Name: LAD-Ligation: A Murine Model of Myocardial Infarction (Video) | JoVE Journal
Uploaded: 2009-10-14T23:30:00.000+00:00
Duration: 8 min 23 s
Description: Learn the LAD ligation surgical technique for inducing myocardial infarction in mice. This method enables researchers to study ischemic heart disease pathophysiology and test therapeutic interventions.

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5 minutes

The authors thank Christiane Pahrmann for her technical assistance and performing histology.

Balb/C mice weighing a minimum of 20g at an age of 8 to 12 weeks are purchased from Charles River (Sandhofer Weg 7, D-97633 Sulzfeld). 
 Mice are housed under conventional conditions, fed standard mouse pellets and water ad libitum. Anesthetize mouse with isoflurane (2%) using an induction chamber. 
<ol>
 <li>Shave the neck area and the left side of the ribcage and disinfect using 80% ethanol.</li>
 <li>Place the mouse on its back and place a facemask over its nose and mouth to keep up the anesthesia.</li>
 <li>Check the reflexes pinching the tail and hind feet to be sure that the mouse is sufficient anesthesized. </li>
 <li>Under microscopic view perform a midline cervical incision separating the skin, muscle and tissue covering the trachea.</li>
 <li>When the trachea is exposed cut a small hole into the tissue between two cartridge rings below the glottis to insert the endotracheal tube (Figure 1). Insert the endotracheal tube holding the cranial part of the trachea using micro surgical forecepts. Check the thoracic movement to be sure that both lungs are well ventilated.</li>
 <li>The respiration rate (RR) should be approximately 110 per minute, with an inspiratory pressure of 17 to 18cm H2O. Turn the mouse carefully, lying on its right side, facing its left side. Perform a leftsided thoracotomy between the 3rd and the 4th rib, and dissect the tissue and muscle carefully, using a cauter to prevent bleeding. </li>
 <li>Open the thorax carefully, once the thorax is opened, find the heart, without touching the lung with any sharp object. Now remove the part of the pericardial sac that is covering the heart.</li>
 <li>The LAD is located between the pulmonary artery and the left auricle. Use an 8-0 Prolene suture (Ethicon, Norderstedt, Germany) to ligate the LAD proximal with one single suture (Figure 2).</li>
 <li>Place a chest tube (28G, venal catheter), between the 4th and the 5th rib. </li>
 <li>Close the thoracic incision in layers, using 6-0 Prolene running sutures (Ethicon, Norderstedt, Germany) to adapt the ribs and 4-0 Prolene running sutures (Ethicon, Norderstedt, Germany) to close the skin. </li>
 <li>Drain the thorax with the help of a 2ml syringe carefully (Figure 3). Place the mouse on its back. Now take the endotracheal tube out and adapt the tracheal cartridge rings with one single stitch using 7-0 Prolene sutures (Ethicon, Norderstedt, Germany).</li>
 <li>Place the face mask on the mouse and close the skin using 4-0 Prolene running sutures (Ethicon, Norderstedt, Germany).</li>
</ol>
Validation
There are different possibilities to confirm the success of the LAD-ligation.
The troponin test can be performed 6 to 18 hours after surgery, using only 150 l blood without the need to euthanize the animal. The blood is applied to a customized troponin test kit (TROP T Sensitive, Roche, Mannheim) (Figure 4). Troponin is a regulative protein in the actin filaments of the muscle cell, there are different isotypes in the three types of muscle. The isotypes cTnI and cTnT are found in the heart and are released in tissue injury. The standardized test for troponin T (cTnT) is based on two heart specific monoclonal antibodies, one traps the troponin in the blood sample, the second one is a marker.
The infarcted area can be identified macroscopically after 3 days (Figure 5).
TTC (2,3,5-Triphenyltetrazolium chloride) staining measures tissue viability used to evaluate infarct size. Evans blue dye (1,5%, 1.0mL) in phosphate-buffered saline (PBS) is injected into the left ventricular cavity to measure the myocardial ischemic area. The mouse is euthanized and the heart is harvested and sectioned into slices. The tissue slices are incubated in 1% TTC PBS solution, pH 7.4 at 37°C for 20min. Tissues are fixed in 10% PBS-buffered formalin overnight at 2-8°C. TTC is administered ex vivo to dye Evans blue-negative areas (Figure 6).
For histology the mouse has to be euthanized and the heart has to be embedded for further processing. After performing paraffin sections, slides are stained with H&amp;E (hematoxylin and eosin) or trichrome to visualize fibrotic tissue (Figure 7).
<img src="/files/ftp_upload/1438/1438fig1.jpg" alt="Surgical procedure series, bone exposure, mechanical manipulation, incision experiment." data-altupdatedat="1787630408000" data-alt="Figure 1"> 
 Figure 1: Tracheal tube. 
1A: Exposed trachea, after ventral cervical incision. 
1B: Trachea is opened two cartilage rings caudal to the larynx. 
1C: Tracheal tube is inserted into the trachea.

