| Purpose:To investigate the effects of triptolide?T10?on the cellular activity of cryopreserved rat sciatic nerves and nerve regeneration after allotransplantation.Materials and methods:Schwann cells?SCs?in the logarithmic growth phase were collected,and the effects of different concentrations of the T10 solution on the proliferation of SCs was determined using the C-terminal octapeptide of cholecystokinin?CCK-8?.Sciatic nerves from Sprague-Dawley?SD?rats were pretreated with different concentrations of the T10 solution at either 4°C or 37°C for 24 h.The levels of nerve growth factor?NGF?,glial cell line-derived neurotrophic factor?GDNF?and brain-derived neurotrophic factor?BDNF?were detected using western blotting to determine the optimal T10concentration.Sciatic nerve fragments from SD rats were randomly divided into 5 groups,namely,the fresh nerve group?group A?,Dulbecco's modified Eagle's medium?DMEM?preservation group?group B?,T10 preservation group?group C?,DMEM preservation after T10 pretreatment group?group D?,and T10 preservation after T10 pretreatment group?group E?,and preserved at 4°C for 4 w.The conditions of live and dead nerve cells after preservation were detected by flow cytometry and observed under a laser confocal microscope after calcein-AM/propidium iodide?PI?double staining.The major histocompatibilitycomplexclassI?MHC-I?,major histocompatibility complex class II?MHC-II?,and intercellular adhesion molecule-1?ICAM-1?proteins were detected using western blotting.The sciatic nerves were cultured at 37°C in a5%CO2 atmosphere for 7 d.The levels of the NGF,GDNF,BDNF proteins were detected using western blotting.The cryopreserved SD rat sciatic nerves?4 w?or fresh nerves were used to repair 10 mm sciatic nerve defects in corresponding Wistar rats?group A',group B',group C',group D',and group E'?;in addition,one fresh homologous transplantation group?group F'?was established.Sixteen weeks after transplantation,the compound muscle action potential?CMAP?and the motor nerve conduction velocity?MNCV?were determined using electrophysiology,and histological observations of the regenerating nerve in the transplanted segment and the morphology of the gastrocnemius muscle were performed.Results:At concentrations ranging from 10-9-10-77 M,the T10solution was not obviously toxic to SCs.Pretreatment of rat sciatic nerves with different concentrations of the T10 solution at 37°C exerted significantly greater effects on the expression of neurotrophic factors compared to the pretreatment at 4°C.At the same temperature,NGF was expressed at significantly higher levels in the group treated with the 10-8 M T10 solution than in groups treated with other concentrations?P<0.05?.After rat sciatic nerves were cryopreserved for 4 w,group E exhibited a greater number of live nerve cells and higher levels of NGF,GDNF,and BDNF than groups B,C,and D?P<0.05?but lower levels of MHC-I,MHC-II,and ICAM-1?P<0.05?.Sixteen weeks after transplantation,CAMP,MNCV,and the number of regenerated myelinated nerve fibers in group E'were all increased compared with groups A',B',C',and D'?P<0.05?.Based on the ultrastructure of regenerated nerves,compared with groups A',B',C',and D',groups E'and F'displayed greater numbers of myelinated nerve fibers with a uniform thickness,extensive distribution and thick myelin sheath.The cross-sectional areas and diameters of gastrocnemius muscle fibers in groups E'and F'were obviously larger than those in groups A',B',C',and D'.Conclusions:The application of certain concentrations of T10in vitro induced the expression of neurotrophic factors in rat sciatic nerves,increased the biological activity of cryopreserved nerves,reduced the immunogenicity,and promoted recipient nerve regeneration after allotransplantation.In addition,better effects were observed when rat sciatic nerves were pretreated with certain concentrations of T10 before cryopreservation. |
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