【作者】 谭蕾；

【导师】 罗爱林；

【作者基本信息】 华中科技大学 ， 麻醉学， 2009， 博士

【摘要】 研究背景氯胺酮被广泛用于婴幼儿手术，为N-甲基-D-天冬氨酸（N-methyl-D-aspartate，NMDA）受体离子通道非选择性拮抗剂，文献报道其能够导致发育期神经元广泛的剂量依赖性的凋亡。一些文献显示，暴露于NMDA受体拮抗剂MK801等，实验动物呈现远期海马突触功能以及持续性的学习记忆功能受损。这显示突触NMDA受体介导的膜去极化通过激活激酶信号介导的促存活基因蛋白表达产物在触发以及维持神经营养支持中起着非常重要的作用。NMDA受体是一种离子通道型谷氨酸能受体，能够特异性地被其选择性的激动剂NMDA激活。NMDA受体的激活会导致允许Na⁺、K⁺以及Ca²⁺离子流动的通道的开放。通过NMDA受体的钙内流会导致下游多种信号分子的激活，构成许多生理过程，例如突触可塑性、学习和记忆的基础。体外研究证实，突触NMDA受体的神经营养作用主要是通过细胞外信号调节蛋白激酶（extracellular signal-regulated protein kinase，ERK1／2），钙／钙调蛋白依赖性激酶IV(Ca²⁺／CaMK)以及cAMP反应原件结合蛋白（cAMP response-binding protein，CREB）发挥作用的。CREB是介导cAMP以及钙依赖性基因转录的中心转录因子。其Ser-133位点的磷酸化对启动转录非常重要，突变这一位点至丙氨酸会导致其转录活性的缺失。CREB调节大量促进神经元存活的基因的表达，其中神经营养因子基因BDNF以及抑凋亡基因Bcl-2为其研究最多的下游基因。神经营养因子在中枢神经系统的生长、发育以及突触可塑性中具有多种作用。钙内流诱导BDNF的转录能力由钙进入细胞内的路径不同而不同，通过诱导CREB磷酸化的方式不同进行调节。各种刺激主要通过升高细胞内cAMP以及Ca²⁺来激活CREB（见图1）。Ca²⁺／CREB／CREB结合蛋白（CREB banding protein，CBP）依赖性基因调节同时为长时程突触可塑性以及神经存活的重要机制。激活CREB信号通路涉及到调节神经元功能的两个主要的基因表达级联通路。第一，CREB作为分子开关的重要组成部分，通过调节形成包括突触素I在内的新突触所必需的基因的表达来控制神经可塑性。第二，与神经存活及保护有关的基因表达级联通路通过控制神经营养因子以及抗凋亡基因的转录。CREB依赖性的基因表达为中枢神经系统发育期神经细胞数量的维持以及随后突触发生以及神经分布所必需。因此本研究拟通过在体动物实验以及离体细胞培养从影响神经元存活以及突触形成、突触可塑性及远期学习记忆功能两方面来探讨氯胺酮致发育期海马神经元的神经毒性。本研究检验了以下假说（1）氯胺酮可诱导体内外发育期海马神经元的凋亡（2）氯胺酮会破坏体内外发育期海马神经元的突触形成（3）氯胺酮会导致远期学习记忆功能的损害（4）氯胺酮可通过阻断NMDA受体偶联钙离子通道使钙离子内流减少（5）氯胺酮下调CREB Ser-133位点的磷酸化（6）氯胺酮会下调其下游基因BDNF以及Bcl-2的表达减少突触素I的表达。研究方法与结果1．常用静脉麻醉药对发育期海马神经元细胞内钙浓度的影响方法：将原代培养第5d的大鼠海马神经元用10μmol／L的Ca²⁺指示剂Fluo-4共孵育30min，洗涤后，分别加入150μmol／L氯胺酮，3μmol／L咪达唑仑及10μmol／L丙泊酚在激光共聚焦显微镜下选定多个细胞分别测定荧光强度的变化。结果：氯胺酮使体外培养第5d的海马神经元代表钙浓度的荧光强度明显下降，由给药前987±307下降至给药后（766±226）（P＜0．05），咪达唑仑以及丙泊酚均使体外培养5d的海马神经元荧光强度明显升高：咪达唑仑由作用前（1707±514）升高至（2663±572）（P＜0．05），而丙泊酚由（1057±353）升高至（1749±708）（P＜0．05）。2．氯胺酮对发育期海马神经元神经存活的影响方法：1日龄新生SD大鼠进行原代海马神经元培养，体外培养至第5d，随机分对照组（C组）和氯胺酮组（K组），每组6孔。K组在含1000μmol／L氯胺酮的Neurobasel培养基中孵育3 h，采用Annexin V-PI凋亡试剂盒在流式细胞仪上检测两组神经元凋亡率的改变。结果：与对照组相比，氯胺酮组神经元早晚期凋亡率均增高（P＜0．05）3．氯胺酮对体外培养大鼠发育期海马神经元突触发生的影响方法：分组同前，氯胺酮共培养3h后两组神经元全量换液，体外培养至第14d，采用免疫荧光双标检测两组神经元突触素I的表达。使用FITC标记激发光下呈绿色荧光代表突触素I染胞浆，以及激发光下呈蓝色荧光的DAPI染核，观察体外培养第14d，神经元存活以及突触形成。结果：与对照组相比，氯胺酮组神经元细胞密度低，形成的突触连接少，突触素I的表达减少。4．氯胺酮对体外培养大鼠发育期海马神经元CREB信号的影响方法：体外培养第5d的海马神经元随机分为对照组（C组）以及氯胺酮组（K组）具体处理同上，采用免疫细胞化学检测两组神经元p-CREB的表达。免疫组化法测定神经元Ser-133位点磷酸化的CREB（p-CREB）表达，RT-PCR法半定量测定CREB下游基因脑源性生长因子（BDNF）和抑凋亡基因Bcl-2的表达。结果：与C组相比，p-CREB、BDNF、Bcl-2表达均下调（P＜0．05）。5．NMDA预处理能拮抗氯胺酮对体外培养大鼠发育期海马神经元磷酸化的影响方法：体外培养第5d的原代海马神经元随机分为6组即C组为对照组，K组为氯胺酮组即与1000μmol／L氯胺酮共培养3h，N₁组为2μoml／L NMDA共培养4h，N₂组为与10μoml／L NMDA共培养4h，N₁+K组2μoml／L NMDA共培养1h后与1000μmol／L共培养3h，N₂+K组10μoml／L NMDA共培养1h后再与1000μmol／L共培养3h，采用Western-blot检测各组神经元CREB以及p-CREB的表达，使用平均光密度值代表各组蛋白的表达量。结果：与对照组相比，各组神经元CREB的表达差异均无显著性（P＞0．05），N₂组p-CREB的表达增加，N₁+K组、N₂+K组以及K组p-CREB的表达均减少，与K组相比N₂+K组p-CREB的表达增加。6．氯胺酮对发育期大鼠海马神经元凋亡率的影响方法：7日龄SD大鼠随机分对照组（C组）、临床浓度氯胺酮组（K₁组）及超临床浓度氯胺酮组（K₂组）每组5只。K₁组和K₂组分别为皮下注射10mg／kg氯胺酮及皮下注射20mg／kg氯胺酮，给药重复7次，每次间隔90min。给药后1d，断头处死乳鼠，制成大脑冰冻切片，采用Tunel试剂盒检测各组大鼠海马神经元的凋亡率。