【作者】 成丹丹；

【导师】 熊利泽； 朱正华；

【作者基本信息】 第四军医大学 ， 麻醉学， 2007， 硕士

【摘要】 全麻问世并广泛应用于临床已有160余年。然而,全麻药物是如何引起机体意识丧失、遗忘、制动等全麻效应的?全麻药物的作用靶点是什么?至今仍是未解之谜,对全麻药物本质及作用机理认识的局限,极大制约了全身麻醉安全、可控的临床应用及新型麻醉药物的研发。应该肯定的一点是:全麻药物是作用于中枢神经系统而产生意识丧失、遗忘、制动等麻醉效应的。一般认为,全麻药物是作用于前额皮层、顶皮层、海马等区域产生镇静和遗忘作用,而脊髓和低位脑干在临床全麻药物的制动效应中起了主导作用。可见,全麻药物对中枢神经系统的作用是多方位、多靶点的,单一的膜脂溶性、离子通道、受体及基因等学说无法完全解释全麻(尤其是吸入麻醉)的复杂效应。既往全麻机制研究中已发现大量受体、分子与全麻效应可能相关,但很难将这些分散的结果有机地联系起来。细胞内信号转导系统具有变化迅速,能将大量信号有机串联和整合的特点。本研究中我们拟以目前临床常用的吸入性麻醉剂七氟醚为例,从麻醉时脑内信号转导分子活化水平的变化入手,为探讨吸入麻醉的中枢机制寻找突破口。目的观察七氟醚麻醉-苏醒过程中脑内ERK1/2、CREB和PKCγ磷酸化水平的变化,以了解这些信号转导分子在全麻机理中的可能作用,为探索吸入麻醉药的中枢神经系统作用靶位和相关信号通路提供资料。方法第一部分1.小鼠七氟醚麻醉-苏醒过程中脑内磷酸化ERK1/2的表达变化BALB/c小鼠60只,平均分为5组:正常对照组(Con)、七氟醚麻醉5min组(Sevo-1)、麻醉1h组(Sevo-2)、麻醉1h停药2min组(E-1)和麻醉1h停药1h组(E-2)。各组动物在相应时间点脱臼处死,取脑制样或经灌注固定后取脑切片,行Western blotting或免疫组织化学染色,观察七氟醚麻醉-苏醒过程不同脑区磷酸化ERK1/2 (pERK1/2)水平的变化。2.七氟醚麻醉下小鼠的血气电解质状况雄性BALB/c小鼠12只,随机分为2组:对照组(Con)和七氟醚麻醉1h组(Sevo-1h),相应时间点开胸心脏采集动脉血,了解麻醉过程中的血气及电解质情况。3. MEK1/2拮抗剂SL327对七氟醚麻醉行为的影响雄性BALB/c小鼠18只,随机分为3组:Con组;DMSO组:麻醉前1小时腹腔注射25%DMSO,6 ml·kg-1;SL327组:麻醉前1小时腹腔注射溶解于25%DMSO的SL327,30 mg·kg-1。各组小鼠以4%七氟醚诱导,记录翻正反射消失时间,然后以1MAC七氟醚维持麻醉30min后,停止麻醉,高流量氧气洗脱,观察夹尾反射恢复时间。待夹尾反射恢复后,动物断头,取脑制样,Western blotting检测脑内pERK1/2表达水平。第二部分小鼠七氟醚麻醉-苏醒过程不同脑区磷酸化CREB的表达变化雄性BALB/c小鼠30只,随机分为5组:Con组、Sevo-1组、Sevo-2组、E-1组和E-2组。处理措施同实验1.1。在相应时间点断头,取感觉皮质、海马、脑干行Western blotting测定脑内pCREB表达情况。第三部分1.磷酸化PKCγ在正常小鼠脑内的免疫组织化学定位雄性BALB/c小鼠8只,脱臼处死,灌流取脑,免疫组织化学染色,观察pPKCγ阳性反应产物在脑内的分布。2.小鼠七氟醚麻醉-苏醒过程中脑内磷酸化PKCγ的表达变化雄性BALB/c小鼠48只,随机分为4组:对照组(Con)、七氟醚麻醉5min组(Sevo-1)、七氟醚麻醉1h组(Sevo-2)和麻醉1h后停药1h组(E-1h)。在相应时间点断头取样行Western blotting,灌注取脑切片行免疫组织化学染色,以观察七氟醚麻醉-苏醒过程中小鼠脑内pPKCγ的表达变化。结果第一部分1.小鼠七氟醚麻醉-苏醒过程中脑内磷酸化ERK1/2的表达变化Western blotting结果:与Con组相比,麻醉后各组动物感觉皮质、海马pERK表达水平下降,苏醒后回升至对照组水平(E-1, E-2),而脑干在麻醉后pERK表达水平上升,且苏醒后仍未降至对照组水平。免疫组织化学染色发现外侧网状核、孤束核、EW核、臂旁外侧核背侧亚核(LPBD)、视上核(SO)、下丘脑室旁核(PVN)、杏仁中央核和终纹床核在Con组弱表达,Sevo-1、Sevo-2及E-1三组表达显著增强(P<0.05),而在E-2组表达减弱,除LPBD、SO、PVN外在其余各核团均恢复至对照组水平。而室周灰质腹外侧部、丘脑室旁核、弓状核则表达相反:Con组高表达,Sevo-1,Sevo-2及E-1三组阳性神经元数量显著减少(P<0.05),E-2组恢复至Con组水平。此外,海马、边缘前皮质、扣带回、运动皮质、感受皮质则表现为Con组高表达,Sevo-1组和Sevo-2组低表达,E-1和E-2组恢复至对照组水平。2.七氟醚麻醉下小鼠的血气电解质状况1MAC七氟醚麻醉1h,小鼠血气及电解质情况与对照组相比无改变。3. MEK1/2拮抗剂SL327对七氟醚麻醉行为的影响SL327组与对照组相比,翻正反射消失时间及夹尾反射恢复时间均缩短,而DMSO组与Con组无差异。Western blotting显示:SL327组动物的感觉/运动皮质、海马pERK1/2表达水平均较对照组显著降低(P<0.05),而DMSO组与Con组无差异。第二部分小鼠七氟醚麻醉-苏醒过程不同脑区磷酸化CREB的表达变化各组间感觉皮质、海马、脑干在麻醉-苏醒过程中pCREB表达无统计学差异。第三部分1.磷酸化PKCγ在正常小鼠脑内的免疫组织化学定位pPKCγ阳性产物主要见于神经元的胞浆和突起中,在正常小鼠脑内表达广泛。仅有少量胶质细胞被染色,着色浅淡。海马CA1区锥体细胞,小脑的浦肯野细胞,大脑新皮质V层细胞,边缘前皮质、岛皮质的深层细胞以及前梨皮质的颗粒层细胞,外侧嗅束核、杏仁基底外侧核和杏仁外侧核的神经元,以及穹窿、锥体束纤维等均有强或较强的pPKCγ表达。吻侧丘脑大多数核团,尾侧丘脑的内侧膝状体核及外侧膝状体的背侧核,脑干耳蜗背侧核的表浅部位,三叉神经感觉核簇及巨细胞网状结构神经元中均有弱阳性表达。2.小鼠七氟醚麻醉-苏醒过程中脑内磷酸化PKCγ的表达变化Western blotting示海马pPKCγ表达在对照组低表达,麻醉后表达增强,在E-1h组回落至Con组水平。免疫组织化学染色显示:在Con组,锥体束和穹窿中pPKCγ呈高水平表达,而Sevo-1和Sevo-2组表达显著减弱,E-1h组恢复至对照组水平。杏仁基底外侧核pPKCγ表达则不同:在Con组低水平表达,而Sevo-1、Sevo-2和E-1h组均呈现高水平表达(P<0.05)。结论1.在麻醉-苏醒过程中,脑内某些核团、脑区其pERK1/2水平出现与麻醉表象一致的变化。根据已有知识和资料分析,部分pERK1/2表达升高的核团如LPBD、PVN等可能与麻醉造成的应激反应有关;而另一些pERK1/2水平下调的核团如M、S、Hip等,可能与麻醉引起意识与感觉认知缺失有关。七氟醚麻醉-苏醒过程中,总ERK1/2表达未发生改变,主要是ERK磷酸化水平的上调或下调,提示麻醉主要影响ERK通路的活化水平,而不涉及新的(de novo)蛋白质合成。2. MEK-ERK通路特异阻断时,全麻诱导加快,苏醒迅速,从功能行为学上进一步证实了ERK可能在全麻不同时程中介导不同效应,其确切机制尚待进一步深入研究。3.作为ERK通路的下游分子之一,pCERB在七氟醚麻醉-苏醒过程中各组间表达无差异,提示CREB通路可能不参与此过程的调节或CREB介导的核内转录机制在所观察的时间点尚未激活。4.在正常状态及麻醉-苏醒过程中pPKCγ表达变化的观察显示,传导束中的pPKCγ在七氟醚麻醉时磷酸化水平减弱,提示全麻可能抑制了pPKCγ在轴突中的功能,是否进而影响了轴突转运或兴奋传导尚待进一步研究。5.结合本实验室既往研究,七氟醚麻醉-苏醒过程中引起的脑内pERK1/2表达部位及变化趋势与异氟醚十分相似,提示氟醚类麻醉的中枢机制可能有相似之处,ERK可能是其共同的作用分子之一。更多还原

