ABSTRACT
ObjectiveTo observe the effect of ginsenoside Rg1 （G-Rg1） on the biological activity of cryopreserved Schwann cells （SCs） of the rat sciatic nerve and explore the feasibility of G-Rg1 in reducing the cryopreservation-induced injury in SCs. MethodBilateral sciatic nerves of SD rats were randomly divided into a fresh group， a blank group， and five G-Rg1 groups of different doses （1×10-7， 1×10-6， 1×10-5， 1×10-4， and 1×10-3 mol·L-1）. The nerves in the blank group and the G-Rg1 groups were preserved in liquid nitrogen solutions containing 0， 1×10-7， 1×10-6， 1×10-5， 1×10-4， and 1×10-3 mol·L-1 G-Rg1 for four weeks. The apoptosis of SCs was detected by TdT-mediated dUTP-biotin nick end labeling （TUNEL）/S100 immunofluorescence staining. The expression of cysteinyl aspartate-specific protease （Caspase）-9， Caspase-3， major histocompatibility complex （MHC）-Ⅰ， and MHC-Ⅱ was detected by Western blot. Subsequently， all nerves were cultured in the incubator at 37 ℃ with 5% CO2 for 7 days. The expression of glial cell line-derived neurotrophic factor （GDNF） and nerve growth factor （NGF） was detected by Western blot. In addition， the above cryopreserved nerves in the blank group and the 1×10-6， 1×10-5， and 1×10-4 mol·L-1 G-Rg1 groups were transplanted to the Wistar rats by allografting （blank transplantation group and the 1×10-6， 1×10-5， and 1×10-4 mol·L-1 G-Rg1 transplantation groups）， and fresh sciatic nerve allograft and isograft control group were set up. Sixteen weeks after transplantation， compound muscle action potential （CMAP） and nerve conduction velocity （NCV） were measured by electrophysiology. Nerve filament （NF）200 immunofluorescence staining， transmission electron microscopy， and toluidine blue staining were used to analyze the histology of the regenerated nerves. ResultCompared with the fresh group， the blank group and the G-Rg1 groups showed increased expression of Caspase-9， Caspase-3， and the apoptosis of SCs （P<0.05，P<0.01） and decreased expression of GDNF， NGF， MHC-Ⅰ， and MHC-Ⅱ （P<0.01）. Compared with the results in the blank group， the expression of Caspase-9 and Caspase-3 decreased in the 1×10-7， 1×10-6， 1×10-5，1×10-4 mol·L-1 G-Rg1 groups （P<0.01）， and the apoptosis of SCs was reduced in the 1×10-7-1×10-4 mol·L-1 G-Rg1 groups（P<0.05，P<0.01） and increased in the 1×10-3 mol·L-1 group （P<0.05）， while the expression of GDNF and NGF increased in the 1×10-7， 1×10-6， 1×10-5，1×10-4 mol·L-1 G-Rg1 groups and decreased in the 1×10-3 mol·L-1 group （P<0.05）. There was no statistical significance in the expression of MHC-Ⅰ and MHC-Ⅱ between the blank group and the G-Rg1 groups. Compared with the 1×10-7 mol·L-1 and 1×10-3 mol·L-1 G-Rg1 groups， the 1×10-6 1×10-5， 1×10-4 mol·L-1 G-Rg1 groups showed decreased expression of Caspase-3 and the apoptosis of SCs （P<0.05，P<0.01） and increased expression of GDNF and NGF （P<0.05，P<0.01）. There was no statistical significance in MHC-Ⅰ and MHC-Ⅱ expression among G-Rg1 groups. Sixteen weeks after transplantation， compared with the isograft group， the blank transplantation group and the G-Rg1 transplantation groups showed decreased CMAP， NCV， myelin sheath thickness， and number of myelinated nerve fibers （P<0.01）， and the 1×10-6 and 1×10-4 mol·L-1 G-Rg1 transplantation groups showed decreased NF200 （P<0.01）. Compared with the allograft group， the blank transplantation group and the G-Rg1 transplantation groups showed increased CMAP， NCV， NF200， myelin sheath thickness， and number of myelinated nerve fibers （P<0.05，P<0.01）. Compared with the blank transplantation group， the G-Rg1 transplantation groups showed increased CMAP， NCV， NF200， myelin sheath thickness， and number of myelinated nerve fibers （P<0.05，P<0.01）. Among all groups of G-Rg1 transplantation， each index of the 1×10-5 mol·L-1 G-Rg1 transplantation group was superior to that of the 1×10-4 and 1×10-6 mol·L-1 G-Rg1 transplantation group （P<0.05）. ConclusionG-Rg1 at a certain centration can maintain the biological activity of cryopreserved SCs of rat sciatic nerve， alleviate the cryopreservation-induced injury of rat sciatic nerve， and promote nerve regeneration after allograft.