结果：与C组相比，K₁、K₂组大鼠海马神经元凋亡率均有明显升高，分别由（0．82±1．66）％升高至（1．55±2．25）％（P＜0．05）和（2．77±2．42）％（P＜0．05）。7．氯胺酮致发育期大鼠海马突触形成的影响方法：7日龄SD大鼠随机分为对照组（C组）和临床浓度氯胺酮组（K₁组），超临床浓度氯胺酮组（K₂组），给药方式同前，每组5只。用药后均仔细观察乳鼠的皮肤颜色，有无缺氧表现。在麻醉期间均低浓度给氧（氧流量：2L／min）。麻醉清醒后，返回母鼠身边继续喂养。给药后7d，断头处死大鼠，分别采用免疫组织化学以及Western-blot检测突触前膜标记物突触素I以及突触后膜标记物PSD95的表达。分别采用IOD值以及相对灰度比值代表突触素素I以及PSD95的表达量。结果：与C组相比，K₂组突触素I以及PSD95的表达减少（P＜0．05），K₁组突触素I以及PSD95的表达差异无统计学意义（P＞0．05）。8．氯胺酮致发育期大鼠海马CREB信号通路的影响方法：7日龄SD大鼠随机分为对照组（C组）和临床浓度氯胺酮组（K₁组），超临床浓度氯胺酮组（K₂组），给药方式同前，每组10只。给药方式同前，麻醉清醒后，返回母鼠身边继续喂养，至给药后1d，断头处死大鼠，采用免疫荧光双标检测各组乳鼠p-CREB的表达，采用RT-PCR检测各组乳鼠BDNF、Bcl-2的表达。结果：与C组相比，K₁、K₂组大鼠代表p-CREB表达的IOD值分别由（2627．86±345．29）下降至（1607．98±97．63）（P＜0．05）和（967．58±61．24）（P＜0．05）、K₁、K₂组大鼠代表BDNF表达的相对灰度值分别由（1．355±0．147）下调至（1．241±0．004）（P＜0．05）和（0．863±0．009）（P＜0．05），K₁、K₂组大鼠代表Bcl-2表达的相对灰度值分别由（0．790±0．008）下调至（0．740±0．034）（P＜0．05）和（0．560±0．014）（P＜0．05）。9．氯胺酮对发育期大鼠成年后学习记忆功能的影响方法：7日龄SD大鼠随机分为对照组（C组）和临床浓度氯胺酮组（K₁组），超临床浓度氯胺酮组（K₂组），给药方式同前，每组10只。给药方式同前，麻醉清醒后，返回母鼠身边继续喂养，至给药后6周，采用Morris水迷宫检测各组大鼠学习记忆功能的改变，连续训练7d，7d后采用定位航行逃逸潜伏期以及空间探索实验中的潜伏时间、穿越原平台的次数以及目标想象所占探索时间比值和探索距离比值来评价各组大鼠空间学习记忆功能。结果：与对照组相比超临床浓度组大鼠Morris水迷宫潜伏时间由（5．12±3．24）延长至（10．83±6．48）（P＜0．05）。空间探索实验各组大鼠差异无统计学意义。统计学分析计量资料以均数±标准差（（?）±s）表示，采用SPSS 13．0软件进行数据处理，钙荧光强度采用配对t检验，离体试验中凋亡率、p-CREB、BDNF、Bcl-2采用成组t检验，其余结果比较采用单因素方差分析，P＜0．05为差异有统计学意义。研究总结一、主要研究结果1．通过离体原代神经元培养以及在体动物实验从神经元存活以及神经突触发生两个方面证实了氯胺酮致发育期海马神经元神经毒性。2．通过离体原代神经元培养证实氯胺酮对发育期海马神经元CREB上游NMDA受体的阻滞及对钙离子内流的影响。同时在在体动物以及离体细胞实验中均证实了氯胺酮对CREB信号通路即对p-CREB、BDNF、Bcl-2表达的影响。3．通过对临床浓度以及超临床浓度氯胺酮对发育期大鼠远期空间学习记忆功能的影响的研究。证实只有超临床浓度的氯胺酮才能够导致学习记忆功能部分受损。二、研究结论氯胺酮致发育期海马神经元神经毒性的作用可能主要通过非选择性的阻断NMDA受体钙离子通道，下调CREB Ser-133位点磷酸化，从而使得其下游对神经存活以及突触发生起重要作用的Bcl-2、BDNF以及突触素I表达减少来实现。更多还原

【Abstract】 BackgroundKetamine, a noncompetitive N-methyl-D-aspartate （NMDA） receptor ion channelblocker, has been used as a general pediatric anesthetic for surgical procedures ininfants and toddlers.Ketamine may cause widespread and dose-dependent apoptoticneurodegeneration during development. After been exposed to the NMDA receptorantagonists, the animals are found to exhibit deficits in hippocampal synaptic functionand persistent memory and learning impairments at a later age.It is recognized that synaptic NMDA receptor-induced membrane depolarizationplays a vital role in providing and sustaining neurotrophic support by activation ofpro-survival protein kinase signaling pathways. In particular, there is compelling invitro evidence for the trophic role of synaptic NMDA receptors via coupling toextracellular signal-regulated protein kinase （ERK1/2）, calcium/calmodulin-dependentkinaseⅣ（CaMKⅣ） and cAMP-responsive element binding protein （CREB）. CREB isa central transcription factor that mediates cyclic AMP （cAMP） and calcium-dependentgene expression and Ser-133 phosphorylation is considered to be a critical event thatmediates the initiation of transcription, since mutation