Name: left coronary artery ligation: a surgical murine model of myocardial infarction
Uploaded: 2022-08-09T14:45:00.000+00:00
Duration: 5 min 48 s
Description: Watch this Scientific Journal Video about Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction at JoVE.com

This work was supported by National Institute of Health grants (R01HL143967, R01HL142629, R01AG069399, and R01DK129339), AHA Transformational Project Award (19TPA34910142), AHA Innovative Project Award (19IPLOI34760566), and ALA Innovation Project Award (IA-629694) (to PD).

The present study protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh. Eight (sham n = 4 and MI n = 4) 1-year-old female C57BL/6J mice weighing 24-30 g were used for these experiments. Approximately 100% and at least 80% of mice survived in the first 24 h and 28 days, respectively.
1. Preparation and endotracheal intubation of the mice
<ol>
	<li>Preheat a bead sterilizer (see Table of Materials) to 250 &#176;C and place autoclaved surgical instruments in it for a few minutes.</li>
	<li>Anesthetize the mouse in an induction chamber with 3% isoflurane and 1 L/min oxygen for 5 min.</li>
	<li>Ensure the depth of anesthesia in the mouse by checking the response to a firm toe pinch.</li>
	<li>Weigh the mouse to estimate the dosage of the pre-operative analgesic drug, buprenorphine (0.1 mg/kg). Inject the drug intraperitonially.</li>
	<li>Trim the fur on the left side of the thorax using an electric razor.</li>
	<li>Disinfect the surgical site with povidone-iodine and 70% ethanol thereafter three times.</li>
	<li>Place the mouse in the supine position on an inclined board. Secure the head and limbs of the mouse using an elastic band attached to the upper incisors and adhesive tape, respectively. Apply sterile ophthalmic lubricant on the eyes to prevent dryness while under anesthesia.</li>
	<li>Open the jaw and gently pull the tongue out of the oral cavity.</li>
	<li>Identify the opening of the larynx by illuminating the oropharynx using a laryngoscope (see Table of Materials).</li>
	<li>Cut off about 0.5 cm from a 24 G catheter needle and insert the blunt needle into the plastic shield. Direct the blunt needle with the plastic shield into the trachea. Take out the needle, leaving the plastic shield into the trachea.</li>
	<li>Set the ventilator (see Table of Materials) to a respiratory rate of 137 beats per min (optimized for the mice used in this study) and tidal volume 0.18 cc. Connect the respirator tubes to the catheter shield and confirm correct intubation by looking for a synchronized chest movement with the ventilator.</li>
	<li>Disconnect the respirator tube from the catheter shield and place the animal in the supine position on a preheated temperature-controlled surgical board. Reconnect the mouse to the ventilator.</li>
</ol>
2. Permanent ligation of the left coronary artery
<ol>
	<li>Disinfect the surgical site with&#160;povidone-iodine and 70% alcohol.&#160;Apply&#160;a sterile drape having a quarter-sized hole in the center&#160;to secure the surgical site. Gently lift the skin using a pair of forceps and make a small (1.5-2 cm) cutaneous transverse incision along the line between the left pectoralis major and minor muscles using a pair of surgical scissors. 
	NOTE: Scissors were used to make the incision as it provides&#160;the required control over the depth and direction of the cut.</li>
	<li>Separate the underlying pectoralis muscles with forceps and dissecting scissors. The muscles were separated using retractors attached to elastic bands.</li>
	<li>Make an incision in the third intercostal space with a pair of micro scissors following the natural angle of the ribcage. At this phase, extreme caution must be exercised to prevent injury to the heart and lungs.</li>
	<li>Gently stretch the ribs apart using retractors to expose the left ventricle. Move the pericardial fat aside and locate the LCA, which runs from the edge of the left atrium towards the apex of the heart.</li>
