Name: left anterior descending coronary artery ligation for ischemia-reperfusion research: model improvement via technical modifications and quality control
Uploaded: 2022-12-16T12:40:00
Description: Watch this Scientific Journal Video about Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control at JoVE

This model was developed with financial support from the Ministry of Science and Technology, Taiwan (MOST 109-2320-B-030-006-MY3).

All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The study protocol was approved by and in accordance with the guidelines of the Institutional Animal Care and Use Committee at Fu-Jen Catholic University.
1. Preparation before surgery
<ol>
	<li>Preparation of saline wet cotton balls
	<ol>
		<li>Put on a surgical mask and gl...

The proposed protocol has several distinctive features, such as identifying the exact position for LAD ligation, creating a gadget to control a snare loop in a single suture, and supporting a modified surgical maneuver to reduce tissue damage, thus enabling researchers to ligate the LAD accurately, securely, and consistently, as well as control the state of the snare loop instantly for acute myocardial IR research.
The location of LAD ligation influences the area and size of the myocardial inf...

Ischemic heart disease is a leading cause of death worldwide1,2. In addition to the control of modifiable risk factors for preventing the development of coronary heart disease, therapeutic strategies are crucially required for acute coronary syndrome3,4. Cardiogenic shock and fatal arrhythmia in acute ST-segment elevation myocardial infarction (STEMI) have been found to increase the likelihood of in-hospital mortality5,6,7,8. Primary percutaneous coronary intervention (PCI) is the preferred treatment for STEMI9,10,11; however, the therapeutic effects have a ceiling when the door-to-balloon time is &#60;90 min12,13. Additional strategies are required for further improving the clinical outcomes of the disease14,15,16,17,18,19.
An acute myocardial ischemia- reperfusion (IR) experiment involving left anterior descending artery (LAD) ligation in rats is one of the animal models comparable to the clinical scenario wherein short door-to-balloon times are required for patients with STEMI to rescue the heart from ischemic damage. However, surgery-induced STEMI in small animals is often technically challenging because it is a complex operation associated with high mortality and high variation in infarction size20,21,22,23,24. To overcome the technical challenge, the present study developed a comprehensive and effective animal model in rats (because they are larger than mice) to establish a reliable and reproducible acute myocardial IR research protocol through technical modification. The proposed protocol results in fewer surgical complications, less tissue damage, and less likelihood of mortality during surgery. Additionally, a procedure was used to measure the infarct size and area at risk (AAR) and, thus, verify the quality of the study results. The proposed protocol can be used to investigate the pathophysiological processes of acute myocardial IR stress to develop new therapeutic strategies against the damage.

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	<tbody>
		<tr>
			<td>Evan&#8217;s blue</td>
			<td>Sigma Aldrich</td>
			<td>E2129</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Shinva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital</td>
			<td>Sigma Aldrich</td>
			<td>1507002</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades</td>
			<td>Shinva</td>
			<td>s2646</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handles</td>
			<td>Shinva</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silk sutures</td>
			<td>SharpointTM</td>
			<td>DC-2150N</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical needle</td>
			<td>AnchorTM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triphenyltetrazolium chloride (TTC) solution</td>
			<td>Solarbio</td>
			<td>T8170-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilator</td>
			<td>Harvard Rodent Ventilator</td>
			<td></td>
			<td></td>
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left anterior descending coronary artery ligation for ischemia-reperfusion research: model improvement via technical modifications and quality control

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation myocardial infarction (STEMI), resulting in cardiogenic shock and fatal arrhythmia, which are associated with high mortality. Primary coronary intervention (PCI) for recanalizing the coronary artery significantly improves the outcomes of STEMI, but advancements made in shortening the door-to-balloon time have failed to reduce in-hospital mortality, suggesting that additional therapeutic strategies are required. Left anterior descending coronary artery (LAD) ligation in rats is an animal model for acute myocardial IR research that is comparable to the clinical scenario in which rapid coronary recanalization through PCI is used for STEMI; however, PCI-induced STEMI is a technically challenging and complicated operation associated with high mortality and great variation in infarction size. We identified the ideal position for LAD ligation, created a gadget to control a snare loop, and supported a modified surgical maneuver, thereby reducing tissue damage, to establish a reliable and reproducible acute myocardial ischemia-reperfusion (IR) research protocol for rats. This is a non-survival surgery. We also propose a method for validating the quality of study results, which is a critical step for determining the accuracy of subsequent biochemical analyses.

