线粒体动力学异常在异氟烷致发育期大鼠神经元毒性中作用及机制研究
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摘要
研究背景
据2006年报道,仅美国每年约有600万婴幼儿在全身麻醉(全麻)下接受手术,其中婴儿约150万；尽管中国目前尚无相关数据,但基于国内的人口规模,每年接受全麻手术的婴幼儿应比美国多。由于人类神经系统发育始于妊娠4-5w,而止于出生后若干年后；一般认为人类大脑发育高峰期在妊娠后期至出生后6m。因此全麻是否对患儿智力发育及其长期认知功能产生不良影响是近年来备受关注的热点问题。
已有临床研究表明,婴幼儿时期,特别是两岁以内的婴儿接受全身麻醉会导致较长时期的行为改变,提示全麻药可能损害婴幼儿中枢神经系统。近年来基础研究发现,在神经系统发育关键期(啮齿类动物为出生前两天到出生后1～2w),全麻药(异氟烷、七氟烷、地氟烷、氯胺酮及异丙酚)影响动物发育期神经系统突触可塑性,并导致发育期神经元凋亡及其它退行性神经病变,最后影响成年期动物的学习记忆功能。
吸入麻醉药在临床麻醉中应用非常广泛。新近研究表明,吸入麻醉药异氟烷致发育期神经元内钙稳态失衡,最终触发线粒体途径凋亡。但是吸入麻醉药影响线粒体结构和功能的确切机制尚不清楚。如果小儿全麻不可避免,那么阐明吸入麻醉药导致线粒体结构和功能异常的准确机制,并探索有效的防治手段,是亟待解决的难题之一。
线粒体是一个不断进行分裂、融合等动态变化的细胞器,这种动态变化称为线粒体动力学。线粒体分裂／融合主要受一下两组蛋白的调控：(1)调节线粒体分裂的drp-1(dynamin-related protein)、 mff (mitochondrial fission factor)、 FIS1;(2)调节线粒体融合的mfn (mitofusionl)1mfn2、OPA1(optic atrophy1)。生理状况下,线粒体分裂/隔合既决定线粒体形状和大小,也调节线粒体的分布和功能,对维持细胞生理功能发挥重要作用。已有研究发现,神经元内线粒体分裂/融合异常影响神经突生长、突触形成、长时程增强,甚至神经元凋亡等。
调节线粒体分裂、融合的蛋白参与了很多神经退行性疾病过程中神经元凋亡和突触联系障碍等病理变化。在凋亡早期,哺乳动物细胞中drp-1活性增加,促进线粒体碎片化和分裂,增加线粒体膜的通透性,促使细胞色素C(cytochrome C, cyto-C)释放,最终触发线粒体途径凋亡。在阿尔茨海默病和亨廷顿舞蹈症等研究中发现,p淀粉样蛋白(Aβ)、亨廷顿蛋白((huntingtin, HTT)及帕金蛋白(Parkin)都可促使神经元内dr-1表达上调且与drp-1相互作用,激活drp-1,促进线粒体过度分裂甚至碎片化,进而导致线粒体动力学异常,甚至神经元凋亡并损害突触联系的建立,最终导致患者长期认知功能障碍。由此可见,线粒体异常分裂是凋亡的早期表现；神经元线粒体分裂、融合失衡是退行性神经病变的病理生理基础。
神经元胞浆Ca2+可通过多种信号通路调控线粒体动力学蛋白活性,继而调节线粒体动力学,影响神经元存活及其他功能。在伤害性刺激下,神经元胞浆Ca2+浓度([Ca2+]C)持续升高,继而激活钙调神经磷酸酶(calcineurin, CaN),引起drp-1第637丝氨酸去磷酸化,导致drp-1活性增强并向线粒体移位,促进线粒体分裂,进而引起线粒体片段化甚至神经元凋亡,最终干扰突触联系及神经环路形成。本项目组的前期研究证实,临床相关浓度的异氟烷可致神经元[Ca2+]。升高。但是,异氟烷是否通过这条信号通路,引起线粒体分裂／融合失衡并最终导致发育期神经元毒性有待于进一步研究。
因此本研究拟从离体细胞和在体动物水平,根据异氟烷作用后,线粒体形态及线粒体动力学调节蛋白表达的变化,选择对线粒动力学起重要作用的调节蛋白,阐述线粒体动力学失调在异氟烷抑制发育期神经元毒性作用中的机制。
研究方法与结果
1.异氟烷对发育期神经元线粒体形态学和膜电位的影响
方法：体外培养第5d (DIV5)海马神经元随机分为对照组(C组)以及异氟烷组(I组)。C组在5%CO2-8%O2-87%N2三气培养箱中处理6h；I组以5%CO2-8%O2-87%N2作为载体气,1.5%异氟烷处理6h,采用电镜观察线粒体形态学的变化。DIV3海马神经元,转染CellLight(?) Mitochondria-RFP BacMan2.0,异氟烷处理后在激光共聚焦显微镜下观察线粒体的形态学改变
结果：与C组比较,I组线粒体直接明显减小(P<0.05)。在转染了CellLight(?) Mitochondria-RFP BacMan2.0的神经元中,与C组比较,I组神经元变圆变小。
2.异氟烷对发育期神经元线粒体膜电位的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。1.5%异氟烷处理6h。选用JC-1染色后在激光共聚焦显微镜和细胞流式仪下观察线粒体膜电位变化。
结果：与C组标胶,I组神经元线粒体膜电位明显去极化(P<0.05)。
3.异氟烷对发育期神经元内线粒体分裂/融合调节蛋白mRNA和蛋白表达水平的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。异氟烷结束后,提取总RNA和总蛋白,分别进行实时定量PCR和Western Blot (WB)方法检测线粒体动力学调节蛋白的mRNA和蛋白水平的表达变化
结果：与C组相比,I组线粒体动力学调节蛋白在mRNA和蛋白水平的表达差异均无显著性(P>0.05)。
4.异氟烷对发育期神经元p-drp-1表达的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。异氟烷麻醉结束后提取总蛋白,采用WB方法检测drp-1第637丝氨酸磷酸化的表达(p-drp-1(ser637))。选用免疫荧光双标检测p-drp-1(ser637)的表达变化。
结果：与C组比较,I组p-drp-1(ser637)表达明显下调(P<0.05)。
5.抑制drp-1对异氟烷诱发发育期神经元线粒体膜电位去极化的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)、异氟烷组(I组)、mdivi-1预给药后异氟烷处理组(M+I组)及mdivi-1处理组(M组)。M+I组和M组在异氟烷处理2h前在无血清培养基中加入5μMmdivi-1,而后C组和M组载体气处理6h,I组和M+I组1.5%异氟烷处理6h。异氟烷处理结束后,进行JC-1染色,在激光共聚焦显微镜和细胞流式仪下检测线粒体膜电位。
结果：与C组比较,M+I组和M组线粒体膜电位没有明显变化(P>0.05),I组线粒体膜电位明显去极化(P<0.05)；与I组比较,M+I组和M组线粒体膜电位水平较高(P<0.05)。
6. Mdivi-1对异氟烷诱发发育期神经元线粒体形态学的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、M+I组、M组,处理同上。异氟烷处理结束后,采用电镜观察线粒体形态学的变化,采像后的图片在Imaris下进行处理统计。采用CellLight(?) Mitochondria-RFP BacMan2.0在海马神经元体外培养3d时转染,在异氟烷处理结束后,固定、封片后在激光共聚焦显微镜下观察线粒体形态学改变。
结果：与C组比较,M组及M+I组线粒体直径差异无统计学意义(P>0.05)；I组线粒体直径减小(P<0.05)；线粒体密度增加,但差异无统计学意义(P>0.05)。与I组比较,M+I组线粒体直径增大(P<0.05)；线粒体密度差异无统计学意义(P>0.05)。
7. Mdivi-1对异氟烷诱发发育期神经元存活率及凋亡相关蛋白表达的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、M+I组、M组,处理同上。采用WB检测细胞色素C (cytochrome C,cyto-C)的表达；采用免疫荧光双标检测active caspase3染色阳性细胞数目。
