Functional Outcomes and Reinnervation of Vascularized Composite Auto- and Allotransplantations following ex-vivo Perfusion of Rat Hindlimbs for Reduction of Ischemia-Reperfusion-Injury

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Traumatic amputations and severe destruction of functional body parts like face or hands are extremely life-changing events mostly affecting young patients. Replantation of amputated limbs is possible in selected trauma centers during a short time frame after injury. If unsuccessful or in the case of severe destruction of face or hands, allogenic transplantation (“Vascularized Composite Allotransplantation”, VCA) is an increasingly popular therapeutic option achieving superior results compared to prosthetic replacement. However, tissue damage through ischemia-induced accumulation of toxic metabolites resulting in ischemia-reperfusion damage is a limiting factor for both, re- and transplantation. Ex-vivo perfusion of amputated limbs or transplants using cell-free solutions or microbubbles showed promising results in reducing hypoxic tissue damage. To date, long-term effects of ex-vivo perfusion on functional outcomes and transplant rejection have not been investigated.The aim of the proposed study is to investigate the effects of ex-vivo perfusion using microbubbles or cell-free solutions on functional outcomes as well as transplant rejection in the setting of VCA and autologous replantation. By extending the tolerable time of tissue ischemia, clinical success of these therapeutic approaches could potentially be significantly improved.For this study, we will apply a hindlimb trans- and replantation model of the rat including 6 experimental and 5 control groups. Lewis or Brown Norway rats will serve as hindlimb donors. After removal, hindlimbs will be perfused for 12 hours with two different concentrations of microbubbles or a cell-free solution, depending on the study group. Following, hindlimbs will be transplanted syngeneic (BN/BN) or allogeneic (Lewis/BN). Hindlimb storage in a cold-water bath, immediate re- and transplantation or sole surgical transection of hindlimb nerves with subsequent coaptation serve as controls. The follow-up duration is 16 weeks postoperatively. Evaluation of results includes clinical gait analysis and monitoring of graft rejection, histologic assessment of ischemic tissue damage, rejection as well as nerve regeneration, immunohistochemistry and immunofluorescence. Additionally, qRT-PCR analysis of gene expression will be performed.