Effects Of Inhibition Of Rac1on Neurological And Behavioral Outcomes After Cardiac Arrest GCI Model In Rats

Extensive research has been aimed at finding effective strategies and drugs to ameliorate or prevent brain ischemia-reperfusion injury.However,few have been successfully applied in clinical practice,although several strategies and drugs have been shown to decrease ischemic damage in the brain in animal models.In clinical practice,many accidents lead to global ischemia,such as drowning,cardiac arrest,and marked hypotension during cardiac surgery,and the brain is intrinsically more vulnerable to ischemia than other organs. Rapid initiation of reperfusion is the most effective treatment to reduce injury and the behavioral deficits caused by ischemia. However, reperfusion also has the potential to introduce additional injury; many studies have shown that overproduction of reactive oxygen species (ROSs) occur in the early reperfusion period and lead to reperfusion injury.NADPH oxidase is a major complex that produces reactive oxygen species during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear.Racl is the cytosolic components of NADPH oxidase and this subunit activation contributes to the increased NADPH oxidase activity.Recent studies demonstrated inhibition of Rac1protects neuronal injury after global cerebral I/R injury and spinal cord ischemia in rat. However,the molecular mechanisms underlying this phenomenon are not completely understood.In the current study,we estimated the protective effect of inhibition of Rac1against neuronal loss and behavioral deficits after global cerebral I/R using an animal model modified from transoesophageal electrical stimulation induced cardiac arrest global ischemia model in rats. Furthermore,we also determined whether the change of the expression of thioredoxin system in attenuation of global ischcmia-reperfusion injury are involved to explore the GCI mechanisms.This study suggests that Rac1can be a direct molecular target for modulation of NADPH oxidase activity after ischemic brain injury.Part I Transoesophageal electrical stimulation induced cardiac arrest model in ratsObjective Establish the Global Cerebral Ischemic Model in Rats and investigate Racl inhibition protective effect on Global Cerebral could protect brain against global cerebral I/R injury.Method Adult male Sprague-Dawley rats weighing250-300g were randomly divided into four groups:Sham group, CA group,NSC group treated with NSC23766,a highly selective inhibitor of Rac1and Vehicle group.Transoesophageal electrical stimulation induced cardiac arrest in rats so as to obtain Global cerebral ischemia reperfusion model.The rats brains were removed at48h of cerebral reperfusion were measured by wet-dry weight method and the coronal frozen sections were determined neuronal apoptosis using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) in hippocampal CA1region. Neuronal injury was quantified by Nissl staning counting the neuronal density (ND) to campare the neuronal damage induced by global cerebral I/R in the hippocampal CA1layer at9d after reperfusion.Results The rats in CA group,in comparison with the sham and NSC group,had a higher brain water content and short survival rate.Extensive neuronal death after GCI was significantly attenuated by NSC23766inactivate Racl activity reduced neuronal loss and the number of TUNEL positive cells (p<0.05).Conclusion Transoesophageal electrical stimulation induced cardiac arrest in rats can establish the global cerebral ischemia model.Inhibition of Rac1activty improve the achievement ratio of the model and protect brain against global cerebral I/R injury by reducing delayed neuronal death and apoptosis. Part Ⅱ Effects of Inhibition of Racl on neurological and behavioral outcomes after GCI.Objective To investigate whether inhibition of Rac1activity attenuated the spatial learning and memory deficit and related to oxidative stress.Methods Adult male Sprague-Dawley rats weighing250-300g were randomly divided into four groups:sham group, CA group,NSC group treated with NSC23766,a highly selective inhibitor of Racl and Vehicle group. Transoesophageal electrical stimulation induced cardiac arrest in rats so as to obtain Global cerebral ischemia reperfusion model.Rat brains of hippocampal CA1region were removed at30min、3h、6h、1d、3d of cerebral reperfusion for determination of Rac1activity by Western-Blot.Neurological severity scores (NSS) were tested at6h,1d,2d,4d after reperfusion.Morris water maze task was used to test the learning and memory function and behaviorally by evaluating the spatial learning and memory deficit from7d to9d of reperfusion.Furthermore, we examined the activities of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) at48of reperfusion to detect oxidative stress injury in the hippocampal CA1region.Results Our results show low, baseline Rac1activity levels in sham non-ischemic animals,while there was a robust elevation of Racl activity in ischemic animals at6h post-reperfusion, whereas treatment with the Racl inhibitor, NSC23766resulted in50%reduction in Racl activity as compared to Vehicle groups.Compared to the sham group,the NSSs increased in CA group and NSC group,but the change was relieved in NSC group.Cognition impairment was found in the CA and NSC groups,and the statistic analysis showed that there were significant differences between CA and NSC groups in ANOVA.Shortened mean escape latency was detected in the NSC group compared with the CA group during the same trial times.Significant differences were noted in the level of SOD and MDA among sham,CA group and NSC group(p<0.05).Conclusion Inhibition of Rac1attenuates spatial learning and memory deficit and this neuroprotective effect against global cerebral I/R injury through reducing oxidative stress levels. ParⅢ The changes of thioredoxin system Trx2and Prx3on cardiac arrest ischemia-reperfusion injury in ratsObjective To investigate the relationship between the effects of Racl inhibition protection global ischemia-reperfusion injury and thioredoxin system in rats.Method Adult male Sprague-Dawley rats weighing250-300g were randomly divided into four groups:sham group, CA group,NSC group treated with NSC23766,a highly selective inhibitor of Rac1and Vehicle group. Transoesophageal electrical stimulation induced cardiac arrest in rats so as to obtain Global cerebral ischemia reperfusion model.Rat brains of hippocampal CA1region were removed at6h、1d、3d、5d after cerebral reperfusion for Western-blot detection of Trx2、 Prx3.Results Compared with Sham group, CA group decreased Trx2protein expression at6h and increased at1d, Prx3protein expression increased at1d(p<0.05).Compared with CA group,NSC group didn’t increased Trx2、Prx3protein expression.Conclusion Inhibition of Racl neuroprotective effect against global cerebral I/R injury didn’t involved Trx2,Prx3protein expression.