线粒体ATP敏感性钾离子通道及活性氧介导七氟醚迟发型脑保护作用机制探讨
摘要
临床上常见的严重颅脑外伤手术、控制性降压、颅内动脉瘤夹闭术和冠状动脉旁路移植术以及颈动脉内膜剥脱术等的病人都具有潜在脑缺血的可能。例如:为便于脑动脉瘤手术实施和减少术中出血,常将脑动脉血管短暂夹闭,然而随着受阻血管血流的恢复,往往会导致局部性脑组织缺血/再灌注(ischemic-reperfusion injury,I/R)损伤。而在实际工作中,临床医生不仅关心术中的神经系统的保护,更关心术后神经功能的恢复。探求脑缺血损失的发病机制、减轻脑缺血性损伤、降低脑缺血性疾病的致残率和死亡率,已成为近年来神经科学领域广大工作者致力的研究目标。与脑缺血预处理相似,吸入麻醉药预处理与缺血损伤之间也有短暂的重要联系,包括两个重要时期:预处理早期阶段,即中断预处理刺激后5 min～2 h出现;第二个保护时期,延迟预处理期,出现在预处理刺激停止后12～24 h,并可持续24至72 h。已有研究证实七氟醚具有早期预处理的脑保护效应,在大鼠海马脑片模型中,反复吸入七氟醚(每天一次持续30 min,连续4天)能够诱导迟发型脑保护作用。目前,仍未知单次给予七氟醚是否能够诱导迟发型脑保护效应,值得进一步研究。大量研究证实,线粒体内膜ATP敏感钾通道(mitochondrial ATP-sensitive potassium channels,mitoK_(ATP)),是吸入麻醉药预处理产生心肌保护作用的重要机制之一。而脑组织的mitoK_(ATP)通道蛋白浓度是心肌含量的6～7倍。因此我们有理由推测mitoK_(ATP)通道参与了七氟醚的迟发型脑保护作用。开放后的mitoK_(ATP)通道如何进一步介导脑保护作用?现在认为活性氧(reactive oxygen species,ROS)可能作为一种关键的细胞内信息物质,介导缺血缺氧预处理和mitoK_(ATP)开放剂预处理的心肌及脑保护作用。因此本课题探讨脑遭受缺血再灌注损伤前24 h,单次给予60 min七氟醚预处理是否产生迟发型脑保护效应,以及mitoK_(ATP)通道及ROS的作用。
本课题首先通过观察缺血后大鼠的神经功能及测定脑梗塞容积百分比,并通过HE染色评定CA1区神经元形态学改变,明确不同浓度的七氟醚对SD大鼠产生迟发型的脑保护作用;其次第二部分通过加入特异性mitoK_(ATP)通道阻断剂及ROS清除剂,观察缺血后大鼠的神经功能及测定脑梗塞容积百分比和缺血侧项叶皮层神经元PKC-ε和-δ两种同功酶的膜转位变化,探讨七氟醚迟发型脑保护作用可能是通过mitoK_(ATP)通道和ROS所介导的,且PKCε和δ是否作为mitoK_(ATP)通道的下游靶点参与了七氟醚迟发型脑保护效应;之后,进一步研究和探讨p38MAPK信号通路可能也为mitoK_(ATP)通道的下游蛋白信号分子,参与了七氟醚诱导迟发型脑保护中的作用;随后,通过检测缺血后胞浆细胞色素C(Cytochrome C,Cyt C)的释放和Caspase-3活性变化,以及TUNEL染色等证实七氟醚迟发型预处理可以通过mitoK_(ATP)通道及ROS介导,抑制缺血再灌注损伤后神经元的凋亡,明确七氟醚迟发型脑保护机制;最后通过用紫外分光光度计测定各组缺血侧皮层神经元线粒体通透转运通道(MPTP)开放程度,Western-blot检测Bcl-2/Bax蛋白表达情况,探讨七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响及可能机制,证实了激活和开放mitoK_(ATP)通道在七氟醚抑制MPTP开放发挥迟发型脑保护效应中起到重要作用。结果证实:(1)缺血前24h,短暂的予以2.4%及4.0%的七氟醚预处理可增强大鼠耐受局灶性脑缺血损伤,产生显著的迟发型脑保护效应,但短时间高浓度(4.0%)吸入七氟醚有引起动脉血pH值及MAP降低的趋势。(2)mitoK_(ATP)通道和活性氧介导七氟醚对局灶性脑缺血再灌注损伤的迟发型保护作用,其机制可能与调控PKC-ε转位激活有关,但PKC-ε转位激活仅发生于脑保护的早期相,提示可能有其他的信号通路机制参与七氟醚迟发型脑保护作用。(3)七氟醚可以诱导大脑皮层磷酸化p38MAPK表达的增加,且p38MAPK可能做为线粒体ATP敏感性钾离子通道抗脑缺血再灌注损伤中的下游通路,参与了七氟醚预处理的迟发型脑保护作用。(4)七氟醚通过抑制缺血后细胞色素c释放、Caspase-3的激活以及神经元的凋亡发挥迟发型脑保护作用,其作用可能与线粒体ATP敏感性钾离子通道(mitoK_(ATP))以及活性氧有关。(5)七氟醚预处理能通过激活mitoK_(ATP)通道上调bcl-2蛋白的表达,抑制MPTP开放发挥迟发型脑保护作用。
