神经甾体对氯胺酮所致发育期皮层神经元凋亡的影响及机制研究
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摘要
全世界每年有150万婴幼儿因需进行外科手术或介入治疗等原因而接受全身麻醉，长期以来全身麻醉药是否影响婴幼儿的大脑发育一直是患儿家属及麻醉学者十分关注的问题。传统观念认为只要麻醉过程中不存在大脑缺氧等因素就不会影响婴幼儿大脑的发育，但近年来大量的动物实验研究表明全身麻醉药可对发育期大脑产生损伤，并引起远期学习记忆损伤。因此全身麻醉药的发育期神经毒性已引起了众多学者的关注。
氯胺酮为N-甲基-D-天冬氨酸（N-methyl-D-aspartate, NMDA)受体拮抗剂，被广泛应用于婴幼儿手术的麻醉。NMDA受体是一种离子通道型谷氨酸能受体，具有许多不同变构调控位点并对Ca2+高度通透，在中枢神经系统主要分布在大脑皮层及海马。研究发现NMDA受体与中枢神经系统的发育密切相关，控制神经元的分化、迁移和存活，并在学习、记忆的形成和维持过程中发挥着重要作用。动物出生前后时间阶段是中枢神经系统生长发育的高峰期，有大量神经细胞增殖、大量树突生成和大量突触连接形成，进而形成复杂的神经网络。许多研究已证实在大脑发育的关键时期NMDA受体保持适度兴奋对大脑神经元的存活具有重要的意义，如果改变NMDA受体的活性可能会影响中枢神经系统的发育和功能。
目前大量研究发现氯胺酮可引起发育期动物大脑广泛脑区神经元的凋亡，同时也发现氯胺酮可引起原代培养皮层神经元的大量凋亡。关于氯胺酮引起发育期大脑神经元凋亡与神经功能损害的确切机制目前还不清楚。既往研究表明在大鼠大脑快速发育期注射氯胺酮会导致皮层组织的NMDA受体表达上调，引起神经元兴奋性毒性增加。此外，氯胺酮作为NMDA受体的非竞争性拮抗剂，可以通过作用于NMDA受体PCP结合位点，阻断与NMDA受体耦联的钙通道，减少Ca2+内流，降低细胞内Ca2+浓度，从而拮抗谷氨酸、天冬氨酸等兴奋性氨基酸对神经系统发育的调节。目前有研究认为氯胺酮通过抑制神经元细胞内钙震荡而对发育期神经元产生神经毒性。
针对全身麻醉药引起的发育期大脑损伤，NIH、FDA以及IARS要求研究学者不仅要研究其发生机制而且要寻找安全有效的措施来防治麻醉药所引起的发育期大脑损伤。近年来国内外学者对如何防治氯胺酮引起的发育期大脑损伤进行了一系列研究，结果表明维生素D3、锂剂、促红细胞生成素、肉毒碱、烟酰胺、可乐定等可对氯胺酮引起的发育期神经元损伤产生保护作用，但它们在婴幼儿时期应用的安全性还有待于进一步研究。
神经甾体（neurosteroid）是一类由神经系统的神经元或神经胶质细胞合成、代谢产生的具有神经活性的甾体物质的总称，主要包括：孕烯醇酮（pregnenolone，PREG）、孕烯醇酮硫酸酯（pregnenolone sulfate，PREGS）、脱氢表雄酮（dehydroepiandrosterone，DHEA）、脱氢表雄酮硫酸酯（dehydroepiandrosterone sulfate，DHEAS）、孕酮（progesterone，PROG）、别孕烯醇酮（allopregnanolone，AP）以及雌二醇（estradiol，E2）等。大量研究表明神经甾体不仅参与调节神经系统的发育并对神经系统的功能产生广泛影响，对神经系统还具有营养保护以及促进神经发生和神经元存活的作用。
雌二醇作为一种内源性神经甾体，在神经元发育过程中发挥重要的调节作用，促进神经元的存活以及功能维护。最近有研究表明氯胺酮可引起斑马鱼发育期17β雌二醇水平的下降，睾酮水平升高，同时还有研究表明氯胺酮对发育期斑马鱼具有神经损伤作用。PI3K-Akt信号通路是介导多种生长因子促进细胞存活的重要通路，有研究表明氯胺酮通过抑制NMDA受体的活性进而抑制pAkt的表达对原代培养的皮层神经元产生损伤。有研究表明17β雌二醇可通过激活PI3K-Akt信号通路对NMDA受体拮抗剂MK-801以及麻醉药咪达唑仑、笑气、异氟醚联合应用所引起的发育期大鼠大脑广泛脑区神经元凋亡产生保护作用。另外Ramezani等研究表明17β雌二醇可对酒精引起的发育期大鼠大脑神经损伤以及远期行为学异常产生保护作用。本实验室前期研究发现Aβ25-35对原代培养皮层神经元产生损伤时伴随神经甾体代谢通路的变化。氯胺酮引起发育期皮层神经元损伤的同时是否伴随神经甾体合成代谢的变化，以及神经活性甾体17β雌二醇是否通过PI3K-Akt信号通路对氯胺酮引起的皮层神经元损伤产生保护作用，国内外未见报道。
本课题首先以原代培养的大鼠皮层神经元为研究对象采用不同浓度的氯胺酮建立氯胺酮体外诱导皮层神经元损伤的细胞模型。在该模型的基础上采用液液萃取结合高效液相色谱-质谱联用（HPLC-MS/MS）方法对氯胺酮引起皮层神经元损伤时主要神经甾体PREG、PROG以及17β雌二醇合成代谢水平进行了研究。随后以氯胺酮损伤皮层神经元时合成代谢水平明显降低的17β雌二醇进行外源性给药，从离体细胞和在体动物两方面研究17β雌二醇对氯胺酮诱导的原代培养皮层神经元损伤以及发育期大鼠皮层区神经细胞损伤的保护作用。最后应用原代培养皮层神经元进一步研究17β雌二醇是否通过PI3K-Akt信号通路保护发育期皮层神经元免受氯胺酮的损伤。本实验将为神经活性甾体17β雌二醇防治氯胺酮引起的发育期大脑损伤提供理论依据及实验基础。
本研究采用的试验方法包括：
1大鼠大脑皮层神经元的原代培养
大鼠大脑皮层神经元进行原代培养7天用于实验。倒置显微镜下观察皮层神经元的形态学变化。
2分组与药物处理
①观察氯胺酮对神经元的损伤作用时分为：不同浓度氯胺酮（0、1、10、100、1000μΜ）作用神经元24h100μΜ氯胺酮作用神经元不同时间（0、6、12、24、48h）
②观察氯胺酮对神经元神经甾体合成的影响分为：对照组、氯胺酮组、胆固醇组、氯胺酮+胆固醇组
③观察17β雌二醇对氯胺酮诱导皮层神经元凋亡的影响分为：对照组，氯胺酮组，17β雌二醇+氯胺酮组
④观察17β雌二醇对氯胺酮引起发育期大鼠大脑皮层区神经细胞凋亡和远期学习记忆损伤的影响分为：对照组，溶剂对照组，17β雌二醇组，氯胺酮组，17β雌二醇+氯胺酮组
⑤研究17β雌二醇保护原代培养皮层神经元免受氯胺酮损伤的机制分为：
对照组，氯胺酮组，17β雌二醇+氯胺酮组，17β雌二醇+氯胺酮+LY294002组
3细胞样品的收集与蛋白定量
给予各种药物处理的皮层神经元，加入细胞裂解液，低温离心收集上清，应用多功能酶标仪测定蛋白质浓度。
4MTT法测定皮层神经元的存活率
5Hoechst33258核染色法检测神经元凋亡
在荧光显微镜下，随机选取5个视野进行形态学观察和计数，计算凋亡率，凋亡率=（凋亡细胞数/细胞总数）×100%。
6TUNEL法检测原代培养皮层神经元凋亡
细胞爬片于倒置显微镜下任意选取5个视野观察阳性细胞并计数，计算神经元的凋亡率，凋亡率=（凋亡细胞数/细胞总数）×100%。
7免疫组织化学染色分析大脑皮层区cleaved-caspase-3表达：
以细胞中出现棕褐色浓染颗粒作为阳性细胞的判定标准。在光镜下观察皮层区的凋亡神经细胞，以个/0.01mm2计数。
8Western-blot法检测p-Akt、Akt、cleaved-caspase-3、Bcl-2的蛋白表达
9HPLC-MS/MS测定细胞培养液中主要神经甾体的浓度
10Morris水迷宫行为学测试
11统计学处理
各组数据均以均数±标准差（±s）表示，应用SPSS13.0统计软件进行处理，水迷宫行为学测试中定位航行实验数据采用重复测量方差分析进行统计学处理，其他数据采用单因素方差分析（one way ANOVA）继以LSD post hoc test进行统计学处理,以P<0.05为差异有统计学意义。
结果：
1氯胺酮对原代培养皮层神经元的损伤作用
1.1氯胺酮呈浓度和时间依赖性降低原代培养皮层神经元存活率
不同浓度的氯胺酮作用于体外培养7天的皮层神经元24h后，神经元存活率下降，其中100、1000μΜ的氯胺酮使神经元存活率显著下降（P<0.01，P<0.01）。100μΜ氯胺酮作用于神经元不同时间后神经元存活率下降，其中作用24h及48h后，神经元存活率显著下降（P<0.05，P<0.01）。
1.2氯胺酮损伤大鼠皮层神经元时的细胞形态变化
倒置显微镜下观察刚种植的皮层神经元呈圆形，单个均匀分布，折光性强，胞体透亮，接种8h后细胞贴壁，24h后细胞贴壁牢固并可见短小的突起，胞体呈梭型或雏形，第三天后神经元胞体进一步增大，部分细胞突起延长，神经元培养至第7天，胞体进一步增大，多呈梭型或雏形，胞体饱满，部分神经元集聚成团，突起增多并延长，相互交织形成网络。培养7天的神经元经100μΜ氯胺酮处理24h后，神经元细胞数量较对照组明显减少，折光性差，胞体明显缩小，轴突断裂，部分神经元细胞膜破裂成细胞碎片。
1.3氯胺酮对原代培养皮层神经元凋亡的影响
TUNEL结果显示，对照组仅见少量的TUNEL阳性神经元，100μΜ氯胺酮组TUNEL阳性神经元较对照组显著增加（P<0.01）。Hoechst33258荧光染色结果显示，对照组细胞核内呈均匀分布的淡蓝色荧光，内有较深的蓝色颗粒，有少量凋亡细胞呈亮蓝色。100μΜ氯胺酮组皮层神经元染色质高度凝聚、边缘化，细胞核甚至裂解为碎块，有大量凋亡细胞呈亮蓝色(P<0.01)。
2氯胺酮对原代培养皮层神经元神经甾体水平的影响
2.1不同处理对皮层神经元存活率的影响
MTT结果显示，与对照组比较，氯胺酮组皮层神经元的存活率显著下降（P<0.01），胆固醇单纯处理组神经元存活率未见显著改变（P>0.05）。与氯胺酮组比较，胆固醇与氯胺酮共处理组神经元存活率显著升高（P<0.01）。
2.2不同处理对神经元凋亡的影响
