Role Of Reactive Oxygen Species In The Neuroprotection Of Ischemic Brain

Ischemic brain injury,which is caused by partial or total interruption of the blood supply to the brain due to permanent or prolonged occlusion of a cerebral artery or to cardiac arrest with resuscitation,is one of the leading causes of mortality and morbidity in developed countries,and is the second leading cause of death and the leading source of serious and long-term disability in China.Therefore,it is a hotspot in neuroscience to study the mechanisms of ischemic brain injury and protection against it.Reactive oxygen species(ROS)are highly reactive molecules generated predominantly during cellular respiration and normal metabolism;they include the superoxide anion radical,hydrogen peroxide and the hydroxyl radical.A large body of evidence indicates that enhanced ROS and the subsequent oxidative stress play a pivotal role in cerebral ischemia/reperfusion(I/R)-induced damage.Increase in ROS not only causes oxidative damage to lipids,proteins,and DNA and induces the destruction of cell membranes and other cellular structures,but also perturbs mitochondrial function and activates apoptotic pathways,leading to delayed neuronal death(DND)in vulnerable brain regions such as the hippocampal CA1 region,where DND occurs days after the initial ischemic insult.However,it has recently been shown that ROS function as key intracellular messengers and participate in the development of neuronal tolerance against lethal ischemia after ischemic preconditioning.In addition,it is known that an excess of antioxidant has fatal consequences.Thus,it is important to study further the role of ROS in ischemic brain injury and neuroprotection.Mitochondrial ATP-sensitive potassium channels(mitoK_ATP)are located in the inner mitochondrial membrane of various types of cells and regulate matrix volume, which is related to mitochondrial and cell function.Recent studies have shown that mitoK_ATPchannels play an important role in neuroprotection;their activation protects the brain against ischemic or chemical challenge.Recently ROS have been found to function as key intracellular messengers to mediate the protective effects of ischemic preconditioning,prolonged oxygen exposure preconditioning or ethanol preconditioning in brain.However,whether ROS are involved in the neuroprotective effects of mitoK_ATPopeners is unknown.Genistein is a phytochemical that occurs naturally in a variety of plant-derived foods.It not only has tyrosine kinase inhibitor activity,but also has chemoprotectant activity against cancers and cardiovascular disease,as well as antioxidant and phytoestrogen activity.Several studies have indicated that administration of genistein prevents DND after transient global cerebral ischemia.Although genistein inhibits the lipid peroxidation induced by pro-oxidant agents in cultured cortical neurons,little is known about whether it diminishes oxidative damage after cerebral ischemia in vivo.Therefore,the present study was undertaken to explore the role of ROS in the neuroprotection of ischemic brain by examining the neuroprotective effects of diazoxide,a mitochondrial ATP-sensitive potassium channel opener,and genistein against ischemia-induced damage. Ⅰ.Role of ROS in the neuroprotection of diazoxide,a mitochondrial ATP-sensitive potassium channel opener, against ischemia-induced damage in the rat hippocampal sliceThe work was conducted to examine whether diazoxide,a mitoK_ATPchannel opener,protects neurons against ischemia-induced damage in rat hippocampal slices, and if it does,whether ROS are involved in this neuroprotection.Ischemia was simulated by oxygen and glucose deprivation.The direct current potential and population spike(PS)were recorded in the stratum pyramidale of the CA1 region,and lactate dehydrogenase(LDH)efflux into the medium was assayed.ROS generation was measured spectrophotofluorometrically.The results were as follows:1.Electrophysiology1.1.Latencies,amplitudes and durations of ischemic depolarizationUnder ischemic conditions,a rapid drop in the direct current potential,called ischemic depolarization(ID),occurred in all slices.The latencies of ID in the group pretreated with diazoxide(DIA,300μM)were longer than in controls(P＜0.01).In the groups treated with 5-hydroxydecanoic acid(5-HD,200μM),a mitoK_ATPchannel blocker or N-2-mercaptopropionyl glycine(MPG,500μM),a ROS scavenger,prior to treatment with DIA,the ID latencies were shorter than in the DIA group(P＜0.01). No differences in ID latencies were found among control,5-HD and MPG groups. The ID amplitudes in the DIA group were smaller than in controls(P＜0.01).In the 5-HD + DIA and MPG + DIA groups,the ID amplitudes were larger than in the DIA group(P＜0.01),while no differences in ID amplitudes were found among control, 5-HD and MPG groups.No differences in ID durations were found among any groups.1.2.Loss and recovery of the population spikeThe PS amplitudes did not differ among the six groups either during preincubation or following the application of drugs,as measured 1-2 min before the onset of ischemia.Under ischemic conditions,the PS decreased gradually and then disappeared.The