Effects Of Endogenous Neurotrophic Factors On Nerve Regeneration After Cryopreserved Sciatic Nerve Allograft In Rats

Objective:To investigate the effect of endogenous neurotrophic factors?ENTFs?on nerve regeneration after cryopreserved sciatic nerve allograft in rats.Methods:The 15-mm sciatic nerves from female Sprague-Dawley rats were placed in DMEM solution and pretreated in vitro for 1 day,3 days,7 days,14 days,and 21 days at 37°C with 5%CO₂?group A,group B,group C,group D and group E?respectively.The fresh nerve group?group F?was set up.The protein expression of glial cell line-derived neurotrophic factor?GDNF?,nerve growth factor?NGF?,Bcl-2,Bax,Caspase-3,major histocompatibility complex?MHC?-?and MHC-?was detected by Western Blot.The pretreated different time sciatic nerves and the fresh sciatic nerves were cryopreserved in liquid nitrogen for 4 weeks.The living cells and dead cells of the nerves after cryopreservation were examined by Calcein-acetoxymethyl/Propidium iodide staining.After cryopreservation,these pretreated-cryopreservation sciatic nerves,the cryopreserved fresh sciatic nerves and the fresh sciatic nerves?group G?were cultured in vitro for 7 days,the protein expression of GDNF and NGF was detected by Western Blot.In addition,these pretreated-cryopreservation sciatic nerves,the cryopreserved fresh sciatic nerves and the fresh sciatic nerves were used to repair sciatic nerve 10-mm-long defects of the female Wistar rats by allografting?groups A',B',C',D',E',F'and G'?.The isograft group?group H'?was set up.One week after surgery,the invasion of CD8⁺T cells and macrophages of the grafts were observed by immunofluorescence staining,and the plasma levels of interleukin?IL?-2,interferon?IFN?-?,and tumor necrosis factor?TNF?-?of the recipients were detected by ELISA.At twenty weeks postoperation,the compound muscle action potential?CMAP?and nerve conduction velocity?NCV?were examined by electrophysiology.The wet weight ratio of gastrocnemius muscle was calculated by the operational side compared with the contralateral side.The expression of neurofilament 200?NF200?of the transplanted nerves was observed by immunofluorescence staining.The density of regenerated myelinated nerve fibers was examined by toluidine blue staining.The myelin sheath thickness of regenerated myelinated nerve fibers was examined by transmission electron microscopy.Results:The protein expression of GDNF and NGF were higher in groups C,D and E of the pretreated different time sciatic nerves than in group F?P<0.05?.Compared with group F,the protein expression of Bcl-2decreased and the protein expression of Bax and Caspase-3 increased in groups B,C,D,and E?P<0.05?.Compared with group F,the protein expression of MHC-?and MHC-?decreased in all the pretreated groups?P<0.05?.Four weeks after cryopreservation of the pretreated different time sciatic nerves and the fresh sciatic nerves,compared with groups F and G,the number of living cells decreased in groups C,D,and E.These pretreated-cryopreservation sciatic nerves,the cryopreserved fresh sciatic nerves and the fresh sciatic nerves were cultured in vitro for 7 days,compared with group G,the protein expression of GDNF decreased in groups A,B,C,D,E and F?P<0.05?,and the protein expression of NGF decreased in groups A,B,C,E and F?P<0.05?,but there was no significant difference in NGF protein expression between group D and group G?P>0.05?.Compared with group F,the protein expression of GDNF increased in groups D and E?P<0.05?,the protein expression of NGF increased in groups A,B,C and D?P<0.05?.One week after surgery,the invasion of CD8⁺T cells and macrophages of the nerve grafts,compared with groups F'and G',there were fewer CD8⁺T cells and macrophages in groups C',D'and E'.One week after surgery,the plasma levels of IL-2,IFN-?and TNF-?of the recipients,compared with groups F'and G',the plasma levels of IL-2 and TNF-?decreased in groups C',D',and E'?P<0.05?,the plasma levels of IFN-?decreased in group E'?P<0.05?.At twenty weeks postoperation,CMAP,NCV,the wet weight ratio of gastrocnemius muscle,the density and thickness of regenerated myelinated nerve fibers were significantly better in groups C',D',and E'than in groups F'and G'?P<0.05?,as well as the expression of NF200.Conclusion:In conclusion,ENTFs was induced by pretreated in vitro in the sciatic nerve.And after cryopreservation,although the number of surviving SCs of the sciatic nerves with high-expression ENTFs was decreased,they can continue to express highly ENTFs in vitro,and the immune response after allograft was weak,which promoted recipient nerve regeneration and functional recovery.