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
<ol>
	<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 cage under local IACUC guidelines) until the day of surgery for temperature telemetric device implantation. House them in standard 7.25&#34; (W) x 11.75&#34; (L) x 5&#34; (H) cages containing corncob bedding. Maintain the light cycle on a 12 x 12 light cycle (on: 7 AM; off: 7 PM). Maintain the housing temperature at 20-22 &#176;C and relative humidity (RH) at 30%-60%. Provide the standard chow diet and water ad libitum until the EHS protocol. 
	NOTE: The rationale for individual housing is to avoid frequent fighting injury in male C57bl/6J mice and to provide ample opportunity for spontaneous wheel running for each mouse.</li>
	<li>For placement of the telemetry devices, anesthetize the mouse with isoflurane (4%, 0.4-0.6 L/min of O2 flow) in an induction chamber. Then, place the mouse under continuous anesthesia via a nose cone (1.5%, 0.6 L/min).</li>
	<li>Use eye lube, such as a vet ointment, to protect the animal&#39;s eyes from damage or injury during surgery.</li>
	<li>To prepare the surgical site, shave the lower abdomen with small animal hair clippers or use a commercially available hair remover. Administer the first dose of subcutaneous buprenorphine (0.1 mg/kg) during this time.</li>
	<li>Scrub the area with three washes of povidone-iodine (or similar germicidal scrub) followed by 70% isopropyl alcohol rinse (or sterile saline depending on local veterinary requirements). Then, transfer the mouse to the surgical area.</li>
	<li>Use an adhesive drape to isolate the surgical site on the mouse. Using sterile instruments and aseptic technique, make a ~1 cm incision on the midline along the linea alba, about 0.5 cm from the costal margin. Then, separate the skin from the muscle layer and make a slightly smaller incision on the linea alba, careful not to damage the bowels or internal organs.</li>
	<li>Once the muscle layer is open, place the sterile telemeter (miniature reusable battery-free radiotelemetry device; 16.5 x 6.5 mm) into the intraperitoneal cavity in front of the caudal arteries and veins and dorsal to the digestive organs to allow it to float freely. 
	NOTE: All telemeters are cleaned with soap and water, thoroughly rinsed and gas sterilized with ethylene oxide between usages.&#160;If gas sterilization is not available, immersion in sterilization solutions (following manufacturer&#8217;s recommendation for dilution and immersion time) is accepted to&#160; disinfect and sterilize the telemeters.</li>
	<li>Close the abdominal opening with a sterile 5-0 absorbable suture, and close the skin using a simple interrupted stitch with 5-0 proline suture. 
	NOTE: Allowing the telemeter to float in the abdominal compartment without tying it to the abdominal wall (a method recommended by the manufacturer) has been demonstrated to be successful and preferred by the authors to eliminate excess tension in the abdominal wall during healing. Further, this has no impact on the receiver&#39;s ability to obtain the signal from the emitter.</li>
	<li>Place the mouse in its clean cage with a portable heating pad under the cage. Monitor the mouse every 15 min during the first hour of recovery from anesthesia, and then return to the animal housing facility.</li>
	<li>Provide mice with subcutaneous buprenorphine injections every 12 h for 48 h during recovery and continue to monitor for signs of distress. If available, give slow-release buprenorphine subcutaneously every 24 h (1 mg/kg) for 48 h. Allow the mice to recover for ~2 weeks following surgery before introducing a voluntary wheel running.</li>
</ol>
2. Familiarization: Voluntary and forced wheel running
<ol>
	<li>Following recovery from surgery, place the voluntary running wheels in the cage for free access to the wheel. Other running wheel selections may be equally effective, but ensure it fits within the limited cage sizes available. 
	NOTE: The running wheels had to be slightly reduced in dimension to fit in a standard cage.</li>
	<li>Acclimate the mouse to the voluntary wheel in the cage for 2 weeks. Once acclimated, the mouse is ready for training with familiarization procedures for the forced running wheels.</li>
	<li>Perform the four training sessions (one/day) in the environmental chamber at room temperature (~25 &#176;C, 30% relative humidity). 
	NOTE: Although this is ideal, mice were also successfully trained in identical forced running wheels outside the chamber. Several mice can then be trained simultaneously without interfering with the use of the chamber.</li>
	<li>To begin the first training session, allow the mouse to free wheel in the modified running wheel for 15 min by removing or loosening the motor drive belt to allow the mouse to determine the speed of the wheel and acclimatize to it in a non-stressful manner. 
	NOTE: Protocols can be run with software and hardware supplied by the running wheel manufacturer or may be substituted by an external programmable power supply that is wired directly to the wheel motor, which allows for automation of the incremental exercise protocol.</li>
	<li>Calibrate the system for each running wheel to determine the relationship between the power supply voltage and meters/minute (m/min) of each wheel. 
	NOTE: The forced running wheels were also modified to elevate the motor 15 cm, invert and move the pulley driving the wheel down to 5 cm above the telemetry receiver platform. This ensured that the receiver platform obtained accurate telemetry data during the running protocol without interference from the motor.</li>
	<li>After a brief rest period (&#60;5 min), initiate the forced running wheel protocol. Start the wheel at 2.5 m/min and increase 0.3 m/min every 10 min for a total of 1 h to mimic the first hour of the actual EHS trial, but at room temperature. Return the mouse to its home cage and allow for 24 h recovery. Conduct the subsequent three forced running sessions in the same manner on consecutive days. After day 1, the free-wheeling acclimation portion is unnecessary.</li>
	<li>Allow the mouse 2-3 days of wash-out or recovery from the stress of the forced running wheel practice, but allow the mouse free access to the home cage voluntary wheel. The mouse is now prepared to undergo the EHS protocol.</li>
</ol>
3. EHS protocol
<ol>
	<li>The night before the EHS protocol, place the mouse in the environmental chamber at room temperature (~25 &#176;C, &#8776;30% relative humidity) to acclimate to the chamber.</li>
	<li>Use a data acquisition system to collect continuous Tc, averaged over 30-s intervals overnight.</li>
	<li>On the morning of the EHS protocol, make sure the mouse is at or below a normal range of diurnal temperature before increasing the chamber temperature (i.e., 36-37.5 &#176;C). This ensures the mouse does not have a fever and&#160;has not experienced undue stress during this period.</li>
	<li>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 &#176;C and 40%-50% relative humidity, or the desired environmental temperature and humidity19. Verify the chamber temperature and humidity with a calibrated temperature and humidity monitor.</li>
	<li>Surround the chamber with a black-out curtain to keep light and disturbances minimal during the protocol. Monitor the mouse continuously during the protocol via remote IR illuminated cameras. Focus a second camera on the temperature and humidity monitor, placed close to the running wheel. Make any adjustments to the controller for the environmental chamber set-point to ensure accurate temperature readings near the animal.</li>
	<li>Once the chamber has reached its target temperature as measured by the second camera on the temperature monitor (this can take ~30 min), quickly open the chamber door and place the mouse in the forced running wheel.</li>
	<li>Initiate the forced running wheel protocol at a speed of 2.5 m/min and increase the speed 0.3 m/min every 10 min until the mouse reaches a Tc of 41 &#176;C. Once the mouse has reached this core temperature, allow the speed to remain constant until symptom limitation, characterized by an apparent loss of consciousness, a backward fall or fainting, and the inability to continue to run or hold on to the wheel. Confirm this time point when the mouse has three backward rotations on the wheel without signs of a physical response. Alternatively, identify a humane endpoint following local IACUC rules to determine when to stop the protocol (e.g., when Tc ~43 &#176;C). This endpoint is slightly above symptom limitation in essentially all mice.</li>
	<li>To perform the Rapid Cooling protocol (R), once the mouse reaches symptom limitation, stop the wheel, and remove it 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&#160;and return the incubator set point to room temperature to allow the chamber to cool rapidly. This procedure results in &#62;99% long-term survival.</li>
	<li>To perform a more Severe (S) EHS exposure, keep the animal&#39;s home cage within the 37.5 &#176;C chamber during the EHS 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 (~5-9 min).</li>
	<li>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 (Figure 1A, red dashed line), essentially eliminating the EHS hypothermic phase. Remove the filter top from the cage during this time to improve equilibration with the chamber.</li>
	<li>Use a recovery cage precooled to room temperature to perform a less severe alternative procedure to result in a suppressed hypothermic phase but with a 100% survival rate20.</li>
	<li>For the S protocol, carefully monitor the mouse&#160;during recovery and check continuously for humane endpoints. Although it is difficult to remotely test for commonly used humane endpoints (e.g., righting reflex), observe the mice remotely for normal movements during recovery such as grooming, normal breathing, licking, etc. Monitor the Tc during this time.</li>
	<li>Mice are unlikely to recover if their core temperature reverses direction during the recovery phase, eventually exceeding 40 &#176;C; at this time, terminate the experiment and evaluate the mouse for standard humane endpoints.</li>
</ol>