TUNEL结果显示，与对照组比较，胆固醇单纯处理组TUNEL阳性神经元未见显著变化（P＞0.05），氯胺酮组TUNEL阳性神经元显著增加（P<0.01）。与氯胺酮比较，胆固醇与氯胺酮共处理组TUNEL阳性神经元显著降低（P<0.01）；Hoechst33258荧光染色结果进一步证实了以上变化。
2.3氯胺酮对皮层神经元神经甾体水平的影响
对照组及氯胺酮处理组因未加入神经甾体合成所必需的底物胆固醇，均未测得各种神经甾体。与胆固醇单纯处理组相比，氯胺酮共处理后神经甾体PREG水平未见显著变化，而17β雌二醇水平显著下降(P<0.01)，PROG水平显著升高（P＞0.05）。
317β雌二醇对氯胺酮诱导的发育期皮层神经元凋亡以及发育期大鼠远期学习记忆损伤的影响
3.117β雌二醇对氯胺酮导致的原代培养皮层神经元损伤的影响
MTT结果显示，与对照组比较,氯胺酮组原代培养皮层神经元存活率显著下降（P<0.01）；与氯胺酮组比较，不同浓度（0.001、0.01、0.1、1μΜ）的17β雌二醇与氯胺酮共处理24h后神经元的存活率显著升高，其存活率分别为(54.8±5.1)%，(75.1±9.3)%，(88.3±8.5)%，(79.5±10.3)%，其中0.1μM的17β雌二醇保护效果最好。
3.217β雌二醇减轻氯胺酮诱导的原代培养皮层神经元凋亡
TUNEL结果显示，对照组可见少量的凋亡神经元，氯胺酮组神经元凋亡较对照组显著增加（P<0.01），0.1μM的17β雌二醇使氯胺酮导致的神经元凋亡显著降低（P<0.01）。Hoechst33258核染色法进一步证实了17β雌二醇减轻氯胺酮诱导的皮层神经元凋亡。对照组细胞核多呈弥散均匀分布的淡蓝色荧光，内有较深的蓝色颗粒，有少量的凋亡细胞呈亮蓝色。氯胺酮组凋亡细胞较对照组显著增加(P<0.01)，17β雌二醇与氯胺酮共处理组神经元凋亡较氯胺酮组显著降低（P<0.01）。
3.3不同处理对发育期大鼠大脑额叶皮层区cleaved-caspase-3表达的影响
与对照组比较，溶剂对照组和17β雌二醇组cleaved-caspase-3阳性表达细胞数/0.01mm2未见统计学差异（均为P>0.05），氯胺酮组阳性表达显著增加（P<0.01）。与氯胺酮组比较，17β雌二醇与氯胺酮共处理组阳性表达显著降低（P<0.01）。
3.4Morris水迷宫测试SD大鼠学习记忆能力
SD幼鼠饲养至60天时，进行Morris水迷宫5天训练，一天四次。把SD大鼠逃逸潜伏期（s）和游泳距离（cm）作为学习能力的指标。在5天的训练期中，前2天实验各组逃逸潜伏期和游泳距离未见统计学差异。与对照组比较，氯胺酮组第3天、第4天、第5天逃逸潜伏期和游泳距离均显著增加（均为P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组逃逸潜伏期和游泳距离均显著降低（均为P<0.01）；第6天撤去平台，进行60s空间探索试验作为对记忆能力的评估。与对照组比较，氯胺酮组穿越平台的次数和靶象限的时间比率均显著减少（均为P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组穿越平台的次数和靶象限的时间比率均显著增加（均为P<0.01）。
417β雌二醇保护原代培养皮层神经元免受氯胺酮损伤的机制研究
4.1不同处理对原代培养皮层神经元pAkt表达水平的影响
结果显示，与0h相比，0.1μM的17β雌二醇单独处理皮层神经元0.5、1、2、4h, pAkt表达水平时间依赖性显著升高（P<0.01）；17β雌二醇与氯胺酮共处理皮层神经元0.5、1、2、4h， pAkt表达水平随时间延长显著升高（P<0.01）。为了进一步证实17β雌二醇通过PI3K-Akt信号通路对氯胺酮引起的皮层神经元损伤产生保护作用。给予PI3K抑制剂LY294002，观察对皮层神经元pAkt表达水平的影响。与对照组比较，17β雌二醇培养皮层神经元2h引起pAkt表达水平的显著升高（P<0.01）,氯胺酮培养皮层神经元2h引起pAkt表达水平的显著降低（P<0.01）。与氯胺酮组比较，17β雌二醇与氯胺酮共培养2h pAkt表达水平显著升高（P<0.01），加入PI3K抑制剂LY294002后培养2h，LY294002抑制了17β雌二醇对pAkt表达水平的上调作用（P<0.01）。
4.217β雌二醇对原代培养皮层神经元Bcl-2表达水平的影响
Bcl-2作为一种抗凋亡分子，对多种损伤产生保护作用。Akt可通过激活CREB磷酸化，提高Bcl-2的表达水平，从而对各种应激提供神经保护作用。为了探讨17β雌二醇是否有可能通过PI3K-Akt信号通路影响Bcl-2的表达水平对氯胺酮引起的神经元损伤产生保护作用，我们将皮层神经元给予100μM氯胺酮和（或）0.1μM的17β雌二醇以及10μMLY294002处理24h，结果显示与对照组比较，氯胺酮组Bcl-2表达水平显著下降（P<0.01）；与氯胺酮组比较，17β雌二醇与氯胺酮共处理组Bcl-2表达水平显著增加（P<0.01）；与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组Bcl-2表达水平显著下降（P<0.01）。
4.317β雌二醇对原代培养皮层神经元cleaved-caspase-3表达水平的影响
Caspase-3是各种细胞凋亡通路的最终执行者。为了探讨17β雌二醇是否有可能通过PI3K-Akt信号通路影响cleaved-caspase-3的表达水平对氯胺酮引起的神经元损伤产生保护作用，我们将皮层神经元给予100μM氯胺酮和（或）0.1μM的17β雌二醇以及10μM LY294002处理24h，结果显示与对照组比较，氯胺酮组cleaved-caspase-3表达水平显著增加（P<0.01）；与氯胺酮组比较，17β雌二醇与氯胺酮共处理组cleaved-caspase-3表达水平显著下降（P<0.01）；与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组cleaved-caspase-3表达水平显著增加（P<0.01）。
4.4LY294002对17β雌二醇神经保护作用的影响
MTT结果显示,与对照组比较，氯胺酮组皮层神经元存活率显著降低（P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组神经元存活率显著增加（P<0.01），与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组皮层神经元存活率显著降低（P<0.01）。TUNEL结果显示对照组可见少量的凋亡神经元，与对照组比较，氯胺酮组神经元凋亡显著增加（P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组神经元凋亡显著降低（P<0.01），与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组神经元凋亡显著增加（P<0.01）。Hoechst33258核染色法进一步证实了TUNEL试验所见。
结论：
1氯胺酮能浓度和时间依赖性的引起原代培养皮层神经元损伤。
2氯胺酮在导致原代培养皮层神经元损伤的同时伴随某些神经甾体水平的变化，其中PROG水平显著升高，17β雌二醇水平显著降低。
317β雌二醇对氯胺酮引起的原代培养皮层神经元损伤具有保护作用。
4外源系统性给予17β雌二醇对氯胺酮引起的发育期大鼠皮层区神经细胞凋亡以及成年后学习记忆损伤产生保护作用。
5激活PI3K-Akt信号通路，促进抗凋亡分子Bcl-2的表达，抑制促凋亡分子cleaved-caspase-3的表达可能是17β雌二醇对氯胺酮引起的皮层神经元损伤产生保护作用的机制之一。
Globally, there were about1.5million infants and young childrenundergoing general anesthesia because of various reasons such as surgery orinterventional therapy each year. For a long time, whether or not generalanesthesia would affect the development of infant's brain has been the focus ofattention of family members and anesthesiologists. The conventional belief isthat, as long as there was no cerebral hypoxia or other relevant consequencesduring the anesthesia, it would not affect the brain development of infants andyoung children. However, neuronal cell death after general anesthesia hasrecently been demonstrated in neonatal animal models. The possibility ofanesthesia-induced neurotoxicity in human neonates or infants has led toserious questions about the safety of pediatric anesthesia.
Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, iswidely used in surgical anesthesia of infants and young children now. TheNMDA receptor is a kind of central nervous system excitatory amino acidionotropic receptors, with many different allosteric regulatory sites and Ca2+high degree of permeability. In the central nervous system, NMDA receptorsare mainly distributed in the cerebral cortex and hippocampus. Studies havefound that the NMDA receptor is closely related to the development of centralnervous system, controlling neuronal differentiation, migration and survival,and playing an important role in the formation and maintenance of learningand memory. Recent studies have found that developing neurons can beinduced to activate pathways resulting in cell death at the peak of the centralnervous system growth and development, when their synaptic development,dendritic branching, and remodeling activities are interfered with. Manystudies have confirmed that it is of great significance for the NMDA receptorto maintain moderate excitement for the survival of neurons in the critical period of brain development. If the activity of NMDA receptors was changed,it would affect the development and function of the central nervous system.
An increasing number of animal studies have suggested that ketaminecauses neurodegeneration during early development. Studies in vitro have alsoshown that ketamine administration induces apoptosis in cultured neurons.The exact mechanism triggering ketamine-induced apoptosis has not yet beenfully elucidated by now. Previous studies have shown that injection ofketamine could cause up-regulation of the NMDA receptor in the rat brainduring its developmental stages, resulting in a wide range of dose-dependentapoptosis. In addition, as a noncompetitive antagonist of the NMDA receptor,ketamine can act on PCP binding sites of the NMDA receptor, blocking theNMDA receptor coupled calcium channels, reducing calcium influx, andreducing the concentration of intracellular calcium. This can antagonize theregulation of nervous system development by glutamate, aspartate and otherexcitatory amino acids. Recent studies showed that ketamine inducedneuroapoptosis by inhibiting Ca2+oscillations in neurons.
With respect to anesthesia-induced brain injury in neonates, the NIH, theFDA, and the International Anesthesiology Research Society (IARS) haveurged researchers not only to define the scope of the problem but also todevelop therapeutic strategies that target prevention. Although many strategies,including the use of activity-dependent neuroprotective protein peptidefragment NAPVSIPQ (NAP), vitamin D, lithium, erythropoietin, L-carnitine,nicotinamide, and Clonidine, are known to have neuroprotective effectsagainst ketamine-induced apoptosis both in vivo and in vitro, their efficacyand safety need to be further verified for uses targeting the developing brain.
Steroids hormones and their metabolites within the central nervous system(CNS) are commonly defined as neuroactive steroids or neurosteroids. Theycan be synthesized de novo in the CNS by glial cells and neurons. Neuroactivesteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG), 5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/AP), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. The studyshowed that neurosteroids played an important role as endogenous modulatorsin the development of CNS and exerted neuroprotection.
Estradiol is a kind of neurosteroids, which has the potential for promotingsurvival and function of neurons. Recently, Trickler et al reported thatketamine attenuated17β-estradiol level and increased testosterone level andketamine had been shown to be neurotoxic in early life stages of zebrafish.PI3K-Akt signaling is very important for promoting neuronal survival.Ketamine induces apoptosis by blocking NMDA receptor activity anddecreasing levels of p-AKT. Previous studies reported that estradiol treatmentof neonatal rats inhibited neuroapoptosis induced by the NMDAR antagonistMK-801and anesthetic cocktails including midazolam, isoflurane, and nitrousoxide. More recently, Ramezani and colleagues suggested that17β-estradiolcould protect the developing rat cerebellum from ethanol-inducedneurotoxicity and improve behavioral deficits. The results from our laboratoryhave shown that Aβ25-35induces apoptosis in primary cultured cortical neuronsaccompanying the change of the methabolism of neurosteroids. However,whether ketamine induces apoptosis in primary cultured cortical neuronsaccompanied by the change of the methabolism of neurosteroids and whether17β-estradiol can protect rat cortical neurons from ketamine-induced apoptosisremain relatively elusive. In our study, an in vitro ketamine-induced cellapoptosis model was established based the primary cultured rat corticalneurons. Based on this model, the HPLC-MS/MS assay was applied toinvestigate the changes of chief neurosteroids, PREG, PROG and17β-estradiol levels in cultured neurons induced by ketamine cytotoxicity.Afterwards,17β-estradiol, whose level found obviously decreased byketamine treatment, was chosen as intervening substances to investigateitsprotective effects against ketamine-induced neuroapoptosis. Then whetherPI3K-Akt signaling takes part in neuroptotection of17β-estradiol against ketamine-induced neuroapoptosis was studied. The study is to add valuableefforts for the protective effects of17β-estradiol against ketamine-inducedneuroapoptosis.
The following methods were applied in this thesis:
1Cortical neurons of newborn SD rat were cultured.Cortical neurons were grown for7days before experimentation. Invertedmicroscope was used to observe morphological changes of cultured neurons.
2Grouping and drug treatments
①Ketamine induces injuries in primary cultured cortical neurons:different concentrations of ketamine treatment for24h100μM ketamine treatment for different time
②Effect of ketamine on neurosteroidogenesis in primary rat corticalneurons:control, ketamine, cholesterol, cholesterol+ketamine
③Effects of17β-estradiol treatment on neuroapoptosis induced byketamine exposure:control, ketamine,17β-estradiol+ketamine
④17β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis and long-term behaviour deficts in developing brain:control, veichle-control,17β-estradiol, ketamine,17β-estradiol+ketamine
⑤Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons:control, ketamine,17β-estradiol+ketamine,17β-estradiol+ketamine+LY294002
3Sampling and quantitation
Treated neurons were collected, after cell lysis, supernatant proteinconcentrations were quantitatived by multifunctional enzyme markinstrument.
4Neuron viability assay
5Apopyosis of neurons indentified by Hoechst233258dying
Apoptosis were identified by nuclear morphometry under fluorescencemicroscope. The apoptosis was calculated by5randomly selected eye fields.
6Apoptotic neurons were detected using the TUNEL assay
Apoptotic neurons were identified by TUNEL assay under invertedicroscopy. The apoptosis was calculated by5randomly selected eye fields.
