Delayed Neuroprotection Induced By Sevoflurane Preconditioning Depends On Mitochondrial ATP-sensitive Potassium Channel And Reactive Oxygen Species

Partâ… :The delayed neuroprotection induced by sevoflurane following focal cerebral ischemia-reperfusionObjective To investigate the delayed protective effects of 2.4%and 4.0%sevoflurane inducing 24 h before focal cerebral ischemia-reperfusion injury in rats.Methods Adult male Sprague-Dawley rats(250～280 g) were randomly assigned into four groups:Sham,Ischemia-reperfusion(I/R),2.5%Sevoflurane(Sevol), 4.0%Sevoflurane(Sevo2) groups,and subjected to right middle cerebral artery occlusion(MCAO) for two hours expect sham group.Sevoflurane preconditioning was induced 24 h before brain ischemia by exposing the animals to 2.4%or 4.0%sevoflurane + oxygen for 60 min.In the Sham, I/R:animal were exposed to 100%oxygen 60 min at 24h before MCAO. Neurological deficit scores and brain infarct volumes were evaluated 6, 24 and 72 h,respectively after reperfusion.In a separate experiment,we measured the physiological variables in twelve additional rats weighing 220 to 280g during oxygen or sevoflurane treatment.The animals were randomly assigned into one of three groups(n=4 each): Ischemia-reperfusion(I/R),2.4%sevoflurane(Sevol),4.0%sevoflurane (Sevo2).Anesthesia was induced with 4%sevoflurane and was maintained with 2.4%sevoflurane delivered by a mask.The right femoral artery was cannulated for continuous monitoring of blood pressure and for arterial blood sampling.A rectal probe was inserted to monitor core temperature.Then the animals were put into the container for oxygen or sevoflurane treatment(100%oxygen,2.4%or 4.0%sevoflurane in oxygen for one hour).Arterial blood gases and plasma glucose were measured five minutes before sevoflurane treatment and at the end of the treatments.Result Animals in the Sevol and Sevo2 groups developed smaller brain infarct volumes than I/R group after 2 h of MCAO followed by 6,24 and 72 h reperfusion,respectively.Neurological deficit scores were also significantly improved in rats pretreated with sevoflurane.No differences were found in rectal temperature,mean arterial blood pressure, arterial pH,PaCO₂,PaO₂,and blood glucose levels during treatment with oxygen or 2.4%sevoflurane.However,mean arterial blood pressure and arterial pH decreased and PaCO₂ increased in Sevo 2 group,compared with I/R and Sevol groups.Concision Sevoflurane could induce the delayed neuroprotection against cerebral ischemia-reperfusion injury. However,blood gas alterations and arterial hypotension might have been present during 4.0%sevoflurane exposure.Partâ…¡:Sevoflurane delayed preconditioning stimulated PKCεcellular translocation through mitochondrial ATP-sensitive potassium channel and reactive oxygen speciesObjective This study tested inhaled sevofiurane is capable of producing a delayed neuroprotection and investigated the effect of sevoflurane on the cellular translocation of PKCεandδand identified mitoK_ATP channel and ROS as mediators in this neuroprotection. Methods One hundred and fourty healthy adult male Sprague-Dawley rats weighing 220～300 g were randomly assigned into seven groups: Sham,Ischemia-reperfusion(I/R),Sevoflurane(Sevo),5-Hydroxydecanoate (5-HD,a selective antagonist for mitoK_ATP channel) + sevoflurane (5-HD+Sevo),2-mercaptopropionylglycine(2-MPG,a selective ROS scavenger) + sevoflurane(MPG+Sevo),5-HD and MPG groups,and subjected to right middle cerebral artery occlusion(MCAO) for two hours expect sham group.Sevoflurane preconditioning was induced 24 h before brain ischemia in sevoflurane,5-HD+Sevo and MPG+Sevo groups by exposing the animals to 2.4%sevoflurane + 97.6%oxygen for 60 min.In the Sham,I/R,5-HD and MPG groups:animals were exposed to 100% oxygen 60 min at 24 h before MCAO.A selective ROS scavenger, 2-mercaptopropionylglycine(20mg/kg,i.v.) or a selective antagonist for mitoK_ATP channel,5-HD(40 mg/kg,i.p.) was administrated 30 min before