总之,脑缺血缺氧所致的神经元损伤并非是单一的病理生理过程,而是一系列复杂的生化级联反应。七氟醚的脑保护作用也并非单一作用于上述的某一环节,而是多位点、多途径、交互作用的结果。
第一部分七氟醚对大鼠缺血再灌注损伤的迟发型脑保护作用
目的观察缺血前24 h单次予以2.4%或4.0%的七氟醚(sevoflurane,Sevo)60 min是否产生迟发型的脑保护作用。方法健康SD雄性大鼠60只,体重250～280 g,随机分为4组:假手术组(S组),单纯缺血再灌注组(IR组),2.4%七氟醚组(Sevol组),4.0%七氟醚组(Sevo2组)。七氟醚预处理组在制备MCAO模型前24h,吸入浓度为2.4%或4.0%七氟醚+氧气60 min;而S组和IR组在制备MCAO模型前24 h,吸入浓度为100%氧气60 min。24 h后用10%水合氯醛(300～350mg/kg)麻醉大鼠后,分离右颈总动脉、颈内动脉和颈外动脉,结扎颈外动脉和颈总动脉近端,经颈总动脉向颈内动脉插入栓线,放置到大脑前动脉起始部,阻断大脑中动脉。S组只分离血管,不阻断大脑中动脉的血流。缺血2 h,再灌注6,24,72 h后,用神经功能缺陷评分观察动物神经行为学改变、TTC染色法测大鼠脑梗死容积百分比。额外取12只雄性成年SD大鼠随机分为IR组,Sevo1组,Sevo2组。均用4.0%七氟醚(氧流量4.0 L/min)动物麻醉面罩吸入诱导麻醉,2.4%七氟醚(氧流量1.5～2.0 L/min)面罩持续吸入维持麻醉深度。分离、暴露右侧股动脉,动脉置管后缝合局部皮肤。停用七氟醚,待大鼠清醒后,放入七氟醚麻醉预处理箱内,接好持续动脉测压,体温测量装置。Sevo1组,Sevo2组通过麻醉气体挥发罐持续输入含有2.4%或4.0%七氟醚的O_2(4L/min),而IR组持续输入100%的纯氧。三组大鼠均保留自主呼吸。在七氟醚或氧气预处理前5 min以及结束时抽取动脉血检测动脉血气、血糖并记录平均动脉压(MAP),心率(HR),体温(T)等参数。结果IR组缺血2 h,再灌注6,24,72 h后,神经功能缺陷评分分别为4(2～6),4(3～6)及4(3～6),脑梗死容积百分比分别为22.6±4.0%,33.7±4.3%,31.1±3.4%。与IR组相比,缺血2 h,再灌注6,24,72h后,Sevo1组和Sevo2组均明显改善神经功能及显著减少脑梗死面积(P＜0.05),神经功能缺陷评分Sevo1组分别为2(1～5),3(1～4),3(2～5),Sevo2组分别为2(1～4),3(2～4),3(1～5)。脑梗死容积百分比Sevo1组分别为:14.3±3.5%,23.4±4.7%,25.7±3.0%;Sevo2组分别为:15.6±4.5%,23.8±3.7%,27.1±4.0%。但Sevo1组与Sevo2组之间神经功能缺陷评分及脑梗死容积差异无统计学意义(P＞0.05)。与IR组相比较,Sevo1组,pH值,动脉血二氧化碳分压(PaCO_2),动脉血氧分压(PaO_2),血糖(BS)以及血流动力学参数(MAP,HR),体温(T)均无显著差异;但Sevo2组pH值较IR组及Sevo1组有所降低,差异有统计学意义。结论缺血前24 h,短暂的七氟醚预处理可增强大鼠耐受局灶性脑缺血损伤,产生显著的迟发型脑保护效应。短时间高浓度吸入七氟醚有引起动脉血pH值及MAP降低的趋势。
第二部分PKCε参与七氟醚预处理诱导的迟发型脑保护作用及mitoK_(ATP)通道和ROS的影响
