Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

Heart transplantation in mouse is a valuable model for studying alloimmune response Here, we introduce a new surgical technique of heterotopic abdominal heart transplantation in mouse. In traditional mouse heart transplantation the donor aorta and the recipient abdominal aorta are anastomosed. The pulmonary artery is anastomosed to the recipient inferior vena cava.

In this new mouse heart transplantation. The donor aorta is anastomosed to the recipient abdominal aorta and the donor intrathoracic inferior vena cava is anastomosed to the recipient inferior vena cava. Now we start the experimental demonstration.

After put mouse in box for isoflurane inhalation and anesthesia, weigh the mouse. According mouse's weight, to give intraperitoneal injection of ketamine xylazine acepromacine to anesthetize mouse. Fix and disinfection mouse on the operation table when anesthesia have effected.

Cut the skin from the neck to the lower abdomen. Cut the muscles of the abdominal wall and gently removed the viscera to the left. Next, expose the inferior vena cava.

Inject 0.4 milliliter of the heparin solution slowly into the lower vena cava and wait for one minute before pulling out the needle. Pull out the needle and cut both the inferior vena cava and the abdominal aorta with scissors to accelerate exsanguination. Open the chest cavity by performing a u-shaped cut.

Completely expose the heart, lungs, and all chest blood vessels. Expose the thoracic aorta. Cut half of the lumen.

Insert the irrigation tube into the opening of the thoracic aorta. Cut the pulmonary vein to facilitate irrigation and drainage. Inject at least two millimeter of four degree HTK solution.

The heart completely stops beating until the pulmonary vein outflow is completely clear. Pull out the irrigation tube and detach the sternum. Remove the thymus.

Lightly strip the fat around the aortic arch. Expose and ligate the trunk of arteria pulmonalis with a 10 zero suture. Separate the fact and connective tissue attached on the intrathoracic inferior vena cava Expose and ligate the superior vena cava with seven zero suture and cut it behind the ligation.

Make a seven zero suture around the base of the heart underneath the aortic arch. Inferior vena cava and the both auricle, then ligate the pulmonary artery branches and venous lung vessels. Transect the aortic arch as distal as possible but make sure you have one clear lumen without the openings of side branches.

The lung vessels under the ligature and intrathoracic inferior vena cava near the diaphragm. Now, remove the heart out of the chest. Place the explanted donor's heart into 4 degree cold HDK solution and to preserve temporarily.

Be sure not to extend a cold ischemia time longer than 60 minute. Now, we begin to demonstrate receptor operation. The initial operation steps are similar to those previously shown for the donor mouse, including anesthesia and sterilization.

Abdominal skin cut in a transverse manner. After intestinal dissection, cover the abdominal organs with a wet gauze using saline solution. Expose the inferior vena cava and abdominal aorta and free them from surrounding fat tissue.

If necessary, ligate or electrocauterize side branch vessels under the renal vessels. Insert the first micro hemostatic forceps from the right in the upper part of the brain and aorta underneath the renal vessels. Insert the second micro hemostatic forceps from the right in the lower part of the brain and aorta.

There are more than one centimeter for both aorta and vein space for anastomosis between two hemostatic forceps. Make an incision into the aorta, a little nearer to the lower clamp than to the upper clamp. Also, you can use a 30G needle to make a small hole and open it it up with micro scissors.

Place the heart into the abdominal cavity. The aorta in vision and the vena cava on the other side and cover it with a small wet gauze pad. Adapt and stitch the donor aorta to the recipient aorta, starting caudally.

Make a knot and proceed with a running suture to the top of this incision, about four to five stitches. Next, flip the heart over to the right, cover it again, and continue the suture on the left side until reaching the caudal end and knot it. Use micro scissors to cut a round hole on the abdominal inferior vena cava of recipient, which should have the same size of the donor's intrathoracic inferior vena cava lumen.

The incision should be located cranial to the planned aortic anastomosis. Make the vein incision larger than the aortic incision. Flush donor's intrathoracic inferior vena cava.

Sew the donor intrathoracic inferior vena cava to the recipient vena cava starting caudally. Tie a knot and perform a running suture until the top of the incision is reached. Use five stitches and continue the suture on the left.

Finally, tie a knot in a tail corner and carefully tighten. Be careful not to pull too tight. Lay small parts of the hemostatic sponge around the vein and aortic anastomosis.

Remove first the lower and then the upper micro hemostatic clamps and rinse the abdominal cavity with warm 0.9%sodium chloride. Take away the hemostatic sponge. Observe the heartbeat of the transplanted heart.

If everything is correct, the heart will start contracting again. Put the intestines back into the abdominal cavity and close it with four zero suture. Provide a warm and oxygen rich environment for the transplanted mice to recover.

In our previous research work, we reported on a comparison of overall 40 cases of vena cava to vena cava versus 40 cases of the traditional pulmonary artery to vena cava anastomosis procedure. The operation time for vessel anastomosis in a vena cava to vena cava group was significantly shorter compared to the conventional pulmonary artery to vena cava group. The surgical technique of heterotopic abdominal heart transplantation in mice represents a standard model for research in transplantation immunology.

This novel technique for the blood circuit reconstruction presented here is facilitating abdominal heart transplantation in mice. And this is called concomitant with reduced warm ischemia and rebeating time of the grafted heart, thus improving the success rate of this model.