The author is grateful for the assistance of Mrs. Sharon Spana (University of Sydney) in rodent monitoring, handling and experimentation. The authorwould like to thank Prof. Jonathan Stone and Dr. Dan Johnstone for the assistance in the preparation of this manuscript. PhD funding support has been provided by University of Sydney and Australian Center for Excellence in Vision.

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
<ol>
	<li>Use real time skin temperature tracking. Switch on computer, and data acquisition hardware.</li>
	<li>Open temperature recording software and adjust temperature setting to between 30-35 &#176;C and the frequency of sampling to every 100 msec.</li>
	<li>Optional: Insert rectal thermometer to ensure core temperature remains stable at 37.5 &#176;C.</li>
</ol>
2. Calibration of the Manual Sphygmomanometer
<ol>
	<li>Connect neonatal arm cuff to sphygmomanometer.Use a size 2 cuff for a 250-550 g rat. An adaptor may be necessary to connect the cuff tubing to the sphygmomanometer.</li>
	<li>Deflate the cuff by either loosening the air release valve or disconnecting the cuff tubing from the adaptor. Ensure no pressure remains in the tubing and the manometer needle rests at zero inside the oval/rectangle.</li>
	<li>Check the pressure between tubing, manometer and cuff. Inflate the cuff with gentle pumps of the inflation bulb until it reads 100 mmHg on the manometer. Ensure the pressure remains constant. Deflate the cuff by slowly opening the air release value.</li>
</ol>
3. Animal Preparation
Note: Animals that are to undergo light injury require dark adaption the night before remote ischemia. Animals undergoing light damage require dark rearing (12 hr light:dark cycle (5 lux))
<ol>
	<li>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.</li>
	<li>Anesthetised preparation for RIP
	<ol>
		<li>Inject rats with an intraperitoneal injection of 60 mg/kg ketamine and 5 mg/kg xylazine. Check the depth of anaesthesia by extending the leg and pinching the skin on the underside of the foot. The animal has no reflex if it is deeply anesthetised. Apply artificial tears to avoid corneal dryness whilst under anaesthesia.</li>
		<li>Place rats on either a heat pad or circulating water heater tubing to maintain a constant body temperature of 37.5 &#176;C. Position the rat in the prone position with the lower limbs&#8217; foot pads facing up. Either the right or left limb may undergo remote ischemia.</li>
	</ol>
	</li>
	<li>Awake preparation for RIP Note: Awake animal experimentation requires two people. One person restrains the animal and the second person operates the manual&#160;sphygmomanometer. The experimenters must be confident to perform the procedure as restraint increases the risk of injury to handlers. The rats undergoing remote ischemia must be conditioned to manual restraint. Depending on intuitional guidelines manual restraint should be progress from 30 sEC to a maximum of 5 min&#160;over a number of weeks. Timid animals that fail to acclimatise to manual restraint should be excluded from awake experimentation. Lastly, manual restraint is likely to cause stress (and potentially introduce confounds to the study) to animals and a sham cohort (placement of cuff without inflammation) must be used to accurately interpret RIP study results.
	<ol>
		<li>Cut a towel into a 15 cm x 30-50 cm piece and place the short edge perpendicular to the rat&#8217;s spine, covering the head to the top of the hind limbs.</li>
		<li>Tuck the short edge under the rat&#8217;s torso tightly and begin to wrap the rat with the remaining long edge of towel. Secure the wrapped animal under the arm in a supine position. If the rat is held under the left arm, free the rat&#8217;s right limb from the towel.</li>
	</ol>
	</li>
</ol>
4. Application of Skin Temperature Probe
<ol>
	<li>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.</li>
	<li>Check the skin probe placement by tracking the temperature on temperature recording software. Ensure that the skin temperature is between 30-34 &#176;C and remains stable. Track the skin temperature for 1-2 min. Adjust the skin probe if the temperature is unstable or below 30 &#176;C.</li>
</ol>
5. Remote Ischemia
<ol>
	<li>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.</li>
