Development of an Ex-Situ Limb Perfusion System for a Rodent Model

Ex-situ perfusion (ESP) is a promising method in preserving vascularized composite tissue allografts (VCAs) with potential to widen donor procurement to larger geographic areas. To optimize the method of preservation, we developed a small animal model to conduct biomolecular investigations. Twenty rat hind limbs (18.2 ± 1.3 g) were procured and connected to our custom-made ESP system. Perfusion pressure and flow parameters were measured with hourly blood gas analysis under near-normothermic (30-35˚C) conditions. Perfusate was prepared with swine hemoglobin (6-9 g/dL) and STEEN Solution. After 6 hours of perfusion, gastrocnemius muscles were evaluated for their histology and metabolomic profiling. Following 3 sets of experiments, perfusion was maintained at an average flow of 0.9 ± 0.24 mL/min and resulted in lactate levels of 3.78 ± 1.02 mmol/L. Metabolomic analysis revealed maintained cellular energy stores (total adenylates perfusion 0.698 ± 0.052 versus baseline 0.685 ± 0.091 umols/ug, p = 0.831), and histologic analysis revealed no evidence of barotrauma or myodegeneration. Rat hind limbs were viable after 6 hours of ESP on our miniaturized ESP system. This study is the first to document the ex-situ hind limb perfusion platform on a rodent model. These experimental findings have potential to guide future research to extend the viable duration of VCA preservation.