7Immunohistochemical Staining for cleaved caspase-3
Apoptotic cells were identified by immunohistochemical Staining forcleaved caspase-3under inverted icroscopy. The apoptotic cells (per0.01mm2)in the prefrontal cortex were counted
8Western Blot Analysis for p-Akt, Akt, cleaved-caspase-3, Bcl-2expression
9Determination of the concentrations of neurosteroids in culture mediausing HPLC-MS/MS assay
10Morris water maze test
11Statistical Analysis
The data were expressed as mean±standard deviation (SD). The meanescape latency of the Morris water maze behavioral tests collected during thetraining days were analyzed by repeated-measure analysis of variance(ANOVA). Other data were statistically analyzed by one-way ANOVA,followed by between-group comparisons using LSD post hoc test. Statisticalsignificance was concluded with a value of P<0.05for all analyses. Dataanalyses were performed with the SPSS software version13.0
Results:
1Ketamine induces injuries in primary cultured cortical neurons
1.1Ketamine decreased neuron viability dose and time dependently
The MTT assay was used to evaluate the viability of neurons. Ketaminedecreased cell viability dose and time dependently. Compared withvehicle-control group, the neuron viability of ketamine treated group (24h)significantly (P<0.01for ketamine100μM and1000μM) decreased.Compared with control group, the neuron viability of100μM ketaminesignificantly decreased (P<0.05for24h, P<0.01for48h).
1.2Morphoolgical alteration of neurons
The newly inoculated cortical neurons were round, small, translucent,and in a single suspension uniform distribution. After8h, the cells started toadhere to the culture plates, and most of neurons had adhered in24h. Theirmorphologies were fusiform, triangular, with different length of slenderprotrusions. On the3rd day, the cells were well-rounded and full, and theprominences were elongated and enlarged more significantly and connected tothe network, sparse connections had come into being. Cortical neuronscultured for7days, were generally bipolar or multipolar cells under invertedmicroscopy, most of them with triangular soma,and usually aggregated withtheir branching dendrites interconnected as network. Compared with controlgroup,24h after100μM ketamine treatment, numbers of neurons decreasedobviously, with reduced refraction, axons break, and granuliform pieces.
1.3Ketamine influences on neuroapoptosis identified by TUNEL assay andHoechst33258staining.
Compared with control group, ketamine treatment increased neuronsapoptosis signficantly (P<0.01).
2Effect of ketamine on neurosteroidogenesis in primary rat cortical neurons
2.1Influence on neurons viability
Compared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while cholesterol treatment made nodifference with controls (P>0.05). But, in ketamine+cholesterol group, neuronviability was obviously higher than ketamine treatment alone (P<0.01)
2.2Influence on neuron apoptosis
We use TUNEL assay and Hoechst33258staining to identified neuronapoptosis in different treatment group. Compared with control group,apoptosis rate increased significantly in ketamine-treated group (P<0.01),while cholesterol treatment made no difference with controls (P>0.05). But, inketamine+cholesterol group, apoptosis rate was obviously lower thanketamine treatment alone (P<0.01).
2.3Influence on neurosteroidgenesis
Absence of the necessary substance, cholesterol, no neurosteroids found in control and ketamine alone groups. Compared with cholesterol treatmentalone, no changes in PREG level were observed after treatment with ketamine,while PROG increased significantly (P<0.05) and17β-estradiol decreasedgreatly (P<0.01).
3Effects of17β-estradiol treatment on neuroapoptosis induced by ketamineexposure
3.1Protective effects of17β-estradiol against ketamine-induced cell death incultured cortical neurons.
We used the MTT assay to assess the changes in neuron viability.Compared with the control group, neurons exposed to100μM ketamine for24h exhibited a significant decrease in neuron viability (P<0.01). Toinvestigate the possible neuroprotective effects of17β-estradiol againstketamine-induced cell death, we used an MTT assay to study the changes inviability after the neurons were co-incubated with ketamine and differentconcentrations (0.001,0.01,0.1and1μM) of17β-estradiol for24h.Co-incubation with17β-estradiol resulted in a significant andconcentration-dependent enhancement of survival. The maximal rescueoccurred at a17β-estradiol concentration of0.1μM.
3.217β-estradiol treatment reduces neuroapoptosis induced by exposure toketamine.
To determine the amount of apoptosis that occurs after differenttreatments, we carried out TUNEL assays, in the control group, only a fewapoptotic cells were seen, but the number of apoptotic cells increased afterexposure to100μM ketamine for24h (P<0.01). This increase was preventedby the addition of17β-estradiol (P<0.01). To further investigate theantiapoptotic effect of17β-estradiol, we used Hoechst33258staining todetermine the amount of neuron apoptosis, and similar results were obtained.
3.317β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis in brain PFC.
We use immunohistochemical staining for cleaved caspase-3toinvestigate the neuroprotective effects of17β-estradiol against ketamine-induced neuroapoptosis in PFC. Compared to control group, arobust degenerative reaction was detected in PFC of ketamine-treatedgroup(P<0.01). Administration of17β-estradiol only had no influence on theamount of ongoing physiological apoptosis. However, when17β-estradiol wascoadministered with ketamine, it significantly amelioratedneuroapoptosis-induced by ketamine exposure (P<0.01).
3.417β-estradiol improved long-term behavioral deficits in Morris water mazetest
At60day of age, all rats were trained in the Morris water maze to testlearning and memory abilities. Briefly, rats received four training trials dailyfor five consecutive days. All animals showed a progressive decline in theescape latency and path length with training. The escape latency and pathlength of ketamine treatment group were more than the vehicle controls on thethird, fourth and fifth training days (P<0.01), while those of the17β-estradiolin combination with ketamine treatment group were significantly lower thanketamine treatment group (P<0.01). On test day6, rats were subjected to aprobe trial, ratio of time spent in the target quadrant and the number ofcrossings over previous platform locations in ketamine group were fewer thanveichle-control group (P<0.01), while those of17β-estradiol in combinationwith ketamine treatment group were more than ketamine treatment group(P<0.01).
4Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons
4.1Effects of different treatments on pAkt expression
We measured the changes in pAkt in neurons exposed to17β-estradiolalone, pAkt level remained elevated for at least4h when only17β-estradiolwas added to the culture.When17β-estradiol and ketamine were added tocultures simultaneously, p-Akt level remained elevated for at least4h.Wesuspect that17β-estradiol can attenuate the ketamine-induced down-regulationof pAkt by activating the PI3K pathway. To ascertain this, we treated cellswith10μM LY294002, a PI3K inhibitor. Compared with control group, pAkt level decreased when neurons were exposed to ketamine alone for2h(P<0.01). Compared with ketamine-treated group, pAkt increasedsignificantly in cells treated with both ketamine and17β-estradiol for2h(P<0.01), while a1-hr pre-treatment with LY294002abolished the17β-estradiolinduced up-regulation of p-Akt (P<0.01).
4.217β-estradiol enhances Bcl-2expression through the PI3K/Akt pathway
Western blot analysis detected decreased Bcl-2expression in neurons ofthe ketamine group compared with those in the control group (P<0.01).However, Bcl-2expression increased in cortical neurons treated with17β-estradiol after24hr of ketamine exposure (P<0.01). The effect of17β-estradiol treatment was inhibited by LY294002pre-treatment (P<0.01).
4.317β-estradiol prevents apoptosis in ketamine-treated neurons by activatingthe PI3K pathway and blocking caspase-3activity
Compared with control group, the expression of cleaveded caspase-3incells exposed to ketamine for24h increased significantly (P<0.01).Co-incubation of cells with both17β-estradiol and ketamine significantlydecreased the up-regulation of cleaved caspase-3induced by exposure toketamine alone (P<0.01). Consistent with the critical role of PI3K inmediating17β-estradiol induced neuroprotection, the addition of LY294002tothe medium reversed the effect of17β-estradiol on cleaved caspase-3expression in neurons (P<0.01).
4.4Effect of LY294002on neuroprotection of17β-estradiol
Compared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while only LY294002treatment made nodifference with controls (P＞0.05). But, in ketamine+17β-estradiol group,neuron viability was obviously higher than ketamine treatment alone (P<0.01),the addition of LY294002to the medium reversed the effect of17β-estradiolon neuron viability (P<0.01).We used TUNEL assay and Hoechst33258staining to identified neuron apoptosis in different treatment groups.Compared with control group, apoptosis rate increased significantly inketamine-treated group (P<0.01), while ketamine+17β-estradiol treatment group, apoptosis rate was obviously lower than ketamine treatmentalone(P<0.01). The addition of LY294002to the medium reversed the effectof17β-estradiol on neuron apoptosis (P<0.01).
Conclusions
1The inhibitions of ketamine to the immature cortical neurons areconcentration-and time-dependent.
2Ketamine-induced neuron apoptosis is accompanied by increasedlevel of PROG and decreased level of17β-estradiol.
317β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis.
417β-estradiol attenuates ketamine-induced neuroapoptosis and long-term behavioral deficits in developing rats.
517β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis, in part through the PI3K/Akt/signalling pathway,with the increase of Bcl-2expression and the decrease of cleaved-caspase-3expression.
