This publication was supported by the South Carolina Clinical &#38; Translational Research (SCTR) Institute, with an academic home at the Medical University of South Carolina, NIH/NCATS Grant Number UL1 TR000062. Further support was provided by VA merit award BX002327-01 to DRM. DJH was supported by NIH/NCATS Grant Number TL1 TR000061 and by NIH Grant Number T32 GM008716. SEA was supported by NIH Grant Number T32 HL07260.

All procedures listed here have been approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. The experiments described here are acute, non-survival experiments. As such, there is no use of eye ointment and a sterile operating suite is not required. Euthanasia is achieved by exsanguination during harvesting of the heart.
1. Experimental Preparation
<ol>
	<li>Set up either a constant pressure or constant flow Langendorff Perfusion Apparatus4.</li>
	<li>Prepare 4 L of modified Krebs Henseleit Buffer (in mM: 112 NaCl, 5 KCl, 1.2 MgSO4, 1 K2HPO4, 1.25 CaCl2, 25 NaHCO3, 11 D-glucose, 0.2&#160;octanoic acid, pH = 7.4).
	<ol>
		<li>Make 10x stock buffers.
		<ol>
			<li>Dissolve NaCl, KCl, MgSO4, and K2HPO4 in 3 L ultrapure water.</li>
			<li>Dissolve CaCl2 in 500 ml ultrapure water with 40 ml of 1 N HCl.</li>
			<li>Slowly add CaCl2 solution to the buffer made in 1.2.1.1. Add enough ultrapure water to total 4 L. Allow to stir for 1 hr before filtering through a 0.8 &#181;m filter. Store at 4 &#176;C.</li>
			<li>Dissolve NaHCO3 in 4 L ultrapure water. Allow to stir for 1 hr before filtering through at 0.8 &#181;m filter. Store at 4 &#176;C</li>
		</ol>
		</li>
		<li>Combine 400 ml 10x Krebs Henseleit buffer with 400 ml 10x NaHCO3 buffer. Add enough ultrapure water to total 4 L. Dissolve glucose and octanoic acid while stirring. Filter through 0.8 &#181;m filter.</li>
	</ol>
	</li>
	<li>Prepare system for heart.
	<ol>
		<li>Add 2 L of filtered buffer to a 4 L bottle placed 38 in&#160;above the stopcock attached to the aortic cannula. This will deliver a perfusion pressure of 70-80 mmHg to the heart, which is the recommended perfusion pressure4.</li>
		<li>Insert gas dispersion bubbler into the buffer in the bottle.</li>
		<li>Run the buffer through the system, being sure to eliminate any bubbles from inside the tubing.</li>
		<li>Turn on gas (95% O2/5% CO2) at least 30 min before heart is to be placed on the system. Use a gas flow probe and blood gas analyzer to verify oxygen saturation.</li>
	</ol>
	</li>
	<li>Prepare surgical supplies and balloon.
	<ol>
		<li>For harvesting of heart, prepare one pair of 6.75&#8221; surgical scissors, one pair of 4.5&#8221; scissors, two pairs of hemostats, two sets of caramalt forceps, two syringes with 27 G needles, 2 pieces of 0-guage silk suture (about 15 cm in length), one 1/16 inch barbed cannula, and one tracheal cannula.</li>
		<li>To assemble tracheal cannula, glue a short piece of 1/16 inch diameter plastic tubing to a Y-connector.</li>
		<li>To assemble balloon apparatus: attach a gavage needle to a piece of 1/16 inch tubing, then attach tubing to pressure transducer housing. A small syringe attached to the other side of the pressure transducer housing will allow inflation and deflation of the balloon.
		<ol>
			<li>Cut a square of plastic wrap and place tip of a gavage needle in the center. Wrap the plastic wrap around the gavage needle.</li>
			<li>Fill balloon with saline and tie off with two sutures. Inflate balloon to ensure there are no leaks. The volume of saline within the inflated balloon should be roughly 200 &#181;l, but can vary depending on the size of the rat.</li>
			<li>Alternatively, use a latex finger cot in place of the plastic wrap. Place gavage needle within finger cot, fill with saline and tie off.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Harvest the heart
<ol>
	<li>Restrain a Sprague Dawley rat using a decapicone or other manual restraint device. Weigh rat.</li>
	<li>Administer ketamine/xylazine mixture (0.85 mg/kg ketamine and 0.15 mg/kg xylazine) via intraperitoneal injection to anesthetize rat.&#160; This dose of anesthetic will provide sufficient anesthesia for 20-40 min, but the procedure should be completed as quickly as possible after anesthesia is confirmed.</li>
	<li>Confirm proper degree of anesthesia by testing toe pinch reflex.</li>
	<li>Tracheotomy
	<ol>
