evidence of diastolic dysfunction associated with age has also been demonstrated, including increased isovolumic relaxation time in dogs >10 years of age and decreased mitral inflow E to A wave ratios (E:A) in dogs >6 years of age when compared to dogs <2 years of age (5). On a cellular level, upregulation of inflammatory cytokines that promote cardiac fibrosis and hypertrophy, decreased macroautophagy and increased oxidative stress are all proposed mechanisms of cardiac aging (3, 6).
+
+Rapamycin is a macrocylic lactone molecule first identified from soil samples obtained on Easter Island. It is naturally produced by the bacterium *Streptomyces hygroscopicus*, and was first studied for its potent antifungal activity. It was later found to also inhibit growth of mammalian cells and was developed clinically for use as an anti-cancer and immune suppressive agent. Rapamycin (sirolimus) was approved by the FDA in 1999 to prevent renal transplant rejection and, along with rapamycin derivatives (rapalogs), has been used in organ transplant medicine for more than two decades. Additionally, rapamycin or rapalogs have been FDA approved for use in cardiac stents to prevent restenosis, for use in tuberous sclerosis complex disorders, and for use in some forms of cancer (7, 8).
+
+Rapamycin is a mechanistic target of rapamycin complex I (mTORC1) inhibitor. mTOR signaling plays a major role in regulation of cell growth, nutrient response, proteostasis, and survival (9–11). Recent work suggests that rapamycin impacts the aging process at a cellular level by inducing macroautophagy, altering mRNA translation, modifying nutrient sensing, decreasing sterile inflammation, and improving stem cell function (11–13). Researchers have increased lifespan in yeast (14), *Caenorhabditis elegans* (15), and *Drosophila melanogaster* (16) through modification of mTOR signaling by various means. Rodent rapamycin studies have shown numerous benefits, including increased lifespan (17–23), improved cognitive and muscle function (22, 24, 25), reduced tumorigenesis (26), prevention of retinopathy (27), restoration of stem cell function (9, 28), reversal of immune declines (9), reversal of periodontal disease (29, 30), improved kidney function (31), preservation of tendon (32), improved intestinal function and reduced gut dysbiosis (22, 28), preservation of ovarian function (33) protection against hearing loss (34), and reversal of age-related cardiac dysfunction, specifically by improving indices of systolic function [ejection fraction (EF), fractional shortening (FS), and left ventricular end systolic dimensions] and decreasing age related cardiac hypertrophy (6, 35, 36). Rapamycin was recently accessed in a clinical trial as an adjuvant therapy for appendicular osteosarcoma, but no significant improvement in median disease-free interval or overall survival were observed (37).
+
+At dosages intended to mitigate age-related changes, adverse events associated with rapamycin are uncommon in rodents, and include hyperlipidemia, gonadal atrophy, and cataract formation, as well as altered glucose homeostasis with chronic use (19, 38). In studies of low-dose rapamycin and its derivatives in elderly humans, adverse events are generally mild and transient, and include mouth ulcers, diarrhea, elevated cholesterol, and headache (39, 40). The few *in vivo* studies in dogs evaluating rapamycin at non-immunosuppressive doses have shown it is well-tolerated with low incidence of adverse events (41, 42).
+
+Additional work is needed to fully elucidate the cellular mechanisms by which rapamycin exerts these reported effects. Previously, our group investigated low-dose rapamycin and placebo treatments in healthy, middle-aged dogs over a 10-week period, with the primary goal of establishing the safety of low-dose rapamycin in healthy pet dogs. Cardiac endpoints were also assessed because of the robust evidence of rapamycin’s mitigation of age-related cardiac functional decline in mice (6, 36, 43). The study period was only 10 weeks, and all dogs had normal echocardiograms at enrollment. Interestingly, this trial found that FS improved in treated dogs, although a statistically significant change in EF was not detected, over the 10-week study period. Additionally, the E/A ratio was statistically significantly higher in treated dogs compared to placebo, and no clinically meaningful adverse events were observed over the 10-week treatment period (44). The results of this small and short-term trial prompted the decision to undertake a follow-up study over a longer period of time.
+
+The advantages of further evaluating the potential benefits of rapamycin and other geroscience therapeutics in companion dogs are numerous. These studies could validate therapies that reduce or delay age-related disease and increase healthspan in companion dogs. Furthermore, conclusions from geroscience investigations in dogs have high translational potential to human geroscience, as companion dog aging draws close parallels to human aging; humans and companion dogs share common environments, receive comparable medical care, and experience many similar age-related diseases (43, 45, 46).
+
+The major objectives of this study were to assess the effect of 6 months of low-dose rapamycin treatment on baseline indices of diastolic and systolic cardiac function and to document occurrence of adverse events in healthy, middle-aged companion dogs. We hypothesized that rapamycin would improve echocardiographic indices of diastolic function in treated dogs compared to placebo-treated controls over a 6-month period. We also hypothesized that no treated dogs would demonstrate adverse events requiring medical intervention. Assessment of the impact of low-dose rapamycin on routine clinical pathology was a secondary objective.
+
+2.
+
+## Materials and methods
+
+2.1.
+
+### Trial design
+
+This study was a prospective, randomized, placebo-controlled double-masked trial conducted at the Texas A&M University (TAMU) Veterinary Medical Teaching Hospital (VMTH).
+
+2.2.
+
+### Dogs
+
+2.2.1.
+
+#### Inclusion criteria
+
+To be eligible, dogs were required to be between the ages of 6 and 10 years and have a body weight > 18 and < 36 kg. A negative heartworm antigen test was required, as was documentation of consistent heartworm prevention administration for the preceding 6 months and the owner’s willingness to continue heartworm prevention. Documentation of appropriate vaccination status in concordance with the 2017 AAHA Canine Vaccination Guidelines (47) was also required, as well as the owner’s willingness to continue vaccination. On physical examination, a grade II/VI or softer systolic murmur was permitted.
+
+2.2.2.
+
+#### Exclusion criteria
+
+Dogs were excluded from the study if any of the following systemic diseases were present in their history or identified at the screening/baseline examination: diabetes mellitus, hyperadrenocorticism, hypothyroidism, renal disease, liver disease, chronic gastrointestinal disease, respiratory disease, neurological disease, systemic hypertension, cardiac arrhythmia other than sinus arrhythmia, and cardiac disease other than ACVIM Stage B1 myxomatous mitral valve degeneration (48). A diagnosis of occult dilated cardiomyopathy was made if the normalized left ventricular internal diameter at end-systole was >1.26 (49) in combination with a FS <20% and/or an EF <40% (50). Dogs were also excluded if they exhibited poor tolerance of examination and venipuncture, had a SBP greater than 160 mmHg, had a poor echocardiographic scanning window, or if another dog in the same household was enrolled.
+
+2.2.3.
+
+#### Enrollment process
+
+Owners nominated their dogs through an online eligibility questionnaire that requested basic demographic, health history, and temperament information and that also provided background on the study design and commitments required. Owners who indicated understanding of the trial and availability for four VMTH visits, and whose dogs were the correct age (6–10 years), correct weight (18–36 kg), and described as being in good health and cooperative for veterinary visits and medication administration, were invited to submit their dogs’ comprehensive veterinary medical records. Records in any format were accepted, and a minimum of 3 years of records was required. Submitted records were evaluated for the presence of exclusion criteria.
+
+Owners of dogs with no exclusionary findings in their medical records were invited to schedule a screening examination. For those dogs who were enrolled, this also served as the baseline examination. At the time of the appointment, the study was described in detail to the owners, and they were allowed to ask questions before reviewing and signing an Informed Owner Consent form. All procedures and forms were reviewed and approved by the TAMU College of Veterinary Medicine and Biomedical Sciences’ Institutional Animal Care and Use Committee and Clinical Research Review Committee under protocol number 2017–0125.
+
+The screening evaluation included a physical examination, complete blood count, serum chemistry profile, urinalysis, total thyroxine level (TT4) plus free thyroxine and thyroid stimulating hormone if TT4 was low, heartworm antigen test, systolic arterial blood pressure (SBP) measurement, electrocardiogram (ECG), and echocardiogram. If no exclusion criteria were identified after this process, dogs were considered eligible for the study.
+
+2.3.
+
+### Randomization and masking
+
+Dogs who fulfilled all enrollment criteria were enrolled and randomized using a schedule designed and administered by the TAMU VMTH pharmacist. A block randomization schedule was used with blocks of four and a 1:1 allocation ratio to either the rapamycin group or placebo group.
+
+2.4.
+
+### Trial medication
+
+Rapamycin [Dr. Reddy’s (1 mg), Princeton, NJ; ZyGenerics (0.5 mg), Pennington, NJ] was formulated into capsules containing whole or half tablets to create capsules containing 0.5, 0.75, or 1.0 mg of rapamycin. Placebo (lactose; PCCA, Houston, TX) capsules were created to be identical in appearance. Dogs in the treatment group were given 0.025 mg/kg rapamycin per dose, rounded to the nearest 0.25 mg capsule. Owners were instructed to give the study medication on Monday, Wednesday, and Friday mornings. Owners were provided with study medication logs to document administration. Administration of rapamycin and placebo was continued for a period of 6 months. Owners returned pill vials to the TAMU VMTH pharmacy at each visit, and leftover capsules were counted.
+
+2.5.
+
+### Clinical evaluation
+
+Following the screening/baseline examination, dogs were examined at 3 and 6 months during the treatment period. A final examination was performed at 12 months (6 months after discontinuation of study medication) to determine whether any changes induced by rapamycin therapy persisted beyond the treatment period. The 3-month examination included history, physical examination, complete blood count, serum chemistry profile, urinalysis, and SBP measurement, without cardiology examination. The 6- and 12-month examinations were identical and included history, physical examination, complete blood count, serum chemistry profile, urinalysis, SBP measurement, ECG, and echocardiogram.
+
+2.6.
+
+### Systolic arterial blood pressure
+
+All SBP measurements were taken by a trained clinician or technician prior to physical examination and other diagnostics. Dogs were allowed 10–15 min of acclimatization in a quiet environment before measurements were taken. The SBP measurements were taken on the antebrachium with a Doppler unit, utilizing a cuff with a diameter approximately 40% of the extremity circumference. Body position, cuff size, and cuff location were recorded. The first measurement was discarded, and three consecutive values were recorded and averaged. If consecutive readings varied by >20%, the cuff was repositioned, and the measurements repeated until ≤20% variation was observed.
+
+2.7.
+
+### Electrocardiography
+
+Standard six-lead ECGs were performed with dogs positioned in right lateral recumbency. Three-minute ECG traces were recorded. All dogs were conscious and unsedated for the recording. One of three board-certified veterinary cardiologists (SW, SG, AS) evaluated the ECG traces for the presence of arrhythmias. Because drug-induced QT prolongation is a potential adverse event associated with diverse medications (51), three corrected QT intervals (QTc) were also calculated and averaged (52).
+
+2.8.
+
+### Echocardiography
+
+Standard echocardiography including two-dimensional, M-mode and Doppler imaging was performed by a board-certified cardiologist using a Vivid E95 Vet 2.0 (GE Healthcare, Chicago, IL, United States) with dogs unsedated and gently restrained in right and left lateral recumbency. Each measurement was taken in triplicate and the averages of these measurements were recorded. The following measurements were obtained: left atrial to aortic root ratio (LA:Ao) (53), left atrial major axis diameter obtained at end ventricular systole from a right parasternal long axis view (54), left ventricular internal dimension at end-diastole and end-systole obtained from short axis M-mode images at the level of the papillary muscles which were then normalized to body weight as LVIDdN and LVIDsN (49), normalized left ventricular free wall thickness at end diastole (LVPWdN) (49), left ventricular FS, left ventricular EF from a left apical four-chamber view using Simpson’s method of discs, peak E wave velocity, mitral inflow E to A wave ratio, isovolumic relaxation time (IVRT), peak aortic and pulmonic velocities (AV Vmax, PV Vmax) and aortic velocity time integral (AV VTI), as well as color Doppler investigation of all valves with presence and severity of any valvular insufficiencies noted. Each dog was grouped into a diastolic functional class for baseline, 6 and 12 month time points which were defined as: normal pattern (Class 1), relaxation delay pattern (Class 2), pseudonormal pattern (Class 3), and restrictive pattern (Class 4) (55).
+
+2.9.
+
+### Clinicopathologic evaluation
+
+Peripheral blood, urine and fecal samples were collected from all dogs at each visit. Urine was collected by cystocentesis or catheterization if free catch was not successful. The TAMU VMTH Clinical Pathology Laboratory performed complete blood counts (Advia 120, Siemens Healthineers, Malvern, PA), serum chemistry profiles (Vitros 4,600, Ortho Clinical Diagnostics, Raritan, New Jersey), and urinalyses (Multistix 10SG urine strips, read on a Clinitek strip reader, both by Siemens Healthineers, Malvern, PA). At the baseline examination only, TT4 was measured by the TAMU Gastrointestinal Laboratory, and if that value was below the reference interval (<1.7 μg/dL), free thyroxine and thyroid stimulating hormone assays were performed (Immulite 2000 Xpi, Siemens Healthineers, Malvern, PA). The TAMU Clinical Immunology Service performed heartworm antigen testing (DiroCHEK Canine Heartworm Antigen Test Kit, Zoetis, Parsippany-Troy Hills, NJ) according to manufacturer instructions. Because all enrolled dogs resided in Texas, samples were submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory to screen for exposure to the agent that causes Chagas disease (*Trypanosoma cruzi*) with immunofluorescent antibody tests. For this assay, serum samples were diluted 1:20 in phosphate buffered saline (PBS) and incubated on substrate slides (Trinity Biotech, Jamestown, NY) containing fixed *Trypanosoma cruzi* epimastigotes. The slides were then washed with PBS, and an anti-dog IgG secondary antibody conjugated with FITC (SeraCare/KPL, Milford, MA) was added to all sample wells. Following incubation with the secondary antibody, the slides were washed and examined under a fluorescent microscope. Sample wells exhibiting bright fluorescence of the fixed epimastigotes were considered positive for *T. cruzi* antibodies at the 1:20 dilution. Any samples testing positive at the 1:20 dilution were serially diluted twofold and tested again; the reciprocal of the highest dilution exhibiting a positive reaction was reported as the antibody titer. Blood and fecal samples were banked from each dog at each examination. Banked baseline and 6-month samples were analyzed by the Michigan State University Veterinary Diagnostic Laboratory to determine fasting insulin (Insulin RIA, Millipore Sigma, Burlington, MA), and glucose (AU680, Beckman Coulter, Brea, CA) concentrations and results were used in the homeostatic model assessment for insulin resistance (HOMA-IR) to estimate the current level of insulin resistance, as well as beta cell function (56, 57). At the conclusion of the study, banked baseline and 6-month samples were analyzed by the TAMU Gastrointestinal Laboratory to determine cardiac troponin (cTnI) concentrations using a three-site sandwich direct chemiluminometric immunoassay (TnI-Ultra platform Advia Centaur CP, Siemens Healthineers, Malvern, PA) (58).
+
+2.10.
+
+### At-home observation
+
+Each owner was sent an automated online questionnaire every 2 weeks for the duration of the study. This questionnaire included forced-choice items addressing specific adverse events over the prior two-week period, forced-choice items addressing aspects of canine cognitive dysfunction, and free-text items addressing general negative or positive changes in behavior.
+
+2.11.
+
+### Statistical analysis
+
+Baseline characteristics of the dogs were summarized by assigned treatment group (rapamycin vs. placebo). The primary intent-to-treat analysis method was calculation of the absolute difference in echocardiographic outcome measures between the treatment groups at each follow-up time point (6 and 12 months). The null hypothesis of no difference for each outcome measure was tested using two-sample *t*-tests. Due to the small sample size, only univariable comparisons between treatment groups were performed. The two-sided type 1 hypothesis testing threshold was set at 0.05 for assessments of treatment effect on each parameter. For echocardiographic parameters, the *p-*value was adjusted using the Holm-Bonferroni step-down method to account for multiple comparisons (59).
+
+HOMA-IR scores were compared between treatment groups at baseline and 6 months using two-sample *t*-tests.
+
+Owner responses on questionnaires were inspected for reports of adverse events; too few adverse events occurred to permit formal analyses. Owners answered questions about positive changes in behavior an average of 23 ± 3.1 times (mean ± 1 SD). We calculated the average positivity score for each owner, converting yes/no responses to the question regarding observed positive changes in behavior to 1/0, respectively, and taking the numeric average across all answers. We then compared mean positivity scores of rapamycin-treated and control groups using a two-sample two-tailed t-test. Free-text explanations of any observed changes were inspected informally for common themes.
+
+3.
+
+## Results
+
+The owners of 79 dogs contacted the study investigators by email to inquire about the study and were sent an email reply including an invitation to the initial online eligibility questionnaire. Twenty-three owners either did not respond to the email or did not complete the initial online eligibility questionnaire. Fifty-six owners nominated their dogs by completing the initial online eligibility questionnaire. Of these, 39 dogs were excluded as previously reported (60) and 17 dogs were enrolled and randomly assigned to rapamycin or placebo groups. A flow diagram representing the enrollment and randomization process is depicted in Figure 1. There were no differences between groups in key characteristics at baseline (Table 1).
+
+*Figure: Figure 1 Flow diagram of enrollment and randomization process.*
+
+**Table 1 Baseline characteristics of dogs in the two treatment groups in the trial population.**
+
+|  |  | Treatment groups |
+| --- | --- | --- |
+| Characteristic |  | Rapamycin (*n* = 9) | Placebo (*n* = 8) |
+| Age at enrollment (years) |  | 7.8 (1.0) | 8.5 (1.7) |
+| Sex and castration status | Male/castrated | 4 (44.4) | 2 (25.0) |
+|  | Female/spayed | 4 (44.4) | 6 (75.0) |
+|  | Female/intact | 1 (11.1) | 0 (0.0) |
+| Breed category | Herding | 2 (22.2) | 4 (50.0) |
+|  | Sporting | 4 (44.4) | 2 (25.0) |
+|  | Terrier | 1 (11.1) | 0 (0.0) |
+|  | Working | 2 (22.2) | 0 (0.0) |
+|  | Other | 0 (0.0) | 2 (25.0) |
+| Body condition score | 4 | 3 (33.3) | 0 (0.0) |
+|  | 5 | 4 (44.4) | 4 (50.0) |
+|  | 6 | 2 (22.2) | 4 (50.0) |
+| Left atrial to aortic root ratio |  | 1.16 (0.12) | 1.19 (0.17) |
+| Ejection fraction (%) |  | 58.3 (11.3) | 58.6 (8.1) |
+| Fractional shortening (%) |  | 30.1 (9.7) | 32.2 (8.5) |
+| E to A wave ratio |  | 1.15 (0.30) | 0.98 (0.27) |
+
+3.1.
+
+### Clinical evaluation
+
+Over the 12-month period of the study, the body weights of each dog varied by an average of 2.0 kg across the four visits. There was no association between treatment group and net gain or net loss. Over the 12-month period of the study, the SBP of each dog varied by an average of 31 mmHg across the four visits. Most dogs (12/17) showed an overall decline in SBP with subsequent visits, and no dog developed sustained hypertension (SBP ≥ 180 mmHg) during the study. One placebo-treated dog had SBP of 190 mmHg at the 3-month visit, which declined to 150 mmHg without treatment at the 6-month visit.
+
+Abnormalities reported in the medical history by owners of dogs in the rapamycin group over the 12-month study period included decreasing willingness to exercise vigorously at agility competitions in one dog; apparent false pregnancy in one intact female dog; acute moist pyoderma in one dog; and diagnosis of a cutaneous mast cell tumor that was removed by the primary care veterinarian in one dog. Abnormalities reported in the medical history by owners of dogs in the placebo group over the 12-month study period included improvement in long-standing bilious vomiting syndrome in one dog; unilateral conjunctivitis that resolved with topical therapy in one dog; intermittent anal sacculitis in one dog; and declining vision in two dogs.
+
+New abnormalities detected on physical examination over the 12-month study period included development of subcutaneous masses compatible with lipomas in eight dogs (*n* = 4 rapamycin); increasing stiffness on gait observation associated with pain and decreased range of motion on physical examination [hips (*n* = 2) or lumbosacral region (*n* = 1)] in a total of three dogs (*n* = 2 rapamycin); and a focal pruritic, crusted lesion on the nasal planum in one dog in the rapamycin group that persisted for 3 months and then resolved with no therapy.
+
+One owner of a dog in the rapamycin group administered the study medication incorrectly (daily instead of three times per week) for a two-week period between the baseline visit and the 3-month recheck. There were no apparent adverse events from this dosing error. No other dosing errors were detected through inspection of owner medication logs or returned pill vials.
+
+3.2.
+
+### Electrocardiography
+
+No new arrhythmias were diagnosed in any dogs during the 12-month study period. When analyzing the change in QTc from baseline to both the 6- and 12-month time points, there appeared to be a > 1% decrease associated with rapamycin treatment (Table 2). However, this result was not statistically significant at either time point (*p* = 0.304, and *p* = 0.800, respectively).
+
+**Table 2 Mean (standard deviation) echocardiographic values for rapamycin and placebo groups at baseline, 6  months, and 12 months into the study.**
+
+| Outcome measure | Treatment group | Baseline | 6 month mean (SD) | 6 month effect* | *p*-value** | Adjusted *p*-value*** | 12 month mean (SD) | 12 month effect* | *p*-value** | Adjusted *p*-value*** |
+| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
+| Left atrial to aortic root ratio | Rapamycin | 1.159 (0.117) | 1.132 (0.144) | −0.18 (−0.32, 0–0.04) | 0.014 | 0.196 | 1.138 (0.112) | −0.166 (−0.321, −0.012) | 0.037 | 0.481 |
+| Placebo | 1.195 (0.168) | 1.315 (0.127) |  |  |  | 1.304 (0.182) |  |  |  |
+| Left atrial major diameter^ (cm) | Rapamycin | 3.58 (0.41) | 3.69 (0.42) | 0.06 (−0.33, 0.44) | 0.757 | 1 | 3.74 (0.43) | 0.292 (−0.113, 0.697) | 0.145 | 1 |
+| Placebo | 3.42 (0.35) | 3.63 (0.30) |  |  |  | 3.45 (0.34) |  |  |  |
+| Ejection fraction (%) | Rapamycin | 58.27 (11.86) | 62.33 (13.96) | 1.63 (−6.41, 9.67) | 0.692 | 1 | 58.77 (7.29) | 1.98 (−9.10, 12.99) | 0.707 | 1 |
+| Placebo | 58.55 (8.10) | 59.15 (10.00) |  |  |  | 56.79 (13.47) |  |  |  |
+| Fractional shortening (%) | Rapamycin | 30.08 (9.75) | 30.98 (7.65) | −2.91 (−9.60, 3.78) | 0.524 | 1 | 31.85 (6.12) | −0.513 (−7.062, 6.037) | 0.87 | 1 |
+| Placebo | 32.21 (8.50) | 33.89 (4.74) |  |  |  | 32.37 (6.55) |  |  |  |
+| LVIDdN | Rapamycin | 1.463 (0.093) | 1.420 (0.093) | 0.003 (−0.103, 0.110) | 0.948 | 1 | 1.444 (0.131) | −0.026 (−0.180, 0.128) | 0.721 | 1 |
+| Placebo | 1.456 (0.171) | 1.417 (0.113) |  |  |  | 1.470 (0.166) |  |  |  |
+| LVIDsN | Rapamycin | 0.953 (0.146) | 0.916 (0.133) | 0.04 (−0.08, 0.16) | 0.489 | 1 | 0.920 (0.145) | −0.014 (−0.055, 0.102) | 0.856 | 1 |
+| Placebo | 0.921 (0.151) | 0.876 (0.093) |  |  |  | 0.934 (0.157) |  |  |  |
+| Peak E-wave velocity (m/s) | Rapamycin | 0.65 (0.11) | 0.70 (0.12) | −0.05 (−0.19, 0.01) | 0.511 | 1 | 0.67 (0.10) | −0.004 (−0.113, 0.105) | 0.938 | 1 |
+| Placebo | 0.66 (0.20) | 0.74 (0.16) |  |  |  | 0.68 (0.11) |  |  |  |
+| E to A wave ratio | Rapamycin | 1.15 (0.30) | 1.10 (0.22) | −0.05 (−0.29, 0.19) | 0.688 | 1 | 1.19 (0.12) | 0.201 (0.054, 0.347) | 0.011 | 0.154 |
+| Placebo | 0.98 (0.27) | 1.15 (0.24) |  |  |  | 0.99 (0.16) |  |  |  |
+| IVRT (ms) | Rapamycin | 83.08 (12.28) | 79.39 (13.01) | −9.32 (−21.35, 2.72) | 0.119 | 1 | 85.33 (13.42) | 2.232 (−11.523, 15.988) | 0.734 | 1 |
+|  | Placebo | 84.10 (9.74) | 88.71 (9.77) |  |  |  | 83.10 (13.12) |  |  |  |
+| AV Vmax (m/s) | Rapamycin | 1.45 (0.26) | 1.57 (0.25) | 0.02 (−0.31, 0.28) | 0.89 | 1 | 1.44 (0.20) | 0.003 (−0.203, 0.196) | 0.972 | 1 |
+|  | Placebo | 1.49 (0.24) | 1.59 (0.32) |  |  |  | 1.44 (0.19) |  |  |  |
+| AV VTI (cm) | Rapamycin | 17.76 (4.66) | 16.50 (1.87) | −1.14 (−7.45, 0.82) | 0.018 | 0.234 | 17.85 (4.60) | 0.743 (−3.200, 4.685) | 0.694 | 1 |
+|  | Placebo | 17.63 (3.21) | 20.64 (4.24) |  |  |  | 17.10 (2.62) |  |  |  |
+| PV Vmax (m/s) | Rapamycin | 0.89 (0.22) | 1.02 (0.24) | 0.08 (−0.19, 0.34) | 0.544 | 1 | 0.95 (0.17) | 0.068 (−0.091, 0.226) | 0.375 | 1 |
+|  | Placebo | 0.83 (0.22) | 0.94 (0.27) |  |  |  | 0.88 (0.13) |  |  |  |
+| LVPWdN | Rapamycin | 0.462 (0.073) | 0.485 (0.044) | −0.010 (−0.044, 0.062) | 0.7 | 1 | 0.452 (0.042) | −0.016 (−0.059, 0.026) | 0.418 | 1 |
+|  | Placebo | 0.473 (0.061) | 0.475 (0.057) |  |  |  | 0.469 (0.039) |  |  |  |
+| QTc (ms) | Rapamycin | 206.57 (6.48) | 206.99 (13.82) | −7.19 (−21.06, 7.22) | 0.304 | 1 | 196.13 (15.62) | −3.22 (−23.38, 29.82) | 0.8 | 1 |
+|  | Placebo | 216.10 (17.32) | 214.17 (14.01) |  |  |  | 192.91 (33.67) |  |  |  |
+
+3.3.
+
+### Echocardiography
+
+Table 2 displays the results for each echocardiographically-defined outcome measure recorded at the 6-month follow-up visit. While several outcomes appeared to show that treatment with rapamycin was associated with increases (EF) or decreases (FS, IVRT, AV VTI) in certain parameters by >1%, none of these results were statistically significant when accounting for the large number of outcomes being compared.
+
+Table 2 also displays the results for each echocardiographically-defined outcome measure at 12 months. Again, while two outcomes appeared to show that treatment with rapamycin yielded increases (EF) or decreases (IVRT) in certain parameters by >1% neither of these were statistically significant after accounting for multiple comparisons. Mean values for each treatment group for all echocardiographic parameters collected are displayed in Table 2. The number of dogs in each diastolic functional class for each treatment and time point is displayed in Table 3.
+
+**Table 3 Diastolic functional class of the rapamycin and placebo groups at baseline, 6 months, and 12 months into the study.**
+
+| Treatment group | Diastolic functional class* | Baseline | 6 months | 12 months |
+| --- | --- | --- | --- | --- |
+| Rapamycin | Class 1 | 4 (44.4) | 4 (44.4) | 6 (66.7) |
+| Class 2 | 3 (33.3) | 4 (44.4) | 0 (0.0) |
+| Class 3 | 2 (22.2) | 0 (0.0) | 3 (33.3) |
+| Class 4 | 0 (0.0) | 0 (0.0) | 0 (0.0) |
+| Placebo | Class 1 | 0 (0.0) | 2 (25.0) | 2 (25.0) |
+| Class 2 | 4 (50.0) | 2 (25.0) | 4 (50.0) |
+| Class 3 | 4 (50.0) | 4 (50.0) | 2 (25.0) |
+| Class 4 | 0 (0.0) | 0 (0.0) | 0 (0.0) |
+
+3.4.
+
+### Clinicopathologic evaluation
+
+There were no clinically significant laboratory abnormalities among enrolled dogs. All enrolled dogs were seronegative for *Trypanosoma cruzi*. A total of nine dogs (*n* = 5 rapamycin) had serum lipemia detected at one or more visits with a total of 17 instances, including four dogs (*n* = 1 rapamycin) with lipemia at baseline. A total of nine dogs (*n* = 5 rapamycin) had a platelet count below the reference interval detected at one or more visits, with a total of 15 instances. There were only three instances (*n* = 2 rapamycin) at which a platelet count <100,000/μL was documented, and in each instance 4+ clumping was noted on the blood smear.
+
+Mean HOMA-IR scores were not significantly different between control and rapamycin treated groups at baseline (2.35 ± 2.1 and 2.14 ± 1.4, respectively; *p* = 0.82), nor at 6 months (2.61 ± 3.1 and 1.99 ± 1.3; *p* = 0.59). Additionally, the mean change in HOMA-IR over 6 months was not significantly different between rapamycin and control groups (+0.26 ± 2.4 and − 0.15 ± 1.5; *p* = 0.67).
+
+Ultrasensitive cardiac troponin I values for each dog at the baseline and 6 month time points are presented in Supplementary Table 1. Seven of eight placebo dogs were within normal reference ranges for aging dogs at baseline and 6 months (0.0–0.12 ng/mL), as were seven of nine rapamycin-treated dogs. One dog in the rapamycin group had missing troponin samples. One placebo and one rapamycin dog had mildly elevated troponin levels at baseline that were unchanged at 6 months. Finally, one rapamycin dog that originally had a troponin value in the normal range for aging dogs (0.088 ng/mL) had a mildly elevated troponin value at 6 months (0.164) with no observable cardiac change or outward morbidity observed. This change was outside of the established reference change interval for the assay (61).
+
+3.5.
+
+### At-home observation
+
+A total of 442 biweekly at-home observation surveys were requested, and 392 were received for an overall response proportion of 88.7% (94.0% in the rapamycin group; 82.6% in the placebo group). There were no clinically significant owner-reported adverse events among enrolled dogs. Among dogs in the rapamycin group, a total of one dog (11.1%) experienced one episode of self-limiting diarrhea, five dogs (55.5%) experienced a total of eight episodes of self-limiting vomiting, and two dogs (22.2%) experienced a total of three episodes of poor appetite throughout the study. One dog (11.1%) in the rapamycin group appeared mildly constipated during most of the treatment period and bowel movements returned to normal after the treatment period. This same dog had a change in hair coat during the treatment period characterized by decreased shedding, leading to a thick undercoat and dull topcoat; within the first month following the treatment period she had a period of increased shedding, and her coat became glossy again. Among dogs in the placebo group, a total of three dogs (37.5%) experienced four episodes of self-limiting diarrhea, two dogs (25%) experienced a total of 11 episodes of self-limiting vomiting (one of these dogs had been known to have bilious vomiting intermittently over her life prior to the trial), and no dogs experienced poor appetite throughout the study. In response to the question, “Has your dog experienced any positive changes in behavior or other things that you would associate with good health within the past week? (please explain)” owners of dogs in the rapamycin group replied with a “yes” response in 26.8% of all surveys [*n* = 59 out of 220 submissions, representing six of the nine dogs (66%) in this group] while owners of dogs in the placebo group replied with a “yes” response in 8.1% of all surveys [*n* = 14 out of 172 submissions, representing five of the eight dogs (62.5%) in this group]. This frequency of positive owner-reported outcomes in the rapamycin treatment group was significantly greater than in the placebo group (*p* = 0.04). When asked to describe the nature of the observed changes, owners of two dogs in the rapamycin group (*n* = 0 placebo) described increased activity levels and playfulness throughout the treatment period that did not persist into the post-treatment period.
+
+4.
+
+## Discussion
+
+The major objectives of this study were to assess the impact of 6 months of treatment with low-dose rapamycin on baseline indices of diastolic and systolic cardiac function and to document occurrence of adverse events in healthy, middle-aged companion dogs. No clinically or statistically significant difference in echocardiographic parameters between the rapamycin and placebo groups was observed in the dogs in this study at the 6-month or 12-month recheck. Additionally, no statistically or clinically significant adverse events were observed in the rapamycin-treated dogs in this study. A single rapamycin treated dog developed a mildly elevated cardiac troponin I level at the 6 month timepoint (0.164 ng/mL), however the significance of this remains unknown. The dog exhibited no outward cardiac change, nor any obvious clinical morbidity. Additionally, elevations in cardiac troponin I can also be seen in association with non-cardiac disease (62).
+
+The echocardiography results differ from previous work by our group, which showed significantly higher FS and E/A ratio in dogs treated with a shorter duration but higher dose of rapamycin compared to a placebo group in a randomized controlled trial (44). Dogs in the previous trial received 0.05 mg/kg three times weekly in one rapamycin treatment group and 0.1 mg/kg three times weekly in another rapamycin treatment group (both compared to a placebo group in that trial), whereas dogs in this trial’s rapamycin treatment group received 0.025 mg/kg three times weekly. Previous studies with mice have highlighted that dosing can greatly influence the effects of rapamycin (20). Because the treatment duration was substantially extended from 10 weeks in the prior trial to 6 months in the current trial, we chose the lower dose of 0.025 mg/kg three times weekly out of caution for potential adverse events. Clinically significant adverse events were not seen in the dogs of this report, even over the longer duration of the study. Additionally, dogs in the previous trial had a mean age of 9.7 years, while the mean age in the present trial was 7.8 years for the treatment group and 8.5 years for the placebo group. The failure to demonstrate differences in echocardiographic parameters between treatment groups in the study reported here could indicate that the dose in the present study was too low or could relate to the prior trial enrolling an older cohort of dogs with more potential for pre-existing age-related cardiac dysfunction. Additionally, the small number of dogs enrolled decreased the statistical power of the study. The diversity of the breeds enrolled may have been a contributing factor as well.
+
+The sensitivity of different echocardiographic measurements to detect between-group differences in cardiac structure and function should also be considered when interpreting the lack of significant differences in echocardiographic parameters in this study. For instance, while most age-related cardiac declines relate to impaired diastolic function, a concurrent decline in systolic function is not traditionally expected (3, 5). This is evidenced by preserved echocardiographic measurements such as EF in aging human populations (4, 63). More subtle assessments of systolic function such as strain, strain rate, twist or torsion of the left ventricle, however, have demonstrated statistically significant age-related declines in humans (4, 63, 64). Through this lens, the lack of significant differences in EF and FS between treatment groups in this study may be expected. Further study using more sensitive assessments of systolic function, and additional markers of diastolic function beyond E/A ratio and IVRT, may be indicated.
+
+A total of five dogs in the rapamycin group (and four in the placebo group) were reported to have mild and transient thrombocytopenia at least once during the study. A total of five dogs in the rapamycin group (and four in the placebo group) were reported to have visible lipemia at least once during the study. Increased serum cholesterol concentrations were not associated with lipemia in these dogs and serum triglycerides were not measured as part of the routine chemistry profile in this study. As thrombocytopenia (65) and hyperlipidemia (40, 66) are known adverse events in humans treated with rapamycin and other mTOR inhibitors, continued monitoring of these parameters in future studies of rapamycin in dogs is indicated.
+
+Two separate mTOR complexes, mTORC1 and mTORC2, are essential in nutrient-sensing pathways responsible for cell growth and proliferation, and their inhibition can lead to changes in metabolism including glucose regulation (67). Although rapamycin is a specific inhibitor of mTORC1, chronic treatment with rapamycin can lead to indirect inhibition of mTORC2, which has been proposed to cause insulin resistance and impaired response to a glucose tolerance test (68). Surprisingly, however, long-lived mice treated with rapamycin throughout life do not show evidence of impaired glucose homeostasis such as elevated fasting glucose levels or HbA1c, leading to the hypothesis that, on balance, the metabolic changes associated with rapamycin are neutral or beneficial (69, 70). It is difficult to accurately predict what effect rapamycin administration may have on a dog’s glucose regulation, as it likely depends on a combination of factors such as age, duration of treatment, and genetics. For this reason, it was important to investigate potential changes in glucose tolerance in the dogs in this study, and no overt impact on HOMA-IR scores was detected. Continued attention to glucose regulation is warranted in future studies of rapamycin in dogs.
+
+One of the most notable differences in the collected data was that significantly more of the biweekly at-home surveys from rapamycin-treated dogs indicated a “yes” response to the question, “Has your dog experienced any positive changes in behavior or other things that you would associate with good health within the past week? (please explain),” versus a “yes” response to this question in the biweekly at-home surveys from placebo-treated dogs. Despite the increased frequency with which owners reported positive changes in dogs in the rapamycin group, it is worth noting that the number of dogs whose owners reported “positive changes” was similar between groups (six out of nine rapamycin-treated dogs, and five out of eight placebo-treated dogs). Owners who indicated “yes” were given a free-text box in which to describe the observed positive changes. Because of the subjectivity associated with free-text responses, it is difficult to specifically characterize these positive changes. However, given the statistically significant difference in frequency with which this response was given between groups, it will be of interest to investigate the specific positive changes in behavior or health that owners are perceiving as associated with rapamycin treatment in future studies. Interestingly, owners had reported similar subjective benefits to their dogs in our prior masked, placebo-controlled, randomized clinical trial of rapamycin, including increases in owner-reported affection and activity (44).
+
+One of the key challenges of this clinical trial was the process of recruiting dogs that simultaneously met the size and age inclusion criteria without displaying any of the health-related and other exclusion criteria. This was reflected in our high proportion of excluded dogs, with only 17/79 dogs whose owners received the initial online eligibility questionnaire, and only 17/56 dogs whose owners submitted medical records, proceeding to randomization. Because the study population was intended to include “normal” older dogs, it becomes important to define what normal aging is. Many have attempted to clearly define the characteristics of both normal and abnormal aging in dogs (71, 72). These distinctions become increasingly important for this study and others like it that seek to recruit a “normal” aging dog population and to evaluate the ability of novel interventional therapies to mitigate age-related physical and/or pathophysiologic changes. It is possible that our inclusion and exclusion criteria led to the enrollment of an atypically healthy cohort of dogs in this size and age range, and that our results may not be generalizable to a more typical population of dogs.
+
+Another limitation of this study is that left atrial volume was not consistently obtained. In humans, volumetric measurements of left atrial size are considered standard due to their ability to assess for enlargement in multiple planes, rather than the single-dimension LA:Ao ratio that is commonly used in veterinary medicine (1, 2, 73). Because left atrial volume was not analyzed for the dogs in this study, it is possible that subtle changes in left atrial size could have been missed. Additionally, echocardiographic strain measurements were also not consistently obtained in this study. Had it been possible to analyze strain data, more subtle alterations in systolic function may have been identified. Further work is needed to better define the spectrum of expected age-related changes in cardiac structure and function in older dogs, as well as the ideal echocardiographic measurements to detect and monitor those changes over time.
+
+The initial study design sought to enroll 50 dogs (25 placebo, 25 rapamycin) based on power calculations. Several factors caused us to cease enrollment early, limiting the power to detect significant changes in the primary endpoint. These factors included difficulty identifying eligible dogs near the clinical site and the launch of the larger, multi-center clinical trial described below.
+
+In order to further evaluate the effects of rapamycin in the aging dog, our group is conducting a larger masked, placebo-controlled, randomized clinical trial (the Test of Rapamycin in Aging Dogs) in which the rapamycin treatment group will receive a higher cumulative dose (0.15 mg/kg) administered once weekly. There is evidence that intermittent administration of rapamycin yields beneficial effects of lifespan extension in mice (74) and reduces the incidence of adverse events in people (40).
+
+In conclusion, the present study did not show evidence that rapamycin, at the dose and duration given, resulted in any clinically or statistically significant improvement in cardiac function over 12 months as measured echocardiographically. At the same time, there were no clinically significant adverse events associated with rapamycin detected in medical histories, physical examinations or clinical pathology parameters. Some owners reported subjective “positive changes” associated with rapamycin therapy. If rapamycin truly has benefit for cardiac function, the relatively small sample size or lower dose compared with our previous study could be responsible for the lack of statistically significant findings. Further assessment evaluating a higher dosing regimen with a larger study population may be more effective at identifying a beneficial effect of rapamycin on cardiac health in dogs.
+
+## Data availability statement
+
+The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
+
+## Ethics statement
+
+The animal study was reviewed and approved by the Texas A&M University School of Veterinary Medicine and Biomedical Sciences Institutional Animal Care and Use Committee (IACUC) and Clinical Research Review Committee protocol number 2017–0125. Written informed consent was obtained from the owners for the participation of their animals in this study.
+
+## Author contributions
+
+BB, KC, and SW wrote the initial draft of the manuscript. KC, DP, MK, SW, SG, AS, LC, and JE developed the experimental design and were involved in data collection. SS performed analysis of the study data. All authors had the opportunity to participate in editing both the form and content of the manuscript and approved the final version.
+
+## Funding
+
+This study was funded by the William H. Donner Foundation. Additional support for some authors was provided by the Dog Aging Project (NIA U19 AG057377; BB, DP, MK, JE, LC, and KC).
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10234096.md

@@ -0,0 +1,120 @@
+---
+pmcid: "PMC10234096"
+pmid: "37259166"
+doi: "10.1186/s40575-023-00129-w"
+title: "Demography, common disorders and mortality of Boxer dogs under primary veterinary care in the UK."
+journal: "Canine medicine and genetics"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Demography, common disorders and mortality of Boxer dogs under primary veterinary care in the UK
+
+## Abstract
+
+### Background
+
+The Boxer is a popular dog breed with a distinctive appearance. However, the breed has been linked with several health conditions, some of which have been associated with its moderately brachycephalic conformation and its white colouration. Anonymised primary-care veterinary clinical records were explored to extract data on the demography, common disorders and mortality of Boxers in the UK in 2016.
+
+### Results
+
+The study population of 336,865 dogs included 3,219 (0.96%) Boxers, of which 10.71% were recorded as white. The mean adult bodyweight was 30.43 kg (SD 5.73 kg). Annual disorder counts did not differ statistically between the sexes or between white and non-white Boxers. The most prevalent fine-level precision disorders were otitis externa (*n* = 230, 7.15%), epulis (188, 5.84%), corneal ulceration (161, 5.00%) and periodontal disease (149, 4.63%). Of the 34 most common fine-level disorders, none differed in prevalence between white and non-white dogs. The most prevalent disorder groups were skin disorder (*n* = 571, 17.74%), neoplasia (457, 14.20%) and ear disorder (335, 10.41%). White Boxers had higher prevalence than non-white Boxers for two disorder groups: dental disorder and brain disorder. The median longevity of 346 Boxers that died during the study was 10.46 years (IQR 9.00–11.98, range 2.76–18.00). Median longevity did not differ statistically between the sexes or between white and non-white Boxers. The most common grouped causes of death were death – unrecorded cause (*n* = 73, 21.10%), neoplasia (43, 12.43%) and brain disorder (33, 9.54%).
+
+### Conclusions
+
+There was minimal evidence of substantial health differences between white and non-white Boxers. Among the four most common disorders recorded in Boxers, two were typically common across all types of dogs (otitis externa and periodontal disease) while two suggested strong predispositions for the Boxer breed (epulis and corneal ulceration), showing the value of eliciting breed-specific disorder patterns for insights for potential health reforms. The overall longevity of Boxer dogs was consistent with other breeds of similar body size.
+
+## Background
+
+The Boxer was developed in late nineteenth century Germany, through the deliberate cross-breeding of a traditional large hunting breed, the Bullenbeisser, with English Bulldogs and other breeds, to produce an athletic working dog with a stable temperament [1]. The breed was brought to the U.K. during the 1930s, and first registered with The Kennel Club (UK) in 1939 [2]. After the Second World War, the breed’s popularity in the UK rapidly increased, peaking at over 7,000 annual registrations in 1959, when they were the fourth most popular breed (approximately 6% of all dogs registered that year with The Kennel Club). Their numbers then roughly halved during the following two decades, before climbing to a second peak of over 10,000 annual registrations in 1997 (the eighth most popular breed in the UK; 4% of total registrations). In recent years, the breed has averaged 3–4,000 annual UK registrations and was the fourteenth most popular breed in the UK in 2020 [3].
+
+The Boxer is a medium-large sized, active dog with a short coat and a moderately brachycephalic skull shape [4]. The Kennel Club (UK) categorises the breed in the working group, and currently classifies it in Group 2 on its Breed Watch scheme, citing ‘pinched nostrils’ as a point of possible concern to be monitored by judges in the show ring [5]. The breed standard calls for a muzzle one-third the total length of the head, with a broad upper jaw and an undershot lower jaw (mandibular prognathism). The Boxer’s limbs are long and straight. The breed standard colours are fawn or brindle, with or without limited white markings that should not exceed one-third of the ‘ground’ colour [4]. However, some Boxers are completely or mostly white, and these individuals, like genetically similar white dogs in other breeds such as Bull Terriers, are thought to be at higher risk of congenital sensorineural deafness, although little research has specifically investigated the incidence of deafness in Boxers [6, 7].
+
+Over the last sixty years, several breed-related Boxer health problems have been reported in the Anglophone literature. The first epidemiological study of disease in UK pedigree dogs was carried out in 1962 [8]. In that study, Boxers were the second most common breed reported with ectropion, the seventh most common breed reported with entropion, the third most common breed reported with ‘prolonged soft palate’, and the seventh most common breed reported with skin fold dermatitis. However, these figures were not definitive for breed predispositions for these conformational issues, given the high popularity of the breed at that time and the basic methodology of this pioneering survey. In 1964, a leading human dentist alerted the veterinary profession to a ‘disastrous’ incidence of dental malocclusion in Boxers and other brachycephalic breeds, as a conformational issue which he attributed to selective breeding for a shorter face [9].
+
+In 1980, progressive axonopathy, a novel autosomal recessive neurological disease, was described in Boxers in the UK [10]. Over the following decade, this was eliminated from the breed through collaborative selective breeding, and is no longer considered a problem in the UK Boxer population today [11]. This successful intervention has left a relatively proactive and transparent cultural legacy within the Boxer breed community, which maintains an active web page dedicated to tracking investigations into certain breed health problems [11]. The first high-quality full genome sequence of the domestic dog was published in 2005 and used a female Boxer dog called Tasha [12]. This specific animal was selected for sequencing because it had the lowest heterozygosity at a small set of loci suggesting high levels of inbreeding that would make the sequencing easier. However, the researchers also chose the Boxer breed for their sequencing because of their belief that a predisposition to cancers in Boxers would enable them to combine information from the canine and human genome sequences to improve their searches for genetic contributors to cancer in man [13].
+
+In recent years, The Kennel Club, together with breed community representatives, has produced a Breed Health and Conservation Plan for Boxers, which includes a review of the recent literature on breed predispositions to disease. It collates the results of breed health surveys and other sources of information to provide an evidence-based overview of currently evidenced major health concerns in Boxers. This document lists cancer, cardiac disease (particularly aortic stenosis and arrhythmogenic right ventricular cardiomyopathy (ARVC)), juvenile kidney disease and skin disease as current key health priorities for the Boxer [14].
+
+Previous epidemiological reports by The Kennel Club [14], the Boxer Breed Council Health Committee [11] and the insurers Agria (U.K. and Sweden) [15] have provided insight into the incidence of various health problems in Boxers. In addition, a review of the wider literature in 2018 identified evidence of some predisposition to 86 disorders in Boxer dogs [16]. The breed has also featured in previous VetCompass publications, which have, for example, demonstrated a high Boxer breed predisposition for hypothyroidism [17] and corneal ulceration [18]. However, no single previous study has specifically investigated the health of Boxers across the full range of disorders using data harvested from primary-care veterinary clinics rather than owner reports (where the data may be distorted through information or social desirability bias) or insurance data (which are biased towards disorders that are not excluded from insurance cover) [19].
+
+Using veterinary clinical data from the VetCompass Programme [20], this study aimed to characterise the demography, common disorders and mortality of the general population of Boxers under primary veterinary care in the UK, with a special comparative focus between males and females. Focus was also placed on exploring potential health effects for white Boxers. The results from the current study could provide a reliable framework to assist reforms in breeding practices and ultimately contribute to improved health and welfare of Boxers.
+
+## Materials and methods
+
+The study population included all dogs under primary veterinary care at clinics participating in the VetCompass™ Programme during 2016. Dogs under veterinary care were defined as those with either a) at least one electronic patient record (EPR) (free-text clinical note, treatment or bodyweight) recorded during 2016 or b) at least one EPR recorded both before and after 2016. The VetCompass Programme collates de-identified EPR data from primary-care veterinary practices in the UK for epidemiological research [20]. Data fields available for VetCompass researchers include a unique animal identifier along with species, breed, date of birth, colour, sex, neuter status and bodyweight, and clinical information from free-form text clinical notes and treatments with relevant dates.
+
+A cohort study design with a cross-sectional analysis of disorder data on dogs registered at participating practices was used to estimate the one-year period prevalence of the most commonly diagnosed disorders. Sample size calculations estimated that 2,903 dogs were needed to report the prevalence for a disorder having a frequency of 2.0% to a precision of 0.5% [21]. Ethics approval was obtained from the RVC Ethics and Welfare Committee (reference number 2015/1369).
+
+Dogs recorded as Boxer breed were categorised as Boxer and all remaining dogs were categorised as non-Boxer. *Adult Bodyweight* (Kg) described the mean bodyweight recorded from all bodyweight data for dogs aged over 18 months. *Neuter* described the status of the dog (entire or neutered) at the final available EPR. *Age* described the age at December 31^st^ 2016, the final date by which dogs were categorised as cases or non-cases for each disorder.
+
+The list of unique Boxer animal identification numbers was randomly ordered and the clinical records of all animals were reviewed manually in detail to extract the most definitive diagnoses recorded for all disorders recorded in the clinical records as existing during 2016. Disorders were recorded to an ontology extended from the VeNom Code list as previously described [22]. Elective (e.g. neutering) or prophylactic (e.g. vaccination) clinical events were not included. No distinction was made between pre-existing and incident disorder presentations. Disorders described within the clinical notes using presenting sign terms (e.g. ‘vomiting’ or 'vomiting and diarrhoea'), but without a formally recorded clinical diagnostic term, were included using the first clinical sign listed (e.g. vomiting). Mortality data (recorded cause, date and method of death) were extracted on all deaths at any date during the available EPR data.
+
+The extracted diagnosis terms were mapped to a dual hierarchy of precision for analysis: fine-level precision and grouped-level precision as previously described [22]. Briefly, fine-level precision terms described the original extracted terms at the maximal diagnostic precision recorded within the clinical notes (e.g. *inflammatory bowel disease* would remain as *inflammatory bowel disease*). Grouped-level precision terms mapped the original diagnosis terms to a general level of diagnostic precision (e.g. *inflammatory bowel disease* would map to *enteropathy*).
+
+Following data checking for internal validity and cleaning in Excel (Microsoft Office Excel 2016, Microsoft Corp.), analyses were conducted using Stata Version 16 (Stata Corporation). The sex, neuter status, colour, age and adult bodyweight for Boxers under veterinary care during 2016 were described. Continuous variables that were normally distributed were described using mean (standard deviation [SD]); otherwise the median (interquartile range [IQR] and range) was reported [23]. One-year (2016) period prevalence values were reported for common disorders along with 95% confidence intervals (CI) that described the probability of diagnosis at least once during 2016. Binomial CI were calculated using the Wilson method. The median age was reported for each common disorder. Prevalence values were reported overall and separately for males and females. The chi-square test was used to compare categorical variables and the Students t-test or Mann–Whitney U test to compare continuous variables as appropriate [23]. Statistical significance was set at the 5% level.
+
+## Results
+
+### Demography
+
+The study population of 336,865 dogs from 438 clinics in the VetCompass database under veterinary care during 2016 included 3,219 (0.96%) Boxers. Twelve of these Boxers (0.37%) had unrecorded sex and neuter status. Of the Boxers with information available, 1,542 (48.08%) were female and 1,435 (44.75%) were neutered overall. The probability of neutering did not differ between the sexes (female 44.75% vs male 44.74%, *P* = 0.999). Adult bodyweight data were available for 2306/3219 dogs (71.64%). The mean adult bodyweight overall was 30.43 kg (SD 5.73 kg). The mean adult bodyweight of males (32.99 kg, SD 5.42 kg) was heavier than for females (27.56 kg, SD 4.57 kg) (*P* < 0.001). Age data were available for 3156/3219 dogs (98.04%). The median age of Boxers overall was 5.81 years (IQR 3.10–8.79, range 0.18–18.37). The distribution of ages (years) did not differ statistically between females (median 5.92, IQR 3.23–5.92, range 0.10–18.37) and males (median 5.72, IQR 2.96–8.68, range 0.38–16.21) (*P* = 0.071). Colour information was recorded for 2940/3219 (91.33%) dogs. Of these, 1,821 (61,94%) were recorded as solid colour and 1,119 (38.06%) were recorded as multi-colour. The most common colours overall were brindle (*n* = 671, 22.82%), dark red (665, 22.62%) and dark red multi-colour (615, 20.92%). There were 315 (10.71%) Boxers recorded as white (Table 1).Table 1Colour of Boxers under primary veterinary care at practices participating in the VetCompass™ Programme in the UK from January 1^st^, 2016 to December 31^st^, 2016 (*n* = 2,940)Colour^a^No%Brindle67122.82Dark red66522.62Dark red multicolour61520.92Brindle multicolour33211.29White31510.71Light fawn1575.34Light fawn multicolour1454.93Black multicoloured170.58Black130.44Tricolour80.27Blue multicoloured20.07Total2,940100
+
+### Disorder prevalence
+
+Manual examination of the EPRs of all 3,219 Boxers yielded 5,022 unique disorder events recorded during 2016. There were 2,381 (73.97%) Boxers with at least one disorder recorded during 2016 while the remaining 26.03% had no disorder recorded and either presented for prophylactic management only or did not present at all during 2016. The median annual disorder count per Boxer during 2016 was 1 disorder (IQR 0–2, range 0–12). Annual disorder counts did not differ statistically between female (median 1, IQR 0–2, range 0–12) and male Boxers (median 1, IQR 1–2, range 0–9) (*P* = 0.654). Annual disorder counts did not differ statistically between white (median 1, IQR 1–2, range 0–8) and non-white Boxers (median 1, IQR 0–2, range 0–12) (*P* = 0.414).
+
+There were 420 distinct fine-level disorders across the study dogs. The most prevalent fine-level precision disorders recorded were otitis externa (*n* = 230, 7.15%), epulis (188, 5.84%), corneal ulceration (161, 5.00%), periodontal disease (149, 4.63%), heart murmur (138, 4.29%) and skin mass (138, 4.29%). The median age for dogs with individual fine-level disorders varied from 2.70 years (thin) to 10.75 years (death—unknown cause). Among the 34 most common fine-level disorders, females were more likely to be diagnosed with three disorders (periodontal disease, skin mass, urinary incontinence) while males were more likely to be diagnosed with three disorders (heart murmur, aggression, aural discharge). Among the 34 most common fine-level disorders, there were no disorders with differing prevalence between white and non-white dogs (Table 2). Only two dogs were recorded with deafness during the year of the study; one was white and one was non-white.Table 2Prevalence of the most common disorders at a *fine-level of diagnostic precision* recorded in Boxers (*n* = 3,219) under primary veterinary care at UK practices participating in the VetCompass™ Programme from January 1^st^, 2016 to December 31^st^, 2016. Disorders with 40 or more events includedFine-level DisorderNoOverall prevalence %95% CI^a^Median age (years)Female prevalence %Male prevalence %Sex *P*-value*White prevalenceNon-white prevalenceColour *P*-value**Otitis externa2307.156.31–8.095.507.007.270.7727.307.350.974Epulis1885.845.08–6.708.756.365.350.2235.715.830.935Corneal ulceration1615.004.30–5.818.774.605.350.3366.035.140.503Periodontal disease1494.633.96–5.418.665.583.72**0.012**6.984.530.054Heart murmur1384.293.64–5.047.553.504.98**0.038**4.134.380.835Skin mass1384.293.64–5.048.705.323.36**0.006**4.764.380.756Obesity1233.823.21–4.546.644.093.540.4233.173.810.575Overgrown nail(s)1183.673.07–4.376.774.023.240.2393.173.810.575Hypersensitivity allergy disorder952.952.42–3.594.713.112.820.6282.863.090.824Aggression902.802.28–3.425.961.563.96**< 0.001**2.542.930.694Diarrhoea852.642.14–3.252.872.332.940.2842.862.670.843Osteoarthritis822.552.06–3.159.972.662.460.7253.492.550.327Post-operative complication682.111.67–2.673.611.882.280.4271.272.210.273Urinary incontinence652.021.59–2.579.913.700.48**< 0.001**1.902.020.891Aural discharge641.991.56–2.534.141.492.46**0.049**2.542.060.573Claw injury631.961.53–2.504.821.951.920.9611.271.980.380Mast cell tumour601.861.45–2.397.242.011.740.5752.541.900.444Alopecia591.831.42–2.366.212.141.560.2231.901.870.962Pyoderma591.831.42–2.365.271.751.920.7192.861.790.191Lameness571.771.37–2.295.871.561.980.3622.541.710.298Hypothyroidism541.681.29–2.188.841.561.800.5890.631.830.122Thin531.651.26–2.152.701.621.680.8932.861.520.081Conjunctivitis521.621.23–2.114.981.561.680.7792.221.600.415Skin disorder501.551.18–2.045.471.751.380.3991.591.640.946Limb mass491.521.15–2.018.141.491.560.8721.271.640.622Death—unknown cause471.461.10–1.9410.751.491.380.7932.541.260.067Infectious canine tracheobronchitis471.461.10–1.944.351.171.740.1760.951.450.479Seizure disorder471.461.10–1.949.591.431.500.8602.221.410.262Vomiting471.461.10–1.944.341.491.440.9062.221.490.320Anal sac impaction461.431.07–1.905.511.101.740.1280.951.600.376Cruciate disease431.340.99–1.796.150.971.680.0810.951.410.508Atopic dermatitis421.300.97–1.765.011.491.140.3832.221.260.162Acrochordon411.270.94–1.727.421.041.040.3031.591.260.624Stiffness411.270.94–1.7210.401.231.320.8221.271.220.938
+
+There were 59 distinct disorder groups recorded across the study dogs. The most prevalent disorder groups were skin disorder (*n* = 571, 17.74%), neoplasia (457, 14.20%), ear disorder (335, 10.41%), mass lesion (325, 10.10%) and ophthalmological disorder (320, 9.94%). The median age for dogs with individual disorder groups varied from 3.11 years (underweight) to 9.78 years (brain disorder). Among the 22 most common grouped disorders, females were more likely to be diagnosed with three disorders (mass lesion, dental disorder, urinary system disorder) while males were more likely to be diagnosed with one disorder (behavioural disorder). Among the 22 most common grouped disorders, white Boxers had higher prevalence than non-white Boxers for two disorders: dental disorder and brain disorder (Table 3).Table 3Prevalence of the most common disorders at a *grouped-level of diagnostic precision* recorded in Boxers (*n* = 3,219) under primary veterinary care at UK practices participating in the VetCompass™ Programme from January 1^st^, 2016 to December 31^st^, 2016. Disorders with 50 or more events includedGrouped-level Disorder^a^NoOverall prevalence %95% CI*Median age (years)Female prevalence %Male prevalence %Sex *P*-value**White prevalenceNon-white prevalenceColour *P*-value**Skin disorder57117.7416.46–19.105.3317.7717.780.99517.1418.480.563Neoplasia45714.2013.03–15.458.1514.8513.450.25615.2414.320.663Ear disorder33510.419.40–11.515.349.6011.170.14510.7910.780.995Mass lesion32510.109.10–11.198.7511.878.47**0.001**9.5210.550.573Ophthalmological disorder3209.948.95–11.028.379.6610.150.64510.4810.290.916Musculoskeletal disorder2868.887.95–9.928.248.119.670.12110.168.910.467Enteropathy2507.776.89–8.743.867.268.230.3089.847.810.210Heart disease2046.345.55–7.237.935.517.090.0676.356.440.952Nail disorder1956.065.28–6.945.926.295.710.4865.086.210.428Dental disorder1705.284.56–6.118.646.164.44**0.030**7.945.14**0.039**Urinary system disorder1314.073.44–4.818.926.232.10**< 0.001**4.134.150.983Obesity1233.823.21–4.546.644.093.540.4233.173.810.575Traumatic injury1123.482.90–4.173.282.923.960.1062.543.470.389Complication associated with clinical care procedure1083.362.79–4.034.713.573.120.4853.173.500.762Behavioural disorder1063.292.73–3.975.962.084.44< 0.0012.863.430.595Upper respiratory tract disorder1043.232.67–3.904.222.853.600.2314.133.120.341Female reproductive disorder832.582.08–3.194.555.320.06~1.902.700.401Brain disorder822.552.06–3.159.782.202.820.2655.082.25**0.003**Underweight712.211.75–2.773.112.142.280.7843.492.020.090Anal sac disorder662.051.61–2.605.512.082.040.9471.592.290.426Endocrine system disorder662.051.61–2.608.952.012.100.8550.632.250.058Male reproductive system disorder561.741.34–2.254.65~3.36~2.861.680.137
+
+### Mortality
+
+There were 346 deaths recorded in Boxers during the study. Information on age at death was available for 335 of these deaths. The median longevity of Boxers overall was 10.46 years (IQR 9.00–11.98, range 2.76–18.00). The median longevity of females (10.41 years, IQR 9.16–11.86, range 1.34–18.00, *n* = 171) did not differ statistically to males (10.53 years, IQR 8.60–12.00, range 1.62–15.43, *n* = 164) (*P* = 0.920). Coat colour was recorded for 310 of the deaths. The median longevity of white Boxers (10.55 years, IQR 9.00–11.70, range 3.65–14.51, *n* = 43) did not differ statistically to non-white Boxers (10.38 years, IQR 8.88–11.94, range 1.34–15.46, *n* = 262) (*P* = 0.791). The median age at death for dogs with individual disorder groups varied from 3.11 years (thin) to 9.78 years (brain disorder). The method of death was recorded for 334 deaths. Of these, 311 (93.11%) deaths were by euthanasia while 23 (6.89%) deaths were unassisted. The probability of death by euthanasia did not differ statistically between females (92.90%) and males (93.29%) (*P* = 0.887). The probability of death by euthanasia did not differ statistically between white (90.70%) and non-white Boxers (93.77%) (*P* = 0.454). The most common causes of death described at a grouped-precision level were death – unrecorded cause (*n* = 73, 21.10%), neoplasia (43, 12.43%), brain disorder (33, 9.54%) and mass lesion (29, 8.38%). The probability of death did not differ statistically between females and males for any of the 12 most common grouped causes of death. White Boxers had a higher probability of death than non-white Boxers for two of the 12 most common grouped causes of death: brain disorder and enteropathy (Table 4).Table 4Common grouped causes of mortality in Boxers under primary-care veterinary at UK practices participating in the VetCompass™ Programme from January 1^st^, 2016 to December 31^st^, 2016 (*n* = 346). Disorders with 8 or more events includedGrouped-level disorderNoOverall %95% CI^a^Median age at death (years)% Female% Male*P*-Value*White prevalenceNon-white prevalenceWhite *P*-value**Death – unrecorded cause7321.1017.13–25.7010.5823.7317.860.18020.4520.900.947Neoplasia4312.439.36–16.329.4511.8613.100.7299.0912.690.499Brain disorder339.546.87–13.0911.019.0410.120.73318.187.84**0.028**Mass lesion298.385.90–11.7810.958.478.330.9624.559.700.267Collapsed236.654.47–9.7811.616.217.140.7304.557.460.484Heart disease164.622.87–7.3810.133.955.360.5360.005.220.121Poor quality of life113.181.78–5.6010.552.264.170.3142.273.730.627Renal disease113.181.78–5.607.923.952.380.4062.273.730.627Spinal cord disorder102.891.58–5.2410.841.694.170.1714.552.610.478Musculoskeletal disorder92.601.37–4.8712.012.262.980.6772.272.610.895Behaviour disorder82.311.18–4.507.001.692.980.4290.002.240.316Enteropathy82.311.18–4.508.773.391.190.1756.821.49**0.027**
+
+## Discussion
+
+This study of over 3,000 Boxers among the wider domestic dog population in the UK has demonstrated an ongoing popularity of the breed, comprising around 1% of dogs in the UK in 2016. The study also identifies the most common disorders recorded in the breed, showing that some of these, such as otitis externa and periodontal disease, occur at levels typical of dogs overall while others, such as epulis and corneal ulceration, are quite breed predisposed. There was only limited evidence for important health differences between the sexes and very little evidence supporting reduced health in white Boxers. With an overall median longevity of 10.46 years, the typical lifespans of Boxers appeared to be largely consistent with other breeds of this body size. This study highlights the value of Big Data resources such as VetCompass to provide breed health profiles that can support improved health plans for individual breeds.
+
+### Demography
+
+Boxers comprised just under 1% of the total canine population surveyed in this 2016 dataset. In the same year, Boxers comprised 1.6% of dogs registered at The Kennel Club [3]. This higher proportion among the registered pedigree dogs is expected, given that the wider canine population also includes designer crossbreeds, mixed breeds and other breeds of dog that are not registered with or recognised by The Kennel Club. There were no statistically significant demographic differences between males and females except for bodyweight. Mean bodyweight for males (32.99 kg) averaged almost 5.5 kg more than females (27.56 kg). This mirrors the sexual dimorphism encoded in the breed standard, which suggests a male bodyweight of 30–32 kg and a female bodyweight of 25–27 kg [4]. In both sexes, the mean bodyweights reported through VetCompass analysis exceeded the upper limit recommended by the breed standard, suggesting that either the wider general population is truly comprised of structurally larger dogs than the idealised view of the breed or that a population tendency to overweight/obesity is skewing the ideal bodyweight results.
+
+Just over 10% of the Boxers in this study cohort were recorded as white. Although this colour is not accepted under the breed standards of The Kennel Club (UK) or the Fédération Cynologique Internationale (FCI) [4, 24], white animals have existed in the breed since its foundation [25]. Gene mapping has shown a shared genetic mutation for the white colour in Boxers and Bull Terriers, and white colouring has been linked to congenital deafness for over a century [26, 27]. For this reason, white Boxers have long been considered undesirable by breeders and historically were often culled [28]. Consequently, little reliable information has been available until now on the proportion of white dogs within the Boxer breed or on associations of white colour with deafness and other health problems in the wider population. In a study in the Netherlands in 1994–5, 16.9% of a cohort of 2,629 Boxer puppies were euthanized at birth because they were white [29], and it is likely that similar culling practices prevailed elsewhere at that time. However, changing attitudes towards such large-scale culling among pedigree dogs now mean that more white Boxers are sold as pets and fewer are euthanized as puppies. A more recent study in Italy reported that 17.5% of a Boxer population in Italy were reported by their owners as white, with none of these white dogs recorded as deaf [28]. In the current VetCompass study, 10.71% of Boxers were recorded as white in the UK. This lower UK proportion of white Boxers could reflect a genuine difference in allele frequency between different national dog populations or might indicate some ongoing level of neonatal culling of white dogs in the UK. For comparison, The Kennel Club registration data for all Boxers recorded as born between 2015 and 2018 identified that 6.8% (1,022 from a total of 15,124) were white [3]. Although these Kennel Club data refer to births during these years rather than to the adult population that was reported in the VetCompass data, this even lower figure could reflect ongoing reluctance by breeders to register non-standard white dogs, particularly if they perceive these puppies as deaf or likely to be deaf. An alternative explanation could be that genetic mutations for white dogs are genuinely more common in the wider non-Kennel Club population of Boxers in the UK. The current study also failed to show any predisposition for deafness in the white Boxers. This may reflect previous culling of fully deaf dogs to mainly leave white Boxers with at least unilateral hearing in the living population. An alternative explanation could be that some of these living white Boxers were indeed deaf but that neither the owner or veterinary teams were aware of their deafness, which can be challenging to identify, or that this information was not recorded in the clinical notes [30].
+
+### Disorder prevalence
+
+Neither males versus females, nor white versus non-white Boxers differed statistically in their annual disorder counts, suggesting little or no overall health differences between these categories of dog. The two most prevalent disorder groups both overall and in each sex were skin disorder (overall 17.74%) and neoplasia (overall 14.20%). This finding provides supporting evidence for the breed health priorities of neoplasia and skin disease previously determined by The Kennel Club’s Breed Health and Conservation Plan [14], with the finding of mass lesion in the current study (overall 10.10%) as another common disorder group that was likely to also include many cases of neoplasia that had not been worked up to full diagnoses. There is a long and deep literature worldwide on predispositions to a range of neoplasia in Boxer dogs based on data sources including referral veterinary records [31], post-mortem studies [32], histopathology sample analyses [33], cancer registries [34, 35] and mortality studies [36]. The current study provides further supporting evidence of cancer predisposition in Boxers that is based on primary-care veterinary data. At a disorder group level, 14.2% of the Boxers in the current study were diagnosed with neoplasia, which is substantially higher than results using the same data source for other similar sized breeds such as German Shepherd Dog 4.82% [37], Greyhound 5.5% [38] Labrador Retriever 7.4% [39] and Rottweiler 7.96% [40]. The evidence for a cancer predisposition in the current study also supports the choice of Boxer dogs as comparative oncology models for translational research to better understand the origins of cancer and translate these findings to novel therapies, in line with the thinking of the team that chose the Boxer for the first full genomic sequencing in dogs [13, 41].
+
+Delving deeper into health differences between the sexes, there were differences detected for some disorders at both grouped and fine levels of analysis. For example, at the grouped level, females were almost three times more likely than males to be diagnosed with a urinary system disorder: a difference that reflects an even more marked sex difference at the fine level of analysis, where females were almost eight times more likely than males to be diagnosed with urinary incontinence. This female predisposition for urinary incontinence in Boxers concurs with the relative risk of over 3 times for females compared to males that has previously been recorded in dogs overall in the UK [42, 43]. At the grouped disorder level, females were also significantly more likely than males to be diagnosed with mass lesion and dental disorder, while at the fine level of disorder, females were 1.5 times as likely as males to be diagnosed with skin mass and 1.5 times as likely to be diagnosed with periodontal disease. However, despite this sex predisposition, the prevalence of 5.58% for periodontal disease in females was still lower than the overall prevalence of 12.5% recorded across all dogs in a previous VetCompass study using the same methodology, suggesting that Boxers may be a relatively protected breed against periodontal disease overall [44]. This relative protection is also reflected in other brachycephalic breeds such as the French Bulldog and English Bulldog [45, 46]. Paradoxically, it is possible that the open mouthed breathing, dental malocclusion, mandibular prognathism, drooling and widely spaced teeth seen in many Boxers and other brachycephalic breeds, which have been considered problematic for over seventy years, are nevertheless somehow protective against gingivitis: perhaps by reducing food trapping and by increasing saliva flow [9].
+
+At the grouped disorder level, males were over twice as likely (× 2.1) as females to be diagnosed with behavioural disorder. At the fine level of diagnostic precision, males were 2.5 times as likely to show aggression, which was recorded in 3.96% of males compared with 1.56% of females. Somewhat surprisingly, despite promotion by The Kennel Club as ‘much loved as a family companion for his intelligence and character’, aggression was the fifth most common fine-level disorder in male Boxers, reported more frequently than anecdotally ‘common’ disorders in primary care practice such as diarrhoea and osteoarthritis. Given that behavioural disorder was also the fourth commonest cause of mortality in this male cohort and showed the lowest median age of death, this suggests that behavioural problems, particularly aggression and especially in males, may have a significant impact on the welfare of Boxers and the people they live with, which potentially merits further attention [47]. This finding further corroborates reports of relatively high levels of aggression in Boxers compared to otherwise behaviourally similar dogs, identified via large-scale analysis of C-BARQ data on over 32,000 dogs in the USA [48]. However, although the 2.80% prevalence of aggression reported overall in Boxers in the current study was higher than the 1.09% aggression in Labrador Retriever [49] and 1,7% in Greyhounds [38] previously reported, it was substantially lower than the 4.76% aggression reported in German Shepherd Dogs [37] and 7.46% reported in Rottweilers [40]. This wide variation in the prevalence of aggression both between breeds, and also between sexes within breeds, suggests that improved information on behavioural and temperament attributes of individual breeds is needed to help to owner make appropriate decisions when selecting breeds as companion and family dogs.
+
+At the fine level of disorders, males were also statistically more likely than females to be diagnosed with heart murmur, with a male prevalence of 4.98% compared to a female prevalence of 3.50%. Although heart murmur was the fifth most commonly recorded fine-level disorder overall, it jumped to the fourth most common fine-level disorder in males compared to only the ninth most common in females. While specific individual cardiological diagnoses, such as aortic stenosis and atrial septal defects, which can cause murmurs, have been recognised for many years as breed health concerns in Boxers, many true cases of these conditions in typical primary care caseloads may not be clinically worked up to this level of diagnostic precision [14]. Supporting the idea that clinical signs are often used in lieu of formal biomedical diagnoses in general veterinary care, previous work within VetCompass has reported that while just 0.36% of dogs under primary veterinary care were recorded with the specific diagnosis of degenerative mitral valve disease, there were almost nine times as many (3.18%) recorded and clinically managed for a health murmur diagnosis that was clinically consistent with the existence of degenerative mitral valve disease [50]. Nevertheless, the finding that heart murmurs are recorded more frequently in Boxer males is consistent with previous research that reported a male predisposition for both aortic and pulmonic stenosis in Boxers [51].
+
+In addition to some disorder predispositions where the prevalence in Boxers is higher than in other types of dogs, the current study also highlights that the Boxer also has some disorders with concerningly high prevalence that are similarly common in dogs that are not Boxers. Although otitis externa was the most prevalent fine-level precision disorder in Boxers (7.15%), this prevalence was very similar to the 7.30% prevalence for otitis externa reported across all UK dogs in a previous VetCompass study that used a similar methodology [22]. None the less, it could be argued that the high frequency of otitis externa is suggestive that this disorder should be considered as a key health priority for the Boxer. There has been a tendency for many decades to focus breed health reforms on predispositions within breeds or on disorders where there is a genetic or other diagnostic test available, no matter how rare the actual disorders are, rather than on also considering common disorders that may not even have a predisposition [14]. It may be that veterinary and breeding communities need to give greater future emphasis in breed health plans to the sum of the health and welfare impacts at an overall breed level so that these health schemes truly deliver health and welfare gains at a population level.
+
+However, that said, many of the most frequently reported fine-level disorders in the current study do show the double welfare impact of both high prevalence and high breed predisposition which marks these as very strong candidates for consideration as key disorder priorities for Boxers. Strong historical support for these disorders as breed-related problems in Boxers only further supports their prioritisation. Predisposition of Boxers to dental epulis (also known as peripheral odontogenic fibroma or fibromatous epulis of periodontal ligament origin) has been recognised since at least the 1960s [52]. More recently, this predisposition has been highlighted in a UK owner survey [53], in Swedish insurance data [14] and in UK diagnostic laboratory data [54]. In the current study, epulis was the second most prevalent fine-level disorder in both sexes, with an overall prevalence of 5.84%.
+
+As the third most common disorder in Boxers in the current study, with a prevalence of 5.00%, corneal ulceration is another condition that is flagged by the current work with both high prevalence and predisposition. Conformational predisposition to corneal ulceration among dogs with brachycephaly has been recognised for over a century [55] with a breed-specific problem of non-healing (indolent) corneal ulceration formally described in Boxers in the 1960s [56]. Previous VetCompass work on corneal ulceration across multiple breeds identified the Boxer to have the second highest breed prevalence of corneal ulceration (4.98%) (exceeded only by the Pug at 5.42%), with the Boxer prevalence being 13 times higher than the 0.38% prevalence recorded in crossbreeds [18]. Such a high prevalence and predisposition for a painful condition that is linked to extreme body conformation suggests a very strong rationale for prioritising this condition within reforms that could include breeding towards conformational moderation to improve innate health in the breed [14, 57]. To build on this concept of evaluating disorder prioritisation within breeds based on both prevalence and predisposition, the current work also highlighted a relatively high prevalence for hypothyroidism, which affected 1.68% of the Boxer dogs in this study. Although this was only around one third of the prevalence for corneal ulceration, previous VetCompass work has reported the Boxer breed with the third highest prevalence of hypothyroidism and to have over 10 times the risk compared with crossbred dogs [17]. This comparison between corneal ulceration and hypothyroidism in Boxers highlights that even when a breed is highly predisposed to a certain disorder, a more prevalent disease leads to a higher disease burden and therefore may be considered as having a greater breed welfare impact. Other factors such as the duration and the severity of each disorder should also be considered with making decisions about breed health prioritisation [58].
+
+There is growing awareness that identification of disorders that are protected against may also offer useful information for breed health plans. For best effect, studies that identify protections should include clinical data on both the breed of interest as well as on a wider comparator group e.g. all remaining dogs in the population that are not of this breed. To date, such comparative studies have been reported for breeds including French Bulldog [45], Pug [59], English Bulldog [46], Labrador Retriever [49] and Staffordshire Bull Terrier [60]. However, although the current study included only results on Boxer dogs, it is still possible to identify some evidence on disorder protections by comparing the current results to prevalence data on dogs overall that were derived from the same VetCompass data source [22]. By comparing the current results to the prevalence values for dogs overall in the UK, there was some evidence to suggest protection in Boxers to periodontal disease (prevalence 4.63% in Boxers vs 12.52% in dogs overall), obesity (3.82% vs 7.07%), overgrown nails (3.67% vs 5.52%) and diarrhoea (2.64% vs 3.81%). The existence of both protections as well as predispositions to differing disorders within breeds adds to the nuances needed to fully understand overall breed health and promotes the value of holistic approaches to promoting breed health such as are taken by the Kennel Club’s Breed Health and Conservation Plans [61].
+
+Boxers are widely regarded as a breed with a moderately brachycephalic head shape [4]. However, current research increasingly draws nuanced distinctions between the typical attributes and pathologies from the brachycephalic conformation across different breeds in dogs [62-64]. Despite the strong predisposition in Boxers to corneal ulceration, which is typical for the brachycephalic conformation, it is important to consider whether every disorder predisposition linked to brachycephaly will also show predisposition in the Boxer. Brachycephalic airway obstructive syndrome (BOAS) is widely considered as a common disorder in dogs with extreme brachycephaly [65, 66]. A previous VetCompass study examining associations between brachycephaly and upper respiratory tract disorder across extreme, moderate and non-brachycephalic breeds of small and medium size reported a higher prevalence of upper respiratory tract disorders in extreme brachycephalic dogs (22%) than in the moderate and non-brachycephalic group (9.7%). It is therefore, somewhat surprising that the current Boxer study reveals an unexpectedly low grouped level prevalence for upper respiratory tract disorder of only 3.23%, suggesting that upper respiratory tract disorder does not carry a major welfare burden in Boxers and that perhaps its larger body size may be protective for the Boxer, despite its moderately brachycephalic features. The relatively higher craniofacial ratio (muzzle to overall skull length ratio) of the Boxer compared to more extreme brachycephalic breeds (Boxer: 0.31, vs. Pug: 0.08; French Bulldog: 0.19; English Bulldog: 0.22) has previously been reported to be associated with markedly lower predisposition to upper respiratory tract disorders in Boxers with a conclusion that the more moderate facial conformation of the Boxer likely results in reduced risk of anatomical abnormalities and subsequent overcrowding of the airways [67]. Other predispositions that are recorded in dogs overall with brachycephaly but that had lower relative prevalence in Boxers included umbilical hernia, pododermatitis, patellar luxation and anal sac impaction [64]. These results highlight the dangers of ecological fallacy whereby the disorder risks of a group (e.g. all breeds with brachycephaly) are attributed to an individual (e.g. the Boxer as a single breed with brachycephaly) and underlines the value of breed-specific epidemiological and clinical investigation such as the current study [68].
+
+One aim of the current study was to investigate the health of white Boxers compared to non-white Boxers. The Kennel Club breed standard currently excludes white dogs, which have long been anecdotally considered to have an elevated risk of congenital sensorineural deafness and, possibly, other diseases [4, 28]. However, despite this, very little empirical research has been reported on the incidence of congenital sensorineural deafness in Boxers. One small study in Brazil carried out brainstem auditory-evoked response (BAER) testing on 43 Boxers, of which 14 were white, showing that two white dogs were bilaterally deaf and one was unilaterally deaf, while all the non-white dogs had normal hearing [7]. However, as those authors pointed out, the study sample size was too small to draw meaningful statistical conclusions. Another approach here is to extrapolate results from Bull Terriers, which have the same genetic cause for colour-related deafness as Boxers [6, 7], but have been studied in more detail, with these studies benefitting from a higher proportion of white dogs among Bull Terriers because white dogs are allowed within The Kennel Club breed standard [4]. A recent BAER study of 1060 Bull Terriers in the UK reported an overall 10.19% prevalence of congenital sensorineural deafness, with 8.21% unilaterally deaf and 1.98% bilaterally deaf [69]. When divided by colour, 19.29% of white dogs were deaf in one or both ears, whereas only 0.77% of the non-white dogs were deaf in one or both ears. Although these data refer to Bull Terriers, all other things being equal, a similar prevalence of deafness should be expected in white Boxers, given the common underlying cause. However, in the current VetCompass study, only two dogs (one white and one non-white) were identified as deaf, suggesting the potential for substantial under-reporting of deafness within primary care caseloads, perhaps because the owner and/or clinician were unaware of the problem, particularly in the case of unilateral deafness. Alternatively, the true proportion of deaf white dogs in the adult dog population may be low because culling as a default decision among breeders may still be common in the UK [28]. Overall, the current study identified only weak evidence for any significant difference in health between white and non-white Boxers. None of the 34 fine-level disorders showed prevalence differences between white and non-white Boxers while just two of the grouped-level disorders differed, with white Boxer having higher prevalence of brain disorders and enteropathy. Given the multiple testing of 66 individual tests across the fine-level and group disorders, it would not be unexpected for two tests to return false positive (type I) errors, so even these two findings should be treated with caution [70]. An absence of wider health differences between white and non-white Boxers apart from hearing issues is not surprising because, despite the long-standing prejudice against white colour in this breed, the causative mutation is not generally thought to be linked to health problems other than congenital deafness [71] and vulnerability to sunburn as in all animals with little pigmentation [72].
+
+### Mortality
+
+The median age at death for Boxers in the current study was 10.46 years, which is midway across the range for some other large-sized breeds previously reported using a similar methodology; Rottweiler (9.0 years) [40], German Shepherd Dogs (10.3 years) [37] and Labrador Retrievers (12.0 years) [39]. The longevity of Boxers reported here is shorter than the median of 12.0 years reported across all dogs in the UK but it is well recognised that smaller dogs have a longevity advantage over larger dogs [73]. Therefore, these results do not suggest that the longevity overall of Boxers is particularly shorter than might be expected for a pure breed of this body size. This is especially noteworthy given that there is strong evidence that extreme brachycephaly is linked to substantially shortened lifespan [74]. It may be that the moderate rather than extreme degree of brachycephaly alone of many Boxers does not impose meaningful lifespan reductions on this breed, which does not show extreme conformation in other parts of the body [4].
+
+Among the common causes of mortality, four disorder groups were identified with markedly earlier ages at death than the overall mean age of 10.46 years described for this Boxer population: neoplasia (9.45 years); renal disease (7.92 years); behavioural disorder (7.00 years); and enteropathy (8.77 years). Given these early deaths, it could be considered that these disorders exert an important impact on Boxer health and welfare. It is noteworthy that of these, neoplasia and renal disease map onto two priority health concerns reported by the breed community, cancer and juvenile kidney disease [14]. Given the strong prior evidence base on predisposition to neoplasia in Boxers [32, 36, 75], the current findings of high rates of mortality from neoplasia are not surprising. However, even these may be a substantial underestimate of the true overall neoplasia mortality as, in addition to the 12.43% of deaths ascribed directly to neoplasia, large components of the deaths ascribed to brain disorder (9.54%), mass lesion (8.38%) and death with an unrecorded cause (21.10%) may also have involved neoplasia. There were no differences detected between male and female for disorders as a cause of mortality, but white Boxers showed evidence of higher probability of death related to brain disorder and enteropathy than non-white Boxers. Despite the possibility of false positive findings in the current study, given the neurological component to the aetiological pathway for sensorineural deafness linked to white colour in dogs [71] and given the increased prevalence for brain disorder as a grouped disorder already reported in the current study, further work on an association between white colouration and neurological disorders is warranted.
+
+This study had some limitations in addition to those previously reported for the application of veterinary primary-care clinical records as a research resource [19, 22]. The current study aimed to report the annual prevalence of common disorders in Boxers and therefore did not extract information about the date of first diagnosis, which would have allowed additional reporting of annual incidence. Additionally, information was not extracted on the severity or duration of these disorders, which would have been needed for a fuller evaluation of the welfare impact [58]. The current study reported results from multiple analyses so there are risks of false positive findings (type I error) as discussed above [76]. Consequently, the individual analyses in the current study should be considered as ‘hypothesis-generating’ rather than ‘hypothesis-confirmatory’ [77] and the results reported here should be considered in the light of other studies and incorporated within each of our individual understandings to optimise interpretations and applications.
+
+## Conclusions
+
+In conclusion, the current study identifies that the Boxer comprises around 1% of dogs in the UK, with around 10% of these Boxers being white. There was minimal evidence of noteworthy health differences between male and female Boxers, or between white and non-white Boxers. Among the four most common disorders recorded in Boxers, two are typically common across all types of dogs (otitis externa and periodontal disease) while two showed strong predisposition in the Boxer breed (epulis and corneal ulceration), suggesting the value of understanding breed-specific disorder patterns to offer insights for potential health interventions. The overall longevity of Boxer dogs was consistent with dogs of other breeds of similar body size.