of Ser-133 to alanine abolishes transcription.The NMDA receptor （NMDAR） is an ionotropic glutamate receptor that isspecifically activated by the selective agonist, NMDA （N-methyl d-aspartate）. NMDAreceptor activation leads the opening of an ion channel which allows the flow of Na⁺and K⁺ and Ca²⁺. Calcium influx through NMDARs drives the activation of manydownstream signaling molecules, which underlie many physiological processes such assynpatic plasticity, and learning and memory.In vitro the trophic role of synapticNMDA receptors couples with the cAMP-responsive element binding protein（CREB） .The purposes of this study are to determine whether treatment of developinghippocampus neurons cultures with ketamine results in increase of neurotoxicity andthe contribution of CREB signaling pathway. CREB regulates transcription of manypro-survival genes, including BDNF and Bcl-2. The ability of calcium influx to inducetranscription of brain-derived neurotrophic factor （BDNF） is regulated by the route ofcalcium entry into the cell, by the pattern of phosphorylation induced on thetranscription factor cAMP-response element （CRE） binding protein （CREB）.Stimulations activate CREB mainly through increased intracellular cAMP and Ca²⁺（Figure 1）. Ca²⁺/CREB/CBP-dependent gene regulation might be a shared mechanismcriticalin both long-term synaptic plasticity and neuronal survival.ThecAMP-responsive element-binding protein （CREB） pathway has been involved in 2major cascades of gene expression regulating neuronal function. The first one presentsCREB as a critical component of the molecular switch that controls long-lasting formsof neuronal plasticity and learning. The second one relates CREB to neuronal survivaland protection.In this paper we studied neurotoxicity to developing hippocampal neuronscaused by ketamine in vivo and in vitro from the effects on neuronal survival, synapseformation, synaptic plasticity and long-term learning and memory.We tested hypothesises that （1） ketamine induces the the apoptosis of developing hippocampusneurons in vitro and in vivo （2） ketamine spoiled synaptogenesis in vitro and in vivo（3） ketamine disrupted memory and learning at a later age. （4） ketamine decreased thecalcium influx of developing neurons by antagonized NMDA receptor calcium channel（5） ketamine down regulated the phosphorylation of CREB on Ser-133 and （6）ketamine reduced expressions of downstream genes of CREB （ BDNF ,Bcl-2 andsynaptophysinI）. Methods and Results1. Effect of intravenous general anesthetics on intracellular Ca²⁺ of ratdeveloping hippocampal neurons in vitroMethods: On the fifth day of cultured ,hippocampal neurons were co-incubated with10μmol/L Fluo-4 AM for 30 min at 37℃. Excess dye was washed out with threerinses of DMEM. Fluorescence imaging of intracellular Ca²⁺ was performed to anumber of neurons selected when