sevoflurane/oxygen exposure in the MPG+Sevo,MPG groups and 5-HD+Sevo,5-HD groups to evaluated the role of ROS and mitoK_ATP channel on sevoflurane late preconditioning.Neurological deficit scores (NDS) and infarct volumes were evaluated 6,24 and 72 hours of reperfusion after 2-hour MCAO,respectively.Cellular translocation of PKC-εand -δalso was determined by western-blot analysis at 6 and 24 hours after reperfusion.Results:Animals in the sevoflurane group developed lower neurological deficit scores and smaller brain infarct volumes than I/R group(P＜0.05).This protection was reversed by administration of 5-HD and 2-MPG,but no distinguished difference among I/R,5-HD and MPG groups(P＞0.05).Compared with the I/R group,PKC-ε,not PKC-δ,was activated and translocated to the membrane fraction only at 6 h but not 24 h after reperfusion,induced by sevoflurane late preconditioning(P＜0.05),and this effect was also abolished by 5-HD and 2-MPG.Conclusion:Delayed neuroprotection of sevoflurane is mediated by mitoK_ATP channel and ROS.Furthermore, PKC-εactivation probably occurs downstream of mitoK_ATP channel and ROS in the sevoflurane late preconditioning signaling cascade.Partâ…¢:Sevoflurane preconditioning induces a time-dependent change in the phosphorylated p38 and the role of mitochondrial K_ATP -sensitive channelObjective To investigate the effect of sevoflurane late preconditioning to the phosphorylated p38 in the cerebral cortex of the rats and to observe the role of mitochondrial K_ATP-Sensitive channel. Methods This experiment was invidided into two parts:A and B.A):The samples were taken at the points of 0,2,6,12,24,72 h and 3 d after sevoflurane exposure and the expression of the phosphorylated p38 were measured with Western-bolt.B):50 healthy adult male Sprague-Dawley rats weighing 220～300 g were randomly assigned into six groups: Ischemia-reperfusion(I/R),Sevoflurane(Sevo),5-Hydroxydecanoate (5-HD,a selective antagonist for mitoK_ATP channel) + sevoflurane (5-HD+Sevo),SB203580(a p38MAPK inhibitor) + sevoflurane (SB+Sevo),5-HD and SB groups,and subjected to right middle cerebral artery occlusion(MCAO) for two hours.Sevoflurane preconditioning was induced 24 h before brain ischemia in sevoflurane,5-HD+Sevo and SB+Sevo groups by exposing the animals to 2.4%sevoflurane + 97.6% oxygen for 60 min.In the I/R,5-HD and SB groups:animals were exposed to 100%oxygen 60 min at 24 h before MCAO.A selective p38MAPK inhibitor,SB203580(0.2mg/kg,i.p.) or a selective antagonist for mitoK_ATP channel,5-HD(40 mg/kg,i.p.) was administrated 30 min before sevoflurane/oxygen exposure in the SB+Sevo,SB groups and 5-HD+Sevo,5-HD groups to evaluated the role of p38MAPK and mitoK_ATP channel on sevoflurane late preconditioning.Neurological deficit scores(NDS) and infarct volumes were evaluated 24 hours of reperfusion after 2-hour MCAO.Phosphorylated p38 also was determined by western-blot analysis at 24 hours after reperfusion.Result We found that cerebral necortex of SD rats,after being exposure to 2.4% sevoflurane for 60 min,had a higher level of the phosphorylated p38MAPK than that of control rats.This increase in the active p38MAPK was statistically significant at 2 h after sevoflurane exposure,and peaked at 24 h after the exposure,and lasted for 3 d at least.In the part B, although SB203580 and 5-HD(alone or 30 min before the sevoflurane exposure) did not affect the neurological deficit scores or brain infarct sizes compared with those of MCAO,it abolished the sevoflurane preconditioning-induced neuroprotection.Moreover,the amount of the phosphorylated p38 was decreased in the 5-HD +Sevo and SB+Sevo group,suggesting that p38MARK may be a downstream target of mitoK_ATP channel and pay a critical for sevoflurane late preconditioning-induced neuroprotection.Conclusion Sevoflurane preconditioning may improve delayed neuroprotection after focal brain ischemia and that effects may be mediated by mitoK_ATP channel and its downstream