【作者】 夏萍萍；

【导师】 郭曲练；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 临床上脑动脉粥样硬化、严重的颅脑外伤、脑血管栓塞、动脉畸形等病人都有发生脑缺血的可能。脑缺血会严重影响病人的健康,给患者本人、家属及社会造成严重的负担。早期恢复血流是治疗脑缺血的关键。但随着受阻血管血流的恢复,往往会导致局部性脑组织发生缺血再灌注(ischemic-reperfusion, I/R)损伤。大量的研究表明在发生脑缺血前进行预处理能减少缺血再灌注损伤。但由于脑缺血的发生具有不可预知性,故预处理的临床应用受到很大的局限。相对于脑缺血的不可预见性,再灌注发生的时机较易控制。所以人们越来越关注于脑缺血发生后进行的后处理的研究。现已有研究证明了缺血后处理(ischemic postconditioning, IPSC)能降低脑缺血再灌注损伤,促进神经功能的恢复。但缺血后处理毕竟是有创的、伤害性操作,虽然很多动物实验都证实反复多次短暂的缺血并不会加重损伤,但真正应用于临床时,临床医生还是存在顾虑的。相对于缺血后处理,七氟醚后处理临床医生更易接受。七氟醚是目前比较理想的吸入麻醉药之一,临床上使用具有苏醒快、刺激小,不良反应少,对器官有保护作用等优点。在对其脑缺血再灌注机制的研究中发现,它能降低颅内压,减少梗死容积,抑制神经细胞凋亡,能起到良好的脑保护作用。目前在对心脏的研究中也发现七氟醚后处理能减少再灌注损伤,抑制细胞凋亡,促进功能恢复。但七氟醚后处理的脑保护机制目前仍不清楚,值得我们进行研究。线粒体渗透性转化孔(Mitochondrial permeability transition pore,MPTP)是吸入性麻醉药预处理产生脑保护作用的机制之一。人们已经在心脏的缺血再灌注模型中证实了七氟醚后处理能通过抑制MPTP的开放发挥心肌保护效应。因此我们推测MPTP有可能参与了七氟醚后处理的神经保护机制。蛋白激酶B (Protein kinase B,PKB),也被称为Akt。磷脂酰肌醇-3激酶(Phosphoinositide-3 kinase,PI3K)/Akt细胞内信号途径具有促进细胞增殖、分化,调节细胞存活、内皮生长、血管生成等广泛的生物学效应,是一种重要的神经保护机制。现在也已经证实PI3K/Akt途径参与了七氟醚后处理的心肌保护机制。因此,本课题主要是探讨脑缺血再灌注的同时吸入60min的七氟醚进行后处理是否能产生脑保护效应,以及MPTP与PI3K/Akt途径是否参与其中。本课题首先通过观察缺血后大鼠的神经功能恢复情况、测定脑梗死容积百分比、HE染色评价缺血侧皮层的神经元形态学改变及Nissl染色进行缺血半影区存活神经细胞计数,明确不同浓度的七氟醚后处理是否对Sprague-Dawley大鼠脑缺血再灌注损伤具有保护作用；其次,第二部分通过加入特异性MPTP开放剂和PI3K/Akt通道阻断剂,观察缺血后大鼠的神经功能恢复情况、测定脑梗死容积百分比,分光光度法测定各组缺血皮层半影区神经元MPTP的开放度,测定半影区磷酸化的Akt、磷酸化的糖原合成激酶3β(Glycogen synthase kinase 3β, GSK-3β)的表达,探讨七氟醚后处理的脑保护效应可能是通过激活PI3K/Akt/GSK-3β途径,增加p-Akt, p-GSK-3β的表达,抑制MPTP的开放而介导的。随后,第三部分通过计数缺血半影区的凋亡细胞和Caspase-3,8,9阳性细胞,证实七氟醚后处理可能通过对PI3K/Akt途径和MPTP进行调控,抑制线粒体凋亡途径,从而减少神经细胞的死亡。最后通过检测半影区脑源性神经营养因子(Brain-derived neurotrophic factor, BDNF)和血管内皮生长因子(Vascular endothelial growth factor, VEGF)的表达,推测七氟醚后处理有可能通过上调BDNF和VEGF的水平,进而促进神经干细胞(Neural stem cell, NSC)的激活和新生血管的形成,有利于神经功能的重建和修复。结果证实：(1)脑缺血再灌注的同时吸入2.4%及4.0%的七氟醚进行后处理具有脑保护作用,但是未发现七氟醚后处理在本实验模型中的保护效应具有剂量依赖性。(2)七氟醚后处理可通过激活PI3K/Akt/GSK-3β途径,促进Akt的活化,增加p-GSK-3β的表达,进而抑制MPTP的开放,降低脑缺血再灌注损伤。(3)七氟醚后处理可通过抑制线粒体凋亡途径,减少脑缺血再灌注造成的神经元死亡,发挥显著的脑保护作用。其机制可能与激活PI3K/Akt途径,抑制MPTP的开放有关。(4)七氟醚后处理可通过上调BDNF和VEGF的水平,参与神经保护作用。总之,脑缺血及再灌注所致的神经元损伤是一系列极为复杂的病理生理过程,而七氟醚后处理也是一个多分子参与的多信号通路交织的复杂级联反应。七氟醚后处理的脑保护作用并非仅仅局限于某一环节或某一方面,它与脑缺血交织在一起,是多位点、多途径、相互作用的结果。目的：观察脑缺血再灌注的同时吸入2.4%或4.0%的七氟醚60min是否会产生脑保护效应。方法：健康雄性SD大鼠,体重300-350g,随机分为四组：假手术组(Sham),缺血再灌注组(工／R),缺血再灌注+2.4%七氟醚1组(Sevol),缺血再灌注+4.0%七氟醚2组(Sevo2)。