We have developed a model to reduce complications associated with normothermic ex-situ perfusion and increase the safe preservation time for donor hearts. Compared to the alternative animal models, this model is simple and cost-effective, and can be executed by a single experimenter. This model can be useful in testing the long-term effectiveness of pharmacologic and genetic interventions on the donor hearts after transplantations.
The surgical procedure requires microsurgical skill. The key factor in achieving success in this model is to practice and focus on the critical steps outlined in the manuscript. To begin, assemble the Langendorff apparatus, including the oxygenator, pump, and perfusion lines.
Fill the perfusion circuit with 20 milliliters of saline solution, and circulate it until it is primed with autologous blood. On the anesthetized donor rat, make a 7-centimeter mid-line abdominal incision, and then bilateral incisions measuring 3 centimeters from the xiphoid process to the mid-clavicle. Remove the pelt from the thoracic region.
Inject 1, 000 international unit heparin dissolved in 0.3 milliliters of isotonic saline through the inferior vena cava using a 1-milliliter syringe Stop any bleeding from the needle hole by gently compressing it with a cotton swab. Insert a 5-French femoral catheter into the abdominal aorta. Ensure that the catheter tip reaches the aortic arch, and confirm the catheter location by assessing the approximate length of the inserted part of the catheter.
Collect around 10 milliliters of blood via the catheter inserted in the abdominal aorta. Connect the cardioplegic solution perfusion line to the abdominal catheter, and start the cardioplegic solution administration with the syringe pump at a rate of 800 milliliters per hour. Open the thoracic cavity from the diaphragm, and cut the inferior vena cava close to the diaphragm to prevent ventricular distension.
Cut the ribs bilaterally along the thoracic spine up to the thoracic inlet. Reflect the mobilized ventral chest wall superiorly with mosquito forceps. After administering all the cardioplegic solution, isolate the aortic arch from the surrounding tissues, and carefully dissect it just below the left subclavian artery.
Transect the brachiocephalic and left common carotid arteries at a distant position, leaving the longer stumps of the aortic arch for easy handling during aorta cannulation. Then transect the main pulmonary artery as close as possible to the bifurcation. Carefully ligate the superior vena cava and inferior vena cava with 5-0 silk sutures, preventing the obstruction of the right atrium and coronary sinus.
Cover the left margins of the thorax with wet gauze, place the heart onto it, and gently retract the superior vena cava and inferior vena cava ligatures to expose the hilum. Ligate the pulmonary and azygos veins together with a 5-0 silk suture. Sever the tissue dorsal to the ligature and extract the heart.
Weigh the heart after examining it for any injury. Replace the saline-primed circuit with blood priming, and start the perfusion at a flow rate of two to three milliliters per minute, allowing the perfusate to leak from the cannulation site to remove any air bubbles. After placing the permanent ligature, ensure the heart begins to contract within a few seconds, and reaches a normal rhythm in 60 seconds.
A mean perfusion pressure of 55 to 65 millimeters of mercury with a coronary flow rate of three to four milliliters at 37 degrees Celsius indicates adequate perfusion. Collect 0.15 milliliters of blood from the reservoir, and check the blood gas analysis at the beginning of perfusion and every 20 minutes thereafter. After 120 minutes of perfusion, administer three milliliters of Custodiol through the syringe pump at a rate of 250 milliliters per hour to arrest the heart.
Perform the mid-line laparotomy and insert a retractor to widen the abdominal cavity. Mobilize the abdominal organs to the left side of the recipient using cotton swabs to make space for the procedure. Utilizing a surgical microscope with a 10x magnification, mobilize the duodenum and proximal jejunum by blunt dissection with cotton swabs.
to expose the abdominal aorta and inferior vena cava. Place 5-0 silk ligatures on the mesenteric branches of both the cranial and caudal sides of the major vessels. Elevate the abdominal vessels and coagulate or ligate the lumbar branches with 5-0 silk sutures.
Remember to spare the testicular arteries and veins, and do not clamp them. Use ligatures to lift the vessels and position the micro-clamps to the mesenteric branches, caudal, and cranial sides of the major vessels to stop the blood flow at the anastomosis site. Puncture the aorta using a 27-gauge needle, and elongate the incision with micro scissors to a length equal to or slightly larger than the opening of the donor ascending aorta.
Make a longitudinal incision of the inferior vena cava in the same way as the aortotomy, but make it three millimeters closer to the caudal side compared to the aorta incision. Starting the anastomoses, place the donor heart on the right side of the recipient's abdomen, and attach the donor ascending aorta to the recipient's abdominal aorta with one simple interrupted stitch at the cranial corner of the longitudinal incision. Move the heart to the left side of the recipient's abdomen, and perform anastomosis of the donor's ascending aorta with the recipient's abdominal aorta, using a running 9-0 polypropylene suture.
Fixate the donor pulmonary artery to the inferior vena cava with two interrupted sutures at the caudal and cranial corners of the longitudinal incision. After completion of the anastomosis, remove the caudal clamp and the cranial clamp. Insuring adequate hemostasis, replace the abdominal organs in a meander-like manner, and close the layers of the abdominal incision using continuous 5-0 polypropylene sutures.
The parameters used to assess the viability of the heart during ex-situ perfusion, such as lactate, potassium, and mean aortic pressure are shown in this figure. Here, the use of normothermic ex-situ preservation decreased the total ischemic time of six successful cases to 46.2 plus-minus 4.7 minutes, while the total out-of-body time was 166.2 plus-minus 4.7 minutes. The extraction of the heart from the donor and preparation for ex-situ perfusion and heterotopic transplantation required was 5.8 plus-minus 1.3 minutes.
The overall success rate of the surgery was 70%and the mean anastomosis time of the six successful cases was 38.4 plus-minus 3.4 minutes. The electrophysiologic performance of the donor heart before procurement and after implantation is presented in this figure. In all the experiments, the heart rate significantly decreased immediately after implantation, but it eventually recovered over time.
The macroscopic and microscopic appearance of the donor heart before normothermic ex-situ preservation, after normothermic ex-situ preservation, and after two hours of heterotopic implantation are shown here. When doing this procedure, it's important to take enough blood for perfusion, carefully cut the aorta without damaging the ascending part, and closely watch the physiological parameters during preservation. Following this procedure, we can test the effectiveness of novel preservation solutions and the recipient's immune response to newly-introduced preservation methods.
With the combination of ex-situ perfusion and heterotopic transplantation, we are able to investigate novel techniques through elongate preservation time, and observe the long-term impact on the recipient immune response.
