This protocol targets the limited transplantation organ supply issue by facilitating the evaluation of different cardioprotective conditioning strategies to allow the use of hearts donated after circulatory death. Place the animal on a heating pad set to medium. After confirming a lack of response to toe pinch in an anesthetized adult rat, insert a rectal temperature probe.
Attach a transdermal pulse oximeter sensor to a hind paw, and cover the rat with an absorbent pad to maintain body temperature. Make a three-to four-centimeter midline skin incision in the neck, and use blunt tip curved scissors to blunt dissect the subcutaneous tissue, to expose the right sternohyoid muscle. Using nontraumatic forceps, move the muscle laterally until the right carotid artery, jugular vein, and vagus nerve are identified.
Use scissors to carefully separate the vagus nerve from the carotid artery. Inject 2, 000 international units per kilogram of heparin into the right jugular vein, applying pressure to the injection site after needle retraction to avoid blood leakage. Use curved forceps to pass two 5-0 silk sutures around the carotid artery.
Firmly attach the distal suture to occlude the carotid artery at the superior aspect of the exposed artery, keeping the proximal suture untied, and place the rat under a stereo microscope. Then use microsurgery scissors to carefully make a one-millimeter incision over the anterior wall of the carotid artery, and insert a 22-gauge, one-inch closed intravenous catheter toward the aortic arch. The use of magnification and microdissection tools is highly recommend for these steps.
A clean field can be achieved by tensioning the proximal suture to avoid bleeding. Cardiac donation is necessary to assemble the ex vivo cardioplegia apparatus. To initiate the DCD protocol, first extubate the animal, then use mosquito forceps to clamp the trachea, starting the agonal phase.
Begin counting the functional WIT when the peak systolic blood pressure drops below 30 millimeters of mercury or asystole or ventricular fibrillation appears, whichever comes first. At the end of WIT, perform a medial sternotomy, keeping the thorax open with an Alm retractor if necessary. Separate the heart from the pulmonary veins and other thoracic structures to complete the cardiectomy.
Immediately submerge the heart in ice-cold Krebs solution for a rapid transportation to the ex vivo system. Use a 2-0 silk suture to tightly fix the aorta to the Langendorff apparatus, and then open the stopcock. Now fully open the flow to the cannula, and start the initial reperfusion and stabilization time.
Rotate the heart so the atria is facing the pressure sensor, and dissect the pulmonary veins to widen the left ventricular atrial opening if necessary. Next, insert a latex balloon connected to a pressure sensor, making sure that the balloon is fully positioned inside the ventricle. Slowly fill the balloon with saline until the end diastolic pressure reaches 15 millimeters of mercury.
Insert the pacing electrode in the anterior face of the heart without puncturing the coronary vessels. Once spontaneous beating is observed, set the pacing to 300 beats per minute. After 10 minutes of stabilization, initiate the continuous intraventricular pressure measurement recording to begin the reconditioning and assessment phase.
After one hour, remove the heart from the Langendorff apparatus and use a straight high-carbon steel blade to remove the atria. With the right ventricle facing down, cut one-to two-millimeter-thick transverse ventricular slices. Excise the right ventricle from the third slice of tissue, and snap freeze the left ventricle for downstream biochemical analyses.
Submerge the remaining sections in freshly prepared 5%TTC solution in PBS for 10 minutes at 37 degrees Celsius. The next morning, wash the samples two times in fresh PBS and submerge the samples in fresh PBS for imaging. Viable tissues will appear brick-red in color.
Following extubation, the blood pressure rapidly drops in a predictable pattern and the expected time to death is less than five minutes. Here, average pressure versus time curves at the start of reconditioning following zero, 10, and 15 minutes of WIT are shown. The contractile function will improve over time, the use of short periods of WIT will allow contractility to return to normal, and morphological damage will not be detectable.
The use of a conditioning agent added with the cardioplegia and at the stabilization phase showed that the damage generated by 15 minutes of WIT in this model are amenable to modulation by cardioprotective agents. This technique allows full control of all the relevant variables and pharmacological and nonpharmacological intervention testing and can be reproduced in larger models, facilitating clinical translation.