【Abstract】 General anesthetics have been applied into clinical practice for more than 160 years. However, until now, it is still not fully understood how these general anesthetics induce their complex clinical effects including unconsciousness, amnesia, immobility and which areas and what molecules they are targeted at in the central nervous system. This situation leads to a limitation for the security and controllability of the clinical application of general anesthesia and the development of novel drugs.It is well known that general anesthetics induce the effect of unconsciousness, amnesia, immobility, etc, by activating on the central nerve system (CNS). It is believed that the sedation and amnesia effect of inhaled anesthetics may be related to forecortex, parietal cortex and hippocampus, while immobility may be related to spinal cord and lower brain stem. Thus, for the mechanism of general anesthesia, especially for inhalation anesthesia, multiple-target-hypothesis becomes more favorable in contrast to one-target-theory such as lipid theory, ion channel theory, receptor theory and gene theory, etc. A large amount of receptors and molecular have been reported to be involved in the production of general anesthesia, but what role they play and how they interact among these molecules is still unclear. Intracellular signal transduction system, which can be actived quickly to transform the information from extramembrane to the intramembrane, even to nucleus, has drawn much attention as to the study on the mechanism of general anesthesia. In this study, the alternation of action of some signal transduction moleculars under sevoflurane anesthesia was investigated to explore the central mechanism of inhalation anesthesia.ObjectiveAlternations of the expression of phosphorylated ERK1/2, CREB, PKCγduring sevoflurane anesthesia-emergence course were investigated to clarify the role of these signal transduction molecules in mechanism of general anesthesia and to provide basis for the targets of inhaled anesthetics.MethodsPart 11. Male BALB/c mice at 8 weeks of age were randomly divided into five groups: group Sevo-1 and Sevo-2, undergoing sevoflurane anesthesia for 5min and 1h respectively; group E-1 and E-2, receiving sevoflurane anesthesia for 1h and emerged from anesthesia for 2min or 1h, respectively; Animals in group Con, were left untreated. At the end of treatments, the brain of all animals were taken out and processed for determination of phosphorylated ERK1/2 expression by Western blotting and the immunohistochemical staining.2. Twelve Male BALB/c mice were randomly divided into two groups: control (Con) and Sevo-1h which was exposured under sevoflurane anesthesia for 1h. At the end of treatments, artery blood samples were collected through left ventricular puncture following opening of the thoracic cavity for the blood gas analysis and assays of electrolytes.3. Eighteen Male BALB/c mice were randomly divided into 3 groups: Con, DMSO and SL327. Animals in the latter two groups were given an intraperitoneal injection of 25%DMSO,6 ml·kg-1 and SL327,30ml·kg-1, respectively, 1h before sevoflurane anesthesia. All the mice were anesthetized with 4% sevoflurane and maintained for 30min under 1.0 MAC sevoflurane. After recording the time of loss of righting reflex (LORR) and emergence of clamp-tail reflex, the animal was decapitated and processed for determination of phosphorylated ERK1/2 expressions by Western blotting.Part 2BALB/c mice at 8 weeks of age were randomly divided into five groups: Con, Sevo-1 and Sevo-2, E-1 and E-2. The treatments were as same as that in part 1. At the end of treatments, the brain of all animals was taken out and processed for Western blotting for determination of phosphorylated CREB expressions.Part 31. Eight normal BALB/c mice were sacrificed by cervical dislocation and fixed with perfusion. The brains were sectioned and immunohistochemically stained for pPKCγ.2. Forty-eight Male BALB/c mice at 8 weeks of age were randomly divided into four groups: group Sevo-1 and Sevo-2, receiving sevoflurane anesthesia for 5 min and 1h, respectively; E-1h, sevoflurane anesthesia for 1h and emergence for 1h; Con, normal animals without being treated. At the end of treatments, the brain of all animals were taken out and processed for Western blotting and the immunohistochemical staining for determination of phosphorylated PKCγexpressions.ResultsPart 11. As shown by Western blotting, the pERK expressions in the mice sensory cortex and hippcampus were decreased after sevoflurane inhalation and restored to the level of control after emergence from anesthesia (E-1, E-2); whereas the expression of pERK in brainstem was increased after sevoflurane inhalation and gradually restored but not back to the level of control after stopping of inhalation. For the immunohistochemistry staining, except LPBD, SO and PVN, the numbers of pERK1/2-like immunoreactive (pERK1/2-ir) neurons in lateral reticular nucleus (LRt), solitary tract nucleus (Sol), Edinger-Westphal nucleus (EW) and dorsal part of lateral parabrachial nucleus (LPBD), supraoptic nucleus (SO), paraventricular thalamic nucleus (PVN), central amygdaloid nucleus (Ce), bed nucleus of the stria terminalis (BST) were significantly increased during sevoflurane anesthesia and then restored to the level of control after emergence compared to the control group in which fewer pERK1/2-ir cells were observed in these nuclei; whereas the numbers of pERK1/2-ir neurons in ventrolateral periaqueductal gray (VLPAG), paraventricular thalamic nucleus (PV), arcuate hypothalamic nucleus (Arc), hippocampus (Hip), prelimbic cortex (PreL), cingulate cortex (Cg), motor cortex (M) and sensory cortex (S), where numerous pERK1/2-ir cells were seen in control group, were significantly decreased during sevoflurane anesthesia and restored to the level of control group again at 2min or 1h after stopping of sevoflurane inhalation.2. There is no statistic difference between control and Sevo-1h in each index of blood gas analysis and electrolytes examination.3. The time of LORR and the time of emergence of clamp-tail reflex were shorter than that in control after injection of SL327 but not DMSO. Western blotting showed that the pERK expressions in the mice sensory/motor cortex and hippcampus were significantly decreased in SL327 group compared to the control.Part 2There is no significant difference in the expression of pCREB in sensory cortex, hippcampus and brain stem among different groups.Part 31. Phosphorylated PKCγ-like immunoreactivities were widely distributed in discreted area in the mouse brain. The positive products were mainly