罗哌卡因致幼鼠中枢神经毒性惊厥对其突触可塑性及学习记忆的影响
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摘要
局麻药中枢神经毒性惊厥系其全身毒性的常见毒副反应。由于婴幼儿特殊的解剖和发育特点,局麻药中枢神经毒性惊厥在婴幼儿中更为常见。有研究证实在幼年时期经历长程或反复惊厥,对其中枢神经突触可塑性及学习记忆有影响。
婴幼儿或幼年动物神经系统发育处于突触爆发期,是神经细胞快速分化、迁徙、树突快速形成神经网络的关键时期,此期对各种伤害性刺激抵御能力差易对其中枢神经系统生理发育和功能形成产生影响。众多研究报道其它非局麻药因素致发育期反复或长程惊厥可导致动物成年认知功能和学习记忆能力下降,其机制与离子通道、氨基酸受体、神经递质、突触发育及学习i记忆相关的蛋白质合成、基因表达、信号转导有关。局麻药致发育期婴幼儿中枢神经系统毒性惊厥,是否会对其神经系统发育造成损害,对其学习记忆能力的形成是否有影响尚无研究定论。
早在上世纪20年代已有关于局麻药中枢神经毒性的相关报道。此后麻醉领域学者们展开了大量关其机制和防治的研究。局麻药中枢神经毒性以惊厥发作为主要临床表现,其机制主要与兴奋性—抑制性氨基酸学说、NMDA-Ca2+-NOS通路学说、多巴胺、乙酰胆碱、M型钾通道等有关。而以上受体、神经递质、及离子通道等均不同程度参与中枢神经系统神经突触发育及学习记忆能力的形成。海马作为与学习记忆最为相关的解剖部位,也是局麻药中枢神经毒性惊厥的发生形成关键部位。。
学习记忆是大脑的高级神经功能。学习和记忆是两个相互联系和影响的神经活动过程,学习是指人或生物依赖经验来改变自身环境的神经活动过程,而记忆则是将通过学习得到的信息储存和输出的一种神经活动。学习记忆的神经基础是中枢神经系统的高度可塑性,包括神经网络连接形态及与神经突触发育相关的各种神经递质、蛋白、离子通道、受体等多种不同层次不同阶段的可塑性。而其中突触连接是神经可塑性的关键部位,也是实现神经元之间传递信息、实现生理功能的关键部位。
突触可塑性是指在外环境改变时,神经元突触发生适应性改变从而维持其相对稳定的状态,包括形态结构和功能上的改变。研究认为在反复或长程惊厥后,海马突触形态结构遭到不同程度的破坏,从而影响学习记忆的维持和神经功能的发挥,其破坏程度与学习记忆功能的减退呈正相关。以往局麻药中枢神经毒性研究发现,局麻药抑制突触轴突生长,并减弱神经冲动传导,从而削弱了突触维持正常生理功能的能力。突触素是一种钙结合糖蛋白,特异性位于轴突末梢的突触前膜上。突触素参与谷氨酸、乙酰胆碱等神经递质的释放过程,与突触可塑性关系密切。突触素作为突触囊泡的一种特异性标志蛋白,其密度和分布可间接反应单位体积内突触数量和分布情况,与突触重建及认知过程密切相关。另一方面突触素调节神经元的发生、发育及成熟,还可通过调节细胞骨架蛋白组装而影响神经元的分化。突触素在神经轴突延伸、突触连接形成和突触成熟中具有重要的意义。
钙/钙调素依赖性蛋白激酶Ⅱ(Calcium/calmodulin dependent protein kinase Ⅱ, CaMK Ⅱ) CaMK Ⅱ是海马内含量最高的一种蛋白质,是钙离子的主要结合蛋白,且与突触长时程增强密切相关,有“学习记忆分子开关”之称。环磷酸腺苷反应单元结合蛋白(cAMP-responsive element binding protein,CREB) CREB是一种真核生物细胞核内转录转导因子,具有调节基因转录的功能,是细胞内多种信号转导通路的关键成分之一。研究发现CREB在长期记忆的维持中起关键作用。在以往对可卡因急性或反复应用研究中发现,可卡因通过影响CREB磷酸化,对学习记忆产生影响。Ca2+-CaMK Ⅱ-CREB通路是中枢神经系统突触可塑性和学习记忆能力形成与维持的主要信号通路之一。以往研究发现,利多卡因、可卡因、普鲁卡因抑制海马长时程增强,与影响钙调素有关。局麻药中枢神经毒性惊厥时引发钙超载,严重钙超载与CaMK Ⅱ、CREB之间影响如何?是否会对突触可塑性和学习记忆产生影响目前尚不明了
本课题通过测定0.5%罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥ED95值,分别制备幼鼠单次惊厥和反复惊厥实验模型。应用Morris水迷宫测定实验鼠惊厥后近期及成年后学习i记忆能力变化；采用透射电镜观察中枢神经毒性惊厥后实验鼠在不同时间海马突触超微结构变化；以免疫组织化学染色和Western Blot免疫印迹技术观察海马CA1区突触素蛋白的表达；应用Western Blot和荧光实时定量PCR技术测定CaMK Ⅱ和p-CREB蛋白和基因表达水平变化。本研究从行为学、形态学、蛋白和基因水平检测局麻药致发育鼠中枢神经毒性惊厥对其突触可塑性及学习记忆的影响,并探讨和研究其可能涉及的机制。