fulltext/md/PMC10240686.md

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+---
+pmcid: "PMC10240686"
+pmid: "37277858"
+doi: "10.1186/s40575-023-00130-3"
+title: "Comprehensive analysis of geographic and breed-purpose influences on genetic diversity and inherited disease risk in the Doberman dog breed."
+journal: "Canine medicine and genetics"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Comprehensive analysis of geographic and breed-purpose influences on genetic diversity and inherited disease risk in the Doberman dog breed
+
+## Abstract
+
+### Background
+
+Publicly available phenotype data and genotyping array data from two citizen science projects: “Doberman Health Surveys” and “The Doberman Diversity Project” were analyzed to explore relative homozygosity, diversity, and disorder risk according to geographical locale and breeding purpose in the Doberman.
+
+### Results
+
+From the phenotypic data cohort, life expectancy of a Doberman at birth is 9.1 years. The leading causes of death were heart disease (accounting for 28% of deaths) and cancers (collectively accounting for 14% of deaths).
+
+By genotyping, the world Doberman population exists as four major cohorts (European exhibition-bred, Americas exhibition-bred, European work, Americas pet/informal). Considering the entire Doberman population, four genomic regions longer than 500 Kb are fixed in 90% or more of 3,226 dogs included in this study. The four fixed regions reside on two autosomal chromosomes: CFA3:0.8–2.3 Mb (1.55 Mb); CFA3: 57.9–59.8 Mb (1.8 Mb); CFA31:0–1.2 Mb (1.2 Mb); and CFA31:4.80–6.47 Mb (1.67 Mb). Using public variant call files including variants for eight Doberman pinschers, we observed 30 potentially functional alternate variants that were evolutionarily diverged relative to the wider sequenced dog population within the four strongly homozygous chromosomal regions.
+
+Effective population size (*Ne*) is a statistical measure of breed diversity at the time of sampling that approximates the number of unique individuals. The major identified sub-populations of Dobermans demonstrated *Ne* in the range 70–236. The mean level of inbreeding in the Doberman breed is 40% as calculated by the number of array variants in runs of homozygosity divided by the assayed genome size (excluding the X chromosome). The lowest observed level of inbreeding in the Dobermans assayed was 15% in animals that were first generation mixes of European and USA bred Dobermans. Array variant analysis shows that inter-crossing between European and USA-bred Dobermans has capacity to re-introduce variation at many loci that are strongly homozygous.
+
+### Conclusions
+
+We conclude that efforts to improve breed diversity first should focus on regions with the highest fixation levels, but managers must ensure that mutation loads are not worsened by increasing the frequencies of rarer haplotypes in the identified regions. The analysis of global data identified regions of strong fixation that might impact known disorder risks in the breed. Plausible gene candidates for future analysis of the genetic basis of cardiac disease and cancer were identified in the analysis.
+
+### Supplementary Information
+
+The online version contains supplementary material available at 10.1186/s40575-023-00130-3.
+
+## Background
+
+The Doberman dog breed was founded in the late nineteenth century by German individual Karl Friedrich Louis Dobermann and was initially developed as a dog for personal protection in Mr Dobermann’s work as a tax collector. The breed foundation is said to include Pinscher, Weimaraner, Rottweiler and German Shepherd dog breeds and was recognised by the German Kennel Club in 1899 (The Kennel Club https://www.thekennelclub.org.uk/search/breeds-a-to-z/breeds/working/dobermann/ accessed September 7 2021). Since its founding, the intelligence and trainability of the breed has been valued by military and police forces. The breed was first exported from Germany in the 1920s and since then, the breed has had a purpose divided between the original breed working function and as a family companion. As is common in working dog breeds, division of purpose has resulted in differences in breeding objectives between breeders producing animals fit for each purpose. To enhance the protective demeanour of the animal, conformation showing rewards an upright head and neck carriage (specifically referred to as having a general appearance of “proud carriage”), and breeders in some countries have employed tail and or ear docking. Docking is used on the breed until the current time in the USA and Canada but has been eschewed by Federation Cynologique Internationale (FCI) and The Kennel Club (KC) standards since 2015. Differences in breed standards between countries have the potential to affect genes underlying traits in the standards, including conformation of the head carriage, ears and tail in dogs bred for exhibition and temperament for working dogs. Over time the name of the breed has been simplified from Dobermann pinscher to Doberman.
+
+The breed standards and breeding practices of many pedigreed dog breeds have been closely scrutinized in recent times. This scrutiny has revealed potential negative impacts of cosmetic procedures on dog health and welfare (for example, through tail-docking or ear-cropping, or alternatively breeding for tail-less) and negative impacts of breeding practices relating to desirable phenotypes on dog health and longevity [1]. Breeding for novel phenotypes excluded from the breed standard (for example rare coat color or variation in size e.g., giant or miniature) is more common in animals bred for purposes other than exhibition.
+
+Genome-wide homozygosity has been negatively associated with breed mean lifespan (after correction for body-size) and positively associated with disease risk [2–6]. In a study of Kennel Club registered dog breeds in the UK, of 25 breeds with age at death registered for more than 50 individuals, the Doberman had the shortest observed lifespan [7]. Breed mean statistics do not consider geographic, or purpose-bred cohort differences in fitness traits. For example, it is commonly presumed that animals bred for exhibition suffer more negative health consequences due to cosmetic breeding practices and inbreeding than other breed-purpose groups. However, analyses that test these common presumptions are currently lacking.
+
+Breed enthusiasts come from all walks of life, and many are highly educated in scientific findings that are relevant to the health of their breeds. Public declarations may be made regarding the health and genetics of particular breeds with little or no hard data. When data are available, they may be scant or reflect the influences of locale of the reporting researchers or be affected by the individual animals to which researchers have access for DNA collection or phenotypic information. Citizen science projects initiated by breed enthusiasts seek to address deficiencies in relevant information pertaining to the breed of interest by gaining the participation of a large number of interested study participants from many regions of the world.
+
+The current analysis thoroughly investigates the locale and breed-purpose population genomics (disease-associated variant allele frequency, genomic heterozygosity, and genomic sweep) and co-occurrence with disease risk across the Doberman pinscher dog breed. Information generated in this analysis can be used to design measures to improve breed genetic health in the future and may provide some information to identify disorder risk loci including those that might differentially affect individual breed sub-groups.
+
+## Results
+
+### Locale-purpose cohort-based phenotype data
+
+A life table (Table S1) was constructed using 1,738 reported ages at death. The table reveals that at birth, the life expectancy for a Doberman is approximately 9.1 years. The oldest reported age at death was 17.25 years. In general, dogs born in Europe have shorter life expectancy than those born elsewhere (Fig. 1). At least some of this reduction in life expectancy appears to be attributable to inbreeding depression, since dogs that are of mixed European exhibition and working lines exhibit extended lifespans over the contributing populations.Fig. 1Survival curves for locale-purpose cohorts phenotyped
+
+After data cleaning and restricting analysis to the leading causes of death, 1,653 reported causes of death remained. The leading causes of death according to dog age in years at the time of death across all locales are described in Fig. 2. Major identifiable disorders leading to death in the reported animals are dilated cardiomyopathy, congestive heart failure, osteosarcoma, cervical spondylomyelopathy (wobblers) – here abbreviated to CS, lymphoma, gastric dilation volvulus (bloat), spondylosis and haemangiosarcoma. Percentages of dogs in each locale-purpose group succumbing to leading causes of death are listed in Table 1. Locale-purpose groups with statistically different frequencies of leading causes of death are highlighted for individual cohorts that were identifiable in our genotyping data.Fig. 2Leading causes of death by age in the Doberman dog breed (causes contributing more than 50 deaths from 1,653 reports)Table 1Percentage of deaths attributable to leading causes normalised for regional representation. Grayed text locale-purpose cohorts are not explicitly identifiable in genotyped sample
+
+The phenotypic survey data reports that the American and European populations have differential risk of cardiac disease and gastric dilatation volvulus (Table 1). In general, the European exhibition and working populations appeared to have similar disorder risk. No consistent pattern of disorder risk was observed to differentiate the exhibition purpose from other purpose types, although the Americas pet sub-population was reported as having a significantly increased risk of cervical spondylopmyelopathy (CS) and the European working population had significantly lower risk of osteosarcoma than other sub-groups.
+
+### Genotyping array data
+
+The DNA based analysis using Illumina canine genotyping arrays included 216,184 variants and 3,226 dogs (1,489 males, 1,737 females). The total genotyping rate was 0.986 and all dogs passed quality filtering.
+
+### Locale-purpose based DNA profiles
+
+The multi-dimensional scaling (MDS) plot is shown in Fig. 3 and Figure S1. Genotype-based clustering in PLINK using the maximum cluster size option identified five clusters Fig. 3, Table S2.Fig. 3Multi-dimensional scaling of de-identified study participants (*N* = 3,226) colored by cluster with shaded regions identifying zones of refinement for cluster 2 (2A shaded in green) and cluster 3 (3A shaded in gray)
+
+The dispersion of 48 known locale-purpose individuals among the genotype-based clusters is described in Table 2. Certain dogs with known origins fell discretely into two clusters that clearly separated European exhibition (Cluster 0, N~identified~ = 11, N~unidentified~ = 886) and American non-exhibition/pet populations (Cluster 4, N~identified~ = 9, N~unidentified~ = 625). Cluster 1 identified animals that were likely hybridized between the European and American exhibition-bred groups (N~identified~ = 5, N~unidentified~ = 386) and could not be formally identified using our known purpose-locale animals (all known individuals informally bred from unknown or mixed locale). Cluster 2 (N~identified~ = 13, N~unidentified~ = 561) comprised both informally-bred and European working-line dogs. Cluster 3 (N~identified~ = 10, N~unidentified~ = 768) clearly comprised animals bred for exhibition. Most of the known-locale animals in Cluster 3 were located in the Americas with a further two located in Australia (Table 2).Table 2Dispersion of known locale-purpose individuals among DNA-based clusters identified by applying unsupervised DNA genotype clustering to 3226 unknown and 48 known locale-purpose dogs. Shaded cells represent the accepted region and purpose for each cluster
+
+As a result of combining information from the MDS analysis with the newly defined clusters, we could readily observe that C1 from the MDS broadly describes the transition from European to American locale, while the C2 axis in the MDS broadly describes the transition from dogs bred for exhibition to those bred for other purposes including work or pet.
+
+For DNA comparative analyses, Cluster 2 was refined to include only animals with MDS C1 < -0.05, C2 < 0 (N~identified~ = 10, N~unidentified~ = 309) to restrict the new cluster (Cluster 2A) to animals co-located with known European working dogs in the multi-dimensional scaling analysis Fig. 3.
+
+Likewise, the initial Cluster 3 clearly comprised dogs bred for exhibition with most from the Americas. We further refined this cluster to exclude dogs known to be bred elsewhere (e.g., Australia) by restricting this group to those with MDS C1 > 0.09 (N~identified~ = 7, N~unidentified~ = 687) where the dogs based in the Americas were placed in the analysis. In later analyses we labelled this group for comparative purposes as Cluster 3A.
+
+### Effective population size (Ne)
+
+According to Waples et al. (2016) effective population size (Ne) determines the rate of evolutionary change due to genetic drift and informs the equilibrium level of genetic variation and the effectiveness of selection. The approximation of Ne provides population managers with a metric that can enable the population to be monitored to manage the loss of diversity and rate of inbreeding.
+
+Initial filtering of the genotyping data for the 3,226 Dobermans resulted in the retention of 103,286 autosomal array marker loci. Genotyping success of less than 99% (–geno 0.01) removed 17,548 variants, while a further 87,007 variants were removed for low minor allele frequency (–maf 0.05). Local LD pruning resulted in the removal of a further 101,088 variants, resulting in 2,198 variants considered as unlinked in the primary data resource (*r*^*2*^ < 0.2). Later filtering to retain only variants polymorphic across all sub-cohorts resulted in the ultimate retention of 1,673 variants for analysis of *Ne*. During analysis, further filtering of resulting marker-pair LD values removed results where markers co-occurred on the same autosome, or where the observed r^2^ was higher than 0.2 (linked). This final filtering was unique to each locale-purpose cohort.
+
+The estimated *Ne* from analysis of unlinked marker pairs for the entire sample cohort was *Ne* = 44. Values for *Ne* in locale-purpose cohorts are reported in Table 3. The refined formal locale-purpose cohorts (including European exhibition, Cluster 0; European work, Cluster 2A; Americas exhibition, Cluster 3A) defined by the multi-dimensional scaling analysis all demonstrated higher *Ne* relative to the entire data set providing strong evidence that the entire global cohort *Ne* value is under-estimated using the method applied.Table 3Effective population sizes for locale-purpose cohorts based on sample size adjusted inter-chromosomal linkage-disequilibriumLocale-purposeDNA ClusterCluster SizeMean ^a^ r^2^*Ne*AllGlobal3,2260.007944Europe exhibition08860.0030177Informal13860.006389Europe working25610.006570Europe working (refined)^b^2A3090.007872Americas exhibition37680.0034158Americas exhibition (refined)^c^3A6870.0036155Americas informal46250.0030236
+
+### Inbreeding and runs of homozygosity
+
+The homozygosity analysis identified 551,586 individual runs of homozygosity (ROH). For any random region of the autosomal genome, the average percentage of dogs exhibiting a ROH including any random marker was 35.6% (range 0–98.5%, standard deviation 14.17%).
+
+Figure 4 depicts regions on eight chromosomes where either more than 80%, or more than 90% of the 3,226 Doberman samples had a concurrent ROH. Locale-purpose ROH regions for four sub-populations (Americas exhibition: cluster 3A, Americas informal: cluster 4, European exhibition: cluster 0, and European working: cluster 2A) are shown on the same image. Across the entire global Doberman population including the pet population, four genomic regions of larger than 1 Mb were fixed in 90% or more of the 3,226 dogs, comprising 5.2 Mb (0.23% of 2.2 Gb autosomal genome). The genomic regions were CFA3:0.8–2.3 Mb (1.55 Mb); CFA3: 57.9–58.7 Mb (0.8 Mb); CFA31:0–1.2 Mb (1.2 Mb); and CFA31:4.80–6.47 Mb (1.67 Mb). A total of 31 Mb (1.4% of genome) were included in ROH for more than 80% of individuals in the population including 11 loci on eight chromosomes.Fig. 4Eight Doberman chromosomes that exhibit regions with < 80% (light gray), >  = 80% homozygosity (medium gray) or >  = 90% homozygosity (dark blue) by locale-purpose cohort. Manual observation of the canine assembly suggests that uncoloured areas are likely reference assembly anomalies
+
+Markers where at least one locale-purpose cluster showed more than 90% of members in a concurrent ROH are provided in Table S3. Of 7,517 array markers in such regions, 681 markers affected all locale-purpose clusters (Fig. 4, Tables S3 and S4). Later analyses considering fine-scale gene annotation focused on regions of >  = 500 Kb.
+
+To test whether exhibition-bred animals had additional fixation relating to selection for breed standards, we focused on the merged exhibition group (cluster 0,3A). This analysis identified three further regions of strong fixation longer than 500 Kb: CFA2:60.6–64.3 Mb; CFA23:47.0 Mb-48.0 Mb; and CFA27:13.3 Mb-14.3 Mb. Of 7,047 array marker loci included in ROH for more than 90% of either European exhibition-bred (cluster 0) or Americas exhibition-bred groups (cluster 3A), only 777 markers were strongly homozygous in both group clusters (Table S4).
+
+To test whether migration-based bottlenecks might influence fixation, we next focused on European animals derived from merged European exhibition (cluster 0) and the refined European working group (cluster 2A). This analysis identified no additional fixed regions but shared new regions identified in the exhibition-bred group, other than the CFA27 locus (Table 4). Of the 7,517 markers in regions with more than 90% of individuals from any locale-purpose cohort in an ROH, 5,734 markers were in ROH for more than 90% of either European working or European exhibition-bred groups. Of these, 2,340 array markers were in ROH for more than 90% of both the European exhibition-bred and European working bred Dobermans (Table S4). The number of markers with ROH for more than 90% of the merged European (exhibition and working) cohort (4,045 markers) was considerably greater than the equivalent number for the merged Americas and European exhibition cohort (1,844 markers). Thus, there is lower diversity within the European locale than within the exhibition purpose (Table S4).Table 4Regions with more than 90% of Dobermans in ROH^a^ by locale-purpose cohortChromosomeROH startROH endArray markers (N)ROH Size (Mb)Locale-purpose cluster(s)^b^260,704,38464,336,9941683.6303A, 02A3778,9392,325,9411261.55Global357,930,62258,718,298680.79Global1532,931,14033,274,592330.3403A, 02A1744,229,68444,259,33730.0303A, 02A2346,996,10248,025,418901.0303A,02A2713,280,57813,840,836460.5603A319,5671,211,0401051.20Global314,803,6756,469,3091381.67GlobalTotal77710.80
+
+The strongest regions of differentiation between European (high-fixation) and American dogs (lower-fixation) affected CFA4:16.3-17 Mb, CFA11:63–64.8 Mb, and CFA16:40.5–41.2 Mb. Fine-scale variation in these regions was not assessed as the divergence was intermittent in the regions.
+
+### High-resolution variant function analysis
+
+Merging of the public variant call files yielded 1,257 unique dog identifiers and 8 unique Dobermans.
+
+Regional fine-scale analysis follows. Where smaller genomic ROH regions for the global cohort are expanded within locale-purpose cohorts, results for the wider region are reported. The number of possible functional variants identified was 2,727 after multiple entries for the same locus were removed. Restricting attention to functional variants with Doberman FST of 0.7 or greater resulted in only 30 variants remaining. Further details relating to 2,727 variant sites are listed in Table S5. Attribute details for all observed variants in the regions based on 1,527 dogs represented in the public variant call files from whole genome sequencing are provided in Table 5. Attributes for the same variant set in eight unique Dobermans are reported separately in the same table.Table 5Attributes of variants in regions larger than 500 Kb observed as homozygous in 90% of Dobermans in any locale-purpose cohort. Variants are not filtered for FSTChromosome23323273131Region (Mb range)60–641–257–5946–4813.3–13.81–1.24–6Regional Variants ^a^ (1,527 dogs)167,34953,23970,77751,35023,99357,97668,953Polymorphic Variants (1,527 dogs)166,00852,62970,30351,03523,87157,49268,445Array ^b^ Markers (1,527 dogs)4,3758124,7653,6153154,230934Observed Functional Variants ^c^ (1,527 dogs)7134298543125729864Polymorphic Variants (8 Dobermans)4,3758124,7653,6153154,230934Polymorphic Array ^b^ markers (8 Dobermans)325764813-Functional Polymorphic Variants ^c^ (8 Dobermans)65108443291Monomorphic, Alternate allele ^d^, Functional ^c^ (8 Dobermans)91911-5Locale-purpose clusters that have regions of concern0, 2A, 3AAllAll0, 2A, 3A2AAllAll
+
+#### CFA2
+
+A large region on CFA2, CFA2: 60.7–64.3 Mb (~ 3.63 Mb) affected 90% ROH for clusters 0 (exhibition-bred Europe), 3A (exhibition-bred Americas) and 2A (working Dobermans from Europe). This genomic region accounted for 21 of the high FST variant alleles. Of these, 18 were missense variants, while a further three were variants in either 5-prime or 3-prime untranslated regions (UTR). All missense variants affected the gene *Carboxylesterase 1* (*CES1*) and were predicted to have moderate impact. Among the sequenced Dobermans, all 21 variant sites were polymorphic (Doberman DO263 heterozygous for 21/21 loci and DO159 heterozygous for 20 of 21 loci). All high FST *CES1* variant sites were very rare in the broader sequenced dog population.
+
+Three variant sites affected the untranslated regions of genes *Iroquois Homeobox 6* (*IRX6*), *Iroquois Homeobox 3* (*IRX3*), and *Chromodomain Helicase DNA Binding Protein 9* (*CHD9*). The variants were predicted to be modifiers of gene action. At these sites all sequenced Dobermans were homozygous for the alternate allele.
+
+Variant site coverage for Dobermans across the CFA2 region did not show any regions with differences in coverage that were suggestive of duplication or deletion relative to the broader sampled canine population (Table S6).
+
+#### CFA3
+
+Two regions were assessed on CFA3, CFA3:0.8–2.3 Mb (~ 1.55 Mb) and CFA3: 57.9–58.7 Mb (~ 0.8 Mb) affected the global Doberman population. The first region is close to the centromere. Seven high FST variant sites all occurred within the second region (CFA3: 57.9–58.7 Mb). Three missense variants were predicted to have moderate gene impacts on the genes *Mannosidase Alpha Class 2B Member 2* (*MAN2B2*) (2 variants) and *TBC1 Domain Family Member 14* (*TBC1D14*) (1 variant). The remaining variants affected the UTRs of *Ras Protein Specific Guanine Nucleotide Releasing Factor 1* (*RASGRF1*) (2 variants), *Cathepsin H* (*CTSH*), and *Protein Phosphatase 2 Regulatory Subunit B gamma* (*PPP2R2C*), each with a single variant. All UTR variants were predicted to be gene modifiers.
+
+Marker coverage from the CFA3 regions showed no significant deviation between Doberman and other breeds (Figure S2, Table S6), other than one small region of potential deletion CFA3:1,204,000–1,205,000 that lies within a repeat element cluster that is intronic in the gene *Calcium/Calmodulin Dependent Protein Kinase IV* (*CAMK4*).
+
+#### CFA23
+
+The region taken forward on this chromosome (CFA23:46,996,102–48,025,418) affected all formally bred cohorts (0, 2A, 3A). Two putatively functional variants demonstrated FST values of higher than 0.7. These were a 3’-UTR variant in *LOC102151520* (polymorphic in Doberman) and a 3’-UTR variant in the gene *Ras-Related Protein Rap-2b* (*RAP2B*). Both variants are predicted to be modifiers. Doberman regional coverage was unremarkable.
+
+#### CFA27
+
+The region (CFA27: 13,280,578–13,840,836) was uniquely enriched for the exhibition-bred Dobermans (cohorts 0 and 3A) contained no putative functional mutations with strong FST (> 0.7). Low diversity in the CFA27 region may indicate differential selection for desirable traits or may result from genetic drift. Three potential functional variants were identified in the gene *Leucine-Rich Repeat Serine/Threonine-Protein Kinase 2* (*LRRK2*). One monomorphic alternate variant was a SNP in the 5’-UTR. Two further Doberman polymorphic variants affected a splice-region (FST = 0.3) and the 3’-untranslated region (FST = 0.38) of *LRRK2* (Table S5). Sequence alignment coverage over the region was not unusual.
+
+#### CFA31
+
+Marker coverage data from the CFA31;1–1.2 Mb region in the variant call file for eight sequenced Dobermans suggests that this part of the genome may be subject to a significant structural variant with copy number expansion of perhaps up to 3.6-fold (mean regional coverage of 56 reads at CFA31:30,000–69,000 compared with a mean cover of ~ 18 reads elsewhere (Figure S2, Table S6). The importance of the apparent copy number expansion was not determined or further explored in this analysis. Since the region is close to the centromere the higher level of cover may signify centromeric satellite sequence expansion. The Doberman excess read coverage in the region was unremarkable in relation to the wider sequenced dog population. The broader CFA31 ROH region includes the potential behaviour gene *5-hydroxytryptamine receptor 1F* (*HTR1F*) (serotonin receptor) at CFA31:272,358–274,597 (canFam 3.1) where Dobermans exhibit the reference allele at an annotated variant site.
+
+## Discussion
+
+Purebred dog populations and, thus, their population management are influenced by the registry in each region. In certain countries, registries allow for controlled importation and breeding of foreign animals. The American Kennel Club allows such importation, breeding, and registration of subsequent puppies depending on a foreign dog’s country of origin. This has allowed for relatively free flow of foreign dogs into the Americas. However, other registries, such as Germany’s Dobermann-Verein, have in past times adopted policies that effectively disallow breeding of imported dogs and prevent recognition of resulting puppies [9]. Additionally, the American Kennel Club designates all dogs descended from an albino female Doberman with a unique “WZ” registration, regardless of the puppy’s coloring or genetic status. This designation allows for easy identification of possible albino ancestry and presumably discourages breeding [10]. Thus, interbreeding among the Americas-exhibition, European-exhibition, and some European-working, and Americas-pet cohorts is technically allowed but not commonly performed for various reasons. Despite this, there is no functional barrier preventing the interbreeding of any of these groups and resulting puppies may be registrable depending on country and individual registry regulations, which are subject to change.
+
+### Population dynamics
+
+The higher *Ne* within cohorts is a result of lower mean r^2^ in the within-cohort groups and was not driven by the correction applied for sample size. This phenomenon is recognised as a signature of population admixture and suggests that the global cohorts of Doberman pinschers can be regarded as genetically distinct populations for analytical purposes [11]. The sub-cohort effective population sizes are therefore expected to better reflect the true characteristics of those individual populations.
+
+It is rare that such a large single breed cohort is available for population analysis. The outcomes of this analysis challenge our perceptions of accepted methods for determining *Ne* using genotypes, and in particular the common belief that DNA-based assessment is always superior in accuracy to pedigree-based estimates. The reduced estimated *Ne* in the global cohort relative to the individual sub-groups using the analysis of unlinked inter-chromosomal markers shows that the estimation of *Ne* using admixed population samples can generate significant analytical biases that manifest as an underestimation of *Ne* in the admixed group. The within-cohort *Ne* values are likely closer to the true values. A previous pedigree analysis of UK exhibition-bred Doberman pinschers revealed an *Ne* of 133.4 but with declining rate of inbreeding since the year 2000 that is suggestive of increasing *Ne* [12]. The observed UK value supports the values reported for the exhibition-bred cohorts in the current study that possibly originate from the Americas gene pool. Unlike pedigree-based population genetics analysis, DNA-based analyses represent a snapshot in history at the time of sample collection. The lower *Ne* observed in the European working line dogs may well relate to the historical population bottlenecks occurring around the time of World-War II as reported by the Veterinary Genetics Laboratory (VGL), University of California, Davis report [13]. Using microsatellite analysis, the VGL report showed very low values for *Ne*. However, because the values for *Ne* were calculated separately for each locus, it is uncertain how the VGL reported *Ne* statistics relate to measures based on LD between unlinked loci. Another possible reason for lower *Ne* in the European cohort may relate to region of origin biases in breeding programs. It is possible that because the breed originated in Europe, European breeders may be reluctant to include genetics from foreign-bred animals thus reducing the capacity for maintaining diversity. Different breeding norms between Europe and the Americas may also contribute to this, since it is likely that European breeders will include consideration of tail and ear phenotypes that are less important in countries where dogs are routinely docked.
+
+### Genomic regions diverged by locale
+
+Geographic locale was observed to exert a greater influence on genomic fixation than breed purpose in the Doberman (Table S3 and S4). Regions on CFA4, CFA11 and CFA16 were particularly diverged by geographic locale.
+
+The CFA4 region that has low fixation in the Americas and high fixation in the European animals includes potential long non-coding RNAs and is near the gene *catenin alpha 3* (*CTNNA3*), while the region on CFA11 that is similarly diverged between European and American-bred dogs also contains a gene from the catenin family: *catenin alpha-like 1* (*CTNNAL1*). A related gene *catenin alpha 2* (*CTNNA2*) was previously identified as a potential candidate for obsessive behaviour in the breed [14]. The CFA16 region contains genes that may influence cardiac and neoplastic disorder risk including *microtubule-associated tumor suppressor 1* (*MTUS1*) and *fibrinogen-like protein 1* (*FGL1*). Fine-scale variation was not assessed in the diverged regions.
+
+### External phenotype and locale-purpose
+
+Using array markers, rather than whole genome sequencing data, it was not possible to directly ascertain the locale-purpose prevalences for the mutations with genetic tests that are offered for the breed. However, an approximation of the prevalence of off-standard white/cream coat color related to genotype at the gene *solute carrier family 45 member 2* (*SLC45A2*) could be made through observation of allelic calling of two markers contained within the deletion mutation (CFA4:73,867,685C > Del and CFA4:73,870,657G > Del) and heterozygosity could be assessed through observation of two further loci in strong LD (CFA4: 73817338A > G and CFA4:73912726G > A). Of 44 animals observed to have two copies of the deletion haplotype, 30 occurred in Cluster 4 (Americas informal/Pet), one in Cluster 3A (Americas exhibition) and the remainder had initially been included with the Americas exhibition cluster (Cluster 3) but were excluded by refinement based on the multi-dimensional scaling.
+
+Short-tailed phenotype (Natural bobtail) dogs [OMIA 000975–9615] could be desirable among exhibition competitors in countries that do not allow tail-docking. One mutation in the *T*-gene is known to code for the phenotype as a semi-dominant gene that is lethal as a homozygote [15, 16]. It has been suggested that there may be further undescribed mutations affecting the existence of the phenotype [16]. We observed low frequency heterozygous genotype calls and no alternate homozygous calls for one marker in the vicinity of the *T*-gene (marker BICF2S23215117 at CFA1: 54,199,967). The concordance of this genotype with any tail phenotype could not be directly assessed but the heterozygous calls occurred predominantly in the informally bred clusters (Cluster 1: 25 heterozygotes, and Cluster 4: 15 heterozygotes). Four heterozygous dogs fell into Cluster 0 (European exhibition) and three in Cluster 3A (Americas exhibition—refined).
+
+### Disease genomics
+
+The Doberman has been identified as a breed that has an elevated risk of premature death related to cardiac disease (particularly dilated cardiomyopathy or DCM) [OMIA 000162–9615], cancer (particularly lymphoma [OMIA 001733–9615] or osteosarcoma [OMIA 001441–9615]), and CS (wobbler syndrome) [OMIA 001894–9615] [17–19]. The analysis of the public phenotype data accessed by the current study supports that the named conditions are truly important reasons for mortality in the breed.
+
+*Cardiac disease – congestive heart failure (CHF) and dilated cardiomyopathy (DCM):* Cardiac disease is by far the most significant cause of premature death in the breed, accounting for an average of 28% of deaths in the sampled cohort (Table 1). In the sampled phenotypic data, significantly elevated mortality from cardiac disease was demonstrated by European dogs bred for both work and exhibition relative to the global mean. From the phenotypic analysis, the European dogs had a two-fold higher risk of death from cardiac disease than dogs bred in either Australia or South Africa, although samples from the latter two countries were limited and so this finding should be interpreted with caution.
+
+Commercial genetic testing is currently offered for DCM in Dobermans at two loci. The first tested locus considers a mutation that is a 16-base pair deletion in the 5' donor splice site of intron 10 of the *pyruvate dehydrogenase kinase 4* (*PDK4*) gene (NM_002612.4 chr14:20,827,965–20,838,970). Functional evidence [20, 21] suggests that the mutation affects the response to mitochondrial stress in affected cells. In-vitro, cells that are heterozygous or homozygous for the *PKD4* deletion demonstrate evidence of mitochondrial stress and activation of the intrinsic apoptotic pathway following cell line starvation [21]. To date, further studies have failed to validate the *PKD4* association in the breed [22]. A second locus was identified by the same research group in an affected family of Dobermans that was clear for the *PKD4* mutation. This family segregated a coding mutation in the gene titin (*TTN*, NM_001329611 chr36:22,146,870–22,417,858) which is a major candidate gene for the condition based on human studies [20]. The percentage of dogs with ROH at the *PDK4* locus was 24% and was 23–31% at the *TTN* locus (Table S3). Both loci have lower than average numbers of dogs exhibiting ROH. Therefore, *fixation* at these loci is unlikely to be the reason for high levels of DCM in the breed. However, the *PDK4* and *TTN* loci may segregate to add to DCM risk in individuals and families. The results of the current analysis provide no information as to the importance of the two loci in the population levels of DCM.
+
+The current analysis has identified promising targets for further research into heart disease in the breed. Among the locations identified as having low diversity and high FST in the breed, the low diversity region on CFA2 contains multiple genes with potential cardiovascular function. According to the Genecards resource [23] *CES1* has a demonstrated role in cardiac conduction and cardiac drug metabolism while *IRX6* is a potential hub gene in the pathogenesis of heart failure [24]. *IRX3* is potential modulator of cardiac copper metabolism [25]. On CFA3, the gene *MAN2B2* in Doberman populations contains two potentially damaging missense variants that are very rare in the general dog population. *MAN2B2* has been reported as a potential early biomarker for cardiac hypertrophy and heart failure in an isoproterenol-induced model of cardiac hypertrophy in the mouse [26].
+
+*Cervical spondylomyelopathy (CS):* It has been reported that neonatal Doberman pups are born with abnormal structure of their cervical vertebrae and that the breed is over-represented among those affected by CS [18]. However, to date no study has shown a conclusive heritability for CS risk in the breed [17]. In the phenotypic data analyzed here, CS was responsible for approximately 6% of deaths reported. The observation of breed elevated risk of CS without obvious within-population heritability may suggest the enrichment of predisposing genetic factors in the breed by selection or genetic drift. It is possible, although not discussed in the literature, that the upright stature and head carriage of the breed may be associated with underlying genes that predispose to the condition. However, if the desirable breed external phenotype increased risk for CS, we would expect to observe an increased prevalence of Wobbler syndrome among animals bred for exhibition. That trend was not observed in the phenotypic data. Of the major locale-purpose cohorts represented in the phenotypic data, the risk of CS was highest in Australian Dobermans with 10.5% of mortality attributed to CS (perhaps influenced by sampling error from poor group representation in the survey) and in USA informally bred Dobermans with 10.3% of mortality due to CS. To date, there is no commercial genetic test that predicts CS risk in the breed. European bred dogs, regardless of breed purpose, exhibit lower than expected CS-mortality. No obvious predisposing candidate genes for CS were identified in the strong ROH regions, however, the region on CFA4 that is diverged between American and European dogs contains a retrocopy of the gene *Single Stranded DNA Binding Protein 3* (*SSBP3*). According to the University of Santa Cruz genome browser, the primary *SSBP3* gene on CFA5 may play a role in the development of spinal interneurons. If retaining any function, the retrogene may have potential to influence geographically linked risk of CS. If the retrotransposed gene has relevance to this disorder, then it is more likely that a deleterious haplotype in the region has been selected against in the European dogs.
+
+*Neoplasm (Osteosarcoma, Lymphoma, and Hemangiosarcoma):*In the phenotypic data, the different locale-purpose cohorts appeared to segregate different predispositions to cancer (Table 1) which collectively accounted for around 14% of deaths. European dogs exhibited lymphoma more frequently than the Americas derived dogs, whilst hemangiosarcoma was unreported in Europe and highest among exhibition-bred dogs in the Americas. The occurrence of osteosarcoma was lowest in the European working bred dogs. Recent evidence suggests that hemangiosarcoma risk may be linked to persistent infection with the bacterium *Bartonella spp* that is transmitted via blood sucking parasites including ticks and fleas [27]. The relative prevalence of canine infection with *Bartonella spp* in Europe versus the Americas is not known so it is not possible to determine whether a genomic or local environmental difference may underlie the differential risk for hemangiosarcoma.
+
+On CFA16, the geographically diverged region contains multiple genes that may influence geographically diverged disorder risk. According to the mouse phenome browser [28], the gene *MTUS1* is linked to both cardiac hypertrophy and B-cell lymphoproliferative disease in the mouse [29]. In the same region of geographic divergence,*FGL1* is a major ligand of *Lymphocyte-activation gene 3* (*LAG3*) that inhibits antigen-specific T cell activation. In mice, knock out of *FGL1* promotes T cell immunity [30] and this might have relevance to T-cell based lymphoma.
+
+### Lifespan
+
+The phenotypic data used in this analysis suggest that the life expectancy of a Doberman at birth is 9.14 years. An earlier analysis of UK companion dog data showed that the mean life expectancy for companion dogs (across breeds and mixes) under primary veterinary care in the UK was 11.2 years [5]. Eighteen breeds were included in the Teng analysis excluding Dobermans. The 2014 British study [7] reported a lifespan of 7.67 years for the Doberman breed, however, that result was obtained from far fewer dogs (90 participants) and so may have been subject to sampling error.
+
+Growing evidence suggests that organismal lifespan is shortened by deficits in autophagy [31, 32]. The second ROH region on CFA3 (CFA3: 57.9–58.7 Mb) includes a missense variant in the gene *TBC1D14*. *TBC1D14* controls regulation of autophagosome assembly, the recycling of endosome to Golgi transport; and is a negative regulator of autophagy. Several further variants affecting the gene and this region did not meet the FST cut-off but may contribute to complex phenotype interactions arising from this locus.
+
+The phenotypic data suggest that inbreeding depression almost certainly plays a role in reducing lifespan in this breed. The European born animals, regardless of breeding purpose, exhibited lower life expectancy than other locale-purpose cohorts. The European working bred Dobermans have the shortest life expectancy and the lowest effective population size of all groups. Based on observations of animals in the phenotypic analysis, crossing European working line dogs with the dogs from elsewhere (European exhibition bred or Americas bred dogs) is expected to improve life expectancy (Fig. 1). There may be reluctance to use outcrossing if it is considered that out-breeding will diminish the working potential of the resulting pups. This dilemma is common in breeds that have breed splits based on breed purpose. If the major impacts on lifespan are being generated by the genes and genomic loci observed as regions of concern in this study, then it may be possible to use targeted crossing programs to maintain the desirable working characteristics while alleviating the major negative influences of the identified loci. Similar programs have been applied in other breeds (e.g., Dalmatians to alleviate hyperuricosuria risk [33]. Gene targeted crossing programs that apply out-crossing followed by back-crossing have potential to maintain the essential or highly desirable characteristics of the animals while improving welfare outcomes. This might be possible without using other breed contributions but is likely to be more difficult.
+
+It is also possible that work-purpose bred dogs may have reduced life expectancy due to wastage (election for euthanasia at the end of work life). There was little evidence to support this as a potential cause in the phenotypic data. The major causes of death in the group were: cardiac disease 36%, cancer (various) 32% and other (13%). Figure 2 shows that the “other” category for death appears to be reasonably constant through dog ages.
+
+### General discussion
+
+The analysis of gene function applied here relies on previous functional annotation provided by the Dog Biomedical Variant Database Consortium [34] and may not be exhaustive. However, already we have identified target genes and variants that might play a role in the expression of complex disorders that have high prevalence in the breed and impact observed longevity.
+
+Typically, genotyping array markers are polymorphic in many populations of animals, meaning that the included variants arose prior to breed development. Variants arising at later times may not be visible using the older variants as markers. For this reason, although we have identified four regions with strong ROH across the global Doberman population, fine-scale genomic analysis shows that variation (including potential functional variation) exists in the polymorphic state within the breed within most of these regions. Although marker-based evaluation in such regions is unlikely to be effective unless it is based on the direct genotyping of functional mutant sites, phenotype-based selection can still act on hidden allelic frequencies of younger variants.
+
+Where potential deleterious variants are identified in low-diversity regions, it is critical to identify which haplotype harbors the deleterious allele. It is possible that where phenotypic selection has reduced diversity in the region to remove a deleterious gene variant, the rarer haplotype might be damaging to health. Population managers must ensure that the frequencies of deleterious variants are not increased in attempts to improve statistical diversity for a given region.
+
+In many cases in this analysis, the fixed or most common allele was the global main variant (likely the ancestral normal variant). An example where attempts to improve diversity may inadvertently create problems is the gene *MAN2B2*. At this locus we identified two missense variants that have FST > 0.7. The ancestral haplotype is CFA3:58673093C, CFA3:58696797 T (CT). Observed Doberman haplotypes were: CFA3:58673093C, CFA3:58696797 T (CT); CFA3:58673093C, CFA3:58696797 T > C (CC); and CFA3:58673093C > G, CFA3:58696797 T (GT). In Dobermans the most common haplotype observed was CC (one missense variant), the second most common haplotype was GT (two-missense variants). Thus, selecting to increase the frequency of the lower frequency haplotype in this instance may inadvertently increase the deleterious mutation load on the gene. At this gene locus, the wild-type haplotype was present only as a single heterozygote in the sequenced Dobermans. Table S5 provides information on many putative functional variant sites across the ROH regions of concern. Careful navigation will be required to understand the individual and combined impacts of the variants on gene function and clinical outcomes in the breed. That depth of analysis was not attempted in this study.
+
+## Conclusions
+
+The analysis here agrees with other published results that demonstrate high levels of homozygosity for genotyping array markers in the Doberman breed. Despite high general levels of homozygosity in the breed, surprisingly few regions of the genome are “fixed” (denoted as regions of concern). Less than 1% of the autosomal genome is depauperate of variation as defined by more than 90% of individuals with a concurrent ROH and less than 2% of the genome (31 Mb) has fixation at the 80% ROH level.
+
+Genetic divergence between the Americas-exhibition and European-exhibition locale-purpose groups means that hybridization between the North American and European exhibition-bred animals has potential to alleviate fixation and improve heterozygosity at many (though not all) problematic gene loci in regions of concern. This should be possible given current accepted breeding practices in the registered dog world. The same opportunity is less likely to generate substantive improvement for hybrids between the European working and European-exhibition groups as there is strong genetic concordance among the European locale-purpose groups in this analysis. Animals within the Americas pet/informal population may provide a further reservoir of diversity that can be used to alleviate diversity loss in genomic regions of concern.
+
+Functional analysis of regions of concern has provided a rich resource for further research that may improve clinical outcomes in the breed. Genes within the regions have potential to impact important disease and life outcome processes such as cardiac health, cancer and lifespan as influenced by autophagy.
+
+## Methods
+
+### Geographic cohort-based phenotyping data
+
+A citizen-science based breed survey (dobehealth.org accessed 7/9/2021) collected cause of death for 1,744 Doberman dogs between years 2010–2021. The data are freely available in the public domain and include dogs identified by participating owners as being from Europe, North America, Africa, and Australia. It is possible that some dogs are represented in both genotyping and phenotyping data but as the animals are de-identified it is not possible to effectively match individual participants.
+
+All dogs with recorded age of death contributed to a life table constructed using the age at death data reported [5, 35]. Where animals had two concurrent disorders at the time of death, the primary cause of death was the disorder requested.
+
+Causes of death contributing more than 50 deaths in the data resource—hereafter termed leading causes of death, were identified. The data were first cleaned to exclude individual reports with missing or impossible age of death and counts of leading causes of death at each whole year of age were identified. Animals less than one year old were attributed an age of zero. Animals older than 18 years were excluded. Deaths caused by “old-age”, “other” or “cancer-other” were excluded from further analysis.
+
+Data relating to remaining leading causes of death were next analysed to determine the percentage of animals from each geographic region affected by any leading cause of death. The percentage occurrences of individual and grouped leading causes of death in each region were compared. Geographic regions exhibiting over- or under-representation for the cause of death were identified. Over- or under-representation was based on the mean geographic region percentage of animals affected by the individual or grouped leading cause and the 95% confidence interval (i.e. ± 1.96*s.e).
+
+### Genotyping array data
+
+The Doberman Diversity Project is a citizen science-based project devised by Sophie Liu and Robin Loreth in collaboration with Embark Veterinary. The project obtains data from Illumina Canine HD genotyping arrays that are gathered through routine genetic testing of collaborating dogs with the consent of the owners. The de-identified data are released for analysis. With owner consent, genotyping array data from a further 48 individuals with known geographic and breed-purpose origins were released to enable the attribution of geographic cluster to the wider genotyping array sample. These known samples were used only to assign geographic location and purpose (locale-purpose) to DNA-based clusters and were then excluded from the broader analyses.
+
+### Locale-purpose DNA profiles
+
+Locale-purpose attribution was determined by two methods, first, individual multi-dimensional scaling coordinate proximity to 48 animals with known geographic location and breed purpose (locale-purpose) was determined. To do this, the first and second principal components were derived from multi-dimensional scaling analysis in PLINK following standard quality filtering of autosomal array data: [minor allele frequency (maf) –maf 0.05; genotyping fail rate –geno 0.1]. Clustering (–cluster) and multi-dimensional scaling plots (–mds-plot 2) were generated for autosomal chromosomes (–chr 1–38). The resulting variance component coordinates (C1 and C2) were used to ascertain two-dimensional coordinate values. Animals from the de-identified set (*N* = 3,226) were assigned to groups based on common clustering with the animals of known-origin in an analysis that included the 48 known-origin samples and with the maximum cluster size set at 900 to force separation [ –geno 0.01 –cluster –mc 900].
+
+### Effective population size
+
+Assessment of effective population size (*Ne*) can be made using the observation of residual pairwise-linkage disequilibrium (LD) in otherwise unlinked SNP markers. We generated a set of unlinked markers that could be used to estimate *Ne* following previously described methods [8, 36, 37]. To achieve this, array variants for the entire sample group were manipulated in PLINK [38] to prune markers with LD of *r*^*2*^ > 0.2 in 100 kilobase windows with a single marker step-size (–indep-pairwise 100 1 0.2), and to include only autosomal markers (–chr 1–38). Minor allele frequency (maf) (–maf 0.05) and stringent genotyping rate (–geno 0.01) filtering were employed to further reduce the size of the marker set. The allele frequency estimation option in PLINK was used to ascertain the minor allele frequency of the “prune.in” variants that were deemed as unlinked in the first pass analysis for each individual cohort group.
+
+Next, PLINK was used to assess the pair-wise LD (–r2 inter-chr) on a reduced set of markers retaining those from the “filename.prune.in” list with maf > 0.05 in every cohort. The allowable observable LD in the scanning window was reduced to zero (–ld-window-r2 0). Resulting pairwise r^2^ values from the presumptively unlinked marker pairs were averaged across the data but restricted to marker pairs located on different chromosomes and marker pairs with *r*^*2*^ < 0.2 to avoid artefacts caused by possible reference genome assembly errors. Only markers that were polymorphic in all locale-purpose groups were retained to prevent biased r^2^ estimates resulting from having one marker in the marker pair fixed.
+
+The equation of Waples (2006) [36] was used to ascertain *Ne*:\documentclass[12pt]{minimal}
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+ \begin{document}$$Ne=\frac{1}{3({{r}_{adj}}^{2}-\frac{1}{S})}$$\end{document}Ne=13(radj2-1S)where \documentclass[12pt]{minimal}
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+ \begin{document}$${r}^{2}$$\end{document}r2:\documentclass[12pt]{minimal}
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+ \begin{document}$${r}_{adj}^{2}={r}^{2}{[\frac{S}{\left(S-1\right)}]}^{2}$$\end{document}radj2=r2[SS-1]2following the equation of Weir (1979) cited in [8]. In the equation, \documentclass[12pt]{minimal}
+ \usepackage{amsmath}
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+ \begin{document}$${r}^{2}$$\end{document}r2 is the unadjusted mean inter-marker association, \documentclass[12pt]{minimal}
+ \usepackage{amsmath}
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+ \usepackage{amsfonts} 
+ \usepackage{amssymb} 
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+ \begin{document}$${r}_{adj}^{2}$$\end{document}radj2 is the adjusted mean \documentclass[12pt]{minimal}
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+ \begin{document}$${r}^{2}$$\end{document}r2 and, S is the sample size (e.g. S = 3,226 for the global cohort).
+
+The same files and parameters were re-analyzed for individual locale-purpose subsets of the data (European exhibition, European work, American exhibition, and American pet populations) to assess intra-population *Ne*.
+
+### Inbreeding and runs of homozygosity (ROH)
+
+The Doberman Illumina canine high density array genotypes were used to conduct genome-wide analysis of breed homozygosity and to obtain any evidence for selective sweep in the breed. The main analysis was conducted using PLINK employing the running parameters [–homozyg-snp 50 –homozyg-window-het 1 –homozyg-window-missing 1 –homozyg-gap 200 –chr 1–38]. These parameters define the number of markers in the scanning window frame (–homozyg-snp), the maximum allowable number of heterozygous markers in a segment to accept it as homozygous (–homozyg-window-het), the maximum allowable number of missing markers in a homozygous segment (–homozyg-window-missing), and the maximum allowable gap between adjacent markers in a homozygous segment in kilobases (–homozyg-gap 200. Diversity statistics are reported for autosomal markers only (–chr 1–38). The same analysis was repeated for the defined locale-purpose subsets of the data.
+
+### Identification of regions of concern
+
+Genomic regions exhibiting strong levels of homozygosity suggesting selective sweep or paucity of variation (*regions of concern*) were explored in detail. Analysed population cohorts included the global cohort, individual locale-purpose cohorts, and two merged cohorts. For each analysed cohort, the arbitrary cut-off for further analysis of an individual locus was having 90% of cohort individuals within a common run of homozygosity (ROH) and a region size of > 500 kilobases (Kb). The region size was defined by the inter-marker distance between the first and last markers in an ROH exhibiting at least 90% cohort individuals homozygous. All ROH coordinates are reported relative to canFam3.1 [39]. Genotype frequencies for each individual array marker (as opposed to ROH segments) were calculated using the –freq-x option in PLINK [38]. Note that ROH segments for individual animals can include occasional heterozygous or missing markers according to the running parameters.
+
+In addition to the global merged group, two merged locale-purpose groupings (*co-managed clusters*) were considered. First, we focused on loci with more than 90% ROH in locale-purpose groups that commonly allow interbreeding (i.e., the merged locale-purpose cluster 03A that combines European animals bred for exhibition with exhibition animals from the Americas). This grouping was chosen to enable better management of individuals that can be freely interbred using standard international conventions. The second merged locale-purpose group considered was that of the European animals (comprising animals bred for exhibition and for work in Europe). This grouping enabled the potential evolutionary influences of geographic breed bottlenecks to be better examined.
+
+### High-resolution variant function analysis in regions of concern
+
+Regions of concern (defined as at least 90% of cohort dogs in a concurrent ROH) were explored in higher resolution using public variant call file (vcf) data resources [34, 40]. To do so, ROH region variant calls were first extracted from the vcf for 722 diverse dogs and wild canids from [40] using the TABIX tool [tabix -h 722 g.990.SNP.INDEL.chrAll.vcf.gz chrN:region_start-region_end > 722_region_vcf] [41]. The same procedure was used to extract regional data from 590 dogs and canids from [34] [tabix -h 590_220K_array.vcf.gz N:region_start-region_end > 590_region.vcf] where N is the chromosome number.
+
+The regional vcf files from Jagannathan et al. (2019) and Plassais et al.(2017) were next merged using PLINK [38] and converted to ped and map format. The map file was recoded to show each marker identifier as a combination of the chromosome and base position. This avoided many markers having the same marker identifier (otherwise they were recorded as typically either “.” or their rs-code).
+
+The vcf data from the [34] study is coded relative to the canFam3.1 reference assembly and includes functional prediction as a component of the vcf file information field for each variant called. The “INFO” field within the vcf was interrogated to identify variant positions that generated potentially functional variants using the command-line tool *grep* [grep (missense, codon, splice, frame, prime) 590_region.vcf > 590_region_functional.vcf]. From the resulting functional variant file, variant positions and functional annotations were extracted.
+
+To test the hypothesis that strongly diverged Doberman breed alleles in the regions of low diversity are damaging, pairwise *FST* (Doberman versus non-Doberman) was calculated using PLINK (options –fst case–control). The *FST* compared whole-genome sequenced identified Doberman genotypes with those of all other dogs represented in the large merged vcf for each individual ROH region. Variants annotated as putatively functional that demonstrated high frequency derived alleles (pairwise *FST* >  = 0.7) were analyzed further. Potential error regions exist when all sequenced animals (including non-Dobermans) have the alternate variant when successfully genotyped. The likelihood of Doberman relevant phenotypic expression for individual identified genes was evaluated through literature review of the gene name in relation to the leading causes of death in the breed.
+
+Regional sequence coverage was assessed for biallelic markers in the ROH regions with >  = 90% individuals in the ROH. Read coverages for the eight whole-genome sequenced Doberman individuals were determined by the allele depths derived from the individual genotype calls in the combined vcf. Allelic coverage values for the eight sequenced Doberman individuals were averaged to generate a breed mean sequence depth metric for the breed at each biallelic marker. The per-variant coverage was assessed in ten kilobase windows.
+
+## Supplementary Information
+
+**Additional file 1.**

fulltext/md/PMC10243369.md

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+---
+pmcid: "PMC10243369"
+pmid: "37288441"
+doi: "10.29374/2527-2179.bjvm004922"
+title: "Linear CT-scan measurements of cerebral ventricles in senile Poodle dogs."
+journal: "Brazilian journal of veterinary medicine"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Linear CT-scan measurements of cerebral ventricles in senile Poodle dogs
+
+## Abstract
+
+Breed traits seem to influence the dimensions of the cerebral ventricles in dogs. The ratios between the ventricles and the brain are crucial diagnostic criteria for suspected canine cognitive dysfunction (CCD). This study aimed to establish linear computed tomography (CT)-scan measurements of the cerebral ventricles in 55 Poodle dogs aged >7 years. To this end, cross-sectional CT images were evaluated. The measurements in the whole sample were: height of the right ventricle, 6.0 ± 1.6 mm; height of the left ventricle, 5.8 ± 1.6 mm; width of the right ventricle, 6.9 ± 1.4 mm; width of the left ventricle, 7.0 ± 1.3 mm; height of the third ventricle, 3.4 ± 0.8 mm; height of the right cerebral hemisphere, 39.5 ± 2.0 mm; and height of the left cerebral hemisphere, 40.2 ± 2.6 mm. The average ventricular measurements were higher in dogs older than 11 years (p < 0.05). However, the average ratio of the ventricle height to the height of the brain did not reveal differences between age groups, sex, or antimeres. In addition, none of the images showed fused lateral ventricles. Thus, these data can assist in interpreting ventricle size in senile Poodle dogs (aged >7 years).
+
+## Introduction
+
+There is diagnostic interest in the ratios between the ventricles and the brain in dogs, especially in cases with suspected canine cognitive dysfunction (CCD). This disease typically affects senile dogs (≥8 years) and resembles human Alzheimer's disease (Dewey et al., 2019; Fast et al., 2013; Schütt et al., 2015). Macroscopically, CCD presents with structural abnormalities such as cerebral atrophy, enlargement of the ventricles, and thickening of the meninges (Dewey et al., 2019; Schütt et al., 2016). A combination of a thorough patient history, owner-reported behaviors, clinical cognitive tests, and assessment of ventricle size using imaging examinations may support the diagnosis of CCD (Pilegaard et al., 2017).
+
+Although magnetic resonance imaging (MRI) is the best method for visualizing structural changes in the brain, many diagnostic centers have no devices available for animals, and examination costs are prohibitive for many pet owners. Thus, computed tomography (CT) scans may represent an accessible alternative for evaluating the shape, symmetry, and size of the lateral and third ventricles (Doiche et al., 2022). In a comparative study of methods for measuring ventricle sizes, linear measurements were efficient in detecting simple dilations. Furthermore, the ventricular height is positively correlated with the ventricular area and volume (Woo et al., 2010).
+
+Despite the availability of CT scans, standardized measurements of brain structures and their ventricular systems in different skull morphotypes and breeds need to be made available (Doiche et al., 2022; Kii et al., 1997). In addition, previous studies using CT and MRI demonstrated that the adoption of a single pattern of ventricular measurements cannot be applied to all domestic dogs because of the wide variety of skull sizes and shapes and, consequently, that of the brain and its related structures (Doiche et al., 2022; Esteve-Ratsch et al., 2001; Schröder et al., 2006; Vite et al., 1997).
+
+For linear analysis of the ventricular system using CT scans, the heights of the lateral and third ventricles are routinely measured with the "distance" tool by drawing a straight line parallel to the longitudinal fissure at the interthalamic adhesion. These measurements are broadly applied, regardless of the wide variation in the canine skull shapes and sizes (Doiche et al., 2022; Esteve-Ratsch et al., 2001; Schröder et al., 2006; Vite et al., 1997; Woo et al., 2010).
+
+Poodles are the most common breed of dogs in many countries (Federation Cynologique Internationale, 2013; Yamato et al., 2006). Thus, this study aimed to present the average linear CT-scan measurements and indices from the cerebral ventricles in senile Poodles.
+
+## Material and methods
+
+This study analyzed cross-sectional images of 55 Poodle dogs aged ≥7 years at the level of interthalamic adhesions. Animals that presented structural brain lesions or gross asymmetries in CT images were excluded. This study consisted of a retrospective analysis of images and clinical histories in a database; therefore, it was exempted from approval by the Ethics Committee on the Use of Animals.
+
+Patient images were divided into four groups: **M1**, male dogs aged 7–10 years; **M2**, male dogs aged ≥11 years; **F1**, female dogs aged 7–10 years; and **F2**, female dogs aged ≥11 years. The scans were done in a Siemens^®^ Somatom AR Star CT scanner from a veterinary diagnostic center. The cross-sections were acquired with 2 mm thickness and 2 mm increments between them, using an 83 mA/110 kVp technique. The dogs were always positioned in ventral/sternal recumbency with the hard palate parallel to the gantry table and the thoracic limbs positioned caudally to the scanning field. A scout radiograph was obtained, and the cross-sections were programmed from the foramen magnum level to the rostral limit of the nasal cavity. In addition, a “CT brain” filter (window level 35 and width 150) was used to visualize nervous tissue.
+
+The measurements were performed using the “distance” tool of the MedWork^®^ software (Table 1 and Figure 1). The ratios between the heights of the ventricles and cerebral hemispheres and between the heights of the third ventricle and the medial encephalon (RVH: RHH, LVH: LHH, and TVH: MBH) were calculated and expressed as percentages.
+
+**Table 1 List of measurements in cross-sectional images at the level of interthalamic adhesion of Poodle dogs ≥7 years.**
+
+| **Measurement** | **Description** |
+| --- | --- |
+| **RVH** and **LVH** | Height of the right (RVH) or left (LVH) ventricle measured from the most ventral to the most dorsal point of the ventricle wall |
+| **RVW** and **LVW** | Width of the right (RVW) or left (LVW) ventricle measured from the most lateral to the most medial point of the ventricle wall |
+| **TVH** | Height of the third ventricle measured from the most ventral to the most dorsal point of the ventricle wall |
+| **RHH** and **LHH** | Height of the right (RHH) or left (LHH) cerebral hemisphere at the level of the ventricle |
+| **MBH** | Height of the encephalon at the level of the third ventricle (median fissure) |
+
+*Figure: Figure 1 CT scan cross-section at the level of interthalamic adhesion of a 16-year-old female Poodle dog. (A) RVH, height of the right ventricle; LVH, height of the left ventricle; RVW, width of the right ventricle; LVW, width of the left ventricle; TVH, height of the third ventricle; (B) RHH, height of the right hemisphere; LHH, height of the left hemisphere; MBH, height of the brain at the midline.*
+
+The averages and standard deviations of the lateral ventricle, third ventricle, and encephalon measurements were calculated. The Kolmogorov–Smirnov (KS) test was used to evaluate the normality of the distribution of each variable. An unpaired Student's t-test was used to compare measurements between antimeres from the same group and between sexes and ages. One-way ANOVA (complemented by Tukey's test) was used to compare the ratios between the four groups. In both tests, statistical significance was set at p < 0.05. Statistical analyses were performed using GraphPad Prism 5 software.
+
+## Results
+
+The number of animals evaluated and the average age of the dogs in each category are shown in Table 2. A total of 55 dogs were included in the study. Considering the entire sample (n = 55), the average measurements of the cerebral ventricles and cerebral hemispheres were: RVH 6.0 ± 1.6 mm; LVH 5.8 ± 1.6 mm; RVW 6.9 ± 1.4 mm; LVW 7.0 ± 1.3 mm; TVH 3.4 ± 0.8 mm; RHH 39.5 ± 2.0 mm; and LHH 40.2 ± 2.6 mm. The measurement values by group are listed in Table 3. No differences were observed between the left and right lateral ventricles or the heights of the left and right hemispheres (p > 0.05).
+
+**Table 2 Group division according to age and sex and the average age in each group**
+
+| **Group** | **Age/Sex** | ***n*** | ***Average age*** |
+| --- | --- | --- | --- |
+| **M1** | 7–10-year-old male dogs | 9 | 8.4 y/o |
+| **M2** | ≥11-year-old male dogs | 17 | 13.5 y/o |
+| **F1** | 7–10-year-old female dogs | 6 | 7.5 y/o |
+| **F2** | ≥11-year-old female dogs | 23 | 12.9 y/o |
+| **Total** |  | 55 | 11.8 y/o |
+
+**Table 3 Measurements from cross-sectional CT images in Poodle dogs, separated by groups**
+
+| **Groups** | **Average ± standard deviation (mm)** | ***p*-value** |
+| --- | --- | --- |
+|  | **RVH** | **LVH** |  |
+| **M1** | 5.2 ± 0.7 | 4.7 ± 1.3 | 0.39 |
+| **M2** | 6.1 ± 1.5 | 6.1 ± 1.3 | 0.98 |
+| **F1** | 6.2 ± 1.8 | 5.6 ± 2.0 | 0.46 |
+| **F2** | 6.2 ± 1.7 | 6.0 ± 1.8 | 0.78 |
+|  | **RVW** | **LVW** |  |
+| **M1** | 6.2 ± 1.6 | 6.1 ± 1.4 | 0.86 |
+| **M2** | 7.0 ± 1.3 | 7.4 ± 1.0 | 0.36 |
+| **F1** | 6.5 ± 1.5 | 6.9 ± 1.2 | 0.58 |
+| **F2** | 7.2 ± 1.7 | 7.2 ± 1.5 | 0.46 |
+|  | **RHH** | **LHH** |  |
+| **M1** | 39.4 ± 2.3 | 39.3 ± 2.3 | 0.86 |
+| **M2** | 39.8 ± 2.3 | 40.2 ± 2.2 | 0.63 |
+| **F1** | 39.0 ± 1.6 | 39.4 ± 1.6 | 0.73 |
+| **F2** | 39.2 ± 1.7 | 39.5 ± 1.8 | 0.68 |
+|  | **TVH** | **MBH** |  |
+| **M1** | 3.1 ± 0.6 | 40.4 ± 2.4 | - |
+| **M2** | 3.3 ± 0.9 | 41.5 ± 2.2 | - |
+| **F1** | 3.7 ± 0.3 | 40.1 ± 1.2 | - |
+| **F2** | 3.6 ± 0.8 | 40.7 ± 1.8 | - |
+
+On average, the ventricle measurements in older dogs (groups M2 and F2) were higher than those in younger dogs (groups M1 and F1). However, only LVH and LVW were significantly higher in older males (M2) than in younger males (M1) (p = 0.02 and p = 0.01, respectively) (Table 4).
+
+**Table 4 P-values of Student's t-test comparing the average of measurements taken between the four groups**
+
+| **Measurements** | **M1 x M2** | **F1 x F2** | **M1 x F1** | **M2 x F2** |
+| --- | --- | --- | --- | --- |
+| RVH | 0.11 | 0.98 | 0.14 | 0.83 |
+| RVW | 0.20 | 0.26 | 0.79 | 0.67 |
+| LVH | 0.02* | 0.43 | 0.47 | 0.96 |
+| LVW | 0.01* | 0.70 | 0.27 | 0.60 |
+| TVH | 0.56 | 0.71 | 0.06 | 0.42 |
+| RHH | 0.46 | 0.80 | 0.99 | 0.38 |
+| LHH | 0.41 | 0.92 | 0.98 | 0.25 |
+
+In the whole sample (n = 55), the average RVH: RHH ratio was 15.3 ± 4.1%, LVH: LHH was 14.8 ± 4.3%, and TVH: MBH was 8.4 ± 2.1%. These ratios did not differ according to age and sex (Figure 2 and Table 5).
+
+*Figure: Figure 2 The boxplot shows the average and standard deviation of the RVH:RHH and LVH:LHH percentage ratios separated by age and sex groups (M1, F1, M2, F2). RVH, height of the right ventricle; RHH, height of the right hemisphere; LVH, height of the left ventricle; LHH, height of the left hemisphere.*
+
+**Table 5 Measurement ratios expressed in percentages**
+
+| **Groups** | **Average** ± **Standard deviation (%)** |
+| --- | --- |
+|  | **RVH:RHH** | **LVH:LHH** | **TVH:MBH** |
+| **M1** | 13.3 ± 2.0 | 12.2 ± 4.0 | 7.8 ± 1.7 |
+| **M2** | 15.3 ± 3.0 | 15.1 ± 3.4 | 7.8 ± 2.5 |
+| **F1** | 16.0 ± 5.2 | 14.8 ± 4.7 | 9.1 ± 0.9 |
+| **F2** | 15.8 ± 4.5 | 15.5 ± 4.8 | 8.8 ± 2.1 |
+| ***p*-value** | 0.46 | 0.26 | 0.30 |
+
+The ventricular system was clearly identified at the level of the interthalamic adhesions in all cases. Visualization of the septum pellucidum was possible in all animals and none of the images showed fused lateral ventricles. Of the 55 animals evaluated, only eight (14.5%) exhibited slight asymmetry between the lateral ventricles; five had a larger right lateral ventricle; and three demonstrated a larger left lateral ventricle.
+
+## Discussion
+
+On average, Poodles older than 11 years had slightly larger ventricles than younger dogs. Particularly, LVH and LVW were significantly higher (p < 0.05) in older than in younger males. At first glance, older dogs seem to have decreased cerebral tissue and, consequently, ventricles become proportionally larger with age, as previously described in a study on German Shepherd dogs (González-Soriano et al., 2001). However, the ratios between ventricles and cerebral heights did not support this inference in Poodle dogs, because there were no significant differences between these ratios in terms of sex, antimeres, or age. Similarly, other authors have calculated the ratios between ventricular and cerebral heights to interpret the ventricular sizes of different breeds and skull morphotypes (Doiche et al., 2022; Esteve-Ratsch et al., 2001; Kii et al., 1997; Vite et al., 1997; Woo et al., 2010).
+
+A positive correlation among ventricular height, area, and volume has been previously reported in dogs (Woo et al., 2010). Thus, the values presented here (especially the ratios) can assist in interpreting ventricle sizes in Poodle dogs older than 7 years. However, because the transition zone between the dural extension and bone represents an area and not a specific landmark, slight differences in the measurements between distinct evaluators must be considered.
+
+Doiche et al. (2022) conducted linear measurements of ventricular height using CT scans in 45 healthy dogs aged 2–5 years. In that study, the averages of RVH, LVH, and TVH were 3.8, 4.2, and 2.3 mm in German Shepherd; 3.9, 4.3, and 2.6 mm in Rottweiller; and 8.3, 8.3, and 2.6 mm in Boxer dogs, respectively. The present study identified that the mean RVH, LVH, and TVH sizes were 6.0, 5.8, and 3.4 mm for Poodle dogs, respectively. The RVH: RHH ratios were 11.35 mm, 9.56 mm, and 20.75%, and LVH:LHH ratios were 12.30 mm, 12.10 mm, 20.75% for German Shepherd, Rottweiller, and Boxer dogs, respectively (Doiche et al., 2022). In contrast, the RVH: RHH and LVH: LHH ratios in Poodles (15.3 and 14.8, respectively) were higher than those in German Shepherds and Rottweilers, but smaller than those in Boxer dogs.
+
+Kii et al. (1997) examined 21 clinically normal Beagle dogs aged 1–2 years and identified that the average RVH and LVH were 4.7 and 4.6 mm, respectively. Since the four breeds analyzed by Doiche et al. (2022) and Kii et al. (1997) were larger than Poodles, it may be hypothesized that certain breed traits, rather than body mass or size, are more closely associated with differences in ventricle measurements in dogs. These results reinforce the necessity of establishing ventricular dimensions by breed, especially in those most commonly affected by neurological or behavioral problems.
+
+Schröder et al. (2006) compared the measurement of ventricles in various breeds with different weights and skull conformations and reported that the differences were minor. Both Labradors and Poodles, who are mesaticephalic with varying body sizes, presented absolute linear measurements without statistical difference. Similar results were obtained when comparing dolichocephalic breeds (Dachshunds and German Shepherds). In another study, Pilegaard et al. (2017) inferred that the skull shape influences lateral ventricle volumes.
+
+The RVH and LVH dimensions measured in ten clinically normal Yorkshire Terriers, aged 2–8 years, using MRI instead of CT-scan, were 4.06 ± 0.98 mm, and 4.47 ± 0.98 mm, respectively. The same study reported an average RVH: RHH of 15.6% and LVH: LHH of 17.1%. These values are very close to those obtained in Poodles (15.3% and 14.8%, respectively), possibly because they are both mesaticephalic breeds. Similarly, there were no significant differences in these ratios according to sex, age, and antimeres. In other mesaticephalic breeds, the average RVH: RHH was 9.6% and LVH: LHH was 12.1% in Labradors and Poodles (Schröder et al., 2006) and 12.2% and 12.1%, respectively, in Beagles (Kii et al., 1997).
+
+Few studies have reported the width of the lateral ventricles in dogs. However, concurrent to the observations of Pilegaard et al. (2017), no significant differences were observed in the widths of the right and left lateral ventricles in this study.
+
+An MRI study reported significant volumetric enlargement for both lateral ventricles in seven normal dogs rescanned after four years. These results suggested that ventricular enlargement during aging did not necessarily reflect observable pathological cognitive or behavioral changes (Gunde et al., 2020).
+
+A slight to mild ventricular asymmetry is a common finding and may represent anatomical variations without clinical relevance in many cases. However, the frequency of asymmetry between the lateral ventricles in the Poodle dogs in our sample was low (14.6%) when compared with data from other studies (20% to 53.3%) (De Haan et al., 1994; Doiche et al., 2022; Kii et al., 1997; Pivetta et al., 2013; Schröder et al., 2006).
+
+## Conclusion
+
+Even though the averages of ventricular measurements tend to be higher in Poodles ≥11 years, the proportionality ratios indicate that this difference is insignificant. Therefore, ventricular measurements in senile Poodle dogs with symptoms of neurocognitive dysfunction may clarify whether these pathologies can change the size and ratios between the ventricles and brain in this breed.

fulltext/md/PMC10249473.md

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+---
+pmcid: "PMC10249473"
+pmid: "37304080"
+doi: "10.3389/fnagi.2023.1128521"
+title: "Activated gliosis, accumulation of amyloid β, and hyperphosphorylation of tau in aging canines with and without cognitive decline."
+journal: "Frontiers in aging neuroscience"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Activated gliosis, accumulation of amyloid β, and hyperphosphorylation of tau in aging canines with and without cognitive decline
+
+## Abstract
+
+Canine cognitive dysfunction (CCD) syndrome is a well-recognized naturally occurring disease in aged dogs, with a remarkably similar disease course, both in its clinical presentation and neuropathological changes, as humans with Alzheimer’s disease (AD). Similar to human AD patients this naturally occurring disease is found in the aging canine population however, there is little understanding of how the canine brain ages pathologically. It is well known that in neurodegenerative diseases, there is an increase in inflamed glial cells as well as an accumulation of hyperphosphorylation of tau (P-tau) and amyloid beta (Aβ~1-42~). These pathologies increase neurotoxic signaling and eventual neuronal loss. We assessed these brain pathologies in aged canines and found an increase in the number of glial cells, both astrocytes and microglia, and the activation of astrocytes indicative of neuroinflammation. A rise in the aggregated protein Aβ~1-42~ and hyperphosphorylated tau, at Threonine 181 and 217, in the cortical brain regions of aging canines. We then asked if any of these aged canines had CCD utilizing the only current diagnostic, owner questionnaires, verifying positive or severe CCD had pathologies of gliosis and accumulation of Aβ~1-42~ like their aged, matched controls. However uniquely the CCD dogs had P-tau at T217. Therefore, this phosphorylation site of tau at threonine 217 may be a predictor for CCD.
+
+1.
+
+## Introduction
+
+Age is the number one risk factor for cognitive decline in canines and humans; however, the etiology is not known. Studying aging dogs with and without canine cognitive dysfunction (CCD) syndrome shows promise for a better understanding of human age-related neurodegeneration. Canine cognitive dysfunction is considered an age-related disease, with the prevalence ranging from 14–35% of the senior canine population, exponentially increasing with age (Neilson et al., 2001; Azkona et al., 2009; Salvin et al., 2010). In a two-year longitudinal study of 51 dogs over the age of 8 years, 33% of dogs with normal cognitive status progressed to mild cognitive impairment and 22% of dogs with mild cognitive impairment progressed to CCD (Schutt et al., 2015). In another study of 180 geriatric dogs (aged 11–16 years), 28% of dogs 11–12 years old and 68% of dogs 15–16 years old exhibited signs of cognitive impairment (Neilson et al., 2001). Unlike commonly used transgenic, canines age and develop CCD naturally, making them an ideal avenue for studying brain aging and disease. With the aging canine population steadily increasing, it is essential to study these aging dogs, especially given the implications and translational value for their human counterpart. Therefore, a clearer understanding of the neuropathogenesis of aging canine brains is essential for the future development of diagnostic tests and to facilitate timely intervention, for human patients and canines alike.
+
+Although research has shown neuropathology within the aged canine brain, information on CCD-specific pathology, outside of those associated with age, is needed. The aging canine brain, similar to humans, correlates with declined cognition and neuronal pathology (Cummings and Cotman, 1995; Head et al., 1998; Rofina et al., 2006; Mormino and Papp, 2018). One common pathology in brain aging is an increase in glial inflammation, which usually affects the cortex and hippocampus (Beach et al., 1989). Although glia, especially microglia and astrocytes, are critical for normal brain function, neuroinflammation in the central nervous system (CNS) induces reactive cell phenotypes. Microglial proliferation and reactivity play an important role in neurodegenerative responses. Studies show that aged canines exhibit greater levels of the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) in the hippocampus (Hwang et al., 2008; Ozawa et al., 2016; Smolek et al., 2016). Microglia are also critical mediators of astrocyte activation through their release of proinflammatory chemokines and cytokines (Liddelow et al., 2017). Like microglia, activated, or A1, astrocytes secrete pro-inflammatory molecules and promote neuronal death over time. They are also a source of complement system proteins, especially C3, which mediates neuronal damage in models of AD (Wu et al., 2019; Pekna and Pekny, 2021).
+
+In addition to glial inflammation, the aggregation and deposition of amyloid-β~1-42~ (Aβ~1-42~) as insoluble, extracellular plaques is a common brain pathology seen in humans with neurodegenerative disease, especially in the cerebral cortex. Aβ~1-42~ is also present in canines with cognitive decline, and cognitive impairment in aged canines has been strongly associated with the accumulation of Aβ~1-42~ (Pugliese et al., 2005; Rofina et al., 2006; Barnes et al., 2016). The amino acid sequence of β-amyloid is identical between humans and dogs (Johnstone et al., 1991) allowing for the use of similar reagents across species, further establishing the canine as an appropriate model. In addition to Aβ~1-42,~ hyperphosphorylation of the tau protein (P-tau) is also commonly found in humans with AD and has also been recently discovered in CCD dogs (Abey et al., 2021). Thus, we investigated the occurrence of P-tau and Aβ~1-42~ in our aged canine brains, using antibodies for multiple phosphorylation sites of tau and techniques to verify the role of these misfolded proteins in the aging canine.
+
+2.
+
+## Materials and methods
+
+2.1.
+
+### Sample collection and preparation
+
+Forty-seven canine brains were obtained from privately owned dogs by owner consent after canines were euthanized and necropsied. The brains were collected at the James L. Voss Veterinary Teaching Hospital at Colorado State University. Canine brains were fixed in 10% neutral buffer formalin for a minimum of 72 h. After fixation, brains were processed using a tissue processor (Leica TP 1020) and embedded in paraffin wax using a tissue embedder (Leica EG 1160). Tissue blocks containing the cortex were cut from the frontal lobe when possible, however due to the condition of brain tissue, this was not always possible. Hippocampus blocks were selected by finding the most intact area of hippocampus available. Using a microtome (ThermoFisher HM1030), sections were cut at 5 μm and mounted on a charged slide. The details for euthanasia, age, and weights of the canines are listed in Table 1.
+
+**Table 1 Aged canine cause of death, age and weight information.**
+
+| Canine # | Age (years) | Weight (kg) | Reason for Euthanasia |
+| --- | --- | --- | --- |
+| Ca110 | 2.2 years | N/A | N/A |
+| Ca111 | 1.2 years | 14.6 | N/A |
+| Ca112 | 4.7 years | 25.6 | N/A |
+| Ca113 | 4.2 years | N/A | N/A |
+| Ca152 | 15 years | 44 | Severe OA, inability to rise |
+| Ca153 | 9 years | 7.8 | Nasal carcinoma |
+| Ca154 | 8 years | 39.4 | Hemiangiosarcoma |
+| Ca155 | 12 years | 7.23 | Severe lethargy, mental obdundation |
+| Ca156 | 11 months | 5.79 | GME |
+| Ca159 | 15 years | 21.1 | Nasal carcinoma with multifocal metastisis |
+| Ca160 | 8 years | 8.41 | Hind limb paralysis |
+| Ca169 | 8 years | 30 | Cardiac mass |
+| Ca170 | 9 years | 18.7 | Severe cushing’s disease |
+| Ca171 | 13 years | 26.8 | Disseminated lymphoma (stage V) |
+| Ca172 | 10 years | 26.1 | Left temporal osteosarcoma |
+| Ca173 | 14 years | 8 | DOA – cardiac arrest |
+| Ca174 | 11 years | 19.6 | Multifocal carcinomas and sarcomas |
+| Ca175 | 13.5 years | N/A | N/A |
+| Ca177 | 16 years | N/A | N/A |
+| Ca178 | 15 years | N/A | N/A |
+| Ca183 | 14 years | 11.3 | Decline, unable to walk, possible bladder stones |
+| Ca186 | 9 years | N/A | N/A |
+| Ca187 | 13yo | N/A | N/A |
+| Ca188 | 9 years | 54 | Pericardial effusion, peritoneal effusion, acute lethargy and anorexia |
+| Ca189 | 11.5 years | 43.3 | Metastatic mast cell tumor |
+| Ca192 | 10 years | N/A | Hemoabdomen |
+| Ca193 | 8 years | 15.7 | Severe nasopharyngeal obstruction with fibrous scar tissue |
+| Ca194 | 10 years | 10.1 | Labored breathing |
+| Ca195 | 12.5 years | 23.6 | DCM due to grain-free diet |
+| Ca196 | 12 years | 20 | Abdominal mass, difficulty defecating and urinating |
+| Ca197 | 12 years | 30 | Bicavity effusion |
+| Ca198 | 13.5 years | 35 | Anorexia, lethargy, obtunded on presentation, abdominal effusion, hypovolemic, diffuse hepatic nodules |
+| Ca199 | 13 years | 8.4 | Decline due to age |
+| Ca256 | 8 years | 20 | Hemangiosarcoma |
+| Ca264 | 13 years | 9.7 | Respiratory distress |
+| Ca289 | 15 years | 4.9 | Chronic cough, hepatomealgy, degenerative heart disease |
+| Ca351 | 13.6 years | N/A | Degenerative mitral disease, bilateral adrenomegaly, hypoadrenocorticism |
+| Ca800 | 12.4 years | N/A | Severe emaciation, general decompensation |
+
+2.2.
+
+### Immunohistochemical staining and microscopy imaging analysis
+
+Immunohistochemistry was used to visualize a variety of neurological antibodies including: GFAP (DAKO Cat. #20334), Iba1 (Abcam 5,076), S100β (Abcam 41,428), AT270 (Thermo Fisher MN1050), T217 (Abclonal AP1233) and Aβ~1-42~ (Thermo Fisher 44344). Sections were dewaxed in xylene and rehydrated through graded ethanol’s, before undergoing antigen retrieval in 0.01 M sodium citrate for 20 min at 95°C. Endoperoxides were removed by incubating sections in 0.30% hydrogen peroxide and water solution for 30 min. Blocking of non-specific labeling was performed with 10% hose serum in Tris-A in 2% BSA [2% bovine serum albumin (BSA) and 2% Triton-X in tris buffered saline (TBS)]. Sections were incubated overnight at 4°C in primary antibodies diluted with Tris-A in 2% BSA [GFAP 1:400, Iba1 1:400, Aβ~1-42~ 1:200, S100β 1:750, T217 1:100, T181 1:500]. Sections were then washed with Tris-A in 2% BSA and incubated for an hour against their corresponding biotinylated secondary antibodies, goat anti-rabbit or horse anti-mouse (Vector labs), diluted in 10% horse serum in Tris-A 2% BSA 1:250. Slides were counterstained with hematoxylin, rinsed in bluing reagent and dehydrated through graded ethanol followed by xylene, mounted with mounting media and cover slipped with #1 cover glass.
+
+Slides were visualized using Olympus BX51 microscope and Olympus DP70 camera and saved as virtual slide images at 20X objective. Quantitative analysis was performed using Olympus CellSens Dimension Desktop 3.1 using Count and Measure quantification application. After scanning the tissue on the slide, a representative area was selected for quantification by cell counting using CellSens software. The representative area was chosen after looking at the full area of cortex or hippocampus. The size of the area chosen was calculated in μm^2^, and the number of positive cells in that area were then normalized over an area of 1 mm^2^.
+
+2.3.
+
+### Immunofluorescent staining and microscopy imaging analysis
+
+Immunofluorescence was used to visualize co-expression of certain proteins in glial or neuronal cell types: C3(Abcam 181,147) [1:250] and S100β (Abcam 41,428) [1:750]. Slides were visualized using BX63 fluorescence microscope equipped with a Hamamatsu ORCA-flash 4.0 LT CCD camera and collected using Olympus CellSens software version 3.1. Images were taken as virtual slide images at 10X objective to be analyzed on Olympus Cell Sense technology using Count and Measure quantification application.
+
+2.4.
+
+### Analysis of canine owner questionnaires for canine cognitive decline
+
+Two commonly used veterinary scales for CCD were given to client owners following the necropsy of their canine, Canine Cognitive Dysfunction Rating Scale (CCDR) (Salvin et al., 2011a) and the Canine Dementia Scale (CADES) (Madari et al., 2015). These questionnaires were either collected in person by veterinary staff or distributed online, when in person communication was not an option.
+
+2.5.
+
+### Statistical analysis
+
+All data is presented as mean ± SEM. Differences between experimental groups were analyzed using an unpaired *t*-test using Prism GraphPad. Significance is denoted as * = *p* ≤ 0.05.
+
+3.
+
+## Results
+
+3.1.
+
+### Increased glial inflammation in the cortex and hippocampus of aged canines
+
+Young (1–4 years of age) and aged (over 8 years of age) canine brains were stained for glial markers and quantified in two brain regions related to neurodegenerative disease, the cortex and hippocampus. Canines between the ages of 5–7 years of age were excluded from analysis due to information in current literature about canine aging occurring significantly after the age of 8.
+
+Representative images of the cortex of a 3-, 9- and 12-year-old canine stained for GFAP (Figures 1A–C), Iba1 (Figures 1E–G) and S100β (Figures 1I–K) is shown. No significant increase is seen in any of the glial cells, however a trending increase is present in all three. GFAP staining cells show a mean of 156.2 GFAP positive cells per mm^2^ in young canines compared to 187.8 GFAP positive cells per mm^2^ in aged canines (Figure 1D; *p* value = 0.4348; *t* = 0.7882; df = 44). In Iba1 staining, the mean for young canines is 164.4 Iba1 positive cells per mm^2^ while in aged canines it increases to 199.6 Iba1 positive cells per mm^2^ (Figure 1H; *p* value = 0.5634; *t* = 0.5822; df = 44). Finally, in S100β staining, the mean is 98.2 S100β positive cells per mm^2^ in young canines and 176.0 S100β positive cells per mm^2^ in aged canines (Figure 1L; *p* value = 0.0580; *t* = 1.946; df = 44).
+
+*Figure: Figure 1 Increased glial reactivity in the cortex of aging canines. Increase no significant change was detected in GFAP+ cells **(A–D)**, IBA1+ cells **(E–H)** or S100β positive cells **(I–L)** compared to young canines. *n* = 5 young; *n* = 41 aged. Scale bar = 20 μM Unpaired *t*-test (**D**: *t* = 0.8882; **H**: *t* = 0.5822; **L**: *t* = 1.946. df = 44), error bars = SEM, *p* < 0.05. *p* values: *D* = 0.4348, *H* = 0.5634, L = 0.0580, ns = non-significance.*
+
+All available brains with a hippocampus, 27 of the 46 dogs included in this study, were also analyzed. Due to the availability and nature of tissue received for dissection, not all canines had hippocampus present. Representative images of the hippocampus of 3-, 9- and 12-year-old canines stained for GFAP (Figures 2A–C), Iba1 (Figures 2E–G) and S100β (Figures 2I–K). A significant increase in S100β positive cells is also seen with a mean of 159.6 S100β positive cells per mm^2^ in aged canines compared to 69.5 S100β positive cells per mm^2^ in young canines (Figure 2L; *p* value = 0.0237; *t* = 1.464; df = 25), the difference means being 90.09 ± 37.29. No significant change, although a trend of an increase, was identified in the number of GFAP + and Iba1 + cells in the hippocampus (Figure 2D; *p* value = 0.1557; *t* = 0.2122; df = 25, Figure 2H; *p* value = 0.8336; *t* = 2.416; df = 25). Raw quantitative values of GFAP+, Iba1+, and S100b+ cells for each canine is listed in Table 2.
+
+**Table 2 Quantitative values of GFAP, Iba1 and S100beta for each canine in the coretex and hippocampus.**
+
+| Canine ID | Cortex | GFAP + cells per mm^2^ | Iba1 + cells per mm^2^ | S100β + cells per mm^2^ | Hippocampus | GFAP + cells per mm^2^ | Iba1 + cells per mm^2^ | S100β + cells per mm^2^ |
+| --- | --- | --- | --- | --- | --- | --- | --- | --- |
+| Ca110 |  | 109 | 76 | 234 |  | 77 | 188 | 69 |
+| Ca111 |  | 266 | 116 | 105 |  | 70 | 181 | 132 |
+| Ca112 |  | 146 | 190 | 59 |  | 124 | 227 | 42 |
+| Ca113 |  | 117 | 140 | 75 |  | 136 | 368 | 35 |
+| Ca152 |  | 241 | 517 | 133 |  | N/A | N/A | N/A |
+| Ca153 |  | 238 | 523 | 378 |  | N/A | N/A | N/A |
+| Ca154 |  | 339 | 485 | 199 |  | 220 | 251 | 253 |
+| Ca155 |  | 264 | 175 | 222 |  | N/A | N/A | N/A |
+| Ca156 |  | 143 | 300 | 18 |  | N/A | N/A | N/A |
+| Ca157 |  | N/A | N/A | N/A |  | 328 | 331 | 123 |
+| Ca159 |  | 227 | 209 | 236 |  | N/A | N/A | N/A |
+| Ca160 |  | 232 | 239 | 110 |  | 156 | 351 | 110 |
+| Ca163 |  | 169 | 312 | 111 |  | 58 | 546 | 148 |
+| Ca164 |  | 182 | 389 | 90 |  | 179 | 218 | 184 |
+| Ca169 |  | 218 | 150 | 162 |  | 242 | 274 | 146 |
+| Ca170 |  | 236 | 223 | 131 |  | N/A | N/A | N/A |
+| Ca171 |  | 140 | 326 | 171 |  | 309 | 237 | 95 |
+| Ca172 |  | 128 | 295 | 56 |  | N/A | N/A | N/A |
+| Ca173 |  | 126 | 168 | 92 |  | 66 | 239 | 117 |
+| Ca174 |  | 121 | 307 | 182 |  | N/A | N/A | N/A |
+| Ca175 |  | 53 | 329 | 147 |  | 102 | 280 | 119 |
+| Ca176 |  | 341 | 213 | 259 |  | N/A | N/A | N/A |
+| Ca178 |  | 388 | 207 | 126 |  | N/A | N/A | N/A |
+| Ca183 |  | 203 | 105 | 71 |  | 266 | 370 | 6 |
+| Ca184 |  | 167 | 61 | 294 |  | N/A | N/A | N/A |
+| Ca185 |  | 75 | 57 | 122 |  | N/A | N/A | N/A |
+| Ca186 |  | 203 | 84 | 49 |  | N/A | N/A | N/A |
+| Ca187 |  | 240 | 39 | 137 |  | N/A | N/A | N/A |
+| Ca188 |  | 90 | 169 | 83 |  | 573 | 95 | N/A |
+| Ca189 |  | 44 | 141 | 146 |  | 85 | 316 | 143 |
+| Ca192 |  | 147 | 395 | 102 |  | 257 | 298 | 248 |
+| Ca193 |  | 130 | 123 | 191 |  | 117 | 292 | 175 |
+| Ca194 |  | 122 | 212 | 219 |  | N/A | N/A | N/A |
+| Ca195 |  | 339 | 106 | 240 |  | 210 | 179 | 224 |
+| Ca196 |  | 292 | 274 | 299 |  | N/A | N/A | N/A |
+| Ca197 |  | 225 | 88 | 312 |  | N/A | N/A | N/A |
+| Ca198 |  | 295 | 238 | 251 |  | N/A | N/A | 275 |
+| Ca199 |  | 212 | 234 | 208 |  | 192 | 303 | 299 |
+| Ca250 |  | 205 | 81 | 47 |  | N/A | N/A | N/A |
+| Ca253 |  | 181 | 147 | 58 |  | 87 | 56 | 90 |
+| Ca256 |  | 40 | 111 | 206 |  | 104 | 70 | 125 |
+| Ca264 |  | 87 | 77 | 239 |  | 59 | 203 | 127 |
+| Ca289 |  | 182 | 2 | 253 |  | 417 | 67 | 624 |
+| Ca290 |  | 82 | 112 | 320 |  | N/A | N/A | N/A |
+| Ca294 |  | 69 | 84 | 92 |  | 191 | 63 | 196 |
+| Ca351 |  | 219 | 122 | 280 |  | 50 | 128 | 220 |
+| Ca800 |  | 208 | 55 | 190 |  | 295 | 5 | 88 |
+
+*Figure: Figure 2 Increase glial reactivity in the hippocampus of aging canines. Increase in astrocyte reactivity detected by increase in S100β + (**J–L**; *n* = 25) in the hippocampus of aged canines when compared to young canines (**A,E,I**; *n* = 4). No significant change was detected in GFAP+ **(A–D)** or Iba1 + cells **(E–H)**. *n* = 4 young; *n* = 25 aged. Scale bar = 20 μM Unpaired *t*-test (**D**: *t* = 1.464; **H**: *t* = 0.2122; **L**: *t* = 2.416. df = 25), error bars = SEM, *p* < 0.05. *p* values: D = 0.1557, H = 0.8336, L = 0.0237 ns = non-significance.*
+
+3.2.
+
+### A1 Astrocytes increase in the cortex of aged canines
+
+Young and aged canine brains were analyzed to identify co-localization of S100β and C3 staining using co-immunofluorescence. Representative images show colocalization of S100β and C3 staining of the aged canine in the cortex (Figures 3A,B). A significant increase in the percent of S100β to C3 was found (Figure 3C), with *p* < 0.05 and the difference between means being 22.94 ± 10.95.
+
+*Figure: Figure 3 A1 reactive astrocytes increase within the cortex of the aging canine. An increase in C3 (red) co-localized with pan astrocyte marker S100b (green) is seen in aged canines **(B)** compared to young animals **(A)**. Quantification of these cells in the cortex reveals a significant increase in A1 astrocytes with age in this brain region **(C)**. (*n* = 22). Scale bar = 20 μM Unpaired *t*-test, *p* = 0.0486, error bars = SEM, *p* < 0.05.*
+
+3.3.
+
+### Phosphorylated tau and amyloid beta plaques increase in the cortex of aging canines
+
+Representative images of hyperphosphorylated tau and Aβ~1-42~ in the cortex from 3-, 9- and 12-year-old canines show possible deposits and tangles in the cortex (Figure 4). Two different phosphorylation sites of tau (P-tau) were analyzed by immunohistochemistry, including threonine 181 (P-tau-Thr181) (Figures 4A–C) and threonine 217 (P-tau-Thr217) (Figures 4D–F). An increase in intracellular accumulation and extracellular fibrils of P-tau is found in canines aged 9- and 12- years old compared to the young canine brain, aged 3 years. A possible formation of an extracellular neurofibrillary tangle with P-tau T217 antibody in the 9-year-old canine (see inset in Figure 4E) and intracellular P-tau aggregates in the 12-year-old canine (see inset Figure 4F) are identified. Intracellular amyloid-β~1-42~ aggregation was increased in the cortex of 9- and 12-year-old canines compared to young dogs, although no extracellular plaques are detected (Figures 4G–I).
+
+*Figure: Figure 4 Hyperphosphorylation of Tau and Amyloid β~1-42~ accumulation in the cortex of aging canines. Representative images of young (3 years of age; *n* = 5) and aged (8 years of age and older; *n* = 41) canines are shown. Phosphorylated threonine 181 tau is increased at 9 **(B)** and 12 years of age **(C)** compared to young dog **(A)**. Phosphorylated threonine 217 tau is increased at 9 **(E)** and 12 years of age **(F)** with P-tau positive plaque like structure in (**E**, see inset) the 9-year-old dog and an intracellular phosphorylation in the 12-year-old canine (**F**, see inset) compared to young dog **(D)**. Aβ~1-42~ accumulates in the 9 **(H)** and 12 **(I)** year old dogs more than the young 3-year-old **(G)**. Scale bar = 20 μM.*
+
+3.4.
+
+### Phosphorylated tau and amyloid beta plaques are present in the hippocampus of aging canines
+
+Representative images of hyperphosphorylated tau and Aβ~1-42~ in the hippocampus of 3-, 9- and 12-year old canines possible tau hyperphosphorylation at two sites as well as amyloid beta~1-42~ aggregation. Possible tau fibrils are seen at tau phosphorylation site threonine 181 (Figures 5A–C) and at threonine 217 (Figures 5D–F), intracellular accumulation is seen in both 3- and 12-year old canines. Amyloid beta~1-42~ (Figures 5G–I) aggregation is seen (see inset in Figure 5H) in the 9-year old canine.
+
+*Figure: Figure 5 Hyperphosphorylation of Tau and Amyloid β~1-42~ accumulation in the hippocampus of young and aging canines. Representative images of young (3 years of age; *n* = 5) and aged (8 years of age and older; *n* = 41) canines are shown. Phosphorylated threonine 181 tau is increased at 9 **(B)** and 12 years of age **(C)** compared to young canines **(A)**. Phosphorylated threonine 217 tau is present at 3 years of age **(D)** and at 12 years of age with distinct intracellular deposits at 12 years of age **(F)** and very little staining at a different dog at 9 years old **(E)**. Aβ~1-42~ accumulates in the 9 **(H)** year old dog, more than the 3 year old **(G)** and 12 year old dog **(I)**. Scale bar = 20 μM.*
+
+3.5.
+
+### Questionnaires determined canine cognitive dysfunction in aging dogs and correlated to neuropathological results
+
+The Canine Cognitive Dysfunction Rating Scale (CCDR) is based on a scale of 0–80 points, ranging from no abnormal behavior to severe behavioral disturbances (Salvin et al., 2011a). The CCDR has a diagnostic accuracy of 99.3% in dogs. In accordance with previously published data, we used a score of 50 for the CCDR questionnaire as an optimal diagnostic threshold, meaning it is expected that dogs scoring more than 50 points were likely to have CCD (Salvin et al., 2011a). The second questionnaire, Canine Dementia Scale (CADES), has been established as a validated screening tool for CCD, as well as a long-term assessment tool for progression and treatment efficacy (Madari et al., 2015). Dogs were classified as having either mild (1–7), moderate (scores of 24–44), or severe cognitive impairment (scores of 45–95). Of the 19 owner questionnaires we received from the aged canines, 26% were positive according to CCDR scoring and 32% of the dogs were classified as having severe cognitive impairment from the CADES questionnaire (Table 3). Another 26% of the dogs were classified as either mild or moderate for cognitive impairment using the CADES (Table 2). Critically, canines with either positive CCDR scores or severe impairment according to the CADES show an increase in both gliosis and accumulation of the misfolded proteins P-tau and Aβ~1-42~ compared to aged canines without CCD (Figure 6). We also through H&E staining were able to see an increase in pyknotic neurons within the aged brains with CCD positive surveys. However, some of the aged canines with a negative score for CCDR or CADES were not distinguishable from the CCD brains (Figures 6E–H). Canines with known comorbidities with the potential of impacting cognitive function were excluded from further analysis (Table 2).
+
+**Table 3 Scores of canines with CADES and CCDR owner questionnaires.**
+
+| Canine ID | Age | CADES | Cognitive impairment | CCDR | CCD ± |
+| --- | --- | --- | --- | --- | --- |
+| Ca152 | 15yo | 34 | Moderate | 49 | Negative |
+| Ca163 | 10yo | 19 | Mild | 55 | Positive |
+| Ca169 | 8yo | 0 | None | n/a | N/A |
+| Ca170 | 9yo | 56 | Severe | 48 | Negative |
+| Ca173 | 14yo | 4 | None | 36 | Negative |
+| Ca174 | 11yo | 0 | None | 35 | Negative |
+| Ca183 | 14yo | 0 | None | 34 | Negative |
+| Ca188 | 9yo | 26 | Moderate | 34 | Negative |
+| Ca256 | 8yo | 0 | None | 34 | Negative |
+| Ca264 | 13yo | 0 | None | 35 | Negative |
+| Ca289 | 15yo | 55 | Severe | 58 | Positive |
+| Ca351 | 13.6yo | 49 | Severe | 51 | Positive |
+| Ca800 | 12.4yo | 34 | Moderate | 45 | Negative |
+
+*Figure: Figure 6 Gliosis and accumulation of misfolded proteins correlates to canines with positive cognitive dysfunction scores. **(A)** Aged canines with a CCDR/CADES score of being negative for CCD **(A–J)** does show an increase in gliosis and Aβ~1-42~ accumulation but no P-tau at T-217. **(B)** Aged canines with a moderate or severe CCD score **(K–T)** show gliosis and Aβ~1-42~ and P-tau positive cells. Canines negative or mild CADES *n* = 7; canines moderate or severe CADES *n* = 6. Scale bar = 20 μM.*
+
+4.
+
+## Discussion
+
+Aged dogs with CCD show promise for modeling human age-related neurodegeneration, including Alzheimer’s disease (AD) and other AD related dementias (ADRDs), and their respective hallmarks of disease, such as brain atrophy, cognitive decline, and Aβ in the CSF and brain (Pugliese et al., 2006; Yu et al., 2011; Schutt et al., 2015; Ozawa et al., 2016; Smolek et al., 2016). However, there is incomplete knowledge about how age-associated neuropathological progression relates to cognitive phenotypes in canines. This data is valuable from not only a veterinary perspective, but also a clinical one, as the canine is potentially a better model of age-related disease progression. The aged canine shares similar environmental factors to humans, unlike laboratory murine models, making them an ideal path to study the aging brain process. They also demonstrate critical elements of human disease, including neuroinflammation and aggregations of misfolded proteins. Here, we correlate gliosis to the accumulation of the neurotoxic proteins Aβ~1-42~ and P-tau in aged canines with and without CCD.
+
+Microglia are essential for CNS homeostasis and play a role in age related neurodegenerative disease pathogenesis by contributing to an inflammatory brain state (Wang et al., 2015; Sobue et al., 2021). Our study is consistent with previous research that shows aged canines exhibit greater levels of the Iba1 protein in the hippocampus and canine cortical brain region compared to adult canines (Ozawa et al., 2016; Thomsen et al., 2021; Figures 1, 2). Similar to microglia, activated astrocytes produce numerous reactive and proinflammatory molecules in the AD brain (Heppner et al., 2015). Further, it is also known that microglial reactivity is followed by the progressive activation of astrocytes which are characterized by co-expression of C3 and S100β in AD human brains (Liddelow et al., 2017; Wu et al., 2019). In this study, we identify that C3 and S100β positive astrocytes are significantly increased in the cortex of aged canines. Additionally, quantifications of S100β^+^ cells show a significant increase in both the hippocampus and cortex. Although no significant increase of GFAP^+^ astrocytes is seen, these results are likely due to discrepancies in cellular expression of these proteins. Although more recent data has differentiated astrocytic populations into subtypes based on their transcriptional expression, data such as this is extremely limited in the canine. Murine studies show that there are populations of astrocytes, particularly in the hippocampus, that stain GFAP^−^. Considering the existence of this unique cell population that lacks the common glial marker, as well as evidence that S100β plays critical roles in astrocyte activation and migration, our data demonstrates the presence of gliosis in our canine model. Co-localization of C3 and S100 also establish the presence of reactive glial phenotypes in the canine brain, irrespective of their subtype (Figure 3).
+
+Additionally, hippocampal and cortical astrocytes highly express genes related to glutamate receptor activity and synaptic transmission compared to populations in other brain regions (Lozzi et al., 2020). Reactive astrocytic phenotypes, such as those found in the cortex in aged canines, reduce glutamate uptake and subsequent conversion into its inactive form glutamine. Excessive accumulation of glutamate in the synaptic cleft overstimulates NMDA receptors, which are often upregulated in the aged brain to overcompensate for loss of function, especially in the cortex. Overstimulation eventually causes excitotoxicity of the neurons in those regions, contributing to cognition loss and overall neurodegeneration (Rothstein et al., 1996). Chronic activation of the glial cells in the hippocampus and cortex of CCD positive canines may cause dysregulation of glutamate uptake and synaptic transmission that exacerbates neuronal damage in addition to age-related mechanisms.
+
+Two characteristic features of AD in humans are the presence of neurofibrillary tangles, composed of abnormally hyperphosphorylated tau protein, and Aβ plaques. Canines share tight AD gene sequence homology with humans (Sharman et al., 2013) and have alterations in amyloid precursor protein (APP) processing and tau immunoreactivity with age (Bates et al., 2014). Aβ deposits have been observed in aging dog brains, with the load of Aβ often correlated with CCD clinical signs (Head et al., 2010). The astrocyte marker S100β is also highly expressed by reactive astrocytes in close vicinity of beta amyloid deposits, and immunotherapies for Aβ in dogs reduces glial inflammation (Neus Bosch et al., 2015). Similarly, phosphorylated tau protein has been detected in the aging dog brain, although with less frequency and density than human AD brains (Smolek et al., 2016; Abey et al., 2021). Physiological tau is mostly confined to neuronal axons, where it is involved in microtubule stabilization. Microtubule binding, among other tau functions, are regulated by numerous serine, threonine, and tyrosine amino acid sites within the protein. Phosphorylation of these sites results in protein dysfunction and microtubule instability. Cytoplasmic deposits of tau phosphorylated at Threonine 181 have been previously recognized in the cortex of canines, but no neurofibrillary tangles (NFTs) have been identified (Smolek et al., 2016). In our study, we revealed fibril formation of tau phosphorylated at Thr181 and intracellular accumulation of tau phosphorylated at Thr217 in the cortices and hippocampus of aged canines compared to young animals (Figures 4, 5). Threonine 217 phosphorylation in the nine-year-old dog shows a unique, plaque-like structure or NFT. Although, to our knowledge, extracellular aggregation at this phosphorylation site has not been shown, this phosphorylation site is found in the proline-rich domain of the tau protein in close proximity to other amino acid residues commonly implicated in NFT formation (Xia et al., 2021). We were able to detect this finding in five of our aged canine brains. Different structures, conformations, and misfolds are associated with tauopathies, even within the same disease classification, which further highlights the variety on hyperphosphorylation and aggregation that may occur, especially in canines (Moloney et al., 2021; Wennstrom et al., 2022).
+
+Lastly, it is critical that we are able to determine if these aging canines have CCD syndrome through the only current clinical diagnostic tool available, owner questionnaires (Salvin et al., 2010, 2011b; Ozawa et al., 2019; O'Brian et al., 2021). To determine this, we asked owners to complete both the CCDR and CADES questionnaires on necropsied dogs, as none were diagnosed with CCD at the time of death (Table 1). All owners of the canines we have performed pathological analysis on were contacted and 19, or 40%, of the surveys were returned for analysis (Table 2). However, pathology of the dogs with either severe criteria from the CADES questionnaire or CCDR positive for dementia show pathologies that were not always distinguishable from age matched canine brains. We found no significant change in the accumulation and aggregation of P-tau and Aβ (Figures 6N,P) in canines with CCD compared to aged, matched canines without CCD in all of our canine brains. Although misfolded protein aggregates are characteristic of neurodegenerative disease, their presence alone does not equate to declined cognitive state and neuropathological damage. Aggregates themselves do not cause neuronal death, rather, they contribute to neuronal dysfunction and exacerbate damaging, chronic neuroinflammatory signaling. The presence of misfolded proteins in correlation with increased numbers of phenotypically neurotoxic glia, as seen in the CCD positive animals, may intensify neuronal damage and loss. This is seen by an increased number of punctate neurons (Figure 6M), indicative of apoptotic or dying neurons, identified in aged canines with CCD compared to aged canines without CCD. Together, these data address the potential cause of cognition loss associated with CCD outside of normal aging.
+
+4.1.
+
+### Study limitations
+
+The variability of our data indicates that there are a few limitations to our study that could explain variability within our findings. The small sample size of owner questionnaires that we received may not be entirely accurate, as these were performed after their dog had passed, and in some cases even years afterword. We were also unable to perform a medical review to rule out other causes of clinical disease, such as structural abnormalities in the brain or infection. Therefore, we are unsure if the absence of significance is due to the lack of power for the clinical questionaries, the lack of a full medical review, or that there is no true difference between aspects of aging pathology and CCD clinical diagnosis. The tissues analyzed was also a limitation of this study. We were only able to use available necropsied tissue, which meant that not all canine brain sample had the hippocampus brain regions. Also due to our small sample size we are unable to assess the role of breed differences in aging currently. We therefore chose to use eight years of age as our cutoff irrespective of the breed to determine in general the neuropathological changes induced. We are award this is an extreme limitation of our study and hope with time we will be able to expand these findings to better understand breed differences. Future studies that are well powered, include a complete medical review (to rule out other medical conditions that could cause the neuropathology) and have timely owner questionnaires are necessary for future development of this research.
+
+4.2.
+
+### Conclusion
+
+The aging brain and the factors that lead to susceptibility to diseases like cognitive decline is still unknown. To better understand this, we have studied thirty-eight naturally aged canine brains for pathological markers of glial inflammation, morphological changes, accumulation of amyloid β~1-42~ and hyperphosphorylation of tau. Both microglial and astrocyte number are increased, making this the first report to our knowledge detecting activation of reactive astrocytes defined by co staining with complement 3 (C3) and S100β in naturally aging canines, a similar phenotype found in Alzheimer’s disease laboratory rodent models and patients. We also detect Aβ~1-42~ accumulation and T181 and T217 for hyperphosphorylation of tau in most of the aged brain samples in this cohort. Following owner questionnaires, 19 of these aged dogs, 26% were positive for CCD based on the CCDR; however, there was no correlation between these biomarkers and CCD. However, future studies with timely clinical medical review and questionaries may allow us to determine differences between the two aging populations.
+
+## Data availability statement
+
+The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
+
+## Ethics statement
+
+The animal study was reviewed and approved by College of Veterinary and Biomedical Sciences College Regulatory Broad (CRB) and the Colorado State University IACUC committee. Written informed consent was obtained from the owners for the participation of their animals in this study.
+
+## Author contributions
+
+JM and SM conceived and designed the project. AH, AL, and MR performed the pathology experiments. BK and LM collected canine samples and owner questionnaires. AH, AL, SM, and JM contributed to data analysis. JM, AH and AL wrote the manuscript. All authors reviewed and edited the manuscript.
+
+## Funding
+
+This research received funding from the Colorado State University Translational Medicine Institute and Office of Vice President of Research Catalyst Innovative Program- for the Center for Healthy Aging.
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10251977.md

