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身体功能部位的严重破坏是极端改变生活的事件，主要影响年轻患者。在受伤后的短时间内，在选定的创伤中心进行肢体再植是可能的。如果不成功或在面部或手部严重受损的情况下，同种异体移植（“血管化复合异体移植”，VCA）是一种日益流行的治疗选择，与假体置换相比，其效果更好。然而，缺血诱导的毒性代谢物积累导致的组织损伤导致缺血-再灌注损伤是再灌注和移植的限制因素。用无细胞溶液或微泡对断肢或移植物进行体外灌注，在减少缺氧组织损伤方面显示出良好的效果。迄今为止，离体灌注对功能结局和移植排斥反应的长期影响尚未得到研究。本研究的目的是研究微泡或无细胞溶液离体灌注对VCA和自体再植的功能结局和移植排斥反应的影响。通过延长组织缺血的耐受时间，这些治疗方法的临床成功率可能会显著提高。本研究采用大鼠后肢移植再植模型，分为6个实验组和5个对照组。刘易斯或布朗挪威鼠将作为后肢供体。切除后，根据研究组的不同，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，将后肢移植为同基因（BN/BN）或异基因（Lewis/BN）。后肢冷水浴保存，立即再移植或手术切除后肢神经并随后适应作为对照。术后随访16周。评估结果包括临床步态分析和移植物排斥反应监测，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.