Purpose: The safe maximum cold preservation time remains unknown in Vascularized Composite Allotransplantation (VCA), yet is of critical clinical significance. VCAs with a large muscular component, such as upper and lower limbs, may be uniquely susceptible to ischemia-reperfusion injury (IRI), an innate immune response leading to primary graft non-function, acute/chronic rejection, and graft loss. This study investigated how cold ischemia affects VCA innate immune response and survival. Methods: Syngeneic (C57BL6→C57BL6) orthotopic hindlimb transplants were performed with cold ischemia times of 1, 4 or 6h. Skin, muscle and vessel biopsies were collected at day 1, 3 and 7 to assess cellular infiltration/cytokine expression by histology/immunohistochemistry. Blood was collected for biochemical analysis.

Results: Mice receiving VCAs exposed to 6h of cold storage demonstrated significantly reduced survival, with 14/20 recipients (70%) surviving <48h. A severe reperfusion syndrome developed within 24h, with visible edema and overall poor recovery. Necropsies confirmed no evidence of technical failure contributing to death. Hematologic analysis revealed massive elevations in serum creatinine, BUN and creatine kinase, as compared to 1h cold storage, with some readings beyond the detectable range (n=3). By contrast, VCAs performed with 1 or 4h of cold storage demonstrated 100% graft and animal survival (n=20). Histology and immunohistochemistry demonstrated progressive increase in CD3⁺ and F4/80⁺ inflammatory cell infiltration in VCA's skin/muscle, and vascular endothelial disruption with increasing cold storage time. Control hindlimb grafts subjected to cold storage alone demonstrated intact vascular endothelium and insignificant inflammatory cell infiltration. Conclusion: IRI resulting from prolonged cold storage contributes to a vigorous immune response in VCAs with large muscle volumes that impacts graft and recipient survival. Systemic complications, including remote organ dysfunction, are an important consideration and these data indicate shorter cold ischemia may confer improved outcomes. Our data suggests that even moderate cold ischemia time may significantly damage the vascular endothelium, contributing to graft vasculopathy and remote organ dysfunction. As IRI has not been investigated in VCA, this animal model although technically most challenging, may be useful in guiding the clinical practice.

CITATION INFORMATION: Datta N, Devaney S, Allen A, Busuttil R, Azari K, Kupiec-Weglinski J. Prolonged Cold Ischemia Time Negatively Impacts Graft and Recipient Survival in a Murine Orthotopic Hindlimb Transplant Model. Am J Transplant. 2016;16 (suppl 3).

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