D81. Gene Expression as Biomarkers of Tissue Viability in Ex Vivo Normothermic Limb Perfusion (EVNLP)

PURPOSE: Muscle viability remains the primary focus of ex vivo limb preservation. We hypothesized that genes associated with tissue injury pathophysiology are differentially expressed during EVNLP and static cold storage (SCS). METHODS: Bilateral porcine forelimbs were procured and randomly allocated to EVNLP or SCS. Skeletal muscle specimens were collected before limb procurement and during EVNLP or SCS at 0, 12, and 24h. Analysis of differential gene expression was performed with reverse transcriptase PCR to obtain fold changes. Pathway analysis was performed using Panther Classification System.The dream method from R package variance Partition (version 1.24.0) was used for differential gene expression analysis. Plots were made using log2-transformed normalized counts per million mapped reads. Statistical analyses were conducted using Bonferroni correction; the critical P value (α) was determined by the number of comparisons. RESULTS: A significantly different gene expression pattern, between EVNLP and SCS, was detected in pathways involved in inflammation, response to hypoxia, apoptosis, muscle and tissue regulation, coagulation, and other cellular signaling. At 12h, genes related to inflammation were transcribed at a higher rate in the EVNLP group, aside from macrophage inhibiting factor (MIF) which was downregulated at 12h and EVNLP endpoint. IL-10 was downregulated in both groups.Genes in pathways for coagulation, plasminogen activating cascade, presenilin pathway, cholecystokinin receptor signaling, and nicotine degradation were differentially expressed in SCS and EVNLP limbs at 12h. CONCLUSION: We identified genes differentially expressed in EVNLP compared to SCS. Increased transcription during EVNLP aligned with preserved metabolic activity.