目的探讨七氟醚预处理对大鼠局灶性缺血再灌注损伤的迟发型脑保护作用和对PKC-ε,δ转位激活的影响以及与mitoK_(ATP)通道和活性氧(ROS)生成的关系。方法健康雄性SD大鼠,体质量220～300 g,采用线栓法制备大鼠局灶性脑缺血模型(middle cerebral arteryocclusion,MCAO)并随机分为7组:假手术组(S组)、缺血再灌注组(I/R组)、七氟醚预处理组(Sevo组)、mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)+七氟醚(5-HD+Sevo组)、ROS清除剂2-硫基丙酰氨基乙酸(2-MPG)+七氟醚(MPG+Sevo组)和单纯5-HD组及MPG组。除假手术组外,其余各组大鼠阻闭右侧大脑中动脉2 h,再灌注6,24和72 h。假手术组(S);缺血再灌注组(I/R):吸入100%纯氧60min,24 h后行大脑中动脉阻闭(MCAO),缺血2 h,再灌注6,24,和72 h;七氟醚组(Sevo):制备MCAO模型前24 h,吸入浓度为2.4%七氟醚+97.6%氧气60 min;5-HD+Sevo组:七氟醚预处理之前30 min,腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)40mg/kg,余同七氟醚组;MPG+七氟醚组(MPG+Sevo):七氟醚预处理之前30 min,经尾静脉予以ROS清除剂MPG 20 mg/kg,余同七氟醚组;单纯5-HD组:吸入氧气前30 min,腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)40 mg/kg,余同I/R组;单纯MPG组(MPG):吸入氧气前30 min,尾静脉给予MPG 20 mg/kg,余同I/R组。缺血2h,再灌注6,24和72 h后进行神经功能缺陷评分(NDS)并取脑组织,运用TTC染色测算脑梗死容积百分比,再灌注6,24 h后运用Western-Blot法测定PKC-ε,δ膜转位水平。结果I/R组相比,七氟醚能明显改善缺血后的神经功能,减小脑梗死面积,而发挥迟发型脑保护作用(P＜0.05),七氟醚预处理前30 min腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)(40 mg/kg)或尾静脉给予ROS清除剂2-硫基丙酰氨基乙酸(2-MPG)(20 mg/kg)能拮抗七氟醚迟发型脑保护效应(P＜0.05),单独使用5-HD及MPG则无明显影响(P＞0.05)。I/R组,MPG+Sevo组,MPG组之间脑梗死容积以及神经功能评分差异无统计学意义(P＞0.05)。此外,与I/R组相比,七氟醚显著促进PKC-ε,而非PKC-δ的膜转位激活,且仅发生于再灌注后6 h(P＜0.05),七氟醚的此效应同样可以被mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)或ROS清除剂2-MPG所废止。结论mitoK_(ATP)通道和活性氧介导七氟醚对局灶性脑缺血再灌注损伤的迟发型保护作用,其机制可能与调控PKC-ε转位激活有关。
第三部分p38MAPK参与七氟醚预处理迟发型脑保护效应及线粒体ATP敏感性钾离子通道的作用
目的研究七氟醚迟发型预处理对大鼠皮层脑组织p38蛋白磷酸化激活的影响以及线粒体ATP敏感性钾离子通道的作用。方法实验分两部分,A)40只SD实验大鼠结束七氟醚预处理前0 h及七氟醚预处理后2 h、6 h、12 h、24 h、3 d,7d通过Western-bolt法检测p-p38MAPK表达情况;B)50只SD大鼠随机分为6组:缺血再灌注组(I/R)、七氟醚组(Sevo)、5-HD+七氟醚组(5-HD+Sevo)、SB 203580+七氟醚组、单纯SB 203580(SB)和单纯5-HD组(5-HD)。缺血2 h,再灌注24后进行神经行为学评分,TTC法检测脑梗死容积百分比以及Western-bolt法检测缺血侧顶叶皮层神经元磷酸化p38MAPK(p-p38MAPK)的水平。结果A):与对照组(七氟醚预处理前,即0 h)相比较,吸入七氟醚后2 h,p38MAPK磷酸化(p-p38MARK)明显增加,随着再灌注时间延长,p-p38MARK表达也逐渐增强,于再灌注后24 h达到高峰,并可至少持续3 d,再灌注7 d后基本回落至初始水平。B)与I/R组相比较,七氟醚预处理组能显著改善大鼠缺血后神经功能和明显减小脑梗死面积,但是七氟醚预处理前使用5-HD及SB203580干预,可以取消七氟醚预处理的脑保护作用。而与I/R组相比较,单独使用5-HD及SB203580对实验结果无明显影响。结论七氟醚可以诱导大脑皮层磷酸化p38MAPK表达的增加,且p38MAPK可能做为线粒体ATP敏感性钾离子通道抗脑缺血再灌注损伤中的下游通路,参与了七氟醚预处理的迟发型脑保护作用。
