Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

I am Martin Ryll from the research laboratories of the Department of General, Visceral and Transplant Surgery of the LMU in Munich. Today we are going to show you a murine heterotopic aortic transplantation model using a modified non-suture cuff technique. First, anesthetize the recipient animal with an intraperitoneal injection of midazolam, medetomidine and fentanyl.

Clip all hair of the cervical lateral region with an electric hair clipper for small animals and apply ophthalmic ointment with cotton swabs to prevent the eyes from drying out during the procedure. Place the animal in the supine position on a heating pad under the microscope and gently tape its legs to the operating table with skin sensitive plaster strips. Tilt its head back and scrub the operative field several times with alcohol.

Make a skin incision from the jugular incision to the right lower mandible with small scissors. Remove the right lower lobe of the submandibular gland via bipolar cautery of the vessel pedicle and subsequent excision with micro scissors. Remove the right sternocleidomastoid muscle via bipolar cautery of the upper and lower portion and subsequent excision with micro scissors to gain access to the common carotid artery.

Mobilize the common carotid artery as far distally and proximally as possible by pulling the surrounding connective tissue apart with fine forceps. Tie two 7-0 silk ligatures with minimal distance between each other around the middle of the common carotid artery and transect the common carotid artery with fine micro scissors between the ligatures. Pass the proximal ligated end through the cuff and fix it with a small artery clamp.

Remove the ligature with fine micro scissors as close to the ligature as possible and flush the lumen with heparinized saline with a 30 gauge needle while taking care not to damage the vessel walls. Distend the open lumen using fine vascular dilatators and invert the carotid stump over the cuff by pulling it gently over the polyamide tube. Immediately fix the inverted carotid with a loosely pre-tied 7-0 silk loop.

Perform the same procedure on the other end of the carotid artery with the other cuff. Pass the distal ligated end through the cuff and fix it with a small artery clamp. Remove the ligature with fine micro scissors as close to the ligature as possible and flush the lumen with heparinized saline with a 30 gauge needle while taking care not to damage the vessel walls.

Distend the open lumen using fine vascular dilatators and invert the carotid stump over the cuff by pulling it gently over the polyamide tube. Immediately fix the inverted carotid with a loosely pre-tied 7-0 silk loop. Set the recipient animal aside and moisturize the upper two-third with saline until the aortic segment is explanted.

Anesthetize the donor mouse in the same fashion as the recipient animal. Clip all hair of the abdominal and thoracic region with an electric hair clipper for small animals and apply ophthalmic ointment with cotton swabs to prevent the eyes from drying out during the procedure. Place the animal in the supine position on a heating pad under the microscope and gently tape its legs to the operating table with skin sensitive plaster strips.

Scrub the operative field several times with alcohol. Perform a midline abdominal laparotomy with small scissors and push the intestines slightly upward to expose the inferior vena cava. Inject the IVC with one cc of heparinized saline using a 30 gauge needle.

Cut the abdominal aorta and IVC below the renal arteries with small scissors to exsanguinate the donor animal. Loosely place a compress into the abdomen to absorb the blood. Perform a thoracotomy via bilateral diversion of the ribs with scissors and tilt the anterior chest wall cranially with a surgical clamp to expose the mediastinum.

Cut the IVC and the esophagus directly above the diaphragm with micro scissors. Remove the heart and the lungs by titling them upward with forceps holding the cut IVC and esophagus and then excising them with micro scissors from the base to gain access to the thoracic aorta and the dorsal mediastinum. Mobilize the thoracic aorta from its surrounding tissue by pulling apart the surrounding connective tissue and fat with fine forceps while being careful not to damage any intercostal arteries.

Cauterize all branches from the thoracic aorta with bipolar cautery forceps and excise the aortic segment between the diaphragm and the aortic arch using micro scissors. Flush the excised aortic segment with heparinized saline with a 30 gauge needle while taking care not to damage the vessel walls to remove any remaining blood or clots and transfer the graft to the recipient animal. Pull the proximal end of the donor aortic segment over the proximal cuff on top of the inverted carotid artery with fine forceps and immediately fix it with a loosely pre-tied 7-0 silk loop.

Trim the distal free end of the aortic graft with micro scissors so that the graft length fits the distance between the two cuffs. Pull the distal end of the donor aortic segment over the distal cuff on top of the inverted carotid artery with fine forceps and immediately fix it with a loosely pre-tied 7-0 silk loop. Remove the distal clamp first to allow retrograde perfusion.

After achieving hemostasis, remove the proximal clamp to complete the anastomosis. Finally, close the wound with 6-0 continuous suture. After 28 days, the animals are sacrificed and the grafts preserved for histological staining.

As shown in these Van-Gieson's stainings of aortic sections, fully MHC mismatched allografted aortas from Balb/c donors to black six recipients developed a significant neointimal hyperplasia resulting in luminal narrowing. On the other hand, syngenic aortic transplants from black six donors to black six recipients developed only little neointimal hyperplasia. Immunofluorescence staining against SM22, a marker for vascular smooth muscle cells, as shown in this picture in green fluorescence, demonstrates that the neointima mainly consists of vascular smooth muscle cells.

The murine aortic transplantation serves as a model for chronic transplantation vasculopathy which is the most common cause of late graft failure. In comparison to the orthotopic abdominal model, our modified cuff technique is less microsurgical demanding leading to a high success rate. In addition, due to the constant diameter of the cuff, variations of the vascular anastomosis as seen in other microsurgical transplant models can be neglected.

Furthermore, the warm ischemic time is kept constant throughout the experimental groups as the recipient animal is prepared first before the aortic segment is explanted. Our modified model should help other laboratories to establish murine aortic transplantation.