		<li>Remove pelt from mid-forepaw to base of jaw, using hemostats to elevate pelt and scissors to make incision. Keep the pelt elevated to separate it from the underlying muscle, allowing one to cut along the pelt and remove it.</li>
		<li>Using hemostats, lift glandular tissue and make a horizontal incision in fascia inferior to the glandular tissue. Use hemostats to spread incision open, taking care not to nick the jugular veins.</li>
		<li>Using hemostats, pinch muscle overlying trachea, and using scissors make a transverse incision in the muscle just below the tip of the hemostats. Using hemostats, spread tissue apart to reveal trachea.</li>
		<li>Carefully insert hemostats under trachea, clearing fascia while progressing by gently advancing and retracting hemostats.</li>
		<li>Once hemostats are successfully placed behind the trachea, pinch 0-0 silk suture with hemostats and hemisect the trachea with a small, sharp pair of scissors.</li>
		<li>Insert tracheal cannula into the hemisected trachea, pull suture behind the trachea, and tie the suture around the cannulated trachea, anchoring the suture to the Y-groin of the cannula with a surgeons knot.</li>
		<li>Using a 27 G needle, inject 1,000 U/kg heparin into the jugular vein and allow to circulate for 30 sec.</li>
	</ol>
	</li>
	<li>Thoracotomy
	<ol>
		<li>Remove pelt from mid-abdomen to mid-forepaw region by pinching with hemostats and cutting with scissors.</li>
		<li>Make a &#190; inch transverse incision into the abdominal muscle wall, just below the diaphragm.</li>
		<li>Make a vertical incision up the abdominal wall and chest wall, cutting along the sternum.</li>
		<li>Cut the diaphragm back bilaterally, then spread ribcage to reveal heart, clamp ribcage with hemostats to ensure it remains spread.</li>
		<li>Using hemostats, gently remove thymus, exposing the ascending aorta.</li>
		<li>Tease hemostats through aortic loop, advancing and retracting hemostats and gently spreading fascia to allow tip of hemostats through.</li>
		<li>Use hemostats to pull 0-0 silk suture behind ascending aorta, tie suture into a loose half-square knot.</li>
		<li>Turn stopcock to start flow of perfusion buffer, hemisect aorta and immediately insert cannula into aortic lumen. Rapidly cinch a half-square knot and complete the knot, tightening thoroughly.</li>
		<li>Use scissors to dissect heart away from the great vessels, remove heart from chest and attach to perfusion column.</li>
		<li>Trim away excess lung tissue and allow heart to equilibrate on column for about 15 min before insertion of balloon. Use a normal flow rate of buffer through the heart of 10-20 ml/min.</li>
	</ol>
	</li>
</ol>
3. Langendorff Perfusion and Ischemia Reperfusion Injury
<ol>
	<li>Placement of LV pressure balloon
	<ol>
		<li>Deflate balloon while rolling into a cone shape and turn stopcock to ensure balloon remains deflated.</li>
		<li>Excise left atrium with a small pair of scissors to expose access to mitral valve.</li>
		<li>Insert balloon through left atrium and mitral valve into left ventricle.</li>
		<li>Start pressure monitoring software (LabChart Pro).</li>
		<li>Inflate balloon by opening stopcock and gently increasing amount of saline in balloon until diastolic pressure reading from micromanometer exceeds zero.</li>
		<li>To check length-tension response, inflate balloon slowly to a diastolic pressure of 75 mmHg, deflate slowly, repeat once.</li>
		<li>Set diastolic pressure to 10 mmHg by modulating the amount of saline in the balloon. Turn stopcock to seal balloon at this level of inflation, and maintain closed pressure system between LV balloon and pressure transducer.</li>
		<li>Lower heart into heated chamber and cover chamber with plastic.</li>
	</ol>
	</li>
	<li>Administration of Ischemia Reperfusion Injury
	<ol>
		<li>Plug the bottom of the heated chamber and allow chamber to fill with effused buffer.</li>
		<li>When buffer has submerged heart, turn stopcock to stop the flow of buffer into the heart, inducing global ischemia. Ischemia time can vary depending on the goals of the experiment.</li>
		<li>After 30 min of ischemia, turn stopcock to restore flow of buffer into heart, initiating reperfusion.</li>
		<li>Allow reperfusion of heart for 1 hr before terminating the experiment. Use variable reperfusion times, depending on the goals of the experiment.</li>
	</ol>
	</li>
	<li>Termination of Experiment and Harvesting of Tissue
