异氟烷对发育期大鼠海马神经元电生理及兴奋性神经毒性的影响
摘要
随着现在医学的发展进步,相当一部分小儿,特别是婴幼儿和孕妇不得不在全身麻醉下接受手术治疗。最新统计资料表明仅美国每年约有600万儿童患者接受手术和麻醉,其中包括150万婴儿患者。由于人类神经系统从妊娠第5W开始发育生长,持续至婴幼儿,其中妊娠后期以及新生儿期是神经元轴突生长、突触形成及可塑性的关键时期,因此吸入麻醉药(如异氟烷.七氟烷等)对发育期神经元的毒性作用越来越受到人们的广泛关注。最近研究表明,吸入麻醉药以剂量时间依赖性导致发育期神经元毒性。然而具体机制尚未完全明确。因此本研究探讨全身麻醉药对发育期神经系统生长发育、突触形成及可塑性是否存在毒性作用。
与成熟神经元不同,发育期神经元展现很多独特的特征。其中,最为重要的一个特征是GABA (Gamma aminobutyric acid, GABA)介导的兴奋性。在发育期由于升高细胞内氯离子浓度的Na+-K+-2Cl-同向转运体(Na+-K+-2Cl-cotransporter, NKCC)和降低内氯离子浓度的K+-Cl-同向转运体(K+-Cl-cotransporter, KCC)的相互作用导致发育期神经元胞内处于高浓度氯离子状态。因此GABA这一成熟期中枢神经系统的重要抑制性神经递质,在神经元发育早期是作为一种主要的兴奋性神经递质而发挥作用。N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid, NMD A)受体即具有配体门控的特点又具有电压依赖性激活特点。在成熟神经元中NMDA受体激活需要借助α-氨基羟甲基恶唑丙酸受体(a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor, AMPAR)去除Mg2+对NMDA受体的阻滞,激活NMDA受体引起细胞外Ca2+内流,继而激活CK2, PLC-PKC和PKA等细胞信号通路,促进神经元轴突生长、突触形成及可塑性。而在发育期神经元胞膜上,AMPA受体尚不具备完整离子运输功能,NMDA受体的激活依赖于激活其胞膜表达的GABAA受体(Gamma aminobutyric acid type A receptor, GABAA),使细胞内Cl-外流,细胞膜去极化,代替或者协同AMPA受体的功能,去除Mg2+对NMDA受体的阻滞作用,从而激活NMDA受体。从出生后第二天AMPA才逐渐开始发挥其在成熟神经元中的神经传递功能。亦有研究报道GABA能够直接激活电压依赖性钙通道(Voltage dependent calcium channels, VDCC)引起钙离子从细胞外内流。而过分的激活VDCC会引起细胞内自由钙离子水平升高。研究表明高细胞内自由钙离子会导致细胞产生病理性改变,甚至凋亡。因而与成熟神经元不同,在胚胎期和出生后早期,GABAA受体介导突触后兴奋效应,在轴突生长、突触形成和神经元可塑性方面起着重要作用。
图1发育期神经元膜受体与成熟期神经元之间的差异(A)成熟期神经元,激活AMPA受体使细胞膜去极化,可以移除镁离子对NMDA受体阻滞作用。而GABA受体激活则细胞膜超极化,可以抑制NMDA受体的激活,形成一个兴奋和抑制平衡。(B)发育期神经元,激活AMPA受体和(或者)GABA受体都可以致细胞膜去极化,促进NMDA受体的激活
异氟烷(Isoflurane)在体内代谢少,大部分以原形从肺排出体外,具有可控性好、比较安全、有效的特点,是一种广泛应用于临床的吸入麻醉药。异氟烷主要通过直接激活或者增强中枢神经系统内的γ-氨基丁酸A (Gamma aminobutyric type A, GABAA)受体、谷氨酸(Glutamine, Glu)受体和甘氨酸(glycine, Gly)受体的电生理功能发挥作用。最近研究证实吸入性全身麻醉药具有通过GABAA受体激活NMDA受体和VDCC等引起钙离子内流,进而增加细胞内自由钙离子浓度,导致cytochrome C释放及Caspase-3的激活,最终导致内源性和外源性细胞内凋亡。但是具体机制尚未明确。图2麻醉药致发育期神经毒性机制线路图(A)生理状态下,内源性GABA作用于突触后膜的GABAA受体,细胞膜去极化,易化NMDA受体和VDCC,导致钙离子内流,激活Ca2+/CAM等一系列激酶,导致CREB磷酸化,启动促细胞生长因子和抗凋亡因子。(B)麻醉药如异氟烷作用后,直接激活GABAA受体和增强内源性GABA反应性,过度激活NMDA受体和VDCC,导致大量细胞外钙离子内流,钙离子内流触发细胞内钙释放,导致钙超载,引起细胞色素C释放,启动内源性和外源性细胞凋亡。
因此本研究拟选择离体培养海马神经元培养,从神经元电生理特性,神经元内自由钙离子浓度、神经元存活等方面探讨异氟烷在发育期海马神经元导致的神经毒性。本研究拟检验以下假说：(1)异氟烷可影响发育期海马神经元的电生理功能；(2)临床安全浓度的异氟烷在安全时间内使用并不导致发育期海马神经元凋亡；(3)临床安全浓度的异氟烷通过增强海马神经元的自由钙水平而双向调节神经元的生长(如图2所示)。
研究方法与结果
1.异氟烷对发育期神经元GABAA受体电流的影响
方法：用出生后1d的SD乳鼠分离培养海马锥体神经元,全细胞膜片钳技术记录锥体神经元GABAA受体介导的Cl-电流(GAB A电流,Igaba),不同浓度的GABA和1μmol/L GABAA受体特异性拮抗剂荷包牡丹碱(Bicuculline)诱发并确定IGABA;用不同浓度的异氟烷(0.25、0.5、0.75、1MAC)对50μmol/LGABA诱发IGABA的影响
结果：GABA诱发内向电流(IGABA),其EC50为23.73μmol/L,荷包牡丹碱阻断IGABA;异氟烷(0.25、0.5、0.75、1MAC)剂量依赖性增强IGABA,其EC为0.53MAC。
2.异氟烷对发育期神经元NMDA受体电流的影响
方法：原代培养1d龄SD大鼠海马神经元5天(DIV5)。运用全细胞膜片钳技术记录暴露不同浓度异氟烷下NMDA受体电流(NMDA电流,INMDA),100μmol/L NMDA和10μmol/L NMDA受体特异性拮抗剂MK801诱发并确定INMDA;用不同浓度的异氟烷对100μmol/LNMDA诱发INMDA的影响
结果：异氟烷(0.25、0.5、0.75、1MAC)增强NMDA受体电流幅度分别为116±8.8,122±11.7,135±14.3和132±14.6%。10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的NMDA受体电流(P<0.05)。而40μM MK801预处理海马神经元可完全逆转异氟烷增加的NMDA受体电流(P<0.05)
3.异氟烷对发育期神经元VDCC电流的影响
方法：原代培养1d龄SD大鼠海马神经元5天(DIV5)。利用全细胞膜片钳技术记录暴露不同浓度异氟烷(0.25、0.5、0.75、1MAC)下电压依赖性钙通道电流(IVDCC)。运用L-钙通道拮抗剂Nicadipine和GABAA受体拮抗剂Bicuculline探讨临床相关浓度异氟烷对电压依赖性钙电流增强机制
结果：表明异氟烷(0.25、0.5、0.75、1MAC)增强IVDCC电流幅度分别为109.11±9.03,120.56±11.46,141.33±13.87和146.78±15.87%。10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的IVDCC (P<0.05)。而10μM Nicardipine预处理海马神经元可完全逆转异氟烷增加的IVDCC (P<0.05)。
4.异氟烷对发育期海马神经元胞内自由钙离子浓度[Ca2+]的影响
方法：1d龄SD大鼠海马神经元原代培养5天(DIV5)。运用Calcium Imaging测定异氟烷(0.25、0.5、0.75、1MAC)对100μmol/L GABA荧光强度的变化。Nicadipine和Dantrolene探讨临床相关浓度异氟烷增强GABA对细胞内钙浓度的机制。
结果：异氟烷呈浓度依赖性增强体外培养第5d的海马神经元GABA诱发的钙浓度增加(P<0.05),10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的GABA诱发的钙离子浓度升高(P<0.05)。而10μM Dantrolene预处理海马神经元可完全逆转异氟烷增强GABA钙离子浓度升高(P<0.05)。
5.异氟烷对发育期海马神经元caspase-3表达的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于培养至第5d(DIV5),运用Western blot和实时定量PCR技术,检测不同浓度异氟烷(0.25、0.5、0.75、1MAC)暴露6h后发育期神经元中caspase-3表达变化。
结果：与对照组相比,异氟烷组呈剂量依赖性增加海马神经元nRNA caspase-3表达(P<0.05)；在蛋白水平上,1MAC异氟烷可以明显增强海马神经元cleaved caspase-3表达(P<0.05)。
6.异氟烷对发育期海马神经元cytochrome c表达的影响