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+---
+pmcid: "PMC10251977"
+pmid: "37889770"
+doi: "10.3390/ani13111869"
+title: "Psychological Stress Is Associated with Increased Cancer Risk in Dogs."
+journal: "Animals : an open access journal from MDPI"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Psychological Stress Is Associated with Increased Cancer Risk in Dogs
+
+## Abstract
+
+### Simple Summary
+
+It is widely accepted that long term mental stress increases the risk of cancer. However, it has been impossible to show this conclusively in any species due to the complexities of genetic variation and the difficulty of inducing or measuring levels of psychological stress. Here, we report evidence that heritable canine temperament that increases psychological stress in dogs is somehow linked to overall risk of multiple cancer types. We propose that studying this link in dogs would result in a new understanding of the relationship between psychological stress and risk of cancer and lead to new preventive and therapeutic veterinary medicine advances that could be tested in clinical trials in pet dogs.
+
+### Abstract
+
+Although there is evidence that psychological stress may be associated with increased cancer risk, the effect of stress on cancer risk is difficult to study, both in humans, due to socioeconomic factors, and in animal models, due to questionable biological relevance. Here, we test whether heritable canine temperament that increases psychological stress is associated with cancer risk. The study data are breed-specific averages of incidences of multiple cancer types and of temperament classes. The latter are derived from a latent class analysis of behavioral questionnaires completed by owners (C-BARQ). We thus classified the dogs according to whether they are calm vs. reactive within and across breeds. Using meta-analysis approaches, we modeled the risk of multiple cancer types in calm vs. reactive dogs. We adjusted for breed averages of body mass and lifespan, which are common confounders that impact cancer. Our study confirms that body size has a significant effect of on risk of multiple types of cancers in dogs and shows for the first time that temperament also has a moderate effect. These findings suggest dog models of heritable psychological stress are suitable for molecular epidemiological and translational studies on its effects on cancer risk.
+
+## 1. Introduction
+
+There is a longstanding and popular belief that mental stress in animals is positively associated with cancer risk. However, due to challenges to the evolutionary, epidemiological, and physiological approaches, it has been impossible to conclusively test this possibility. For instance, a shared ecological trait of long-lived invertebrate and vertebrate species is reduced risk of predation (and mortality in general [1]), and lifespan has been shown to be negatively associated with cancer risk (e.g., in sea urchins, Greenland sharks, Brandt’s bats, bowhead whales, and naked mole-rats). As predation risk can be reduced through diverse adaptations, such as large body size, flight, and group living, it is not straightforward to isolate the cancer effects of metabolism, mental stress, etc. [2].
+
+Given the high levels of genetic and phenotypic heterogeneity, and the socioeconomic differences in humans, it is difficult to isolate and study the effects of stress and behavior that lead to cancer [3,4,5]. The large body of scientific studies has not yielded consistent evidence that specific behaviors, including psychological stress, can increase the risk of cancer and its development in humans [4,6,7,8,9]. The evidence for and against was recently reviewed by Falcinelli et al. [4], who examined 21 case-control or population studies on the effects of common types of acute and chronic psychological stress on cancer risk. Of these, 15 considered breast cancer alone, and only 3 tested for many or all cancer types; 13 of the 21 studies yielded positive results and 8 were negative (for breast cancer, 7 were positive and 9 were negative).
+
+Despite the lack of clear evidence from prospective studies, and with a single study showing inconclusive findings [10], it is commonly believed that adverse effects of psychological stress on cancer risk and development are pervasive. This is reflected in the long history of investigation into the pathophysiology of stress related to all major aspects of cancer, an investigation that is currently active [4,11]. General pathways that are strongly affected by stress include the hypothalamic–pituitary axis and the sympathetic nervous system, which induce glucocorticoid and adrenaline/noradrenaline signaling. The duration of stress physiology can result in dramatically different effects. Chronic psychological stress has adverse consequences in multiple tissues through immune and inflammatory mechanisms. These can affect rates of bacterial/viral infections and DNA mutations, respectively. Many other cancer initiation mechanisms are also affected by stress. Moreover, psychopathology is associated with behaviors that are risk factors for cancer, such as smoking.
+
+Since the optimal method for evaluating these factors in humans remains elusive and is complicated due to sampling and confounding issues, here we use a canine model to test for the association of psychological stress and cancer risk. Dogs are the most popular companion animal across the globe [12], and in many countries, such as the United States, they often receive highly sophisticated medical care [13]. In these societies, dogs commonly share environments with their human counterparts and thus are outstanding translational models. Additionally, due to the combination of extreme selection pressure for meeting breed standards and little to no selection for addressing health concerns, dogs have greatly reduced heterogeneity and polygenicity for diverse, complex traits [14]. Put another way, traits such as cancer risk and problematic behaviors tend to be explained by relatively very few variations with moderate to large effects, while in humans, these same traits can be explained by a great many variations with minute-to-small effects [15].
+
+Like cancer traits, dog breed-specific behavioral profiles have been shown to have a genetic basis [16,17]. This has been carried out, in large part, through owner questionnaires mostly concerning problem behaviors such as different types of anxiety, fear, and aggression [18]. Breed averages of such temperament traits were recently used for genetic mapping [16,17]. Many of those mapped alleles were (i) shown to be associated with brain structure differences [19] and (ii) subsequently supported in a population sample of pedigree and mixed-breed dogs that included dogs with clinical behavioral diagnoses [20]. Dog temperament is associated with body size. As breed body size decreases, the risk of most problem behaviors—including reactive traits such as fear, anxiety, and aggression—increases [21,22]. This pattern can also be applied to lifespan since both dog size and lifespan are correlated [23]. Behavioral assessment batteries enable the broad classification of temperament profiles using latent class analysis (LCA) [22]. Rather than giving each breed an overall score for each temperament factor, this approach stratifies behavior into calm vs. fearful or aggressive classes and measures the percentage of each breed in each of these classes. Dog owners typically prefer dogs that are less prone to problematic behaviors, and this can be determined using C-BARQ, which evaluates problematic behavior, and LCA, which can stratify dogs into temperament profiles.
+
+The combination of such canine cancer and temperament resources allows us to test whether problematic behavior that causes psychological stress in dogs is associated with cancer incidence. The relatively simple genetics of dogs presents the opportunity to quantify psychological stress across many breeds in a normalized way. Using this heritable predisposition for psychological stress obviates having to induce or measure environmental stress stimuli, as is typically the case in such studies on rodents and humans. While cancer effects could be confounded by breed-specific cancer risks, we mitigate that risk by using latent class analysis-derived temperament phenotypes, using an interbreed study design, and controlling for body size. Our findings have major implications for companion animal welfare and provide a powerful new insight into the links between stress and cancer risk in mammals in general.
+
+## 2. Materials and Methods
+
+### 2.1. Study Design
+
+This is an exploratory study of breed stereotype association that combines multiple types of data related to stressful behavior, i.e., temperament profiles [22] derived from C-BARQ breed summaries [18], breed-specific genome-wide genotype data and derived genetic associations with behavioral traits relevant to stressful behavior (anxiety, fear, aggression, etc.) [17,19,20], and lifespan and body mass covariates. These data were modeled to detect an association between psychological stress and cancer incidence. The overall approach was to exploit breed stereotypes of behavior and cancer incidence to test for their association. A major advantage of studying domesticated animals with many breeds is the ability to measure association at loci which are fixed in some or most breeds. We mitigated possible sources of type 1 error caused by biased and internal population structures in the sample by using many breeds and multiple cohorts of genotype and phenotype data, as well as multiple types of cancer, and by running models with and without the inclusion of osteosarcoma data and breeds that are singletons across cancer types (e.g., see the effects of lymphoma risk in Fox Hounds and Irish Terriers in the Results section). Breed stereotype studies have provided a powerful discovery tool that has been used for many purposes in the past, such as morphological [24,25,26] and behavioral [16,17,20,25,26] genome-wide association studies.
+
+### 2.2. Sources of Dog Breed-Specific Data for Cancer, Behavior, Lifespan, and Body Mass
+
+A comprehensive data set was generated by combining multiple data from different sources, and these data included cancer incidence, behavior and associated genetic markers (temperament profiles from C-BARQ owner questionnaires, allele frequencies of genetic markers associated with behavioral traits), lifespan, and dog body mass. Data from a total of seven studies that included multi-breed cancer incidence data were included in this study. These studies presented data per breed for mast cell carcinoma [27], lymphoma [28,29], osteosarcoma [30,31,32], and melanoma [33]. A breakdown of the breeds represented in these studies is presented in Supplementary Table S1. The behavioral data, temperament group classification, and group distributions for each breed were sourced from LCA profile data generated from the C-BARQ questionnaire data of 57,454 dogs from 350 purebred and mixed-breed dogs [22]. The allele frequency data for the genetic markers associated with problematic behavior were previously reported [20] and were derived from a multi-breed dataset generated by Parker et al. [34]. The rationale for selecting this set of genetic markers for follow-up of our interbreed genetic mapping was that most were quasi-replicated across cohorts, while the others implicated genes prioritized for biological relevance (*ESR1*, *SHISA6*, *PRKG2*, *RASGEF1B*, and *IGF2BP2*) [17,19,20]. Breed lifespan and body mass data were obtained from the American Kennel Club (www.akc.org, accessed on 5 February 2023) and the Kennel Club (www.thekennelclub.org.uk accessed on 5 February 2023).
+
+### 2.3. Statistical Analysis
+
+Associations between cancer incidence and behavioral data were evaluated using generalized linear mixed models. All models were simultaneously assessed with lifespan and body mass as covariates. Since the sources of incidence data varied in sample size, the total sample size of each cancer incidence was introduced as a random effect adjustment in the model. Due to the nature of the data, residual distributions were assumed to be log normal. This assumption was based on residual panel assessments (residual plots, QQ plots, and residual histograms) of the models.
+
+The analysis was run in two main modes: all breed data included, and singleton breeds removed. Singleton breeds are defined as those that are only represented in the study by a single assessment value in the comprehensive dataset. Removing these breeds reduces the risk of single breed observation bias. In addition, a set of models was run in the same modes, but excluding osteosarcoma. This was carried out because dog osteosarcoma is known to be closely associated with dog body size [31] (see Suppl. Text, [35]). All models were run using PROC GLIMMIX in SAS/STAT v.9.4 (SAS Institute Inc., Cary, NC, USA).
+
+## 3. Results
+
+We established previously that LCA categories distinguish between dogs that are more or less desirable to humans [22]. Dogs of higher desirability have temperament profiles that compile lower CBARQ scores. We thus use the LCA desirability classes to refer to the proportions of desired scores in behavioral profiles. In other words, behavioral desirability or calmness represents the absence, or the lowest level, of heritable predisposition to psychological stress, whereas the reactive classes are predisposed to moderate or high levels of psychological stress and are less desirable to dog owners. The evaluation of the models that included the LCA-generated temperament profiles and osteosarcoma data showed that cancer type and body size were consistently associated (Table 1, “Including Osteosarcoma”). In this first set of models, the effect of the cancer type dropped out once singleton breeds were removed. We speculate that this was due to rare breeds being included in such studies due to increased cancer incidence. The inclusion in lymphoma studies of Fox Hounds, which have an odds ratio of 34.37, and Irish Terriers, which have an odds ratio of 22.91 [29], are examples of this, and both are singleton breeds that were removed. After the removal of singleton breeds, only the effect of body size remained consistently associated.
+
+When the temperament models were evaluated in the same modes (all breeds included and singleton breeds removed), but without the osteosarcoma data, only a few associations with temperament were detected (Table 1, “Not Including Osteosarcoma”). These were only found in comparisons in which the best, or lower stress, group was compared with all the others. The effect of body size was not relevant, except when evaluating osteosarcoma incidence. In all the significant cases, the risk of lymphoma was significantly higher in breeds within the best desirability group, i.e., those with the least stressed temperaments (*p* < 0.0001). This effect dropped once the single breeds were removed. An association between lifespan and cancer incidence was never detected when temperament was assessed.
+
+We next evaluated the genetic markers associated with stressful behavior, as describes in the Materials and Methods section, and their association with cancer incidence. We observed in the first set of models, including osteosarcoma (Table 2), that three loci were positive (and that the most consistent effect was association with body size). Two loci on chromosome 10 (positions 2,431,382 for the first locus; 8,059,173, 8,397,696, and 8,454,499 for the second locus) and one marker on chromosome 32 (5,421,210) were associated with increased cancer risk. In contrast to the temperament profiles, the removal of singleton breeds did not change the overall pattern of association effects. The removal of singletons resulted in the loss of the chromosome 32 association but added a single association to the cancer type effect for the chr10:2,431,382 marker. In each model, as body size increased, the effect was one of increased incidence risk.
+
+Next, we evaluated the same genetic markers associated with behavior, but we excluded the osteosarcoma data (Table 3). This resulted in a different pattern in which the body size effect was lost, but this replaced by a cancer type effect. In these models, variants on chromosomes 10 and 32 were again associated, as were new ones on chromosomes 1 and X. After the removal of singleton breeds, only the associations with variants in chromosomes 32 and X dropped out. Again, the risk of lymphoma was significantly higher (*p* < 0.0001). Lifespan was never found to be associated in any instance in which genetic markers were associated with stressful behavior and cancer incidence.
+
+## 4. Discussion
+
+Our results suggest that temperaments which increases psychological stress—including reactive states of fear, anxiety, and aggression—are positively associated with cancer incidence in dogs. Simply put, this is to say that a temperament that causes psychological stress is linked with a higher incidence of cancer in dogs. We first showed this through associations with behavioral profiles derived from a latent class analysis of high-powered data that included all common problem behaviors from 350 purebreds and mixed-breed dogs [22]. Among the positive findings, osteosarcoma and lymphoma had the strongest signal. For osteosarcoma, this is at least in part due to its association with larger body size, but more studies are necessary to understand cancer type effects. Given the great diversity of biological mechanisms involved in different cancer types, it would not be surprising if psychological stress greatly contributed to a subset of pathways (e.g., glucocorticoid and adrenergic signaling, immunological, and inflammatory) and thus cancer types [4,11]. Body size had a consistent effect associated with cancer incidence, but we were able to isolate or remove, as we explain below.
+
+Dog breeds can have an increased or decreased germ line risk for diverse cancers and numbers of cancer types [36]. This has been established through epidemiological, genetic, and translational approaches that have identified oncogenic mechanisms and potential therapies, which in turn have led to veterinary clinical trials [35,37,38]. In a study relevant to the present approach and involving a cancer type included in this work, dogs were instrumental in identifying a cancer risk factor that was long suspected but difficult to determine in humans. Unlike in dogs, osteosarcoma is rare in humans. Due to its low incidence, its mostly sporadic nature, and socioeconomic and genetic confounding, it would be complicated to conclusively establish whether human osteosarcoma risk is associated with height [39]. However, in dogs it was possible to measure the effect of body mass on osteosarcoma risk [31] (see Suppl. Text, [35]). In this study, we mitigated breed effects due to body size, lifespan, and cancer predispositions by using many breeds and multiple cohorts of genotype and phenotype data, by controlling for body mass and lifespan in association models, and by separately running models with and without the inclusion of osteosarcoma data and singleton breeds across cancer types.
+
+We next showed the effects of genetic markers associated with problem behaviors presumed to increase psychological stress [17,19,20]. Specifically, we found that genetic markers on chromosomes 1, 10, 32, and X were associated with increased cancer risk. Of these, the chromosome 10 and X markers were not only associated with canine behavior but also body size. Previous studies of individual genetic markers associated with canine body size have not detected associations with cancer risk, but it has been proposed that this is because these markers are fixed (homozygous) in many or most breeds [40]. A main advantage of our interbreed design is that it can detect associations of markers which are fixed in some or most breeds. These marker associations are important starting points for future studies. One possible use would be to identify breeds segregating both alleles to test for associations with cancer risk. Another possibility would be to conduct a focus study of these markers for canine cancers, such as osteosarcoma, with polygenic risk data [35], and this could feasibly include gene–gene and gene–environment study designs. Unlike the generally minute effect sizes of common individual variations associated with human psychopathology, these dog variations have moderate to large effect sizes and, thus, clinical utility [20]. Following further studies, it seems likely that genetic testing for these markers will be useful for dog selection and breeding, preventative veterinary medicine, and the development of therapies which would be validated in clinical trials in pet dogs.
+
+The findings of this study have important implications for understanding the genetic and environmental factors that contribute to cancer incidence in dogs. It has long been assumed that stress physiology is likely to increase the risk of cancer and its development [4,11]. We favor that possibility, but we cannot rule out others, and the effects could be complex, multifactorial, and variable across cancer types. By identifying temperament profiles and genetic markers associated with increased cancer risk, this study provides valuable avenues through which the mechanisms involved can be studied. This will, in turn, produce insights into potential strategies for cancer prevention and early detection in dogs, and subsequently in other mammals. There are many ways to take advantage of the unique evolutionary history of dogs. Arguably, the most efficient approaches include studying specific large effect variations in individual breeds segregating both alleles. In this way, the overall genetic variation may be reduced one hundred-fold compared with the overall dog population. However, the functional locus can be tested for cancer risk in the homozygotes of both alleles and in heterozygotes (which show low, high, and intermediate effects, respectively). Once such genetic factors are established, they can, in turn, be used for measuring environmental effects and testing preventative interventions. Alternatively, higher-powered studies of mixed-breed dogs could be carried out using an admixture approach. Dogs are common (e.g., they number approximately 80 million in the USA, roughly half-and-half pedigree and mixed-breed), share the environments of their owners, frequently receive a high level of health care, and are generally considered family members. This presents a powerful opportunity to leverage observational and translational studies in pet dogs. Because psychological stress and cancer are widely understood to result in devastating morbidity and mortality in dogs and humans, dog owner interest in study participation is likely to be high.
+
+It is important to note the limitations of this study. Due to the scarcity of canine epidemiological data, the study only examined a limited number of cancer types and did not evaluate other potential risk factors, such as environmental exposures. The future development of canine epidemiological data and cancer tissue repositories would have a great impact on this field of study [40,41]. In our study, we evaluated temperament, cancer type, dog size, and lifespan simultaneously in our models; all these factors are relevant to cancer risk, but they are also challenging because of confounding factors. Specific breeds are more likely to develop specific cancers, and they have defined weigh and lifespan ranges. To reduce this concern, we included as many breeds and cancers as the available data would permit. It is important to mention that even when dog size and lifespan are correlated, these effects did not display the same association in the models, suggesting that although there is some overlap, they have a component that is still distinct. Other confounders that may have a relevant association to cancer risk [42] cannot be evaluated from aggregate data. Additionally, rather than using phenotype and genotype data from the same dogs, the study relied on breed averages of owner-reported temperament data and the latent class analysis classes that were derived from these. It is unclear how robustly these measures reflect relative levels of psychological stress. However, it should be noted that we successfully performed diagnostic predictive modeling in a cohort of 397 dogs that included 122 dogs with a clinical behavioral diagnosis. Of the markers highlighted above, we correctly predicted diagnoses of anxiety for chr1, aggression for the second locus on chr10, and “any diagnosis” for the same chr10 locus and for chrX. It is also worth noting that the genetic mapping signal was compared using both the body size measurements of individual dogs and breed averages, and it was found that breed stereotypes had far greater power with the same number of dogs (in most cases, several to many orders of magnitude [15]).
+
+## 5. Conclusions
+
+The results of this study suggest that temperament associated with psychological stress is a factor in cancer incidence in dogs. This finding is important because it is difficult to conduct similar investigations in humans due to confounding socioeconomic and genetic factors, and it is difficult in rodents due to the requirement for inducible models of chronic stress. Further research is needed to fully understand the complex interplay between temperament and other factors, and to develop effective strategies for cancer prevention and treatment in dogs.

fulltext/md/PMC10280140.md

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+---
+pmcid: "PMC10280140"
+pmid: "36869852"
+doi: "10.1111/cpr.13435"
+title: "TGF-β-induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease."
+journal: "Cell proliferation"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# TGF‐β‐induced PI3K/AKT/mTOR pathway controls myofibroblast differentiation and secretory phenotype of valvular interstitial cells through the modulation of cellular senescence in a naturally occurring in vitro canine model of myxomatous mitral valve disease
+
+## Abstract
+
+PI3K/AKT/mTOR signalling contributes to several cardiovascular disorders. The aim of this study was to examine the PI3K/AKT/mTOR pathway in myxomatous mitral valve disease (MMVD). Double‐immunofluorescence examined expression of PI3K and TGF‐β1 in canine valves. Valve interstitial cells (VICs) from healthy or MMVD dogs were isolated and characterized. Healthy quiescent VICs (qVICs) were treated with TGF‐β1 and SC‐79 to induce activated myofibroblast phenotypes (aVICs). Diseased valve‐derived aVICs were treated with PI3K antagonists and expression of RPS6KB1 (encoding p70 S6K) was modulated using siRNA and gene overexpression. SA‐β‐gal and TUNEL staining were used to identify cell senescence and apoptosis, and qPCR and ELISA to examine for senescence‐associated secretory phenotype. Protein immunoblotting was used to examine expression of phosphorylated and total proteins. TGF‐β1 and PI3K are highly expressed in mitral valve tissues. Activation of PI3K/AKT/mTOR and increased expression of TGF‐β are found in aVICs. TGF‐β transitions qVICs to aVICs by upregulation of PI3K/AKT/mTOR. Antagonism of PI3K/AKT/mTOR reverses aVIC myofibroblast transition by inhibiting senescence and promoting autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with reduced capacity for apoptosis and autophagy. Selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. TGF‐β‐induced PI3K/AKT/mTOR signalling contributes to MMVD pathogenesis and plays crucial roles in the regulation of myofibroblast differentiation, apoptosis, autophagy and senescence in MMVD.
+
+1
+
+## INTRODUCTION
+
+Myxomatous mitral valve disease (MMVD) is one of the most devastating heart valve diseases in humans (syndromic and non‐syndromic forms) and dogs and a major cause of heart failure and sudden cardiac death, leading to significant morbidity and mortality in both species.^1^, ^2^, ^3^ It accounts for 7% of deaths in dogs before 10 years of age and its prevalence is estimated to be between 30% and 70% of all elderly dogs.^4^, ^5^ MMVD affects 2%–3% of the human global population with approximately 15% of those affected requiring surgical valve replacements.^6^ These treatments are invasive, costly, carry a risk for elderly adults and may lead to more severe complications including thrombosis, post‐operative infections and heart attack.^7^ Currently, there are no medications to prevent, slow progression or reverse valve pathology associated with MMVD. An improved understanding of the pathogenesis of MMVD is necessary for the development of novel therapeutic strategies for MMVD both in humans and dogs.
+
+Accumulating evidence indicates that MMVD is a progressive and degenerative disease regulated by growth factors, in particular, members of the transforming growth factor β (TGF‐β) superfamily.^1^, ^8^ TGF‐β has been shown to have an important role in myxomatous degeneration in human MMVD and the associated end‐stage valve fibrosis.^9^, ^10^, ^11^, ^12^, ^13^ Aberrant upregulation of TGF‐β signalling has been reported in the various forms of mitral valve prolapse where myxomatous degeneration is found, including an X‐linked filamin‐A (FLNA) mutation, Marfan syndrome (MFS) and Barlow's Disease (BD).^1^ Similar observations are found in spontaneously occurring canine MMVD, although dogs lack end‐stage fibrosis, with transcriptomic data supporting the pivotal role of TGF‐β.^14^, ^15^ In human and canine myxomatous mitral valves, there is an increased number of valve interstitial cells (VICs) expressing α‐smooth muscle actin (α‐SMA), indicating an activated myofibroblast phenotype (aVICs).^16^, ^17^ TGF‐β has been shown to induce differentiation of cultured human and canine VICs to this myofibroblast phenotype, with associated excess extracellular matrix (ECM).^10^, ^18^, ^19^ Pharmacological antagonism of the TGF‐β receptor complex reverses aVICs back to a normal quiescent phenotype.^18^ Taken together, these studies indicate that the TGF‐β‐induced myofibroblast differentiation of VICs plays an important role in the pathogenesis of MMVD.
+
+The role of canonical TGF‐β mediated Smad2/3 signalling in controlling VIC phenotype and ECM synthesis has been identified in human valve tissue, cultured primary VICs and transgenic mouse models.^9^, ^10^, ^11^, ^16^ However, a large‐scale clinical trial in children and young adults with MFS showed no effect by abolishing the Smad2/3 cascade using the angiotensin II receptor blocker losartan.^20^ This suggests that further investigation of the non‐canonical parts of the TGF‐β signalling pathway would be beneficial. Of particular interest is the phosphoinositide 3‐kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway which is recognized to regulate multiple cellular processes including cell differentiation, survival and death.^21^ Dysregulation of PI3K/AKT/mTOR signalling is associated with a variety of degenerative disorders. In pulmonary fibrosis, TGF‐β‐induced differentiation of human lung fibroblasts to fibrogenic myofibroblasts is repressed by inhibiting the PI3K/AKT/mTOR pathway.^22^ The antagonism of PI3K/AKT/mTOR signalling has also been reported to promote autophagy of articular chondrocytes and attenuate the inflammatory response in rats with osteoarthritis.^23^ Recently, it has been shown that pharmacological inhibition and knockdown of mTOR/p70 S6 kinase (p70 S6K) signalling protects against intervertebral disc cell senescence and ECM catabolism in human intervertebral disc disease.^24^, ^25^ PI3K signalling has been widely reported to play a crucial role in the pathogenesis of atherosclerosis, thrombosis and myocardial infarction, but not in valvulopathies.^26^ Considering the importance of this pathway in a range of degenerative diseases, the data from our own preliminary studies and the lack of data on PI3K/AKT/mTOR signalling pathway in the development of MMVD, we believe investigating this pathway would be beneficial.
+
+Examining the pathogenesis of MMVD in dogs will have relevance to understanding the same disease in humans. Studying the pathogenesis of human MMVD has relied on the use of transgenic mouse models and examining surgically‐resected mitral valve samples from patients with end‐stage disease. Although genetically modified mice give useful insights into many molecular signalling events, they are limited in modelling the triple layer structure of human valves and the chronicity of this disease and are not able to generate mitral‐valve specific myxomatous pathology.^27^, ^28^, ^29^, ^30^ Furthermore, human patient‐derived tissues (end‐stage disease) typically have extensive secondary fibrosis that hampers the examination of molecular events controlling the much earlier development and progression of the pre‐fibrosis myxomatous changes.^11^, ^12^, ^31^ The dog with the same triple‐layer valvular structure, but lacking the end‐stage fibrosis, has shared pathological and molecular characteristics of human MMVD and can be examined as the disease appears and progresses. This naturally occurring analogous disease in dogs is now well recognized as a credible large animal model to investigate human MMVD.^1^, ^3^, ^14^ By examining cell and molecular events in the dog, we can gain insights into MMVD in both species.
+
+To that end, in the present study, we have performed in vitro mechanistic studies on cultured VICs isolated from healthy dogs and dogs with spontaneously developed mid‐stage MMVD to examine the role of PI3K/AKT/mTOR signalling in MMVD.
+
+2
+
+## MATERIALS AND METHODS
+
+2.1
+
+### ETHICS STATEMENT
+
+All tissue collection procedures were performed under the approval and guidance of the Veterinary Ethics Research Committee (Institutional Care and Use Committee; project number 96/21) at The Royal (Dick) School of Veterinary Studies, University of Edinburgh. Written informed consent was obtained from each dog owner and no dogs were euthanized for the purpose of this study.
+
+2.2
+
+### Clinical samples
+
+Six mitral valves from diseased dogs of various breeds with MMVD and six mitral valves from healthy young adult dogs of various breeds were collected at the Hospital for Small Animals, The Royal (Dick) School of Veterinary Studies, University of Edinburgh. Collected resected valves were graded according to their gross pathological appearance normal (grade 0) or diseased (grades 1–4) using the Whitney classification, and graded independently by two observers.^32^ For this study, all six affected dogs were Whitney grade 2 (moderate disease).
+
+2.3
+
+### Cell isolation, culture and phenotyping
+
+Diseased canine VICs were isolated from the whole valves from dogs with grade 2 MMVD (moderately affected), and healthy VICs were isolated similarly from healthy dogs' valves. Briefly, canine mitral valve leaflets were rapidly removed, dissected, phenotyped and prepared for cell culture as previously described.^18^ Dissected valves were then incubated with 1 mg/mL trypsin (Gibco) for 10 min and washed in HBSS buffer (Gibco) to remove valve endothelial cells.^33^ The valve tissues were then digested in 250 U/mL type II collagenase solution (Worthington) at 37°C for 18 h. The cells subsequently obtained were resuspended in a low‐serum DMEM medium (Gibco) supplemented with 2% fetal bovine serum (FBS), 100 U/mL of penicillin and 100 mg/mL streptomycin (Gibco).^34^ Cells were cultivated using standard tissue culture techniques and used between 3 and 5 passages to ensure in vitro cultures maintain in vivo phenotype.^10^, ^11^ Cell phenotypes were determined by protein‐immunoblotting (Western blotting; WB) and quantitative PCR for the myofibroblast markers α‐SMA (*ACTA2*),^10^, ^11^ SM‐22 (*TAGLN*)^35^ and Smemb (*MYH10*).^18^, ^36^ All disease cell samples were positive for these markers and all normal cell samples were negative, confirming the accurate phenotype of the two groups.
+
+2.4
+
+### Cell viability assay
+
+Cell viability was measured with a commercial alamarBlue assay (Invitrogen). Briefly, cells were plated in a 96‐well plate for 24 h and then treated with a test compound before proceeding with the assay. The alamarBlue reagent was added directly to each well and incubated at 37°C for 3 h to allow cells to convert resazurin to resorufin. The absorbance at 570 nm for each well was measured using a microplate reader. The average 600 nm absorbance values of the background control were subtracted from the 570 nm absorbance values of experimental wells. The results were evaluated as background subtracted 570 nm absorbance versus concentration of the compounds.
+
+2.5
+
+### siRNA transfection
+
+aVICs were seeded at the density of 1.0 × 10^6^ cells/well in six‐well plates and transfected with 1.0 μM mouse p70 S6K siRNA (Santa Cruz Biotechnology), human p70 S6K siRNA (Santa Cruz Biotechnology), or scrambled control siRNA (Santa Cruz Biotechnology) using Lipofectamine 3000 (Invitrogen) in Opti‐MEM (Gibco) medium according to the manufacturer's instructions. aVICs transfected with Lipofectamine 3000 without p70 S6K siRNA were used as a mock control. aVICs with the expected density were treated with 10 ng/mL TGF‐β1 for 3 days to ensure the completion of senescent myofibroblast transition. These cells were then transfected with siRNAs. The siRNA sequences for gene silencing are listed in Table S1. RPS6KB1 genes responsible for the translation of p70 S6K protein is highly evolutionarily conserved in mouse, human and canine and therefore these siRNAs were used in this study.
+
+2.6
+
+### Gene overexpression
+
+Extraction of p70 S6K cDNA plasmids was performed with a Plasmid Plus Kit (QIAGEN) according to the manufacturer's instructions. qVICs were seeded into six‐well plates with a density of 1 × 10^6^ per well and cultured overnight. About 10 μg p70 S6K cDNA ORF plasmid (Genescript) was incubated with 50 μg Lipofectamine 3000 and 500 μL Opti‐MEM medium and then DNA‐Lipofectamine 3000 complexes were transferred to each well. Cells transfected with pcDNA3.1‐C‐(k) DYK vectors (Genescript) without p70 S6K cDNA were used as a negative control and cells transfected with Lipofectamine 3000 served as mock controls. The p70 S6K cDNA ORF clone sequences for gene overexpression are summarized in Table S2. Human and mouse p70 S6K cDNA ORF were used in this study because RPS6KB1 genes in these species share high homology.
+
+2.7
+
+### Histology and immunohistochemistry
+
+Canine mitral valve tissues were fixed with 10% (v/v) neutral buffer formalin (NBF) for 24 h, dehydrated and embedded in paraffin wax before sectioning at 3–5 μm using standard procedures. For evaluation of valve pathology, sections were dewaxed in xylene and ethanol and then stained with haematoxylin and eosin (H&E; Sangon Biotech). Light microscopy images were obtained by a scanning light microscope (Leica CS2) and histological analysis was performed. For immunohistochemistry, sections were subjected to sodium citrate buffer (pH 6.0) for antigen retrieval for 5 min at 95°C. Endogenous peroxidase activity was blocked using 1% hydrogen peroxide for 30 min at room temperature (RT). The blocking for non‐specific antibodies was performed with 10% normal goat serum (NGS) for 1 h at RT before overnight incubation at 4°C with rabbit anti‐PI3K 110α antibody (1:100, A94027, Antibodies), rabbit anti‐TGF‐β1 antibody (1:50, 21898‐1‐AP, Proteintech) and mouse anti‐α‐SMA antibody (1:300, #48938, Cell Signalling Technology) or mouse anti‐p21^CIP1^ antibody (1:100, 67362‐1‐Ig, Proteintech), mouse anti‐ATG7 antibody (1:100, 67341‐1‐Ig, Proteintech) and rabbit anti‐α‐SMA antibody (1:300, #19245, Cell Signalling Technology). PI3K 110α was selected as it is widely distributed in multiple tissues and p110 is the key catalytic subunit of the PI3K enzyme to trigger the downstream ATK/mTOR pathway.^37^ After washing in PBS slides were treated with Alexa Fluor 488 anti‐rabbit (1:500, Life Technologies), Alexa Fluor 645 anti‐mouse antibody (1:500, Life Technologies) or polymerized horseradish peroxidase (HRP) conjugated goat anti‐rabbit/mice antibody (1:1000, P0047/P0048, Dako) for 1 h at RT. Slides were then washed with PBS and finally stained with DAPI (1:5000, D9542, Sigma‐Aldrich) or 3,3′‐diaminobenzidine (DAB, SK‐4100, Vector Labs) followed by haematoxylin counterstain (H‐3401‐500, Vector Labs). Glass coverslips were mounted onto slides with Prolong Gold Anti‐Fade Reagent (Life Technologies). Control sections were incubated with equal concentrations of normal rabbit (ab172730, Abcam) and mouse IgG (ab37355, Abcam) in place of the primary antibody. The images were detected under an inverted confocal microscope (Zeiss LSM 710). Mean fluorescence intensity (MFI) and co‐localization analysis of TGF‐β1, PI3K 110α and α‐SMA were processed using ImageJ analysis software (National Institutes of Health).
+
+2.8
+
+### Immunofluorescence staining
+
+Cells seeded on glass coverslips were fixed with 10% (v/v) NBF at 4°C for 10 min, permeabilized in 0.1% (v/v) triton X‐100 (Sigma‐Aldrich) for 15 min and washed with PBS, followed by blocking in 5% NGS for 1 h at RT. Glass coverslips were washed in PBS and then incubated with rabbit anti‐LC3 antibody (1:300, PM036, MBL) at 4°C overnight. After washing, the cells were incubated with Alexa Fluor 488 anti‐rabbit antibody (1:500, A11034, Life Technologies) in NGS at 37°C for 1 h in the dark. Coverslips were then stained with Hoechst (1:10,000, 62249, Sigma‐Aldrich) and fluorescence signal was detected under an inverted confocal microscope (Zeiss LSM 710). Negative controls were carried out simultaneously by incubating with equivalent concentrations of normal rabbit IgG (ab172730, Abcam) in place of primary antibody.
+
+2.9
+
+### Western blotting
+
+VICs were collected with radioimmunoprecipitation assay (RIPA) lysis buffer (Thermo Fisher Scientific) supplemented with Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific) and total protein concentration was determined (Thermo Fisher Scientific). Immunoblotting was performed as previously described.^38^ Equal amounts of protein lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). After blocking with 5% (v/v) skimmed milk in Phosphate Buffered Saline Tween‐20 (PBST), membranes were incubated overnight at 4 °C with primary antibodies (Table S3) diluted in 5% skimmed milk. Subsequently, membranes were incubated with HRP‐conjugated anti‐mouse (1:1000, P0047, Dako) or anti‐rabbit (1:1000, P0048, Dako) secondary antibodies at RT for 1 h. Membranes were developed using the GeneGenome system (Syngene). Semi‐quantitative assessment of band intensity was performed using ImageJ analysis software (National Institutes of Health).
+
+2.10
+
+### Terminal dUTP nick‐end labelling (TUNEL) staining
+
+Apoptotic activities of canine VICs were identified using a fluorescein‐labelled TUNEL assay kit (ab252888, Abcam) following the manufacturer's instructions. In brief, coverslip‐seeded VICs were cultured with 60 μM LY294002 (Cayman Chemical), 5 μM copanlisib (Cayman Chemical), 50 μM alpelisib (Cayman Chemical) and DMSO (Sigma‐Aldrich) vehicle control for 3 days. Cells were then fixed and permeabilized followed by incubating with the TUNEL reaction cocktail overnight at RT. After washing in PBS, coverslips were treated with the click reaction cocktail and incubated for 30 min at RT in the dark. Cells were finally analysed for red fluorescence generated by TUNEL‐positive cells and green fluorescence by total DNA using an inverted confocal microscope (Zeiss LSM 710).
+
+2.11
+
+### Flow cytometry
+
+VICs treated with PI3K inhibitors and DMSO vehicle was harvested by trypsinization, washed with PBS and detached using trypsin. Cells are then resuspended with medium and counted using trypan blue to ensure dead cells are excluded. Cells were then stained with TUNEL reaction cocktail and the click reaction cocktail using TUNEL assay kit (ab252888, Abcam). Cell suspensions were finally transferred into flow cytometry vessels and 10,000 cell events were recorded in FL‐2 channel using a BD FACS Calibur Flow Cytometer (Becton, Dickinson & Company) for signals generated by TUNEL positive cells during click reaction.
+
+2.12
+
+### BrdU cell proliferation assay
+
+Cell proliferation was assessed using the BrdU Cell Proliferation ELISA Kit (Ab126556, Abcam) following the manufacturer's instructions. Briefly, 6 × 10^3^ cells were plated in a 96‐well plate and BrdU was added to the cells for 3 h. Subsequently, cells were subjected to fixation, permeabilization and DNA denaturation. Cells were then incubated with anti‐BrdU antibody for 1 h, washed and incubated with peroxidase‐conjugated secondary antibody. Finally, the coloured reaction that indicates cell proliferation was quantified at a wavelength of 450 nm.
+
+2.13
+
+### Cell cycle analysis
+
+For cell cycle determination, 4 × 10^5^ cells were rinsed with PBS and harvested by trypsinization. Pelleted cells were resuspended in 1 mL Hoechst‐solution containing 2 μg/mL Hoechst 33342 (H3570, Invitrogen) and incubated for 30 min. Subsequently, 1 μL 1 mg/mL 7‐aminoactinomycin D (7‐AAD) was added to the tube and incubated for 5 min to exclude apoptotic and dead cells.^39^ Fluorescent intact single nuclei were analysed for DNA content using cell analyser BD FACS Calibur Flow Cytometer (Becton, Dickinson & Company). Cell cycle was assessed through analysis of the proportion of cells in the G1, S and G2/M fraction of the cell cycle using FlowJo v10.8.
+
+2.14
+
+### Senescence‐associated β‐galactosidase staining
+
+Senescence‐associated β‐galactosidase (SA‐β‐gal) staining was performed according to the manufacturers' instructions (Merk Millipore). Briefly, canine VICs seeded in 12‐well plates were fixed in 0.25% glutaraldehyde and SA‐β‐gal staining was performed at pH 6.0. The percentage of SA‐β‐gal‐positive cells was quantified relative to the number of total cells, which were both counted in six random low‐power fields (×100) using the image analysis software ImageJ.
+
+2.15
+
+### Quantitative real‐time PCR
+
+Total RNAs were extracted from canine VICs using an RNeasy Kit (Qiagen) according to the manufacturer's instructions. RNA was quantified and reverse transcripted and the target gene expressions were evaluated by quantitative RT‐PCR in a 700 Fast Real‐Time PCR Systems (ViiA7 Real‐time PCR, ABI) using the SYBR™ Green PCR Master Mix (Thermo Fisher Scientific). Each PCR was run in triplicate. The relative expression levels of mRNAs were determined by a comparative 2^−Ct (ΔΔCt)^ method and normalized against glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). The control values were expressed as 1 to indicate a precise fold change value for each gene of interest. The primers used in this study were synthesized by Sigma‐Aldrich and the sequences for target genes are shown in Table S4.
+
+2.16
+
+### Enzyme‐linked immunosorbent assay
+
+The supernatants were collected from canine VIC cultures and centrifuged at 16,000 rpm at 4°C for 20 min to remove debris. The purified supernatants were diluted accordingly and examined by enzyme‐linked immunosorbent assays (ELISAs) using human TGF‐β1, interleukin‐6 (IL‐6) and matrix metalloproteinase‐9 (MMP‐9) ELISA kits (Invitrogen) according to the manufacturer's instructions. The genes responsible for the protein translation of TGF‐β1, IL‐6 and MMP‐9 are conserved in human and canine species.
+
+2.17
+
+### Statistical analysis
+
+All experiments were performed in three technical replicates with six biological replicates and the representative results are shown. All data are presented as mean ± SEM. Statistical analyses were analysed by one‐way analysis of variance (ANOVA) followed by Tukey's range test using GraphPad Prism software. *p* < 0.05 was considered to be significant, and *p* values are represented as: **p* < 0.05; ***p* < 0.01; ****p* < 0.001.
+
+3
+
+## RESULTS
+
+3.1
+
+### Expression of TGF‐β1 and PI3K 110α are significantly increased in α‐SMA positive aVICs in canine myxomatous mitral valves
+
+To investigate the role of TGF‐β and PI3K in MMVD expression of TGF‐β1 and PI3K 110α was assessed in six canine healthy and six myxomatous mitral valves using IHC. Healthy valves were characterized by the normal triple‐layer valvular structure with no evidence of myxomatous lesions. Valves from dogs diagnosed with grade 2 MMVD had moderate myxomatous degeneration and no fibrotic changes (Figure 1A). Immunohistochemical assessment revealed TGF‐β1 and PI3K 110α expression within areas of ECM disorganization in myxomatous mitral tissues (Figure 1B). Double‐staining confocal immunofluorescence showed that TGF‐β1 and PI3K 110α expression were significantly increased in myxomatous valves, with a high density of α‐SMA+ cells, compared to healthy valves (Figure 1C,D).
+
+*Figure: FIGURE 1 Histopathological assessment of canine healthy and myxomatous mitral valves. (A) Representative images (two biological replicates) of H&E staining in canine healthy and myxomatous mitral valves, scale bar 200 μm (magnified 100 μm). (B) Representative immunohistochemistry images of TGF‐β1 and PI3K 110α expressions (yellow/brown) in canine healthy and myxomatous mitral valves, scale bar 100 μm. (C) Representative confocal immunofluorescent images of TGF‐β1, PI3K 110α and α‐SMA expressions in canine healthy and myxomatous mitral valves, scale bar 20 μm. (D) Quantitative analysis of mean fluorescence intensity (MFI) of TGF‐β1, PI3K 110α and α‐SMA in canine healthy and myxomatous mitral valves. (E) Representative images of co‐localization ratio analysis of TGF‐β1 (green) and α‐SMA (red), PI3K 110α (green) and α‐SMA (red) fluorescence signals in canine mitral valve tissues. (F) Quantitative analysis of co‐localization parameters (Pearson's correlation and overlap coefficient) of TGF‐β1 (green) and α‐SMA (red), PI3K 110α (green) and α‐SMA (red) fluorescence signals. Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.01 compared to control. ANOVA, analysis of variance; H&E, haematoxylin and eosin.*
+
+To further confirm TGF‐β1 and PI3K 110α are expressed in α‐SMA positive aVICs, co‐localization analysis of PI3K 110α, TGF‐β1 and α‐SMA was performed. There was a high level of PI3K 110α and TGF‐β1 co‐localization with α‐SMA, but not between TGF‐β1 and α‐SMA in myxomatous valves (Figure 1E,F). These data indicate aVICs highly express PI3K 110α and TGF‐β1, and TGF‐β1 was secreted into the valvular matrix in myxomatous degeneration.
+
+3.2
+
+### PI3K/AKT/mTOR/p70 S6K signalling is upregulated in α‐SMA positive aVICs
+
+Initially, studies were performed to validate the canine VIC in vitro 2D low‐serum culture model as previously reported.^18^ aVICs stained positive for the myofibroblast marker α‐SMA (Figure 2A). In addition, western blotting identified significantly increased expression of myofibroblast‐related cytoskeletal proteins including α‐SMA and SM22‐α (Figure 2B,D). The synthesis of the ECM proteins versican, collagen type I and collagen type III was significantly increased in aVICs, as was the expression of TGF‐β (Figure 2B,D). All these data are consistent with previous reports and confirm the phenotype of the normal and diseased samples.^16^, ^19^, ^40^ To investigate the mechanisms controlling VIC phenotype transition, we investigated the activation of PI3K/AKT/mTOR signalling. The baseline expression of PI3K signalling was evaluated by western blotting. PI3K 110α expression was significantly increased as were the phosphorylated forms of the downstream signalling molecules Akt Ser473, mTOR Ser2448 and the downstream mTOR transcriptional factor p70 S6 kinase (S6K) Thr389, which most closely correlates with its p70 kinase activity (Figure 2C,E,F).^41^ Considering the phosphorylation of p70 S6K can be controlled by insulin‐mediated insulin receptor (IR)/PI3K signalling, we assessed the baseline expression of insulin receptor substrate 1 (IRS‐1), the main substrate of IR kinase which activates PI3K/AKT/mTOR signalling.^42^ Protein expression of total IRS‐1 and its phosphorylated form at Ser636/639 were significantly reduced in aVICs (Figure 2C,E). Furthermore, proline‐rich AKT substrate of 40 kDa (PRAS40) interacts with raptor in mTOR complex 1 (mTORC1) and inhibits the activation of the mTORC1/S6K pathway, while phosphorylation of PRAS40 at Thr246 by AKT relieves this PRAS40 inhibition of mTORC1.^43^, ^44^ Interestingly we identified increased expression of phosphorylation of PRAS40 (Thr246) in aVICs but no significant difference in total PRAS40 expression comparing aVICs and qVICs (Figure 2C,F). These data indicate activation of PI3K signalling is associated with the abnormal VIC phenotype transition and ECM protein expression.
+
+*Figure: FIGURE 2 PI3K/AKT/mTOR/p70 S6K signalling is upregulated in activated myofibroblasts (aVICs). (A) Representative confocal images of α‐SMA (red) staining in canine aVICs and qVICs (two biological replicates), scale bar 20 μm. (B, D) Representative western blot of ECM and VIC phenotype protein expression and quantification of the relative protein expression (two biological replicates shown in blots, *n* = 6). (C, E, F) Representative western blot of PI3K 110α, total AKT, phosphorylated AKT (p‐AKT), total mTOR, phosphorylated mTOR (p‐mTOR), p70 S6K, phosphorylated p70 S6K (p‐p70 S6K), PRSA40, phosphorylated PRSA40 (p‐PRSA40), IRS1 and phosphorylated IRS1 (p‐IRS1) protein expression and quantification of the relative protein expression (two biological replicates shown in blots, *n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; ECM, extracellular matrix; VIC, valve interstitial cell.*
+
+3.3
+
+### TGF‐β‐induced PI3K signalling activation regulates VIC phenotype differentiation and ECM protein synthesis
+
+To determine if TGF‐β mediated PI3K signalling controls myofibroblast activation qVICs were treated with TGF‐β1 (10 ng/mL) for 3 days and then exposed to the selective Akt activator SC‐79 (300 nM) for 2 h.^45^ SC‐79 is known to increase the cell's responsiveness to TGF‐β by inducing the transport of TGFBRs (TGF‐β receptors) to the cell surface.^46^, ^47^ The appropriate concentration of SC‐79 was determined by cell viability assays (Figure S1). α‐SMA immunostaining showed that TGF‐β1 in the presence or absence of SC‐79 treatment induced α‐SMA expression and largely increased α‐SMA positive VICs by Day 3 (Figure 3A,B). Myofibroblast‐related genes, including *ACTA2* (α‐SMA), *TAGLN* (SM22) and *MYH10* (Smemb), were dramatically upregulated after TGF‐β1 induction in qVICs cultured with low‐serum media supplemented with SC‐79 (Figure 3C). The protein expression of α‐SMA, SM22, collagen type III and TGF‐β in qVICs was significantly increased by TGF‐β1 treatment, again in both the presence or absence of SC‐79 (Figure 3D,E). In addition to the induction of canonical Smad‐mediated signalling, TGF‐β is known to initiate the non‐canonical PI3K/AKT/mTOR pathway.^48^ Western blotting revealed that TGF‐β1 treatment activated PI3K signalling by increasing PI3K 110α, phosphorylated AKT, phosphorylated mTOR, p70 S6K, phosphorylated p70 S6K expressions in qVICs (Figure 3F–H) in the presence or absence of SC‐79. These data show that TGF‐β‐induced PI3K signalling activation results in an aberrant transformation of VIC phenotype and ECM protein synthesis.
+
+*Figure: FIGURE 3 TGF‐β‐induced PI3K activation controls VIC phenotype and ECM protein production. Canine qVICs were exposed to DMSO (Control), TGF‐β1 (10 ng/mL) and SC‐79 (300 nM) treatment. (A, B) Representative confocal images of α‐SMA immunostaining and quantitative analysis of the percentage of α‐SMA positive cells treated with DMSO, TGF‐β1 (10 ng/mL) and SC‐79 (300 nM), scale bar 20 μm (*n* = 12 microscopic fields). (C) Quantitative RT‐PCR for α‐SMA, SM22 and Smemb mRNA expression in qVICs treated with DMSO, TGF‐β1 (10 ng/mL) and SC‐79 (300 nM) (*n* = 6). (D, E) Representative western blot of α‐SMA, SM22, collagen type III and TGF‐β and quantitative analysis of the relative protein expression (*n* = 6). (F, G, H) Representative western blot of PI3K p110α, AKT, phosphorylated AKT (p‐AKT), mTOR, phosphorylated mTOR (p‐mTOR), p70 S6K, phosphorylated p70 S6K (p‐p70 S6K) protein expression and quantitative analysis of the relative protein expression (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; ECM, extracellular matrix; TGF‐β, transforming growth factor β; VIC, valve interstitial cell.*
+
+3.4
+
+### aVICs exhibit a senescent‐associated secretory phenotype with a reduced capacity for autophagy
+
+Cell senescence has been shown to be modulated by PI3K signalling.^49^, ^50^ Autophagy impairment is considered as an important characteristic of cell senescence.^51^ To determine whether aVICs are in a senescent phenotype, p21^CIP1^ and ATG7 expression profiles were examined in mitral valve tissues by double‐staining confocal immunofluorescence p21^CIP1^ expression was significantly increased while conversely ATG7 expression was significantly decreased in myxomatous valves compared to healthy valves (Figure 4A,B). To further confirm whether ATG7 and p21^CIP1^ are expressed in α‐SMA positive aVICs, co‐localization analysis of ATG7, p21^CIP1^ and α‐SMA was performed. There was a high level of p21^CIP1^ co‐localization with α‐SMA, but not between ATG7 and α‐SMA in myxomatous valves (Figure 4C,D). These data indicate aVICs are in a senescent state but with a reduced autophagy flux in myxomatous valves.
+
+*Figure: FIGURE 4 Activated myofibroblasts (aVICs) exhibit a senescent‐associated secretory phenotype (SASP) with a reduced capacity for autophagy. (A) Representative confocal immunofluorescent images of ATG7, p21^CIP1^ and α‐SMA expressions in canine healthy and myxomatous mitral valves, scale bar 20 μm. (B) Quantitative analysis of mean fluorescence intensity (MFI) of ATG7, p21^CIP1^ and α‐SMA in canine healthy and myxomatous mitral valves. (C) Representative images of co‐localization ratio analysis of ATG7 (red) and α‐SMA (green), p21^CIP1^ (red) and α‐SMA (green) fluorescence signals in canine mitral valve tissues. (D) Quantitative analysis of co‐localization parameters (Pearson's correlation and overlap coefficient) of ATG7 (red) and α‐SMA (green), p21^CIP1^ (red) and α‐SMA (green) fluorescence signals. (E) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (*n* = 12 microscopic fields/treatment). (F) Cell cycle analysis (left panel) and BrdU incorporation assay (right panel) of canine aVICs and qVICs (*n* = 6). (G) Representative western blot of p16^INK4A^, p21^CIP1^, p53 and β‐actin protein expression and quantification of the relative protein expression in VICs (two biological replicates shown in blots, *n* = 6). (H) Quantitative RT‐PCR for SASP cytokine expression (left panel) and secreted TGF‐β1, IL‐6 and MMP‐9 (right panel) in collected supernatant from VIC cultures (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; VIC, valve interstitial cell.*
+
+To further confirm aVICs are senescent, SA‐β‐gal staining was performed on canine in vitro VIC cell cultures. A larger number of SA‐β‐gal positive cells were observed in aVICs compared with qVICs (Figure 4E). Cell cycle analysis showed that a higher percentage of aVICs accumulated in the G1 phase, together with a reduced number of S and G2/M phase cells compared with qVICs (Figure 4F). In addition, cell proliferation was determined by the measurement of newly synthesized DNA using the thymidine analogue BrdU. qVICs exhibited a significantly increased capacity for BrdU incorporation compared to aVICs (Figure 4F). p53/p21^CIP1^ and p16^INK4A^ tumour suppressor signalling has been reported as the key pathways involved in the activation of cellular senescence.^52^ p16^INK4A^, p53 and p21^CIP1^ protein expressions were significantly increased in aVICs (Figure 4G). Taken together, these data indicate aVICs are in a senescent state.
+
+Senescent cells can develop a senescence‐associated secretory phenotype (SASP), which allows them to secrete a complex mixture of factors causing continual ECM disorganization and alter the behaviour of nearby non‐senescent cells. The main components of SASP include multiple pro‐inflammatory cytokines, chemokines, growth modulators, ECM components, and MMPs.^52^ To examine SASP in aVICs, a series of SASP members, including IL‐6, IL‐1β, MMP‐9, TNF‐α, TGF‐β1, TGF‐β2 and TGF‐β3, were selected for quantitative RT‐PCR analysis. The upregulation of the selected SASP mRNAs was observed in aVICs (Figure 4H). Detected by ELISA TGF‐β1, IL‐6 and MMP‐9 levels in the culture supernatant from aVICs were significantly increased compared to qVICs (Figure 4H). Taken together, these data suggest that senescent aVICs exhibit a SASP.
+
+3.5
+
+### Pharmacological inhibition of PI3K signalling reverses myofibroblast activation and normalized ECM production
+
+To attenuate the aberrant activation of PI3K signalling in aVICs, we treated aVICs with 60 μM LY294002 (highly selective pan‐inhibitor of PI3K), 5 μM copanlisib (pan‐PI3K inhibitor) and 50 μM alpelisib (isoform‐selective PI3K p110α inhibitor) for 3 days.^37^ The optimal concentrations of the three PI3K inhibitors were determined by cell viability assays (Figure S1). LY294002, copanlisib and alpelisib attenuated α‐SMA expression and significantly reduced the number of α‐SMA positive VICs (Figures 4B and 5A,B). The upregulation of *ACTA2* (α‐SMA), *TAGLN* (SM22‐α) and *MYC10* (Smemb) mRNA expression in aVICs were significantly attenuated by LY294002 treatment (Figure 5C). Similar results were observed with copanlisib or alpelisib treatment (Figure 5C). Moreover, western blotting revealed that expression of α‐SMA, SM22‐α, TGF‐β and ECM protein collagen type I, collagen type III and versican was reduced by LY294002, copanlisib and alpelisib (Figure 5D,E). As expected, LY294002, copanlisib and alpelisib significantly reduced the expression of PI3K 110α, p70 S6K, phosphorylated p70 S6K, phosphorylated AKT and phosphorylated mTOR expression in aVICs (Figure 5F,G).
+
+*Figure: FIGURE 5 Pharmacological inhibition of PI3K signalling reverses VIC phenotype and reduces ECM protein expression in canine aVICs. aVICs were exposed to DMSO (Control), LY294002 (60 μM), copanlisib (5 μM) and alpelisib (50 μM) treatment for 3 days. (A, B) Representative confocal images of α‐SMA (red) staining in canine aVICs and quantitative analysis of the percentage of α‐SMA positive cells, scale bar 20 μm (*n* = 12 microscopic fields/treatment). (C) Quantitative RT‐PCR for α‐SMA, SM22 and Smemb mRNA expression in aVICs (*n* = 6) at Day 3. (D, E) Representative western blot of ECM and VIC phenotype protein expression and quantification of the relative protein expression (*n* = 6). (F, G) Representative western blot of PI3K 110α, AKT, phosphorylated AKT (p‐AKT), mTOR, phosphorylated mTOR (p‐mTOR), p70 S6K, phosphorylated p70 S6K (p‐p70 S6K) protein expression and quantification of the relative protein expression (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; ECM, extracellular matrix; VIC, valve interstitial cell.*
+
+3.6
+
+### Antagonism of PI3K pathway promotes VIC apoptosis
+
+PI3K signalling has been previously reported to play a crucial role in the regulation of cell proliferation and apoptosis.^21^ To determine if PI3K antagonism can affect apoptosis aVICs were treated with 60 μM LY294002, 5 μM copanlisib and 50 μM alpelisib for 3 days, as described earlier, followed by TUNEL staining and examination by confocal microscopy. The number of TUNEL‐positive (apoptotic) cells was markedly increased, with nuclear fragmentation, chromatin condensation, chromatin and apoptotic body formation observed (Figure 6A,B). Flow cytometry confirmed that the TUNEL‐positive cells were increased after treatment with one of the three antagonists (Figure 6C). Since caspase‐3 has been shown as the key terminal executioner of caspase‐activated both by extrinsic and intrinsic apoptosis pathways, Western blotting and quantitative PCR were performed to examine the expression of caspase‐3 and cleaved caspase‐3.^53^ Treatment by all three antagonists significantly increased the expression of caspase‐3 and cleaved caspase‐3 in aVICs (Figure 6D–G). Taken together, these data confirm pharmacological inhibition of the PI3K signalling pathway promotes aVIC cell apoptosis, and conversely that apoptosis is repressed in aVICs and in MMVD.
+
+*Figure: FIGURE 6 Antagonism of PI3K pathway promotes apoptosis in canine aVICs. aVICs were treated with DMSO (Control), LY294002 (60 μM), copanlisib (5 μM) and alpelisib (50 μM) for 3 days. (A, B) Representative confocal images of TUNEL (red) staining and quantitative analysis of the percentage of TUNEL positive (apoptotic) cells (arrowhead), scale bar 20 μm (*n* = 12 microscopic fields/treatment). (C) Flow cytometry analysis of TUNEL staining and quantification of the percentage of apoptotic cells at Day 3 (*n* = 3). (D–F) Representative western blot of caspase‐3, cleaved caspase‐3 and β‐Actin protein expression and quantification of the relative protein expression (*n* = 6). (G) Quantitative RT‐PCR for caspase‐3 mRNA expression in aVICs treated with PI3K inhibitors after 3 days (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; aVIC, activated myofibroblast phenotype.*
+
+3.7
+
+### Suppression of PI3K signalling enhances VIC autophagy
+
+In addition to cell apoptosis, PI3K signalling plays a key role in the control of autophagy through the modulation of the downstream effects mTOR/p70 S6K.^54^ aVICs were pre‐treated with 60 μM LY294002, 5 μM copanlisib and 50 μM alpelisib for 24 h, and then treated with 5 μM baflomycin‐A1 for 16 h to inhibit autophagy flux by blocking autolysosomal degradation.^55^ The appropriate concentrations of baflomycin‐A1 were assessed in VICs by cell viability assay (Figure S1). LC3‐II immunostaining was performed to examine the formation of autophagosomes in aVIC cytoplasm. LY294002, copanlisib and alpelisib treatment induced LC3‐II labelled autophagosome formation and LC3‐II puncta numbers were significantly increased in PI3K suppressed aVICs (Figure 7A,B). The formation of autophagosomes from phagophores has been reported to require the participation of the evolutionarily conserved autophagy‐related (ATG) genes; we therefore examined the expression of ATG3, ATG5, ATG7 and LC3‐II by Western Blotting (Figure 7C). Inhibition of PI3K signalling by the three antagonists significantly increased the expression of ATG3, ATG5, ATG7 and LC3‐II (Figure 7D–G). These data indicate that pharmacological inhibition of the PI3K signalling pathway promotes aVIC cell autophagy, and that autophagy is suppressed in aVICs and in MMVD.
+
+*Figure: FIGURE 7 Inhibition of PI3K signalling enhances autophagy in canine aVICs. aVICs were exposed to DMSO (Control), 60 μM LY294002, 5 μM copanlisib and 50 μM alpelisib treatment with 5 μM baflomycin‐A1 (Baf‐A1) for 16 h. (A, B) Representative confocal images of LC3‐II marked autophagosomes (green) and quantitative analysis of the number of LC3‐II puncta, scale bar 20 μm (*n* = 98 cells/treatment). (C) Representative western blot of ATG3, ATG5, ATG7 and LC3‐II protein expression in aVICs exposed to DMSO and PI3K inhibitors. (D–G) Quantification of the relative protein expression of ATG3, ATG5, ATG7 and LC3‐II (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; aVIC, activated myofibroblast phenotype.*
+
+3.8
+
+### Pharmacological antagonism of PI3K signalling reverses VIC cellular senescence and secretory phenotype
+
+To investigate whether PI3K signalling antagonism has an effect on cell senescence, senescence associated‐β‐galactosidase (SA‐β‐gal) staining was performed to detect the status of senescence in aVICs treated with LY294002, copanlisib and alpelisib for 24 h.
+
+There was a decrease in SA‐β‐gal positive staining after the inhibition of PI3K in aVICs (Figure 8A,B). Since DNA damage is also recognized as an important characteristic of senescent cells, we decided to examine the presence of discrete nuclear γ‐H2AX foci using immunofluorescence‐based confocal microscopy.^52^ The formation of γ‐H2AX foci was markedly reduced by LY294002, copanlisib and alpelisib treatment of aVICs (Figure 8C,D). p16^INK4A^, p53 and p21^CIP1^ protein expression were significantly decreased in treated aVICs (Figure 8E–H). However, the majority of treated cells accumulated in G1 phase, together with a decreased BrdU incorporation (Figure S2). The upregulation of the selected SASP mRNAs in aVICs was significantly attenuated by PI3K antagonism (Figure 8I). TGF‐β1, IL‐6 and MMP‐9 levels in the culture supernatant from aVICs were significantly reduced showed by ELISA (Figure 8J–L). Taken together, these data suggest that pharmacological inhibition of the PI3K signalling pathway abolishes cell senescence and SASP in aVICs returning cells to a more normal phenotype.
+
+*Figure: FIGURE 8 Pharmacological antagonism of PI3K signalling attenuates cellular senescence in canine aVICs. aVICs were exposed to DMSO (Control), 60 μM LY294002, 5 μM copanlisib and 50 μM alpelisib treatment for 24 h. (A, B) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (*n* = 12 microscopic fields/treatment). (C, D) Representative confocal images of γ‐H2AX (green) immunostaining in nuclei and quantitative analysis of the number of γ‐H2AX puncta, scale bar 20 μm (*n* = 50 cells/treatment). (E) Representative western blot of p16^INK4A^, p21^CIP1^, p53 and β‐actin protein expression and (F–H) quantification of the relative protein expression (*n* = 6). (I) Quantitative RT‐PCR for SASP cytokine expression in aVICs exposed to DMSO and PI3K inhibitors (*n* = 6). (J–L) Quantification of secreted TGF‐β1, IL‐6 and MMP‐9 in collected supernatant from aVIC cultures (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; aVIC, activated myofibroblast phenotype; SASP, senescent‐associated secretory phenotype.*
+
+3.9
+
+### Upregulation of mTOR signalling by overexpressing p70 S6K induces activated myofibroblast differentiation and cellular senescence with a reduced capacity for apoptosis and autophagy
+
+As a major downstream target of PI3K/AKT signalling mTOR occupies a pivotal position in the regulation of cell apoptosis, autophagy and senescence.^56^ Activation of the mTOR pathway initiates senescent p53/p21^CIP1^ signalling, inhibits caspases‐mediated apoptosis and inactivates ATG‐associated autophagy.^50^, ^57^ mTOR complex 1 (mTORC1) regulates these cellular activities at the transcriptional level by modulating the phosphorylation of the key downstream transcriptional factor ribosomal protein p70 S6K.^58^, ^59^ To elucidate the role of p70 S6K in aberrant qVIC myofibroblast differentiation and the modulation of these important cellular activities, human and mouse p70 S6K were overexpressed in canine qVICs separately using the DNA‐Lipofectamine method. Overexpression of p70 S6K resulted in the transition of qVICs to aVICs by inducing a significant increased protein expression of α‐SMA and SM22‐α. The synthesis of ECM proteins (collagen type I, collagen type III, versican), TGF‐β and MMP‐9 was markedly increased with VIC phenotype transformation (Figure 9A).
+
+*Figure: FIGURE 9 Overexpression of p70 S6K induces activated myofibroblast differentiation and ECM production through downregulating apoptosis and autophagy while enhancing cellular senescence. Canine qVICs were transfected with human p70 S6K cDNA ORF plasmids, mouse p70 S6K cDNA ORF plasmids, pcDNA3.1 plasmids (vectors) and Lipofectamine 3000 (Mock) with or without 5 μM of baflomycin‐A1 (Baf‐A1). (A) Representative western blot of p70 S6K, phosphorylated p70 S6K (p‐p70 S6K), VIC phenotype and ECM protein expression and quantification of the relative protein expression (*n* = 6). (B) p70 S6K overexpressed qVICs were exposed to 5 μM Baflomycin‐A1. Representative western blot of ATG7 and LC3‐II protein expression and quantification of the relative protein expression (*n* = 6). (C) Representative confocal images of LC3‐II labelled autophagosomes (green) and quantitative analysis of the number of LC3‐II puncta, scale bar 20 μm (*n* = 98 cells/treatment). (D) Representative western blot of caspase‐3, cleaved caspase‐3, p16^INK4A^, p21^CIP1^ and p53 protein expression and quantification of the relative protein expression (*n* = 6). (E) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (*n* = 12 microscopic fields). (F) Quantitative RT‐PCR for senescence‐associated secretory phenotype (SASP) cytokine expression (left panel) in qVICs transfected with p70 S6K cDNA ORF plasmids (*n* = 6). Quantification of secreted TGF‐β1, IL‐6 and MMP‐9 (right panel) in collected supernatant from qVIC cultures overexpressing p70 S6K (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ANOVA, analysis of variance; ECM, extracellular matrix; VIC, valve interstitial cell.*
+
+To clarify the status and role of autophagic flux p70 S6K overexpressed qVICs were treated with 5 μM baflomycin‐A1. The autophagic flux was compromised with the downregulation of ATG7 and LC3‐II expression (Figure 9B). LC3‐II immunostaining identified the LC3‐II puncta formation was largely inhibited by overexpression of p70 S6K (Figure 9C). p70 S6K overexpressed qVICs showed significantly increased expression of the senescent markers p16^INK4A^, p53 and p21^CIP1^ with a reduced caspase‐3 and cleaved caspase‐3 expression level (Figure 9D). Overexpression of p70 S6K also increased SA‐β‐gal positive cells (Figure 9E), significantly reduced BrdU incorporation (Figure S3B) and enhanced cells in G1 phase (Figure S3A), with concomitantly upregulated gene expression of SASP (genes for IL‐6, IL‐1β, MMP‐9, TNF‐α, TGF‐β1, TGF‐β2 and TGF‐β3) (Figure 9F). There was significantly increased expression of IL‐6, MMP‐9 and TGF‐β1 detected by ELISA (Figure 9F). These data show that overexpression of p70 S6K induces the activated myofibroblast differentiation, ECM disorganization and cellular senescence, while a reducing VIC capacity for apoptosis and autophagy.
+
+3.10
+
+### Knockdown of p70 S6K revives VIC phenotype and alleviates cellular senescence with a promoted autophagic state
+
+To further examine the central regulatory effects of p70 S6K and its potential as a novel therapeutic target for MMVD, gene expression of p70 S6K was silenced using human and mouse siRNA in aVICs. The upregulation of α‐SMA and SM22‐α was dramatically attenuated by the downregulation of p70 S6K in aVICs in the presence or absence of TGF‐β1 treatment. As expected, the ECM protein synthesis (collagen type I, collagen type III, versican) and TGF‐β expression were significantly decreased with VIC phenotype recovery. In the presence of 10 ng/mL TGF‐β1, the knockdown of p70 S6K reduced the expression of the senescent transcription factors p16^INK4A^ and p53/p21^CIP1^ (Figure 10A), while there was a concurrent inactivation of caspase‐3 and cleaved caspase‐3 mediated apoptotic activities (Figure 10B), together with a significant increased capacity for BrdU incorporation (Figure S4B).
+
+*Figure: FIGURE 10 Knockdown of p70 S6K reverses VIC phenotype through upregulation of autophagy and inhibition of cellular senescence while protecting against apoptosis. Canine aVICs were transfected with Lipofectamine 3000 (Mock), scramble control, human p70 S6K siRNA, mouse p70 S6K siRNA with or without 10 ng/mL of TGF‐β1 or 5 μM of baflomycin‐A1 (Baf‐A1). (A) Representative western blot of p70 S6K, phospho‐p70 S6K (p‐p70 S6K), p16INK4A, p21CIP1 and p53, VIC phenotype and ECM protein expression and quantification of the relative protein expression (*n* = 6). (B) Representative western blot of p70 S6K, phospho‐p70 S6K (p‐p70 S6K), phospho‐IRS1 (p‐p70 IRS1), phospho‐AKT (p‐AKT), caspase‐3, cleaved caspase‐3 and quantification of the relative protein expression (*n* = 6) (C) Representative western blot of ATG7 and LC3‐II protein expression in qVICs treated with 5 μM baflomycin‐A1 with or without 10 ng/mL of TGF‐β1 and quantification of the relative protein expression (*n* = 6). (D) Representative confocal images of LC3‐II labelled autophagosomes (green) and quantitative analysis of the number of LC3‐II puncta, scale bar 20 μm (*n* = 98 cells/treatment). (E) Representative images of SA‐β‐gal (blue) staining and quantitative analysis of the percentage of SA‐β‐gal positive cells, scale bar 50 μm (*n* = 12 microscopic fields). (F) Quantitative RT‐PCR (left panel) for senescence‐associated secretory phenotype (SASP) cytokine expression in aVICs transfected with p70 S6K siRNA with or without TGF‐β induction (*n* = 6). Quantification of secreted TGF‐β1, IL‐6 and MMP‐9 (right panel) in collected supernatant from aVIC cultures (*n* = 6). Results are presented as mean ± SEM. ANOVA followed by Tukey's range test. **p* < 0.05, ***p* < 0.01, ****p* < 0.001 compared to control. ECM, extracellular matrix; VIC, valve interstitial cell.*
+
+To further investigate the mechanism of unexpected decreased apoptosis, the phosphorylated forms of p70 S6K, IRS1 and AKT were examined considering their roles in the negative feedback loop from p70 S6K to IRS1 and the key regulatory functions of AKT in cell apoptosis though anti‐apoptotic proteins.^60^, ^61^ The decreased phosphorylated level of p70 S6K, caused by silencing p70 S6K, resulted in an increase in phosphorylated expression of IRS1 and the downstream effector AKT (Figure 10B). In the presence of 5 μM baflomycin‐A1, autophagy flux was promoted as shown by significantly increased expression of ATG7 and LC3‐II (Figure 10C). LC3‐II puncta were significantly increased by silencing p70 S6K (Figure 10D). p70 S6K downregulated aVICs showed less SA‐β‐gal staining (Figure 10E), more accumulation in S and G2/M phases (Figure S4A) and significant downregulation of SASP mRNA expression and the level of secreted IL‐6, MMP‐9, TGF‐β1 (Figure 10F). These data indicate that p70 S6K knockdown transitions cells to a more quiescent and normal phenotype, induces normal ECM homeostasis and abolishes cellular senescence and SASP, while inhibiting apoptosis and promoting a more autophagic state.
+
+4
+
+## DISCUSSION
+
+In this study, we have identified TGF‐β‐induced PI3K/AKT/mTOR/p70 S6K signalling controls mitral VIC differentiation, function and cellular activities in canine MMVD. A significantly increased expression of PI3K and TGF‐β was observed in α‐SMA positive aVICs in canine myxomatous mitral valves. Activation of PI3K/AKT/mTOR/p70 S6K signalling was shown to promote the transformation of activated myofibroblast. Pharmacological inhibition of PI3K signalling restored the normal quiescent VIC phenotype by suppressing senescence and SASP and promoting apoptosis and autophagy. Upregulation of mTOR/S6K induces transformation of senescent aVICs, with compromised apoptotic activity and impaired autophagy flux. Conversely, selective knockdown of p70 S6K reverses cell transition by attenuating cell senescence, inhibiting apoptosis and improving autophagy. These findings provide novel evidence that the PI3K signalling pathway antagonism is a promising target to inhibit the pathological processes of MMVD, by preventing myofibroblast transition, counteracting cellular senescence, SASP and restoring apoptosis and autophagy, with potential as a therapeutic target for MMVD in the dog, and by extension for the analogous human disease. Furthermore, the dysregulation of the PI3K/AKT/mTOR pathway in MMVD supports the concept that this degenerative disease is associated with tissue ageing.
+
+Smad‐mediated canonical TGF‐β signalling has been shown to contribute to the pathogenesis of human MMVD, but to what extent this is driving fibrosis and/or myxomatous degeneration cannot be stated with certainty.^10^, ^11^, ^12^, ^16^ We have now shown that one of the non‐canonical pathways (PI3K) also likely contributes to MMVD pathogenesis. TGF‐βs are recognized as an important initiator of signalling pathways that contribute to the development of human and canine MMVD.^1^, ^62^ TGF‐β signalling‐dependent VIC phenotypic transformation and myxomatous degeneration are widely reported in both species.^10^, ^18^, ^63^ TGF‐β‐induced PI3K/AKT/mTOR activation has been widely reported in multiple degenerative disorders, including idiopathic pulmonary fibrosis (IPF), cardiac and renal fibrosis.^22^, ^64^, ^65^ The upregulation of PI3K/AKT/mTOR signalling in fibroblasts induces the aberrant transition of myofibroblastic phenotype and ECM remodelling, and therefore serves as a primary driver for the development and progression of these diseases. However, the role of these signalling pathways in both human and canine MMVD is not fully understood, in particular, the further downstream effects on transcription factors that control interstitial cell (VIC) phenotype, survival and ECM synthesis. To our knowledge, the current study is the first report showing that TGF‐β‐induced PI3K/Akt/mTOR signalling controls the phenotypical transitions of mitral VICs and their functional roles in ECM remodelling in MMVD. The PI3K/AKT/mTOR pathway is well characterized as an intracellular signalling pathway important in regulating cellular quiescence, differentiation and survival.^48^ We have shown that activation of PI3K/AKT/mTOR pathway promotes the transformation of mitral VICs into myofibroblasts and enhances the ECM protein synthesis. These data are consistent with observations in many other types of cells and diseases.^66^, ^67^, ^68^ The persistence of activated PI3K signalling depends on several regulatory mechanisms including AKT activation through a negative feedback loop from p70 S6K to PI3K and the upregulated phosphorylation of PRAS40.^43^, ^69^ In the present study, the high level of phosphorylated AKT, PRAS40, mTOR and p70 S6K observed in aVICs suggests AKT activation enhances PRAS40 phosphorylation and thereby reduces the inhibitory effects of PRAS40 on mTORC1, intensifying the mTOR/p70 S6K signalling pathway.^43^ Augmented mTOR/p70 S6K can inhibit the upstream IRS1 mediated signals initiating from other growth factor receptors rather than TGF‐β receptor I/II and thereby intensify the TGF‐β‐induced PI3K signalling.^69^ This could explain the persistence and survival of aVICs resulting from aberrantly activated PI3K/Akt/mTOR/p70 S6K signalling in the development of MMVD. However, other growth factors, such as insulin, fibroblast growth factor (FGF), insulin‐like growth factor (IGF) and epidermal growth factor (EGF), have been shown to also trigger PI3K/AKT/mTOR signalling.^70^, ^71^ Furthermore, the antagonism of canonical Smad2/3‐mediated TGF‐β signalling has similar effects on mitral VIC transformation and ECM remodelling.^10^ To what extent one of these might be a dominant pathway for the disease, and the interplay between these signalling pathways, are still unknown and requires further study.
+
+As a direct downstream target of PI3K, AKT is at the molecular junction controlling cell death and survival. AKT promotes cell survival by blocking apoptosis through the inactivation of pro‐apoptotic proteins such as Bcl‐2.^60^ In our study, AKT inactivation by pharmacological inhibition of PI3K signalling induced cell apoptosis in aVICs. This may in part explain aVIC persistence in MMVD.^72^ As a form of programmed cell death, apoptosis is a key factor causing target cells to be cleared from tissues. However, VICs play a crucial role in the maintenance of a balanced ECM in mitral valves and directly removing them may also result in further damage to the ECM.^73^, ^74^ In the current study inhibiting the downstream AKT effectors mTOR/S6K by selective silencing p70 S6K improved autophagy flux and attenuated cell senescence and SASP, while inhibiting apoptosis. Considering the apoptosis inhibition induced by AKT activation through the negative feedback loop between mTOR/p70 S6K and IRS1/AKT,^60^, ^61^ selectively targeting the downstream effector mTOR may be a better way to restore cell transitions and functions, minmising undesirable effects.
+
+Recently mTOR/p70 S6K signalling has received wide attention due to its key roles in controlling cell transition, apoptosis, autophagy and senescence. Small‐molecule modulators based on the manipulation of mTOR have been investigated in a variety of cardiovascular diseases.^75^ Activation of mTOR is known to result in myofibroblast differentiation, inhibition of apoptosis and autophagy while enhancing cellular senescence. This is consistent with our observations where we have induced p70 S6K overexpression qVICs. Conversely, inhibition of mTOR signalling in aVICs by global pharmacological manipulation of PI3K signalling or selective knockdown of p70 S6K reversed cell phenotype, revived ECM protein synthesis and promoted apoptosis and autophagy, while reversing cell senescence. This suggests a pivotal role for mTOR/p70 S6K signalling in the regulation of important VIC activities in MMVD. p70 S6K has been shown to regulate mRNA translation initiation and thereby protein synthesis.^75^ Increased α‐SMA expression induced by activation of mTOR/p70 S6K signalling has been observed in pulmonary artery smooth muscle cells (SMCs), while the mTOR inhibitor rapamycin suppressed the proliferation of α‐SMA positive SMCs.^76^ In addition, the accumulation of SM22 in myocytes with a contractile phenotype can be reduced by PI3K/mTOR/p70 S6K inhibition by LY294002 and rapamycin treatment.^77^ This again is consistent with our observations in VIC transition being regulated by PI3K signalling in MMVD. On that basis, it would be reasonable to presume that p70 S6K regulates the function and differentiation of VICs through the phosphorylation of its substrate S6 ribosomal protein, and this might be considered as a potential therapeutic target. Pharmacological inhibition or knockdown of mTOR/p70 S6K has been shown to protect against human intervertebral disc apoptosis, cellular senescence and ECM catabolism, through autophagy induction.^24^, ^25^ Mice with hypomorphic mTOR have increased lifespan and reduced senescent marker p16^INK4A^ expression, demonstrating the link between tissue degeneration and ageing, while mice with articular cartilage‐specific mTOR deletion are protected against osteoarthritis through enhancement of autophagy.^49^ The complex interplay between these cellular and molecular responses needs to be further investigated.
+
+In the present study, the typical characteristics of cellular senescence were noticeable in aVICs as shown by decreased apoptotic activities, nuclear γ‐H2AX foci formation, cytoplasmic positive SA‐β‐gal staining, activation of p53/p21^CIP1^ and p16^INK4A^ pathways, and increased intracellular and extracellular SASP expression. Senescence is now considered to be a highly dynamic process associated with multiple cellular, molecular changes and distinct phenotypic alterations. Senescent cells resist elimination from tissues by apoptosis through the upregulation of anti‐apoptotic pathways.^52^ Considering the activation of PI3K signalling in aVICs and its effects on promoting cell proliferation and transformation, we can reasonably speculate that due to PI3K activation some transformed aVICs can undergo senescent changes while others retain the proliferative ability, and so contribute to more senescent aVICs as MMVD develops and progresses. Since senescent cells remain metabolically active, despite being in a growth‐arrested state, they will affect the events inside cells, the behaviour of neighbouring non‐senescent cells and the remodelling of the surrounding microenvironment by secreting a complex mixture of secreted factors (SASP). This special secretome includes inflammatory, pro‐apoptotic, insulin resistance‐inducing cytokines, such as IL‐6, IL‐1β and TNF‐α, MMPs and relevant regulators such as MMP‐9 and TIMP1 that cause ECM remodelling, and finally TGF‐β family members that contribute to fibrosis, myxomatous degeneration and dysregulated trans‐differentiation of VICs.^52^ In the current study, the accumulation of senescent cells and expression of SASP suggests that as the disease develops TGF‐β‐induced activation of PI3K/AKT/mTOR/p70 S6K signalling triggers the proliferation and abnormal transition of qVICs to senescent aVICs with reduced ability of apoptosis and autophagy. Subsequent induction of SASP formation intensifies TGF‐β signalling, causing abnormal ECM remodelling and continual valve tissue damage. SASP itself may be caused, in part, by senescence‐associated mitochondrial dysfunction (SAMD), closely associated with the dysregulation of mTOR/p70 S6K signalling. Increased pro‐inflammatory cytokines (IL‐6, IL‐1β and TNF‐α) can be induced by activated danger‐associated molecular patterns, such as reactive oxygen species and mtDNA fragments, released by dysfunctional mitochondria, which may then contribute to ECM damage.^78^ Further studies are required to examine the possible contribution of mitochondrial dysfunction to MMVD pathogenesis.
+
+In targeting the highly expressed Senescent Cell Anti‐Apoptotic Pathway networks, senolytic compounds have been shown to clear senescent cells by promoting apoptosis through the upregulation of multiple pro‐apoptotic pathways.^79^ The naturally occurring flavonoid quercetin has been reported to remove senescent cells through apoptosis by inhibiting PI3K/AKT/mTOR signalling, in a similar fashion to LY294002, copanlisib and alpelisib used in this study.^80^ Flavonoids might serve as better potential therapeutic candidates to treat MMVD due to their wide availability and moderate toxicity. However, clearance of functional VICs in myxomatous valves may cause unexpected valvular outcomes and it is likely reversing aVICs to a non‐senescent qVICs using autophagy activators inhibiting mTOR/p70 S6K signalling might be a more promising therapeutic approach to control MMVD in both the human and the dog.^75^
+
+The findings of this study will require in vivo validation. Although in vitro primary cells isolated from clinical samples are largely able to reflect the disease parthenogenesis, in vivo validations are needed considering the drug metabolism, individual variance and clinical transitional research. However, there are a limited numbers of animal models available to study the pathogenesis of MMVD. A recently developed spontaneously occurring FVB/NJ mouse model of MMVD rapidly exhibits disease progression and pathology and would appear to be a good candidate.^81^ Specifically, to further validate whether PI3K/AKT/mTOR signalling controls the MMVD progression in vivo, a novel genetically modified VIC‐specific p70 S6K knockout or overexpression mouse model is required.
+
+In conclusion, TGF‐β‐induced PI3K/AKT/mTOR/p70 S6K signalling controls the phenotypic transformation and functions of VICs in canine MMVD (Figure 11). Pharmacological inhibition of PI3K signalling reverses diseased senescent VICs, with improved capacity for apoptosis and autophagy. Furthermore, downstream mTOR/p70 S6K signalling plays an important role in the regulation of VIC transformation, ECM protein synthesis, apoptosis, autophagy and senescence in MMVD. This work informs the naturally occurring disease in dogs as a novel large animal model to investigate human early stage MMVD and warrants further investigations of senolytic compounds or autophagy activators as a potential novel therapeutic strategy for the treatment of MMVD and other age‐related degenerative disorders.
+
+*Figure: FIGURE 11 Schematic illustration of TGF‐β‐induced PI3K/AKT/mTOR/p70 S6K pathway in the regulation of cell phenotype, ECM synthesis, apoptosis, autophagy and senescence in aVICs in canine MMVD. aVIC, activated myofibroblast phenotype; ECM, extracellular matrix; MMVD, myxomatous mitral valve disease; TGF‐β, transforming growth factor β.*
+
+## AUTHOR CONTRIBUTIONS
+
+Qiyu Tang, Kanchan Phadwal, Vicky E. MacRae and Brendan M. Corcoran conceptualized the study and and experiments. Qiyu Tang, Greg R. Markby, Andrew J. MacNair, Keyi Tang and Michal Tkacz performed the experiments. Qiyu Tang and Kanchan Phadwal accomplished data analysis and prepared the manuscript. Maciej Parys, Kanchan Phadwal, Vicky E. MacRae and Brendan M. Corcoran evaluated the manuscript and data reliability. All authors have read and agreed to the published version of the manuscript.
+
+## CONFLICT OF INTEREST STATEMENT
+
+The authors declare no conflict of interest.

