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Immediate induction of varicosities by transverse compression but not uniaxial stretch in axon mechanosensation
Acta Neuropathologica Communications volume 10, Article number: 7 (2022)
Uniaxial stretch is believed to drive diffuse axonal injury (DAI) in mild traumatic brain injury (mTBI). Axonal varicosities are enlarged structures along axonal shafts and represent a hallmark feature of DAI. Here we report that axonal varicosities initiate in vivo immediately after head impact and are mainly induced by transverse compression but not uniaxial stretch. Vertical and lateral impacts to the mouse head induced axonal varicosities in distinct brain regions before any changes of microglial markers. Varicosities preferentially formed along axons perpendicular to impact direction. In cultured neurons, whereas 50% uniaxial strain was needed to rapidly induce axonal varicosities in a nanowrinkled stretch assay, physiologically-relevant transverse compression effectively induced axonal varicosities in a fluid puffing assay and can generate large but nonuniform deformation simulated by finite element analysis. Therefore, impact strength and direction may determine the threshold and spatial pattern of axonal varicosity initiation, respectively, partially resulting from intrinsic properties of axon mechanosensation.
Traumatic brain injury (TBI), a worldwide health problem, contributes to about a third of all injury-related deaths in the United States, while most of the 1.7 million TBIs that occur each year belong to mild TBI (mTBI) or concussion . Among the mTBIs, those showing long-term deficits (~ 225,000) approximate to the combined number of patients diagnosed with breast cancer, multiple sclerosis or spinal cord injury each year . However, the prognosis of long-term deficits among mTBIs remains extremely poor, in part due to the gap of knowledge regarding the primary injury that often involves a combination of different mechanical forces to the brain tissue, such as tension, compression, torsion and shear. Diffuse axonal injury (DAI) is the most common neuropathology in mTBI, in which uniaxial stretch is believed to play a central role. It has been postulated that uniaxial stretch induces microtubule breakage to disrupt axonal transport of various organelles and other cargos, causing axonal dysfunction and hence classical symptoms of decreased processing speed and memory impairment [3,4,5,6,7]. Whereas DAI has been extensively described using animal models and postmortem human tissues, mechanistic insights into axonal injury by uniaxial stretch have been provided by elegantly designed in vitro assays using primary neuron cultures combined with compartment chamber, stretchable membrane and/or magnetic tweezers [3, 8,9,10,11]. Despite progress, our understanding of mTBI primary injury remains highly limited, in contrast to more extensive studies of mTBI secondary injury.
Mechanosensation underlying the senses of touch, hearing and balance, and pain, has been well studied, but little is known about how central nervous system (CNS) neurons convert mechanical stimuli into changes in morphology and function. One possible mechanism underlying CNS neuron mechanosensation involves axonal varicosities, enlarged structures along axonal shafts [12, 13]. The geometric irregularity alone in axonal varicosities can profoundly affect axonal conduction and synaptic transmission [13, 14]. Our recent studies showed that fluid mechanical pressure through puffing rapidly (≤ 5 s) and reversibly (≥ 20 min half recovery) induced axonal varicosity formation in cultured CNS neurons . Using Thy1-YFP transgenic mice with a subset of projection neurons expressing YFP, we further visualized axonal varicosity formation immediately after the second impact that took place 24 h after the first impact in a repeated closed-skull impact model (rcTBI) . Consistent with our findings, several groups independently reported that axonal varicosities were observed from 3 h to 3 days after one impact in the brain of Thy1-YFP transgenic mice in different closed-skull mTBI models [15,16,17], as well as in an open-skull TBI model combined with immunostaining for amyloid precursor protein . However, it remains unknown whether axonal varicosities can be induced in vivo immediately but not hours after one mechanical impact, just like rapid axonal varicosity formation in cultured neurons induced by fluid puffing shown in our recent study .
Many in vivo and in vitro model systems have been developed to study various aspects of TBI [19, 20], but cross-system studies are still very limited, hindering a better understanding of the core mechanism underlying CNS mechanosensation and injury. In the present study, by comparing two mTBI mouse models, we discovered that mechanical impact indeed induced axonal varicosity formation in mouse brains immediately after mechanical impact, representing the earliest subcellular event known in mTBI. Our results indicate that whereas impact strength sets the threshold of axonal varicosity formation, impact site and direction likely determine the spatial pattern of axonal varicosity initiation in the brain. Axonal varicosity induction preferentially formed along axons perpendicular to the impact direction, which is consistent with our in vitro results that transverse compression but not uniaxial stretch efficiently induced axonal varicosities using two biomechanical assays with primary neuron culture. Taken together, our results suggest that transverse compression may play a predominant role in mechanical stress-induced axonal varicosity formation, contributing to its spatial pattern in the brain during mTBI primary injury.
Materials and methods
Repetitive closed-skull TBI (rcTBI) model
The detailed procedure was previously described [12, 21]. In brief, male C57BL/6 J and Thy1-YFP-H transgenic mice (8 weeks old; Stock # 003,782 from the Jackson Laboratory) anesthetized with 5% isoflurane and placed in a stereotaxic frame with rounded Kopf head holders (David Kopf Instruments, Tujunga, CA, USA). Temperature was controlled at 37 °C using a feedback temperature controller (Cell Microcontrols, Norfolk, VA). Isoflurane was delivered by nose cone at 2% in air. The heads were shaved and prepped with Betadine. A midline skin incision was made and the skull was exposed. A rubber tip (Precision Associates, Inc., Minneapolis, MN, USA) was mounted on an electromagnetic stereotaxic impact device. The rubber tip was 9 mm in diameter and the rubber had a spring constant of 3.01 N/mm. The tip was fully extended and lowered at a 20-degree angle until the vertex touched the skull at 1.8 mm caudal to bregma and 3.0 mm left of midline. This was confirmed with a hand lens in all cases. The tip was then retracted automatically. The stereotaxic device was moved down by 3.3 mm, and the electromagnetic device was triggered, driving the tip 3.3 mm into the exposed skull at 5.0 m/s with a dwell time of 100 ms. The deformation of the rubber tip spread the impact force over the skull. There were < 3% skull fractures and no immediate fatalities after these injuries. Mice with skull fractures were killed and were not used in any experiments and mice with hemorrhages were excluded from the analyses. After impact, the skin was sutured and the mice were allowed to recover from anesthesia on a warming pad and then returned to their home cages. After 24 ± 1 h, a second identical closed-skull TBI procedure was performed, and then the mice were immediately perfused and fixed. For sham injuries, the same procedure was performed except that the impact device was discharged in the air; the handling of the mice and duration of anesthesia were the same for rcTBI and sham procedures.
Closed-head impact model of engineered rotational acceleration (CHIMERA)
We adopted the CHIMERA system and performed the model as previously described [22, 23]. In brief, the system included an accumulator air tank, pressure regulator, digital pressure gauge, two-way solenoid valve, trigger button and a 50 g free-floating chrome-coated steel piston. We adjusted the pressure regulator to reach 5.36 psi shown in digital pressure gauge, which enabled reliable delivery of piston velocity (~ 6.0 m/s) and hence 0.9 Joule (0.9 J) impact energy. All animal handling procedures in the current study were approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC) and conformed to the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. C57BL/6 J and Thy1-YFP-H transgenic mice (8–12 weeks old; from the Jackson Laboratory) were used. We did not detect any difference between male and female mice in axonal varicosity induction, so their results have been combined in this study. Mice were anaesthetized with isoflurane (induction: 4%, maintenance: 1.5%) in oxygen (0.9 L/min), which stopped right before impact. Lubricating eye ointment was applied to prevent corneal drying. Meloxicam (1 mg/kg) and saline (1 mL/100 g body weight) were administered by subcutaneous injections for pain control and hydration, respectively. The piston strikes the vertex of the head covering a 5 mm area at the midpoint between bregma and lambda. Impacted mice with regained breathing and heartbeat (usually < 15 s after impact) proceeded to cardiac perfusion immediately (from impact to cardiac perfusion < 2 min). Sham mice underwent all of these procedures, except for the impact. Approximately 3% of mice did not regain breathing after impact, and were thus euthanized and excluded from our analysis.
Brain tissue fixation, sectioning, and immunostaining
The procedures of cardiac perfusion, fixation, sectioning, staining and imaging were described in our previous papers [24,25,26,27]. In brief, all mice were deeply anesthetized with Euthasol (Virbac; 100 mg/kg) and perfused transcardially with 20 ml of PBS followed by 20 ml of a 4% formaldehyde/PBS solution. Brains were removed and post-fixed for 1 h in 4% formaldehyde/PBS solution, and then were cut into 3-mm blocks using an acrylic brain matrix (Braintree Scientific, Braintree, MA, USA) and cryoprotected in a 30% sucrose/PBS solution for > 24 h. All parts from the same brain was arranged into one block, embedded in optimal cutting temperature (OCT) media (Sakura Finetek USA, Inc., Torrance, CA, USA), and stored at − 80 °C until sectioning. The tissue blocks were cut with a Microm HM550 cryostat (Thermo Scientific, Waltham, MA, USA) and the 40-µm sections were collected on Superfrost Plus microscope slides (FisherScientific, Pittsburgh, PA, USA) for storage at − 20 °C. After immunostaining, all sections were coverslipped using tris-buffered Fluoro-Gel mounting media (Electron Microscopy Sciences, Hatfield, PA, USA).
