The Biological Mechanism Of Secondary Brain Injury And Pharmacological Intervention On Intracerebral Hemorrhage

OBJECTIVES To investigate the time course of Keap1/Nrf2 pathway and anti-apoptosic protein phosphorylation of Akt expression after acute intracerebral hemorrhage in rats,with respect to the relationship with brain water content and neurological behavior score.Thereafter,the therapeutic effect of edaravone for attenuating neurologic deficit,brain edema,cell apoptosis,and perihematomal cerebral metabolism in experimental intracerebral hemorrhage(ICH)rat was investigate,the underlying biological mechanism was explored via detecting the activities of Keap1/Nrf2 pathway and phosphorylation of Akt.METHODS The ICH rat models were established by stereotaxic microinjection of autologous whole blood into the right striatum.The expression of Keap1/Nrf2,the downstream antioxidative enzymes,and phosphorylation of Akt were detected by realtime PCR,western bloting,and immunohistochemistry staining at 2h,8h,24 h,3d,5d,or 10 d after blood infusion.Meanwhile,the neurological behavior test and brain water content were evaluated at every time point.Systemic administration of edaravone for three days in ICH rats,the brain water content and neurological behavior score were evaluated.The perihematomal glucose metabolism was detected by positron emission tomography/computed tomography(PET/CT).To access the cellular apoptosis after ICH,TUNEL assays were performed in perihematomal region.The expression of Keap1/Nrf2 pathway and phosphorylation of Akt were detected by realtime PCR,western bloting and immunochemistry staining at both transcriptional and translational levels.RESULTS After blood infusion,the Keap1 showed a decremental expression started at 8 h,whereas the Nrf2 began to show a significant increase at 2 h with a peak at 24 h.Both Keap1 and Nrf2 are mainly expressed in neuronal cells but not in glia cells.The downstream antioxidative enzymes such as hemeoxygenase-1(HO-1),glutathione(GSH),thioredoxin(TRX),and glutathione-S-transferase(GST-?1)increased in different degree within early stage of ICH.Among these enzymes,the HO-1 showed a significant increase in a time dependent manner from 8 h after ICH.There was a positive correlation between HO-1 level and brain water content.P-Akt activity in sham control and ICH 2h was weak.However,p-Akt expression showed a gradual increase from 8 h after ICH,and peaked at day 5.After blood infusion,the rats treated with edaravone showed significant improvement in both forelimb placing and corner turn tests compared to those treated with vehicle.Moreover,the brain water content of the edaravone-treated group was significantly decreased compared to that of the vehicle group on day 3 after ICH.PET/CT images of ICH rats exhibited obvious decreases in FDG uptake in perihematomal region on day 3,and the relative cerebral metabolic rate of glucose(r CMRgluc)of the edaravone-treated ICH rats was significantly increased compared to that of the control rats.Calculation of the brain injury volumes from the PET/CT images revealed that the volumes of the blood-induced injuries were significantly smaller in the edaravone group compared to the vehicle group.TUNEL assays performed 3 days after ICH revealed that the numbers of apoptotic cells in perihematomal region of edaravone-treated ICH rats were decreased relative to the vehicle group.Edaravone activated Keap1/Nrf2 pathway and upregulated antioxidative proteins,such as hemeoxygenase-1(HO-1),thioredoxin(TRX),glutathione S-transferase-?1(GST-?1).The phosphorylation of Akt was significantly unregulated in edaravone treated ICH rats.CONCLUSIONS The present study provides a quantitative information regarding Keap1 and Nrf2 activity in the perihemotomal regions,which suggest that Keap1/Nrf2 signaling pathway participate in the mechanism of antioxidative damage induced by ICH,activation of Nrf2 in the acute period may represent a potential target for treatment of secondary brain injury of ICH;It is noteworthy that HO-1 expression is rapidly associated with brain edema and neurological deficit produced by blood infusion,suggesting that HO-1 is a sensitive marker of brain injury after ICH.Edaravone has scavenging properties that attenuate neurological behavioral deficits and brain edema in the early period of ICH.Additionally,edaravone may improve cerebral metabolism around the hematoma and attenuating apoptotic cell death after ICH,possibly through upregulating Keap1/Nrf2 pathway and phosphorylation of Akt expression.