fulltext/md/PMC10284893.md

@@ -0,0 +1,177 @@
+---
+pmcid: "PMC10284893"
+pmid: "37344566"
+doi: "10.1038/s42003-023-05034-3"
+title: "Going beyond established model systems of Alzheimer's disease: companion animals provide novel insights into the neurobiology of aging."
+journal: "Communications biology"
+year: "2023"
+license: "cc by"
+article_type: "review-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Going beyond established model systems of Alzheimer’s disease: companion animals provide novel insights into the neurobiology of aging
+
+## Abstract
+
+Alzheimer’s disease (AD) is characterized by brain plaques, tangles, and cognitive impairment. AD is one of the most common age-related dementias in humans. Progress in characterizing AD and other age-related disorders is hindered by a perceived dearth of animal models that naturally reproduce diseases observed in humans. Mice and nonhuman primates are model systems used to understand human diseases. Still, these model systems lack many of the biological characteristics of Alzheimer-like diseases (e.g., plaques, tangles) as they grow older. In contrast, companion animal models (cats and dogs) age in ways that resemble humans. Both companion animal models and humans show evidence of brain atrophy, plaques, and tangles, as well as cognitive decline with age. We embrace a One Health perspective, which recognizes that the health of humans is connected to those of animals, and we illustrate how such a perspective can work synergistically to enhance human and animal health. A comparative biology perspective is ideally suited to integrate insights across veterinary and human medical disciplines and solve long-standing problems in aging.
+
+## Introduction
+
+One Health is the concept that cross-disciplinary approaches that bring humans, animals, and their shared environments together can synergistically resolve problems afflicting humans and animals^1^. One Health recognizes that the health of humans is connected to those of animals and the environment. One Health is frequently discussed in the context of infectious diseases. For example, environmental degradation and ecological disruption can promote zoonotic diseases (i.e., diseases transmissible across species), which negatively impact human health. A One Health approach has much broader implications for human health than simply reducing zoonotic transmission^2–4^. A One Health perspective can positively impact our understanding of non-transmissible diseases shared by humans and other species (Fig. 1a).Fig. 1Companion animals share many commonalities with humans.**a** Humans and companion animals suffer from many overlapping diseases. Those include cognitive aging, obesity, cancer, and hypertrophic cardiomyopathy. Age is one of many factors in the development of these diseases. A One Health perspective advocates that individuals from veterinary and human medicine working collaboratively across fields will synergistically enhance the health of humans and animals. **b** Phylogeny of model systems and associated brains from each Order. Rodents, marsupials, and NHPs (e.g., macaques, marmosets) are used as model systems to understand human aging. Although companion animals (i.e., cats, dogs) are more distantly related to primates than rodents are to primates, companion animals share many similarities with humans. **c** Carnivores and primates both possess gyrencephalic brains. A close-up of the cortical gray matter highlights similarities in cytoarchitecture (e.g., an expansion in upper layers) in cats and macaques relative to rodents^36,39^.
+
+The fields of evolutionary and comparative biology are ideally suited to bridge veterinary and human medicine disciplines to improve our understanding of diseases affecting humans and animals^3^. Companion animals, which are domesticated dogs and cats maintained as household pets (Table 1 and Supplementary Data 1), suffer from many diseases also observed in humans. Like humans, companion animals suffer from cancer, aging, obesity, heart disease, diabetes, and kidney disease^5^. Veterinary and human medicine are focused on identifying and treating similar diseases, but these two fields rarely interact. Nevertheless, there are ground-breaking examples where treatments developed for companion animals have yielded concrete benefits to human patients^6–11^. Obesity, brain aging, retinal aging, and cancer are a few examples of the many areas in biology that stand to gain from increased exchanges between the fields of human and veterinary medicine (Fig. 1a). For example, cats have proven to be excellent models for ophthalmological conditions because of similarities with humans, including high genomic diversity, with the Abyssinian cat breed proving a ‘naturally’ occurring model of retinal degeneration^12^. We illustrate how a One Health approach could expand our toolkit to solve enduring challenges in the neurobiology of aging.Table 1Keywords used for purposes of this article.Keywords:Companion animal models: are domesticated cats and dogs, which are species typically maintained as household pets. Some studies referenced in this review are from animals housed in laboratory colonies.Nonhuman primate (NHP) models: are defined as any primate species used as primate model systems, such as macaques, marmosets, and lemurs.Traditional mice and rat models: are laboratory domesticated rats and mice (genera: *Rattus, Mus*), which are most frequently used for study in biomedical sciences (see Fig. 3).Carnivore: are any wild or domesticated animal classified as belonging to the order Carnivora (e.g., cats, dogs, ferrets, polar bears, cheetahs, and red pandas).Domesticated animals: is defined as any animal that is maintained as a companion, pet, or is raised on a farm.
+
+Alzheimer’s disease (AD) is a disease of aging and is characterized by brain plaques and tangles, brain atrophy, and cognitive dysfunction^13–15^. AD was traditionally thought to be unique to humans though this interpretation is based on observations made from a handful of model systems^16,17^. This limited range of species spans rodents (i.e., rats, mice; Table 1), which are widely used in biomedical sciences (Fig. 1b; See Supplementary Methods). We also consider nonhuman primates (i.e., NHPs^18^) used in research (e.g., marmosets, mouse lemurs, African green monkeys, macaques, and chimpanzees) and companion animal models (Fig. 1b). NHPs have commonly been considered the model organisms that most closely resemble human behavior and disease profiles due to their psychosocial and genetic similarities^19^. Rodent and NHP model systems recapitulate limited aspects of AD, but companion model systems (i.e., carnivores used as companion animals) are emerging as valuable models to understand human aging in health and disease.
+
+Companion animal model systems are here defined as domesticated cats (*Felis catus*) and dogs (*Canis lupus familiaris*), which are species that are maintained as common household pets though they are sometimes used in the laboratory (Table 1 and Supplementary Data 1)^20^. Companion animals are rarely considered model systems to probe human biology predominantly *because they are our companions*, and there are additional regulations for using cats and dogs (and also primates) as model systems in research compared to rodent models. For example, the U.K. Government’s Home Office classifies them as “Specially protected species” which can only be used for research cases in the absence of any other suitable model system^21^. The U.S. Government’s Animal Welfare Act was put in place largely to protect cats and dogs to which it applies (and also primates), but this does not apply to common laboratory strains of rodents^22^ (Supplementary Data 1). Cats and dogs encapsulate a diverse group of animal breeds; they are thought to have been domesticated over the past 4–30,000 years^23,24^, with a conservative review suggesting dogs were domesticated 15,000 years ago^25^ in Europe or Asia and cats were domesticated 4000 years ago^25^ in the eastern Mediterranean Basin of Northern Africa and the Near East.
+
+A similarity between domesticated animals and humans is that they occupy the same (built) environments that humans do. The term domestic comes from domus, which is Latin for house. Yet until recently domesticated animals have lived around, but outside of, human homes. One study found that although dogs lived among humans in 53 of 60 cultures studied, they were allowed inside human houses only in seven of them^26^. Practices have been changing in recent decades, however. In the 1960s a quarter of dogs in the USA (owned and unowned) roamed the streets, but this was reduced when animal sheltering started in the 1970s^27^. While cats and dogs have been domesticated for millennia, they previously endured much work and/or hostility from humans, but they are now typically house-dwelling and treated as members of the family^26^. This factor might link companion animals to humans more than other domesticated species.
+
+On the other hand, Darwin^28^ suggested that domesticated species (including both companion animals and farm animals) share a suite of anatomical, physiological, and behavioral traits, to the exclusion of their wild ancestors. This so-called *domestication syndrome* may be caused by selection for reduced aggression in mammals, which may result in changes in the expression of genes that direct neural crest cell development^29^. It has further been suggested that the domestication of some species, including dogs and cats, cannot be attributed entirely to human actions. Instead, it has been suggested that they were *self-domesticated* and had autonomously come to occupy domesticated niches^30^. Dogs may have participated in their own domestication from wolves by benefiting from being near human habitation sites^31^. Cats may have been active in their own domestication by lurking near human habitations, while maintaining contact with the wild population and having control of their own resource supply, a process dubbed *semi-domestication*^32^. It has even been suggested that humans may have self-selected for tameness and that the same genetic factors contributed to reduced craniofacial features compared to our closest relatives the Neanderthals^30^. It remains uncertain to what extent the domestication *process* in cats and dogs was different than in livestock species and akin to something that took place in recent human evolution. However, here we propose that the similar environments of humans and companion animals make them especially well-suited to study human biology.
+
+Comparative biology is an important area of study because it considers the natural variation that has arisen through evolutionary pressures. We can use information from comparative neuroscience research to identify the model systems best suited to aging^33–35^. Companion animals share many similarities in brain organization (Fig. 1c) and possess age-related changes in biology and behavior that resemble humans^36–40^. Companion animals also share very similar environments (e.g., shared sleeping quarters, diets, experiences, toxins, stressors) with their owners. The close relationship of companion animals to humans combined with their relatively shortened life makes them well-suited to monitor how the environment impacts health and rates of aging^41–43^. Companion animals are also advantageous to study because some of them are genetically homogenous populations in purebreds. Others are genetically heterogeneous outbred populations in mixed breeds^44^. The pattern of variation across these different populations means that we can investigate the interaction of genotypes and toxins, stressors, or diseases on rates of aging. Similarities in these traits point to companion animal models as valuable models to study aging.
+
+We discuss the structural, molecular, and behavioral changes in companion animal models during aging. More specifically, we discuss brain atrophy, plaques, tangles, and memory impairments across species. We evaluate which model systems demonstrate age-related changes in behavior and biology^45–48^. Our comparative analyses showcase that companion animal models spontaneously accrue many of the hallmarks of human aging. These observations prompt the need for increased communication across human and veterinary medicine. These synergies may resolve challenges in the study of aging and other long-standing biomedical issues.
+
+## What is Alzheimer’s disease?
+
+Alzheimer’s disease (AD) is a progressive, fatal brain and behavioral disease that impairs memory and cognitive skills. AD also includes non-cognitive psychiatric symptoms such as irritability, and loss of normal sleep patterns^49–51^. Historically, AD has been considered unique to humans because it has been elusive in a handful of studied model systems. AD accounts for 50–80% of dementia cases and, accordingly, is one of the most common types of dementia^52^. Some records indicate ancient Egyptians may have documented AD^53^. Still, it was not until 1906 that Alois Alzheimer discerned neuritic senile plaques and neurofibrillary tangles in the autopsied brain of 51-year-old Auguste Deter, who suffered from amnesia and dementia^54^. In humans, AD is classified as early-onset if the disease emerges earlier than 65 and late-onset if it arises at 65 and over^55^, and the latter makes up around 95% of cases^56^. Clinically, individuals with AD experience irreversible and progressive behavioral disabilities, including cognitive dysfunction, memory loss, and brain pathologies^57^. We describe each of these dysfunctions in AD and consider how age-related changes map onto rodent, primate, and companion model systems.
+
+## Cognitive loss in AD
+
+The most salient aspect of the disease is cognitive loss. The disease usually begins subtly and progresses sequentially (stages 1–3). Initial symptoms include forgetting names, locations, events, and misplacing objects (i.e., mild stage 1^54^). Individuals then become disoriented and eventually fail to recognize family members (i.e., moderate stage 2^54,58^). Finally, AD patients have difficulty writing, reading, speaking, and concentrating. They become restless, depressed, withdrawn, and lose basic functioning abilities in their final years (severe stage 3^54,58^). Cognitive aging has also been reported in diverse nonhuman animal species and includes memory loss, decreased attention span, reduced performance on spatial tasks, anxiety, locomotor disabilities, as well as changes in vocalizing^46,48,59–71^.
+
+## Neuropathologies and brain atrophy in AD
+
+Specific neuropathologies characterize AD though it is still unknown whether these pathologies are side effects of the disease process or their cause^72,73^. The accumulation and deposition of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs), which are formed by hyper-phosphorylation of tau (pTau) proteins (Fig. 2^73^), are hallmarks of the disease^74^. The Aβ peptides are dense deposits of 38, 40, or 42 long amino acid peptides called plaques^73,75^. These extracellular plaques are formed from the breakdown of Aβ precursor protein (*APP*^72,73,76,77^). NFTs are another hallmark of AD and consist of abnormal buildups of twisted strands of intracellular tau (tubulin-associated unit^73,78,79^). In health, tau binds to tubulin, which stabilize microtubules^73,75^. In AD, tau disaggregates from microtubules and bonds to other taus, forming C-shaped paired helical filaments that form relatively long intracellular tangles^73,78,79^, which impact neuronal communication^73,78–80^. These plaques and tangles are first observed in the olfactory and hippocampal regions^72,81–83^ (Fig. 2^81–83^). These neuropathologies subsequently spread through the frontal cortex, the cingulate gyrus, and the rest of the cortex^82^. One of the first symptoms of AD is a loss of smell, which coincides with the presence of neuropathologies (Fig. 2c). Evidence points to the presence of NFTs as a better marker of cognitive decline and disease progression compared to the accumulation of Aβ plaques^84,85^ (Fig. 2c). While extracellular plaques and NFTs characterize the disease, other pathologies are present in AD.Fig. 2Alzheimer’s disease is associated with a specific range of pathologies, which emerge at different ages in different species.**a** Major hallmarks of AD include brain atrophy, plaques, and tangles (NFTs). **b** The disease is characterized by a leaky blood-brain barrier. Normally, astrocytes form tight junctions, so much so that these barriers formed by astrocytes prevent the entry of materials into the brain. In AD, the blood-brain barrier is compromised, and the disease is also characterized by vacuoles and neuronal death. **c** There is a stereotypic sequence in the accumulation of brain plaques and tangles starting in the olfactory cortex and hippocampus, which then spread through most of the brain. The early accumulation of plaques and tangles in the olfactory cortex is concomitant with the loss of olfactory abilities. We compare the timeline of brain plaques and tangles in cats (**d**) and macaques (**e**). Cats may accumulate plaques and tangles for a relatively long percentage of their life. Many reports indicate that cats can develop plaques and tangles and cognitive dysfunction before the age of 10, but similar deficits are more likely expected to occur in macaques in their 20s^38,174^. Cats recapitulate the hallmarks of aging relatively early and so may be an ideal model system of aging. Dashed lines represent a probability of occurrence and are based on qualitative reports from the literature (see Supplementary Data 2)^125^.
+
+Many aged humans suffer from neuropathologies. Aged humans suffer from brain atrophy, which consists of brain volume decrease, neuronal loss, inflammation, cerebral vascular dysfunction, and neuronal vacuoles^15^. Microglia have also been highlighted as an important cell type for study^13,14,73,76,86,87^. Some plaques, including Aβ40 and Aβ42, are especially toxic because they cause surrounding amyloid groups to form clumps called intermediate oligomers and protofibrils, which are polypeptide masses^72,73,76,88–90^. Plaques and vascular problems may lead to additional diseases, including cerebral amyloid angiopathy (CAA), hemorrhage, atherosclerosis, strokes, and ischemic brain damage^72,73^. Although CAA is highly associated with AD being present in approximately 90% of cases, it also affects between 20–40% of the elderly population with no cognitive impairment^91,92^. Whether these neuropathologies represent a disease that emerges in old age or whether they represent an emergent property of aging remains an open question.
+
+## Potential causes of AD
+
+Many studies have sought to identify genetic and environmental factors, but age remains the top predictor for AD. The likelihood of developing AD doubles every five years after 65^57,93–96^. Nearly half of those over 85 are at risk of AD^54,97^. Besides age, biological sex is a leading predictor for AD in humans, with females at a higher risk of developing AD^98,99^. Past work has not identified strong environmental factors that predict AD^100^. However, it has been suggested that environmental toxins (e.g., formaldehyde from paint, some furniture, and flooring), drug use (e.g., tobacco, alcohol use), diet, a weakened immune system, and hormonal changes impact the likelihood of developing AD^54,58,73,101–104^. AD is also, to some extent, comorbid with hypertension, diabetes, hyperlipidemia (i.e., a disease characterized by an excess of fat in the blood), stroke, sleep apnea, cardiovascular disease, and metabolic syndrome^95^. Interestingly, there is an inverse relationship between educational attainment and AD risk^54,105^. However, this may, in part, be due to cognitive decline measures not being sensitive enough for those who are highly practiced at assessments, potentially leading to a late diagnosis^106,107^. Thus, the threshold set to diagnose the disease could be adjusted depending on the individual’s background^107,108^.
+
+After the age of 80, females are at higher risk of developing AD than males^109,110^, and several factors are likely influencing biological sex differences in AD incidence risk. Females live longer than men, and the relatively extended lifespan in females may elevate the risk of developing AD^111^. Other factors are likely important in dictating sex differences in disease risk. For example, females are at higher risk of developing diseases such as obesity and diabetes that predispose them to AD^112^. Alternatively, or in addition, hormonal changes during menopause may predispose females to contract AD^109,112,113^. Several female-specific biological processes may increase the likelihood that females contract AD.
+
+Family history elevates the risk of AD^54,93,114^. Mutations in key genes predispose individuals to AD. Those include amyloid beta precursor protein (*APP*), presenilin 1 (*PSEN1*), and presenilin 2 (*PSEN2*), which contribute to early-onset types of AD. Other gene variants (e.g., *SORL1*, *TREM2*, *ABCA7*, and apolipoprotein E4: *APOE4*) and long non-coding RNA gene variants contribute to AD^54,57,58,73,93,104,115–118^. One gene called *APOE* comes in several different alleles, including *APOE2*, *APOE3*, and *APOE4*. *APOE3* is neutral and is the most common allele (observed in 75% of the population); *APOE2* reduces the risk of AD, but *APOE4* is associated with an increased risk of AD^119^ and late onset of the disease^16,120^. Female carriers of *APOE4* have a higher risk of developing AD than male carriers^121^. *A**POE*4 is the ancestral *A**PO**E* allele and is similar in sequence to the common *A**POE* allele in companion animals and non-human primates^17,122,123^. A commonly studied gene called *APP* plays an integral role in forming plaques and tangles because *APP* produces a transmembrane protein, which gives rise to amyloid-β (Aβ) peptides. *APP* mutations lead to abnormal amounts of Aβ^54^ and brain plaque formation^54,86,93,116,124^. Much effort has focused on manipulating *APP* levels in rodent models to characterize how *APP* leads to plaque formation and cognitive deficits.
+
+## Down syndrome informs the etiology of AD
+
+Individuals with Down syndrome are at high risk of developing AD. Down syndrome patients have a third copy of chromosome 21 and a concomitant third copy of the *APP* gene^93^, which produces large amounts of amyloid-β (Aβ) peptides. Individuals with Down syndrome exhibit many of the AD phenotypes present in the general population, but they do so on an accelerated timeline relative to general AD patients (Supplementary Data 2, 3^125^; Supplementary Methods). Variation amongst individuals with Down syndrome informs the role of *APP* in the development of AD.
+
+Individuals with Down syndrome accumulate relatively mature Aβ deposits (Aβ42 > Aβ40), microgliosis, astrocytosis, neuritic dystrophy, and neurofibrillary tangles early on, and they possess brain atrophy, especially in the amygdala, hippocampus, and the white matter^126^. Together, these phenotypes resemble those of AD patients from the general population^126,127^. Unlike general AD patients, however, individuals with Down syndrome and AD demonstrate behavioral and personality changes not clearly observed in other AD populations^126^. What is most striking is the early timeline of AD in Down syndrome (Supplementary Data 2, 3^125^).
+
+Individuals with Down syndrome are at much higher risk of developing AD, and at relatively early ages. The average onset age of AD in Down syndrome is in the mid 50s with a mean age of death set to late 50s^128^. As much as 80% of Down syndrome patients with AD have plaques and tangles in their brain by their 40s and these can appear in the teens^126^ (Supplementary Data 2). The third copy of the APP gene on chromosome 21 is associated with increased and accelerated rates of plaques and tangles.
+
+Some individuals with Down syndrome have a translocation that eliminates the *APP* gene in the replicated portion of the gene, and these individuals are less likely to develop the neuropathologies associated with AD than those who do not have such a translocation. Together, these data strongly indicate that the triplication of the APP gene is responsible for the development of AD in Down syndrome^127,129^, and more generally that APP gene is a factor in the development of AD.
+
+### The established model systems: limitations for the study of AD
+
+Mice have been the model system of choice for studying AD (Fig. 3). In support of this claim, we text-mined NCBI and we extracted the number of articles with keywords containing species names (e.g., *Mus musculus* Fig. 3a, b). We also performed another search, including the species name and the term Alzheimer’s disease, to track the preferred species of study in AD (Fig. 3c). According to these data, mice have been the preferred model system of AD for decades. Indeed, much effort is focused on genetic manipulations of APP in mice to recapitulate aspects of AD. Genetic manipulations are needed to make mice suitable for AD study because mice do not naturally exhibit many aspects of AD. In the following sections, we explore the history of rodents, primates, and companion animals as model systems of human biology. We evaluate how each of these model systems age, whether they age similarly to humans, and we note that companion animal models spontaneously show many similarities to AD.Fig. 3Mice and rats are the most frequently studied model systems.On NCBI, we used species names as keywords and we extracted the number of articles linked to each species. **a** In 2021, rats and mice were utilized substantially more than other model systems. **b** This has been the case for multiple decades. **c** We repeated the search with species name and Alzheimer’s disease as keywords. These data show that mice and rats are the dominant models to understand AD.
+
+## A review of mice, rats, and primate model systems
+
+### Traditional mice and rat model systems: strengths and limitations
+
+Rodents have traditionally been used as model systems to investigate the molecular basis of AD. The Norway rat was the first mammal bred as a laboratory model over 170 years ago^130^. Rodent species have been selected as models because they are mammals (rodents shared a common ancestor with humans approximately 87 million years ago^131^) with similar brain features, including a six-layered neocortex (Fig. 1c). Further advantages in research include a short reproductive cycle, thriving in captivity, and being easy to manage^130,132^. In the wild, rats and mice occupy a wide diversity of environments and live among humans. Mice and rats are common household pests. Norway rats, in particular, are associated with the spread of diseases such as the Bubonic plague, so the use of these species has been seen as a redemption for lost human lives^133,134^. While any species could serve as a laboratory model^135^, rats and mice are the only mammals specifically mentioned by the NIH as model organisms^136^. Past research efforts focused on the use of traditional model organisms to facilitate studies on the same species. Our understanding of the role of the hippocampus and entorhinal cortex in spatial memory was founded on research in rodents to identify how specific environmental coordinates correspond to the firing of specific hippocampal place cells^137^ and geometrically positioned entorhinal grid cells^138^. Rodent models have also implicated the role of the hippocampus in social memory^139^.
+
+## Neuropathologies in mice and rat model systems
+
+Despite the widespread usage of rodents, naturally occurring plaques and tangles have generally been challenging to detect in these species so far, suggesting they could be absent^70,140,141^, nor do they exhibit dramatic gross brain loss in old age as humans do^142^. Transgenic mice have been engineered to overexpress beta and tau to recapitulate the biological hallmarks of the disease in humans^143,144^. Some of these models produce human Aβ above physiological levels, leading to a great abundance of senile plaques^143^; however, many fail to develop NFTs and neuronal loss unless mutant tau is also introduced. This limitation is true for the McGill transgenic rat model of AD since NFTs and the component tauopathy are not present^70^. Other models include Aβ-overexpressing Tg344-AD rat models, which do express NFTs formed from endogenous pTau^144^, as well as recurrent HSV-1 infections in mice, which induce both plaques and pTau^145^. Many of the rodent models studied are either adults or at a relatively early stage of life, so age-related changes are sometimes neglected from consideration in these investigations^146^.
+
+## Nonhuman primates as closely related model systems: strengths and limitations
+
+Non-human primates (NHPs) have also been studied in the context of AD^147,148^. The reason for selecting NHPs as model species is because their brain structure and function more closely resemble humans than rodents (Fig. 1c^19,149^). Humans share a common ancestor with all other primates approximately 74 million years ago, with rhesus macaques approximately 29 million years ago, and with our closest relatives – the chimpanzees and bonobos – approximately 6.5 million years ago^131^. Several aspects of the cerebral cortex are shared between humans and rhesus macaques (Fig. 1c; who are both catarrhine primates) to the exclusion of rodent models. Their cerebral cortex is large and folded (gyrified; Fig. 1c). NHPs and humans share similarities, which include their visual systems with an enlarged visual cortex and differentiated frontal and posterior parietal association areas (related to social cognition and dexterity). In addition, NHPs are useful biomedical models of humans due to their behavioral similarities to humans^19^. Among NHPs, rhesus monkeys have been used as models for 130 years, with advantages, including easy maintenance in captivity and rapid development. They thrive in urban areas and have the largest range of any NHP^150^. Their behavior is better studied than any other NHP, particularly through the documentation of a free-ranging island colony that has been studied for 85 years^150^.
+
+There are certainly some disadvantages to using primates as model organisms. No primate species — unlike cats, dogs, and rodents — have been domesticated^151^; however, most primates used in UK research are usually born in captivity^21^. Since these animals have genetic and behavioral adaptations to wild versus domesticated conditions, they have more complex living requirements that create practical (and ethical) issues when keeping them in captivity^151,152^. Despite being the species most closely related to humans, chimpanzees and other great apes (e.g., gorillas, orangutans) are not conventional model organisms and are challenging to maintain in captivity. Nonhuman primates can carry zoonotic diseases (e.g., B virus^153^). Moreover, nonhuman primates share with humans slow development, large bodies, and a dependency on social relationships^154^. Indeed, chimpanzees who experience social and sensory deprivation can develop aimless motions and self-injurious behavior, which can be summarized as stereotypical behaviors^155^. These behaviors are aggravated by physical restraints^156,157^. Self-injurious behavior has also been linked to early separation from mothers in infancy in apes and macaques^157^; this behavior can also develop spontaneously in macaques, whereas this idiopathic development is not typical in rodents^158^. Other monkeys, such as African green monkeys, can display aggressive behaviors, creating challenges when housing them with conspecifics^152^. Furthermore, there are heightened ethical issues with using great apes as models due to their proposed ‘personhood’^159^, which is partly based on their cognitive abilities, such as self-awareness. Unlike dogs and cats, they recognize their reflection in a mirror^160^. Also, great apes have advanced verbal and non-verbal communication^161^ as well as good working and long-term memory^148,154,162,163^. Also, wild populations are not readily available due to their endangered status^164^.
+
+## Neuropathologies in nonhuman primates
+
+Similar to aged humans, NHPs develop Aβ deposits; nevertheless, they lack the profuse manifestation of NFTs typical of AD^16,165^. Among NHPs, cercopithecid species develop senile plaques but rarely develop neurofibrillary tangles^16^. However, this depends on the age of the individual. Specifically, rhesus macaques have an age-related increase in tau phosphorylation (pTau)^166^. Further aggregation of this pTau into NFTs has been observed in particularly aged rhesus macaques, as was the case for a 38-year-old individual who developed NFTs in their entorhinal cortex^147^. Although other cercopithecid species, such as African green monkeys^167^ and cynomolgus monkeys^168^, have occasionally demonstrated NFT-like structures, these have been revealed not to exhibit the biochemical and morphological traits of AD.
+
+Only a few individual NHPs have been observed to have deposits and co-occurring tangles, and this has largely been restricted to chimpanzees. The first case was an obese 41-year-old chimpanzee with high cholesterol who displayed both Aβ plaques and NFTs^169^. Also, only a few have been found to have pathology to the degree observed in humans with AD. For example, a 57-year-old chimpanzee was found to have phase 4 Aβ plaques as well as stage V NFTs^82,170^. That individual was part of a study of twenty chimpanzees aged 39–62 years old, of which only three of the other chimpanzees had NFTs and Aβ deposits co-occurring, but these were within the first phase of the pathology^170^. In contrast, humans clinically diagnosed with AD would typically have phase 3 Aβ deposits and above^171^. These chimpanzees were all younger than the 57-year-old male, suggesting that age, as well as obesity/high cholesterol, are large risk factors in the emergence of the disorder.
+
+One hypothesis is that macaques rarely develop neuropathologies associated with AD because they do not live sufficiently long for these neuropathologies to become manifest. AD pathology, as well as brain atrophy, is difficult to observe in NHPs^16^. Age-related brain atrophy occurs in primates but appears to be relatively modest (e.g., macaques, chimpanzees^142^), though variation in atrophy exists across primate species (e.g., macaques, chimpanzees). Some authors report small (e.g., 5% change) to no significant difference in brain size across the lifespan in chimpanzees^172–174^, but brain atrophy may be pronounced in aged mouse lemurs^67^. Developing new approaches to extend the lifespan of NHPs and other animals falls within the realm of veterinary medicine and may inform what biological programs are products of an extended lifespan and which are unique to humans.
+
+## Companion animal model systems: suitability for AD study
+
+Human brain aging is characterized by brain atrophy, memory, locomotor impairments, and an increased risk of developing certain diseases (e.g., AD). Still, many age-related changes have been observed in rodent and primate models, but some of them have been elusive in rodent and primate models^175^. Rodents and NHPs may not live sufficiently long for some age-related diseases to manifest. Accordingly, rodent and NHP models may not clearly show plaques, tangles, and brain atrophy because their lifespans are shorter than humans. In contrast, companion animals have relatively extended lifespans and exhibit evidence of brain pathologies, atrophy, and cognitive dysfunction. We overview the evidence supporting companion animals as models of aging.
+
+## Evidence of lifespan extension in companion animals
+
+Humans exhibit an extended lifespan contrasted to great apes. Accordingly, biological processes evident in humans may not be observed in great apes simply because the extended lifespan in humans unmasks biological processes not seen in great apes^176^. A recent study aligned ages across the lifespan of humans and chimpanzees^176,177^ (Fig. 4a–c). Age alignments were possible in humans and chimpanzees across prenatal and postnatal stages, but there was no clear age alignment between humans and chimpanzees past 50 years of age; this is because most chimpanzees die in their 40s, which roughly equates to humans in their 50s (Fig. 4c). Accordingly, chimpanzees would not live sufficiently long to recapitulate biological processes that occur in humans in their 50s or later. Broad surveys across mammals are needed to inform which model systems may live sufficiently long lifespans to be useful as model systems of geriatrics.Fig. 4There are evolutionary modifications in lifespans across species.A past study extracted 137 time points from multiple scales of organization, including (**a**) anatomy, transcription, and growth, to generate cross-species age alignments across the lifespan of (**b**) humans and chimpanzees^176,177^. These analyses showcase a surprising degree of similarities between humans and chimpanzees. **c** Shortly after birth, humans are similar in age to chimpanzees in their first year of life. Similarly, a teen human roughly equates to a teen chimpanzee, and a human in their 50s roughly equates to a chimpanzee in their 40s. Most chimpanzees do not live past their 40s such that there is a phase of life in humans with no clear counterpart in chimpanzees. The extended human lifespan relative to other NHPs calls for the need to find species that show a relatively extended lifespan akin to humans to be useful models of geriatry. Figures are modified from^176,177^.
+
+Many age-related changes occur in elderly humans. Past work identified nine biological processes as hallmarks of aging^178^. Some of these are genetic changes that emerge from genomic instability leading to the increased tendency for mutations and telomere attrition (i.e., the graduate loss of caps at the end of chromosomes). Epigenetic changes, the loss of proteostasis (i.e., the process of regulating proteins), dysregulations in nutrient sensing, mitochondrial dysfunction, cellular senescence, exhaustion in stem cells, and altered intercellular communication are all important factors in aging^178^. More work is needed to compare the biology of humans and other animals at these different scales and to identify how rates of aging in these biological processes have changed across species^179–181^.
+
+We compared the maximum lifespan of different species to assay which, if any, of the model systems possess an extended lifespan relative to their close counterparts (Fig. 5). We used the AnAge database to compare maximum lifespans across hominids, carnivores, traditional model systems, and farm animals (Fig. 5). We compared the maximum lifespan of the domesticated species relative to others within their genus. We pooled the average maximum lifespan per genus, and we divided the domesticated animal’s lifespan relative to the average lifespan within their genus. A score greater than 1 indicates that the model system has an extended lifespan relative to other members of their genus (Fig. 5a). The lifespan is extended in humans relative to great apes, as expected given studies translating ages across these primate species^176^. Of interest, companion animals, especially cats, exhibit a large relative increase in maximum lifespan compared to others in their genus (Fig. 5c). The relative extension in cat lifespan rivals that observed in humans. Commonly studied model systems such as mice, macaques, marmosets, and farm animals such as sheep and cattle exhibit, at best, a modest increase in relative maximum lifespan measured against members within their genera (Fig. 5d–g^182^). Broad comparative analyses such as these highlight that companion animals, especially cats, may be ideal model systems for human aging because of their relatively extended lifespan and the likelihood of an extended period in which to age. In line with this, demographic research has demonstrated that the life expectancy of pet companion animals is increasing^75^, which could explain why a substantial number of cats have been reported to have NFTs compared with NHPs^75^.Fig. 5Maximum recorded lifespans across great apes, companion animals, and other domesticated animals.**a** We pooled the average maximum lifespan per genus and divided the lifespan of each species of interest versus the average lifespan per genus. A score above 1 indicates that the domesticated species has an extended lifespan relative to other species in their genus (**a**). Evidently, humans (**b**) possess an extended lifespan relative to others in their genus (**b**). Interestingly, cats (**c**) have also evolved a relatively extended lifespan compared to others in their genus. Macaques (**d**), rodents (e.g., mice) (**e**), and farm animals (**f**, **g**) have not evolved such long lifespans relative to their wild counterparts. These data are from the AnAge database^182^.
+
+## Evidence for brain atrophy in companion animals
+
+Emerging evidence shows that brain atrophy in dogs is as extensive as it is in humans. Here, we considered lateral ventricles (Fig. 6a), which comprise a space that houses cerebrospinal fluid, to measure brain atrophy. The size of the lateral ventricles relative to brain volume expands in beagles with old age, especially beyond 12 years of age^183^ (Fig. 6a, b). In humans, lateral ventricles also enlarge. These age-related changes become particularly evident at around 60 years of age and beyond (Fig. 6c^184^). Although the relative increase in lateral ventricles observed in dogs resembles humans, we have yet to rigorously quantify brain atrophy across humans and companion animals after aligning ages. This work is important because it will enable us to assay the extent to which age-related changes in the brain volume of companion animals map onto humans.Fig. 6Brain atrophy is evident in aged dogs and in humans.We used the relative volume of lateral ventricles as an index of brain atrophy in dogs (**a**, **b**) and humans (**c**^184^). **b** Plots of the relative lateral ventricles volume versus chronological age show that the lateral ventricles expand with age, but that this largely occurs at 12 years of age and beyond in dogs and at (**c**) 60 years and beyond in humans^183^. The relative lateral ventricle volumes in humans were computed by dividing the lateral ventricle volumes by the cerebral cortex gray, white matter, and lateral ventricle volumes^184^. The dog brains are redrawn from a structural MRI scan of a 2- and 15-year-old beagle available at: https://veteriankey.com/the-effects-of-aging-on-behavior-in-senior-pets/.
+
+## Cognitive dysfunction syndrome in companion animals
+
+Normal aging is linked to diminished sensory, memory, and cognitive capacities in humans as in companion animals^61,64,65,185–188^. Some aged cats and dogs demonstrate a decline in exploratory behavior and social interaction, and engage in excessive vocalizations^61^. If behavioral and cognitive deficits sufficiently impact daily activities, companion animals can be diagnosed with cognitive dysfunction syndrome (CDS)^64^. Among client-owned pets, the prevalence of CDS may be as high as 22% in geriatric dogs^60,63^ and 50% in geriatric cats^189,190^. These studies further report that CDS is likely underdiagnosed, so the prevalence of age-related cognitive deficits may be higher than that which is frequently reported. These relatively high rates of CDS suggest that it is commonplace for companion animals to suffer from a suite of behavioral and cognitive changes at later stages of life.
+
+Companion animals share many similarities to humans, but cats and dogs also belong to two taxonomic groups with different cognitive and behavioral capabilities^59,69,191–194^. Accordingly, several assessment tools have been designed to monitor age-related impairments in humans and companion animals, but some behavioral and cognitive domains are species-specific. A CDS checklist is used to assess age-related impairments in companion animals^46^. The CDS evaluates whether animals vocalize excessively, house soil, possess abnormal sleep-wake cycles, are anxious, show decreased exploratory behavior, and whether time spent grooming is unusual^46,60,62,64,71,187,195,196^. The rate of occurrence and the number of these behavioral deficits are used to assess the amount of cognitive deficit in companion animals^46,62,71,187,195^. Assessment tools such as the Montreal Cognitive Assessment in humans focus on capturing impairments in emotion, sleep, memory, language, and cognitive capacities^69^. Many of these assessments focus on language and thus assay human-specific cognitive capacities. There are clear behavioral and cognitive impairments at late stages of life in companion animals as in humans, but some of the age-related impairments are species-specific.
+
+## Brain pathology in companion animals
+
+Aged companion animals exhibit brain pathologies that mirror those found in humans, including plaques, tangles, vascular cerebral amyloid angiopathy (CAA), myelin disruption, lipofuscin, neuronal vacuoles, axonal degeneration, and abnormalities in cholinergic neurons^3,47,71,75,88,197^. There are discrepancies in the conclusions drawn from studies, likely due to the small sample sizes and variation in ages. We document the presence of plaques and tau pathologies in the brain and blood vessels in humans, companion animals, and wild carnivores to identify common themes across studies (Supplementary Data 2, 3^125^). Our broad survey shows that the brains of aged companion animals, as well as wild carnivores and humans, harbor Aβ plaques, tau pathology, and CAA (Supplementary Data 2^125^; Fig. 7)^45,48,75,197–202^. Despite these similarities, there are important species differences in the composition and spatial patterns of plaques and tangles (Supplementary Data 3^125^). We discuss the composition, location, and time course of plaques and tau pathology in different species.Fig. 7AD associated neuropathologies are found in cats and dogs.We computed the relative number of studies reporting a pathology in humans (**a**, **b**) cats (**a**), and dogs (**b**). These percentages show that many AD-related neuropathologies in humans are also observed in companion animals. Only studies explicitly searching for the presence of **a** neuropathology are considered in these statistics. **a**, **b** Studied pathologies include phosphorylated tau (pTau), NFTs, CAA (i.e., meningeal, and cortical), as well as early and mature plaques. A relatively large percentage of studies report plaques, pTau, and NFTs in cats and dogs. **a** However, no mature plaques are reported in cats. These data are from Supplementary Data 2^125^. Age distribution of neuropathologies onset across humans and carnivore species (**c**–**e**), including companion animals and **e** other carnivore species. We considered the age of onset of early (**d**) and Amyloid-β e as well as tau pathology (**f**) and tau (**g**) from data provided in Supplementary Data 2. We included all listed ages (i.e., ≥ and >), as well as data from individuals with Down syndrome. The age of early and mature plaques, as well as neurofibrillary tangles onset in Down syndrome is relatively early and their ages are highlighted with a star (Supplementary Data 2). These graphs show that companion animals and humans possess plaques and tangles. **e** Interestingly, mature plaques are not observed in cats and other feliforme species (see names in light grey), but they are observed in dogs and their close relatives (e.g., polar bears, sea lions). These boxplots show upper and lower quartiles, maximums, and minimums. We used adult (and not juvenile) ages to assess neuropathology onset for wild Tsushima leopard cats.
+
+There are similarities but also differences in plaque composition across species. Cats produce mostly small and granular Aβ deposits composed of Aβ1–42 peptides, which are similar to early-stage plaques^47,200^. In comparison, humans most often produce larger Aβ plaques composed of Aβ1–42 as well as Aβ1–40^71,88^ residues, particularly in the dense cores of mature plaques. In humans, the deposition of Aβ1–42 (but not Aβ1–40) increases with age in AD^203,204^. Dogs also have more Aβ1–42 than Aβ1–40 in their plaques, but mature plaques, with their dense Aβ1–40 cores, appear less commonly than in humans^205–211^. However, like humans, both cats and dogs have Aβ1–40 deposits in cerebral and meningeal blood vessels (Supplementary Data 2^125,200,212^) that may lead to CAA^213^.
+
+Not all species exhibit mature plaques (Fig. 7). Dogs, but not cats, possess mature plaques. Sea lions^211,212^ and bears^206,214^, which are wild caniforme species and are more closely related to dogs than to cats, exhibit mature plaques (Fig. 7a). In contrast, wild feliforme species, including Tsushima leopard cats and cheetahs, which are more closely related to cats than to dogs, lack mature plaques^215,216^ (Fig. 7). Therefore, there may be a phylogenetic effect for the existence of plaque maturity between dogs and other caniformes versus cats and other feliformes.
+
+Although some studies have found that Aβ deposits in the vasculature system (e.g., meningeal vessels) in advance of the neocortex in dogs^211^ and cats^217^, other studies have found the opposite order for dogs^207,209^ and cats^211^. This suggests that CAA and neocortical plaques may have different pathways^218^. Furthermore, the occurrence of CAA in feliforme species appears sparse compared with primates and dogs^200,210,216,219^. For example, there was no Aβ42 deposited in the cerebral vasculature of Tsushima leopard cats (Fig. 7). Some elderly cheetahs have Aβ42 and Aβ40 deposits in cerebral capillary vessels, but these deposits are not observed in meningeal vessels^216^. Variation in deposits within the meningeal systems could indicate species variation in the amyloid clearance system^216^.
+
+It is unclear whether the temporal progression of plaques is similar across humans and companion animals. In humans, it is typical for AD plaques to cluster in the hippocampus before spreading to the entorhinal cortex and temporal cortex (part of the neocortex^91^), but the opposite order can occur in Down Syndrome and in non-AD neurodegenerative diseases^220,221^. Cheetahs^216^, sea lions^80^, and dogs show an early pattern of progression in the neocortices – including the frontal, temporal, and parietal cortices. Cats initially have diffuse plaques in the neocortex as well as intracellular Aβ deposits in the hippocampus^143^. Similar to humans, dogs have plaque deposits starting in a few layers of the neocortex (layers IV, V) before reaching outer layers across the cerebral cortex^8,75,88,202,207^. The spatial pattern of progression in plaques has yet to be worked out in more detail.
+
+Plaques generally emerge at late stages of life in humans as in companion animals (Fig. 7 and Supplementary Data 2^125^). Plaques usually first occur in humans between the ages of 50–70 though they may occur earlier, as is the case in Down syndrome (Fig. 7d, e). In dog studies, half or more of the samples possess early-stage plaque pathology by age 15^206,208,210,211,222,223^. Similarly, early-stage plaques are frequently reported by age 16 in cats^71,76,88,200,217,224^ (Supplementary Data 2^125^) though cats or other feliformes do not possess mature plaques^50,59,156^ (Fig. 7e and Supplementary Data 2^125^). Despite these species’ differences, a common theme is that the onset of plaques occurs at relatively late stages of life in different species.
+
+The presence of hyperphosphorylated tau (pTau) and NFTs has been controversial in non-human species, but a large percentage of studies report tau pathology (Fig. 7a, b). These pathologies have been found in older cats and dogs. Tau pathology can be detected by AT8 and PHF1 antibodies. There have been mixed findings when testing the AT8 antibody (Supplementary Data 2^200,225–228^). NFTs and pTau emerge by age 40–60 s in humans (Fig. 7 and Supplementary Data 2^125^). Around half of the dog and cat studies found aggregates of pTau forming NFTs in cats from 15 years or older^229^ and in dogs from as early as 13 years^207,223^ (Fig. 7 and Supplementary Data 2^125^). Tau pathology occurs in similar regions across species. In humans, tau pathology spreads across the entorhinal cortex, hippocampus, and neocortex^206,220,221^. A similar spatiotemporal pattern occurs in dogs^223,225^, cats^143,229^ and wild carnivores, including cheetahs^216^ and polar bears^230^. Collectively, our broad survey highlights that pTau and NFTs, as well as plaques, occur in cats and dogs.
+
+## Insights from veterinary medicine for human health in old age
+
+There are many variables to consider in selecting a model system. The study of human diseases has relied extensively on mice as model systems (Fig. 3). Rodents are easy to maintain and breed. However, many rodent studies fail to translate into meaningful treatments for humans because of the large gap that exists across the biology of mice and humans and because they are used to model human biological processes that are not clearly observed in humans. Rodents do not recapitulate many aspects of the aging process, and this is particularly pernicious in an age-related disease such as AD.
+
+The characteristics that make mice ideal for studies (e.g., a short lifespan, large litter size, genetic manipulations) are those characteristics that sometimes make them inappropriate for expanding our understanding of human biology, physiology, and disease. Standardizing studies across human and veterinary animal work would enhance our ability to advance both fields. While experimental work on laboratory dogs is sometimes used in biomedical research and has provided important information on neurobiological mechanisms in controlled conditions^231^, our primary aim is to point to the many untapped opportunities to synergize with veterinarians who are focused on enhancing the quality of care in companion animals. Investigating how companion animals age with us in our homes could provide many insights into the field of aging. Our focus on companion animals is in line with initiatives such as the Dog Aging Project^232^.
+
+For many years, animals have been used to detect health hazards. For example, miners in the early 20th century brought canaries into coal mines to determine whether there were toxic exposure levels to gasses (e.g., carbon monoxide, methane) that would impact the health of miners. Similarly, companion animals have been used as indicators of toxins (e.g., mercury, asbestos, lead) in our diet or in the household, also referred to as ‘animal sentinels’^204,233–236^. Companion animals are particularly useful for studying the environmental impact on human aging because they have relatively short lifespans, and they share similar environments with humans. They can therefore provide many insights into the long-term consequences of environmental hazards. Large-scale projects such as the Dog Aging Project will offer many opportunities to study how lifestyle, toxins, and medications impact the biology and cognition of aged companion animals^237–239^.
+
+### New possibilities from the integration of veterinary and human medicine
+
+The integration of human and veterinary medicine offers new possibilities for studying a range of biomedical problems, including aging. Companion animals consist of a pool of diverse genetic breeds, raised in a wide range of different environments, and span a wide range of habitats. This diverse pool of systems permits studying natural variation, which can be integrated with manipulations in the laboratory and in client-owned homes. For example, we can track companion animals with similar genotypes (e.g., siblings) raised in different environments (e.g., indoor versus outdoor cats) to evaluate the impact of the environment on health and disease incidence. Studying siblings raised in different environments would permit parcellating the impact of environment and the interaction between genotype and environment on health. We can also harness the broad variation across cat and dog breeds to find markers of CDS and identify conserved and species-specific risk alleles of aging. The variation and diverse range of possibilities in the laboratory and in client-owned homes stand in contrast with the limited variation within inbred mouse lines, which are constrained to be housed in the laboratory. Clinical trials in companion animals are already implemented to evaluate the impacts of drugs on companion animals, and we can integrate these findings with human medicine.
+
+Dogs have been bred from relatively small founder populations, and they have been selected based on appearance, leading to high levels of inbreeding. Many breeds are also predisposed to inherited diseases^240–243^, but this can be less the case in some mixed breeds^244^. Evidently, the variation in allele frequency and differential predisposition to disease across dog populations makes them well-suited to uncover the genetic basis of diseases. Less recognized, however, is that dogs would be well-suited to investigate the genetic basis of individual variation in aging. The shortened lifespan of dogs relative to humans means that they are well-suited to study the interaction of genetics and environment on rates of aging, and that they can act as animal sentinels for human health.
+
+## Insights from experiments in veterinary medicine
+
+There are clinical trials focusing on dietary modifications and drugs aimed to improve the standard of living in aged companion animals^62^. Dietary modifications have led to improvements in animal cognition (e.g., disorientation, changes in social interactions, reduced house-soiling behavior), with improvements observed within a few weeks after the onset of treatment^245,246^. Drugs, including antidepressants (e.g., selegiline hydrochloride), are given to animals suffering from cognitive dysfunction. These drugs are effective in alleviating sleep patterns and other cognitive traits^247^. Although more work is needed to evaluate how these interventions can improve cognitive capacities integrating these experimental interventions in humans and companion animals could accelerate progress in characterizing and treating diseases of aging.
+
+## Diverse model systems reform AD research
+
+There is an open question as to whether AD is a disease that is an emergent property of aging or is a distinct condition that occurs during aging^16^. The field of comparative neuroscience is ideally poised to tackle these challenging possibilities. In our broad survey, we identified many commonalities in lifespan and age-related brain and behavioral changes in humans and companion animals^248^. Companion animals and humans share similar environments; they exhibit relatively extended lifespans and possess evidence of brain atrophy, plaques, and tangles in aging. Rodents, NHPs, and species from other taxonomic groups may show some of these traits, but our study highlights that companion animals and humans represent an extreme end of this spectrum in possessing a relatively extended lifespan and neuropathologies like those observed in AD.
+
+The relative lifespan in humans and some companion animals (i.e., cats) is considerably extended relative to their wild counterparts. Several traits, including lifespan extension, similar environments, brain plaques and tangles, and other neuropathologies, are evident in companion animals and humans. These correlated traits are interesting because they may indicate the existence of underlying relationships between lifespan, brain atrophy, and neuropathologies. In this case, lifespan extension in companion animal models and humans is concomitant with brain atrophy and neuropathologies. Perhaps, lifespan extension and other traits are linked together in these distant taxonomic groups.
+
+A broad taxonomic survey across mammals is needed to assess general links between lifespan, aging, and the emergence of AD-like phenotypes. While humans and companion animals display a constellation of similar traits such as lifespan extension and aging, there are also cases where lifespan is not necessarily concomitant with the emergence of brain atrophy and neuropathologies. Indeed, some species are long-lived but may not necessarily exhibit AD-related brain pathologies as observed in humans. For example, naked mole rats have some resistance to the toxic effects of plaques, which is unlike that in humans^249^. On the other end of the spectrum, spawning salmon, which are not known to be a long-lived species, exhibit brain pathologies that resemble those observed in AD^250^. Accordingly, aging and longevity may stimulate the likelihood of developing AD-like neuropathologies, but each species may age differently. The environment plays a role in the development of disease incidence. Shared environments across humans and companion animals, with increased medical care and subsequent survival to old age, as well as more sedentary lifestyles, may provide the conditions for the development of these neuropathologies at late stages of life.
+
+## Conclusion
+
+AD is sometimes considered uniquely human, in part because the hallmarks of this disease are seldom naturally occurring in rodent models and NHPs. Also, these traditional model systems tend not to survive into old age. The presence of neuropathologies in companion animals casts doubt on the notion that AD is unique to humans. Our broad survey highlights instances of convergent evolution with humans and companion animals, which have evolved a relatively extended lifespan and concomitant age-related changes in brain atrophy and neuropathologies. Therefore, companion animals are well-suited as models of human aging. The issue of identifying the most appropriate model system for a disease is one of many examples where increased integration across veterinary and human medicine could synergize to address long-standing problems in biomedical fields. A One Health perspective can enhance our comprehension of neurodegenerative diseases shared by humans and our companion animals as they age alongside us.
+
+### Reporting summary
+
+Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
+
+## Supplementary information
+
+Supplementary Information
+Description of Additional Supplementary Files
+Supplementary Data 1
+Supplementary Data 2
+Supplementary Data 3
+Reporting Summary

fulltext/md/PMC10291811.md

@@ -0,0 +1,112 @@
+---
+pmcid: "PMC10291811"
+pmid: "37365660"
+doi: "10.1186/s12917-023-03633-0"
+title: "Postural stability measures in healthy miniature Dachshunds obtained using a pressure mat and a force platform: a validity and reliability study."
+journal: "BMC veterinary research"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Postural stability measures in healthy miniature Dachshunds obtained using a pressure mat and a force platform: a validity and reliability study
+
+## Abstract
+
+### Background
+
+Miniature Dachshunds have a high prevalence of neurological and musculoskeletal diseases potentially affecting their balance. The postural stability of dogs in quiet standing is an indicator of postural control and can aid in diagnosing and monitoring lameness and other pathologies affecting balance. Measures of centre of pressure (CoP) can be obtained from force and pressure platform systems to evaluate postural stability, however the two systems have not been compared and the latter has not been validated in dogs. The aims of this study were to assess the validity and reliability of using a pressure mat compared to a force platform and report normative values of CoP measures in healthy miniature Dachshunds. Forty two healthy miniature Dachshunds of smooth, long and wire-haired breed types stood still on a pressure mat (Tekscan MatScan®) placed on a force platform and the two systems were synchronised. Maximum anterior-posterior (AP) and medial-lateral (ML) ranges, sway path and 95% area of a best-fit ellipse were computed. Bland-Altman plots and coefficients of correlation assessed validity; intra-class correlation coefficients (ICC) assessed inter-test reliability for both systems. Non-linear regression analyses were used to describe the relationship between CoP and demographic measures.
+
+### Results
+
+Strong correlations for AP range, ML range and 95% ellipse area and moderate correlation for sway path were found between the two devices. ICC showed good reliability (0.75–0.90) for AP range and moderate (0.5–0.75) for ML range and the 95% ellipse area for both devices. Sway path reliability was excellent (> 0.90) with the force platform but moderate with the pressure mat. Age was positively correlated with balance (inversely correlated with all measures except sway path), while weight explained 94% (force platform) and 27% (pressure mat) of the variance in sway path.
+
+### Conclusions
+
+Pressure mats can be used to obtain valid and reliable measures of CoP and replace use of force platforms. Older (non-senior) and heavier (non-obese) dogs show better postural stability. Clinical examinations should include the use of a range of CoP measures when assessing postural balance, while accounting for the effects of age and body weight.
+
+## Introduction
+
+Interest in studying postural control and stability of dogs date back to the early 1900s [1]. Studying postural stability can reveal important information about the biological mechanisms responsible for postural control [2] and the responses to visual and other perturbations [3]. More recently, measures of postural stability have been used clinically to diagnose lameness in dogs [4, 5, 6] and ponies [7], and to measure the effects of ageing on postural control using adults and senior dogs [8].
+
+Postural stability can be assessed during quiet standing using measures of the centre of pressure (CoP) in bipeds [9] and quadrupeds [4]. CoP is the point at which the ground reaction force is applied. Movements of the CoP are related to accelerations of the body’s centre of mass and are how movements of the centre of mass are controlled [10]. Force platforms are considered to be the gold standard measurement tool in human medicine to obtain measures of CoP and their reliability have been assessed in multiple studies [11, 12, 9]. Previous studies have used metrics obtained from the movements of the CoP (position, velocity and acceleration) in studies concerned with the assessment of balance [13].
+
+Pressure mat systems such as Tekscan MatScan® are portable and easy to use, making them a preferred measurement tool in clinical settings. Recently, these systems have been used to obtain CoP measures in quiet standing in humans [14, 15]. However, there is limited evidence of their validity in obtaining these measures, with only a single study testing its validity against force platforms in humans [16] and a preliminary study in lame dogs [4]. The main difference between the two measurement systems, is that whilst force platforms measure the three components of the ground reaction force, pressure mats typically only measure the vertical component. Therefore, it is important that measurements of CoP obtained from pressure systems are assessed for their validity and reliability before their use in clinical studies.
+
+The aim of this study was to assess the validity and reliability of obtaining CoP measures of postural stability in sound dogs using a pressure mat (Tekscan MatScan®) as compared to force platform measurements. A secondary aim was to collect normative measures of CoP from a non-lame group of miniature Dachshunds for use of future studies that include clinical populations. This is a population of clinical interest because of their high prevalence of spinal problems.
+
+## Methods
+
+A sample size calculation using G*Power 3.1.9.7, with α level of 0.05, a power of 0.8, effect size of 0.4, and based on a correlation analysis suggested that a minimum of 34 dogs were needed for the analysis. Forty two healthy miniature Dachshunds dogs (30 females) of different breed type (16 smooth-haired, 16 long-haired and 10 wire-haired) participated in the study after being recruited through the Dachshund Breed Council UK. Dogs had to be above 6 months of age to be included and they were excluded if they had an existing problem that could cause pain, lameness or postural imbalance.
+
+Owners completed a health form for their dogs and the dogs were examined by a veterinary surgeon blinded to the information provided by the owners. Dogs were found to be free of lameness or other signs of pain based on a visual lameness assessment by the veterinary surgeon and confirmed by force platform data of their gait. The recruited dogs had a mean age of 4.3 (± 3.2) years old, weight of 5.4 (± 1.4) kg, Body Condition Score (BCS) of 3.4 (± 0.9) out 5 [17], length of 26.9 (± 2.8) cm, height at the withers of 19.2 (± 1.9) and a length-to-height ratio of 1.40 (± 0.11).
+
+Measurement trials of the dogs in quiet standing were obtained in the presence of their owners by one of the researchers (AS) in the Human Movement Laboratory of the University of Surrey, as the dogs stood still on a pressure mat (MatScan®, Tekscan, South Boston MA, USA) running at 60 Hz placed on top of a force platform (AMTI, Watertown MA, USA) running at 100 Hz. The two systems were synchronised and calibrated as per the instructions of the manufacturers, the force platform was zeroed at the start of the session and the pressure mat was calibrated using the body weight (50 kg) of one of researchers at the start of each session. Five standing trials of 10 s durations were collected. Trials where the dog was seen to move the body or the head, moved its tail, unloaded one of its limbs or attempted to sit were discarded before analysis. Figure 1 shows one of the dogs in quiet standing during a measurement.
+
+Fig. 1One of the dogs in quiet standing during a measurement on a synchronised pressure mat (Tekscan Matscan®) on top of a force platform
+
+Measures of CoP were computed from the data obtained using the two measurement systems, these measures were; maximum anterior-posterior (AP) range, maximum medial-lateral (ML) range, sway path and 95% area of a best-fit ellipse. Data from the pressure mat was resampled to 100 Hz and a re-analysis of these measures was conducted and compared to data obtained using the original frequency of 60 Hz.
+
+Bland-Altman plots and Pearson’s coefficient of correlation were used to assess the validity of the CoP measures obtained using the pressure mat compared to those obtained using the force platform. Intra-class correlation coefficients (ICC) were used to assess the inter-test reliability of the CoP measures obtained using the two devices [18]. ICC estimates and their 95% confidence intervals were calculated based on a mean-rating (k = 5), absolute agreement, 2-way mixed effects model [19].
+
+ANOVA tests were used to investigate differences in age, weight, height, length-to-height ratio and BCS between males and females and between the three breed types (smooth-haired, long-haired and wire-haired) and Pearson’s correlations were used to look at relationships between these demographic measures. ANOVA tests were also used to investigate differences in the CoP measures obtained using both devices with sex and breed type as independent factors and age, weight, height, length-to-height ratios and BCS used as covariates. Linear and non-linear regression (logarithmic, quadratic, exponential, power) analysis was used to assess the relationship between CoP measures and demographic measures (age, weight, height, BCS and length-to height ratio).
+
+SPSS statistical package version 26 (SPSS Inc, Chicago, IL) was used to run all statistical tests.
+
+This study has been checked against the ARRIVE 2.0 Essential 10 list.
+
+## Results
+
+### Validity and reliability of CoP measures obtained using a pressure mat (***Tekscan Matscan®)***
+
+Bland-Altman plots (Fig. 2) show a good level of agreement between the two devices for AP range, ML range and the 95% ellipse area but a large difference in sway path. There is a positive bias for all measures, with greater values for measures obtained from force platforms. This difference is relatively small with relatively narrow agreement limits for AP range (0.99 ± 0.41 cm), ML range (0.69 ± 0.62 cm) and the 95% ellipse area (0.83 ± 1.61 cm^2^), but is quite large for sway path (670 ± 167 cm). There is also an obvious trend in the sway path measure that suggests that the difference between the measures increases with an increase in the mean. The resampling of the pressure mat data to 100 Hz resulted in a negative effect on the correlations between the devices and therefore the data with the original sampling frequency are presented here. The correlation analyses showed that there was a strong correlation for AP range, ML range and 95% ellipse area, but only moderate correlation for sway path between the two devices as shown in Table 1.
+
+Fig. 2Bland-Altman plots showing the difference in the centre of pressure (CoP) measures (Anterior-Posterior AP range, Medial-Lateral ML range, 95% area of best-fit ellipse and sway path) between the force platform and pressure mat (Tekscan Matscan®). A positive bias indicates a greater mean for the measure obtained from the force platform
+
+Table 1Means and standard deviations of centre of pressure (CoP) measures obtained using a force platform and a pressure mat; and Pearson correlation coefficients between the two systems and their p-valuesCoP MeasureForce PlatformPressure MatPearson CorrelationMean (SD)Mean (SD)**R**p-value**Anterior-posterior (AP) range (cm)**2.45 (0.75)1.46 (0.74)**0.843****< 0.001****Medial-lateral (ML) range (cm)**3.45 (0.98)2.76 (1.07)**0.816****< 0.001****95% area of best-fit ellipse (cm**
+^**2**^
+**)**4.64 (3.56)3.80 (3.70)**0.900****< 0.001****Sway path (cm)**816.38 (186.61)146.78 (44.89)**0.490****0.001**
+
+The ICC reliability of the two measurement systems was similar for the AP range, ML range and the 95% ellipse area, with both systems showing a good reliability (between 0.75 and 0.90) for AP range and a moderate reliability (between 0.5 and 0.75) for the ML range and 95% ellipse area. The reliability of the sway path measure was excellent (above 0.90) when obtained from the force platform but only moderate with the pressure mat measures (Table 2).
+
+Table 2Intra class coefficient (ICC) values and the lower and upper bound of the 95% confidence interval for the centre of pressure (CoP) measures obtained using a force platform and a pressure mat. AP (anterior-posterior), ML (medial-lateral)CoP MeasureForce PlatformPressure MatICC95% confidence intervalICC95% confidence intervalLower boundUpper boundLower boundUpper bound**AP range****0.792**0.6570.885**0.768**0.6810.871**ML range****0.704**0.5100.837**0.618**0.3680.788**95% area of best-fit ellipse****0.707**0.5190.836**0.726**0.5480.848**Sway path****0.986**0.9770.992**0.718**0.5340.844
+
+### Normative CoP measures for healthy miniature dachshunds
+
+The results show that there was a difference in height and weight between sex, with males having a greater weight (p = 0.011, 3.54±1.0 kg vs. 3.41±0.90 kg) and height (p = 0.003, 20.50±2.62 cm vs. 18.64±1.15 cm). For breed type, there was a difference between the groups in age (p = 0.021) and height (p = 0.017), where the smooth-haired dogs were older and taller than dogs in the other two groups (Table 3). Therefore, any differences in CoP measures between breed types would need to be interpreted in light of an existing difference in these measures.
+
+Table 3Showing the means for the demographic measures and the computed centre of pressure (CoP) measures obtained using the force platform and the pressure platform Tekscan Matscan® for males and females, and the different breed types (smooth, long and wire-haired) and the results of the ANOVA tests, significance set at p < 0.05. AP (anterior-posterior), ML (medial-lateral). *p < 0.05, **p < 0.01, ***p < 0.001SexBreed typeMalesFemalesp-valueSmooth-hairedLong-hairedWire-hairedp-value**Age (years)**2.98 (3.83)4.72 (3.14)0.1693.20 (3.25)6.17 (3.19)3.23 (2.42)**0.021*****Weight (kg)**3.54 (1.01)3.41 (0.90)**0.011***5.67 (1.07)5.43 (1.87)4.88 (0.94)0.374**Height at the withers (cm)**20.5 (2.62)18.64 (1.15)**0.003****20.22 (2.28)18.46 (1.01)18.60 (1.54)**0.017*****length-to-height ratio**1.42 (0.15)1.40 (0.11)0.6931.38 (0.12)1.46 (0.08)1.37 (0.14)0.088**Body condition score - BCS**3.54 (1.01)3.41 (0.90)0.6873.56 (0.85)3.64 (1.08)3.00 (0.71)0.204**CoP Measure****Force Platform (AMTI)****AP range (cm)**2.91 (1.11)2.27 (0.46)0.1172.74 (0.84)2.22 (0.66)2.38 (0.66)0.246**ML range (cm)**3.83 (1.41)3.30 (0.73)0.2543.71 (1.16)3.16 (0.75)3.48 (0.99)0.347**95% area of best-fit ellipse (cm**
+^**2**^
+**)**7.01 (5.27)3.69 (2.02)0.3106.37 (4.71)3.12 (1.79)4.29 (3.56)0.089**Sway path (cm)**753 (191)842 (182)**0.026***758 (138)848 (236)860 (155)0.103**CoP Measure****Pressure platform (Tekscan Matscan®)****AP range (cm)**1.95 (0.90)1.26 (0.57)0.1791.78 (0.88)1.27 (0.57)1.26 (0.61)0.367**ML range (cm)**3.23 (1.32)2.57 (0.91)0.1582.93 (1.22)2.68 (0.90)2.61 (1.14)0.904**95% area of best-fit ellipse (cm**
+^**2**^
+**)**5.74 (4.64)3.02 (3.00)0.6885.36 (5.06)2.67 (2.13)3.11 (2.25)0.387**Sway path (cm)**137 (57)151 (40)0.166126 (39)170 (45)142 (39)**0.006****
+
+There were no differences between males and females and between different breed types for all CoP measures obtained with both measurements systems with the exception of sway path (Table 3). The results for sway path were inconsistent between the force platform measurements and the pressure mat. Force platform measures suggested that females had lower sway path values than males (adjusted p = 0.026) but this was not shown by the measures obtained by the pressure mat (adjusted p = 0.103). On the other hand, the pressure platform mat measures for CoP sway path suggested a difference by breed type (adjusted p = 0.006), whilst force platform CoP sway path measures did not show a difference (adjusted p = 0.166).
+
+Correlational analysis between the demographic measures showed that age and weight are independent variables, but that height (r = 0.582, p = < 0.001) and BCS (r = 0.363, p = 0.018) were correlated with weight. Regression analysis showed that there were relationships between age and the AP range, ML range and 95% Ellipse area measures obtained by both devices. These relationships were best described using non-linear equations (see Fig. 3). An increase in age resulted in a decrease in these three measures but had no effect on sway path. Suggesting that older adult dogs were more stable. Interestingly, weight had a relationship with sway path but not with the other CoP measures, where an increase in weight resulted in a reduced sway (Fig. 4). Weight was found to explain 94% of the variance in sway path measured by the force platform (p < 0.001, using a quadratic regression model) and 27% in sway path measured by the pressure mat (p < 0.001, using a power regression model), these relationships and the expressions are shown in Fig. 4.
+
+Fig. 3Regression equations showing the relationship between Anterior-Posterior range, Medial-Lateral range, 95% ellipse area and age for the CoP measures obtained using the force platform (FP) (black circles and continuous black line) and pressure mat (Tekscan Matscan®) (PM) (grey triangles and dashed line)
+
+Fig. 4Regression equations showing the relationship between sway path and weight for the CoP measure obtained using the force platform (black circles and continuous line) and pressure mat (Tekscan Matscan®) (grey triangles and dashed line)
+
+## Discussion
+
+The study aimed to assess the validity and reliability of postural stability parameters measured with the pressure mat (Tekscan Matscan®) compared to the force platform - commonly used to obtain CoP measures - as well as to present a normative database for CoP measures of miniature Dachshunds from both systems which can be used in future canine studies with patient populations.
+
+The results showed that CoP measures obtained from the pressure mat system have good agreement and excellent correlations with those obtained using the force platform, with the exception of sway path. Sway path measures were different in magnitude and there was only a good correlation between the two systems. The large difference seen in the sway path measure is expected because sway path is largely dependent on the frequency of capture, which was different for the two devices. Although the difference in the frequencies of capture may go some way in explaining this difference, it does not seem to be the only cause; resampling of the pressure data to the same frequency did not improve the correlations. It is therefore likely that differences in sway path were not just due to the frequency of capture but are also related to inherent differences in the sensitivity of the devices and the type of data obtained (3D for force platform with CoP accuracy of < 0.5 mm vs. vertical force for pressure mat of a measuring resolution of 1.4 sensel/cm^2^ and a range of 34.5–86.2 N/cm^2^).
+
+Both devices had comparable reliabilities (as assessed using ICC) for CoP measures except for sway path. This measure when obtained using a force platform has an excellent between-trial reliability, whilst all other measures (including sway path measured by the pressure mat) had moderate or good reliabilities between trials. Whilst the study showed comparable reliabilities of the two systems, one advantage of using the pressure system is that it allows the researcher to easily spot when the animal off-loads one of the limbs which would have a considerable effect on CoP measures. This is possible because the system provides measures of the force cells loaded by each paw, whilst a force platform only provides a measure of the movement of the overall ground reaction force vector, making these measurement errors more difficult to spot.
+
+Sway path was the only measure that was found to be positively related to the dog’s weight. Regression equations used to describe this relationship showed that weight can explain 94% of the difference in sway path when obtained using the force platform and 27% when obtained using the pressure mat. The results are in line with findings in horses [20] and humans [21], and with a study on dogs [8] where weight and height are found to be positively correlated with stability when participants are not obese, as in our study. One way to reduce the effect of this on clinical interpretation is to normalise the measure to the weight of the animal, this has also been suggested by Chiari et al. [21].
+
+The other CoP measures had significant relationships with age. Regression analysis show that age can explain between 17 and 30% of the difference in these CoP measures (using non-linear equations). Similarly, age was positively correlated with stability (reduction in AP range, ML range and 95% Ellipse area). The positive relationships between stability (as assessed by CoP measures) and age may appear to contradict the current knowledge based on human research that suggests that CoP measures are sensitive to changes in stability caused by age, such as deficits in vision, proprioception or muscle strength [9] and based on senior dogs defined as being older than 75% of their lifespan [8]. In the latter study, some of the tested senior dogs also presented with a degree of joint pain. It is important to note that in our study, we used a healthy adult population, not necessarily senior dogs, and excluded dogs with known lameness or pain. Furthermore, the study did not aim to study the effect of senior age on CoP measures and stability.
+
+The results showed that on the whole, there were very little to no differences in the CoP measures between males and females and between the breed types. Where there were differences, these were only found in sway path and were inconsistent between the two measurement systems.
+
+Finally, there were a number of limitations to data presented in this study. Firstly, the CoP measures reported here are based on standing on the pressure mat directly with no cover. Previous canine studies have demonstrated that ground reaction force data is affected by the surface [22, 23] and human studies have also suggested that a different balancing mechanism is employed on different surfaces [24]. Therefore, this should be taken into account when using the normative data from this study. Another limitation of the study is related to the length of the trials. Measurement trials were kept to 10 s only for convenience, this is similar to the study by Manera et al. [6] but is shorter than the 20 s used in other canine studies [4, 5]. Future canine studies should assess the effect of trial length on the reliability of the CoP measures in order to reach a consensus within the scientific community.
+
+## Conclusions
+
+Pressure mat systems can be used to obtain valid and reliable measures of CoP and can replace the use of force platforms. However, care should be taken in interpreting differences between populations where these measurements are not obtained using the same technology, this is particularly true for sway path (or measures derived from sway path). It is important to include a range of CoP measures in clinical assessments when evaluating balance in dogs, and comparisons to healthy/normative values should account for expected variations caused by age and weight.

fulltext/md/PMC10298135.md

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+---
+pmcid: "PMC10298135"
+pmid: "37372311"
+doi: "10.3390/genes14061131"
+title: "Genome-Wide Integrative Transcriptional Profiling Identifies Age-Associated Signatures in Dogs."
+journal: "Genes"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Genome-Wide Integrative Transcriptional Profiling Identifies Age-Associated Signatures in Dogs
+
+## Abstract
+
+Mammals experience similar stages of embryonic development, birth, infancy, youth, adolescence, maturity, and senescence. While embryonic developmental processes have been extensively researched, many molecular mechanisms regulating the different life stages after birth, such as aging, remain unresolved. We investigated the conserved and global molecular transitions in transcriptional remodeling with age in dogs of 15 breeds, which revealed that genes underlying hormone level regulation and developmental programs were differentially regulated during aging. Subsequently, we show that the candidate genes associated with tumorigenesis also exhibit age-dependent DNA methylation patterns, which might have contributed to the tumor state through inhibiting the plasticity of cell differentiation processes during aging, and ultimately suggesting the molecular events that link the processes of aging and cancer. These results highlight that the rate of age-related transcriptional remodeling is influenced not only by the lifespan, but also by the timing of critical physiological milestones.
+
+## 1. Introduction
+
+The dog’s significant role as a model species for comprehending genome evolution, population genetics, and the genes responsible for complex phenotypic characteristics can be attributed to its abundant genetic resources and advancement in genome sequencing [1]. Studies have shown that over 11 years of age, 28% of dogs have one or more diseases [2], and olfactory epithelial cells degenerate from 14 years of age or older [3]. Aging in dogs also leads to cognitive abnormalities and similar aging lesions and treatment methods as humans [4], which illustrates that dogs are an important model for studying aging and other related diseases [5]. The longevity of different dog breeds varies significantly, with larger breeds such as the Great Danes and Bouviers typically having a shorter lifespan of around 6 or 7 years, while smaller breeds such as the Chihuahuas and Toy Poodles can live up to 15 years. Dog breeds also show variation in age-associated diseases. For instance, Bernese Mountain Dogs exhibit a cancer mortality rate over 10 times higher than that of other dog breeds, even after adjusting for age and sex [6]. Further investigations specific to this breed have revealed variations in the *CDKN2A/B* gene region that are associated with histiocytic sarcoma, the most prevalent type of tumor observed in Bernese Mountain Dogs [7]. Dalmatians display an elevated risk of urolithiasis, characterized by the formation of urinary tract stones due to the increased excretion of uric acid [8]. Pedigree analyses of Dalmatians have confirmed the heritability of this trait [9], and linkage mapping has identified the *SLC2A9* gene as the locus responsible for hyperuricemia in Dalmatians [10]. Similar to humans, as dogs age, their susceptibility to various diseases and mortality rates increases exponentially [11]. The observed variations in mortality risk and causes of death among these dog breeds highlight the valuable role of dogs as a robust model for investigating both the genetic and environmental factors that influence the aging process, as well as the underlying molecular mechanisms involved. These valuable insights have the potential to enhance our comprehension of aging, not only in canines but also in all mammals, including humans [12].
+
+Biological aging should not be perceived solely as a gradual deterioration of organisms. Rather, organisms possess inherent mechanisms of self-repair that aim to counteract physical decline and maintain homeostasis [13]. However, for reasons that remain poorly elucidated, these self-repair mechanisms gradually diminish over time, rendering aged organisms susceptible to damage, deterioration, and mortality [14]. Aging is a complex process that involves the accumulation of harmful changes in cells and tissues [15]. The elderly are at an increased risk of developing severe illnesses such as cancer, cardiovascular disease, senile dementia, and type-II diabetes due to the gradual deterioration of several physiological processes that occur with aging [16]. In the last decade, a mounting body of literature has indicated that aging is associated with gradual alterations to epigenetic information in both dividing and nondividing cells [17]. These modifications manifest at different levels, including reductions in the bulk levels of the core histones, changes in the patterns of histone posttranslational modifications and DNA methylation, the substitution of canonical histones with histone variants, and modifications in noncoding RNA expression, throughout organismal aging, and replicative senescence [18]. Ultimately, the cumulative effects of these epigenetic changes during aging is a shift in the local accessibility to the genetic material, leading to dysregulated gene expression, reactivation of transposable elements, and genomic instability [19]. With the advent of the Next-Generation Sequencing (NGS) technique, the high-throughput study of DNA methylation and gene expression has become feasible, providing valuable insights into the biology of aging and associated diseases [20].
+
+More specifically, although the DNA sequence is subject to the forces of natural selection over many generations, certain aspects of the genome can undergo changes within shorter timeframes, including within an individual’s lifespan. One notable type of change involves chemical modifications of the DNA sequence itself, specifically patterns of DNA methylation, which have been recognized as a characteristic feature of aging [21].
+
+The initial investigations into the relationship between DNA methylation changes and aging were conducted on various organs and life stages of humpback salmon [22]. These studies revealed a significant decrease in levels of 5-methylcytosine (5mC) during ontogenesis, highlighting the evolutionary significance of DNA methylation. Subsequently, these findings were extended to mammals [23]. Notably, the highest levels of 5mC were observed in embryos, followed by a gradual decline. In rodents, it was demonstrated that the decrease in 5mC was inversely correlated with their lifespan [24]. Additional support for the association between global DNA hypomethylation and senescence was provided by experiments conducted on fibroblast cultures [25]. Furthermore, it was observed that the DNA methylation inhibitor 5-aza-2′-deoxycytidine substantially shortened the lifespan of cells [26], further strengthening the link between global DNA hypomethylation and the aging process. Through the application of epigenetic clocks, these DNA methylation patterns can accurately predict the chronological age and capture elements of the biological age that have implications for mortality risk beyond what chronological age alone can reveal [27]. In model organisms, these epigenetic clocks have demonstrated responsiveness to interventions aimed at extending lifespan, such as caloric restriction, making them valuable biomarkers for assessing how interventions, genotypes, and environmental exposures influence the rates of aging [28]. For example, Thompson et al. found that the age-related correlation between DNA methylation level and age remains conserved across syntenic regions in the genomes of these canid species, as well as in more distantly related mammalian genomes, including humans [29]. A study by Wang et al. further reported the enrichment of epigenetic alterations on extensively conserved modules of developmental genes, where methylation levels revealed a prevailing correlation with advancing age [30].
+
+The transcriptome refers to the entirety of mRNA molecules produced by an individual cell or a group of cells [31]. It serves as a repository of information regarding cell populations, which is encoded in the genome. Consequently, profiling the transcriptome plays a crucial role in deciphering functional genomic elements [32]. Transcriptomics, or the examination of the transcriptome, aims to achieve three main objectives: (1) categorizing all transcripts, (2) analyzing gene expression variations, and (3) quantifying transcript levels across different conditions [33]. Subsequently, the functional characterization of these genes and the transcripts of interest derived from these transcriptome analyses thereby contribute to validating their biological significance [34]. The analysis of age-related alterations in gene expression has been conducted in various organisms, including yeast, flies, worms, rats, mice, and humans [35,36]. These studies have consistently demonstrated a decline in gene expression is significantly associated with the electron transport chain, protein translation, and growth signaling during aging. Conversely, there is an increase in the expression of genes related to innate immunity, inflammation, and DNA damage [37,38]. Nevertheless, it is important to acknowledge that changes in these pathways account for only a fraction of the numerous genes that exhibit altered expression with age, and most observed changes remain unexplained [39]. In human studies, the transcriptional profiles of peripheral blood samples obtained from large cohorts have been utilized to develop expression signatures that exhibit a high correlation with chronological age, regardless of ethnic backgrounds [35]. These expression signatures can serve as transcriptional clocks, effectively indicating an individual’s age [40]. It is worth noting that the construction of these signatures involved iterative correlations with chronological age, aiming to create molecular clocks, rather than unraveling the mechanisms by which gene expression influences the aging process [41]. Notably, different research groups have generated signatures that exhibit limited overlaps with each other and with epigenetic clocks, which are also derived from correlation analyses [42]. However, the strong correlation between these signatures and the chronological age suggests that transcriptomes contain age-related information that remains elusive to our current understanding [43].
+
+In this work, our primary goal was to identify the molecular signatures of aging using transcriptomic profiles. Such signatures, which we define as distinguishing features of molecular changes with age, may be associated with, and play a biological role in the physiological decline that characterizes aging. Therefore, we collected publicly available RNA-sequencing data and investigated the differentially expressed genes involved in aging from various breeds of dogs to find the global genetic markers that could be applied across different dog breeds. Previous research by Wang et al. compared methylation status as an indicator of aging, but it is unclear whether methylation differences affect gene expression levels, and it may only apply to Labrador retrievers [30]. At present, the impact of age-related alterations in DNA methylation on the transcriptome or functionality of a given tissue or cell remains largely unknown due to the limited available data. Therefore, we also collected the whole-genome bisulfite sequencing data to navigate the genetic markers that could be used in combination with the methylation index genes. We contextualized our findings within the backdrop of established age-related physiological and biochemical alterations. Our results additionally expose an array of previously unidentified transcriptional changes in genes, processes, and functions that may serve as prospective targets for future investigations and provide enhanced insights into the relationship between transcriptional changes and the aging process at varying levels.
+
+## 2. Materials and Methods
+
+### 2.1. RNA-Sequencing Data and Analysis of Differentially Expressed Genes
+
+RNA-sequencing data from 15 healthy dogs (15 different purebreds) and 25 healthy mice (C57BL/6J strain) with age information provided were sourced from the NCBI (National Center for Biotechnology Information) [44] and are presented in the Supplementary Tables S1 and S2. For both studies, RNA was extracted from whole blood following the manufacturer’s instructions. For dogs, the age of each sample was standardized in accordance with the life expectancy of the breed detailed by the American Kennel Club (AKC). Trimmomatic (version 0.39) [45] was employed to eliminate low-quality reads. We used the STAR alignment tool to map the reads to the Canfam3.1 (dog) and GRCm38.p6 (mouse) reference genomes, respectively [46] and the read count per gene was estimated using featureCounts [47]. RSEM (ver. 1.3.1) [48] software was used to estimate gene expression levels (counts per million, CPM). The gene expression profile data were then analyzed using the “limma-voom” and “affy” packages of R software (version 4.1.0) [49,50]. By treating normalized age as a continuous variable, differential expression levels were then quantified to identify genes with *p*-value < 0.05, |log2FC| > 1, which were considered statistically significant. Genes with maximal TPM values less than 1 were subsequently excluded.
+
+### 2.2. Age-Dependent DNA Methylation
+
+Whole-genome bisulfite sequencing data from 104 Labrador retrievers’ whole blood samples were retrieved to estimate the methylation values for all the CpG sites [30]. The sequencing reads were demultiplexed and subjected to quality assessment using FastQC [51]. Next, TrimGalore [52] was employed to remove the first 4 bp of the reads, and the trimmed reads were then aligned to a bisulfite-converted reference genome using Bismark (version 0.14.3) [53] under the parameters “-score min L, 0, −0.2”. The methylation status of the CpG sites was determined using MethylDackel (version 0. 2. 1) (Ryan, 2017) [54].
+
+### 2.3. Gene Set Enrichment Analysis
+
+The FUMA GWAS program [55] was used to identify a significant over-representation of genes with particular functional categories, such as the biological process of gene ontology [56] The adjusted *p*-value of 0.05 was used as the criterion for statistical significance.
+
+### 2.4. Figure Visualization
+
+The Manhattan plot was created using the ‘qqman’ and the ‘ggplot2’ package of the R program (version 4.2.2).
+
+## 3. Results and Discussion
+
+### 3.1. Differentially Expressed Genes and Enriched Pathways Associated with Aging
+
+To investigate whether certain genomic regions have a differential impact on changes in transcript profiles during aging, and to examine whether there is a global dysregulation of gene expression, we leveraged the publicly available RNA-sequencing data of 15 dog samples with their age information. These dogs represented 15 different breeds (Rat Terrier, Rottweiler, Miniature Pinscher, Dachshund, Shih Tzu, Collie, Dachshund, Cocker Spaniel, Staffordshire terrier, Australian Shepherd, Standard Poodle, Pekingese, Boxer, Shih Tzu, and Labrador Retriever, respectively), ranging from small to big dog breeds. As the common and surrogate measures of chronological ages from different dog breeds, we thereby weighted the age of each dog with the lifespan for the corresponding breed (Supplementary Table S1), using data from the American Kennel Club. We aimed to find a significant correlation between the gene expression level and the measure of aging.
+
+As a result, a total of 154 genes (77 downregulated and 77 upregulated DEGs, respectively) were found to have changed expression levels significantly with age in dogs (Figure 1 and Supplementary Table S2). Analysis of gene ontology (GO) enrichment for DEGs indicated a significant overrepresentation of the categories associated with “hormone transport” (*SLCO1C1*, *EXOC3L1*, *NOS2*, *FKBP1B*, *PRKCE*, *SPP1*, *TNF*, *SLC44A4*, and *SLC16A2*, respectively) and “regulation of hormone levels” (*SLCO1C1*, *EXOC3L1*, *NOS2*, *FKBP1B*, *PRKCE*, *SPP1*, *LRAT*, *TNF*, *SLC44A4*, *PCSK5*, and *SLC16A2*, respectively) (Table 1). Various factors that impact the rate of aging in mammals have been identified [57]. These include well-known endocrine signals such as insulin, growth hormone (GH), insulin-like growth factor-I (IGF-I), and thyroid hormones (THs). Each of these signals play a crucial role in regulating metabolism and influences an organism’s ability to adapt to different environmental conditions [58]. In the process of aging, there is a notable alteration in the secretion patterns of hormones generated by the hypothalamic–pituitary axis [59]. Additionally, the axis becomes less responsive to negative feedback mediated by end hormones. Numerous population studies have reported that serum thyroid-stimulating hormone (TSH) levels increase with normal aging [60,61]. These pathways together may explain the aging mechanisms, particularly the hormonal response to aging, which is governed by altered gene expression and regulation.
+
+Initial observations have demonstrated a specific link between reduced thyroid function and an extended lifespan in both small and large mammals, including humans [62,63]. As a result, higher levels of serum thyroid-stimulating hormone (TSH) and/or low levels of serum free thyroxine (T4) have been associated with an increased life expectancy, indicating a significant role of thyroid hormones (THs) in the aging process [64,65]. Older rats with similar thyroid-stimulating hormone (TSH) levels as younger rats were found to have a lower DIO1 activity, and research in rodents has indicated that levels of the thyroid hormone (TH) transporter MCT8 in the liver are decreased in aging individuals, suggesting a change in TH responsiveness with age [66]. Additionally, there is evidence suggesting that THs may have a decreased ability to activate certain post-receptor mechanisms involved in thyroid function in older individuals [67]. Therefore, it is noteworthy to mention that genes associated with thyroid hormones *(SLCO1C1*, *SLC7A5*, and *SLC16A2)* showed differential expression patterns (as shown in Supplementary Table S2) in the experiment. For example, the TH-activating deiodinase (DIO2) and the transmembrane transporter of thyroxine (*SLCO1C1*) are the primary thyroid hormone transporters at the blood-brain barrier. Reduced thyroid hormone transport across the blood-brain barrier contributes to neurological deficits [68].
+
+It has been observed that epigenetic modifications during the aging process target extensively preserved developmental gene pathways, wherein methylation levels typically increase with age [69,70]. Our findings also revealed that age-dependent genes were significantly overrepresented in terms such as “embryo development” (*GPR161*, *ETNK2*, *ITGA8*, *PDZD7*, *CDON*, *PRICKLE1*, *HOXC4*, *HOXC4*, *PAF1*, *APOB*, *ADA*, *PDGFC*, *FBN2*, *TNF*, *SLC44A4*, and *PCSK5*, respectively) and “embryonic organ development” (*ITGA8*, *PDZD7*, *HOXC4*, *HOXC4*, *ADA*, *PDGFC*, *FBN2*, *TNF*, *SLC44A4*, and *PCSK5*, respectively) (Table 1). Although the biology of aging has traditionally been regarded as distinct from that of development [71], the compelling correlation presented in this study suggests that they are intricately linked. This finding supports the notion that certain elements of the aging process may represent a continuation of these developmental processes, rather than as a discrete phenomenon.
+
+In dogs, there is a wide range of cancer incidence rates, with the group aged 9–11 years or older exhibiting rates 14 to 32 times greater than those aged 3 years or younger [72]. Most epigenetic changes that occur during the aging process are believed to be programmed and restricted to a subset of tissue cells, potentially making them susceptible to tumorigenesis [73]. Methylation is thought to hinder the plasticity of cell differentiation mechanisms, and likely contributes to the development of tumors [74]. We note that the most significant DEG from our analysis was found in *PARD6* (*p*-value = 2.62 × 10^−4^), as the expression level was found to have increased with age (Figure 2). It is typically found in many cancer tissues, including breast and epithelial cancer, and is considered to play a role in preventing the development of cancer cells by inhibiting cell proliferation and restricting the epithelial cell cycle [75,76].
+
+### 3.2. Conserved Signatures across Mammals
+
+To investigate the conserved age-dependent transcripts across these species, we leveraged the publicly available RNA-sequencing data from mice and found 346 significant DEGs (146 downregulated and 200 upregulated genes, respectively) (Supplementary Tables S3 and S4), where we found a modest overlap of five genes (*ITGA8*, *KCNJ1*, *NCR1*, *SLC44A4*, and *PRICKLE1*) between two species. We noted that the common differentially expressed genes have been elucidated for their roles in tumorigenesis. The downregulation of *ITGA8* has been linked to unfavorable overall survival (OS) outcomes in individuals with clear cell renal cell carcinoma [77]. Conversely, in patients with an early relapse of multiple myeloma (MM), high levels of *ITGA8* expression can prompt epithelial–mesenchymal transition (EMT), resulting in increased migratory and invasive properties of the MM cells [78]. The *KCNJ1* gene plays an essential role in potassium balance, and its mutations are associated with several diseases such as prenatal Bartter syndrome and diabetes. The expression of *KCNJ1* suppresses tumor cell growth and promotes cancer cell apoptosis [79]. Studies have shown that patients with prostate cancer exhibit decreased levels of *NCR1*, and that prostate cancer cells themselves suppress the expression of *NCR1*, thus impeding their ability to identify tumor cells. Hypoxic conditions lead to a reduction in the expression of NKp46, ultimately compromising the ability of natural killer cells to effectively eliminate tumor cells [80]. When the expression decreases, the toxicity of natural killer cells against prostate cancer increases, and when the expression increases, it plays the opposite role [81]. In this study, we found the consistent directionality of expression changes between two species during aging in *ITGA8*, *SLC44A4*, and *KCNJ1*, while *PRICKLE1* and *NCR1* showed the opposite patterns. It is not necessarily the case that different species follow the same trajectory of changes as they age. Specifically, dogs exhibit rapid methylome remodeling during early life when compared to humans. Further investigations into this matter suggests that the rate of remodeling is not solely influenced by the lifespan of a species, but also by the timing of the crucial physiological milestones [70].
+
+### 3.3. Differentially Expressed Genes with Age-Related DNA Methylation Patterns
+
+Wang et al. characterized the methylomes of 104 Labrador retrievers to report the epigenetic modifications that occurred during the aging process [30]. Regarding gene promoters, hypomethylated CpGs are typically correlated with actively expressed genes that are constitutively expressed, while hypermethylated CpGs tend to be associated with genes that are either silenced or have low levels of expression [82]. We thus integrated the DNA methylation profiles with the transcriptome data to identify the genes that may have altered expression due to DNA methylation, those changes in gene expression that are linked to age-related changes in DNA methylation, and ultimately elucidate the epigenetic regulation of gene expression across a wider range of breeds. Only four genes (*SLC44A4*, *PLCB4*, *HSPB1*, and *PREX2*, respectively) showed concordant results between two independent analyses, and we noted that *SLC44A4* also revealed conserved gene expression changes during aging in mice analysis. *SLC44A4* has been demonstrated to cause deafness [83], and is significantly upregulated in various epithelial tumors, particularly in prostate and pancreatic cancers, resulting in antitumor responses [84]. Methyl groups bind to CpG islands and consequently contribute to the downregulation of gene expression [85]. Older dogs were found to encompass significantly hypomethylated CpG sites within *SLC44A4*, which may have been attributed to the increased gene expression level and a higher risk of developing cancer in aged dogs (Figure 2 and Figure 3). Age-associated DNA methylation changes in canids implicated similar gene ontology categories as those that are observed in humans, suggesting an evolutionarily conserved mechanism underlying age-related DNA methylation in mammals.
+
+Overall, however, we observed a limited correlation between the age-dependent DNA methylation patterns of Labrador retrievers and gene expression levels across the 15 breeds assessed, which may be possibly attributed to the distinct dog breeds exhibiting widely heterogeneous molecular mechanisms of aging and varying lifespans. The application of recent genomic methodologies has unveiled a more intricate relationship between methylation and gene expression [86]. It is now evident that genes linked to unmethylated CpG islands (CGIs) do not always exhibit expression, while genes associated with methylated islands can indeed be expressed. To further elucidate this complexity, future genomic investigations can be directed toward exploring the involvement of chromatin structures, as well as activators and repressors of gene transcription [87,88]. These experimental endeavors may be required for unraveling the underlying intricacies in the association between methylation and gene expression.
+
+### 3.4. Limitations
+
+Initially, our study employed RNA extracted from whole blood samples, a widely utilized source in which age-related alterations in leukocyte populations have been well-documented. Notably, prior investigations have established that certain T and B lymphocytes exhibit a decline in numbers as individuals age, which may have impacted our results. In a recent investigation conducted by Day et al., age-associated alterations in CpG DNA methylation discovered in various human tissues showed that hypomethylated CpG sites exhibited tissue-specific age-related changes [89]. Another study focusing on blood DNA also indicated a lack of predictive capacity for other tissues [86], emphasizing the necessity of studying more tissues to understand global methylation status throughout the lifespan. Perhaps more critically, it should be acknowledged that the sample size employed in this study was relatively modest, consisting of 15 samples. Undoubtedly, the construction of a more precise and reliable epigenetic and transcriptomic aging clock could be facilitated by utilizing larger sample sizes.
+
+The emergence of “-omics” technologies, which facilitate investigations into gene expression, chromatin modifications, metabolomes, and proteomes, offers a promising avenue to comprehensively characterize the molecular patterns associated with aging. In order to gain deeper insights into the contrasting factors that either promote or counteract the aging process, numerous studies have examined gene expression alterations across various age groups. Hence, rather than solely examining the biological function of genes exhibiting expression drift, it is perhaps more important to investigate their association with chromatin modifications or chromosomal location. Such analyzes can provide valuable insights into the underlying mechanisms that lead to the disruption of gene regulation. Consequently, future research endeavors should focus on developing novel analytical strategies for transcriptomes that consider the distinct biology of aging. These strategies should incorporate co-expression values, chromosomal location information, and histone modification data to distinguish between gene expression changes that are appropriately regulated and those that are dysregulated [39].
+
+## 4. Conclusions
+
+Our study conducted a comprehensive meta-analysis of aging systematically with the aim of identifying molecular mechanisms of aging. From analyzing gene expression data from various dog breeds, we have identified genes and cellular processes that are globally affected by aging at the transcriptional level. Our research has uncovered previously unknown transcriptional changes that occurred during the aging process. These genes and cellular processes can be targeted for future studies to develop aging biomarkers, and to better understand the transcriptional regulation that occurs during aging. Our results suggest that these molecular patterns represent degenerative processes and reflect the transcriptional responses of healthy cells to degeneration. However, the field of epigenomic studies focusing on aging in dogs, as well as other species, is still in its early stages, and further investigations involving larger cohorts of dogs are needed. By gathering more data from these expanded cohorts, researchers can potentially identify novel molecular mechanisms of aging that could be subjected to modifications or interventions. As our knowledge of the aging transcriptome continues to grow, integrating and analyzing data using meta-profiling and integrative approaches will become increasingly valuable for understanding the aging process.