结果：与C组比较,M组所检测指标差异无显著性(P>0.05)；I组AC3(active caspase3)和cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组比较,M+I组cyto-C蛋白表达降低,AC3阳性神经元数目减少(P<0.05)。
8.异氟烷对钙神经磷酸酶的表达和活性的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。采用试剂盒检测CaN活性；提取总蛋白,用WB方法检测CN表达。
结果：与C组比较,CaN表达明显上调,活性明显增强(P<0.05)。
9.CaN抑制剂FK506对异氟烷诱导的p-drp-1表达和线粒体形态学改变的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)、异氟烷组(I组)FK506预给药后异氟烷处理组(F+I组)及FK506处理组(F组)。F+I组和F组在异氟烷处理2h前在无血清培养基中加入5μMFK506,而后C组合F组载体气处理6h,I组和F+I组1.5%异氟烷处理6h。异氟烷处理结束后,WB检测p-drp-1的表达。采用CellLight(?)Mitochondria-RFP BacMan2.0在海马神经元体外培养3d时转染,在异氟烷处理结束后,固定、封片后在激光共聚焦显微镜下观察线粒体形态学改变,采像后的图片在Imaris下进行处理统计。
结果：与C组比较,F组及F+I组所检测指标差异无显著性(P>0.05)；I组p-drp一1表达降低,线粒体直径减小(P<0.05)。与I组比较,F+I组p-drp-1表达升高,线粒体直径增加(P<0.05)。
10.FK506对异氟烷诱导的发育期神经元凋亡的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、F+I组、F组,处理同上。采用WB检测cyto-C的表达；采用免疫荧光双标检测AC3的表达。
结果：与C组相比,F组及F+I组所检测指标差异无显著性(P,0.05)；I组cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组相比,F+I组cyto-C蛋白表达降低,AC3阳性神经元数目减少(P<0.05)。
11.异氟烷对新生5d (postnatal5day, PND5)大鼠海马线粒体形态学的影响
方法：PND5大鼠,随进分为C组合I组。C组吸入30%O2-70%N26h, I组以30%02-70%N2作为载气1.5%异氟烷处理6h,然后电镜灌注液灌注后,取标本放入2.5%戊二醛固定后,进行电镜检测。
结果：与C组相比,I组海马线粒体直径减小(P<0.05),甚至有部分线粒体破裂。
12.异氟烷对PND5大鼠海马线粒体动力学调节蛋白及p-drp-1表达的影响
方法：PND大鼠1.5%异氟烷处理6h后,取海马组织,提取总蛋白和总RNA,采用WB和实时定量PCR检测线粒体动力学调节蛋白及p-drp-1的表达
结果：与C组相比,I组海马线粒体动力学调节蛋白表达在mRNA和蛋白水平差异无统计学意义(P>0.05),但是p-drp-1表达明显降低(P<0.05)。
13. Mdivi-1对异氟烷诱导的PND5大鼠海马神经元凋亡的影响
方法：PND大鼠随机分为四组：C组、I组、M+I组及M组。C组和I组处理同前；M+I组在麻醉2h前腹腔注射5μg/kg mdivi-1,然后1.5%异氟烷处理6h；M组腹腔注射5μg/kg mdivi-1然后吸入30%O2-70%N2。麻醉结束后,尽取海马组织,提取总蛋白和总RNA,采用WB和实时定量PCR检测cyto-C的表达。1.5%异氟烷处理6h后,4%多聚甲醛固定,取大脑切片,采用免疫荧光双标检测AC3阳性神经元数目。
结果：与C组相比,M+I组和M组所检测指标无明显差异(P>0.05)；I组cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组相比,M+I组cyto-C表达降低,AC3阳性神经元数目减少(P<0.05)。
14.Mdivi-1对异氟烷诱导的PND5大鼠长期认知障碍的影响
方法：PND大鼠1.5%异氟烷处理6h后,待其完全清醒,送回原笼饲养至60d,进行水迷宫检实验检测其空间学习记忆功能。实验完成后,麻醉电镜固定液灌注固定后,取海马组织进行电镜检测线粒体形态的变化。
结果：与C组比较,I组第3-5天时逃避潜伏期延长,探索时间缩短(P<0.05),M+I组和M组逃避潜伏期和探索时间差异无统计学意义(P>0.05)；与I组比较,M+I组第3-5d时逃避潜伏期缩短,探索时间延长(P<0.05)。电镜检测发现,与C组比较,I组线粒体直径减少(P<0.05),M+I组和M组差异无统计学意义(P>0.05)；与I相比,M+I组线粒体直径增加(P<0.05)。
15.统计学分析
采用SPSS20统计软件对数据进行分析,计量资料表示为均数±标准差(x±s),组间比较采用单因素方差分析,组内比较采用独立样本t检验；Morris水迷宫大鼠训练期间数据采用连续测量的方差分析,P<0.05为有统计学意义。
研究总结
一、主要研究结果
1.通过体外原代海马神经元培养以及在体动物实验证明临床相关浓度的异氟烷导致发育神经元线粒体动力学失衡。
2.体外及体内实验证实,异氟烷激活发育期神经元CaN,导致drp-1第637位丝氨酸去磷酸化,继而促使drp-1活性增加并向线粒体移位,导致线粒体过度分裂甚至碎片化,从而触发线粒体途径凋亡。
3.体外及体内实验证实,drp-1抑制剂mdivi-1可减轻异氟烷发育期神经元凋亡,改善异氟烷所致长期认知功能障碍。
二、研究结论
异氟烷引起胞浆Ca2+浓度升高,激活CaN,促进drp-1(ser637)去磷酸化,引起drp-1活性增强并向线粒体移位,从而导致线粒体过度分裂甚至碎片化,继而导致线粒体膜通透性增加,cyto-C释放,触发线粒体凋亡途径,最终导致发育期神经系统损伤；而drp-1抑制剂mdivi-1则可减轻异氟烷所诱导神经元凋亡和长期认知功能障碍。
Background
It was estimated that6million children (including1.5million infants) undergo surgery and anesthesia each year in the USA alone. Although there was no relevant data in China, the number of Chinese children received general anesthesia and surgery was much larger than that in USA on the fact of large number of Chinese population. The development of human brain begins in the early pregnant stage and ends several years after birth. It is generally accepted that brain growth spurt in humans was between the last month of gestation and first6months after birth. Therefore it is a hot issue whether general anesthesia could exert adverse effect on children's intelligence and long-lasting deficit in learning and memory.