	<li>Pass an 8-0 nylon suture under the LCA with the help of a needle holder. Ligate the LCA with a double knot followed by a second knot (a modified surgeon&#39;s knot). 
	NOTE: Blanching of the lower left ventricle confirms a successful LCA ligation.In addition to this, troponin measurement, ECG monitoring (ST elevation), echo/in vivo cardiac-gated MRI, or micro-CT images are also advised to confirm the comparable MI lesions.</li>
	<li>Remove the retractors and insert a 22 G catheter needle into the chest cavity. Remove the needle, leaving the tip of the plastic shield in the thoracic cavity. Close the ribcage using a 4-0 nylon suture.</li>
	<li>Connect a syringe to the 22 G plastic shield and slowly remove excess air trapped in the thoracic cavity by gently pressing the chest to establish a negative air pressure. Remove the plastic shield.</li>
	<li>Close the skin with a 4-0 nylon suture.</li>
	<li>Switch off the isoflurane supply. At this stage, the mouse is on the ventilator supplying oxygen.</li>
</ol>
3. Post-operative care
<ol>
	<li>Switch off the ventilator once spontaneous breathing starts. 
	NOTE: The procedure takes about 30-35 min per animal from preparation of the mice up to this step.</li>
	<li>Keep the mouse under a heat lamp and monitor it until it is awake. The animal should not be left unattended until it has recovered enough consciousness to maintain sternal recumbency.</li>
	<li>After surgery, place the animal is in a separate cage and return it to the original cage with other animals only after it fully recovers.</li>
	<li>Monitor the mouse daily for any sign of pain or discomfort.</li>
	<li>Continue intraperitoneal injection of buprenorphine (0.1 mg/kg) every 6-8 hours&#160;for an additional 2 days following the surgery.</li>
</ol>
4. Echocardiographic Evaluation
NOTE: Echocardiography was performed to evaluate the parameters of heart failure on day 28 after MI.
<ol>
	<li>After 28 days following the surgery, anesthetize the mice with 3% isoflurane and 1 L/min oxygen, apply sterile ophthalmic lubricant on the eyes, and remove chest hair using hair removal cream. Disinfect the chest area with povidone-iodine and 70% ethanol three times.</li>
	<li>Secure the anesthetized mice atop the imaging platform (see Table of Materials) in the supine position and maintain a steady level of anesthesia throughout the procedure using a nose cone connected to the anesthetic system (1%-2% isoflurane and 1 L/min oxygen).</li>
	<li>Tape the four paws to the ECG electrodes with electrode gel (see Table of Materials). Monitor the temperature of the animal by inserting a rectal probe (see Table of Materials).</li>
	<li>Apply the scanning gel (see Table of Materials) to the chest, place the transducer vertically, lower it to the parasternal line (parallel to the thorax), and rotate 35&#176; anticlockwise to obtain the parasternal long axis view of the left ventricle.</li>
	<li>Tap the B-mode imaging button on the imaging software (see Table of Materials) to get a complete long axis view of the heart. Adjust the gate size and brightness and save the images using Save Clip or Save Frame for later measurements16.</li>
	<li>Switch to M-mode (motion-mode) and place the M-mode axis at the level of the papillary muscle. Adjust the gate size and tap the M-mode Start button. Save the images using Save Clip or Save Frame16,17.</li>
	<li>As the 4D mode image acquisition process is automated, verify that the ECG and respiration signals are active (Figure 1) before acquiring the data.</li>
	<li>Start acquiring the data in B-Mode. Open the 4D scan panel and initiate the 3D motor. Set the image parameters in the 4D scan panel and tap the Scan button to begin the scanning. After reviewing the images in the 2D view, load the images into 4D mode using the Load into 4D button.</li>
</ol>