	<li>Raise the anesthetised animal&#8217;s cuff pressure to 160 mmHg, and in awake animals increase the cuff pressure to 180 mmHg. 
	Note: The anesthetised 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 &#176;C after 5 min of constant pressure.</li>
	<li>Maintain the position of the cuff above the animal&#8217;s &#8220;knee&#8221; throughout the ischemia. Cuff pressure will begin to drop after a few minutes, or if the rat&#8217;s limb is moving.</li>
	<li>Repeatedly pump the inflation bulb in short bursts to maintain the desired cuff pressure repetitive short burst of pumping</li>
	<li>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.</li>
	<li>Deflate the cuff pressure by loosening the air pressure valve. Check the temperature change over the course of the ischemia protocol. Release the cuff.</li>
	<li>Continue with injury experimentation. Animals under the effect of anaesthesia will need to be placed on a heat pad. Continue to monitor animals until ambulatory. Animals cannot be returned to housing until walking.</li>
</ol>
6. Light Injury - Retinal Degeneration Model
<ol>
	<li>Dark adapt the animals overnight (12-15 hr). Immediately following remote ischemia conditioning or sham remote ischemia (animal restraint) place animals in Perspex housing with food and water.</li>
	<li>Switch on fluorescent lighting (1,000 lux) located above Perspex housing at 9 am for 24 hr.&#160;Following light exposure, return the animals to dim cyclic lighting for 7 days. </li>
</ol>
7. Post-remote Ischemia Procedures
<ol>
	<li>Vision assessment with Electroretinogram (ERG): 
	Note: The ERG set-up and flash protocol followed Brandli and Stone26.
	<ol>
		<li>Dark adapt animals overnight (12-15 hr). Under dim red illumination anesthetize the animals by intraperitoneal injection of ketamine and xylazine (60 mg/kg and 5 mg/kg, respectively). Mydriatic (atropine sulphate 1.0%), corneal anaesthetic (proxymetacaine 0.5%).</li>
		<li>Apply corneal hydration (Carbomer polymer) eye drops immediately to the cornea. Apply eye gel at 20 min intervals to maintain corneal hydration.</li>
		<li>Draw a loosely tied thread around the eyeball to aid stable ERG recordings. Monitor temperature using a rectal probe and maintain animal&#8217;s body temperature at 37-37.5 &#176;C.</li>
		<li>Position the head inside a Ganzfeld integrating sphere. 
		Note: The Ganzfeld is a fully programmable light stimulus that delivers uniform whit flashes from LEDs to the eye.</li>
		<li>Record the electroretinogram using a custom-made 4 mm platinum positive electrode lightly touching the cornea and a 2 mm diameter Ag/AgCl pellet electrode inserted into the mouth. Reference both electrodes to a stainless steel needle inserted subcutaneously into the rump.</li>
		<li>Record signals with band-pass setting of 0.3-1,000 Hz (-3 dB), with a 2 kHz acquisition rate (AD Instruments). After a stable ERG recording is established subject the animal to a 10 min dark adaptation before commencing recordings.</li>
		<li>Follow the flash protocol as previously described by Brandli and Stone26.
		<ol>
			<li>Program the duration of the flash (we used flashes 1-2 msec in duration), and set its intensity to -4.4 to 2.0 log scot cd.s.m-2. Use bright flashes (2.0 log scot cd.s.m-2, 1 ms) to measure the retinal function. In this study it is the comparison between control, light injury and light injury with RIP.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
8. TUNEL Assay
<ol>
	<li>Euthanize animals by intraperitoneal injection of phenobarbital overdose (100 mg/kg). Enucleate the eyes and fix in 4% paraformaldehyde.</li>
	<li>Wash the eyes in PBS before cyroprotecting eyes overnight in sucrose 30% (w/v). Embed the eyes in OCT compound and cut into 20 &#181;m sagittal sections using a cryostat.</li>
	<li>Perform the TUNEL assay on retinal sections with DAPI staining following the protocol of Maslim et al.27</li>
	<li>Use fluorescence microscopy for TUNEL counts of the retina. TUNEL cells were recorded from the outer nuclear layer (ONL); the outermost layer of the retina which contains photoreceptor nuclei. In this study, TUNEL counts were made in triplicate for each eye, with 5 eyes for each treatment group.</li>
	<li>Use one-way ANOVA to statistical compare group means of control, light injury, and light injury + RIP rats.</li>
</ol>

