Background
Limb allotransplantation is largely limited by the allowable ischemia time in limbs Muscle makes up most of the volume of isolated limbs, and its maximum allowable ischemia time is currently in the order of 4–6–h Extracorporeal perfusion devices may support safe extension of the allowable ischemia time for limb tissues The aim of this study was to assess the effect of different storage modalities on gene expression in porcine forelimbs.
Methods
Porcine forelimbs were amputated and either perfused with oxygenated acellular supplemented perfusion solution (Perfadex®) for 12 h at 10°C (treatment; n = 3) or stored on ice slurry at 4°C (control; n = 2) before replantation Gene expression profiles in muscle tissues from the amputated limb were analyzed after 2 h on ice as well as after 2–h and 12–h perfusion using a hypoxia gene expression qPCR array of 84 selected genes.
Results
At the 2 h time point muscle tissues from the perfusion group showed significant (at least 2-fold) up-regulation of 12 genes and down-regulation of 20 genes compared with muscle tissues from the 2 h ice storage group Up-regulated genes were part of the inflammatory pathway, whereas down-regulated genes belonged to glycolytic and glucose transport pathways, as well as angiogenesis and cell damage pathways Eight of the genes upregulated in the perfusion group at 2 h remained upregulated after 12 h of perfusion In contrast, the profiles of downregulated genes changed after 12 h of perfusion with regards to 2 h perfusion, such that there was further downregulation in expression of genes pertaining to glycolytic, angiogenesis and cell damage pathways.
Conclusion
Muscle tissues in limbs stored for up to 12 h using our extracorporeal perfusion device showed a significantly reduced expression of genes related to glycolytic pathways and glucose transportation This suggests that muscle perfusion may protect muscle in the limbs from non-oxidative glucose metabolism when compared with storage on ice In addition, the perfusion group showed decreased gene expression of angiogenetic pathway genes, which may indicate improved tissue oxygenation in the perfusion group This is concordant with findings of reduced tissue damage and expression of genes related to cellular damage processes.