氯胺酮为N-甲基-D-天冬氨酸（N-methyl-D-aspartate, NMDA)受体拮抗剂，被广泛应用于婴幼儿手术的麻醉。NMDA受体是一种离子通道型谷氨酸能受体，具有许多不同变构调控位点并对Ca2+高度通透，在中枢神经系统主要分布在大脑皮层及海马。研究发现NMDA受体与中枢神经系统的发育密切相关，控制神经元的分化、迁移和存活，并在学习、记忆的形成和维持过程中发挥着重要作用。动物出生前后时间阶段是中枢神经系统生长发育的高峰期，有大量神经细胞增殖、大量树突生成和大量突触连接形成，进而形成复杂的神经网络。许多研究已证实在大脑发育的关键时期NMDA受体保持适度兴奋对大脑神经元的存活具有重要的意义，如果改变NMDA受体的活性可能会影响中枢神经系统的发育和功能。
目前大量研究发现氯胺酮可引起发育期动物大脑广泛脑区神经元的凋亡，同时也发现氯胺酮可引起原代培养皮层神经元的大量凋亡。关于氯胺酮引起发育期大脑神经元凋亡与神经功能损害的确切机制目前还不清楚。既往研究表明在大鼠大脑快速发育期注射氯胺酮会导致皮层组织的NMDA受体表达上调，引起神经元兴奋性毒性增加。此外，氯胺酮作为NMDA受体的非竞争性拮抗剂，可以通过作用于NMDA受体PCP结合位点，阻断与NMDA受体耦联的钙通道，减少Ca2+内流，降低细胞内Ca2+浓度，从而拮抗谷氨酸、天冬氨酸等兴奋性氨基酸对神经系统发育的调节。目前有研究认为氯胺酮通过抑制神经元细胞内钙震荡而对发育期神经元产生神经毒性。
针对全身麻醉药引起的发育期大脑损伤，NIH、FDA以及IARS要求研究学者不仅要研究其发生机制而且要寻找安全有效的措施来防治麻醉药所引起的发育期大脑损伤。近年来国内外学者对如何防治氯胺酮引起的发育期大脑损伤进行了一系列研究，结果表明维生素D3、锂剂、促红细胞生成素、肉毒碱、烟酰胺、可乐定等可对氯胺酮引起的发育期神经元损伤产生保护作用，但它们在婴幼儿时期应用的安全性还有待于进一步研究。
神经甾体（neurosteroid）是一类由神经系统的神经元或神经胶质细胞合成、代谢产生的具有神经活性的甾体物质的总称，主要包括：孕烯醇酮（pregnenolone，PREG）、孕烯醇酮硫酸酯（pregnenolone sulfate，PREGS）、脱氢表雄酮（dehydroepiandrosterone，DHEA）、脱氢表雄酮硫酸酯（dehydroepiandrosterone sulfate，DHEAS）、孕酮（progesterone，PROG）、别孕烯醇酮（allopregnanolone，AP）以及雌二醇（estradiol，E2）等。大量研究表明神经甾体不仅参与调节神经系统的发育并对神经系统的功能产生广泛影响，对神经系统还具有营养保护以及促进神经发生和神经元存活的作用。
雌二醇作为一种内源性神经甾体，在神经元发育过程中发挥重要的调节作用，促进神经元的存活以及功能维护。最近有研究表明氯胺酮可引起斑马鱼发育期17β雌二醇水平的下降，睾酮水平升高，同时还有研究表明氯胺酮对发育期斑马鱼具有神经损伤作用。PI3K-Akt信号通路是介导多种生长因子促进细胞存活的重要通路，有研究表明氯胺酮通过抑制NMDA受体的活性进而抑制pAkt的表达对原代培养的皮层神经元产生损伤。有研究表明17β雌二醇可通过激活PI3K-Akt信号通路对NMDA受体拮抗剂MK-801以及麻醉药咪达唑仑、笑气、异氟醚联合应用所引起的发育期大鼠大脑广泛脑区神经元凋亡产生保护作用。另外Ramezani等研究表明17β雌二醇可对酒精引起的发育期大鼠大脑神经损伤以及远期行为学异常产生保护作用。本实验室前期研究发现Aβ25-35对原代培养皮层神经元产生损伤时伴随神经甾体代谢通路的变化。氯胺酮引起发育期皮层神经元损伤的同时是否伴随神经甾体合成代谢的变化，以及神经活性甾体17β雌二醇是否通过PI3K-Akt信号通路对氯胺酮引起的皮层神经元损伤产生保护作用，国内外未见报道。
本课题首先以原代培养的大鼠皮层神经元为研究对象采用不同浓度的氯胺酮建立氯胺酮体外诱导皮层神经元损伤的细胞模型。在该模型的基础上采用液液萃取结合高效液相色谱-质谱联用（HPLC-MS/MS）方法对氯胺酮引起皮层神经元损伤时主要神经甾体PREG、PROG以及17β雌二醇合成代谢水平进行了研究。随后以氯胺酮损伤皮层神经元时合成代谢水平明显降低的17β雌二醇进行外源性给药，从离体细胞和在体动物两方面研究17β雌二醇对氯胺酮诱导的原代培养皮层神经元损伤以及发育期大鼠皮层区神经细胞损伤的保护作用。最后应用原代培养皮层神经元进一步研究17β雌二醇是否通过PI3K-Akt信号通路保护发育期皮层神经元免受氯胺酮的损伤。本实验将为神经活性甾体17β雌二醇防治氯胺酮引起的发育期大脑损伤提供理论依据及实验基础。
本研究采用的试验方法包括：
1大鼠大脑皮层神经元的原代培养
大鼠大脑皮层神经元进行原代培养7天用于实验。倒置显微镜下观察皮层神经元的形态学变化。
2分组与药物处理
①观察氯胺酮对神经元的损伤作用时分为：不同浓度氯胺酮（0、1、10、100、1000μΜ）作用神经元24h100μΜ氯胺酮作用神经元不同时间（0、6、12、24、48h）
②观察氯胺酮对神经元神经甾体合成的影响分为：对照组、氯胺酮组、胆固醇组、氯胺酮+胆固醇组
③观察17β雌二醇对氯胺酮诱导皮层神经元凋亡的影响分为：对照组，氯胺酮组，17β雌二醇+氯胺酮组
④观察17β雌二醇对氯胺酮引起发育期大鼠大脑皮层区神经细胞凋亡和远期学习记忆损伤的影响分为：对照组，溶剂对照组，17β雌二醇组，氯胺酮组，17β雌二醇+氯胺酮组
⑤研究17β雌二醇保护原代培养皮层神经元免受氯胺酮损伤的机制分为：
对照组，氯胺酮组，17β雌二醇+氯胺酮组，17β雌二醇+氯胺酮+LY294002组
3细胞样品的收集与蛋白定量
给予各种药物处理的皮层神经元，加入细胞裂解液，低温离心收集上清，应用多功能酶标仪测定蛋白质浓度。
4MTT法测定皮层神经元的存活率
5Hoechst33258核染色法检测神经元凋亡
在荧光显微镜下，随机选取5个视野进行形态学观察和计数，计算凋亡率，凋亡率=（凋亡细胞数/细胞总数）×100%。
6TUNEL法检测原代培养皮层神经元凋亡
细胞爬片于倒置显微镜下任意选取5个视野观察阳性细胞并计数，计算神经元的凋亡率，凋亡率=（凋亡细胞数/细胞总数）×100%。
7免疫组织化学染色分析大脑皮层区cleaved-caspase-3表达：
以细胞中出现棕褐色浓染颗粒作为阳性细胞的判定标准。在光镜下观察皮层区的凋亡神经细胞，以个/0.01mm2计数。
8Western-blot法检测p-Akt、Akt、cleaved-caspase-3、Bcl-2的蛋白表达
9HPLC-MS/MS测定细胞培养液中主要神经甾体的浓度
10Morris水迷宫行为学测试
11统计学处理
各组数据均以均数±标准差（±s）表示，应用SPSS13.0统计软件进行处理，水迷宫行为学测试中定位航行实验数据采用重复测量方差分析进行统计学处理，其他数据采用单因素方差分析（one way ANOVA）继以LSD post hoc test进行统计学处理,以P<0.05为差异有统计学意义。
结果：
1氯胺酮对原代培养皮层神经元的损伤作用
1.1氯胺酮呈浓度和时间依赖性降低原代培养皮层神经元存活率
不同浓度的氯胺酮作用于体外培养7天的皮层神经元24h后，神经元存活率下降，其中100、1000μΜ的氯胺酮使神经元存活率显著下降（P<0.01，P<0.01）。100μΜ氯胺酮作用于神经元不同时间后神经元存活率下降，其中作用24h及48h后，神经元存活率显著下降（P<0.05，P<0.01）。
1.2氯胺酮损伤大鼠皮层神经元时的细胞形态变化
倒置显微镜下观察刚种植的皮层神经元呈圆形，单个均匀分布，折光性强，胞体透亮，接种8h后细胞贴壁，24h后细胞贴壁牢固并可见短小的突起，胞体呈梭型或雏形，第三天后神经元胞体进一步增大，部分细胞突起延长，神经元培养至第7天，胞体进一步增大，多呈梭型或雏形，胞体饱满，部分神经元集聚成团，突起增多并延长，相互交织形成网络。培养7天的神经元经100μΜ氯胺酮处理24h后，神经元细胞数量较对照组明显减少，折光性差，胞体明显缩小，轴突断裂，部分神经元细胞膜破裂成细胞碎片。
1.3氯胺酮对原代培养皮层神经元凋亡的影响
TUNEL结果显示，对照组仅见少量的TUNEL阳性神经元，100μΜ氯胺酮组TUNEL阳性神经元较对照组显著增加（P<0.01）。Hoechst33258荧光染色结果显示，对照组细胞核内呈均匀分布的淡蓝色荧光，内有较深的蓝色颗粒，有少量凋亡细胞呈亮蓝色。100μΜ氯胺酮组皮层神经元染色质高度凝聚、边缘化，细胞核甚至裂解为碎块，有大量凋亡细胞呈亮蓝色(P<0.01)。
2氯胺酮对原代培养皮层神经元神经甾体水平的影响
2.1不同处理对皮层神经元存活率的影响
MTT结果显示，与对照组比较，氯胺酮组皮层神经元的存活率显著下降（P<0.01），胆固醇单纯处理组神经元存活率未见显著改变（P>0.05）。与氯胺酮组比较，胆固醇与氯胺酮共处理组神经元存活率显著升高（P<0.01）。
2.2不同处理对神经元凋亡的影响
TUNEL结果显示，与对照组比较，胆固醇单纯处理组TUNEL阳性神经元未见显著变化（P＞0.05），氯胺酮组TUNEL阳性神经元显著增加（P<0.01）。与氯胺酮比较，胆固醇与氯胺酮共处理组TUNEL阳性神经元显著降低（P<0.01）；Hoechst33258荧光染色结果进一步证实了以上变化。