电凝法进行左侧大脑中动脉永久性阻断(Permanent middle cerebral artery occlusion, pMCAO),双侧颈总动脉阻断60min后松开,造成脑缺血再灌注,气管插管,再灌注的同时,机械通气,吸入2.4%或4%的七氟醚+氧气,连续吸入60min。Sham组开颅,暴露大脑中动脉,但不烧灼,分离两侧颈总动脉,但不夹闭,气管插管,吸入100%纯氧60 min; I/R组再灌注的同时吸入100%纯氧60 min。各组在麻醉后5min、大脑中动脉永久性阻断后15min及再灌注后15min分别记录平均动脉压、心率、体温,并抽动脉血,行血气分析。于再灌注1d、3d、7d,用神经功能评分观察动物神经行为学的变化、HE染色评价缺血侧皮层神经元形态学的改变及Nissl染色进行缺血半影区存活神经元计数。于再灌注3d,运用2,3,5-三苯基氯化四氮唑(2,3,5-triphenyltetrazolium chloride, TTC)法测定大鼠脑梗死容积百分比。结果：与Sham组相比,除了Sevo2组在再灌注15min的MAP值降低(P<0.05)之外,其余各组pH值、动脉血二氧化碳分压(PaCO2)、动脉血氧分压(Pa02)、平均动脉压(MAP)及体温(T)差异无统计学意义(P>0.05)。与I／R组相比,再灌注1d、3d、7d,神经功能评分显示Sevol组和Sevo2组均能明显改善神经功能(P<0.05), Nissl染色显示Sevol组和Sevo2组均能显著增加缺血半影区存活神经细胞的数量(P<0.05),再灌注3d, TTC染色法显示,Sevol组和Sevo2组脑梗死容积百分比较I／R组显著减少(P<0.05)。但Sevol组和Sevo2组的神经功能评分、半影区存活神经细胞数目及脑梗死容积比之间的差异无统计学意义(P>0.05)。结论：七氟醚后处理能明显改善脑缺血后的神经功能,减少梗死容积,增加神经细胞的存活,产生有效的脑保护效应。但未证实七氟醚后处理在本模型中的保护效应具有剂量依赖性。目的：探讨PI3K/Akt/GSK-3β信号途径及MPTP在七氟醚后处理脑保护效应中的作用。方法：健康雄性SD大鼠,体重300-350g,随机分为八组：假手术组(Sham),缺血再灌注组(I／R),缺血再灌注+七氟醚组(Sevo),缺血再灌注+DMSO组(DMSO)组,缺血再灌注+七氟醚+MPTP特异性开放剂Atractyloside组(Sevo+Atr),缺血再灌注+七氟醚+PI3K特异性抑制剂LY294002组(Sevo+LY),缺血再灌注+Atractyloside组(Atr),缺血再灌注+LY294002组(LY)。缺血再灌注模型为pMCAO+夹闭双侧颈总动脉60min。再灌注的同时,予2.4%的七氟醚持续吸入60min,进行七氟醚后处理。Sham组开颅,暴露大脑中动脉,但不烧灼,分离两侧颈总动脉,但不夹闭,气管插管,吸入100%纯氧60 mmin；I／R组再灌注的同时吸入100%纯氧60 min。Atractyloside 1.6mg/mL,30μL,溶于DMSO中,再灌注的前10min侧脑室给药。LY294002 1.7mg/mL,30μL,溶于DMSO中,再灌注的前10min侧脑室给药。于再灌注1d、3d、7d,用神经功能评分观察动物神经行为学的变化。再灌注3d,运用TTC法测定大鼠脑梗死容积百分比,分光光度法测定各组大鼠缺血皮层半影区神经元MPTP的开放度。再灌注6h、1d、3d、7d取大鼠缺血侧顶叶皮层半影区,Western Blot检测p-Akt、P-GSK-3β的表达。结果：与I/R组相比,七氟醚后处理能缓解大鼠脑缺血再灌注损伤后Ca2+诱导的(max A520 min A520)的下降,促进神经功能的恢复,减少脑梗死容积(P<0.05)。而MPTP特异性开放剂Atractyloside和PI3K特异性抑制剂LY294002能拮抗七氟醚的脑保护效应(P<0.05),单独使用DMSO、Atractyloside及LY294002则无明显影响(P>0.05)。与I／R组相比,七氟醚后处理能增加各时点Akt-Ser473和GSK-3β-Ser9磷酸化的表达(P<0.05)。七氟醚的此效应也可以被PI3K特异性抑制剂LY294002所废止。结论：七氟醚后处理可通过激活PI3K/Akt/GSK-3β途径,促进Akt的活化,增加P-GSK-3β的表达,进而抑制MPTP的开放,降低脑缺血再灌注损伤。目的：观察七氟醚后处理对脑缺血再灌注缺血半影区Caspase-3,8,9的影响以及MPTP与细胞凋亡的关系。方法：健康雄性SD大鼠,体重300-350g,随机分为七组：假手术组(Sham),缺血再灌注组(I/R),缺血再灌注+七氟醚组(Sevo),缺血再灌注+七氟醚+MPTP特异性开放剂Atractyloside组(Sevo+Atr),缺血再灌注+七氟醚+PI3K特异性抑制剂LY294002组(Sevo+LY),缺血再灌注+Atractyloside组(Atr),缺血再灌注+LY294002组(LY)。缺血再灌注模型为pMCAO+夹闭双侧颈总动脉60min。再灌注的同时,予2.4%的七氟醚持续吸入60min,进行七氟醚后处理。于再灌注1d、3d、7d对缺血半影区进行观察,原位末端标记(Terminal deoxynucleotidyl transferase mediated Biotin-dUTP niek-end labeling,TUNEL)法检测神经元的凋亡情况,免疫组化法计数Caspase-3,8,9的阳性细胞数量。