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>

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 infarction. Ligation is generally suggested at a certain distance on the proximal LAD27,28. Overlooking the variance in coronary artery branching patterns may increase the variability in myocardial infarction23,24. In this study, the LAD was ligated immediately distal to the 1st branch of the LMCA, thus preventing accidental ligation of the left circumflex artery or septal artery and leading to consistent infarct size and a lower likelihood of fatal arrhythmia29,30,31.
Secure LAD ligation is essential for LAD occlusion. Experts have recommended that the LAD should be ligated with tiedto create one to three knots or with a small piece of tubing to compress the coronary artery32,33,34,35,36. In this paper, we suggest a controllable gadget with a snare loop to ligate the LAD in a single suture; this approach enables secure LAD ligation and instant control of loop closure and release while preventing tissue laceration, bleeding, and breaking of the strength of the suture during repeat myocardial puncture, arterial ligation, and ligature release. The approach is, thus, useful for experimental and validation procedures in acute myocardial IR research.
Recognizing anatomical features and histological properties during surgery is helpful for reducing tissue damage and improving study replication. Regarding the opening of the thorax, scholars have suggested separating the pectoral and 3rd or 4th intercostal muscles by using scissors, a retractor, forceps, blunt tweezers, or stitches to pull aside the thoracic muscles and rib cage32,33,35,37,38. The present study suggests an incision along the skin tension lines (the connective tissue framework of the skin)39,40, cutting a single rib&#39;s cartilage, which contains avascular flexible connective tissue41, and hooking the thoracic muscle and rib cages to open the chest wall. This approach helps maintain tissue integrity and reduces the bleeding risk. Additionally, starting the approach by identifying reliable surface makers through touch means that the surgical procedure utilizing a skin incision is highly repeatable and consistent.
Confirming the quality of myocardial infarction induced by LAD ligation is a critical step before the investigation of pathophysiological changes in acute myocardial IR research. In the literature, the occurrence of myocardial infarction after LAD ligation is confirmed by observing sudden regional paleness of the myocardium28,33; an acute ST segment of electrocardiogram elevation from the baseline33; elevated serum cardiac enzyme levels such as CK-MB, troponin I, and troponin T28,32,42; or infarcted regions macroscopically42. The consistency of LAD ligation should further be validated by determining the AAR for infarction by using Phthalo or Evan&#39;s blue dye32,35,37,38. Low variability in AAR percentage among samples proves the consistency and quality of the procedure for acute myocardial IR research. Moreover, the infarct area can be distinguished from the AAR by demarcating myocardial infarction regions with TTC28,36. Evans blue/TTC double staining was previously employed to evaluate the quality of a myocardial IR study ex vivo37. Compared with the requirement in ex vivo evaluations that the isolated heart be perfused under the Langendorff apparatus, this study supports the animal protocol of in vivo evaluation in which the results are obtained, and the quality of the study is validated instantly and directly.
More importantly, the use of Evan&#39;s blue and TTC to define the infarct area to AAR precludes the use of the infarcted myocardium for biochemical analyses, which is a requirement to exclude confounding factors and obtain accurate results in acute myocardial IR research.

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

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 infarction.
The accuracy of the location for LAD ligation was further evaluated by quantifying the variation in AAR percentage among the study animals. After the heart was sliced horizontally, the AAR percentage was determined by dividing the AAR by the whole myocardial mass (Figure 1B). A low variation in AAR percentage among the study animals indicated accurate location of the LAD ligation.
The myocardial infarct size is the primary result in acute myocardial IR research. To quantify this parameter, the sliced heart sections were incubated in 1% 2,3,5-triphenyltetrazolium chloride (TTC) in normal saline at 37 &#176;C for 30 min and then in 10% formaldehyde for 3 days. The infarct area was white. The infarct size percentage was calculated as the ratio of the infarct area to the AAR (Figure 2).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63921/63921fig1v2.jpg" /> 
Figure 1: Validation of LAD ligation quality with Evan&#39;s blue. (A) The AAR for myocardial infarction was the non-perfused myocardial mass that remained red even after Evan&#39;s blue was injected, thereby confirming secure LAD ligation. (B) The AAR percentage was calculated by dividing the AAR (red area) by the whole myocardial mass (red and blue area); a low variation in AAR percentage among the study animals demonstrated accurate location of the LAD ligation. A smaller infarct size was demonstrated in drug-treated groups as compared to&#160;non-treated groups.&#160;AAR, area at risk; LAD, left anterior descending artery. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63921/63921fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63921/63921fig2v2.jpg" /> 
Figure 2: Quantification of myocardial infarct size in TTC treatment. Infarct size was estimated as the ratio of the infarct area (white area) to the AAR (red area) in the LAD ligation group. AAR, area at risk; TTC, triphenyltetrazolium chloride. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63921/63921fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Medicine