In clinical trials, children who received general anesthesia in infant period represented cognitive impairment in adulthood. This indicated that general anesthetics could damage central nervous system in infants'period. In rodents, exposure to general anesthetics such as isoflurane, sevoflurane, devoflurane, ketamine and propofol in the period of peak brain growth induced damage of synaptic plasticity, neuroapoptosis and other neurodegenerative changes, finally impaired learning and memory in adulthood.
Inhalational anesthetics are extensively used in clinical anesthesia. In recent reports, isoflurane induced imbalance of cytoplasmic Ca2+and subsequently lead to neuroapoptosis in the mitochondrial pathway. However, the mechanism involved in the effect of isoflurane mitochondrial structure and function is unclear. Supposed that infants exposed to general anesthetics was indispensable, it is a challenge to elucidate the exact mechanism involved in isoflurane-induced abnormal structure and function of mitochondria and investigate the effective interventions for the prophylaxis and treatment of this disorder.
Mitochondria undergo dynamical fusion and fission events which maintain steady mitochondrial morphology. This process is also called mitochondrial dynamics. The rates of fusion and fission are regulated by several large GTPase molecules (OPA1, mfn1, mfn2, drp-1, mff and FIS1). Drp-1binds to the mitochondrial fission factor (mff) located on the outer membrane of mitochondrial and then promotes mitochondrial division, while OPA1, mfnl and mfn2potentiate mitochondrial fusion. Under physiological condition, the events of fusion and fission not only determine the shape and size of mitochondria but also regulate mitochondrial distribution and function. It was reported that dysregulation of fusion and fission of mitochondria in neurons affected the neurite growth, synaptic plasticity, long-term potentiation and even induced neuroapoptosis.
The molecules regulated mitochondrial dynamics were associated with neuroapoptosis and synaptic mis-interconnection in neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. In the early stage of apoptosis, the activity of drp-1was upregulated and promoted mitochondrial fragmentation in mammalian cells. These changes lead to enlargement of mitochondrial cristae and increase of mitochondrial membrane permeability which facilitated the release of cytochrome C from mitochondrial cristae to the cytoplasm and initiated the mitochondrial apoptotic pathway. Amyloid-beta protein and huntingtin induced upregulation of drp-1and downregulation of mfiil and mfn2and promoted excessive mitochondrialfission and fragmentation which contributed to neuroapoptosis, damage of synaptic connections and long-lasting cognitive disorder. In summary, abnormal mitochondrial fission is an early demonstration of apoptosis; imbalance of mitochondrial fusion and fission underlie the pathophysiological mechanism of neurodegenerative diseases.
Ca2+in neuronal cytoplasm regulated the mitochondrial dynamics in several signaling pathway by the way that influence the expression and activity of the regulating proteins.With the nocuous stimuli, the level of cytoplasmic Ca2+was elevated, and then calcineurin was activated which dephosphorylated the637serine of drp-1. This change enhanced the activity of drp-1and promoted the fragmentation of mitochondrial and ultimately disturbed the interconnection of synapse and formation of neural circuit. Our previous results demonstrated that clinically relevant concentration of isoflurane elevated the level of cytoplasmic Ca2+and triggered mitochondrial apoptotic pathway in developmental neurons. However, it remains to be elucidated that isoflurane induced dysregulation of mitochondria dynamics via calcineurin signaling pathway.
In this study, the dynamic changes of mitochondrial morphology the regulating proteins were detected in neurons and rats. Also, pathways of cell signaling transduction relevant to the imbalance of mitochondrial dynamics induced by isoflurane were investigated. All the designed experiments are conducted to elucidate the role of mitochondrial dynamics disruption in the developmental neurotoxicity triggered by isoflurane and provide a new prospective for prophylaxis and medication of the neurotoxitiy.
Methods and Results
1. Effects of isoflurane on the mitochondrial morphological changes and mitochondrial membrane potential of rat developmental hippocampal neurons in vitro
Methods Neurons cultured5day in vitro(DIV) were randomly assigned to control group (group C) and isoflurane treated group (group I). Group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours, while group C was exposed to carrier air for6h. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and then treated by a series of special steps. And then eletron microscope was used to detect mitochondrial morphology. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed with4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended.
Results Compared with group C, the length of mitochondria in group I was significantly shorter (P<0.05).
2. Effects of isoflurane on mitochondrial membrane potential ((?)m) of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).
Results Compared with group C,(?)m in group I was significantly depolarized with detection of CLSM and flow cytometer(p<0.05).
3. Effects of isoflurane on proteins that regualted mitochondrial dynamics of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Immediately at the end of isofluane exposure, total RNA and protein was extracted respectively for Western Blot (WB) and real-time PCR detection.
Results Compared with group C, the expression of drp-1, mff, FIS1, OPA1, mfn1and mfn2was no staticstical difference in group I at the level mRNA or protein (P>0.05).
4. Effects of isoflurane on the expression of phospholyrated drp-1(ser637) of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. At the end of isoflurane anesthesia, total protein was extracted by RIPA for detection p-drp-1(ser637). And also the expression of p-drp-1(ser637) was assayed by double-immunofluorescence methods. Red fluorescence represented p-drp-1(ser637).
Results Compared with the group C, the expression of p-drp-1(ser637) significantly decreased and the positive stained neurons and red fluorescence drammatically declined (P<0.05).
5. Effects of inhibition of drp-1on isoflurane-induced(?)m depolarization of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), mdivi-1pretreated and isoflurane expousre group (group M+I) and only mdivi-1treated group (group M). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6h; group M+I treated with5μM mdivi-1for2h and then exposed to1.5%isoflurane; group M was treated by5uM mdivi-1and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).
Results Compared with the group C, the (?)mingroup M and group M+I did not declined significantly(P>0.05); while the (?)m in group Idecreased significantly (P<0.05). Compared with groupI, the (?)ningroup M+I depolarized significantly (P<0.05).
