The overall goal of this protocol is to introduce a novel murine model of solid organ and vascularized composite allotransplantation to investigate the effects of donor-derived vascularized hematopoietic and primary lymphoid tissue on immune tolerance and prolonged allograft survival. This method can help answer key questions in the field of reconstructive transplantion such as the role of vascularized bone and thymus on tolerance induction. The main advantage of this technique is that for the first time, combined vascularized bone marrow and thymus is being transplanted with a solid organ, the heart and the immunologically versatile mass model system.
The implications of this technique extend from the basic immunological research toward therapeutic translation into the clinical arena to treat complex tissue defects and simultaneously improve long-term survival in solid organ transplantion. Demonstrating this procedure will be Dr.BC Oh and Dr.Georg Furtmuller, who are post-doctoral fellows in the department of plastic and reconstructive surgery at the Johns Hopkins University. To begin, after anesthetizing a male donor mouse according to the text protocol, while wearing sterile gloves, use mechanical clippers to remove the hair from the cervical, thoracic and abdominal region.
On a sterile surgical surface, place the animal in a supine position. Attach a nose cone and maintain isoflurane at 1 to 2%Prior to the skin incision, widely prepare the incision site by using a sterile cotton swab to apply povidone iodine antiseptic followed by isopropyl alcohol. Using scissors, make superficial transfer of skin incisions across the cervical and abdominal skin.
Connect both incisions bilaterally, along the midaxillary lines. With microforceps, dissect the cervical region bilaterally to identify the external jugular veins. And with 6-0 silk suture, ligate the vessels before using scissors to divide them.
Then with electrocautery, divide the sternocleidomastoid muscles to bilaterally expose the internal jugular veins and common carotid arteries. Pass a 6 0 silk suture under the left and right common carotid arteries and internal jugular veins, respectively, in bulk fashion. Using bipolar electrocautery and sharp dissection, divide the pectoralis major muscles and clavicles to expose the subclavian vessels.
Used 6-0 silk suture to ligate and divide the vessels proximally. Next, gently grasp and withdraw the animal's penis. Along the dorsum of the penis, locate the dorsal vein and use isopropyl alcohol to disinfect the region.
Then, using a 30-gauge needle, inject 30, 000 units of heparin intravenously through the dorsal vein and allow the penis to recoil back to its original position. With the previously placed bulk ties around the common carotid artery and internal jugular vein, ligate and divide the structures bilaterally. Now use scissors to make a transverse incision to access the abdominal cavity.
Eviscerate the intestines to expose the intrahepatic inferior vena cava and inject two milliliters of cold EuroCollins cardioplegia solution into the intrahepatic inferior vena cava. Ensure proper injection by visualizing liver discoloration and cessation of the heartbeat. Using scissors, access the intrathoracic cavity via a bilateral diaphragmatic incision from the exposed abdomen, extending the incision cephalad through the intercostal muscles and ribs.
Flossing the pulmonary trunk and heart by using EuroCollin solution is critical to prevent micro thromboembolism from potential remaining inside of the coronary circulation. After injecting cold EuroCollins solution into the super hepatic inferior vena cava according to the text protocol, identify the root of the aorta and trace distally to the descending aorta. Sharply cut the descending aorta, preserving maximal length.
Now identify the pulmonary trunk and divide just proximal to its branch point, preserving maximal length. Then using two milliliters of cold EuroCollins cardioplegia solution, flush the pulmonary trunk and heart by placing a soft plastic tape catheter into the lumen of the pulmonary trunk. Using 6-0 silk suture, ligate and divide the inferior vena cava, confluence of pulmonary veins and accessory branches of the bilateral superior vena cava.
Then evaluate and dissect the heart cephalad from the attachments along the main stem bronchi and trachea with care, so as not to enter the airway. With sharp and bipolar electrocautery, dissect the chest wall, thymus and heart, completely liberating it from the donor mouse. Finally, using scissors, trim the allograft chest wall ex vivo to a smaller size along the sternum and lateral costi, taking care not to disrupt the internal thoracic vessels.
