Name: a preclinical model of exertional heat stroke in mice
Uploaded: 2021-07-01T14:30:00
Description: Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

This work was funded by the Department of Defense W81XWH-15-2-0038 (TLC) and BA180078 (TLC) and the BK and Betty Stevens Endowment (TLC). JMA was supported by financial aid from the Kingdom of Saudi Arabia. Michelle King was with the University of Florida at the time this study was conducted. She is currently employed by the Gatorade Sports Science Institute, a division of PepsiCo R&#38;D.

All procedures have been reviewed and approved by the University of Florida IACUC. C57BL/6J male or female mice, ~4 months old, weighing within a range of 27-34 g and 20-25 g, respectively, are used for the study.
1. Surgical implantation of the telemetric temperature monitoring system
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	<li>Upon arrival from the vendor, allow the animals to rest in the vivarium for at least 1 week prior to surgery to minimize the stress of transportation.</li>
	<li>Group house the mice (maximum of 5 per...

<ol>
	<li>Leon, L. R., Bouchama, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stroke.">Heat stroke.</a> Comprehensive Physiology. 5 (2), 611-647 (2015).</li><li>Laitano, O., Leon, L. R., Roberts, W. O., Sawka, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controversies+in+exertional+heat+stroke+diagnosis,+prevention,+and+treatment.">Controversies in exertional heat stroke diagnosis, prevention, and treatment.</a> Journal of Applied Physiology. 127 (5), 1338-1348 (2019).</li><li>King, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+prior+illness+on+exertional+heat+stroke+presentation+and+outcome.">Influence of prior illness on exertional heat stroke presentation and outcome.</a> PLOS One. 14 (8), 0221329 (2019).</li><li>Carter, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+hospitalizations+and+deaths+from+heat+illness+in+soldiers.">Epidemiology of hospitalizations and deaths from heat illness in soldiers.</a> Medicine and Science in Sports and Exercise. 37 (8), 1338-1344 (2005).</li><li>Howe, A. S., Boden, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat-related+illness+in+athletes.">Heat-related illness in athletes.</a> The American Journal of Sports Medicine. 35 (8), 1384-1395 (2007).</li><li>Wallace, R. F., Kriebel, D., Punnett, L., Wegman, D. H., Amoroso, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prior+heat+illness+hospitalization+and+risk+of+early+death.">Prior heat illness hospitalization and risk of early death.</a> Environmental Research. 104 (2), 290-295 (2007).</li><li>Wang, J. -. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 288 (1), 197-204 (2005).</li><li>Leon, L. R., Gordon, C. J., Helwig, B. G., Rufolo, D. M., Blaha, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermoregulatory,+behavioral,+and+metabolic+responses+to+heatstroke+in+a+conscious+mouse+model.">Thermoregulatory, behavioral, and metabolic responses to heatstroke in a conscious mouse model.</a> American Journal of Physiology. Regulatory, Integrative and Comparative Physiology. 299 (1), 241-248 (2010).</li><li>King, M. A., Leon, L. R., Morse, D. A., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+cytokine+and+chemokine+responses+to+exertional+heat+stroke+in+mice.">Unique cytokine and chemokine responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 122 (2), 296-306 (2016).</li><li>Costa, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=l-Arginine+supplementation+prevents+increases+in+intestinal+permeability+and+bacterial+translocation+in+Male+Swiss+mice+subjected+to+physical+exercise+under+environmental+heat+stress.">l-Arginine supplementation prevents increases in intestinal permeability and bacterial translocation in Male Swiss mice subjected to physical exercise under environmental heat stress.</a> The Journal of Nutrition. 144 (2), 218-223 (2014).</li><li>Hubbard, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+exercise+in+the+heat+on+predisposition+to+heatstroke.">Effects of exercise in the heat on predisposition to heatstroke.</a> Medicine and Science in Sports. 11 (1), 66-71 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+model+of+acute+heatstroke+mortality.">Rat model of acute heatstroke mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 42 (6), 809-816 (1977).