Effects Of Cold-inducible RNA Binding Protein On Cryopreserved Sciatic Nerve Regeneration After Allograft

ObjectiveTo investigate the effect of cold-inducible RNA binding protein(CIRP)on the viability of cryopreserved sciatic nerve and nerve regeneration after allograft.MethodsSciatic nerve segments of 15 mm were placed from male Sprague-Dawley rats in DMEM solution and pretreated with 4?,15? and 32? for 24 h(group A,group B and group C)respectively.Fresh nerve group(group D)without pretreatment.The mRNA and protein level of CIRP was detected by Real-time PCR and Western blotting,respectively.The above nerves were cryopreserved in liquid nitrogen for 4 weeks.The viable cells of the nerve segments after cryopreservation were observed by calcein-AM/propidium iodide staining.The expression of Bax and Bcl-2 was detected by Western blotting.After cryopreservation,the nerve segments were cultured in vitro for one week,the protein level of NGF and GDNF were detected by Western blotting.In addition,the above four cryopreserved groups transplanted to the Wistar rats by allografting(A',B',C',D').Fresh nerve allograft group(E')and isograft group(F')were set up.At 4-week postoperation,the expression of CD4 of the nerve and plasma level of IL-6 and IFN-? were detected by immunohistochemistry and ELISA,respectively.At 20-week postoperation,muscle compound action potential(CMAP)and motor nerve conduction velocity(MNCV)were examined by electrophysiology.The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining.The ultrastructure of the sciatic nerve by electron microscopy were observed.ResultsThe mRNA and protein of CIRP in group C were significantly higher than those in groups A and B(P<0.05).After 4 weeks of cryopreservation,compared with groups A,B and D,the more viable cells,the decreased Bax and increased Bcl-2,the increased NGF and GDNF in group C were observed.At 4-week postoperation,the expression of CD4 and plasma concentration of IL-6 and IFN-? were significantly decreased in C' group compared with E' group(P<0.05),but there was no significance when compared with D' and F' group(P>0.05).At 20-week postoperation,CMAP,MNCV,the number of axon and thickness of myelin sheath in C' group were significantly better than those in A',B',D' and E 'groups(P <0.05).Compared with A',B',D' and E' group,the more myelinated nerve fibers,the uniform fiber thickness,the wide fiber distribution and the higher thickness of myelin sheath in C' and F' groups were observed.ConclusionCIRP was induced at 32?in the sciatic nerve.It exerted a significant protective effect on the viability of the nerves during cryopreservation and promote nerve regeneration and functional recovery after transplantation.