结果：Sham组大脑皮质中未见明显凋亡细胞,神经元中Caspase-3,8,9的表达也极少。与Sham组相比,缺血再灌注组(I/R)半影区的凋亡细胞、Caspase-3,8,9阳性细胞数目明显增多(P<0.05)。与I/R组相比,七氟醚后处理能减少各时点半影区的神经元凋亡,降低Caspase-3,9的表达(P<0.05),但对Caspase-8的表达无明显影响(P>0.05)。同样,MPTP特异性开放剂Atractyloside和PI3K特异性抑制剂LY294002可以废除七氟醚后处理抑制凋亡的效应。结论：七氟醚后处理可通过抑制线粒体凋亡途径,减少脑缺血再灌注造成神经元的死亡,发挥显著的脑保护作用。其机制可能与激活PI3K/Akt途径,抑制MPTP的开放有关。目的：观察七氟醚后处理对脑缺血再灌注后缺血侧顶叶皮层半影区BDNF和VEGF的影响。方法：健康雄性SD大鼠,体重300-350g,随机分为三组：假手术组(Sham),缺血再灌注组(I／R),缺血再灌注+七氟醚组(Sevo)。缺血再灌注模型为pMCAO+夹闭双侧颈总动脉60min。再灌注的同时,予2.4%的七氟醚持续吸入60min,进行七氟醚后处理。于缺血再灌注的6h、1d、3d、7d,用RT-PCR和Western blot检测缺血侧顶叶皮层半影区BDNF和VEGF的表达。结果：Sham组脑皮质中仅有少量的BDNF和VEGF表达。与Sham组相比较,缺血再灌注组(I/R) BDNF和VEGF的表达均于再灌注后6h开始增加,1d达到高峰,BDNF蛋白和VEGFmRNA的表达于3d逐渐衰减,7d与Sham组无明显差别。BDNFmRNA的表达衰减较快,3d就已经衰减到与Sham组无明显差异。而VEGF蛋白的表达可持续到7d。与I/R组相比,七氟醚后处理能明显上调各时点半影区BDNF和VEGF的水平(P<0.05)。结论：七氟醚后处理可通过上调BDNF和VEGF的表达,发挥神经保护作用。更多还原

【Abstract】 Objective:To investigate the protective effects of 2.4% and 4.0% sevoflurane postconditioning against cerebral ischemia-reperfusion injury in rats. Methods:Adult male Sprague-Dawley rats (300-350g) were randomly assigned into four groups:Sham group, Ischemia reperfusion group (I/R), Ischemia-reperfusion+2.4% Sevoflurane group (Sevol), Ischemia reperfusion+4.0% Sevoflurane group (Sevo2). The left distal middle cerebral artery was occluded permanently by electrocogulation; subsequently both commom carotid arteries were exposed and occluded with miniature clips for 60 mins. Lossening the clips resulted in the cerebral ischemia-reperfusion injury. Tracheal intubated, at the same time of reperfusion, the animals were inhaled of 2.4% or 4% sevoflourane +oxygen or 100% oxygen for 60 mins. The Sham group was exposed the left distal middle cerebral artery and the commom carotid arteries, but did not operated. Physiological variables (mean arterial blood pressure, heart rate, temperature, plasma glucose and arterial blood gases)were measured 5 min after induction of anesthesia,15 min after ischemic,15 min after reperfusion. Results:Compared with Sham group, the mean arterial blood pressure was lower in Sevo2 group 15 min after reperfusion. No difference was found in rectal temperature, mean arterial blood pressure, arterial pH, PaCO2, PaO2 and blood glucose level in other groups. Compared with I/R group, the