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

创伤性截肢和面部或手部等功能性身体部位的严重破坏是极其改变生活的事件，主要影响年轻患者。断肢再植是可能的，在选定的创伤中心在短期内受伤后。如果不成功或在面部或手部严重破坏的情况下，同种异体移植（“血管化复合同种异体移植”，VCA）是越来越受欢迎的治疗选择，与假体置换相比，其实现了上级结果。然而，通过缺血诱导的毒性代谢物积累导致缺血-再灌注损伤的组织损伤是再次移植和移植的限制因素。使用无细胞溶液或微泡对截肢或移植物进行离体灌注在减少缺氧组织损伤方面显示出有希望的结果。到目前为止，离体灌注对功能结果和移植排斥反应的长期影响还没有被调查，提出的研究的目的是调查使用微泡或无细胞溶液的离体灌注对功能结果的影响，以及在设置VCA和自体再植的移植排斥反应。通过延长组织缺血耐受时间，可以显著提高这些治疗方法的临床成功率。刘易斯或Brown Norway大鼠将作为后肢供体。取出后，将根据研究组，用两种不同浓度的微泡或无细胞溶液灌注后肢12小时。随后，将移植同基因（BN/BN）或同种异体（刘易斯/BN）后肢。将后肢储存在冷水浴中，立即重新移植或后肢神经单独手术横断并随后接合作为对照。术后随访时间为16周。结果的评价包括临床步态分析和移植排斥反应的监测，缺血性组织损伤的组织学评估，排斥反应以及神经再生，免疫组织化学和免疫荧光。此外，将进行基因表达的qRT-PCR分析。

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.