目的
采用序贯法(up-down method)结合单位概率回归法(probit法)测定SD幼鼠(P21日龄)清醒状态下,0.5%罗哌卡因腹腔注射致其惊厥ED50及ED95。
方法
选用健康Sprague Dawley(SD)P21日龄幼鼠40只。用生理盐水配置浓度为0.5%盐酸罗哌卡因备用。罗哌卡因腹腔注射剂量分为5组,起始剂量为24mg/kg,相邻剂量间隔等比系数为0.8。其余剂量组依次为15.4mg/kg、19.2mg/kg、30mg/kg、37.5mg/kg。
序贯法操作：第一只实验鼠腹腔注射剂量为24mg/kg,如出现惊厥则下一只实验鼠腹腔注射剂量下调至较低相邻剂量,即19.2mg/kg,如未出现惊厥则下一只实验鼠腹腔注射剂量上调至较高相邻剂量,即30mg/kg,依次类推,直至所有动物均接受一次注射。统计每个剂量组阳性例数、阴性例数及总例数。记录发生惊厥实验鼠惊厥潜伏时间及持续时间。
实验鼠行为依照Racine分级法进行分级,0级：无任何反应,呈常态表现；Ⅰ级：点头样抖动、面部肌肉痉挛表现如节律性眨眼、咀嚼、胡须抖动等；Ⅱ级：痉挛性表现,点头或甩尾；Ⅲ级：一侧前肢体阵挛性抖动；Ⅳ级：双侧前肢体阵挛,但仍可站立；V级：全身阵挛性发作,失去平衡,无法站立,翻正不能。
纳入标准：鼠发生惊厥以Racine分类V级(翻正不能、直至全身强直-阵挛性发作)为标准。惊厥结束以抽搐停止,恢复自主行动(翻正恢复)为标准。统计学处理
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。用单位概率回归法(probit法)测定罗哌卡因腹腔注射ED50及ED95。
结果
发生惊厥实验鼠表现为自由活动减少,逐渐转变为躁动、无目的性奔跑、肢体挛缩倒地、翻正不能、直至全身强直-阵挛性发作。腹腔注射罗哌卡因后从注射到出现惊厥时间(潜伏期)为4.7±0.9min,自出现惊厥到惊厥结束(持续时间)为26.4±6.0mmin。经probit法计算得出方程式为：y=-17.89+12.75(1g剂量),得出0.5%罗哌卡因P21日龄SD幼鼠腹腔注射致惊厥剂量ED50为25.12mg/kg(95%可信区间为22.38-27.54mg/kg)。ED95为33.79mg/kg(95%可信区间为29.99-48.38mg/kg)。
结论
采用序贯法结合单位概率回归法(probit法)测定清醒状态下,SD大鼠(P21日龄)腹腔注射0.5%罗哌卡因致惊厥ED50及ED95值分别为25.12mg/kg和33.79mg/kg。
目的
采用第一章测得的0.5%罗哌卡因致P21日龄SD幼鼠惊厥ED95值,制备中枢神经单次和反复毒性惊厥模型。用Morris水迷宫测定惊厥后实验鼠的近期和成年期学习记忆能力变化。
方法
选用21日龄SD幼鼠,采用第一章实验测得的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,纳入实验鼠30只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=10)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=10)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=10)：腹腔注射注射等量的生理盐水。各组分别注射后1h～第3d、第5d～第7d、第60日龄(P60)～第62日龄进行行为学测试。
测试内容：①定位航行实验：测试经历3天,每天上、下午各测试1次。实验鼠由不同落水点面朝桶壁入水,软件自动记录大鼠自入水至找到平台(在平台停留时间为5s)的图像,并分析其路径、游泳速度和所用时间(逃避潜伏期)。如实验鼠在120s内仍未寻找到平台,则由操作者引导至平台并停留5s,记录其潜伏期为120s。②空间探索实验：最后一次定位航行实验结束后(训练第4d、8d、P62),撤除水池中的平台。将实验鼠由3个落水点面壁放入水中,记录大鼠在120s内穿越原平台所在象限的次数及在此象限内的游泳时间。③观察寻找平台潜伏期探索策略。统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。大鼠寻找平台逃避潜伏期采用重复测量的方差分析进行统计；同一时点不同组间,组内不同时间点间均采用One-Way ANOVA方差分析。穿越平台次数、在平台所在象限游泳时均采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05差异有统计学意义。
结果
定位航行实验寻找平台潜伏期