target- p3 8MARK.Partâ…£:Effect of sevoflurane preconditioning on neuronal apoptosis after cerebral ischemic reperfusion in ratsObject T o study the effect of sevoflurane on neuronal apoptosis, Cyt C and caspase-3 protein expression after focal cerebral ischemia in rats,and to disclose the mechanism of neuroprotection by sevoflurane late preconditioning.Methods One hundred and fourty healthy adult male Sprague-Dawley rats weighing 220～300 g were randomly assigned into seven groups:Sham,Ischemia-reperfusion(I/R),Sevoflurane(Sevo), 5-Hydroxydecanoate(5-HD,a selective antagonist for mitoK_ATP channel) + sevoflurane(5-HD+Sevo),2-mercaptopropionylglycine(2-MPG,a selective ROS scavenger) + sevoflurane(MPG+Sevo),5-HD and MPG groups,and subjected to right middle cerebral artery occlusion(MCAO) for two hours expect sham group.Sevoflurane preconditioning was induced 24 h before brain ischemia.After survival period of 6h,24 h and 3d,the rats were anesthetized and the brains were removed.Cyt C release and caspase-3 activation were determined by an enzyme activity assay, immunohistological staining and Western blot.Apoptosis was also determined by terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-biotin nick end-labeling(TUNEL) staining after 24 h reperfusion among groups.Result Se voflurane preconditioning reduced the release of cytochrome c and the activation of caspase-3 in the penumbra,compared with I/R group,after survival period of 6 h,24 h and 3d.This protection was reversed by administration of 5-HD and 2-MPG,but no distinguished difference among I/R,5-HD and MPG groups.TUNEL-positive cells were observed 24 h after reperfusion in cortex and striatum respectively.Sevoflurane reduced the number of TUNEL-positive cells,and this effect was also abolished by 5-HD and 2-MPG.Conclusion Sevoflurane play a neuroprotective role by inhibiting the neuronal apoptosis and the release of cytochrome c and the activation caspase-3,which were induced by cerebral ischemia reperfusion injury.The delayed neuroprotection of sevoflurane may be mediated by mitoK_ATP channel and ROS.Partâ…¤:The delayed neuroprotection of sevoflurane preconditioning by activating the mitochondtial ATP-sensitive K⁺ channel is mediated by inhibiting the mitochondrial permeability transition poreObject In order to further explore the sevoflurane-induced delayed neuroprotective mechanisms by which activation of mitochondtial ATP-sensitive K⁺ channel,we investigate the role of mitochondrial permeability transition pore(MPTP) in in vivo models.Methods Male Sprague-Dawley rats weighing 220～300 g were randomly assigned into five groups:Ischemia-reperfusion(I/R),Sevoflurane(Sevo), 5-Hydroxydecanoate(5-HD,a selective antagonist for mitoK_ATP channel) + sevoflurane(5-HD+Sevo),5-HD and subjected to right middle cerebral artery occlusion(MCAO) for two hours followed by reperfusion 24 h. Sevoflurane preconditioning was induced 24 h before brain ischemia in sevoflurane,5-HD+Sevo and by exposing the animals to 2.4% sevoflurane + 97.6%oxygen for 60 min.A selective antagonist for mitoK_ATP channel,5-HD(40 mg/kg,i.p.) was administrated 30 min before sevoflurane/oxygen exposure in the 5-HD+Sevo and 5-HD groups to evaluate the role of mitoK_ATP channel on sevoflurane late preconditioning.Spectrophotometry was used to determine the effect of the mitochondrial ATP-sensitive potassium channel agonists on the swelling of ischemic brain mitochondria and the expression of Bcl-2/Bax were determined by Western blot,respectively.Result Compared with I/R group,sevoflurane could inhibit the decrease of calcium induced mitochondrial absorbance at 520 nm(A520),which were blocked by 5-HD.Sevoflurane could still upregulate the expression of Bcl-2,but had no effect of Bax,which were also blocked by 5-HD.Conclusion Sevoflurane could probably inhibit the mitochondrial permeability transition via activation of mitochondrial ATP sensitive potassium channel and upregulation Bcl-2 expression.