The author has nothing to disclose.

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 injury9,28.
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...

The survival of most, perhaps all, tissues in the face of metabolic stress can be improved by prior conditioning with a period of sublethal ischemia1,2. 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 lungs3-6. Correspondingly, observations in stroke patients showed a link between previous transient ischemic attacks and better clinical outcomes7,8. IP also protects retinal photoreceptors from non-ischemic injuries9.
The effectiveness of IP in diverse tissues and injuries suggests that it is activating an innate mechanism of cell survival present in all tissue. Ischemic preconditioning of the myocardium has been suggested to have protective effects through the upregulation of hypoxia inducible factor (HIF), known to regulate many metabolic pathways through the release of adenosine or through the opening of mitochondrial ATP potassium channels10,11. Adenosine release and ATP potassium channels are implicated in cerebral ischemia but, investigations into the neuroprotective mechanisms of ischemic conditioning to date have been focused on modifications to anti-excitotoxicity, anti-apoptotic and anti-inflammatory pathways12,13. Overall, understanding of the molecular process of ischemic conditioning for protecting neurons is limited.
Remote ischemic preconditioning attempts to condition distant critically important organs (heart, brain, lung) by generating ischemia in less critical tissues. Remote ischemic preconditioning (RIP) using the hind limb has been demonstrated to be neuroprotective in rodent models of stroke14-17. The method described by us provides a simple, reliable and non-invasive protocol for inducing RIP.
The vast majority of RIP protocols involve the hind limb, presumably because the femoral artery located in the upper hind limb can be easily identified and accessed for surgical clamping and tourniquet application. In invasive limb ischemic studies for the study of brain and skin protection, ischemia is induced by separating the femoral artery from the groin ligaments and clamping the femoral artery2,15,18.
The ischemia resulting from either limb cuffing or femoral artery clamping has been confirmed by changes at the limb including a loss of pulse, decrease in oxygenation and a drop in skin temperature. Remote ischemia can be confirmed by the loss of pulse by using laser Doppler or ultrasound Doppler17-19. Skin temperature can be used as alternative to Doppler although the relationship is non-linear20,21. Accurate temperature recordings are commonplace in laboratories and can be easily incorporated into remote ischemic studies.
An alternative to femoral clamping surgery is the induction of ischemia using a tourniquet. Tourniquet application produces comparable ischemia to that achieved with vessel clamping; Kutchner et al. compared invasive femoral artery clamping to a non-invasive tourniquet and found both methods halted blood flow to the limb and reduced skin damage in a plastic surgery model of skin flap ischemia18. Cuffing either the leg or arm and raising the cuff pressure to above systolic blood pressure has been found to be protective against ischemic damage in pigs and humans17,19,22.
Different tourniquet approaches to inducing remote ischemic include the use of a blood pressure cuff or an elastic band17,22,23. However, the use of an elastic band to induce ischemia is an unsafe method, potentially giving rise to an unregulated amount of pressure in the limb, with pressure rises above 500 mmHg being recorded in humans24. Further, limb ischemia using an elastic band leads to muscle damage in rats following the removal of the band23, as assessed by Evans Blue Dye, an in vivo marker of myofiber permeability25. In contrast, delivery of a controlled pressure to the tourniquet can be achieved using a blood pressure cuff connected to a sphygmomanometer17,19,22,26.
In this study, a light injury model of photoreceptor degeneration was used to demonstrate the neuroprotective efficacy of remote ischemic preconditioning. Remote ischemia was induced immediately before light injury, and prevented subsequent photoreceptor degeneration as confirmed by retinal function testing. The accompanying video will demonstrate the application of non-invasive remote ischemia.

<table border="0" cellpadding="0" cellspacing="0">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td>Gold series DuraShock Hand Aneroid Sphygmomanometer</td>
			<td>Welch Allyn</td>
			<td>DS56</td>
			<td>Manual Sphygmomanometer</td>
		</tr>
		<tr>
			<td>Neonate [size 2] 1 Tube, 10 Pack</td>
			<td>Welch Allyn</td>
			<td>5082-102-1&#160;</td>
			<td>Limb blood pressure cuff</td>
		</tr>
		<tr>
			<td>Luer lock adaptor</td>
			<td>Welch Allyn</td>
			<td>5082-178</td>
			<td>Adaptor for neonatal cuff</td>
		</tr>
		<tr>
			<td>Thermistor pod</td>
			<td>AD Instruments</td>
			<td>ML 309</td>
			<td>skin tempertature unit</td>
		</tr>
		<tr>
			<td>Skin temperture probe</td>
			<td>AD Instruments</td>
			<td>MLT 422/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Skin temperature recorder</td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Towel</td>
			<td></td>
			<td></td>
			<td>optional: awake remote ischemia</td>
		</tr>
		<tr>
			<td>Isoflo - 100% Isoflurane -250 ml</td>
			<td>Abbot Animal Health</td>
			<td>05260-05</td>
			<td>optional: inhaltion anaesthetic remote ischemia</td>
		</tr>
		<tr>
			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>optional: injectable anaesthetic remote ischemia</td>
		</tr>
		<tr>
			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
			<td></td>
			<td>optional: injectable anaesthetic remote ischemia</td>
		</tr>
	</tbody>
</table>

remote limb ischemic preconditioning:  a neuroprotective technique in rodents

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 &#8211; specifically the hind limb of a rat &#8211; 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.

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&#8217;s foot temperature for an ischemia-reperfusion protocol (Figure 2). The foot temperature (33 &#176;C) was lower than core temperature and reliably reduced during cuff pressure elevation (31 &#176;C) rising when the cuff was deflated (32 &#176;C). A single 1,000 lux light injury was delivered to dim raised a...

Watch this Scientific Journal Video about Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents at JoVE.com

Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

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.

Sublethal ischemia protects tissues against subsequent, more severe ischemia through the upregulation of endogenous mechanisms in the affected tissue. ...

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10.9K Views.  University of Sydney. 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.