The overall goal of this procedure is to deliver ischemia in the hind limb that conditions remote tissues against subsequent stress. For example, the retina against light injury. This is accomplished by first calibrating the manual Ingle mom manometer.
Next, the animal is anesthetized and placed on a heating pad. Then the animal's hind leg is extended and the cuff is placed into position. Finally, remote ischemia is performed by inflating the cuff with the pump, deflating the cuff, and repeating the process a second time.
Ultimately, when applied prior to light injury, remote ischemia is used to show an improvement in retinal function and the number of photoreceptors undergoing apoptosis. This method can answer key questions in the neuroscience field, such as how self-protection is induced with remote ischemic preconditioning For carrying out real-time skin temperature tracking. Open the temperature recording software, adjust the temperature setting to between 30 to 35 degrees Celsius, and the frequency of sampling to every 100 milliseconds using a size two neonatal arm cuff for a 250 to 550 gram rat, connect the cuff to the fome manometer using an adapter if necessary.
Next, deflate the cuff by either loosening the air release valve or disconnecting the cuff tubing from the adapter. Ensure no pressure remains in the tubing and the manometer needle rests at zero inside the oval rectangle. Then to check the pressure between the tubing, manometer, and cough.
Use gentle pumps of the inflation bulb to inflate the cuff until the manometer reads 100 milligrams of mercury. After ensuring the pressure remains constant, deflate the cuff by slowly opening the air release valve after dark, adapting the animal according to the text protocol. Confirm that it has good muscle tone by pinching the upper hind limb to confirm that there is adequate muscle present.
After sedating the animal according to the text protocol, check the depth of anesthesia by extending the leg and pinching the skin on the underside of the foot. Then apply artificial tears. Place the rat on either a heating pad or circulating water heater tubing in a prone position with the foot pads of the lower limbs facing up.
To apply the skin temperature probe, extend the leg of the rat that will undergo ischemia and firmly place the skin probe on the foot pad, positioning it to maximize contact between the temperature probe and the skin. Then use paper tape to affix the probe. Check the skin probe placement by tracking the temperature on the temperature recording software for one to two minutes.
Ensure that the skin temperature is between 30 to 34 degrees Celsius and remains stable. Otherwise, adjust the probe to carry out remote ischemia. Begin with a deflated 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. Now, raise the cuff pressure to 160 millimeters of mercury.
Once the correct pressure is reached, start the timer and foot temperature recordings. The foot temperature should drop by two degrees Celsius after five minutes of constant pressure, repeatedly pump the inflation bulb in short bursts to maintain the desired cuff pressure and maintain the position of the cuff above the animal's knee throughout the ischemia. Continuously deliver remote ischemia for five minutes.
Then release the air pressure valve for a five minute reperfusion before repeating the remote ischemia. When the procedure is complete, deflate the cuff pressure by loosening the air pressure valve. Check the temperature change over the course of the ischemia protocol.
Then release the cuff. Place the animal on a heating pad and continue to monitor until ambulatory. When the animal is walking, return it to housing.
Finally carry out light injury and perform the tunnel assay. According to the text protocol as seen clearly here, a blood pressure cuff placed above the knee elevated to above 160 millimeters of mercury halts blood flow to the hind limb. The lack of blood flow resulted in a reduction in foot temperature that was lower than the animal's core temperature, and that rose after the cuff was deflated consistent with the condition necessary for RIP.
In this figure, the dark adapted electroretinogram or ERG from a normal dim raised rat showed a large photoreceptor and inner retinal response to a bright flash one week after light injury. The ERG recordings showed a severe reduction in amplitude relative to controls, reflecting the loss of photoreceptors, however, preconditioning the hind limb with ischemia using a reperfusion protocol of two by five minutes immediately before ischemia protected the photoreceptors from light injury. This panel shows that the R-I-P-E-R-G amplitudes were greater than light injury alone with a slight reduction to the A wave.
Finally, a cuff placed below the knee does not protect photoreceptors from light injury as reflected in the reduced ERG amplitudes seen here. Methods like remote ischemic preconditioning can be performed in order to answer additional questions such as what are the mechanisms of endogenous neuroprotection and how can remote ischemia be applied to improve function from CNS injury and degeneration.
