Heterotopic Hindlimb-to-Neck Transplantation in the Mouse: A Supermicrosurgical Model of Vascularized Composite Allotransplantation
April 10th, 2026
We present a reproducible mouse model of heterotopic hindlimb-to-neck vascularized composite allotransplantation. This supermicrosurgical protocol, involving 0.3-0.5 mm vessel anastomoses, integrates nylon filament guidance and venous cuffing. It provides a reliable platform for translational studies on tolerance, ischemia-reperfusion injury, and immunological mechanisms in vascularized composite allotransplantation.
This study presents a supermicrosurgical mouse hindlimb-to-neck vascularized composite allotransplantation model to study immune mechanisms and graft injury. Currently, murine VCA protocols are scarce and difficult to reproduce. This detailed video protocol enhances consistency and reduces technical failures.
To begin, make a quadrangular skin incision in an anesthetized mouse just proximal to the knee joint. Elevate the cutaneous flap beginning from its perforator, including the superficial inferior epigastric artery and vein, and skeletonize as needed. Transect the inguinal ligament cranially and proceed with dissection caudally.
Ensure that branches that are exposed are coagulated with bipolar cautery. Dissect the surrounding adipose tissue to achieve full exposure of the external iliac vessels extending to the level of the common iliac vessels. Dissect the surrounding tissues of the external and common iliac arteries using smaller monopolar cautery.
Flex the knee joint to identify its location and open it using bipolar cautery. Extend the knee joint again and transect the biceps femoris, quadriceps femoris, and other associated muscles with bipolar cautery while coagulating. Next, carefully coagulate any remaining branches arising from the external and common iliac vessels as well as the surrounding muscular tissue, and dissect with bipolar cautery while maintaining hemostasis.
Perforate the membrane between the artery and vein at a point distant from the graft. Then hook an ophthalmic retractor onto the artery. Then carefully separate the vein and artery as distally as possible using curved Potts scissors.
Ligate the artery with 8-0 prolene suture and place a stay suture on the artery as a marker to prevent twisting during vascular anastomosis. Next, ligate the vein with 8-0 prolene. Place stay sutures at two sites on the vein to facilitate its passage through the cuff.
Insert a 4-0 Nylon into the lumen to facilitate placement of the stay sutures as the vein is very thin, and the vessel wall can occasionally be difficult to distinguish. Pass the vein through the cuff using stay sutures and clamp the cuff with a vascular clip. Evert the vessel wall and secure it to the cuff with 9-0 Nylon.
After restoring the cuff, obtain the partial hindlimb allograft. Make a 10-millimeter wide circular skin incision in the cervical region of an anesthetized mouse. Insert the scissors caudally through the incision and gently open them to dissect the subcutaneous tissue of the chest, thereby creating a pocket for graft placement.
Expose the external jugular vein by cauterizing and dividing its branches with a larger monopolar cautery. Identify and carefully dissect the common carotid artery. Perform an end-to-end arterial anastomosis with 11-0 or 12-0 Nylon using six interrupted sutures.
Begin with four anterior stitches, flip the artery, then complete the posterior wall. Insert 5-0 Nylon into both vessels, and use it as a stent. Insert the cuff into the external jugular vein and secure it with 9-0 Nylon, taking care to avoid twisting of the pedicle.
Finally, position the graft carefully in the subcutaneous chest pocket, avoiding displacement of the trachea or restriction of forelimb movement, and ensure the perforator is untwisted. Close the skin with 6-0 Monofilament Nylon sutures. The graft was assessed daily after the procedure.
On the day of surgery, the graft showed good perfusion. By postoperative day seven, erythema had resolved and the skin color had stabilized. The skin paddle remained viable and showed visible hair regrowth by postoperative day 14.
A total of 13 animals completed the study following successful postoperative recovery, and the median total operative time was around 169 minutes. One case of graft infection occurred, which was associated with subsequent graft necrosis. Four cases of partial necrosis of the skin paddle were observed, two of which required a single debridement and resuturing.
One case of wound dehiscence occurred, requiring resuturing three times before acute rejection developed. This protocol enables the study of immune tolerance, rejection, ischemia-reperfusion injury, and graft survival in mice. Precise supermicrosurgical technique and careful handling of the pedicle are essential for successful graft perfusion.
Future studies can optimize tolerance induction strategies and translate findings toward clinically-applicable immunomodulatory therapies.
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Overview
This article presents a detailed protocol for supermicrosurgical mouse hindlimb-to-neck vascularized composite allotransplantation (VCA), enabling the study of immune mechanisms, graft injury, and transplantation outcomes. The protocol emphasizes reproducibility and technical precision, providing stepwise guidance and practical tips to improve surgical success and minimize complications in murine VCA models.
Key Study Components
Area of Science
- Transplantation biology
- Immunology
- Microsurgery
- Preclinical animal models
Background
- VCA offers reconstructive options for severe tissue loss, such as facial and hand injuries.
- Clinical application is limited by surgical complexity and the need for long-term immunosuppression.
- Preclinical models are essential for refining surgical techniques and testing immunomodulatory strategies.
- Mouse models provide advantages in cost, accessibility, and compatibility with advanced immunological assays.
Purpose of Study
- To establish a reproducible, stepwise protocol for mouse hindlimb-to-neck VCA.
- To provide technical tips and highlight common pitfalls in supermicrosurgical procedures.
- To enable the study of immune tolerance, rejection, ischemia/reperfusion injury, and graft survival in mice.
Methods Used
- Quadrangular skin incision and elevation of the cutaneous flap in donor mice.
- Dissection and skeletonization of external iliac vessels, with careful coagulation of branches.
- Separation and ligation of artery and vein, with insertion of nylon filament to stabilize anastomosis.
- End-to-end arterial anastomosis and cuff technique for venous anastomosis in the recipient's neck region.
- Postoperative monitoring of graft perfusion, viability, and complications.
Main Results
- Thirteen animals completed the study with a median operative time of 169 minutes.
- Most grafts showed good perfusion and viability, with visible hair regrowth by day 14.
- Complications included one graft infection with necrosis, four cases of partial skin paddle necrosis, and one wound dehiscence.
- The protocol enabled consistent study of immune and graft injury mechanisms.
Conclusions
- This protocol provides a reliable and reproducible murine VCA model for translational research.
- Technical precision and careful handling are critical for successful outcomes.
- The model supports investigation of immune tolerance, rejection, and ischemia/reperfusion injury, and can facilitate the development of novel immunomodulatory therapies.
