Objective To investigate the effect of cold-inducible RNA binding protein (CIRP) on the viability of cryopreserved sciatic nerve and nerve regeneration after allograft.Methods Sciatic nerve segments of 15 mm from male Sprague-Dawley rats were placed in DMEM solution and pretreated with 4 ℃, 15 ℃ and 32 ℃ for 24 hours (group A, group B and group C, respectively). Fresh nerve group (group D) without pretreatment was set up. The mRNA and protein level of CIRP was detected by RT-PCR and Western blotting, respectively. The above nerves were cryopreserved in liquid nitrogen for four 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 nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) was detected by Western blotting. In addition, the above four cryopreserved groups were transplanted to the Wistar rats by allografting (groups A', B', C' and D'). Fresh nerve allograft group (E') and isograft group (F') were set up. At four-week post operation, the expression of CD4 of the nerve and plasma level of interleukin (IL)-6 and interferon (IFN)-γ were detected by immunohistochemistry and ELISA, respectively. At 20-week postoperation, the muscle compound action potential (CMAP) and motor nerve conduction velocity (MNCV) were examined by electrophysiological examination. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve was observed by electron microscopy. Results The mRNA and protein of CIRP were significantly higher in group C than in groups A and B (P<0.05). After 4 weeks of cryopreservation, compared with groups A, B and D, the viable cells increased, the expression of Bax decreased and the expression of Bcl-2 increased in group C (P<0.05). The expression of NGF and GDNF increased in group C than in groups A and B (P<0.05). At four-week postoperation, the expression of CD4 and plasma concentration of IL-6 and IFN-γ significantly decreased in group C' than in group E' (P<0.05), however, no significant difference was found in group C' compared with groups D' and F' (P>0.05). At 20-week postoperation, CMAP, MNCV, the number of axon, and thickness of myelin sheath were significantly better in group C' than in groups A', B', D' and E' (P<0.05). Compared with groups A', B', D' and E', the myelinated nerve fibers were more, the fiber thickness was more uniform, the fiber distribution was wider, and the myelin sheath was thicker in groups C' and F'.Conclusion CIRP was induced at 32 ℃ in the sciatic nerve, which exerted a significant protective effect on the viability of the nerves during cryopreservation, and promoted nerve regeneration and functional recovery after transplantation.
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