A Standardized Murine Model with a Three-Person Workflow for Studying Acute Cellular and Antibody-Mediated Rejection in Xenotransplantation

Our research focuses on standardized small animal xenotransplantation models to study mechanisms of acute cellular and antibody mediated rejection. Existing models often suffer from operator vulnerability and lung procedures. This protocol provides a rapid, standardized, and reproducible workflow.

This figure illustrates the standardized three-person workflow for murine xenatransplantation, in which operators simultaneously perform recipient preparation, donor heart procurement, and donor heart anastomosis. To begin, obtain 10 to 12 day old neonatal sprayed dolly rats and seven to eight week old C57 Black six or BALB/c mice. House all donor rats and recipient mice in a specific pathogen-free environment.

With povidone iodine-soaked cotton balls, disinfect the thoracoabdominal area of the anesthetized rat. Incise the abdomen with scissors. Then use a cotton swab to gently move the intestines aside to expose the inferior vena cava.

Use an insulin syringe to inject 0.2 to 0.5 milliliters of heparinized saline into the inferior vena cava. Gently compress the injection site with a cotton ball to prevent bleeding. Incise the diaphragm.

Bilaterally cut the chest wall and retract it to fully expose the heart. Then remove the thymus and sever the superior vena cava to relieve pressure. Perfuse one milliliter of University of Wisconsin solution into the aorta to induce cardiac arrest.

Continue perfusion until the heart changes from red to pale. Place a gauze pad soaked in ice-cold saline over the heart to lower its temperature and prevent dehydration. Ligate the superior and inferior vena cava with an 8.0 silk suture.

Transect both vessels. Then cut the aorta at the aortic arch. Sever the pulmonary artery at its bifurcation.

Ligate all pulmonary veins with the 8-O silk suture. Then excise the donor heart. Place the harvested heart in ice cold saline for temporary preservation.

Using a depilatory cream, remove hair from the neck region of the anesthetized recipient mouse. Disinfect the skin with alternating rounds of povidone iodine and alcohol three times each. Make a two centimeter longitudinal incision approximately one centimeter to the right of the midline on the neck with a surgical blade.

Perform blunt dissection to remove subcutaneous fat and muscle layers. Excise the submandibular gland. After exposing the external jugular vein, use an electrocautery pen set to low coagulation mode to individually cauterize the branches.

Place two 8.0 silk sutures near the distal end of the vein for ligation. Then use microsurgical scissors to transect the external jugular vein between the two ligatures. Insert the external jugular vein through the venous cuff and push the cuff to the base of the vein.

Then secure it with a vascular clamp. Use microsurgical scissors to remove the ligatures. Then flush the residual blood from the vein using heparinized saline.

With a pair of microsurgical forceps, evert the external jugular vein over the cuff and secure it with an 8.0 silk suture. Perform blunt dissection posterior to the sternocleidomastoid muscle to locate the carotid sheath. Open the carotid sheath.

Carefully separate the common carotid artery from the internal jugular vein and vagus nerve. Next, ligate and transect the carotid artery. Then insert the lower end of the transected artery through the arterial cuff.

Evert the artery over the cuff and secure it in place. Slip the donor aorta over the arterial cuff on the recipient carotid artery. Secure it with the 8.0 silk ligature.

Create a loose knot loop around the donor pulmonary artery. Slip the pulmonary artery over the venous cuff on the recipient external jugular vein. Then secure it with the silk ligature.

Release the vascular clamps on the recipient carotid artery and external jugular vein to restore blood flow. Observe the donor heart resume strong contractions and rapidly turn red within 10 seconds. Once the donor heart regains a stable rhythm, adjust its position.

Using 4.0 sutures, close the skin incision on the neck. Disinfect the surgical site with povidone iodine. Place the mouse on a warm, soft pad maintained at 37 to 38 degrees Celsius to preserve body temperature.

Monitor the mouse until it regains consciousness. House the mouse individually in a cage with adequate access to food and water once fully awake. The median graft survival time in BALB/c recipients was four days.

Whereas in C57 Black 6 recipients, it was significantly prolonged to 6.5 days. In the SD-to-BALB/c group, hematoxalin and eosin staining showed severe myocardial destruction with widespread erythrocyte extravasation. In the SD-to-C57 Black 6 group, hematoxalin and eosin staining showed myocardial damage characterized predominantly by inflammatory infiltrates.

In the SD-to-BALB/c group, immunofluorescent staining showed minimal CD3 positive T-cell infiltration, minimal CD68 positive macrophage infiltration, and intense widespread C4D deposition. In the SD-to-C57 Black 6 group, immunofluorescent staining showed prominent CD3 positive T-cell infiltration, moderate CD68 positive macrophage presence, and moderate C4D deposition. The average success rate was 93.3%in the three-person group, compared with 86.7%in the single person group.

The main challenge in team correlation, earning practice, circularization and the assure stable and fashioned modern establishment. Future studies can use this standardized model to investigate xenograft immunomechanisms and evaluate normal enumosuppressive or tolerance-inducing stretches.