Ischemia heart disease is a leading cause of death worldwide. This procedure can be performed to induce ischemia-reperfusion injury in the heart. This protocol is to ligate the LAD accurately, securely, and consistently.
To prepare saline wet cotton balls, pinch off a small portion of sterile cotton and roll it repeatedly to form a ball. Dip the cotton balls in sterile 0.9%saline and squeeze out excess saline. Store the cotton balls in a clean box sterilized with 75%ethanol.
To prepare holding hooks, sterilize clips and rubber bands with 75%ethanol. To prepare a ligation loop, place the middle of a 7-0 silk stitch in the half circle size three spring eye of a tapered non swaged surgical needle. To prepare a snare loop controller, cut a five millimeter polyethylene 10 tube using scissors.
Next, for the experiment, anesthetize an eight week old Sprague Dawley male rat and check the reflexes by pinching the tail and hind feet. Then open the tissue between the two cartilage rings below the glottis using scissors and insert a three centimeter polyethylene 50 tube to act as an endotracheal tube. Connect the endotracheal tube to a ventilator.
Touch the chest and find the manubrium and sternal angle. Then identify the left side rib that connects with the sternal angle. Identify the intercostal space below rib A.Use fine tipped forceps to gently lift the skin close to the intercostal space.
Then use scissors to create a one centimeter oblique incision along the skin tension lines from the point approximately five millimeters to the left of the sternal body. Separate the skin and muscle layers from the incision using scissors. Then hook the muscle layers outside the left anterior chest wall downward with bent clips to expose the ribs underneath.
After identifying rib B below rib A, cut rib B with a blunt scissor from the middle of the rib cartilage. Gently touch and compress the wound with a saline wet cotton ball for several seconds if bleeding occurs. Next, hook the four bent clips to the intercostal muscle and ribs to gently spread the chest wall in four directions and create a rectangular surgical window to open the thorax carefully from the cut of rib B.Hook gently against the left lung and adjacent tissues covering the pericardium with another bent clip to prevent accidental tissue damage during the procedure.
Expose the heart by removing the thin pericardium with forceps. Then identify the first branch of the left main coronary artery, or LMCA, between the pulmonary artery and the left auricle. Using a surgical needle, create an open ligation loop by inserting and passing the silk stitch under the LAD at a location immediately distal to the first branch of the LMCA in the direction from the left toward the right side of the LAD to avoid accidentally puncturing the left auricle.
If the LAD is invisible because of fluid or blood covering the surface of the heart, gently swab the surface of the heart to visualize the coronary arteries. Insert the two ends of the silk suture on one side of the open loop into the circle of the other side to form a snare loop. Then insert the two ends of the silk suture of the snare loop into the prepared snare controller before closing the loop.
Slide the snare loop controller along the silk suture while gently stretching the silk to close the snare loop. Cease the coronary flow of the LAD to induce myocardial ischemia for one hour. Once the loop has been tied securely, hold the silk to fix the position of the snare loop controller with Kelly forceps.
Place the other end of the Kelly forceps on the surgical table. Cover the surgical window with saline wet cotton balls during LAD ligation. Open the Kelly forceps then release the snare loop controller for reperfusion of the coronary flow for two hours.
Validation of LAD ligation quality with Evans blue is shown. The myocardium with coronary perfusion was stained blue compared with a non perfused region, which remained red. A low variation in the area at risk, or AAA percentage among the study animals, indicated the accurate location of the LAD ligation.
Further, the infarct size was estimated as the ratio of the infarct area to the AAR in control and treated groups. The infarct size percentage of the treatment group is lowered than the controlled LAD ligation group. This technique paves the way for researchers to explore novel therapeutic approaches against ischemic heart disease.