fulltext/md/PMC10306367.md

@@ -0,0 +1,240 @@
+---
+pmcid: "PMC10306367"
+pmid: "37388194"
+doi: "10.1093/emph/eoad011"
+title: "Social determinants of health and disease in companion dogs: a cohort study from the Dog Aging Project."
+journal: "Evolution, medicine, and public health"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Social determinants of health and disease in companion dogs: a cohort study from the Dog Aging Project
+
+## Abstract
+
+Exposure to social environmental adversity is associated with health and survival across many social species, including humans. However, little is known about how these health and mortality effects vary across the lifespan and may be differentially impacted by various components of the environment. Here, we leveraged a relatively new and powerful model for human aging, the companion dog, to investigate which components of the social environment are associated with dog health and how these associations vary across the lifespan. We drew on comprehensive survey data collected on 21,410 dogs from the Dog Aging Project and identified five factors that together explained 33.7% of the variation in a dog’s social environment. Factors capturing financial and household adversity were associated with poorer health and lower physical mobility in companion dogs, while factors that captured social support, such as living with other dogs, were associated with better health when controlling for dog age and weight. Notably, the effects of each environmental component were not equal: the effect of social support was 5× stronger than financial factors. The strength of these associations depended on the age of the dog, including a stronger relationship between the owner’s age and the dog’s health in younger as compared to older dogs. Taken together, these findings suggest the importance of income, stability and owner’s age on owner-reported health outcomes in companion dogs and point to potential behavioral and/or environmental modifiers that can be used to promote healthy aging across species.
+
+## INTRODUCTION
+
+The social environment is one of the strongest predictors of health and mortality in many mammals, where exposure to less social adversity is associated with a lower risk of disease and death [1]. In humans, higher socioeconomic status (SES) can lead to access to more resources, including healthcare, and a more predictable, less stressful environment that is associated with lower morbidity and mortality [2, 3]. Additionally, social isolation—another component of the social environment—has a stronger association with mortality than heavy smoking, heavy drinking and lack of exercise, when controlling for SES [3, 4]. These sociality-health links appear to be deeply rooted in evolutionary history: having been identified in species ranging from mice to primates [5–7].
+
+How social adversity impacts older individuals is less clear and may vary based on timing, severity and perceived impact of exposure to adversity [8]. Environmental stability, which is composed of positive socioeconomic, built and natural environmental factors—such as higher household income, more social connections and access to greenspace, patterns overall health and disease risk in both early and late life [9]. In humans, urban environments with high residence turnover, high pollution levels and low economic status are associated with reduced physical mobility, increased disease risk and poor health outcomes [10] that can also worsen with age [11]. However, researchers are only beginning to explore how health and mortality associations with environmental factors vary across the lifespan in humans [12, 13]—knowledge that is necessary to optimize the distribution of resources and interventions to those who would benefit most. This gap in our knowledge is largely due to the long lives of humans and confounding factors that may be linked to other environmental factors (e.g. smoking).
+
+To address this gap, we can turn to animal systems with similar biology, recent co-evolutionary history and environments to humans, but compressed into a much shorter lifespan. Companion dogs (*Canis familiaris*) offer a unique opportunity to look across the lifespan at how and when aspects of the social and physical environment may alter aging, health and survival. Dogs have been shown to be a powerful comparative model due to our shared biology, shared environment and significantly shorter lifespan [14–18]. Similar to humans, the major causes of death and disease in older dogs are cancer, cardiovascular incidences and immune-mediated diseases—making them a particularly important model for understanding the biology of aging in mammals [18–20]. Dogs are also genetically and phenotypically diverse due to hundreds of years of selective breeding [21, 22], and this genetic history and architecture have also made them a powerful model for investigating the genetic factors underlying complex traits, including many human diseases—many of which are associated with aging [23, 24].
+
+Yet, despite our shared environments and passion for our companion dogs, we still know relatively little about the health impacts of the social environmental factors on companion animals, such as dogs. This gap in our knowledge is in large part due to a dearth of detailed social-environmental data available for large samples of dogs of all ages. We, therefore, have a limited understanding of how lived experiences might affect health and aging in dogs, which could: (1) inform how environmental factors impact human health and aging across the lifespan, and (2) help our companion animals live longer, healthier lives.
+
+In humans, older individuals are often more vulnerable to adverse risk exposures due to a decreased ability to recover from exposures (‘resilience’), and a limited capacity for withstanding these exposures (‘reserve’) [25, 26]. For example, older individuals who experience a stressor such as infection or surgery are at increased risk of adverse outcomes later in life [25]. This association extends to companion dogs, who have been shown to have poorer outcomes for relatively common conditions at older ages as compared to younger ages even when medical and surgical treatments are provided [27, 28]. Additionally, in humans, social adversity such as social isolation and undernourishment early in life can have far-reaching effects on development and, subsequently, on adult health, reproduction and survival [29–33]. It is likely that social adversity such as reduced social companionship, less time spent outdoors and other exposures to environmental risk factors identified in human populations can impact adult dog health and aging as well. In fact, it has been shown that early-life malnutrition in dog populations can lead to long-term behavioral effects, like showing increased levels of fear and aggression, in addition to affected physical health in adulthood [34]. Thus, broader research into the impacts of social adversity on health and aging in companion animals is warranted.
+
+Here, we examined factors related to the social environment of companion dogs to determine if there were associations with health and disease outcomes, as well as a measure of frailty (mobility), using data collected by the Dog Aging Project (DAP). We focused on these outcome measures because they all exhibit age-related changes (declines in health and mobility and increases in disease occurrences) in humans and other animals. In particular, mobility is a key phenotypic signature of frailty—the age-related increase of vulnerability to adverse outcomes. Mobility is associated with overall health [35, 36] and declines with age [37, 38] in both human and dog populations, suggesting that it can be used as a potential biomarker for aging and age-related disease risk. We hypothesized that exposure to higher levels of social environmental adversity in early adulthood would predict poorer age-associated health outcomes in this population of dogs, and that the strength of this effect would vary across the lifespan. Specifically, we predicted that dogs living in environments associated with adversity (i.e. low social integration, less stable environments and lower SES) would be less mobile, in poorer health and have more disease than more socially advantaged dogs within the population.
+
+## METHODS
+
+### Study population and recruitment
+
+The Dog Aging Project (DAP) is a community science project that aims to understand how genes, lifestyle and the environment influence aging and disease outcomes [39, 40]. Study participants are recruited to DAP through word-of-mouth, mainstream media and social media. For this study, we drew on the first year of survey data released on 10 May 2021, from 27,541 dogs completed by owners on or before 31 December 2020. Study data were collected and managed using REDCap (Research Electronic Data Capture) electronic data capture tools hosted through the DAP [41, 42].
+
+### Dog Aging Project survey data
+
+The Health and Life Experiences Survey (HLES) includes a number of small surveys that collect information about dog demographic characteristics, physical activity, environment, dog behavior, diet, medications and preventatives, health status and owner demographic characteristics. The DAP Environment Dataset contains geographically defined data from secondary sources pertaining to the dog’s external environment. Environmental data are based on respondents’ primary and (where applicable) secondary address information, provided in the HLES owner contact form. Environmental metadata was generated from the US Census Bureau (2019) and the 2015–2019 American Community Survey 5-year Public Use Microdata Samples. Respondent addresses are geocoded and linked to existing data that characterize various aspects of the dog’s external environment. A total of 56,285 Environment data records (two per dog) are included in the 2020 Curated Data Release, reflecting all study participants who became DAP pack members on or before 31 December 2020, and released on 10 May 2021 (*n* = 27,541). We selected only the data corresponding to the primary residence information for the year 2020. We removed participants under 2 years of age in order to only include dogs that are fully grown, following the American Animal Hospital Association life stages guidelines [43]. After applying all these criteria, 21,410 dogs remained for downstream analysis. We excluded variables that were less than 98% complete and redundant variables that captured similar metrics (e.g. swimming/aerobic activity which was captured by overall activity level). Variables that were excluded due to missingness either lacked census data or the question did not apply to the owner (i.e. potential follow-up questions associated with a specific type of home heating method).
+
+### Mobility
+
+We generated a composite metric that could roughly capture age-related decline without someone carrying out an in-person physical examination of the dog. This metric was calculated by taking the z-score for each activity-related variable in the HLES (activity level, average activity intensity, on-leash walk frequency, aerobic activity frequency, physical games frequency, average daily activity and on leash walk average) then assigning each dog a score that corresponded to the average of their z-score on the activity-related variables.
+
+### Disease instances
+
+The disease instance outcome metric was generated by summing the number of owner-reported diseases for each dog. We excluded diseases that did not have a severe effect on overall dog health as described in [44] and detailed in SI Table 5.
+
+### Stability
+
+The stability metric is a combined variable that encompasses the percentage of the population in a census tract that was in the same home 1 year ago, the percent of homes that are owned by the occupant, and the percentage of the population born in the USA. Each dog received a stability score calculated from taking the z-score of these variables and averaging them. These data were collected from the US Census Bureau (2019). Some of the variables included in this metric are human-centric measures that will also capture the stability of a dog’s local environment.
+
+### Factor analysis
+
+For this study, we focused our analysis on the responses to 33 survey questions in the HLES. These 33 questions were selected a priori based on their link to at least one of the axes of the social determinants of health (e.g. financial, environmental, social connection; see SI Table 2 and methods). We then reduced the 33 survey questions into a smaller set of latent variables using exploratory factor analysis and following well-established guidelines [45]. Briefly, we first performed parallel analysis to determine the number of factors to retain then conducted factor analysis using the Psych package (version 2.0.8) in R [46]. After determining the optimal number of factors to retain, we then performed exploratory factor analysis using the default settings for the fa() function.
+
+### Modeling of health-related variables
+
+We modeled each of our health metrics (owner-reported health, disease instances and mobility score) as a function of all environmental factors, age and weight, as well as the interaction between age and each factor (Supplementary Fig. 4). We included weight in the model to control for variance in the health-related variables associated with breed ancestry, due to large inter-breed differences in dog size [20, 47]. We do not have genotypic data from the mixed-breed dogs, so could not control for breed ancestry directly. Due to their distributions and variable types, each health metric required a different linear modeling framework. We used an ordinal regression to model owner-reported health, which was scored on a Likert scale from 1 to 6. We modeled the count-based owner-reported disease instances using a generalized linear model with a Poisson link function. Mobility was modeled using a standard linear model. For all outcomes, we conducted sensitivity analyses that included modeling a dataset that only included mixed-breed dogs which was modeled in a mixed-effects framework that included breed as a random effect (SI Table 4, Supplementary Fig. 4).
+
+### SEM confirmatory factor analysis
+
+Confirmatory factor analysis was performed on a manually curated set of variables (described below) for use in the structural equation modeling analysis. This analysis was used to verify the structure of our observed set of variables. This was performed using the cfa() function under the Lavaan package (version 0.6-9) in R [48].
+
+### Model specification
+
+The latent factor structure was defined based on known relationships in the literature studied primarily in humans ([49, 50], SI Table 6). To specify the model for this study, we chose measured variables based on the following criteria, they: (1) had clear individual interpretability, (2) were likely to capture different aspects of the latent factor, (3) were continuously distributed, (4) did not have missing data and (5) together maximized sample variance as recorded in factor analysis. Our latent factor of finance was informed using measures from the census median income, percent living below the poverty line and percent of households making less than $100,000 US per year as well as owner-reported annual income range. The latent factor of the local environment was informed by census-tract population density, neighborhood walk score, percent of individuals in the same household 1 year ago, and percent of the population that was born in the USA. These variables were selected as measures that capture unique information about the dog’s neighborhood environment. Lastly, our social environment latent variable was informed by the measurable variables of routine hours per day with dogs, children and adults. We defined the structural equation model (SEM) using the Lavaan (version 0.6-9) package in R [48]; structural equation meta-models and model syntax were adapted from guidelines for a graph-theoretic implementation of structural equation modeling [51]. Prior to the implementation of the SEM, thirteen variables were standardized to a mean of zero and standard deviation of 1 to account for scale differences among variables, and models were run using unweighted least squares. Two models were tested, with the difference being the addition of a pathway in model two from finance to the environment. Fit for each of the two models was assessed through several metrics and associated cutoffs for ‘good’ agreement between data and model: root mean squared error of approximation (RMSEA, <0.08), comparative fit index (CFI, >0.9), standardized root-mean-squared residual (SRMR, <0.08) and the Tucker–Lewis index (TLI, >0.9) [52]. Comparative model fit was assessed using the corrected Akaike information criterion (AICc). Confirmatory factor analysis on both latent and indicator variables was conducted using the cfa function in R. All structural model visualizations were generated using the TidySEM and SEMplot packages in R. We generated two models; the first model (M1) describes direct paths from the latent factor of finance, environment and social interactions to our outcome of health (Supplementary Fig. 2). The second model (M2) describes all direct paths indicated in M1 but with the added indirect path between finance and environment (Supplementary Fig. 3). These two models were both hypothesized to reliably describe the system and were tested for model fit before interpreting the structural equation modeling analysis.
+
+## RESULTS
+
+### Owner survey data from a large cohort of companion dogs
+
+Our dataset included survey and health data from 21,410 dogs between the ages of 2 and 25.5 years old (Fig. 1a), that weighed between 2 and 210 lbs (Fig. 1b). We had a similar proportion of male (50.1%) and female dogs (49.8%; Fig. 1c), as well as purebred (52.3%) and mixed-breed dogs (47.7%; Fig. 1d). Given the low percentage of intact male and female dogs (4% and 2%, respectively), we chose to include them in our analyses. Additional demographic information on the dogs and their owners can be found in Supplementary information (SI Table 1, Supplementary Fig. 1).
+
+*Figure: Figure 1. Demographic characteristics of dogs included in this study. Distribution of (a) age and (b) weight of dogs included in this study. The sample was roughly balanced by (c) sex and (d) mixed vs. purebred ancestry. Data are from the Dog Aging Project Health and Life Experience (HLES) Survey, 2019–2020*
+
+### Social, financial and demographic factors
+
+We identified five factors, based on parallel analysis (see Methods), that collectively explained 33.7% of the variance in responses to the 33 survey questions, and then labeled the factors based on the survey questions that primarily contributed to each factor (loading >|0.4|): neighborhood stability (factor 1: 10.6% variance explained), total household income (factor 2: 9.7% variance explained), social-children (factor 3: 5.1% variance explained), social-animals (factor 4: 4.4% variance explained) and owner age (factor 5: 3.9% variance explained; Fig. 2).
+
+*Figure: Figure 2. Five factors capture much of the social-environmental variation in the Dog Aging Project cohort. (a) Exploratory factor analysis revealed five factors that together explained 33.7% of the variance in survey responses. We named each factor based on the survey questions that loaded the strongest onto each factor (loading > |0.4|). (b) Distribution of factor scores for individual dogs for each of the five factors in our sample*
+
+### Household environment is associated with dog health, disease and mobility
+
+#### Owner-reported dog health.
+
+Owner-reported dog health was reported as an integer on a scale from 1 (‘very poor health’) to 6 (‘excellent health’). Since this is an unrefined measure of health, we first assessed its internal validity to capture dog health. As expected based on previous literature, older and heavier dogs were rated by their owners as being in the poorest health (β~age~ = −1.01, *P* ≤ 2 × 10^−16^; β~weight~ = −0.13, *P* = 6.45 × 10^−23^; Fig. 3 [53, 54]).
+
+*Figure: Figure 3. Owner-reported health is worse in older dogs. (a) Dog age was significantly associated with owner-reported health, such that older dogs were reported to be in poorer health compared to younger dogs controlling for dog weight (β~age~ = −0.421, *P* ≤ 2 × 10^−16^)*
+
+We found that all five environmental factors were significantly associated with health outcomes (controlling for dog age and weight). Higher income was associated with better health (β~income~ = 0.037, *P* = 5.99 × 10^−3^), indicating that owners of higher SES rated their dogs as being healthier. Dogs with other dogs and/or other pets in their house were also rated as significantly healthier than dogs with fewer household companions (β~social-animals~ = 0.044, *P* = 1.20 × 10^−3^; Fig. 4a). Dogs who lived in households with a higher stability score were reported to be healthier than those that did not (β~stability~ = 0.044, *P* = 1.07 × 10^−45^; Fig. 4a). Dogs that lived in households with more children were reported to be less healthy than those with fewer children (β~children~ = −0.041, *P* = 2.72 × 10^−3^; Fig. 4a). Finally, older owners reported their dogs were in better health compared to younger owners (β~owner_age~ = 0.23, *P* = 5.60 × 10^−41^; Fig. 4a).
+
+*Figure: Figure 4. Social environment factors are associated with health, disease and mobility in companion dogs. Effect sizes of each environmental factor on (a) owner-reported dog health (ordinal model), (b) disease prevalence (cumulative number of diseases reported) (generalized linear model with Poisson link function) and (c) mobility (composite measure of activity level) (linear model). Points depict the effect sizes (β) of each factor and lines represent the 95% confidence intervals*
+
+#### Disease diagnoses.
+
+We found similar associations between environmental factors and health when using an arguably more objective measure of health: the total number of disease diagnoses per dog over their lifetime (the total count of all reported diagnosed diseases; see methods). All five factors were significantly associated with disease diagnoses, controlling for age and weight. Specifically, income was positively associated with the number of reported diseases, while stability, time with other animals, owner age and time with children were negatively associated with the number of diseases reported. Dogs in more stable households or with older owners were reported to have fewer diseases (β~stability~ = −0.03, *P* ≤ 2 × 10^−16^; β~owner_age~ = −0.16, *P* ≤ 2 × 10^−16^). For the two measures of social integration and connectedness, we found that dogs with more nonhuman companions as well as those with more children in the house had significantly fewer reported diseases (β~social-animals~ = −0.03, *P* = 9.09 × 10^−6^, β~social-children~ = −0.09, *P* ≤ 2 × 10^−16^) (Fig. 4b). Paradoxically, we observed a positive association between income and reported disease instances (β~income~ = 0.04, *P* = 2.27 × 10^−7^), which stood in contrast to the negative association between higher income and better owner-reported health.
+
+#### Mobility.
+
+Given that mobility declines with age and can be used as a marker for exposures that might alter the rate of aging, we investigated associations between the environmental factors and composite mobility score that captured measures of physical activity duration, intensity and frequency [55–57]. In this metric, a higher score reflected higher levels of activity and mobility (see Methods for details on the calculation of the composite mobility score). As expected, older dogs were less mobile (β~age~ = −0.12, *P* ≤ 2 × 10^−16^; SI Table 3), and, when controlling for age, we found that dogs with higher mobility were in better health compared to less mobile dogs of the same age (β = 0.35, *P* ≤ 2 × 10^−16^; SI Table 3). This suggests that, similar to humans, a more active lifestyle is associated with better health in dogs [58, 59]. We next found that the dog’s social environment—specifically stability, time spent with children and owner age—were significantly associated with dog mobility. Mobility was lower in dogs that lived in households with more children and higher for dogs that lived in households with older owners (β~children~ = −0.04, *P* ≤ 2 × 10^−16^, β~owner age~ = 0.03, *P* = 1.15 × 10^−14^, respectively), while it was higher in dogs in households that were more stable (β~stability~ = 4.55 × 10^-3^, *P* = 4.52 × 10^−12^; Fig. 4c, SI Table 3).
+
+### Effects of environmental determinants of health can vary across the lifespan
+
+To investigate whether associations between these factors and health, disease and mobility vary across the lifespan, we extended each regression model by including an interaction term between each environmental factor and age (Supplementary Fig. 4). We found five significant interactions between dog age and our environmental factors on owner-reported dog health (SI Table 3). The positive association between household stability and dog health was strongest in the younger dogs, and weakest in older dogs (Fig. 5a, β = −0.03, *P* = 5.33 × 10^−17^). Time spent with other animals was more strongly associated with health when the dog was older rather than younger (Fig. 5b, β = 0.03, *P* = 4.18 × 10^−2^). We also found that owner age was more impactful for younger dogs compared to older dogs (Fig. 5c, β = −0.18, *P* = 5.91 × 10^−26^). Lastly, for older dogs, increased time spent with children was more impactful to overall general health than for younger dogs (Supplementary Fig. 6, β = −0.04, *P* = 1.01 × 10^−2^). We saw no significant interactions between environmental factors and age on disease instances.
+
+*Figure: Figure 5. Age-dependent associations between environmental factors and health. (a) Household stability was more strongly associated with health for younger dogs compared to older dogs. (b) The presence of more social companions was more strongly associated with health for older dogs compared to younger dogs. (c) The factor of owner age was more strongly associated with health for younger dogs compared to older dogs. (d) More time spent with children was more strongly associated with health for older dogs compared to younger dogs. All graphs show predicted trends using a linear model framework with 95% confidence intervals for a hypothetical population of dogs aged 4 (−1 SD, lightest purple), 7.9 (mean age, intermediate purple) and 11.8 (+1 SD, darkest purple) years old*
+
+We did find a significant interaction between dog age and four environmental factors on mobility. First, a significant interaction between neighborhood stability and dog age on mobility revealed that neighborhood stability is most strongly associated with the mobility of younger dogs compared to older dogs (β = −0.002, *P* = 6.0 × 10^−4^; Supplementary Fig. 5a; SI Table 3). Second, the effect of spending time with other animals on mobility significantly interacted with dog age, where more time was considerably more important for the mobility of older dogs (β = −0.01, *P* = 8.2 × 10^−4^, Supplementary Fig. 5b: SI Table 3). Third, the effect of spending time with children on mobility significantly interacted with dog age, where more time with children was more important for the mobility of older dogs (β = −0.01, *P* = 0.01, Supplementary Fig. 5c; SI Table 3). Lastly, having an older owner had a stronger effect on a younger dog’s mobility (β = −0.01, *P* = 7.9 × 10^−5^, Supplementary Fig. 5d).
+
+### Pathways linking social-environmental variables and owner-reported dog health
+
+Environmental factors could have both direct and indirect effects on health. To examine these putative paths, we took an orthogonal modeling approach, structural equation modeling (SEM), which can capture more complex relationships among the environmental variables and health. This approach can have more flexibility, the capacity to represent indirect relationships, and supports the integration of results into broader theory [51]. To do so, we first organized our variables into latent factors that represent concepts known to fundamentally influence mammalian health and that were chosen to maximize the interpretability of the latent factors [60]. We chose the variables (i.e. survey items) that were identified as most informative in our exploratory factor analysis described above. The latent factor of health was informed by the same variables that we used as outcome variables in our linear models: health, disease instances and mobility.
+
+Both showed good model fit according to established parameters [52] (M1: CFI = 0.84, RMSEA = 0.06, SRMR = 0.05, TLI = 0.80; M2: CFI = 0.84, RMSEA= 0.06, SRMR = 0.05, TLI = 0.80). Here we will present results for M2, which includes a pathway from the financial factor to the environmental factor, which is a better representation of the system given the established literature [61, 62]. In humans, we know that the quality of the environment is influenced by the finances of the household thus providing support for evaluating M2 as the more generalizable model [63].
+
+As with our linear models, we found evidence for all three latent variables associated with the latent factor capturing health. However, the relative importance of the latent factors varied: the effect of the social factor was ~5x larger than the effect of finance and the local environment on our health outcome (β~Social~ = 0.107, β~Finance~ = 0.015, β~Environment~ = −0.023; Fig. 6). In line with our linear models, we found that increased income and more social interaction with humans and other dogs both predicted better health. In contrast, a more urban environment with less stability was associated with worse health, pointing to the complex relationship between overall health and the environment. These data capture a previously hypothesized indirect pathway from finance through the local environment that our linear models failed to identify, suggesting that the local environment might be an important mediator between finance and health.
+
+## DISCUSSION
+
+Using the largest observational dataset of companion dog environment and health, we identified measures of the social and lived environment that were linked to age-associated changes in health, mobility and disease. Unsurprisingly, age was the strongest predictor of health deficits. However, not all dogs exhibited the same age-related changes in health, disease and mobility, and some of this inter-individual variation was linked to the environment. Health, mobility and disease measures were also associated with variations in household social environmental determinants. We also identified key changes in the environment that were associated with the health and mobility of dogs in an age-dependent manner within this cross-sectional dataset.
+
+Similar to humans, we found that measures of income and social connectedness (i.e. presence of more potential social companions) predicted better health in dogs, although dogs from wealthier owners had more diagnosed diseases [64]. This counterintuitive association does not necessarily mean that dogs of wealthier owners had more diseases, especially given the fact that dogs from higher SES households were reported as healthier by their owners. Rather, this finding points to the role that finance plays in the opportunities for disease diagnosis, while also generating new questions on the directionality of the relationship between income and health as well as what we ultimately consider ‘health’ and how we diagnose diseases [60]. Dogs who live in households with wealthier owners might seek veterinary care more frequently and have the funds to pay for additional tests, resulting in more disease diagnoses even though the diseases are unlikely to be more prevalent in higher SES households. Owners may perceive their dogs to be in good health because they have more information and are more confident that any disease would have been detected and treated or managed. Dogs who lived in households with other pets had higher reported health scores and fewer disease diagnoses than dogs who had fewer household companions, suggesting that this social enrichment confers health benefits, which outweighed the effects of finances five-fold (Fig. 6). These other companions are often other dogs—of homes with other animals present, 70% of them had another dog—but could be other pets (e.g. cats, SI Table 7). This suggests that similar to humans, increased social enrichment in companion dogs is associated with health benefits, and also provides an example of an easily modifiable environmental intervention to improve dog welfare. These health benefits may also impact the owners. Indeed, a recent study found that interspecies companionship lowered frailty among older humans and may point to an effective intervention to improve the health of aging humans [65]. Dogs who lived in households with more children had fewer diseases, but tended to be in poorer health. This observation could be due to modified resource allocation (e.g. time and money) in households with more children. Mobility was also associated with environmental factors and showed unique age dependencies. This recapitulates what we see in humans, where individuals who live in lower-income neighborhoods and experience adverse community-level factors, such as decreased access to green space, unequal resource distribution and access (e.g. healthcare or food), have poorer health outcomes [66, 67].
+
+*Figure: Figure 6. Structural equation modeling captures a complex network of environmental effects on health. Structural equation model of how the social environment can influence general health. Path coefficients explain the effect sizes and directionality of each major path. Latent factors (circles) are informed by measured variables (squares) that were chosen via factor analysis, and the direction of the effect of each measured variable on the latent variables is indicated next to or below the variable (+: positive association; −: negative association)*
+
+We found that owner age was significantly associated with our health-related outcomes, but why this pattern occurs is unknown. One possible mechanism is the link between owner age and dog activity levels, where a recent analysis in the DAP found that older owners had more active dogs, which is linked to better health. Further answers to this question could elucidate the complex relationships between owner age and our other factors and their associations with health [68]. This suggests that as the DAP continues, investigators will need to pay particular attention to owner age in our dataset, as it has a potential association with physical activity, cognitive function and now perceived health.
+
+An important outstanding question is identifying the path through which the social environment impacts health. In humans, it has been hypothesized (and there is some support for the fact) that exposure to psychosocial stressors from unpredictable environments (e.g. work-related stress, low SES, unpredictable environments and low social integration) lastingly alter the stress-response pathway and, consequently, immune function and health [69]. Our findings fit within this framework and show that the negative health effects of unpredictable social environments can extend to companion dogs. However, more work is needed to identify whether or not the physiological pathways (e.g. stress pathway) are shared among these two cohabitating species.
+
+We also unexpectedly found that younger dogs with older owners experienced better health outcomes compared to older dogs who had older owners. These data suggest the importance of understanding how age impacts lifestyle, which could have knock-on effects for companions and companion animals. Specifically, the human social environment might contribute to dog health indirectly through variables like owner age that affect how often a dog might engage in social enrichment activities due to their owner’s activity budget and lifestyle. We also acknowledge that the distribution of dog age is highly non-random with respect to owner age, which may explain some of this association. We found that younger dogs who lived in less stable households were in worse health than older dogs who lived in stable homes, suggesting that, similar to humans, environmental adversity plays a particularly important role in early life [70–72]. Older dogs who lived in higher-income households were found to be in better health compared to older dogs in lower-income households, while the health of younger dogs was less impacted by income variation, suggesting that dogs may be more resilient to variation in this form of adversity earlier in life [73, 74]. Younger dogs’ mobility experienced a larger age-associated increase when living in more stable neighborhoods compared to older dogs, suggesting that the benefits of living in a more stable household, while important across the lifespan, are more impactful in earlier life. Older dogs who lived in less stable neighborhoods were much less mobile than older dogs in more stable neighborhoods, suggesting that, similar to older people in lower SES neighborhoods, dogs experience a reduction in activity that could ultimately be linked with poorer health outcomes [75, 76].
+
+Our environmental and health measures are not independent of one another, suggesting a more complex network among environmental factors and their association with health outcomes. We used SEM to capture direct and indirect effects of each variable in one model. The results of our SEM supported the independent models of each health variable, but allowed us to propose a holistic model for the environmental impacts on companion dog health and aging that is generalizable to similar work done on humans. The model points to direct effects of finance and the local environment on dog health, in addition to an indirect contribution of finance on health through its effect on the local environment. This indirect path is supported in the human literature, where we see that SES dictates where a person will live and thus their environment as well as access to healthcare and both of these contribute to both positive and negative health outcomes [68, 77]. This indicates the latent structures affecting health outcomes in dogs and humans are similar, and may be a product of aspects of modern society as well as the deeper evolutionary history of dogs being selectively bred alongside humans for thousands of years.
+
+While the strengths of this dataset include the large sample size (*n* = 21,410) of the study cohort and the breadth of the HLES survey data, there are some limitations. First, our dataset comprises both mixed-breed and purebred dogs, and genetic background and dog breed are both known to be associated with dog health and disease [20, 53]. However, our results remained largely consistent when we examined pure and mixed breed dogs independently, as well as when we controlled for individual breeds based on owner reports (SI Table 4). The Dog Aging Project is currently sequencing the genomes of 10,000 dogs in the project [16], which will allow us to more accurately control for the genetic background as well as identify putative gene-by-environment interactions that impact health, disease, or mobility.
+
+Second, all data in the HLES are owner-reported and can thus be affected by subjective error, bias and/or misinterpretation of survey questions. Further investigation of biases in owner-reported responses will be needed to verify our findings in the future. The strong association between dog age and all three of our outcome measures—health, disease incidence and mobility—suggests that the survey is an instrument that, on average, accurately captures the variables we cannot objectively measure. All measures could be improved by an owner-independent measure or veterinarian-informed measure of frailty and health. In future research, we will draw on electronic veterinary medical records, molecular and immunological measures and at-home physical tests to generate more accurate measures of health and frailty in the companion dog.
+
+Third, there is a limitation due to the average household income being well above the U.S. median income (U.S. median: $67,521 in 2020, DAP median: $100,000–$119,999 [78]) and biases our knowledge away from lower-earning households. This underscores the need to recruit a diverse set of human participants to better account for the variation of environmental exposures a dog is subject to [16].
+
+Fourth, given the observational nature of the data, we were not able to determine causality or directionality among variables; we instead drew a causal structure in the SEM based on a critical evaluation of the literature (SI Table 6). For instance, owners who have dogs in poorer health might limit exposures to demanding activities or social enrichment compared to owners who have dogs who are in excellent health, which would also lead to an association between health and social companionship and activity.
+
+Given the above limitations, the results of this analysis should not be used to influence medical decisions, behavioral interventions, or policy changes related to the care of companion dogs, but rather encourage the continued investigation by veterinary professionals and research scientists on best practices for increasing companion welfare. Some of these relationships can be evaluated in the future with randomized, controlled experiments.
+
+Overall, our study provides further evidence for the strong link between the social environment and health outcomes that reflects what is known for humans. From this knowledge, we propose the companion dog to be a novel model for human aging. This highlights the need for more attention to the role of the social environment on health and disease and continued investigation of how each environmental factor can contribute to more years of healthy living (i.e. ‘healthspan’) in both companion dogs and humans. A holistic understanding of environmental effects on both humans and dogs may offer insight into what factors are important for health—independent of confounding factors that complicate human studies (e.g. smoking). Results highlight the need to study how differences in resource allocation among households contribute to the health and well-being of all members of the household. A future use of longitudinal data will allow a stronger inferential capacity to identify putative causal factors that affect trajectories of health and aging in both dogs and humans, driving new insights into what factors promote healthy aging and potential lifestyle modifications that can increase time spent free of disease.
+
+Dog Aging Project Consortium Authors (as of March 2022)
+
+Joshua M. Akey^1^, Brooke Benton^2^, Elhanan Borenstein^3^, Marta G. Castelhano^4^, Amanda E. Coleman^5^, Kate E. Creevy^6^, Kyle Crowder^7^, Matthew D. Dunbar^8^, Virginia R. Fajt^9^, Annette L. Fitzpatrick^10^, Unity Jeffery^11^, Erica C Jonlin^12^, Matt Kaeberlein^13^, Elinor K. Karlsson^14^, Kathleen F. Kerr^15^, Jonathan M. Levine^16^, Jing Ma^17^, Robyn L McClelland^18^, Daniel E.L. Promislow^19^, Audrey Ruple^20^, Stephen M. Schwartz^21^, Sandi Shrager^22^, Noah Snyder-Mackler^23^, Katherine Tolbert^24^, Silvan R. Urfer^25^, Benjamin S. Wilfond^26^
+
+^1^ Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, USA
+
+^2^ Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
+
+^3^ Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel
+
+^4^ Cornell Veterinary Biobank, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA
+
+^5^ Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
+
+^6^ Department of Small Animal Clinical Sciences, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA
+
+^7^ Department of Sociology, University of Washington, Seattle, WA, USA
+
+^8^ Center for Studies in Demography and Ecology, University of Washington, Seattle, WA, USA
+
+^9^ Department of Veterinary Physiology and Pharmacology, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA
+
+^10^ Department of Family Medicine, University of Washington, Seattle, WA, USA
+
+^11^ Department of Veterinary Pathobiology, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA
+
+^12^ Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
+
+^13^ Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
+
+^14^ Bioinformatics and Integrative Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA
+
+^15^ Department of Biostatistics, University of Washington, Seattle, WA, USA
+
+^16^ Department of Small Animal Clinical Sciences, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA
+
+^17^ Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
+
+^18^ Department of Biostatistics, University of Washington, Seattle, WA, USA
+
+^19^ Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
+
+^20^ Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA
+
+^21^ Epidemiology Program, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
+
+^22^ Collaborative Health Studies Coordinating Center, Department of Biostatistics, University of Washington, Seattle, WA, USA
+
+^23^ School of Life Sciences, Arizona State University, Tempe, AZ, USA
+
+^24^ Department of Small Animal Clinical Sciences, Texas A&M University College of Veterinary Medicine & Biomedical Sciences, College Station, TX, USA
+
+^25^ Department of Laboratory Medicine and Pathology, University of Washington School of Medicine, Seattle, WA, USA
+
+^26^ Treuman Katz Center for Pediatric Bioethics, Seattle Children’s Research Institute, Seattle, WA, USA
+
+## Supplementary Data
+
+Supplementary data is available at *EMPH* online.
+
+**Supplementary Figure 1.** Distribution of owner ages (a), distribution of owner education level (b), distribution of owner income range (c), distribution of neighborhood median income (d) and distribution of neighborhood unemployment rate (e)
+
+**Supplementary Figure 2.** M1
+
+**Supplementary Figure 3.** M2
+
+**Supplementary Figure 4.** Ordinal, linear, and generalized linear models for each outcome and analyses
+
+**Supplementary Figure 5.** (a) The association between dog mobility and household stability varied with dog age. (b) The association between dog mobility and time spent with dogs varied with dog age. (c) The association between dog mobility and time spent with children varied with dog age. (d) The association between dog mobility and owner age varied with dog age
+
+**Supplementary Figure 6.** The association between overall dog health and time spent with children varied with dog age
+
+**SI Table 1.** Dog and owner demographics
+
+**SI Table 2.** Variables
+
+**SI Table 3.** Main effects models
+
+**SI Table 4.** Models for pure/mixed
+
+**SI Table 5.** Disease metric
+
+**SI Table 6.** SEM variables
+
+**SI Table 7.** Other animals
+
+**SI Table 8.** Ordinal model vs linear model
+
+**SI Table 9.** Ordinal model with dogs ≤ 20yrs
+
+**SI Table 10.** SEM model output

fulltext/md/PMC10309205.md

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+---
+pmcid: "PMC10309205"
+pmid: "37396989"
+doi: "10.3389/fvets.2023.1150590"
+title: "Winning the race with aging: age-related changes in gait speed and its association with cognitive performance in dogs."
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Winning the race with aging: age-related changes in gait speed and its association with cognitive performance in dogs
+
+## Abstract
+
+### Introduction
+
+In humans, gait speed is a crucial component in geriatric evaluation since decreasing speed can be a harbinger of cognitive decline and dementia. Aging companion dogs can suffer from age-related mobility impairment, cognitive decline and dementia known as canine cognitive dysfunction syndrome. We hypothesized that there would be an association between gait speed and cognition in aging dogs.
+
+### Methods
+
+We measured gait speed on and off leash in 46 adult and 49 senior dogs. Cognitive performance in senior dogs was assessed by means of the Canine Dementia Scale and a battery of cognitive tests.
+
+### Results
+
+We demonstrated that dogs' food-motivated gait speed off leash is correlated with fractional lifespan and cognitive performance in dogs, particularly in the domains of attention and working memory.
+
+### Discussion
+
+Food-motivated gait speed off leash represents a relatively easy variable to measure in clinical settings. Moreover, it proves to be a more effective indicator of age-related deterioration and cognitive decline than gait speed on leash.
+
+## Introduction
+
+Canine geriatric syndrome has been proposed as a new concept that describes “the constellation of key physical, functional, and metabolic changes that characterize aging in dogs and predispose to age-related dysfunction, disease, and death” (1). Some of the most remarkable age-related changes associated with this syndrome occur in motor and cognitive function (1, 2). Physical impairments and slowing of movement reflect the diminished performance of numerous systems, such as musculoskeletal system, central and peripheral nervous system, and sensory organs (3–5).
+
+In humans, gait speed is a valid, reliable, and simple measure of physical function. This is considered a crucial component of geriatric evaluation because it is associated with disability, hospitalization, mortality and cognitive decline (6–9). In fact, gait speed is considered a “sixth vital sign” in older adults together with blood pressure, respiration, pulse, temperature, and pain (7). The link between gait speed and cognition has become particularly interesting since it has been demonstrated that slow gait speed can be a harbinger of cognitive decline and dementia (10–12).
+
+Dogs are an excellent animal model for studying aging (13) because they suffer from age-related impairments in mobility (14, 15) and cognition (16, 17) and may develop canine cognitive dysfunction syndrome (CCDS) which has several similarities with human Alzheimer's disease (18). Therefore, a similar association between gait speed and cognitive performance is expected in dogs as in humans. Age-related changes in gait speed do happen in dogs; Morgan et al. (15) showed a weak negative correlation between age and gait speed. They found that senior dogs (older than their 75% of their expected lifespan) can be up to 63% slower than younger dogs (15). However, these authors did not evaluate whether there is an association between gait speed and cognitive performance.
+
+In dogs, cognitive performance can be assessed in several ways. Owner-based questionnaires such as the Canine Dementia Scale (CADES) have undergone psychometric validation (19) and represent an accessible way to detect the occurrence of behaviors associated with CCDS. Additionally, our group has recently shown the feasibility and utility of a battery of cognitive tests in senior dogs. These tests allow us to evaluate different domains of cognition such as sustained attention, memory, executive function and response to social cues (16).
+
+In this study, we first investigated age-related changes in gait speed on and off leash in adult and senior dogs. Secondly, in the group of senior dogs, we evaluated the association between gait speed and cognitive performance (evaluated by CADES questionnaire and the cognitive tests battery) as well as the role of pain in gait speed.
+
+## Materials and methods
+
+### Study population
+
+All procedures were approved by the North Carolina State University (NCSU) Institutional Animal Care and Use Committee, protocol number: 21–303 and 21–306. Two groups of client-owned dogs were included in this study, a group of senior dogs (older than 75% than their expected lifespan) and a group of adult dogs (older than 1 year and younger than 75% of their expected lifespan)(20). Since expected lifespan in dogs is highly associated with the dogs' body size, we calculated it for each individual dog using the formula proposed by Greer et al. (21) which takes into consideration dogs' height and weight. The group of senior dogs are a population of dogs participating in a longitudinal study of neuro-aging at the North Carolina State University (NCSU) College of Veterinary Medicine [described elsewhere (16, 22, 23)]. To be included, dogs had to be free of co-morbidities that could impede their ability to perform the tests such as inability to walk or blindness. The dogs' most recent visit in the study was used for this cross-sectional analysis. The group of adult dogs was recruited through emails and flyers sent to the NCSU community to establish gait speed in a younger cohort of dogs. We selected dogs from a range of different breeds, sizes, and ages. Dogs with abnormal orthopedic or neurological examination and blind dogs were excluded. Owners dropped the dogs off in the morning and picked them up in the afternoon. In a screening visit, the dogs' behavior toward the experimenters was evaluated by bringing the dogs in the cognitive testing room and offering them treats. Dogs that displayed signs of fear, aggression or anxiety, or wouldn't approach the experimenters to get treats (or showed no food motivation), were not included in the study. We collected demographic information for every dog and health status was evaluated by veterinarians (AM, GF, WP, and NO). We performed physical, orthopedic, and neurological examinations. During physical examination, height to the dorsal border of the scapula (withers) was measured. In the population of senior dogs, urinalysis, biochemistry panel and complete blood cell count were also performed to identify co-morbidities. Dogs with active, severe disease processes that could affect their ability to participate in the longitudinal study of Neuro-Aging were excluded.
+
+### Gait speed evaluation
+
+On leash and off leash gait speed in dogs was determined by performing 3 consecutive trials and calculating the average speed. For on leash gait speed, each trial consisted of walking the dog on their own leash over a 5 m long indoor flat course. A stopwatch was started when the dog crossed the start line and stopped when it crossed the finish line with one of the thoracic limbs. The experimenter let the dog set the pace and was careful not to put pressure on the leash, the leash was always held loosely (Supplementary Video S1). For the off leash trials, a handler held the dog at the starting line of the 5-meter course. The experimenter walked to the dog and showed the dog a treat, then the experimenter stood at the finish line, called the dog by their name and said “Look”. The handler said “Ready, set, go” and released the dog on “Go” (Supplementary Video S2). At the same time the experimenter started the stopwatch. The stopwatch was stopped when the dog crossed the finish line with one of their thoracic limbs. If in any of the trials (either on or off leash) the dog was distracted and stopped walking, the trial was discarded and repeated. The average of the time recorded for each test was divided by 5 to calculate the speed in m/s.
+
+### Cognitive evaluation in senior dogs
+
+Behavioral changes associated with cognitive dysfunction syndrome were assessed by a validated clinical metrology instrument, the Canine Dementia Scale (CADES) sent to owners to complete on-line. The first time the owners completed the questionnaires in the Longitudinal Study of Neuro-Aging the questionnaires were reviewed with the owners to ensure they understood the questions. Owners were encouraged to reach out to the investigators if they had any questions about the completion of the questionnaire. This questionnaire asks the owners about the frequency of behaviors related to spatial disorientation, social interactions, sleep-wakefulness cycle and house soiling (19). The score range for this questionnaire goes from 0 (completely normal) to 95 (severely affected) and allows classification of dogs into 4 different categories: normal (0–7), mild cognitive impairment (MiCI, 8–23), moderate cognitive impairment (MoCI, 24–44) and severe cognitive impairment (SCI, 45–95).
+
+Cognitive testing was also performed as described by Fefer et al. (16) to evaluate attention (sustained gaze test), social cues (pointing test), working memory, executive control (inhibitory control cylinder task) and problem solving (detour cylinder task). The sustained gaze test measures the time the dog holds the gaze with the experimenter holding a treat near their face. The pointing test determines the percentage of correct trials in which dogs can retrieve a treat in a two-choice task when the experimenter points to where it was hidden. For the working memory task, the dog is allowed to see where the experimenter hides a treat in a two-choice task and the upper threshold of time that dogs are able to remember and select where the treat was hidden is measured. In inhibitory control, test dogs are asked to retrieve a food reward from a transparent cylinder open on both ends without touching the cylinder. The detour task is similar but adds an additional difficulty–the dog's preferred side for entering the cylinder is covered, and the dog needs to retrieve the treat using the opposite side. The percent of correct trials was calculated for these two tasks. Tests were performed in the morning, between 8 AM and 12 PM, in a quiet room designated for this type of study. Dogs were provided with short breaks when they seemed tired or to be losing interest (failing to make choices in the tasks). Pre-determined abort criteria were used to terminate testing if dogs were not making choices during the tasks after the breaks.
+
+### Pain evaluation in senior dogs
+
+We evaluated pain in the population of senior dogs to determine its relationship with gait speed.
+
+Owners' perception of dogs' pain was assessed by means of the Canine Brief Pain Inventory (CBPI) questionnaire. This questionnaire can be divided into two subscales, a pain severity subscale (which assesses the severity of the dog's pain according to the owners) and a pain interference subscale (which assesses how pain interferes with dog's activities) (24). As with CADES questionnaire, owners underwent initial training on the questionnaire and were encouraged to ask questions if further clarification was needed. Additionally, we scored joint pain during orthopedic examination. Each appendicular joint was assessed, with all toes considered as one single site for each paw, so the joints evaluated were manus, carpus, elbow, shoulder, pes, hock, stifle and hip. Joint pain was quantified by scoring each joint from 0 to 4 during flexion and extension as follows: 0: Does not notice; 1: Orients to site, does not resist or mild resistance; 2: Orients to site, slight objection to manipulation; 3: Withdraws from manipulation, may vocalize, may turn to guard area; and 4: Tries to escape/prevent manipulation, may bite or show aggression. Total joint pain was determined as the sum of the score in each joint on a scale of 0–64 (25).
+
+### Statistical analysis
+
+Statistical analyses were performed using JMP Pro, Version 16.0 SAS Institute Inc., Cary, NC and IBM SPSS Statistics, Version 27.0. IBM Corp. Armonk, NY. Normality was assessed in all variables by means of Shapiro-Wilk test. Variables that were not normally distributed were presented as median (range), while normally distributed variables were presented as mean ± standard deviation. As most variables were not normally distributed, correlation between two variables was assessed using the non-parametric Spearman's ρ test. For each group of dogs, we assessed the potential impact of height on gait speed by calculating the correlation between gait speed on leash and gait speed off leash with the height of the dogs. In the group of senior dogs, we tested whether there was a correlation between gait speed on leash and gait speed off leash (independent variables) with CADES scores and each cognitive test result (dependent variables). We have demonstrated that CADES scores and performance in cognitive tests are correlated with fractional lifespan, with older dogs having more cognitive impairment than younger dogs. Therefore, for variables that were associated with fractional lifespan, fractional lifespan was included as a covariate in multivariate analyses. For these analyses, variables that were not normally distributed were transformed by log(x+1). We also evaluated, in the group of senior dogs, whether pain was associated with gait speed on and off leash by means of Spearman's correlation analyses. In these analyses we included the two CBPI subscales scores and total joint pain measured by orthopedic examination as independent variables and gait speed on and off leash as dependent variables. Finally, we determined the test-retest reliability comparing the three measurements of both gait speed on and off leash using an intraclass correlation coefficient (ICC) with a two-way random effects, absolute agreement, single rater/measurement, i.e., ICC~(2, 1)~ (26).
+
+Due to the exploratory nature of this study, we did not perform correction for multiple comparisons (27). Results were considered statistically significant if *p* < 0.05. All the collected data are provided in Supplementary File 1.
+
+## Results
+
+### Study population
+
+A total of 109 dogs were screened for this study. Nine dogs were excluded for being too anxious and refusing to approach the experimenter in order to obtain treats. One dog was excluded because he was blind in his left eye and had severe cachexia, one dog was excluded for having antibiotic resistant urinary tract infection, three dogs were excluded for abnormalities in their bloodwork; one had marked liver enzymes (alkaline phosphate, alanine transaminase and aspartate aminotransferase) elevation, one had leukocytosis, and was therefore tested for infectious disease and was positive for Rocky Mountain Spotted Fever and the other one showed electrolyte imbalances (hypocalcemia, hypomagnesemia and hyperkalemia), and had also a history of seizures. Forty six adult dogs [4.7 ± 2.30 years old (Range: 1.04–9.12)] and 49 senior dogs [13.01 ± 1.70 years old (Range: 9.8–16.2] were finally enrolled. Their mean fractional lifespan was 0.38 ± 0.19 (Range 0.08–0.71) and 1.04 ± 0.12 (Range 0.77–1.23) for each group respectively.
+
+Twenty-four of the adult dogs were females (18 spayed) and 22 were males (17 neutered). The most represented breeds were Border collie with 7 dogs, German Shepherd with 6 dogs, and golden retriever and Siberian husky with 5 dogs each. There were 4 each of Labrador retriever, Jack Russell terrier, Maltese terrier and pit bull terrier, 2 Scottish terriers and 1 dog of each of the following breeds: beagle, Boston terrier and chihuahua. The median height of the group of adult dogs was 55 cm (Range 23–78 cm).
+
+Thirty-three of the senior dogs were spayed females and 16 were neutered males. There were eighteen mix breed dogs, 7 Labrador retrievers, 5 pit bull terriers, 3 beagles, 2 golden retrievers and Border collies and one of each of the following breeds: Basset hound, Bernese Mountain dog, Brittany spaniel, Cairn terrier, Pembroke Welsh corgi, dachshund, German shepherd dog, German short-haired pointer, great Dane, Jack Russell terrier, Pomeranian, and Irish setter. The median height of these dogs was 51.53 cm (Range 19.89–89.5 cm). There was no significant difference in height between the adult and senior dogs (*t* = 0.64, *p* = 0.523).
+
+### Speed on and off leash
+
+Gait speed on and off leash were not normally distributed. The median speed for adult and senior dogs on leash was 0.96 m/s (range 0.66–1.90 m/s) and 0.77 m/s (range 0.41–1.46) and the median speed off leash was 2.06 m/s (range 0.83–3.77) and 1.26 m/s (range 0.43–2.84), respectively.
+
+In the group of adult dogs, there was a significant correlation between height and gait speed off leash (ρ = 0.340, *p* = 0.017) but no correlation was found with gait speed on leash (ρ = 0.198, *p* = 0.173). In contrast, speed on leash but not off leash was correlated with height in the group of senior dogs (ρ = 0.367, *p* ≤ 0.009 and ρ = 0.113, *p* = 0.442 respectively).
+
+Gait speed on leash had a moderate test-retest reliability (ICC~(2, 1)~ = 0.72 (0.63–0.79) *p* ≤ 0.001) and gait speed off leash had a good test-retest reliability [ICC~(2, 1)~ = 0.82 (0.74–0.87) *p* ≤ 0.001].
+
+### Effect of age on speed
+
+Both speed on and off leash were negatively correlated with age (ρ = −0.501, *p* ≤ 0.001 and ρ = −0.459, *p* ≤ 0.001, respectively). However, when looking at the scatter plot, the correlation between gait speed off leash and age did not follow a linear trend; speed remained stable up to approximately 12 years and rapidly declined in older dogs. Therefore, we evaluated the role of age in each group of dogs separately (Figure 1A), and we found that while speed on leash was not correlated with age in either adult or senior dogs, speed off leash was negatively correlated with age in the group of senior dogs, but not in adult dogs. Similar results were obtained when looking at age as fractional lifespan (Figure 1B), but the correlation with gait speed off leash was slightly higher than with chronological age. Therefore, fractional lifespan was used instead of chronological age in further analyses.
+
+*Figure: Figure 1 Role of age in gait speed. Correlation between age **(A)** [and fractional lifespan **(B)**] and gait speed on leash (left), gait speed off leash (right). The analyses were performed for each group of dogs (adult vs. senior) independently. For easier visualization both groups are shown in the same graph separated by a black dashed line. Asterisks indicate significant differences.*
+
+### Relationship between gait speed and cognition in senior dogs
+
+Senior dogs' median CADES score was 12 (Range 0–74). Fifteen dogs were classified as normal, 14 as having MiCI, 11 as MoCI and 9 as SCI. Not all 49 dogs completed all cognitive tasks; the number of dogs who completed each task was: sustained gaze = 49, working memory = 47, pointing task = 40, inhibitory control task = 48, and detour task = 46. Median sustained gaze time was 18.46 s (Range 0–60) and median memory threshold was 20 s (Range 0–120). The median % of correct attempts at pointing was 83.33% (Range 33–100%), at inhibitory control was 87.5 % (Range 0–100%) and at detour was 44% (Range 0–100%). Fractional lifespan was correlated with CADES score (ρ = 0.641, *p* ≤ 0.001), sustained gaze (ρ = −0.521, *p* ≤ 0.001), memory (ρ = −0.547, *p* = 0.001), inhibitory control (ρ = −0.480, *p* ≤ 0.001) and detour (ρ = −0.493, *p* ≤ 0.001). No significant correlation was found between fractional lifespan and score in the pointing test (ρ = −0.275, *p* = 0.085).
+
+Table 1 provides the correlation between gait speed results and the performance in cognitive testing. Gait speed off leash was positively correlated with sustained gaze, memory, and detour. Additionally, gait speed off leash was correlated with CADES score and inhibitory control test.
+
+**Table 1 Correlation between gait speed and cognitive performance in senior dogs.**
+
+| **Cognitive performance** | **Gait speed on leash** | **Gait speed off leash** |
+| --- | --- | --- |
+|  | **Spearman's** ρ | ***p***
+**-value** | **Spearman's** ρ | ***p***
+**-value** |
+| CADES | −0.109 | 0.457 | −0.289 | 0.046^*^ |
+| Sustained gaze | −0.053 | 0.716 | 0.438 | 0.002^*^ |
+| Pointing | 0.263 | 0.100 | 0.379 | 0.016^*^ |
+| Memory | 0.075 | 0.616 | 0.307 | 0.005^*^ |
+| Inhibitory Ctrl | 0.213 | 0.146 | 0.388 | 0.006^*^ |
+| Detour | 0.037 | 0.806 | 0.347 | 0.018^*^ |
+
+As shown in Table 2, after adjustment for fractional lifespan, gait speed off leash remained significantly associated with performance in the sustained gaze, memory and pointing tests suggesting dogs who moved faster also performed better on cognitive testing independent of their life stage. Additionally, there was a significant effect of the interaction between gait speed off leash and fractional lifespan on CADES score, sustained gaze and inhibitory control tasks.
+
+**Table 2 Multivariate analysis evaluating the relationship between gait speed off leash and cognitive performance including fractional lifespan as a covariate.**
+
+|  | **Intercept** | **Fractional lifespan** | **Gait speed off leash** | **Fractional lifespan** × **Gait speed off leash** |
+| --- | --- | --- | --- | --- |
+|  | ***p*** | ***Std B*** | ***p*** | ***Std B*** | ***p*** | ***Std B*** | ***p*** |
+| CADES | R^2^ = 0.442 | 0.013^*^ | 0.598 | < 0.001^*^ | 0.023 | 0.854 | −0.320 | 0.007^*^ |
+| Sustained gaze | R^2^ = 0.327 | 0.001^*^ | −0.284 | 0.045^*^ | 0.316 | 0.026^*^ | 0.277 | 0.030^*^ |
+| Pointing | R^2^ = 0.268 | < 0.001^*^ | −0.214 | 0.173 | 0.316 | 0.048^*^ | 0.289 | 0.051 |
+| Working memory | R^2^ = 0.311 | 0.019^*^ | −0.285 | 0.049^*^ | 0.437 | 0.017^*^ | 0.217 | 0.098 |
+| Inhibitory control | R^2^ = 0.365 | < 0.001^*^ | −0.350 | 0.012^*^ | 0.263 | 0.056 | 0.320 | 0.011^*^ |
+| Detour | R^2^ = 0.153 | 0.009^*^ | −0.320 | 0.048^*^ | 0.105 | 0.509 | 0.135 | 0.346 |
+
+### The relationship between pain and speed
+
+Owners of 47 of the 49 senior dogs included in this study completed the CBPI pain questionnaire. The median score was 1 (Range 0–6.75) for the CBPI pain severity subscale and 1.5 (Range 0–7.8) for the CBPI pain interference subscale. No correlation was found between the CBPI pain severity subscale and gait speed on leash, or gait speed off leash (Figure 2A) while CBPI pain interference subscale was negatively correlated with gait speed off leash, but not with gait speed on leash (Figure 2B). However, when we adjusted for fractional lifespan, these associations did not remain statistically significant (fractional lifespan *p* = 0.002, CBPI pain interference *p* = 0.122 and fractional lifespan × CBPI pain interference *p* = 0.573).
+
+*Figure: Figure 2 Role of pain on gait speed. Correlation between CBPI-pain scores **(A)** [and CBPI-interference scores **(B)**] and gait speed on leash (left), gait speed off leash (right). Asterisks indicate significant differences.*
+
+Additionally, complete orthopedic examination was performed in 47 of the 49 dogs. The examination was aborted in two dogs who behaved aggressively. Median score for total joint pain was 5 (range 0–17). There was no correlation between total joint pain and gait speed on leash, or off leash (ρ = 0.115, *p* = 0.450 and ρ = −0.235, *p* = 0.120 respectively).
+
+## Discussion
+
+In people, gait speed has been proven to be a reliable measure of physical function and to be associated with frailty, disability, mortality, and cognitive decline (7, 9, 28). In this work we demonstrated that dogs show age-related changes in gait speed and that gait speed is associated with cognitive performance, independent of pain scores. We measured gait speed on leash while being walked by a handler, and gait speed off leash by allowing the dog to walk, trot or run toward a treat. We found that taller dogs were significantly faster on leash (in the group of senior dogs) and off leash (in the group of young dogs). Interestingly, in senior dogs, height was not associated with gait speed off leash. This could suggest that in senior dogs age-related changes might have a much stronger effect than height. We posit that gait speed off leash may capture the effects of both physical ability and motivation (food motivation in particular). In people, a role for motivation in gait speed is also suspected, as apathy (reduction in goal directed activity) is associated with slower walking speed and with risk of dementia (29).
+
+### Role of age
+
+Age was correlated with gait speed off leash in the group of senior dogs. These results agree with a previous study by Morgan et al. (15); however, those authors also found age-related variations in gait speed on leash. A potential reason for this difference is that in the previous study, the experimenters trotted the dog instead of walking them. Additionally, they measured gait speed over a longer distance (10 m instead of 5) and longer distances might be able to capture the effect of exhaustion in older dogs more easily. Nevertheless, their study also found that the strength of the correlation with age was higher when gait speed was measured off leash than on leash. It is important to take into consideration that speed on leash might be also influenced by the experimenter; while the dog was allowed to set the pace, pet dogs tend to match the speed of the handler when walking on leash (30). In addition to this, our study shows that gait speed on leash has a lower test-retest reliability than gait speed off leash.
+
+Noteworthy in our study, the association between speed and age was only seen in the group of senior dogs, but no association was found in the group of adult dogs (defined as from 1 year old to 75% of expected lifespan) (20). These results demonstrate that in dogs, gait speed decreases with age in a non-linear fashion. The same has been shown to occur in humans and mice in whom decline in gait speed starts to occur at approximately 70% of their expected lifespan (31). Hence, our results further support the dog as a translational model of aging.
+
+We found that gait speed off leash had a stronger association with fractional lifespan (the life stage) than with the chronological number of years. This is particularly relevant in dogs, since their lifespan is highly dependent on body size, and this species is characterized by a huge size diversity (32, 33). Scientists have been investigating the impact of aging rates in specific health domains in different breeds of dog (34–36). In this regard two hypotheses have been proposed: the compression hypothesis, which states that age-related changes occur earlier in larger dogs than in smaller dogs and are related to life stage rather than chronological age, and the truncation hypothesis, which purports that aging is related to chronological age rather than lifestage, thus larger dogs frequently die prior to developing certain age-related changes (37). Current evidence suggests that not all health-domains behave the same way; some studies have shown that larger dogs age at an accelerated rate (38, 39), however, Watowich et al. (37) demonstrated that age-related changes in cognition are largely independent of the expected lifespan (37). Our study suggests that variations in gait speed may fit best with the compression hypothesis and are related to lifestage rather than chronological age. Similarly, Morgan et al. (15) showed that dogs older than 75% of their expected lifespan had slower gait speeds than younger dogs (15).
+
+### Association between gait speed and cognitive performance
+
+Gait speed off leash was correlated with several domains of cognitive performance. For instance, it was negatively correlated with CADES score and positively correlated with performance in all the cognitive tests, showing that the speed at which dogs are willing to move in order to obtain a food reward is associated with specific domains of cognition. When correcting for fractional lifespan, gait speed off leash remained correlated with sustained gaze and memory; both tasks that require sustained attention. Studies in humans have shown similar results; gait speed was more strongly associated with attention than with other measures of executive function (40, 41). However, another study found that gait slowing was associated with poorer performance in several different cognitive tasks, and they propose that there is no specific domain in cognitive decline that accounts for the reduction in gait speed (42). As we were measuring gait speed toward a treat, we consider that we are not measuring just the physical ability of the dogs, but also their motivation. Future studies should evaluate the relationship between motivation and different cognitive domains in dogs, and if this could explain why speed off leash are correlated with only some of the cognitive domains.
+
+Studies in people have shown that specific areas of the brain are greatly atrophied in those with declines in memory and gait speed (known as dual decliners) in comparison with people with decline in only one domain (41). Some of these areas are the frontal gyrus, cerebellum, precuneus and thalamus. The last two are critical for sensorimotor integration (43). While the cerebellum is mainly known for its role in control and integration of motor activity, it also engages in cognitive processing (44). In fact, in people, cerebellar lesions can impair cognitive function (45) and neuroimaging studies have demonstrated cerebellar activation in executive and working-memory processes (46). In dogs with CCDS brain atrophy, reduction of the inter-thalamic adhesion and hippocampal volume has been demonstrated (47, 48). Moreover, aging is associated with a loss of Purkinje and granular cells and dogs with CCDS showed a further reduction in Purkinje cells (49) than age-matched healthy dogs. Further studies are needed to determine whether dogs with dual decline (in gait speed and cognition) are characterized by specific brain changes on MRI.
+
+We did find an association between gait speed and CADES score but this association was lost when correcting for fractional lifespan. This could be explained by the fact that while the CADES questionnaire has been validated (19), it relies on the sensitivity of the owner to detect behavioral changes. Moreover, this questionnaire evaluates cognitive decline by asking about several different behavioral domains, without looking at particular domains of cognition.
+
+### Association between gait speed and pain
+
+Finally, since prevalence of chronic pain conditions such as osteoarthritis increase with age in dogs (50), we wanted to investigate the relationship between pain and gait speed. Regarding this, a recent study has shown that according to the owner's perception, dogs with osteoarthritis walk slower than normal physically sound dogs (51). However, in our study, neither the CBPI pain severity subscale nor total joint pain were correlated with speed on or off leash. We did find an association between the CBPI pain interference subscale and gait speed off leash, but this association was lost when correcting for fractional lifespan. However, these results do not rule out the role of pain in declining speed in dogs, since our population of dogs were not particularly painful, (i.e., the highest joint pain score was 17, which is only 27% of the highest possible score for this scale). Further studies in dogs with diagnosed osteoarthritis are warranted.
+
+Our study had some limitations. Firstly, gait speed measurements and cognitive tests were conducted without the presence of the dogs' owners, who serve as their attachment figure. This may have influenced the behavior and performance of dogs with separation anxiety (52). We did not use any clinical metrology instrument to measure separation anxiety in these dogs, however, we excluded dogs that displayed signs of anxiety and avoided approaching the experimenters for treats, even though we did not use any clinical metrology instrument to measure separation anxiety. Additionally, while the presence of the owner may be beneficial for some specific dogs, it could potentially interfere with testing results (53) or cause dogs to exhibit more aggression (54). Nonetheless, we ensured that the experimental conditions were consistent within and across dogs, which we believe is more crucial than the presence or absence of the owner during testing. Secondly, since this was an exploratory study, we did not adjust for multiple comparisons. Therefore, future studies should investigate the correlation between gait speed off leash and various cognitive domains in greater detail and with a larger number of dogs to confirm our findings.
+
+## Conclusion
+
+In conclusion, our study shows that gait speed off leash is a relatively easy variable to measure in clinical settings and is correlated with fractional lifespan and cognitive performance in dogs. Moreover, gait speed off leash seems to be a better measure than gait speed on leash as an indicator of age-related deterioration and cognitive decline. This simple and non-invasive measurement is an impactful addition to assessment of senior and geriatric dogs.
+
+## Data availability statement
+
+The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.
+
+## Ethics statement
+
+The animal study was reviewed and approved by the Institutional Animal Use and Care Committe, NCSU. Written informed consent was obtained from the individual(s) for the publication of any identifiable images or data included in this article.
+
+## Author contributions
+
+AM: conceived and designed work, acquired and interpreted data, and wrote manuscript. MK and BC: acquired and interpreted data, and reviewed manuscript. GF and WP: designed work, acquired and interpreted data and reviewed manuscript. MG: designed work, reviewed and interpreted data, and reviewed manuscript. NO: conceived and designed work, reviewed and interpreted data, and reviewed manuscript. All authors contributed to the article and approved the submitted version.

fulltext/md/PMC10315540.md

@@ -0,0 +1,208 @@
+---
+pmcid: "PMC10315540"
+pmid: "37405168"
+doi: "10.3389/fmicb.2023.1209158"
+title: "Gut microbiome dysbiosis is associated with host genetics in the Norwegian Lundehund."
+journal: "Frontiers in microbiology"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Gut microbiome dysbiosis is associated with host genetics in the Norwegian Lundehund
+
+## Abstract
+
+A group of diseases have been shown to correlate with a phenomenon called microbiome dysbiosis, where the bacterial species composition of the gut becomes abnormal. The gut microbiome of an animal is influenced by many factors including diet, exposures to bacteria during post-gestational growth, lifestyle, and disease status. Studies also show that host genetics can affect microbiome composition. We sought to test whether host genetic background is associated with gut microbiome composition in the Norwegian Lundehund dog, a highly inbred breed with an effective population size of 13 individuals. The Lundehund has a high rate of a protein-losing enteropathy in the small intestine that is often reported as Lundehund syndrome, which negatively affects longevity and life-quality. An outcrossing project with the Buhund, Norrbottenspets, and Icelandic sheepdog was recently established to reintroduce genetic diversity to the Lundehund and improve its health. To assess whether there was an association between host genetic diversity and the microbiome composition, we sampled the fecal microbiomes of 75 dogs of the parental (Lundehund), F1 (Lundehund x Buhund), and F2 (F1 x Lundehund) generations. We found significant variation in microbiome composition from the parental Lundehund generation compared to the outcross progeny. The variation observed in purebred Lundehunds corresponded to dysbiosis as seen by a highly variable microbiome composition with an elevated Firmicutes to Bacteroidetes ratio and an increase in the prevalence of *Streptococcus bovis/Streptococcus equinus* complex, a known pathobiont that can cause several diseases. We tracked several other environmental factors including diet, the presence of a cat in the household, living in a farm and the use of probiotics, but we did not find evidence of an effect of these on microbiome composition and alpha diversity. In conclusion, we found an association between host genetics and gut microbiome composition, which in turn may be associated with the high incidence of Lundehund syndrome in the purebred parental dogs.
+
+1.
+
+## Introduction
+
+The gut microbiome of an animal is influenced by many factors including diet (De Filippo et al., 2010), exposures to bacteria during post-gestational growth (Torrazza and Neu, 2011; Dowling and Levi, 2014), lifestyle (Clemente et al., 2015; Tun et al., 2017), disease status (Deng and Swanson, 2015), and host genetics (Hughes et al., 2020). Studies suggest a genetic effect on the microbiome composition (Blekhman et al., 2015; Weissbrod et al., 2018; Hughes et al., 2020; Bubier et al., 2021), and differences in gut microbiome composition among dog breeds (Hooda et al., 2012; You and Kim, 2021), although the mechanisms through which specific genes modulate microbiome composition is still unclear. Bubier et al. (2021) describes three mechanisms through which genes could control diseases and link to the microbiome: (1) they might cause the disease phenotype, and microbiome is altered as consequence of disease; (2) they might affect gene expression in the host and indirectly alter the microbiome, which causes the disease; (3) they might affect the microbiome directly and cause the disease through the microbiome. Unraveling these mechanisms requires that we have good models where we can consider host genetics, microbiome, disease phenotype and their relationships simultaneously (Bubier et al., 2021).
+
+Several diseases occurring in humans and dogs, such as Inflammatory Bowel Disease (IBD, including Crohn’s disease and ulcerative colitis), are characterized by an imbalance in the gut microbiota, called “dysbiosis,” where an overgrowth of harmful bacteria, a loss of beneficial bacteria or lowered alpha diversity can occur simultaneously (DeGruttola et al., 2016). It is, however, still unclear whether the dysbiosis is a risk factor or a consequence of the disease (DeGruttola et al., 2016). In both dogs and humans, an association between IBD and dysbiosis has been reported, as seen by an increase of Bacteroidetes (Suchodolski et al., 2012). Additionally, Firmicutes have been found to be decreased in dogs with IBD (Minamoto et al., 2015). However, the dysbiosis in dogs and humans differs in some key bacterial groups (Vázquez-Baeza et al., 2016), possibly due to profound morphological and physiological differences and a relatively recent human adaptation to a more carnivorous diet (Price et al., 2012). For example, *Fusobacterium* appears to be associated with IBD and colorectal cancer in humans, but no association has been established in dogs (V��zquez-Baeza et al., 2016). In humans and mice, obesity has been found to be associated with decreased microbial diversity and an increased Firmicutes/Bacteroidetes (F/B) ratio, whereas in dogs this relationship was not confirmed (Chun et al., 2020; You and Kim, 2021).
+
+The Norwegian Lundehund is a small spitz dog breed that was used to fetch nesting Atlantic puffins *Fratercula arctica* on steep cliffs in the coast of northern Norway (Melis et al., 2013). Towards the end of the 19th century, using nets to hunt puffin became more common than using dogs. Thus, the breed lost its economic importance and was confined to the small fisherman’s village of Måstad on the island of Værøy in the Lofoten archipelago. Two bottlenecks, the first caused by an outbreak of canine distemper in the 1940s, and the second caused by the abandonment of the village of Måstad in the 1960s, left only five highly related individuals. Currently, the breed counts more than 1,500 individuals, which all descend from these five dogs. For this reason, the Lundehund has an extremely low level of heterozygosity, which is around 5% as estimated by high-density SNP arrays (Melis et al., 2022), and an effective population size of only 13 individuals (Melis et al., 2013; Kropatsch et al., 2015). The low genetic diversity is associated with low fertility and with high rates of a protein-losing enteropathy localized to the small intestine, often reported as the Lundehund syndrome, but also as intestinal lymphangiectasia and IBD (Berghoff et al., 2007). Chronic atrophic gastritis and gastric neoplasms are also common in dogs with Lundehund syndrome (Kolbjornsen et al., 1994a,b; Qvigstad et al., 2008). Lundehund syndrome is usually treated by administration of immunosuppressant and anti-inflammatory drugs such as prednisone, prednisolone, or azathioprine and with antibiotics to reduce bacterial overgrowth (Berghoff et al., 2007). However, the extremely low genetic diversity also makes the Lundehund a possible genetic model of gut and autoimmune disease. A mortality study estimated that about 40% of deaths before 11 years of age occurs as a consequence of Lundehund syndrome or other gastrointestinal diseases (Norwegian Lundehund Club, 2014). Although a study found an association between Lundehund syndrome and a missense mutation in the gene LEPREL1 (Metzger et al., 2016), the inheritance mechanism of the Lundehund syndrome is not clear, and it is likely that several genes are involved in the development of this illness (Metzger et al., 2016). Very few individuals in the population do not carry the LEPREL1 mutation, making it impossible to use this information to select breeding individuals, without further reducing the already depauperate gene pool.
+
+Because of the extremely low genetic diversity, the associated disease, and low fecundity, the Norwegian Lundehund Club started an outcrossing project in 2014 with three Nordic dog breeds, the Buhund, the Norrbottenspets, and the Icelandic sheepdog. We focus this study on the Buhund outcross, which started earlier and has, so far, produced the most individuals. The outcrossing project started by mating two unrelated females of Buhund with male Lundehunds, in order to avoid problems with gestation and delivery, due to the difference in relative size, the Buhund being about 30% larger than the Lundehund. The first-generation crossings (F1) were then backcrossed to pure Lundehunds, resulting in the second-generation crossings (F2). All dogs included in the project were screened for hip dysplasia and hereditary ocular pathologies before including them in breeding. We previously analyzed the genetic diversity of the parental, F1, and F2 animals from the Lundehund x Buhund outcross and found a restoration of genetic diversity through outcrossing, with F1 animals having highest diversity (Melis et al., 2022). The mean heterozygosity (estimated from 8,184 linkage-disequilibrium-pruned loci) of Lundehund, F1 and F2 dogs was 0.043, 0.272 and 0.153, respectively (Melis et al., 2022). None of the F1 and F2 progeny has, so far, developed Lundehund syndrome.
+
+To ask whether there might be a microbiome basis for Lundehund syndrome, we sampled stool from parental, F1 and F2 dogs. We analyzed the 16S rRNA gene diversity of the fecal microbiome of purebred Lundehund (P), F1 and F2 individuals (Table 1; Figure 1) with the following aims:Characterize the fecal microbiome composition of Lundehund (P) and first (F1) and second (F2) generation of outcrossings with Buhund.Test whether a range of factors including diet type, presence of a cat in the household, administration of probiotics, and living on a farm correlate with microbiome diversity.Test whether microbiome composition clusters according to cohort (P, F1, F2).Explore whether the ratio between Firmicutes and Bacteroidetes (F/B, an index of dysbiosis) differs between dogs who had Lundehund syndrome and those who did not.
+
+**Table 1 Phenotypical traits and environmental characteristics of the dogs included in this study.**
+
+|  | Lundehund (P) | F1 | F2 |
+| --- | --- | --- | --- |
+|  | *n* | % | *n* | % | *n* | % |
+| Gender |
+| Females | 28 | 56 | 6 | 75 | 10 | 45 |
+| Males | 22 | 44 | 2 | 25 | 12 | 55 |
+| Age (years) |
+| Mean | 5.7 | – | 5.6 | – | 3 | – |
+| SD | 2.7 | – | 1.06 | – | 1.34 | – |
+| Weight (kgs) |
+| Mean | 7.7 | – | 9.62 | – | 9.41 | – |
+| SD | 1.3 | – | 2.13 | – | 2.17 | – |
+| Lundehund syndrome history |
+| Yes | 11 | 22 | 0 | 0 | 0 | 0 |
+| No | 39 | 78 | 8 | 100 | 22 | 100 |
+| Probiotics in the last six months |
+| Yes | 20 | 40 | 1 | 13 | 1 | 5 |
+| No | 30 | 60 | 7 | 88 | 21 | 95 |
+| Prevalent diet type |
+| Home made | 26 | 52 | 2 | 25 | 4 | 18 |
+| Industrial dry | 16 | 32 | 4 | 50 | 13 | 59 |
+| Raw | 8 | 16 | 2 | 25 | 5 | 23 |
+| Presence of a cat in the household |
+| Yes | 9 | 18 | 3 | 38 | 9 | 41 |
+| No | 41 | 82 | 5 | 63 | 13 | 59 |
+| Antibiotics in the last six months |
+| Yes | 5 | 10 | 0 | 0 | 0 | 0 |
+| No | 45 | 90 | 8 | 100 | 22 | 100 |
+| Home environment |
+| Farm | 3 | 6 | 3 | 38 | 7 | 32 |
+
+*Figure: Figure 1 Study design to test whether outcrossing Norwegian Lundehund with Buhund is associated with fecal microbiome composition. The dataset included stool samples from 73 dog individuals: 45 Lundehunds (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2).*
+
+2.
+
+## Materials and methods
+
+2.1.
+
+### Stool samples collection and analysis
+
+In April to August 2021, we collected stool samples from Lundehund (P, *n* = 50), Lundehund x Buhund crosses (F1, *n* = 8), and F1 × Lundehund crosses (F2, *n* = 22). The lower number of individuals in the F1 cohort is due to the challenge in finding owners of Buhund females that were willing to let their dog being paired with a Lundehund. Owners were instructed in how to collect and handle fresh naturally deposited samples, avoiding contamination. The stool samples were stored at room temperature in Stool Nucleic Acid Collection and Preservation Tubes (Norgen BioTek Corp, Cat. 45,660). In September 2021, the samples were analyzed with the ZymoBIOMICS® Targeted Sequencing Service (Zymo Research, Irvine, CA). The ZymoBIOMICS®-96 MagBead DNA Kit (Zymo Research, Irvine, CA) was used to extract DNA using an automated platform. Bacterial 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16S™ NGS Library Prep Kit (Zymo Research, Irvine, CA). The bacterial 16S rRNA primers amplified the V3-V4 region of the 16S rRNA gene. The final PCR products were quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned with the Select-a-Size DNA Clean & Concentrator™ (Zymo Research, Irvine, CA), then quantified with TapeStation® (Agilent Technologies, Santa Clara, CA) and Qubit® (Thermo Fisher Scientific, Waltham, MA). The ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research, Irvine, CA) was used as a positive control for each targeted library preparation. Negative controls (i.e., blank extraction control, blank library preparation control) were included to assess the level of bioburden carried by the wet-lab process. The final library was sequenced on an Illumina® MiSeq™ with a v3 reagent kit (600 cycles). The sequencing was performed with a 10% PhiX spike-in. Unique amplicon sequences variants (ASVs) were inferred from raw reads using the DADA2 pipeline (Callahan et al., 2016). Potential sequencing errors and chimeric sequences were also removed with the DADA2 pipeline. ASVs that were present in only one sample, i.e., singletons, were examined for each cohort and removed from the dataset for clustering analysis. Taxonomy assignment was performed using Uclust from Qiime v.1.9.1 (Caporaso et al., 2010) with the Zymo Research Database. Any taxa that were not represented at over 1% relative abundance in at least one sample were removed. Five purebred Lundehund dogs older than 9 years and two F2 dogs that were under medication at sampling were also removed from further analyses, in order to obtain a sample more homogenous in age and without the influence of antibiotics. Normalization of the data was performed by calculating the relative abundance for each sample by library scaling.
+
+2.2.
+
+### Microbiome statistical analyses
+
+The microbiome analyses were done with a dataset including 306 bacterial species and 73 individual samples in R version 4.1.3 (R Core team, 2021) with RStudio version 2022.07.2 (RStudio Team, 2022) and with the R packages *Phyloseq* (McMurdie and Holmes, 2013) and *Microbiome* (Lahti et al., 2017). We explored the microbiome composition by plotting the relative abundance of bacterial phyla and of the genera present at >1% relative abundance of which there were 15. These plots were produced with the function *comp_barplot* from the package *microViz* version 0.10.8 (Barnett et al., 2021). The R package *plyr* (Wickham, 2011) was used to calculate richness and diversity of bacteria according to the different categories reported in Table 1. We tested for a statistical difference in relative abundance of all phyla and the subset of 15 most abundant genera with the function *xdc.sevsample* in the Human Microbiome Project (*HMP*) R package version 2.0.1 (La Rosa et al., 2012). This function performs a multivariate test for differences in composition between groups assuming Dirichlet-multinomial distribution by testing for a difference in the mean distribution of each taxon across groups and also account for the overdispersion in the count data (Wilks, 1938). Differences in relative abundances of specific phyla between groups were compared by the R package *Maaslin2* (Mallick et al., 2021) with Benjamini-Hochberg correction to control for false discovery rate. For this analysis, due to the low number of F1 individuals, the F1 and the F2 generations were pooled together and compared to the purebred Lundehund generation (P).
+
+Alpha diversity, as calculated by the Shannon index, was compared across groups according to Table 1. As a measure of beta diversity, a principal component analysis at species level was performed with the R package *Phyloseq* (McMurdie and Holmes, 2013) and the command *ordinate* and “RDA” method (the distance method on the Bray Curtis distance). Comparisons between Shannon indices and F/B ratios were performed by Wilcoxon tests with Bonferroni correction. We also tested whether the samples clustered to a higher degree than expected by sampling variability using permutational multivariate analysis of variance (PERMANOVA). For the permutational analysis the distance method was set to “Euclidean.”
+
+2.3.
+
+### Environmental variables
+
+A questionnaire was also sent to the dog owners, together with the stool sampling tubes, to obtain information about environmental factors such as the typical diet, Lundehund syndrome history, antibiotics and probiotics administration (in the previous 6 months), presence of a cat in the household and whether the dog lived on a farm or in a more urban environment (Table 1). Many of the purebred Lundehunds (40%) used a combination of two to three different types of probiotics, whereas probiotics administration was less common among F1 and F2 dogs.
+
+3.
+
+## Results
+
+3.1.
+
+### Microbiome compositional variation is correlated with dog genotype
+
+To assess whether Lundehund syndrome is associated with gut dysbiosis, we collected stool samples and metadata (Table 1) from 73 dogs, comprising 45 purebred Lundehunds, 8 Lundehund × Buhund F1 animals, and 20 F1 x Lundehund crosses, which are the F2 generation. To examine the microbiome of the Norwegian Lundehund and F1 and F2 outcrossing generation, we assessed the bacterial taxonomic composition of the stool by 16S rRNA gene amplicon library sequencing. After assigning taxonomy to the bacteria in each sample based on ASV and filtering the data to remove singletons and rare taxa, the final microbiome data set included 306 bacteria species and 73 individual samples. Five ASVs, with taxonomic assignments to *Collinsella intestinalis-stercoris*, *Blautia hansenii-producta*, *Lachnoclostridium* sp32341-sp32430, Clostridiales (no species), and *Fusobacterium mortiferum*, were present in all samples. A loss of alpha diversity has also been shown by various studies to correlate with microbiome-associated disease. We detected no significant differences in alpha diversity, as calculated by Shannon index (Appendix S1), as a function of dog generation, Lundehund syndrome history, diet type, administration of antibiotics, administration of probiotics, presence of a cat in the household, or living on a farm versus in a suburban environment (Wilcoxon rank sum test, all *p >* 0.05), although we note that the potential influence of these factors on microbiome composition cannot be ruled out due to the relatively small sample size.
+
+To further assess the microbiome compositional variation between dogs (beta diversity), we performed a principal component analysis at the species level (Figure 2). By defining the centroid of variation for each of the dog generations, we found that purebred Lundehunds were clearly differentiated from F1 and F2 dogs based on principal component #1 (PC1), which explains 33% of the variation in the data (Figure 2A). Whereas there was wide variation in PC1 for purebred Lundehunds, the F1 and F2 generations were much more similar to one another (Figure 2A). The distance-based test of homogeneity of multivariate dispersions showed that the samples clustered beyond the expectation from sampling the total variability (*F* = 7.3, df = 2, *p* = 0.002, Figure 2B). We also performed a principal component analysis with a dataset including only purebred Lundehunds and plotted the ordination by Lundehund syndrome history (Appendix S2). The ellipses overlapped almost totally, showing that there is no difference in beta diversity between purebred Lundehunds which had a Lundehund syndrome history and those which did not.
+
+*Figure: Figure 2 **(A)** Ordination analysis performed with *Phyloseq* on gut microbiome of 73 dog individuals, including 45 Lundehunds (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2). **(B)** Ordination centroids and dispersion measured by Aitchison distance on gut microbiome composition of the same dataset.*
+
+Overall, our results indicate that microbiome composition is associated with the genetic background of the dogs. The majority of the variation occurred in purebred Lundehunds, suggesting that these dogs lack some control mechanism regulating their microbiome composition. We found no evidence that other known factors associated with microbiome disease contribute to the microbiome composition in Lundehunds.
+
+3.2.
+
+### Lundehunds have a microbiome compositional signature at the phylum and the genus levels
+
+The most abundant bacterial phyla across all samples were Firmicutes (57%), Bacteroidetes (23%), and Fusobacteria (10%), followed by Proteobacteria (4%), and Actinobacteria (4%; Figure 3A). To assess which were the compositional differences based on the genetic background of the dogs, we examined the composition of bacteria at the phylum level. We expected that any taxa that are associated with the Lundehund genetic background should be highest in Lundehund, lowest in the F1 generation, and intermediate between Lundehunds and F1 in the F2 backcrosses. Phylum-level differences in relative abundances were evident based on purebred versus outcrossed status of the dogs (Figure 3A).
+
+*Figure: Figure 3 **(A)** Relative abundance of bacterial phyla identified in stool samples from 73 dog individuals, including 45 Lundehunds (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2). **(B)** Box and whiskers plots of the relative abundance of bacterial phyla identified in stool samples from 73 dog individuals. Whiskers represent 1.5 times the interquartile range past the low and high quartiles. Points outside whiskers range are outliers.*
+
+We plotted the relative abundance of each phylum for each generation of dogs (Figure 3B). The relative abundance at the phylum level was significantly different between the three generations (X ~several sample test~ = 53.65, *p* = 6 × 10^−6^). Overall, the F1 and F2 progeny were more similar to each other in their microbiome composition than to the purebred dogs. Due to the low number of F1 dogs, we combined the F1s and F2s and compared the phylum-level microbiome abundances for these with the purebred Lundehunds. The abundances of Actinobacteria and Firmicutes were higher in purebred Lundehunds than in the F1 and F2 progeny (both *p* = 0.007). The relative abundance of Fusobacteria showed the opposite pattern, with a lower abundance in purebreds than in the F1 and an intermediate abundance in the F2 progeny (*p* = 0.04). The F1 and F2 dogs also had lower variance in composition at the phylum level (Figure 3B), consistent with the variation observed by principal component analysis (Figure 2A).
+
+To further delineate bacterial taxa associated with the different dog genetic backgrounds, we performed the same analyses as in Figure 3 at the genus level on a subset including the genera present at >1% relative abundance across all samples, amounting to 15 highest abundance genera (Figure 4A). These analyses also revealed some differences between cohorts. To examine more in detail the genus-level variation in relative abundance in the purebred Lundehunds versus F1 and F2 progeny, we plotted the relative abundance of the 15 most abundant genera for each generation of dogs (Figure 4B). The relative abundances of genera were overall significantly different between the three cohorts (X ~several sample test~ = 110.39, *p* = 4 × 10^−11^). We next made pairwise statistical tests comparing the relative abundance of each genus between purebred and outcrossed dogs (F1 and F2). We detected significant differences for 7 genera by doing pairwise tests, indicating these specific genera are associated with the genetic background of the dogs. *Streptococcus* (*p =*3 × 10^−7^), *Lactobacillus* (*p =* 0.0005), and *Holdemanella* (*p =* 0.01) were at higher abundance in the purebred Lundehunds whereas *Alloprevotella* (*p =* 0.003), *Blautia* (*p =* 0.005), *Lachnoclostridium* (*p =* 0.02) and *Fusobacterium* (*p =* 0.02) were at higher abundance in the outcrossed dogs. Overall, we detected significant associations between specific bacterial taxa and the Lundehund genetic background, indicating a host genetic basis for the gut microbiome compositional differences.
+
+*Figure: Figure 4 **(A)** Relative abundance of the 10 most abundant bacterial genera identified in stool samples from 73 dog individuals, including 45 Lundehunds (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2). **(B)** Box and whiskers plots of the relative abundance of the 10 most abundant bacterial genera identified in stool samples from 73 dog individuals. Whiskers represent 1.5 times the interquartile range past the low and high quartiles. Points outside whiskers range are outliers.*
+
+Since changes in the ratio of Firmicutes to Bacteroidetes have been associated with microbiome dysbiosis in numerous studies of microbiome-associated diseases (e.g., Suchodolski et al., 2012; Minamoto et al., 2015; Vázquez-Baeza et al., 2016; Chun et al., 2020; You and Kim, 2021), we computed the Firmicutes to Bacteroidetes ratio for each dog and compared between the generations. We found that purebred Lundehunds have a higher Firmicutes to Bacteroidetes ratio than F1 and F2 progeny (Wilcoxon test, W = 943, *p* = 0.0003). We note that no Lundehund syndrome has been detected to date in any of the F1 or F2 progeny.
+
+3.3.
+
+### The Lundehund microbiome is not indicative of Lundehund syndrome
+
+To examine whether Lundehund syndrome is also associated with microbiome composition, we plotted the relative abundance of each phylum as a function of Lundehund syndrome status (Figure 5) within the purebred Lundehunds. The relative abundance of phyla did not differ significantly between purebred Lundehund who had a diagnosis of Lundehund syndrome at some point in their life versus those who did not (X ~several sample test~ = 7.56, *p* = 0.48).
+
+*Figure: Figure 5 Box and whiskers plots of the relative abundance of bacterial phyla identified in stool samples from dogs which Y = had Lundehund syndrome (LS), and N = which did not have LS. Whiskers represent 1.5 times the interquartile range past the low and high quartiles. Points outside whiskers range are outliers. The data set includes 45 purebred Lundehunds (P).*
+
+To further examine whether there is a Lundehund syndrome microbiome composition, we calculated the Firmicutes to Bacteroidetes ratio for purebred Lundehunds with and without a history of Lundehund syndrome. The median ratio of Firmicutes to Bacteroidetes was 4.41 (*n* = 9) in dogs who had Lundehund syndrome versus 2.25 (*n* = 36) in dogs who did not. While there is a trend of a higher ratio in dogs with a history of Lundehund syndrome, these medians were not significantly different between the two groups (Wilcoxon test, W = 215, *p* = 0.1).
+
+To further delineate the microbiome associated with Lundehund syndrome, we plotted the relative abundance of the 15 most abundant genera as a function of Lundehund syndrome status (Figure 6). Purebred Lundehunds who had a diagnosis of Lundehund syndrome at some point in their life did not differ significantly in the relative abundance at genus level compared to healthy dogs (X ~several sample test~ = −10.11, *p* = 1). Overall, we found no compositional differences that were significantly associated with Lundehund syndrome within the purebred dogs, consistent with the genotype of the dogs driving the compositional differences we observed between generations.
+
+*Figure: Figure 6 Box and whiskers plots of the relative abundance of the 15 most abundant bacterial genera identified in stool samples from Lundehunds which Y = had Lundehund syndrome (LS), and N = which did not have LS. Whiskers represent 1.5 times the interquartile range past the low and high quartiles. Points outside whiskers range are outliers. The data set includes 45 purebred Lundehunds.*
+
+3.4.
+
+### *Streptococcus equinus-Infantarius-lutetiensis* is more abundant in purebred Lundehund
+
+In examining the variation in microbiome composition (Figure 2) and the phylum- and genus-level differences in abundances between purebred dogs and the F1 and F2 progeny (Figures 3, 4), we noticed that the F1 and F2 generations appeared similar to one another and had overall more consistent abundances of each bacterial phylum and genus, whereas purebred Lundehunds have much wider variation in microbiome composition.
+
+To assess whether any specific taxa corresponded to the differences in the variation based on principal component analysis, we examined the major contributing taxa to principal component 1, which clearly differentiated the purebred dogs from the F1 and F2 generations (Figure 2). Based on the loadings (Table 2), we found that *S. equinus-infantarius-lutetiensis* was by far the most important species for principal component #1. We next examined the abundance of *S. equinus-infantarius-lutetiensis* in the individual samples and found that this bacterium is much more abundant in purebred Lundehunds and almost absent in F1 and F2 dogs (Figure 7), indicating that, in addition to the dysbiosis signature in the microbiome in the principal component analysis (Figure 2), and the Firmicutes/Bacteroidetes ratio, Lundehunds also have a characteristic species, *S. equinus-infantarius-lutetiensis*, which is associated with the dysbiosis. When examining the relative abundance of *S. equinus-infantarius-lutetiensis* within the pure bred Lundehunds, we did not find any pattern related to Lundehund syndrome (Appendix S3), indicating that the association is not a potential causative agent in accord with Koch’s first postulate on infectious disease (Evans, 1978) which says that the microorganism must be found in the diseased animal, and not found in healthy animals. Rather, *S. equinus-infantarius-lutetiensis* is a species associated with dysbiosis in Lundehunds.
+
+**Table 2 Most important species dominating the first component of the PCA on microbiome composition of 73 dog individuals, including 45 Lundehund (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2).**
+
+| Sequence | PCA1 | Species |
+| --- | --- | --- |
+| 102 | 0.9304039 | *Streptococcus equinus-infantarius-lutetiensis* |
+| 34 | −0.1612259 | *Bacteroides* sp12209 |
+| 274 | −0.1372722 | *Fusobacterium mortiferum* |
+| 278 | −0.1278976 | *Fusobacterium* sp37464 |
+| 53 | −0.1040231 | *Alloprevotella* sp13496-sp13497 |
+| 195 | −0.0764320 | *Clostridiales* sp. |
+| 147 | −0.0737325 | *Lachnoclostridium* sp32341-sp32430 |
+| 88 | 0.0693046 | *Lactobacillus reuteri-vaginalis* |
+| 72 | 0.0663620 | *Lactobacillus* sp. |
+| 124 | −0.0635586 | *Blautia hansenii-producta* |
+
+*Figure: Figure 7 Relative abundance of *S. equinus-infantarius-lutetiensis* in gut microbiome of 73 dog individuals, including 45 Lundehunds (P), eight first-generation crosses Lundehund × Buhund (F1) and 20 first-generation backcrosses F1 × Lundehund (F2).*
+
+4.
+
+## Discussion
+
+This study examined the microbiome of purebred Lundehund dogs compared with first and second-generation outcrossings with the Buhund. Consistent with previous studies on the dog fecal microbiome, the dominant phyla in all cohorts were Firmicutes, followed by Bacteroidetes and Fusobacteria (e.g., Swanson et al., 2011; You and Kim, 2021).
+
+We sampled privately owned dogs that had a range of diet and probiotic regimes, therefore our dataset included several potentially confounding factors, such as diet, probiotics regime, different living conditions and age classes. Moreover, Lundehund syndrome is an acute life-threatening disease, which requires immediate treatment with anti-inflammatory drugs and antibiotics. For this reason, we could not collect samples from sick dogs before antibiotics administration and we thus compared the fecal microbiome of healthy dogs (which never had a diagnosis for Lundehund syndrome) with that of dogs which had recovered from the illness.
+
+Despite these limitations, we detected a signature of purebred Lundehund status in the microbiome composition when comparing the different generations of dogs. Given the expectation that Lundehund genetic background-associated taxa should be high in purebred dogs, low in F1s, and intermediate in F2, Firmicutes and Actinobacteria appear Lundehund-associated, whereas this was not the case for Fusobacteria and Bacteroidetes. Our results are consistent with several studies which showed that sequences belonging to the phylum Bacteroidetes decreased in dogs with acute diarrhea compared to healthy dogs (Chaban et al., 2012; Guard et al., 2015). Fusobacteria have also been found to be decreased in dogs with clinically active inflammatory bowel disease (IBD; Suchodolski et al., 2012) and are generally associated with a healthy microbiota (Vázquez-Baeza et al., 2016; Pilla and Suchodolski, 2020).
+
+An increased or decreased F/B ratio is considered as a sign of imbalance in the intestine, or dysbiosis. An increased F/B ratio is often observed in humans with obesity (Abenavoli et al., 2019), although there are contradictory results (Chun et al., 2020; Magne et al., 2020; You and Kim, 2021), whereas a decreased F/B ratio is observed in the intestine of humans with IBD (Shen et al., 2018). We observed a significantly increased F/B ratio in purebred Lundehunds. However, contextualizing this result, we might not necessarily expect to be able to compare the relative abundances of functional bacterial groups in humans and dogs, since they have evolved under different pressures, such an omnivorous diet in humans versus a carnivorous diet in dogs (Vázquez-Baeza et al., 2016).
+
+We could not observe any correlation between environmental factors and microbiome alpha diversity as calculated by the Shannon index, but the small size of the dataset and the coexistence of several factors, could make it difficult to disentangle their effects. Despite that, microbiome composition (beta diversity) clustered according to cohort, revealing a signature of the genome in the microbiome. When purebred Lundehunds were compared with F1 and F2 crosses, the variance in microbiomes was larger within the purebred Lundehunds than within F1 and F2 animals. Higher microbiome disparity was evident in purebred Lundehunds when examining the PC1 in the principal component analysis (Figure 2), suggesting the loss of a control mechanism over microbiome composition in Lundehunds.
+
+Interestingly, we found that a single taxon, *S. equinus-infantarius-lutetiensis* was the most important species driving PC1 (explaining 33% of variation in the data). *S. equinus-infantarius-lutetiensis* was much more abundant in the purebred Lundehund, than in the F1 or F2. Several studies suggest an association between gut diseases in humans and bacteria belonging to the *S. bovis* group, which includes *S. equinus-infantarius-lutetiensis*. *S. equinus-infantarius-lutetiensis* was for instance isolated in stool samples of children with diarrhea of unknown origin, suggesting its pathogenic potential (Jin et al., 2013). This bacterium has also been linked to colorectal carcinogenesis in humans, since it could be found at higher rates in the stools of patients with colorectal tumors (Chirouze et al., 2013; Kaindi et al., 2018). In 2005, Vanhoutte et al. (2005) conducted a study on the stability of the gut microbiome after administration of prebiotics, and recommended investigation of *S. equinus-infantarius-lutetiensis* ecology and its role in the gut of healthy dogs, since it was the streptococcal group with the most pronounced population growth observed after administration of the prebiotic, fructan. *S. equinus-infantarius-lutetiensis* was also isolated in a cat with intestinal lymphoma (Piva et al., 2019) and in the equine hindgut, in conjunction with oligofructose-induced laminitis (Milinovich et al., 2008). All of these studies point towards *S. equinus-infantarius-lutetiensis* being a pathobiont, i.e., a commensal bacteria normally present in the gut of healthy humans and other animals, with the potential to either cause serious infections or activate the immune system, causing inflammatory diseases (Chow et al., 2011; Jans and Boleij, 2018). Thus, by being more permissive of this strain, the Lundehund genetic background may increase the risk of *S. equinus-infantarius-lutetiensis* causing pathology.
+
+The fact that the principal component analysis performed on a subset of purebred Lundehunds did not show any difference in fecal microbiome composition between the healthy individuals and that with a history of Lundehund syndrome might indicate that all purebred Lundehunds are genetically predisposed to dysbiosis.
+
+By comparing the microbiome of Lundehunds with that of first and second-generation outcrossings with Buhund, we concluded that Lundehunds have a highly varied microbiome and that Lundehund syndrome is characterized by a dysbiotic state, similar to what is observed in humans and dogs with IBD, which is consistent with the disease etiology. However, the F/B ratio was higher in Lundehunds which have had Lundehund syndrome, whereas a lower F/B ratio is observed in humans and dogs with Crohn’s disease (an IBD type) (Minamoto et al., 2015), a difference that might be affected by many factors including both genetic and environmental differences between dogs and humans.
+
+We propose that the loss of microbiome consistency in purebred Lundehunds is due to the loss of genetic loci that are needed for microbiome colonization stability. We further propose that these genetic loci were regained by outcrossing with Buhund in the F1 and F2 dogs, leading to a more stable gut microbiome. Further studies linking microbiome traits with genetic markers at individual level will help elucidate the mechanisms behind gut microbiome specificity in vertebrates.
+
+## Data availability statement
+
+The data presented in this study are available on Dryad, at the URL: https://datadryad.org/stash/dataset/doi:10.5061/dryad.tb2rbp05m.
+
+## Ethics statement
+
+Ethical review and approval were not required for this study because we only collected stool samples and this non-invasive procedure does not require evaluation by an ethics committee in Norway. The owners of the animals were provided with written information about the study and agreed to participate to it by sending stool samples of their dogs.
+
+## Author contributions
+
+CM and WL contributed to conception and design of the study and wrote the first draft of the manuscript. CM and P-AW did the data collection. CM and AB performed the statistical analyses. All authors contributed to manuscript revision and approved the submitted version.
+
+## Funding
+
+This study was supported by Queen Maud University College, the Norwegian Agriculture Agency (grant Agros 1135359), the Peder Sather Center for Advanced Study, and the Norwegian Lundehund Club. WL was supported by NIH DP5OD017851, NSF IOS 2144342, and the Carnegie Institution for Science endowment.
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10317228.md