第四部分七氟醚预处理对局灶性脑缺血再灌注损伤神经元凋亡的影响及机制
目的探讨七氟醚预处理对局灶性脑缺血再灌注损伤后神经元凋亡、Caspase-3激活以及细胞色素c释放的影响。方法利用MACO法建立大鼠局灶性脑缺血模型,以酶活性测定、Western Blot免疫组化和TUNEL法对Caspase-3活性变化和激活、细胞色素c释放以及神经元凋亡进行规律性观察。结果缺血2 h,再灌注6,24,72 h后,假手术组(Sham)胞浆未见明显的细胞色素C释放及活性Caspase-3。与Sham组相比较,缺血再灌注组(I/R)细胞色素C胞浆释放及活性Caspase-3明显增加。与I/R组相比较,七氟醚预处理显著减少细胞色素C的胞浆释放及活性Caspase-3增加。七氟醚预处理前给与线粒体ATP敏感性钾离子通道(mitoK_(ATP))抑制剂5-HD以及活性氧(ROS)清除剂2-MPG,可以废除七氟醚抑制细胞色素C释放及活性Caspase-3增加的效应(P＜0.05),但与I/R组相比较,单纯给与5-HD及2-MPG,对缺血后各时点细胞色素C的胞浆释放无明显影响。缺血2 h,再灌注24 h后使用TUNEL法检测凋亡神经元显示,与I/R组相比,七氟醚能明显减少缺血后神经元的凋亡,同样线粒体ATP敏感性钾离子通道(mitoK_(ATP))抑制剂5-HD以及活性氧(ROS)清除剂2-MPG,可以废除七氟醚抑制神经元凋亡的效应。
结论七氟醚通过抑制缺血后细胞色素c释放、Caspase-3的激活以及神经元的凋亡发挥迟发型脑保护作用,其作用可能与线粒体ATP敏感性钾离子通道(mitoK_(ATP))以及活性氧有关。
第五部分七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响
目的探讨七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响及可能机制。方法雄性SD大鼠随机分为5组:假手术组(Sham组);缺血损伤组(I/R组):吸入纯氧60 min,24h后行大脑中动脉阻塞2 h,再灌注24 h造成局灶性脑缺血再灌注;七氟醚预处理组(Sevo组):缺血前24 h吸入2.4%七氟醚60 min;5-HD+Sevo组:腹腔内注射5-HD 40 mg/kg,30 min后同Sevo组。5-HD组:腹腔内注射5-HD 40 mg/kg,30 min后同I/R组,用紫外分光光度计测定各组缺血侧皮层神经元线粒体通透转运通道(MPTP)开放程度,Western-blot检测Bcl-2/Bax蛋白表达情况。结果I/R组线粒体(_(max) A_(520)—_(min) A_(520))呈现快速下降,且比假手术组更为显著(P＜0.05);与I/R组相比较,七氟醚预处理组(Sevo)能缓解Ca~(2+)诱导的(_(max)A_(520)—_(min)A_(520))的下降(P＜0.05);但七氟醚预处理前腹腔内注射线粒体ATP敏感性钾离子通道特异性抑制剂5-HD 40mg/kg,可取消七氟醚的此作用,而单独使用5-HD则无影响。同样,与I/R组比较,七氟醚可明显增加bcl-2蛋白表达(P＜0.05);七氟醚预处理前使用5-HD,可以取消七氟醚增加bcl-2蛋白表达的效应(P＜0.05);除假手术组外,各组间Bax的表达差异无统计学意义。结论七氟醚预处理能通过激活mitoK_(ATP)通道上调bcl-2蛋白的表达,抑制MPTP开放发挥迟发型脑保护作用
PartⅠ:The delayed neuroprotection induced by sevoflurane following focal cerebral ischemia-reperfusion
Objective 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,PaCO_2,PaO_2,and blood glucose levels during treatment with oxygen or 2.4%sevoflurane.However,mean arterial blood pressure and arterial pH decreased and PaCO_2 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.