方法：1d龄新生SD大鼠海马神经元原代培养至第5d (DIV5),运用Western blot和实时定量PCR技术,检测不同浓度异氟烷(0.25、0.5、0.75、1MAC)暴露6h后发育期神经元中cytochrome c表达变化。
结果：与对照组相比,异氟烷组呈剂量依赖性增加海马神经元cytochrome c mRNA表达(P<0.05)；在蛋白水平上,0.75MAC和1MAC异氟烷可以明显增强海马神经元cytochrome C的表达(P<0.05)
7.异氟烷对体外培养大鼠发育期海马神经元CREB信号的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于体外培养至第5d(DIV5),利用实时定量PCR技术,检测临床相关浓度异氟烷暴露6h后发育期神经元中CREB表达变化及其下游基因脑源性生长因子(BDNF)、生长相关蛋白(GAP43)、PSD-95和抑凋亡基因Bcl-2的表达。
结果：与对照组相比,异氟烷组呈剂量依赖性增加神经元CREB mRNA、GAP43mRNA、PSD-95mRNA、BDNF mRNA和Bcl-2mRNA表达(P<0.05)。
8.异氟烷对体外培养发育期大鼠海马神经元MEF2信号的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于体外培养至第5d(DIV5),利用用Real-time PCR进行定量分析不同浓度异氟烷暴露6h后发育期神经元MEF2信号通路(MEF2mRNA、synGAPI mRNA和Arc mRNA)。
结果：与C组相比,临床相关浓度异氟烷干预6h后呈时间依赖性上调海马神经元MEF2mRNA、synGAP I mRNA、Arc mRNA (P<0.05)。
9.异氟烷对发育期大鼠海马神经元凋亡的影响
方法：将新生5d SD大鼠放入相对密闭的自制器皿,用麻醉挥发罐1MAC异氟烷干预6h。对照组不做任何处理。干预后提取出海马神经元总mRNA,运用Real-time PCR检测异氟烷干预后发育期海马神经元凋亡率。
结果：与对照组相比,1MAC处理组异氟烷剂量依赖性增加神经元凋亡(P<0.05)。
10.异氟烷对发育期大鼠海马神经元MEF2信号的影响
方法：新生5d龄SD大鼠(PSD5),随机分为6h和麻醉停止后24h异氟烷组和对照组(C组)。将新生5d SD大鼠放入相对密闭的自制器皿,用麻醉挥发罐1MAC异氟烷干预。对照组不做任何处理。干预后在各时间点取海马神经元提取RNA和组织总蛋白质,运用Real-time PCR进行定量分析MEF2信号通路(MEF2mRNA、 synGAPI mRNA和Arc mRNA)。
结果：与C组相比,1MAC异氟烷干预后呈时间依赖性上调海马神经元MEF2mRNA、synGAPI mRNA、Arc mRNA (P<0.05),麻醉停止后24h恢复正常。
统计学分析
采用SPSS13.0统计软件进行分析,计量资料以均数±标准差(x±s)表示,组内比较采用one-way ANOVA,组间比较采用独立样本或者配对t检验,P<0.05为有统计学意义
研究总结
一、主要研究结果
1.通过离体1d龄SD大鼠海马神经元原代培养5天(Day in vitro, DIV5d)以及整体动物实验从神经突触形成以及神经元存活两个方面证实了临床相关浓度吸入麻醉药异氟烷剂量时间依赖性致发育期海马神经元神经毒性。
2.通过离体1d龄SD大鼠海马神经元原代培养证实临床相关浓度吸入性麻醉药异氟烷对发育期海马神经元VDCCs, NMDA受体和GABAA受体的激活及升高细胞内自由钙离子浓度的影响。
3.在体动物以及离体细胞实验中均证实了临床浓度异氟烷(0.25、0.5、0.75、1MAC)对MEF2和CREB信号通路的影响。
二、研究结论
异氟烷致发育期海马神经元神经毒性的作用可能主要通过增强GABAA受体,细胞膜去极化,从而易化NMDA受体和VDCC,触发细胞外钙离子内流,引起钙触发钙释放(calcium-induced calcium release, CICR)从而导致细胞内钙离子超载,caspase-3和cytochrome C表达上调,导致细胞凋亡。同时钙离子内流可以上调MEF2和CREB Ser-133位点磷酸化,调节其下游对突触发生以及神经存活起重要作用的BDNF、Bcl-2以及突触素Ⅰ (synapsin Ⅰ)表达。
Background
With the increasing sophistication of surgical and anesthetic techniques, increasingly pregnant patients and fetal surgeries are being undertaken. Statistics showed that in the U.S. alone, about6million children annually undergo surgery and anesthesia, including1.5million infant patients. In this view, the study that the inhalation anesthetics isoflurane causes neurotoxicity in the immature brain arouses wide-spread social concern. Recent experiments suggest that volatile anesthetics cause excitotoxicity in a concentration-and time-dependent manner in neuronal models in vitro. And neurons in the developing brain are specifically vulnerable to isoflurane hyperexcitability. However, the mechanisms for isoflurane cytotoxicity remain enigmatic. Because most fetal surgeries in humans are performed during mid-gestation, it is important and urgent to know if the anesthetics used cause damage to the developing brain and subsequent postnatal memory problems and learning disabilities.
Developing neurons exhibit some characteristics different from those of adult neurons. GAB A mediates excitability in the immature brain. In this respect, a major finding is that the GABAA receptor, activated by its agonist, induces a depolarizing membrane in neonatal neurons compared with the hyperpolarizing one in adult neurons, and results in a cytosolic Ca2+increase. The probable mechanism through which GABA elicits cytosolic Ca2+increases in the neurons seems to involve activation of the GABAa receptors; subsequent Cl-efflux attributable to a developmental depolarized Cl-reversal potential causes membrane depolarization and Ca2+entry via voltage-activated Ca2+channels (VDCC) During the early period of synapse development, the immature neuron has a relatively high intracellular cell concentration of Cl-because of developmental-specific expression of the Na+-K+-2Cl-co-transporters (NKCCs) and the K+-Cl-co-transporters (KCCs) and GABA depolarizes and excites neuronal membranes by a reverse chloride gradient. Animal experiments suggest that activation of GABAergic synapses