	<ol>
		<li>Using side port of stopcock, administer 10 ml of phosphate buffered saline at 4 &#176;C into the heart at a rate of 10 ml/min to arrest the heart.</li>
		<li>Remove heart from perfusion column, trim away remaining atrial tissue.</li>
		<li>Place heart into a stainless steel tissue slicing matrix, cover with Parafilm, and place at -80 &#176;C for 8 min, or until the heart has a marshmallow-like consistency.</li>
		<li>Remove block from freezer and insert razorblades into slicing matrix to slice heart into 2 mm slices. Tissue blocks can also be purchased with slice sizes other than 2 mm.</li>
		<li>Remove slices one at a time and drop into liquid nitrogen to flash freeze for biochemical studies. Retain the third ventricular slice from the apex for infarct staining.</li>
		<li>Remove tissue from liquid nitrogen and store at -80 &#176;C until use in biochemical experiments.</li>
	</ol>
	</li>
	<li>Infarct Staining
	<ol>
		<li>Incubate the third ventricular slice in 10 ml of 1% w/v triphenyltetrazolium chloride (TTC) at 37 &#176;C for 15 min. 
		Note: Incubation in TTC can be performed for 15 or 30 min with no change in effectiveness15.</li>
		<li>Transfer tissue slice from TTC to 10% buffered formalin and allow to incubate at room temperature overnight. For optimal results, hearts should be taken out of formalin and imaged within 24 hr4.</li>
		<li>Photograph stained slice and use software such as ImageJ to estimate infarct size according to manufacturer&#8217;s protocol. Take photographs as soon as possible after fixation, as the dye will fade over time.</li>
	</ol>
	</li>
</ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+perspectives+on+reperfusion+injury+in+acute+myocardial+infarction.">Clinical perspectives on reperfusion injury in acute myocardial infarction.</a> American Heart Journal. 167 (5), 637-645 (2014).</li><li>Beyersdorf, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+controlled+reperfusion+strategies+in+cardiac+surgery+to+minimize+ischaemia/reperfusion+damage.">The use of controlled reperfusion strategies in cardiac surgery to minimize ischaemia/reperfusion damage.</a> Cardiovascular Research. 83 (2), 262-268 (2009).</li><li>Langendorff, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchugen+am+&#252;berlebenden+S&#228;ugethierherzen.">Untersuchugen am &#252;berlebenden S&#228;ugethierherzen.</a> Pfl&#252;gers Archives. 61, 291-307 (1895).</li><li>Bell, R. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+phase+acute+myocardial+infarct+size+quantification:+Validation+of+the+triphenyl+tetrazolium+chloride+tissue+enzyme+staining+technique.">Early phase acute myocardial infarct size quantification: Validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.</a> American Heart Journal. 101 (5), 593-600 (1981).</li><li>Aune, S. E., Herr, D. J., Mani, S. K., Menick, D. 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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protective+role+of+heat+stress+in+the+ischaemic+and+reperfused+rabbit+myocardium.">The protective role of heat stress in the ischaemic and reperfused rabbit myocardium.</a> Journal of Molecular and Cellular Cardiology. 24 (8), 895-907 (1992).</li><li>Khaliulin, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+preconditioning+of+isolated+rat+hearts--a+potent+cardioprotective+mechanism+involving+a+reduction+in+oxidative+stress+and+inhibition+of+the+mitochondrial+permeability+transition+pore.">Temperature preconditioning of isolated rat hearts--a potent cardioprotective mechanism involving a reduction in oxidative stress and inhibition of the mitochondrial permeability transition pore.</a> The Journal of Physiology. 581 (Pt 3), 1147-1161 (2007).</li><li>Molojavyi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+ketamine+and+its+isomers+on+ischemic+preconditioning+in+the+isolated+rat+heart).">Effects of ketamine and its isomers on ischemic preconditioning in the isolated rat heart).</a> Anesthesiology. 94 (4), 623-629 (2001).</li><li>Asfour, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADH+fluorescence+imaging+of+isolated+biventricular+working+rabbit+hearts.">NADH fluorescence imaging of isolated biventricular working rabbit hearts.</a> The Journal of Visualized Experiments. 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The authors declare that they have no competing financial interests.

