This model was developed with the support of the National Institute of General Medical Sciences (NIGMS)/the National Institute of Health (NIH) grant 1P20GM103652 (Project# 3) (to MRA) and the American Heart Association (AHA) Grant-in-Aid 14GRNT20460291 (to MRA); the Brazilian government grant CAPES (to KR and FR); and a Brown University LINK award (to IM). We also acknowledge the outstanding technical support from our veterinarians and animal facility staff.

This animal protocol has been reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Rhode Island Hospital.
1. Anesthesia and Intubation
<ol>
	<li>Weigh the mouse to calculate the dosage of post-operative pain medication.</li>
	<li>Place the mouse in an induction chamber and deliver 4% isoflurane for 9 - 10 min, monitoring the animal throughout. Turn on a hot bead sterilizer so that the apparatus can preheat to approximately 250 &#176;C. Preheating will take 15 -...

<ol>
	<li>Hausenloy, D. J., Yellon, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+directions+for+protecting+the+heart+against+ischaemia-reperfusion+injury:+targeting+the+Reperfusion+Injury+Salvage+Kinase+(RISK)-pathway.">New directions for protecting the heart against ischaemia-reperfusion injury: targeting the Reperfusion Injury Salvage Kinase (RISK)-pathway.</a> Cardiovasc Res. 61 (3), 448-460 (2004).</li><li>Roffi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2015+ESC+Guidelines+for+the+management+of+acute+coronary+syndromes+in+patients+presenting+without+persistent+ST-segment+elevation.">2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation.</a> Eur Heart J. 37 (3), 267-315 (2015).</li><li>Kumar, A., Cannon, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Coronary+Syndromes:+Diagnosis+and+Management,+Part+I.">Acute Coronary Syndromes: Diagnosis and Management, Part I.</a> Mayo Clin Proc. 84 (10), 917-938 (2009).</li><li>Eitan, A., Nikolsky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+therapy+in+patients+with+acute+coronary+syndromes:+how+to+make+the+right+choice.">Antithrombotic therapy in patients with acute coronary syndromes: how to make the right choice.</a> Minerva Med. 104 (4), 357-381 (2013).</li><li>Abbate, A., Bussani, R., Amin, M. S., Vetrovec, G. 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E., Tetens-Woodring, J., Belicha-Villanueva, A., Wilding, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+Efficacy+of+Various+Combinations+of+Injectable+Anesthetics+in+BALB/c+Mice.">Safety and Efficacy of Various Combinations of Injectable Anesthetics in BALB/c Mice.</a> J Am Assoc Lab Anim Sci. 47 (1), 11-17 (2008).</li><li>Kolk, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LAD-Ligation:+A+Murine+Model+of+Myocardial+Infarction.">LAD-Ligation: A Murine Model of Myocardial Infarction.</a> J Vis Exp. (32), e1438 (2009).</li><li>Wu, Y., Yin, X., Wijaya, C., Huang, M. H., McConnell, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+myocardial+infarction+in+rats.">Acute myocardial infarction in rats.</a> Journal of visualized experiments : J Vis Exp. (48), (2011).</li><li>Ferrera, R., Benhabbouche, S., Bopassa, J. C., Li, B., Ovize, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hour+reperfusion+is+enough+to+assess+function+and+infarct+size+with+TTC+staining+in+Langendorff+rat+model.">One hour reperfusion is enough to assess function and infarct size with TTC staining in Langendorff rat model.</a> Cardiovasc Drugs Ther. 23 (4), 327-331 (2009).</li><li>Zeng, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+5-ethynyl-2'-deoxyuridine+staining+as+a+sensitive+and+reliable+method+for+studying+cell+proliferation+in+the+adult+nervous+system.">Evaluation of 5-ethynyl-2'-deoxyuridine staining as a sensitive and reliable method for studying cell proliferation in the adult nervous system.</a> Brain Res. 1319, 21-32 (2010).</li></ol>

With an increasing use of the MI model in laboratories, the described procedure seeks to increase the efficiency and survival rate of the mice while minimizing their post-operative pain and discomfort. This protocol strives to minimize mortality by making numerous improvements to various aspects of the LAD ligation procedure. There are a few distinctions. Some murine intubation studies that utilize ketamine and xylazine along with isoflurane for induction, due to the benefit of their longer duration of a...