generates action potentials, removes the voltage-dependent Mg2+block from NMDA channels in the hippocampus and in other brain structures. These effects may underlie the well-characterized modulation by GABA of activity-dependent developmental processes including neuronal growth, neuronal differentiation, proliferation and migration, growth rates of neuronal processes and synapse formation and clustering. GABA through the activation of GABAA receptors triggers spontaneous giant depolarizing potentials. These large depolarizations give rise to fast action potentials, which are synchronous over the entire hippocampus and are associated with spontaneous calcium transients. These highly correlated calcium signals are thought to be essential for consolidation of synaptic connections and development of the adult neuronal network. Fig.l The different actions of GABA and AMPA receptors in developing neurons.(A)The equilibrium between excitation (glutamate-releasing synapses) and inhibition (GABA-releasing synapses) in the adult mammalian brain (B) The disequilibrium between glutamate-mediated excitation and GABA-mediated inhibition.The activation of GABAA receptors in neonatal slices generated a depolarization that was sufficient to remove the voltage-dependent Mg2+block of NMDA receptors. Several experiments in vitro confirm that a conversion of "silent" glutamatergic synapses to conductive AMPA synapses starts gradually after P2in the CA3-CA1pyramidal neurons.
Isoflurane, one of the most widely used inhalation anesthetics for various fetal surgeries or procedures in pregnant patients, has been shown previously to induce caspase activation and apoptosis when applied in concentration in the immature brain. Recently, it was reported that isoflurane might induce apoptosis by increasing [Ca2+]i. Isoflurane has been shown to potentiate GABAA receptor current and directly open GABAA receptor and glycine receptor channels even in the absence of agonist. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitation of NMDA receptors and opens VDCC, elevates [Ca2+]i, and promotes the activation of caspase-3. However, up until recently, the exact mechanism to induce apoptosis remains enigmatic.
Fig.2During development, when the GABAA receptors are activated, there is an efflux of chloride, leading to a depolarization that can remove the voltage-dependent Mg2+block from NMDA receptors, generate sodium and calcium action potentials, directly activates VDCC and elevates the [Ca2+]i, leads further CICR from the ryanodine-sensitizing Ca2+store in the hippocampus and in other brain structures. The influx of GABA-induced Ca2+through extracellular medium linked to the binding of Ca2+to CaM and the rapid translocation of CaM to the cell nucleus. This process leads to the activation of the transcription factors, CREB. When activated, CREB promoted the transcription of a set of genes, such as the expression of c-fos, BDNF, PSD-95, synapsinl, Bcl-2and so on, which regulate expression of axon growth and synaptic plasticity in the developing hippocampal neurons and results in the stimulation of new gene expression
In the present research, we evaluated the effect of clinically relevant concentrations of isoflurane on electrophysiological properties and gene expression in developing rat hippocampal neurons. We tested the following hypotheses:Ⅰ isoflurane could affect neuronal electrophysiological function in the immature brain; Ⅱ clinically relevant concentrations of volatile anesthetic isoflurane have no effect on the neuronal apoptosis in short-term; Ⅲ Bidirectional effect of isoflurane on regulation of neuronal development in the immature brain
Methods and Results
1. Isoflurane potentiates IGABA in primary rat hippocampus neuronal culture at day in vitro (DIV)5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of IGABA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on IGABA. bicuculline was employed.