PartⅡ:Sevoflurane delayed preconditioning stimulated PKCεcellular translocation through mitochondrial ATP-sensitive potassium channel and reactive oxygen species
Objective 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.
PartⅢ:Sevoflurane preconditioning induces a time-dependent change in the phosphorylated p38 and the role of mitochondrial K_(ATP) -sensitive channel
Objective 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.
PartⅣ:Effect of sevoflurane preconditioning on neuronal apoptosis after cerebral ischemic reperfusion in rats
Object 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.
PartⅤ:The delayed neuroprotection of sevoflurane preconditioning by activating the mitochondtial ATP-sensitive K~+ channel is mediated by inhibiting the mitochondrial permeability transition pore
Object 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.
本课题首先通过观察缺血后大鼠的神经功能及测定脑梗塞容积百分比,并通过HE染色评定CA1区神经元形态学改变,明确不同浓度的七氟醚对SD大鼠产生迟发型的脑保护作用;其次第二部分通过加入特异性mitoK_(ATP)通道阻断剂及ROS清除剂,观察缺血后大鼠的神经功能及测定脑梗塞容积百分比和缺血侧项叶皮层神经元PKC-ε和-δ两种同功酶的膜转位变化,探讨七氟醚迟发型脑保护作用可能是通过mitoK_(ATP)通道和ROS所介导的,且PKCε和δ是否作为mitoK_(ATP)通道的下游靶点参与了七氟醚迟发型脑保护效应;之后,进一步研究和探讨p38MAPK信号通路可能也为mitoK_(ATP)通道的下游蛋白信号分子,参与了七氟醚诱导迟发型脑保护中的作用;随后,通过检测缺血后胞浆细胞色素C(Cytochrome C,Cyt C)的释放和Caspase-3活性变化,以及TUNEL染色等证实七氟醚迟发型预处理可以通过mitoK_(ATP)通道及ROS介导,抑制缺血再灌注损伤后神经元的凋亡,明确七氟醚迟发型脑保护机制;最后通过用紫外分光光度计测定各组缺血侧皮层神经元线粒体通透转运通道(MPTP)开放程度,Western-blot检测Bcl-2/Bax蛋白表达情况,探讨七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响及可能机制,证实了激活和开放mitoK_(ATP)通道在七氟醚抑制MPTP开放发挥迟发型脑保护效应中起到重要作用。结果证实:(1)缺血前24h,短暂的予以2.4%及4.0%的七氟醚预处理可增强大鼠耐受局灶性脑缺血损伤,产生显著的迟发型脑保护效应,但短时间高浓度(4.0%)吸入七氟醚有引起动脉血pH值及MAP降低的趋势。(2)mitoK_(ATP)通道和活性氧介导七氟醚对局灶性脑缺血再灌注损伤的迟发型保护作用,其机制可能与调控PKC-ε转位激活有关,但PKC-ε转位激活仅发生于脑保护的早期相,提示可能有其他的信号通路机制参与七氟醚迟发型脑保护作用。(3)七氟醚可以诱导大脑皮层磷酸化p38MAPK表达的增加,且p38MAPK可能做为线粒体ATP敏感性钾离子通道抗脑缺血再灌注损伤中的下游通路,参与了七氟醚预处理的迟发型脑保护作用。(4)七氟醚通过抑制缺血后细胞色素c释放、Caspase-3的激活以及神经元的凋亡发挥迟发型脑保护作用,其作用可能与线粒体ATP敏感性钾离子通道(mitoK_(ATP))以及活性氧有关。(5)七氟醚预处理能通过激活mitoK_(ATP)通道上调bcl-2蛋白的表达,抑制MPTP开放发挥迟发型脑保护作用。
总之,脑缺血缺氧所致的神经元损伤并非是单一的病理生理过程,而是一系列复杂的生化级联反应。七氟醚的脑保护作用也并非单一作用于上述的某一环节,而是多位点、多途径、交互作用的结果。
