Effect Of ROCK Inhibitor-Fasudil On The Spinal Cord Regeneration In Rat After SCI

Objective To observe the effect of ROCK inhibitor and RhoA silencing on the Spinal Cord Regeneration in rat after Spinal Cord injury(SCI), and determine whether the ROCK inhibitor RhoA gene silencing in neural stem cells can optimized neural stem cells cultures.Methods In vitro experiment:neural stem cells and Schwann cells were cultured by the suspension culture in vitro, and were divided into 5 groups:A group was culture cells with 0μmol/L Fasudil; B group was culture cells with 5μmol/L Fasudil;C group was culture cells with 10μmol/L Fasudil; D group was culture cells with 15μmol/L Fasudil;E group was culture cells with 20μmol/L Fasudil;F group was RhoA gene silencing group.Three days later, RT-PCR and western blot were used to assess the expression of RhoA mRNA and RhoA protein. Cellular proliferation was determined by the cell growth curve (five days) and MTT assay. The cell cycle was analyzed by flow cytometry. In vivo experiment:Sixty adult Wistar female rats weighing (200±30)g,were randomly divided into three groups, A group was single SCI group, B group was SCI group treated with ROCK inhibitor Fasudil, C group was SCI group treated with RhoA gene silencing. Atl,2,4,6,8 weeks post-injury, all animals were evaluated of the hind limb behavior with Basso, Beatlie, Bresnahan (BBB) score and inclined plane test. Four weeks post-operation, histopathology was performed. RhoA mRNA and RhoA protein expression were determined by RT-PCR and Western Blot. Eight weeks post-transplantation, horseradish peroxidase (HRP) nerve trace and Somatosensory evoked potential (SEP) testing were performed.Results In vitro experiment:three days later, neural stem cells D, E, F group A, B, C group compared with RhoA gene and protein expression was significantly lower (P <0.05), cell growth rate significantly increased, cell cycle G0/G1 reduce, S phase cells increased. There were significant differences among the groups (P<0.05). E group compared with the F group showed no significant difference (P> 0.05).3 days after treatment of Schwann cells D, E, F group A, B, C group compared with RhoA gene and protein expression was significantly lower (P<0.05), cell growth rate significantly increased, cell cycle G0/G1 phase reduced, S phase cells increased. There were significant differences among the groups (P<0.05). E group compared with the F gene and protein expression of RhoA were significantly different (P<0.05). In vivo experiment:The outcome of C group surpasses the B group's. There was significant difference between B and C group (P<0.05).The A group's outcome were similar to B and C, but it's degree was smaller than the others'.There was significant difference between B,C and A group after 6 weeks (P<0.05). The A group was reported to have no neuroaxonal in the part of lesions but the B group and C group was reported to have little. RhoA mRNA and RhoA protein expression in B and C group were lower than in A group (P<0.05), and B group was lower than in C group (P<0.05). HRP-labeled neurofibra were found in spinal cord of group B, some in group C but little in group A(P<0.05). Somatosensory evoked potential (SEP) testing was B and C group surpass the A group's (P<0,05),and There was significant difference between B and C group (P<0.05)Conclusion Neural stem cells in vitro to be given Rho kinase inhibitor fasudil and RNAi-mediated RhoA gene silencing can promote the proliferation of neural stem cells. However, given fasudil treatment and RNA interference treatment there is no statistical difference.After spinal cord injury in rats, given Rho-kinase inhibitors, fasudil or RNAi-mediated RhoA gene silencing can promote the injured spinal cord nerve function recovery.8 weeks after injury, fasudil treatment group, the degree of recovery is better than RhoA gene silencing group.