Results:GABA evoked inward current (IGABA), the EC50was23.73μmol/L, bicuculline blocked IGABA; isoflurane dose-dependent increase IGABA, the EC50was23.73μmol/L.
2. Isoflurane potentiates INMDA in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and MK801were employed.
Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated INMDA peak current amplitude by116±8.8,122±11.7,135±14.3and132±14.6%, respectively. Application of bicuculline partially inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05), and application of MK801completely inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05)
3. Isoflurane potentiates IVDCC in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and nicardipine were employed.
Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated IVDCC peak current amplitude by109.11±9.03,120.56±11.46,141.33±13.87and146.78±15.87%, respectively. pplication of nicadipine partially inhibited the amplitude of IVDCC to the63.11±8.48%of control (P<0.001) and application of nicadipine inhibited the amplitude of isoflurane-mediated IVDCC to the79.33±7.57%of control, but application of bicuculline had no effect on the amplitude of IVDCC (P>0.05) and application of bicuculline inhibited the amplitude of isoflurane-mediated IVDCC from146.78±15.87%to116.56±10.31%of control
4. Isoflurane potentiates GABA-triggered [Ca2+]i transient in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used.[Ca2+]i was monitored using Fluo-4AM fluorescence imaging. To evaluate whether intracellular calcium release was involved in the potentiation effect of isoflurane, Nicardipine and Dantrolene was employed.
Results:The enhancement effect of isoflurane was in a concentration-dependent manner. The EC50for enhancement effect was0.61±0.05MAC. Consistent with previous reports, dantrolene significantly decreased the GABA-triggered [Ca2+]i responses of control (P<0.001, n=11). Furthermore, preconditioning with dantrolene significantly decreased the potentiation of GAB A induced [Ca2+]i transients mediated by1MAC isoflurane in developing hippocampal neurons. Pre-application of nicadipine partially inhibited GABA-tiggered [Ca2+]i transients of control (P<0.001) and isoflurane-mediated potentiation of the GABA-evoked [Ca2+]i rise of GABA (P=0.001).
5. Effect of isoflurane on caspase-3levels in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of caspase-3is regulated by high [Ca2+]i, the possibility of increased caspase-3 levels after isoflurane in dose-and time-dependent manner was evaluated.
Results:The effect of treatments with isoflurane on caspase-3activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal caspase-3mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.001).
6. Effect of isoflurane on the levels of cytochrome C in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of cytochrome C is regulated by high [Ca2+]i, the possibility of increased caspase-3levels after isoflurane in dose-and time-dependent manner was evaluated.
Results:The effect of treatments with isoflurane on cytochrome C activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal cytochrome C mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.05).
7. Effect of isoflurane on the levels of transcriptional factor CREB in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the CREB signaling pathway (GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA) after intervention.
Results:Compared with the group C, isoflurane increased in neuronal GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA expression in dose-dependent manner (P<0.05).
8. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA、synGAPI mRNA and Arc mRNA) and Synapsin I mRNA after intervention. Western blot was used to detect the expressions of Synapsin I at the protein level.
Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and Synapsin I mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)
9. Effect of Isoflurane on the development of hippocampal neurons apoptosis in vivo
Methods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane6h. And a control group which received no treatment. Pups were killed by decapitation to extract total mRNA after intervention. Real-tine PCR was used to detect the expressions of caspase-3.
Results:Compared with the group C, isoflurane increased in neuronal caspase-3expression (P<0.05)
10. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in vivo
Methods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane2,4,6h, respectively. And a control group which received no treatment. Pups were killed by decapitation to extract total RNA from hippocampal neurons after intervention. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA、synGAPI mRNA and Arc mRNA) and SynapsinI mRNA after intervention. Western Blot was used to detect the expressions of synapsinl at the protein level.
Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and SynapsinI mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)
Main Results
1. Isofluane-induced developmental neurotoxicity in developing hippocampal neurons was confirmed by researches in vitro (5-day cultured primary neurons) and in vivo (whole animal experiments) through two aspects of survival and neurogenesis of neurons, neurotoxicity.
2. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitates NMDA receptors and opens VDCC, elevates [Ca2+]i. Effects of isoflurane on MEF2and CREB signal pathway were confirmed both in vitro and in vivo.
3. From the investigation of the effect of isoflurane on the synaptic plasticity in developing hippocampal neurons, we can draw a conclusion that bidirectional effects of isoflurane on regulation of neuronal development in the immature brain.
Conclusions
Clinical relevant concentrations of inhaled anesthetics isoflurane-mediated potentiation of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCC and further CICR from the ryanodine-sensitizing Ca2+store. An increase in [Ca2+]i, due to activation of GABAA receptor, facilitation of NMDA receptor and opening of VDCC, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.