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+significance+of+selected+serum+enzymes+in+a+rat+heatstroke+model.">Diagnostic significance of selected serum enzymes in a rat heatstroke model.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 46 (2), 334-339 (1979).</li><li>Hubbard, R. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+physical+effort+in+the+etiology+of+rat+heatstroke+injury+and+mortality.">Role of physical effort in the etiology of rat heatstroke injury and mortality.</a> Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology. 45 (3), 463-468 (1978).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex-dependent+responses+to+exertional+heat+stroke+in+mice.">Sex-dependent responses to exertional heat stroke in mice.</a> Journal of Applied Physiology. 125 (3), 841-849 (2018).</li><li>Garcia, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Ibuprofen+during+Exertional+Heat+Stroke+in+Mice.">Effects of Ibuprofen during Exertional Heat Stroke in Mice.</a> Medicine and Science in Sports and Exercise. 52 (9), 1870-1878 (2020).</li><li>King, M. A., Leon, L. R., Mustico, D. L., Haines, J. M., Clanton, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomarkers+of+multi-organ+injury+in+a+pre-clinical+model+of+exertional+heat+stroke.">Biomarkers of multi-organ injury in a pre-clinical model of exertional heat stroke.</a> Journal of Applied Physiology. 118 (10), (2015).</li><li>Murray, K. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exertional+heat+stroke+leads+to+concurrent+long-term+epigenetic+memory,+immunosuppression+and+altered+heat+shock+response+in+female+mice.">Exertional heat stroke leads to concurrent long-term epigenetic memory, immunosuppression and altered heat shock response in female mice.</a> The Journal of Physiology. 599 (1), 119-141 (2021).</li><li>Leon, L. R., DuBose, D. A., Mason, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+stress+induces+a+biphasic+thermoregulatory+response+in+mice.">Heat stress induces a biphasic thermoregulatory response in mice.</a> American Journal of Physiology-Regulatory, Integrative and Comparative Physiology. 288, 197-204 (2005).</li><li>Laitano, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+metabolic+dysfunction+in+myocardium+following+exertional+heat+stroke+in+mice.">Delayed metabolic dysfunction in myocardium following exertional heat stroke in mice.</a> The Journal of Physiology. 598 (5), 967-985 (2020).</li><li>Iwaniec, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+phase+response+to+exertional+heat+stroke+in+mice.">Acute phase response to exertional heat stroke in mice.</a> Experimental Physiology. 106 (1), 222-232 (2020).</li><li>He, S. -. X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+a+rhabdomyolysis+model+in+mice+with+exertional+heat+stroke+mouse+model+of+EHS-rhabdomyolysis.">Optimization of a rhabdomyolysis model in mice with exertional heat stroke mouse model of EHS-rhabdomyolysis.</a> Frontiers in Physiology. 11, (2020).</li><li>Lopez, J. R., Kaura, V., Diggle, C. P., Hopkins, P. M., Allen, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malignant+hyperthermia,+environmental+heat+stress,+and+intracellular+calcium+dysregulation+in+a+mouse+model+expressing+the+p.G2435R+variant+of+RYR1.">Malignant hyperthermia, environmental heat stress, and intracellular calcium dysregulation in a mouse model expressing the p.G2435R variant of RYR1.</a> British Journal of Anaesthesia. 121 (4), 953-961 (2018).</li><li>Laitano, O., Murray, K. O., Leon, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+mechanisms+of+exertional+heat+stroke+and+malignant+hyperthermia:+evidence+vs.+conjecture.">Overlapping mechanisms of exertional heat stroke and malignant hyperthermia: evidence vs. conjecture.</a> Sports Medicine. 50 (9), 115-123 (2020).</li><li>Casa, D. J., Armstrong, L. E., Kenny, G. 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This technical review aims to provide guidelines for the performance of a preclinical model of EHS in mice. Detailed steps and materials required for the execution of a reproducible EHS episode of variable severities are provided. Importantly, the model largely mimics the signs, symptoms, and multi-organ dysfunction observed in human EHS victims11,19. Furthermore, this model allows for the examination of the mechanism underlying short- and long-term EHS recovery<...