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The authors do not have any conflicts of interest to disclose.

The murine model of MI is gaining popularity in cardiovascular research laboratories, and this study describes a reproducible and clinically relevant MI model. This protocol improves the LCA ligation process in several ways. To begin with, the use of injectable pre-operative anesthetics such as xylazine/ketamine or sodium pentobarbital14,15 is avoided. Only isoflurane anesthesia was used, which helps enhance animal survival rates (&#62;80% survival 28 days after surgery), minimize drug-induced complications, and has minimal cardiac effects as compared to other agents19.&#160;However, isoflurane does also slow the heart, albeit at a lower degree compared to other anesthetic agents20. This protocol involves less invasive endotracheal intubation avoiding tracheostomy21, which reduces post-operative pain and discomfort. Previous murine LCA ligation studies have recommended making a mid-neck incision to improve the visualization of endotracheal intubation; however, the current protocol uses a laryngoscope instead to illuminate the oropharynx15. Lugrin et al. recently demonstrated a murine LCA MI model without thoracentesis14; however, the current protocol includes an effective thoracentesis, which will help remove excess blood and air from the chest cavity, preventing pneumothorax19. In addition, this method uses sterile gauze for bleeding management in place of a cauterizer, as using a cauterizer to reduce bleeding can result in iatrogenic burns and may alter inflammatory readings21.
One of the critical steps in this surgical model is identification and ligation of the LCA. The location of the coronary artery may vary depending on mouse strains and genotypes9. In most cases, the artery is not visible under a microscope. From experience, ligating the myocardial tissue 2-4 mm below the edge of the left atrium results in efficient blanching of the left ventricular wall. Furthermore, the procedure can be simply modified to induce temporary myocardial ischemia followed by reperfusion (I/R) by removing the ligation22. This animal model mimics the restoration of coronary blood flow in MI patients following percutaneous coronary intervention23,24. Since the permanent LCA occlusion model differs from the I/R model in several aspects, such as the size of the infarcted area, location of the infarct, and infiltration of inflammatory cells, researchers must be cautious while selecting the relevant model depending on the study7,14,25.
There are multiple approaches to ensure successful ligation of the LCA and subsequent development of MI. Observing immediate blanching of the lower left ventricle is the earliest confirmation of successful LCA ligation. Apart from this, the extent and location of the myocardial infarct can be visualized by staining the whole heart with Evan&#39;s blue or 2,3,5-triphenyl tetrazolium chloride (TTC)26. Measurement of circulating cardiac troponin can further validate the myocardial tissue injury21. Electrocardiography can be used as a non-invasive method of confirming elevation of the ST segment after MI17. The degree of cardiac fibrosis associated with MI can be evaluated by Masson&#39;s trichrome staining and cardiac MRI27,28,29. Echocardiography can be used to evaluate the parameters of heart failure on days 1 and 28 after MI. To examine cardiac remodeling following the MI, Masson&#39;s trichome staining and echocardiography can be utilized17. It is also possible to use qPCR and immunoblot to further investigate and confirm the expression of the genes and proteins implicated in fibrosis, inflammation, and heart failure after MI14.
The major limitation of LCA ligation is the high incidence of mortality, which could be due to post-operative cardiac arrhythmias, ventricular rupture, hemorrhage, pneumothorax, and post-operative discomfort19,30. However, a successful thoracentesis, minimizing the non-target tissue damage, and proper post-operative pain and temperature management may help reduce the death of the animal. As with any other surgical model, exact reproducibility is another limitation of this surgical model. Researchers can, however, reproduce MI, control infarct size, and improve post-surgery survival by rigorous practice and experience.

Cardiovascular disease is a major public health concern that claims 17.9 million lives each year, accounting for 31 percent of global mortality1. The most prevalent type of cardiovascular anomaly is coronary heart disease, and myocardial infarction (MI) is one of the major manifestations of coronary heart disease2. MI is usually caused by thrombotic occlusion of a coronary artery due to the rupture of a vulnerable plaque3. The resulting ischemia causes profound ionic and metabolic changes in the affected myocardium, as well as a rapid decrease in systolic function. MI results in the death of cardiomyocytes, which can further lead to ventricular dysfunction and heart failure4.
Research on MI in patients is limited due to the scarcity of tissues obtained from patients with MI5. As such, murine models of MI are useful in both studying disease mechanisms as well as developing potential therapeutic targets. Currently available murine models of MI includeirreversible ischemia models (LCA and ablation methods) and reperfusion models (ischemia/reperfusion, I/R)6. Permanent ligation of the left coronary artery (LCA) in mice is the most used method, and it imitates the pathophysiology and immunology of MI in patients7,8,9. Permanent MI can also be induced by ablation methods, which involve electrical damage or cryoinjury. Ablation methods are able to generate uniform-sized infarction at the precise location10. On the other hand, scar formation, infarct morphology, and molecular signaling mechanisms may vary among the ablation methods10,11. The murine I/R method is another important MI model as it represents the clinical scenario of reperfusion therapy12. The I/R model is associated with challenges such as a variable infarct size, difficulty in distinguishing responses of initial injury, and reperfusion6.
Although widely used, LCA ligation methods are associated with low survival rates and post-operative pain13. This protocol demonstrates the murine surgical MI model of LCA ligation that involves the preparation and intubation of mice, LCA ligation, post-operative care, and validation of MI. Rather than using an invasive tracheotomy14, this method employs endotracheal intubation. The animal is intubated by illuminating the oropharynx using a laryngoscope, making the procedure easier, safer, and less traumatic15. The mouse is kept on ventilator support and under isoflurane anesthesia throughout the procedure. Further, echocardiography and Masson&#39;s trichrome staining are performed to evaluate heart function and cardiac fibrosis after MI, respectively. Overall, this method provides a reliable and reproducible surgical murine model of MI that can be used to study pathophysiology and inflammation after MI.

