| Objective:To explore the effect of the10%hypertonic saline (HS) on the blood brain barrier (BBB) permeability in brain edema caused by cerebral ischemia/reperfusion injury. To further analyze the relations between the BBB permeability and the effect of10%HS on regulating the expression of VEGF in astrocytes and the expression of VEGFR2.Method:1.Experiment animals and groups:180healthy adult Sprague-Dawley male rats were randomly divided into three groups:sham operation+normal saline treatment group(Sham, n=60),cerebral ischemia+normal saline treatment group(Ischemia, n=60),and cerebral ischemia+10%hypertonic saline treatment group (10%HS, n=60). At2h following MCAO, the occluded middle cerebral artery was reperfused.Rats in the Sham group and Ischemia group were treated with a continuous intravenous infusion (0.3ml/h) of NS via the tail vein. Rats in the10%HS group were treated with10%HS (0.3ml/h). The three groups were further subdivided into three groups according to different treatment times:6,12and24h subgroups. 2.Mesures:①The brain water content(BWC) of the peri-ischemic brain tissue was detected by proportion of dry and wet method at different time points in each group.②At the beginning of reperfusion, the2mg/kg of2%Evens Blue(EB) solution was administered through the caudal vein, the contain of EB was detected by spectrophotometer at different time points to compare the differences of the BBB permeability in every groups.③VEGF and VEGFR2mRNA were detected by real-time fluorescence quantitative PCR in the peri-ischemic brain tissue of rats in each group.④VEGF and VEGFR2protein were detected by immunoblotting (western blot) in the peri-ischemic brain tissue of rats in each group.⑤Double immunofluorescence staining in the peri-ischemic brain tissue was carried out to detect VEGF expression and cellular localization.3.Date analysis and statistics:All data were analyzed by the SPSS13.0statistical software and different statistical methods were applied according to different types of data. Normal distribution measurement data was expressed with mean±standard deviation(x±s). Two factor measurement data was analyzed by two way classification ANOVA. Univariate factor measurement data was analyzed by one way ANOVA if the date was homogeneity of variance. Multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. The difference was statistically with P<0.05.Results:1. The BWC in the peri-ischemic brain tissue:The difference of the BWC of the peri-ischemic brain tissue was significantly different among the sham-operated group, the Ischemia group, and the10%HS group(F=1032.195, P=0.000). The BWC in the Ischemia group is higher than sham-operated group and10%HS group, but the difference of the BWC of the ischemic hemisphere was not significantly different in each treatment time (F=0.026, P=0.975). There was interaction between treatment times and treatment factors (F=45.54, P=0.000). To further analyze the separated effects of the treatment factors: at the different treatment times, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, the BWC of the peri-ischemic brain tissue was higher in Ischemia group and10%HS group(P<0.01); Compared with Ischemia group, the BWC in the10%HS group was lower(P<0.01). This suggest that ischemia hypoxia can lead to brain edema, and10%HS intervention can effectively relieve cerebral edema.2. The content of Evans Blue in the peri-ischemic brain tissue:The difference of the content of Evans Blue in the peri-ischemic brain’tissue was significantly different among the sham-operated group, the Ischemia group and the10%HS group(F=351.702, P=0.000). The content of Evans Blue in Ischemia group is higher than sham-operated group and10%HS group, and the difference of the content of Evans Blue in the ischemic hemisphere was significantly statistical in each treatment times (F=3.428, P=0.041. There was interaction between treatment times and treatment factors (F=23.81, P=0.000). To further analyze the separated effects of the treatment factors:at the different treatment times, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, the content of Evans Blue of the peri-ischemic brain tissue was higher in Ischemia group and10%HS group(P<0.01); Compared with Ischemia group, the content of Evans Blue in the10%HS group was lower(P<0.01). This suggest that ischemia hypoxia can increase BBB permeability, and10%HS intervention can effectively reduce ischemia hypoxia BBB permeability caused by ischemia hypoxia.3.The expression of VEGF mRNA and protein in the peri-ischemic brain tissue:①The difference of the expression of VEGF mRNA in the peri-ischemic brain tissue was significantly different among the sham-operated group, the Ischemia group and the10%HS group(F=510.700, P=0.000). The expression of VEGF mRNA is higher in Ischemia group than in sham-operated group and10%HS group, and the difference VEGF mRNA expression in the peri-ischemic brain tissue was significantly different in each treatment times (F=48.651, P=0.000. There was interaction between treatment times and treatment factors (F=31.028, P=0.000). To further analyze the separated effects of the treatment factors:at the different treatment times, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, the expression of VEGF mRNA in the peri-ischemic brain tissue was higher in Ischemia group and10%HS group(P<0.01); Compared with Ischemia group, VEGF mRNA in the10%HS group was lower(P<0.01). This suggest that ischemia hypoxia can increase VEGF mRNA expression, and10%HS intervention can effectively reduce VEGF mRNA expression.