与成熟神经元不同,发育期神经元展现很多独特的特征。其中,最为重要的一个特征是GABA (Gamma aminobutyric acid, GABA)介导的兴奋性。在发育期由于升高细胞内氯离子浓度的Na+-K+-2Cl-同向转运体(Na+-K+-2Cl-cotransporter, NKCC)和降低内氯离子浓度的K+-Cl-同向转运体(K+-Cl-cotransporter, KCC)的相互作用导致发育期神经元胞内处于高浓度氯离子状态。因此GABA这一成熟期中枢神经系统的重要抑制性神经递质,在神经元发育早期是作为一种主要的兴奋性神经递质而发挥作用。N-甲基-D-天冬氨酸(N-methyl-D-aspartic acid, NMD A)受体即具有配体门控的特点又具有电压依赖性激活特点。在成熟神经元中NMDA受体激活需要借助α-氨基羟甲基恶唑丙酸受体(a-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor, AMPAR)去除Mg2+对NMDA受体的阻滞,激活NMDA受体引起细胞外Ca2+内流,继而激活CK2, PLC-PKC和PKA等细胞信号通路,促进神经元轴突生长、突触形成及可塑性。而在发育期神经元胞膜上,AMPA受体尚不具备完整离子运输功能,NMDA受体的激活依赖于激活其胞膜表达的GABAA受体(Gamma aminobutyric acid type A receptor, GABAA),使细胞内Cl-外流,细胞膜去极化,代替或者协同AMPA受体的功能,去除Mg2+对NMDA受体的阻滞作用,从而激活NMDA受体。从出生后第二天AMPA才逐渐开始发挥其在成熟神经元中的神经传递功能。亦有研究报道GABA能够直接激活电压依赖性钙通道(Voltage dependent calcium channels, VDCC)引起钙离子从细胞外内流。而过分的激活VDCC会引起细胞内自由钙离子水平升高。研究表明高细胞内自由钙离子会导致细胞产生病理性改变,甚至凋亡。因而与成熟神经元不同,在胚胎期和出生后早期,GABAA受体介导突触后兴奋效应,在轴突生长、突触形成和神经元可塑性方面起着重要作用。
图1发育期神经元膜受体与成熟期神经元之间的差异(A)成熟期神经元,激活AMPA受体使细胞膜去极化,可以移除镁离子对NMDA受体阻滞作用。而GABA受体激活则细胞膜超极化,可以抑制NMDA受体的激活,形成一个兴奋和抑制平衡。(B)发育期神经元,激活AMPA受体和(或者)GABA受体都可以致细胞膜去极化,促进NMDA受体的激活
异氟烷(Isoflurane)在体内代谢少,大部分以原形从肺排出体外,具有可控性好、比较安全、有效的特点,是一种广泛应用于临床的吸入麻醉药。异氟烷主要通过直接激活或者增强中枢神经系统内的γ-氨基丁酸A (Gamma aminobutyric type A, GABAA)受体、谷氨酸(Glutamine, Glu)受体和甘氨酸(glycine, Gly)受体的电生理功能发挥作用。最近研究证实吸入性全身麻醉药具有通过GABAA受体激活NMDA受体和VDCC等引起钙离子内流,进而增加细胞内自由钙离子浓度,导致cytochrome C释放及Caspase-3的激活,最终导致内源性和外源性细胞内凋亡。但是具体机制尚未明确。图2麻醉药致发育期神经毒性机制线路图(A)生理状态下,内源性GABA作用于突触后膜的GABAA受体,细胞膜去极化,易化NMDA受体和VDCC,导致钙离子内流,激活Ca2+/CAM等一系列激酶,导致CREB磷酸化,启动促细胞生长因子和抗凋亡因子。(B)麻醉药如异氟烷作用后,直接激活GABAA受体和增强内源性GABA反应性,过度激活NMDA受体和VDCC,导致大量细胞外钙离子内流,钙离子内流触发细胞内钙释放,导致钙超载,引起细胞色素C释放,启动内源性和外源性细胞凋亡。
因此本研究拟选择离体培养海马神经元培养,从神经元电生理特性,神经元内自由钙离子浓度、神经元存活等方面探讨异氟烷在发育期海马神经元导致的神经毒性。本研究拟检验以下假说：(1)异氟烷可影响发育期海马神经元的电生理功能；(2)临床安全浓度的异氟烷在安全时间内使用并不导致发育期海马神经元凋亡；(3)临床安全浓度的异氟烷通过增强海马神经元的自由钙水平而双向调节神经元的生长(如图2所示)。
研究方法与结果
1.异氟烷对发育期神经元GABAA受体电流的影响
方法：用出生后1d的SD乳鼠分离培养海马锥体神经元,全细胞膜片钳技术记录锥体神经元GABAA受体介导的Cl-电流(GAB A电流,Igaba),不同浓度的GABA和1μmol/L GABAA受体特异性拮抗剂荷包牡丹碱(Bicuculline)诱发并确定IGABA;用不同浓度的异氟烷(0.25、0.5、0.75、1MAC)对50μmol/LGABA诱发IGABA的影响
结果：GABA诱发内向电流(IGABA),其EC50为23.73μmol/L,荷包牡丹碱阻断IGABA;异氟烷(0.25、0.5、0.75、1MAC)剂量依赖性增强IGABA,其EC为0.53MAC。
2.异氟烷对发育期神经元NMDA受体电流的影响
方法：原代培养1d龄SD大鼠海马神经元5天(DIV5)。运用全细胞膜片钳技术记录暴露不同浓度异氟烷下NMDA受体电流(NMDA电流,INMDA),100μmol/L NMDA和10μmol/L NMDA受体特异性拮抗剂MK801诱发并确定INMDA;用不同浓度的异氟烷对100μmol/LNMDA诱发INMDA的影响
结果：异氟烷(0.25、0.5、0.75、1MAC)增强NMDA受体电流幅度分别为116±8.8,122±11.7,135±14.3和132±14.6%。10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的NMDA受体电流(P<0.05)。而40μM MK801预处理海马神经元可完全逆转异氟烷增加的NMDA受体电流(P<0.05)
3.异氟烷对发育期神经元VDCC电流的影响
方法：原代培养1d龄SD大鼠海马神经元5天(DIV5)。利用全细胞膜片钳技术记录暴露不同浓度异氟烷(0.25、0.5、0.75、1MAC)下电压依赖性钙通道电流(IVDCC)。运用L-钙通道拮抗剂Nicadipine和GABAA受体拮抗剂Bicuculline探讨临床相关浓度异氟烷对电压依赖性钙电流增强机制
结果：表明异氟烷(0.25、0.5、0.75、1MAC)增强IVDCC电流幅度分别为109.11±9.03,120.56±11.46,141.33±13.87和146.78±15.87%。10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的IVDCC (P<0.05)。而10μM Nicardipine预处理海马神经元可完全逆转异氟烷增加的IVDCC (P<0.05)。
4.异氟烷对发育期海马神经元胞内自由钙离子浓度[Ca2+]的影响
方法：1d龄SD大鼠海马神经元原代培养5天(DIV5)。运用Calcium Imaging测定异氟烷(0.25、0.5、0.75、1MAC)对100μmol/L GABA荧光强度的变化。Nicadipine和Dantrolene探讨临床相关浓度异氟烷增强GABA对细胞内钙浓度的机制。
结果：异氟烷呈浓度依赖性增强体外培养第5d的海马神经元GABA诱发的钙浓度增加(P<0.05),10μM Bicuculline预处理海马神经元能够部分抑制异氟烷增加的GABA诱发的钙离子浓度升高(P<0.05)。而10μM Dantrolene预处理海马神经元可完全逆转异氟烷增强GABA钙离子浓度升高(P<0.05)。