time to loss of the PS in the DIA group was longer than in control(P＜0.01).No differences in the time to PS loss were found among control,5-HD and MPG groups.The times to PS loss in the 5-HD + DIA and MPG + DIA groups were shorter than in the DIA group(P＜0.01).After the supply of oxygen and glucose was re-established,greater recovery of the PS was found in the DIA group.However,the recovery of PS was poor in the other groups.2.Lactate dehydrogenase release during ischemiaAfter 30 min ofischemia,LDH efflux in the DIA group was decreased compared with control(P＜0.01).No statistically significant differences of LDH efflux were observed among control,5-HD and MPG groups.LDH efflux in both the 5-HD + DIA group and the MPG + DIA group was higher than in the DIA group(P＜0.01).3.Generation of reactive oxygen speciesDCF fluorescence in the DIA group was increased compared with control(P＜0.01).No statistically significant differences of DCF fluorescence were found among control,5-HD and MPG groups.DCF fluorescence in the 5-HD + DIA group and the MPG + DIA group was lower than in the DIA group(P＜0.01). These results indicate that DIA conferred neuroprotection against ischemia-induced damage in rat hippocampal slices.The neuroprotection is attributed to the opening of mitoK_ATPchannels and ROS generation.Ⅱ.Effect of genistein on oxidative stress and neuronal damage following global cerebral ischemia/reperfusion in rat hippocampusThis work was carried out to investigate the effects of genistein on DND,ROS generation and lipid peroxidation following transient global cerebral ischemia in rat hippocampus.The results were as follows:1.Brain damage after global cerebral ischemiaIn sham-operated controls,there were no histopathological changes in the hippocampal CA1 region as assessed by the cresyl violet-staining.At 1 day of reperfusion,there was no detectable damage in the CA1 region of ischemic rats. However,cells with irregular shapes and dense staining and cell loss were progressively severe at 3,5 and 7 days of reperfusion.The neuronal densities in the CA1 region were decreased,compared to the level in sham-operated animals(p＜0.01).2.Effect of genistein on I/R-induced neuronal damageAt 5 days of reperfusion,no cell damage was evident by cresyl violet-staining in the CA1 region of sham-operated rats.In ischemic animals,extensive neuronal loss was found in the CA1 region.Hippocampal neuronal damage was decreased by treatment with genistein(p＜0.01).3.Effect of genistein on I/R-induced cytochrome c releaseCytosolic cytochrome c levels in the CA1 region increased in the ischemic group after 48 and 72 h of reperfusion over the sham-operated group(p＜0.01),and treatment with genistein suppressed the I/R-induced release of cytochrome c(p＜0.05 or 0.01).4.Effect of genistein on I/R-induced neuronal apoptosisCaspase-3 like activity in the CA1 region had not changed at 48 h of reperfusion. However,the caspase-3 like activity increased at 72 h of reperfusion,compared to the level in sham-operated animals(p＜0.01).Treatment with genistein inhibited caspase-3 activation(p＜0.01).No TUNEL-positive cells were found in the CA1 region of sham-operated rats.In ischemic animals,most of the morphologically damaged neurons were also positive for TUNEL staining at 5 days of reperfusion.The number of TUNEL-positive cells was reduced by treatment with genistein(p＜0.01).5.Effect of genistein on the generation of ROSIn ischemic animals,ROS detection in isolated hippocampal mitochondria increased at 4 h,returned to basal levels at 24 h,but increased again at 48 h of reperfusion.Treatment with genistein decreased the elevated ROS levels(p＜0.01).6.Effect of genistein on lipid peroxidationIn ischemic animals,malondialdehyde(MDA)levels in hippocampus increased at 4 h,returned to lower levels at 24 h,but increased again at 48 h of reperfusion. Treatment with genistein decreased the elevated MDA levels in the hippocampus(p＜0.01).These results suggest that transient global cerebral ischemia results in DND in the hippocampal CA1 region.Genistein protects neurons from cerebral I/R injury in rat hippocampus by attenuating oxidative stress,lipid peroxidation and the signaling cascade leading to apoptotic cell death.Conclusion1.eretreatment of slices with DIA(ⅰ)conferred neuroprotection against ischemia-induced damage in rat hippocampal slices and(ⅱ)increased ROS levels. The effects induced by DIA were attenuated by a mitoKATP blocker.eretreatment with a ROS scavenger also decreased the effects induced by DIA, while treatment with MPG alone had no effect during normoxia and ischemia. These results indicate that ROS participate in the neuroprotection conferred by a mitoKATP opener during ischemia.2.Treatment with genistein attenuated ischemia-induced DND in the hippocampal CA1 region.Genistein administration also decreased ROS generation,MDA concentration,and the apoptotic indices.These results suggest that genistein protects neurons from transient global cerebral I/R injury in rat hippocampus by attenuating oxidative stress,lipid peroxidation and the signaling cascade leading to apoptotic cell death which is caused by excessive production of ROS during cerebral I/R.