Antibodies and immunofluorescence staining
Antibodies used in this study include rabbit polyclonal anti-neurofilament 200 antibody (Cat. #: N41421; Sigma, St. Louis, MO, USA), rabbit polyclonal anti-Iba1 antibody (Wako Pure Chemical Industries, Ltd., Osaka, Japan), rat monoclonal anti-CD68 antibody (Cat. #: MCA1957GA; Bio-Rad; Hercules, CA, USA), Cy3- and Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). All antibodies were used in a 1:200 dilution. The nuclear dye Hoechst 33,342 and Fluorescein-conjugated Wisteria Floribunda Agglutinin (WFA) for labeling perineuronal nets were purchased from Invitrogen (Cat. #: H3570; Carlsbad, CA, USA) and Vector Laboratories (Cat. #: FL-1351–2; Burlingame, CA, USA), respectively. The procedure of immunofluorescence staining was previously described [26,27,28]. Sections were permeabilized in 1% Triton X/PBS for 1 h at room temperature (RT) and then blocked with 2.5% normal donkey or goat serum for 1 h at RT. Sections were then incubated overnight at 4 °C with the primary antibodies in blocking buffer. Twenty-four h later, sections were rinsed for 5 min seven times, incubated with the appropriate secondary antibodies in blocking buffer for 3 h at RT, counterstained with Hoechst 33,342 (1:2500) and/or WFA (1:500) for 10 min, and again rinsed for 5 min seven times. Stained slides were coverslipped with tris-buffered Fluoro-Gel mounting media (Electron Microscopy Sciences, Hatfield, PA, USA). Multiple staining rounds were carried out with at least one slide from each experimental group.
Conventional fluorescent microscopy and spinning-disc confocal microscopy
Fluorescence microscopy and image analyses were carried out as described in previous publications from our laboratory [12, 26, 28,29,30,31,32]. Low magnification images were captured using a Spot CCD camera RT slider (Diagnostics Instruments, Sterling Heights, MI, USA) on a Zeiss Axiophot upright microscope with a 20X/0.50 Plan Apo objective and saved as 12-bit TIFF files. Exposure times were adjusted to ensure that pixel intensity in targeted tissue samples were below saturation, and kept constant across all experimental conditions for each of the specific fluorophores utilized within each round. Representative high magnification images were captured with an Andor Revolution WD spinning disk confocal system (Oxford Instruments, Abingdon-on-Thames, UK) based on a Nikon TiE inverted microscope using a 60X CFI Plan Apo VC water immersion objective with a numerical aperture of 1.40. Z-stack images (8-bit TIFF files) were taken for each region of interest at ~ 0.25 µm steps and flat images were generated using a maximum intensity projection. An YFP + axon with varicosities was defined as having the beads-on-a-string morphology (> 1 varicosity in every 20 μm length) and bead’s diameter > 200% the diameter of its adjacent shafts. All continuous YFP + axonal segments that were longer than 50 μm in length in an image stack (20X) were included to give rise to the number of total axons. For each brain region of each mouse, we captured 1–2 representative and non-overlapping image stacks in addition to random 2D images for quantification. We focused on the following brain regions, corpus callosum (CC) (Bregma between − 1.82 and − 2.30 mm), external capsule (EC) and cortical layer VI (Bregma between + 1.98 and + 1.78 mm), cortical layers I–V (Bregma between + 1.98 and + 1.78 mm), and hippocampus (Bregma between + 1.98 and + 1.78 mm). Closed-skull impacts, staining of brain slides and fluorescent microscopy were carried out in a blinded fashion.
Mouse cortical neuron culture systems and transfection
Cortical neuron culture was prepared from mouse pups at postnatal days 1–3 (P1-P3) using the same procedure as previously described [29, 33]. Rat tail collagen and poly-D-Lysine were used to coat glass coverslips for neuron culture. In briefly, 2 d after neuron plating, 1 μM cytosine arabinose (Sigma-Aldrich, St Louis, MO, USA) was added to neuronal culture media to inhibit glial growth for the subsequent 2 d, then replaced with normal neuronal culture media. Culture media were replenished twice a week by replacing half volume. For transient transfection, neurons in culture at 5–7 DIV were incubated in Opti-MEM containing 0.8 μg of cDNA plasmid and 1.5 μl of Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C.
The nanowrinkled stretchable device and uniaxial stretching assay
An array of microscale rectangular cell loading membranes was fabricated using soft lithography. Linear nanoscale wrinkled structures are patterned on the top surface of the membranes and align to the longitudinal axis of the membranes. The expansion degree of loading membranes was calibrated with the air pressure. Loading membranes were coated with the same procedure as glass coverslips. Mouse cortical neurons were seeded on the membranes and their processes were often partially aligned due to the topographic cues provided by the nanowrinkles. Different air pressures provided by an air pump were used to induce different levels of uniaxial strain, which was further confirmed with the light microscope. In engineering, mechanical strain represents the relative displacement between particles in the body, which can be expressed by a tensor which 6 independent strain components (three normal strains and three shear strains). For the uniaxial stretching assay in this paper, the normal strain along the stretching direction can be expressed by ΔL/L, where L is the original length of the subject and ΔL is the change in length along the stretching direction upon loading.
Fluid micromechanical pressure provided by local puffing
The fluid puffing system was previously described [12, 34]. To provide local fluid puffing, the glass pipette (tip diameter ~ 50 μm) was connected to a syringe via tubing filled with 20 ml Hank’s buffer and elevated at 190 mm above the tip of the pipette. The vertical distance between the pipette tip and cultured neurons was set at 0.4 mm. The formation of axonal swelling was considered a varicosity when it was ≥ 200% width of its adjacent axonal shafts. The onset time is defined as the time for an axonal segment to reach 10 varicosities per 100 μm length during puffing. Of note, under normal conditions without puffing, axon diameters are not perfectly uniform with the presence of a low level of varicosities, similar to the in vivo situation. Thus, the baseline of varicosity density along axons is not absolute zero.
Live-cell timelapse imaging and transmission electron microscopy (TEM)
Neurons growing on 25 mm coverslips or loading membranes were incubated with Hank’s buffer at room temperature 15 min before imaging experiments. The time-lapse imaging setup was built on a Nikon TE2000 inverted microscope. Images were captured with a CCD camera Coolsnap HQ (Photometrics, Tucson, AZ, USA) through yellow fluorescent protein (YFP) or other filter sets with 1 s exposure time. The filters were changed through filter wheels controlled via Lambda 10–3 (Sutter Instrument, Novato, CA, USA) by the MetaMorph software (Molecular Devices). Time-lapse imaging was performed with indicated intervals for hundreds of frames. This procedure was described in our previous papers [24, 35]. For TEM, cultured cortical neurons with or without puffing were fixed with 4% paraformaldehyde and 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. Embedding, sectioning, and imaging with TEM were done in the Campus Microscopy and Imaging facility at The Ohio State University as described previously [12, 31].
Finite element modeling
The deformation and mechanical strain of the axon was estimated using finite element analysis (COMSOL Multiphysics 5.5) (Analyzing the Mechanical Behavior of Cells for Biological Applications | COMSOL Blog by Fallqvist 2018) . The axon is assumed a homogenous and hyperelastic material. The properties of cytoplasm are used since cytoplasm occupies the majority of the cell body and the entire axon. The axon is modeled as a semi-cylindrical body fixed on the bottom substrate. The fluid puffing is simplified as a uniform pressure applied to the half surface of the semi-cylinder. A 2D model is established with the puffing pressure ranges from 0 to 250 Pa. The first principal strain was plotted on the deformed axon (side view). We use first principal strain to represent the strain magnitude. The principal strains refer to the strains that are normal to the planes where the shear strains are zero. The first principal strain is the algebraically largest principal strain.
Results were presented as the mean ± SEM. Two-tailed Student’s t-test was used for comparisons between two groups. One-way ANOVA followed by Dunnett’s test was used for comparing two or more groups to one control group. (*) p < 0.05 and (**) p < 0.01 or 0.001 were considered statistically significant.
Immediate induction of axonal varicosities in closed-skull mTBI models
To determine whether axonal varicosities form in vivo immediately but not hours after a mechanical impact, we adopted the Closed-Head Impact Model of Engineered Rotational Acceleration (CHIMERA), which closely mimics concussion [22, 23]. Both rcTBI and CHIMERA are closed-skull mouse models for mTBI. Their major differences are impact position and head movement post impact, besides the impact number (Fig. 1A,B). In rcTBI, lateral impact took place on one side of the head (3 mm left of the midline and 1.8 mm caudal of bregma). The mouse head was immobilized and impacted twice with 24 h (h) interval. Mice were immediately perfused and fixed after the 2nd impact. In CHIMERA (0.9 J), mice received only one vertical impact on the top of the head (at the midpoint between bregma and lambda) and the head moved after impact. Thus, there were additional linear and rotational loadings from acceleration and deceleration of head movement in CHIMERA. We used Thy1-YFP transgenic mice, in which a subset of projection neurons express YFP, and perfused the mice immediately after CHIMERA impact. YFP fluorescence allowed clear visualization of morphological changes in axons and dendrites of these neurons, including axonal varicosities. A substantial amount of axonal varicosities formed in the corpus callosum (CC) and external capsule (EC) in CHIMERA, whereas no significant increase in CC and a relatively small but significant increase in EC in terms of axonal varicosity formation were found in rcTBI (Fig. 1C,D,F–H and Additional file 2-5). Actually, the highest level of CHIMERA-induced axonal varicosities was found in the EC (Fig. 1G-I). In the cerebral cortex, rcTBI induced axonal varicosities in a multifocal fashion in the superficial cortical layers with a bias around the impacted side, whereas CHIMERA induced axonal varicosity formation throughout all cortical layers (Fig. 1E,I and Additional file 6, 7). Thus, different closed-skull impacts can induce distinct spatial patterns (partially overlap) of axonal varicosities in the brain. More importantly, since there are normally less than 2 min between impact and cardiac perfusion, our results show that mechanical impact can indeed very rapidly, or immediately, induce axonal varicosity formation in vivo, representing the earliest subcellular event that is known in mTBI primary injury.