第一部分七氟醚对大鼠缺血再灌注损伤的迟发型脑保护作用
目的观察缺血前24 h单次予以2.4%或4.0%的七氟醚(sevoflurane,Sevo)60 min是否产生迟发型的脑保护作用。方法健康SD雄性大鼠60只,体重250～280 g,随机分为4组:假手术组(S组),单纯缺血再灌注组(IR组),2.4%七氟醚组(Sevol组),4.0%七氟醚组(Sevo2组)。七氟醚预处理组在制备MCAO模型前24h,吸入浓度为2.4%或4.0%七氟醚+氧气60 min;而S组和IR组在制备MCAO模型前24 h,吸入浓度为100%氧气60 min。24 h后用10%水合氯醛(300～350mg/kg)麻醉大鼠后,分离右颈总动脉、颈内动脉和颈外动脉,结扎颈外动脉和颈总动脉近端,经颈总动脉向颈内动脉插入栓线,放置到大脑前动脉起始部,阻断大脑中动脉。S组只分离血管,不阻断大脑中动脉的血流。缺血2 h,再灌注6,24,72 h后,用神经功能缺陷评分观察动物神经行为学改变、TTC染色法测大鼠脑梗死容积百分比。额外取12只雄性成年SD大鼠随机分为IR组,Sevo1组,Sevo2组。均用4.0%七氟醚(氧流量4.0 L/min)动物麻醉面罩吸入诱导麻醉,2.4%七氟醚(氧流量1.5～2.0 L/min)面罩持续吸入维持麻醉深度。分离、暴露右侧股动脉,动脉置管后缝合局部皮肤。停用七氟醚,待大鼠清醒后,放入七氟醚麻醉预处理箱内,接好持续动脉测压,体温测量装置。Sevo1组,Sevo2组通过麻醉气体挥发罐持续输入含有2.4%或4.0%七氟醚的O_2(4L/min),而IR组持续输入100%的纯氧。三组大鼠均保留自主呼吸。在七氟醚或氧气预处理前5 min以及结束时抽取动脉血检测动脉血气、血糖并记录平均动脉压(MAP),心率(HR),体温(T)等参数。结果IR组缺血2 h,再灌注6,24,72 h后,神经功能缺陷评分分别为4(2～6),4(3～6)及4(3～6),脑梗死容积百分比分别为22.6±4.0%,33.7±4.3%,31.1±3.4%。与IR组相比,缺血2 h,再灌注6,24,72h后,Sevo1组和Sevo2组均明显改善神经功能及显著减少脑梗死面积(P＜0.05),神经功能缺陷评分Sevo1组分别为2(1～5),3(1～4),3(2～5),Sevo2组分别为2(1～4),3(2～4),3(1～5)。脑梗死容积百分比Sevo1组分别为:14.3±3.5%,23.4±4.7%,25.7±3.0%;Sevo2组分别为:15.6±4.5%,23.8±3.7%,27.1±4.0%。但Sevo1组与Sevo2组之间神经功能缺陷评分及脑梗死容积差异无统计学意义(P＞0.05)。与IR组相比较,Sevo1组,pH值,动脉血二氧化碳分压(PaCO_2),动脉血氧分压(PaO_2),血糖(BS)以及血流动力学参数(MAP,HR),体温(T)均无显著差异;但Sevo2组pH值较IR组及Sevo1组有所降低,差异有统计学意义。结论缺血前24 h,短暂的七氟醚预处理可增强大鼠耐受局灶性脑缺血损伤,产生显著的迟发型脑保护效应。短时间高浓度吸入七氟醚有引起动脉血pH值及MAP降低的趋势。
第二部分PKCε参与七氟醚预处理诱导的迟发型脑保护作用及mitoK_(ATP)通道和ROS的影响
目的探讨七氟醚预处理对大鼠局灶性缺血再灌注损伤的迟发型脑保护作用和对PKC-ε,δ转位激活的影响以及与mitoK_(ATP)通道和活性氧(ROS)生成的关系。方法健康雄性SD大鼠,体质量220～300 g,采用线栓法制备大鼠局灶性脑缺血模型(middle cerebral arteryocclusion,MCAO)并随机分为7组:假手术组(S组)、缺血再灌注组(I/R组)、七氟醚预处理组(Sevo组)、mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)+七氟醚(5-HD+Sevo组)、ROS清除剂2-硫基丙酰氨基乙酸(2-MPG)+七氟醚(MPG+Sevo组)和单纯5-HD组及MPG组。除假手术组外,其余各组大鼠阻闭右侧大脑中动脉2 h,再灌注6,24和72 h。假手术组(S);缺血再灌注组(I/R):吸入100%纯氧60min,24 h后行大脑中动脉阻闭(MCAO),缺血2 h,再灌注6,24,和72 h;七氟醚组(Sevo):制备MCAO模型前24 h,吸入浓度为2.4%七氟醚+97.6%氧气60 min;5-HD+Sevo组:七氟醚预处理之前30 min,腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)40mg/kg,余同七氟醚组;MPG+七氟醚组(MPG+Sevo):七氟醚预处理之前30 min,经尾静脉予以ROS清除剂MPG 20 mg/kg,余同七氟醚组;单纯5-HD组:吸入氧气前30 min,腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)40 mg/kg,余同I/R组;单纯MPG组(MPG):吸入氧气前30 min,尾静脉给予MPG 20 mg/kg,余同I/R组。