Objective To explore the role of intracellular Ca~(2+) in inhibition of axonal outgrowth,which could learn more about the signal transduction mechanism of Nogo-A inhibiting axonal outgrowth. Methods Using laser confocal microscope,intracellular calcium level in cerebellar granule neurons after Nogo-A added in different times was measured and the effect of Nogo-A inhibiting axonal outgrowth when priming nimodipine was observed. Results The concentration of Ca~(2+) increased in 5 minutes after Nogo-A added,and reached to peak in 30 minutes,then decreased gradually,90 minutes later dropped to normal level.When priming with nimodipine,the axonal inhibition by Nogo-A was partly abrogated compared with control.Conclusion Ca~(2+) may participate in the signal transduction of Nogo-A inhibiting axonal outgrowth.

Objective: To establish a method using non-ionic detergent for extracting lipid rafts. Methods: Because lipid rafts can resist solubilization by non-ionic detergents under 4℃, we use non-ionic detergent (Triton X-100) to treat with epicyte fractions, the non-raft membrane would be solubilized. Then we utilize sucrose gradient centrifugation, preparations enriched in lipid rafts could be obtained.caveolin-1 was used as markers of lipid-raft structures. Results:A white light-scattering band under light illumination located at the interface between 15%-20% sucrose was detectable, and a brown stripe which comparative molecular quantity is 24 000 was identified by Western-Blot analysis. Conclusion: The method using non-ionic detergent is simple and useful for extracting lipid rafts, extracting lipid rafts would be prerequisite in studying the function of lipid rafts.

In order to evaluate the neuroprotective effect of Rosiglitazone Maleate (RSG) against brain ischemic injury, the effects of Rosiglitazone Maleate on the inflammation following cerebral ischemia/reperfusion were investigated. Focal cerebral ischemia was induced by the intraluminal thread for cerebral middle artery (MCA) occlusion. Rosiglitazone Maleate at concentrations of 0.5, 2 and 5 mg/kg was infused by intragastric gavage twice immediately and 2 h after MCA occlusion, respectively. The effects of Rosiglitazone Maleate on brain swelling, myeloperoxidase and interleukin-6 mRNA level in brain tissue after MCA occlusion and reperfusion were evaluated. The results showed that as compared with the model control group, RSG (0.5 mg/kg) had no significant influence on brain swelling (P>0.05), but 2 mg/kg and 5 mg/kg RSG could significantly alleviate brain swelling (P<0.05). All different doses of RSG could obviously reduce MPO activity in brain tissue after MCA occlusion and reperfusion in a dose-dependent manner. RSG (0.5 and 2 mg/kg) could decrease the expression levels of IL-6 mRNA in brain tissue after MCA occlusion and reperfusion to varying degrees (P<0.05) with the difference being significant between them. It was concluded that RSG could effectively ameliorate brain ischemic injury after 24 h MCA occlusion and inhibit the inflammatory response after ischemia-reperfusion in this model.

Objective:To explore the expression of Nogo-A in membrane of mature oligodendrocyte, and obtain these living cells. Methods:Mature oligodendrocytes were stained by indirect immuno-fluorescent technique.Stained unpermeable oligodendrocytes were sorted by flow cytometry. Results: The percentage of mature oligodendrocytes with Nogo-A expression in membrane was 3.36%. Conclusion:Nogo-A could be expressed in membrane of mature oligodendrocytes,and the ratio of positive cells was about 3.36%.

The changes in the tau protein phosphorylation and expression of bcl-2, and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion (I/R) were explored, and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease. The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method. The level of tau protein phosphorylation at Ser396, Ser404, Tyr231, Ser199/202 sites and the expression of bcl-2, bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot. The relationship between the tau protein phosphorylation and the expression of bax, or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method. The results showed that the level of tau hyperphosphorylation at Ser199 / 202, Ser396, Ser404, Tyr231 sites and the expression levels of bcl-2, and bax were significantly higher in I/R group than in the sham group, but the ratio of bcl-2/bax was decreased. Neuronal apoptosis, bax expression and the tau protein hyperphosphorylation were co-localized. It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R. The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis. The cerebral infarction may contribute to Alzheimer's disease occurrence and development.

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P>0.05), while the expression level in shRNA-transfected group decreased significantly (P<0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC12 cells.
Cloning, Molecular ; Gene Knockdown Techniques/*methods ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Myelin Proteins/*genetics ; Myelin Proteins/metabolism ; PC12 Cells ; Plasmids ; RNA Interference ; RNA, Messenger/genetics ; RNA, Messenger/metabolism ; RNA, Small Interfering ; Transfection