In CHIMERA, we tested two additional levels of impact energy, 0.5 J and 0.7 J. Both impact energies caused significant increase of axonal varicosities and 0.5 J was most likely the threshold energy, consistent with earlier threshold studies by examining mouse behaviors and staining intensities of endogenous markers . There was a clear correlation between impact strength and abundance (& size) of induced axonal varicosities, but the spatial pattern of varicosity formation largely remained the same, mainly in EC, CC and cortical layers (data not shown). For consistency, we used 0.9 J CHIMERA throughout this paper. Taken together, our results of rcTBI and CHIMERA have further suggested that whereas impact strength sets the threshold, impact direction/site may be a key determinant of brain-region specificity in mTBI-induced axonal varicosity formation at the initial stage of primary injury.
Varicosities are preferentially initiated along the axons perpendicular to impact direction
The spatial pattern of axonal varicosity formation and injury may contribute to predictions of injury-induced neurological symptoms and long-term outcomes. However, it remains unknown what is the impact variable(s) associated with the spatial pattern of primary injury. Potential variables include the impact strength, number, site and direction. In rcTBI, no significant formation of axonal varicosities was observed in CC, in stark contrast with massive formation of axonal varicosities in CC in CHIMERA (Fig. 1C). CC axons are highly parallel to each other, but their orientations to the impact direction of rcTBI and CHIMERA are drastically different. Thus, these CC axons, as well as axons in many other brain regions, received different amounts and types of mechanical forces in the two mTBI models with different impact directions.
In this Thy1-YFP transgenic mouse line, EC had the highest percentage of axons that developed varicosities in CHIMERA (Fig. 1G–I). Some axons in cortical layer VI can be observed in the same focal plane to make 90 degree turn and merge into the EC. Within these axons, we observed the following three types in sham and CHIMERA mice, (1) smooth segment in layer VI and smooth segment in EC (mostly in sham), (2) smooth segment in layer VI and varicosities in axonal segment in the EC in CHIMERA, (3) varicosities in both axonal segments in layer VI and EC in CHIMERA (Fig. 2A). Thus, there were axons with varicosities only formed within a segment that was perpendicular to the impact direction. This is consistent with our recent in vitro results that puffing-induced axonal varicosity formation is a highly localized event and only occurs within the puffing region . This in vivo result suggests that mechanical stress-induced axonal varicosity formation is a highly localized event, at least at the early stage, and those axons perpendicular to impact direction are more likely to contain varicosities.
To further determine whether axon orientation under specific impact direction correlates with the level of axonal varicosity formation, we thoroughly analyzed different brain regions in CHIMERA and rcTBI (Fig. 2B). CHIMERA caused transverse compression onto CC axons and most EC axons, while rcTBI only directly caused transverse compression onto ipsilateral EC axons, but not CC axons. However, it became more complicated in the cortex due to the axons with mixed orientations (Fig. 2C). YFP + axons in cortical layers I-V confirmed with staining for neurofilaments (NFs; an axonal marker) can be perpendicular or parallel (or somewhere in between) to the impact direction, and a significant higher percentage of perpendicular axons contained varicosities in CHIMERA, but not in sham or rcTBI (Fig. 2C–F). Of note, NF staining signals in YFP + axons were present but not highly enriched in axonal varicosities (Fig. 2E).
Cortical layer VI (gray matter) is adjacent to EC (white matter) at the junction of gray and white matter. Whereas EC contained the highest percentage of axons with varicosities in CHIMERA (Fig. 1G-I), the level of induced-axonal varicosities in the cortical layer VI was relatively lower, and comparable to the level in layers I-V (Fig. 2G–K). Thus, axons in the cortical layer VI adjacent to the gray-white-matter interface did not contain more varicosities after CHIMERA impact. There were varicosities formed along axons parallel to the impact direction, but there were significantly more axons perpendicular to the impact direction that contained varicosities (Fig. 2H,I). In rcTBI, although some axonal varicosities can be observed in EC, there was no clear increase of axonal varicosity formation in the layer VI (Figs. 1H and 2K). Similar to layers I–V, significant higher percentage of axons perpendicular to than those parallel to the impact direction in the layer VI contained varicosities in CHIMERA, but not in sham or rcTBI (Fig. 2K). Those axons in the EC also sometimes contained NF staining, and with a low or no enrichment in axonal varicosities (Fig. 2J). Therefore, NF is a widely used marker for axons, but unlikely useful for revealing axonal varicosities in mTBI.
Axonal varicosity formation precedes adjacent microglial activation after closed-skull impact
Due to relatively low levels of axonal varicosity formation in CC and EC in rcTBI compared to CHIMERA, we wondered whether this might result from an overall lower level of brain injury in rcTBI, compared to that in CHIMERA. As a key pathological feature of mTBI, microglial activation is commonly indicated by upregulation of ionized calcium-binding adaptor protein-1 (Iba1) and morphological transition from the ramified resting state into the hypertrophied bushy phenotype . Reactive microglia appeared mainly proinflammatory at the early stage of the mTBI injury . Seven days after the first impact in rcTBI, early studies showed microglial activation in cortex, hippocampus, EC and CC with some bias on the impact side . On the other hand, extensive microglial activation was seen 2 days after impact in multiple white matter regions in CHIMERA [22, 23]. In the present study, we used immunofluorescent imaging and found that in sham mouse brain, the overall expression level of Iba1 was quite low throughout various brain regions including the hippocampus, EC, CC and cortical layers II-VI, except the cortical layers I-II where Iba1 staining was relatedly higher (Fig. 3A–C). Moreover, these Iba1-positive cells displayed ramified morphology, consistent with that of resting microglia. Right after the second impact in rcTBI, extensive increases of Iba1-positve cell density and hypertrophied morphology were present in those brain regions (Fig. 3A–C and Fig. S1A,B), highly consistent with the early report . Importantly, the increases occurred on both sides of the brain, though being biased on the impacted side (Fig. S1A). Immediately after CHIMERA (a single impact), we did not observe any significant change of Iba1 staining, compared to sham, consistent with early results . Overall, rcTBI brains appeared to clearly have higher level of neuroinflammation than CHIMERA brains right after a single impact (Fig. 3A–C), indicating significant brain injury, despite the lower level of axonal varicosity formation in CC and EC in rcTBI.
To further determine whether there was any correlation between axonal varicosity formation and adjacent microglial activation, we performed confocal microscopy on brain slides stained for two different microglial markers. In rcTBI, clear increase of Iba1 + cell density was observed in EC, where smooth axons without any clear varicosities were often found nearby (Fig. 3D, E). In contrast, axonal varicosities in EC, CC and cortical layer VI induced by CHIMERA did not colocalize with Iba1 + cells or significant Iba1 signals nearby (Fig. 3F). Of note, Iba1 is a pan-microglial marker. To verify the increase of neuroinflammation, we performed the staining against CD68, which a transmembrane glycoprotein highly expressed by cells in the monocyte linage and often used as a marker for proinflammatory reactive microglia. The CD68 staining results were consistent with those from Iba1 staining. For instance, CD68 + microglia were often found near smooth axons in EC in rcTBI, but not in Sham or CHIMERA (Fig. 3G). Taken together, despite relatively less axonal varicosity formation in CC and EC, rcTBI induced a significantly high level of microglial activation throughout many brain regions. Therefore, initial formation of axonal varicosities appears to precede and not to require local microglia activation. A caveat here is that since only a subset of axons expressed YFP in this transgenic mouse line, it remains to be determined whether these features can be applicable for all axons.
To determine whether the different patterns of microglial activation resulted from single impact in CHIMERA versus two impacts in rcTBI, we performed repeated CHIMERA with 24 h interval similar to rcTBI. Indeed, there was a significant increase of Iba1 staining and deramified microglia overall in repeated CHIMERA in the CC, EC, cortical layers I-V and cerebellar cortex, compared to single CHIMERA, whereas Iba1 staining density remained relatively low in the hippocampus in repeated CHIMERA, in contrast to rcTBI (Fig. S2). Actually, throughout later time points in single CHIMERA (up to 1 month), significant microglial activation by Iba1 density was present in CC, EC and cortex, but not in the hippocampus, which is consistent with earlier reports [22, 23, 39]. For Iba1 staining of the resting microglia, it is important to note that the DAB staining used in those studies is more sensitive, whereas immunofluorescence staining used here is suitable for double labeling and intensity quantification. Thus, lateral impact but not vertical impact appeared to more effectively induce microglia activation in the hippocampus in the mouse brain, consistent with our hypothesis that the injury pattern in the brain correlates with impact site/direction. Taken together, our results suggest that each suprathreshold impact with particular site and direction induces a distinct pattern of axonal varicosities followed by a delayed and partially overlapping pattern of microglial activation in mTBI.