The isolated perfused rat heart can be successfully used to study the effect of pharmacological intervention on cardiac preconditioning in ischemia reperfusion injury9. However, there are some essential steps to the procedure that must be standardized in order to ensure reproducible results. Maintaining a temperature of 37.4 &#176;C within the system is critical, as even mild hypothermia and hyperthermia can cause cardiac preconditioning10,11. The overall time that elapses from injection of anesthet...

Elucidation of the events underlying the cardiac response to both ischemia and reperfusion are essential in improving the treatment of myocardial infarction1 and cardiac surgical procedures that require aortic cross-clamping2. While in vivo models of ischemia reperfusion injury allow very useful endpoint analysis, they are not as effective for studying the functional effects of ischemia reperfusion injury acutely in real time. Additionally, in vivo ischemia reperfusion models generally produce significant variability in infarct size, and direct delivery of drug to the heart at the time of reperfusion is challenging. The utilization of a Langendorff isolated heart system for studying ischemia reperfusion injury allows for real-time functional assessment of pharmacological treatments, uniform area of infarcted tissue, and instantaneous delivery of drug directly to the myocardium.
First described by Oscar Langendorff in 18953, the Langendorff isolated heart is a robust model for studying ischemia reperfusion injury, having been used in ischemia reperfusion research for the last 40 years4,5. Here, some modifications are made to optimize the isolated heart for functional analysis. In situ cannulation of the aorta while the heart is beating ensures that the heart does not experience ischemic preconditioning, which would alter the results of ischemia reperfusion trials6. To facilitate this, a tracheotomy is performed, allowing ventilation and ensuring physiological stability of the rat during surgery. The heart is then attached to a glass water-jacketed spiral column through which Krebs Henseleit buffer is delivered via retrograde perfusion directly into the aorta. A saline-filled balloon is inserted into the left ventricle and attached to a pressure transducer, which allows for real time measurement of pressures from within the ventricle and calculation of multiple functional parameters. At the conclusion of the experiment, the heart is flushed with cold saline to arrest contraction and flash frozen in liquid nitrogen to enable downstream analysis of DNA, RNA and protein levels. Thus modified, the Langendorff perfused heart serves as an effective system for direct monitoring of the physiological effect of pharmacological interventions at any time acutely during the ischemia reperfusion injury.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The left ventricular balloon apparatus allows for real-time monitoring of the pressure developed by the contracting left ventricle (Figure 1). As described previously7, this pressure trace can be used to calculate many of the parameters of ventricular function. These calculations can be made in the baseline phase as well as the reperfusion phase, averaged over multiple traces within each group, and compared in order to determine whether the pharmacological intervention resulted in cardiac prec...

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Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

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22.7K Views.  Medical University of South Carolina. The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

Video: Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused by whole-body hypoperfusion.5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with 
difficulty.7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger 
animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.11,12 However, in recent 
years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.13 This model reliably reproduces 
physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical 
model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience 
with use of this model to assess a number of organ-specific outcomes in AKI.14,15

A powerful model for perioperative and critical care related acute kidney injury is presented. Using whole body hypoperfusion induced by cardiac arrest it is possible to nearly replicate the histologic and functional changes of clinical AKI.

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality1-4 and which is most 
frequently caused ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...