Protection Of Vascular Endothelial Growth Factor To Brain Edema Following Intracerebral Hemorrhage And Its Involved Mechanisms:Effect Of Aquaporin-4

Intracerebral hemorrhage (ICH) is a severe stroke type, which always leads to severe neurological deficits and poor prognosis. Brain edema formation after ICH is one of the major causes of these consequences. Vascular endothelial growth factor (VEGF) has protective effects on various neurological diseases, including ICH. But no related research on how VEGF acting on brain edema after ICH has been reported. Our previous study indicated that as the most abundant water channel in brain, aquaporin-4(AQP4) played an important role in edema resolution after ICH. In addition, it was reported that VEGF and AQP4colocalized on astrocyte processes after cerebral hypoxia and blood-brain barrier (BBB) disruption and intracerebral VEGF injection can highly up-regulate AQP4mRNA and protein. Since there is close connection between them, we speculated that the effect of VEGF on brain edema following ICH may result from regulating AQP4expression. We first studied the effects of recombinant human VEGF165(rhVEGF165) to wild type mice on neurological deficits, brain water contents, BBB permeability and cell apoptosis after ICH and the influence of rhVEGF165on AQP4expression in normal state and ICH. We also compared the parameters between AQP4+/+and AQP4-/-mice to indicate whether the above roles played by rhVEGF165resulted from regulating AQP4expression. We further explored the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression through astrocyte cultures. Our results can provide a theoretical basis to downstream signal transduction pathways of VEGF, regulation of AQP4expression and the mechanisms involved in the effects of VEGF on ICH. Double targets can be supplied to treatment of brain edema following ICH, which can present new insights for new drugs development.Part Ⅰ Effect of VEGF on brain edema after ICHObjective:To study the effect of intracerebroventricular rhVEGF165injection on brain edema after ICH and its influence on AQP4expression in normal state and ICH.Methods:CD1mice were randomly divided into six groups:control, control plus VEGF (3μg/kg), sham operation, ICH, ICH plus VEGF, ICH plus SU5416(60μg/kg), a kind of VEGFR inhibitor. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of Evans blue (EB) and evaluated cell apoptosis through TUNEL staining in the first5groups3d after drug given. We also detected the expression of AQP4protein at striatum or perihemotoma using Western blot and immunofluorescence in each group at the same time point.Results:1. Intracerebroventricular rhVEGF165injection significantly reduced neurological deficits (p<0.05), brain water contents (p<0.01) and TUNEL-positive cells (p<0.01) perihemotoma in AQP4+/+mice, but had no change of EB extravasation.2. Western blot showed an increase of AQP4protein at striatum (p<0.01) after rhVEGF165injected intracerebroventricularly. Furthermore, AQP4protein expression perihemotoma was increased by intracerebroventricular injection of rhVEGF165after ICH (p<0.01), but was decreased by SU5416(p<0.01). The intensity and number of fluorescence expression were in accord with Western blot.Conclusion:1. RhVEGF165injected intracerebroventricularly can reduce brain edema, alleviate neurological deficits and reduce apoptotic cells in AQP4+/+mice after ICH, but has no influence on BBB permeability.2. Intracerebroventricular rhVEGF165injection can increase AQP4protein expression perihemotoma after ICH. Endogenous VEGF may be involved in AQP4protein up-regulation after ICH. Part Ⅱ Roles played by AQP4in effects of VEGF to brain edema following ICHObjective:To investigate the effects of rhVEGF165to AQP4-/-mice on brain edema after ICH. We also studied whether the above roles played by rhVEGF165resulted from regulating AQP4expression by comparison of AQP4+/+and AQP4-/-mice.Methods:AQP4-/-mice were randomly divided into five groups:control, control plus VEGF, sham operation, ICH, ICH plus VEGF. We scored for neurological deficits, measured brain water contents by dry-wet weight ratio method, determined BBB permeability with the use of EB and evaluated cell apoptosis through TUNEL staining.Results:1. Neurological deficits were more severe in AQP4-/-mice than AQP4+/+mice after ICH (p<0.05). Injection of rhVEGF165intracerebroventricularly alleviated neurological deficits of AQP4-/-mice (p<0.05), but the difference between them was the same as that mentioned above (p<0.05). Moreover, the percentage of neurological deficits decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice (p<0.01).2. An increase of brain water contents in normal AQP4-/-mice after intracerebroventricular injection of rhVEGF165was observed (p<0.01). AQP4-/-mice had more severe brain edema after ICH than AQP4+/+mice (p<0.05). Injection of VEGF intracerebroventricularly had no effect on AQP4-/-mice.3. The amount of EB extravasation was markedly increased by intracerebroventricular rhVEGF165injection in AQP4-/-mice (p<0.01), which was much more than AQP4+/+mice (p<0.01). Injection of VEGF intracerebroventricularly after ICH developd an increased effect in AQP4-/-mice (p<0.05).4. There were more TUNEL-positive cells found perihemotoma after ICH in AQP4-/-mice than AQP4+/+mice (p<0.01). RhVEGF165injected intracerebroventricularly reduced TUNEL-positive cells in AQP4-/-mice (p<0.01), but the difference between them was the same as that mentioned above (p<0.01). In addition, the percentage of TUNEL-positive cells decreased by rhVEGF165after ICH was significantly higher in AQP4+/+mice than AQP4-/-mice(p<0.01).Conclusion:1. Increase of AQP4expression after ICH may have protective effects on neurological functions, brain edema, BBB integrity and cell apoptosis.2. Up-regulation of AQP4expression is one of the possible mechanisms by which rhVEGF165significantly alleviates neurological deficits after ICH.3. AQP4has an effect in normal state on eliminating brain edema potentially induced by VEGF. However, rhVEGF165can reduce brain edema following ICH, which has close relationship with enhancement of edema clearance by AQP4up-regulation induced by rhVEGF165.4. RhVEGF165can damage BBB in normal mice, which can be worsened by AQP4knock-out. The effect of rhVEGF165on BBB after ICH can be both protective and destructive and increase of AQP4expression may participate in the positive aspect.5. Apoptotic cells perihemotoma after ICH can be markedly reduced by exogenous rhVEGF165. Up-regulating AQP4expression is one of the possible mechanisms. Part Ⅲ The signal transduction pathways through which VEGF regulates AQP4expressionObjective:To explore the possible signal transduction pathways activated by rhVEGF165to regulate AQP4expression.Methods:The subcultured and determined astrocytes were randomly divided into six groups:control; VEGF (50ng/ml); VEGF plus SP600125(20μM), a kind of JNK inhibitor; VEGF plus U0126(10μM), a kind of ERK inhibitor; VEGF plus SB239063(20μM), a kind of p38-MAPK inhibitor; VEGF plus Ly294002(20μM), a kind of PI3K inhibitor. We detected the expression of AQP4protein using Western blot and immunofluorescence2d after drug given.Results:1. Western blot showed that rhVEGF165increased AQP4protein expression of astrocytes (p<0.01), which was inhibited by SP600125and U0126(p<0.01), but not SB239063or Ly294002.2. The intensity and number of fluorescence expression were in accord with Western blot.Conclusion:1. RhVEGF165can make cultured astrocytes increase expression of AQP4.2. RhVEGF165up-regulates AQP4expression via activating JNK and ERK pathways, but not p38-MAPK or PI3K pathways.