Sevol and Sevo2 groups improved the neurological functions and increased the numbers of the surviving nerve cells in ischemic penumbra after pMCAO followed by reperfusion on 1d, 3d and 7d (P<0.05). Animals in Sevol and Sevo2 groups developed smaller brain infarct volumes than I/R group after reperfusion on 3d (P<0.05). But No difference was found in neurological scores, the numbers of the surviving nerve cells and the infarct volumes between the Sevol and Sevo2 groups (P>0.05). Conclusion:Sevoflurane postconditioning could induce the neuroprotection against cerebral ischemia-reperfusion injury, but the protective effect was not dose-dependent in this model.Objective:To investigate the effects of sevoflurane on PI3K/Akt/ GSK-3βpathway and MPTP in this neuroprotection against cerebral ischemia reperfusion. Methods:Adult male Sprague-Dawley rats (300-350g) were randomly assigned into eight groups:Sham group, Ischemia-reperfusion group (I/R), Ischemia-reperfusion+Sevoflurane group (Sevo), Ischemia-reperfusion+DMSO group (DMSO), Ischemia reperfusion+Sevoflurane+a selective mitochondrial permeability transition pore (MPTP) opener Atractyloside group (Sevo+Atr), Ischemia reperfusion+Sevoflurane+a selective PI3K inhibitor LY294002 group (Sevo+LY), Ischemia reperfusion+Atractyloside group (Atr), Ischemia reperfusion+LY294002 group (LY). Atractyloside (1.6mg/mL,30μL, in DMSO) and LY294002 (1.7mg/mL,30μL, in DMSO) were right intracerebroventricularly injected before the reperfusion. Result:Compared with I/R group, sevoflurane inhibited the decrease of calcium induced mitochondrial absorbance at 520 nm (A520), improved neurological functions and developed smaller brain infarct volumes (P<0.05). This protection was reversed by administration of Atractyloside and LY294002, but no distinguished difference was found among I/R, DMSO, Atr and LY groups (P>0.05). Compared with I/R group, sevoflurane postconditioning could not only increase the phosphorylated Akt and GSK-3βin ischemic penumbra, but also inhibit the MPTP opening (P<0.05). This effect was also abolished by LY294002. Conclusion:The sevoflurane postconditioning induced neuroprotective effect could be exerted via the activation of the PI3K/Akt/GSK-3p pathway and inhibition of MPTP.Objective:To study the effects of