In-depth assessment of ischemia/reperfusion injury in a murine model of hindlimb transplantation
Franka Messner1, Kentaro Kadono2, Yinan Guo1, Byoung C. Oh1, Jerzy Kupiec-Weglinski2, Gerald Brandacher1.
1Department of Plastic and Reconstructive Surgery, VCA Laboratory, Johns Hopkins University School of Medicine, Baltimore, MD, United States; 2Dumont-UCLA Transplantation Center, Department of Surgery, Division of Liver and Pancreas Transplant, David Geffen School of Medicine at UCLA, Los Angeles, CA, United States
Background: Static cold storage (SCS) still represents the gold standard for organ and tissue preservation in transplantation. Ischemia/reperfusion injury that occurs due to the lack of oxygen and nutrient supply and subsequent generation of oxygen radicals contributes crucially to the 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/6àC57BL/6) and allogeneic (Balb/càC57BL/6) orthotopic hindlimb transplants. Hindlimb grafts were subjected to 6, 12, or 24h of SCS. In the allogeneic setting, animals received rapamycin 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 did correlate with ischemic time as well as the number of apoptotic cells. Compared to skin, muscle tissue 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 animals. In 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, that 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.
Conclusion: Ischemia/reperfusion injury still poses a major obstacle in transplantation substantially limiting the time between tissue procurement and implantation. The current study demonstrates the susceptibility of muscle tissue and displays different dynamics of RNA expression depending SCS duration and MHC barrier.
This study was supported by the Department of Defense (Award Number W81XW1810794)..
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