*Purpose: Static cold storage (SCS) still represents the gold standard for organ and tissue preservation in transplantation. Ischemia/reperfusion injury (IRI) that occurs due to the lack of oxygen and nutrient supply and subsequent generation of oxygen radicals contributes crucially to the innate immune-driven cellular damage occurring during this time period. In this study, we investigated tissue damage at different durations of SCS in a sophisticated murine VCA model.

*Methods: 6 to 10-week-old mice were used for syngeneic (C57BL/6 to C57BL/6) and allogeneic (Balb/c to C57BL/6) orthotopic hindlimb transplants. Hindlimb grafts were subjected to 6, 12, or 24h of SCS prior to transplantation. In the allogeneic setting, animals received rapamycin (RPM; 1mg/kg i.p.) after recovery from surgery. Biopsies of the different components of the VCA graft were taken 24h after reperfusion and further analyzed by histology, qRT-PCR and ELISA.

*Results: All animals tolerated up to 24h of SCS without increased mortality. Histologic analysis revealed that the amount of cellular infiltration (macrophages/neutrophils) did correlate with ischemic time as well as the number of apoptotic cells (TUNEL assay). Compared to skin, muscle VCA tissue component was more susceptible to ischemic injury. In syngeneic animals, expression of IL-1a, IL-1b, MCP-1, CD-28, and FoxP3 in the skin increased with duration of SCS, while a trend toward decrease was seen in allogeneic transplant combination. In the muscle tissue, an increase in expression of CXCL-1, CXCL-2, MCP-1, IL-1b, IL-6, and IL-10 was seen for both, the syn- and allogeneic group, which correlated with the SCS duration. Compared to baseline, serum levels of ALT, AST, CK and creatinine were elevated, however, no clear trend toward an increase after prolonged storage was seen.

*Conclusions: Ischemia/reperfusion injury poses a major obstacle in transplantation substantially limiting the time between tissue procurement and implantation. Currently, there is no cut-off threshold for acceptable cold ischemia time in clinical VCA. This study demonstrates the susceptibility of the VCA muscle tissue to ischemic stress and displays divergent dynamic pattern of RNA expression and functional phenotype depending on SCS duration and MHC barrier.

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