The overall goal of this procedure is to transplant a heart from a donor to a recipient mouse. This is accomplished by first removing the heart from the donor thorax and storing it in cold saline. The next steps are to transplant the heart into the abdomen of the recipient and then monitor the transplanted heartbeat.
Finally, transplant rejection is confirmed by visual inspection and pathology analysis. Ultimately, results show that hearts transplanted between unrelated strains of mice are rejected while hearts transplanted between the same strain are accepted. Long term Video demonstration of this measured is critical at the microsurgical step are difficult to learn because of the very small size of donor and the recipient animals.
The complicated surgical procedure involved and the need for boost accuracy and the speed in performing the recipient operations Always make certain that all the instruments are thoroughly cleaned and sterilized in an 80%ethanol bath. Use of disposable sterile equipment is highly advisable. After anesthetizing the donor mouse in an isof fluorine chamber position, its supine on the operating board.
Then quickly connect the nose cone, delivering anesthetic. Be sure to test the quality of the anesthesia with a hind foot pinch. If there is no flinch response, commence with shaving the skin using a surgical blade.
Then sterilize the skin with three alternating wipes of 2%Chlorhexidine in 70%isopropyl alcohol throughout the surgery. Visually monitor the animal's breathing and heartbeat. Adjust the delivery of isoflurane accordingly.
Begin by performing a thoracotomy to expose the heart and vessels open the chest through both sides of the rib cage. Cut from the rib edge up the axilla and transversely level with the zaphy sternum. This makes a chest wall flap.
Pin the flap open to the operating board near the head. Then remove the pericardium from around the heart. Lift the inferior vena CVA with forceps and inject a milliliter of cold heparinized saline to the IVC proximal to the heart.
Then attach a small artery clamp to the IVC to prevent solution from flowing back through the needle hole with gauze and a cotton applicator. Move the heart down to expose the aorta and pulmonary artery. The transverse sinus is the channel posterior to the aorta and pulmonary artery bundle.
Next, cut the aorta and pulmonary artery as distally as possible to maximize the length of the vessels for the anastomosis. Now divide the IVC, the right SVC, the left SVC and the pulmonary veins. Each should be tied apart using 6.0 silk thread.
First, tie the IVC and the right SVC. Then tie off the left SVC and the pulmonary veins. Now remove the heart by cutting all the vessels distal to the ties.
Store the heart in cold, sterile saline at four degrees Celsius until transplantation. After preparing the animal for surgery as described for the donor, start by performing a laparotomy. Cut through the midline from the pubis to the zaphy sternum.
Using paperclips bent into retractors. Open the abdomen, wrap the bowels in warm saline soaked gauze, and retract them to the upper right of the abdomen. Next, expose the infrarenal aorta and IVC between the iliac bifurcation and left renal artery and vein.
Free the bundled segment of aorta and IVC. Then divide the aorta and IVC from the lumbar vessels with a calibrated cautery device. Apply small atraumatic clamps to the proximal and distal ends of the freed aorta IVC segment.
Using a 30 gauge needle, punch a hole into the front wall of the aorta from the hole cut vertically making a hole the size of the donor's aorta. Take this opportunity to remove any blood clots from the aorta lumen with a rinse of heparinized saline. Now introduce the donor heart in cold saline.
Position the donor heart next to the incision on the recipient's aorta. Position the donor pulmonary artery next to the recipient's IVC using 10 oh running nylon sutures. Anastomosis the aortas end to side.
Start at the proximal corner and suture along the left side to the distal corner. Then rotate the animal 180 degrees. Then move the heart over and continue the sutures through the right side of the aortic wall distal to proximal.
Before closing the anastomosis, gently flush the lumen with heparin eyes saline to remove clots and air. Then finish the suturing. Next, make a vertical incision in the front wall of the IVC where the pulmonary artery will be anastos.
Second, use 10 oh running sutures to anastomosis. The vessels starting at the distal end of the left wall within the lumen of the IVC at the proximal end. Continue along the front right side wall and suture up to the distal end.Again.
Give the lumen a good flushing before completing the anastomosis. Next place pieces of gel foam around the anastomosis. Hemostasis is achieved with gentle pressure from cotton applicators.
To revascularize the recipient. Release the distal clamp first, then the proximal clamp. During the recipient operation, it's vital to optimize the accuracy and the speed of the surgery.
So the cross clamp time is ideally less than 30 minute. To aid the recovery, apply warm saline at 37 degrees Celsius to the graft site. Fibrillation should be immediate and revert spontaneously to the sinus rhythm within a few minutes.
Before closing the surgical site, an injection of buprenorphine should be given intramuscularly. Finish the operation by closing the wound with one five oh absorbable running suture for all the tissue layers. After closing the surgical wound, inject the recipient animal with ampicillin and a subcutaneous injection of 0.6 milliliters of saline.
To help hydration place the animal on a heating pad set to 37 degrees Celsius A until it recovers consciousness. Most animals are drinking and even eating within three hours of the surgery. If after eight hours, the mouse still shows signs of distress, provide injections of buprenorphine every 12 hours until the symptoms resolve.
If the mouse remains in distress for more than 48 hours, contact a veterinarian for the first 10 postoperative days. Directly monitor the grafted heartbeat by abdominal palpation. On a daily basis, record the strength of beat as four plus for a healthy graft to one plus for a weak beat due to advanced rejection.
Record a non beating graft as a negative one due to complete rejection of the graft after 10 days. Continue monitoring the grafted heart pulse three times per week after 227 heterotopic heart transplants, 90.3%were successful for 24 hours, and 86.8%were successful for 48 hours. Failure included hind limb paralysis, a non beating heart due to graft, ischemic injury and or thrombosis and death graft survivors were cataloged.
The cause of heart transplant failures was established by pathology. Analysis of the transplanted heart here marked by an arrow thrombosis and infarction of the heart tissue was the cause of this grafted heart's failure in the strain combination of C 57 Black six donor and recipient, no heart was rejected and the transplanted hearts survived for more than 100 days. In this case, the heart had reduced slightly in size due to muscle atrophy secondary to its non-life sustaining status.
In contrast, in the rejecting strain combination of DBA two donor to C 57 Black six recipient, this transplanted heart had ceased to beat by day seven and showed evidence of rejection by 100 days. The same heart was completely fibrotic and shrunken Once marketed. This technique can be done in 90 million or less if it's performed properly, and don't forget that working with US flooring gas can be extremely hazardous.
The use of an anesthetic gas scavenger is highly available.