<table><tbody><tr><td>22 G catheter needle</td><td>Exel INT</td><td>26741</td><td>Thoracentesis</td></tr><tr><td>24 G catheter needle</td><td>Exel INT</td><td>26746</td><td>Endotracheal intubation</td></tr><tr><td>4-0 nylon suture</td><td>Covetrus</td><td>29263</td><td>Suturing of muscles and skin</td></tr><tr><td>8-0 nylon suture</td><td>S&amp;amp;T</td><td>3192</td><td>Ligation of LAD</td></tr><tr><td>Anesthetic Vaporizers</td><td>Vet equip</td><td>VE-6047</td><td>Anesthetic support</td></tr><tr><td>Animal physiology monitor</td><td>Fujifilm</td><td>VEVO 3100</td><td>Monitor heart rate,respiration rate and body temperature</td></tr><tr><td>Betadine solution</td><td>PBS animal health</td><td>11205</td><td>Antispetic</td></tr><tr><td>Buprenorphine</td><td>Covetrus</td><td>55175</td><td>Analgesic</td></tr><tr><td>Disecting microscope</td><td>OMANO</td><td>OM2300S-V7</td><td>Binocular</td></tr><tr><td>Electric razor</td><td>Wahl</td><td>79300-1001M</td><td>Shaving</td></tr><tr><td>Electrode gel</td><td>Parker Laboratories</td><td>W60698L</td><td>Electrically conductive gel</td></tr><tr><td>Ethanol</td><td>Decon Laboratories</td><td>22-032-601</td><td>Disinfectant</td></tr><tr><td>Forceps</td><td>FST</td><td>11065-07</td><td>Stainless Steel</td></tr><tr><td>Gauze</td><td>Curity</td><td>CAR-6339-PK</td><td>Sterile</td></tr><tr><td>Heat lamp</td><td>Satco</td><td>S4998</td><td>Post surgery care</td></tr><tr><td>Heating pad</td><td>Kent scientific</td><td>Surgi-M</td><td>Temperature control</td></tr><tr><td>Hot Bead sterilizer</td><td>Germinator 500</td><td>11503</td><td>Sterilization of surgical instrument</td></tr><tr><td>Isoflurane</td><td>Covetrus</td><td>29405</td><td>Anesthesia</td></tr><tr><td>Masson&amp;rsquo;s trichrome staining kit</td><td>Thermoscientific</td><td>87019</td><td>Measurement of cardiac Fibrosis</td></tr><tr><td>Micro Needle Holder</td><td>FST</td><td>12500-12</td><td>Stainless Steel</td></tr><tr><td>Micro scissors</td><td>FST</td><td>15000-02</td><td>Stainless Steel</td></tr><tr><td>Ophthalmic ointment</td><td>Dechra</td><td>Puralube Vet</td><td>Sterile occular lubricant</td></tr><tr><td>Scanning Gel</td><td>Parker Laboratories</td><td>Aquasonic 100</td><td>Aqueous ultrasound transmission gel</td></tr><tr><td>Scissors</td><td>FST</td><td>14060-11</td><td>Stainless Steel</td></tr><tr><td>Small Animal Laryngoscope</td><td>Penn-Century</td><td>Model LS-2-M</td><td>Illuminating the oropharynx</td></tr><tr><td>Small animal ventilator</td><td>Harvard apparatus</td><td>557058</td><td>Ventilator support</td></tr><tr><td>Surgical light</td><td>Cole parmer</td><td>41723</td><td>Illuminator Width (in): 7</td></tr><tr><td>Vevo 3100 preclinical imaging platform</td><td>Fujifilm</td><td>VEVO 3100</td><td>Echocardiography</td></tr><tr><td>VevoLAB software</td><td>Fujifilm</td><td>VevoLAB 3.2.6</td><td>Echocardiography data analysis</td></tr></tbody></table>

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.

