Short-Duration Hypothermia Induction in Rats using Models for Studies examining Clinical Relevance and Mechanisms

All experimental procedures were in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and were approved by the Animal Care and Ethics Committee of the University of Newcastle (A-2013-343 and A-2020-003). In addition to the hypothermia induction methods described below, the following protocols are routinely done in conjunction with hypothermia: femoral line cannulation to monitor blood pressure and heart rate²¹, and experimental stroke²².

1. Datalogger implantation

NOTE: The datalogger device used in this protocol was not capable of providing real time read-out of body temperature. Read out is possible once the datalogger has been removed from the animal and connected back to the computer. As a result, the rectal temperature probe is used to provide real time information during the cooling and rewarming process. Additionally, the rectal probe is also vital to this method because the surgical heat mat on which the animal is placed during the procedure is regulated by the rectal probe system. The datalogger also serves a valuable purpose of providing temperature data in freely moving, awake rats and is important for ensuring that normal body temperature is maintained after rewarming. Therefore, both temperature-monitoring devices are important for this protocol.

1. Anesthetize 10-12-week-old male outbred Wistar rat with isoflurane (5% for induction and 2-2.5% for maintenance) in a 50% N₂ and 50% O₂ mixture.
2. Following induction, place the rat in the prone position on a surgical heat mat.
3. Position the rat so that the nose is sitting in the nose cone. Secure the nose with surgical tape to ensure no gases are escaping.
4. Shave the fur from the lower right abdomen and inject the site subcutaneously with a local anesthetic, Bupivacaine 0.2 mL, 0.05%.
5. Apply antiseptic solution to the freshly shaved region.
6. Using sterilized surgical tools, make a 2 cm longitudinal incision along the right abdominal region, proximal to the right thigh. Make the incision deep enough to expose the space at the ventral thigh crease.
7. Use hemostats and forceps to create a 'pocket' under the skin that is large enough to hold the device.
8. Insert the temperature monitoring datalogger device into the pocket and close the muscle and skin using 5-0 silk sutures. The subcutaneous method described here is preferred to the intra-peritoneal method as it is less invasive and allows for better recovery after the procedure.
9. Ensure datalogger and rectal probe are cross calibrated for temperature monitoring (see Discussion).
10. Ensure the datalogger is not resting against the animal's heat mat after insertion, as this will influence temperature readings.

Figure 1: Implantation of datalogger device. (A) Panels left to right show an incision of approximately 2 cm being made on the right side of the lower abdomen of the rat. (B) Temperature monitoring datalogger was inserted subcutaneously into the pocket incision. (C) The incision was closed with nylon sutures. Please click here to view a larger version of this figure.

2. Induction of active (fast) hypothermia for mechanistic studies

1. Set up for hypothermia (see Figure 2). Place two retort stands with clamps at either side of the rat's body.
2. Attach a 60 mm 12 v/130 mA fan to each retort stand ensuring that the fans are aimed towards the lower back of the rat. The distance between the clamp and the rat is approximately 20 cm. The fan used must have a speed of 4,000 rpm.
3. Have an animal heat lamp ready either at the side or on a third retort stand.
4. Commence hypothermia by adjusting the animal heat mat to the desired target temperature. In this example, 32.5 °C is the target temperature (3.75 on the temperature control unit).
5. Turn both fans on and apply three to four sprays of 70% ethanol (standard plastic spray bottle) on the lower back of the rat. Ruffle animal fur when spraying for faster cooling induction.
   NOTE: Ethanol is used as a preferred solution over water because it has a faster rate of evaporation and therefore results in more rapid hypothermia induction.
6. Take care not to oversaturate the fur, since this can contribute to overshooting the target temperature.
   NOTE: The fans will accelerate ethanol evaporation and the cooling process.
7. Allow short intervals between ethanol applications while keeping a close eye on the rectal temperature of the rat.
8. Cease any further application of ethanol once rectal temperature reaches within 1 °C of target temperature.
9. Turn off both fans once the temperature has reached within 0.5 °C of target temperature (33 °C in this case).
   ​NOTE: Turning off fans before the target temperature is reached helps prevent the rat from overcooling beyond required temperature.
10. Allow the temperature to drop to 32.5 °C.
    1. If overcooling does occur, use animal heat lamp to mildly warm the animal back up to target. The assistance of one fan may be used to prevent a rewarming overshoot.
11. Once target temperature is reached and has stabilized, continue to monitor temperature. Temperature will usually remain very stable for the remainder of the hypothermia period without the need for spraying, use of fans or use of heat lamp.
12. To rewarm the animal at the end of hypothermia, adjust the heat mat temperature back to 37 °C (6 on the temperature control unit used in this example) and allow the animal to thermoregulate over a 30 min period.
    ​NOTE: Temperature settings on temperature control units may vary and it may, therefore, be necessary to determine settings for target hypothermia and normothermia on individual devices.

3. Induction of clinically achievable gradual onset hypothermia without active skin cooling

1. Achieve hypothermia by reducing the temperature of the core temperature regulated homeothermic heat mat in small increments to the required target temperature. In the illustrated example (Figure 3B), a 1 °C increment every 30 min was used.
2. Cool the animal to target temperature over the desired period (2 h in the example described). Once cooled, maintain at target for the desired interval. Usually, no further intervention is required if they are maintained on the homeothermic heat mat set for the desired target temperature.
3. No external cooling is necessary with this protocol as anesthesia prevents the normal regulation of core body temperature.
   ​NOTE: Isoflurane requirements decline with hypothermia. In most animals, a starting isoflurane concentration of 2% can be reduced in 0.1% increments every 20-30 min to 1.5% to maintain stable respiratory rate (>50 breaths/min), heart rate and blood pressure, and preserve suppression of reflex responses.
4. To rewarm the animal after hypothermia, adjust the heat mat to allow the animal to rewarm over the desired interval. In the example, rewarming with a single adjustment to 37 °C (6 on the FHC temperature control unit used in the example) was achieved over a 30 min period.
   1. For longer-term studies that require animal recovery, keep animals in a cage that is placed half over a heat mat to allow the animal to thermoregulate and to avoid inadvertent post-operative hypothermia.
   2. Rectal paracetamol (250 mg/kg) can be administered for recovery and ovrenight pain relief. Subcutaneous saline injections (2 x 1.5 mL) can also be given to prevent dehydration from anesthesia and surgical procedures.