This protocol is the first to describe how therapeutics can be effectively administered to a whole cardiac allograft in the large animal model of cardiac transplantation. The main advantage of this protocol is that it allows investigators to test the effects of a therapeutic on a whole cardiac allograft over extended periods of time following transplantation. This technique can be used to investigate interventions that prevent cardiac allograft rejection, primary graft dysfunction, minimize the need for postoperative immunosuppression or extend the lifetime of a cardiac allograft.
Optimizing exposure is the most challenging part of the transplant procedure and requires multiple team members to complete. Similarly, experienced perfusionists are needed to coordinate and monitor ex vivo perfusion. To begin, make a 20 to 30 centimeter long incision using the number 10 blade from the manubrium down to the xiphoid, depending on the size of the pig.
Use electrocautery to divide the pectoralis major down from the sternum to the xiphoid being careful to divide along the midline of the sternum. Once down to the sternum, score the midline and begin the sternotomy from the xiphoid by dividing it with heavy scissors. Then extend the sternotomy cephalad after each cut.
Bluntly separate the heart from the sternum using finger sweeps and complete the sternotomy through the manubrium. After optimizing the surgical field exposure using a sternal retractor, identify and remove the thymus with electrocautery by entering the pericardium longitudinally from the diaphragm to the aorta and creating a pericardial cradle using silk sutures. Fully divide the tissue between the aorta and pulmonary artery and visualize the location of the aortic arch and the brachycephalic trunk to facilitate proper placement of the aortic cross clamp.
Using scissors and blunt dissection, circumferentially free the superior vena cava and tie two silk sutures of size zero around the superior vena cava. Apply size 4-0 polypropylene suture to the ascending aorta in a U-stitch manner and to the right atrium in a purse-string manner. Insert a pediatric aortic root cannula secured by the previously placed U-stitch.
And then after de-airing the cannula, secure in place with a Rummel tourniquet. Connect the aortic root cannula to the cardioplegia tubing after the tubing has been flushed with del Nido cardioplegia. Create a right atriotomy within the previously placed purse-string.
Insert a venous cannula into the right atrium and secure with a Rummel tourniquet. Connect the venous cannula to a sterile suction line connected to the cell saver cardiotomy and collect approximately 1 to 1.3 liters of blood. Then apply the aortic cross clamp, carefully ensuring that the clamp completely occludes the ascending aorta.
After placing sterile ice slush on the heart, divide the inferior and superior vena cava just proximal to the azygos vein. The aorta at the level of the arch just distal to the innominate artery and the main pulmonary artery at the bifurcation. Identify and ligate the pulmonary veins, then remove the heart from the chest and place it in a container with sterile ice.
To prepare the aorta for placement of the aortic connector, place four pledgeted size 4-0 polypropylene sutures in a simple horizontal mattress fashion around the inside of the distal aorta at a depth of five millimeters below the cut edge, and then tie them down. While holding up the pledgeted aortic sutures, insert the aortic connector into the aorta and tie an umbilical tape around the aorta to secure the connector. Place a size 4-0 polypropylene purse-string around the distal cut edge of the main pulmonary artery.
Insert the pulmonary artery cannula and tie down the ends of the purse-string to secure the cannula. Then connect to the pulmonary artery cannula to the pulmonary artery connector on the device and secure it with the tie. Take the prepared graft from the back table to the ex vivo perfusion device and connect the aortic connector to the device place.
The left ventricle vent drain through the untied pulmonary vein into the left atrium and across the mitral valve into the left ventricle. Using the number 10 blade, make a 20 to 30 centimeter long skin incision and then continue dissection down to fascia using electrocautery. After lifting the fascia and peritoneum with two Kocher clamps, carefully make a small incision into the peritoneal cavity using Metzenbaum scissors.
Extend the peritoneal opening for the full length of the incision using electrocautery. Placing a finger underneath to protect the underlying viscera. Place a Balfour retractor to optimize exposure and retract to the small bowel cranially using a wet towel.
Carry the dissection down to the abdominal aorta in inferior vena cava, then ligate the lymphatics with medium and large clips. On the back table, when the pulmonary vein left atriotomy where the left ventricle ventricle vent had been inserted is oversewed, trim the distal aspect of the aorta and pulmonary artery where attachment to the cannulas may have crushed the tissue. Place a Satinsky clamp on the inferior vena cava and create a longitudinal venotomy measuring approximately 1.5 centimeters using the number 11 blade and Pott's scissors.
Anastomose the pulmonary artery graft to the recipient's infrarenal inferior vena cava in an end-to-side fashion using a running size 4-0 polypropylene suture. Then anastomose the aorta graft to the recipient's infrarenal aorta in end-to-side fashion using a running size 4-0 polypropylene suture. Carefully place the heart into the right retroperitoneal space, preventing tension on the anastomosis and kinking of the vessels.
Then replace the small bowel. Close the fascia with loop size zero Maxon suture in a running fashion starting from both ends of the incision and tying in the middle, avoiding any injury to the bowel. Close the deep dermal layer with size 2-0 Vicryl in a running fashion and the skin with size 4-0 Monocryl in a running fashion.
Several different perfusion parameters were acquired. Circulatory flow rates were measured from the pulmonary artery, the aorta, and the coronary arteries. The aortic pressure including mean pressure, systolic pressure, diastolic pressure were also measured.
During ex vivo perfusion, the heart rate and temperature of the cardiac allograft were also measured. During the perfusion period, mixed venous oxygen saturation and the hematocrit values were measured from the perfusate. The viability of the intrabdominal heterotopic heart in situ 35 days indicate successful transplantation using this protocol.
The most important thing to remember is to select a site for the allograft that minimizes the potential for tension and kinking of the anastomoses. By following this procedure, investigator will be able to test several different types of therapeutics to determine their effects on immunologic responses against the allograft, as well as determine the longevity of a therapeutics effect on the allograft. After developing this protocol, our group has been able to test different viral vector vehicles for gene delivery, as well as a model for acute allograph rejection to gain an understanding of the pathophysiology of rejection and efficacy of gene delivery using viral vectors.