Coronary disease, or ACS, is the most prevalent cardiovascular event and will be considered the main cause of morbidity and mortality worldwide in 20201. The cause of ACS is the presence of a myocardial thrombosis due to the rupture of a coronary atherosclerotic plaque that blocks or reduces blood flow to the heart tissue2. Therefore, there are clinical signs consistent with the presence of acute myocardial ischemia, such as myocardial infarction (MI)3,4. MI leads to a loss in mass of the cardiomyocytes and a progression to pathological ventricular remodeling, which can lead to ventricular dysfunction and heart failure5,6.
One of the most effective ways to study IHD has been to mimic human myocardial infarction in an animal model. This is achieved by occluding the LAD in mice. Using this model, we study how the heart can be protected from the damage resulting from IHD.
Over the last decade, researchers have shifted from using larger animal models to smaller animals, including the shift from rats to mice. The smaller mouse model is starting to be preferred for many reasons, including their small size, large litter size, low cost to maintain, and short gestation period, as well as for the expansive availability of transgenic and gene knockout models7. Although mice are small in size, new surgical instruments specifically designed for them have aided in this development. Our method utilizes these new surgical instruments.
While several methods implement an invasive tracheotomy, we use a less invasive method of endotracheal intubation. Using overhead illumination of the oropharynx, we intubate without creating any incisions, providing a safer and less traumatic experience for the animal. The mouse is then placed on a ventilator and kept on isoflurane during the entire procedure. Due to the short duration of anesthesia produced by the drug, it only takes a few minutes for the animal to recover from the anesthetic once it is discontinued. Our surgical model also includes a minimally invasive thoracentesis. The careful removal of blood and excess air from the chest cavity using thoracentesis through the original thoracotomy incision has addressed a common post-operative complication of the LAD ligation: the tension pneumothorax. This method, which eliminates the need for the two additional incisions used in other methods-one for the tracheotomy and another for the thoracentesis-has yielded fewer post-surgical complications and has drastically reduced mortality.

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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.

The mice are euthanized twenty-eight days after surgery, and the hearts are harvested and examined. The mice are anesthetized with 50 - 75 mg/kg ketamine and 5 - 10 mg/kg xylazine. When the animal is under adequate anesthesia, the thoracic cavity is opened, and using a 23-gauge needle, cold potassium chloride (KCL, 30 mM) is injected into the posterior basal region of the heart. The heart is arrested in diastole. For further validation of the ligation, the heart is removed from the animal...

Watch this Scientific Journal Video about Murine Left Anterior Descending LAD Coronary Artery Ligation: An Improved and Simplified Model for Myocardial Infarction at JoVE.com

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).

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 ...