Figure 1 demonstrates the representative active ECG and respiration signals during the echocardiographic evaluation of sham (Figure 1A) and MI (Figure 1B) mice. Verification of active ECG and respiration signals are important before acquiring the echocardiographic data. Figure 2 shows echocardiographic measurement of cardiac functional parameters following 28 days after LCA ligation. Figure 2 shows M-mode images of the para sternal short axis view of sham (Figure 2A) and MI (Figure 2B) hearts. Figure 2B shows defective heart wall movement following LCA ligation. Indicators of heart failure, such as increased LV mass (Figure 2C), decreased ejection fraction (Figure 2D), and decreased cardiac output (Figure 2E), were observed in the MI group as compared to the sham group.
All the animals were euthanized according to standard protocols using an excessive dose of CO2 gas. The hearts were fixed and frozen in optimal cutting temperature (OCT) compound. Masson&#39;s trichrome staining18 was performed for three different ventricle sections (lower, middle, and upper) and images were taken using a research slide scanner under 10x magnification to examine the degree of cardiac fibrosis. Figure 3 shows increased collagen staining (blue) in the infarcted heart, indicating augmented fibrosis.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64387/64387fig01.jpg" /> 
Figure 1: Active ECG and respiration signals during the echocardiographic evaluation. Representative active ECG and respiration signals during the echocardiographic evaluation of (A) sham&#160;and (B) MI&#160;mice. Green = ECG signals, yellow = respiration signals. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64387/64387fig01large.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/64387/64387fig02.jpg" /> 
Figure 2: Echocardiographic evaluation of cardiac functional parameters following LCA ligation on day 28 after the surgery. Representative parasternal short axis (PSAX) M-mode echocardiographic images of (A) sham and (B) MI mice. Assessment of (C) left ventricular mass (mg), (D) ejection fraction (%), and (E) cardiac output (mL/min) of sham and mice with MI. LVAW;d = left ventricular anterior wall thickness in diastole; LVAW;s = left ventricular anterior wall thickness in systole; LVPW;d = left ventricular posterior wall thickness in diastole; LVPW;s = left ventricular posterior wall thickness in systole; LVID;d = left ventricular internal diameter in diastole; LVID;s = left ventricular internal diameter in systole. Data are shown as Mean &#177; SD. * P &#60; 0.05, ** P &#60; 0.01, **** P &#60; 0.0001. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64387/64387fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64387/64387fig3v2.jpg" /> 
Figure 3: Assessment of fibrosis after LCA ligation on day 28 after the surgery. Representative images showing Masson&#39;s trichrome staining of (A) sham and (B) MI hearts 28 days after the surgery. The fibrotic regions in the infarcted heart are characterized by collagen deposition and stained blue after Masson&#39;s trichrome staining. Scale bar = 500 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64387/64387fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Left Coronary Artery Ligation: A Surgical Murine Model of Myocardial Infarction at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

4.2K Views.  University of Pittsburgh. Het chirurgische model permanente ligatie van linkerslagader bij muizen kan worden gebruikt om de pathofysiologie en bijbehorende ontstekingsreactie na een hartinfarct te onderzoeken. Deze methode biedt een betrouwbaar en produceerbaar muismodel van MI, dat eenvoudig, veilig en minder invasief is. Verwarm om te beginnen een kraalsterilisator voor op 250 graden Celsius en plaats er een paar minuten geautoclaveerde chirurgische instrumenten in.Zorg voor de diepte van de anesthesie in de ...

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

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 ...

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 ...

Biology

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 ...

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

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

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

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.

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

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 ...

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

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

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 ...

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

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