the hippocampal neurons were exposed to 150μmol /L ketamine, 3μmol / L midazolam or 10μmol / L propofol respectively by confocallaser microscope.Results: After exposed to 150μmol/L ketamine, the intracellular calcium intensity ofthe neurons decreased from （987±307） to （766±226）（P＜0.05）. While exposed to3μmol/ L midazolam or 10μmol/ L propofol, the cytosolic Ca²⁺ of the neuronsincreased significantly ,fluorescence intensity of the neurons exposed to midazolamincreased from （1707±514）to （2663±572） （P＜0.05）, fluorescence intensity of theneurons exposed to propofol increased from （1057±353） to （1749±708） （P＜0.05）.2. Effects of ketamine on neuronal survival of rat developinghippocampal neurons in vitroMethods After cultured primary hippocampal neurons for 5 days, the neurons weredevided into two groups, each group had six well of culture plates: group C （controlgroup） and the group K （co-culture the neurons with the neuronbasel mediumincluding 1 000μmol/L ketamine for 3 h）. Detected the apoptosis of the neurons byAnnexin V-PI Apoptosis Kit on flow cytometryResults Compared to group C,the apoptosis of the neurons from group K increased significantly.3. Effects of ketamine on synaptogenesis of rat developing hippocampalneurons in vitroMethods After been cultured for 5 days , primary hippocampal neurons weredevided into group C and the group K.After been intervented, the neurons werecultured to the 14th day continuously then expressions of SynaptophysinI of neuronsin both groups were detected by double-immunofluorescence methods. Greenfluorescence the expression of Synaptophysin I and blue fluorescence representedDAPI.Results Compared with the group C, density and synaptic connections of neurons ingroup K declined significantly and expression of synapsin I reduced statistically.4. Effects of ketamine on CREB signaling pathway of rat developinghippocampal neurons in vitroMethods After been cultured for 5 days, primary hippocampal neurons were devidedinto group C and the group K .After been intervented, expression of p-CREB in bothgroups were measured by immunocytochemistry ,then expressions of the BDNF andBcl-2 in both groups were detected by RT-PCR.Results Compared with the group C,expressions of p-CREB, Bcl-2 and BDNF of theneurons from K proup all decreased statistically （P＜0.05） .5. Preconditioning with NMDA antagonised the effects of ketamine onphosphorylation of CREB on Set-133 of rat developing hippocampalneurons. Methods DIV5 hippocampal neurons were divided into six groups: group C（Control group）, group N₁ （ the neurons were co-culture with the neuronbaselmedium including 2μmol/L NMDA for 4 h ）, group N₂ （ the neurons were co-culturedwith the neuronbasel medium including 10μmol/L NMDA for 4 h ）, group N₁ +K （ theneurons were co-culture with the neuronbasel medium including 2μmol/L NMDA for1 h then the neurons