3.3K Views.  Fu Jen Catholic University. Ischemie hartziekte is wereldwijd een belangrijke doodsoorzaak. Deze procedure kan worden uitgevoerd om ischemie-reperfusieletsel in het hart te induceren. Dit protocol is om de LAD nauwkeurig, veilig en consistent te ligateren.Om zoute natte wattenbollen te bereiden, knijp je een kleine portie steriel katoen af en rol je het herhaaldelijk op om een bal te vormen. Doop de wattenbolletjes in een steriele zoutoplossing van 0,9% en knijp overtollige zoutoplossing eruit. Bewaar de wattenbo...

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

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

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

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.

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

Modified Technique for Coronary Artery Ligation in Mice

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI we need better knowledge about pathophysiology of myocardial ischemia. This knowledge may be valuable to define new therapeutic targets for innovative cardiovascular therapies4. Experimental MI model in mice is an increasingly popular small-animal model in preclinical research in which MI is induced by means of permanent or temporary ligation of left coronary artery (LCA)5. In this video, we describe the step-by-step method of how to induce experimental MI in mice.
The animal is first anesthetized with 2% isoflurane. The unconscious mouse is then intubated and connected to a ventilator for artificial ventilation. The left chest is shaved and 1.5 cm incision along mid-axillary line is made in the skin. The left pectoralis major muscle is bluntly dissociated until the ribs are exposed. The muscle layers are pulled aside and fixed with an eyelid-retractor. After these preparations, left thoracotomy is performed between the third and fourth ribs in order to visualize the anterior surface of the heart and left lung. The proximal segment of LCA artery is then ligated with a 7-0 ethilon suture which typically induces an infarct size ~40% of left ventricle. At the end, the chest is closed and the animals receive postoperative analgesia (Temgesic, 0.3 mg/50 ml, ip). The animals are kept in a warm cage until spontaneous recovery.

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.

Myocardial infarction (MI) is one of the most important causes of mortality in humans1-3. In order to improve morbidity and mortality in patients with MI ...

A Murine Closed-chest Model of Myocardial Ischemia and Reperfusion

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ischemia and reperfusion. Immune response includes cytokine expression and release or secretion of endogenous ligands of innate immune receptors. Activation of innate immunity can potentially modulate infarct size. We have modified an existing murine closed-chest model using hanging weights which could be useful for studying myocardial pre- and postconditioning and the role of innate immunity in myocardial ischemia and reperfusion. This model allows animals to recover from surgical trauma before onset of myocardial ischemia.
Volatile anesthetics have been intensely studied and their preconditioning effect for the ischemic heart is well known. However, this protective effect precludes its use in open chest models of coronary artery ligation. Thus, another advantage could be the use of the well controllable volatile anesthetics for instrumentation in a chronic closed-chest model, since their preconditioning effect lasts up to 72 hours. Chronic heart diseases with intermittent ischemia and multiple hit models are other possible applications of this model.
For the chronic closed-chest model, intubated and ventilated mice undergo a lateral blunt thoracotomy via the 4th intercostal space. Following identification of the left anterior descending a ligature is passed underneath the vessel and both suture ends are threaded through an occluder. Then, both suture ends are passed through the chest wall, knotted to form a loop and left in the subcutaneous tissue. After chest closure and recovery for 5 days, mice are anesthetized again, chest skin is reopened and hanging weights are hooked up to the loop under ECG control.
At the end of the ischemia/reperfusion protocol, hearts can be stained with TTC for infarct size assessment or undergo perfusion fixation to allow morphometric studies in addition to histology and immunohistochemistry.

Surgical trauma induces an inflammatory response. Cytokines and endogenous ligands are known to modulate myocardial infarct size following ischemia and reperfusion. We present a modified closed-chest model of murine ischemia and reperfusion using hanging weights to minimize effects of thoracotomy.

Surgical trauma by thoracotomy in open-chest models of coronary ligation induces an immune response which modifies different mechanisms involved in ...

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

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

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.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

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

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by reduced blood supply to the heart, resulting in the loss of oxygen to and the ensuing necrosis of the heart muscle. The MI model has gained popularity for its use as a short-term ischemia-reperfusion model and a long-term permanent ligation model. Below, we describe a reliable method for the permanent ligation of the LAD. With mouse genetic engineering technology becoming more advanced, and with an increasing availability of quality murine surgical instruments, the mouse has become a popular model for MI surgeries. Our surgical model incorporates the use of an easily reversible anesthetic for the rapid recovery of the mouse; a minimally invasive endotracheal intubation without involving a tracheotomy; and a thoracentesis through the original thoracotomy site without creating an additional incision in the chest, as is done in some other methods, to effectively remove excess blood and air from the chest cavity. This method is comparatively less invasive than other methods, which dramatically reduces surgical and post-surgical complications and mortality and improves reproducibility.