Heat stroke is characterized by central nervous system dysfunction and subsequent organ damage in hyperthermic subjects1. There are two manifestations of heatstroke. Classic heat stroke (CHS) affects mostly elderly populations during heat waves or children left in sun-exposed vehicles during hot summer days1. Exertional heat stroke (EHS) occurs when there is an inability to thermoregulate adequately during physical exertion, typically, but not always, under high ambient temperatures resulting in neurological symptoms, hyperthermia, and subsequent multi-organ dysfunction and damage2. EHS occurs in recreational and elite athletes as well as military personnel and in laborers with and without concomitant dehydration3,4. Indeed, EHS is the third leading cause of mortality in athletes during physical activity5. It is extremely challenging to study EHS in humans as the episode can be lethal or lead to long-term negative health outcomes6,7. Therefore, a reliable preclinical model of EHS could serve as a valuable tool to overcome the limitations of retrospective and associative clinical observations in human EHS victims. Preclinical models of CHS in rodents and pigs have been well characterized8,9,10. However, preclinical models of CHS do not directly translate into EHS pathophysiology due to the unique effects of physical exercise on the thermoregulatory profile and innate immune response11. In addition, previous attempts to develop preclinical EHS models in rodents posed significant restrictions, including superimposed stress stimuli induced by electric shock, insertion of a rectal probe, and predefined maximum core body temperatures with high mortality rates12,13,14,15,16 that do not match current epidemiological data. These represent significant limitations that may confound data interpretation and provide unreliable biomarker indexes. Therefore, the protocol aims to characterize and describe the steps of a standardized, highly repeatable, and translatable preclinical model of EHS in mice that is largely free from the limitations mentioned above. Adjustments to the model that can result in graded physiological outcomes from moderate to fatal heat stroke are described. To the authors&#39; knowledge, this is the only preclinical model of EHS with such characteristics, making it possible to pursue relevant EHS research in a hypothesis-driven manner11,17,18.

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a preclinical model of exertional heat stroke in mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The typical thermoregulatory profiles during the entirety of the EHS protocol and early recovery of a mouse is illustrated in Figure 1A. This profile comprises four distinct phases that can be defined as the chamber heating stage, incremental exercise stage, steady-state exercise stage, and a recovery stage by either a rapid cooling (R) or severe (S) method17. The main thermoregulatory outcomes include maximum Tc achieved (Tc,max) and&#160;the time required to reach T...

Watch this Scientific Journal Video about A Preclinical Model of Exertional Heat Stroke in Mice at JoVE.com

A Preclinical Model of Exertional Heat Stroke in Mice

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...