located in neurons, both perikaria and processes. Strong immunoreactivity staining for pPKCγwas observed in the pyramidal neurons in CA1 of hippocampus, Purkinje cells in cerebellum, the neurons in layer V of neocortex, the deep layers of prelimbic and insular cortex, granular layer of prepiriform cortex, basolateral amygdaloid nucleus, lateral amygdaloid nucleus, as well as fornix and pyramidal tract. In contrast, neurons in most nuclei of rostral thalamus, medial and dorsolateral geniculate nuclei, superficial area of dorsal cochlear nucleus, nucleus of spinal trigeminal tract and gigantocellular reticular nucleus were weakly stained. Few glial cells were faintly stained.2. Results of Western blotting showed that the pPKCγexpression in the mice hippcampus was increased after sevoflurane inhalation and down to the level of control after emergence (E-1h). The numbers of pPKCγ-like immunoreactive neurons (pPKCγ-ir) were significantly increased during sevoflurane anesthesia in basolateral amygdaloid nucleus where fewer pPKCγ-ir cells were found in control group, but not down to the level of control after emergence. On the contrary, the positive staining of pPKCγwas significantly decreased during sevoflurane anesthesia in pyramidal tract and fornix where strong pPKCγ-ir staining was found in control group, but restored to the level of control after emergence.Conclusion1. A significant alternation of expression of pERK1/2 in some brain areas/nuclei in mice was observed during sevoflurane anesthesia and emergence process. As to our knowledge, the increase of the expression of pERK1/2 in certain nuclei, such as LPBD and PVN, may be involved in anesthetics stress; the decrease of the expression of pERK1/2 in other brain areas, such as motor cortex, sensory coxtex and hippocampus, may be involved in the loss of consciousness, sensorium and cognition. During anesthesia and emergence course, only the phosphorylation level of ERK, but not the expression of total ERK1/2, was changed, indicating that general anesthesia probably induces activation of ERK pathway but not the synthesis of protein de novo.2. The time of induction and emergence (as shown by LORR and clamp-tail reflex) under sevoflurane anesthesia was shorten after the pathway of MEK-ERK was blocked with intraperitoneal injection of SL327, indicating that ERK perhaps played different roles in different course of general anesthesia course. The precise mechanisms need to be further studied.3. There was no significant difference in expression of pCREB among different groups during anesthesia and emergence course, showing that CREB perhaps wasn’t involved in the intracellular events of anesthesia, or, it has not be activated during the time observe.4. The expression of pPKCγin neural fiber tracts was decreased during sevoflurane anesthesia and emergence course, indicating that general anesthesia probably suppressed the action of pPKCγin axons. Further studies were needed to clarify whether general anesthesia affect the axonal transport or the conduction of firings on the fibers.5. The alternation of the expression of phosphorylated ERK1/2 during sevoflurane anesthesia was similar to that in isoflurane anesthesia in our previous studies, indicating that common mechanisms may be shared by different volatile anesthetics, such as activation of phosphorylated ERK1/2.更多还原