6. Effects of mdivi-1on isoflurane-induced mitochondrial morphological changes of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divied into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed by4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and sent for detection mitochondrial morphology with eletron microscope.
Results Compared with the group C, the diameter of mitochondria was not significantly different in group M and group M+I (P>0.05), while it was much smaller in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group M+I was significantly larger(P<0.05).
7. Effects of mdivi-1on neuronal viability, theexpression of cyto-C and AC3of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. The expression of cyto-C was detected at the levels of protein after isoflurane treatment. The expression of AC3was detected in a double-stained immunocytochemistric way.
Results Compared with the group C, all the detected parameters were not significantly different in group M and group M+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group M+I (P<0.05).The number of AC3-positive neurons was reduced in group M+I (P<0.05).
8. Effects of isoflurane on activity and expression of calcineurin(CaN) of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned in Part.1. Immediately at the end of isofluane exposure, the total protein was extracted for WB and the activity of CaN was assayed by the commecially kit following the manufacture's instructions.
ResultsAfter1.5%isoflurane treatment, the activity and expression of CaN increased significantlly in neurons(P<0.05).
9. Effects of FK506(inhibitor of CaN) on the isoflurane-induced downregualtion of p-drp-1(ser637), abnomal mitochondrial morphology and depolarized (?)mof rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), FK506pretreated and isoflurane expousre group (group F+I) and only FK506treated group (group F). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours; group F+I treated with5nM FK506for2hours and then exposed to1.5%isoflurane; group F was treated by5nMFK506and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and CLSM.
Results Compared with the group C, diameter was not significantly different in group F and group F+I (P>0.05), while it significantly changed in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group F+I was significantly larger (P<0.05); the (?)mingroup F+I increased significantly (P<0.05); the downregulation of p-drp-1(ser637) was significantly upregulated (P<.05).
10. Effects of FK506on the expression of cyto-C and AC3of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part9. The expression of cyto-C was detected at the protein level after isoflurane treatment.The expression of AC3was detected in a double-stained immunocytochemistric way.
Results Compared with the group C, all the detected parameters were not significantly different in group F and group F+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group F+I (P<0.05); The number of AC3-positive neurons was reduced in group M+I (P<0.05).
11. Effects of isoflurane on the mitochondrial morphological changes in CA1of PND5rats
Methods PND5rats were randomly assigned to group C and group I. Group I was exposure to1.5%isoflurane for6h carried by30%O2-70%N2,while group C exposed to carrier air. At the end of anesthesia, CA1in hippocampus was gently dissected and transferred to2.5%glutaraldehyde for2h fixation and send for eletron microscopy imaging and analysis.
Results Compared with the group C, the diameter of mitochondria was significantly reduced in group I and mitochondrial in group I was significantly condensed(P<0.05).
12. Effects of isoflurane on proteins that regualted mitochondrial dynamics and p-drp-1in hippocampi of PND5rats
MethodsPND5rats were randomly assigned to group C and group I. The detailed treatment information was refered to Part.11. At the end of anesthesia, hippocampi were dissected for total protein and RNA extraction.
ResultsCompared with group C, the expressions of drp-1, mff, FIS1, OPA1, mfn1and mfn2were no staticstical difference in group I at the level mRNA (P>0.05);but the expression of p-drp-1(ser637) significantly decreased(P<0.05).
13. Effects of mdivi-1on isoflurane-induced neuronal apoptosis and the expression of cyto-Cin hippocampi of PND5rats
Methods PND5rats were randomly divided into group C,group I, group M+I and group M. Group C and group I were treated as delineted Part.11. Group M+I and group Mreceived a dose of5μg/kg mdivi-1by intraperitoneal injection2h before isoflurane treatment. After anesthesia, hippocampi was dissociated for detection ofcyto-C. And ratswere decapitated and frozen sections of brainwere made to stain the positive active caspse3neurons.
Results Compared to group C, there were no statistical difference in expression of cyto-C and the amount of active caspase3-positive neurons in group M+I and group M(P>0.05). Compared with group I, the expression cyto-C was downregulated significantly in group M+I (P<0.05) and the number of active caspase3-positive neurons was smaller(P<0.05).
14. Effects of mdivi-1on isoflurane-induced long-term mitochondrial changes and deficit in learning and memory
Methods PND5rats were randomly divided into group C,group I, group M+I and group M.The detailed treatment processure was refered to setion Methods in Part13. After isofluane exposure, rats were sent to cages until they were fully awake and raised to60days after their birth when Morris water maze was used to assess spatial learning and memory.
Results Compared with group C, the latency was significantly longer and the probe time wasshorter in group I from the third training dayto the lasttraining day (P<0.05). However, these changes were reversed in groupM+I (P<0.05).
15. Statistical analyses
All datawerepresentedandgraphedas the mean±EM. The Statistical Package for the Social Sciences20software was used for the statistical analyses. Data acquired fromthe detection of protein and mRNA was analyzed with an analysis of variance (ANOVA), followed by a least square difference (LSD) multiple comparison test. Data collected from the spatial acquisition trials were analyzed using a repeated measures ANOVA (the different treatments were the between groups factors and time was the repeated measures factor), followed by a post-hoc test to compare four groups. Differences were deemed statistically significant if P<0.05.
Summaries
1. It was verified that clinical relevant concentration isoflurane induced imbalance of mitochondrial dynamics in in vitro and in vivo.
2. It was confirmed that isoflurane activated calcineurin which dephosphorylated drp-1at serine-637. The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation.
3. Pretreatment with mdivi-1, an inhibitor of drp-1, mitigated isoflurane-induced neuroapoptosis and long-lasting cognitive impairment in in vitroandin vivo.
Conclusions
Elevated cytoplasmic Ca2+induced by Isoflurane via activating GABAA or facilitating IP3and RyR receptors increased the activity of calcineurin which dephosphorylated drp-1at serine-637.The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation, which increased the permeability of mitochondrial transition pore. Thus cyto-C tethered in mitochondrial cristae released and initiated the mitochondrial apoptototic pathway of developmental neurons. However, mdivi-1, an inhibitor of drp-1, alleviated isoflurane-induced neuroapoptosis in vitro and in vivo and improved long-lasting cognitive function.
