Tert-butylhydroquinone Alleviates Early Brain Injury And Cognitive Dysfunction After Experimental Subarachnoid Hemorrhage: Role Of Keap1/Nrf2/ARE Pathway

Objective: To establish subarachnoid hemorrhage model and to investigateneuroprotection of tBHQ on early brain injury in male rats after subarachnoidhemorrahage.Methods: Forty-eight health male Sprague-dawley(SD) rats were assigned randomlyinto following groups: Control group (n=12), SAH group (n=12), SAH+vehiclegroup(n=12), SAH+tBHQ group (n=12). All SAH animals were subjected to injection ofautologous blood into the prechiasmatic cistern once in20sec. In SAH+tBHQ group,tBHQ was administered via oral gavage at a dose of12.5mg/kg at2h,12h,24h and36hafter SAH. Brian samples were extracted and evaluated at48h after SAH. The brian tissueadjacent to the clotted blood was taken to analysis in our study.The cerebral edema andblood-brain barrier permeability were evaluated at48h after experimental SAH.Results: Significant increase (P <0.05) in water content was detected in the brainsamples at48h after SAH when compared with rats in control group. The mean value ofbrain water content in the cortex was decreased by tBHQ administration (P <0.05) ascompared with SAH+vehicle group. Rats in SAH+vehicle group demonstrated asignificant increase (P <0.01) in BBB permeability to Evans blue relative to rats of controlgroup. Administration of tBHQ significantly inhibited Evans blue extravasation (P <0.01),indicating a reduced BBB opening in response to tBHQ treatment.Conclusions:1. Significant increase in water content was detected in the brainsamples at48h after SAH when compared with rats in control group. And SAH groupattenuate blood-brain barrier(BBB).2. The brain edma, and blood-brain barrier(BBB) were significantly released in TBHQ group. Objective: To establish subarachnoid hemorrhage model and to observe the cognitivefunction effect of tBHQ.Methods: Forty health male Sprague-dawley(SD) rats were assigned randomly intofollowing groups: Control group (n=10), SAH group (n=10), SAH+vehicle group(n=10),SAH+tBHQ group (n=10). All SAH animals were subjected to injection of autologousblood into the prechiasmatic cistern once in20sec. In SAH+tBHQ group, tBHQ wasadministered via oral gavage at a dose of12.5mg/kg at2h,12h,24h and36h after SAH.Then we observed the behavior changes of SD rats, and cognitive and memory changeswere investigated in the Morris water maze.Results: As compared with control group, clinical behavior function impairmentcaused by SAH was evident in SAH subjects. For all behavioral measurements,swimming speed and thigmotaxis (percent time spent in the perimeter of the pool) wereevaluated and found no significant difference among the four groups. Rats usually can findthe visible platform. It was usually on the first trial even they failed, which happenedequally across the groups. There was no difference in the escape latency and swimmingspeed in the cued learning procedure between the four groups (P>0.05). Spatial learningwas also the same for all four groups during the second and third days after blood injection.Spatial learning deficits appeared during the fourth and fifth days in the SAH group ascompared to controls. The tBHQ group exhibited significantly shorter escape latency thanthe vehicle group during the4th and5th days. Animals of all four groups learned to findthe platform to escape from water within each testing day. But the SAH group wassignificantly impaired compared to controls (the4th and5th days), which was alleviated bytBHQ compare to the vehicle group. Repeated measures ANOVA indicated a significantdifference in escape latency (P<0.01) and in swimming distance (P<0.01) between theSAH group and control groups, which was also improved by tBHQ compare to the vehiclegroup. When the escape latency and swimming distance from all of the testing days wasseparated into the four daily trials, the escape latency of the SAH group exhibited significantly longer compared to the control group. The tBHQ group’s exhibitedsignificantly shorter than the vehicle group (P<0.01). But in swimming distance only thetBHQ group exhibited significantly compared to the vehicle group on the5th day (P<0.01).The percent improvement in latency of the SAH group decreased significantly by dayfive (P<0.05), while rats in the control groups had gradual, non-significant decreases inpercent improvements. However, the percent improvement of the tBHQ group appearednegative growth by day four. There was no significant difference between the groups inreference memory as measured by the percent time spent in the target quadrant in the probetrial, although there was a trend towards fewer annulus crossings between the four groups(data