At the end of myocardial ischemia and reperfusion, the quality of LAD ligation should be assessed before further biochemical or molecular analyses.
The sufficiency of LAD occlusion through ligation was determined by injecting 1 mL of 2% Evan&#39;s blue dye through the central venous catheter. Then, the myocardium with coronary perfusion was stained blue compared with the non-perfused region, which remained red (Figure 1A). The red region is the AAR of myocardial i...

Watch this Scientific Journal Video about Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control at JoVE

Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control

Herein, we present a protocol focusing on the quality control of the left anterior descending coronary artery ligation by technically modifying the traditional procedure in rats for acute myocardial ischemia-reperfusion research.

Coronary heart disease is the leading cause of death globally. Complete cessation of blood flow in coronary arteries causes ST-segment elevation ...

Ischemia heart disease is a leading cause of death worldwide. This procedure can be performed to induce ischemia-reperfusion injury in the heart. This protocol is to ligate the LAD accurately, securely, and consistently.To prepare saline wet cotton balls, pinch off a small portion of sterile cotton and roll it repeatedly to form a ball. Dip the cotton balls in sterile 0.9%saline and squeeze out excess saline. Store the cotton balls in a clean box sterilized with 75%ethanol.To prepare holding hooks, sterilize clips and rubber bands with 75%ethanol. To prepare a ligation loop, place the middle of a 7-0 silk stitch in the half circle size three spring eye of a tapered non swaged surgical needle. To prepare a snare loop controller, cut a five millimeter polyethylene 10 tube using scissors.Next, for the experiment, anesthetize an eight week old Sprague Dawley male rat and check the reflexes by pinching the tail and hind feet. Then open the tissue between the two cartilage rings below the glottis using scissors and insert a three centimeter polyethylene 50 tube to act as an endotracheal tube. Connect the endotracheal tube to a ventilator.Touch the chest and find the manubrium and sternal angle. Then identify the left side rib that connects with the sternal angle. Identify the intercostal space below rib A.Use fine tipped forceps to gently lift the skin close to the intercostal space.Then use scissors to create a one centimeter oblique incision along the skin tension lines from the point approximately five millimeters to the left of the sternal body. Separate the skin and muscle layers from the incision using scissors. Then hook the muscle layers outside the left anterior chest wall downward with bent clips to expose the ribs underneath.After identifying rib B below rib A, cut rib B with a blunt scissor from the middle of the rib cartilage. Gently touch and compress the wound with a saline wet cotton ball for several seconds if bleeding occurs. Next, hook the four bent clips to the intercostal muscle and ribs to gently spread the chest wall in four directions and create a rectangular surgical window to open the thorax carefully from the cut of rib B.Hook gently against the left lung and adjacent tissues covering the pericardium with another bent clip to prevent accidental tissue damage during the procedure.Expose the heart by removing the thin pericardium with forceps. Then identify the first branch of the left main coronary artery, or LMCA, between the pulmonary artery and the left auricle. Using a surgical needle, create an open ligation loop by inserting and passing the silk stitch under the LAD at a location immediately distal to the first branch of the LMCA in the direction from the left toward the right side of the LAD to avoid accidentally puncturing the left auricle.If the LAD is invisible because of fluid or blood covering the surface of the heart, gently swab the surface of the heart to visualize the coronary arteries. Insert the two ends of the silk suture on one side of the open loop into the circle of the other side to form a snare loop. Then insert the two ends of the silk suture of the snare loop into the prepared snare controller before closing the loop.Slide the snare loop controller along the silk suture while gently stretching the silk to close the snare loop. Cease the coronary flow of the LAD to induce myocardial ischemia for one hour. Once the loop has been tied securely, hold the silk to fix the position of the snare loop controller with Kelly forceps.Place the other end of the Kelly forceps on the surgical table. Cover the surgical window with saline wet cotton balls during LAD ligation. Open the Kelly forceps then release the snare loop controller for reperfusion of the coronary flow for two hours.Validation of LAD ligation quality with Evans blue is shown. The myocardium with coronary perfusion was stained blue compared with a non perfused region, which remained red. A low variation in the area at risk, or AAA percentage among the study animals, indicated the accurate location of the LAD ligation.Further, the infarct size was estimated as the ratio of the infarct area to the AAR in control and treated groups. The infarct size percentage of the treatment group is lowered than the controlled LAD ligation group. This technique paves the way for researchers to explore novel therapeutic approaches against ischemic heart disease.

Research

JoVE Journal

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