@@ -0,0 +1,219 @@
+---
+pmcid: "PMC10317228"
+pmid: "37399166"
+doi: "10.1371/journal.pone.0288088"
+title: "The effect of age on the induction dose of propofol for general anesthesia in dogs."
+journal: "PloS one"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# The effect of age on the induction dose of propofol for general anesthesia in dogs
+
+## Abstract
+
+### Objective
+
+In people, the dose of propofol (DOP) required for procedural sedation and anesthesia decreases significantly with age. The objective of this study was to determine if the DOP required to perform endotracheal intubation decreases with age in dogs.
+
+### Study design
+
+Retrospective case series.
+
+### Animals
+
+1397 dogs.
+
+### Methods
+
+Data from dogs anesthetized at referral center (2017–2020) were analyzed with three multivariate linear regression models with backward elimination using a combination of either absolute age, physiologic age, or life expectancy (ratio between age at the time of anesthetic event and expected age of death for each breed obtained from previous literature) as well as other factors as independent variables, and DOP as the dependent variable. The DOP for each quartile of life expectancy (<25%, 25–50%, 50–75%, 75–100%, >100%) was compared using one-way ANOVA. Significance was set at alpha = 0.025.
+
+### Results
+
+Mean age was 7.2 ± 4.1 years, life expectancy 59.8 ± 33%, weight 19 ± 14 kg, and DOP 3.76 ± 1.8 mg kg^-1^. Among age models, only life expectancy was a predictor of DOP (-0.37 mg kg^-1^; *P* = 0.013) but of minimal clinical importance. The DOP by life age expectancy quartile was 3.9 ± 2.3, 3.8 ± 1.8, 3.6 ± 1.8, 3.7 ± 1.7, and 3.4 ± 1.6 mg kg^-1^, respectively (*P* = 0.20). Yorkshire Terrier, Chihuahua, Maltese, mixed breed dogs under 10 kg, and Shih Tzu required higher DOP. Status of neutered male, ASA E, and Boxer, Labrador and Golden Retriever breeds decreased DOP, along with certain premedication drugs.
+
+### Conclusions and clinical relevance
+
+In contrast to what is observed in people, an age cut-off predictive of DOP does not exist. Percentage of elapsed life expectancy along with other factors such as breed, premedication drug, emergency procedure, and reproductive status significantly alter DOP. In older dogs, the dose of propofol can be adjusted based on their elapsed life expectancy.
+
+## Introduction
+
+Propofol is a widely used hypnotic drug able to induce and maintain general anesthesia in people and animals [1, 2]. Propofol inhibits the N‐methyl‐D‐aspartate (NMDA) receptor and activates the ionotropic subtype of the γ‐aminobutyric acid receptor-A (GABA~A~), causing hyperpolarization of the postsynaptic cell membrane and a dose-dependent depression of the central nervous system (CNS) [3]. In dogs, as in people, propofol is commonly used for procedural sedation, and induction and maintenance of general anesthesia [4].
+
+Dogs are heavily relied upon as comparative biomedical research models for several human physiologic and pathophysiologic processes, including aging [5, 6]. In fact, several literature reviews have been published in regard to the usefulness of pet dogs as longitudinal research model for aging and age-associated morbidity and mortality [5–9]. In the last decades, the average life span of dogs has progressively increased. Roughly 30% of the population of client-owned dogs is nowadays considered geriatric [10]. Lower anesthetic requirements of many analgesic and hypnotic drugs in elderly patients, including propofol, have been observed in people [11–15].
+
+There is a large body of literature investigating the relationship between the dose of propofol (DOP) and age in people. It has been shown that, not only is the DOP for induction of a person 60 years of age and older (1.5–1.75 mg kg^-1^) significantly lower than for people under 60 years (2.25–2.5 mg kg^-1^), but also that elderly patients receiving a DOP above 1.75 mg kg^-1^ would manifest significant hypotension and apnea [15]. In 1998 and 1999, two capstone works published in *Anesthesiology* demonstrating the influence of age on the pharmacokinetics and pharmacodynamics of propofol showed that aging affects the intercompartmental distribution of propofol and increases brain sensitivity to propofol [16, 17]. More recent clinical studies have reported that elderly patients (≥65 year-old) receiving propofol in the emergency department required a significantly lower dose for induction and maintenance of procedural sedation compared to young adults [18], and that when doses higher than the recommended one were used for this age group, the incidence of post-induction hypotension was greater [19]. Furthermore, age is one of the factors included in a published formula that predicts the dose of propofol to prevent post-induction hemodynamic fluctuations in human patients [20].
+
+Even though this pharmacological variance is well documented in the geriatric human population, studies have yet to elucidate if a comparable phenomenon occurs in elderly dogs (*Canis lupus familiaris*). The assumption that aging may also affect DOP in the canine elderly population as observed in people with similar adverse effects is not an unreasonable one. The incidence and magnitude of adverse events after propofol administration, such as post-induction decrease in systolic arterial pressure [21], hypotension [22], and apnea [23], are dose-dependent and more accentuated in the human elderly, and the anesthesiologist may modify dosing with the goal of optimizing post-operative outcomes [19]. In elderly canine patients, the incidence of adverse events after propofol administration is unknown. Post-induction dose-dependent apnea [24] and decrease in mean arterial pressure [25–27] are common occurrences in dogs administered propofol for induction of general anesthesia. Hence, it is logical to infer that the lowest possible DOP should be used to accomplish induction of general anesthesia in order to minimize these potential adverse effects.
+
+The primary goal of this study was to determine the effect of aging on the intravenous (IV) DOP required to induce general anesthesia and facilitate endotracheal intubation in dogs, whereas the secondary goal was to identify other predictors of DOP. The tested null hypothesis stated that the DOP per unit of mass required to perform endotracheal intubation in elderly dogs would not differ from that required for young and adult subjects, and that none of the investigated variables would constitute a predictor of DOP.
+
+## Materials and methods
+
+This study was designed as a retrospective case series study including demographic and perianesthetic data collected from dogs that underwent general anesthesia from January 2017 to June 2020 at the Anesthesiology Service of the Louisiana State University, Veterinary Teaching Hospital. Informed owner consent to general anesthesia and for data collection was obtained from all owners prior to the anesthetic event. The Louisiana State University Institutional Animal Care and Use Committee (LSU IACUC) was consulted prior to the start of this study about the need for their oversight. We obtain verbal and written confirmation from the LSUIACUC that retrospective studies using historically collected data or tissues does not require their oversight in keeping with national and international standards.
+
+### Data collection
+
+A digital anesthetic record (DAR) software (VetDAR, Dimple Hill Software LLC, Corvallis, OR) was used to collect and store canine patient demographic and anesthetic-related data. According to the anesthesiology service’s standard operating procedure, a senior veterinary student created and maintained an electronic anesthetic record for each anesthetic event and dog, generating a unique record. After dogs had recovered from anesthesia and records were completed by senior students, these were checked for accuracy by the technician assigned to the clinical case, and subsequently by the anesthesiologist on duty. Aesthetic records were then approved and locked to prevent *post-hoc* modification and tampering. An Excel (Version 16.65 2022; Microsoft Corporation, Redmond, WA) spread sheet including all anesthetic cases contained into the database of DAR was generated. All anesthetic records collected between January 2017 and June 2020 were included in the study. Further case selection and filtering was carried out by including only cases containing the terms “Canine” under the category “Species”, and “Propofol” under the “Induction Agent” used. Dogs receiving propofol (Propoflo; 10 mg mL^-1^, Zoetis Inc., Kalamazoo, MI) as part of their induction regimen were included in the studied cohort (*n* = 2346). If demographic data were missing from the anesthetic record file, they were retrieved from the digital hospital managing software (Cornerstone, IDEXX, Westbrook, ME). If data was irretrievable or inaccurate, the anesthetic record was excluded from the study (*n* = 924). The same investigator performed data gathering and filtering to handle the data uniformly. Dogs receiving remifentanil as a constant rate infusion as a co-induction agent with propofol were excluded from the study (*n* = 25).
+
+### Propofol administration
+
+Sedation prior to induction of general anesthesia was subjectively scored by a veterinary student based on a 4-point descriptive scale (0 = no sedation; 1 = mild sedation; 2 = moderate sedation; 3 = profound sedation). Administration of propofol prior to endotracheal intubation was performed by manual injection, starting from a standard estimated dose of 4 mg kg^-1^, which was then slowly (at an approximate rate of 1 mg kg^-1^/30 seconds) titrated with the end points of loss of medial palpebral reflex and of jaw tone before performing endotracheal intubation. Propofol injections were performed by senior veterinary students closely supervised by a veterinary anesthesia technician, anesthesia intern, anesthesia resident, or anesthesiologist on duty. After intubation was performed and maintenance of general anesthesia started, the total DOP (mg) was recorded on DAR. The dose per unit of mass was automatically calculated by dividing the total administered dose by the weight of the dog at the time of anesthesia.
+
+#### Independent variables
+
+*Age*. For statistical analysis purposes, three variants of the age concept were investigated as the independent variables of the statistical analysis model:
+
+- Absolute age (year): Calculated in days by subtracting the date of birth from the date of procedure and then converted into year by dividing the number of days by 365.
+- Elapsed percentage of life expectancy (percentage): Life expectancy was defined as the average period of time that a dog of a specific breed is expected to live. As substantial breed variation in longevity exists in the canine species, despite apparent similarities in physiologic, dietary, and environmental factors [28–30], life expectancy of each breed (in days) was calculated by averaging the median values of life expectancy for each breed from the available literature [30–34], and percentage of elapsed life expectancy calculated for each subject based on the age at the time of the anesthetic episode. Dogs were then allocated in 5 interquartiles representing 0–25%, 26–50%, 51–75%, 76–100%, >100% of their expected life expectancy. Toy and miniature sizes of Poodles and Schnauzers were grouped together, and average median life expectancy was calculated for the two groups.
+- Physiologic age (year): Defined as the chronological age of a canine subject corresponding to human equivalent based on a polynomial curves relating human age to canine age constructed for different sizes of dogs (weight ranges: 6.8–13.6, 13.7–22.7, 22.8–34, 34.1–45.3, >45.4 kg) [35].
+
+#### Primary outcome measure
+
+*Dose of propofol*. mass of propofol (mg) required to perform endotracheal intubation per unit of mass (kg) of the dogs included in this study.
+
+#### Secondary outcome measures
+
+*Demographic and physiologic variables*. From the subset of dogs receiving propofol, alone or in combination with other IV hypnotic, dissociative, and/or sedative agents for induction of anesthesia, the following data were extracted from DAR: body weight, body condition score (BCS), sex, reproductive status, and breed. If the words “Mix”, “Mixed”, or “Cross” was present in the assigned breed name, dogs were allocated into two mixed-breed groups according to their body weight (< 10 kg and >10 kg, respectively) according to previously published data for purpose of data analysis [32].
+
+*Perianesthetic data*. Date and type of procedure (5 categories: dental, soft tissue surgery, orthopedic surgery, imaging, minimally invasive procedure), American Society of Anesthesiologists physical and emergency status (ASA E) [36], premedication regimen used, level of sedation after premedication, route of administration of premedication were obtained as defined in Table 1.
+
+**Table 1 Variables and score system for demographic and pre-anesthetic data evaluating the influence of age on the induction dose of propofol in dogs.**
+
+| Variable | Score | Description |
+| --- | --- | --- |
+| Body Condition Score | 1 | Emaciated |
+| 2 | Very thin |
+| 3 | Thin |
+| 4 | Underweight |
+| 5 | Ideal weight |
+| 6 | Overweight |
+| 7 | Heavy |
+| 8 | Obese |
+| 9 | Severely obese |
+| Level of sedation after premedication | 1 | None |
+| 2 | Mild |
+| 3 | Moderate |
+| 4 | Severe |
+| Physical status | 1 | Clinically healthy |
+| 2 | Mild systemic disease |
+| 3 | Severe systemic disease |
+| 4 | Severe systemic disease that is a constant threat to life |
+| 5 | Moribund and not expected to survive more than 24 hours without surgery |
+| E | Emergency or non-emergency procedure |
+| Sex | Male or Female |
+| Reproductive status before the procedure | Neutered or Intact |
+
+### Statistical analysis
+
+Statistical analysis was carried out via a commercially available software (IBM SPSS Statistics for Windows, Version 26.0. Armonk, NY: IBM Corp). If an individual subject had been anesthetized on multiple occasions, only the first anesthetic episode was included in the analysis to avoid carry-over effect. Data were analyzed using multivariate linear regression with backward elimination. Breed was included as an independent variable if there were 40 or more subjects representing that breed. Three models were built. The first used the dose per mass unit of propofol (mg kg^-1^) as the dependent variable and absolute age, breed (for breeds represented by more than 40 subjects), sex, weight, BCS, ASA status, level of sedation, and mg kg^-1^ dose of intramuscular (IM) hydromorphone, morphine, methadone, butorphanol, buprenorphine, acepromazine, dexmedetomidine, midazolam, alfaxalone, ketamine, and sub-cutaneous or IM maropitant, and IV acepromazine, hydromorphone, morphine, methadone, butorphanol, fentanyl, dexmedetomidine, midazolam, diazepam, alfaxalone, ketamine, and lidocaine as independent variables. The second model was identical to the first except percentage of life expectancy was substituted for age as an independent variable. The third model was identical to the first two except breed was excluded as an independent factor, and physiologic age in relation to humans based on body weight, calculated as per a previously published equation [37], was used as the independent variable in lieu of absolute age or elapsed life expectancy. For this model, dogs were divided into weight groups (6.8 kg-13.6 kg, 13.7–22.7 kg, 22.8–34 kg, 34.1–45.3 kg, >45.4 kg) and excluded if their weight was below 6.8 kg. The cut-off age in canine years of age corresponding to 65 years of age of a human subject was identified for each canine weight group based on previously published data [35]. If significance was found, a paired t test was used to test the DOP for dogs within the same weight range between subjects under and above the calculated age cut off (<65 and ≥65 in human years). The DOP for each interquartile of life expectancy (<25%, 25–50%, 50–75%, 75–100%, >100%) was compared using one-way ANOVA. An additional analysis was made using a subgroup of 56 subjects that did not receive premedication drugs before propofol administration, and the DOP from the 20 youngest and the 20 oldest dogs was compared using a 2-way unpaired t-test. Significance was set at α = 0.025.
+
+## Results
+
+The initial selection process generated a sample of 2346 dogs, which was further screened and filtered by a board-certified anesthesiologist for accuracy of data. A total of 1397 dogs were included for the final analysis (Table 2). The sample size included in the elapsed percentage of life expectancy model was *n* = 1276 due to the lack of available data in the literature on the expected life span of certain breeds. Average age and weight (mean ± SD) were 7.2 ± 4.1 years (95% CI: 7–7.4) and 19 ± 14 kg (95% CI 18–20), respectively. Average percentage of elapsed life expectancy of the studied population was 59.8 ± 33% (95% CI: 58–62; based on 1276 subjects). The mean DOP needed to perform endotracheal intubation was 3.7 ± 1.8 mg kg^-1^. A synopsis of the drugs administered in a multimodal anesthetic protocol regimen along with propofol is provided in Table 3. Fifty-six subjects received no drugs other than propofol. There were 101 represented breeds with the most represented being: Boxer (*n* = 46), Chihuahua (*n* = 55), Dachshund (n = 108; long-haired and short-haired combined), German Shepherd dog (*n* = 44), Golden Retriever (*n* = 42), Labrador Retriever (*n* = 185), Maltese (*n* = 54), Pitbull (*n* = 69), Shih Tzu (*n* = 72), Yorkshire terrier (*n* = 56). In addition, subjects of mixed breed (<10kg; *n* = 61. >10kg; *n* = 180) were also largely present. Cut-off ages for the weight groups used in the physiologic age statistical model were 13 (6.8 kg-13.6 and 13.7–22.7 kg), 12 (22.8–34 kg), 11 (34.1–45.3 kg), and 10 (>45.4 kg) years, respectively [35]. Absolute and physiologic ages failed to be significant predictors of DOP in the respective statistical models, whereas percentage of elapsed life expectancy was significant. There was no difference between DOPs from the five life expectancy interquartiles (*P* = 0.20; Table 4), and between youngest and oldest subjects (*P* = 0.86) which received no premedication in this cohort for DOP. Significant predictors for reduced or increased DOP for the three models are presented in Table 5.
+
+**Table 2 Summary of demographic and perianesthetic data from 1397 dogs.ASA = American Society of Anesthesiologists; BCS = Body Condition Score; CI = Confidence Interval; DOP = Dose of propofol; E = Emergency; MI = Minimally Invasive; SD = Standard Deviation; ST = Soft Tissue Surgery.**
+
+|  | Number of Subjects | Female | Male | Body Weight Mean ± SD (kg) | Age Mean ± SD (years) | Median BCS | Frequency of ASA Status | Frequency of E Status | Frequency of Sedation Scores | Frequency of procedural categories | Mean ± SD DOP (mg kg^-1^) |
+| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
+|  |  | Intact | Neut. | Intact | Neut. |  |  |  |  |  |  |  |  |
+| **All Breeds** | 1397 | 136 | 567 | 186 | 508 | 19 ± 14 (95% CI: 18.3–19.8) | 7.2 ± 4.1 (95% CI: 7.0–7.4) | 5 (4–6) | I | 217 | 211 | 0 | 120 | Dental | 51 | 3.7 ± 1.8 (95% CI: 3.6–3.7) |
+| II | 792 | 1 | 570 | Imaging | 527 |
+| III | 326 | 2 | 531 | MI | 45 |
+| IV | 59 | 3 | 164 | Orthopedic | 253 |
+| V | 3 |  |  | ST | 520 |
+
+**Table 3 Synopsis of the drugs administered in a multimodal anaesthetic protocol regimen along with propofol.**
+
+| Drug | Route | Number of Subjects | Dose (Mean ± SD) mg kg^-1^ |
+| --- | --- | --- | --- |
+| Acepromazine | IM | 125 | 0.03 ± 0.03 |
+| Acepromazine | IV | 3 | 0.02 ± 0.02 |
+| Alfaxalone | IM | 36 | 1.3 ± 0.6 |
+| Alfaxalone | IV | 4 | 1.2 ± 0.7 |
+| Atropine | IM/IV | 20 | 0.03 ± 0.01 |
+| Buprenorphine | IM | 3 | 0.03 ± 0.01 |
+| Butorphanol | IM | 239 | 0.34 ± 0.1 |
+| Butorphanol | IV | 13 | 0.4 ± 0.2 |
+| Dexmedetomidine | IM | 361 | 0.004 ± 0.005 |
+| Dexmedetomidine | IV | 59 | 0.002 ± 0.004 |
+| Diazepam | IV | 14 | 0.26 ± 0.11 |
+| Fentanyl | IV | 175 | 0.004 ± 0.002 |
+| Glycopyrrolate | IM/IV | 8 | 0.01 ± 0.01 |
+| Hydromorphone | IM | 86 | 0.12 ± 0.05 |
+| Hydromorphone | IV | 5 | 0.12 ± 0.04 |
+| Ketamine | IM | 1 | 5 |
+| Ketamine | IV | 119 | 1.9 ± 1.3 |
+| Lidocaine | IV | 104 | 1.4 ± 0.6 |
+| Maropitant | SQ/IV | 46 | 1.0 ± 0.04 |
+| Methadone | IM | 578 | 0.35 ± 0.13 |
+| Methadone | IV | 18 | 0.30 ± 0.11 |
+| Midazolam | IM | 167 | 0.22 ± 0.08 |
+| Midazolam | IV | 262 | 0.24 ± 0.07 |
+| Morphine | IM | 190 | 0.41 ± 0.17 |
+| Morphine | IV | 6 | 0.32 ± 0.04 |
+
+**Table 4 Summary of the number of subjects and dose propofol for each life expectancy interquartile (*n* = 1276).**
+
+| Life Expectancy Interquartile | Number of Subjects | Dose of Propofol (mean ± SD) mg kg^-1^ | *P*-value |
+| --- | --- | --- | --- |
+| 0–25% | 249 | 3.9 ± 2.3 | 0.20 |
+| 25–50% | 243 | 3.8 ± 1.8 |
+| 50–75% | 317 | 3.6 ±1.8 |
+| 75–100% | 341 | 3.7 ±1.7 |
+| >100% | 126 | 3.4 ± 1.6 |
+
+**Table 5 Summary of predictors that increase (+ UC) and decrease (- UC) the dose of propofol for the three statistical models used for data analysis.CI = Confidence Interval; NA = Non-Applicable; NS: non-significant; SD = Standard Deviation; UC = Unstandardized Coefficient. Note: non-significant *P*-values for absolute age and physiologic age are found under the respective models.**
+
+| Variable | Dose (mg kg ^-1^) Mean ± SD | Model 1 –Absolute Age | Model 2 –Life Expectancy | Model 3 –Physiologic Age |
+| --- | --- | --- | --- | --- |
+|  |  | UC | 95% CI | *P*-value | UC | 95% CI | *P*-value | UC | 95% CI | *P*-value |
+| (Constant) |  | 4.29 | 4.0–4.5 | <0.001 | 4.4 | 4.2–4.7 | <0.001 | 4.4 | 4.1–4.7 | <0.0001 |
+| Absolute Age (year) | NA | N/A | N/A | N/A | NA | NA | NA | NA | NA | NA |
+| Life Expectancy (%) | NA | NA | NA | NA | -0.37 | -0.65 to -0.08 | 0.013 | NA | NA | NA |
+| Physiologic Age (year) | NA | NA | NA | NA | NA | NA | NA | -0.004 | -0.009 to 0.001 | .098 |
+| Neutered Male | NA | -0.33 | -0.5 to -0.1 | 0.0001 | -0.31 | -0.5 to -0.1 | 0.002 | -0.3 | -0.5 to -0.1 | .001 |
+| Chihuahua | NA | +1.43 | 0.9 to 1.9 | 0.0001 | +1.32 | 0.8 to 1.8 | 0.0001 | +1.4 | 0.9 to 1.9 | .000 |
+| Maltese | NA | +1.28 | 0.8 to 1.8 | 0.0001 | +1.15 | 0.6 to 1.7 | 0.0001 | +1.3 | 0.8 to 1.8 | .000 |
+| Yorkshire Terrier | NA | +2.05 | 1.6 to 2.5 | 0.0001 | +1.88 | 1.4 to 2.3 | 0.0001 | +2.0 | 1.6 to 2.5 | .000 |
+| Shih Tsu | NA | +0.60 | 0.2 to 1.0 | 0.006 | NS | NS | NS | +0.6 | 0.2 to 1.1 | .006 |
+| Mixed breed <10kg | NA | +1.06 | 0.6 to 1.5 | 0.0001 | +0.88 | 0.4 to 1.4 | 0.0001 | +1.1 | 0.6 to 1.5 | .000 |
+| Labrador Retriever | NA | -0.40 | -0.7 to -0.1 | 0.006 | -0.58 | -0.9 to -0.3 | 0.0001 | -0.4 | -0.7 to -0.1 | .007 |
+| Golden Retriever | NA | NS | NS | NS | -0.59 | -1.1 to -0.03 | 0.038 | NS | NS | NS |
+| Boxer | NA | -0.58 | -1.1 to -0.1 | 0.029 | -0.73 | -1.3 to -0.2 | 0.008 | -0.6 | -1.1 to -0.1 | .030 |
+| ASA “E” Status | NA | -0.33 | -0.6 to -0.1 | 0.014 | -0.35 | -0.6 to -0.1 | 0.017 | -0.3 | -0.6 to -0.1 | .017 |
+| Dental Procedure | NA | NS | NS | 0.61 | 0.1 to 1.1 | 0.017 | NS | NS | NS | NS |
+| IM Butorphanol (mg kg ^-1^) | 0.35 ± 0.11 | -1.27 | -2.0 to -0.5 | 0.001 | -0.76 | -1.5 to -0.1 | 0.034 | -0.9 | -1.6 to -0.3 | .006 |
+| IM Dexmedetomidine (μg kg ^-1^) | 0.004 ± 0.005 | -0.079 | -0.1 to -0.05 | 0.0001 | -0.10 | -0.1 to -0.6 | 0.0001 | -0.08 | -0.1 to -0.05 | .000 |
+| IV Dexmedetomidine (μg kg ^-1^) | 0.002 ± 0.004 | NS | NS | NS | -0.32 | -0.6 to -0.02 | 0.037 | NS | NS | NS |
+| IM Hydromorphone (mg kg ^-1^) | 0.12 ± 0.05 | -5.89 | -8.8 to -3.0 | 0.0001 | -4.73 | -7.7 to -1.7 | 0.002 | -5.2 | -8.0 to -2.4 | .000 |
+| IV Fentanyl (μg kg ^-1^) | 0.004 ± 0.002 | -0.10 | -0.2 to -0.04 | 0.002 | -0.07 | -0.1 to -0.003 | 0.039 | -0.09 | -0.2 to -0.02 | .007 |
+| IV Midazolam (mg kg ^-1^) | 0.24 ± 0.07 | -2.68 | -3.6 to -1.8 | 0.0001 | -2.65 | -3.6 to -1.7 | 0.0001 | -2.7 | -3.6 to -1.7 | .000 |
+| IV Ketamine (mg kg ^-1^) | 1.9 ± 1.3 | -0.32 | -0.5 to -0.2 | 0.0001 | -0.31 | -0.5 to -0.2 | 0.0001 | -0.3 | -0.5 to -0.2 | .000 |
+| IV Lidocaine (mg kg ^-1^) | 1.4 ± 0.6 | -0.40 | -0.6 to -0.1 | 0.002 | -0.43 | -0.7 to -0.2 | 0.002 | -0.4 | -0.6 to -0.1 | .002 |
+| IV Alfaxalone (mg kg ^-1^) | 1.2 ± 0.7 | NS | NS | NS | -2.34 | -4.4 to -0.3 | 0.023 | NS | NS | NS |
+
+## Discussion
+
+During the process of determining the effective dose of sedatives and anesthetics, patient related factors such as age, sex, and weight are usually considered. In this large retrospective study, we have investigated the hypothesis that the DOP per unit of mass required to perform endotracheal intubation in elderly dogs would not differ from that required for young and adult subjects. Based on our results, we must accept the null hypothesis as true for the models that used raw age as a predictor, as DOP was not affected by absolute and physiologic age. This finding is in striking contrast with what occurs in people [18, 20, 38], in which a significant reduction in DOP occurs in the elderly after 60 years of age. This phenomenon has been attributed to pharmacokinetic and pharmacodynamic differences of juvenile and adult individuals, such as a reduction in central compartment and clearance [39], and increased sensitivity to the hypnotic effects of propofol [15, 17, 40] in the elder. These pharmacokinetic and pharmacodynamic changes have also been observed in a study with a limited canine population of geriatric dogs, in which a lower induction dose and a slower clearance of propofol were observed [24]. Based on absolute and physiologic age, our results are also in contrast with those from Faggella and Aronsohn, in which young and healthy dogs required a larger induction and maintenance dose of propofol compared with adult dogs [41]. In fact, absolute age is not a reliable predictor of perianesthetic mortality in dogs. However, other factors such as obesity may contribute to the absolute age at death. For example, a study found that median age at death was negatively correlated with average ideal bodyweight with 40% variability in age at death explained by bodyweight in 81 breeds [31]. This seems to reinforce the idea that dogs should be used carefully as aging models in pharmacokinetic and pharmacodynamic studies involving propofol, and potentially other drugs.
+
+Percentage of elapsed life expectancy was a statistically significant predictor of DOP. The interpretation of the significant unstandardized coefficients should be carried out by attributing a decrease (-UC) or an increase (+UC) of the parameter of interest by the unit of measure of that parameter. The interpretation of the statistical model predicts a decrease of DOP by 0.37 mg kg^-1^ for each 100% change in life expectancy. In other words, a dog at 1.2 times their life expectancy would need 0.37 mg kg^-1^ less than a dog at 0.2 times their life expectancy, which represent a clinically unimportant effect.
+
+We rejected the null hypothesis stating that none of the investigated variables in this cohort would constitute a predictor of DOP as several predictors were identified. Likely not coincidentally, subjects from several small breeds, Yorkshire, Maltese, Chihuahua, Shih Tzu, and mixed-breed dogs under 10 kg have been found to have increased requirements of propofol for induction of anesthesia, whereas large breeds such as Boxer, Golden Retriever, and Labrador Retriever have been found to require a lower DOP. This finding has never been documented previously on a large sample of dogs but is not surprising. Evidence to support these findings is provided by Martinez *et al*. who demonstrated that drug clearance is not directly proportional to body weight of dogs of different breeds, and that pharmacokinetics of a drug could markedly differ in large or small dogs from concentrations estimated from studies using beagle-sized dogs as their model [42]. Furthermore, smaller dogs have been shown to have increased mass-specific metabolic rate and higher metabolically active organs, whereas larger breeds have significantly smaller liver, kidney, and brain relative size compared to smaller breeds [43, 44], which could explain differences in mass specific metabolism based on dog size [43], independent of BCS. Furthermore, potential breed and sex differences in propofol hydroxylation by CYP2B11 could be resulting from differences in the liver content of this CYP, although these conclusions were derived from a limited sample size, age (1–3 year of age), and body weight (15–31 kg) [45]. Propofol disposition may be different in Greyhound dogs compared to mixed-breed dogs with increased recovery time [46, 47]. This cohort included only 3 Greyhound dogs; therefore, data is insufficient to detect any statistical difference with other breeds. Based on our results, we recommend increasing the calculated DOP by 2, 1.4, 1.3, 1.1, and 0.6 mg kg^-1^ for Yorkshire Terrier, Chihuahua, Maltese, mixed breed dogs under 10 kg, and Shih Tzu subjects, respectively, and to reduce it by 0.58 to 0.73 mg kg^-1^ in Boxer, 0.6 mg kg^-1^ in Golden Retriever, and 0.4 to 0.58 mg kg^-1^ in Labrador Retriever.
+
+In our analysis, the status of neutered male has been found to be a significant predictor of reduced DOP by 0.3 to 0.33 mg kg^-1^ compared to other sex status depending on the statistical model. This is in contrast with the conclusions from other studies [41, 45, 48, 49] in which male dogs required a slightly higher induction dose than female dogs. However, studied samples included intact and gonadectomized subjects, so the role of the reproductive status on these results is difficult to determine. The available information on the role of gonadectomy in the development of obesity in dog is conflicting [50, 51].
+
+The emergent nature of the anesthetic procedure was significant predictor for reduced DOP. The average decrease in DOP for patients assigned an ASA E in the present study ranged from 0.3 to 0.35 mg kg^-1^ when compared to non-E ASA patients. It has been speculated that critically ill patients may require lower doses of induction agents due to their compromised health status and decreased mentation [52]. However, the impact of this dose reduction on hemodynamic and respiratory performances in critically ill dogs is unknown, and DOP should always be tailored to the individual subject and to their health status.
+
+As reported previously in the literature [48, 53, 54], several premedication drugs reduced DOP. In the context of this analysis, the reader should be advised to interpret the UCs in the following manner: “For each mg of each drug used, a linear decrease (-UC) or increase (+UC) in mg kg^-1^ of propofol is predicted”. In this cohort of dogs, some drugs administered intramuscularly prior to induction reduced DOP. Only two opioid compounds used intramuscularly reduced DOP: butorphanol and hydromorphone. While the sparing effect on DOP of butorphanol was modest (0.76 to 1.27 mg kg^-1^ per mg used), hydromorphone produced a decrease in DOP by 0.47 to 0.59 mg kg^-1^ per 0.1 mg kg^-1^, which is a commonly used dose. We speculate that the lack of sparing effect by other intramuscular opioids and the greater observed sparing effect of hydromorphone are due to its higher potency. The alpha~2~-adrenergic agonist dexmedetomidine also exerted a significant sparing effect as it reduced DOP when administered both intramuscularly and IV, as reported in previous publications [53].
+
+The technique of co-induction consists in the practice of administering one or more opioid, sedative, hypnotic, or dissociative compounds to reduce the dose of the main anesthetic employed [54]. In this cohort, IV fentanyl, midazolam, ketamine, lidocaine, and alfaxalone administration alone with propofol, or in various combinations with other co-induction agents, reduced DOP, which agrees with the existing literature. Notably IV midazolam in the context of the mean dose used in this study (0.24 mg kg^-1^), produced a reduction in DOP by 2.65 to 2.7 mg kg^-1^ of propofol. The magnitude of this sparing effect is greater than previously reported, which ranged from 17.6 to 42.1% (1.9 mg/kg to 1.1 mg/kg with 0.3 mg/kg of midazolam in ill dogs, and from 3.4 mg/kg to 2.8 mg/kg with 0.2 mg/kg of midazolam in healthy dogs, respectively) [52, 55]. We speculate that some pre-analytical bias may have occurred in this cohort, as midazolam is often prescribed to juvenile or elderly dogs, or those that present with co-morbidities and may be severely ill. This decisional bias may cause the reduction of DOP seen with midazolam appear greater than what it would be if the same dose was used on a healthy population.
+
+Interestingly, weight, BCS, and level of sedation were not found to be a predictor of DOP. The lack of reduction in DOP with increasing weight is in contrast with what observed in human medicine [56]. In fact, adjustments in the DOP based on lean body weight for obese patients have been implemented for people [56, 57]. In veterinary medicine, a prospective clinical study investigating the effect of body condition on propofol requirements in dogs, overweight (BCS = 6/9) and obese dogs (BCS (7-9/9) have been found to require a lower DOP (1.83 ± 0.36 mg kg^-1^) compared to dogs with normal body condition (2.24 ± 0.53 mg kg^-1^; Boveri et al., 2013). Subjects included (ASA I-III), premedication regimen, and propofol delivery technique were standardized in this prospective study [58]. In contrast, our results reflect a much larger population of dogs, with a wider variety of health status, comorbidities, and preanesthetic regimen. These factors may have contributed to these contrasting results. In the present study, the effect of body condition, actual body fat composition, and pure effect of BCS on the UCs are unknown. Therefore, based on our results, we do not recommend adjusting DOP in overweight and obese subjects, but based on breed.
+
+Several limitations to this study should be considered. This study is retrospective in nature and, as such, carries the inevitable risk of inaccuracies and inconsistencies in the medical and anesthetic records. However, significant attempts at reducing bias by strict filtering of incomplete records by the same boarded anesthesiologist (CEH) were made in addition to the routine triple check-point system used as a standard operating procedure at this anesthesiology service. Different combinations of sedative agents have been used in this study, and it is important to note that the effects of time of administration and peak of sedation of these sedatives on DOP is unknown. Since multiple individuals administered propofol during induction, bias may have occurred due to inconsistent speed of injection, or due to excessive administration of propofol before waiting the proper amount of time for attempting intubation which have been shown to affect DOP [59]. Breed classification was based on the phenotype of the subject and not on DNA profiling or pedigree certificate from the American Kennel Club; Therefore, misclassification of subjects based on breed may have occurred. Finally, the pharmacodynamic effects of adjusting DOP based on the coefficients determined in this study are unknown.
+
+In conclusion and in contrast to what is observed in people, an age cut-off predictive of DOP does not exists in dogs. Only percentage of elapsed life expectancy along with other factors such as breed, premedication drug, emergency procedure, and reproductive status significantly altered DOP. Based on these findings, absolute age can be excluded as a confounding factor determining DOP. In contrast with absolute age, elapsed life expectancy may hide unexpected value as a predictor and further research incorporating it in future study designs is warranted. This information is relevant not only to researchers, but to the veterinary practitioner who routinely uses propofol for induction of general anesthesia as adjustments in the amount of propofol drawn should be made based on significant predictors of DOP such as breed, premedication drugs, reproductive and emergency ASA status and not based on absolute or physiologic age. Although the reduction in DOP was statistically significant in the model accounting for percentage of life expectancy elapse at the time of the anesthetic episode, this has minor clinical importance as this reduction was minimal. As the magnitude and incidence of the post-induction side effects of propofol in clinical practice is unknown, we emphasize the practice of individualizing the calculated induction dose based on a holistic approach that includes these predictive factors along with the overall health status of the canine patient to minimize anesthetic risk and optimize perianesthetic outcome.

fulltext/md/PMC10321594.md

@@ -0,0 +1,180 @@
+---
+pmcid: "PMC10321594"
+pmid: "37415700"
+doi: "10.3389/fpubh.2023.1214127"
+title: "Examining human-animal interactions and their effect on multidimensional frailty in later life: a scoping review."
+journal: "Frontiers in public health"
+year: "2023"
+license: "cc by"
+article_type: "review-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Examining human-animal interactions and their effect on multidimensional frailty in later life: a scoping review
+
+## Abstract
+
+This scoping review sought to compile outcomes associated with any human-animal interaction study regarding adults aged 50 and older in any living context and concerning a multidimensional (i.e., physical, psychological, cognitive, and social) perspective of frailty. Despite our best attempts at incorporating the broadest inclusion criteria possible, only four articles were relevant to this review. Participants across the included studies were rural, community-dwelling Japanese or Chinese individuals aged 60 years and older. Thematic analysis of reported results includes dog ownership as a protective factor regarding frailty, the interconnected health effects of pet ownership, and increased meaning and purpose through pet ownership implications. More research is needed globally to determine how human-animal interactions may moderate frailty comprehensively, as well as the efficacy and appropriateness of these interactions or interventions in older adult populations and across cultural boundaries.
+
+1.
+
+## Introduction
+
+Physical, psychological, social, and cognitive health trajectories have been widely studied, but these concepts as domains of frailty are rarely assessed concurrently. Frailty can hinder well-being and quality of life (QOL), yet the lack of a universal definition (1) of this health challenge impedes assessment of its prevalence and identification of appropriate interventions. Some have focused their work on physical (2, 3) or cognitive elements of frailty (4), but significant gaps remain regarding an exhaustive understanding of this phenomenon. A comprehensive definition may yield better understandings of how frailty occurs, within what health domains, and steps to prevent or mitigate it to uphold well-being and QOL over time.
+
+Cumulative deficit perspectives have been proposed but rarely incorporate a comprehensive list of variables to account for frailty risks. Like cognitive health trajectories (5), frailty risk often increases gradually over time (6) with strong positive correlations existing between risk and old age. Frailty is also associated with decreased autonomy, which can result in poorer physical and psychological health for older adults (6) *via* hindered positive affect and feelings of self-efficacy (7).
+
+As global demographics continue to age and the pertinence of independence endures, we call for researchers, practitioners, and policymakers to effectively identify and intervene against frailty risks. One such possible intervention is human-animal interaction (HAI). Interactions with the same animal over time can reduce cardiovascular activity and cortisol levels (8), and may also contribute to stress reduction, perceptions of adequate social support (9), and improved mobility (10). Existing reviews suggest potential associations between dog ownership, social contact, and well-being (11). Moreover, improved symptoms of depression, anxiety, cognitive impairment, and even dementia are reportedly associated with HAIs (12).
+
+Human-animal interactions (HAIs) constitute the many modalities that facilitate exchanges between humans and other animals. Animal-assisted therapy is a type of HAI intervention where “an animal that meets specific criteria is an integral part of the treatment process” (13, p. 34). These therapeutic interactions require service delivery by a professional with specialized expertise and in specific settings (13). Alternatively, animal-assisted activities “[provide] opportunities for motivational…and/or therapeutic benefits to enhance [QOL]” (13, p. 34), which can be delivered in different settings and with varying levels of expertise. Other forms of HAI include visiting animals, service animals, and emotional support animals. Pet ownership (PO) is the primary HAI driving this work and includes interactions with domesticated pets and farm animals.
+
+We aimed to compile outcomes on any HAI study with older adults regarding a multidimensional perspective of frailty. We defined frailty as a dynamic process involving both losses and increased vulnerability regarding one’s psychological, physical, cognitive, and social functioning that are correlated with chronological age. The guiding research question was, “What is known about HAI studies available to 50+ adults regarding their effect on frailty?.” To our knowledge, a relevant scoping review does not yet exist. A related systematic review has been conducted and is included here (14) but incorporated superfluous exclusion criteria that necessitated this expanded literature review (e.g., only searched PubMed and Google Scholar for articles published between 2000 and 2020; excluded randomized controlled trials, reviews, editorials, dissertations, and conference abstracts; only included healthy participants).
+
+2.
+
+## Methods
+
+2.1.
+
+### Search strategy
+
+This review followed established guidelines (15) and the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for scoping reviews (PRISMA-ScR) (16). We consulted with an expert librarian from our university in May 2022 for feedback on search terms, truncation, Boolean phrasing, and databases best suited for this review. The final search terms are included in Supplementary Table S1a. Six databases were searched in June 2022 using EBSCOhost (APA PsycInfo, PubMed [includes MEDLINE], Sociological Abstracts, SocINDEX with Full Text, Social Science Full Text, and Psychology and Behavioral Sciences Collection) as well as one publicly available platform (HABRI Central). Google Scholar and Web of Science Collection searches were attempted but inaccessible due to imposed character limits. The reference lists of assessed articles were also screened for inclusion. Our PRISMA-ScR Flow diagram is provided in Figure 1.
+
+*Figure: Figure 1 PRISMA Flow diagram of the scoping review search and selection process.*
+
+2.2.
+
+### Inclusion and exclusion criteria
+
+Articles were required to provide a frailty definition and relevant instrumentation or otherwise consider it an outcome. Those that only addressed human-animal observations (i.e., aviaries, fish tanks) were excluded due to the notable emphasis on physical conceptualizations and measurements of frailty in the literature (17). All forms of academic manuscripts published in English were considered. Participants must be 50+ but could have reported any form of housing or living arrangement (e.g., multigenerational; retirement community residents), any health status (i.e., healthy, unhealthy) or any type of physical, cognitive, or psychiatric diagnosis or comorbidity (e.g., cognitive impairment; mental illnesses).
+
+2.3.
+
+### Study selection
+
+After downloading full texts to Zotero for screening, core data was extracted (e.g., frailty instrumentation; animal species) and further assessed for eligibility. From the articles included for review *via* database searches, the first author reviewed each reference list and screened titles and abstracts to identify other relevant articles. Reported key findings and implications were charted and thereupon analyzed for inductive thematic analysis.
+
+3.
+
+## Results
+
+3.1.
+
+### Search outcomes
+
+Overall, 3,407 sources were retrieved. After 289 duplicates were automatically removed and titles and abstracts of the remaining 3,118 unique articles were assessed, the first two authors independently read through 34 full texts to determine eligibility. Thereafter, 43 articles identified *via* reference reviews were screened and three were assessed for eligibility. Any inclusion uncertainty was resolved through discussion among the first two authors. A total of four articles are included: two were retrieved from PubMed (includes MED-LINE) (4, 14), one from HABRI (Human Animal Bond Research Institute) Central (18), and one from reference reviews (19).
+
+3.2.
+
+### Study characteristics
+
+3.2.1.
+
+#### Settings and participants
+
+All four studies focused on the effects of self-selected pet ownership—predominantly dogs and cats—among community-dwelling individuals. Participants across the included studies were predominantly women, lived in East Asia (*n* = 3 Japan, *n* = 1 China), and averaged 60+ years. Additional characteristics (e.g., household size) were inconsistently collected.
+
+Of the three original research articles, one interviewed older adults (*N* = 2,638) in rural environments who experienced the “social loss” of either divorce or widowhood to determine whether PO influenced cognitive frailty trajectories (4), but excluded individuals 85+, those not fluent in Chinese, and with dementia or psychiatric diagnoses. Two studies surveyed 65+ adults on the experience of dog or cat ownership (*N* = 11,233) (18, 19), with one requiring participants to be non-frail (*N* = 6,197). For additional attributes of included publications, see Supplementary Table S1b.
+
+3.2.2.
+
+#### Study design, procedures, and materials
+
+Two of the included studies were cross-sectional (4, 19), one was cross-sequential (18), and one was a systematic review (14). Of the original research articles, two utilized self-administered mail surveys for data collection (18, 19) and one used structured face-to-face interviews (4). Each article asked a variation of the dichotomous question, “Do you live with a pet?” (4, 18, 19). Two asked whether one lived with a pet currently, in the past, or never (18, 19), whereas another asked if one *owned* a pet (4). For those who either currently or had lived with a pet, two articles asked whether it was a “dog, cat, or other” (18, 19), and one asked whether the pet was a “dog, cat, bird, [or] other” (4). One article did not analyze data by way of PO type, nor was it concerned with past PO (4).
+
+3.2.3.
+
+#### Defining frailty
+
+Various conceptualizations of frailty were uncovered. Two of the four studies defined frailty using outcome measures (14, 19). One article specifically concerned with “incident frailty” conceptualized it as a combination of “physical frailty” and “social frailty” (18). Another homed into the effects of PO on one’s “cognitive frailty**,”** or the combination of physical frailty and cognitive impairment (4). Additional concepts, like “psychological frailty” and environmental health (20), were encountered but did not meet inclusion criteria (21). Notably, only one article spoke to the concept of frailty as potentially reversible (4).
+
+3.2.4.
+
+#### Measuring frailty
+
+Three articles (14, 18, 19) assessed frailty using the Kaigo-Yobo Checklist (CL15) (22), where a score of four or more (≥4) typically indicates frailty. However, one of those studies reported “a score *higher* than [four] was defined as frailty” (>4) (19, p. 3), deriving confusion as to what the appropriate cutoff score is. One article included in the reported systematic review measured frailty using a modification of the Cardiovascular Health Study (23), which was foundational to the development of the frailty phenotype (3). Another measured “cognitive frailty” by defining it as the presence of both cognitive impairment and a score of ≥3 on the frailty phenotype (4). This phenotype is a five-item scale to determine frailty risk through a combination of physical domain variables (shrinking, weakness, exhaustion, slowness, low physical activity level). Respectively, frailty is defined as a clinical syndrome made up of the presence of ≥3 of the included criteria, with the risk of frailty defined as a score of one or two (3).
+
+3.2.5.
+
+#### Additional measures
+
+The Mini-Mental State Examination (24) and frailty phenotype (3) were used to evaluate the presence of cognitive frailty (4). All additional measures exclusively pertained to covariates (e.g., functional impairment).
+
+3.3.
+
+### Reported results of included studies
+
+The following results are organized by article and in chronological order.
+
+Survey responses of Ota Genki Senior Project participants (*N* = 11,233) were analyzed regarding their experiences of “dog/cat ownership” to examine physical, psychological, and social functioning among current, past, and never pet owners (19). While no significant differences in frailty (i.e., score > 4) were found among current, past, and never owners through this scoring mechanism, compared to never owners, past and current ownership was associated with benefits in physical, social, and psychological functioning domains (19). This work suggests that never owning a dog/cat is associated with lower levels of walking, lesser degrees of light physical activity, and poorer social functioning (i.e., feelings of trust in neighbors) (*p* < 0.001) (19). Finally, certain health characteristics were reported as associated with either current or past dog ownership [Motor Fitness Scale (*p* < 0.001), interaction with neighbors (*p* < 0.001), trust in neighbors (*p* < 0.001), social isolation (*p* < 0.001)] and cat ownership [interaction with neighbors (*p* < 0.001), trust in neighbors (*p* < 0.005), social isolation (*p* < 0.002)] (19).
+
+The study concerned with incident frailty (*N* = 6,197) found greater reduced risks among past dog/cat owners (OR = 0.85, CI: 0.71–0.99, *p* < 0.05) than current (OR = 0.90, CI: 0.72–1.13) or never (OR = 1) owners (18). When separating dog from cat owners, past dog owners were significantly less likely to experience incident frailty (OR = 0.84, CI: 0.70–0.99, *p* < 0.05) than never (OR = 1) or current (OR = 0.86, CI: 0.65–1.13) dog owners. No statistically significant associations were found between incident frailty and cat ownership, but past cat owners (OR = 0.89, CI: 0.70–1.12) experienced slightly lower incident frailty risks than never (OR = 1) or current (OR = 1.04, CI: 0.77–1.40) owners. Compared to those who never owned a dog/cat, social function (i.e., interaction with neighbors) had a stronger negative association with incident frailty than those who currently or previously owned either pet species. They also found current dog/cat owners to be significantly younger than past and never owners and more often married [*p* < 0.001] with higher education [*p* < 0.001] and income rates [*p* < 0.001], obtained higher Motor Fitness Scale scores [*p* < 0.001] and frequencies of going outside [*p* < 0.001], and interacted with neighbors more often [*p* < 0.003] than past or never owners (18).
+
+While the systematic review reported no statistically significant differences in frailty percentages among never, past, and current dog or cat owners, calculated odds ratios across the three included studies suggest that cat and/or dog ownership may be associated with benefits in the physical, psychological, and social health domains, which constitutes frailty (14). Additionally, an article concerned with psychological frailty (i.e., depressed mood and phenotypic frailty) (21) found the risks to be 40% lower for participants rearing grandchildren or pets (OR = 0.60, 95% CI: 0.47–0.76, *p* < 0.001) than those who did not (14, p. 4).
+
+Finally, significant differences in cognitive frailty risk were uncovered among those (*N* = 2,638) who experienced social loss and did or did not own pets (4). Regardless of gender, experiencing either form of social loss had higher cognitive frailty rates than those who did not, but women with social loss and did not own a pet experienced the highest risk (15.5%) (OR = 2.06, 95% CI: 1.20–3.54, *p* < 0.01), followed by men with social loss who *did* own a pet (13.5%) (OR = 4.20, CI: 1.38–12.77, *p* < 0.05) (4). Conversely, rates were lowest among those who did not experience social loss and did own a pet [women: OR = 1.63 (CI: 0.99–2.71); men: OR = 1.50 (CI: 0.72–3.11)] (4). Overall, the prevalence of cognitive frailty was higher for women (9.3%) than for men (6.1%), suggesting sex differences may be at play and warrant consideration. Those deemed cognitively frail were older [*p* < 0.001], had lower education [*p* < 0.001] and income [*p* < 0.001] rates, higher psychological distress scores [*p* < 0.001], did not drink alcohol [*p* < 0.01], and did not own a pet [*p* < 0.01] (4).
+
+4.
+
+## Discussion
+
+To the best of our knowledge, this is the first scoping review to explore the influence HAIs may have on multidimensional frailty for 50+ adults. While only four articles are included, thematic analysis ensued despite this restriction and yielded three overarching constructs.
+
+4.1.
+
+### Interconnected health effects of pet ownership
+
+Physical, psychological, cognitive, and social health benefits can result from PO (4, 18, 25, 26) and yet, how these health domains affect one another are rarely assessed through the interdisciplinary perspectives of gerontology and anthrozoology. Each included article spoke to benefits in the physical, psychological, and social domains, but only one spoke to the influence of PO on cognitive health. Understanding the directionality behind the interdependent effects of these health domains is bound to result in more appropriate instrumentation and interventions regarding this incipient topic (27). One article spoke to social frailty presaging physical frailty (18). Another argued that dog ownership is associated with greater degrees of walking and social functioning, suggesting physical activity can promote social interaction (19). Relatedly, poor social health can result in social isolation, consequently impeding psychological health. The conceptualizations of cognitive frailty (4) and psychological frailty (14) build this argument further.
+
+One article examining frailty differences among men and women spoke to the possibility of social roles, like gender norms, being influential to differences in cognitive frailty rates (4), further highlighting the interconnectedness of psychological, social, cognitive, and physical health. Cultural differences, including but not limited to social norms, warrant increased attention to better understand how these concepts might influence individual health domains and, consequently, frailty risk.
+
+4.2.
+
+### Current and past pet ownership as a protective factor
+
+Evidence of PO as a protective mechanism against frailty was found, with dog ownership possibly having more influence than cat ownership. This may be because current and past dog owners reported greater degrees of physical activity *via* walking compared to those who never owned a dog (19). Or this finding could be due to the phenomenon known as the “pet ownership effect,” which argues that physically active individuals are more likely to own a pet than those who are not (18). Despite these causality concerns, this work argues that older adults who never owned a pet are consistently more likely to be frail than those who currently or previously owned a pet (18, 19). The varied effectiveness of past and current dog/cat ownership on frailty risk should be further explored.
+
+4.3.
+
+### Increased meaning and purpose through pet ownership
+
+This work supports the notion that PO can result in increased meaning and purpose, which can positively influence psychological health (26, 28). Experiencing a sense of meaning and purpose in life is similarly associated with lower risks of ill-being (29). This is especially true for older adults with low socioeconomic status or weak social networks (28). The included review (14) revealed psychological frailty as significantly less prevalent in older individuals who rear either grandchildren or pets (21). Feeling intrinsically valued and purposeful through PO can afford “social situatedness” to older adults even in light of fluctuating autonomy (28). Interventions to increase meaning and purpose in older individuals should be developed and assessed for appropriateness and effectiveness.
+
+4.4.
+
+### Limitations
+
+Meta-analysis was unattainable due to the inconsistent collection of frailty-based variables. Although we aimed to synthesize the largest body of literature possible, only incorporating articles published in English likely limited the number of hits, particularly considering the limited geographical diversity identified in this review. The generalizability of these findings is further tethered by all articles pertaining to older individuals in East Asia, predominantly Japan, potentially limiting the applicability of these findings to other older adult populations.
+
+Varied findings across the included articles necessitate investigation into the heterogeneity of effects that PO may have on frailty for 50+ adults. Further, whether said associations are direct or indirect, and the specific mechanisms by which older adults benefit from HAIs regarding frailty risk remain unclear. Improved development and assessment of relevant studies and interventions may determine whether (and which) HAIs are successful in averting frailty in older adults, what health domains are affected when, and the directionality between any interconnected health effects.
+
+4.5.
+
+### Future directions
+
+Future investigation should consider longitudinal analysis, companion animals outside of dogs and cats, and HAI studies and interventions outside of PO. Other studies and interventions that could glean meaning and purpose for older adults should be explored, like interacting with visiting animals or caring for houseplants.
+
+More research is needed on PO in various housing environments. While most prefer to age in place (28), retirement community enrollment will likely increase as global societies continue aging. Related housing policies that prevent PO for older adults—in and outside of retirement communities—require review to ensure these regulations foster autonomy, avoid ageist and ableist language and intentions, and to eliminate the possibility of one choosing between housing or their pet (28). When older adults are better supported at the individual, communal, and policy levels to age in place the beneficial effects of PO are likely to ensue, including increased degrees of well-being and QOL (26).
+
+Social determinants of health, like one’s built environment, are known to significantly impact QOL (30). These multifaceted health determinants likely contribute to frailty risk over time but are not yet adequately integrated into frailty assessments. Future research should also integrate the environmental health domain when assessing and defining multidimensional frailty.
+
+5.
+
+## Conclusion
+
+We reported outcomes associated with HAI studies and multidimensional frailty in 60+ older adults. The importance of identifying effective frailty interventions cannot be understated, as such may be integral to upholding individual autonomy, well-being, and QOL throughout the lifespan. While only a few relevant HAI studies exist, synthesized findings suggest PO has interconnected and protective health effects for older adults, including heightened feelings of meaning and purpose.
+
+Researchers could behoove current and future older adult populations by developing explicit definitions, instruments, and interventions that encompass the interconnected health variables related to frailty. Policymakers could suggest housing guidelines that prioritize the human-animal bond between older adults and their animal companions. Moving forward, interdisciplinary experts should reach consensus on a multidimensional frailty definition and appropriate HAI interventions for older adults as this often-overlooked population continues to expand worldwide.
+
+## Author contributions
+
+AT and KM administered the project and led the conceptualization as well as the design of this review. All articles were searched, charted, and assessed for eligibility by AT and MC. Thematic analysis and its validation were completed by AT, MC, KW, and KM. AT and MC wrote the original first draft, and AT took the lead on revisions. All authors contributed to the article and approved the submitted version.
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10333704.md

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+---
+pmcid: "PMC10333704"
+pmid: "37441560"
+doi: "10.3389/fvets.2023.1139308"
+title: "A review of frailty instruments in human medicine and proposal of a frailty instrument for dogs."
+journal: "Frontiers in veterinary science"
+year: "2023"
+license: "cc by"
+article_type: "review-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# A review of frailty instruments in human medicine and proposal of a frailty instrument for dogs
+
+## Abstract
+
+Over the last few decades, frailty has become a pillar of research and clinical assessment in human gerontology. This complex syndrome, characterized by loss of physiologic reserves leading to decreased resilience to stressors, is of critical importance because it predicts higher risks of poor health outcomes, including mortality. Thus, identifying frailty among the elderly human population has become a key focus of gerontology. This narrative review presents current scientific literature on frailty in both humans and animals. The authors discuss the need for an accessible frailty instrument for companion dogs suitable for general use in veterinary medicine and the advances that would be facilitated by this instrument. A phenotypic frailty instrument for companion dogs, utilizing components that are easily collected by owners, or in the general practice setting, is proposed. The authors elaborate on the domains (physical condition, physical activity, mobility, strength, cognitive task performance, and social behavior), factors that will be included, and the data from the Dog Aging Project that inform each domain.
+
+1.
+
+## Introduction
+
+1.1.
+
+### Definition of frailty
+
+Age carries a widely known association with increased risk for disease and adverse health outcomes. However, chronological age alone is neither a sensitive nor specific predictor of morbidity (1) when assessing health at the level of the individual. In lieu of chronological age, the phenomenon of frailty is now recognized in human gerontology as a superior way to assess the manifestations of aging and associated risks of disease and death within individuals and communities (1–5). Recognition of frailty adds a valuable dimension to the assessment of health and prognosis by documenting states of decline or loss of function which can emerge without any corresponding diagnosis of disease (6, 7). While frailty is associated with age, that association is complex in that individuals manifest frailty to varying degrees and at varying ages (8–11). Furthermore, frailty, unlike chronological age, can sometimes be reversed (12–14).
+
+Frailty, an evolving concept in human gerontology, is a complex, multidimensional syndrome characterized by a loss of resilience to stressors associated with aging (2, 5, 15, 16). Resilience itself is also a multifaceted characteristic which is affected by patient-specific and environmental factors, and which confers both physiological and emotional reserves (4, 17). The underlying etiology of frailty is thought to be the dysregulation of multiple physiologic systems resulting in loss of these reserves (2, 3, 18). Underlying mechanisms of frailty are not yet fully elucidated, and possibilities include sarcopenia (19, 20), low-grade inflammation (2), immune dysfunction (20), endocrinopathies (20, 21), and genetic risk factors (2). Increased resilience has been shown to predict recovery from illness and/or injury and improved quality of life in individuals living with chronic conditions (17). By contrast, frailty is associated with an increased risk of health problems including falls/fractures (15, 20, 22), hospitalization (15, 22, 23), functional decline/disability (15, 20, 22), the requirement for long-term care or institutionalization (3, 24, 25), and death (15, 16, 22, 24). Because frailty leads to adverse health outcomes, including mortality, identifying frailty among elderly human individuals and populations has become an important focus of gerontology.
+
+1.2.
+
+### Human frailty applications
+
+In 2001, Fried et al. proposed a phenotype of frailty to be applied in community-dwelling populations to identify at-risk individuals. It included components of unintentional weight loss, weakness, exhaustion, slowness, and low physical activity (15). Frailty, defined as the presence of three or more of these five characteristics, was predictive of adverse outcomes in relatively functional geriatric adults (15). Individuals identified as frail were six times more likely to die in the subsequent 3 years compared to their non-frail counterparts (15). The field of frailty has expanded dramatically over the last 20 years, with a multitude of instruments currently available to measure frailty in humans. These instruments typically can be categorized as Frailty Phenotype (FP) or Frailty Index (FI) instruments (25–27) (Table 1). Broadly, the FP model is based on the presence or absence of components that can be physically evaluated, while the FI model includes medical and laboratory findings and assigns numeric scores to those components. FP and FI instruments have been found to be comparable in the prediction of mortality (25, 27) and can be considered complementary in certain situations (26).
+
+**Table 1 Frailty phenotype (FP) and frailty index (FI) comparison.**
+
+| Frailty phenotype (FP) | Frailty index (FI) |
+| --- | --- |
+| Presence or absence of specific components, most commonly: | Cumulative score of health deficits, detected by: |
+| Weakness (often based on grip strength) | Overall disease count, |
+| Low mobility (often based on slow walking speed) | Tally of specific diseases, |
+| Low physical activity (kilocalorie expenditure per week) | and/or |
+| Unintentional weight loss | Laboratory abnormalities |
+| Fatigue or exhaustion (self-reported) |  |
+| Score is a sum of binary classifications | Score is either continuous, or a ratio of deficits detected within components assessed |
+| Considers frailty to be a decline in physical function | Considers frailty to be an accumulation of health deficits within multiple domains |
+
+Frailty models have evolved over the years from physical-only models to ones that include frailty factors from psychological (28, 29), social (1, 29, 30), and environmental domains (31, 32). Currently, there are a plethora of different instruments to detect and measure frailty. A 2019 systematic review of studies evaluating frailty instruments used in human populations found a total of 51 diverse instruments, including numerous instruments using the FP model and many using the FI model (33). These instruments varied greatly in length/duration, use, interpretive categories, information collected, collection method, need for additional diagnostics, areas of investigation, etc. (Table 2). This diversity of frailty instruments is the product of different needs depending on the setting, administrator, available time, and aim of the measurements (42). However, these different instruments classify different subsets of the population as frail (43) which means that investigators must carefully select instruments for target applications. Across studies, the highest prevalence of frailty in human populations is observed in studies using multidimensional instruments (44), suggesting they may be more sensitive. There is also evidence that an increased number of included variables tends to result in a higher agreement between instruments and narrow prediction intervals, and that multidimensional instruments result in less error at the median point of frailty (43).
+
+**Table 2 Design and intended uses of a sample of frailty instruments in human gerontology.**
+
+| Number of interpretive categories within the instrument |
+| --- |
+|  | Frail vs. non-frail (34, 35) |
+| ≥3 Levels of frailty (15, 23) |
+| Scale type |
+|  | Dichotomous scale (36–38) |
+|  | Ordinal scale (1, 15, 39) |
+|  | Continuous scale (28, 29) |
+| Domains included, with most commonly used domains listed first: |
+|  | Physical (15, 23, 37, 39) |
+| Psychological (28, 29) |
+| Social (1, 28, 30) |
+| Environmental (31, 32) |
+| Settings in which instrument is used |
+|  | Community (1, 15, 28, 39) |
+|  | Primary care/geriatric practice (29) |
+| Emergency departments (40) |
+| General hospitals (41) |
+| Long-term care facilities/nursing homes (30, 36, 41) |
+
+Along with the number of instruments and the domains they cover, the possible settings for the use of frailty assessment are also an expanding area of investigation. The identification of frail individuals informs prognosis and medical options and allows for several types of intervention directed at overall health and frailty itself. In human medicine, frailty can be used to stratify risk profiles and inform decision-making (16, 45). In a study of geriatric people who were treated for pelvic fractures, those identified as frail were at greater risk of mortality at the time of discharge and one-year post-discharge, and were also at risk of having reduced functionality and needing greater assistance at discharge (46). Frailty was associated with an increased risk of serious complications in geriatric trauma patients (47), and frailty status increased the risk of long-term mortality in COPD patients by 80% (48). This prognostic information is valuable for both patients and their families to understand prior to surgery or other medical treatment.
+
+The identification of frail patients is also necessary to take any protective or preventive action, such as adjustments in the typical geriatric medical or surgical protocols to improve outcomes for a specific frail individual, or enrollment in programs targeted at altering the frailty state itself. In human geriatric patients, screening instruments can be utilized to identify frail patients simply to inform their healthcare team so that they can make treatment alterations as needed. One study looking at postoperative survival for human patients undergoing major elective surgical procedures found that simply implementing a frailty screening initiative resulted in improved survival (49). When a patient was identified as frail, anesthesia, surgery, critical care, and palliative care clinicians were informed and, if needed, a modified perioperative plan was developed (49). Decreased mortality using this approach was attributed to multiple factors, including increased vigilance for complications leading to earlier treatment and improved family involvement, leading to improved post-discharge care and social support (49).
+
+As frailty is a dynamic process, influenced by both individual and environmental factors, it is susceptible to intervention. Frailty studies in human medicine have shown that early identification and targeted intervention can delay (50, 51), prevent (52), or reverse the progression of frailty (12–14). Early diagnosis is often noted to be important for a more positive outcome (12, 14, 51). Interventions are wide-ranging, including nutritional supplementation, medications, and exercise programs, among others (2). In a study of frailty scores, life-space (a measure of the geographical space in which a person’s life takes place) and quality of life, Chitalu and colleagues found that among people with high frailty scores, those with high life-space had better quality of life than similarly frail people with lower life-space; by contrast, life-space was a less relevant factor to quality of life among non-frail individuals (53). This finding suggests that specific interventions such as increasing life-space, may preferentially benefit frail individuals compared to their potential benefit on the greater geriatric population overall. An exploratory analysis of the MoveStrong exercises suggested improvement in frailty indicators (gait speed, balance, sit-to-stand functioning) and health-related quality of life in both frail and pre-frail individuals who used these exercises (54).
+
+It is clear that instruments deployed in the assessment of human frailty fulfill a variety of roles. Some are used for screening large community populations for both pre-frailty and frailty (15), some are used within long-term care facilities (30, 36, 41) and others are used in healthcare facilities (29, 40, 41) and may include components of diagnostic data (34, 55, 56). The diverse instruments range from simple to complex and include assessment of numerous and varying domains. There are challenges inherent to the design of some instruments, and the mere fact that so many instruments exist creates challenges. The ability of a clinician or investigator to select the ideal human frailty instrument for the setting and goals is valuable, however, the existence of so many instruments and lack of consistency in use precludes large-scale assessment of their performance or comparison between groups of individuals or intervention techniques.
+
+1.3.
+
+### Frailty in other species
+
+The importance and relevance of defining positive and negative trajectories of aging have been recognized widely. Outside of human gerontology, frailty scales developed in the laboratory setting for mice and rats have been shown to predict adverse outcomes (57–60), confirming that frailty is relevant and applicable to other species. By contrast, companion animal assessments rely heavily on owner-reported information. In this vein, veterinary specialties have developed quality of life (QoL) tools that are specific to a condition or body system [e.g., Canine Owner Reported QoL Questionnaire (61), Canine Symptom Assessment Scale (62), and questionnaires for QoL in patients with spinal cord injuries (63), patients with pain secondary to neoplasia (64), and patients undergoing chemotherapy (65)]. QoL questionnaires frequently capture a range of domains that are closely aligned with frailty and demonstrate the feasibility of companion animal frailty assessment by owner-reported metrics. Taking it a step further, McKenzie et al. proposed a Canine Geriatric Syndrome as a framework to evaluate physical, functional, behavioral, and metabolic changes in aging dogs to better understand and investigate the biological aging process; frailty is one of the proposed components of the syndrome (66). Despite this, the study of frailty in veterinary medicine, and specifically in dogs, is still in its infancy.
+
+Hua et al. developed a FP instrument, evaluating five phenotype components derived from those used in human gerontology (67). They extrapolated findings relevant to these five components from the records of physical examinations performed on geriatric service dogs, and found that the presence of more than two of these components was significantly associated with decreased time until death, independent of age, health status, or subclinical and clinical diseases (67). Banzato et al. developed a questionnaire-based FI instrument including 33 health deficits, meant to reflect data collected in a routine health exam (45). Banzato et al.’s FI was shown to have moderate accuracy in predicting short-term mortality (45). In both Hua et al. and Banzato et al.’s work, the prevalence of frailty in dogs was noted to be similar to that of humans (15, 43–45, 67), and the overall mean FI calculated by Banzato et al. was similar to the overall FI for community-dwelling individuals (45, 57). In human gerontology, it has been reported that a FI of 0.7 is considered to be the “threshold when the accumulation of deficits becomes incompatible with life” (45, 68); Banzato et al. noted a similar cut-off point of 0.7 in their FI for dogs (45). Additionally, a new frailty phenotype based on owner responses to simple questions has shown strong predictive power for short term (6 months) mortality in a cohort of healthy senior dogs (69). Collectively, these studies demonstrate that a frailty syndrome similar to that described in human gerontology is also present, and can be assessed, in dogs. A standardized frailty assessment could become as important a tool in geriatric canine health as it is in human healthcare.
+
+2.
+
+## The need for frailty assessment in veterinary medicine
+
+The importance of frailty assessment in human geriatric medicine has expanded as the elderly proportion of the human population has grown (22, 70, 71). Similarly, as the geriatric veterinary patient population expands, due to progress in diagnostic capability and therapeutic options that improve the overall management of pet health (72), the need for a clinically applicable frailty score in veterinary medicine is becoming clear. Age is the greatest risk factor for the development of frailty and, perhaps because of this strong association, the development of frailty and other age-related diseases within an aging individual is often viewed as inevitable (66). The belief that age alone is the cause of poor health outcomes may lead owners to forego opportunities to investigate actual underlying causes and provide potential interventions. This concept of a ‘normal’ state of poor health inevitably associated with aging is a barrier to the progress of geriatric veterinary medicine. The development and wide use of validated, objective frailty instruments in veterinary medicine are needed to advance the study of companion animal gerontology. Specifically, care of the geriatric companion dog is becoming a more important facet of veterinary general and specialty practice (73–75).
+
+The ability to stratify risk profiles within the geriatric companion dog population, as was described above for human gerontology, could significantly impact prognostication and medical care for dogs. Humane euthanasia of companion dogs is a common manner of death, elected by owners due to a variety of reasons including old age, devastating illness or injury, the perception that recovery is unlikely, lack of access to or affordability of needed care, and poor quality of life, among others (76–78). Thus, veterinary medicine is uniquely impacted by the need to provide accurate prognostic information and to combat assumptions of poor prognosis based on age alone, because perceived poor prognosis can lead to elective euthanasia. Owners’ assessments of their dogs’ health-related quality of life (HRQL) have been shown to be heavily influenced by age itself, rather than survivability (79), and perception of poor HRQL could also promote the owner’s choice to euthanize (17, 18, 42, 80). By contrast, the deployment of a validated companion dog frailty instrument would enable studies to determine the actual stratified assessment of the risk of negative outcomes for geriatric dogs as a function of their frailty. Such a tool could then better support owner decision-making, avoiding the assumption of a poor prognosis simply based on the age of the dog. For example, a retrospective study of the outcomes of over 6,000 dogs entered into the Veterinary Committee on Trauma registry found that among moderately injured dogs, geriatric dogs had a significantly higher risk of death despite intervention, or of experiencing euthanasia due to grave prognosis, than their nongeriatric counterparts (81). The higher rate of deaths despite intervention among geriatric dogs suggests that they may have been frailer; by contrast, the higher rate of euthanasia among these dogs suggests that their owners or attending clinicians may have believed their prognosis was worse than the prognosis of younger dogs with similar injuries.
+
+A frailty screening tool for veterinarians to use to easily identify frail dogs would also allow for adjustments in management protocols, from anesthesia to treatment, similar to the previously mentioned study in human geriatric elective surgical patients (49) – and ideally in the future allow veterinarians to more accurately predict recovery from specific events or illnesses. The first step in the ability to make frailty-specific protocol adjustments to improve frail patients’ outcomes is the ability to identify those patients. The identification of frail dogs would also allow for direct intervention in the frailty state as is seen in human medicine (2, 13, 14, 51, 52, 54). For instance, programs to target strength, balance, or maintenance of muscle mass could be implemented in veterinary medicine to directly improve the lives of geriatric companion dogs. Furthermore, there is a growing body of research into strategies to extend lifespan and healthspan in companion dogs (82–85). Lifespan is a challenging target to deploy in such clinical trials because it can be modified by owner-elected euthanasia in response to owner perception of the dog’s status, access to care, and other factors. Widespread adoption of a validated frailty instrument would enable frailty – physiologically meaningful, aging-associated phenotype – to replace lifespan as a valuable endpoint for such clinical trials. The ability to evaluate the effect of individual frailty on outcomes in specific situations and disease conditions as well as to appraise the utility and impact of an interventional program hinges on the ability to recognize and measure frailty. A widely used frailty assessment for dogs would also allow for comparison between medical institutions and different studies, longitudinal monitoring for individual patients, and an endpoint in clinical trials that assess interventions into aging and age-related decline.
+
+3.
+
+## Dog Aging Project proposed frailty instrument for dogs
+
+The ideal companion dog frailty instrument would maximize the benefits while minimizing or eliminating the weaknesses associated with the diverse frailty instruments used in human gerontology (Table 3). As described above, diverse instruments available in human gerontology may satisfy the needs of different users but also make the collection of comparable data among sites or populations challenging. If frailty is to become a common component of the assessment of companion dogs, it would be beneficial to avoid this fragmentation of data by ensuring that a single tool is widely accessible and used in all dimensions of veterinary practice. The Dog Aging Project (DAP) is an open-data, long-term, longitudinal study of the genetic and environmental determinants of healthy aging in companion dogs (100, 101) and the ability to assess frailty in this population is paramount to fulfill the goals of the study. Enrolled dogs represent diverse household environments in all 50 US States and receive medical management from their primary care veterinarians throughout the study. A companion dog frailty instrument that can be used in all the practices in which these dogs receive routine medical attention will create an opportunity for the collection and clinical application of comparable frailty information not just from DAP-enrolled dogs, but from all companion dogs. The DAP has been previously described (100). Briefly, the DAP collects comprehensive information about dietary and exercise management, home environments, and health histories of enrolled dogs and it is likely that some of these experiences could influence, or be influenced by, frailty. The utility of a proposed frailty instrument would be assessed among these diverse groups of dogs, to better describe the manner in which varying attributes and experiences impact the trajectory of frailty in companion dogs.
+
+**Table 3 Maximizing strengths and avoiding weaknesses of human gerontology frailty assessment in a proposed companion dog frailty instrument.**
+
+| Strengths |
+| --- |
+| Human frailty assessment | Proposal |
+| Several key phenotypic factors (unintentional weight loss, self-reported exhaustion, weakness, slow walking speed, poor endurance, and low physical activity) are included in the most commonly used instruments (15, 39, 41, 56, 86–91). | Adapt these key dimensions to dogs |
+| Instruments that include multiple physiologic domains such as illness or comorbidities, functional status for daily activities, use of medications, decreased appetite, nutrition, balance, flexibility, falls, mobility disability, and vision loss (1, 23, 29, 30, 34, 37, 40, 41, 55, 87, 89, 90, 92–94). | Include assessments of physiologic domains relevant to dogs to broaden the assessment |
+| Instruments that include multiple functional (non-medical) domains such as activities of daily living (ADL)/instrumental activities of daily living (IADL), communication (speech/hearing), mood alteration, low motivation, reclusion, cognition, and social support (1, 23, 28–31, 34, 41, 55, 92) | Include assessments of functional domains relevant to dogs to broaden the assessment |
+| Multidimensional instruments (31, 36, 95). | Ensure multi-dimensional components are included to maximize predictive value |
+| Short instruments (15, 39, 87). | Minimize the number of components to decrease the burden on users |
+| Instruments utilizing self-reporting (29, 89, 92). | Select components apparent to dog owners at home |
+| Assessment that can be performed at home, by phone or mail (35, 91–93). | Select components readily assessed by dog owners at home |
+| Instruments that can be performed by individuals without medical training (31, 32, 88). | Select components that can be performed by owners |
+| Instruments that can be performed by primary care providers (29). | Select components that can quickly be performed by veterinary general practitioner |
+| Weaknesses |
+| Human frailty assessment | Proposal |
+| Lengthy/time-consuming instruments (28, 96, 97). | Ensure that owner or primary care veterinarian can complete in less than 30 min to minimize burden on users |
+| Instruments that include complex measurements including the Progressive Romberg Test, Brachial Ankle Index, and tests using wireless motion sensors (56, 98). | Limit to simple, low-tech measurements to facilitate wide use |
+| Instruments that include medical or laboratory data (albumin, blood pressure – systolic or arterial, red blood cells/hemoglobin counts, etc.) which may be unavailable or expensive to acquire (23, 28, 34, 37, 55, 56, 99). | Limit to externally apparent phenotypic data to avoid invasiveness or added cost |
+| Instruments that require specifically trained personnel to administer (17). | Ensure that minimally trained owners or veterinary personnel can obtain each measurement to facilitate wide use |
+
+As discussed above, frailty instruments in human gerontology may be optimized for different settings, and ultimately a three-tiered structure (Screening, Assessment, Triage) for frailty evaluation in dogs seems valuable. The Screening step would enable owners to identify dogs likely to benefit most from frailty assessment, whereas the Triage step could be deployed in urgent medical settings to enable appropriate frailty-based protocol modifications for dogs who had not been previously assessed. Here we propose the design and rationale for an Assessment instrument, the Frailty Instrument for Dogs (FIDo).
+
+Development of the FIDo is guided by several overarching goals, derived from observations about the strengths and weaknesses of various human frailty instruments (Table 3). To ensure that this instrument will be accessible to most veterinarians and owners, it must be easy to use, low-tech, low-cost, and of reasonable length (ideally less than 15 min for completion). To capture the complexity and multifactorial essence of frailty, the instrument must be a multi-dimensional tool, including physical, social, and psychological components. As previously discussed, there are two major models for frailty instruments utilized in human gerontology (FP and FI), however, there is no consensus on the better model. Both have been found to provide comparable predictions of mortality (27), and they may be complementary (26) in providing a more robust understanding of an individual. To achieve the goal that the FIDo is low-tech, low-cost, easily implemented, and widely available and utilized for the assessment of frailty, the DAP elected to build a phenotypic frailty model that includes externally apparent components. Use of a phenotypic instrument will allow the identification of frail patients using information that can be readily collected by owners and veterinarians without the need for medical or diagnostic interactions, that may hinder implementation due to financial or other barriers to care. A phenotypic approach also facilitates the identification of frail individuals that may be free of diagnosed disease (9, 26). Thus, in contrast to Banzato et al.’s FI instrument (45) diagnostic data and multimorbidity will be components of a separate assessment tool in the DAP framework. This multimorbidity tool would be available to use as a complementary assessment tool with FIDo for geriatric care in the future. In contrast to the FP instrument previously reported by Hua et al. (67), the components of the DAP FIDo will be prospectively collected by both owners and veterinarians.
+
+Domains for the DAP FIDo were carefully chosen to reflect those most often used in human gerontology which can be assessed in dogs. Multiple factors within each domain will be included in the initial analysis to determine which are the most relevant phenotypic markers of frailty. The domains that will be included in the DAP FIDo are physical condition, physical activity, mobility, strength, cognitive task performance, and mood and social relations (Table 4). In previous studies of congestive heart failure, diabetes mellitus, and osteoarthritis it was demonstrated that owners are sensitive and accurate reporters of their dog’s health status (102–104), thus owners will serve as primary data sources for most domains. Specific owner-provided information will be extracted from the DAP’s Health and Life Experience Survey (HLES), Annual Follow-Up Survey (AFUS), Canine Social and Learned Behavior Survey (CSLB) (105), Measurement and Mobility Activities (M&M), and Cognitive Tasks (100). Certain factors will also be extracted from veterinary electronic medical records (VEMRs) uploaded by participating owners. These components will initially be evaluated separately to determine which are predictive of mortality in dogs. As each component will be collected longitudinally, we will also assess whether the rate of change of each component is more strongly predictive of mortality than a discrete value at a single time point. All predictive components will be included in the first comprehensive model. Components that are not found to have a relationship with frailty will be removed. Stepwise analysis will allow removal of components that provide redundant information; remaining components will be weighted by a coefficient proportional to the association of each parameter with mortality.
+
+**Table 4 Dog Aging Project (DAP) proposed companion dog frailty instrument.**
+
+| Domain | Factor | Source of data | Nature of data |
+| --- | --- | --- | --- |
+| Physical condition | Body condition score (BCS) | VEMR | Ordinal numeric value extracted from records |
+| Thigh girth deterioration | M&M | Continuous numeric value (centimeters) obtained by owners |
+| Unintentional weight lossVeterinarian-reportedOwner-reported | HLESVEMR | Continuous numeric value (kilograms) extracted from records and reported by owners |
+| Physical activity | Customary activity | HLESAFUS | Ordinal qualitative description reported by owners |
+| Mobility | Gait speedOn-leashOff-leash | M&M | Continuous numeric value (seconds) obtained by owners |
+| Strength | Stair climb | M&M | Continuous numeric value (seconds) obtained by owners |
+| Cognitive performance | Working memory | Cognitive task | Continuous numeric value (number of accurate trials) obtained by owners |
+| Learned behavior | CSLB | Ordinal qualitative description reported by owners |
+| Mood and social relations | Anxiety/nervousness | HLES(C-BARQ) | Ordinal numeric score reported by owners |
+| Social avoidance | CSLB | Ordinal qualitative description reported by owners |
+
+3.1.
+
+### Physical condition
+
+Three different metrics of physical condition, body condition score (BCS), thigh girth deterioration, and unintentional weight loss, will be included in the initial DAP FIDo. Body condition score, a commonly used 1–9 scale used for the evaluation of an animal’s body fat from emaciated to obese, will be extracted from VEMRs. Increased BCS has been shown to be predictive of disease and mortality risk in canines (106), but a ‘protective effect’ associated with increased BCS has also been reported in the study of a canine frailty index model (45). Sarcopenia is an increasingly important factor in the assessment of human frailty and the mechanism by which it occurs is receiving increasing scrutiny (19, 107–109). As a component of frailty or an isolated finding, sarcopenia has been shown to be a marker for increased risk for disease and death (109). Sarcopenia has been described in the dog (110, 111) and a clinical assessment, the Muscle Condition Score (MCS) (111, 112), is available in veterinary practice. However, MCS is not yet a widely utilized tool in general practice and is found in only 5% of VEMRs submitted to the DAP. By contrast, it has been shown that minimally trained owners can obtain measurements of their dogs’ thigh circumference with strong agreement to those obtained by veterinary researchers (113). Thus, the DAP FIDo will use serial thigh circumference measurements to assess canine sarcopenia. Unintentional weight loss is a consistent factor in human frailty instruments. DAP participating owners provide their dogs’ weights annually (once in HLES, and subsequently in AFUS). We will also extract weights recorded at veterinary visits from VEMRs. As is the case for people, intentional weight loss in adult dogs is difficult for most owners to achieve (114, 115) the assumption will be that any detected weight loss was unintentional. However, the variable intervals at which weights are recorded at veterinary visits, and the difficulty of confirming whether any detected weight loss was truly unintentional, may preclude the value of this factor; validation analysis will determine whether it remains in the final instrument.
+
+3.2.
+
+### Physical activity
+
+A variety of metrics of physical activity including objective kilocalorie expenditure, as well as self-reported activity, inactivity, exhaustion, and fatigue, are heavily utilized in human frailty instruments due to their predictive value. Importantly, these metrics are designed to detect or assess the individual’s customary amount and vigor of activity (15, 56, 86, 116), rather than the peak performance of which an individual is capable. Companion dogs often depend upon their owners for physical activity, such as being taken on leashed walks, or being given access to a park for play. For this reason, a simple assessment of the frequency of activity may reflect owner preferences more than dog ability. By contrast the dog’s interest in activity, vigor of activity, and change in activity patterns over time may be better suited to detect the onset of frailty. These topics of dog activity are addressed in HLES and AFUS, and we will derive a measurement of physical activity from owner responses to questions including, “Please choose the best description of your dog’s lifestyle over the past year. [not active/moderately active/very active],” “Over the past year, on average how much time per day is your dog physically active? Number of hours active: [0–8+], Number of minutes active: [0/10/20/30/40/50],” and “Over the past year, when your dog is active, what is the average intensity level of that activity? [low(walking)]/moderate [jogging]/[vigorous (sprinting)].” Other follow-up items identify the nature of the activity, including environmental conditions, on vs. off-leash, type of activity, etc. The use of multiple questions surrounding owner-reported activity allows for us to find the components that most accurately reflect the change in a dog’s activity as well as its relation to frailty.
+
+3.3.
+
+### Mobility
+
+Despite significant variability between instruments used in human medicine, there is an overarching consensus that a measure of physical function is needed (117–119). A common measurement of mobility in human frailty instruments is walking speed, where an individual’s time to walk, a specified distance is compared to the lowest 20% of the population (15). Slower walking speeds in people have been shown to be predictive of worse postoperative outcomes (120–122), morbidity, and mortality (122–124); similar results were seen in rodents (125–127). Several small studies of mobility have shown a general trend of decline in functional capacity and spontaneous activity with but results also varied by location, breed and sex (128–130). Mobility scales for use in companion dog populations have previously been proposed, but they have not been validated or implemented in large populations (131, 132).
+
+When developing a canine mobility scale Gonçalves et al. found a statistically significant difference in mobility scale scores between age quartiles (131), however, the presence of orthopedic and neurologic diseases was also noted to produce a statistically significant difference in scores (131). Banzato et al.’s frailty index study also reported poor mobility and low physical activity were significantly associated with time to death independently (45).
+
+Morgan et al. morphologic and mobility trials found age was a weak but significant predictor of a dog’s speed for a given height (113), suggesting speed could be a useful frailty factor. They also showed that owners were able to perform low-tech assessments of their dogs’ speed with minimal training and were able to obtain measurements that strongly agreed with those obtained by investigators (113). Consequently, the DAP FIDo will include times to traverse a measured distance on-leash and off-leash over a flat-surface from our Measurement and Mobility instrument. A greater magnitude of change in off-leash speed with increasing age was reported (109), but this metric may not be a significant indicator of frailty among dogs of the same age. Thus, both on- and off-leash flat-surface speed will be evaluated separately in the initial model to determine whether either or both add predictive value to the frailty model independently of age alone.
+
+3.4.
+
+### Strength
+
+Strength is also frequently included in human frailty instruments. In humans, strength can be assessed utilizing a variety of measurements including the ability to rise from a chair (39) and stair climbing (133); grip strength (15, 87) is the most popular measure. Grip strength is not a feasible metric in dogs, but stair climbing is a routine and accessible activity for many dogs. Elderly people often become reluctant to climb stairs as they age (127–129) and the authors’ clinical experience suggests that the same is true among companion dogs. Morgan et al.’s morphologic and mobility trials also assessed timed stair ascent and found that age was a weak but significant predictor of speed in this task as well (113). Other options that were considered as strength assessments, such as pressure-sensing chew toys (134), or systems that measure the strength with which a dog can pull against a measurement device (135, 136), are expensive, have limited availability, depend upon the dog’s training and interest, or some combination of those flaws. The DAP FIDo will use timed stair ascent as our measurement of canine strength.
+
+3.5.
+
+### Cognitive task performance
+
+Cognitive decline is an important factor in many human frailty instruments (33, 137–139). There is an incompletely understood relationship among frailty, mild cognitive impairment, and the ability to perform the Instrumental Activities of Daily Living (IADL) in humans (55, 92, 140–142). While IADLs cannot readily be extrapolated to dogs, simple cognitive tasks that mimic daily activities have been developed and validated in dogs. Dogs, like humans, display evidence of mild cognitive decline with age, and their performance on these tasks also declines with age (143, 144). We will use performance on a purpose-built at-home cognitive task designed to assess working memory within our DAP FIDo. The purpose-built cognitive task is easily administered at home and does not require specialized equipment or dog training. Dogs are tested for their ability to (1) recall the location of a food item after varying delays [delayed search], or to recall which location still contains a food item after consuming food items from all other locations [radial array]. Instructions are provided through online video tutorials and owners respond to simple questions about their dog’s behavior through an interactive online survey.
+
+Like humans, some dogs also develop dementia, called Canine Cognitive Dysfunction Syndrome (CCDS) (145, 146). The Canine Cognitive Dysfunction Rating Scale (CCDRS) is a validated survey used to diagnose CCDS among dogs with a cumulative score of 50 or greater (104, 105, 147). We have deployed this instrument within our DAP population as an annual survey, rebranded the Canine Social and Learned Behavior (CSLB) instrument, to avoid potential negative connotations of the diagnostic term, “cognitive dysfunction.” However, the purpose of a frailty instrument is to detect early, mild change in function, before an overt diagnosis, such as CCDS, is made. Thus, we will use individual items from the CSLB, rather than the total score, within our DAP FIDo. Specifically, we will use responses to the items “How often does your dog stare blankly at the walls or floor?” and “Compared with 6 months ago, does your dog have difficulty finding food dropped on the floor?” to detect changes in at-home cognitive performance over time. The total CSLB score, as well as owner-reported diagnoses in the Health Section of HLES, will be evaluated to ensure that the selected CSLB items do not reflect a diagnosis of CCD or another neurologic disease that may have its own impact on mortality.
+
+3.6.
+
+### Social behavior
+
+Recent work in human frailty has promoted the importance of including social components in frailty assessments (1, 148). While questions about happiness and social support networks cannot be readily extrapolated to dogs, there is a large body of research describing normal companion dog behavior, including socially interactive behavior (149–152). The Canine Behavioral Assessment and Research Questionnaire (C-BARQ) is a validated survey used to objectively document companion dog behavior in a variety of settings (153–155). We have deployed the C-BARQ as the Behavior component of HLES and AFUS, so that it is completed annually by DAP participants. We will use responses to specific items from both C-BARQ and CSLB to represent mood and social relations in the DAP FIDo. Specifically, we will use the cumulative score from the Fear and Anxiety section of C-BARQ and the item on social avoidance, “How often does your dog walk away while, or avoid, being petted?” from CSLB.
+
+These items addressing physical condition, physical activity, mobility, strength, cognitive task performance, and mood and social relations will be included in the first version of the DAP FIDo to identify those domains and specific factors that most strongly predict mortality among companion dogs. The model will be revised to include the fewest, but most informative elements, to facilitate wide deployment among companion dog owners and veterinary practices.
+
+4.
+
+## Discussion and conclusion
+
+The assessment of frailty plays a central role in human gerontology as a superior means to understand, describe, and mitigate certain manifestations of aging and the associated risks of poor health outcomes, including death within individuals and communities. The numerous frailty instruments for humans that have been developed include diverse domains and factors within each domain, leading to challenges in comparing the efficacy and utility of these instruments across populations and settings. As companion dogs increasingly survive into geriatric ages, the ability to document their frailty, and to understand its impact on health outcomes, becomes increasingly valuable. The Dog Aging Project is collecting targeted data to enable the construction and validation of a companion dog frailty instrument designed to be uncomplicated, quick to complete, low-tech, low-cost, and accessible to dog owners and veterinary general practitioners. Use of this tool, once finalized, will enhance both research opportunities, and the ability to provide excellent veterinary care, to aging companion dogs.
+
+## Author contributions
+
+AF, AR, KC, NO, and RM worked together to construct an outline of the manuscript. RM wrote the first draft of the manuscript. AR, NO, AF, and KC revised and edited sequential drafts of the manuscript. EP contributed to the visualization and revision. All authors have read and approved the final manuscript.
+
+## Funding
+
+The Dog Aging Project is supported by U19 grant AG057377 from the National Institute on Aging, a part of the National Institutes of Health, and private donations. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
+
+## Conflict of interest
+
+The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
+
+The reviewer BM declared a past co-authorship with the author NO to the handling editor.
+
+## Publisher’s note
+
+All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

