A Preclinical Model of Exertional Heat Stroke in Mice

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.