第1-3d定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时间点寻找平台潜伏期有显著差异(F=288.96,P<0.001)；对照组、单次组及反复组均随训练时间的增加,潜伏期逐渐缩短(F=225.094,P<0.001；F=152.614,P<0.001；F=46.603,P<0.001)；第1天、第2天和第3天3组间训练潜伏期均有显著差异(F=7.371,P<0.005；F=111.73,P<0.001；F=172.096,P<0.001)进一步用LSD两两比较分析各组之间的差异：第1、2天潜伏期时间,对照组分别与单次组、反复组比较组间均有显著差异(P<0.05),单次组和反复组潜伏期时间明显长于对照组；而单次组和反复组间无统计学差异(P>0.05)；第3天潜伏期时间分析,对照组与单次组间潜伏期时间无统计学差异(P>0.05),对照组、单次组分别与反复组比较,均有显著性差异(P<0.05),反复组潜伏期时间明显长于对照组和单次组。
第5-7d定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时点寻找平台潜伏期有显著差异(F=19.361,P<0.001)；对照组和单次惊厥组均随训练时间增加潜伏期逐渐缩短(F=14.75,P<0.001；F=17.194,P<0.001)反复惊厥组随训练时间延长寻找平台潜伏期无明显变化(F=2.777,P>0.05)第5、6、7天,3组间寻找平台潜伏期有显著差异(F=169.044,P<0.001：F=235.824,P<0.001； F=285.290,P<0.001),进一步用LSD两两比较分析各组间的差异：第5、6、7天寻找平台潜伏期,对照组、单次惊厥组与反复惊厥组比较,组间均有显著差异(P<0.05),反复组潜伏期时间明显长于对照组；而对照组和单次惊厥组间无统计学差异(P>0.05)
P60-P62天定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时点寻找平台潜伏期有显著差异(F=80.667,P<0.001)；3组寻找平台潜伏期均随训练天数增加逐渐缩短(F=16.718,P<0.001；F=20.580,P<0.001；F=14.214,P<0.001)；寻找平台潜伏期相同时点组间存在显著差异(F=177.218,P<0.005；F=353.348,P<0.001；F=269.125,P<0.001),进一步用LSD两两比较分析各组之间的差异：在P60-P62天,反复惊厥组与对照组、单次惊厥组比较均有显著差异(P<0.05),反复惊厥组潜伏期时间明显长于对照组；而对照组和单次惊厥组无统计学差异(P>0.05)
空间探索实验
第4天空间探索实验：①穿越原平台所在象限次数：3组在第4天穿越原平台所在象限次数有显著差异(F=43.246,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。②在原平台所在象限游泳时间：3组在原平台所在象限的游泳时间有显著差异(F=129.646,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
第8天空间探索实验：①穿越原平台所在象限次数：第8天3组穿越原平台所在象限次数有显著差异(F=57.474,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。②在原平台所在象限游泳时间：3组在前3天测试结束后,在原平台所在象限的游泳时间有显著差异(F=124.692,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
P62空间探索实验：①穿越原平台所在象限次数：3组在P62时间穿越原平台所在象限次数有显著差异(F=25.327,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。
②在原平台所在象限游泳时间：3组在前3天测试结束后,在原平台所在象限的游泳时间有显著差异(F=120.061,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
平台潜伏期探索策略
3组大鼠寻找平台潜伏期探索策略：对照组大鼠多采用直线和趋向式策略；单次组大鼠1～3d多采用边缘式和趋向式,5～d和P60～62多以直线式和趋向式为主；反复组多以边缘式和随机式为主。
结论
1.罗哌卡因致SD幼鼠(P21日龄)单次中枢神经毒性惊厥后学习记忆能力有一过性减弱,至惊厥后3天即恢复至正常水平,对其成年后学习记忆能力无明显损害。
