Remote Limb Ischemic Preconditioning: A Neuroprotective Technique in Rodents

Summary

Remote ischemic preconditioning (RIP) is a method of conditioning tissues against damaging stress. We have established a method of remote ischemia at the hind limb, by inflating a sphygmomanometer cuff for 5-10 min. The neuroprotective capabilities of RIP have been demonstrated in a model of retinal degeneration in rodents.

Abstract

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. Sublethal ischemia has also been shown to upregulate protective mechanisms in remote tissues. A brief period of ischemia (5-10 min) in the hind limb of mammals induces self-protective responses in the brain, lung, heart and retina. The effect is known as remote ischemic preconditioning (RIP). It is a therapeutically promising way of protecting vital organs, and is already under clinical trials for heart and brain injuries. This publication demonstrates a controlled, minimally invasive method of making a limb – specifically the hind limb of a rat – ischemic. A blood pressure cuff developed for use in human neonates is connected to a manual sphygmomanometer and used to apply 160 mmHg pressure around the upper part of the hind limb. A probe designed to detect skin temperature is used to verify the ischemia, by recording the drop in skin temperature caused by pressure-induced occlusion of the leg arteries, and the rise in temperature which follows release of the cuff. This method of RIP affords protection to the rat retina against bright light-induced damage and degeneration.

Introduction

The survival of most, perhaps all, tissues in the face of metabolic stress can be improved by prior conditioning with a period of sublethal ischemia^1,2. Ischemic preconditioning (IP) in practical terms is the exposure of tissue to sublethal ischemia, before the tissue experiences more severe stressors, such as a subsequent ischemic insult. In animal models, IP provides striking protection to the brain, retina, heart and lungs^3-6. Correspondingly, observations in stroke patients showed a link between previous transient ischemic attacks and better clinical outcomes^7,8. IP also protects retinal photoreceptors from non-ischemic injuries

Protocol

Ethics statement: The protocol follows the animal care guidelines of University of Sydney, AEC #5657. Anaesthesia was approved by animal ethics committee (University of Sydney, AEC #5657).

1. Equipment Preparation

1. Use real time skin temperature tracking. Switch on computer, and data acquisition hardware.
2. Open temperature recording software and adjust temperature setting to between 30-35 °C and the frequency of sampling to every 100 msec.
3. Optional: Insert rectal thermometer to ensure core temperature remains stable at 37.5 °C.

2. Calibration of the Manual Sphygmomanom....

Results

A blood pressure cuff elevated to above 160 mmHg halts blood flow to the hind limb as seen clearly in Figure 1B. The lack of tissue oxygenation resulted in a reducing the animal’s foot temperature for an ischemia-reperfusion protocol (Figure 2). The foot temperature (33 °C) was lower than core temperature and reliably reduced during cuff pressure elevation (31 °C) rising when the cuff was deflated (32 °C). A single 1,000 lux light injury was delivered to dim raised albino rats with or wi.......

Discussion

Rodent hind limb ischemia was successfully induced with a manual sphygmomanometer and cuff delivering neuroprotection to the photoreceptors of the retina. A finding consistent with ischemic conditioning induced protection of photoreceptors from light injury^9,28.

Essentially, remote ischemia causes brief oxygen deprivation to tissues. Hence, remote ischemic preconditioning has many similarities to ischemic conditioning or alternatively termed ischemic tolerance, hypoxic preconditioni.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Gold series DuraShock hand aneroid sphygmomanometer	Welch Allyn	DS56	Manual Sphygmomanometer
Neonate [size 2] 1 tube, 10 pack	Welch Allyn	5082-102-1	Limb blood pressure cuff
Luer lock adaptor	Welch Allyn	5082-178	Adaptor for neonatal cuff
Thermistor pod	AD Instruments	ML 309	skin tempertature unit
Skin temperture probe	AD Instruments	MLT 422/A
Powerlab, 4 channel acquistion hardware	AD Instruments	PL 35044
Homeothermic blanket system with flexible probe	Harvard Appartus	507222F
Towel			optional: awake remote ischemia
Isoflo - 100% Isoflurane (250 ml)	Abbot Animal Health	05260-05	optional: inhaltion anaesthetic remote ischemia
Ketamil - ketamine 100 mg/ml (50 ml)	Troy Laboratories Pty Ltd		optional: injectable anaesthetic remote ischemia
Xylium - Xylazine 100 mg/ml (50 ml)	Troy Laboratories Pty Ltd		optional: injectable anaesthetic remote ischemia