2.3氯胺酮对皮层神经元神经甾体水平的影响
对照组及氯胺酮处理组因未加入神经甾体合成所必需的底物胆固醇，均未测得各种神经甾体。与胆固醇单纯处理组相比，氯胺酮共处理后神经甾体PREG水平未见显著变化，而17β雌二醇水平显著下降(P<0.01)，PROG水平显著升高（P＞0.05）。
317β雌二醇对氯胺酮诱导的发育期皮层神经元凋亡以及发育期大鼠远期学习记忆损伤的影响
3.117β雌二醇对氯胺酮导致的原代培养皮层神经元损伤的影响
MTT结果显示，与对照组比较,氯胺酮组原代培养皮层神经元存活率显著下降（P<0.01）；与氯胺酮组比较，不同浓度（0.001、0.01、0.1、1μΜ）的17β雌二醇与氯胺酮共处理24h后神经元的存活率显著升高，其存活率分别为(54.8±5.1)%，(75.1±9.3)%，(88.3±8.5)%，(79.5±10.3)%，其中0.1μM的17β雌二醇保护效果最好。
3.217β雌二醇减轻氯胺酮诱导的原代培养皮层神经元凋亡
TUNEL结果显示，对照组可见少量的凋亡神经元，氯胺酮组神经元凋亡较对照组显著增加（P<0.01），0.1μM的17β雌二醇使氯胺酮导致的神经元凋亡显著降低（P<0.01）。Hoechst33258核染色法进一步证实了17β雌二醇减轻氯胺酮诱导的皮层神经元凋亡。对照组细胞核多呈弥散均匀分布的淡蓝色荧光，内有较深的蓝色颗粒，有少量的凋亡细胞呈亮蓝色。氯胺酮组凋亡细胞较对照组显著增加(P<0.01)，17β雌二醇与氯胺酮共处理组神经元凋亡较氯胺酮组显著降低（P<0.01）。
3.3不同处理对发育期大鼠大脑额叶皮层区cleaved-caspase-3表达的影响
与对照组比较，溶剂对照组和17β雌二醇组cleaved-caspase-3阳性表达细胞数/0.01mm2未见统计学差异（均为P>0.05），氯胺酮组阳性表达显著增加（P<0.01）。与氯胺酮组比较，17β雌二醇与氯胺酮共处理组阳性表达显著降低（P<0.01）。
3.4Morris水迷宫测试SD大鼠学习记忆能力
SD幼鼠饲养至60天时，进行Morris水迷宫5天训练，一天四次。把SD大鼠逃逸潜伏期（s）和游泳距离（cm）作为学习能力的指标。在5天的训练期中，前2天实验各组逃逸潜伏期和游泳距离未见统计学差异。与对照组比较，氯胺酮组第3天、第4天、第5天逃逸潜伏期和游泳距离均显著增加（均为P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组逃逸潜伏期和游泳距离均显著降低（均为P<0.01）；第6天撤去平台，进行60s空间探索试验作为对记忆能力的评估。与对照组比较，氯胺酮组穿越平台的次数和靶象限的时间比率均显著减少（均为P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组穿越平台的次数和靶象限的时间比率均显著增加（均为P<0.01）。
417β雌二醇保护原代培养皮层神经元免受氯胺酮损伤的机制研究
4.1不同处理对原代培养皮层神经元pAkt表达水平的影响
结果显示，与0h相比，0.1μM的17β雌二醇单独处理皮层神经元0.5、1、2、4h, pAkt表达水平时间依赖性显著升高（P<0.01）；17β雌二醇与氯胺酮共处理皮层神经元0.5、1、2、4h， pAkt表达水平随时间延长显著升高（P<0.01）。为了进一步证实17β雌二醇通过PI3K-Akt信号通路对氯胺酮引起的皮层神经元损伤产生保护作用。给予PI3K抑制剂LY294002，观察对皮层神经元pAkt表达水平的影响。与对照组比较，17β雌二醇培养皮层神经元2h引起pAkt表达水平的显著升高（P<0.01）,氯胺酮培养皮层神经元2h引起pAkt表达水平的显著降低（P<0.01）。与氯胺酮组比较，17β雌二醇与氯胺酮共培养2h pAkt表达水平显著升高（P<0.01），加入PI3K抑制剂LY294002后培养2h，LY294002抑制了17β雌二醇对pAkt表达水平的上调作用（P<0.01）。
4.217β雌二醇对原代培养皮层神经元Bcl-2表达水平的影响
Bcl-2作为一种抗凋亡分子，对多种损伤产生保护作用。Akt可通过激活CREB磷酸化，提高Bcl-2的表达水平，从而对各种应激提供神经保护作用。为了探讨17β雌二醇是否有可能通过PI3K-Akt信号通路影响Bcl-2的表达水平对氯胺酮引起的神经元损伤产生保护作用，我们将皮层神经元给予100μM氯胺酮和（或）0.1μM的17β雌二醇以及10μMLY294002处理24h，结果显示与对照组比较，氯胺酮组Bcl-2表达水平显著下降（P<0.01）；与氯胺酮组比较，17β雌二醇与氯胺酮共处理组Bcl-2表达水平显著增加（P<0.01）；与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组Bcl-2表达水平显著下降（P<0.01）。
4.317β雌二醇对原代培养皮层神经元cleaved-caspase-3表达水平的影响
Caspase-3是各种细胞凋亡通路的最终执行者。为了探讨17β雌二醇是否有可能通过PI3K-Akt信号通路影响cleaved-caspase-3的表达水平对氯胺酮引起的神经元损伤产生保护作用，我们将皮层神经元给予100μM氯胺酮和（或）0.1μM的17β雌二醇以及10μM LY294002处理24h，结果显示与对照组比较，氯胺酮组cleaved-caspase-3表达水平显著增加（P<0.01）；与氯胺酮组比较，17β雌二醇与氯胺酮共处理组cleaved-caspase-3表达水平显著下降（P<0.01）；与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组cleaved-caspase-3表达水平显著增加（P<0.01）。
4.4LY294002对17β雌二醇神经保护作用的影响
MTT结果显示,与对照组比较，氯胺酮组皮层神经元存活率显著降低（P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组神经元存活率显著增加（P<0.01），与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组皮层神经元存活率显著降低（P<0.01）。TUNEL结果显示对照组可见少量的凋亡神经元，与对照组比较，氯胺酮组神经元凋亡显著增加（P<0.01），与氯胺酮组比较，17β雌二醇与氯胺酮共处理组神经元凋亡显著降低（P<0.01），与17β雌二醇与氯胺酮共处理组比较，LY294002预处理组神经元凋亡显著增加（P<0.01）。Hoechst33258核染色法进一步证实了TUNEL试验所见。
结论：
1氯胺酮能浓度和时间依赖性的引起原代培养皮层神经元损伤。
2氯胺酮在导致原代培养皮层神经元损伤的同时伴随某些神经甾体水平的变化，其中PROG水平显著升高，17β雌二醇水平显著降低。
317β雌二醇对氯胺酮引起的原代培养皮层神经元损伤具有保护作用。
4外源系统性给予17β雌二醇对氯胺酮引起的发育期大鼠皮层区神经细胞凋亡以及成年后学习记忆损伤产生保护作用。
5激活PI3K-Akt信号通路，促进抗凋亡分子Bcl-2的表达，抑制促凋亡分子cleaved-caspase-3的表达可能是17β雌二醇对氯胺酮引起的皮层神经元损伤产生保护作用的机制之一。
Globally, there were about1.5million infants and young childrenundergoing general anesthesia because of various reasons such as surgery orinterventional therapy each year. For a long time, whether or not generalanesthesia would affect the development of infant's brain has been the focus ofattention of family members and anesthesiologists. The conventional belief isthat, as long as there was no cerebral hypoxia or other relevant consequencesduring the anesthesia, it would not affect the brain development of infants andyoung children. However, neuronal cell death after general anesthesia hasrecently been demonstrated in neonatal animal models. The possibility ofanesthesia-induced neurotoxicity in human neonates or infants has led toserious questions about the safety of pediatric anesthesia.
Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, iswidely used in surgical anesthesia of infants and young children now. TheNMDA receptor is a kind of central nervous system excitatory amino acidionotropic receptors, with many different allosteric regulatory sites and Ca2+high degree of permeability. In the central nervous system, NMDA receptorsare mainly distributed in the cerebral cortex and hippocampus. Studies havefound that the NMDA receptor is closely related to the development of centralnervous system, controlling neuronal differentiation, migration and survival,and playing an important role in the formation and maintenance of learningand memory. Recent studies have found that developing neurons can beinduced to activate pathways resulting in cell death at the peak of the centralnervous system growth and development, when their synaptic development,dendritic branching, and remodeling activities are interfered with. Manystudies have confirmed that it is of great significance for the NMDA receptorto maintain moderate excitement for the survival of neurons in the critical period of brain development. If the activity of NMDA receptors was changed,it would affect the development and function of the central nervous system.
An increasing number of animal studies have suggested that ketaminecauses neurodegeneration during early development. Studies in vitro have alsoshown that ketamine administration induces apoptosis in cultured neurons.The exact mechanism triggering ketamine-induced apoptosis has not yet beenfully elucidated by now. Previous studies have shown that injection ofketamine could cause up-regulation of the NMDA receptor in the rat brainduring its developmental stages, resulting in a wide range of dose-dependentapoptosis. In addition, as a noncompetitive antagonist of the NMDA receptor,ketamine can act on PCP binding sites of the NMDA receptor, blocking theNMDA receptor coupled calcium channels, reducing calcium influx, andreducing the concentration of intracellular calcium. This can antagonize theregulation of nervous system development by glutamate, aspartate and otherexcitatory amino acids. Recent studies showed that ketamine inducedneuroapoptosis by inhibiting Ca2+oscillations in neurons.
With respect to anesthesia-induced brain injury in neonates, the NIH, theFDA, and the International Anesthesiology Research Society (IARS) haveurged researchers not only to define the scope of the problem but also todevelop therapeutic strategies that target prevention. Although many strategies,including the use of activity-dependent neuroprotective protein peptidefragment NAPVSIPQ (NAP), vitamin D, lithium, erythropoietin, L-carnitine,nicotinamide, and Clonidine, are known to have neuroprotective effectsagainst ketamine-induced apoptosis both in vivo and in vitro, their efficacyand safety need to be further verified for uses targeting the developing brain.
Steroids hormones and their metabolites within the central nervous system(CNS) are commonly defined as neuroactive steroids or neurosteroids. Theycan be synthesized de novo in the CNS by glial cells and neurons. Neuroactivesteroids mainly include pregnenolone (PREG), dehydroepiandrosterone(DHEA), their sulfate derivatives pregnenolone sulfate (PREGS) anddehydroepiandrosterone sulfate (DHEAS), and progesterone (PROG), 5α-dihydroprogesterone (5α-DHP),3α,5α-tetrahydroprogesterone(allopregnanolone/AP), deoxycorticosterone (DOC),tetrahydrodeoxycorticosterone (THDOC), estradiol (E2), etc. The studyshowed that neurosteroids played an important role as endogenous modulatorsin the development of CNS and exerted neuroprotection.
Estradiol is a kind of neurosteroids, which has the potential for promotingsurvival and function of neurons. Recently, Trickler et al reported thatketamine attenuated17β-estradiol level and increased testosterone level andketamine had been shown to be neurotoxic in early life stages of zebrafish.PI3K-Akt signaling is very important for promoting neuronal survival.Ketamine induces apoptosis by blocking NMDA receptor activity anddecreasing levels of p-AKT. Previous studies reported that estradiol treatmentof neonatal rats inhibited neuroapoptosis induced by the NMDAR antagonistMK-801and anesthetic cocktails including midazolam, isoflurane, and nitrousoxide. More recently, Ramezani and colleagues suggested that17β-estradiolcould protect the developing rat cerebellum from ethanol-inducedneurotoxicity and improve behavioral deficits. The results from our laboratoryhave shown that Aβ25-35induces apoptosis in primary cultured cortical neuronsaccompanying the change of the methabolism of neurosteroids. However,whether ketamine induces apoptosis in primary cultured cortical neuronsaccompanied by the change of the methabolism of neurosteroids and whether17β-estradiol can protect rat cortical neurons from ketamine-induced apoptosisremain relatively elusive. In our study, an in vitro ketamine-induced cellapoptosis model was established based the primary cultured rat corticalneurons. Based on this model, the HPLC-MS/MS assay was applied toinvestigate the changes of chief neurosteroids, PREG, PROG and17β-estradiol levels in cultured neurons induced by ketamine cytotoxicity.Afterwards,17β-estradiol, whose level found obviously decreased byketamine treatment, was chosen as intervening substances to investigateitsprotective effects against ketamine-induced neuroapoptosis. Then whetherPI3K-Akt signaling takes part in neuroptotection of17β-estradiol against ketamine-induced neuroapoptosis was studied. The study is to add valuableefforts for the protective effects of17β-estradiol against ketamine-inducedneuroapoptosis.
The following methods were applied in this thesis:
1Cortical neurons of newborn SD rat were cultured.Cortical neurons were grown for7days before experimentation. Invertedmicroscope was used to observe morphological changes of cultured neurons.
2Grouping and drug treatments
①Ketamine induces injuries in primary cultured cortical neurons:different concentrations of ketamine treatment for24h100μM ketamine treatment for different time
②Effect of ketamine on neurosteroidogenesis in primary rat corticalneurons:control, ketamine, cholesterol, cholesterol+ketamine
③Effects of17β-estradiol treatment on neuroapoptosis induced byketamine exposure:control, ketamine,17β-estradiol+ketamine
④17β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis and long-term behaviour deficts in developing brain:control, veichle-control,17β-estradiol, ketamine,17β-estradiol+ketamine
⑤Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons:control, ketamine,17β-estradiol+ketamine,17β-estradiol+ketamine+LY294002
3Sampling and quantitation
Treated neurons were collected, after cell lysis, supernatant proteinconcentrations were quantitatived by multifunctional enzyme markinstrument.
4Neuron viability assay
5Apopyosis of neurons indentified by Hoechst233258dying
Apoptosis were identified by nuclear morphometry under fluorescencemicroscope. The apoptosis was calculated by5randomly selected eye fields.
6Apoptotic neurons were detected using the TUNEL assay
Apoptotic neurons were identified by TUNEL assay under invertedicroscopy. The apoptosis was calculated by5randomly selected eye fields.
7Immunohistochemical Staining for cleaved caspase-3
Apoptotic cells were identified by immunohistochemical Staining forcleaved caspase-3under inverted icroscopy. The apoptotic cells (per0.01mm2)in the prefrontal cortex were counted
8Western Blot Analysis for p-Akt, Akt, cleaved-caspase-3, Bcl-2expression
9Determination of the concentrations of neurosteroids in culture mediausing HPLC-MS/MS assay
10Morris water maze test
11Statistical Analysis
The data were expressed as mean±standard deviation (SD). The meanescape latency of the Morris water maze behavioral tests collected during thetraining days were analyzed by repeated-measure analysis of variance(ANOVA). Other data were statistically analyzed by one-way ANOVA,followed by between-group comparisons using LSD post hoc test. Statisticalsignificance was concluded with a value of P<0.05for all analyses. Dataanalyses were performed with the SPSS software version13.0
Results:
1Ketamine induces injuries in primary cultured cortical neurons
1.1Ketamine decreased neuron viability dose and time dependently
The MTT assay was used to evaluate the viability of neurons. Ketaminedecreased cell viability dose and time dependently. Compared withvehicle-control group, the neuron viability of ketamine treated group (24h)significantly (P<0.01for ketamine100μM and1000μM) decreased.Compared with control group, the neuron viability of100μM ketaminesignificantly decreased (P<0.05for24h, P<0.01for48h).
