Name: induction and assessment of ischemia-reperfusion injury in langendorff-perfused rat hearts
Uploaded: 2015-07-27T15:00:00
Description: Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

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 Apparatus...

<ol>
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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...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

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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