据2006年报道,仅美国每年约有600万婴幼儿在全身麻醉(全麻)下接受手术,其中婴儿约150万；尽管中国目前尚无相关数据,但基于国内的人口规模,每年接受全麻手术的婴幼儿应比美国多。由于人类神经系统发育始于妊娠4-5w,而止于出生后若干年后；一般认为人类大脑发育高峰期在妊娠后期至出生后6m。因此全麻是否对患儿智力发育及其长期认知功能产生不良影响是近年来备受关注的热点问题。
已有临床研究表明,婴幼儿时期,特别是两岁以内的婴儿接受全身麻醉会导致较长时期的行为改变,提示全麻药可能损害婴幼儿中枢神经系统。近年来基础研究发现,在神经系统发育关键期(啮齿类动物为出生前两天到出生后1～2w),全麻药(异氟烷、七氟烷、地氟烷、氯胺酮及异丙酚)影响动物发育期神经系统突触可塑性,并导致发育期神经元凋亡及其它退行性神经病变,最后影响成年期动物的学习记忆功能。
吸入麻醉药在临床麻醉中应用非常广泛。新近研究表明,吸入麻醉药异氟烷致发育期神经元内钙稳态失衡,最终触发线粒体途径凋亡。但是吸入麻醉药影响线粒体结构和功能的确切机制尚不清楚。如果小儿全麻不可避免,那么阐明吸入麻醉药导致线粒体结构和功能异常的准确机制,并探索有效的防治手段,是亟待解决的难题之一。
线粒体是一个不断进行分裂、融合等动态变化的细胞器,这种动态变化称为线粒体动力学。线粒体分裂／融合主要受一下两组蛋白的调控：(1)调节线粒体分裂的drp-1(dynamin-related protein)、 mff (mitochondrial fission factor)、 FIS1;(2)调节线粒体融合的mfn (mitofusionl)1mfn2、OPA1(optic atrophy1)。生理状况下,线粒体分裂/隔合既决定线粒体形状和大小,也调节线粒体的分布和功能,对维持细胞生理功能发挥重要作用。已有研究发现,神经元内线粒体分裂/融合异常影响神经突生长、突触形成、长时程增强,甚至神经元凋亡等。
调节线粒体分裂、融合的蛋白参与了很多神经退行性疾病过程中神经元凋亡和突触联系障碍等病理变化。在凋亡早期,哺乳动物细胞中drp-1活性增加,促进线粒体碎片化和分裂,增加线粒体膜的通透性,促使细胞色素C(cytochrome C, cyto-C)释放,最终触发线粒体途径凋亡。在阿尔茨海默病和亨廷顿舞蹈症等研究中发现,p淀粉样蛋白(Aβ)、亨廷顿蛋白((huntingtin, HTT)及帕金蛋白(Parkin)都可促使神经元内dr-1表达上调且与drp-1相互作用,激活drp-1,促进线粒体过度分裂甚至碎片化,进而导致线粒体动力学异常,甚至神经元凋亡并损害突触联系的建立,最终导致患者长期认知功能障碍。由此可见,线粒体异常分裂是凋亡的早期表现；神经元线粒体分裂、融合失衡是退行性神经病变的病理生理基础。
神经元胞浆Ca2+可通过多种信号通路调控线粒体动力学蛋白活性,继而调节线粒体动力学,影响神经元存活及其他功能。在伤害性刺激下,神经元胞浆Ca2+浓度([Ca2+]C)持续升高,继而激活钙调神经磷酸酶(calcineurin, CaN),引起drp-1第637丝氨酸去磷酸化,导致drp-1活性增强并向线粒体移位,促进线粒体分裂,进而引起线粒体片段化甚至神经元凋亡,最终干扰突触联系及神经环路形成。本项目组的前期研究证实,临床相关浓度的异氟烷可致神经元[Ca2+]。升高。但是,异氟烷是否通过这条信号通路,引起线粒体分裂／融合失衡并最终导致发育期神经元毒性有待于进一步研究。
因此本研究拟从离体细胞和在体动物水平,根据异氟烷作用后,线粒体形态及线粒体动力学调节蛋白表达的变化,选择对线粒动力学起重要作用的调节蛋白,阐述线粒体动力学失调在异氟烷抑制发育期神经元毒性作用中的机制。
研究方法与结果
1.异氟烷对发育期神经元线粒体形态学和膜电位的影响
方法：体外培养第5d (DIV5)海马神经元随机分为对照组(C组)以及异氟烷组(I组)。C组在5%CO2-8%O2-87%N2三气培养箱中处理6h；I组以5%CO2-8%O2-87%N2作为载体气,1.5%异氟烷处理6h,采用电镜观察线粒体形态学的变化。DIV3海马神经元,转染CellLight(?) Mitochondria-RFP BacMan2.0,异氟烷处理后在激光共聚焦显微镜下观察线粒体的形态学改变
结果：与C组比较,I组线粒体直接明显减小(P<0.05)。在转染了CellLight(?) Mitochondria-RFP BacMan2.0的神经元中,与C组比较,I组神经元变圆变小。
2.异氟烷对发育期神经元线粒体膜电位的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。1.5%异氟烷处理6h。选用JC-1染色后在激光共聚焦显微镜和细胞流式仪下观察线粒体膜电位变化。
结果：与C组标胶,I组神经元线粒体膜电位明显去极化(P<0.05)。
3.异氟烷对发育期神经元内线粒体分裂/融合调节蛋白mRNA和蛋白表达水平的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。异氟烷结束后,提取总RNA和总蛋白,分别进行实时定量PCR和Western Blot (WB)方法检测线粒体动力学调节蛋白的mRNA和蛋白水平的表达变化
结果：与C组相比,I组线粒体动力学调节蛋白在mRNA和蛋白水平的表达差异均无显著性(P>0.05)。
4.异氟烷对发育期神经元p-drp-1表达的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。异氟烷麻醉结束后提取总蛋白,采用WB方法检测drp-1第637丝氨酸磷酸化的表达(p-drp-1(ser637))。选用免疫荧光双标检测p-drp-1(ser637)的表达变化。
结果：与C组比较,I组p-drp-1(ser637)表达明显下调(P<0.05)。
5.抑制drp-1对异氟烷诱发发育期神经元线粒体膜电位去极化的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)、异氟烷组(I组)、mdivi-1预给药后异氟烷处理组(M+I组)及mdivi-1处理组(M组)。M+I组和M组在异氟烷处理2h前在无血清培养基中加入5μMmdivi-1,而后C组和M组载体气处理6h,I组和M+I组1.5%异氟烷处理6h。异氟烷处理结束后,进行JC-1染色,在激光共聚焦显微镜和细胞流式仪下检测线粒体膜电位。
结果：与C组比较,M+I组和M组线粒体膜电位没有明显变化(P>0.05),I组线粒体膜电位明显去极化(P<0.05)；与I组比较,M+I组和M组线粒体膜电位水平较高(P<0.05)。
6. Mdivi-1对异氟烷诱发发育期神经元线粒体形态学的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、M+I组、M组,处理同上。异氟烷处理结束后,采用电镜观察线粒体形态学的变化,采像后的图片在Imaris下进行处理统计。采用CellLight(?) Mitochondria-RFP BacMan2.0在海马神经元体外培养3d时转染,在异氟烷处理结束后,固定、封片后在激光共聚焦显微镜下观察线粒体形态学改变。
结果：与C组比较,M组及M+I组线粒体直径差异无统计学意义(P>0.05)；I组线粒体直径减小(P<0.05)；线粒体密度增加,但差异无统计学意义(P>0.05)。与I组比较,M+I组线粒体直径增大(P<0.05)；线粒体密度差异无统计学意义(P>0.05)。
7. Mdivi-1对异氟烷诱发发育期神经元存活率及凋亡相关蛋白表达的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、M+I组、M组,处理同上。采用WB检测细胞色素C (cytochrome C,cyto-C)的表达；采用免疫荧光双标检测active caspase3染色阳性细胞数目。
