To begin clamp the 3 by 8 inch tubing right before the aortic port to stop perfusate flow. Position the prepared pig's heart with its posterior wall facing the operator and angle the organ chamber at roughly 20 degrees. Then, place the aortic cannula at a 90 degree angle from the aortic port, and unclamp the aortic line slowly.
De-air the aortic cannula by allowing perfusate to flow into it gradually. Now, slowly decrease the angle of the aortic cannula until it aligns with the aortic port, then fully connect it to the aortic line. Once fully connected, gently massage the heart intermittently to prevent distension due to left ventricular filling.
Vent the left ventricle through the open left atrium during this period. Start data acquisition and initiate the adenosine drip at 333 microliters per minute. Then, connect the pacing wires to the pacing box and set it to 60 beats per minute for backup pacing.
If fibrillation is present, defibrillate the heart using 30 JUULs paddles. Once an organized rhythm is present, discontinue manual venting and place EKG leads directly on the heart using hook needles. Connect the right angle cannula to the atrial port of the organ chamber.
Once connected, clamp the cannula and release the Hoffman clamp in the line between the oxygenator and the loading reservoir to allow fluid into the loading reservoir. Then, fill the loading reservoir until the pressure reaches 15 to 20 millimeters of mercury. Increase the pump output to maintain both aortic and atrial pressure.
Next, insert half of the right angle metal tip into the left atrium with the tip pointing toward the appendage to promote mitral valve competence. Insert the pressure sensor inside the left ventricle to record left ventricular pressure. After that, release the clamp on the cannula to allow the left atrium to fill.
Once de-aired, use the previously placed sutures and tourniquet snares to close the left atrium opening completely. Adjust the cannula and snares as needed to minimize fluid leakage. After securing the cannula in the left atrium, clamp the line from the oxygenator to the windkessel bag to completely stop retrograde perfusion to the aorta.
Finally, move the adenosine drip from the line leading to the windkessel bag to the line between the loading reservoir and the atrial port. Vascular resistance, oxygen uptake rate, lactate accumulation, glucose consumption, and potassium accumulation showed no statistical difference throughout the perfusion period. Similarly, no difference was seen in the weight gained by the grafts.
However, failing grafts heart rate steadily declined after two hours of loaded time with the area under the curve showing no statistically significant difference until three hours of perfusion. Additionally, loading dependent metrics suggested a loss of cardiac function as early as 30 minutes into loading time with the measure for contractility being the first metric to indicate a loss of function. Like heart rate, left ventricular pulse pressure steadily declined after two hours of loaded perfusion with statistically significant differences between grafts, pulse pressure, and relaxation seen after 1.5 hours of perfusion.
