Role Of Nischarin In Neural Regeneration After Spinal Cord Injury And The Underlying Mechanism

Objectives:We will determine the role of Nischarin in axonal regeneration after SCI and its potential molecular mechanism.Methods:(1) Spinal cord injury on Sprague-Dawley rats is established by Allen’s impact method.(2) Expression of nischarin is inhibited by transfection of Nis-shRNA plasmids or Nis-siRNA fragments in injured area of spinal cord.(3) The efficiency of shRNA is evaluated by the fluorescence of GFP in spinal cord.(4) The copies of Nischarin gene after injecting the shRNA plasmids or siRNA fragments are measured by real time PCR.(5) The motor function of rats was assessed by BBB scores in an open field.(6) The motor function of rats was assessed by FAS (foot stepping angles), RHI (rump height indices) and pass time in beam walking test.(7) Axonal regeneration was evaluated by injection of retrograde tracer hydroxystilbamidine (Fluoro-Gold).(8) GAP43expression was detected by western blot and immunohistochemistry.Results:(1) About80%of rats were subjected to SCI successfully and keep alive for6weeks longer.(2) The weight of rat in all the groups is not significantly different in6weeks after SCI.(3) The fluorescence of GFP confirmed that shRNA plasmid was transfected successfully into the neurons of spinal cord.(4) The gene copies of Nischarin were inhibited by shRNA or siRNA transfection.(5) During6weeks after SCI, the locomotor function of rats evaluated by BBB score were better in nischarin inhibition groups (Nis-shRNA or Nis-siRNA transfection) than control groups (Ctl-shRNA or Ctl-siRNA transfection) or untreated SCI group.(6) During6weeks after SCI, FSA was significantly less in Nischarin inhibition groups (Nis-shRNA or Nis-siRNA transfection) than control groups (Ctl-shRNA or Ctl-siRNA transfection) and SCI group, RHI was significantly higher in Nischarin inhibition groups (Nis-shRNA or Nis-siRNA transfection) than control groups (Ctl-shRNA or Ctl-siRNA transfection) and SCI group. However, the pass time in beam walking test was not significantly different among all the groups.(7) After the retrograde tracer Fluoro-Gold was injected for1week, the numbers of Fluoro-Gold positive neurons in red nucleus were increased in nischarin inhibition groups (Nis-shRNA or Nis-siRNA transfection) compared with the control groups (Ctl-shRNA or Ctl-siRNA transfection) or SCI group.(8) At the time of1week or6week after SCI, the expression of GAP43is significantly elevated in Nischarin inhibition groups (Nis-shRNA or Nis-siRNA transfection) compared with the control groups (Ctl-shRNA or Ctl-siRNA transfection) or SCI group.Conclusion:The inhibition of nischarin facilitates the motor recovery and promotes the axonal regeneration after SCI, which may be associated with increased GAP43expression.