Previously, only passive models of heatstroke have been well-established. This model of exertional heat stroke is free from major limitations and more closely mimics the human pathophysiology. It is not possible to effectively study heat stroke in humans because it is a life-threatening condition.This preclinical model retains the clinical characteristics of human exertional heat stroke. This technique helps to test the effects of cooling rate on recovery from exertional heat stroke, comorbidities from drug ingestion, as well as long-term consequences of heat stroke exposure. Demonstrating the procedure will be Jamal Alzahrani, a senior PhD student from Dr.Clanton's lab.After anesthetizing the mouse, use eye lube such as the Vet Ointment to protect the animal's eyes from damage or injury during surgery. To prepare the surgical site, shave the lower abdomen with small animal hair clippers or a commercially available hair remover. Administer the first dose of subcutaneous 0.1 milligrams per kilogram buprenorphine.Scrub the area with three washes of povidone-iodine or Chlorhexidine, followed by 70%isopropyl alcohol rinse, then transfer the mouse to the surgical area, use an adhesive drape to isolate the surgical site on the mouse. Using sterile instruments in aseptic technique, make an approximately one centimeter incision on the midline along the linea alba about 0.5 centimeters from the costal margin, then separate the skin from the muscle layer and make a slightly smaller incision on the linea alba being careful not to damage the bowels or internal organs. Once the muscle layer is open, place the sterile telemeter into the intraperitoneal cavity in front of the caudal arteries and veins and dorsal to the digestive organs to allow it to float freely.Close the abdominal opening with a sterile 5-O absorbable suture and close the skin using a simple interrupted stitch with 5-O proline suture. Place the mouse in its clean cage with a snuggle microwave heating pad under the cage. Monitor the mouse every 15 minutes during the first hour of recovery from anesthesia and then return to the animal housing facility.The night before the exertional heat stroke protocol, place the mouse in the environmental chamber at room temperature to acclimate to the chamber. Use the data acquisition system to collect continuous core temperature or TC averaged over 30-second intervals overnight. On the morning of the exertional heat stroke protocol, make sure the mouse is at or below the normal range of diurnal temperature of 36 to 37.5 degrees Celsius before increasing the chamber temperature to ensure that the mouse does not have a fever or has experienced undue stress during this period.Once the mouse is stable and within a range of normal resting core temperature, remove the food and water and weigh the animal. Shut the chamber door and increase the chamber temperature to a target of 37.5 degrees Celsius and 40 to 50%relative humidity or the desired environmental temperature and humidity. Verify the chamber temperature and humidity with a calibrated temperature and humidity monitor.Surround the chamber with a blackout curtain to keep light and disturbances minimal during the protocol. Monitor the mouse continuously during the protocol via remote IR illuminated cameras. Focus the second camera on the temperature and humidity monitor placed close to the running wheel.Make adjustments to the controller for the environmental chamber set point to ensure accurate temperature readings near the animal. Once the chamber has reached its target temperature as measured by the second camera on the temperature monitor, quickly open the chamber door and place the mouse in the forced running wheel. Initiate the forced running wheel protocol at a speed of 2.5 meters per minute, and increase the speed by 0.3 meters per minute every 10 minutes until the mouse reaches a core temperature of 41 degrees Celsius.Once the core temperature has been achieved, allow the speed to remain constant until symptom limitation occurs, characterized by an apparent loss of consciousness, a backward fall or fainting, and the inability to continue running or holding onto the wheel. Confirm the time point when the mouse has three backward rotations on the wheel without signs of a physical response. Alternatively, identify humane endpoint following local IACUC rules to determine when to stop the protocol.This endpoint is slightly above symptom limitation in essentially all mice. To perform the rapid cooling protocol, stop the wheel once the mouse reaches symptom limitation and remove the mouse immediately from the forced running wheel. Weigh the mouse and place it back in its home cage to recover at room temperature.During this time, leave the chamber door open and return the incubator set point to room temperature to allow the chamber to cool rapidly, which results in greater than 99%long-term survival. To perform a more severe exertional heat stroke exposure, keep the animal's cage within the 37.5 degrees Celsius chamber during the exertional heat stroke protocol. When the animal reaches symptom limitation, allow them to remain in the running wheel until they return to consciousness as observed by the remote camera, then quickly remove the mouse from the running wheel and return it directly to its pre-warmed cage to result in a much slower cooling profile, essentially eliminating the exertional heat stroke hypothermic phase.Remove the filter top from the cage during this time to improve equilibration with the chamber. A recovery cage pre-cooled to room temperature can be used to perform a less severe alternative procedure to result in a suppressed hypothermic phase with a survival rate of 100%For the severe exertional heat stroke protocol, carefully monitor the mice during recovery and check continuously for humane endpoints, including normal movements during recovery, such as grooming, normal breathing, and licking. Monitor the core temperature during this time.The mice are unlikely to recover if their core temperature reverses direction during the recovery phase, eventually exceeding 40 degrees Celsius. In such a case, terminate the experiment and continue to evaluate the mouse for standard humane endpoints. The typical thermoregulatory profiles during the entirety of the exertional heat stroke protocol in early recovery of a mouse comprise of four distinct phases that can be defined as the chamber heating stage, incremental exercise stage, steady state exercise stage, and a recovery stage by either a rapid or slow cooling method.For the severe model, the recovery temperature profile indicated that the core temperature stayed above 37 degrees Celsius throughout the two-hour recovery period. Female mice were more resistant to heat stroke in this model and ran nearly twofold longer distances than male mice. No difference was observed in the time required to recover to 39.5 degrees Celsius in the two models.However, the time to cool to the environmental temperature of 37.5 degrees Celsius was greatly prolonged. The most important thing is the careful monitoring of the animals at all times post-surgery and post-exertional heat stroke. This method has helped study the long-term effects of heat stroke exposure up to several months, and also has allowed us to measure the effects of additional factors in heat stroke illness, such as ingestion of nonsteroidal antiinflammatory drugs and epigenetics.

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A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

PET Imaging of Neuroinflammation Using [11C]DPA-713 in a Mouse Model of Ischemic Stroke

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide ...