Reduced Hypoxia-Related Genes in Porcine Limbs in Ex Vivo Hypothermic Perfusion Versus Cold Storage

Ischemia-reperfusion injury remains the major limiting factor for limb replantation and transplantation. Static cold storage (SCS) on ice currently represents the standard mode of preservation but is limited to 6 h of duration. Ex vivo machine perfusion has evolved as a potential alternative to safely extend the duration of ex vivo preservation by providing continuous supply of oxygen and nutrients. This study aims to evaluate underlying molecular mechanisms of both preservation modalities. We assessed molecular changes in amputated porcine forelimbs stored on ice at 4°C for 2 h (n = 2) and limbs perfused with Perfadex solution at 10°C for 2 h (n = 3) or 12 h (n = 3) before replantation. Muscle biopsies were examined for histological changes and gene expression levels using H&E staining and a hypoxia-related PCR gene array, respectively. Histology revealed only minor differences between the ice (SCS) and perfusion groups after 2 h of preservation, with decreased muscle fiber disruption in the perfusion groups compared with the ice (SCS) group. Perfused limbs demonstrated downregulation of genes coding for glycolytic pathways and glucose transporters after 2 h and 12 h when compared with SCS after 2 h. Similarly, genes that induce angiogenesis and those that are activated on DNA damage were downregulated in both perfusion groups as compared with SCS. Perfusion of porcine limbs resulted in less activation of hypoxia-related gene families when compared with SCS. This may indicate a state more closely resembling physiological conditions during perfusion and potentially limiting ischemic injury. Our study confirms ex vivo perfusion for up to 12 h as a viable alternative for preservation of vascularized composite tissues.