5.异氟烷对发育期海马神经元caspase-3表达的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于培养至第5d(DIV5),运用Western blot和实时定量PCR技术,检测不同浓度异氟烷(0.25、0.5、0.75、1MAC)暴露6h后发育期神经元中caspase-3表达变化。
结果：与对照组相比,异氟烷组呈剂量依赖性增加海马神经元nRNA caspase-3表达(P<0.05)；在蛋白水平上,1MAC异氟烷可以明显增强海马神经元cleaved caspase-3表达(P<0.05)。
6.异氟烷对发育期海马神经元cytochrome c表达的影响
方法：1d龄新生SD大鼠海马神经元原代培养至第5d (DIV5),运用Western blot和实时定量PCR技术,检测不同浓度异氟烷(0.25、0.5、0.75、1MAC)暴露6h后发育期神经元中cytochrome c表达变化。
结果：与对照组相比,异氟烷组呈剂量依赖性增加海马神经元cytochrome c mRNA表达(P<0.05)；在蛋白水平上,0.75MAC和1MAC异氟烷可以明显增强海马神经元cytochrome C的表达(P<0.05)
7.异氟烷对体外培养大鼠发育期海马神经元CREB信号的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于体外培养至第5d(DIV5),利用实时定量PCR技术,检测临床相关浓度异氟烷暴露6h后发育期神经元中CREB表达变化及其下游基因脑源性生长因子(BDNF)、生长相关蛋白(GAP43)、PSD-95和抑凋亡基因Bcl-2的表达。
结果：与对照组相比,异氟烷组呈剂量依赖性增加神经元CREB mRNA、GAP43mRNA、PSD-95mRNA、BDNF mRNA和Bcl-2mRNA表达(P<0.05)。
8.异氟烷对体外培养发育期大鼠海马神经元MEF2信号的影响
方法：1d龄新生SD大鼠进行原代海马神经元培养,于体外培养至第5d(DIV5),利用用Real-time PCR进行定量分析不同浓度异氟烷暴露6h后发育期神经元MEF2信号通路(MEF2mRNA、synGAPI mRNA和Arc mRNA)。
结果：与C组相比,临床相关浓度异氟烷干预6h后呈时间依赖性上调海马神经元MEF2mRNA、synGAP I mRNA、Arc mRNA (P<0.05)。
9.异氟烷对发育期大鼠海马神经元凋亡的影响
方法：将新生5d SD大鼠放入相对密闭的自制器皿,用麻醉挥发罐1MAC异氟烷干预6h。对照组不做任何处理。干预后提取出海马神经元总mRNA,运用Real-time PCR检测异氟烷干预后发育期海马神经元凋亡率。
结果：与对照组相比,1MAC处理组异氟烷剂量依赖性增加神经元凋亡(P<0.05)。
10.异氟烷对发育期大鼠海马神经元MEF2信号的影响
方法：新生5d龄SD大鼠(PSD5),随机分为6h和麻醉停止后24h异氟烷组和对照组(C组)。将新生5d SD大鼠放入相对密闭的自制器皿,用麻醉挥发罐1MAC异氟烷干预。对照组不做任何处理。干预后在各时间点取海马神经元提取RNA和组织总蛋白质,运用Real-time PCR进行定量分析MEF2信号通路(MEF2mRNA、 synGAPI mRNA和Arc mRNA)。
结果：与C组相比,1MAC异氟烷干预后呈时间依赖性上调海马神经元MEF2mRNA、synGAPI mRNA、Arc mRNA (P<0.05),麻醉停止后24h恢复正常。
统计学分析
采用SPSS13.0统计软件进行分析,计量资料以均数±标准差(x±s)表示,组内比较采用one-way ANOVA,组间比较采用独立样本或者配对t检验,P<0.05为有统计学意义
研究总结
一、主要研究结果
1.通过离体1d龄SD大鼠海马神经元原代培养5天(Day in vitro, DIV5d)以及整体动物实验从神经突触形成以及神经元存活两个方面证实了临床相关浓度吸入麻醉药异氟烷剂量时间依赖性致发育期海马神经元神经毒性。
2.通过离体1d龄SD大鼠海马神经元原代培养证实临床相关浓度吸入性麻醉药异氟烷对发育期海马神经元VDCCs, NMDA受体和GABAA受体的激活及升高细胞内自由钙离子浓度的影响。
3.在体动物以及离体细胞实验中均证实了临床浓度异氟烷(0.25、0.5、0.75、1MAC)对MEF2和CREB信号通路的影响。
二、研究结论
异氟烷致发育期海马神经元神经毒性的作用可能主要通过增强GABAA受体,细胞膜去极化,从而易化NMDA受体和VDCC,触发细胞外钙离子内流,引起钙触发钙释放(calcium-induced calcium release, CICR)从而导致细胞内钙离子超载,caspase-3和cytochrome C表达上调,导致细胞凋亡。同时钙离子内流可以上调MEF2和CREB Ser-133位点磷酸化,调节其下游对突触发生以及神经存活起重要作用的BDNF、Bcl-2以及突触素Ⅰ (synapsin Ⅰ)表达。
Background
With the increasing sophistication of surgical and anesthetic techniques, increasingly pregnant patients and fetal surgeries are being undertaken. Statistics showed that in the U.S. alone, about6million children annually undergo surgery and anesthesia, including1.5million infant patients. In this view, the study that the inhalation anesthetics isoflurane causes neurotoxicity in the immature brain arouses wide-spread social concern. Recent experiments suggest that volatile anesthetics cause excitotoxicity in a concentration-and time-dependent manner in neuronal models in vitro. And neurons in the developing brain are specifically vulnerable to isoflurane hyperexcitability. However, the mechanisms for isoflurane cytotoxicity remain enigmatic. Because most fetal surgeries in humans are performed during mid-gestation, it is important and urgent to know if the anesthetics used cause damage to the developing brain and subsequent postnatal memory problems and learning disabilities.