②The difference of VEGF protein expression in the peri-ischemic brain tissue was significantly different among the sham-operated group, the Ischemia group, and the10%HS group(F=479.866, P=0.000). The expression of the VEGF protein in Ischemia group is higher than in sham-operated group and10%HS group, and the difference of VEGF protein expression in the peri-ischemic brain tissue was significantly different in each treatment times (F=24.217, P=0.000. There was interaction between treatment times and treatment factors (F=54.566, P=0.000). To further analysis the separated effects of the treatment factors:at the different treatment time, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, the expression of VEGF protein in the peri-ischemic brain tissue was higher in Ischemia group and10%HS group(P<0.01); Compared with Ischemia group, VEGF protein in the10%HS group was lower(P<0.01). This suggest that ischemia hypoxia can increase VEGF protein expression, and10%HS intervention can effectively reduce VEGF protein expression.4.The expression of VEGFR2mRNA and protein in the peri-ischemic brain tissue:①The difference of VEGFR2mRNA expression in the peri-ischemic brain tissue was significantly different among the sham-operated group, the Ischemia group and the10%HS group(F=783.791, P=0.000). The expression of the VEGFR2mRNA in Ischemia group is higher than sham-operated group and10%HS group, and the difference of VEGFR2mRNA expression in the peri-ischemic brain tissue was significantly different in each treatment times (F=47.556, P=0.000. There was interaction between treatment times and treatment factors (F=26.035, P=0.000). To further analysis the separated effects of the treatment factors:at the different treatment time, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, the expression of VEGFR2mRNA in the peri-ischemic brain tissue was higher in Ischemia group and HS group(P<0.01); Compared with Ischemia group, VEGFR2mRNA in the HS group was lower(P<0.01). This suggest that ischemia hypoxia can increase VEGFR2mRNA expression, and10%HS intervention can effectively reduce VEGFR2mRNA expression.②The difference of VEGFR2protein expression in the peri-ischemic brain tissue was significantly different among the sham-operated group, the Ischemia group and the10%HS group(F=831.850, P=0.000). The expression of VEGFR2protein in Ischemia group is higher than in sham-operated group and10%HS group, and the difference of the VEGFR2protein expression in the ischemic hemisphere was significantly different in each treatment times (F=31.734, P=0.000. There was interaction between treatment times and treatment factors (F=76.745, P=0.000). To further analyze the separated effects of the treatment factors:at the different treatment times, multiple comparisons were analyzed by LSD method if the data was homogeneity of variance, otherwise analyzed by Dunnett’s T3method. Compared with sham-operated group, VEGFR2protein expression in the peri-ischemic brain tissue was higher in Ischemia group and10%HS group(P<0.01); Compared with Ischemia group, VEGFR2protein in the10%HS group was lower(P<0.01). This suggest that ischemia hypoxia can increase VEGFR2protein expression, and10%HS intervention can effectively reduce VEGFR2protein expression.5. The coexpression of VEGF and astrocytes in the peri-ischemic brain tissue:At the time of the24h treatment, compared with the sham-operated group, very intense immunoreactivity of VEGF was detected in the astrocytes in the peri-ischemic brain tissue in Ischemia group and10%HS group. When compared with Ischemia group, the immunoreactivity of VEGF was weakened in10%HS group. This shows that10%HS may reduce the VEGF expression in astrocytes caused by ischemia-reperfusion injury in the peri-ischemic brain tissue.Conclusion:①The injury of ischemia-reperfusion in MCAO model of SD rat may upregulate the expression of VEGF in the astrocytes which located in the peri-ischemic brain tissue, and the expression of VEGFR2.At the same time, the increased permeability of BBB, and the brain water contain were found, which resulted in cerebral edema.②10%HS may downregulate the permeability of BBB resulted from ischemia, reduce the brain water content and the cerebral edma by mitigating the expression of VEGF in the astrocytes which was located in the peri-ischemic brain tissue, and mitigate the expression of VEGFR2. |
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