sevoflurane on neuronal apoptosis, caspase-3,8,9 expressions after cerebral ischemia in rats, and to disclose the relationship between MPTP and neuronal apoptosis. Methods:Adult male Sprague-Dawley rats (300-350g) were randomly assigned into seven groups:Sham group, Ischemia-reperfusion group (I/R), Ischemia-reperfusion+Sevoflurane group (Sevo), Ischemia-reperfusion +Sevoflurane+a selective MPTP opener Atractyloside group (Sevo+Atr), Ischemia reperfusion+Sevoflurane+a selective PI3K inhibitor LY294002 group (Sevo+LY), Ischemia-reperfusion+ Atractyloside group (Atr), Ischemia-reperfusion+LY294002 group (LY).At the 1d,3d and 7d after reperfusion, the rats were anesthetized and the brains were removed. Caspase-3,8,9 expressions were determined by immunohistological staining. Apoptosis was also determined by terminal deoxynucleotidyl transferase-mediated uridine 5’-triphosphate-biotin nick end-labeling (TUNEL) staining. Result: Compared with I/R group, sevoflurane decreased the numbers of TUNEL and caspase-3,9 positive cells in ischemic penumbra (P<0.05). This effect was also abolished by Atractyloside and LY294002. No distinguished difference was found among I/R and Sevo groups about caspase-8. Conclusion:Sevoflurane postconditioning could inhibit the neuronal apoptosis by inactivating the mitochondrial pathway of apoptosis in cerebral ischemia reperfusion injury. The neuroprotection of sevoflurane may be mediated by PI3K/Akt pathway and MPTP.Objective:To investigate the effects of sevoflurane postconditioning on BDNF and VEGF in ischemic penumbra after cerebral ischemic reperfusion. Methods:Adult male Sprague-Dawley rats (300-350g) were randomly assigned into three groups:Sham group, Ischemia-reperfusion group (I/R), Ischemia-reperfusion+Sevoflurane group (Sevo). The expressions of BDNF and VEGF in ischemic penumbra were determined by RT-PCR and Western blot after reperfusion on 6h, 1d,3d and 7d. Result:Compared with Sham group, the expressions of BDNF and VEGF in I/R group were increased. Compared with I/R group, sevoflurane postconditioning could significantly increase the levels of BDNF and VEGF. Conclusion:Sevoflurane postconditioning could play a neuroprotective role by increasing the levels of BDNF and VEGF in penumbra after cerebral ischemic reperfusion.更多还原