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Abstract
Extensive peripheral nerve injury requires reconstruction with interpositional grafts. Nerve autografting has the drawbacks of donor site morbidity and limited nerve material and nerve allografting requires systemic immunosuppression with potential significant side effects (Mackinnon 2001). Alternatives to immunosuppression include manipulation of the patient's immune system to induce tolerance or graft pretreatment to render it less antigenic (Evans 1994, Trumble 2000). Cold preservation of nerve allografts in University of "Wisconsin solution has emerged as a promising strategy for minimizing antigenicity, thereby creating an acellular scaffold that is conducive to nerve regeneration. Schwann cells are the primary antigenic target in nerve grafts (pollard 1981). Increasing duration of cold preservation diminishes the expression of Schwann cell antigens (Atchabahian 1999) and reduces Schwann cell viability (Levi 1994 ) while preserving a scaffold of Schwann cell tubes. Part I of the study investigates the effects of cold preservation on nerve antigenicity. Part II examines how these changes in antigenicity affect nerve regeneration and functional recovery. In Part I of the study, a mouse model is employed to characterize the immune response to cold preservation. Mice will be randomly assigned to 4 groups of 8 animals each and will undergo corresponding inlay sciatic nerve grafting. Peripheral blood, spleen, and nerve material will be harvested on day 10. Cellular immune responses will be quantified using the enzyme-linked immunosorbent spot assay (Elispot). This assay allows for detection of T cell sub-populations in the recipient mice spleens that have been stimulated to produce cytokines (Hutchings 1989). Both interferon gamma secretors, which represent the T helper 1 subset involved in the inflammatory response, and interleukin 4 secretors, which represent the T helper 2 subset involved in regulation of the cellular immune response, will be measured (Benichou, 1999). The humoral immune response will be evaluated by measuring for donor specific antibodies in the recipient animal's serum (Whitley 1989) using flow cytometry (Rodey2000). In Part II of the study, a rat model is used to optimize for functional and histomorphometric analysis (Dellon 1989, Mackinnon 1985). As in Part I, animals will be randomly assigned to 4 groups of 8 animals each. Rats will undergo 2.5 cm posterior tibial nerve grafting according to group. Walking track analysis, a functional assay previously described (Bain 1989), will be performed preoperatively, at 6 weeks and pre-sacrifice. At the 9-week end-point, chosen to maximize differences between groups (Calvert 2001), animals will be sacrificed and the posterior tibial nerve harvested. After tissue processing, cross sections of nerve will be analyzed for cellular infiltration, Wallerian degeneration and quality of nerve regeneration. The immunologic, functional and histomorphometric data generated in this study will provide a more complete understanding of the effects of cold preservation on nerve allografts. These findings will serve as a basis for future studies combining nerve allograft cold preservation with autologous Schwann cell injection in a large animal model. The long-term goal of this research is to provide a safe alternative to immunosuppression-dependent allografting that can be readily applied to human nerve reconstruction .