The overall goal of this instructional video is to introduce a murine model of Orthotopic Hind Limb Transplantation using a non-suture cuff technique for super-microvascular anastomosis. Animal models are paramount to the understanding and treatment of human disease and have paved the way for reconstructive transplantation to become a clinical reality over the past two decades. This particular model can help answer key questions related to the path of physiology of an alloimmune response specific to vascularized composite allografts.

The main advantage of the technique is that the non-suture cuff technique significantly reduces the complexity and increases the success rate of this super-microvasular procedure. After confirming the appropriate level of sedation by toe pinch shave the hind limb and the groin of the donor animal. Next disinfect the skin with 10%povidone iodine and place the mouse under a disecting microscope.

Using scissors begin the donor limb harvest with a groin skin incision proximal to the mid thigh area. Circumstantially connecting the incision to demarcate the hind limb from the rest of the rest of the body. Identify and dissect the femoral artery, vein, and nerve.

Then use forceps and microscissors to separate the three structures. Preventing microtrauma to the vessel wall by using a no touch technique is essential for ensuring the success of the procedure. Once the vascular pedicle has been dissected use the microscissors to divide the vessels at the inguinal ligament.

Continue the division of the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the graft from the donor animal. Then, transect the femur cutting at the middle of the femoral shaft. Using a syringe equipped with a 33 gauge needle, flush the harvested limb with two ml of heparinized four degree Celsius saline.

Then, place individual polyimide cuffs on the femoral vein and artery. And store the graft in wet cotton gauze in a petri dish at four degrees Celsius until inset. Mounting the pesis onto the polyimide cuffs is the single most critical step of the procedure.

As malnotation may impair the bloodflow and improper tish pressmon may expose some antibiotic tissues increasing the likelihood of thrombosis formation. To remove the recipient hind limb, inject 0.3 ML of warm saline intraperitoneally. Make a circumferential incision around the hind limb and identify and dissect the femoral artery, vein, and nerve as just demonstrated.

Once the vascular pedicle has been dissected clamp the femoral vessels at the inguinal ligament. And cut the vessels distally at the superficial epigastric artery. Continue to divide the individual ventral and dorsal muscle groups approximately at the mid thigh to separate the native hind limb from the recipient animal.

Then transect the femur in the middle of the femoral shaft. Then using a 20 gauge spinal needle as a intramedullary rod and align the femur bones of the recipient and the graft in the native hind limb position. Connect the donor and recipient femur bones.

Use absorbable suture material to coapt the ventral and dorsal muscle groups. Performing this alignment accurately and quickly prevents extended blood loss from the bone cavity. To connect the femoral vessels, pull the recipient side of the vessel over the polyimide cuffs.

And use a 10-0 nylon suture to fix the recipient vessel onto the cuff with a circumferential tie. When the vessel has been stabilized release the clamps. And visually verify that the cuffs are not misrotated.

Visually verify homeostasis and use electrocautery to stop any source of bleeding. Then close the skin with non-absorbable nylon sutures. And monitor the animal until it is fully recovered.

Performing vascularized composite allotransplantation in a mouse model using this non-suture cuff technique facilitates the achievement of excellent and long-term graft and animal survival. Moreover, this technique represents a reliable method for obtaining reproduceable outcomes of gradual allograft rejection and a vascularized composite allotransplantation model as documented by the images. Indeed, histological analysis of tissues obtained from animals undergoing rejection further underscores the reproducible dynamics of allograft rejection in this murine model.

Once mastered, this technique can be completed in 90 to 120 minutes, if it is performed properly. While attempting this procedure it's important to remember to pay the utmost attention to the handling of the tissue and to avoid any sort of bleeding. After its development, this technique can pave the way for researchers in the field of reconstructive transplantation.

To explore basic mechanistic questions related to the alloimmunity of vascularized composite allografts. After watching this video, you should have a good understanding of how to perform all the critical steps of an Orthotopic Hind Limb transplantation in the mouse.