Name: Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control (Video) | JoVE Journal
Uploaded: 2022-12-16T12:40:00.000+00:00
Duration: 5 min 41 s
Description: Learn left anterior descending coronary artery ligation in rats for ischemia-reperfusion research. This modified protocol improves accuracy and reproducibility for studying myocardial injury and therapeutic approaches.

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5 minutes

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 gloves.</li>
		<li>Pinch off a small portion of sterile cotton and roll it to form a ball. Repeat this procedure.</li>
		<li>Dip the cotton balls in sterile 0.9% saline and squeeze out excess saline.</li>
		<li>Store the cotton balls in a clean box sterilized with 75% ethanol.</li>
	</ol>
	</li>
	<li>Preparation of holding hooks.
	<ol>
		<li>Put on a surgical mask and gloves.</li>
		<li>Sterilize clips and rubber bands with 75% ethanol.</li>
		<li>Bend the clips into the shape of a hook for the chest wall and tissue.</li>
		<li>Connect the bent clips with one, two, or three rubber bands to ensure the tension from the wound of the surgical window is wide enough for LAD ligation.</li>
		<li>Prepare and store at least five homemade hooks in a clean box sterilized with 75% ethanol.</li>
	</ol>
	</li>
	<li>Preparation of a ligation loop.
	<ol>
		<li>Place the middle of a 7-0 silk stitch in the 1/2 circle size 3 spring eye of a tapered nonswaged surgical needle.</li>
	</ol>
	</li>
	<li>Preparation of a snare loop controller
	<ol>
		<li>Cut a 5-mm polyethylene (PE)-10 tube using scissors.</li>
		<li>Heat and soften the tube under flame to smooth both of its edges.</li>
	</ol>
	</li>
	<li>Preparing the rats
	<ol>
		<li>Select 8-week-old Sprague-Dawley male rats with a minimum weight of 250 g.</li>
		<li>House and maintain the rats under a 12 h light/dark cycle at a controlled temperature (21 &#176;C &#177; 2 &#176;C) with free access to food, standard mouse pellets, and tap water.</li>
		<li>Anesthetize the rats with pentobarbital (50 mg/kg, administered intraperitoneally). 
		NOTE: Additional anesthetic (pentobarbital, 30 mg/kg) should be administered after every hour.</li>
		<li>Check the rats&#39; reflexes by pinching the tail and hind feet to ensure that the animal is sufficiently anesthetized.</li>
		<li>Open the tissue between two cartridge rings below the glottis using scissors and insert a 3 cm PE-10 tube to act as an endotracheal tube25.</li>
		<li>Connect the endotracheal tube to a ventilator manually.</li>
		<li>Inspect the animal&#39;s thoracic movement synchronized with the respiration cycle to ensure the lungs are adequately ventilated.</li>
		<li>Open the neck region and cannulate the jugular vein26.</li>
	</ol>
	</li>
</ol>
2. LAD ligation
<ol>
	<li>Put on a surgical mask and gloves.</li>
	<li>Touch the chest and find the manubrium and sternal angle (the junction of the manubrium and sternum body).</li>
	<li>Identify the left-side rib that connects with the sternal angle (rib A) by touching manually.</li>
	<li>Identify the intercostal space immediately below rib A. Use fine-tip forceps to gently lift the skin close to the intercostal space, and then use a surgical scalpel with a blade to create a 1 cm oblique incision along the skin tension lines from the point approximately 5 mm to the left of the sternal body.</li>
	<li>Use curved forceps to gently separate the skin and muscle layers from the incision. Hook the muscle layers outside the left anterior chest wall downward with bent clips to expose the ribs underneath.</li>
	<li>Identify the rib below rib A (rib B). Cut rib B with a blunt scissor from the middle of the rib cartilage (approximately 2-3 mm from the sternal body). Gently touch and compress the wound with a saline wet cotton ball for several seconds if bleeding occurs.</li>
	<li>Open the thorax carefully from the cut of rib B with four bent clips. Each bent clip should hook the intercostal muscle and ribs to gently spread the chest wall in four directions (namely, upper then left, upper then right, left downward, and right downward) and create a rectangular surgical window.</li>
	<li>Hook gently against the left lung and other adjacent tissues covering the pericardium with another bent clip to prevent accidental tissue damage during the procedure.</li>
	<li>Expose the heart by gently removing the thin pericardium with forceps. Identify the 1st branch of the left main coronary artery (LMCA), which is usually between the pulmonary artery and left auricle. The LMCA and LAD present as a superficial bright red line that runs from the edge of the left auricle toward the apex.</li>
	<li>Use the prepared surgical needle to create an open ligation loop by inserting and passing the silk stitch under the LAD at a location immediately distal to the 1st branch of the LMCA in the direction from the left toward the right side of the LAD to avoid accidentally puncturing the left auricle. With a single suture, the open loop is created. Gently swab the surface of the heart to visualizethe coronary arteries if the LAD is invisible because of fluid or blood covering the surface of the heart.</li>
	<li>Hold one side of the suture and gently separate the needle from the suture using a needle holder.</li>
	<li>Insert the two ends of the silk suture on one side of the open loop into the circle on the other side to form a snare loop.</li>
	<li>Insert the two ends of the silk suture of the snare loop into the prepared snare controller before closing the loop.</li>
	<li>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 temporary myocardial ischemia for 1 h.</li>
	<li>Hold the silk to fix the position of the snare loop controller with Kelly forceps once the loop has been tied securely. Place the other end of the Kelly forceps on the surgical table during LAD ligation.</li>
	<li>Cover the surgical window with saline wet cotton balls during LAD ligation.</li>
	<li>Open the Kelly forceps.</li>
	<li>Release the snare loop controller for reperfusion of the coronary flow for 2 h.</li>
	<li>Resect the heart along the base and vascular borders carefully and avoid grabbing the tissue. 
	NOTE: Euthanize the rat with CO2&#160;at a flow rate of 40% &#160;cage volume/min.</li>
</ol>