were co-culture with the neuronbasel medium including 1000μmol/L ketamin for 3 h）, group N₂ +K （the neurons were co-cultured with theneuronbasel medium including 10μmol/L NMDA for 1 h then the neurons wereco-cultured with the neuronbasel medium including 1000μmol/L ketamin for 3 h） andgroup K （ the neurons were co-cultured with the neuronbasel medium including 1 000μmol/L ketamine for 3 h） and expressions of CREB and p-CREB in each groupwere detected by Western-blot.Results Compared to group C, there were no statistically difference in expression ofCREB among six groups,expression of p-CREB in group N₂ increased significantly,expressions of p-CREB in group N₁+K, group N₂+K and group K all decreasedstatistically. Compared to group K, expressions of p-CREB in group N₂+K increasedstatistically.6. Effects of ketamine on apoptosis rate in hippocampus of developingratMethods Fifteen 7-day-old rats were randomly devided into three groups, group C（control group）, group K₁, clinically relevant concentrations of ketamine （10mg/kgsubcutaneous injection） and group K₂ , superclinical concentration of ketamine（20mg/kg subcutaneous injection） . Repeat Subcutaneous administrations for 7 times,each time interval 90min. After administration for 1d, rats were decapitated and frozensections of brain were made.TUNEL Assay Kits was used to detecte the apoptosis of the neurons.Results Compared to group C, apoptosis of neurons in both groups increasedstatistically, from （0.82±1.66） %to （1.55±2.25） % （P＜0.05） and （2.77±2.42）% （P＜0.05） respectively.7.Effects of ketamine on synaptogenesis of rat hippoeampus during developingMethods Thirty 7-day-old rats were randomly derided into three groups, group C,K₁ andK₂, rats were administrated as former. Skin color of rats and whether had performance ofhypoxia were observed carefully after treatment of neonatal rats with ketamine. Duringanesthesia low concentration of oxygen （oxygen flow: 2L/min） were given. Afterrecovery from anesthesia, the rats were returned to mother rats for continue feeding.Afterbeen administrated for 1 week,immuno -histochemistry was used to measur theexpression of Synaptophysin I （the marker of Presynaptic membrane） and Western-blotwas used to detecte the expression of PSD95 （marker of postsynaptic membrane ）. IODand relative gray value represented the expressions of synapsin I and PSD95 respectively.Results Between group C and K₁ the expressions of Synaptophysin I and PSD95 had nostatistically difference. Compared to group C ,the expressions of Synaptophysin I andPSD95 in group K₂ both decreased significantly. （P＜0.05） .8. Effects of ketamine on CREB signaling pathway of rat hippocampus duringdevelopingMethods Thirty 7-day-old rats were randomly devided into three groups, group C,K₁and K₂, rats were administrated as former. Double-immunofluorescence methods wereused to