Axonal varicosities can be rapidly induced in vitro by uniaxial stretch with 50% or more strain
Currently still lacking is a clear understanding of the transmission of mechanical loads from the scale of tissues to axons. A simple head impact can generate heterogeneous mechanical stresses in different brain regions (e.g. with some regions compressed but others stretched). In both mTBI models described above, especially CHIMERA, axon orientation highly correlated with the induction level of axonal varicosities—axons perpendicular to the impact direction contained more varicosities compared to the axons parallel to the impact direction (Fig. 2). One possibility is that depending on their location and orientation, some axons are compressed while others were stretched during mechanical impact. Among various types of mechanical forces, uniaxial stretch was postulated as the major force to induce DAI [3,4,5,6,7]. In fact, uniaxial stretch was the most frequently used way of load application among all TBI in vitro models published in the past decade or so .
To determine whether uniaxial stretch can effectively induce axonal varicosity formation in cortical neurons, we developed a nanowrinkled stretch assay to study cultured cortical neurons. An array of microscale rectangular cell loading membranes was fabricated using soft lithography (Fig. 4A–C). Linear nanoscale wrinkled structures were patterned on the top surface of the membranes and aligned to the longitudinal axis of the membranes. Mouse cortical neurons were seeded on the membranes and cultured for up to 2 weeks. Their processes were often partially aligned due to the topographic cues provided by the nanowrinkles (Fig. 4D). In-plane strain was applied to the neurons via the membranes and the strain direction was along the nanowrinkles. For the axons growing strictly along the nanowrinkles, uniaxial stretch was the only type of mechanical force that they received (Fig. 4D). The relationship between the air pressure and nanowrinkled membrane strain was calibrated. The cortical neurons transfected with YFP were imaged with a conventional fluorescent microscope before stretching. The nanowrinkled and stretchable membrane moved out of the focal plane of the microscope during stretching (2–9 s depending on the target strain, or a similar strain rate ~ 0.06 s−1), and quickly returned back to the original focal plane after reaching the target strain. Then, the neurons were imaged again post stretching.
We tested the effects of a variety of target strains on both axonal and dendritic morphology of cultured cortical neurons. 10–35% strain did not cause any clear morphological changes in axons, including varicosity induction, and the minimal strain to reliably induce axonal varicosities was 50% (Fig. 4E–H and Fig. S3). Similar to our puffing results , dendrites were relatively more resistant to form varicosities compared to axons, but could develop varicosities in distal branches after repeated stretching with 50% strain (Fig. 4F). The relationship of linear strain and stretch time was plotted (Fig. 4G). Therefore, in our nanowrinkled stretch assay, the minimal strain to induce varicosity formation from axons or dendrites was around 50% (Fig. 4H). However, combining tagged magnetic resonance imaging (MRI) and digital image analysis, an early study provided a dense set of displacement measurements in the human brain during mild frontal skull impact and showed that the maximum principal strain was only around 5% . A more recent computation modeling suggested that material heterogeneities at the gray-white interface could lead to a highly nonuniform distribution of stress in axons, reaching the maximal strain around 25% near the interface . Similar maximal strain was reported from another computation modeling for multi-axial acceleration loading in human brain . Therefore, our results from nanowrinkled stretch assay suggest that the maximal strain of uniaxial stretch in the brain during mTBI may be too small to induce axonal varicosities. Of note, axons are viscoelastic, so a caveat here is the strain rate. The strain rate is a key factor in uniaxial stretch-induced axonal injury and will be discussed in a later section.
Physiologically-relevant transverse compression efficiently induces axonal varicosities
Using fluid puffing assay, we recently showed that axonal varicosity formation induced by mechanical stress was unexpectedly rapid (≤ 5 s) and partially reversible (≥ 20 min for half recovery) in cultured hippocampal neurons . This puffing system was modified based on a local drug perfusion system (Fig. 5A), in which the exact same solution was used for both the puffing pipette and the bath each time. Using this system, we tested multiple levels of puffing pressures and found that varicosity induction speed correlated with the pressure value . Our micromeasurement showed that the minimal pressure that rapidly and reliably induced axonal varicosities was 0.25 ± 0.06 nN/µm2 (or 250 ± 60 Pa) onto cultured neurons at the center of puffing area, while the static pressure at the tip of puffing pipette was 190 mmH2O (~ 1.863 nN/μm2 or ~ 1863 Pa) [12, 34]. This pressure value onto the neurons is well within the estimated physiological range in the brain [12, 43]. Here, by using this system, we examined the effect of fluid puffing onto cultured mouse cortical neurons and found that axonal varicosity induction in a similar fashion to our previous in vitro results (Fig. 5B,C). Axonal varicosities were induced rapidly (~ 5 s) and could slowly and incompletely recover (Fig. 5C). We also stained for endogenous NF and found that NF was relatively enriched in some axonal varicosities but not in others (Fig. 5D). This moderate level of NF present in induced axonal varicosities is consistent with our in vivo staining results. To further determine the ultrastructure of induced axonal varicosities, we performed transmission electron microscopy (TEM). Our TEM results show that among puffing-induced axonal varicosities: ∼50% of them with a few MTs remaining, ∼30% without any visible microtubule filament, and ∼20% with extensive vesicular structures (some with mitochondria- and multivesicular body-like structures) (Fig. 5E). Taken together, transverse compression appeared to more effectively induce axonal varicosities, compared to uniaxial stretch.
How do we quantitatively compare the results from the nanowrinkled stretch and fluid puffing assays? The pressure and strain values carry different meaning. However, both types of mechanical forces, uniaxial stretch and transverse compression, induce local deformation in neurons, leading to neuronal mechanosensing and adaptive responses. Thus, to further understand why fluid puffing was more effective in axonal varicosity induction than uniaxial stretch, we performed a 2D finite element analysis for puffing induced local deformation, in order to convert pressure values into deformation or strain to allow comparison between the two biomechanical assays. The mechanical strain within the cells was estimated using COMSOL software bundle. For the sake of simplicity, the axon was assumed to have a semicircular cross-section and firmly bonded to the substrate. The axon was modelled as a hyperelastic body, with the shear modulus of 0.155 kPa and the bulk modulus of 1000 kPa, which were adopted from a relevant study . A distributed pressure was loaded on the right half side (Fig. 5F), with the magnitude ranging from 0 to 250 Pa. The results showed that even a modest distributed pressure could induce considerably large, albeit nonuniform, strains, or local deformation. In particular, a 250 Pa pressure could generate 50% or more strain in more than half of the axonal area, and up to 80% strain in a small axonal area (Fig. 5G). Assuming the strain distribution was uniform in the nanowinkled stretch assay, the 50% strain (50% longer than the original length) would be identical across the entire axon (Fig. 4). Therefore, transverse compression with the physiological-relevant value (250 Pa) can cause nonuniformly distributed local deformation, which is at least on average comparable to, if not bigger than, the one induced by uniaxial stretch with 50% strain.
In the present study, using two different closed-skull impact mouse models, we have shown that mechanical impact can immediately induce axonal varicosity formation in mouse brains, representing the earliest subcellular event known in mTBI. The spatial pattern of initial formation of axonal varicosities in the brain appears to correlate with impact site/direction, which is partially explained by our in vitro results that transverse compression is more effective than uniaxial stretch in axonal varicosity induction. Our simulation has further indicated that transverse compression produces nonuniform but huge local deformation.
CHIMERA induced extensive formation of axonal varicosities in multiple brain regions, which happened immediately after impact (< 2 min between impact and cardiac perfusion) (Fig. 1). To our knowledge, this is the first report for such rapid induction. This in vivo result is highly consistent with our in vitro finding using cultured CNS neurons with the onset time ≤ 5 s . Among published studies using closed- and open-skull models, the earliest observation of axonal varicosity formation so far was 3 h after impact [16, 44]. Moreover, our results confirmed extensive but delayed microglial activation in both mTBI models (Fig. 3), and further showed that axonal varicosities could initiate without nearby microglial activation revealed by markers (Iba1 and CD68), whereas microglial activation could happen adjacent to the axons without any varicosity (Fig. 3D–G). Thus, these results also suggest that microglial activation in mTBI may not require adjacent axonal varicosity formation. Whether and how activated microglia can promote the formation of axonal varicosities during the secondary injury remains to be determined. Moreover, it is possible that certain form of significant axonal injury does not involve varicosity formation or any other morphological change, and therefore cannot be detected by our imaging. Such form of axonal injury remains to be identified in future studies. Nonetheless, axonal varicosity formation is the earliest known subcellular event so far in neuronal injury in mTBI, and can become a valuable biomarker for studying the primary injury in mTBI.
Could different patterns of axonal varicosity formation in rcTBI and CHIMERA result from their difference in head movement post impact? Using the similar setup and procedure as our rcTBI model, a recent study showed that one impact onto the bregma (similar impact site and direction as our CHIMERA model) led to extensive axonal varicosity formation in the CC and EC . A caveat here is that this study used Thy1-YFP-16 mouse line, in which slightly more neurons express YFP compared to Thy1-YFP-H mice used in our studies . Nonetheless, a vertical impact like CHIMERA without head movement post impact like rcTBI caused a similar spatial pattern of axonal varicosities in the brain as CHIMERA but not rcTBI, suggesting that impact site and direction but not head movement post impact, play a key role here. Furthermore, using microglial markers, we found that rcTBI appeared to cause more wide-spread neuroinflammation across brain regions than CHIMERA, indicating that the impact strength of our rcTBI was adequate and not likely the cause of the difference (Fig. 3). Therefore, whether the head moves or not after impact appears not important in terms of the pattern of axonal varicosity formation. Of note, this notion does not argue against that acceleration and/or deceleration loading itself can be sufficient to cause brain injury. Taken together, whereas impact strength dictates the threshold, impact site and direction likely contribute to the spatial pattern of axonal varicosity formation.
