This protocol provides a simple, reliable, and reproducible methodology to induce in situ acute regional myocardial ischemia and reperfusion injury in a rabbit model for non-survival and survival experiments. This technique has minimal mortality and morbidity via two different surgical approaches. The methodology is cost-effective and has low technical demand and thus can be performed by investigators without surgical expertise.
Exposing the LAD through a mini-thoracotomy can be challenging. We recommend making a fourth intercostal space anterior parasternal incision extending five to six centimeters laterally and practicing such exposure and rabbit necropsy prior. To perform a left mini-thoracotomy for survival studies, elevate the left side of the anesthetized rabbit approximately 30 degrees using a pillow.
Palpate and outline the bony landmarks with a felt-tipped marking pen. Using a number 10 blade, incise the skin overlying the fifth rib, extending from adjacent to the lateral border of the sternum to five to six centimeters laterally. Using electrocautery, divide the pectoralis major muscle medially, followed by the serratus anterior muscle laterally.
Then divide the intercostal muscles above the fifth rib to preserve the neurovascular bundle. Using sharp dissection, incise the parietal pleura with Metzenbaum scissors, carefully enter the pleural space through the fourth intercostal space, and extend the initial pleural incision in both directions parallel to the rib until a rib spreader or sternal retractor can be inserted. Place a rib spreader or sternal retractor within the rib space and widen it to visualize the heart and pericardial sac.
Use DeBakey forceps to lift the pericardium. Open it with Metzenbaum scissors and proceed with LAD artery isolation. This will provide optimal visualization of the anterolateral surface of the heart with adequate exposure to the LAD and its branches.
To isolate the LAD artery, encircle its third diagonal branch with a 3-0 polypropylene suture on a taper needle, then remove the needle. In survival cases, trim the three 3-0 polypropylene thread used to isolate the LAD and loosely tie the ends together. The three 3-0 polypropylene thread is used to identify the area at risk and the infarct zone upon harvesting the heart.
Post-surgery, tie two 2-0 Polyglactin 910 figure-of-eight stitches around the ribs. Close the muscular and subcutaneous layers with a 3-0 polydioxanone suture in a running fashion. Then close the skin in a subcuticular fashion using a 5-0 monofilament suture.
Finally, perform a needle thoracentesis to evacuate the pleural error. For non-survival studies, immediately proceed with midline sternotomy using curved Mayo scissors. Using a sternal retractor to visualize the heart and pericardial sac, open the pericardium as previously demonstrated.
Visualize the 3-0 polypropylene thread loop placed during the previously performed thoracotomy portion of the procedure. To isolate the LAD artery, encircle its third diagonal branch with a 3-0 polypropylene suture on a taper needle. Then remove the needle and thread both ends of the polypropylene filament through a small vinyl tube to form a snare.
Using DeBakey forceps, place a rectangular PTFE pledget between the two polypropylene filaments, sandwiching it between the isolated LAD artery and the vinyl tube. Tighten this snare by pressing down on the vinyl tube and pulling up on the polypropylene suture filaments to occlude the coronal artery. Clamp the tube using a mosquito clamp to maintain the desired tightness.
Confirm myocardial ischemia by observing regional cyanosis of the epicardium. Remove the snare after 30 minutes of ischemia induction. At the end of the experiment, harvest the heart for biochemical and tissue analysis.
The recorded ECGs at pre-ischemia, during ischemia, and reperfusion demonstrated tachycardia, arrhythmias, conduction system defects, the development of infarct-related Q waves, and ST segment deviation. The 2D echo readings showed that the fractional shortening decreased during ischemia and post-ischemia compared to pre-ischemia. After 30 minutes of induced regional myocardial ischemia, evidence of myocardial necrosis was observed.
The most critical protocol steps are carefully encircling the LAD without damaging the artery or causing venous bleeding, appropriately placing the PTFE pledget to prevent vasospasm, and including the LAD to create a consistent area at risk. This methodology is useful in vascular injury, chronic myocardial ischemia, and acute myocardial stunning models to unveil cardiovascular molecular mechanics, pathophysiological mechanisms, and therapeutic targets, demonstrating usability and developing diagnostic and treatment algorithms.
