93. The Effects of Subnormothermic Plasma and Red Blood Cell Based Perfusion on the Function and Viability of Limb Allografts

Abstract

Purpose: Vascularized composite allotranplantation (VCA) is an evolving area of modern transplantation medicine to reconstruct composite tissue defects such as abdominal wall, face, and limb. One of the challenges in VCA is the narrow window of tolerable ischemia time which limits the number of available donors and imposes a single center transplantation approach. Current standard of care, static cold storage, is only effective for short periods and may result in severe tissue damage and acute rejection when prolonged. Ex-situ perfusion has the potential to overcome these challenges by extending allowable pre-transplantation ischemia time. However, optimum perfusate composition, preservation temperature, and hemodynamic parameters of perfusion are still debated. Over the past decade, the benefits of addition of fresh plasma in hemorrhagic shock resuscitation and attenuation of subsequent reperfusion injury have been recognized. Whether these observations are due to high or low molecular weight (HMW and LMW, respectively) plasma proteins require further investigation. This study investigated the effects of the addition of plasma on the viability of perfused limb allografts with prolonged ischemia time. Methods: All limb transplantations were performed orthotopically between male Lewis rats (200-250 g). In group 1, amputated limbs were transplanted immediately to determine the effects of surgical trauma. In group 2, amputated limbs were preserved at 4°C simulating the clinical standard of care. In other groups, amputated limbs were perfused for 6 hours at 20° to 22°C using one of the following perfusates groups and transplanted to another recipient (in less than 90 minutes): RBC only (G3), RBC + whole plasma (G4), RBC + LMW plasma (G5), RBC + HMW plasma (G6). At 3 months post-transplantation, surviving recipients had end-point assessment using electromyography where compound muscle action potentials (CMAP) and extensor digitorum longus (EDL) muscle force generation were compared to contralateral healthy limb. Muscle and nerve histology samples were collected at the end of EMG testing. Results: Recipients in the group perfused with RBC and HMW plasma proteins had severe reperfusion injury and did not survive beyond 3 days. Among surviving recipients, the addition of whole plasma did not appear to improve amplitude and latency on gastrocnemius and EDL muscles compared to immediately transplanted limbs. Groups perfused with RBC only and RBC with LMW plasma demonstrated excellent amplitude and latencies on both muscle groups, with no significant difference compared to immediately transplanted limbs (p>0.05). The EDL muscle in these groups also generated similar tetanic and twitch forces compared to the immediate transplantation group (p>0.05). Conclusion: Ex-situ perfusion at 20° to 22°C for 6 hours using RBC and LMW plasma appears to protect both fast and slow twitch muscles, and improve axonal regeneration and myelin sheath thickness to a degree comparable with the immediately transplanted group.