| Part IExpression of Nogo-A and NgR gene in cerebral cortex of rats after cerebral ischemia-reperfusion and The effect of Nogo-A antisence oligodeoxynucleotides1, Expression of Nogo-A and NgR mRNA and protein in rats after cerebral ischemia-reperfusionObjective To investigate the expression of a myelin-associated inhibitory molecule Nogo-A and its receptor NgR mRNA and protein in rats with cerebral ischemia-reperfusion and its significance.Methods The focal cerebral ischemia and reperfusion model was induced by nylon monofilament in rats. The rats were randomly assigned to eight groups, sham operated (control) group and groups at 6h, 12h, 24h, 48h, 72h, 7d and 14d after reperfusion following middle cerebral artery occlusion(MCAO). Histological features were identified by hematoxylin and eosin (HE) staining. RT-PCR was used to determine the change of Nogo-A and NgR mRNA expression while the immunohistochemistry technique was used to detect the Nogo-A and NgR protein in cerebral cortex both the core and the periphery of infarction.Results The expression level of Nogo-A mRNA and protein in control group were 0.801±0.029 and 0.616±0.022 respectively, in the core of the infarction it was decreased in 6h group (0.731±0.050 and 0.544±0.034, p<0.05) ,it was decreased with the time reperfusion after ischemia and weakest in 24h group(0.502±0.013 and 0.454±0.037, p<0.01). However, in the periphery of infarction, the expression of Nogo-A mRNA and protein was no change until 72h reperfusion after ischemia. It increased significantly both in 72h group (0.895±0.056 and 0.668±0.092, p<0.05) and 7d group (1.036±0.079 and 0.690±0.063, p<0.01) and declined to control level in 14d group. The expression of NgR mRNA and protein in control group were 0.916±0.024 and 0.565±0.027 respectively, it decreased in all cerebral ischemia-reperfusion groups in the core and it was lowest in 48h group(0.604±0.051 and 0.449±0.022, p<0.01), no significant change was detected in the periphery of infarction.Conclusion Increasing of The expression of Nogo-A mRNA and protein in the periphery of ischemia lasted for more than 7 days. Treatments for cerebral infarction focus this characteristic of Nogo-A expression in time window may be an effective way for axonal regeneration. The expression of NgR had not significant change suggested that the inhibitory effect of NogoA mediated not only by NgR but also by other receptor. 2, The effect of antisence oligodeoxynucleotides on expression of Nogo-A mRNA and protein in rats by intra-cerebroventricular injectionObjective To observe the effect of intra-cerebroventricular administration of antisence oligodeoxynucleotides (ASODN) on expression of Nogo-A mRNA and protein in rats.Methods Fifty three adult Wistar rats were randomly divided into three groups: group A (control), group B (received antisence oligodeoxynucleotides by intracerebroventricular injection), group C (a random sequence oligodeoxynucleotides was given by intra- cerebroventricular perfusion ). At 12h, 24h, 48h, 72h after intra-cerebroventricle injection the level of Nogo-A mRNA in cerebral cortex was detected by RT-PCR while the level of Nogo-A protein was determined by immunohistochemistry technique.Results The expression level of Nogo-A mRNA and protein in the control group were 0.832 ± 0.050 and 0.615 ± 0.022 respectively, Nogo-A mRNA was decreased at 12h (0.519 ± 0.378, p<0.01) and the protein was decreased at 24h after injection ASODN. The decreasing both of Nogo-A mRNA and protein came to a peak at 24h after injection ASODN (0.227 ± 0.305 and 0.461 ±0.025, p<0.01) and the level of Nogo-A mRNA and protein recovered at 72h.Conclusion Antisence oligodeoxynucleotides by intra-cerebroventricle injection can inhibit the expression of Nogo-A mRNA and protein. These results suggest that maybe Nogo-A antisence oligodeoxynucleotides can promote axonal regeneration after CNS injury. Part IIThe effects of Nogo-A antisence oligodeoxynucleotides on signal pathway of PKC-RhoA in rats with cerebral ischemia-reperfusionObjective To investigate the change of PKC y and RhoA gene expression after cerebral ischemia-reperfusion on rats and the effects of Nogo-A antisence oligodeoxynucleotides on signal pathway of PKC-RhoA.Methods Fourty rats were randomly assigned to eight groups, sham operated (control) group and groups at 6h, 24h, 72h, 1w and 2w after reperfusion following middle cerebral artery occlusion(MCAO) and groups at 72h, lw reperfusion with intracerebroventricular injection of Nogo-A antisence oligodeoxynucleotides. RT -PCR was used to determine the change of PKC and RhoA mRNA expression while the Western blot technique was used to detect the PKC y and RhoA protein expression in the periphery of infarction cerebral cortex.Results The expression level of PKCy and RhoA mRNA protein in control group were 0.768±0.067 and 0.852±0.045 respectively in the periphery of infarction. It was increased