Developing neurons exhibit some characteristics different from those of adult neurons. GAB A mediates excitability in the immature brain. In this respect, a major finding is that the GABAA receptor, activated by its agonist, induces a depolarizing membrane in neonatal neurons compared with the hyperpolarizing one in adult neurons, and results in a cytosolic Ca2+increase. The probable mechanism through which GABA elicits cytosolic Ca2+increases in the neurons seems to involve activation of the GABAa receptors; subsequent Cl-efflux attributable to a developmental depolarized Cl-reversal potential causes membrane depolarization and Ca2+entry via voltage-activated Ca2+channels (VDCC) During the early period of synapse development, the immature neuron has a relatively high intracellular cell concentration of Cl-because of developmental-specific expression of the Na+-K+-2Cl-co-transporters (NKCCs) and the K+-Cl-co-transporters (KCCs) and GABA depolarizes and excites neuronal membranes by a reverse chloride gradient. Animal experiments suggest that activation of GABAergic synapses generates action potentials, removes the voltage-dependent Mg2+block from NMDA channels in the hippocampus and in other brain structures. These effects may underlie the well-characterized modulation by GABA of activity-dependent developmental processes including neuronal growth, neuronal differentiation, proliferation and migration, growth rates of neuronal processes and synapse formation and clustering. GABA through the activation of GABAA receptors triggers spontaneous giant depolarizing potentials. These large depolarizations give rise to fast action potentials, which are synchronous over the entire hippocampus and are associated with spontaneous calcium transients. These highly correlated calcium signals are thought to be essential for consolidation of synaptic connections and development of the adult neuronal network. Fig.l The different actions of GABA and AMPA receptors in developing neurons.(A)The equilibrium between excitation (glutamate-releasing synapses) and inhibition (GABA-releasing synapses) in the adult mammalian brain (B) The disequilibrium between glutamate-mediated excitation and GABA-mediated inhibition.The activation of GABAA receptors in neonatal slices generated a depolarization that was sufficient to remove the voltage-dependent Mg2+block of NMDA receptors. Several experiments in vitro confirm that a conversion of "silent" glutamatergic synapses to conductive AMPA synapses starts gradually after P2in the CA3-CA1pyramidal neurons.
Isoflurane, one of the most widely used inhalation anesthetics for various fetal surgeries or procedures in pregnant patients, has been shown previously to induce caspase activation and apoptosis when applied in concentration in the immature brain. Recently, it was reported that isoflurane might induce apoptosis by increasing [Ca2+]i. Isoflurane has been shown to potentiate GABAA receptor current and directly open GABAA receptor and glycine receptor channels even in the absence of agonist. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitation of NMDA receptors and opens VDCC, elevates [Ca2+]i, and promotes the activation of caspase-3. However, up until recently, the exact mechanism to induce apoptosis remains enigmatic.
Fig.2During development, when the GABAA receptors are activated, there is an efflux of chloride, leading to a depolarization that can remove the voltage-dependent Mg2+block from NMDA receptors, generate sodium and calcium action potentials, directly activates VDCC and elevates the [Ca2+]i, leads further CICR from the ryanodine-sensitizing Ca2+store in the hippocampus and in other brain structures. The influx of GABA-induced Ca2+through extracellular medium linked to the binding of Ca2+to CaM and the rapid translocation of CaM to the cell nucleus. This process leads to the activation of the transcription factors, CREB. When activated, CREB promoted the transcription of a set of genes, such as the expression of c-fos, BDNF, PSD-95, synapsinl, Bcl-2and so on, which regulate expression of axon growth and synaptic plasticity in the developing hippocampal neurons and results in the stimulation of new gene expression
In the present research, we evaluated the effect of clinically relevant concentrations of isoflurane on electrophysiological properties and gene expression in developing rat hippocampal neurons. We tested the following hypotheses:Ⅰ isoflurane could affect neuronal electrophysiological function in the immature brain; Ⅱ clinically relevant concentrations of volatile anesthetic isoflurane have no effect on the neuronal apoptosis in short-term; Ⅲ Bidirectional effect of isoflurane on regulation of neuronal development in the immature brain
Methods and Results
1. Isoflurane potentiates IGABA in primary rat hippocampus neuronal culture at day in vitro (DIV)5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of IGABA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on IGABA. bicuculline was employed.
Results:GABA evoked inward current (IGABA), the EC50was23.73μmol/L, bicuculline blocked IGABA; isoflurane dose-dependent increase IGABA, the EC50was23.73μmol/L.
2. Isoflurane potentiates INMDA in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and MK801were employed.
Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated INMDA peak current amplitude by116±8.8,122±11.7,135±14.3and132±14.6%, respectively. Application of bicuculline partially inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05), and application of MK801completely inhibited the amplitude of isoflurane-induced potentiation of INMDA (P<0.05)
3. Isoflurane potentiates IVDCC in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. The peak of INMDA was recorded by means of the whole cell patch clamp technique with use of EPC10exposed to isoflurane. To further explore the mechanism of the potentiation effect of isoflurane on INMDA, bicuculline and nicardipine were employed.