fulltext/md/PMC10340034.md

@@ -0,0 +1,113 @@
+---
+pmcid: "PMC10340034"
+pmid: "37443999"
+doi: "10.3390/ani13132203"
+title: "The Relationship between Signs of Medical Conditions and Cognitive Decline in Senior Dogs."
+journal: "Animals : an open access journal from MDPI"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# The Relationship between Signs of Medical Conditions and Cognitive Decline in Senior Dogs
+
+## Abstract
+
+### Simple Summary
+
+As dogs age, they develop conditions associated with old age, which include pathologies of the brain and the body. One of these conditions is canine cognitive dysfunction syndrome, a neurodegenerative disorder similar to the early stages of human Alzheimer’s disease. The goal of the current study was to investigate the relationship between cognitive dysfunction syndrome and other medical conditions in geriatric dogs, as reported by their owners, using an online questionnaire. The results indicated that dogs with greater cognitive dysfunction also had more signs associated with musculoskeletal and neurological problems, including pain and sensory decline. The relationship was similar, but weaker, for symptoms of digestive or metabolic disorders, and it was weakest for dermatological symptoms. The take-home message of the current study is that, in older patients, medical conditions should be carefully screened, and in cases of suspected cognitive dysfunction, the underlying cause should be cautiously assessed, especially for pain, sensory decline, and digestive and metabolic conditions.
+
+### Abstract
+
+Canine cognitive dysfunction syndrome (CCDS) is a progressive age-related neurodegenerative disorder in dogs. Minimal research has been performed to investigate how clinical signs may be impacted by other medical conditions. A cross-sectional study was performed using the Canine Cognitive Assessment Scale (CCAS) to evaluate cognitive impairment as reported by owners. Owner-reported health-related measures included behaviour changes, the body condition score, and veterinary diagnoses of disease. The responses from 804 dogs in the last 25% of their expected lifespan were analysed. Factors were identified in the owner-reported behavioural signs of disease representing pathologies in four body systems: musculoskeletal–neurological, digestive, metabolic, and dermatological, with the items comprising these factors also compiled into a cumulative measure of health. The results showed a strong correlation between the CCAS score and both the musculoskeletal–neurological factor and the overall cumulative measure of health. Moderate correlations between the CCAS score and the digestive factor and metabolic factor were also observed. The correlation between the dermatological factor and the CCAS score was weak. This study highlights the need to screen dogs for concurrent diseases when using scales to assess cognitive impairment and to monitor dogs who have health conditions, particularly those that are painful, for the onset of cognitive impairment.
+
+## 1. Introduction
+
+Canine cognitive dysfunction syndrome (CCDS) describes an age-related disorder featuring cognitive impairment, which is analogous to the early stages of Alzheimer’s disease (AD) in humans [1]. The prevalence of cognitive impairment in older dogs, as reported in the literature, varies widely, with the comparison between studies being hampered by the different scales used and populations studied. The published figures range from 14.2% in dogs over 10 years of age [2] to 22.5% in dogs over 9 years of age [3] to as high as 74% in dogs over 7 years of age [4]. Age is a major risk factor for the increasing prevalence and severity of CCDS [2,3,5]. The authors observed that over a period of 24 months, 33% of dogs over eight years of age with normal cognitive function progressed to mild cognitive impairment (MCI), and 22% with MCI progressed to severe cognitive impairment [6]. A similar pattern of findings was reported by Madari et al. [7]. The true prevalence of CCDS in the canine population may be even higher than it was estimated to be due to under-diagnosis, with a discrepancy reported between cognitive impairment as measured using a scale, 14.2%, and that diagnosed by a veterinarian, 1.9%, in a population of dogs over 10 years of age [8]. This was, in part, attributed to under-reporting by owners, who may consider the behavioural changes to be a part of normal ageing [9].
+
+CCDS is also important due to its impact on the well-being of the dogs’ human guardians. The caregiver burden is the negative impact on a caregiver of having to care for an individual with a chronic condition [10], which, in veterinary medicine, is linked to making a decision about euthanasia [11]. The caregiver burden associated with owning a dog with CCDS has not been investigated directly; however, it is likely to be high due to owners having to alter their routines, accommodate the dog’s changing requirements, and the impact of the behavioural symptoms associated with CCDS [12]. Increasing age alone is a risk factor for euthanasia, while behavioural changes are the fourth most commonly reported reason for canine euthanasia [13], reflecting the negative impact of unwanted behaviours on the human–animal bond.
+
+The diagnosis of CCDS relies on the identification of suggestive behavioural symptoms and the exclusion of other possible causes. Magnetic resonance imaging (MRI) allows the assessment of some gross intracranial changes that may be associated with CCDS, including cortical atrophy [14], leukoaraiosis (periventricular changes to white matter) [15], and the reduced size of the interthalamic adhesion [16]; however, these abnormalities are not specific to CCDS and are also found in senior and geriatric dogs without cognitive impairment [1]. MRI does serve a role in the exclusion of other intracranial pathologies, such as space-occupying lesions and cerebellar infarction [17]; however, the benefit of MRI often does not outweigh the cost and risks associated with general anaesthesia [1]. Authors have demonstrated the relationship between the accumulation of beta-amyloid plaques and CCDS’ cognitive deficit [18,19]. However, there is not yet sufficient evidence that beta-amyloid or other markers, such as TAU protein or Neurofilament Light Chain (NFL), could be used as markers of cognitive decline in dogs [20]. Due to the absence of disease-specific changes, an ante-mortem presumptive diagnosis relies on the identification of behavioural changes. An acronym, DISHA, is used to describe areas of behaviour that might be impacted, standing for disorientation, interaction, sleep–wake cycle, house soiling, and activity levels [21], and has gone through several iterations, with the addition of AAL (activity-apathy and depression; anxiety; learning and memory) [22]. These behavioural changes are non-specific, and so may be displayed as a result of many different pathological processes, including sensory decline and musculoskeletal disease [23,24], making CCDS a diagnosis of exclusion. To aid veterinarians in identifying these behaviours and the extent to which they are displayed, several scales have been developed, with some also allowing the categorisation of the degree of cognitive impairment [2,7,25]. Researchers are also trying to develop behavioural tests to assess the cognitive state of pet dogs as they age, with the aim of aiding the diagnosis of CCDS [26,27,28,29]. However, these still need further validation [25]. It is notable that although the scales may indicate the presence of cognitive impairment, it is still incumbent on the veterinarian to rule out other possible causes of the behavioural changes reported.
+
+As an organism ages, the risk factors for disease increase, and the likelihood of multimorbidities rises [30]. As a result, there is often a complex clinical picture with the presence of behavioural signs that might be suggestive of CCDS accompanied by other physical disease states. This interplay between physical and cognitive health was the focus of the current study, considering how cognitive impairment, measured by a CCDS assessment scale, related to the presence of other disease states. This study aimed to understand: (1) how different disease states correlate with the degree of cognitive impairment in dogs; (2) what relationship exists between a cumulative measure of health and the degree of cognitive impairment in dogs. It was anticipated that there would be common pathological processes linking the different behavioural signs of the disease assessed and that these would be associated with greater cognitive impairment. It was hypothesised that greater cognitive impairment would also be associated with worsening overall health and with the presence of specific disease states.
+
+## 2. Materials and Methods
+
+### 2.1. Subjects
+
+A cross-sectional study design was used to assess the relationship between owner-reported behavioural signs of disease and veterinary diagnoses, with canine cognitive impairment as indicated by the Canine Cognitive Assessment Scale (CCAS) [25]. The CCAS was selected due to the ability to categorise the degree of cognitive impairment based on resulting scores, as well as its inclusion of a broad range of behavioural changes, including behaviours relating to anxiety.
+
+A convenience sample of participants was taken via social media platforms. The online questionnaire was active between 4 February 2021 and 22 March 2021. Participants needed to be over 18 years of age. To be eligible, they were also required to have owned their dog for over one year, in order for the participant to have knowledge of the veterinary diagnoses made during that period. The dogs needed to be over five years of age to allow the inclusion of dogs estimated to be in the last 25% of their estimated lifespan, which represents the senior age bracket in dogs [31]. This relatively young age was selected to capture data from large breed dogs that have a shorter estimated lifespan than smaller dogs [32,33]. If the participant owned more than one dog, they were asked to complete the survey considering their oldest dog.
+
+### 2.2. Questionnaire
+
+Participants were invited to complete an anonymous online survey about ageing in dogs hosted on the JISC Online Surveys platform. The survey comprised three sections:(1)Demographic information about the dog—breed, weight, height, age, body condition score (BCS), and how recently it had been examined by a veterinarian;(2)Assessment of the dog’s cognitive health—CCAS scale [25] comprising 17 items which assessed six domains of behaviour (disorientation, sleep–wake cycles, social interactions, learning and memory, activity level, and anxiety). The scale utilises a four-point Likert scale; never (0), once a month (1), once a week (2), almost every day (3), reporting behaviour over the previous six months. If a participant was unsure how to respond, they were asked to select ‘Never’ as opposed to leaving it blank as directed by the original CCAS;(3)An assessment of the subject’s general health—37 questions regarding behaviours that reflect the pathology of different body systems, hereafter referred to as ‘general health questions (GHQs),’ and 12 questions regarding diagnoses made by a veterinarian in the preceding year, hereafter referred to as ‘diagnoses questions’ (see Supplementary Information). Questionnaires published in the literature concerning the assessment of health in older dogs [34,35,36] were used as a basis for the GHQs. Items were phrased in colloquial English to ensure that they were not ambiguous. The GHQs utilised a four-point Likert scale describing the degree of change. The diagnoses questions had binary yes/no response options.
+
+### 2.3. Data Analysis
+
+Statistical analyses were performed on IBM SPSS version 25. The weight and height of the dogs were used to calculate their expected lifespan, in order to account for the fact that dogs of different sizes age at different speeds [34]. The height and body weight of the dogs were used to calculate the estimated lifespan and hence select the data to use for further analysis using the formula [33] below, adjusted from imperial to metric units:Estimated lifespan = 13.620 + (0.0276 H) − (0.1186 W)(1)
+where H indicates the height of the withers (cm), and W indicates the body weight (kg).
+
+The formula provides the theoretical lifespan (in years) for a dog based on its size. We then compared this value with the actual age of the dog to select those that had reached or were above 75% of their lifespan (the marker for senior age, according to the AAHA guidelines for senior dogs and cats).
+
+As the height was collected as categorical information, the midpoint of each range was used to represent the height of the subject (for example, the range 21–40 cm was represented by 31 cm). In addition, two subjects were excluded from the analyses as the participant’s responses to the weight change questions were inconsistent (both weight gain and loss). Therefore, the analysis was run on 802 dogs. The overall CCAS score was calculated by summing single-item scores. In addition, the domain ‘disorientation’ was weighted as double. The final CCAS score indicated degrees of cognitive impairment ranging from normal ageing (scores 0–7), mild (scores 8–40), and severe (scores 41–69). Given that score data was used, non-parametric analyses were employed.
+
+A Spearman’s rank order correlation test was used to measure the correlation between the CCAS scores and both the OHS and mean factor scores. Additionally, a Spearman correlation was also used to assess the relationship between subject weight and age with the overall CCAS score. The strength of correlation was assessed as absent (0–0.09), weak (0.1–0.29), moderate (0.3–0.49), or strong (0.5–1.0). A Kruskal–Wallis test was conducted to test whether there was a difference in the factor means between CCAS categories (normal ageing, mild cognitive impairment, severe cognitive impairment).
+
+Dogs who had received a veterinary examination in the preceding year, n = 710, were included in a Pearson’s chi-square test to analyse the relationship between the CCAS categories and the conditions comprising the ‘Diagnoses’ variable. All expected cell frequencies were required to be over 1, and at least 80% greater than 5. When these criteria were not met, the categories of mild and severe cognitive impairment were merged, resulting in a two-category variable, ‘Presence of cognitive impairment’. The Pearson’s chi-square test was also utilised to assess the relationship between the BCS of the subjects and the presence of cognitive impairment. The categories of BCS 1–2 and 3–4 were merged to give an ‘underweight’ category, while BCS 6–7 and 8–9 were merged, giving an ‘overweight’ category. The strength of association was determined by calculating Cramer’s V, with interpretation adapted from Cohen [37]; small 0.1–<0.3, medium 0.3–<0.5, large ≥0.5.
+
+Finally, we ran a cumulative link regression model with the CCAS score as the dependent variable and the four factors and age as fixed factors without interactions, in order to assess the combined effect of the medical signs and age.
+
+The significance level was set at *p* < 0.05 for all tests. However, when multiple tests were performed, increasing the risk of type 1 errors [38], a Bonferroni correction [39] was applied (0.05/25 = 0.002), resulting in a significance level of *p* < 0.002.
+
+## 3. Results
+
+Data regarding 1374 dogs were collected. Of these, 804 dogs were calculated to be in the last 25% of their lifespan based on body weight and height, and their data were further analysed. The data set included a broad range of breeds, with over 74 named breeds making up 67.7% of the total (n = 544) and mixed breeds forming 32.3% (n = 260). The median age was 12 years (range, 5–19 years), the median bodyweight was 22 kg (range, 2–99), and the median height was 51 cm (range, 10–91). The body condition score (BCS) of the subjects, using a 1–9 scale [40], had a mode of BCS 5.
+
+In this population, about half of the dogs had a CCAS score equal to normal ageing (n = 446, 55% of the population), 43% presented mild cognitive impairment (n = 345), and 2% had a score of severe cognitive impairment (n = 13). Dogs with cognitive impairment represented 45% of the population (n = 358).
+
+There was a negative but weak (r = 0.1–0.29) correlation between the CCAS score and bodyweight (Spearman’s rho, *rs* = −0.210, *p* < 0.001) and a positive, strong (r = 0.5–1.0) correlation with age (Spearman’s rho, *rs* = 0.507, *p* < 0.001), Figure 1.
+
+It was observed that there was a statistically significant relationship between the presence of cognitive impairment (normal ageing and cognitive impairment) and body condition. Specifically, dogs with ideal weight were more common in the normal ageing group (underweight n = 114, ideal weight n = 493, and overweight n = 197), χ^2^ (2) = 21.251, *p* < 0.001. However, this was a small effect (Cramer’s V = 0.163, *p* < 0.001). All expected cell frequencies were greater than 5. There was no significant association detected between breed category (pure breed and mixed breed) and CCAS category (normal ageing, mild cognitive impairment, and severe cognitive impairment), χ^2^ (2) = 0.537, *p* = 0.764. All expected cell frequencies were greater than 1, with 16.7% of cells less than 5.
+
+The GHQ items were assessed before performing the factor analysis. Eight GHQ items (avoids touch, assistance eating, constipation, flinches when eating, urinary incontinence, ear problems, alopecia, syncope) were excluded from the factor analysis due to exhibiting low variance determined by a standard deviation of less than 0.5 or greater than 90% of the dogs not exhibiting a score above 0. Four additional GHQ items (head pressing, circling, seizures, head tilt) with low variance were nevertheless retained as they related to neurological dysfunction and were considered clinically relevant by the authors. A principal axis factor analysis with promax rotation was performed on the remaining 28 items. In this, the minimum amount of data for factor analysis was satisfied, with a ratio of over 20 subjects per item [41]. Several criteria for the factorability of a correlation were used: that the determinant of the correlation matrix was greater than 0.00001; that the Kaiser-Meyer-Olkin (KMO) measure of sampling adequacy was 0.85 which falls into the ‘meritorious’ category [42]; that the KMO for all the individual items was greater than 0.52; and that Bartlett’s test of sphericity was significant (*p* < 0.001). The correlation matrix was visually checked, and none of the items had *r* > 0.9 with one another, thus excluding multicollinearity. Given these indicators, factor analysis was deemed to be suitable for all 28 items. The final correlation matrix can be viewed in Supplementary Information Table S1. An exploratory factor analysis was performed on the GHQ items to identify factor groupings within the data. The scree plot and eigenvalues were considered and used to determine how many factors to extract. Eight factors had an eigenvalue greater than 1; visual inspection of the scree plot revealed points of inflexion at two and three factors. Solutions for four and three factors were each examined, using varimax, oblimin, and promax rotations of the factor loading matrix. The four factors solution was preferred as it met the interpretability criterion, and promax rotation provided the best-defined factor structure. Thereafter factor extraction was an iterative process based on the examination of the pattern matrix, excluding variables that did not load above the cut-off of 0.4. The analysis was repeated until all remaining variables met the criterion level, with the four factors explaining 54.46% of the variance. A total of 19 items were eliminated (avoids touch, assistance eating, constipation, flinches when eating, urinary incontinence, ear problems, alopecia, syncope, vomiting, diarrhoea, halitosis, coughing/dyspnoea, greying, masses, weight change, circling, head pressing, seizures, head tilt). Principal Axis Factoring with Promax rotation was performed with the inclusion of all items. The correlation matrix was visually assessed to check the correlation between variables, before extracting factors on the basis of the eigenvalues using Kaiser’s criteria [43], with a minimum requirement of being greater than 1, and the scree plot [44]. The percentage of non-redundant residuals was checked to ensure that less than 50% were greater than 0.5, indicating that the factor model had a good fit [45]. The pattern matrix resulting from the oblique rotation was assessed, retaining items if their loading was greater than 0.4 on one of the factors. The analysis was repeated after each round of exclusions until all items loaded onto at least one of the factors at a value of greater than 0.4 and cross loading was allowed. The internal consistency of the factors was assessed using Cronbach’s α [46], and items were removed if this would improve the reliability of a factor (explained in the results). For simplicity, the factors were then labelled according to the theme identified within the items, guided by the highest loading item. These were used to calculate the overall health score (OHS), equal to the percentage of the overall sum of scores, divided by the total possible score.
+
+Factor Analysis indicated that two factors had a good level of internal consistency, being greater than 0.7 [47] (Factor 1, α = 0.849, Factor 3, α = 0.758). ‘Quidding’ (dropping food from the mouth whilst eating) had to be deleted from factor 2 to improve Cronbach’s α from 0.626 to 0.682. Factor 4 had the lowest measure of internal consistency, α = 0.551. For all the factors, the correlated item-total correlation was over 0.3, indicating that the items correlated adequately with the total score [45]. The final pattern matrix is illustrated in Table 1.
+
+Factor 1 was labelled ‘Musculoskeletal–neurological conditions’, factor 2 was labelled ‘Digestive conditions’, factor 3 was labelled ‘Metabolic conditions’, and factor 4 was labelled ‘Dermatological conditions’. Composite scores were created for each factor based on the mean of the corresponding items, with higher scores indicating a greater health deficit in that system (Table S2 in the Supplementary Information).
+
+A Spearman’s correlation coefficient test indicated that all mean factor scores and the OHS had a positive correlation with the CCAS score (all *p* < 0.001; Bonferroni adjusted alpha = 0.002 for multiple correlations). The correlation was strong (range r = 0.5–1.0) between musculoskeletal–neurological conditions and the CCAS score (r = 0.69), and between the OHS and the CCAS score (r = 0.71). There were moderate (range r = 0.3–0.49) positive correlations between digestive conditions and the CCAS score (r = 0.32) and between metabolic conditions and CCAS scores (rho = 0.36). The correlation was weak (range r = 0.1–0.29) between dermatological conditions and the CCAS (r = 0.27).
+
+An independent sample Kruskal–Wallis test indicated that all composite factor scores were significantly different between the CCAS categories overall. When looking at pairwise comparisons, the factor scores were, on average, significantly higher, comparing normal ageing with both mild and severe cognitive impairment (all *p* values < 0.001) (Supplementary Information, Table S3).
+
+When considering the veterinary diagnosis of musculoskeletal disease, Pearson’s chi-square test (Table 2) indicated a significant but small association between this and the CCAS category (χ^2^ (2) = 28.551, *p* < 0.001, Cramer’s V = 0.201, *p* < 0.001). For the remaining diagnoses, the variable ‘Presence of cognitive impairment’ was used (mild and severe cognitive impairment were considered together). There was a significant but small relationship between the presence of cognitive impairment and the diagnosis of dental disease (χ^2^ (1) = 18.886, *p* < 0.001, Cramer’s V = 0.163, *p* < 0.001). Specifically, the BCS was higher in the presence of signs of cognitive impairment (mean = 3.12, sd = 0.73, skew = −0.24), compared to normal cognition (mean = 3.12, sd = 0.58, skew = 0.13). Similarly, the dental disease score was higher in the presence of signs of cognitive impairment (mean = 0.4, sd = 0.49, skew = 0.4), compared to normal cognition (mean = 0.25, sd = 0.43, skew = 1.16). There was no significant relationship between the remaining diagnoses (gastrointestinal, dermatological, diabetes mellitus, hypothyroidism, hyperadrenocorticism, chronic kidney disease, epilepsy, hepatic disease, cardiovascular disease, and neoplasia) and the presence of cognitive impairment (all *p* = 0.002).
+
+Finally, the regression model (AIC = 4252.68, χ^2^ (5) = 515.57, *p* < 0.001, R~p~^2^ = 0.52) explained 52% of the variance in the CCAS score. Estimates indicated that higher scores in all four factors and in age predicted a higher CCAS score, i.e., more severe signs compatible with CDS (Table 3).
+
+## 4. Discussion
+
+An online survey was performed to investigate the relationship between canine cognitive impairment, as assessed by a validated questionnaire (CCAS) [25], and symptoms of the disease. Positive correlations were found between CCAS scores (a measure of cognitive impairment) and owner-observed behavioural indicators for health, a measure of body condition, and veterinary diagnoses of musculoskeletal and dental disease. A cumulative measure of health, the OHS (Overall Health Score), also had a strong, positive correlation with the CCAS score. These results support the hypotheses that (i) common pathological processes, in the form of factors, would link behavioural signs of disease and that these would be associated with greater cognitive impairment and (ii) that worsening overall health, assessed by the OHS, would be associated with greater cognitive impairment, as would the presence of specific disease states. This suggests that there might be parallelisms between some aspects of physical health and cognitive impairment.
+
+In the current study, the prevalence of owner-reported signs of moderate cognitive impairment in senior dogs was 43%, and the prevalence of severe cognitive impairment was 2%. The most striking finding of this study is that the cognitive status of the dogs (CCAS score) had a strong positive correlation with both the musculoskeletal–neurological factor and the overall health score (OHS) of the current survey. In particular, the musculoskeletal–neurological health was significantly better in the dogs ageing normally (CCAS—Normal Ageing), compared to those with mild and severe decline (CCAS—Mild cognitive impairment and Severe cognitive impairment), with no difference between the two groups of cognitive impairment. This suggests that there is a relationship between the presence of musculoskeletal–neurological conditions and the development of cognitive decline, but not with the severity of cognitive impairment. A diagnosis of musculoskeletal disease by a veterinarian in the preceding year was also found to be significantly associated with the CCAS category, further adding consistency to the findings. However, considering the relatively small number of dogs within the severe cognitive impairment group compared to the other groups, it cannot be excluded that this limited sample size could affect the statistical power to detect differences between the cognitively impaired groups. All of the composite factor scores and the overall health score (OHS) had significant positive correlations with the CCAS score, indicating that higher measures of disease were associated with greater scores on the measure of cognitive impairment. There was a strong correlation of both musculoskeletal–neurological conditions and OHS with cognitive impairment, compared to moderate and weak correlations for the other factors (digestive, metabolic, and dermatological conditions), suggesting that the measures of musculoskeletal and neurological disease might be of the greatest interest when looking further at the relationship between disease and cognitive decline. Age was also significantly positively correlated with the CCAS score, having a fair strength of association, and with the mean age of subjects in each CCAS category rising with increasing severity of cognitive impairment. Age is the main risk factor for CCDS [48], and a positive correlation between cognitive impairment and age mirrors the findings of other published measures of cognitive impairment [8,23].
+
+The associations identified between physical health and results from the CCAS have three potential mechanisms: (1) the disease process may represent a risk factor for the development of cognitive impairment due to its impact on the pathogenesis of CCDS; (2) both the physical health disorder and the cognitive decline may have a common risk factor, such as age, and as a consequence, individuals may be more likely to have both conditions but without any direct causal link between them; (3) the behaviours assessed on the CCAS are not specific to changes seen with cognitive impairment. It should also be noted that the factors identified following analysis of the GHQ describe underlying latent variables representing pathological processes, such as musculoskeletal disease. As the GHQ items consisted of owner-observed behaviours, disease processes resulting in behavioural change readily identifiable by owners would tend to result in factor formation, such as impaired mobility and house soiling [49,50,51].
+
+Additionally, a high prevalence of the underlying disease processes would increase the likelihood of that pathology being represented in the population sampled. It is likely that some pathology leads to discrete behavioural change that occurs independently of other symptoms (for example, brain pathology might only cause one owner-detected symptom, e.g., head tilt) and does not co-vary, meaning that these conditions would not be represented by the factors identified. The four most prevalent disorder groups in dogs [52] in the UK correspond to three of the factors identified in the current study: dental disease (14.10%) and enteropathy (10.43%), corresponding to the factor “digestive conditions”; skin disorders (12.58%) corresponding to the factor “dermatological conditions”; musculoskeletal disorders (8.64%), corresponding to the “musculoskeletal–neurological conditions” factor. This suggests that the behavioural changes identified in the GHQ items were symptomatic of disease states other than CCDS. However, it should be considered that this is a questionnaire-based piece of research, where owners were aware that the general interest was the welfare and cognition of older dogs. CCDS is a diagnosis of exclusion, and chronic pain diseases can mimic the clinical signs, so while the musculoskeletal–neurological diseases might have been actually occurring, owners could have been confounding the signs and giving dogs a higher score due to confirmation bias.
+
+The OHS had a strong, positive correlation with cognitive impairment (CCAS score). There are similar findings in the human literature, where the concept of frailty has been developed, describing a lack of resilience of an individual to disease processes [53]. Frailty indices are cumulative measures of health that are a means of representing this concept. Although the cumulative health measure in the current study was not a frailty index, which has to meet specific requirements, including a wide breadth of indicators [54], it is nevertheless of interest that the OHS was associated with the degree of cognitive impairment, paralleling findings in human medicine. There is epidemiological evidence in humans that increased frailty is related to greater cognitive impairment [55], potentially due to inflammaging [56] and changes in hormonal, vascular, and intracellular systems [55,57]. The upregulation of inflammation that occurs with increasing age is termed ‘inflammaging’ [56,58]. Frailty indices have been developed for use in dogs [35,59], where the inflammaging processes have also been hypothesised [60], and their relationship with cognitive impairment and CCDS would be an important area of future research. Three of the factors identified in this study are commonly associated with inflammatory processes; musculoskeletal–neurological, digestive, and dermatological conditions. Systemic inflammation has been postulated to mediate age-related cognitive decline in humans [61], with increased levels of inflammatory markers associated with higher measures of cognitive impairment [62,63,64]. In the current study, the digestive conditions factor consisted of two items (assistance feeding, decreased appetite) that could be interpreted as representing impairments in the ingestion of food, potentially associated with inflammation, and was moderately, positively correlated with cognitive impairment (CCAS score). An indication that oral health may be contributing to the digestive conditions factor was the finding that a veterinary diagnosis of dental disease was associated with the presence of cognitive decline, whereas there was no significant association between the diagnosis of gastrointestinal disease and the presence of cognitive impairment. The presence of periodontal pathogens has been found to be a risk factor for Alzheimer’s disease in people [65], and it is possible that this might be a mechanism by which, in this study, the digestive factor relates to cognitive decline. Both musculoskeletal–neurological and digestive conditions can also be associated with chronic pain [66], which could account for their correlation with cognitive impairment (CCAS scores) in the current study. Chronic pain is often maladaptive [67] and can persist after the initiating assault has resolved [68]. Pain has been recently found to be associated with behaviour problems in several cases in veterinary medicine [69,70,71,72]. It is often, though not always, associated with inflammation, and has been demonstrated to be linked with cognitive impairment in human patients [73].
+
+The factor relating to musculoskeletal–neurological conditions, which included items pertaining to sensory function, had a strong positive correlation with cognitive impairment. Human age-related hearing loss is significantly associated with both MCI and Alzheimer’s disease [74]. Hearing loss is a recognised risk factor for dementia [75], although it has been proposed that this relationship may be due to a common underlying pathology, which is yet to be elucidated [76]. Changes to olfaction and vision have also been associated with human cognitive decline, impairments that are related to an increased risk of cognitive impairment [77]. In dogs, sensory impairment has been found to be associated with age-related behavioural change [23], while associations between hearing loss and cognitive decline [78], and between hearing loss and cognitive decline [78], have also been found, highlighting the need for sensory testing prior to cognitive testing [26]. In addition, the presence of cataracts has been found to be a marker of reduced lifespan in dogs [79]. While the underlying mechanisms are yet to be fully understood, in relation to DISHAAL signs, it cannot be excluded that dogs with reduced vision and also hearing manifest disorientation, difficulties in navigating their known surroundings, and/or fearful or aggressive responses when approached suddenly.
+
+The metabolic conditions factor was positively moderately correlated with cognitive impairment (CCAS score). Metabolic diseases can lead to a wide range of behavioural changes, including lethargy, dullness, and house soiling [80,81], which can mimic symptoms of CCDS. Additionally, there could even be a causal link between metabolic conditions and cognitive impairment—there are parallels for this finding in human medicine. Diabetes mellitus is associated with an amnestic mild cognitive impairment through vascular disease mechanisms [82], and chronic kidney disease is also associated with cognitive impairment due to altered cytokine production [83]. This latter mechanism relates back to inflammatory processes and highlights how interconnected the body systems are, meaning that they cannot be considered in isolation. There is currently a lack of research on these mechanisms in the veterinary field, so there is no evidence of causative or even mediation effects, but nevertheless, these should be taken into consideration in clinical settings. In addition, being both underweight and overweight was significantly associated with the presence of cognitive impairment, although this association was small. Due to the low number of dogs with severe cognitive impairment, the two categories of cognitive impairment had to be merged to facilitate analysis. As a result, it is not possible to make considerations between the body condition score and the severity of cognitive decline. According to the human literature, the mechanism of this relationship may be a consequence of the chronic, systemic inflammation associated with obesity [84,85] and accompanying pain [86]. The nature of the association between inflammation and malnourishment is not entirely understood, even in humans, although it is linked as part of the malnutrition-inflammation complex syndrome [87].
+
+Finally, only a weak positive correlation was found between the dermatological conditions factor and cognitive impairment (CCAS score). The factor consisted of only two items (pruritus and licking), both of which are readily perceived by owners, which may have contributed to their forming a factor. The human literature reflects this finding, with a study based on care home residents not finding an association between skin disease and cognitive impairment [88]. When considering dermatological conditions, although pruritus and licking are well-accepted to be a response to skin-related discomfort due to dermatopathies [89], less well-investigated is the tendency for dogs to lick and chew at their own bodies in response to other sources of pain [71]. This latter connection is supported by the fact that the item ‘lameness’ had a relatively high cross-loading (below threshold) on the dermatological conditions factor, although it had a higher loading on musculoskeletal–neurological conditions factor, where it was finally included. This finding further supports the hypothesis that licking behaviour may be important clinically as an indicator of non-dermatological discomfort.
+
+Although the associations identified in the current study may be due to disease acting as a risk factor for cognitive decline, being a cross-sectional study, causality could not be determined between the variables studied. In addition, the regression analysis found a main effect of age alone on the CCAS score. Further large-scale, prospective, longitudinal studies are required to investigate the impact of co-variables (such as neuter status, age at neutering, exposure to environmental factors, and physical activity) on the relationship between physical health and cognitive decline and establish the nature of their interaction. There is high variability in the prevalence of CCDS reported in the literature [2,3,5,7,8,9], which may partly be due to differences in study designs and the scales used, but it is also possible that such differences could be indicative of other confounding factors such as undetected medical conditions (e.g., pain). Given the lack of specificity of behavioural change to underlying causes, it is possible that changes due to disease processes other than cognitive impairment, such as pain, could lead to higher scores on the CCAS. Alternatively, there may be many individual mechanisms by which disease states might lead to increased CCAS scores, with the lack of recognition of these associations potentially resulting in increased scores being mistakenly attributed to cognitive decline. As a consequence, if veterinarians suspect a patient may be suffering from CCDS, this study highlights the need to ensure that the patient is screened for other conditions, particularly those that are painful, that might be confusing the clinical picture.
+
+All in all, the interaction between physical health and cognitive decline is clearly a complex one with nuanced links in pathogenesis. The high prevalence of cognitive impairment in senior dogs highlights the importance of their assessment of the condition. Dogs should be regularly screened once they are considered mature (50–75% of their expected lifespan), facilitating early commencement of treatment and management strategies [1], which can help slow down the pathology and improve the quality of life for the dog and owner. Whilst the presence of medical conditions is useful in differentiating between normal ageing and the presence of cognitive impairment, it does not relate to the progression of cognitive impairment from mild to severe. The findings of the current study suggest that dogs who display behaviours suggestive of cognitive impairment should be screened for other medical diseases, especially musculoskeletal pathology, including the presence of pain and sensory decline.
+
+## 5. Conclusions
+
+In conclusion, this study demonstrated that owner-reported behavioural indicators of musculoskeletal, neurological, digestive, metabolic, and dermatological disease are all positively correlated with signs compatible with cognitive impairment. Similar findings have been found for a cumulative measure of health, being over- and under-weight, and having a current diagnosis of musculoskeletal or dental disease made by a veterinarian. Currently, the mechanism underlying these relationships is not determined and might include mediation and/or moderation effects, for example, disease states acting as risk factors for cognitive impairment, the behavioural signs of disease elevating scores on the CCAS, or other common mechanisms (e.g., inflammaging) underlying both. For veterinarians in practice, the findings draw attention to the importance of screening dogs with these health conditions for evidence of cognitive decline to enable early therapeutic intervention, and also to consider the impact of other disease states, particularly pain, on the interpretation of scales used as an aid in the diagnosis of CCDS.

fulltext/md/PMC10362839.md

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+---
+pmcid: "PMC10362839"
+pmid: "37483958"
+doi: "10.7717/peerj.15718"
+title: "Investigating the relationship between inbreeding and life expectancy in dogs: mongrels live longer than pure breeds."
+journal: "PeerJ"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Investigating the relationship between inbreeding and life expectancy in dogs: mongrels live longer than pure breeds
+
+## Abstract
+
+This study aimed to investigate the establishment of relationship between inbreeding and life expectancy in dogs. A dataset of *N* = 30,563 dogs sourced from the VetCompass™ Program, UK was made available by the Royal Veterinary College, University of London, containing information about breed and longevity and was subject to survival analysis. A Cox regression proportional hazards model was used to differentiate survivability in three groups of dogs (mongrel, cross-bred and pure breed). The model was found highly significant (*p* < 0.001) and we found that mongrel dog had the highest life expectancy, followed by cross-bred dogs with only one purebred ancestor and purebred dogs had the lowest life expectancy. A second Cox regression was also found highly significant (*p* < 0.001) differentiating the lifespan of different dog breed and correlating positively the hazard ratio and the Genetic Illness Severity Index for Dogs (GISID). The results show that survivability is higher in mongrel dogs followed by cross-bred with one of the ancestor only as a pure breed, and pure breed dog have the highest morbidity level. Higher morbidity is associated with higher GISID scores, and therefore, higher inbreeding coefficients. These findings have important implications for dog breeders, owners, and animal welfare organizations seeking to promote healthier, longer-lived dogs.
+
+## Introduction
+
+Dogs have been our faithful companions for thousands of years, their breeding and genetics have been the subject of growing concern in recent times. Dogs, the human’s best friends, have been domesticated from the wolf during the late Pleistocene more than 15,000 years ago (Horard-Herbin, Tresset & Vigne, 2014). Since the beginning of this process, we have understood that dogs could be useful to human activities in many ways, such as hunting, guarding, herding, waste disposal, warfare, entertainment, pest control, transport, clothing, and even food (Janssens et al., 2018). Having so many potential utilitarian roles led to specialization, and humans started to shape dogs accordingly. More recently, in the past 200 to 300 years shape standards were envisaged, and dog’s morphology was manipulated through closed population breeding (Axelsson et al., 2021). It was however, during the late 19th century, in Victorian Britain, that dog breeding standards started to be implemented, and the pedigree and pure blood concepts reached momentum (Worboys, Strange & Pemberton, 2018) by which modern dog had been ‘invented’.
+
+The modern dog has a multitude of shapes, sizes, colors, and hair types, but also behaviors and personalities, adapted to human needs. The closed population breeding of dogs is a modern practice aiming at the fixation of traits of interest (Axelsson et al., 2021). This bottlenecking of gene flow has also, however, undesirable consequences, as each individual carries deleterious genes with the potential to cause harm and affect fitness and health of dogs (Mabunda et al., 2022). Most of these genes are recessive and can affect the phenotype in homozygosity only. Inbreeding decreases the genetic load and creates in the descendants’ genome long homozygous regions, increasing the potential for deleterious genes to express themselves (Bosse et al., 2019).
+
+The deleterious defects in pure-breed dogs have long ago been identified, and the number of problems identified is growing. Hodgman (1963) identified thirteen conditions, having highlighted the most important being hip dysplasia, patella luxation, entropion, retinal atrophy, and the elongated soft palate. More recently Asher et al. (2009) and Summers et al. (2010), in two companion papers, identified almost 400. At present, the Online Mendelian Inheritance in Animals (OMIA) database, Nicholas & Tammen (2023) identifies 856 trait disorders in dogs.
+
+The lifespan of dogs in relation to breeds and types has been presented and discussed (Patronek, Waters & Glickman, 1997; Salvin et al., 2012) and very recently Teng et al. (2022) produced lifetables based on a large UK population above 30,000 dogs. The enormous diversity of dog breeds, ranging between the 1 kg Chihuahua and the 75 kg Saint Bernard, the 30 cm of the Dachshund and the 85 cm of the Great Dane, determines some variation in the different breeds lifespan (Fleming, Creevy & Promislow, 2011). Several factors of variation have been identified by the different epidemiological studies investigating lifespan in dogs: weight (Adams et al., 2010), neutering status (Moore et al., 2001), breed (Teng et al., 2022), and breed purity (Proschowsky, Rugbjerg & Ersbøll, 2003).
+
+Asher et al. (2009) and Summers et al. (2010) in their two companion papers introduced the Generic Illness Severity Index for Dogs (GISID), where the severity of disorders is sensibly scored. In addition, the authors provide an estimate of these scores for the most common breeds in the UK and also classify the different disorders (inherited defects) as conformation related (C), conformation exacerbated (CD), and not previously linked to conformation (D).
+
+The present study used the lifespan data reported by Teng et al. (2022) to relate with the GISID scores obtained by Asher et al. (2009) and Summers et al. (2010). To investigate the establishment of a relationship between inbreeding and life expectancy in dogs. Understanding of such relationship will be helpful in the scientific management of dog breeding for achieving aparent health and welfare of dogs.
+
+## Materials & Methods
+
+Data are open access (CC BY 4.0) and were retrieved from the Royal Veterinary College, University of London, repository (O’Neill, 2022). The sample includes all dogs under primary veterinary care at clinics participating in the VetCompass™ Program, UK during 2016 (*i.e.,* dogs with at least one clinical record in 2016) (Teng et al., 2022). The dog breeds recognized by any of the Kennel Club (KC), the American Kennel Club, and the Australian National Kennel Council were considered purebred, while all others were considered crosses. Crosses with ancestors not belonging to a recognized breed were considered mongrels. A total of *N* = 30,563 entries were found in the original dataset, which after data cleansing and elimination of outliers, resulted in a sample of *N* = 30,470 dogs (*n* = 2,406 mongrel, *n* = 3,962 crossbred, and *n* = 24,102 pure breed).
+
+Outliers were identified after data transformation for a standard normal distribution (a normal distribution with mean with value zero and standard deviation with value one), and when their z score was above 3 or below -3 standard deviations. Data entered a Cox-regression proportional hazards model for survival analysis with ‘Lifespan’ as the ‘time to event’ variable, and ‘Type of Dog’ (levels: ‘Mongrel’, ‘Cross Bred’, and ‘Pure Breed’) as the factor to analyze. There were no censored entries, and the ‘event’ is ‘Age at Death’ (years). An ANOVA with a least significant difference (LSD) test as *post hoc* was also used to differentiate means between ‘Type of Dog’.
+
+The different pure breed dogs were then compared with the inherited defects in pedigree dogs, namely the disorders related (Asher et al., 2009), and not related (Summers et al., 2010) to breed standards. At this stage, some breeds in the original dataset were eliminated from analysis once to compare with Asher et al. (2009) and Summers et al. (2010) as to consider the breeds reported by these authors. The new dataset comprises *N* = 19,466 dogs distributed within breeds as shown in Table 1.
+
+**Table 1 Dog breeds with number (*n*) used for Cox-regression proportional hazards model for survival analysis.**
+
+| Breed | *n* | Breed | *n* |
+| --- | --- | --- | --- |
+| Alaskan Malamute | 50 | Hungarian Vizsla | 44 |
+| Basset Hound | 76 | Irish Setter | 39 |
+| Beagle | 171 | Labrador | 2,501 |
+| Bichon Frise | 338 | Lhasa Apso | 282 |
+| Border Collie | 1,018 | Miniature Schnauzer | 211 |
+| Border Terrier | 294 | Poodle Miniature | 102 |
+| Boxer | 831 | Poodle Standard | 84 |
+| Bull Mastif | 143 | Poodle Toy | 56 |
+| Bull Terrier | 435 | Pug | 196 |
+| Bulldog | 501 | Rhodesian Ridgeback | 68 |
+| Cairn Terrier | 107 | Rottweiler | 505 |
+| Cavalier King Charles Spaniel | 1,004 | Scottish Terrier | 201 |
+| Chihuahua | 405 | Shar Pei | 149 |
+| Cocker Spaniel | 1,063 | Shetland Sheep Dog | 61 |
+| Collie (Rough) | 84 | Shih Tzu | 635 |
+| Dalmatian | 149 | Springer Spaniel | 800 |
+| Doberman | 148 | Staffordshire Bull Terrier | 2,344 |
+| Dogue de Bordeaux | 152 | Tibetan Terrier | 71 |
+| German Shepherd | 1,096 | Weimaraner | 129 |
+| German Short-haired Pointer | 41 | West Highland Terrier | 1,103 |
+| Golden Retriever | 511 | Whippet | 125 |
+| Great Dane | 83 | Yorkshire Terrier | 1,060 |
+
+The number of identified disorders in the different breeds (inherited defects), were divided by the average scores obtained by the application of the GISID (Asher et al., 2009; Summers et al., 2010) for C, CD, D, and Total, to obtain the variables ‘Rate C’, ‘Rate D’, ‘Rate CD’ and ‘Rate Total’ respectively. These new variables giving an average score per disorder entered a Cox-regression proportional hazards model for survival analysis with ‘Lifespan’ as the ‘time to event’ variable, ‘Breed’ as a factor, and the ‘Rate’ variables as covariates. Again, there were no censored entries, and the event considered is ‘Age at Death’ (years).
+
+The models were tested *via* the −2 Log likelihood test and their parameters *via*, the Wald test. A cumulative survival plot was also produced for the first model.
+
+Data were initially entered in a spreadsheet (Microsoft Excel for Microsoft 365 MSO, version 2204 Build 16.0.15128.20240, 64-bit) for cleansing and outlier detection and elimination. Descriptive statistics were also produced with this software. The Cox regression proportional hazards models were produced with the statistical package IBM Corp. SPSS Statistics, Armonk, NY, USA. Version: 28.0.1.1 (15).
+
+## Results
+
+The descriptive statistics of the variables entered in the first model are shown in Table 2. The ANOVA test was highly significant (*F* = 211.84, 2df, *p* < 0.001) and all the means were significantly different (*p* = 0.032) between ‘Pure Breed’ and ‘Cross Bred’, and *p* < 0.001 for the other comparisons. The ANOVA table is presented as Table 3.
+
+**Table 2 Descriptive statistics of the lifespan of the three types of dogs (mongrel, cross-bred, and pure breed).**
+
+| Statistic | Mongrel | Cross bred | Pure breed |
+| --- | --- | --- | --- |
+| Observations | 2,406 | 3,962 | 24,102 |
+| Minimum | 0.008 | 0.003 | 0.003 |
+| 1st Quartile | 11.006 | 8.909 | 9.126 |
+| Median | 13.569 | 12.049 | 11.723 |
+| 3rd Quartile | 15.198 | 14.350 | 13.722 |
+| Maximum | 20.561 | 21.884 | 21.539 |
+| Mean | 12.761 | 11.225 | 11.086 |
+| Standard deviation | 3.762 | 4.276 | 3.729 |
+| Mean CI (95% upper) | 12.838 | 11.293 | 11.110 |
+| Mean CI (95% lower) | 12.684 | 11.158 | 11.062 |
+
+**Table 3 Analysis of variance (ANOVA) used to test significant differences of lifespan between types of dog (mongrel, cross-bred, cross breed).**
+
+| Source | Type III sum of squares | df | Mean square | *F* | Sig. |
+| --- | --- | --- | --- | --- | --- |
+| Corrected Model | 6,140.12 | 2 | 3,070.06 | 211.84 | <0.001 |
+| Intercept | 1,733,638.69 | 1 | 1,733,638.69 | 119,626.32 | <0.001 |
+| Dog group | 6,140.12 | 2 | 3,070.06 | 211.84 | <0.001 |
+| Error | 441,531.33 | 30,467 | 14.49 |  |  |
+| Total | 4,294,503.46 | 30,470 |  |  |  |
+| Corrected Total | 447,671.45 | 30,469 |  |  |  |
+
+The first model differentiating mongrel, cross bred and pure breed dogs was found to be highly significant (-2 Log Likelihood 567,623, *χ*^2^ = 595, 2 df, *p* < 0.001) and the parameter was also significant (Wald 585, with 2 degrees of freedom, *p* < 0.01). The full model parameterization is shown in Table 4.
+
+**Table 4 Cox proportional hazards model, modelling the survivability of three types of dogs (mongrel, cross-bred, and pure breed).**
+
+|  | *β* | Wald | df | *p*-value | *e*
+^*β*^ | *e*^*β*^ 95% CI |
+| --- | --- | --- | --- | --- | --- | --- |
+|  |  |  |  |  |  | Lower | Upper |
+| Cross | −0.177 | 105.474 | 1 | <.001 | 0.838 | 0.810 | 0.867 |
+| Mongrel | −0.494 | 527.621 | 1 | <.001 | 0.610 | 0.585 | 0.637 |
+
+In a Cox proportional hazards model, a negative parameter is indicative of a decrease in the hazard ratio (HR) and an increase in survival in relation to the baseline. The exponential of the parameter (*e*^*β*^) indicates the HR effect size and is interpreted as multiplicative effects on the hazard. Therefore, a cross bred dog has a negative *β*, indicative of a decrease in the HR in relation to a ‘Pure Breed’ of *e*^*β*^ = 0.838 or 17.2%. ‘Mongrel’ dogs also have lower HR in relation to pure breed and the effect size is larger when compared to ‘Cross Bred’. The decrease in the HR in mongrel dogs is 39%. These effects are evident in the survival functions plotted in Fig. 1.
+
+*Figure: Figure 1 Representation of the survival functions of the Cox-regression proportional hazards model, modeling the survivability of three ‘Types of dog’ (mongrel, cross-bred, and pure breed).*
+
+The second model differentiating dog breeds and using the ‘Rates’ calculated from the GISID was also found to be significant (-2 Log Likelihood 335,993, *χ*^2^ = 5,013 with 43 df, *p* < 0.001). The variable ‘Breed’ is significant (Wald 4314, with 42 df, *p* < 0.01), as it is the covariate ‘Total Ratio’ (Wald 64.58, with 1 df, *p* < 0.001). The full model parameterization is shown in Table 5.
+
+**Table 5 Cox-regression proportional hazards model, modeling the survivability of different breeds of dogs with ‘Rate Total’ as a covariate.**
+
+|  | *β* | Wald | df | *p*-value | Exp(*β*) | Exp (*β*) 95% CI |
+| --- | --- | --- | --- | --- | --- | --- |
+|  |  |  |  |  |  | Lower | Upper |
+| Covariate Rate Total | 0.162 | 64.58 | 1 | <0.001 | 1.175 | 1.130 | 1.223 |
+| Breed Dogue de Bordeaux | −12.105 | 44.271 | 1 | <0.001 | 6^−6^ | 1.565^−7^ | 1.960^−4^ |
+| Whippet | −2.419 | 39.009 | 1 | <0.001 | 0.089 | 0.042 | 0.190 |
+| Rhodesian Ridgeback | −1.941 | 24.433 | 1 | <0.001 | 0.144 | 0.067 | 0.310 |
+| Staffordshire Bull Terrier | −1.806 | 40.143 | 1 | <0.001 | 0.164 | 0.094 | 0.287 |
+| Tibetan Terrier | 0.483 | 13.633 | 1 | <0.001 | 1.621 | 1.255 | 2.096 |
+| Bull Mastiff | 0.795 | 20.258 | 1 | <0.001 | 2.214 | 1.566 | 3.129 |
+| Shetland Sheepdog | 1.546 | 61.335 | 1 | <0.001 | 4.691 | 3.186 | 6.907 |
+| Pug | 1.599 | 411.244 | 1 | <0.001 | 4.950 | 4.241 | 5.777 |
+| Bull Terrier | 1.629 | 102.357 | 1 | <0.001 | 5.101 | 3.720 | 6.995 |
+| Poodle Miniature | 1.666 | 38.626 | 1 | <0.001 | 5.292 | 3.129 | 8.950 |
+| Poodle Toy | 1.798 | 41.540 | 1 | <0.001 | 6.037 | 3.494 | 10.429 |
+| Labrador | 1.874 | 57.486 | 1 | <0.001 | 6.514 | 4.013 | 10.575 |
+| West Highland Terrier | 1.983 | 68.114 | 1 | <0.001 | 7.265 | 4.536 | 11.634 |
+| Irish Setter | 2.103 | 78.054 | 1 | <0.001 | 8.187 | 5.135 | 13.053 |
+| Basset Hound | 2.107 | 127.733 | 1 | <0.001 | 8.225 | 5.707 | 11.853 |
+| Cavalier King Chrles Spaniel | 2.205 | 204.593 | 1 | <0.001 | 9.071 | 6.705 | 12.270 |
+| German Shorted-Hair Pointer | 2.210 | 65.239 | 1 | <0.001 | 9.119 | 5.333 | 15.592 |
+| Chihuahua | 2.286 | 182.833 | 1 | <0.001 | 9.832 | 7.059 | 13.693 |
+| Dalmatian | 2.329 | 99.287 | 1 | <0.001 | 10.270 | 6.495 | 16.239 |
+| Springer Spaniel | 2.423 | 85.607 | 1 | <0.001 | 11.278 | 6.750 | 18.841 |
+| Collie (Rough) | 2.431 | 82.005 | 1 | <0.001 | 11.376 | 6.721 | 19.255 |
+| Hungarian Vizsla | 2.444 | 82.177 | 1 | <0.001 | 11.519 | 6.791 | 19.539 |
+| Cairn Terrier | 2.474 | 63.367 | 1 | <0.001 | 11.865 | 6.453 | 21.815 |
+| Border Collie | 2.512 | 73.124 | 1 | <0.001 | 12.334 | 6.935 | 21.938 |
+| Shih Tzu | 2.548 | 83.657 | 1 | <0.001 | 12.779 | 7.402 | 22.059 |
+| Poodle Standard | 2.642 | 68.117 | 1 | <0.001 | 14.036 | 7.496 | 26.285 |
+| Bichon Frise | 2.706 | 92.274 | 1 | <0.001 | 14.963 | 8.615 | 25.987 |
+| German Shepard | 2.746 | 156.512 | 1 | <0.001 | 15.582 | 10.134 | 23.959 |
+| Shar Pei | 2.813 | 349.604 | 1 | <0.001 | 16.662 | 12.407 | 22.377 |
+| Alaskan Malamute | 3.012 | 132.368 | 1 | <0.001 | 20.334 | 12.172 | 33.969 |
+| Cocker Spaniel | 3.030 | 96.623 | 1 | <0.001 | 20.698 | 11.312 | 37.871 |
+| Scottish Terrier | 3.037 | 77.295 | 1 | <0.001 | 20.845 | 10.591 | 41.024 |
+| Lhasa Apso | 3.346 | 89.761 | 1 | <0.001 | 28.381 | 14.205 | 56.705 |
+| Beagle | 3.356 | 112.241 | 1 | <0.001 | 28.661 | 15.406 | 53.320 |
+| Golden Retriever | 3.488 | 83.652 | 1 | <0.001 | 32.730 | 15.499 | 69.118 |
+| Miniature Schnauzer | 3.747 | 87.736 | 1 | <0.001 | 42.381 | 19.351 | 92.823 |
+| Great Dane | 3.912 | 235.326 | 1 | <0.001 | 50.010 | 30.337 | 82.440 |
+| Doberman | 3.935 | 162.363 | 1 | <0.001 | 51.146 | 27.923 | 93.682 |
+| Boxer | 3.983 | 130.334 | 1 | <0.001 | 53.691 | 27.096 | 106.387 |
+| Weimaraner | 4.088 | 99.726 | 1 | <0.001 | 59.640 | 26.734 | 133.048 |
+| Rottweiler | 4.247 | 151.288 | 1 | <0.001 | 69.930 | 35.540 | 137.596 |
+| Bulldog | 4.446 | 201.831 | 1 | <0.001 | 85.273 | 46.178 | 157.466 |
+
+In the second model, the lower the parameter associated with the breed, the higher the HR. Therefore, the breed Dogue de Bordeaux is the breed with the lower HR and higher survivability, while the bulldog has the higher HR and the lower survivability. The breed Yorkshire Terrier is the baseline, therefore negative parameters are associated with lower HR, and positive parameters with higher HR, in relation to this breed. As the model includes forty three breeds any plot of the survival function becomes impossible to read, therefore it is not presented for this model.
+
+The covariate ‘Total Ratio’ indicates that for an additional unit in the ratio, the HR is added by a factor of 17.5% while holding age constant. In other words, the higher the GISID, the higher the death hazard, therefore the lower the survivability. A negative correlation between GISID and survivability becomes, therefore, established within this model.
+
+## Discussion
+
+An increase in dog’s inbreeding coefficients is associated with a lower lifespan. In decreasing order, dogs with longer lifespan are ‘Mongrel’ (mean 12.761 years), followed by ‘Cross Bred’ (11.225 years), and ‘Pure Bred’ (11.086 years). These results were reiterated by the Cox-regression, with a decrease in the HR in relation to pure breed dogs of 17.2% and 39% respectively for cross-bred and mongrel dogs.
+
+Some previous studies have reported this relationship. In a study based on questionnaires filled by members of the Danish Kennel Club and representing a sample of 2,928 dogs, Proschowsky, Rugbjerg & Ersbøll (2003) reported differences between mixed breed (median and interquartile range (IQR) 11 [8, 13]) and several breeds with median varying between 7 and 11. These results were subject to a Kruskal-Wallis test and significant differences were found, however, no *post hoc* tests are reported and, therefore, the statement that differences between pure-breed and cross-bred dogs exist is not robust. Also, a study in Britain (Michell, 1999) using questionnaires, reports that mongrel dogs are between those with larger lifespans (median 14.0). This study, however, does not provide any inferential statistical test and anchors the statement in descriptive statistics only. Patronek, Waters & Glickman (1997) report statistical evidence of significant differences between mixed breeds and pure breeds (medians 8.5 and 6.7 years, respectively), using a large sample (23,535 dogs) of data collected in veterinary hospitals in the USA and Canada. Despite the high credibility of the study, there is no definition of what a mixed breed is; it could eventually be a mongrel, a cross between two pure breeds, or a cross between a mongrel and a pure breed.
+
+Inbreeding is known by agglomerating homozygotic recessive genes in the genome of individuals (Mooney, Yohannes & Lohmueller, 2021), and particularly in pure-breed dogs, it has been shown that higher inbreeding coefficients are associated with higher morbidity (Bannasch et al., 2021; Yordy et al., 2020), due to a larger accumulation of deleterious genes associated with the most common disorders (O’Neill et al., 2014).
+
+The novelty in the present study is the clear differentiation between groups that are not pure breeds. This differentiation allows the definition of three groups with expected different inbreeding coefficients and therefore allows the clear establishment of a relationship between expected inbreeding coefficients and lifespan. Being pure-breed dogs bred in a closed population, they have higher inbreeding coefficients than mongrels; and cross-bred dogs with no more than one pure breed ancestor have, obviously, intermediate inbreeding coefficients. The present study reports results based on a continuous variable allowing the comparison and the report of significantly different means in the three groups of dogs. Previous studies report median values only.
+
+The lifespan differences between breeds have been the objective of study and are well documented, *e.g.*, Bannasch et al. (2021), O’Neill et al. (2013), Teng et al. (2022) and Yordy et al. (2020). The common report in lifespan differences between dog breeds is the size or weight of the breed. Larger dogs are reported to have higher morbidity, *e.g.*, Bannasch et al. (2021), Greer, Canterberry & Murphy (2007), Michell (1999) and O’Neill et al. (2013). This trend is not evident in the present study.
+
+The limitations of this study are associated with the sample. The sample is based on dogs that attended veterinary practices in the UK, and for which both born, and death dates are known. The generalization of the results for other geographies must be done carefully. Also, not all dogs in the UK have records in veterinary practices and many that have records eventually don’t show born and/or death dates. The sample may, therefore, be somehow biased.
+
+Nevertheless, ethical dog breeding must be implemented with respect for dog welfare. Broeckx (2020) revised and elected two points of action: reduce the frequency of disorders and increase genetic diversity. For example in a study (Douglas, Mata & Menem, 2015) of policy comparison between the British Kennel Clube (BKC) and the German Kennel Club, Verband für das Deutsche Hundewesen (VDH) it was demonstrated that it is possible to tackle some of the most common problems faced by pure breeding by reducing the frequency of the disorder. Canine hip scoring can effectively be used in selection programs to reduce the incidence of hip dysplasia. By adopting VDH’s system of mandatory hip scoring of breeding parents and only allowing those with low scores to breed, it was demonstrated that it is possible to reduce faster, hip scores in populations of pedigree dogs when compared with the BKC’s voluntary system.
+
+## Conclusions
+
+It was hypothesized that morbidity in dogs may be associated with inbreeding coefficients. The results of this study confirm the hypothesis, showing that survivability is higher in mongrel dogs followed by cross-bred with one of the ancestor only as a pure breed, and for the last pure breed dogs have the highest morbidity levels. Higher morbidity was associated with higher GISID scores, and therefore, higher levels of homozygotic recessive genes in the genomes of the individuals, and inbreeding coefficients should be reduced for better survivability. Future research may be directed to dog breeding, to decrease inbreeding coefficients and control deleterious genes.

fulltext/md/PMC10383353.md

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+---
+pmcid: "PMC10383353"
+pmid: "37505862"
+doi: "10.3390/vetsci10070457"
+title: "Serum Ferritin in Obese Dogs: Changes and Comparison with Other Analytes."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Serum Ferritin in Obese Dogs: Changes and Comparison with Other Analytes
+
+## Abstract
+
+### Simple Summary
+
+Canine obesity, defined as a weight greater than 15% of the ideal, is associated with decreased quality of life, longevity, and other complications. However, the mechanisms causing these alterations are not fully understood. In obese humans, increased concentrations of ferritin in serum have been found; however, this has been poorly explored in dogs. In this study, ferritin levels from lean/normal weight (CG, *n* = 55) and overweight/obese dogs (OG, *n* = 37) were evaluated, together with complete hemogram and biochemical analyses. Higher ferritin concentrations were observed in the OG group in comparison to the CG. The increases in ferritin, together with higher haematocrit and erythrocyte mean corpuscular volume, could indicate tissue hypoxia in obese dogs.
+
+### Abstract
+
+Canine obesity is the most common nutritional disorder and is associated with decreased quality of life and longevity as well as comorbidities including cardiorespiratory, endocrine, oncologic, or orthopaedic disorders. Ferritin is a major acute-phase protein in dogs, increasing during inflammation; however, it could also be affected by other conditions, including trauma, iron metabolism dysregulations, neoplasia, or hypoxia. Higher ferritin levels have been reported in obese humans, but ferritin has not been explored in canine obesity. To evaluate the possible changes in serum ferritin in canine obesity, ferritin levels from lean/normal weight (CG, *n* = 55) and overweight/obese dogs (OG, *n* = 37) were measured, together with complete hemogram and biochemical analyses. Statistically significant higher ferritin levels (1.2-fold) were found in OG (median, (interquartile range), 204 (166–227.5) µg/L) in comparison to CG animals (172 (137–210) µg/L)), with median levels of ferritin in OG dogs above the reference range for healthy animals in our laboratory (60–190 µg/L). In addition, statistically significant higher mean corpuscular volume (MCV), mean cell haemoglobin concentration (MCHC), total proteins, globulins, haptoglobin, total ferric fixation capacity (TIBC), alkaline phosphatase (ALP), butyrylcholinesterase (BChE), triglycerides, and calcium were observed in OG in comparison to CG. The higher levels in ferritin, together with higher TBIC, haematocrit, and MCV, could indicate tissue hypoxia in obese dogs.
+
+## 1. Introduction
+
+Canine obesity is the most common nutritional disorder [1] characterized by the accumulation of excess adipose tissue in the body and defined in dogs as weight greater than 15% of the ideal. Overweight and obese dogs can reach a prevalence higher than 50% of the population in some places [2,3,4]. Canine obesity is associated with decreased quality of life and longevity and is related to the development of comorbidities such as cardiorespiratory, endocrine, oncologic, or orthopaedic disorders [5,6]. However, although dogs with obesity present insulin resistance, increased blood lipids, altered levels of adipokines, and oxidative stress markers in comparison to control ones, the pathophysiology of canine obesity is still unclear [7].
+
+Ferritin is a protein that stores iron within cells and facilitates its transport between tissues. Circulating levels of ferritin reflect that iron storage levels are being affected by iron metabolism dysregulations [8,9]. Furthermore, ferritin circulating concentrations are increased during inflammatory response [10], due to traumatism which leads to cellular damage and rupture [11], neoplasia [12], or hypoxia [13], among others. In humans, increased ferritin levels cluster with obesity [14] and with a higher risk of metabolic syndrome [15,16,17,18] and its components, such as dyslipidaemia [19], high blood pressure [20], and type 2 diabetes [15,21]. However, to the best of the authors’ knowledge, the relationship between serum ferritin levels and canine obesity is still not elucidated, except for a single article [22] that reported higher, although not statistically relevant, serum ferritin in obese dogs with obesity-related metabolic dysfunction (ORMD) in comparison to non-ORMD obese dogs.
+
+The aim of this study was to evaluate the possible changes in serum ferritin in canine obesity. In addition, a complete blood count and serum biochemistry, including a panel of acute-phase proteins (APPs) with a major (CRP), a moderate (haptoglobin), and negative (albumin, PON-1) APPs, were measured for comparative purposes and to gain information that elucidates possible changes in ferritin levels. Besides the common haematological and biochemical panel, other analytes that could provide additional information were also evaluated. Therefore, iron metabolism was assessed by the measurement of iron, ferritin, total iron-binding capacity (TIBC), unsaturated iron-binding capacity (UIBC), and ferritin saturation. TIBC measures the maximum amount of iron that can be bound to transferrin. UIBC indicates the amount of transferrin available to bind more iron, and ferritin saturation represents the percentage of transferrin that is currently bound to iron. In addition, paraoxonase-1 (PON-1) and butyrylcholinesterase (BChE) were measured. PON-1 is an enzyme with esterase activity that is physically bound to high-density lipoprotein (HDL), and that decreases in inflammatory processes, intestinal tract disorders, or human obesity [23,24,25]. BChE increases significantly in overweight/obese dogs [26].
+
+## 2. Materials and Methods
+
+### 2.1. Animals and Samples
+
+The study was a retrospective study approved by the University of Murcia and the Murcia Region Ethical Committee (Number, A13170503; Date, 27 September 2022).
+
+Serum surplus from lean, normal weight, overweight, and obese client-owned dogs presented at veterinary clinics of the Murcia Region for routine check-ups or vaccinations and submitted to the Clinical Pathology Laboratory Interlab-UMU for CBC and serum biochemistry between January 2020 and January 2022 were assessed for inclusion in the study.
+
+Inclusion criteria for the overweight/obese group (OG) were being an overweight or obese but otherwise healthy adult dog (body condition score (BCS) between 6 and 9 on a 9-point body condition scale chart (BCS 6–9/9) [27] with a history of more than one year, according to clinical examination, CBC, and biochemical analysis. None of the dogs of the OG presented ORMD, according to previous literature [28]. The lean/normal-weight group (control group, CG) was selected to match the OG group in terms of age, breed, and size. In addition, they had to fulfil the following inclusion criteria: to be a slightly lean or normal weight (BCS 4–5/9) adult and present no abnormalities upon clinical examination, CBC, and biochemical analysis.
+
+All animals had a negative serological titter for *Leishmania infantum* and *Ehrlichia canis*, as demonstrated by commercial tests (Canine SNAP tests, IDEXX Europe B.V., Hoofddorp, The Netherlands).
+
+### 2.2. Analysis
+
+Ferritin was measured using commercially available reagents (Olympus, Beckman Coulter, Fullerton, CA, USA) following the manufacturers’ instructions using an automated analyser (Olympus AU600, Beckman Coulter).
+
+Complete blood count analyses were performed. The red fraction was analysed for red blood cells (RBC); haematocrit; haemoglobin; mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean cell haemoglobin concentration calculated from the optically measured cellular haemoglobin (MCHC); and red cell distribution width (RDW). The white blood fraction was analysed for white blood cell count (WBC) and segmented lymphocyte, monocyte, eosinophil, and basophil counts. Platelets were assessed for platelet count (platelets); mean platelet volume (MPV); platelet crit (PCT); mean platelet component concentration (MPC); and platelet component concentration distribution width (PCDW). All analyses were performed using an automated analyser (ADVIA 120 haematology Analyzer; Siemens Healthcare GmbH; Erlangen; Germany).
+
+The following parameters were measured for serum biochemistry using commercially available reagents (Olympus, Beckman Coulter) and following the manufacturer’s instructions: total proteins, albumin, C-reactive protein (CRP), creatinine kinase (CK), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamyl aminotransferase (gGt), total bilirubin, total cholesterol, triglycerides, amylase, urea, creatinine, glucose, calcium, and phosphorus. Globulin concentration was calculated by subtracting the concentration of albumin from the total protein concentration. Serum butyrylcholinesterase (BChE) activity was measured using butyryl thiocholine iodide as a substrate [26]. Serum paraoxonase-1 (PON-1) activity was determined using p-nitrophenyl acetate [29]. Total antioxidant capacity (TAC) and total oxidative status (TOS) were measured using colorimetric assays [30,31,32]. Haptoglobin concentration was measured using a commercially available method (Tridelta Phase Range Haptoglobin Kit; Tridelta Development [33]. All analyses were performed in an automated analyser (Olympus AU600, Beckman Coulter).
+
+### 2.3. Statistical Analysis
+
+The normality of the data’s distribution was assessed for each biomarker using the D’Agostino and Pearson omnibus normality test. Then, since most analytes presented a non-Gaussian distribution, Mann–Whitney U tests were performed to evaluate possible differences between the two groups. A non-parametric Spearman correlation test was used to assess possible correlations between ferritin and the other parameters. Values of *P* < 0.05 were considered as statistically significant. Analyses were performed using the statistical software GraphPad Prism 8.
+
+## 3. Results
+
+The study included a total of 92 dogs. The baseline characteristics of the animals involved in the study are presented in Table 1. There were no statistically significant differences between the groups in terms of sex, age, tartar, gingival inflammation, feed type, meals per day, or whether they were receiving supplements or snacks. However, the OG had a higher percentage of sterilised dogs (73% versus 40%), body weight, and BCS than CG (*P* < 0.05 in all cases). Data in bold highlight statistical significance.
+
+Ferritin was 1.2-fold higher in obese dogs (median, (25–75th interquartile range), 204 (166–227.5) µg/L) in comparison to lean/normal weight animals (172 (137–210) µg/L)) (*P* < 0.05), with median levels of ferritin in OG dogs above the reference range for healthy animals in our laboratory (60–190 µg/L) (Figure 1).
+
+The haematological analyses revealed that dogs from OG had 1.02-fold higher MCV values but 1.03-fold lower MCHC values than lean/normal-weight dogs (Table 2). The biochemical analysis (Table 3) also showed differences between the two groups. OG dogs had 1.2-fold higher levels of haptoglobin, TIBC, and BChE; 1.6-fold higher triglycerides; 1.4-fold higher ALP; and 1.1-fold higher total proteins, albumin, globulins, and calcium in comparison to CG. Haptoglobin, TIBC, and BChE medians were above the reference range of the laboratory in OG.
+
+Correlations between ferritin and the rest of the analytes were calculated for all dogs and for each group separately (CG and OG) and are represented in Table 4. When data from all animals were pooled, a statistically significant correlation was observed between ferritin and AST and gGT. In CG, a positive statistically significant correlation was found between ferritin and MCHC and a negative one between ferritin and gGT. In the OG group, ferritin was positively significantly correlated with MCV and MCH and negatively with RDW, WBC, and eosinophils.
+
+## 4. Discussion
+
+In this study, the effect of obesity on serum concentrations of ferritin was investigated. In addition, a CBC and serum biochemistry that included a panel of acute phase proteins were performed with the aim of providing new insights into canine obesity pathology.
+
+Serum ferritin levels were statistically higher in OG in this report; this agrees with human studies that described an increase in ferritin in obese individuals [18,19] and suggested that this increase is due to different pathways that could be related to inflammation and hypoxia [34,35].
+
+Regarding the possible causes for increased ferritin in OG in our study, there are some that could be ruled out based on the CBC and biochemistry results. Serum iron levels, haemoglobin, UIBC, and ferric saturation did not show increases in the obese dogs of our report; therefore, iron metabolism dysregulation could be ruled out as a possible reason for altered ferritin levels in OG. In a similar manner, it is unlikely that those overweight/obese animals presented muscular damage since the levels of AST and CK were within the reference interval and did not differ significantly from CG. Hepatic enzymes (ALT, AST, and ALP), bilirubin, and hepatic proteins (globulins and albumin) did not show alterations of interest, thus exhibiting adequate hepatic function. None of the dogs were reported to have had recent trauma, and no evidence of neoplasia or immunological disorders was observed in the physical examination, CBC, or biochemistry analysis.
+
+In addition, the presence of active inflammation as a cause of increased ferritin could be discarded based on our results on APPs. Ferritin is a positive moderate APP in dogs [36]. However, there is controversy regarding the presence of inflammation associated with canine obesity. Obesity can cause inflammation through different pathways [34,35,37,38]. The excessive adipose tissue increases the secretion of pro-inflammatory cytokines and APPs, thus promoting a chronic and mild inflammatory state. Additionally, changes in the intestinal microbiome promote the exacerbation of certain bacterial species that produce proinflammatory metabolites. In dogs, there are limited and contradictory data regarding alterations in APPs in canine obesity. One study links obesity with increased levels of CRP, plasma tumour necrosis factor-alpha, and haptoglobin, with a reduction in these parameters after weight loss [39], while others report decreased CRP [40] or no difference in APPs [28,41,42]. In our study, CRP and PON-1 were unaltered, while haptoglobin and albumin were higher in OG. Interestingly, this profile was described in dogs diagnosed with a situation of hypercortisolism such as hyperadrenocorticism [43,44,45]. These studies reported increases in haptoglobin but no significant changes in CRP or albumin in dogs with uncomplicated hyperadrenocorticism in comparison to healthy dogs [43]; in addition, haptoglobin was reduced after hyperadrenocorticism control [45]. Furthermore, OG dogs in our study also presented higher ALP, triglycerides, albumin BChE, and lower lymphocytes (presenting a stress leukogram), thus further supporting the presence of hypercortisolism in dogs with obesity, as described in human obesity [46]. Nevertheless, the profile of APPs highlights that OG dogs did not show signs of active inflammation, which could explain the higher levels of serum ferritin observed in these dogs in comparison to CG ones.
+
+Another possible cause of the increase in serum ferritin could be the presence of tissular hypoxia. In these dogs, the TBIC, which is the maximum ferric concentration fixable by serum proteins, mainly transferrin, was also found to be increased. Previous studies had described elevated haemoglobin, ferritin and erythropoietin concentrations in humans with metabolic syndrome [38], and studies with obese and overweight dogs also support the possible existence of tissular hypoxia in obese dogs. A previous report pointed out a downregulation of haemoglobin subunits alpha- and beta-like genes in obese dogs [7]. Also, a decrease in arterial oxygen partial pressure and inspired oxygen fraction in obese sedated dogs, in comparison to the same animals after a weight loss program, has been described [47]. Therefore, based on our results and previous literature, we hypothesise that OG dogs may present tissue hypoxia, as described for humans, and this condition could have influenced the increase in ferritin serum levels; however, further studies would be needed to confirm this hypothesis.
+
+This study presents some limitations. The use of client-owned dogs increases the variability in terms of breed, feed type and frequency, exercise, and age of onset of obesity, among other factors which may have influenced our results and should be further evaluated. In our study, the OG presented a higher rate of sterilization than the CG. However, this represents the true clinical picture. Second, in this study, data from obese dogs without ORMD were included. In the only study where serum ferritin levels were evaluated in canine obesity by comparing samples obtained from obese dogs with and without ORMD, the ORMD group dogs showed 1.2-fold higher circulating ferritin levels than obese non-ORMD, although this difference was not statistically relevant [22]. Although in our study, non-ORMD OG dogs presented slightly higher ferritin than that reported in the previous study (204 (166–227.5 µg/L) in our study versus 184.3 (145.4–247.1) µg/L in [22]), ferritin levels in non-ORMD are higher than in the normal weight and lower than in the ORMD dogs. Further analyses would be required to determine the possible causes of these differences in ferritin values in non-ORMD obese dogs between both studies, such as the possible presence of dogs that are closer to meeting ORMD diagnosis criteria. In any case, based on the results from both studies, ferritin is lower in lean/normal weight that in overweight/obese dogs without ORMD and obese dogs presenting ORMD, although the differences between the obese dogs with and without ORMD are of a lower magnitude. Finally, since punctual measurements of cortisol in the serum of dogs present high inter- and intra-individual variability, with one of the main reasons being the circadian fluctuations of this analyte [48,49]; it was not used in this study to confirm our hypothesis of hypercortisolism [46]. Other recommended alternatives, such as the ACTH stimulation test, the dexamethasone suppression test, or cortisol in 24 h urine, were not performed due to the retrospective nature of this work, but they could help to clarify the presence of a hypercortisolism state in overweight/obese dogs in future studies.
+
+## 5. Conclusions
+
+In the population of obese dogs of our study, there was an increase in serum ferritin associated with increases in haematocrit, TBIC, and MCV, which could be indicative of tissue hypoxia. In addition, a profile of APPs consisting of normal CRP, increased Hp, BChE, and ferritin is associated with canine obesity and could suggest the presence of a hypercortisolism state in this disease. Further studies should be performed to elucidate the possible application of ferritin alone or in combination with other APPs as biomarkers of obesity in dogs.

fulltext/md/PMC10384417.md

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+---
+pmcid: "PMC10384417"
+pmid: "37505860"
+doi: "10.3390/vetsci10070455"
+title: "Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Detection of Age-Related Somatic Alterations in Canine Blood Using Next-Generation Sequencing-Based Liquid Biopsy: An Analysis of over 4800 Dogs
+
+## Abstract
+
+### Simple Summary
+
+In humans there is a biological phenomenon known as CHIP (clonal hematopoiesis of indeterminate potential) in which somatic (acquired) mutations cause blood cells of a certain type to grow disproportionately by making many copies (or “clones”) of themselves. Although most people who are found to have CHIP do not have cancer, they are known to be at higher risk of developing cancer. CHIP has been studied extensively in humans, where it occurs more frequently with increasing age and is thought to be present in 10–20% of people over the of age 70; however, CHIP has not been well-studied in other species. This study provides the first population-level evidence for the potential existence of CHIP-like findings in dogs. Further research is needed to determine the clinical significance of these findings in canine patients.
+
+### Abstract
+
+Age-related somatic genomic alterations in hematopoietic cell lines have been well characterized in humans; however, this phenomenon has not been well studied in other species. Next-generation sequencing-based liquid biopsy testing for cancer detection was recently developed for dogs and has been used to study the genomic profiles of blood samples from thousands of canine patients since 2021. In this study, 4870 client-owned dogs with and without a diagnosis or suspicion of cancer underwent liquid biopsy testing by this method. Copy number variants detected exclusively in genomic DNA derived from white blood cells (WBC gDNA-specific CNVs) were observed in 126 dogs (2.6%; 95% CI: 2.2–3.1); these copy number variants were absent from matched plasma cell-free DNA, and from tumor tissue in dogs with concurrent cancer. These findings were more common in older dogs and were persistent in WBC gDNA in over 70% of patients, with little to no change in the amplitude of the signal across longitudinal samples. Many of these alterations were observed at recurrent locations in the genome across subjects; the most common finding was a partial loss on CFA25, typically accompanied by a partial gain on the same chromosome. These early findings suggest that age-related somatic alterations may be present at an appreciable frequency in the general canine population. Further research is needed to determine the clinical significance of these findings.
+
+## 1. Introduction
+
+The phenomenon of somatic genomic alterations in hematopoietic cell lines, commonly referred to as clonal hematopoiesis of indeterminate potential (CHIP) or age-related clonal hematopoiesis (ARCH), has been well characterized in humans and refers to somatic alterations that occur as a result of clonal expansion of the blood system, particularly the white blood cell populations. These alterations tend to be associated with advanced age, often exhibit consistent signal over time, and are frequently found in recurrent locations in the genome [1,2,3,4,5]. Most human patients who have these somatic alterations do not have concurrent cancer. In patients who do have concurrent cancer (specifically those with solid tumors), these alterations are typically not found in the corresponding tumor tissue. In humans, CHIP has been shown to be associated with an increased risk of developing a primary or secondary hematologic malignancy and represents an emerging biomarker for cancer risk prediction. Two population-based studies, each involving more than 10,000 people, have independently shown that the presence of CHIP alterations is associated with a roughly 10-fold higher relative risk of developing hematological cancers when compared to age-matched controls, with an absolute risk of around 0.5% per year [2,3]. For this reason, leading medical centers have established “CHIP clinics” for close clinical monitoring of patients with known CHIP findings [6,7,8,9].
+
+In addition to being a well-documented risk factor for malignancy in humans, CHIP has also been associated with an increased risk for cardiovascular disease (HR 1.9; 95% CI, 1.4–2.7) [10] and may be a risk factor for cerebrovascular events (e.g., stroke) [11,12]. Despite the growing body of literature surrounding CHIP in humans, the population-level prevalence and the clinical correlates of CHIP have not been systematically studied in dogs or other species.
+
+Recently, a blood-based liquid biopsy test using next-generation sequencing (NGS) was developed and clinically deployed for cancer detection in dogs. The clinical validation of this test involved 1100 cancer-diagnosed and presumably cancer-free client-owned dogs [13]. The test has been additionally performed in thousands of dogs since it became commercially available in 2021. This recent ability to test large numbers of dogs using liquid biopsy affords an unprecedented opportunity to study the genomic profiles of a broad population of canine patients. During liquid biopsy testing in dogs, cell-free DNA (cfDNA) is extracted from plasma and genomic DNA (gDNA) is extracted from white blood cells (WBCs) present in the buffy coat; extracted DNA is then subjected to NGS to identify genomic alterations. Cell-free DNA comprises DNA shed from a variety of tissues throughout the body, including tumors (if present). When genomic alterations are identified in cfDNA, this indicates the likely presence of cancer in the body. When genomic alterations are identified in gDNA, this could indicate the presence of a constitutional (germline) abnormality in the patient, including mosaicism; certain hematologic malignancies (when corresponding CNVs are also identified in cfDNA); or age-related somatic alterations (e.g., CHIP).
+
+Most studies in humans have focused on characterizing single nucleotide variants (SNVs) in particular genes associated with CHIP, but recent reports have shown that CHIP-associated copy number variants (CNVs) are also an important risk factor for the development of leukemia and cardiovascular disease [4,5,10]. NGS-based liquid biopsy offers an opportunity to study if similar alterations are seen in dogs and whether they may be useful as biomarkers to predict the risk for cancer or other diseases.
+
+The current study aimed to evaluate the population-level frequency of age-related somatic copy number alterations in the WBC gDNA of dogs and to characterize the type of alterations observed in these patients. Additional studies are currently underway to evaluate the clinical significance of these findings; if successful, these studies could pave the way for broad adoption of CHIP-based testing and monitoring approaches in veterinary medicine that are similar to those already employed for human patients.
+
+## 2. Materials and Methods
+
+A total of 4870 client-owned dogs were evaluated as part of this study. Of this total, whole blood samples from 3595 dogs with and without a clinical suspicion of cancer were submitted by the dog’s veterinarian for commercial liquid biopsy testing at the PetDx laboratory in La Jolla, CA (“clinical cohort”); whole blood samples from 1275 dogs with and without a diagnosis of cancer were obtained as part of a larger research collection to support the clinical validation of the NGS-based liquid biopsy test (“research cohort”) [13]. All research subjects were enrolled under protocols that received Institutional Animal Care and Use Committee (IACUC) or site-specific ethics approval [13]. All methods were performed in accordance with the relevant guidelines and regulations and follow the recommendations in the ARRIVE guidelines. All research subjects were client-owned, and written informed consent was obtained from all owners. In a subset of cancer-diagnosed patients from the research cohort, matched tumor tissue samples were also available for analysis. Additionally, a subset of patients across both the clinical and the research cohorts submitted whole blood samples at multiple timepoints, allowing for longitudinal monitoring of genomic alterations.
+
+Whole blood samples were collected from each patient; cfDNA was extracted from plasma, and gDNA was extracted from WBCs present in the buffy coat; in the subset of patients with matched tumor tissue available, DNA was also extracted from tissue [13,14]. All extracted DNA specimens were subjected to proprietary library preparation and NGS as previously described [13]. Sequencing data were analyzed using an internally developed bioinformatics pipeline to determine the presence of genomic alterations.
+
+This study focused on a large cohort of canine patients in which specific CNVs were identified in WBC gDNA but were absent in matched cfDNA samples. These findings will be referred to as “WBC gDNA-specific CNVs”, and one example is shown in Figure 1. It should be noted that a subset of patients with WBC gDNA-specific CNVs also had concurrent CNVs identified in cfDNA (and/or tissue, when available) but those CNVs were different from the CNVs identified in WBC gDNA.
+
+A subset of dogs with WBC gDNA-specific CNVs had clinical evaluations performed to determine the presence of cancer. Though the components of each evaluation varied, they typically comprised a thorough physical examination, laboratory workup (CBC, chemistry panel, and urinalysis), imaging (thoracic radiographs and/or abdominal ultrasound), and tissue sampling (via fine needle aspiration or biopsy) of observed masses, lesions, and/or enlarged lymph nodes.
+
+When the clinical evaluation determined that cancer was present, a definitive or presumptive cancer diagnosis was assigned. “Definitive” diagnoses were those in which cancer was confirmed via tissue-based testing (cytology or histopathology). “Presumptive” diagnoses were based on imaging, direct visualization/exam, or a strong suspicion from cytology or histopathology.
+
+To compare the age profiles of adult dogs with and without WBC gDNA-specific CNVs, a “normalized age” was calculated relative to expected lifespan, where expected lifespan was calculated as a function of weight according to the equation: lifespan = 13.987 − 0.035 × weight [15]. *p*-values were calculated using a two-sided *t*-test. Analyses were performed using R (version 4.0.5).
+
+## 3. Results
+
+### 3.1. Population Characteristics
+
+The study population of 4870 client-owned dogs comprised 3595 dogs from the clinical cohort and 1275 dogs from the research cohort (Table 1).
+
+In the clinical cohort, cancer status was unknown at the time of sample submission. Samples for 962 dogs were submitted due to a clinical suspicion of cancer (liquid biopsy was used as an aid in diagnosis); samples for 2399 dogs were submitted with no reported suspicion of cancer (liquid biopsy was used as a screening test); and samples for 234 dogs were submitted without documentation regarding cancer suspicion. The average age of dogs in the clinical cohort was 9.09 years (SD = 3.06; *n* = 3595) and the average weight was 25.80 kg (SD = 13.48; *n* = 3144).
+
+In the research cohort, 569 dogs had a definitive diagnosis of cancer and 706 dogs were presumably cancer-free. The average age of dogs in the research cohort was 7.17 years (SD = 3.68; *n* = 1275) and the average weight was 28.28 kg (SD = 12.04; *n* = 1275). A wide range of purebred and mixed-breed dogs were represented in both cohorts.
+
+### 3.2. Dogs with WBC gDNA-Specific CNVs and Their Associated Clinical Status
+
+WBC gDNA-specific CNVs were identified in 164 of the 4870 (3.4%; 95% CI: 2.9–3.9) dogs in the overall study population: 129 from the clinical cohort and 35 from the research cohort. For 126 of these dogs (107 from the clinical cohort and 19 from the research cohort), CNVs were identified only in the WBC gDNA, and no CNVs of any kind were observed in plasma-derived cfDNA. (Table 2).
+
+Clinical cancer evaluations were performed in 42 of the 126 dogs with WBC gDNA-specific CNVs: 28 from the clinical cohort and 14 from the research cohort.
+
+Of the 28 patients in the clinical cohort, 18 had samples submitted with no suspicion of cancer at the time of blood draw (i.e., the test was used for cancer screening). Within this group, 78% (14/18; 95% CI: 51.9–92.6) had no evidence of cancer following clinical evaluation, while 22% (4/18; 95% CI: 7.4–48.1) received a definitive or presumptive cancer diagnosis. The four cancer diagnoses in the screening patients included: splenic stromal sarcoma (definitive, diagnosed via histopathology), a splenic mass that was suspected to be cancer on imaging but did not have tissue testing for confirmation (presumptive), a hepatic mass (presumptive), and an adrenal gland tumor (presumptive).
+
+The remaining 10 out of 28 patients in the clinical cohort had samples submitted for testing due to suspicion of cancer (i.e., the test was used as an aid in diagnosis). Within this group, 50% (5/10; 95% CI: 20.1–79.9) had no evidence of cancer following clinical evaluation, while 50% (5/10; 95% CI: 20.1–79.9) received a definitive or presumptive cancer diagnosis. The 5 cancer diagnoses in the aid-in-diagnosis patients included: histiocytic sarcoma (definitive), a mixed germ cell-sex cord stromal tumor (definitive), two bone tumors (both presumptive), and an adrenal gland tumor (presumptive).
+
+All 14 dogs whose samples were submitted as part of the research collection had a definitive diagnosis of cancer at the time of blood collection. The cancer diagnoses in these dogs were as follows: lymphoma/acute lymphoid leukemia (4 cases), osteosarcoma (3 cases), mast cell tumor (2 cases), hemangiosarcoma, anal sac adenocarcinoma, chronic lymphoid leukemia, soft tissue sarcoma, and transitional cell carcinoma.
+
+There were 38 dogs with CNVs identified in plasma-derived cfDNA in addition to WBC gDNA-specific CNVs (22 from the clinical cohort and 16 from the research cohort); 26 of these dogs (11 from the clinical cohort and 15 from the research cohort) had clinical cancer evaluations performed. Among those in the research cohort, all were among the 569 dogs who were enrolled with a definitive diagnosis of cancer. The cancer confirmation rate for these 26 dogs was 100% (26/26; 95% CI: 84.0–100), demonstrating that observations of somatic CNVs in the plasma cfDNA are highly predictive of concurrent cancer.
+
+### 3.3. WBC gDNA-Specific CNVs Not Present in Matched Tumor Tissue When Cancer Is Concurrently Present
+
+Tumor tissue samples (from the same collection timepoint as the blood samples with WBC gDNA-specific findings) were available for testing by NGS from 11 of the 35 cancer-diagnosed dogs in the research cohort. The CNVs observed in gDNA were not present in the matched tumor tissue in 91% of cases (10/11). In one case, the same CNVs observed in WBC gDNA were present in matched tissue (from a fine needle aspirate of the left mandibular lymph node); this subject had a definitive diagnosis of T-zone lymphoma (stage IIa).
+
+Tumor tissue samples (from the same collection timepoint as the blood samples with cfDNA-specific CNV findings) were available for testing by NGS from 105 cancer-diagnosed dogs in the research cohort. In contrast to WBC gDNA-specific CNVs, the CNVs observed in cfDNA did match the CNVs found in tumor tissue in the vast majority of cases (92%; 97/105). Figure 2 shows the matched CNV profiles from one subject with CNVs present in tissue, cfDNA, and gDNA. The WBC gDNA-specific CNVs on CFA25 (canine chromosome 25; Figure 2a) are not visible in the tumor tissue (Figure 2c); however, the cfDNA-specific CNVs on CFA26, CFA32, and CFA35 (Figure 2b) are clearly visible in the tumor tissue.
+
+These results suggest that WBC gDNA-specific CNVs, when present, are typically coincidental (but not biologically related) to the patient’s cancer, while cfDNA-specific CNVs are typically derived from the patient’s cancer.
+
+### 3.4. WBC gDNA-Specific CNVs Persistent across Longitudinal Samples
+
+Longitudinal blood samples were available for a total of 76 patients with WBC gDNA-specific findings. Seventy-one percent of patients (54/76; 95% CI: 60.0–80.0) showed persistence of the gDNA CNVs over time. Figure 3 shows CNV profiles from one of these patients, with persistent WBC gDNA-specific CNVs in CFA25 and CFA36 in three consecutive timepoints spanning 9 months. In the remaining 29% (22/76; 95% CI: 19.4–40.7) of cases, the gDNA CNVs were not persistent across all subsequent timepoints.
+
+### 3.5. WBC gDNA-Specific CNVs at Recurrent Locations in the Genome
+
+Several WBC gDNA-specific CNVs were recurrent across patients; the most common CNV was a partial loss of chromosome 25, typically accompanied by a partial gain on the same chromosome (Table 3; Figure 1a and Figure 2a). This pattern is consistent with human literature, where CHIP has been reported at recurrent locations in the genome [4,5,16].
+
+### 3.6. WBC gDNA-Specific CNVs More Frequently Observed in Older Dogs
+
+The study cohort was analyzed to evaluate the normalized age of patients with and without WBC gDNA-specific CNVs, and their cancer status. Among patients without WBC gDNA-specific CNVs, dogs with a diagnosis of cancer (definitive or presumptive) were significantly older than dogs who submitted samples as part of the clinical cohort for the purpose of screening and had no evidence of cancer (*p* < 0.0001); in addition, patients with WBC gDNA-specific findings (whether or not they had cancer) were significantly older than the cancer-diagnosed dogs without WBC gDNA-specific CNVs (*p* < 0.0001). (Figure 4) No significant difference was observed in the age distribution of patients with WBC gDNA-specific CNVs as a function of cancer status (*p* = 0.5864).
+
+## 4. Discussion
+
+The identification of recurrent CNVs in the WBC gDNA of older dogs, which persist with little to no change in amplitude over time and are absent from cfDNA (and from tumor tissue, in dogs with concurrent cancer), provides early evidence for the existence of age-related somatic alterations in dogs that resemble the phenomenon of CHIP previously described in humans.
+
+Most of the published research on CHIP in humans involves analysis of SNVs [2,3]; recent studies have also reported CHIP-associated CNVs that are likewise age-related and are associated with a higher risk of the development of leukemia in individuals with or without other existing cancer [4,5,16]. Additionally, these studies have shown that the co-occurrence of CHIP-associated CNVs and SNVs was associated with a higher cumulative incidence of leukemia than the exclusive occurrence of either type of genomic alteration. A recent study using blood samples from cancer-diagnosed dogs found that a small percentage (4.3%; 4/93) of subjects had genomic alterations (SNVs) in genes that have been associated with CHIP in human patients; however, no tumor tissue samples were available to confirm that these alterations did not in fact originate from the cancer itself, and no comparisons were made between WBC-derived gDNA and plasma-derived cfDNA [17].
+
+In the current study, analysis was focused on WBC gDNA-specific CNVs in a large cohort of canine patients. Additional studies are underway at PetDx to evaluate the prevalence and distribution of WBC gDNA-specific SNVs in large populations of dogs, as well as the interplay between multiple types of CHIP-associated genomic alterations and clinical outcomes.
+
+The clinical significance of age-related somatic alterations in dogs that do not currently have a diagnosis of cancer is uncertain at present and requires further research. The current study found that 22% (4/18; 95% CI: 7.4–48.1) of dogs with WBC gDNA-specific findings (in the absence of cfDNA findings) but no initial suspicion of cancer (i.e., screening patients) received a definitive or presumptive diagnosis of cancer following a clinical evaluation. It is unclear how this number compares to the actual prevalence of cancer in an age-matched canine cancer screening population. It will be important to prospectively follow cancer-free dogs with WBC gDNA-specific findings to determine whether they are at higher risk of developing cancer in the future compared to their age-matched counterparts. Long-term monitoring studies are ongoing for patients with WBC gDNA-specific findings identified during clinical testing at PetDx.
+
+CHIP in humans is also associated with an increased risk of cardiovascular and potentially cerebrovascular disease [10,11,12]. It is currently unknown whether the presence of WBC gDNA-specific findings confers a similar risk for these or other conditions in canine patients. Though the current study was not designed to address this question, longitudinal monitoring of dogs with these genomic findings may help to provide such information.
+
+In addition to the current lack of longitudinal outcome data from patients with WBC gDNA-specific findings, this study faces other limitations.
+
+CNVs identified in WBC gDNA have not yet been confirmed to originate from a single hematopoietic cell type, which would substantiate their clonal nature; research is ongoing to elucidate the specific biological source(s) of these findings.
+
+Additionally, normalized age calculations were based exclusively on weight for all dogs without consideration of owner-reported breed, which may result in overestimation or underestimation in the case of specific breeds.
+
+Lastly, not all dogs had complete medical records and/or clinical evaluations to determine the presence of cancer.
+
+## 5. Conclusions
+
+The large-scale NGS-based genomic profiling of dogs with and without cancer has uncovered the first potential evidence of age-related somatic copy number alterations in dogs at the population level and is likely to reveal additional novel findings as the use of this type of testing expands. It is not currently known if CHIP-like genomic alterations confer a similar risk of cancer (particularly hematological malignancies) or other conditions (such as cardiovascular disease) in dogs as they do in humans. Continued research, including long-term clinical monitoring of affected canine patients, will be required for proper clinical interpretation of such findings in the context of liquid biopsy testing in dogs. Such studies have the potential to validate CHIP as an independent risk factor for cancer in dogs and pave the way for the development and clinical adoption of NGS-based CHIP testing and monitoring strategies similar to those now being used in people.