结果：与C组比较,M组所检测指标差异无显著性(P>0.05)；I组AC3(active caspase3)和cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组比较,M+I组cyto-C蛋白表达降低,AC3阳性神经元数目减少(P<0.05)。
8.异氟烷对钙神经磷酸酶的表达和活性的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)以及异氟烷组(I组),处理同上。采用试剂盒检测CaN活性；提取总蛋白,用WB方法检测CN表达。
结果：与C组比较,CaN表达明显上调,活性明显增强(P<0.05)。
9.CaN抑制剂FK506对异氟烷诱导的p-drp-1表达和线粒体形态学改变的影响
方法：体外培养第5d的海马神经元随机分为对照组(C组)、异氟烷组(I组)FK506预给药后异氟烷处理组(F+I组)及FK506处理组(F组)。F+I组和F组在异氟烷处理2h前在无血清培养基中加入5μMFK506,而后C组合F组载体气处理6h,I组和F+I组1.5%异氟烷处理6h。异氟烷处理结束后,WB检测p-drp-1的表达。采用CellLight(?)Mitochondria-RFP BacMan2.0在海马神经元体外培养3d时转染,在异氟烷处理结束后,固定、封片后在激光共聚焦显微镜下观察线粒体形态学改变,采像后的图片在Imaris下进行处理统计。
结果：与C组比较,F组及F+I组所检测指标差异无显著性(P>0.05)；I组p-drp一1表达降低,线粒体直径减小(P<0.05)。与I组比较,F+I组p-drp-1表达升高,线粒体直径增加(P<0.05)。
10.FK506对异氟烷诱导的发育期神经元凋亡的影响
方法：体外培养第5d的海马神经元随机分为C组、I组、F+I组、F组,处理同上。采用WB检测cyto-C的表达；采用免疫荧光双标检测AC3的表达。
结果：与C组相比,F组及F+I组所检测指标差异无显著性(P,0.05)；I组cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组相比,F+I组cyto-C蛋白表达降低,AC3阳性神经元数目减少(P<0.05)。
11.异氟烷对新生5d (postnatal5day, PND5)大鼠海马线粒体形态学的影响
方法：PND5大鼠,随进分为C组合I组。C组吸入30%O2-70%N26h, I组以30%02-70%N2作为载气1.5%异氟烷处理6h,然后电镜灌注液灌注后,取标本放入2.5%戊二醛固定后,进行电镜检测。
结果：与C组相比,I组海马线粒体直径减小(P<0.05),甚至有部分线粒体破裂。
12.异氟烷对PND5大鼠海马线粒体动力学调节蛋白及p-drp-1表达的影响
方法：PND大鼠1.5%异氟烷处理6h后,取海马组织,提取总蛋白和总RNA,采用WB和实时定量PCR检测线粒体动力学调节蛋白及p-drp-1的表达
结果：与C组相比,I组海马线粒体动力学调节蛋白表达在mRNA和蛋白水平差异无统计学意义(P>0.05),但是p-drp-1表达明显降低(P<0.05)。
13. Mdivi-1对异氟烷诱导的PND5大鼠海马神经元凋亡的影响
方法：PND大鼠随机分为四组：C组、I组、M+I组及M组。C组和I组处理同前；M+I组在麻醉2h前腹腔注射5μg/kg mdivi-1,然后1.5%异氟烷处理6h；M组腹腔注射5μg/kg mdivi-1然后吸入30%O2-70%N2。麻醉结束后,尽取海马组织,提取总蛋白和总RNA,采用WB和实时定量PCR检测cyto-C的表达。1.5%异氟烷处理6h后,4%多聚甲醛固定,取大脑切片,采用免疫荧光双标检测AC3阳性神经元数目。
结果：与C组相比,M+I组和M组所检测指标无明显差异(P>0.05)；I组cyto-C蛋白表达上调,AC3阳性神经元数目增多(P<0.05)。与I组相比,M+I组cyto-C表达降低,AC3阳性神经元数目减少(P<0.05)。
14.Mdivi-1对异氟烷诱导的PND5大鼠长期认知障碍的影响
方法：PND大鼠1.5%异氟烷处理6h后,待其完全清醒,送回原笼饲养至60d,进行水迷宫检实验检测其空间学习记忆功能。实验完成后,麻醉电镜固定液灌注固定后,取海马组织进行电镜检测线粒体形态的变化。
结果：与C组比较,I组第3-5天时逃避潜伏期延长,探索时间缩短(P<0.05),M+I组和M组逃避潜伏期和探索时间差异无统计学意义(P>0.05)；与I组比较,M+I组第3-5d时逃避潜伏期缩短,探索时间延长(P<0.05)。电镜检测发现,与C组比较,I组线粒体直径减少(P<0.05),M+I组和M组差异无统计学意义(P>0.05)；与I相比,M+I组线粒体直径增加(P<0.05)。
15.统计学分析
采用SPSS20统计软件对数据进行分析,计量资料表示为均数±标准差(x±s),组间比较采用单因素方差分析,组内比较采用独立样本t检验；Morris水迷宫大鼠训练期间数据采用连续测量的方差分析,P<0.05为有统计学意义。
研究总结
一、主要研究结果
1.通过体外原代海马神经元培养以及在体动物实验证明临床相关浓度的异氟烷导致发育神经元线粒体动力学失衡。
2.体外及体内实验证实,异氟烷激活发育期神经元CaN,导致drp-1第637位丝氨酸去磷酸化,继而促使drp-1活性增加并向线粒体移位,导致线粒体过度分裂甚至碎片化,从而触发线粒体途径凋亡。
3.体外及体内实验证实,drp-1抑制剂mdivi-1可减轻异氟烷发育期神经元凋亡,改善异氟烷所致长期认知功能障碍。
二、研究结论
异氟烷引起胞浆Ca2+浓度升高,激活CaN,促进drp-1(ser637)去磷酸化,引起drp-1活性增强并向线粒体移位,从而导致线粒体过度分裂甚至碎片化,继而导致线粒体膜通透性增加,cyto-C释放,触发线粒体凋亡途径,最终导致发育期神经系统损伤；而drp-1抑制剂mdivi-1则可减轻异氟烷所诱导神经元凋亡和长期认知功能障碍。
Background
It was estimated that6million children (including1.5million infants) undergo surgery and anesthesia each year in the USA alone. Although there was no relevant data in China, the number of Chinese children received general anesthesia and surgery was much larger than that in USA on the fact of large number of Chinese population. The development of human brain begins in the early pregnant stage and ends several years after birth. It is generally accepted that brain growth spurt in humans was between the last month of gestation and first6months after birth. Therefore it is a hot issue whether general anesthesia could exert adverse effect on children's intelligence and long-lasting deficit in learning and memory.