at 6h (1.030±0.072, 1.10±0.087, p<0.01) and returned to control group at 24h (p>0.05) after reperfusion. However, the expression of PKCy and RhoA mRNA elevated again at 72h reperfusion after ischemia ( 1.080±0.089 , 1.25±0.091 ) .The increasing of PKCγ mRNA expression was highest at lw (1.216±0.094, p<0.01) . The expression of RhoA mRNA have not difference between 3d and 1w. Both PKCγ and RhoA mRNA returned to control at 2w and decreased significantly at 72h (0.870±0.091, 0.947±0.085, P>0.05 ) and lw (1.216±0.094, 0.984±0.094, p<0.05 ) after reperfusion with intracerebroventricular injection of antisence oligodeoxynucleotides than the ischemia-reperfusion only groups. Moreover, the expression of PKCγ and RhoA membrane protein were increased at 72h and 1w after reperfusion and intracerebroventricular injection of Nogo-A antisence oligodeoxynucleotides can block this increasing.Conclusion The expression of PKCy and RhoA gene have two peaks and the second increasing was coincidence with the change of Nogo-A after cerebral ischemia-reperfusion. Intracerebroventricular injection of Nogo-A antisence oligodeoxynucleotides can inhibit this increasing. It suggests that Nogo-A involved in the activation of PKC/RhoA signal pathway.Part IIIThe effects of NGF and 8-B-cAMP on cAMP-PKA signal pathway and p75NTR protein expression on cerebral ischemia-reperfusion ratsObjective To investigate the effect of intra-cerebroventricular administration of NGF (nerve growth factor) and 8-B-cAMP on axon regeneration and cAMP-PKA signal pathway and the effece of NGF on p75NTR protein expression on cerebral ischemia-reperfusion rats.Methods The focal cerebral ischemia and reperfusion model was induced by nylon monofilament in rats. Seventy five rats were randomly assigned to sham operated (control) group, cerebral ischemia-reperfusion only group and NGF I , II groups(intra-cerebroventricular inject NGF 0.5ug/100mg, 2.5ug/100mg respectively) and 8-B-cAMP group. The contents of cAMP in cerebral cortex of rats was detected by radio immunoassay (RIA) and the change of PKA and GAP-43 mRNA expression was determined by RT-PCR technique. The p75NTR protein expression was detected by immunohistochemistry.Results The contents of cAMP and the expression level of PKA and GAP-43 mRNA decreased at 6h and 24h after cerebral ischemia- reperfusion. NGF and 8-B-cAMP elevated the expression level of GAP-43 mRNA and increased the contents of cAMP and activated PKA. There is a coincidence between the change of GAP-43 and cAMP-PKA. The change induced by NGF was dose dependent. In control group the neurons were negative for p75NTR immunoreactivity, the neuronal cell bodies were positive for p75NTR 6h reperfusion. It increased at 24h reperfusion and NGF had not effect on p75NTR protein expression.Conclusion NGF and 8-B-cAMP can improve axon regeneration. cAMP-PKA signal pathway regulates the effect of NGF on regeneration .NGF by intra-cerebroventricular administration had not effect on p75NTR protein expression.Part IVThe effects of NGF, 8-B-cAMP and Nogo-A antisence oligodeoxynucleotides on neural regeneration after cerebral ischemia-reperfusion on rats.Objective To investigate the effects of NGF, 8-B-cAMP and Nogo-A antisence oligodeoxynucleotides on neural regeneration after cerebral ischemia-reperfusion on rats.Methods Seventy rats were randomly assigned to five groups, control group (A) , cerebral ischemia-reperfusion only group (B) , C, D, E groups accepted NGF 2.5ug/100mg, 8-B-cAMP and Nogo-A antisence oligodeoxynucleotides by intra-cerebroventricular injection respectively. The evaluatioin of neurologic function by neurologic deficit score at lw, 2w and 4w after reperfusion. The length of dendtrite, the amount of branch and spine of dendtrite identified by Golgi stain and the expression of GAP-43 protein detected by immunohistochemistry and Western blot technique. The amount of synapse estimated by transmission electron microscope at 4w referfusion.Results The neurologic deficit score was 18 in control group. In group B, it was decreased at lw, 2w and 4w(12.20±2.49, 13.80±1.30, 14.40±1.82 respectively, p<0.01), in group C, D and E the score increased significantly than group B at 4w reperfusion. The amount of synapse decreased, the length of dendtrite, the amount of branch and spine of dendtrite were degression at lw and 2w. The expression of GAP-43 protein reduced at lw and 2w and increased mildly at 4w. In groups D,E and F, the amount of synapse, the length of dendtrite, the amount of branch and spine of dendtrite and GAP-43 protein expression incresed significantly. In these groups, the effect in group C was most significant.Conclusion NGF, 8-B-cAMP and Nogo-A antisence oligodeoxynucleotides by intra-cerebroventricular injection can promot neutrite regrowth and recovery of neurologic function on rats with focal cerebral ischemia. |
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