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<li>Langer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+anatomy+and+physiology+of+the+skin%5BTitle%5D)+AND+%22British+Journal+of+Plastic+Surgery%22%5BJournal%5D)">On the anatomy and physiology of the skin.</a> British Journal of Plastic Surgery. 31 (4), 277-278 (1978).</li>
<li>Carmichael, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+tangled+web+of+Langer%27s+lines%5BTitle%5D)+AND+%22Clinical+Anatomy%22%5BJournal%5D)">The tangled web of Langer's lines.</a> Clinical Anatomy. 27 (2), 162-168 (2014).</li>
<li>Chang, L. R., Marston, G., Martin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anatomy%2C+Cartilage%5BTitle%5D)+AND+%22StatPearls%22%5BJournal%5D)">Anatomy, Cartilage.</a> StatPearls. , StatPearls Publishing. Treasure Island, FL. (2022).</li>
<li>Kolk, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LAD-ligation%3A+A+murine+model+of+myocardial+infarction%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">LAD-ligation: A murine model of myocardial infarction.</a> Journal of Visualized Experiments. (32), e1438(2009).</li>
</ol>

The authors declare that there is no conflict of interest regarding the publication of this article.

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.

<table><tbody><tr><td>Evan&amp;rsquo;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></tr></tbody></table>

left anterior descending coronary artery ligation for ischemia-reperfusion research: model improvement via technical modifications and quality control

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'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 infar...

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 ...

left-anterior-descending-coronary-artery-ligation-for-ischemia

Research

JoVE Journal

Medicine

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice.  This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

We provide a reliable method for left anterior descending artery (LAD) ligation in a mouse model. This method is comparatively less invasive than other methods, involving endotracheal intubation, a left-sided thoracotomy approach, and thoracentesis. This method can be used as a model for both acute and chronic myocardial infarction (MI).

Murine Left Anterior Descending (LAD) Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Presented here is a surgical procedure for permanent ligation of the left coronary artery in mice. This model can be used to investigate the pathophysiology and associated inflammatory response after myocardial infarction.

Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction

The present study demonstrates a highly reproducible animal model of acute regional myocardial ischemia and reperfusion injury in rabbits using a left mini-thoracotomy for survival cases or a midline sternotomy for non-survival cases.

Model of Ischemia and Reperfusion Injury in Rabbits

This study presents a surgical porcine model of chronic myocardial ischemia due to progressive coronary artery stenosis, resulting in impaired cardiac function without infarction. Following ischemia, animals undergo off-pump coronary artery bypass graft with epicardial placement of stem cells-derived exosomes-laden collagen patch. This adjunctive therapy improves myocardial function and recovery.

Surgical Porcine Model of Chronic Myocardial Ischemia Treated by Exosome-laden Collagen Patch and Off-pump Coronary Artery Bypass Graft

The surgical procedure used to induce experimental myocardial infarction in mice begins with left thoracotomy between the third and the fourth ribs in order to visualize the anterior surface of the heart and left lung. The left coronary artery is ligated, the chest is closed and the mouse is allowed to recover spontaneously.

Modified Technique for Coronary Artery Ligation in Mice

Learn left anterior descending coronary artery ligation in rats for ischemia-reperfusion research. This modified protocol improves accuracy and reproducibility for studying myocardial injury and therapeutic approaches.

Left Anterior Descending Coronary Artery Ligation for Ischemia-Reperfusion Research: Model Improvement via Technical Modifications and Quality Control (Video) | JoVE Journal

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

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