not shown). Animals with control showed significantly longer time saved on6th dayon the working memory task on days6-8as compared to the SAH groups as shown by thetime saved in latency for finding the platform on the second trial compared to the first trial.While there was no difference significantly among the tBHQ group and the vehicle group(P>0.05).Conclusion: As compared with control group, the rats in SAH group obviously sufferthe cognitive dysfunction while tBHQ can significantly alleviate cognitive dysfunction. Objective: The purpose of this part was to find out whether tBHQ administrationcould modulate Keap1-Nrf2-ARE signaling pathway in the early brain injury after SAH.So that we could comprehend the mechanism that tBHQ alleviates early brain injury andcognitive dysfunction after experimental subarachnoid hemorrhage.Methods: Please refer to Part II. Brian samples were extracted and evaluated at48hafter SAH. We measured the cortical apoptosis and necrosis with the method ofTUNEL(TdT-mediated dUTP Nick-End Labeling) and FJB(Fluoro-Jade B),and wemeasured the expressions of Keap1and Nrf2and HO-1by western blot; and theexpressions of Keap1and Nrf2and HO-1by immunohistochemistry; the Nrf2DNAbinding activity with EMSA autoradiography; the mRNA levels of HO-1、NQO1and GST-a1by TR-PCR; NQO1、GST-α1、MDA、SOD and GSH-Px by emzyme activity assay.Results: Few TUNEL-or FJB-positive apoptotic cells were found in the controlgroup rat brains. In SAH and SAH+vehicle groups, the apoptotic and necrotic index in thecortex was found to be significantly increased compared with those in control animals (P<0.01). There was no statistically significant difference between SAH group andSAH+vehicle group (P>0.05). In SAH+tBHQ group, when compared with that in theSAH+vehicle group, the number of TUNEL-positive or FJB-positive cells in the studiedcortex was significantly decreased (P <0.01). Western blot was performed to detect thechanges of Keap1, Nrf2, and HO-1activity,the results showed low levels of Keap1, Nrf2,and HO-1in control group. On day2(48h) after SAH, the levels of Keap1, Nrf2, andHO-1were significantly increased in SAH group and SAH+vehicle group (P <0.05). Therewas no statistically significant difference between SAH group and SAH+vehicle group(P>0.05). After tBHQ administration, the increased activity of Keap1, Nrf2, and HO-1wasmarkedly further induced in animals of SAH+tBHQ group (P <0.05). Low Nrf2bindingactivity (weak EMSA autoradiography) was found in the control group. Compared withcontrol group, Nrf2binding activity in the injured brain was significantly increased(P<0.05) in SAH and vehicle-treated groups. In SAH+tBHQ group, the Nrf2bindingactivity was significantly further up-regulated (P<0.05) in the brain area surrounding theblood clot site after SAH. Immunohistochemical study showed that positive Keap1, Nrf2,and HO-1were mainly located at both of the neurons and glial cells. The immunoreactivityof Keap1, Nrf2, and HO-1was weak in the cortex samples in control group with only afew positive cells in the brain. Compared with control group, more Keap1, Nrf2, and HO-1positively immunostained cells appeared in SAH and SAH+vehicle groups at48hpost-SAH (P<0.05). In SAH+tBHQ group, the number of positive cells was furtherincreased significantly (P<0.05). The mRNA of these proteins expressed at a low level inthe rat brains of control group. The levels of HO-1, NQO1, and GST-α1mRNA weresignificantly increased in the cortex in SAH and SAH+vehicle group as compared with thatof control group (P <0.05). The mRNA expressions had no significant difference betweenSAH group and SAH+vehicle group (P>0.05). The mRNA expressions of HO-1, NQO1,and GST-α1in the brains of SAH+tBHQ group were significantly up-regulated than thoseof the SAH+vehicle group. Compared with control group, cortical levels of theseantioxidant and detoxifying enzymes were greatly induced after SAH. TBHQ administration after SAH could lead to significantly increased NQO1and GST-α1activityin rat brain tissue. SAH significantly increased the tissue MDA levels (P <0.05) andsignificantly decreased the tissue SOD and GSH-Px enzyme activities (P <0.05) whencompared with controls. TBHQ treatment has shown protective effect via decreasingsignificantly (P <0.05) the elevated MDA levels and also significantly increasing thereduced antioxidant enzyme activities (SOD, P <0.01; GSH-Px, P <0.05).Conclusions: The up-regulated cortical levels of these agents related toKeap1/Nrf2/ARE signaling pathway were further activated when treated with tBHQ inlevels of both mRNA and protein synthesis; tBHQ may modulate the SAH-inducedKeap1/Nrf2/ARE signaling pathway activation that may prevent the development of EBIfollowing SAH.