To minimize hemorrhage following revascularization, use bipolar electrocauterization along the borders of the osteomusculocutaneous sternum. After preparing the recipient mouse according to the text protocol, place the mouse in a supine position and angle the right upper limb with a slightly inferior orientation, forming a 110 degree angle between the head and the right upper limb. Make an incision from the midline along the right inferior border of the mandible, and extend the incision infralaterally to the right thoracic region.
With blunt dissection using microvascular forceps, mobilize the external jugular vein by circumferentially freeing the vessel from the soft tissue and adventitia. Use electrocautery to divide all branches and remove the right lobe of the submandibular gland with sharp dissection and electrocautery to free the space for the allograft. Ensure enough length of the jugular vein to evert over a cuff and ligate the external jugular vein using 6-0 silk suture.
Then insert the vein through the lumen of a precut polyamide cuff and use a bulldog microvascular clamp to fix the vessel cuff complex in place. Evert over the cuff and use 10-0 nylon suture to fix in place. While carrying out bipolar electrocautery, divide the right sternocleidomastoid muscle to expose the common carotid artery.
With forceps and blunt dissection, circumferentially mobilize the arteries cephalad to the distal most point within the cervical region by removing soft tissue and surrounding adventitia. Using 6-0 silk suture, ligate and divide the common carotid artery. Pass the artery through the lumen of a precut polyamide cuff and use a bulldog microvascular clamp to fix it in place as close to the thoracic inlet as possible.
Then divide the vessel distally. Use a microsurgical dilator to gently dilate the vessel, evert over the cuff and use 10-0 nylon suture to fix in place. While maintaining sterile conditions, place the donor descending aortic lumen over the arterial cuff construct on the recipient and use 10-0 nylon suture to fix it in place.
Fashion a similar anastomosis between the donor pulmonary artery and the everted external jugular vein cuff construct of the recipient mouse. Then remove the Venus microvascular clamp followed by the arterial clamp. Next, inspect the entire allograft for hemorrhage and, if present, mitigate bleeding according to the text protocol.
Inspect the graft and ensure hemostasis before releasing and completely removing the arterial microvascular clamp. Observe the heart to identify signs of reperfusion, which will be instantaneously apparent with rapid volume expansion of the heart chambers and wait for beading to begin within 30 to 60 seconds. Use warm saline to moisten the heart then place the chest wall into an anatomical position so as not to induce any kinking or tensions on the anastomoses.
Finally, use 6-0 nylon sutures to close the skin of the surgical wound and carry out postoperative care according to the text protocol. Performing end-to-end non-suture cuff anastomosis, maintaining meticulous hemostasis with electrocautery during reperfusion and assuring perfusion of all components of the composite tissue graph are the most critical steps of the procedure. The combined VCA and solid organ allograft is mounted over the previously placed polyamide cuff in the recipient's neck, illustrating optimal graft position and meticulous mounting of the donor vessel onto the cuff's construct.
The design of the allograft demonstrated here approved feasible for sustaining perfusion to the chest wall with minimal impact to the recipient animal. Presented here is survival data for syngeneic transplanted mice. The mean survival time was greater than 109 days, suggesting that the technical aspect of the transplanted allograft is designed to perfuse the entirety of the chest wall, thymus and heart.
Additionally, this mouse model is not only feasible but can be replicated. Once mastered, this technique can be done in 90 minutes if it is performed in a two-team approach. While attempting this procedure, it's important to remember that hemostasis throughout the entirety of the procedure is paramount, and minimization of kinking during the allograft insetting is also very important.
This technique paves the way for researchers in the fields of reconstructive transportation and solid organ transportation to explore the role of vascularized bone marrow and thymus as potential adjuncts to solid organ transportation to improve allograft survival. After watching this video, you should have a good understanding of how to establish a murine model of combined solid organ and vascularized composite allotransportation through a heterotopic unblock chest wall, thymus and heart transplant.