In clinical trials, children who received general anesthesia in infant period represented cognitive impairment in adulthood. This indicated that general anesthetics could damage central nervous system in infants'period. In rodents, exposure to general anesthetics such as isoflurane, sevoflurane, devoflurane, ketamine and propofol in the period of peak brain growth induced damage of synaptic plasticity, neuroapoptosis and other neurodegenerative changes, finally impaired learning and memory in adulthood.
Inhalational anesthetics are extensively used in clinical anesthesia. In recent reports, isoflurane induced imbalance of cytoplasmic Ca2+and subsequently lead to neuroapoptosis in the mitochondrial pathway. However, the mechanism involved in the effect of isoflurane mitochondrial structure and function is unclear. Supposed that infants exposed to general anesthetics was indispensable, it is a challenge to elucidate the exact mechanism involved in isoflurane-induced abnormal structure and function of mitochondria and investigate the effective interventions for the prophylaxis and treatment of this disorder.
Mitochondria undergo dynamical fusion and fission events which maintain steady mitochondrial morphology. This process is also called mitochondrial dynamics. The rates of fusion and fission are regulated by several large GTPase molecules (OPA1, mfn1, mfn2, drp-1, mff and FIS1). Drp-1binds to the mitochondrial fission factor (mff) located on the outer membrane of mitochondrial and then promotes mitochondrial division, while OPA1, mfnl and mfn2potentiate mitochondrial fusion. Under physiological condition, the events of fusion and fission not only determine the shape and size of mitochondria but also regulate mitochondrial distribution and function. It was reported that dysregulation of fusion and fission of mitochondria in neurons affected the neurite growth, synaptic plasticity, long-term potentiation and even induced neuroapoptosis.
The molecules regulated mitochondrial dynamics were associated with neuroapoptosis and synaptic mis-interconnection in neurodegenerative diseases such as Alzheimer's disease and Huntington's disease. In the early stage of apoptosis, the activity of drp-1was upregulated and promoted mitochondrial fragmentation in mammalian cells. These changes lead to enlargement of mitochondrial cristae and increase of mitochondrial membrane permeability which facilitated the release of cytochrome C from mitochondrial cristae to the cytoplasm and initiated the mitochondrial apoptotic pathway. Amyloid-beta protein and huntingtin induced upregulation of drp-1and downregulation of mfiil and mfn2and promoted excessive mitochondrialfission and fragmentation which contributed to neuroapoptosis, damage of synaptic connections and long-lasting cognitive disorder. In summary, abnormal mitochondrial fission is an early demonstration of apoptosis; imbalance of mitochondrial fusion and fission underlie the pathophysiological mechanism of neurodegenerative diseases.
Ca2+in neuronal cytoplasm regulated the mitochondrial dynamics in several signaling pathway by the way that influence the expression and activity of the regulating proteins.With the nocuous stimuli, the level of cytoplasmic Ca2+was elevated, and then calcineurin was activated which dephosphorylated the637serine of drp-1. This change enhanced the activity of drp-1and promoted the fragmentation of mitochondrial and ultimately disturbed the interconnection of synapse and formation of neural circuit. Our previous results demonstrated that clinically relevant concentration of isoflurane elevated the level of cytoplasmic Ca2+and triggered mitochondrial apoptotic pathway in developmental neurons. However, it remains to be elucidated that isoflurane induced dysregulation of mitochondria dynamics via calcineurin signaling pathway.
In this study, the dynamic changes of mitochondrial morphology the regulating proteins were detected in neurons and rats. Also, pathways of cell signaling transduction relevant to the imbalance of mitochondrial dynamics induced by isoflurane were investigated. All the designed experiments are conducted to elucidate the role of mitochondrial dynamics disruption in the developmental neurotoxicity triggered by isoflurane and provide a new prospective for prophylaxis and medication of the neurotoxitiy.
Methods and Results
1. Effects of isoflurane on the mitochondrial morphological changes and mitochondrial membrane potential of rat developmental hippocampal neurons in vitro
Methods Neurons cultured5day in vitro(DIV) were randomly assigned to control group (group C) and isoflurane treated group (group I). Group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours, while group C was exposed to carrier air for6h. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and then treated by a series of special steps. And then eletron microscope was used to detect mitochondrial morphology. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed with4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended.
Results Compared with group C, the length of mitochondria in group I was significantly shorter (P<0.05).
2. Effects of isoflurane on mitochondrial membrane potential ((?)m) of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).
Results Compared with group C,(?)m in group I was significantly depolarized with detection of CLSM and flow cytometer(p<0.05).
3. Effects of isoflurane on proteins that regualted mitochondrial dynamics of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. Immediately at the end of isofluane exposure, total RNA and protein was extracted respectively for Western Blot (WB) and real-time PCR detection.
Results Compared with group C, the expression of drp-1, mff, FIS1, OPA1, mfn1and mfn2was no staticstical difference in group I at the level mRNA or protein (P>0.05).
4. Effects of isoflurane on the expression of phospholyrated drp-1(ser637) of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned. At the end of isoflurane anesthesia, total protein was extracted by RIPA for detection p-drp-1(ser637). And also the expression of p-drp-1(ser637) was assayed by double-immunofluorescence methods. Red fluorescence represented p-drp-1(ser637).
Results Compared with the group C, the expression of p-drp-1(ser637) significantly decreased and the positive stained neurons and red fluorescence drammatically declined (P<0.05).
5. Effects of inhibition of drp-1on isoflurane-induced(?)m depolarization of rat developmental hippocampal neurons in vitro
Methods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), mdivi-1pretreated and isoflurane expousre group (group M+I) and only mdivi-1treated group (group M). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6h; group M+I treated with5μM mdivi-1for2h and then exposed to1.5%isoflurane; group M was treated by5uM mdivi-1and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and confocal laser scanning microscope (CLSM).
Results Compared with the group C, the (?)mingroup M and group M+I did not declined significantly(P>0.05); while the (?)m in group Idecreased significantly (P<0.05). Compared with groupI, the (?)ningroup M+I depolarized significantly (P<0.05).
6. Effects of mdivi-1on isoflurane-induced mitochondrial morphological changes of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divied into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. DIV3neurons were transfected with CellLight(?) Mitochondria-RFP BacMan2.0and fixed by4%paraform and detected mitochondrial changes with confocal laser scanning microscope (CLSM) immediately the anesthesia ended. After isoflurane exposure, neurons were fixed by2.5%glutaraldehyde and sent for detection mitochondrial morphology with eletron microscope.
