Neuroprotective Effect And Molecular Mechanism Of Inhibiting Epidermal Growth Factor Receptor Signaling Against Ischemia-reperfusion Injury

Part oneObjective:To investigated the effects and the mechanism of EGFR blockade in cultured astrocytes exposure to oxygen-glucose deprivation/reoxygenation (OGD/R)Methods:Astrocytes in primary culture were used for OGD/R model, Different concentration Of C225 (2μg/ml,10μg/ml) was applied to cell cultures 1 h before OGD whereas control cultures received only the vehicle. The drug was left in the culture medium during the entire experiment. Cells were harvested after OGD/R at various time points (at 3h,6h,12h and 24h, respectively). Cell cycle progression of astrocytes in vitro was studied by flow cytometric analysis. Expression of p-EGFR, GFAP and cell proliferation related molecules in vitro were evaluated by immunostaining and Western blot analysis.Results:Following exposure to OGD/R, the cultured astrocytes became activated characterized by enhanced GFAP expression, elongated long processes and hypertrophic morphology, accompanied with phosphorylation of EGFR. Double-immunolabeling with GFAP and BrdU demonstrated the percentage of BrdU(+) astrocytes increased nearly two-fold at 12 h as compared to control cultures. Treatment with C225 at 1 h prior to OGD/R attenuated the morphological alterations of astrocytes and decreased the number of BrdU(+) astrocytes. During the early phase of OGD/R (3-6 h), the percentage of astrocytes in S phase increased as compared to the control group. At 12h of OGD/R, the percentage of astrocytes in the S phase reached the highest level, and then declined gradually to the time point of 24 h. When C225 was added to the cultured astrocytes exposed to OGD/R, the percentage of astrocytes in the S phase decreased significantly (P<0.01) at 6,12, and 24 h. In addition, Western blot analysis showed that C225 not only attenuated the increase of GFAP expression but also suppressed the high expression of cyclin D1, ki67 and p-ERK1/2 in cultured astrocytes; furthermore, C225 also up-regulated the expression of p27. Conclusion:EGFR blockade significantly dereased expression of p-EGFR, inhibited cell cycle progression of astrocytes and attenuated reactive astrogliosis in vitro. Part twoObjective:To investigated the effects and the mechanism of EGFR blockade on astrocytes following ischemia reperfusion injury.Methods:Adult male Wistar rats were used for MCAO model. Animals were randomly assigned into experimental groups. Cerebral ischemia was induced by occluding the origin of the middle cerebral artery (MCA) for 1h using the intraluminal thread model. C225(106 ug/day) or vehicle was given into lateral cerebro-ventricle through ALZET microosmotic pumps after 1 h of transient MCAO.Expression of p-EGFR, GFAP and cell proliferation related molecules in Each group animals were evaluated by immunostaining and Western blot analysis.Results:In sham-operated group, GFAP immunohistochemical staining revealed only slim and short astrocytic processes scattered throughout cerebral cortex. After transient MCAO, astrocytes in the cortex boundary zone appeared to be intensively immunoreactive to GFAP with thick, richly branched processes. Furthermore, double immunolabeling showed that cells were GFAP immunoreactive also stained intensely with an antibody to p-EGFR in both vehicle and C225-treated ischemic groups. This phenomenon appeared as early as at day 3 post-injury, increased at day 7, peaked at day 14. As shown by immunolabeling for p-EGFR in the group treated with C225, activation of EGFR is significantly blocked in the ischemic brains of rats. Furthermore, GFAP staining also showed much weaker immunoreactivity than that in the vehicle treated group at the same time points of day 3,7 and day 14, respectively. In addition, change in p-EGFR and total EGFR expression was measured by Western blot analysis, and the results similar with immunofluorescent staining were found. double-labeling of GFAP and Ki-67 (a proliferating cell marker) was performed after MCAO at the time point of day 3,7 and 14. Similarly, Immunostaining of GFAP and Cyclin D1 (a critical marker of cell cycle progression) was also observed. It was shown that the number of Cyclin D1 (+) astrocytes and the number of Ki-67 (+) astrocytes were both grown significantly in the vehicle treatment group. The increase in percentage of proliferative astrocytes after MCAO in the cortex boundary zone was inhibited by C225.Western blots of brain tissues from the cortex boundary zone post ischemia at day 3,7 and 14 in sham, vehicle-treated and C225-treated rats showed that expression of proteins for GFAP, Ki67, cyclin D1 and p-ERK1/2 were highly up-regulated and expression of p27 was down-regulated after MCAO, and C225 treatment significantly reversed these changes.Conclusion:EGFR blockade significantly dereased expression of p-EGFR, inhibited significantly astrocyte proliferation after MCAO and attenuated reactive astrogliosis in vivo. Part threeObjective:To investigated the neuroprotective effects of EGFR blockade on ischemia reperfusion injuryMethods:The MCAO model was established as the part two, Neuronal apoptosis after MCAO was determined by TUNEL method. Neurologic scores and infarct volumes post ischemia were assessed in the rat MCAO model.Results:TUNEL (+) neurons were sparse in sham-operated animals, then increased at day 3 post-ischemia, and the percentage of TUNEL (+) neurons was significantly reduced in the C225 group. Meanwhile, TTC staining was done to determine the volume of lesion at day 7 post-ischemia. The results showed that C225 decreased the infarct volume after MCAO significantly (200.3±21.8 versus 277.3±24.1 mm of vehicle treated group).Neurological deficit scores of vehicle-treated MCAO rats gradually increased and reached the maximum at day 3 after reperfusion, thereafter recovered partly at day 7, and ameliorated spontaneously at day 14 after reperfusion. C225 treatment significantly improved these neurological dysfunctions post ischemia at day 3,7 and 14, respectively (P<0.05, compared to the vehicle treated group).Conclusion:Inhibiting epidermal growth factor receptor signaling by C225 protected against ischemia reperfusion injury in rats.