Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

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In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

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Broader clinical application of reconstructive hand and face transplantation is hindered by the need for lifelong immunosuppression for allograft maintenance. In this review, we summarize various cell-based approaches to tolerance induction currently under investigation in both clinical and pre-clinical models to alleviate the need for chronic immunosuppression. These include strategies to induce mixed hematopoietic chimerism, therapy with T and B regulatory cells, regulatory macrophages, tolerogenic dendritic cells, and mesenchymal stem cells. The vascularized, intragraft bone components inherent to reconstructive transplants serve as a continuous source of donor-derived hematopoietic cells, and make hand and face transplants uniquely well suited for cell-based approaches to tolerance that may ultimately tilt the risk-benefit balance for these life-changing, but not life-saving, procedures.

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Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models. Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

Background:Congenital and acquired chest wall deformities represent a significant challenge to functional reconstruction and may impact feasibility of heart transplantation for patients with end-stage organ failure. In the recent past, the concept of replacing like-with-like tissue by using vascularized composite allografts (VCA) has been enthusiastically employed for reconstruction of complex tissue defects.Methods:In this study, we introduce a novel murine model for en bloc chest wall, heart, and thymus transplantation and thereby the use of complex tissue allografts for reconstruction of both chest wall defects and also end-stage organ failure. Additionally, this model allows us to study the features of combined vascularized bone marrow (VBM), thymus, and heart transplantation on allograft survival and function. Heterotopic chest wall, thymus, and heart transplants were performed in untreated syngeneic and allogeneic combinations and in allogeneic combinations treated with costimulation blockade (CTLA4-Ig and MR-1).Results:Indefinite (ie, 150 d, N = 3) graft survival was observed in syngeneic controls. In untreated recipients of allogeneic grafts, the skin component was rejected after 10 (±1) days, whereas rejection of the heart occurred after 13 (± 1) days (N = 3). Costimulation blockade treatment prolonged survival of the heart and chest wall component (130 d, N = 3) as well as the VBM niche as evidenced by donor-specific chimerism (average: 2.35 ± 1.44%), whereas interestingly, the skin component was rejected after 13 (±1) days.Conclusion:Thus, this novel microsurgical model of VCA combined with solid organ transplantation is technically feasible and results in split tolerance when treated with costimulatory blockade.