缺血2h,再灌注6,24和72 h后进行神经功能缺陷评分(NDS)并取脑组织,运用TTC染色测算脑梗死容积百分比,再灌注6,24 h后运用Western-Blot法测定PKC-ε,δ膜转位水平。结果I/R组相比,七氟醚能明显改善缺血后的神经功能,减小脑梗死面积,而发挥迟发型脑保护作用(P＜0.05),七氟醚预处理前30 min腹腔内注射mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)(40 mg/kg)或尾静脉给予ROS清除剂2-硫基丙酰氨基乙酸(2-MPG)(20 mg/kg)能拮抗七氟醚迟发型脑保护效应(P＜0.05),单独使用5-HD及MPG则无明显影响(P＞0.05)。I/R组,MPG+Sevo组,MPG组之间脑梗死容积以及神经功能评分差异无统计学意义(P＞0.05)。此外,与I/R组相比,七氟醚显著促进PKC-ε,而非PKC-δ的膜转位激活,且仅发生于再灌注后6 h(P＜0.05),七氟醚的此效应同样可以被mitoK_(ATP)通道阻断剂5-羟葵酸盐(5-HD)或ROS清除剂2-MPG所废止。结论mitoK_(ATP)通道和活性氧介导七氟醚对局灶性脑缺血再灌注损伤的迟发型保护作用,其机制可能与调控PKC-ε转位激活有关。
第三部分p38MAPK参与七氟醚预处理迟发型脑保护效应及线粒体ATP敏感性钾离子通道的作用
目的研究七氟醚迟发型预处理对大鼠皮层脑组织p38蛋白磷酸化激活的影响以及线粒体ATP敏感性钾离子通道的作用。方法实验分两部分,A)40只SD实验大鼠结束七氟醚预处理前0 h及七氟醚预处理后2 h、6 h、12 h、24 h、3 d,7d通过Western-bolt法检测p-p38MAPK表达情况;B)50只SD大鼠随机分为6组:缺血再灌注组(I/R)、七氟醚组(Sevo)、5-HD+七氟醚组(5-HD+Sevo)、SB 203580+七氟醚组、单纯SB 203580(SB)和单纯5-HD组(5-HD)。缺血2 h,再灌注24后进行神经行为学评分,TTC法检测脑梗死容积百分比以及Western-bolt法检测缺血侧顶叶皮层神经元磷酸化p38MAPK(p-p38MAPK)的水平。结果A):与对照组(七氟醚预处理前,即0 h)相比较,吸入七氟醚后2 h,p38MAPK磷酸化(p-p38MARK)明显增加,随着再灌注时间延长,p-p38MARK表达也逐渐增强,于再灌注后24 h达到高峰,并可至少持续3 d,再灌注7 d后基本回落至初始水平。B)与I/R组相比较,七氟醚预处理组能显著改善大鼠缺血后神经功能和明显减小脑梗死面积,但是七氟醚预处理前使用5-HD及SB203580干预,可以取消七氟醚预处理的脑保护作用。而与I/R组相比较,单独使用5-HD及SB203580对实验结果无明显影响。结论七氟醚可以诱导大脑皮层磷酸化p38MAPK表达的增加,且p38MAPK可能做为线粒体ATP敏感性钾离子通道抗脑缺血再灌注损伤中的下游通路,参与了七氟醚预处理的迟发型脑保护作用。
第四部分七氟醚预处理对局灶性脑缺血再灌注损伤神经元凋亡的影响及机制
目的探讨七氟醚预处理对局灶性脑缺血再灌注损伤后神经元凋亡、Caspase-3激活以及细胞色素c释放的影响。方法利用MACO法建立大鼠局灶性脑缺血模型,以酶活性测定、Western Blot免疫组化和TUNEL法对Caspase-3活性变化和激活、细胞色素c释放以及神经元凋亡进行规律性观察。结果缺血2 h,再灌注6,24,72 h后,假手术组(Sham)胞浆未见明显的细胞色素C释放及活性Caspase-3。与Sham组相比较,缺血再灌注组(I/R)细胞色素C胞浆释放及活性Caspase-3明显增加。与I/R组相比较,七氟醚预处理显著减少细胞色素C的胞浆释放及活性Caspase-3增加。七氟醚预处理前给与线粒体ATP敏感性钾离子通道(mitoK_(ATP))抑制剂5-HD以及活性氧(ROS)清除剂2-MPG,可以废除七氟醚抑制细胞色素C释放及活性Caspase-3增加的效应(P＜0.05),但与I/R组相比较,单纯给与5-HD及2-MPG,对缺血后各时点细胞色素C的胞浆释放无明显影响。缺血2 h,再灌注24 h后使用TUNEL法检测凋亡神经元显示,与I/R组相比,七氟醚能明显减少缺血后神经元的凋亡,同样线粒体ATP敏感性钾离子通道(mitoK_(ATP))抑制剂5-HD以及活性氧(ROS)清除剂2-MPG,可以废除七氟醚抑制神经元凋亡的效应。
结论七氟醚通过抑制缺血后细胞色素c释放、Caspase-3的激活以及神经元的凋亡发挥迟发型脑保护作用,其作用可能与线粒体ATP敏感性钾离子通道(mitoK_(ATP))以及活性氧有关。