2.罗哌卡因致SD幼鼠(P21日龄)中枢神经反复毒性惊厥对其近期及成年后学习记忆能力均有影响,表现为学习记忆能力减弱。
目的
本章节通过制备惊厥模型,观察发生惊厥SD幼鼠海马突触发育结构变化,以明确罗哌卡因致幼鼠中枢神经毒性惊厥对其突触发育是否会造成影响,以及与学习记忆能力的改变有何联系。
方法
选用21日龄SD幼鼠,采用第一章实验得出0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,共有实验鼠36只纳入实验。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=12)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=12)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=12)：腹腔注射注入等量生理盐水。每组根据处死时间点不同(24h、3d、7d、P60)分为4个亚组(n=3)。
各亚组取大鼠3只,多聚甲醛和戊二醛混合液灌注。灌注结束后快速于冰上剥出脑组织,取出双侧海马组织。按照大鼠脑定位图谱,在解剖显微镜下切取大小约1mm3的海马CA1区组织块,放入2.5%的戊二醛电镜液中固定。每只实验鼠选取2张铜网进行观察测量,观察内容包括神经元超微结构变化。在每张铜网上随机选取10个结构清楚的突触,运用电镜标尺对突触前后膜间隙和突触后致密物厚度进行测量。在透射电镜放大46000倍数下对突触数目进行测量分析。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。突触后致密物厚度、突触间隙宽度、突触数目均采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05有统计学意义。
结果
海马CA1区神经毡一般形态变化
对照组各时间点海马神经元形态规则完整,结构清楚。细胞结构清晰完整,细胞核呈圆形,染色质颗粒分布均匀；胞内线粒体丰富,呈圆形或椭圆形,线粒体内嵴清晰,规则排列：内质网和高尔基小体发达。神经毡树突中线粒体数目较多,轴突内可见神经微管。反复惊厥组在24h、3d、7d时间点神经元数目明显减少；神经元胞浆固缩、浓染,星形胶质细胞高度水肿、液化、核固缩。视野内可见大量细胞核肿胀,形态不规则,核膜肿胀,结构不清晰细胞核内染色质颗粒分布不均；线粒体出现肿胀,可见空泡、固缩现象。在P60时间点电镜下海马神经元、线粒体结构形态基本恢复正常。单次惊厥组电镜下观察,仅在惊厥后24h、3d时间点神经元出现水肿,核膜尚完整,细胞核形态基本正常,少量线粒体出现水肿,偶见线粒体嵴断裂和空泡现象；在7d、P60时间点基本恢复正常。
突触超微结构变化
对照组各时间点突触数目丰富,散在。突触结构清晰,突触前膜、突触间隙及突触后膜致密物清晰可见。在突触前膜靠近突触间隙一侧可见较多形态规则、大小均匀的突触小泡。突触间隙清晰可见,突触后致密结构较厚,散在均匀致密颗粒。反复惊厥组,除P60时间点外,其余各时间点突触数目均较对照组显著减少,尤其在3d、7d时间点。突触结构模糊不清,突触间隙明显增宽,突触小泡散在,形态大小不均,突触后致密物变薄。单次惊厥组在24h、3d时间点,突触数目稍减少,突触结构尚清晰,间隙稍增宽,突触后致密物厚度无明显变化。在7d和P60时间点,突触结构基本正常。
海马CA1区突触数、突触间隙和突触后致密物厚度比较
单次惊厥早期(24h、3d),突触数目较对照组减少,突触间隙较对照组增宽(P<0.05),在惊厥后7d及实验鼠成年期(P60)和对照组比较无统计学差异(P>0.05)；单次惊厥组突触后致密物在惊厥后24h时间点与对照组比较明显变薄,其余时间点与对照组比较无统计学意义(P<0.05)；反复惊厥组在惊厥后24h、3d、7d时间点,突触数目较对照组和单次惊厥组均明显减少,突触间隙明显增宽,突触后致密物明显变薄(P<0.05),成年期(P60)与对照组及单次组比较三者均无统计学差异(P<0.05)。
结论
罗哌卡因致SD幼鼠(P21日龄)中枢神经毒性惊厥影响海马CA1区突触形态结构,表现为线粒体破坏,突触数目减少,突触间隙增宽,突触后致密物变薄。单次及反复惊厥对其成年后海马突触发育无明显影响。
目的
本章节内容采用免疫组化及Western Blot方法检测惊厥大鼠海马不同时点突触素表达,观测局麻药致大鼠惊厥是否影响其表达,以及与突触发育及学习记忆的联系。
方法