fulltext/md/PMC10384453.md

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+---
+pmcid: "PMC10384453"
+pmid: "37505824"
+doi: "10.3390/vetsci10070418"
+title: "Computed Tomographic Features of Thymus in Dogs: Correlation with Age, Gender, Breed and Body Fat Content."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Computed Tomographic Features of Thymus in Dogs: Correlation with Age, Gender, Breed and Body Fat Content
+
+## Abstract
+
+### Simple Summary
+
+The thymus is the first lymphoid organ formed to regulate a newborn’s immunity. It reaches its maximum size during puberty, after which it undergoes an atrophic procedure called involution, but it was observed in humans that its ability to grow again in response to some stresses is maintained. There is no comprehensive study on computed tomographic features of thymus in dogs as a useful modality to evaluate the thoracic organs so this is our aim in the present study. According to our results, there is wide variation in thymic characteristics at different ages and there are some differences between breeds and genders. The present work can provide valuable information for veterinary literature.
+
+### Abstract
+
+Background: The thymus is the first lymphoid organ formed to regulate a newborn’s immunity. It reaches its maximum size during puberty, after which it undergoes an atrophic procedure called involution, but its ability to grow again in response to some stresses, such as infections, neoplasia, surgeries, chemotherapy, and radiotherapy is maintained. There is no comprehensive study on computed tomographic features of thymus in dogs. So, the goal of the present study is to gain better insight into the thymus using computed tomography as a non-invasive method. Methods: One hundred and fifty dogs classified in five age groups and five breed groups were recruited to this study and the thymus was evaluated using a 2-slice computed tomography machine. The inclusion criteria for the present study were having a normal complete blood count, plain and post-contrast CT scan examination of the thoracic region and no history of neoplasia, chemotherapy or radiotherapy. The visibility, density, enhancement, grade, size, volume, shape, borders and lateralization of the thymus were evaluated and statistical analysis was performed. The effect of obesity on thymic grade and volume was also investigated. Results: The visibility, density, dorsal length, volume and grade decreased with increasing age. The thymic shape and lateralization were mostly wedge shaped and left sided, respectively. The borders became concave with aging and increasing body fat content caused an increase in the fatty degeneration of the thymus. Conclusions: Declining thymic density, grade, size and volume with aging are related to thymic involution and fatty degeneration was accelerated by increasing body fat content. Females and males were different only in thymic shape and small and large breeds were different only in thymic volume. The thymus was visible in some geriatric dogs with no underlying disease. We expect that the present work can be used by radiologists in reading thoracic computed tomography but investigation of thymic characteristics in dogs with neoplasia and history of chemotherapy, radiotherapy and thoracic surgeries can complete this study.
+
+## 1. Introduction
+
+The thymus is the first lymphoid organ formed to regulate newborns’ immunity [1,2,3,4]. It reaches its maximum size during puberty, after which it undergoes an atrophic procedure called involution or degeneration, but its ability to grow again in response to some stresses, such as infections, neoplasia, surgeries, chemotherapy, and radiotherapy, is maintained [2,5]. The thymus is a bilobed lymphoid organ with a light pink color in puppies which turns into a light grey color in adult dogs [4,6]. It has an important role in growth and maturation of the immune system especially T cells [3,7]. Anatomically, it is laterally compressed and lies in the cranioventral part of the thoracic cavity in dogs [4]. This organ starts to involute with the changing teeth in dogs. Although the process is initially rapid, the tissue usually does not atrophy completely, even in old age. When it decreases in size and loses its lymphoid structure, it is replaced by fat. However, evidence of thymic tissue can be seen in most dogs, regardless of age [4]. Histologically, thymic shrinkage consists of the emptying of cortical lymphocytes and the substitution of adipose tissue and thymic corpuscles [8].
+
+There are different conditions of the thymus, such as aplasia, hypoplasia, juvenile thymic hematoma, true thymic hyperplasia, rebound hyperplasia, thymic cysts, and thymic tumors such as thymoma and lymphoma [3]. Thymoma and thymic lymphoma are the most common thymic tumors explained in dogs [9,10,11]. Heart-based tumors, ectopic thyroid or parathyroid tumors, thymic branchial cysts, chemodectomas (aortic or carotid body tumors), metastatic neoplasia, granulomas, abscesses, lipomas, and teratomas must also be considered as differential diagnoses [12]. In humans, there is wide variation in thymic characteristics at different ages, making it important for radiologists to reduce unnecessary invasive procedures [2,5]. The recognition of a normal thymus on computed tomography (CT) is easier with knowledge of this normal variation, thereby avoiding false-positive diagnoses of mediastinal neoplasms [2,3]. So, the present study aims to describe the characteristics of the normal thymus in dogs using computed tomography as a non-invasive method and, to the best of our knowledge, this is the first study that evaluates the normal thymus specifically in dogs.
+
+## 2. Materials and Methods
+
+This descriptive and prospective study recruited a total of 150 dogs, 74 females and 76 males, from February 2022 until February 2023. The criteria for inclusion in this study were having a normal complete blood count (CBC), a plain and post-contrast CT scan examination of the thoracic region, and referring for CT scan examination with no thymic abnormality, no neoplasia in the thymus or other organs, and metastasis detection. These patients were referred for computed tomography due to traumatic injuries and orthopedic reasons. All medical records and CT scan images were also evaluated to exclude patients with any pathologies that have a possible effect on the thymus, such as lung nodules or lesions, sternal, mediastinal, or perihilar lymphadenopathy. The patients were of different breeds (Mix breeds, Pomeranian, German Shepherd, Shih Tzu, Maltese, Yorkshire, Terrier, Rottweiler, Spitz, Dachshund, Lhasa Apso, Jack Russel Terrier, Chihuahua, Grate Dane, Poodle, Border Collie, Pekingese, and Cocker Spaniel). They were divided into five age groups, with 30 dogs in each group, according to their ages. Group 1 was a puppy group, including puppies from ages 0 to 6 months old; group 2 was a juvenile group, including 7–12 months old; group 3 was young adults, including 1–2-year-old dogs; group 4 was an old adult group, including dogs aged 3–7 years old; and group 5 was a geriatric group, including dogs more than 7 years old. They were also divided into five size groups based on their size (twenty toy breeds, fifty small breeds, one medium-sized breed, sixty-six large breeds and thirteen giant breeds).
+
+All CT examinations were performed by a 2-slice Somatom-Spirit CT scan machine (Siemens, Munich, Germany) at the department of Diagnostic Imaging of Veterinary Faculty, Tehran University, Tehran, Iran. The contrast media used for all examinations were Omnipaque (240 mg/mL) with a dosage of 4 mL/kg or Visipaque (320 mg/mL) with a dosage of 2 mL/kg. The standard protocol for thoracic CT scan was used (120 kVp, 60–65 mA, acquisition slice thickness of 1–1.5 mm, and reconstructed slice thickness of 1–3 mm). Dorsal and sagittal images were reconstructed and used for better detection of the thymus. All examinations were stored in the PACS system and then free version of radiant viewer was used to evaluating the images. The Leonardo Syngo workstation (Siemens, Germany) was also used for volumetry. Evaluations were performed by a board-certified radiologist in the Osteo window.
+
+The thymus was evaluated in the first step to determine whether it was visible or not. Non-visible thymus was that with complete degeneration seen with fat attenuation and not distinguishable from mediastinal fat. After that, a grading system for thymic tissue appearance was used based on its predominant component (grade 0: totally fatty replacement of the thymus (non-visible) and could not differentiate from mediastinal fat; grade 1: more than 50% fatty replacement of the thymus; grade 2: more than 50% soft tissue component in the thymus; grade 3: totally soft tissue component in the thymus. After grading the thymus, the attenuation (CT number) of thymic tissue in plain and contrast-enhanced images was measured. For this purpose, a round-shaped region of interest (ROI) with a surface equal to 75 mm^2^ was considered in the widest part of the organ for large thymus (Figure 1) and a smaller ROI for small thymus. Then pre- and post-contrast Hounsfield Units (HU) were compared for each patient individually and also between groups for evaluating the pattern of attenuation alteration and enhancement alteration.
+
+The size of the thymus was also measured in transverse and dorsal images. The maximum height (TH) and diameter (TD) in transverse images and maximum diameter (DD) and length (DL) in the dorsal views were measured (Figure 2), as well as thymic volume (TV) in all groups. These indices were measured in the area related to the mediastinal fat at the normal location of the thymus for patients with a completely fatty replacement of the thymus (grade 0). All measurements were performed in contrast-enhanced examinations. To normalize the thickness of the thymus in the transverse view, the height of the second thoracic vertebra midbody was measured and the ratio was calculated. The correlation between thymic size and grade with the body fat content was evaluated by measuring the thickness of subcutaneous fat in the dorsal aspect of the T4 spinous process (Figure 3). Subjective characteristics such as predominant deviation of the thymus to left, midline or right position, the thymic shape (wedge shaped, rectangular shaped, or linear shaped), and the thymic contour (convex and concave) were also evaluated. For patients with fatty degeneration, these characteristics were analyzed in mediastinal fat at the location of the previous thymus. Evaluation of thymic size, volume, attenuation, and grade was performed twice by a single person within a week, and the mean value of the first and second measurements for numerical data and the second results for thymic grading were reported.
+
+### Statistical Analysis
+
+Categorical data were collected as numbers and presented as a percent value. Continuous data were presented as the mean ± SD. Mean density, TD, TH, DD, DL, and TV were compared between groups using a one-way ANOVA and the Tukey method; the *t*-test was used for comparing numerical data between the two groups; and the Pearson Chi-square test was used to analyze the categorical variables such as enhancement, shape, predominant side, and grading. The Pearson correlation coefficient (r value) was calculated for the evaluation of the repeatability and reliability of two-stage measurements (intra-observer agreement). All statistical analyses were performed with SPSS software version 16.
+
+## 3. Results
+
+### 3.1. Visibility
+
+In the present study, the thymus was visible in 104 out of 150 cases. The distinct lobes of the thymus were not distinguishable. The visible thymus was noted with a soft tissue component and homogenous appearance or a combination of soft tissue and fat component based on the degree of involution with heterogenous (mottled) appearance (Figure 4, Figure 5 and Figure 6). We accounted the thymic tissues with complete fat degeneration for non-visible thymus (Figure 7). The anatomical and topographical locations of the thymus are shown in Figure 4, Figure 5, Figure 6 and Figure 7. The thymus’ dorsal border begins at the ventral aspect of the vessels in the cranial mediastinum and ends at the dorsal aspect of the sternebra. In the dorsal and sagittal views, it was extended caudally up to the pericardial region and merged with the pericardium on the left side (Figure 2B). The visibility percentage in group 1 was 100%. The minimum and maximum ages in group 1 were three and six months, respectively. In groups 2 and 3, the thymus was visible in 86.7% of cases and it was not notable in 13.3% of cases. The minimum and maximum ages with visible thymus in group 2 were seven and 12 months, respectively. This group includes one 11-month-old and three 12-month-old cases with a non-visible thymus. In group 3, the youngest and oldest cases with a visible thymus were 1.5 and 2 years old. The patients with a non-visible thymus were two years old. In group 4 the visibility percentage was 53.3% and the thymus was not visible in 46.7% of patients. The youngest and oldest patients with visible thymus in group 4 were three- and six-year-old dogs. The visibility percentage of group 5 was 20% and the maximum age with visible thymus was nine years old (Table 1).
+
+### 3.2. Density (Attenuation)
+
+It was found that the mean density of the thymus decreased with increasing age from group 1 to group 5, but the pattern was not the same between the patients in one group with different ages and the distribution of CT numbers in one group was very wide. The mean, minimum, and maximum densities in plain and post-contrast CT scan examinations in each group were summarized in Table 2 and Table 3. The differences in mean density between plain and post-contrast CT scan examination regarding age groups were significant (*p*-value ≤ 0.05, Table 2 and Table 3). The mean density difference between each group (for example, group 1 with other groups) was evaluated and summarized in Table 4. There was a significant difference between the mean density of group 1 with other groups. Further, there was a significant difference between the mean density of group 2 with groups 1, 4, and 5. The difference in the mean density of group 3 was significant compared to groups 1, 4, and 5, while the difference in the mean density of groups 4 and 5 was not significant. The Pearson correlation coefficient for intra-observer agreement of two-stage measurement of CT numbers was 84.5% and 85% for plain and post-contrast CT scan examinations, respectively (Table 2 and Table 3).
+
+### 3.3. Enhancement
+
+Ninety-four-point seven percent of the thymic tissues in this study were enhanced. According to the results, 0.01 percent of the visible thymus was not enhanced (two patients out of 104). The other non-enhanced thymic tissues were non-visible (six patients out of 46). The percentage of enhancement in groups 1, 2, and 4 was 100%, and in groups 3 and 5, it was 93.3% and 80%, respectively. The *p*-value for comparing enhancement showed a significant difference between age groups (Table 1, *p*-value ≤ 0.05). There was no significant difference between the thymic enhancement of different breeds and genders. The thymus was enhanced in 93% of females and 96% of males. It was also noted that the blood supply of the thymus was mostly by the left and right internal thoracic arteries and veins (Figure 4, Figure 5, Figure 6 and Figure 7).
+
+### 3.4. Shape
+
+Three different shapes of the thymus in the transverse section were noted in the present study: a rectangular shape with convex borders that was visible in younger patients, a wedge-shaped appearance with concave or straight borders in young or adult patients (Figure 4, Figure 5, Figure 6 and Figure 7), and a linear shape that was seen predominantly in older patients (Figure 8). The thymic shape in the sagittal view was variable. Categorization of thymic shape could not be established in the sagittal nor in the dorsal sections. In the dorsal view, the thymus was wider in the cranial and caudal portions and in the mid-part it was narrower and became narrower with aging in all patients (Figure 2B). The shape of the transverse section of the thymus was wedge shaped with a concave border at 74.4%, rectangular with a convex border at 22%, and linear at 3.3%. The difference in shape between age groups and different breeds was not significant, but there was a significant difference in the shape of the thymus between genders (*p*-value = 0.006). The percent values of each kind of shape are summarized in Table 1 and Table 5. The thymus shape in the only medium-sized breed in this study, a seven-year-old female Cocker Spaniel, was wedge shaped.
+
+### 3.5. The Predominant Side
+
+The dominant side of the thymus in the cranial portion was evaluated because the thymus deviated to the left side caudally in all patients, resulting in the well-known ‘sail sign’ in radiographic images. The predominant side of the thymus was left sided in 46% of the cases and mid-line in 53% of the cases. However, there was only one patient with a thymus deviated to the right side in the cranial aspect of the mediastinum (Figure 8). The predominant side percentage of each group was summarized in Table 1. The difference in the predominant side was significant between groups 1, 2, and 3. In group 1, the thymus was mostly in the midline and it was deviated to the left side in groups 2 and 3. There was no significant difference between breeds for this variable.
+
+### 3.6. Volume
+
+The mean, maximum, and minimum thymus volumes of each group are summarized in Table 6. The values of thymic volume decreased with aging, and there was a significant difference between age groups for this variable (*p*-value = 0.000). The mean volume of the thymus in females was 2.3 ± 2.6 cm^3^ and 2.5 ± 3.8 cm^3^ in males, with no significant difference between males and females, but a significant difference in volume between breeds. The mean values of volume in toy breeds, small breeds, large breeds and giant breeds were 1.4 cm^3^, 1.7 cm^3^, 2.9 cm^3^, and 3.6 cm^3^, respectively. The thymic volume in the only medium-sized breed patient was 2.3 cm^3^. The Pearson correlation coefficient was high between the 2-stage measurement of thymic volume (97.5%). There was no correlation between thymic volume and subcutaneous fat content (r = 0.053).
+
+### 3.7. Diameter, Length, and Height
+
+The mean, minimum, and maximum values of the thymic diameter and height in the transverse view and the diameter and length in the dorsal view are shown in Table 6. There is no significant difference in TD, TH, and DD between age groups, but the difference in DL was significant (*p*-value ≤ 0.05). The approximate ratio between TD and the height of the T2 midbody was 1.2 ± 0.7.
+
+### 3.8. Grade
+
+The grade of the thymus has been declining with increasing age. There was no grade 0 thymus in group 1. There was no grade 3 and 2 thymus in group 5. Based on our findings, the correlation between age and grade was significant. The percent value of each grade in each group is summarized in Table 1. Based on the correlation coefficient, there was a reverse correlation between grade and subcutaneous fat content (r: −0.18).
+
+## 4. Discussion
+
+The thymus was visible in all puppies, most of the juveniles and young adults, half of the old adults and some geriatric dogs. According to the results, the thymic component started to atrophy and be replaced by fat from the first months of life, and the fat content was increasing with aging. In one ultrasonographic study of the thoracic region in dogs and cats carried out by Reiche and Wisner (2000) [13], they concluded that the thymus degenerates after puberty. According to our results and the results of this ultrasonographic study, prepuberty period marks the onset of thymic involution but it speeds up after puberty. In one study of the humans, the thymus in 783 patients with no underlying disease was evaluated, and it was recognized that the thymus was present in 100% of patients under the age of 30, 73% of patients between the ages of 30 and 49, and 17% of patients over 49 years of age [14]. Hence, regarding thymus visibility, our results show the same trend as in humans, as it declines by aging.
+
+We observed that the thymic soft tissue component can be preserved in some healthy adult and geriatric dogs. Evans and de Lahunta [4] mentioned that thymus does not atrophy completely and evidence of its remnant can be visible at any age. In humans, it was shown that some disorders, such as infection, tumorigenic conditions, and chronic inflammations accelerate the fatty involution of the thymus [7,15]. It was also shown that the thymus can regrow after recovery from the stress [2]. This phenomenon, which is called ‘rebound hyperplasia’, can happen some months after resolving the stress [2] but this is not confirmed yet in dogs and it is seen rarely in veterinary [16]. Cordella et al. (2023) [16] concluded that the thymus can be visible in adult dogs with different non-thymic neoplasia compared to young dogs and correlated this finding to thymic hyperplasia rather than neoplasia. So, if the thymus is visible with residual soft tissue component in adult and geriatric dogs and there are no clinical signs or suspicion of any neoplasia or infections, it will be a normal variant or it can be hyperplasia related to previous resolved stress. In a human study, Simanovsky et al. (2012) [5] found that soft tissue components in the thymus of four healthy patients aged 40–54, suggesting that the finding of some soft tissue in an asymptomatic patient was occasional, but could be normal. According to another human study, it seems that the shape of the visible thymus can help to differentiate hyperplasia from thymic abnormality. Based on this study by Sklair-Levy et al. (2000) [17], the biconvex margins late in the second decade of life or the focal expansion of a lobe or multilobulation at any age are usually indicative of thymic abnormality. Being aware of all of these, it seems that there is still lack of information about thymus changes in dogs at the time of abnormal conditions such as non-thymic neoplasia, surgeries, radiotherapy and chemotherapy; so, more investigations will be needed in the future.
+
+We hypothesized that the early fatty degeneration of the thymus in some juveniles and young adults in our study can relate to some conditions, such as previous treated infections or inflammations which accelerated the thymic atrophy.
+
+As noticed in the present study, the distinct lobes of the thymus were not seen distinctly in both cranial and caudal portion and our hypothesis was the low accuracy of the 2-slice CT scan machine; but in a most recent study by Cordella et al. (2023) [16] that evaluated the thymus in adult dogs with non-thymic neoplasia in comparison to thymus in young dogs (under 9 months) using a multidetector computed tomography, although they used a 320-row CT scan machine, the bilobed thymus was not noted except in one patient. Evans and de Lahunta [4] also mentioned previously, thymic lobes are not differentiable in cranial portion because of the connective tissue attaching both lobes in this part.
+
+In our study, the same thymic topographical characteristics as Evans and de Lahunta’s study were observed [4]. From an anatomical point of view in a small dog, the caudal aspect of the left thymic lobe is placed between the thoracic wall and the left ventricle and the right lobe of the thymus that usually touches the cranial surface of the pericardial sac is extended laterally [4].
+
+According to the present results, thymic appearance was homogenous with a predominant component of soft tissue, and became heterogenous with mottled appearance by adipose substitution. Cordella et al. (2023) [16] also observed that the thymus appearance was lobulated in adults and homogenous in young dogs. In fact, the combination of soft tissue and fat component makes this lobulated or mottled appearance in thymus.
+
+The CT attenuations of thymus in the present study were in the range of soft tissue and fat based on the predominant component, but there was a wide spectrum of densities in each group and between individuals. CT numbers noted in our study for each patient matched the subjective grade. Declining CT numbers from puppies to geriatric dogs is also due to fat replacement by aging. In fact, there is a reverse correlation between thymus CT attenuation and aging which was also noticed in dogs according to Cordella et al. (2023) [16] and human studies [5].
+
+According to the present findings, only in eight patients, which were young adults and geriatric, no enhancement was notable. In six of these patients, the thymus was also non-visible (grade 0); so non-enhancement could be due to severe atrophy of the thymus. Another reason can be the very small size of the degenerated thymus, which reduces the accuracy of density measurement. Another hypothesis is insufficient injected contrast media, but when the enhancement of other organs was checked, this hypothesis was ruled out. The blood supply of the thymus was by left and right internal thoracic arteries based on our results, although according to Evans and de Lahunta [4], occasionally a thymic branch from brachiocephalic trunk on the right side and subclavian artery on the left side may supply this organ. Evaluating the thymus vasculature in other populations may add more information about thymic blood supply.
+
+The shape of the thymus in the transverse section was variable between dogs, even in the same age and breed group, but we classified the shape. According to our results, it was observed that most of the thymic shapes in the transverse section were wedge shaped with straight and concave borders, perhaps because most of the patients were adults. Opposite of our expectations, most of the puppies’ thymus were wedge shaped with convex border and there were some with rectangular shape and convex border. We hypothesized that this finding was due to premature thymic involution following previous stresses or diseases in puppies. Alteration of thymic borders from convex to straight and concave with aging was also confirmed in human studies [18].
+
+The thymic shape was also significantly different between genders. Females had more rectangular-shaped thymus than males. Contrary to our presumption old adult dogs had more rectangular-shaped thymus than juveniles and young adults; it can be related to the body conformation of dogs in this group. Another reason can be related to the fact that there were more male dogs in the juvenile and young adult groups. The thymic shape was so variable in the sagittal view, making it impossible to classify it in this view. Thymic shape in the dorsal view was the same in all patients. According to Cordella et al. (2023) [16] who evaluate the thymic shape in the dorsal view, the thymus was triangular and elongated in most young and adult patients although there was no shape classification in their study.
+
+The dominant side of the thymus was also variable, but in all of the samples, the caudal aspect of the thymus extends to the left side, which makes a ‘sail sign’ in ventrodorsal thoracic radiographs. This feature in humans is right-sided and the sail sign is notable on the right side in anterior-posterior radiographs [2]. The dominant side was significantly different between puppies, juveniles, and young adults. The thymus was mostly in the midline in puppies and deviated to the left side with age, but this pattern was not repeated in old adults and geriatrics, thus this variable needs more investigation in the future. The predominant side of the thymus was studied by Cordella et al. (2023) [16] and they observed that thymus lateralization was mostly left sided in adults and midline in young dogs, and the same as our finding, they noticed only one right-sided thymus. In one human study evaluating the side predominancy of the thymus, it was also centered at the midline or showed a left-side predominance in most patients [5].
+
+In the present study, the transverse view was mostly used to evaluate the characteristics because it is the main non-reconstructed view with better resolution and because of the best categorization of features such as thymic shape and side predominancy in this view.
+
+In our study population, the volume and thymic length in the dorsal view decreased with aging. The mean values of each index of the thymus were measured with a high reliability coefficient, but there was a wide variation in thymic size between groups and there was no descending pattern in some measurements from puppies to geriatrics. We hypothesized that this finding is due to breed and weight differences. Cordella et al. (2023) [16] also observed a variation in thymic dimensions between different patients with different body weights.
+
+Anatomically, in beagles, it is known that when the thymus is completely developed it is 12 cm long, 6 cm wide, and 3 cm high [4]. Moarrabi et al. (2019) [19] performed radiographic and ultrasonographic examinations of the thymus in five Mongrel puppies every month from birth to five months of age. They reported the thymic width and length at five months of age were about 6.56 ± 0.63 mm and 28.10 ± 2.72 mm, respectively, which were close to the reported measurements of our study.
+
+It was observed that the fat replacement of the thymus increases with an increment in subcutaneous fat content, which can be an index of obesity and body condition in our population. The impact of obesity on the thymic attenuation in human patients between 20 and 30 years old was evaluated in one study that showed overweight patients were more likely to have higher levels of thymic fatty replacement than normal or underweight patients [14]. In the same way Araki et al. (2016) [20] showed that patients with low thymic grades had significantly higher body condition scores.
+
+There are many comprehensive studies on human’s thymus characteristics using imaging modalities which provide valuable data about this organ in newborns, children and adults. Based on these investigations, thymus CT attenuation in plain and contrast-enhanced examinations is variable [2,18]. In all of these studies, the thymic CT attenuation declines with aging due to thymic involution and fat replacement [2,3,5,14].
+
+Ackman et al. (2016) [20] described differences in thymic density between the genders in the 20–30 age groups and they showed increased fatty degeneration of the thymus in young males compared to females [21]. In our study, there is no difference between all males and females in thymic density and the amount of fat replacement. Simanovsky et al. (2012) [5] found no correlation between gender and thymic volume and density in humans.
+
+As we noticed, there was a great variation in thymic shape in dogs, there was also significant variation in the CT scan appearance of a normal thymus in the preadolescent and adolescent age groups according to Heiberg et al. (1982) [18] that investigated the normal and abnormal thymus using CT scan examinations in 40 human cases under 20 years old. They also found that the thymus is larger in younger humans, but the thymic size varied widely in all ages. Thymic size and dimensions were also so diverse between individuals in the present study.
+
+Variation of normal thymus appearance on human CT scans and differences in thymic shape between males and females in the middle and older age was also reported by Araki et al. (2016) [20] which evaluated the impact of physical characteristics such as body mass index (BMI) and cigarette smoking on thymus shape in middle and old-age human patients. Their findings suggested that the start of thymic atrophy in females is delayed by approximately 10 to 20 years in comparison to males. Thymic atrophy is completed before the age of 60 in men and 70 in women, and sexual steroids are considered to affect the thymic shape [20]. We do not report similar findings in our study.
+
+A study by Hussain et al. (2020) [22] investigated how diverse imaging modalities can be used to assess thymus normality and abnormality to differentiate thymic hyperplasia from thymic malignancy in human patients. According to their study, the CT scan is valuable for primary assessment, providing the morphology and density, and MRI is the imaging modality of choice in diseased thymus because of its ability to provide a functional assessment of this organ.
+
+The important limitation in the present study was the 2-slice CT scanner, which causes loss of some details and a decrease in resolution. Human studies for thymus CT scans were performed by different kinds of CT scan machines with different slices [15]. Multidetector row CT (MDCT) scanners can image the thymus with more detail because of thinner slices, unceasing images, and multiplanar reconstructions [5]. Another limitation of the present study was the lack of histopathologic procedures following CT scan examinations to confirm the thymic tissue.
+
+## 5. Conclusions
+
+To the best of our knowledge, this is the first study evaluating the normal thymus in dogs using computed tomography and performing classification and grading. As a conclusion, variable shapes, sizes, and densities of the thymus can be noted in dogs, although some patterns for alteration of these variables with aging are explained through this study. The ratio of thymus thickness in the transverse view to the height of the second thoracic vertebral midbody was approximately 1.2 ± 0.7. A grading system for thymic appearance based on predominant thymic components was also established through this study and can be used by radiologists for reading thoracic CT scans. The thymus was also visible in adult and geriatric dogs with no underlying non-thymic disease, but due to lack of sufficient data in this field, serial examinations were still suggested in the case of suspicions of infiltrative diseases in old dogs owing some remnant thymic tissue. According to the results, thymic atrophy did not significantly affect enhancement. Thymic characteristics were not significantly different between males and females except in the shape of the thymus, which needs more investigation to generalize this finding to other populations. Small and large breed dogs were different only in the volume of the thymus. Further, it can be concluded that increasing body fat content affects thymic fatty degeneration, which can influence the immunity. Finally, further investigation of thymic alterations in the case of infiltrative disease and some stresses such as chemotherapy and radiotherapy can complete the present study.

fulltext/md/PMC10385316.md

@@ -0,0 +1,134 @@
+---
+pmcid: "PMC10385316"
+pmid: "37505818"
+doi: "10.3390/vetsci10070412"
+title: "Effect of Aging on the Immune Response to Core Vaccines in Senior and Geriatric Dogs."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Effect of Aging on the Immune Response to Core Vaccines in Senior and Geriatric Dogs
+
+## Abstract
+
+### Simple Summary
+
+Elderly dogs are increasingly present in households worldwide and have become routine patients in daily veterinary practice, with larger dogs aging earlier than smaller ones. Aging is not a disease but has several negative consequences on the organism in general and the immune system in particular, resulting in a decline in protection over time. Vaccines against parvovirus infection, distemper, and infectious hepatitis are highly recommended for dogs of any age, but their effectiveness in older dogs should not be assumed. The aim of this study was to measure the specific serum antibody titers against these vaccine-preventable diseases in three hundred fifty senior and geriatric dogs with the help of the rapid kit VacciCheck. More than half of the elderly population was well protected from all three diseases. Particularly, 88.6% of aging dogs were protected against parvovirus infection (in this case, larger aging dogs resulted as more protected than smaller ones), 82.3% against infectious hepatitis, and 66.0% against distemper. The advice to vaccinate on a three-year basis, properly adopted for adult dogs, should therefore be carefully considered by veterinarians for older dogs, for whom closer vaccination (every 1 to 2 years) might be more appropriate.
+
+### Abstract
+
+Elderly dogs are steadily increasing worldwide as well as veterinarians’ and owners’ interest in their health and wellness. Aging is not a disease, but a combination of changes negatively affecting the organism in general and the immune system in particular, resulting in a decline in protection over time. The aim of this study was to measure the specific serum antibody titers against the main dangerous and widespread viral diseases preventable by core vaccinations in senior and geriatric dogs using the in-practice test VacciCheck. A cohort of three hundred fifty elderly dogs was analyzed for Protective Antibody Titers (PATs) against CPV-2, CDV and CAdV-1. The age ranged from 5 to 19 years, with two hundred fifty-eight seniors (73.7%) and ninety-two geriatrics (26.3%), and 97.4% of them were vaccinated at least once in their lives. More than half of the entire study population (52.9%) had PATs simultaneously for all three diseases, with 80.5% seniors and 19.5% geriatrics. Specific PATs were found in 88.6% of aging dogs for CPV-2, 82.3% for CadV-1 and 66.0% for CDV, demonstrating that unprotected aging dogs represent a minority. Unexpectedly, the larger elderly dogs resulted as more protected than smaller ones for CPV-2. Protection then decreases over time, with geriatric dogs less protected than senior ones. Veterinary practitioners should therefore always consider whether to maintain core vaccinations in aging dogs as in adults on a three-year basis or opt instead for closer boosters (every 1 or 2 years). PATs for core vaccines could then represent a good biomarker of protection and their titration could become a standard of care, especially in such a sensitive period of the dogs’ life.
+
+## 1. Introduction
+
+In veterinary practices, senior dogs account nowadays for 40–50% of canine patients, with both of their percentages and average ages steadily increasing, resulting in a growing focus on their health and wellness [1,2,3,4,5]. However, when is a dog considered old? The biological age correlates with the chronological one but is not synonymous with it. The latest (2023) guidelines of the American Animal Hospital Association (AAHA) report the recommendations of a task force of experts with decades of collective experience in the care of elderly patients [1] and define as “senior” an older aging pet; however, given the great variability in aging by breed and size, there is no a specific age in defining the senior status. This is especially true in dogs, where there is clear evidence that longevity is inversely related to size (both weight and height); larger dogs not only show the typical negative consequences of aging earlier than smaller ones, but also die younger [6,7,8,9]. Finally, a very recent paper by Montoya et al. [10], which aimed at calculating the life expectancies (LE) of dogs, reported a canine average lifespan of 12.69 years, confirming that larger dogs will age earlier (giant LE 9.51 years, large LE 11.51 years) than smaller dogs (medium LE 12.7, small LE 13.53, toy LE 13.36 years). Accordingly, veterinarians should consider dogs in need of elderly care at differing times depending on their size, starting from 5–6 to 9 years old for giant/large breeds and from 9 to 13 years old for medium/small breeds (Table 1) [11,12,13].
+
+Aging is not a disease, but a combination of physical and dynamic changes in the organism starting from conception, continuing with adulthood and senescence, and ending with death [14,15]. In this process, there are many different factors influencing the aging rate, generating alteration in musculoskeletal, adipose, and cerebral tissue and leading to physical and functional phenotypical changes typical of older patients [16]. These modifications also affect the immune system. Immunosenescence is in fact a dynamic process of immune dysfunction that occurs with age involving a continuous remodeling of lymphoid organs and immune cells, in which some immune parameters increase while others decrease or remain unchanged [17]. These changes undeniably lead to a greater susceptibility to the development of the so-called Age-Related Diseases (ARDs), particularly infectious, autoimmune, and neoplastic diseases [17,18,19,20,21]. While evidence of this phenomenon has been well reported in human medicine, for a long time less was known about dogs, as immunosenescence was one of the less studied and understood areas of veterinary geriatric medicine. In the last 15 years, however, something has changed [7,15,16,22,23,24,25,26]. In addition, the peculiar term “inflammaging”, now used in both human and veterinary medicine, has been coined to describe the effects of a continuous antigenic challenge throughout life leading to the production of inflammatory cytokines triggering the onset of inflammatory conditions in later life [1,7,20,21,27].
+
+Although both cell-mediated and humoral immunity are weakened during the aging process, in both human and veterinary medicine the cell-mediated compartment is the most affected. This explains why elderly patients are more susceptible to infectious diseases and neoplasms than younger ones [7,15,25]. This decline in cell-mediated function is considered a result of the thymic involution. Thymus is a central primary lymphoid organ responsible for the production of T lymphocytes, which represent the pivotal cells in orchestrating both types of immunity (cell-mediated and humoral ones). After birth, the thymus continues its development until puberty, and at that time (in dogs generally at 4–5 months of age) it starts a slow but progressive involution, leading to a significant decrease in production and activity of thymic hormones and T lymphocytes (helper and cytotoxic ones) [15,19,20,28]. This unavoidable thymus involution is regarded as one of the main factors contributing to the loss of immune function typical of the elderly and is considered in turn a genetically programmed event (the so-called “thymus clock”) [15,17,21,29]. In a recent study (2022), Lee et al. proposed both CD4/CD8 ratio and Growth Differentiation Factor 8 (GDF8) levels in canine blood as useful factors to use as an age prediction model for dogs, as these parameters were found to be significantly correlated with age [30].
+
+Clinical evidence in humans and animals suggest that, as age advances, the secondary (anamnestic) immune response against known antigens (for example vaccine boosters) still works well, whereas the ability to mount a primary immune response against new antigens never encountered before (field or vaccinal ones) declines significantly with age for different causes, always leading to a dangerous increased susceptibility to infectious diseases, which may also limit the effectiveness of vaccination in older individuals [15,16,17,18,22,26,31,32]. Few studies, however, have been conducted to date to understand if these age-related changes in the elderly canine patients are able to affect the immune response to vaccines.
+
+Canine and feline vaccines are classified into core and non-core by all the international vaccination guidelines [33,34,35,36,37]. Core vaccines are the recommended ones, i.e., the ones that every dog and every cat should receive almost once in their life, as they are intended for widespread, dangerous, contagious, and even life-threatening diseases. The canine core vaccines protect dogs against canine parvovirus infection (CPV-2), canine distemper (CDV) and infectious canine hepatitis (CAdV-1) [33,34,38]. It has been shown that any dog can be infected regardless of age, and cases of core diseases are reported also in senior dogs [39,40,41]. For this reason, core vaccinations are considered the main tool to control these diseases. For adult dogs the international guidelines recommend core revaccination on a three-year basis using modified live vaccines (MLV), whereas for elderly canine patients only a few indications are available. Indeed, many associations, researchers, and veterinary practitioners have developed detailed canine and feline senior and geriatric care guidelines in order to focus on the main issues regarding elderly patients, but they rarely refer to a particular vaccination protocol for this delicate phase of life or to the need to adapt those already existing for adult pets [1,2,3,5,42,43,44].
+
+The aim of this study was to measure the specific serum antibody titers, and therefore the actual protection, against CPV-2, CDV and CAdV-1 in senior and geriatric dogs by using an in-practice test kit.
+
+## 2. Materials and Methods
+
+### 2.1. Study Population and Study Protocol
+
+Plasma/serum samples analyzed in this study were collected over 9 years (January 2014 to January 2023) to analyze specific antibody titers for core diseases and for other purposes (e.g., diagnostic assays, surgery, health checks) related to patient aging. According to the Ethical Committee position of the University of Milan, residual aliquots of biological samples that were collected under the owner’s informed consent can be freely used for research purposes without any formal approval (EC decision 29 October 2012, renewed with the protocol n. 02-2016). For each patient, different key information was recorded: (1) age: seniors and geriatrics (the age category was determined according to the size of the dogs, following the aforementioned rule that larger dogs age earlier than smaller ones and vice versa [8,9,10]); (2) breed (purebred or crossbred) and breed size: small (<10 kg), medium (≥10–<25 kg), and large (≥25 kg); (3) sex and reproductive status: intact or neutered male or female; (4) health status: healthy or unhealthy, considering as unhealthy only animals with clinical problems that could affect immune function (none of the unhealthy dogs were, however, being treated with drugs that might interfere with the immune system (neither immunosuppressive nor immunostimulant): for example, none of the oncological patients was under chemotherapy) (see below Results section); (5) vaccination history, considering the time elapsed since the last core vaccination: ≤1 year, >1 year–≤3 years, or >3 years.
+
+### 2.2. Detection of Specific Antibodies to Core Vaccines by VacciCheck and Expression of the Results
+
+Each plasma/serum sample was tested with the in-clinic test Canine VacciCheck^®^ (produced by Biogal, Kibbutz Galed, Israel, and supplied in Italy by Agrolabo, Scarmagno, Italy) following the manufacturer’s instruction. VacciCheck^®^ is a rapid semiquantitative dot-ELISA-based kit able to determine specific antibody titers against CPV-2, CDV, and CAdV-1 in serum, plasma, or whole blood of dogs. This test has good specificity and sensitivity for each virus and can be very useful in daily veterinary practice to evaluate the real protection of dogs (and cats) against diseases preventable by core vaccines, as suggested by the international vaccination guidelines and by many other studies [1,33,45,46,47,48,49]. In this test, the antibody concentration is directly proportional to the color intensity of the related spots read by the CombScale and expressed as “S” units on a scale from 1 to 6. The S value of 0 (S0) is equivalent to an antibody titer <1:20 for CPV-2, <1:8 for CDV, and <1:4 for CAdV-1 and is considered negative, while the S value of 3 (S3) is equivalent of the threshold values of 1:80 for CPV-2, 1:32 for CDV, and 1:16 for CAdV-1. Antibody titers equal to or higher than S3 are considered indicative of a significant positive response, corresponding to a good protection against these three core diseases (Table S1). In this study, results were divided into four categories (unprotected, weak positive, medium positive, and high positive) based on the threshold values of each pathogen, and dogs with antibody titers equal to the different threshold values were considered medium positive. Medium to high positive results were expressed as Protective Antibody Titers (PATs) (Table S2), as already done in our recent previous study [50] following the suggestions of the literature [22,51,52,53,54,55,56,57,58].
+
+### 2.3. Statistical Analysis
+
+Statistical analyses were carried out using GraphPad Prism 9 (La Jolla, CA, USA), values at *p*-value < 0.05 were considered statistically significant, and those with *p*-value < 0.1 as a tendence. In order to determine significant differences between protected and unprotected vaccinated dogs, the Chi-square (χ^2^) analysis was performed. All antibody titer data were transformed with log~2~. The Shapiro–Wilk test was used to verify the normal distribution of data, together with the not-parametric Kruskal–Wallis and Mann–Whitney tests.
+
+## 3. Results
+
+### 3.1. Dog Population
+
+A total of three hundred fifty plasma/serum samples of owned older dogs were included in the study. The age ranged from 5 to 19 years, with two hundred fifty-eight seniors (73.7%) and ninety-two geriatrics (26.3%). Considering the breed, two hundred thirty-six were purebred (67.4%) belonging to sixty-four different breeds, while one hundred fourteen were crossbred (32.6%); the most representative breeds were Golden Retriever (18, 5.1%), Labrador Retriever (16, 4.65%), Poodle and Beagle (both 13, 3.7%), and Cocker Spaniel (12, 3.4%). Regarding their size, one hundred twenty-four were small (35.4%), one hundred twenty-three medium (35.1%) and one hundred and three large (29.4%). Considering sex and reproductive status, one hundred eighty-four (52.6%) were females (eighty-nine sexually intact and ninety-five neutered) and one hundred sixty-six (47.4%) were males (one hundred thirty-two sexually intact and thirty-four neutered). Collectively, two hundred twenty-one dogs (63.1%) were intact, while one hundred twenty-nine (36.9%) were neutered. Regarding their health status, two hundred sixty dogs were healthy (74.3%), while ninety (25.7%) had one or more clinical problems including neoplasms (63, 18.0%), endocrinopathies (13, 3.7%), heart problems (7, 2.0%), and others (7, 2.0%), many of which could impact immune function. Finally, for all three hundred fifty patients the vaccination history was known: three hundred forty-one (97.4%) dogs were vaccinated at least once in their lives, while nine (2.6%) had never been vaccinated in their lifetimes. Regarding time elapsed from the last vaccine injection, one hundred fifteen out of the three hundred forty-one vaccinated older patients were vaccinated ≤1 year before sampling (33.7%), one hundred thirty-four received their last vaccination >1–≤3 years earlier (39.3%), and the remaining ninety-two were vaccinated more than 3 years earlier (27.0%).
+
+### 3.2. Antibody Titers and Protection of the Aging Dogs
+
+Antibody titers specific for the three diseases ranged from <1:20 to >1:640 for CPV-2, from <1:8 to >1:256 for CDV, and from <1:4 to >1:128 for CAdV-1. Specific PATs for CPV-2, CDV, and CAdV-1 were found in three hundred and ten (88.6%), two hundred thirty-one (66.0%) and two hundred eighty-eight (82.3%) out of the three hundred fifty older dogs, respectively. Percentages were a little bit higher considering only the three hundred forty-one vaccinated dogs (89.4%, 67,4% and 83.9%, respectively). These percentages were significantly higher if titers just below the threshold values (weak positive) were also considered: 95.7% CPV-2 (335/350 dogs), 89.4% CDV (313/350 dogs), and 95.4% CAdV-1 (334/350 dogs) showing that unprotected aging dogs represent a minority (4.3%, 10.6 and 4.6%, respectively).
+
+PATs found in the elderly dog population both overall and divided into categories (age, size, sex and reproductive status, health status, and time since last vaccination when applicable) are shown in Table 2, Figure 1 and Figure S1.
+
+Results of the Chi square test applied to three hundred fifty dogs are reported in Table 3 (in bold red statistically significant *p*-values are highlighted). Figure 2, Figure 3, Figure 4 and Figure 5 show the statistical results related to the three hundred fifty dogs divided into the different categories.
+
+Finally, results of the nine unvaccinated older dogs are reported in Table 4.
+
+Dogs with PATs simultaneously for all the three diseases were found to be more than half of the entire study population (one hundred eighty-five out of three hundred fifty, 52.9%): of these, one hundred forty-nine were seniors (80.5%) and thirty-six were geriatrics (19.5%). On the contrary, dogs resulting as unprotected for all pathogens simultaneously were sixteen out of three hundred fifty (4.6% of the population), of which twelve were seniors (75%) and four were geriatrics (25%).
+
+### 3.3. Results According to the Different Variables
+
+#### 3.3.1. Age
+
+Comparing elderly dogs, geriatric dogs had statistically lower antibody titers against CDV than senior ones resulting in lower protection both in terms of percentages (56.5% vs. 69.4%, *p* = 0.0254) and PAT values (*p*-value = 0.0295) (Figure 2). In addition, CPV-2 geriatric dogs tended to be less protected than senior ones (83.7% vs. 90.3%, *p* = 0.0869). The same difference was not seen for CAdV-1 (80.4% vs. 82.9%, *p* = 0.5881) (Table 2).
+
+*Figure: Figure 2 Antibody titers against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus type 1 (CAdV-1), considering the variable ages of the three hundred fifty dogs (Mann–Whitney test).*
+
+#### 3.3.2. Size
+
+Considering the variable breed size positivity rates for all three diseases increased as the size raised, and larger elderly dogs tended to be more protected than smaller ones for CPV-2 both in terms of percentages (94.2% large dogs vs. 87.8% medium dogs and 84.7% small dogs, *p* = 0.0772) (Table 2), and PAT values, the latter in a statistically significant way (large vs. small *p =* 0.0041, large vs. medium *p =* 0.0353) (Figure 3).
+
+*Figure: Figure 3 Antibody titers against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus type 1 (CAdV-1), considering the variable sizes of the three hundred fifty dogs (Kruskal–Wallis test).*
+
+#### 3.3.3. Sex and Reproductive Status
+
+Regarding sex and reproductive status, neutered females and intact males had the highest number of protected animals for all the three core vaccines, but no differences were statistically significant except CAdV-1 (*p* = 0.0013), probably due to the higher percentage of unprotected intact females (31.5%) (Table 2). However, no statistical differences were found when comparing PAT values.
+
+#### 3.3.4. Health Status
+
+Considering health status, healthy dogs tended to have a greater number of protected subjects than unhealthy ones for CPV-2 in terms of percentages (90.4% vs. 83.3%, *p* = 0.0835) (Table 2), and in a statistically significant way of PAT values (*p =* 0.0153) (Figure 4).
+
+*Figure: Figure 4 Antibody titers against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus type 1 (CAdV-1), considering the variable health statuses of the three hundred fifty dogs (Mann–Whitney test).*
+
+#### 3.3.5. Time Elapsed since the Last Vaccination
+
+As time passed, vaccine protection has shown a significant decline in the number of protected dogs for CDV (*p* = 0.0007) and CAdV-1 (*p* < 0.0001) (Table 2). Moreover, dogs vaccinated less than 1 year before were more protected than dogs vaccinated more than 3 years before, and these differences were statistically significant for all the three diseases in terms of PAT values (CPV-2 *p* = 0.0002, CDV *p* = 0.0003, CAdV-1 *p* < 0.0001) (Figure 5).
+
+*Figure: Figure 5 Antibody titers against Canine Parvovirus type 2 (CPV-2), Canine Distemper Virus (CDV), and Canine Adenovirus type 1 (CAdV-1), considering the variable time elapsed since the last vaccinations of the three hundred fifty dogs (Kruskal–Wallis test).*
+
+## 4. Discussion
+
+Senior and geriatric pets increasingly represent a reality with which veterinary practitioners worldwide have to deal with daily. Therefore, knowing their immune responses against the most important vaccine-preventable diseases is a great help in their management given the almost total lack of precise indications in the scientific literature.
+
+The results of this study are comforting since they were in line with those of our previous work on a cohort of one thousand and twenty-seven Italian dogs of different ages showing 90.8%, 68.6%, and 79.8% protection against CPV-2, CDV, and CAdV-1, respectively [51]. In fact, specific PATs were found in the majority of elderly dogs, with percentages significantly increasing when the weak positive dogs were considered, which demonstrated that just a few aging dogs were not protected.
+
+CPV-2 was confirmed as the pathogen with the highest values both in terms of the number of protected subjects (310/350, 88.6%) and protective effects (1 or 2 titers higher than the threshold value, Figure S1). This supremacy was likely related to CPV-2 extensive field circulation in rural and urban contexts, and to its well-known environmental resistance and its strong immunogenicity. All of these reasons can explain both the natural booster effect of CPV-2 in vaccinated dogs with high rates of seroprotection, also in the elderly canine population [50,51], and, vice versa, cases of disease in adult and older dogs (sometimes even vaccinated) [39,59]. It should also be considered that cats can be infected with CPV-2 and can shed the virus in the environment with their feces, thus posing a real though uncommon risk to dogs [60,61,62].
+
+The second highest rate of positivity was related to canine infectious hepatitis (288/350, 82.3%), a value that was 2.5 percentage points higher than those of the previous study on a cohort of Italian dogs (79.8%) [51]. In the literature, a higher incidence of CAdV-1 infection has been reported in elderly dogs aged over 10 years [63]. Moreover, in this case the high values of CAdV-1 PATs can be related to their environmental resistance; however, circulation in Italy of this virus is restricted to wild animals and some dogs, especially stray ones in the Southern regions, and the last clinical manifestations of the disease in Italian dogs date back many years [64,65]. Moreover, the presence of CAdV-1 PATs could strengthen the hypothesis of a cross-reaction between CAdV-1 and CAdV-2, pathogens equally resistant in the environment for weeks or even months affecting the canine respiratory tract, circulating in dogs’ populations and spread from dog to dog through coughing and present in canine hepatitis vaccines instead of CAdV-1 due to the adverse effects of the latter [33,66,67].
+
+The lowest positivity rate was associated with CDV (231/350, 66.0%). This was a predictable result since this virus is labile in the environment and its immunogenicity is lower than that of the previous two viruses and even with successful vaccination it tends to develop lower titers if compared with CPV. Moreover, it has immunosuppressive potential and post-vaccination antibody titers are lower than those of CPV-2 [49,67,68].
+
+More than half of the elderly dog population in this study were found to be protected for all three core diseases, while only sixteen (4%) were unprotected for all. Among them, three dogs had never been vaccinated in their lives, and seven dogs had been vaccinated more than 3 years before. These two conditions probably accounted for the total lack of protection found in those ten dogs. On the other hand, the remaining six unprotected dogs were vaccinated more recently (from 1 month to 2.5 years before). This is a critical point to take into serious consideration when planning the vaccination protocol for an elderly dog.
+
+Finally, none of the nine unvaccinated dogs had PATs for all three diseases, neither seniors nor geriatrics, but five out of nine (55.5%) resulted as protected against the most dangerous one (CPV-2), and three out of the remaining four had CPV-2 titers just below the threshold value (1:40). These results confirmed the circulation of this pathogen in the environment and its ability to stimulate a specific immune response without always causing disease but only in subclinical forms, as previously reported [69,70].
+
+Considering the age variable, geriatric dogs were found to have significantly lower protection for distemper compared to senior dogs (*p*-value = 0.00295), and a trend to significance (*p*-value = 0.0869) was found for parvovirus infection when comparing protected and unprotected animals. However, no differences were found for infectious hepatitis. These results were confirmed by analyzing antibody responses by time since the last vaccination, as protection clearly decreases over time. A decrease in serum antibody levels over time is a well-known event reported by many authors, but generally this decline is not significant, since immunity to core vaccines can persist for life after vaccination, in a manner quite similar to what happens after a natural infection [7,24,71]. These results seem to confirm the indications of the WSAVA guidelines and of other experts [24,33,71], which stress that any antibody titer found in an adult (in this case, even an elderly dog) is a marker of protection because it indicates the presence of an underlying immune memory. In fact, experts underlie that old dogs can die from a core vaccine-preventable disease only if they have never been vaccinated [24], since immunization failures, above all for the most dangerous and widespread pathogen (CPV-2), are uncommon today [59]. This difference between senior and geriatric dogs, however, should give pause for thought about the appropriateness of maintaining a three-year basis vaccination protocol, strongly recommended for adult and senior dogs and cats, to geriatric individuals as well, since this vaccination scheme could not confer protection to older dogs [22,26]. In 2016, Ellis et al. [22] compared the serological responses to different vaccinal antigens (and among them the two core CPV-2 and CDV) in lean and obese geriatric dogs, demonstrating that not only age but also nutritional status could affect the vaccine response, with lean and obese geriatric dogs having lower protection to CPV-2 and CDV than young normal-weight dogs, making this another aspect that should be taken into serious consideration when planning a vaccination protocol for older dogs.
+
+Unexpectedly, the larger aging dogs of this study resulted as more protected than smaller ones, and for CPV-2 this difference resulted statistically significant (*p*-value = 0.0041). This result is not in line with other studies in which smaller dogs better respond to antigenic stimulation and vaccination compared to larger sized dogs [38,58,72,73]. Furthermore, in our recent study on a cohort of one thousand and twenty-seven Italian dogs [50], larger dogs have been generally considered less protected than smaller ones. Nevertheless, considering only CPV-2 protection, the peculiarity of more protected larger dogs has been already reported, especially when considering only the nine hundred fifty-one vaccinated animals. CPV-2 is certainly one of the most known immunogenic viruses, which can strongly stimulate a specific immune response in animals with which it comes in contact (for field or vaccine infections). It is also widely distributed in the environment where it is excreted by sick dogs or healthy carriers (and also by infected cats [60,61,62,74,75]) through their feces, and it can persist for very long periods due to its extreme environmental resistance [67,69,70,76,77]. Furthermore, it is closely antigenically and genetically related to Feline Panleukopenia Virus (FPV) (approx. 98% identity [78]), an equivalently resistant and widespread virus [79]. Larger dogs lead less of a home life and are not ideal for small apartments, while they are considered excellent outdoor companions and need more exercise than smaller ones [80]. Furthermore, many large dogs are kept outside (despite the fact that their owners consider them companions and not working dogs) or have an indoor/outdoor life, and many are allowed to roam free going outside with their owners [81]. Finally, larger breeds are generally chosen by owners who prefer to spend time in outside activities [82], and take their dogs outside more often and for longer periods than owners of smaller dogs. Smaller dogs sometimes live exclusively indoors or go outside but in their owners’ arms or bags, and for these reasons the probability of coming into contact with CPV-2 or FPV might be higher for larger dogs.
+
+Regarding sex and comparing protected with unprotected animals, neutered females and intact males showed higher percentages of covered individuals for all of the diseases. On the contrary, intact females were the group with lower protection for infectious hepatitis (result extrapolated from a Chi-square test, *p*-value = 0.0013). This last result agrees with other studies where sterilization is reported to stimulate immune responses [73], and partially also confirms those of our previous study [50] where neutered dogs have been reported to have higher antibody titers compared with intact ones.
+
+Finally, and as expected, healthy older dogs were significantly more and better protected than unhealthy dogs for CPV2 (*p*-value = 0.00153), while for the other two diseases there were no significant differences. It is likely that the diseases carried by the dogs in this study, which were considered unhealthy (especially neoplasms, and among these lymphomas and mastocytomas, and endocrinopathies), did not have a major impact on the immune system and the response to previous vaccination was as might have been expected, probably because they were either at an early stage or kept under control by proper therapies.
+
+This study was able to demonstrate that with aging the specific immune response towards core vaccines undergoes a physiological decline in elderly dogs, but remains at protective levels for most subjects. In fact, dogs vaccinated less than one year before are more protected than those vaccinated than three years before for all three diseases. This study should then make veterinary practitioners think about the appropriateness to maintain core vaccinations on a three-year basis with aging and not opt instead for closer boosters (every 1 or 2 years) in order not to leave already fragile subjects without protection. Further studies on a larger canine cohort will be needed to better elucidate the peculiar aspects of the vaccine immune response in older dogs highlighted in this work, first of all, the high response of larger dogs against CPV-2. Given the known good correlation between antibody titers and protection suggested by the WSAVA guidelines on vaccination of dogs and cats [33], it is possible however to state that the antibody titers for core vaccines represent a good biomarker of protection. Antibody titration (e.g., with VacciCheck) may be a very useful tool in monitoring canine immunity and could become a standard of care in such a delicate period of the dogs’ life. It would also be interesting to extend the study to older cats to confirm whether the specific immunity for core vaccines remains high also in the feline species despite aging or whether it would be necessary to adapt the vaccination protocols to elderly cats in order to avoid dangerous vaccine failures.
+
+This study has some limitations. First of all, the vaccination protocols chosen and applied for each elderly patient was unknown, since only the date of the last core vaccination was available. Therefore, it cannot be determined whether the good protection evidenced in the elderly dogs of this study is the result of a continuous veterinary approach with vaccinations that are too frequent (i.e., on an annual basis and not on a three-year basis despite guidelines and experts’ indications) or whether it is actually related to the excellent stimulation of the immune system by MLV vaccines administered on a triennial basis during the entire dogs’ life. Moreover, not knowing other details of the lives of these elderly patients (e.g., nutritional status, lifestyle, frequency of veterinary visits, etc.) is another limitation of this study, as this additional information would have helped to better understand and interpret the obtained results. The evaluation of specific vaccine immune response is also incomplete, as it is known that it can stimulate also a strong but not easily measurable cell-mediated immunity [50,51,83]. In elderly subjects, however, the aging process particularly affects cell-mediated immunity [7,15,17,19,20,25,28,29], so adding information related to this type of immune response would not add other useful information. One can argue that the sample size of this study was not adequate, however we believe that the sample we analyzed can be considered representative enough of the elderly canine population in Italy and can be helpful to veterinary practitioners in appropriately managing the increasing number of elderly patients they have to visit and provide care for on a daily basis.
+
+## 5. Conclusions
+
+The number of elderly dogs is steadily growing worldwide, now accounting for half of all veterinary pet patients. It is therefore necessary for veterinarians and owners to work together to maintain a high quality of life for their patients and pets by understanding the biological effects of aging and implementing preventive and therapeutic actions to safeguard their delicate health.
+
+Taken together, the results of this study underlie the importance of optimizing vaccination programs and controlling protection in aging dogs, especially in geriatric ones, in which immunosenescence could hamper mounting an adequate immune response after vaccination, increasing the risk of developing dangerous and even life-threatening infectious diseases. Moreover, given the known difficulty of an elderly individual to mount an immune response toward a new antigen never encountered before, the “trick” to try to ensure vaccine protection even in elderly pets is to make the immune system not lose its memory. This would be possible for continuing vaccinations also in the elderly stages, while explaining this rationale to owners who can feel that their dogs are too old for vaccinations.

fulltext/md/PMC10385523.md

@@ -0,0 +1,98 @@
+---
+pmcid: "PMC10385523"
+pmid: "37505873"
+doi: "10.3390/vetsci10070469"
+title: "Quantitative Analysis of Brain CT Perfusion in Healthy Beagle Dogs: A Pilot Study."
+journal: "Veterinary sciences"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [core, extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Quantitative Analysis of Brain CT Perfusion in Healthy Beagle Dogs: A Pilot Study
+
+## Abstract
+
+### Simple Summary
+
+Cerebral perfusion provides important information about capillary-level hemodynamics of the brain parenchyma, especially in conditions such as an ischemic stroke, brain tumors, and inflammation. The assessment of brain perfusion in dogs has been performed using computed tomography (CT), magnetic resonance (MR), sonography, single-photon emission computed tomography (SPECT), and 2-fluoro-2-deoxy-D-glucose positron emission tomography. However, these methods, except for CT, have limitations and difficulties in obtaining quantitative values. In addition, some of them are cost-prohibitive, particularly SPECT and MR imaging. CT scanners are widely available in clinical hospitals, making CT-based perfusion techniques more accessible for studies. The purpose of this study was to investigate the normal range of perfusion determined via CT in the brains of healthy dogs and to compare values between white matter and gray matter, differences in aging, and each hemisphere. The results of our study showed that the cerebral blood volume and cerebral blood flow values of white matter in the frontal, temporal, parietal, and occipital lobes were significantly lower than gray matter values. No significant differences were observed in the age-related variations or between the left and right hemispheres in other brain regions. In addition, quantitative values of CT perfusion parameters in a specific brain region were presented. This information provided by our study might contribute to a better understanding of brain perfusion in healthy dogs, potentially leading to the earlier diagnosis and improved treatment of brain diseases.
+
+### Abstract
+
+Brain computed tomography (CT) perfusion is a technique that allows for the fast evaluation of cerebral hemodynamics. However, quantitative studies of brain CT perfusion in veterinary medicine are lacking. The purpose of this study was to investigate the normal range of perfusion determined via CT in brains of healthy dogs and to compare values between white matter and gray matter, differences in aging, and each hemisphere. Nine intact male beagle dogs were prospectively examined using dynamic CT scanning and post-processing for brain perfusion. Regional cerebral blood volume (rCBV), regional cerebral blood flow (rCBF), mean transit time, and time to peak were calculated. Tissue ROIs were drawn in the gray matter and white matter of the frontal, temporal, parietal, and occipital lobes; caudate nucleus; thalamus; piriform lobe; hippocampus; and cerebellum. Significant differences were observed between the white matter regions and gray matter regions for rCBV and rCBF (*p* < 0.05). However, no significant differences were identified between hemispheres and between young and old groups in brain regions. The findings obtained in this study involving healthy beagle dogs might serve as a reference for regional CT perfusion values in specific brain regions. These results may aid in the characterization of various brain diseases in dogs.
+
+## 1. Introduction
+
+Cerebral perfusion provides important information about the capillary-level hemodynamics of the brain parenchyma, especially in conditions such as an ischemic stroke [1]. Brain tissue flow can be calculated using several parameters, including cerebral blood flow (CBF), cerebral blood volume (CBV), mean transit time (MTT), and time to peak (TTP). CBV refers to the total volume of flowing blood in a given volume of brain tissue, measured in mL/100 g [1]. CBF measures the volume of blood flow through a given brain volume per unit time, measured in mL/100 g/min [1]. MTT represents the average transit time of blood flow through a specific brain region. It is measured in seconds [1]. TTP is defined as the time required to reach peak enhancement according to the time-attenuation curve (TAC) [2,3].
+
+Computed tomography perfusion (CTP) complements non-enhanced computed tomography (NECT) and computed tomography angiography (CTA) in the evaluation of brain parenchyma and other neurovascular disorders [4]. The first application of CTP for human brain imaging was proposed in 1980 [5]. However, due to limitations of CT acquisition and post-processing systems at the time, this method was not readily available [1,6]. Presently, routine CT protocols for imaging ischemic stroke in humans involve a NECT scan of the brain; supra-aortic CTA; and a dynamic, time-resolved CTP scan [7]. In dogs, the first attempt to measure perfusion using CT was made in beagle dogs in 1991 [8]. The assessment of brain perfusion in dogs has also been performed using magnetic resonance (MR) perfusion-weighted imaging [9,10,11], sonography [12], single-photon emission computed tomography (SPECT) [13,14], and 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography (PET) [15]. However, there are limitations and challenges associated with these methods in obtaining quantitative values. They can be less accessible, time-consuming, and cost-prohibitive, especially in the case of SPECT and MR imaging [6]. CT scanners are widely available in clinical hospitals, making CT-based perfusion techniques more accessible for studies [1,6]. However, quantitative studies on brain CT perfusion in veterinary medicine are lacking.
+
+Aging increases cerebral arterial stiffness, which has a negative impact on cerebral blood flow regulation. It is an important risk factor affecting cardiovascular disease [16]. Various perfusion studies have reported differences in humans and dogs according to aging [13,15,17]. Brain perfusion and metabolism are associated with age-related global changes in humans [18] and dogs [19]. However, the reduction in regional frontal and temporal brain lobe perfusion with aging is consistent in human literature [18,20]. Moreover, regionally decreased metabolism has been demonstrated in aging dogs using 2-deoxy-D-glucose [15]. To the author’s knowledge, information regarding regional perfusion parameters for detailed anatomical brain regions in aging dogs using CT units is scarce.
+
+We hypothesize that brain perfusion values might be decreased in older dogs just as in human patients. Thus, the objectives of this study were to determine normal ranges of regional perfusion parameters for individual brain regions and to investigate the differences in perfusion maps between young and old dogs. Data from this study could serve as the basis for future canine brain diseases.
+
+## 2. Materials and Methods
+
+### 2.1. Animals
+
+The study population consisted of nine intact male beagle dogs, with a median weight of 10 kg (range: 7.9–12.5 kg) and a median age of 118 months (range: 32–118 months). These dogs had no evidence of cerebrovascular disease. Before this study, physical and neurologic examinations, blood tests, urinalysis, radiography, echocardiography, abdominal ultrasonography, and MR examinations were performed to ensure the absence of any remarkable findings and underlying diseases.
+
+### 2.2. Preparation for CT
+
+Anesthesia was induced using alfaxalone (2 mg/kg, IV, Alfaxan inj^®^, Careside. Co., Ltd., Seongnam, Korea) administered through an 18-gauge intravenous catheter with a 3-way stopcock in the cephalic vein. General anesthesia was maintained with isoflurane (Ifran^®^, Hana pharm. Co., Ltd., Seoul, Korea) in oxygen (2 L/min) via endotracheal intubation. Throughout these procedures, electrocardiography (ECG), oxygen saturation, and breathing were monitored. The heart rates of the dogs were monitored during scanning to ensure they remained above 100 beats/min. These rates were recorded before induction and after the CT scan.
+
+All procedures were approved by the Institutional Animal Care and Use Committee at Gyeongsang National University. The dogs were cared for according to the Guidelines for Animal Experiments (GNU-220331-D0035) of Gyeongsang National University.
+
+### 2.3. CT Image Acquisition
+
+A 160-slice CT unit (Aquilion lightning 160, Canon Medical Systems, Otawara, Japan) was used for this study. The dogs were positioned in ventral recumbency on the CT table. Non-enhanced scans were initially performed from the nasal concha to the occipital bone, covering the entire brain. Scanning parameters for a non-enhanced helical CT were as follows: 120 kVp, 150 mAs, 512 × 512 matrix, 180 mm field of view (FOV), 0.75 s rotation time, 0.5 mm slice thickness, and 0.5 mm interval. Immediately after the non-contrast helical CT, a dynamic CT series was performed. The target section selected contained the lateral and largest cross section of the third ventricle, including carotid arteries [6]. The perfusion CT allowed for the assessment of four adjacent 10 mm thick slices. This choice was made to ensure a higher signal-to-noise ratio compared to acquiring eight adjacent 0.5 mm thick slices with the same acquisition parameters [17]. The dynamic volume CT scan took a total of 90 s to capture the entire passage of the contrast material bolus within the brain circulation [6]. Each study consisted of 29 sequential images with a 2 s scan interval. The scanning parameters for the dynamic volume CT were as follows: 80 kVp, 100 mAs effective tube current, 512 × 512 matrix, 150 mm FOV, 1 s rotation time, and 10 mm slice thickness.
+
+The amount of contrast medium administered was determined based on the weight of each dog. Each dog was weighed before each contrast injection procedure to calculate the appropriate dosage of contrast medium. The contrast injection procedure was standardized for all experiments, with a total iodine dose of 370 mgI/kg administered at an injection rate of 2 mL/s. Subsequently, saline flushing at a rate of 2 mL/s using normal saline (0.9% NaCl) was performed using a dual injector (Salient^®^, Medrad Inc., Pittsburgh, PA, USA). Dynamic CT scanning was initiated 5 s prior to the injection of contrast media. This delay in the injection of the contrast material allowed for the acquisition of non-enhanced baseline images [6].
+
+### 2.4. Perfusion Data Analysis
+
+CT data were transferred to a workstation (Vitrea software; Vital Images Inc., Minnetonka, MN, USA) for computational analysis with an automated motion correction (in-plane) [21]. TACs were obtained by subtracting the regional mean baseline CT number in images obtained before the administration of the contrast material from the mean Hounds field unit (HU) values in serial contrast-enhanced images [6]. TACs were determined by manually placing circular regions of interest (ROIs) in the rostral cerebral artery (anterior cerebral artery in humans) and at the center of the dorsal sagittal venous sinus (superior sagittal venous sinus in humans) [17]. These data were filtered both spatially and temporally and automatically configured in the software.
+
+The software utilized a Bayesian estimation algorithm [22] to calculate regional cerebral blood volume (rCBV, mL/100 g), regional cerebral blood flow (rCBF, mL/100 g/min), mean transit time (MTT, s), and time to peak (TTP, s) (Figure 1). A manual ROI was drawn using a mouse-guided cursor to outline free curved shapes within CT images. ROIs were defined to obtain tissue curves. A free curved shape of the ROI was adapted to best fit the shape of the structure being evaluated [17]. The following sets of tissue ROIs were selected: gray and white matter in frontal, temporal, parietal, and occipital lobes; bilateral caudate nucleus; thalamus; piriform lobe; hippocampus; and cerebellum [6,9,10,11,14,17] (Figure 2). Major blood vessels were excluded from the ROIs. The areas of ROIs were calculated in mm^2^.
+
+### 2.5. Statistical Analysis
+
+All statistical analyses were performed using a commercially available software package (SPSS 25.0, IBM, Armonk, NY, USA). The normal distribution of the data was confirmed using the Kolmogorov–Smirnov test. The Mann–Whitney U test was used to compare between young and old groups as well as between white matter and gray matter. Left–right differences within the two groups were evaluated using the Wilcoxon signed rank test. A *p*-value of less than 0.05 was considered statistically significant.
+
+## 3. Results
+
+A total of nine series of dynamic CT scans were conducted. The young-age group (*n* = 4) had a mean age of 32 months, and the old-age group (*n* = 5) had a mean age of 118 months, demonstrating a statistically significant difference (*p* = 0.016). Weight was also significantly (*p* = 0.016) different between the young group (mean 11.7 kg) and the old group (mean 9.1 kg). However, there was no significant difference in heart rate (*p* = 0.286) between the young group (mean 106.5 bpm) and the old group (mean 111.4 bpm). ECG and oxygen saturation remained stable throughout the experiments. Complications such as the paravasation of contrast material were not observed. The total mean values and standard deviations (SD) for each of the 13 brain regions are presented in Table 1.
+
+### 3.1. Comparison of CT Perfusion Measurements between White Matter and Gray Matter
+
+A total of four white matter ROIs and four gray matter ROIs were measured. All white matter regions exhibited significantly lower rCBV and rCBF values compared to gray matter regions. Specifically, in the frontal, temporal, parietal, and occipital lobes, the rCBV values in white matter were significantly lower than those in gray matter (*p* = 0.003, 0.003, <0.0001, and 0.001, respectively). Moreover, the rCBF values in the white matter of the frontal, temporal, parietal, and occipital lobes were significantly lower than those in gray matter (*p* ≤ 0.0001, 0.001, <0.0001, and 0.001, respectively). Differences between white matter and gray matter by *p* values are described in Table 2.
+
+### 3.2. Comparison of CT Perfusion Measurements by Aging
+
+There were no significant differences in the mean values for each of the 13 brain regions between the young and old groups, except for the HU value in the occipital gray matter (*p* = 0.034). In terms of the ROI size, the young group had significantly larger sizes in the frontal white matter (*p* = 0.021), frontal gray matter (*p* = 0.021), temporal gray matter (*p* = 0.001), parietal gray matter (*p* = 0.006), and occipital white matter (*p* = 0.034) than the old group. Details of ROIs, HU values, and regional perfusion values within each group are presented in Table 3.
+
+### 3.3. Comparison of CT Perfusion Measurements by the Side of Hemisphere
+
+There were no significant differences in the mean values for any of the 13 brain regions between right and left sides of the hemisphere. Details of ROIs, HU values, and regional perfusion values within each side of the hemisphere are shown in Table 4.
+
+## 4. Discussion
+
+The aim of this study was to establish reference values of perfusion determined using CTP in the brains of healthy beagle dogs and to compare values between white matter and gray matter, differences in aging, and each hemisphere. We evaluated various brain regions and compared the effects of aging in healthy beagle dogs. This study involved nine beagle dogs. Perfusion values were recorded based on detailed brain structures. The brain regions were selected according to the anatomy of their function and on the basis of CT images [6,9,10,11,14,17]. Furthermore, we compared values of CBV, CBF, MTT, and TTP between the young and old age groups.
+
+Brain CTP provides essential information for patients with acute or chronic cerebrovascular diseases [1]. In humans, changes in brain perfusion patterns have been identified in various brain diseases such as ischemic stroke, brain tumors, and inflammation in the brain [23,24,25,26]. Therefore, understanding normal perfusion levels is crucial for diagnosing brain conditions. In human stroke cases, CBV ranges from 0 to 1.5 mL/100 g and CBF decreases from 0 to 10 mL/100 g/min in patients [23]. Another study has reported that CBF is reduced by 34% or more in the infarction region compared to that in a normal hemisphere [24]. Several studies have revealed that CBV, CBF, and permeability are elevated in brain tumors and have used these measurements to differentiate glioma grades [25,26]. In veterinary medicine, studies have revealed alterations in perfusion parameters in cerebrovascular diseases such as ventriculomegaly, epilepsy, and brain tumors using MR perfusion and CTP [9,11,27,28,29,30]. For instance, a study using MR has found that CBF in the periventricular white matter is significantly lower in dogs with ventriculomegaly [9]. Another study using MR has revealed lower CBF and longer TTP in dogs with idiopathic epilepsy than in healthy dogs, with the most pronounced effects observed in the piriform lobe, thalamus, and temporal gray matter [11]. Moreover, brain tumors in animals show higher CBF, CBV, and permeability than surrounding brain tissue, consistent with human studies [27,30]. In our study, general neurological and hematological examinations as well as MR imaging were performed to rule out subclinical diseases in dogs. Consequently, we established reference data for CBV, CBF, MTT, and TTP in various brain structures using dynamic CTP in beagle dogs. Conducting further studies on brain CTP comparing normal and diseased dogs might aid in the diagnosis of various brain diseases in canine patients.
+
+A previous study has found that cerebral white matter has significantly lower CBV and CBF values than gray matter in both dogs and humans [6]. The results of the present study were consistent with this previous finding, showing that the values of white matter in frontal, temporal, parietal, and occipital lobes were significantly lower than those of gray matter.
+
+Normal CBF values in humans vary from 20 mL/100 g/min in white matter to 80 mL/100 g/min in gray matter [31]. The CBF values observed in our study for dogs were much higher than the published CBF values for humans. CBF values in the brains of rats range from 105 ± 16 mL/100 g/min to 139 ± 19 mL/100 g/min [32], which are also greater than those in humans. A recent study using MR perfusion has revealed that the mean CBF value is 146.8 ± 27.3 mL/100 g/min in the semioval center and 257.9 ± 72.4 mL/100 g/min in the temporal cerebral cortex on dorsal perfusion-weighted images of dogs [10]. These findings suggest that CBF is generally higher in dogs than in humans. Another possible reason for the difference could be the use of isoflurane as an anesthetic. In contrast to human medicine, CT imaging techniques for the brains of dogs require anesthesia [10]. Studies in rats have shown that CBF increases when isoflurane is used as an anesthetic compared to pentobarbital or fentanyl. Such an increase is attributed to intracerebral vasodilation, particularly pronounced in the thalamus and subcortical areas [33,34]. Therefore, the influence of anesthesia cannot be excluded from our study. Thus, when comparing results with clinical measurements, the anesthesia method should be standardized among dogs.
+
+Aging has been associated with changes in brain perfusion due to several reasons. Reduced synaptic density and neuronal size can lead to decreased metabolic activity, resulting in reduced perfusion with increasing age [35]. Increased arterial stiffness associated with advanced aging is also related to lower CBF [36]. In human studies, CBF has been found to decrease with age in various brain regions, including the cingulate, parahippocampus, superior temporal white matter, medial frontal white matter, posterior parietal white matter, and left insular cortex [35]. The decrease in CBF ranges from 3.9 mL/min to 4.8 mL/min (0.52%) per year [37]. Reduced CBF associated with aging has also been reported in dogs. In a single photon-emission CT study, dogs older than 96 months showed significantly decreased CBF values in the frontal lobe, temporal lobe, and subcortical nuclei compared to younger control dogs [13]. Another human study has reported a decrease in CBV in gray matter but an increase in MTT in both white and gray matter with increasing age [37]. In our study, we did not find significant differences in any perfusion measurements between young and old dogs. One possible explanation for this might be the small number of dogs in each group used in this study. Another explanation could be that the young-aged group was fully mature or that the old-aged group was not old enough to exhibit significant changes in brain perfusion. Previous studies have defined “senior” as the last 25% of the estimated lifespan until the end of life [38]. One study has reported that the regional cerebellar metabolic rate in beagle dogs over 14 years of age was significantly higher than that in the younger group [39]. These factors might have hindered our ability to detect significant differences. Alternatively, this finding can be interpreted as a result of an age-related study using CTP in healthy dogs.
+
+A previous human study has found significantly greater brain perfusion parameters in the left cerebral hemisphere than in the right hemisphere, attributed to volume differences [20]. Similar findings have been observed in dogs using SPECT [40]. However, other studies using SPECT [14] and MR perfusion [10] did not identify significant differences in perfusion between the left and right cerebral hemispheres of dogs. In our study, the left and right hemispheres showed no significant differences in ROIs for any perfusion measurements.
+
+The present study has several limitations. Firstly, the sample population was small, and only medium-sized male dogs were used. CBF is known to be higher in women than in men, potentially due to differences in hematocrits, blood viscosity, or circulating estrogen concentrations [37]. However, veterinary medicine studies have found no significant differences between male and female dogs [10]. To strengthen our findings, further studies involving a larger and more diverse sample of dogs in terms of size, age, sex, and breed are necessary. Secondly, we only utilized one method for dynamic CT imaging, which involved a total iodine dose of 370 mgI/kg at an injection rate of 2 mL/s with saline flushing. In a pilot study for this research, we found that high-speed contrast medium injection was effective. However, its clinical application was challenging. The high injection flow velocity can increase the pressure on peripheral veins in small dogs [41]. Therefore, we experimented with an optimal protocol that involved a suitably high but manageable injection speed. Thirdly, ROIs were drawn using only CT images, not MR images. If ROIs were not accurately delineated, especially in distinguishing gray matter and white matter, results may differ from expected outcomes, as observed in earlier studies [28]. Hence, it is crucial to carefully select an ROI and consider using high-quality MR images to ensure the accurate differentiation of variables between white matter and gray matter and to delineate detailed anatomy of the brain. Fourthly, we did not collect or compare results with invasive criterion-referenced techniques for direct perfusion measurements. However, the estimation of cerebrovascular variables using CT has been validated in rabbits [42] and dogs [6,28]. The values obtained in our study closely resembled those reported in dogs, rabbits, and humans [6,28,42]. Fifth, a slice thickness of 10 mm, in a medium-sized dog, is a large volume of the brain. If focal and minor perfusion changes were present, they were likely to remain undetected.
+
+## 5. Conclusions
+
+The results of this study demonstrated the use of CT to estimate rCBV, rCBF, MTT, and TTP values in 13 ROIs in the brain regions of nine healthy beagle dogs. Our measurements were consistent with those reported in human and animal studies. Information provided by our study might contribute to a better understanding of brain perfusion in healthy dogs, potentially leading to the earlier diagnosis and improved treatment of brain diseases. Moreover, further studies will need to be more useful and obtain a lot of data using CTP in dogs of more variable sizes and breeds.

fulltext/md/PMC10394911.md

@@ -0,0 +1,154 @@
+---
+pmcid: "PMC10394911"
+pmid: "37533090"
+doi: "10.1186/s12913-023-09790-0"
+title: "Inventory management practices: implications on the pharmaceuticals expenditure of rabies vaccine in public health facilities, Namibia."
+journal: "BMC health services research"
+year: "2023"
+license: "cc by"
+article_type: "research-article"
+tiers: [extended]
+source: "NCBI E-utilities efetch (db=pmc)"
+---
+
+# Inventory management practices: implications on the pharmaceuticals expenditure of rabies vaccine in public health facilities, Namibia
+
+## Abstract
+
+### Background
+
+To achieve well-regulated distribution, storage, and utilization of the rabies vaccine, health facilities should adhere to standard operating procedures. In Namibia, information on inventory management, utilization, monitoring, and reporting of rabies vaccine adherence to standard operating procedures in public healthcare facilities is insufficient. The aim of this study was to assess adherence to rabies vaccine standard operating procedures and inventory management and to compare rabies vaccine expenditure to the number of patients who received rabies vaccination at the Ministry of Health and Social Services’ public healthcare facilities from 2018 to 2020.
+
+### Methods
+
+A cross-sectional, web-based questionnaire consisting of closed-ended questions was sent to 147 pharmacy staff and warehouse managers working in the 14 regions of Namibia during the period of May 1, 2021, to June 2, 2021. The overall expenditure and the total number of patients vaccinated from 2018 to 2020 were obtained from national-level logistic and vaccination program coordinators. Data were coded and transcribed into Microsoft® Excel® 2013 and analyzed using SPSS® version 27.
+
+### Results
+
+One hundred and thirty-three completed questionnaires were received from sixty-nine public health centers and hospitals. The group of respondents consisted of pharmacist assistants (50%), pharmacy technicians (12%), pharmacists (36.8%), senior pharmacists (0.8%), and chief pharmacists (1.5%). Overall, adherence to standard operating procedures was poor (27.1%). Rabies vaccine distributed to public health facilities from 2018 to 2020 was worth N$75,381,419.91 (~ US$4,074,671.46) and was expected to vaccinate 87,269 patients; however, only 95 cases of both rabies and rabid dog-bite patients were reported. The major inventory management challenges for public healthcare facilities include an inadequate number of pharmacy staff, poor adherence to standardized pharmaceutical warehousing, lack of regular supervision, and inadequate staff training.
+
+### Conclusion
+
+Inventory management practices in public healthcare facilities were not in compliance with standard operating procedures. There is a significant discrepancy between rabies vaccine expenditure and the number of patients that were vaccinated. Therefore, there is a need for adequate staff training on inventory management and regular facility supervision to enforce optimal rabies vaccine inventory management practices.
+
+### Supplementary Information
+
+The online version contains supplementary material available at 10.1186/s12913-023-09790-0.
+
+## Background
+
+Effective pharmaceutical inventory management is crucial in ensuring that vaccines are continuously available in the right quantities and quality at every stage of the vaccine supply chain [1]. The Ministry of Health and Social Services (MoHSS) in Namibia has implemented a Standard Operating Procedures (SOP) manual for inventory management of vaccines and other pharmaceutical commodities in healthcare facilities. The SOP manual was designed to standardize stock management practices in public healthcare facilities. The manual further classifies certain classes of pharmaceuticals as security items, including the rabies vaccine, which takes up a significant portion of the annual pharmaceutical budget [2] and remains highly susceptible to theft. The Central Medical Store (CMS) – which is the division of the MoHSS responsible for acquiring and distributing pharmaceutical commodities to respective regional stores and hospitals – has additionally implemented an integrated pharmaceutical logistics system (IPLS) to ensure an effective pharmaceutical supply chain [3].
+
+The World Health Organization (WHO) estimates that more than 59,000 rabies-related deaths occur globally every year, and over 95% of those occur in Asia and Africa [4]. Rabies is a neglected zoonotic disease [5], and up to 99% of human cases are dog mediated. Thus, the most effective measure for rabies control is ensuring high vaccination coverage among the dog population [4]. Rabies can also be prevented in humans through timely and appropriate administration of post-exposure prophylaxis (PEP), which involves immediately administering the rabies vaccine or human or horse-derived rabies immune globulin (HRIG) after a dog bite or other exposures. It has been estimated that without PEP, approximately three million people worldwide would die every year from rabies [5]. Rabies is regarded as a notifiable disease in Namibia according to the Animal Health Act 1 of 2011^6^. Between 2011 and 2017, 113 human rabies cases were reported in Namibia, mainly from the Northern Communal Areas [6]. Recently, there have been several media reports alluding to low stock levels of medicines at state hospital pharmacies, as well as incidences of medicine theft, including rabies vaccine, which was confiscated by the police while being smuggled across the border [7].
+
+To address poor inventory management, theft, and other challenges, Namibia has implemented several measures to build a strong pharmaceutical inventory management system to monitor stock. At the healthcare facility level, these measures include the use of systems and tools such as the National Pharmaceutical Dashboard and the Facility Electronic Stock Card (FESC) [8]. The FESC which was launched in 2016, is now being used in all health centers, district, intermediate and central hospitals in Namibia to manage all pharmaceutical supplies including rabies vaccine. Furthermore, there are significant challenges among healthcare workers regarding following standardized practices and appropriate documentation of pharmaceutical transactions to generate relevant reports [9]. To address these challenges, Management Sciences for Health (MSH), in collaboration with the United States Agency for International Development (USAID), assisted the MoHSS in implementing an inventory management system in public health facilities to strengthen the supply chain for medicines [10].
+
+Poor inventory management practices and limited usage of pharmaceutical information system data for assessing medicine management activities have been reported at both the national and healthcare facility levels in Namibia [9]. This study assessed adherence to rabies vaccine SOPs for inventory management activities and determined the expenditure on the rabies vaccine in comparison to the number of patients who received rabies vaccination at the MoHSS public healthcare facilities. The study provides an empirical snapshot of the current rabies vaccine inventory management practices and baseline information to track progress and improvement in rabies vaccine stock management over time [11].
+
+## Methods
+
+### Study design
+
+This study was a cross-sectional, quantitative, multicenter descriptive study conducted across the 14 regions in all public hospitals and health centers (37 health centers, 27 district hospitals, four intermediate hospitals, and one national central hospital). In this study, pharmacy staff and warehouse managers employed as pharmacy professionals at MoHSS were purposively selected as the study population. The selection of respondents was based on the assumption that pharmacy professionals have skills in using pharmaceutical tools for proper pharmaceutical inventory management. Consequently, the study excluded any nurses involved in pharmaceutical inventory management activities.
+
+### Data collection
+
+Two types of questionnaires were administered in English, one for the national-level logistic and vaccination coordinators and the other for pharmacy heads and warehouse managers. The questionnaire for facility-level staff was structured into six sections consisting of 31 questions. Semi-structured questionnaires were used to collect information on the facility and pharmacy professionals’ background, ordering systems for rabies vaccine by facilities, CMS delivery of rabies vaccine to the facility, storage security, factors influencing inventory management practices, expenditure, monitoring and evaluation activities of rabies vaccine stock movement of the facility under study, adapted from the SOP of MoHSS. The questionnaire for national-level staff was structured into two sections with a total of 11 questions to collect information on the expenditure of rabies vaccine distributed to public health facilities at the national level and the number of clients vaccinated and reported to the national health information system (NHIS). The national-level logistic and vaccination program coordinators completed the questionnaire to obtain the aggregated overall expenditure and the total number of patients vaccinated in the country from 2018 to 2020. The questionnaires were piloted with two pharmacists from the target populations who were not selected as participants to ensure their reliability and appropriateness. The questionnaire was amended following the recommended revisions. The questionnaires were distributed electronically via email to 147 participants between May 1, 2021, and, June 2, 2021.
+
+### Sampling
+
+The number of participants included in the study was calculated using GPower software version 3.1.9.4. An estimated sample size of at least 133 participants was required to detect an effect size of 0.3 about 80% of the time at four degrees of freedom. Therefore, the required minimum sample size was 133. The expected non-response rate was 10%, thus the maximum sample size was 147 participants. The number of participants per region was determined based on the number of health facilities in each region under study. Sampling without replacement was used to draw a sample. All the names of participants per region were put in a cylindrical transparent box and drawn lottery-wise until all allocated sample participants were drawn. The national-level logistic and vaccination program coordinators were included in the study to determine the overall expenditure and the total number of patients vaccinated in the country.
+
+### Data analysis
+
+The data collected from the web-based survey was exported into a Microsoft Excel spreadsheet, and was coded and analyzed accordingly using the Statistical Package for Social Science (SPSS) version 27. Descriptive and inferential statistical analysis methods were used to understand and derive conclusions from the collected data. Categorical variables were described as counts and percentage frequencies, and simple bar charts were used to visually display categorical variables. Chi-square and Fisher’s exact tests (for small frequencies) were used to determine the associations between categorical variables. Factors associated with the effectiveness of inventory management practices were determined using logistic regression (univariate and multiple). Stepwise regression was used to determine the most important factors for effective inventory management. All the inferential statistical analysis tests were conducted at a 5% level of significance.
+
+## Results
+
+### Overview of respondent’s demographics
+
+In total, 133 questionnaires were completed (response rate of 90.5%). The respondents comprised 57.9% females. Pharmacist assistants completed (n = 65, 48.9%) questionnaires, followed by pharmacists (n = 49, 36.8%), pharmacy technicians (n = 16, 12.0%), chief pharmacists (n = 2, 1.5%), and a senior pharmacist (n = 1, 0.8%). The Kavango East and Omusati regions accounted for (n = 14, 10.5% for both regions), while the Omaheke region represented the smallest number of participants (n = 3, 2.3%) (Table 1). Educational qualifications attained by the respondents were as follows: college certificates (n = 61, 45.9%); diplomas (n = 19, 14.3%); bachelor’s degrees (n = 42, 31.6%); and postgraduate degrees (n = 11, 8.3%). There was an equal number of respondents who had less than 5 years of experience and between 6 and 10 years of experience in practice (n = 53, 39.8% for both groups). This was followed by those who had practice experience between 11 and 15 years (n = 20, 15.0%), and lastly, respondents who had above 16 years of practice experience (n = 7, 5.3%).
+
+Table 1Number of respondents per health facility type and regionRegionHealth Facility typeTotalHealth CentreDistrict HospitalIntermediate HospitalCentral HospitalErongo18009(6.8%)Hardap460010(7.5%)Karas370010(7.5%)Kavango East185014(10.5%)Kavango West27009(6.8%)Khomas405211(8.3%)Kunene35008(6.0%)Ohangwena35008(6.0%)Omaheke03003(2.3%)Omusati2120014(10.5%)Oshana415010(7.5%)Oshikoto24309(6.8%)Otjozondjupa27009(6.8%)Zambezi26109(6.8%)**Total**33(24.8%)79(59.4%)19(14.3%)2(1.5%)133(100%)
+
+### Health facilities background information and pharmacy staff
+
+More than half of the respondents (n = 74, 56%) indicated that they had a secured warehouse in compliance with the SOPs. Most of the respondents (n = 111, 83.3%) reported that pharmaceutical warehouses in public healthcare facilities were managed by a pharmacy professional (Table 2). Only 48 respondents (36.1%) reported that they had received training on medicine inventory supply chain management in the last 5 years.
+
+Regarding the adequacy of pharmacy staff, (n = 94, 70.7%) respondents stated that pharmacy providers were not sufficient to effectively offer services in the healthcare facilities. Most respondents reported that they had individual staff members purposely assigned to manage inventory and warehousing activities (n = 111, 83.5%). However, 108 (81.2%) respondents reported not having dedicated supportive staff/data clerks to assist in PMIS for pharmacy services.
+
+Table 2Overview of warehouse security for rabies vaccine storage, warehouse management by pharmacy professionals, and availability of support staff/data clerks to assist PMIS.RegionSecured warehouse in compliance with SOPWarehouse managed by pharmacy professionalSupport staff/data clerk for recording and reporting PMISYesNoYesNoYesNoErongo367209Hardap647328Karas737319Kavango East11395311Kavango West636309Khomas6510192Kunene537117Ohangwena267117Omaheke033003Omusati77140311Oshana649119Oshikoto549009Otjozondjupa637218Zambezi459036**Total**74(55.6%)59(44.4%)111(83.5%)22(16.5%)25(18.8%)108(81.2%)
+
+### Ordering and distribution of rabies vaccine
+
+Most respondents (n = 126, 94.7%) used consumption data from stock cards and Facility Electronic Stock Card (FESC) to quantify and place rabies vaccine orders from CMS, while (n = 5, 3.8%) reported that they used both morbidity data and consumption data from stock cards/FESC (Table 3). Most respondents (n =���111, 83.3%) were satisfied with the rabies vaccine stock quantity received by their facilities from CMS. Among the respondents who did not receive sufficient quantities of rabies vaccine, 17 (out of 22) encountered stockouts (Table 3). Regarding proper documentation (e.g., delivery books, delivery notes, and invoices) required for managing stock, 51.9% (n = 69) of the respondents stated that CMS made deliveries with proper documentation, while 44.4% stated that the stock was sometimes delivered without proper documentation (Table 3). While regarding accountability for receiving stock, (n = 95, 71.4%) of the respondents reported that there was proper handover.
+
+Table 3Key practices and experiences for rabies vaccine ordering and delivery from supplier- CMS by health facility typePractices and experiencesHealth Facility TypeTotalHealth CenterDistrict HospitalIntermediate HospitalCentral HospitalBaseline data used in ordering rabies vaccine from CMSMorbidity Data11002(1.5%)Consumption3174192126(94.7%)Both Methods14005(3.8%)Satisfied with the quantity received from CMSYes2867142111(83.5%)No5125022(16.5%)Encountered any stock out of rabies vaccineYes4114017(12.8%)No11103(2.3%)CMS-delivery of rabies vaccine with-documentsYes14449269(51.9%)No13105(3.8%)Sometimes18329059(44.4%)Handover of rabies vaccine by CMSYes245613295(71.4%)No9236038(28.6%)
+
+### Respondents’ knowledge and perceptions on factors affecting rabies vaccine inventory management
+
+A total of 44 respondents (33.1%) indicated that they were not aware that rabies vaccine is regarded as a security item. Most of the respondents (n = 117, 88%) agreed that a lockable and secure storage area had a positive impact on improving rabies vaccine stock inventory management. Furthermore, (n = 101, 75.9%) of the respondents agreed that a warehouse managed by one responsible person maximizes the efficiency of inventory management and adherence to SOPs. Most respondents (n = 122, 91.7%) agreed that having adequately trained staff in a facility improves inventory management (Table 4).
+
+A total of 105 respondents, comprising 79% of the participants, agreed that the pharmacy head should approve all stock transactions in the facility. A significant portion of the respondents (n = 125, 94%) agreed that capturing any transactions on FESC in a timely manner maximizes accountability and transparency. Moreover, to monitor rabies vaccine usage in the outpatient department, (n = 51, 38.6%) of the respondents agreed that a list of rabies PEP vaccinated patients should be submitted to replenish stock, while 42.1% of respondents agreed that returning all used vaccine vials should be a prerequisite for replenishing stock from the main pharmacy (Table 4).
+
+Approximately 74.4% of the respondents (n = 99) concurred that conducting weekly/monthly reconciliations of distribution data from the pharmacy and the vaccine administering department is crucial for effective inventory stock management. In addition, (n = 87, 65.4%) of the respondents agreed that having standardized guidelines for registering patients for rabies vaccination can have a positive impact on rabies vaccine stock management, and 74.4% (n = 99) agreed that annual auditing for any transaction can maximize accountability and transparency across the system.
+
+Table 4Respondents level of agreement on key factors affecting rabies vaccine inventory management by facility levelKey factorsHealth Facility TypeTotal(Percentage)Health CenterDistrict HospitalIntermediate HospitalCentral HospitalStorage area should only be accessible by one pharmacy professionalAgree2562140101(76%)Neutral7114224(18%)Disagree16108(6%)Adequately trained staff on inventory management is important for rabies inventory managementAgree307416276(91.7%)Neutral31206(4.5%)Disagree04105(3.8%)Pharmacy head approval should be requiredAgree2864130105(79.0%)Neutral4135224(18.0%)Disagree12104(3.0%)Transactions should always be captured on FESCAgree3174182125(94%)Neutral24107(5.3%)Disagree01001(0.8%)Rabies vaccine vaccinated patients list should be a prerequisite for orderingAgree13317051(38.6%)Neutral14319155(41.1%)Disagree6173127(20.3%)Getting back used rabies vaccine vials should be a prerequisite for orderingAgree143110156(42.1%)Neutral12307049(36.8%)Disagree7182128(21.1%)Annual auditing for transaction should be mandatoryAgree276012099(74.4%)Neutral5144225(18.8%)Disagree15309(6.8%)Always track transaction of rabies vaccineAgree296010099(74.5%)Neutral3107222(16.5%)Disagree192012(9.0%)Standardized guidelines for immunization should be used at all timesAgree234914187(65.4%)Neutral9234137(27.8%)Disagree17109(6.8%)
+
+### Factors influencing rabies vaccine inventory management
+
+Respondents reported that the following would positively influence inventory management: adequate training on inventory management (n = 98, 73.7%); availability of trained/skilled staff to effectively use computerized stock management (n = 97, 72.9%); presence of a drug therapeutic committee (n = 86, 64.7%); supportive supervision and on-the-job training (n = 85, 63.9%); regular monitoring of inventory levels (n = 80, 60.2%); adequate personnel (n = 71, 53.4%); sufficient warehouse space (n = 49, 36.8%); and lastly, availability of manual/SOPs at the facility (n = 46, 34.6%) (Table 5).
+
+Table 5Factors influencing the effectiveness of inventory management to adhere SOPs.Factors for the effectiveness of inventory managementFrequency(Percentage)Adequate training on inventory management and use98(73.7%)Trained staff to use effectively computerized stock management97(72.9%)Functional drug therapeutic committee86(64.7%)On-time supervision and on job training by regional and national staff85(63.9%)Inventory levels regularly monitoring80(60.2%)Having adequate personnel71(53.4%)Having sufficient warehouse space for storage49(36.8%)User manual/SOP in place46(34.6%)
+
+### Monitoring and evaluation of rabies vaccine inventory management
+
+Regarding the monitoring of stock movement, all participants (n = 133, 100%) reported that they were using either stock cards/FESC in the pharmacy store. A total of 87 respondents (65.4%) reported recording stock on the stock cards/FESC, which shows the stock balance of rabies vaccine on a weekly, monthly, and yearly basis in the pharmacy store. Meanwhile, (n = 68, 51.1%) respondents reported that there was a rabies vaccine register book in use to register patients vaccinated with rabies vaccine in the outpatient department (Table 6).
+
+Forty-six respondents (n = 46, 34.6%) stated that they did not have stock records in the pharmacy store. On SOP compliance, (n = 65, 48.9%) respondents were non-compliant with SOPs as they did not record vaccinated patients in the rabies vaccine register book in the outpatient department. Among those who reported non-compliance to SOPs, 43 respondents stated that inventory management in their facilities was poor. A total of 97 (72.9%) respondents reported that there was no proper documentation system to compare and contrast rabies vaccine expenditure with the number of patients vaccinated (Table 6).
+
+In Namibia, the acceptable range of pharmaceutical wastage rate (% expenditure on expired and damaged stock) is less than 2% at the facility level [12]. In this study, (n = 68, 51.1%) respondents stated that their facilities were within the acceptable range for expired and damaged rabies vaccine stock, while 46% of the respondents indicated that there was no record at all. All respondents indicated that they submitted PMIS reports to the national level, but only 61 out of 133 respondents received feedback from the national level. Among all respondents (n = 96, 72.2%), indicated that there was no regular supervision by the regional pharmacist. Overall, (n = 93, 69.9%) respondents reported that they were satisfied with the level of rabies vaccine inventory management in their health facilities.
+
+Table 6Analysis of rabies vaccine stock record, register of rabies vaccinated patients, and supervision systemKey areas of analysisHealth Facility TypeTotal(%)Health CenterDistrict HospitalIntermediate HospitalCentral HospitalTools used for recording of inventory managementOnly stock Card11002(1.5%)Only FESC6328248(36.1%)FESC & Stock Card264611071(62.4%)Stock record shows the stock balance regularlyYes224716287(65.4%)No11323046(34.6%)Availability of an outpatient rabies vaccine register bookYes173911168(51.1%)No16408165(48.9%)Expenditure of rabies vaccine and number of patients vaccinated with rabies vaccineExpenditure tally with patients vaccinated7139231(23.3%)Expenditure > actual use14005(3.8%)No documentation to compare the usage256210097(72.9%)Number of vials-damaged and expiredAcceptable range18418168(51.1%)Not acceptable22004(3.0%)Not documented133611161(45.9%)Regular supervision by regional pharmacistYes10225037(27.8%)No235714296(72.2%)Overall satisfaction of participants on rabies vaccine inventory managementGood8293040(69.9%)Bad255016293(30.1%)
+
+### Further analysis to determine significant relationships and differences
+
+To measure the association between categorical variables, chi-square and Fisher’s exact tests (for small frequencies) were used. A statistically significant (p < 0.05) relationship was detected between overall inventory management practice and the following variables: occupation, secured warehouse, baseline-to-order rabies vaccine, vaccine handover, PMIS tools used, stock record, outpatient rabies vaccine patient register, accountability and transparency without patient register, expenditure, number of people vaccinated, PEP vials wastage rate, and regional pharmacist regular supervision.
+
+Multiple logistic regression was conducted to determine the factors associated with the effectiveness of inventory management. A statistically significant difference was noted in the way different respondents responded to certain questions. There was a significant difference (p < 0.05) in overall inventory management practice based on the following variables: professional level of pharmacy cadre, availability of secured warehouse, proper vaccine handover, FESC usage for ordering rabies vaccine, availability of stock record, availability of outpatient rabies vaccine patient register, PEP vials wastage rate, PEP vials wastage documentation system, and active regional pharmacist regular supervision. Furthermore, stepwise regression was used to determine the most influential factors on inventory management. A high PEP vials wastage rate within an acceptable range showed an odds ratio (OR) of 167.33 (95% CI: 2.82-42642.05, p = 0.041) as the most influential determinant factor for poor inventory management.
+
+## Discussion
+
+This study was conducted to ascertain public healthcare facilities’ adherence to pharmaceutical SOPs for inventory management of the rabies vaccine and to compare rabies vaccine expenditure to the number of patients vaccinated with the rabies vaccine in Namibian public healthcare facilities. Adherence to SOPs helps to ensure that there is efficient inventory management practice at all times, clarifies roles and responsibilities, and facilitates the delegation of duties by providing clear and precise instructions [12]. Additionally, SOPs provide standards for monitoring and supervision of pharmacy services and can be used as training tools and a reference document for new staff members [12].
+
+Human resources are a key performance driver for pharmaceutical inventory and supply chain management [13]. Shortage of pharmacy human resources was highlighted by several respondents as a significant challenge that affects the effective implementation of inventory management activities. Similar findings were found in studies from Ethiopia and South Africa, which revealed that medicine management in healthcare facilities is worsened by inadequate availability of experienced pharmacy providers at the facility level [3, 11].
+
+Many factors influence rabies vaccine inventory management and adherence to SOPs. Among these, respondents reported that adequate training on inventory management had the highest positive impact. Pharmacy personnel at different supply chain levels should receive intensive training on inventory management and SOPs to maximize performance in on-time recording of transactions on FESC, placing orders accurately, reducing volumes of expiry, and lowering the rates of stock-outs of pharmaceuticals [3, 9, 11]. A significant number of respondents (72.9%) agreed that the availability of trained staff is an important factor in improving inventory management and SOPs compliance. Most pharmacy staff did not receive adequate training on inventory management at the facility level. For example, some participants did not know that rabies vaccine is a security item. The study also revealed poor handling of rabies vaccine transactions and a lack of accountability and monitoring systems. These challenges could be compounded by inadequate training of pharmacy providers on SOPs and limited inventory management experience among most pharmacy staff with less than five years of experience.
+
+Pharmaceutical supplies need to be stored in a secure and spacious warehouse to enable optimal performance of inventory management activities such as regular stocktaking, inventory reconciliation, first-expired-first-out practices, and traceability of batches [13]. Most of the respondents in the study agreed that having a secure warehouse managed by trained pharmacy professionals has a positive impact on inventory management. The findings of this study underscore the importance of a conducive environment in enabling routine implementation of inventory management activities.
+
+The CMS is responsible for planning and implementing on-time delivery of pharmaceuticals to public healthcare facilities to ensure that the stock demands from all public healthcare facilities are met regularly [14]. The study found that most of the health facilities were utilizing a pull system - receiving rabies vaccine stock according to their demand, although proper documentation was not always done. Using the pull system ensures that healthcare facilities are supplied according to their demand, thus maximizing stock availability and reducing the expiry of vaccines [11].
+
+All pharmaceutical products delivered to the healthcare facilities should be accompanied by appropriate documentation (e.g., delivery books, delivery notes, and invoices) to ensure robust stock movement security and proper handover [15]. Lack of a proper stock handover system by CMS drivers to the facilities was indicated by most respondents. The study confirmed that the current CMS stock delivery handover system is weak due to the incomplete format of the delivery books, as the books only show the total number of boxes for all the items but not individual items as presented in an invoice [3].
+
+Accurate record-keeping is important for inventory management. Health facilities either use stock cards or FESC to record rabies vaccine stock transactions in the pharmacy store. FESC is an electronic PMIS system that automatically calculates health product usage and generates stock status reports, produces an accurate requisition to medical stores for the resupply of health products, and also provides auditable records of transactions for accountability purposes [8]. Rabies vaccine is one of the commodities that are highly vulnerable to theft, due to its high cost. Thus, accurate record-keeping makes it easier to trace and identify missing vials of rabies vaccine. Almost all respondents (94%) agreed that on-time capturing of any transactions on FESC maximizes accountability and transparency. Staff training should emphasize good record-keeping practice.
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+Regular supervision of pharmaceutical warehousing/distribution operations by national-level staff or regional pharmacists helps ensure that inventory management functions are implemented according to the SOPs [13]. Poor facility supervision and a lack of regular feedback on inventory management from the national level could be important contributing factors to rabies vaccine wastage and mismanagement [11]. Regular supervision is important to identify gaps in inventory management and provide necessary corrective education and training. Facilities also need to be furnished with regular feedback from the national level on inventory management so that they can improve inventory management practices. Therefore, the national level should address challenges in regular facility supervision and provision of feedback to the facilities.
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+The national-level study respondents reported that there was no focal person for rabies coordination at the national level, even though rabies is a notifiable disease in Namibia. Thus, the rabies control program should be established at the national and regional level or should be integrated into the expanded program on immunization (EPI) unit. Rabies control programs could learn from the EPI, where SOPs for monitoring and reporting systems are in place and enforced at the facility level [4, 16].
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+The dog-bite data routinely collected at the health facility level and transmitted through the NHIS can improve surveillance data for rabies and provide the basis for budget allocation and monitoring of expenditure for rabies vaccines [17]. The rabies vaccine distributed to all public health facilities from 2018 to 2020 was worth N$75,381,419.91 (~ US$4,074,671.46) and was expected to vaccinate 87,269 patients, which is nine hundred and eighteen times more than what was recorded by NHIS for the 95 cases of both rabies and rabid dog-bite patients reported. While rabies vaccine has a high share of the pharmaceutical budget expenditure at the national level, the estimated annual deaths from rabies in Namibia were only 4 in 2015, with approximately 947 to 6070 PEP administered [18]. Potential cases of misuse and theft would likely explain the high number of rabies vaccines ordered in comparison to the number of rabies cases and deaths estimated. Additionally, poor recording and registration of patients could potentially be a challenge in many facilities, leading to inaccurate monthly and annual estimates. The majority of the respondents (72.9%) confirmed that there was no documentation system to compare and contrast rabies vaccine expenditure with the number of patients vaccinated at the facility level. Similarly, another study done in Namibia has revealed that health providers at the healthcare facility level do not always follow SOPs, nor appropriately document pharmaceutical transactions to generate relevant reports [9]. Standardized guidelines to register patients for rabies vaccination should be in place at each health facility level, and the number of rabies cases and dog-bite patients should be reported on time to the NHIS.
+
+### Limitations
+
+This study solely looked at adherence to SOPs for rabies vaccine inventory management and the national expenditure aggregate for rabies vaccines from all public healthcare facilities. The study did not consider all pharmaceutical supply chain inventory management systems, nor the adherence of clinicians to standard treatment guidelines for vaccine prescribing. Exploration of those factors could provide further insights into rabies vaccine inventory management at the healthcare facility level.
+
+## Conclusion
+
+This study has demonstrated that there are serious deficiencies in rabies vaccine storage security, staff in-service training, and usage of inventory tools to generate relevant reports in most healthcare facilities due to non-adherence to pharmaceutical SOPs. While the results have shown that most healthcare facilities had pharmaceutical inventory management tools in place, most facilities did not have accurate records to generate reports for decision-making. The study revealed that there was a huge gap between rabies vaccine expenditure and the number of patients that were vaccinated and reported at the national level. Therefore, the national supply chain directorate and regional pharmacists need to explore measures to train pharmacy staff on inventory management so that they are better equipped to use and generate relevant on-time reports for decision-making and further improvement. Furthermore, there is a need to establish a management position for the rabies control coordinator at the national level to monitor rabies incidence and vaccine inventory. The public healthcare sector needs to establish measures to ensure rigorous rabies vaccine inventory management practices at all levels of the healthcare system in Namibia.
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+## Electronic supplementary material
+
+Below is the link to the electronic supplementary material.
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+Supplementary Material 1