Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Our research uses an isolated rat heart perfusion circuit to test supplementation strategies for donor heart preservation. We aim to identify preservation strategies that can improve the functional recovery of donor hearts after either long periods of cold storage or long periods of warm ischemia following donation after circulatory death. The isolated working rat heart protocol was used to initially test the current clinical donor heart preservation strategy of using glycerol trinitrate and erythropoietin in the heart preservation solution.

This provided the rationale for testing in a porcine heart transplant study before transitioning to the clinic. The advantage of using an isolated working rat heart perfusion protocol compared to Langendorff perfusion techniques is the ability to obtain functional measurements of the heart, such as aortic flow and cardiac output. To begin, place an anesthetized rat in a supine position.

Secure the limbs of the animal with tape, then fix a nose cone over the animal to administer oxygen. Soak sterile gauze in a container with Krebs-Henseleit buffer. Place the soaked gauze on ice.

After making a lateral incision across the abdomen, move the stomach and intestines to allow access to the left renal vein. Inject heparin and apply pressure to the injection site until the bleeding stops. After letting the heparin circulate for a couple of minutes, cut through the rib cage on either side and across the diaphragm to completely open the chest cavity.

Carefully lift the heart lung block and cut along the descending aorta, keeping the aortic arch intact. Transfer the heart-lung block to the Krebs-Henseleit buffer ice slush to arrest the heart. To begin, place a freshly excised rat heart-lung block on a gauze sheet over ice.

Dissect the excess fat around the aorta, then trim the aorta, leaving about three to five millimeters for cannulation. Make a one to two millimeter long incision in the pulmonary artery. Place the trimmed heart-lung block on ice directly underneath the cannulas.

Next, use a flow control tubing clamp to adjust the flow from the Langendorff side for aorta cannulation. Carefully cannulate the aorta onto the left cannula. Hold it with a black clamp and tie it with a 2-0 silk suture.

Open the flow control tubing clamp completely to increase flow from the Langendorff perfusion. Then open the afterload clamp. Start monitoring heart activity with software.

Adjust the heart-lung block such that the anterior surface of the lungs faces the operator. Tie the left lung lobe with a 2-0 silk suture. Tie the remaining lung lobes in one sweep with a 2-0 silk suture.

Then rotate the heart so that the tied lungs are towards the back. With a pair of small Vannas scissors, trim a two to three millimeter section of the left atrial appendage to create a small opening into the atria. Carefully cannulate the left atrial appendage to the left cannula.

Hold it with a black clamp and tie it with a 2-0 silk suture. Adjust the heart chamber water jacket so that the heart sits within the center of the chamber. Now, open the pressure transducer to air.

Select aortic pressure in the software, then click on bridge amp, followed by zero, and OK to zero the pressure transducer. Perfuse the heart in Langendorff mode for 10 minutes. Next, transfer the air stone from the Langendorff Krebs-Henseleit buffer reservoir into the working Krebs-Henseleit buffer reservoir.

Open the three-way tap leading from working perfusate. Close the large clamp leading to the aortic cannula and open the flow leading to the left atrial appendage to switch to working mode. Perfuse the heart in working mode for 15 minutes.

Fill a cardioplegia chamber with 50 milliliters of the designated cardiac preservation solution. Lower the water jacket and close the three-way tap on the working mode side. Close the clamp to stop the flow towards the left atrial cannula.

Then open the flow to the aortic cannula. Now, close the clamp from the Langendorff side and on the afterload. Open the clamp from the cardioplegia line.

Flush 15 milliliters of the cardioplegia solution out from the pressure line. Flush the heart with cardioplegia via the aortic cannula and collect the coronary effluent. After three minutes, close the cardioplegia clamp and the small blue clamp leading to the aortic cannula.

Place a bulldog clamp on the aortic cannula and another bulldog clamp on the left cannula. Disconnect the heart cannula block from the circuit and place it in a beaker containing cardioplegia in an ice box. To reperfuse the heart after cold static storage, carefully secure the heart-lung block after cold storage onto the perfusion circuit.

Use a blunt-end 18 gauge syringe filled with Krebs-Henseleit buffer to remove any air from the cannula. Connect the aortic cannula first, then open the tubing clamp on the Langendorff side. Reconnect the left cannula and ensure that air has been removed from the cannula before connecting it to the perfusion circuit.

Keep the working side clamp closed until it is time to switch from Langendorff to working mode. An aortic flow of 14 to 22 milliliters per minute resulted in a good Langendorff flow. When switched to working mode, an aortic flow of at least 30 milliliters per minute facilitated heart storage Despite good baseline traces, some hearts did not yield good recovery after prolonged cold static storage.