1.2Morphoolgical alteration of neurons
The newly inoculated cortical neurons were round, small, translucent,and in a single suspension uniform distribution. After8h, the cells started toadhere to the culture plates, and most of neurons had adhered in24h. Theirmorphologies were fusiform, triangular, with different length of slenderprotrusions. On the3rd day, the cells were well-rounded and full, and theprominences were elongated and enlarged more significantly and connected tothe network, sparse connections had come into being. Cortical neuronscultured for7days, were generally bipolar or multipolar cells under invertedmicroscopy, most of them with triangular soma,and usually aggregated withtheir branching dendrites interconnected as network. Compared with controlgroup,24h after100μM ketamine treatment, numbers of neurons decreasedobviously, with reduced refraction, axons break, and granuliform pieces.
1.3Ketamine influences on neuroapoptosis identified by TUNEL assay andHoechst33258staining.
Compared with control group, ketamine treatment increased neuronsapoptosis signficantly (P<0.01).
2Effect of ketamine on neurosteroidogenesis in primary rat cortical neurons
2.1Influence on neurons viability
Compared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while cholesterol treatment made nodifference with controls (P>0.05). But, in ketamine+cholesterol group, neuronviability was obviously higher than ketamine treatment alone (P<0.01)
2.2Influence on neuron apoptosis
We use TUNEL assay and Hoechst33258staining to identified neuronapoptosis in different treatment group. Compared with control group,apoptosis rate increased significantly in ketamine-treated group (P<0.01),while cholesterol treatment made no difference with controls (P>0.05). But, inketamine+cholesterol group, apoptosis rate was obviously lower thanketamine treatment alone (P<0.01).
2.3Influence on neurosteroidgenesis
Absence of the necessary substance, cholesterol, no neurosteroids found in control and ketamine alone groups. Compared with cholesterol treatmentalone, no changes in PREG level were observed after treatment with ketamine,while PROG increased significantly (P<0.05) and17β-estradiol decreasedgreatly (P<0.01).
3Effects of17β-estradiol treatment on neuroapoptosis induced by ketamineexposure
3.1Protective effects of17β-estradiol against ketamine-induced cell death incultured cortical neurons.
We used the MTT assay to assess the changes in neuron viability.Compared with the control group, neurons exposed to100μM ketamine for24h exhibited a significant decrease in neuron viability (P<0.01). Toinvestigate the possible neuroprotective effects of17β-estradiol againstketamine-induced cell death, we used an MTT assay to study the changes inviability after the neurons were co-incubated with ketamine and differentconcentrations (0.001,0.01,0.1and1μM) of17β-estradiol for24h.Co-incubation with17β-estradiol resulted in a significant andconcentration-dependent enhancement of survival. The maximal rescueoccurred at a17β-estradiol concentration of0.1μM.
3.217β-estradiol treatment reduces neuroapoptosis induced by exposure toketamine.
To determine the amount of apoptosis that occurs after differenttreatments, we carried out TUNEL assays, in the control group, only a fewapoptotic cells were seen, but the number of apoptotic cells increased afterexposure to100μM ketamine for24h (P<0.01). This increase was preventedby the addition of17β-estradiol (P<0.01). To further investigate theantiapoptotic effect of17β-estradiol, we used Hoechst33258staining todetermine the amount of neuron apoptosis, and similar results were obtained.
3.317β-estradiol exerts neuroprotective effects against ketamine-inducedneuroapoptosis in brain PFC.
We use immunohistochemical staining for cleaved caspase-3toinvestigate the neuroprotective effects of17β-estradiol against ketamine-induced neuroapoptosis in PFC. Compared to control group, arobust degenerative reaction was detected in PFC of ketamine-treatedgroup(P<0.01). Administration of17β-estradiol only had no influence on theamount of ongoing physiological apoptosis. However, when17β-estradiol wascoadministered with ketamine, it significantly amelioratedneuroapoptosis-induced by ketamine exposure (P<0.01).
3.417β-estradiol improved long-term behavioral deficits in Morris water mazetest
At60day of age, all rats were trained in the Morris water maze to testlearning and memory abilities. Briefly, rats received four training trials dailyfor five consecutive days. All animals showed a progressive decline in theescape latency and path length with training. The escape latency and pathlength of ketamine treatment group were more than the vehicle controls on thethird, fourth and fifth training days (P<0.01), while those of the17β-estradiolin combination with ketamine treatment group were significantly lower thanketamine treatment group (P<0.01). On test day6, rats were subjected to aprobe trial, ratio of time spent in the target quadrant and the number ofcrossings over previous platform locations in ketamine group were fewer thanveichle-control group (P<0.01), while those of17β-estradiol in combinationwith ketamine treatment group were more than ketamine treatment group(P<0.01).
4Study on the mechanism of17β-estradiol against ketamine-inducedneuroapoptosis of the developing cortical neurons
4.1Effects of different treatments on pAkt expression
We measured the changes in pAkt in neurons exposed to17β-estradiolalone, pAkt level remained elevated for at least4h when only17β-estradiolwas added to the culture.When17β-estradiol and ketamine were added tocultures simultaneously, p-Akt level remained elevated for at least4h.Wesuspect that17β-estradiol can attenuate the ketamine-induced down-regulationof pAkt by activating the PI3K pathway. To ascertain this, we treated cellswith10μM LY294002, a PI3K inhibitor. Compared with control group, pAkt level decreased when neurons were exposed to ketamine alone for2h(P<0.01). Compared with ketamine-treated group, pAkt increasedsignificantly in cells treated with both ketamine and17β-estradiol for2h(P<0.01), while a1-hr pre-treatment with LY294002abolished the17β-estradiolinduced up-regulation of p-Akt (P<0.01).
4.217β-estradiol enhances Bcl-2expression through the PI3K/Akt pathway
Western blot analysis detected decreased Bcl-2expression in neurons ofthe ketamine group compared with those in the control group (P<0.01).However, Bcl-2expression increased in cortical neurons treated with17β-estradiol after24hr of ketamine exposure (P<0.01). The effect of17β-estradiol treatment was inhibited by LY294002pre-treatment (P<0.01).
4.317β-estradiol prevents apoptosis in ketamine-treated neurons by activatingthe PI3K pathway and blocking caspase-3activity
Compared with control group, the expression of cleaveded caspase-3incells exposed to ketamine for24h increased significantly (P<0.01).Co-incubation of cells with both17β-estradiol and ketamine significantlydecreased the up-regulation of cleaved caspase-3induced by exposure toketamine alone (P<0.01). Consistent with the critical role of PI3K inmediating17β-estradiol induced neuroprotection, the addition of LY294002tothe medium reversed the effect of17β-estradiol on cleaved caspase-3expression in neurons (P<0.01).
4.4Effect of LY294002on neuroprotection of17β-estradiol
Compared with control group, neuron viability decreased significantly inketamine-treated group (P<0.01), while only LY294002treatment made nodifference with controls (P＞0.05). But, in ketamine+17β-estradiol group,neuron viability was obviously higher than ketamine treatment alone (P<0.01),the addition of LY294002to the medium reversed the effect of17β-estradiolon neuron viability (P<0.01).We used TUNEL assay and Hoechst33258staining to identified neuron apoptosis in different treatment groups.Compared with control group, apoptosis rate increased significantly inketamine-treated group (P<0.01), while ketamine+17β-estradiol treatment group, apoptosis rate was obviously lower than ketamine treatmentalone(P<0.01). The addition of LY294002to the medium reversed the effectof17β-estradiol on neuron apoptosis (P<0.01).
Conclusions
1The inhibitions of ketamine to the immature cortical neurons areconcentration-and time-dependent.
2Ketamine-induced neuron apoptosis is accompanied by increasedlevel of PROG and decreased level of17β-estradiol.
317β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis.
417β-estradiol attenuates ketamine-induced neuroapoptosis and long-term behavioral deficits in developing rats.
517β-estradiol protects primary cultured rat cortical neurons fromketamine-induced apoptosis, in part through the PI3K/Akt/signalling pathway,with the increase of Bcl-2expression and the decrease of cleaved-caspase-3expression.
引文
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