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.

Video: Remote Limb Ischemic Preconditioning:  A Neuroprotective Technique in Rodents

Gene Transfer for Ischemic Heart Failure in a Preclinical Model

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine their efficacy and safety.
Gene therapy using viral vectors is one of the most promising approaches for treating cardiac diseases. Viral delivery of various different genes by changing the carrier gene has immeasurable therapeutic potential.
In this video, the full process of an animal model of heart failure creation followed by gene transfer is presented using a swine model. First, myocardial infarction is created by occluding the proximal left anterior descending coronary artery. Heart remodeling results in chronic heart failure. Unique to our model is a fairly large scar which truly reflects patients with severe heart failure who require aggressive therapy for positive outcomes. After myocardial infarct creation and development of scar tissue, an intracoronary injection of virus is demonstrated with simultaneous nitroglycerine infusion. Our injection method provides simple and efficient gene transfer with enhanced gene expression. This combination of a myocardial infarct swine model with intracoronary virus delivery has proven to be a consistent and reproducible methodology, which helps not only to test the effect of individual gene, but also compare the efficacy of many genes as therapeutic candidates.

A method of gene transfer for the treatment of ischemic heart failure is described using a swine model of myocardial infarction. Our simple and reproducible method enables us to readily evaluate the efficacy of various gene transfers with a very simple and reproducible way.

Various emerging technologies are being developed for patients with heart failure. Well-established preclinical evaluations are necessary to determine ...

A Simplified Technique for Producing an Ischemic Wound Model

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work well in non-ischemic wounds may not work in human diabetic wounds because vasculopathy is one major factor that hinders healing of these wounds. We published an article in 2007 describing a rabbit ear ischemic wound model created by a minimally invasive surgical technique. Since then, we have further simplified the procedure for easier operation. On one ear, three small skin incisions were made on the vascular pedicles, 1-2 cm from the ear base. The central artery was ligated and cut along with the nerve. The whole cranial bundle was cut and ligated, leaving only the caudal branch intact. A circumferential subcutaneous tunnel was made through the incisions, to cut subcutaneous tissues, muscles, nerves, and small vessels. The other ear was used as a non-ischemic control. Four wounds were made on the ventral side of each ear. This technique produces 4 ischemic wounds and 4 non-ischemic wounds in one animal for paired comparisons. After surgery, the ischemic ear was cool and cyanotic, and showed reduced movement and a lack of pulse in the ear artery. Skin temperature of the ischemic ear was 1-10 &deg;C lower than that on the normal ear and this difference was maintained for more than one month. Ear tissue high-energy phosphate contents were lower in the ischemic ear than the control ear. Wound healing times were longer in the ischemic ear than in the non-ischemic ear when the same treatment was used. The technique has now been used on more than 80 rabbits in which 23 were diabetic (diabetes time ranging from 2 weeks to 2 years). No single rabbit has developed any surgical complications such as bleeding, infection, or rupture in the skin incisions. The model has many advantages, such as little skin disruption, longer ischemic time, and higher success rate, when compared to many other models. It can be safely used in animals with reduced resistance, and can also be modified to meet different testing requirements.

We have developed a minimally invasive technique to create a rabbit ischemic ear wound model by dividing the central artery and nerve and the cranial neurovascular bundle. A subcutaneous tunnel then cuts all subcutaneous tissues. This procedure causes minimal skin disruption and can be safely used in diabetic animals.

One major obstacle in current diabetic wound research is a lack of an ischemic wound model that can be safely used in diabetic animals. Drugs that work ...