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

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).

Ischemic heart disease (IHD), or acute coronary syndrome (ACS), is one of the leading causes of death in the United States. IHD is characterized by ...

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

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of 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.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Induction of Myocardial Infarction and Myocardial Ischemia-Reperfusion Injury in Mice

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of heart reflow and induce secondary myocardial injury. A simple and reproducible model of myocardial infarction and myocardial ischemia-reperfusion injury is a good tool for researchers. Here, a customizable method to create a myocardial infarction (MI) model and MIRI by precision ligation of the left anterior descending coronary artery (LAD) through micromanipulation is described. Accurate and reproducible ligature positioning of the LAD helps obtain consistent results for heart injury. ST-segment changes can help to identify model accuracy. The serum level of cardiac troponin T (cTnT) is used to assess the myocardial injury, cardiac ultrasound is employed to evaluate the myocardial systolic function, and Evans-Blue/triphenyl tetrazolium chloride staining is used to measure infarct size. In general, this protocol reduces procedure duration, ensures controllable infarct size, and improves mouse survival.

Here we describe a simple and reproducible method that can induce myocardial infarction or myocardial ischemia-reperfusion injury in mice by precision ligation of the left anterior descending coronary artery through micromanipulation.

Acute myocardial infarction is a common cardiovascular disease with high mortality. Myocardial reperfusion injury can counteract the beneficial effects of ...

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

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.

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

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

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In order to explore the pathophysiological changes after myocardial infarction and design future treatments, research models of MI are required. Permanent ligation of the left coronary artery (LCA) in mice is a popular model to investigate cardiac function and ventricular remodeling post MI. Here we describe a less invasive, reliable, and reproducible surgical murine MI model by permanent ligation of the LCA. Our surgical model comprises of an easily reversible general anesthesia, endotracheal intubation that does not require a tracheotomy, and a thoracotomy. Electrocardiography and troponin measurement should be performed to ensure MI. Echocardiography at day 28 after MI will discern heart function and heart failure parameters. The degree of cardiac fibrosis can be evaluated by Masson's trichrome staining and cardiac MRI. This MI model is useful for studying the pathophysiological and immunological alterations after MI.

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.

Ischemic heart disease and subsequent myocardial infarction (MI) is one of the leading causes of mortality in the United States and around the world. In ...

Adjunctive Exosome Therapy: Enhancing Myocardial Recovery Post-CABG Surgery

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

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts to reduced oxygen availability by reducing metabolic activity, thereby preventing irreversible cardiomyocyte injury and infarction. This is distinct from myocardial infarction, as HIB has the potential for recovery with revascularization. Patients with significant coronary artery disease (CAD) experience chronic ischemia, which puts them at risk for heart failure and sudden death. The standard surgical intervention for severe CAD is coronary artery bypass graft surgery (CABG), but it has been shown to be an imperfect therapy, yet no adjunctive therapies exist to recover myocytes adapted to chronic ischemia. To address this gap, a surgical model of HIB using porcine that is amenable to CABG and mimics the clinical scenario was used. The model involves two surgeries. The first operation involves implanting a 1.5 mm rigid constrictor on the left anterior descending (LAD) artery. As the animal grows, the constrictor gradually causes significant stenosis resulting in reduced regional systolic function. Once the stenosis reaches 80%, the myocardial flow and function are impaired, creating HIB. An off-pump CABG is then performed with the left internal mammary artery (LIMA) to revascularize the ischemic region. The animal recovers for one month to allow for optimal myocardial improvement prior to sacrifice. This allows for physiologic and tissue studies of different treatment groups. This animal model demonstrates that cardiac function remains impaired despite CABG, suggesting the need for novel adjunctive interventions. In this study, a collagen patch embedded with mesenchymal stem cell (MSC)-derived exosomes was developed, which can be surgically applied to the epicardial surface distal to LIMA anastomosis. The material conforms to the epicardium, is absorbable, and provides the scaffold for the sustained release of signaling factors. This regenerative therapy can stimulate myocardial recovery that does not respond to revascularization alone. This model translates to the clinical arena by providing means of physiological and mechanistic explorations regarding recovery in HIB.

Author Spotlight: Enhancing Coronary Artery Revascularization

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.

Chronic myocardial ischemia resulting from progressive coronary artery stenosis leads to hibernating myocardium (HIB), defined as myocardium that adapts ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video