Results Compared with the group C, the diameter of mitochondria was not significantly different in group M and group M+I (P>0.05), while it was much smaller in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group M+I was significantly larger(P<0.05).
7. Effects of mdivi-1on neuronal viability, theexpression of cyto-C and AC3of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part5. The expression of cyto-C was detected at the levels of protein after isoflurane treatment. The expression of AC3was detected in a double-stained immunocytochemistric way.
Results Compared with the group C, all the detected parameters were not significantly different in group M and group M+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group M+I (P<0.05).The number of AC3-positive neurons was reduced in group M+I (P<0.05).
8. Effects of isoflurane on activity and expression of calcineurin(CaN) of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly assigned to control group (group C) and isoflurane treated group (group I). The treatment of neurons was the same as aforementioned in Part.1. Immediately at the end of isofluane exposure, the total protein was extracted for WB and the activity of CaN was assayed by the commecially kit following the manufacture's instructions.
ResultsAfter1.5%isoflurane treatment, the activity and expression of CaN increased significantlly in neurons(P<0.05).
9. Effects of FK506(inhibitor of CaN) on the isoflurane-induced downregualtion of p-drp-1(ser637), abnomal mitochondrial morphology and depolarized (?)mof rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divied into four groups:Control group (group C),isoflurane treated group (group I), FK506pretreated and isoflurane expousre group (group F+I) and only FK506treated group (group F). Group C was exposed to carrier air for6hours;group I was exposed to1.5%isofluane carried by5%CO2-8%O2-87%N2for6hours; group F+I treated with5nM FK506for2hours and then exposed to1.5%isoflurane; group F was treated by5nMFK506and exposure to carrier air for6hours. Neurons was harvested and inbubated with JC-1for30min at37℃and subject to detection with flow cytometer and CLSM.
Results Compared with the group C, diameter was not significantly different in group F and group F+I (P>0.05), while it significantly changed in group I (P<0.05). Compared with groupI, the diameter of mitochondria in group F+I was significantly larger (P<0.05); the (?)mingroup F+I increased significantly (P<0.05); the downregulation of p-drp-1(ser637) was significantly upregulated (P<.05).
10. Effects of FK506on the expression of cyto-C and AC3of rat developmental hippocampal neuronsin vitro
Methods Neurons DIV5were randomly divided into group C,group I, group M+I and group M. The detailed treatment processure was refered to setion Methods in Part9. The expression of cyto-C was detected at the protein level after isoflurane treatment.The expression of AC3was detected in a double-stained immunocytochemistric way.
Results Compared with the group C, all the detected parameters were not significantly different in group F and group F+I (P>0.05). Compared with the group I, the expression of cyto-C was downregulated significantly in group F+I (P<0.05); The number of AC3-positive neurons was reduced in group M+I (P<0.05).
11. Effects of isoflurane on the mitochondrial morphological changes in CA1of PND5rats
Methods PND5rats were randomly assigned to group C and group I. Group I was exposure to1.5%isoflurane for6h carried by30%O2-70%N2,while group C exposed to carrier air. At the end of anesthesia, CA1in hippocampus was gently dissected and transferred to2.5%glutaraldehyde for2h fixation and send for eletron microscopy imaging and analysis.
Results Compared with the group C, the diameter of mitochondria was significantly reduced in group I and mitochondrial in group I was significantly condensed(P<0.05).
12. Effects of isoflurane on proteins that regualted mitochondrial dynamics and p-drp-1in hippocampi of PND5rats
MethodsPND5rats were randomly assigned to group C and group I. The detailed treatment information was refered to Part.11. At the end of anesthesia, hippocampi were dissected for total protein and RNA extraction.
ResultsCompared with group C, the expressions of drp-1, mff, FIS1, OPA1, mfn1and mfn2were no staticstical difference in group I at the level mRNA (P>0.05);but the expression of p-drp-1(ser637) significantly decreased(P<0.05).
13. Effects of mdivi-1on isoflurane-induced neuronal apoptosis and the expression of cyto-Cin hippocampi of PND5rats
Methods PND5rats were randomly divided into group C,group I, group M+I and group M. Group C and group I were treated as delineted Part.11. Group M+I and group Mreceived a dose of5μg/kg mdivi-1by intraperitoneal injection2h before isoflurane treatment. After anesthesia, hippocampi was dissociated for detection ofcyto-C. And ratswere decapitated and frozen sections of brainwere made to stain the positive active caspse3neurons.
Results Compared to group C, there were no statistical difference in expression of cyto-C and the amount of active caspase3-positive neurons in group M+I and group M(P>0.05). Compared with group I, the expression cyto-C was downregulated significantly in group M+I (P<0.05) and the number of active caspase3-positive neurons was smaller(P<0.05).
14. Effects of mdivi-1on isoflurane-induced long-term mitochondrial changes and deficit in learning and memory
Methods PND5rats were randomly divided into group C,group I, group M+I and group M.The detailed treatment processure was refered to setion Methods in Part13. After isofluane exposure, rats were sent to cages until they were fully awake and raised to60days after their birth when Morris water maze was used to assess spatial learning and memory.
Results Compared with group C, the latency was significantly longer and the probe time wasshorter in group I from the third training dayto the lasttraining day (P<0.05). However, these changes were reversed in groupM+I (P<0.05).
15. Statistical analyses
All datawerepresentedandgraphedas the mean±EM. The Statistical Package for the Social Sciences20software was used for the statistical analyses. Data acquired fromthe detection of protein and mRNA was analyzed with an analysis of variance (ANOVA), followed by a least square difference (LSD) multiple comparison test. Data collected from the spatial acquisition trials were analyzed using a repeated measures ANOVA (the different treatments were the between groups factors and time was the repeated measures factor), followed by a post-hoc test to compare four groups. Differences were deemed statistically significant if P<0.05.
Summaries
1. It was verified that clinical relevant concentration isoflurane induced imbalance of mitochondrial dynamics in in vitro and in vivo.
2. It was confirmed that isoflurane activated calcineurin which dephosphorylated drp-1at serine-637. The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation.
3. Pretreatment with mdivi-1, an inhibitor of drp-1, mitigated isoflurane-induced neuroapoptosis and long-lasting cognitive impairment in in vitroandin vivo.
Conclusions
Elevated cytoplasmic Ca2+induced by Isoflurane via activating GABAA or facilitating IP3and RyR receptors increased the activity of calcineurin which dephosphorylated drp-1at serine-637.The dephosphorylated drp-1translocated to mitochondria our membrane and promoted excessive mitochondrial fission even fragmentation, which increased the permeability of mitochondrial transition pore. Thus cyto-C tethered in mitochondrial cristae released and initiated the mitochondrial apoptototic pathway of developmental neurons. However, mdivi-1, an inhibitor of drp-1, alleviated isoflurane-induced neuroapoptosis in vitro and in vivo and improved long-lasting cognitive function.
引文
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