| Objective: Axonal regeneration after injury to the adult mammalian CNS is difficult in part to the inhibitory nature of CNS myelin. To date, three inhibitors have been identified in myelin: Myelin-Associated Glycoprotein (MAG), Nogo-A, and Oligodendrocyte-Myelin glycoprotein (OMgp). All these three of proteins bind to the same receptor: Nogo receptor (NgR). NgR is an axonal surface protein linked to the glycosylphosphatidylinositol (GPI) and binds to the three inhibitory factors and mediates their inhibitory signals by complexing with a signal-transducing coreceptor, the neurotrophin receptor p75NTR, LINGO-1 or TROY that results in the collapse of the growth cone. Consequently, NgR plays a pivotal role in the suppression pathways.In this study, in order to provide a therapeutic approach to promote recovery from ischemic brain injury, we will assess the contribution of NgR to mediate myelin inhibitory signals and regeneration failure by adenovirus-mediated RNAi in ischemia-reperfusion model of rats. Methods: 1.The recombinated adenovious was amplified in 293 cells(human embroyonic kidney cells)and purified by Sartorius Vivapure AdenoPACK 20 kit .The virus titer was determined by TCID50;2. Three different titers of recombinated adenovious(high titer,middle titer,low titer) and adenovious preservation solution(control group)were used to transfect the brain tissue. Histopathological examination was performed to evaluate the local inflammatory reaction and green fluorescent protein (GFP) expression was observed under fluorescence microscope. The optimal transfective virus titer was selected;3. Cerebral ischemia and reperfusion was induced by nylon monofilament. Forty two male SD rats were randomly divided into sham group and ischemic insult groups,adenovious injection groups,negative control groups. Reverse transcriptionpolymerases chain reaction (RT-PCR) was used to determine the changes of NgR mRNA expression at 24h and 2w after cerebral ischemia-reperfusion. Western blotting was used to detect the expression of NgR protein at the same time;4. Twenty two male SD rats were randomly divided into sham group and ischemic insult group,adenovious injection group,negative control group. Enhanced computerized tomography revealed the infarct volume at 24h and 9w.The staircase test evaluated neurofunctional recovery every other week after operation .BDA was used to trace corticospinal tract and corticorubral tract.Results:1.After transfection ,the fluorescence was observed in 293 cells.The titer of amplified recombined adenovirus Ad-shRNA-NgR and Ad-shRNA-HK were 3.16×1010pfu/ml, 2.84×1010pfu/ml respectively;2. Positive expression of transgene was found in all rat brain in the high,middle and low titer group. GFP expression in the high and middle titer group were obviously higher than those in the low titer group and the control group,while there was no obviously difference between the high and middle titer group,also between the low titer group and control group.HE staining showed that inflammatory infiltration was only observed in the high titer group and Nissl's staining showed many neurons were lost in ischemic brain tissue; 3. NgR mRNA and protein in the infarcted cortex and hippocampus were significantly increased at 24h and 2w after cerebral ischemia-reperfusion (p<0.01). NgR mRNA and protein were reduced by 40-60% in the infarcted cortex and hippocampus at 24 h and 2 w after RNAi intervention (p<0.01);4. There was no significantly difference in infarct volume between ischemic insult group,adenovious injection group and negative control group(p>0.05) ,but the behavioral outcomes and the numbers of midline crossing fibers projecting to the contralateral red nucleus were increased in adenovious injection group(p<0.01).Conclusion: Adenovirus can transfect ischemic brain tissue efficiently and low toxicity.NgR-specific shRNA gene transfer can be effectively conducted. The expression of NgR mRNA and protein in infarcted cortex and hippocampus were increased. Adenovirus-mediated RNAi can degrade the expressing level remarkably and enhance neuroanatomical plasticity, promote CNS axonal regeneration.
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