Large-Animal Model of Donation after Circulatory Death and Normothermic Regional Perfusion for Cardiac Assessment

This protocol describes a large animal model of donation after circulatory death followed by thoracoabdominal normothermic regional perfusion that precisely mimics the clinical heart transplantation. This could be used to evaluate potential strategies in translational research. The main advantage of this protocol is that it provides a suitable preclinical model for cardiac transplantation given the anatomical and surgical techniques similarities between the boar swine and the human hearts.

Begin by using a cautery pen to perform a midline incision on an anesthetized animal from the mid cervical region to the xiphoid. Then, divide the subcutaneous fat, layer by layer, along the midline using electrocautery to reach the peristernum. After cutting around the sternal notch, use a finger to retract and sweep soft tissues from under the sternum.

Open the sternum with a bone saw, ensuring that the sternum is completely divided, while being careful with the vein beneath the upper part of the sternal bone. Then, cauterize the sternum or apply bone wax to ensure adequate hemostasis. Next, using electrocautery, dissect and remove the thymus by lifting it from the pericardium and cauterize the vessels that supply the thymus from the aorta and the superior vena cava to prevent bleeding.

Carefully cut the pericardium open with cautery. While cutting, insert fingers under the pericardium to avoid injury to the heart. Then, administer 300 units per kilogram of heparin intravenously to achieve systemic anticoagulation.

If the test is available, ensure the activated clotting time is more than 300 seconds. Administer a bolus of three milligrams per kilogram propofol intravenously. Then, stop mechanical ventilation and disconnect the endotracheal tube.

Monitor arterial pressure and peripheral oxygen saturation. When systolic pressure is less than 50 millimeters of mercury, start functional warm ischemia time. Establish circulatory arrest when there is asystole and an absence of arterial pulsatility.

After the withdrawal of life sustaining therapies and confirmation of death, retract the right ventricle outflow tract inferiorly, the pulmonary artery to the left and the aorta to the right. Then, carefully dissect the aorta pulmonary space using cautery. Apply a large cross clamp over the supraaortic vessels to avoid and exclude cerebral perfusion.

Next, using the Metzenbaum and right angle forceps, dissect delicately between the superior vena cava and the innominate artery and between the inferior vena cava and pericardium. And circle the superior and inferior vena cava using an umbilical tape or a simple zero silk suture and secure them with a tourniquet. Using a 3-0 suture, place two concentric purse-string sutures on the distal ascending aorta adventitia, avoiding full thickness sutures.

Then, place a purse-string suture on the right atrium before securing all sutures with a tourniquet. Next, set up and prime the normothermic regional perfusion system. Then, cannulate the aorta using a 17 to 21 French arterial cannula and secure it in place by tightening the tourniquet, holding the purse-string suture.

Create a five millimeter incision at the center of the purse-string on the right atrium and dilate it using a small angled instrument, like a right angle or snap. Cover the incision with a finger to avoid excessive bleeding. Next, use a double stage venous cannula to cannulate the right atrium.

Then, secure the cannula by tightening the purse-string sutures using a tourniquet. Connect the cannulas to the venous line of the normothermic regional perfusion circuit using a half to three over eight inch connector, ensuring complete de-airing to avoid an airlock in the system. Ensuring that a minimum of 15 minutes has passed between the declaration of death and the initiation of normothermic regional perfusion to encompass the time needed in clinical practice preparation, draping and having access to the heart.

15 minutes after starting functional warm ischemia time, initiate the normothermic regional perfusion and adjust flow rates progressively to reach a perfusion index of 2.5 liters per minute per square meter. Restart mechanical ventilation with a 50%fraction of inspired oxygen and six milliliters per kilogram tidal volume. When weaning off criteria are met, stop normothermic regional perfusion.

Remove the cannula from the right atrium and quickly tighten the purse-string suture to minimize blood loss. Secure the suture with knots. Then follow the same procedure to remove the aortic cannula.

Analyze cardiac function through echocardiography using a standard transesophageal probe and a transthoracic probe placed directly on the heart. Representative results of cardiac function demonstrated a significant decline in heart function following donation after circulatory death. However, these organs demonstrated similar functional recovery post transplantation when compared to conventionally transplanted hearts.

Cerebral oximetry measurements during normothermic regional perfusion with the supraaortic vessels clamped confirmed the absence of adequate cerebral perfusion, while lung compliance measurements during normothermic regional perfusion and after weaning from support shown no significant change from baseline. The development of this technique introduced the importance of assessing the transplantability of other organs like the lungs, the kidneys, and the liver in the context of donation after circulatory death and normothermic regional perfusion.