Little is known regarding the relationship between the nature of impact and neurophysiological impairment. Our present study revealed an interesting correlation between axonal orientation related to impact direction and varicosity level in CHIMERA and rcTBI (Fig. 2). This may result from localized nature of axonal varicosity formation, as shown by our in vivo results (Fig. 2A) and in vitro data . Our studies focused on an early time window after impact. Within this time period, axonal varicosity formation might not yet spread into other brain regions, maintaining a rather specific spatial pattern of axonal varicosities. However, it is important to note that although rcTBI’s effects on CC and EC axons were consistent with our conclusion, in the cortex rcTBI did not induce biased formation of varicosities along axons perpendicular to the impact direction (Fig. 2). This may partially result from several limitations in our analysis, including limited axon representation by YFP + ones, difficulty in group assignment of axons running neither parallel nor perpendicular to impact direction, indirect transverse compression by surrounding cells for axons parallel to impact direction. Currently, it is also technically challenging to analyze induced axonal varicosities in the hippocampus due to the high density of YFP + neurons and their processes. Limited representation of axon species in the present study can be addressed by follow up studies using transgenic mouse lines expressing fluorescent proteins in different types of neurons.
Axon swelling and pathology are commonly found in the deep gyri at the interface between the gray and white matter across all severities of TBI [4, 41]. Computation modeling showed that material heterogeneities lead to a highly uneven distribution of uniaxial strain in axons, with the highest level (~ 25%) in axonal regions from the interface to ~ 10 μm into the gray matter . In contrast, our results from CHIMERA show that axonal varicosities were preferentially within the EC (white matter) at the EC-L6 interface (Fig. 2A). Furthermore, our results using nanowinkled stretch assay show that, to reliably and rapidly induce axonal varicosities, minimal 50% uniaxial strain was required (Fig. 4), in which the strain was evenly distributed in the axons growing along nanowrinkled grooves. This strain amplitude (50%) is not only the twice of the maximal strain in the gray-white matter interface predicted by computation , but also approximately one order of magnitude higher than the local strain measured by imaging . Thus, our results question whether uniaxial stretch is the main mechanical force that drives DAI. However, an important caveat here is the rate of strain. It was reported that at slow strain rates, axons are able to withstand substantial strains, whereas axonal damage occurs at high strain rates for the same magnitude of strain . The strain rates in our nanowrinkled stretch assay were relatively slow (0.05 to 0.06 s−1) and it took about 9 s to reach 50% strain (Fig. 4). However, stretch assay results with much faster strain rates and various amplitudes had been reported before from multiple groups [3, 9, 11]. Under those conditions, even after many repeated fast stretches, it took from 10 min to several hours for axons to develop clear varicosities [3, 9, 11]. It is possible that uniaxial stretch causes microinjuries of some subcellular structures, such as microtubules, before axons develop varicosities. Clearly, those previous results also indicated that uniaxial stretching is not an effective way to induce axonal varicosities, consistent with our results in the present study.
In stark contrast to uniaxial stretch, transverse compression by fluid puffing effectively induced axonal varicosities. The puffing pressure (0.25 ± 0.06 nN/μm2 or 250 ± 60 Pa) onto the neurons in our experimental setting does fall within the range of what previous investigators used in in vitro and in vivo studies. Using scanning force microscopy, the applied stress (0.27 ± 0.04 nN/µm2) altered exploratory behaviors of the leading edge of neurite processes in vitro in a neuronal cell line . Using brain volumetric and physical data extracted from the literature, a recent study estimated 3.43 mmHg (~ 0.46 nN/μm2) in a normal human brain [12, 43]. Although it is still not feasible to accurately measure micromechanical pressures within a living brain, another study used fluorescent cell-sized oil microdroplets with defined mechanical properties to measure the maximal anisotropic stress generated by mesenchymal cells in living embryos at ∼1.6 ± 0.8 nN/µm2 . Despite the small value of the puffing pressure, our simulation has showed that transverse compression can induce huge local strains that are nonuniformly distributed in part due to axon’s elastic properties, and a major portion of the axon is subjected to more than 50% strain under a 250-Pa pressure, leading to a pattern of local deformation at least comparable to that induced by 50% uniaxial strain (Fig. 5). Actually, this result is highly consistent with the notion that material heterogeneities contribute to large and nonuniform strains. This is because, for an axon, the material heterogeneity (e.g. axially aligned microtubules and plasma membranes) is intrinsically higher in the transverse direction than that in the longitudinal direction. Moreover, different types of mechanical forces (e.g. transverse compression versus uniaxial stretch) may preferentially regulate different mechanosensitive ion channels that are responsible for Ca2+ influx leading to axonal varicosity formation. Our recent studies indicated that transient receptor potential V4 (TRPV4) channel functioned as a major mechanosensitive ion channel in this process , and might be preferentially activated by transverse compression, which still needs to be validated in future experiments. Despite advantages for mechanistic studies, in vitro systems usually do not possess the microenvironment similar to the in vivo situation. In particularly, the two in vitro systems used in the present study do not contain myelin. How myelin regulates axonal varicosity formation induced by transverse compression and uniaxial stretch remains to be determined in future studies. Taken together, perhaps in both central and peripheral nervous systems, axons possess robust elastic property in the uniaxial direction and hence are resistant to stretch-induced injury, but are more sensitive to transverse compression-induced damage.
In summary, our findings lay a solid foundation for future mechanistic investigations of potentially distinct mechanosensitive signaling pathways for specific types of mechanical forces (e.g. transverse compression and uniaxial stretch) in the CNS. Moreover, our findings likely have translational or clinical implications in mTBI. For instance, frontal or vertical impact may preferentially damage the CC, whereas lateral impact may damage the hippocampus at the primary injury stage. However, mouse and human brains differ considerably. Whether the two impact directions correlate with behavioral and cognitive alterations related to CC and hippocampal (or any other brain regions) functions in mice and humans remains to be further investigated.
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We thank Campus Microscopy & Imaging facility at OSU for technical assistance in transmission electron microscopy, Drs. David L. Brody and Thomas Esparza for rcTBI mouse brains, Drs. Sheryl L. Wellington and Wai Hang Cheng for helping to set up the CHIMERA system in our laboratory, and Dr. David L. Brody for comments on several versions of the manuscript. This work was supported, in part, by grants from NIH (R01NS093073 and R21AA024873) to CG. All animal experiments have been conducted in accordance with the NIH Animal Use Guidelines. The authors declare no competing financial interests.
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. Supplemental figures and text.
Additional file 2. Video1: YFP+ axons in sham corpus callosum.
Additional file 3. Video2: YFP+ axons in CHIMERA corpus callosum.
Additional file 4. Video3: YFP+ axons in sham external capsule.
Additional file 5. Video4: YFP+ axons in CHIMERA external capsule.
Additional file 6. Video5: YFP+ axons in sham cortex.
Additional file 7. Video6: YFP+ axons in CHIMERA cortex.
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Sun, C., Qi, L., Cheng, Y. et al. Immediate induction of varicosities by transverse compression but not uniaxial stretch in axon mechanosensation. acta neuropathol commun 10, 7 (2022). https://doi.org/10.1186/s40478-022-01309-8

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text:
Raffles boulevard shopping
The 500 meters long Esplanade Park was built in 1943 and underwent upgrading in 1991.
The complex is directly connected to City Hall MRT station by escalators from the lotto and raffle results 27 june 2015 building entrance and to Esplanade MRT station from Basement 2, which leads to the Esplanade Xchange and then to Marina Square.
This article is about the building in Singapore.
There are a myriad of facilities in-house and nearby to cater for all your needs.The nearest educational institutions are Outram Secondary School, Duke Medical School and Singapore Management University.However in 1944 he was captured in Perak of Malaya and was tortured till death.Pan Pacific Singapore is proud to be named Asias Leading Business Hotel by the World Travel Awards from 2006 to 2012, Worlds Leading Business Hotel by the World Travel Awards from 2007 to 20, and Worlds Leading City Hotel by the World Travel Awards.Marina Mandarin Singapore, by Meritus 6 Raffles Boulevard, marina Square, singapore 039594,.Singapore MRT System Map By Public Bus: 97/97E, 106, 133, 502/502A, 518/518A By Taxi: Taxis may access Bayfront Avenue via Raffles Boulevard or Marina Boulevard (ERP charges apply).Lim Bo Seng Memorial is built to commemorate Mr Lim for his brave acts against the Japanese invasion in 1942.The North entrance is located along Bayfront Avenue; and the South entrance is along Bayfront Link.Coordinates : 11737N 1035111E /.29361N 103.85306E /.29361; 103.85306.Gallery edit Interior view The refurbished end Another view of the complex See also edit References edit External links edit.It is also located near to Singapores Downtown Core Central Business District.
If the train ends at Promenade station, you will need to transfer to the next train heading towards Bayfront MRT.
Supermarkets : The nearest supermarkets are Cold Storage at China Square Central, ntuc Fairprice at Tanjong Pagar Plaza, Market Place Supermarket at Raffles City and Sheng Siong Supermarket at 52 Chin Swee Road.
The "long bar " found in Raffles Hotel is where the famous Singapore Cocktail " Singapore Sling " was first mixed.
Rebranding edit On, the two hotels were rebranded as the Swissôtel The Stamford and Raffles The Plaza (now Fairmont Singapore) when frhi Hotels Resorts took over the hotels' management.
