The overall goal of this procedure is to model acute myocardial infarction in rats for the study of the cardiac signaling mechanisms associated with the immediate response of the myocardium to cardiac ischemia, followed by reperfusion injury. These responses include infarct size, area at risk and cell death. The first step is to expose the left ventricle by making a small incision at the fourth intercostal space of the left side of the chest and spreading the ribs once isolated.
The left anterior descending artery or LAD is transiently ligated for 30 minutes following the ischemic period. The ligation of the LAD is released allowing for cardiac reperfusion after a four hour reperfusion period. The LAD is ligated once more before the heart is excised and examined for the size of the infarcted myocardium area at risk and not at risk.
Ultimately, results from the histological evaluation of the infarcted. Myocardium can be used to explore therapeutic strategies for the treatment of heart failure. This technique can provide insight into the signaling mechanisms associated with the immediate responses to ischemia, followed by proficient injuries such as infarct size, area at risk, and cell death.
This model can be used to evaluate novel therapeutic strategies for the treatment of ischemic heart disease. Demonstrating the procedure will be a senior research associate and X, a senior postdoctoral fellow from the laboratory of Dr.Bradley McConnell in the Department of Pharmacological and Pharmaceutical Sciences at the University of Houston, an institutional member of the Texas Medical Center. Before the surgical procedure can begin, the operating surface must be disinfected with 70%ethanol.
Sterilize all surgical instruments with a hot bead sterilizer before each individual surgery. The instruments required include two surgical scissors, one forceps, one curved forceps, two needle holders, and a chest retractor. Adjust the temperature of the homeo themic blanket system so that it will maintain the animal's body temperature at 37 plus or minus one degree Celsius.
The size of an infarct is dependent on the duration of the occlusion as well as the body temperature. Have cotton gauze and applicators readily available to manage bleeding. Only animals that pass a physical examination by visual inspection, measurement of heart rate, respiratory rate, body temperature, and body weight are used in these studies.
Record the weight and identifying characteristics of healthy animals that will undergo surgery the day before surgery. Administer a dose of buprenorphine masks. Gowns, and gloves must be worn throughout the entire procedure, and gloves must be changed between individual surgeries.
To begin anesthetize a rat and prepare the surgical site by thoroughly shaving the fur from the neck and chest areas. Disinfect the shaved areas with Betadine solution, followed by 70%ethanol. Repeat this procedure three times.
Place the animal on a homeo themic blanket system for proper maintenance of body temperature. Lying on its right side. Beveling the edges of a polyethylene tube makes insertion into the larynx easier.
Insert the endotracheal tube with the aid of a surgical stereo microscope. Gently manipulating the tongue as needed and keeping the trachea in view. Connect the endotracheal tube to a ventilator cycling at 80 breaths per minute and a tidal volume of 1.2 milliliters per 100 grams of body weight.
Once mechanical ventilation is achieved, place a sterile drape over the rat leaving only the surgical area exposed. Once steady breathing is established, open the chest at the left fourth intercostal space. Minimize the degree of cutting into the tissue to reduce the risk of bleeding.
Position a chest retractor within the fourth intercostal space and spread the ribs to expose the left ventricle or LV care must be taken not to damage the lung. Next, open the pericardium, leaving it in place. If possible, identify the proximal left anterior descending or LAD artery with the use of a surgical stereo microscope.
Transiently, ligate, or tie with a slip knot the LAD to induce ischemia. Taking care not to exteriorize the heart proper. LAD ligation is confirmed by observing blanching of the myocardial tissue distal to the suture, as well as dysfunction of the anterior wall during the ischemic period.
After a 30 minute ischemic period, begin cardiac reperfusion by cutting or releasing the ligature around the LAD with microsurgical scissors. Reperfusion is verified by the return of red color to the myocardial tissue and the demonstration of some recovery of anterior wall motion observed immediately following the release of the LAD ligation. The procedure is identical for sham control rats, except that the LAD is not transiently ligated.
Remove the chest retractor and draw the ribs together with an interrupted suture pattern. Once the ribs are closed, create negative chest pressure to ensure proper breathing by drawing air from the chest cavity. Finally, close the skin with a continuous suture pattern After surgery, place the rat into a cage with a heating pad and water monitor body temperature by a rectal thermometer, respiratory rate by visual inspection, heart rate by palpitations and abnormal signs of pain until the animal becomes ambulatory.
Sterile saline should be administered. If signs of dehydration are observed, administer buprenorphine immediately following surgery. After a four hour reperfusion period, place the re anesthetized rat back into the surgical area.
Visualize the left ventricle as demonstrated previously and ligate the LADA second time or the slipknot can be retied. Inject one milliliter of 5%Evans blue and saline into the right ventricular chamber and allow to perfuse for two minutes. After this period, inject one milliliter of saturated potassium chloride solution into the right ventricular chamber to stop the heart from beating rapidly excise the heart and embed it in warmed 2%agro phosphate buffered saline.
Once the agro solidifies at room temperature, cut the heart into one to two millimeter sections. Stain the viable myocardium with 2%trien tetra oleum chloride or TTC for 20 minutes and capture an image of each slice seen. Here is a representative image of the rat heart.
After four hours of reperfusion, the area in blue represents the non occluded or not at risk perfused area. The red areas indicate the area at risk for infarction and white yellow areas denote infarcted myocardium within the at-risk area. After watching this video, you should have a good understanding of how to perform in acute myocardial infarction in rats and animal model used to mimic human ischemic heart disease.
This model can be used to study the immediate responses to ischemia, followed by a reperfusion injury that is within the first several hours, such as the infarct size, area risk, and cell death. As you observe in this video, good luck with your experiments.