In order to evaluate the neuroprotective effect of Rosiglitazone Maleate (RSG) against brain ischemic injury, the effects of Rosiglitazone Maleate on the inflammation following cerebral ischemia/reperfusion were investigated. Focal cerebral ischemia was induced by the intraluminal thread for cerebral middle artery (MCA) occlusion. Rosiglitazone Maleate at concentrations of 0.5,2 and 5 mg/kg was infused by intragastric gavage twice immediately and 2 h after MCA occlusion,respectively. The effects of Rosiglitazone Maleate on brain swelling, myeloperoxidase and interleukin-6 mRNA level in brain tissue after MCA occlusion and reperfusion were evaluated. The results showed that as compared with the model control group, RSG (0.5 mg/kg) had no significant influence on brain swelling (P＞0.05), but 2 mg/kg and 5 mg/kg RSG could significantly alleviate brain swelling (P＜0.05). All different doses of RSG could obviously reduce MPO activity in brain tissue after MCA occlusion and reperfusion in a dose-dependent manner. RSG (0.5 and 2 mg/kg) could decrease the expression levels of IL-6 mRNA in brain tissue after MCA occlusion and reperfusion to varying degrees (P＜0.05) with the difference being significant between them. It was concluded that RSG could effectively ameliorate brain ischemic injury after 24 h MCA occlusion and inhibit the inflammatory response after ischemia-reperfusion in this model.

Objective:To analyze the related risk factors for vertebral artery tortuosity, and explore the mechanism of vertebral artery tortuosity.Methods:Two hundred and eighty-two patients accepted head/neck and MR angiography in our hospital from October 2016 to October 2017 were selected. The tortuosity degrees of vertebral artery were measured and calculated by PACS system. The differences of tortuosity degrees of vertebral arteries in different age groups were compared. Correlation analysis was performed to determine the correlation between vertebral artery tortuosity and both clinical data and and biochemical levels, and multivariate linear regression analysis was performed to determine the independent risk factors for vertebral artery tortuosity.Results:The tortuosity degrees of the left and right vertebral arteries in these patients ranged from 5.1% to 72.6%. The tortuosity degrees of vertebral arteries in patients aged 40-49 years were significantly higher than those in patients aged 20-29 years and 30-39 years ( P<0.05). Correlation analysis showed that the tortuosity degree of the right vertebral artery was positively correlated with age and triglyceride level ( r=0.232, P=0.000; r=0.172, P=0.004); the tortuosity degree of the left vertebral artery was positively correlated with triglyceride level ( r=0.123, P=0.043). Multivariate regression analysis showed that age ( 95%CI: 0.059-0.194, P=0.000) and triglyceride level ( 95%CI: 0.173-1.942, P=0.019) were independent risk factors for right vertebral artery tortuosity. Triglyceride level ( 95%CI: 0.041-2.559, P=0.043) was independent risk factor for left vertebral artery tortuosity. Conclusion:There are congenital developmental factors associated with vertebral artery tortuosity; some nurture factor, as triglyceride level, may promote its development.

To study the inhibitory effect of Nogo-A shRNA on cell line PC12, the Nogo-A shRNA (short hairpin RNA, or shRNA) was designed and synthesized. The annealed shRNA template was inserted into plasmid pGenesil-1 containing enhanced green fluorescent protein (EGFP) gene by gene cloning technique to generate eukaryotic expression vector. The recombinant plasmid was transfected into PC12 cells by lipofecamine2000 and the mRNA and protein expression level of Nogo-A gene was detected by RT-PCR and Western blotting 48 h after the transfection. Gene sequencing showed that that the Nogo-A shRNA eukaryotic expression vector was successfully constructed. No significant change was found in the Nogo-A mRNA and protein expression level in empty vector-transfected group as compared with controls (P＞0.05), while the expression level in shRNA-transfected group decreased significantly (P＜0.05). It is concluded that the pGenesil-1/Nogo-AshRNA recombinant plasmid can effectively suppress the expression of Nogo-A gene in PC 12 cells.

The changes in the tau protein phosphorylation and expression of bcl-2,and bax in rat parietal cortex neurons after focal cerebral ischemia-reperfusion(I/R)were explored,and the relationship between the tau protein phosphorylation and the expression of bax or apoptosis was clarified in order to elucidate the relationship between cerebral infarction and Alzheimer's disease.The rat focal cerebral I/R model was induced by occlusion of the right middle cerebral artery using the intraluminal suture method.The level of tau protein phosphorylation at Ser396,Ser404,Tyr231,Ser199/202 sites and the expression of bcl-2,bax and total tau 5 in rat parietal cortex during focal cerebral ischemia/reperfusion were detected by Western blot.The relationship between the tau protein phosphorylation and the expression of bax,or apoptosis was examined by TUNEL method and double-labeling immunofluorenscence method.The results showed that the level of tau hyperphosphorylation at Ser199/202,Ser396,Ser404,Tyr231 sites and the expression levels of bcl-2,and bax were significantly higher in I/R group than in the sham group,bat the ratio of bcl-2/bax was decreased.Neuronal apoptosis,bax expression and the tau protein hyperphosphorylation were co-localized.It is suggested that Alzheimer's disease-like pathological changes occur after cerebral I/R.The highly abnormal phosphorylation of tau protein plays a key role in cerebral I/R-induced apoptosis.The cerebral infarction may contribute to Alzheimer's disease occurrence and development.