The gradual cooling protocol has a slower rate of cooling than current experimental methods. This model is clinically relevant because the time to target temperature is closer to the human range. The main advantage of these techniques is their simplicity and inexpensiveness.
Additionally, these models do not require specialist equipment. The clinical rate of cooling achieved from the gradual model will allow more informed preclinical investigation into the benefits of hypothermia as a treatment measure for stroke patients. Begin by placing an anesthetized rat on a surgical heat mat in the supine position with the nose in a cone.
Secure the nose with surgical tape. Shave the fur from the lower right abdomen and subcutaneously inject 200 microliters of 0.05%bupivacaine as a local anesthetic. Apply antiseptic solution to the freshly shaved region.
Make a two centimeter longitudinal incision along the right abdominal region proximal to the right thigh deep enough to expose the space at the ventral thigh crease and use hemostats and forceps to create a pocket under the skin large enough to hold a data logger device. Insert the temperature monitoring data logger device into the pocket and use 5-0 sutures to close the muscle and skin, making sure that the data logger is not resting against the heating mat. To induce active hypothermia, first place one retort stand with a clamp 20 centimeters from the head of the rat and 120 centimeters from the lower abdomen and attach a 60 millimeter 12 volt fan with an at least 4, 000 rotations per minute setting to each stand with the fans aimed toward the lower back of the rat.
Adjust the heat mat to the desired target temperature for initiating hypothermia and turn on both fans. In the prone position, apply three or four sprays of 70%ethanol every five minutes to the lower back of the rat while ruffling the fur for faster cooling induction and while closely monitoring the rectal temperature. Stop the ethanol application when the rectal temperature is within one degree of the target temperature.
When temperature is within 0.5 degree of the target, turn off the fans to allow the temperature to drop slowly to the target without over cooling. In the case of over cooling, replace one fan with an animal heat lamp and use the heat lamp to mildly warm the animal back up to the target temperature. Once the target temperature has been reached and stabilized, monitor the temperature for the remaining hypothermia period.
At the end of hypothermic period, set the heat mat to 37 degrees Celsius and allow the animal to thermoregulate over a 30-minute period. To induce clinically achievable gradual onset hypothermia, reduce the temperature of the core temperature-regulated homeothermic heat mat in small increments to achieve hypothermia within a specific predetermined time period. When the target temperature has been reached, maintain the target temperature for the desired interval without any external cooling.
To rewarm the animal at the end of the hypothermic period, adjust the heat mat to allow the rat to rewarm to 37 degrees Celsius over the desired time interval. Upon the completion of either type of hypothermia experiment, remove the data logger to allow analysis of the recorded body temperatures. In this analysis, hypothermia was achieved with rapid cooling to a target temperature of 32.5 degrees Celsius in 15 minutes as demonstrated.
A slight temperature overshoot due to failing to stop the cooling from about 0.5 degrees Celsius above the target temperature can be observed. The target temperature was maintained for 30 minutes and the rewarming was initiated at 1.5 hours. In this experiment, the gradual hypothermia induction approach was used to allow the target temperature of 33 degrees Celsius to be reached in two hours.
The target temperature was maintained for 30 minutes before rewarming at 2.5 hours. The gradual cooling technique lead us to investigate whether a more clinically relevant cooling rate would prevent intracranial pressure elevation after ischemic stroke in rats.