Remote Magnetic Navigation for Accurate, Real-time Catheter Positioning and Ablation in Cardiac Electrophysiology Procedures

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac substrates such as left atrial flutter. This protocol describes all the clinical and invasive interventional steps performed during a human electrophysiological study and ablation to assess the accuracy, safety and real-time navigation of the Catheter Guidance, Control and Imaging (CGCI) system. Patients who underwent ablation of a right or left atrium flutter substrate were included. Specifically, data from three left atrial flutter and two counterclockwise right atrial flutter procedures are shown in this report. One representative left atrial flutter procedure is shown in the movie. This system is based on eight coil-core electromagnets, which generate a dynamic magnetic field focused on the heart. Remote navigation by rapid changes (msec) in the magnetic field magnitude and a very flexible magnetized catheter allow real-time closed-loop integration and accurate, stable positioning and ablation of the arrhythmogenic substrate.

This report provides a detailed description of a new remote navigation system based on magnetic driven forces, which has been recently introduced as a new robotic tool for human cardiac electrophysiology procedures.

New remote navigation systems have been developed to improve current limitations of conventional manually guided catheter ablation in complex cardiac ...

The Use of Cystometry in Small Rodents: A Study of Bladder Chemosensation

The lower urinary tract (LUT) functions as a dynamic reservoir that is able to store urine and to efficiently expel it at a convenient time. While storing urine, however, the bladder is exposed for prolonged periods to waste products. By acting as a tight barrier, the epithelial lining of the LUT, the urothelium, avoids re-absorption of harmful substances. Moreover, noxious chemicals stimulate the bladder's nociceptive innervation and initiate voiding contractions that expel the bladder's contents. Interestingly, the bladder's sensitivity to noxious chemicals has been used successfully in clinical practice, by intravesically infusing the TRPV1 agonist capsaicin to treat neurogenic bladder overactivity1. This underscores the advantage of viewing the bladder as a chemosensory organ and prompts for further clinical research. However, ethical issues severely limit the possibilities to perform, in human subjects, the invasive measurements that are necessary to unravel the molecular bases of LUT clinical pharmacology. A way to overcome this limitation is the use of several animal models2. Here we describe the implementation of cystometry in mice and rats, a technique that allows measuring the intravesical pressure in conditions of controlled bladder perfusion.
	After laparotomy, a catheter is implanted in the bladder dome and tunneled subcutaneously to the interscapular region. Then the bladder can be filled at a controlled rate, while the urethra is left free for micturition. During the repetitive cycles of filling and voiding, intravesical pressure can be measured via the implanted catheter. As such, the pressure changes can be quantified and analyzed. Moreover, simultaneous measurement of the voided volume allows distinguishing voiding contractions from non-voiding contractions3.
	Importantly, due to the differences in micturition control between rodents and humans, cystometric measurements in these animals have only limited translational value4. Nevertheless, they are quite instrumental in the study of bladder pathophysiology and pharmacology in experimental pre-clinical settings. Recent research using this technique has revealed the key role of novel molecular players in the mechano- and chemo-sensory properties of the bladder.

Cystometry is an efficient technique to measure bladder function of small animals in vivo. The bladder is continuously infused at rates controlled through an intravesical catheter, whereas the urethra is left free for micturition. This allows for repetitive filling and emptying of the bladder, while intravesical pressure and voided volume are recorded.

	The lower urinary tract (LUT) functions as a  dynamic reservoir that is able to store urine and to efficiently expel it at a  convenient time. While ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Performing Subretinal Injections in Rodents to Deliver Retinal Pigment Epithelium Cells in Suspension

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment epithelium (RPE) cells. RPE provide critical support for photoreceptors and death or dysfunction of RPE cells is characteristic of age-related macular degeneration (AMD), the leading cause of permanent vision loss in people age 55 and older. While no cure for AMD has been identified, implantation of healthy RPE in diseased eyes may prove to be an effective treatment, and large numbers of RPE cells can be readily generated from pluripotent stem cells. Several interesting questions regarding the safety and efficacy of RPE cell delivery can still be examined in animal models, and well-accepted protocols used to inject RPE have been developed. The technique described here has been used by multiple groups in various studies and involves first creating a hole in the eye with a sharp needle. Then a syringe with a blunt needle loaded with cells is inserted through the hole and passed through the vitreous until it gently touches the RPE. Using this injection method, which is relatively simple and requires minimal equipment, we achieve consistent and efficient integration of stem cell-derived RPE cells in between the host RPE that prevents significant amount of photoreceptor degeneration in animal models. While not part of the actual protocol, we also describe how to determine the extent of the trauma induced by the injection, and how to verify that the cells were injected into the subretinal space using in vivo imaging modalities. Finally, the use of this protocol is not limited to RPE cells; it may be used to inject any compound or cell into the subretinal space.

