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All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal Preparation
Note: Animals that are to undergo light injury require dark adaptation the night before remote ischemia. Animals undergoing light damage require dark rearing (12 hr light: dark cycle, 5 lux)
- Perform remote ischemia in either awake or anesthetised rodents. Ensure that animals have a healthy muscle tone. Ensure this by pinching the upper hind limb to confirm there is adequate muscle present. RIP-induced protection against light injury has been tested in sedentary rats up to 6 months of age.
- Anesthetised preparation for remote ischemic preconditioning (RIP)
- Inject rats with an intraperitoneal injection of 60 mg/kg ketamine and 5 mg/kg xylazine. Check the depth of anesthesia by extending the leg and pinching the skin on the underside of the foot. The animal has no reflex if it is deeply anesthetized. Apply artificial tears to avoid corneal dryness whilst under anesthesia.
- Place rats on either a heating pad or circulating water heater tubing to maintain a constant body temperature of 37.5 °C. Position the rat in the prone position with the lower limbs’ foot pads facing up. Either the right or left limb may undergo remote ischemia.
2. Application of Skin Temperature Probe
- Extend the leg of the rat which is to undergo ischemia and place the skin probe on the footpad. Position the skin probe to maximise contact between the temperature probe and the skin. Firmly push the probe into the footpad and affix the probe with paper tape.
- Check the skin probe placement by tracking the temperature on the temperature recording software. Ensure that the skin temperature is between 30-34 °C and remains stable. Track the skin temperature for 1-2 min. Adjust the skin probe if the temperature is unstable or below 30 °C.
3. Remote Ischemia
- Deflate the cuff and ensure the air pressure valve is closed. Extend the leg and loosely encircle the cuff on the upper hind limb. Use the forefinger and thumb to extend the leg and the lower digits to keep the loosened cuff in position.
- Raise the anesthetized animal’s cuff pressure to 160 mmHg, and in awake animals, increase the cuff pressure to 180 mmHg.
Note: The anesthetized blood pressure of rats ranges from 120-140 mmHg and rises to 160 mmHg when conscious. Commence the timer and foot temperature recordings once the correct pressure is reached.
Note: The foot temperature should drop by 2 °C after 5 min of constant pressure. - Maintain the position of the cuff above the animal’s “knee” throughout the ischemia. Cuff pressure will begin to drop after a few minutes or if the rat’s limb is moving.
- Repeatedly pump the inflation bulb in short bursts to maintain the desired cuff pressure repetitive short bursts of pumping
- Remote ischemia can be delivered continuously for between 5 and 15 min. The ischemia reperfusion protocol includes 2 periods of 5 min ischemia with an intervening 5 min reperfusion.
- Deflate the cuff pressure by loosening the air pressure valve. Check the temperature change over the course of the ischemia protocol. Release the cuff.
- Continue with injury experimentation. Animals under the effect of anesthesia will need to be placed on a heat pad. Continue to monitor animals until ambulatory. Animals cannot be returned to housing until walking.