Effect of cold preservation on lymphocyte migration into peripheral nerve allografts in sheep

Lymphocyte migration into fresh and preserved peripheral nerve allografts was quantitated to assess the effect of cold preservation and freeze-thawing pretreatment on the local immunological response to nerve allografts. Out-bred ewes received multiple 1.5-cm sub cutaneous heterotopic peroneal nerve autografts, fresh allografts, and pretreated allografts, implanted within the same recipient. Lymphocyte migration was studied at 7 days by injecting autologous 111indium-labeled lymphocytes intravenously. After 3 hr of recirculation, lymphocyte migration into graft tissue was quantitated by a gamma counter (cpm/g, mean ± SEM). Lymphocyte traffic into fresh nerve allografts (21,623 ± 3783) increased an average 9.4-fold over the autograft value (2918 ± 377, P<0.04). Histologic studies illustrated a marked lymphocytic infiltrate of CD4+ and CD8+ cells and enhanced class I and II MHC expression in fresh allografts, but not in autografts. Short-term cold preservation, for 6 and 12 hr (5°C), enhanced lymphocyte entry into pretreated allograft tissue. Conversely, cold preservation for longer periods (1 and 3 weeks) dramatically reduced lymphocyte migration to values below corresponding autograft levels (783±100 and 1,252 ± 120, respectively, P<0.01). A comparable reduction in lymphocyte migration into nerve allografts was observed after freeze-thawing pretreatment (JP<0.01). Cold preservation of donor allogeneic lymphocytes inhibited their capacity to induce intradermal host lymphocyte migration, implicating passenger lymphocytes as a potential cold-sensitive allogeneic component of the nerve allograft. Assessment of the local response to ovine peripheral nerve allografts, utilizing radiolabeled autologous lymphocytes, demonstrated that cold preservation and freeze-thawing pretreatment significantly reduced lymphocyte migration into nerve allografts. The mechanism(s) of reduced lymphocyte migration may involve inactivation or death of antigen-presenting cells, including passenger lymphocytes.