This is an economical and reproducible model of cardiac transplantation. It can be used both to improve microsurgical skills and allow for the use of genetically modified mouse strains as donors or recipients. The technique broadly includes all the pain management and anesthetic complexity of normal human surgery to limit morbidity following surgery.
This model is critical for studying surgically-induced immunity, the effect of mismatch on transplant rejection, and transplant longevity. The general surgical approaches of this model can be applied in a modified form to other mouse solid organ transplants. Microscopic scale and associated time constraints leave for a small margin of error.
The key to success is regular practice with professional supervision, providing specific feedback on areas of difficulty. Demonstrating the procedure will be Ms.Liu Liu, a research assistant from my laboratory. Assisting the surgical procedures will be Dr.Wen Hua Huang, Ms.Jas Kaur, and Ms.Xiao Zhang.
After anesthetizing and removing the mouse from the induction chamber, remove hair by closely shaving the ventral abdomen using clippers. In the donor's case, shave the area extending from the genitalia to the upper margin of the ventral thorax. Make sure that the area shaved reaches the midaxillary line laterally.
Monitor the temperature by inserting a clean lubricated rectal probe into the animal's rectum and then securing it to the surgical board using micropore tape. Assess the anesthetic depth by observing responses to stimulation of the paw or tail from pressure applied by traumatic forceps, the palpebral reflex, and muscle tone. Measure the respiratory rate by observing the chest wall movement while observing the respiratory effort to assess the tidal volume, and then calculate the respiratory rate as described in the manuscript.
Once the surgical site is disinfected using sterile cotton-tipped applicators, apply chlorhexidine in a circular motion from the surgical site center to the edges thrice, followed by a combination of chlorhexidine and ethanol in the same pattern. Prepare the surgical field by placing sterile surgical drapes on either side of the surgical board as the site to place sterile instruments. After making a small oval-shaped opening in the drape, place the fenestrated drape at the proposed incision site.
After preparing the animal as demonstrated earlier, apply eye lubricant. Using a 29-gauge insulin syringe along the planned incision site, subcutaneously inject a weight-based dose of bupivacaine into the ventral abdomen and look for a straight line of visible blebbing that covers the extent of the planned incision. Then, use a sterile surgical scalpel blade to make a ventral midline skin incision under eight times magnification.
Make sure that the laparotomy spans the lower abdomen to the costal margin. And insert a retractor to maximize the surgical field. Moisten the segment of sterile gauze with warmed 0.9%sodium chloride solution and position it at the superior aspect of the surgical site.
Now, use dampened sterile cotton buds to gently eviscerate the intestines. Position them on top of this gauze and wrap the gauze around the organ. Using a combination of cotton-tipped applicators and round body suture forceps, free and mobilize the abdominal aorta and inferior vena cava from surrounding tissues with a blunt dissection technique.
Ensure that the area of clearance is between the infrarenal aspect of the vessels and above the bifurcation of the aorta. After identifying the posterior abdominal vessels, gently pull the aorta in a direction away from the vertebral column using forceps. Make a channel on either side of these vessels cephalocaudally by passing the curved forceps posterior to the abdominal vessels.
Ligate each vessel identified and mobilized in the planned anastomotic zone using lengths of 10-O nylon tied with instruments into surgeon's knots with one additional throw. Isolate the anastomotic site from circulation by installing a surgical clamp at the abdominal vessel's head and coddle ends. Make sure that the clamps cross both vessels to a sufficient degree to ensure complete occlusion.
Use forceps in the non-dominant hand to steady the aorta. Perform an aortotomy using a 30-gauge needle at the anterior aspect of the aorta. Extend the aortotomy using straight-tipped microsurgical scissors.
To perform adenotomy, use straight forceps and apply gentle anterior traction to the inferior vena cava at the point in line with the middle of the aortotomy. Use curved microsurgical scissors with the concave side facing anteriorly to remove a segment of inferior vena cava of equal length to the aortotomy. Wash the interiors of the open vessels of remaining blood with heparinized sodium chloride solution.
Place the donor heart into the abdomen and ensure that the ascending aorta is directly alongside the abdominal aortotomy and the heart is rotated, so the pulmonary artery can be drawn across for the second anastomosis. Using 10-O nylon, place a stay suture between the 12:00 position of the aortotomy and the corresponding extremity of the lumen of the ascending aorta. Using straight-tipped forceps in a microsurgical needle holder, tie it with three subsequent throws of a surgeon's knot and cut the ends to leave approximately two millimeters of suture.
Place a second stay suture between the 6:00 position of the aortotomy and the corresponding aspect of the ascending aorta. As this suture will also serve as the base for subsequent running sutures, leave at least 10 millimeters of the tail for the ultimate tie off. Place a continuous running suture of 10-O nylon in an ascending manner to oppose the anatomical right edge of the aortotomy and the corresponding free edge of the ascending aorta.
Perform this procedure carefully and use approximately four throws for this line. Pass the free end of the suture around the distal stay suture before placing a second continuous running suture down the anatomical left side to affect apposition with the remaining free edge of the aortotomy. Tie off the suture to the tail using a surgeon's knot with two additional throws.
Place a stay suture between the 12:00 position of the inferior vena cava, venotomy, and the corresponding extremity of the lumen of the pulmonary artery. From this anchor point, place a continuous running suture discerningly between the anatomical left edge of the pulmonary artery and the corresponding edge of the venotomy. Pass the needle through the 6:00 position of the venotomy and the corresponding extremity of the pulmonary artery lumen.
In an ascending manner, pass the needle through the remaining free edge of the pulmonary artery and anatomical right of the venotomy. Use an average of four throws for this line and then draw the final free edges of the pulmonary artery and venotomy together. Tie off the free end of the suture to the anchor and, using an instrument, tie surgeon's knot with two additional throws and cut the extra thread.
Check the vessels for twisting, which would interfere with blood flow. Position gel foam over the suture line. Place two pieces of approximately two-millimeter gel foam around them and mold so that all the visible suture lines are covered.
Release the vascular clamps starting with the coddle clamp and then the cephalo clamp. As a small amount of hemorrhage is expected, preemptively position cotton-tipped applicators over the anastomotic sites to provide pressure. After fixing the visible leaks, check the heart for pulsation.
If it's absent, check the cardiac vessels for twisting, then reposition the intestines over and around the heart, and moisten the peritoneal cavity with warmed sodium chloride solution if appearing dry. Using the continuous non-interrupted technique, close the abdominal cavity using non-absorbable 6-O prolene monofilament by suturing the muscular layer first, followed by the skin. Once the recipient is removed from the surgical board and off the anesthetic, administer one milliliter of warm saline subcutaneously and place the recipient in a prepared cage with warming for observation as per postoperative recovery protocols.
Full recovery and acceptance were observed in the case of syngeneic and congenic heterotopic murine heart transplants for 100 days post-surgery. However, rapid rejection was observed as early as seven days post-surgery in case of major mismatched heterotopic murine heart transplants. During anastomosises, you must maintain awareness of where your suture is within the surgical field.
Failure to do so can lead to critical damage to structures or tangling of the suture. These microsurgical techniques of dissection and anastomosis are highly applicable to other small mammal surgical models, including heterotopic kidney and orthotopic liver transplantation, allowing researchers to study graft rejection.