His remains were then buried at MacRitchie Reservoir.Built on the former site.Civic District within the, downtown Core of the city-state of, singapore.Healthcare : For medical consult, General Practitioner clinics in the area, Outram Polyclinic, National Cancer Centre, Singapore National Eye business gift certificate maker Centre, National Heart Centre, Singapore General Hospital in the area to provide specialist healthcare services for you.Education : There are few renowned local and international schools in the vicinity.The former will take up a 60 percent stake in the complex and the latter taking the remainder 40 percent.For more shopping, dining and entertainment purposes you can drive down to Orchard shopping district which is about 10 herbalife coupon code january 2016 minutes drive away.Pei in one of his earliest works in the city state.Gloria Jean's Coffees has also made a return to the country after exiting the country a few years ago.M/Rendezvous Inspired by the locales vibrant energy, Rendezvous Hotel Singapore has recently transformed by infusing an art-inspired character.

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text:
Where were 'Green Book' approved sites?
Here are the locations of some of the businesses listed in the 1949 edition of The Negro Motorist Green Book, a travel guide for African Americans road-tripping in an era of racial segregation.
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Former Burnley nursery nurse publishes new book for children
and live on Freeview channel 276
Nursery nurse Elaine Mercieca, who swapped Burnley for the sunnier climes of Malta, decided to write a book for children after overcoming endometriosis, allergies and two surgeries for a benign brain tumour.
The book, 'Self Love Star', is a fantasy story but one which teaches children aged three to seven ways in which they can love themselves.
Elaine, who married her Matlese husband Joseph at St Mary Magdalene's Church, has lived on the Mediterranean island for 36 years.
She said: "After seeing many adults seeking alternative therapies for many different illnesses, I learned some important areas of self love, which I garnered from different alternative therapists and reading many books that could change our health for the better.
"We don't always need medication and with some illnesses doctors do not always know enough and have no cure.
"The message is for everyone and especially now in these times of Covid-19 it is more relevent than ever to keep healthy and boost the mood.
"I first read the book to children when it was published in a little school in Jamaica in January when we were there on holiday. It was received very well and I gave them a copy to use and inspire parents.
"I will be giving talks about the book here in Malta and in the UK when it is safe again to do so. I was inspired to write the story for young children so they learn these important lessons of loving and caring for their bodies at a young age, possibly saving much suffering in later life.
"It covers things such as eating well and exercising. I worked at Burnley General on the Special Baby Care Unit when I lived in Burnley and set up pre school playgroups."
The book can be bought on Amazon, Book Deposotory, Olympia Publishers and many more outlets.

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text: Assessments of influenza season severity can guide public health action. We used the moving epidemic method to develop intensity thresholds (ITs) for 3 US surveillance indicators from the 2003-2004 through 2014-2015 influenza seasons (excluding the 2009 pandemic). The indicators were: 1) outpatient visits for influenza-like illness; 2) influenza-related hospitalizations; and 3) influenza- and pneumonia-related deaths. ITs were developed for the population overall and separately for children, adults, and older adults, and they were set at the upper limit of the 50% (IT50), 90% (IT90), and 98% (IT98) 1-sided confidence intervals of the geometric mean of each season's 3 highest values. Severity was classified as low if ≥2 systems peaked below IT50, moderate if ≥2 peaked between IT50 and IT90, high if ≥2 peaked between IT90 and IT98, and very high if ≥2 peaked above IT98. We pilot-tested this method with the 2015-2016 season and the 2009 pandemic. Overall, 4 seasons were classified as low severity, 7 as moderate, 2 as high, and none as very high. Among the age groups, older adults had the most seasons (n = 3) classified as high, and children were the only group to have seasons (n = 2) classified as very high. We will apply this method to classify the severity of future seasons and inform pandemic response.

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Refuel after school with this yummy fruit and dip recipe. Yum-oh!
Fruit, chunked and skewered
Strawberries, grapes, pineapple, apples, melon (really any fruit your child finds irresistible)
1 cup low-fat sour cream (or your go-to non-dairy substitute)
2 tablespoons brown sugar (maple syrup, honey, or agave syrup)
2 tablespoons fresh lime juice (lemon or orange will work too)
Sprinkle of cinnamon, optional (because cinnamon makes everything taste better)
Mix low-fat sour cream or dairy substitute, brown sugar or sugar substitute, fresh lime juice, and optional cinnamon in a bowl and combine. Dip skewers in the dip!
Courtesy of Food Network

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Samsung Galaxy F02s Has Launched In India which sales start on 9 April,12 Noon. Samsung Galaxy F02 S is launching on 5 April. A Samsung launch two smartphones yesterday first one is F02 S and the second is Samsung Galaxy F12. Both are launch yesterday’s live stream at 12 Noon on Samsung India’s Youtube channel. You can watch the live stream click here.
Samsung Galaxy F02 s Specifications
This smartphone has a quad rear camera setup of 13 MP+2 MP+2 MP and a front selfie or video calling camera is 5 MP. As far as the smartphone comes with two storage variants which are 3 GB RAM/32 GB Internal whose price Rs 8,999 and 4 GB RAM/64 GB price Rs 9,999.
The Samsung Galaxy F02s is built on a 6.5 HD+infinity V display with 720 x 1600 pixels. It is powered by a Qualcomm snapdragon 450 soc processor and the operating system is running on Android 10. As the battery fuel is 5000 mah. Herewith Samsung launch a Galaxy F12 both smartphones performance level is very expensive.
A Samsung brand launches new smartphones back to back in the global market. Galaxy F02s and Galaxy F12 sales start on 9 April they will available in Flipkart, Amazon, and Samsung India Store.


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Wonder Woman returns in Injustice 2 as a playable character. She is voiced by the same voice actress whom had voiced her before, Susan Eisenberg. Just like the previous game, she sides with the regime. Later when Supergirl landed on earth, she mentored her. later, her Supergirl Tried to free Superman from his prison, but were stop by Blue Beetle and Firestorm. Because he needed help to fight Brainiac, Batman decide to free Superman himself. When Brainiac invaded the earth, she was among many whom were fighting against his army.
A pantheon of terrible Dark Gods has come to Earth, bringing with them death and destruction. People everywhere are renouncing their own faith in favor of the Dark Gods’, causing riots and worse. The Justice League was supposed to be their cavalry, but with their defeat, Diana and Jason are on their own. The Star Sapphires summon Wonder Woman to help them conquer their own threat, and Jason is left truly alone. Diana is left with no choice but to help the Star Sapphires, while praying Jason can hold out against the dark deities until her return…
Upon arriving, Diana and Hermes found themselves under attack by Hades' minions. Though they fought them off, they were surprised to find that time for Zola had passed much more quickly than for them. Her pregnancy was nearly at the end of its term. When they attempted to leave with her, Hades appeared, warning that one of them must stay behind - and if Diana was unprepared to make Hera his wife as promised - she would do. However, upon Eros' pistols, he instead agreed to let them all go in exchange for them. When Diana's back was turned, he fired them at her, and she fell under love's spell to him.
Wonder Woman’s appearance in the early golden age of comics made her the first prominent female superheroine. The psychologist William Moulton Marston created Wonder Woman somewhat as a counter reaction to the presence of prominent male superheroes (at this time Superman, Batman and Captain America), as well as a counterbalance to the "blood curdling masculinity" that was dominant at the time, with the hopes that the character could serve as an inspiration for young children (though in certain ways it was geared more towards female readers.) Marston had been partially motivated to create this character because of the accomplishments of his own wife, who was also an accomplished academic at a time when it was difficult for women to fulfill this role. As a result, the first Wonder Woman series contained many complementary articles and features which sought to highlight the inner power of women. There were articles for instance on the different career paths that women could pursue (according to the standards of the 1940s) as well as a series of stories on famous and accomplished women, called the Wonder Women of History. Marston introduced the character in All-Star Comics #8 in 1941. She became the lead character in Sensation Comics in 1941, and got her first solo book in 1942.
The New 52 version of Earth 2 was introduced in Earth 2 #1 (2012). In that issue, the Earth 2 Wonder Woman is introduced via flashback. She, along with Superman and Batman, are depicted dying in battle with forces from Apokolips five years in the past. This Wonder Woman worshiped the deities of Roman mythology as opposed to the Greek; the Roman gods perish as a result of the conflict. An earlier version of the Earth-2 Wonder Woman, prior to the Apokoliptian invasion, is seen in the comic book Batman/Superman, where she is seen riding a pegasus.
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Shortly thereafter, Wonder Woman is shown being able to summon it with her tiara, have it hover by the War Department, and extend from it a rope ladder with which she could board it. She uses the plane to fly into outer space, and frequently transports Etta Candy and the Holliday Girls, Steve Trevor, and others. During the 1950s, the plane becomes a jet, and is often shown swooping over Lt. Prince's office; she strips out of her uniform at super speed and bounds to the plane. Though the Plane was depicted as semi-transparent for the reader's convenience, in-story dialogue indicated that it actually was completely invisible, or at least able to become so as the need arose.
Trevor was taken to be interrogated using the Lasso of Hestia. Though he tried to resist the lasso's power at first, unwilling to reveal military secrets, he nonetheless was powerless to withstand it and admitted that he was a spy. He told them that he'd been on a mission to observe General Ludendorff and Doctor Poison, that he'd stolen the latter's research notebook, and that his escape had brought himself and the Germans to Themyscira. He described the Great War, informing them that millions of people had died. Hearing this, Diana deduced that only Ares could be behind a war of that magnitude. She tried to persuade Hippolyta to send the Amazons back with him to face the god, but her mother refused, much to Diana's confusion.
