Medicine
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Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery
Chapters
Summary August 16th, 2019
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.
Transcript
This murine model of myocardial infraction allows the monitoring of pathological processes occurring from coronary occlusion to late-stage heart failure at the local heart tissue and systemic levels. This method is compatible with any readout currently used in cardiac biology and physiology. In addition, it benefits from the immense diversity of murine genetic models.
The acquisition of adequate surgical skills can take time to master. This procedure is best learned by step by step with an experienced researcher. Visual demonstration of this murine model allows the sharing of hints that can help with the surgical procedure and speed up the learning process.
Use an electric razor to quickly shave an anesthetized, 25-to-30-gram, male, C57-Black/6 mouse on the throat and the left side of the rib cage. Confirm a lack of response to toe pinch. Apply ointment to the animal's eyes.
Place the mouse in the supine position on a heating pad, and place a small gauze compress under the head to avoid overheating the eyes. Secure the limbs with adhesive tape. Pass a loop of 5-0 silk suture under the upper incisors, and secure the extremity of the loop onto the heating pad to keep the mouth open during the cannulation.
Apply depilatory cream to the pre-shaved areas, and gently massage with a cotton swab for one minute. Remove the excess fur and cream with gauze, and clean the exposed skin with drops of 0.9%saline solution and gauze. Apply pieces of sterile gauze to the shaved throat and thorax, and soak the gauze with iodopovidone.
Set the ventilator at a tidal volume of seven milliliters per kilogram and a ventilation rate of 140 strokes per minute. Move the mouse under a microsurgery stereo microscope. Holding the skin on the center of the throat, make a 0.5-centimeter incision along the caudal/cephalic line, and separate the lobes of the salivary gland.
Use curved forceps to gently separate the fascia of the sternohyoid muscle until the larynx and trachea are visible. Then secure the edges of the opening with retractors attached to elastic bands. Next, gently pull the tongue sideways, and use forceps to insert the blunted inner needle of a 16-gauge cannula into the trachea.
Visualize a correct insertion into the trachea through the throat incision, and connect the cannula to the ventilator. Place sterile 0.9%saline supplemented with iodopovidone-soaked gauze onto the incision to keep the tissues wet during the operation. Then place the exhaust tubing into water.
The presence of bubbles indicates a successful intubation. For ligation of the left anterior descending coronary artery, or LAD, carefully move the mouse into the right side decubitus position, and resecure the left anterior limb in the new position. Identify the line between the left pectoralis minor and major muscles, and make an oblique one-centimeter skin incision along the line.
Using blunt dissecting micro scissors, separate the fascia of the pectoralis muscles without incising the tissues, and use retractors attached to elastic bands to maintain the separation. Set the ventilator with a positive end-expiratory pressure of three centimeters of water. Use blunt forceps to open the chest cavity at the third intercostal space between the third and fourth ribs.
Place two retractors into the rib cage, one on each rib, and use a curved fine forceps to carefully remove the pericardium without harming the heart and lungs. Locate the LAD as a superficial bright red line running from the edge of the left auricle toward the apex. Use a needle holder to pass a 7-0 silk suture under the LAD two to three millimeters below the left atria.
Pulling the silk slowly to avoid tearing heart tissue, tie the ligature with three knots. The lower left part of the left ventricle will instantly turn pale upon ligation. Now release the rib retractors, and holding the third rib with forceps, make two passes with a 6-0 silk suture under the third and fourth ribs.
Place three drops of 37-degree Celsius 0.9%saline solution onto the opening, and shut the expiration exhaust tube for two or three respiratory cycles to properly inflate the lungs. Tighten and secure the suture with two throws, and release the retractors holding the muscles, helping the muscles return to their correct anatomical location. Then close the thoracic skin with two stitches and two throws of 5-0 suture silk.
Close the throat skin with one stitch of 5-0 suture silk and two throws. At the end of the procedure, remove the tape bands from the limbs, carefully turn the animal to ventral decubitus onto the compress pad, and place a compress on the heating pad on the right side of the animal. Intraperitoneally inject 0.3 milliliters of 37-degree-Celsius warmed 5%glucose solution.
Stop the ventilator. If the mouse spontaneously breathes, cautiously remove the cannula. Subcutaneously inject 0.1 milligrams per kilogram of buprenorphine.
Place the mouse in a 30-degree Celsius cage ventilated with 100%oxygen for at least one hour with monitoring. During the two first days following surgery, provide the mouse with a soft diet and water ad libitum, warming the animal as necessary. Seven days after surgery, the ischemic areas remain unstained by triphenyltetrazolium chloride, whereas the live tissue stains red due to the presence of dehydrogenases.
The ischemic areas can then be calculated as the percentage of white area of the left ventricle in an imaging software program. Western blot analysis for alpha-smooth muscle actin and SMAD2 phosphorylation, which are respectively major readouts of myofibroblasts and of transforming growth factor beta signaling activation, can be used to determine the extent of fibrosis within the myocardial tissue of the infarcted hearts. Real-time polymerase chain reaction analysis of transforming growth factor beta and its downstream target mRNA expression can also be performed to assess the presence of myocardial fibrosis.
Pro-inflammatory signaling pathways and the expression of pro-inflammatory genes is typically found to be activated within the first week following myocardial infarction. mRNA expression of pro-inflammatory genes and monocyte/macrophage markers is also observed. The ligation height will critically influence survival and should be adapted to each strain or genotype, as LAD branching may vary.
Constants in ligation is key to obtain reproducible results. This procedure may be coupled with any analytical method and can be followed by functional analysis, tissue fixation, cell culture, or any technique of interest.
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