ABSTRACT
Objective:To investigate the effect of endogenous neurotrophic factor (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 ℃ with 5% CO2 (groups A, B, C, D and E) respectively. 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)-I and MHC-II was detected by Western blotting. The above six groups were cryopreserved in liquid nitrogen for four weeks. The living cells and dead cells of the nerves after cryopreservation were observed by Calcein-AM/propidium iodide staining. In addition, the above six cryopreserved groups and another fresh nerve group (group G) were transplanted to the Wistar rats by allografting (groups A', B', C', D', E', F' and G'). Isograft group (group H') was set up. One week after transplantation, the expression of CD8+ T cells and macrophages of the graft were observed by immunofluorescence staining, and the plasma levels of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α were detected by ELISA. Twenty weeks after transplantation, 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 protein (NF) 200 of the transplanted nerves was observed by immunofluorescence staining. The number of myelinated nerve fibers was analyzed by toluidine blue staining. The thickness of myelinated was analyzed by electron microscopy. Results:Compared with group F, the protein expression of GDNF and NGF increased in groups C, D and E (P < 0.05); the protein expression of Bcl-2 reduced and the protein expression of Bax and Caspase-3 increased in groups B, C, D, and E (P < 0.05); the protein expression of MHC-I and MHC-II decreased in all the pretreated groups (P < 0.05). Four weeks after cryopreservation, compared with groups F and G, the number of living cells decreased in groups C, D and E. One week after transplantation, compared with groups F' and G', the expression of CD8+ T cells and macrophages decreased, and the plasma concentration of IL-2 and TNF-α decreased in groups C', D' and E' (P<0.05). Twenty weeks after transplantation, CMAP, NCV, the wet weight ratio of gastrocnemius muscle, the number of axon and thickness of myelin sheath were better in groups C', D' and E' than in groups F' and G' (P<0.05), as well as the expression of NF200. Conclusion:ENTFs can be induced by pretreating the sciatic nerve in vitro. Cryopreserved sciatic nerve with high expression of ENTFs could promote nerve regeneration and functional recovery after allograft.
ABSTRACT
In order to investigate the protective effect of tanshinone ⅡA sulfonate on the sciatic nerve activty in rats after cryopreservation as well as the nerve regeneration and functional recovery after allograft and its possible mechanism, Sprague-Dawley (SD) rats were divded into four groups at different doses of tanshinone ⅡA sulfonate (A 0 mg·L⁻¹, B 80 mg·L⁻¹, C 160 mg·L⁻¹, D 480 mg·L⁻¹) cryopreserved at -80 °C for 24 weeks. Fresh control group nerve segments were harvested without cryopreservation. The ultrastructure and the viable cells of the nerve segments after cryopreservation were observed by electron microscopy, calcein-AM/propidium iodide staining, respectively. The expression of Bax and Bcl-2 was detected by Western blot. After cryopreservation, the nerve segments were cultured in vitro for one week, the mRNA and protein level of NGF and GDNF were detected by PCR and Western blot respectively. In addition, the above four cryopreserved groups transplanted to the Wistar rats by allografting (A', B', C', D'). At 16-week postoperation, muscle compound action potential latency and nerve conduction velocity were examined by electrophysiological. The number and the thickness of myelinated nerve fibers were analyzed by toluidine blue staining. The ultrastructure of the sciatic nerve by electron microscopy was observed. According to the results, after the cryopreserved for 24 weeks, compared with groups A and B, the nerve demyelination and vacuolation were weak, and the more viable cells, the decreased Bax and increased Bcl-2, the increased NGF and GDNF in group C and D. At 16-week poseoperation, the results demonstrated that the more larger and thickly regenerated myelinated axons, the shorter latency of muscle compound action potentials and higher nerve conduction velocity in groups C' and D' compared with groups A' and B'. According to these results, tanshinone ⅡA sulfonate exerted a significant protective effect on the viability of the nerves during cryopreservation at -80 °C and promoted nerve regeneration and functional recovery after transplantation especially in middle- and high-dose of tanshinone ⅡA sulfonate.