Significantly Improved Cryopreservation of Rat Hind Limb Vascularized Composite Allografts Using the New PrC-210 Free Radical Scavenger

Abstract

Background Vascularized composite allotransplantation (VCA) represents a promising reconstructive solution primarily conducted to improve quality of life. However, tissue damage caused by cold-ischemia (CI) storage prior to transplant represents a major factor limiting widespread application. This study investigates the addition of the novel free radical scavenger PrC-210 to UW Organ Preservation Solution (UW Solution) to supress CI-induced skeletal muscle injury in a rat hind limb amputation model. Methods Lewis rats received systemic perfusion of UW solution +/- PrC-210 (0mM control, 10mM, 20mM, 30mM, or 40mM), followed by bilateral transfemoral amputation. Limbs were stored in 40mL of the same perfusate at 4°C for 48 hours. Muscle punch biopsies were taken at set times over the 48 hour cold-storage period and analyzed for caspase-3,7 activity, cytochrome C levels, and qualitative histology. Results A single 15 second perfusion of PrC-210-containing UW Solution conferred a dose-dependent reduction of CI-induced muscle cell death over 48 hr. In the presence of PrC-210, muscle cell mitochondrial Cytochrome C release was equivalent to 0 hr controls, with profound reductions in the Caspase 3,7 apoptotic marker that correlated with limb histology. PrC-210 conferred complete prevention of ROS-induced mitochondrial lysis in vitro, as measured by Cytochrome C release. Conclusion The addition of 30mM PrC210 to UW Solution conffered the most consistent reduction in CI limb damage, and it warrants further investigation for clinical application in the VCA setting.