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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. 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C., 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=The+association+between+heat+stroke+and+subsequent+cardiovascular+diseases.">The association between heat stroke and subsequent cardiovascular diseases.</a> PLOS One. 14 (2), 0211386 (2019).</li><li>Leon, L. R., Blaha, M. D., DuBose, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+of+cytokine,+corticosterone,+and+tissue+injury+responses+in+mice+during+heat+strain+recovery.">Time course of cytokine, corticosterone, and tissue injury responses in mice during heat strain recovery.</a> Journal of Applied Physiology. 100 (4), 1400-1409 (2006).</li><li>Leon, L. R., DuBose, D. A., Mason, C. 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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. 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The authors have no conflicts of interest to disclose. All work performed and all support for this project were generated at the University of Florida.

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...

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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, ...

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3.5K Views.  University of Florida. 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.

Video: A Preclinical Model of Exertional Heat Stroke in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

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 ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

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

Neuroinflammation is central to the pathological cascade following ischemic stroke. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and role of certain neuroimmune interactions in stroke. Specifically, Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO), a marker of activated microglia and peripheral myeloid-lineage cells, provides a means to detect and track neuroinflammation in vivo. Here, we present a method to accurately quantify neuroinflammation using [11C]N,N-Diethyl-2-[2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl]acetamide ([11C]DPA-713), a promising second generation TSPO-PET radiotracer, in distal middle cerebral artery occlusion (dMCAO) compared to sham-operated mice. MRI was performed 2 days post-dMCAO surgery to confirm stroke and define the infarct location and volume. PET/Computed Tomography (CT) imaging was carried out 6 days post-dMCAO to capture the peak increase in TSPO levels following stroke. Quantitation of PET images was conducted to assess the uptake of [11C]DPA-713 in the brain and spleen of dMCAO and sham mice to assess central and peripheral levels of inflammation. In vivo [11C]DPA-713 brain uptake was confirmed using ex vivo autoradiography.

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

Positron Emission Tomography (PET) imaging of translocator protein 18 kDa (TSPO) provides a non-invasive means to visualize the dynamic role of neuroinflammation in the development and progression of brain diseases. This protocol describes TSPO-PET and ex vivo autoradiography to detect neuroinflammation 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 ...