Wonder Woman figures were released for Justice League and Justice League Unlimited. Prior to that, there were plans for a show called Wonder Woman and the Star Riders, which similar to Sailor Moon, would have featured Wonder Woman leading a team of teen magical girls. Prototype dolls for the series were made. There was also a statue from the animated Wonder Woman movie and a Wonder Woman action figure for the Justice League War movie. Wonder Woman dolls and figures were also released for the DC Super Hero Girls line.
Due to the format of most golden age comics, the majority of story arcs at the time for all characters were the same, Wonder Woman included. More accurately that is to say that there were not story arcs at all, as issues contained two to three stories, all of which started and concluded within the issue in question. There was therefore not much continuity in Wonder Woman until she reached the silver age. The few exceptions to this were in issues which contained a common theme, such as Wonder Woman meeting some leprechauns and each of the three stories dealing with that. Alternately there were some common themes for the character at the time, one of which was dealing with enemy saboteurs. These were mostly contemporary, and thus started as either the National Socialists in Germany or the Imperial Japanese. Later these became others.
Diana's treacherous paternal half-brother, based on the Greek mythological god of war, who masquerades as a speaker for peace on the Imperial War Cabinet as part of his deceptive master plan of conquest and destruction. Describing the Sir Patrick persona of his character, Thewlis said, "Sir Patrick's entire drive through the other half of the story is to bring about the armistice. That's his whole intention no matter what's going on. He meets Diana and see in her somebody who is sympathetic to his cause, quite vehemently so."
The first of these half-mortal siblings to reveal himself to Wonder Woman was her older half-brother, Lennox Sandsmark, who could transform himself into living, marble-like stone and, before his death, was revealed to be the father of Wonder Girl (Cassie Sandsmark). His killer, the First Born, the eldest progeny of Zeus, would become Wonder Woman's first major super-villain of the New 52.
Mikos delivers Ventouras's dead son to him, presumably killed by the rebel faction. This causes him to seek revenge against the Rebels and Diana. They turn into monsters and attack Diana. Diana fights the witch's forces but is overpowered by the witch herself, but only after retrieving the scroll from Stavros who was already attacked and severely wounded. The scrolls ends up in Julia's hands, and she immediately goes to work decoding it in an effort to save Diana and discover the witch's weakness. Diana is taken to the witch's stronghold, where she is revealed to be Circe, a sorceress possessing the soul of Hecate, the moon god after they had entered into a pact to drive the world into chaos. She tells Diana what became of her aunt Antiope, who was killed by Circe herself after manipulating Antiope's husband Theseus's former wife. Circe explains to Diana that her existence is a threat to her mission, due to Diana's goal of promoting peace and equality among mankind, and therefore she must be eliminated. Circe is about to kill Diana when she is interrupted by Julia and the rebels. Julia had decoded the scroll and knew how to stave off the witch's attacks. But they are beaten by Circe and almost killed when Circe herself is suspiciously summoned off from the island by an unknown force, later revealed to be the god Hermes.
It was later retconned by Gail Simone that Wonder Woman's outfit design had Amazonian roots. During a flashback in Vol. 3, Hippolyta is shown issuing orders to have a garment created for Diana, taking inspiration from the skies on the night Diana was born; a red hunter's moon and a field of stars against deep blue, and the eagle breastplate being a symbol of Athena's avian representations.[volume & issue needed]
Hybrid Physiology: Due to her Amazonian and Old God heritage, Wonder Woman possesses the superhuman abilities typical of these two species, such as superhuman strength, durability, speed, reflexes, agility and stamina, as well as an accelerated regenerative healing factor and the ability to live for thousands of years without visibly aging. In addition, she possesses incredible supernatural powers that allow her to generate and manipulate divine energy in the form of powerful shock waves, as well as possess some mastery over divine electricity. Wonder Woman's amazing abilities far surpass those of any other Amazon, and they are enough to rival the power of an Ancient God, such as Ares or a New God, such as Steppenwolf. Therefore, Wonder Woman is the second most powerful member of the Justice League, second only to Superman.
Gloria Steinem, editor for Ms. magazine and a major supporter of Wonder Woman, stated "... [Marston] had invented Wonder Woman as a heroine for little girls, and also as a conscious alternative to the violence of comic books for boys." Badower described a near-international incident (involving an unnamed Russian general rolling dozens of tanks and munitions through a shady mountain pass) as an outstanding example for standing up to bullies. "She ends up deflecting a bullet back and disarming the general," he says, adding that "she doesn't actually do anything violent in the story. I just think that Wonder Woman is smarter than that."
^ Callahan, Timothy (November 28, 2011). "When Words Collide: The New 52 First Quarter Review". Comic Book Resources. Archived from the original on September 16, 2012. Retrieved September 16, 2012. What is worth reading? "Wonder Woman," definitely. It's the best of the new 52. Brian Azzarello and Cliff Chiang are telling a clean, poetic story with a strong mythological pull and a fierce warrior of a Wonder Woman.
John Byrne's run included a period in which Diana's mother Hippolyta served as Wonder Woman, having traveled back to the 1940s, while Diana ascended to Mount Olympus as the Goddess of Truth after being killed in issue #124. In addition, Wonder Woman's Amazon ally Nubia was re-introduced as Nu'Bia, scripted by a different author. Byrne posited that Hippolyta had been the Golden Age Wonder Woman. Byrne restored the series' status quo in his last issue.
We've already been warned that this outcome will lead to devastating consequences, first of which is the arrival of the Omega Titans, ancient cosmic forces that can hold entire planets in the palm of their hands. The coming of these giant new enemies has been greatly hyped by DC, and the Justice League's new fight to save the world begins in this week's Justice League: No Justice.
In London, they deliver Maru's notebook to the Supreme War Council, where Sir Patrick Morgan is trying to negotiate an armistice with Germany. Diana translates Maru's notes and reveals that the Germans plan to release the deadly gas at the Western Front. Although forbidden by his commander to act, Steve, with secret funding from Morgan, recruits spy Sameer, marksman Charlie, and smuggler Chief Napi to help prevent the gas from being released. The team reaches the front in Belgium. Diana goes alone through No Man's Land and captures the enemy trench, allowing the Allied forces to help her liberate the village of Veld. The team briefly celebrates, taking a photograph in the village, while Diana and Steve begin to develop their own romance.
As the men helped the Amazons prepare for battle against the First Born's army, Diana received news that the First Born had been attacking other gods' realms. With Eros and Artemis, Wonder Woman ambushed the Minotaur at Demeter's home. Unfortunately, the First Born had already defeated Demeter, so Wonder Woman sent her companions to safety while she confronted him by herself.
Despite their displeasure at Diana's capture, Hephaestus was able to bring Lennox and Eros to Hades with him as guests to the wedding, without the aid of Hermes' staff. As the wedding drew nearer, Hades grew annoyed that few of his relatives had agreed to come. Diana's friends were the only attendees, aside from Strife, who merely wanted to cause her namesake emotion. Before the wedding ceremony took place, Hades insisted that Diana should prove her love by wearing his ring. The ring was a noose fashioned with the Lasso of Truth, and if Diana did not truly love him, he would kill her.
Even alongside all of her kindness, compassion, and empathy, Diana is still also a warrior at heart, and when someone she cares about is hurt, becomes far more relentless and ruthless. When seemingly beaten by Ares (who was only growing more powerful from her violence, rage, and hatred), and after having to watch her beloved Steve Trevor die, she flew into an agonizing rage, and she assaulted and brutalized several armed German soldiers with immense speed and ferocity. She even came close to murdering Dr. Poison as vengeance for Steve's death; but after recalling Steve's great love for an undying belief in her and her beliefs, restrained herself and refocused her energies on Ares, harnessing her love for Trevor to overcome her violent emotions and swiftly overpower an angered Ares. Additionally, she resolved to fight even harder after Steppenwolf revealed that he had killed several other Amazons. Outside of battle, however, Diana can also react indigently and negatively towards people who disrespect her, her relatives, or those she cares about. Two such notable instances were when she shot an angry look at Lex Luthor (after he insulted her father Zeus's memory), and when she shoved back Batman (after he had insulted her beloved Steve Trevor's memory). Diana also remains ruthless when facing enemies in battle as Wonder Woman, and she even shows signs of enjoying a good battle when they put up a good fight. This was seen when she briefly smiled and laughed when she was beaten to the ground by Doomsday, and smirked as she stopped the first bullet fired at a group of hostages in London.
Marston was sure he knew what line not to cross. Harmless erotic fantasies are terrific, he said. “It’s the lousy ones you have to look out for—the harmful, destructive, morbid erotic fixations—real sadism, killing, blood-letting, torturing where the pleasure is in the victim’s actual pain, etc. Those are 100 per cent bad and I won’t have any part of them.” He added, in closing, “Please thank Miss Roubicek for the list of menaces.”
While Superman watched out for Zod, Diana visited the deserted Themyscira to speak to her mother, still a clay statue. In her absence, a demon from Tartarus had escaped from its prison and roamed free on the island. Wonder Woman destroyed the creature and sealed the gates to Tartarus with her lasso. Leaving the island, she received word from Batman that Superman was engaging Zod and Faora in battle. Reaching Superman’s side, Wonder Woman steadily overwhelmed Faora until Zod threatened Superman’s life. Wonder Woman was forced to let the criminals go.
Born among the legendary Amazons of Greek myth Princess Diana has a fierce warrior's heart while being an emissary of peace. On a hidden island paradise she was trained in the arts of combat as well as justice and equality. Diana ventured into the 'world of men' armed with magical gifts from the Gods and a message for all men and women - that all the world can be united through compassion strength and understanding.
In May 2017, early tracking had Wonder Woman opening with $65–75 million, and possibly as high as $105 million. The film opened Friday, June 2, 2017, across 4,165 theaters and made $38.7 million on its opening day, including $3.7 million in IMAX. It was the biggest single-day gross for a woman-directed film, ahead of the $35.9 million opening Friday of Catherine Hardwicke's Twilight in 2008 and the biggest opening day for a woman-led comic book superhero film, ahead of Ghost in the Shell ($7 million). This included $11 million it made from Thursday previews, also the best start for a film directed by a woman, surpassing Fifty Shades of Grey's $8.6 million which was directed by Sam Taylor-Johnson, and the third-biggest of the year, behind Beauty and the Beast and Guardians of the Galaxy Vol. 2. Of that, $1.5 million came from IMAX screenings.

domain: entertainment


text:
Sanya doesnâ€™t just have trouble, she has double trouble. Twins Arth and Ansh Sharma, are rich, sexy and sinfully handsome â€“ whatâ€™s a girl to do?
Sanya last saw the twins when she was ten years old. Now, all grown up, she has come looking for gentle Arth, the twin she has loved ever since she can remember. But instead, she is confronted with fiery Ansh, who is hell-bent on seducing her. And whatâ€™s worse, she canâ€™t seem to stop herself from responding to him.
As she chases Arth and is chased by Ansh, Sanya finds herself on a crazy roller coaster ride with no way of getting off. How will she deal with these two very different men in her life? Will she be able to convince one twin that she loves the other?
About the Author
Even as a kid, Sundari VenkatramanÂ she absolutely loved the â€˜lived happily ever afterâ€™ syndrome as she grew up reading all the fairy tales she could lay her hands on. Soon, into her teens, she switched her attention from fairy tales to Mills & Boon. While she loved reading both of these, she kept visualising what would have happened if there were similar situations happening in India; to a local hero and heroine.Â Her imagination took flight and she always lived in a rosy cocoon of romance over the years.
When Sundari Â quit her job at a schoolâ€™s office in the year 2000, she did not know what to do with her life. She was saturated with simply reading books. Thatâ€™s when she got home one evening after her walk and took some sheets of paper and began writing. It was like watching a movie that was running in her head â€“ all those years of visualising Indian heroes and heroines needed an outlet and had to be put into words. Thatâ€™s how her first novel, The Malhotra Bride, took shape.
While she felt discouraged when publishing did not happen, it was her husband who kept encouraging her not to give up. There was no looking back after that. While publishing took a long time happening, Sundari continued to write novels and then short stories and had them published in her blogs. Her luck turned when Indireads approached her to write for them and Double Jeopardy was born.
Expecting Double Jeopardy to be a story of two brothers who fell for the same girl, I snuggled into the bean bag ans settled down to devour this short novella. The book was refreshingly different and it turned out to be something else entirely than what I was expecting. The story beautifully conveys the story of Sanya who is caught between Arth and Ansh, the gorgeous twins from her childhood. The twist in the tale was something we wouldn’t expect and it set the course of the story. In many ways this could even be called as a coming of age story of Sanya.
The way certain topics were handled in the book by Sundari’s characters â€“ subtly and without being judgemental impressed me and I couldn’t help but wish that more and more people were like these characters and not uptight and outright judgemental like how we see many examples in the society.
The writing style is quite lively and unique and it makes you turn the pages till the last page of this 55 page Ebook. I’d definitely recommend this book to you if you’re a lover of short and sweet, somewhat bollywood style stories.


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Energetic, fresh, and entertaining, with an extensive repertoire that spans the decades.
Doctors Orders is fronted by two dynamic vocalists: Kyle Johnson, a veteran of the Washington DC Radio and TV scene as well as a featured performer in regional and national television jingles; and Ms. Heather Rose, who as an Interscope recording artists shared the stage with notables such as Brian McKnight and Tony Bennett along with appearances on Good Morning America and Soul Train. The band’s musicians consist of keyboards, bass, drums, guitar, and a dynamic optional three-piece horn section that will keep your party-goers dancing all night. In addition, two of the musicians in Doctors Orders are experienced DJ’s. If you would like continuous music featuring a DJ on breaks, it’s no trouble for the band to provide that service to you for a nominal charge.
Over the years, Doctors Orders has provided top-notch entertainment for weddings, private events and corporate functions up and down the East Coast. The band’s musical variety and versatility are sure to make your party a memorable one.
Request A Showcase
Please complete the information below and we’ll send you an invitation to the next Doctors Orders showcase.
“Doctors Orders was amazing! They engaged the audience; they were energizing and just truly outstanding! We are already hearing ‘Bring them back next year!’ And, we would like to!” – St. Agnes Foundation Spring Gala
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domain: artistic


How Many Cycles Per Hour In Hot Water Boilers
How long should a boiler run between cycles?
For how long should a central heating boiler run prior to it cycles off? And also how lengthy should it remain off before it cycles back on? The solutions to these concerns will certainly differ from system to system. As a basic regulation, the combination of boiler run time and also off time need to never be any kind of much less than 10 minutes.
How many hours a day should a boiler run?
A 24 kW central heating boiler will utilize 24 kW of energy per hr. If your boiler gets on for a total of five hrs each day, your day-to-day use needs to be around 120 kWh. However if you have actually noticed an increase in your gas bill recently, your central heating boiler can be utilizing even more gas than it should.
Why is my boiler cycling every 2 minutes?
Basically, this suggests that the boiler installed in your house is extra effective than is needed to satisfy need. And you’ll see that the boiler discharges up after that turns off. A boiler that’s as well effective will create more steam than it can condense which will cause brief biking.
How many times should a boiler cycle?
The basic guideline is, anything over 8-10 cycles per hr can be considered excessive. This can differ, depending upon your centers one-of-a-kind scenarios as well as more might be “normal” as well as fewer might be “excessive”. Boilers can cycle for a range of factors.
How do you calculate boiler cycles?
A central heating boiler cycle is the variety of times TDS present in feedwater can focus. For instance, 100 TDS feedwater, cycled five times, would certainly result in 500 TDS. Example: If boiler conductivity = 1,000 and also feedwater conductivity = 100, the boiler is accomplishing 10 cycles.
Should boiler run continuously?
The amount of insulation you have in your home will certainly of training course have some bearing on how much leakage there is, however as a basic regulation The Power Saving Depend on mentions that maintaining home heating running constantly will lead to a continuous loss of power, which it does not advise.
How much electricity does a boiler use per hour?
A 35-kilowatt central heating boiler (which is an average family boiler in the UK) takes in 35 kilowatt-hours well worth of gas. If your 35kW central heating boiler runs for five hours a day, your everyday intake ought to be roughly 175 kWh.
How many hours a day is your heating on?
Policemen coming round to assess the temperature in your spare room is an unusual thought. Typically, UK homes are warmed for about eight hours a day in wintertime– ten hrs a day for houses that have one sustained home heating period as well as 7 hours a day for the more common 2 home heating durations.
How do I know if my boiler is short cycling?
“Brief biking” is when a furnace or central heating boiler keeps transforming on after that off for extremely short periods. A “short duration” in this instance is generally specified as much less than a min approximately a few minutes long. Some short biking furnace will certainly transform on and also off after simply a few secs.
How many times should a boiler fire up?
It must do it when every hr or 2, as well as each time will last a couple of minutes. Once again, this is perfectly typical, so you do not require to do anything. If it’s switching on routinely then switching off, as well as doing this continuously, that’s most likely to signify a fault.


§ 35-3-10. Consideration of bill by senate committee.
The bill, after passage by the house of representatives in its original form as transmitted by the governor, or as amended by the house, shall be transmitted to and received by the senate in the same manner as other bills are transmitted and received, and, upon receipt thereof by the senate, it shall forthwith be referred to an appropriate committee, which shall proceed to the consideration of the bill in the manner as provided in § 35-3-9 for the consideration of the bill by the committee of the house of representatives.
(P.L. 1935, ch. 2187, § 5; G.L. 1938, ch. 7, § 4; G.L. 1956, § 35-3-10.)


Leicester will have to rest striker Jamie Vardy during a "nonsense" festive schedule, says boss Brendan Rodgers.
Vardy, 32, has not missed a Premier League game this season and is the division's top scorer with 16 goals.
Leicester, who are second, play at Manchester City on Saturday, then host leaders Liverpool on 26 December, before trips to West Ham two days later and Newcastle on 1 January.
"The players aren't robots, it's not a computer game," said Rodgers.
Vardy has been key to Leicester's surge since Rodgers took charge in February.
He has 28 league goals in 2019 - five more than any other player - while the Foxes are also in the semi-finals of the Carabao Cup, as well as being the nearest challengers to Liverpool for the title.
On 4 January, three days after the Newcastle match, Leicester host Wigan Athletic in the FA Cup third round.
Asked whether Vardy could cope with the demands of such a schedule, Rodgers replied: "No chance. You cannot risk the fitness of a player.
"You play on the 26th, a high-intensity game against Liverpool, fighting for the result.
"You get that first day of recovery - but it's the second day where it's really tough for a player.
"If we go along that route then the second day, instead of recovering, we're kicking off at 5.30pm at West Ham.
"It's just nonsense really."


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