An Immunological Model for Heterotopic Heart and Cardiac Muscle Cell Transplantation in Rats

Transplant models are valuable tools for immunological research that often require surgical expertise. Here, we present a feasible approach for heterotopic heart and cardiac muscle cell transplantation in rats. By highlighting and simplifying all of the crucial steps of our model, researchers without a surgical background should be able to master this model in a reasonable amount of time.

Both transplant models provide valuable insights into the mechanisms of tolerance and rejection and can be applied in various species. We have a lot of experience in teaching this model to individuals without prior surgical training. After learning the crucial steps, we advice patience and sufficient practice with deceased animals.

For donor heart explantation, after confirming a lack of response to pedal reflex in anesthetized seven to 22 week old rat donor animal, apply eye lubricant and use mechanical clippers to remove the abdominal and thoracic fur. Place the rat in the supine position on a heating base and fix the limbs at the base of the operation table with elastic bands. Disinfect the exposed skin with 70%ethanol and perform a median laparotomy.

Insert retractors into the incision and use sterilized cotton swabs to mobilize the intestine to the left side of the donor to expose the inferior vena cava. Next, puncture the inferior vena cava to allow intravenous injection of 500 International Units of heparin dissolved in one milliliter of ice-cold isotonic saline solution. Stop the bleeding at the punctured site after needle retraction by light compression using a cotton swab.

Incise the diaphragm, perform a lateral thoracotomy to both sides of the donor and pin the thorax wall. Remove the pericardium and the vagal nerve by blunt preparation using two micro-needle holders. For exsanguination, transect the abdominal vessels and insert the blunt branch of a probe pointed scissors into the transverse pericardial sinus.

Use a compress to dissect the ascending aorta and pulmonary artery as distally as possible under light caudal traction of the heart. Place a single 5-0 ligature around the superior and inferior vena cava and the pulmonary veins and tighten the ligature step-wise as dorsally as possible. Sever the tissue dorsal to the ligature and extract the heart.

To perfuse the explanted heart, use an 18 gauge cannula from an intravenous catheter to flush 30 milliliters of ice-cold, isotonic saline solution supplemented with 1000 International Units of heparin through the ascending aorta and the pulmonary artery before placing the heart in a 15 milliliter tube of saline solution on ice. After preparing the 10 to 14 week old recipient rat as just demonstrated, mobilize the intestines to the upper-left side of the recipient onto a warm, wetted compress. And adjust the surgical microscope or a sufficient alternative.

After mobilizing the duodenum and proximal jejunum, respectively, using a five to seven full magnification, use cotton swabs and blunt dissection to expose the abdominal aorta and inferior vena cava. Using two micro needle holders to elevate the abdominal vessels without injuring the lumbar veins, place a Cooley vascular clamp onto the vessels. Use a 30 to 45 degree arched 27 gauge cannula to puncture the abdominal vessels.

And use Potts scissors to enlarge the puncture site into a longitudinal incision that matches the size of the lumen of the donor vessels. To remove clots and to prevent postoperative thrombosis, perfuse the vessels with saline and place the graft into the situs. Using two simple interrupted 8-0 monofilament non-resorbable sutures, fix the donor ascending aorta to the recipient abdominal aorta at the cranial and caudal corners of the longitudinal incision.

To anastomose the ascending aorta of the donor with the abdominal aorta of the recipient place the graft to the right of the recipient vessels while performing the first half of the anastomosis with a running 8-0 monofilament suture. Then place the graft to the left of the recipient vessels and perform the second half of the anastomosis. Fix the donor pulmonary artery to the inferior vena cava in a similar manner with two corner knots at the cranial and caudal corners.

After fixing the donor vessel with knots at each corner, place a stitch from the outside to the inside of the vessel to run an intraluminal anastomosis for the first half of the anastomosis. Before tying down the suture, place another stitch from the inside to the outside. To prevent peripheral embolism after finishing the second half of the anastomosis, flush the anastomosis with saline before tightening the knot of the second half of the anastomosis.

Place a hemostatic gauze around both anastomoses and carefully release the Cooley vascular clamp to allow graft reperfusion. Managing any bleeding with light compression and sterilized cotton swabs. After about 60 seconds, the heart should start beating.

Carefully replace the intestines and close the abdominal fascia and the skin with separate continuous 3-0 polyfilament running sutures. At the appropriate experimental time point, harvest a donor rat heart as demonstrated and use a sterile scalpel or sterile scissors to shred the heart into three by three millimeter fragments. Digest the pieces in 20 milliliters of culture medium supplemented with 0.5 milligrams per milliliter of collagenase for 30 minutes at 37 degree Celsius.

At the end of the digestion, add the tissue to a large-pored sieve, while removing the culture medium. Add five to 10 milliliters of isotonic saline solution and strain the tissue slurry through the sieve. And after centrifuging the cell suspension twice, filter it through a 40 micrometer cell strainer.

Rinse the strainer with five to 10 milliliters of isotonic saline solution to collect any remaining cells. And after centrifugation, resuspend the isolated rat cardiac cells in saline solution at a concentration of five times 10 to the fifth cells per milliliter. Load a one milliliters syringe with the cell solution and place the anesthetized recipient animal in a lateral position with the target ear facing up.

Fix the ear with a finger and double-sided tape, and use a 27 gauge cannula to subcutaneously inject 20 microliters of cardiac muscle cell solution close to the visual capillary vessels into the recipient's ear. After the appropriate experimental observation period, extract the draining cervical lymph nodes and perform the appropriate downstream analyses of interest. Syngenic transplants survived up to 100 days without signs of graft failure.

The specific donor-recipient combination can result in different speeds of heterotopic heart transplant rejection. Cryostat sections of the rejected hearts reveal an increase infiltration of immune effector cells, whereas syngenic grafts are largely free of immune cells. Cervical lymphadenectomy and re-stimulation assays of draining lymph node cells after oral cardiac muscle cell injection reveals distinct strain specific immune responses toward heterotopic cardiac tissue in allogenic transplant recipients, warranting further immunological analyses such as cytokine profiling.

Handle the vessels carefully when inserting and extracting the needle. Don't put too much attention on the sutures to prevent stenosis. Don't place too many stitches for each anastomosis.