measure the expression of p-CREB, then RT-PCR was used to detecte theexpressions of the BDNF and Bcl-2 on the second day after intervention.Results Compared to group C, expressions of the p-CREB、Bcl-2 and BDNF ofneurons in group K1 and K2 both descended statistically （P＜0.05）, IOD of CREB in group Kland K2 decreased from （2627.86±345.29） to （1607.98±97.63） and（967.58±61.24 ） （ P＜0.05 ）,respectively. Relative gray value of BDNF decreased from（1.355±0.147） to （1.241±0.004） （P＜0.05） and （ 0.863±0.009 ） （P＜0.05） respectively.Relative gray value of Bcl-2 decreased from （ 0.790±0.008 ） to （ 0.740±0.034 ） （P＜0.05）and （0.560±0.014） （P＜0.05） respectively.9. Effects of ketamine on learning and memory of developing rat after maturityMethods Thirty 7-day-old rats were randomly devided into three groups, group C,K₁ andK₂, and rats were administrated as former. After recovery from anesthesia, the rats werewere returned to mother rats for continue feeding .After been administrated for 6 weeks,Morris watermaze was used to test learning and memory function of rats in each group.After been trained for 7d continuously, escape latency, times of creossed the formerplatform and the ratio of time and distance in target quadrant of rats were tested by placenavigation test and spatial probe test.Results Compared with group control,latency to find the platform in group K₂increased from （ 5.12±3.24 ） to （ 10.83±6.48 ） （P＜0.05 ） ,whereas between group C andgroup K₁, latency to find the platform had no difference statistically. There are nostatistically difference in times of crossed the former platform and the ratio of time anddistance in target quadrant among three groups.10. Statistical analysisData are presented as mean±standard deviation （SD）, Calcium fluorescence intensityusing paired t test , apoptosis rate ,the expressions of p-CREB,BDNF and Bcl-2 in vitrousing t tests,and the other results using ANOVA by SPSS 13.0. A value of P＜0.05 wasconsidered as statistical significance. CONCLUSIONS and SUMMARY1. Developmental neurotoxicity in hippocampal neurons induced by ketamine wereconfirmed by researches in vitro （cultured primary neurons）and in vivo （animalexperiments） through two aspects of neurotoxicity, survival and neurogenesis ofneurons.2. Effects of ketamine on NMDA receptor and intracellular Ca²⁺ upstream of CREBwere confirmed by researches on cultured primary hippocampal neurons in vitro:ketamine antagonisted NMDA receptor ion channel then reduced calcium influx.Effects of ketamine on expressions of p-CREB,BDNF and Bcl-2 were confirmed bothin vitro and in vivo.3. Researches on long-term learning and memory function of clinically relevantconcentrations of ketamine and superclinical concentration of ketamine conformedthat only superclinical concentration of ketamine can lead to learning and memoryfunction impairment partially.更多还原