<img src="/files/ftp_upload/1438/1438fig2.jpg" alt="Surgical excision process; tissue assessment; clinical procedure; anatomical study context." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2: The heart is exposed through a left sided thoracotomy, the LAD is ligated.
<img src="/files/ftp_upload/1438/1438fig3.jpg" alt="Rodent anatomy study with dissection; illustration of biological research method on cork surface." data-altupdatedat="1787630408000" data-alt="Figure 3"> Figure 3: After the thorax is closed, a pleura drain is performed. The mouse is still under anesthesia.
<img src="/files/ftp_upload/1438/1438fig4.jpg" alt="Rapid antigen test showing positive result; diagnostic method; two visible lines." data-altupdatedat="1787630408000" data-alt="Figure 4"> Figure 4: Two lines appear after a few minutes when the troponin test is positive.
<img src="/files/ftp_upload/1438/1438fig5.jpg" alt="Tissue dissection diagram, images A and B, showing pre- and post-sectioned organ." data-altupdatedat="1787630408000" data-alt="Figure 5"> Figure 5: Shows the explanted heart. 5A: Macroscopic view of the heart with the ischemic area with a faded whitish color. 5B: Cross section of the infarcted heart, the ischemic area shows in the upper right corner.

<ol><li>Atkins, B. Z., Hueman, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+cardiomyoplasty+improves+diastolic+properties+of+injured+Heart%5BTitle%5D)+AND+%22J+Surg+Res%22%5BJournal%5D)">Cellular cardiomyoplasty improves diastolic properties of injured Heart.</a> J Surg Res. 85 (2), 234-242 (1999).</li>
<li>Jaquet, K., KT, K. rause <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reduction+of+myocardial+scar+size+after+implantation+of+mesenchymal+stem+cells+in+rats%3A+what+is+the+mechanism%5BTitle%5D)+AND+%22Stem+Cells+Dev%22%5BJournal%5D)">Reduction of myocardial scar size after implantation of mesenchymal stem cells in rats: what is the mechanism.</a> Stem Cells Dev. 14 (3), 299-309 (2005).</li>
<li>Joung, B., Kim, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bone+marrow+mononuclear+stem+cells+transplanted+in+rat+infarct+myocardium+improved+the+electrical+conduction+without+evidence+of+proarrhythmic+effects%5BTitle%5D)+AND+%22Yonsei+Med+J%22%5BJournal%5D)">Bone marrow mononuclear stem cells transplanted in rat infarct myocardium improved the electrical conduction without evidence of proarrhythmic effects.</a> Yonsei Med J. 48 (5), 754-764 (2007).</li>
</ol>

Funding
The present study was supported by the University Hospital Hamburg (M.K., Woman Habilitation Grant), DFG (S.S., SCHR992/3-1), ISHLT (T.D., Research Award 2009).
Statement
All animals received human care in compliance with the Principles of Laboratory Animal Care, experiments on animals were performed in accordance with the guidelines and regulations set forth by the National Society for Medical research and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (National Institutes of Health publication 85-23, revised 1985).
All animals were obtained from Charles River Laboratories (Sulzfeld, Germany) and were maintained in the animal care facilities of the University Hospital Hamburg Eppendorf. The animals received standard chow and water ad libitum.

In summary, this video visualizes the LAD-ligation model step-by-step and identifies LAD-ligations in mice as easily feasible surgical procedures.

lad-ligation: a murine model of myocardial infarction

LAD-Ligation: A Murine Model of Myocardial Infarction

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes in myocardial ischemia and to develop and optimize future treatment.
Two different methods of creating myocardial ischemia are performed in laboratory rodents. The first method is to create cryo infarction, a fast but inaccurate technique, where a cryo-pen is applied on the surface of the heart (1-3). Using this method the scientist can not guarantee that the cryo-scar leads to ischemia, also a vast myocardial injury is created that shows pathophysiological side effects that are not related to myocardial infarction. The second method is the permanent ligation of the left anterior descending artery (LAD). Here the LAD is ligated with one single stitch, forming an ischemia that can be seen almost immediately. By closing the LAD, no further blood flow is permitted in that area, while the surrounding myocardial tissue is nearly not affected. This surgical procedure imitates the pathobiological and pathophysiological aspects occurring in infarction-related myocardial ischemia.
The method introduced in this video demonstrates the surgical procedure of a mouse infarction model by ligating the LAD. This model is convenient for pathobiological and pathophysiological as well as immunobiological studies on cardiac infarction. The shown technique provides high accuracy and correlates well with histological sections.