The overall goal of this procedure is to induce a myocardial infarction in a mouse by ligating the left anterior descending coronary artery. This method can help answer key questions in the cardiovascular field, such as whether overexpression or knock out of certain genes can affect the recovery of the heart following myocardial infarction. The main advantage of this technique is that the procedure is less invasive than various other myocardial infarction mirroring models, and results in a quick recovery time with fewer complications.Generally, individuals new to this method will struggle because endotracheal intubation and identification of the LAD artery requires practice with a trained instructor. Visual demonstration of this method is critical at identifying and assess the LAD is difficult to learn without any experience or training. Prior to sedating the animal, start heating the bead sterilizer to 250 degrees Celsius which can take up to 20 minutes.Check that the mouse is heavily sedated. The breathing rate will be approximately 32 breaths per minute. Then place the sedated mouse supine on a styrofoam board.Position the mouse at eye level for easier visualization of the oropharynx. Before proceeding, confirm the sedation with a toe pinch. Then, using an elastic band, hold the mouth open by securing the top incisors in an open position.Next, position a high intensity illuminator above the mouse so that the oropharynx is transilluminated. Don surgical loops to take notice of the small structures of the oropharynx. Now, using curved forceps open the jaw.With another pair of forceps move the tongue out of the way and then observe the opening and closing of the vocal chords. When open insert a 20 gauge one inch intravenous catheter with a blunt tip needle introducer. Use the needle to guide the catheter to the tracheal opening, but avoid inserting the needle into the tracheal wall.Then transfer the intubated mouse to an operating surface equipped with a heating device, and there, connect the mouse to a small rodent ventilator. Before proceeding, verify the mouse's plane of anesthesia using a toe pinch. Then tape down the intubation tube at the connecting site between the ventilator and the IV catheter.Follow by taping down all of the extremities and lubricating the eyes. Now, trim the fur from the ventral left side of the thorax. Then apply a small layer of hair removal cream to the exposed skin, and let it penetrate for 30 to 45 seconds.In the mean time, soak sterile cotton swabs in three 1.5 milliliter tubes filled with betadine. Then using distilled water gently wipe away the cream and fur. Follow this by cleaning the surgical field three times with betadine and 70%isopropanol.Now, drape the mouse with a quarter sized hole over the surgical field. Before making the first incision, put the autoclaved surgical instruments in the pre heated hot bead sterilizer at 250 degrees Celsius for approximately 20 seconds. Now, use fine tipped forceps to gently lift the skin at a point approximately five millimeters to the left of the prominent xiphoid cartilage.Then use a number 10 scalpel to create an upward vertical incision to the level of the manubrium. Next, gently separate the dermal layers with curved forceps. To hold open the muscle layer, pass two five zero polypropylene sutures through either side of the incision and secure them with clamps.Now, make an incision in the third intercostal space following the natural angle of the rib cage. Next, remove the tape from the left extremities of the mouse. Then tape together the rear feet.Then reposition the front foot so it is slightly elevated. Be sure to clean the surgical gloves after each time the mouse is handled. Proceed by using a retractor to gently spread apart the third and fourth ribs.Then dip a small piece of gauze in sterile 0.9%saline and squeeze out the excess saline. Using forceps gently insert the wet gauze against the left lung to prevent accidental lung damage. Next, gently remove the thin pericardium with forceps.Then, use a small cotton ball soaked with saline to clean the heart's surface, and make the arteries observable. Gently push the left oracle upwards, and locate the coronary arteries underneath. Now, pass an eight zero nylon suture under the LAD and complete two throws to secure the ligation.If it is successful, the left ventricle distilled from the ligature will blanche. Then, remove the gauze and the retractor. Next, through the thoracotomy opening insert a six inch 25 gauge flexible tube, attached to a 25 gauge needle.Insert it one or two inches into the chest cavity above the left lung. Then return the mouse to a supine position and clean off the surgical gloves. Now, continue to use five zero polypropylene sutures in an interrupted pattern to close the rib cage, keeping the chest tube in place.Then remove the two sutures that hold the muscle layer open and close the muscle layer using five zero polypropylene sutures in a simple continuous pattern. Next, attach a one milliliter syringe to the needle on the chest tube. Pull back the plunger to extract air and blood.If the syringe fills up, detach it, empty it, and reattach it. Now, extract the chest tube, and ensure that the chest is tightly sealed. Proceed by administering analgesics and a subcutaneous bolus of saline.After five minutes remove the mouse from the intubation tube. If the chest doesn't rise bilaterally air is trapped in the chest cavity. Introduce a 25 gauge needle with a one milliliter syringe between the third and fourth ribs, into the thoracic cavity.Then gently draw out the trapped air and the chest will start moving normally. Mice are euthanized 28 days after the described surgery using potassium chloride. Their hearts were arrested in diastole then fixed with paraformaldehyde.This image shows the lack of Evans blue in the ischemic left ventricle. Hearts stained with trichrome dye show that there was an increase in collagen in the infarcted region, indicating fibrosis. Control hearts showed now evidence of increased collagen.After watching this video you should have a good understanding of how to induce a myocardial infarction using a murine model. Utilizing the techniques of endotracheal intubation, a left sided thoracostomy approach to LAD ligation, and total synthesis without any additional incision in the chest wall. Once mastered this technique can be done in 30 minutes, if it is performed appropriately.While attempting this procedure it's important to remember to provide the mangers this around vasculature by ligation the LAD artery. Following this procedure an echo cardiogram can be performed in order to answer additional questions like the extent of functional loss or recovery of the heart after a predetermined post operative period. After its development, this technique paved the way for researchers in the field of cardiovascular surgery to explore the effects of drugs and specific genes on myocardial infarction in mice.

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Research

JoVE Journal

Medicine

23.8K Views.  Rhode Island Hospital, Brown University Warren Alpert Medical School. The overall goal of this procedure is to induce a myocardial infarction in a mouse by ligating the left anterior descending coronary artery. This method can help answer key questions in the cardiovascular field, such as whether overexpression or knock out of certain genes can affect the recovery of the heart following myocardial infarction. The main advantage of this technique is that the procedure is less invasive than various other myocardial infarction mirroring models, and results in a qu...