Here we present a community accepted protocol in multimedia format for subretinally injecting a bolus of RPE cells in rats and mice. This approach can be used for determining rescue potentials, safety profiles, and survival capacities of grafted RPE cells upon implantation in animal models of retinal degeneration.

The conversion of light into electrical impulses occurs in the outer retina and is accomplished largely by rod and cone photoreceptors and retinal pigment ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

A Method for Quantifying Upper Limb Performance in Daily Life Using Accelerometers

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. It has become important to measure a person's activities in daily life, and not just measure their capacity for activity in the structured environment of a clinic or laboratory. A wearable sensor that is now enabling measurement of daily movement is the accelerometer. Accelerometers are commercially-available devices resembling large wrist watches that can be worn throughout the day. Data from accelerometers can quantify how the limbs are engaged to perform activities in peoples' homes and communities. This report describes a methodology to collect accelerometry data and turn it into clinically-relevant information. First, data are collected by having the participant wear two accelerometers (one on each wrist) for 24 h or longer. The accelerometry data are then downloaded and processed to produce four different variables that describe key aspects of upper limb activity in daily life: hours of use, use ratio, magnitude ratio, and the bilateral magnitude. Density plots can be constructed that visually represent the data from the 24 h wearing period. The variables and their resultant density plots are highly consistent in neurologically-intact, community-dwelling adults. This striking consistency makes them a useful tool for determining if upper limb daily performance is different from normal. This methodology is appropriate for research studies investigating upper limb dysfunction and interventions designed to improve upper limb performance in daily life in people with stroke and other patient populations. Because of its relative simplicity, it may not be long before it is also incorporated in clinical neurorehabilitation practice.

This protocol describes a method to quantify upper limb performance in daily life using wrist-worn accelerometers.

A key reason for referral to rehabilitation services after stroke and other neurological conditions is to improve one's ability to function in daily life. ...

Remote Laboratory Management: Respiratory Virus Diagnostics

An uptick in recent pandemics (Ebola, Zika, MERS, influenza, etc.) underlines the need for a more 'nimble,' coordinated response that addresses a multitude of issues ranging from transportation, access, facilities, equipment, and communication to provider training. To address this need, we have developed an innovative, scalable, logistics-enhanced, mobile, laboratory facility for emergencies and epidemics in resource-constrained global settings. Utilizing a background in clinical operations as an academic medical center, we designed a rapidly-deployable, modular BSL-2 and BSL-3 facility with user-friendly software for tracking and management of drugs and supplies in remote regions during epidemics and outbreaks. Here, we present our intermodal, mobile, expandable shipping-container laboratory units. The design of the laboratory facilitates off-grid usage by minimizing power consumption and allowing alternate water sources. The unit's information communication technology (ICT) platform provides (i) user-friendly tablet-based documentation, (ii) enhanced tracking of patients and supplies, and (iii) integrated communication onsite with built-in telehealth capabilities. To ensure quality in remote environments, we have developed a checklist for a basic laboratory workflow and a protocol for respiratory viral diagnosis using reverse-transcription polymerase chain reaction (RT-PCR). As described, this innovative and comprehensive approach allows for the provision of laboratory capability in resource-limited global environments.

A rapidly-deployable, off-grid laboratory has been designed and built for remote, resource-constrained global settings. The features and critical aspects of the logistically-enhanced, expandable, multifunctional laboratory modules are explored. A checklist for a basic laboratory workflow and a protocol for a respiratory viral diagnostic test are developed and presented.