This video demonstrates how to use a fast and reliable model to study pathobiological and pathophysiological processes of myocardial ischemia.

Research models of infarction and myocardial ischemia are essential to investigate the acute and chronic pathobiological and pathophysiological processes ...

A midline cervical incision is performed to expose the trachea and a small hole is cut into the trachea for insertion of an endotracheal tube. The mouse is turned on its right side and a left sided thoracotomy is performed between the third and fourth ribs. The heart is exposed and the LAD is ligated with a single stitch.After placement of a chest tube and closing of the incision, the thorax is drained by aspirating the syringe carefully. Finally, the mouse is turned on. Its back.The tracheal tube is removed and the trachea and cervical incision are closed. Hi, I am Sonya Refa. I'm professor in Immunology and cardiac surgery in the Department of Cardiovascular Surgery at the University Heart Center in Hamburg.My laboratory is focusing on stem cell immunobiology and transplant immunology. Today we'll introduce you to the LID ligation model. We use this model in our lab to study the effect of stem cell therapy to generate the heart after myocardial infarction.And I'm KA Wan, a student at Stanford University and a summer fellow in Drer's TSI Laboratory. And I am Mandy Cooch from the TSI Laboratory of the University Heart Center Hamburg. I will show you how to perform in a lady ligation and later we will show you how to verify the success of a myocardial infarction using different methods.So let's get started. Shave the neck area and the left side of the rib cage of the anesthetized mouse. Place the mouse on its back and place a face mask over its nose and mouth to maintain anesthesia with 2%isof fluorine disinfect.Using beta isod before starting the surgery, pinch the tail and hind feet of the mouse to confirm that it is sufficiently anesthetized. Under microscopic view, perform a midline cervical incision separating the skin muscle and tissue covering the trachea. Once the trachea is exposed, cut a small hole into the tissue between two cartridge rings below the glottis to insert the endotracheal tube.Insert the endotracheal tube while holding the cranial part of the trachea. Using microsurgical forceps, check that thoracic movement to make sure that both lungs are well ventilated. The respiration rate should be approximately 110 per minute with an inspiratory pressure of 17 to 18 centimeters.H2O.Turn the mouse carefully and lay it on its right side while you face its left side. Perform a left sided thoracotomy between the third and the fourth rib and dissect the tissue and muscle. Carefully use a coer to prevent bleeding.Open the thorax carefully. Once the thorax is opened, find the heart without touching the lung with any sharp objects. Next, remove the part of the pericardial sac that is covering the heart.The LAD is located between the pulmonary artery and the left Oracle ligate. The proximal LAD with one single eight oh proline suture. Place an endotracheal tube 28 gauge ven catheter between the fourth and the fifth rib.Close the thoracic incision in layers using six oh proline running sutures to adapt the ribs and four oh proline running sutures. To close the skin, drain the thorax carefully with the help of a two milliliter syringe. After draining the thorax, place the mouse on its back.Place the face mask on the mouse and take the endotracheal tube out. Adapt the tracheal cartridge rings with one single stitch using seven oh prolene sutures and close the skin using four oh prolene running sutures. We have shown that the LAD ligation mouse model is an elegant and reliable technique to create a myocardial infarction.There are different ways to confirm a successful myocardial infarction. One method is the troponin test in which 150 microliters of blood from the mouse is applied to a customized troponin test kit. A positive result where two lines appear after a few minutes indicates the release of troponin T from the heart due to tissue injury.A second approach to confirm a successful myocardial infarction is disdain cross-sections of the heart with 2 3 5 trien tetra oleum chloride or TTC to measure tissue viability and evaluate the infarct size. The heart is covered with TTC and incubated at 37 degrees Celsius for 20 minutes. The viable tissue will appear red while the infarcted area will appear white.Finally, the heart of the mouse can be paraffin, embedded and sectioned for histology. Sections are stained with tri chrome to visualize fibrotic tissue. I have just shown you how to perform an LID ligation in mice afterwards.We have shown you different methods how to confirm the successful myocardial infarction either hours after LID ligation or weeks later in performing in an LID ligation. It is important to stay away from the lung tissue to avoid lung injury. So that's it.Thank you very much for watching the video and good luck with your experiments.

lad-ligation-a-murine-model-of-myocardial-infarction

Research

JoVE Journal

Biology

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Generating Murine Model of Myocardial Infarction: A Surgical Procedure for Permanent Ligation of Left Anterior Descending Coronary Artery in Mouse Model

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

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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

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Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

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

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

Learn the LAD ligation surgical technique for inducing myocardial infarction in mice. This method enables researchers to study ischemic heart disease pathophysiology and test therapeutic interventions.

LAD-Ligation: A Murine Model of Myocardial Infarction

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