第五部分七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响
目的探讨七氟醚预处理对大鼠局灶性脑缺血再灌注损伤后线粒体通透性转换孔的影响及可能机制。方法雄性SD大鼠随机分为5组:假手术组(Sham组);缺血损伤组(I/R组):吸入纯氧60 min,24h后行大脑中动脉阻塞2 h,再灌注24 h造成局灶性脑缺血再灌注;七氟醚预处理组(Sevo组):缺血前24 h吸入2.4%七氟醚60 min;5-HD+Sevo组:腹腔内注射5-HD 40 mg/kg,30 min后同Sevo组。5-HD组:腹腔内注射5-HD 40 mg/kg,30 min后同I/R组,用紫外分光光度计测定各组缺血侧皮层神经元线粒体通透转运通道(MPTP)开放程度,Western-blot检测Bcl-2/Bax蛋白表达情况。结果I/R组线粒体(_(max) A_(520)—_(min) A_(520))呈现快速下降,且比假手术组更为显著(P＜0.05);与I/R组相比较,七氟醚预处理组(Sevo)能缓解Ca~(2+)诱导的(_(max)A_(520)—_(min)A_(520))的下降(P＜0.05);但七氟醚预处理前腹腔内注射线粒体ATP敏感性钾离子通道特异性抑制剂5-HD 40mg/kg,可取消七氟醚的此作用,而单独使用5-HD则无影响。同样,与I/R组比较,七氟醚可明显增加bcl-2蛋白表达(P＜0.05);七氟醚预处理前使用5-HD,可以取消七氟醚增加bcl-2蛋白表达的效应(P＜0.05);除假手术组外,各组间Bax的表达差异无统计学意义。结论七氟醚预处理能通过激活mitoK_(ATP)通道上调bcl-2蛋白的表达,抑制MPTP开放发挥迟发型脑保护作用
PartⅠ:The delayed neuroprotection induced by sevoflurane following focal cerebral ischemia-reperfusion
Objective 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,PaCO_2,PaO_2,and blood glucose levels during treatment with oxygen or 2.4%sevoflurane.However,mean arterial blood pressure and arterial pH decreased and PaCO_2 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.
PartⅡ:Sevoflurane delayed preconditioning stimulated PKCεcellular translocation through mitochondrial ATP-sensitive potassium channel and reactive oxygen species
Objective 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.
PartⅢ:Sevoflurane preconditioning induces a time-dependent change in the phosphorylated p38 and the role of mitochondrial K_(ATP) -sensitive channel
Objective 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.
PartⅣ:Effect of sevoflurane preconditioning on neuronal apoptosis after cerebral ischemic reperfusion in rats
Object 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.
PartⅤ:The delayed neuroprotection of sevoflurane preconditioning by activating the mitochondtial ATP-sensitive K~+ channel is mediated by inhibiting the mitochondrial permeability transition pore
Object 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.
引文
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