Results:Isoflurane (0.25,0.5,0.75and1MAC) potentiated IVDCC peak current amplitude by109.11±9.03,120.56±11.46,141.33±13.87and146.78±15.87%, respectively. pplication of nicadipine partially inhibited the amplitude of IVDCC to the63.11±8.48%of control (P<0.001) and application of nicadipine inhibited the amplitude of isoflurane-mediated IVDCC to the79.33±7.57%of control, but application of bicuculline had no effect on the amplitude of IVDCC (P>0.05) and application of bicuculline inhibited the amplitude of isoflurane-mediated IVDCC from146.78±15.87%to116.56±10.31%of control
4. Isoflurane potentiates GABA-triggered [Ca2+]i transient in primary rat hippocampus neuronal culture at DIV5
Methods:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used.[Ca2+]i was monitored using Fluo-4AM fluorescence imaging. To evaluate whether intracellular calcium release was involved in the potentiation effect of isoflurane, Nicardipine and Dantrolene was employed.
Results:The enhancement effect of isoflurane was in a concentration-dependent manner. The EC50for enhancement effect was0.61±0.05MAC. Consistent with previous reports, dantrolene significantly decreased the GABA-triggered [Ca2+]i responses of control (P<0.001, n=11). Furthermore, preconditioning with dantrolene significantly decreased the potentiation of GAB A induced [Ca2+]i transients mediated by1MAC isoflurane in developing hippocampal neurons. Pre-application of nicadipine partially inhibited GABA-tiggered [Ca2+]i transients of control (P<0.001) and isoflurane-mediated potentiation of the GABA-evoked [Ca2+]i rise of GABA (P=0.001).
5. Effect of isoflurane on caspase-3levels in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of caspase-3is regulated by high [Ca2+]i, the possibility of increased caspase-3 levels after isoflurane in dose-and time-dependent manner was evaluated.
Results:The effect of treatments with isoflurane on caspase-3activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal caspase-3mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.001).
6. Effect of isoflurane on the levels of cytochrome C in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Taking into account that activation of cytochrome C is regulated by high [Ca2+]i, the possibility of increased caspase-3levels after isoflurane in dose-and time-dependent manner was evaluated.
Results:The effect of treatments with isoflurane on cytochrome C activity was dose-and time-dependent, reaching a maximal caspase activity after1MAC of6-h stimulation (P <0.05). Hippocampal cytochrome C mRNA levels began to be significantly increased in isoflurane-treated cultured rat hippocampal neurons after0.25MAC of6-h stimulation (P<0.05).
7. Effect of isoflurane on the levels of transcriptional factor CREB in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the CREB signaling pathway (GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA) after intervention.
Results:Compared with the group C, isoflurane increased in neuronal GAP-43mRNA, PSD-95mRNA, CREB mRNA, BDNF mRNA and Bcl-2mRNA expression in dose-dependent manner (P<0.05).
8. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in primary rat hippocampus neuronal culture at DIV5
Method:The hippocampi were dissected from newborn Sprague-Dawley rats and primary hippocampal neuronal cultures of5days in vitro were used. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA、synGAPI mRNA and Arc mRNA) and Synapsin I mRNA after intervention. Western blot was used to detect the expressions of Synapsin I at the protein level.
Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and Synapsin I mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)
9. Effect of Isoflurane on the development of hippocampal neurons apoptosis in vivo
Methods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane6h. And a control group which received no treatment. Pups were killed by decapitation to extract total mRNA after intervention. Real-tine PCR was used to detect the expressions of caspase-3.
Results:Compared with the group C, isoflurane increased in neuronal caspase-3expression (P<0.05)
10. Effect of isoflurane on the levels of hippocampal transcriptional factor MEF2in vivo
Methods:Forty5-day-old rats which were administrated in relatively airtight container were randomly divided into five groups, treatment groups which received1MAC isoflurane2,4,6h, respectively. And a control group which received no treatment. Pups were killed by decapitation to extract total RNA from hippocampal neurons after intervention. Real-time-PCR was used to detect the expressions of the MEF2signaling pathway (MEF2mRNA、synGAPI mRNA and Arc mRNA) and SynapsinI mRNA after intervention. Western Blot was used to detect the expressions of synapsinl at the protein level.
Results:Compared with the group C, the expressions of MEF2mRNA, synGAPI mRNA, Arc mRNA and SynapsinI mRNA of the neurons from isoflurane groups all increased statistically (P<0.05)
Main Results
1. Isofluane-induced developmental neurotoxicity in developing hippocampal neurons was confirmed by researches in vitro (5-day cultured primary neurons) and in vivo (whole animal experiments) through two aspects of survival and neurogenesis of neurons, neurotoxicity.
2. Isoflurane-mediated activation of GABAA receptors depolarizes the cells, facilitates NMDA receptors and opens VDCC, elevates [Ca2+]i. Effects of isoflurane on MEF2and CREB signal pathway were confirmed both in vitro and in vivo.
3. From the investigation of the effect of isoflurane on the synaptic plasticity in developing hippocampal neurons, we can draw a conclusion that bidirectional effects of isoflurane on regulation of neuronal development in the immature brain.
Conclusions
Clinical relevant concentrations of inhaled anesthetics isoflurane-mediated potentiation of GABA-triggered [Ca2+]i release results from membrane depolarization with subsequent activation of VDCC and further CICR from the ryanodine-sensitizing Ca2+store. An increase in [Ca2+]i, due to activation of GABAA receptor, facilitation of NMDA receptor and opening of VDCC, is necessary for isoflurane-induced calcium overload of immature rat hippocampal neurons, which may be involved in the mechanism of an isoflurane-induced neurotoxic effect in the developing rodent brain.
引文
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