选用21日龄SD幼鼠,采用第一章实验得出的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者予以剔除,共选用实验鼠108只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=36)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=36)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=36)：腹腔注射注射等量生理盐水。每组根据处死时间点不同(24h、3d、7d、P60)分为4个亚组(n=9)。
各亚组取6只大鼠制备免疫组织化标本,灌注后迅速取脑切成薄片置入多聚甲醛固定。进入脑片包埋、切片、染色过程。在光学显微镜下观察切片并拍片,每只动物取6张切片,用IPP图像处理软件,测量海马CA1区免疫反应产物的光密度值。另外每组各时间点再各取3只,给予3%水合氯醛腹腔注射麻醉。迅速断头于冰上取脑,分离出双侧海马组织,用Western Blot免疫印迹法测定大鼠海马突触素蛋白含量。扫描X胶片,采用Quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为突触素蛋白的表达水平。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。不同时间点间各组突触素表达采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05为有统计学意义。
结果海马CA1区突触素表达
单次惊厥组与对照组相比,海马CA1区突触素蛋白水平表达仅在惊厥后24h时间点低于对照组(P<0.05),其余各时间点均无显著差异；反复惊厥组与对照组比较,海马CA1区突触素蛋白表达除P60时间点无统计学差异外,其余各时间点均显著低于对照组(P<0.05)；反复惊厥组与单次惊厥组比较,鼠海马CA1区突触素表达水平除P60时间点无统计学差异外,其余时间点均显著低于单次惊厥组(P<0.05)。
Western Blot检测大鼠海马突触素表达
单次惊厥组与对照组相比,海马突触索蛋白水平表达仅在惊厥后24h时间点显著低于对照组(P<0.05),其余各时间点均无显著差异；反复惊厥组与对照组比较,海马突触素蛋白表达除P60时间点无统计学差异外,其余各时间点均显著低于对照组(P<0.05)；反复惊厥组与单次惊厥组比较,鼠海马CA1区突触素表达水平除P60时间点无统计学差异外,其余时间点均显著低于单次惊厥组(P<0.05)。
结论
1.罗哌卡因致SD幼鼠(P21日龄)单次惊厥对海马突触素表达有一过性影响,在惊厥后第3天基本恢复至正常,对成年期海马突触素表达无影响。
2.SD幼鼠(P21日龄)经历反复惊厥后,海马突触素表达减少至惊厥后第7天,对其成年后表达无影响。
目的
本章通过制备罗哌卡因惊厥模型,运用Western Blot和QT-PCR技术对海马CaMK Ⅱ和p-CREB蛋白和基因表达水平进行检测,观察CaMK Ⅱ和p-CREB在局麻药毒性惊厥对突触发育及学习记忆的影响。
方法
选选用21日龄SD幼鼠,采用第一章实验得出的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,共选用实验鼠120只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=40)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=40)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=40)：腹腔注射注射等量生理盐水。每组根据处死时点不同(24h、3d、7d、P60)分为4个亚组(n=10)。
合成CaMKⅡ和CREB上下游引物。每亚组取5只大鼠提取总RNA、逆转录、PCR扩增,反应条件为：93。C3min,然后93℃45s,55℃1min,共循环40次,对整个升温过程进行全程荧光信号收集,绘制融解曲线并分析。定量的方法以2-AA Ct(Ct代表循环闽值)来表达基因的表达量,计算公式为A△Ct=[Ct(待测组目标基因)—Ct(内参)]—[Ct(对照组目标基因)-Ct(内参)]。
另外每组各时间点再各取5只,给予3%水合氯醛腹腔注射麻醉。迅速断头于冰上取脑,分离出双侧海马组织,用Western Blot免疫印迹法测定大鼠海马CaMKⅡ和p-CREB含量。扫描X胶片,采用Quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为蛋白的表达水平。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。各时间点不同组间CaMKⅡ和p-CREB蛋白和基因表达水平比较采用One-Way ANOVA；两两比较采用LSD法,P<0.05为有统计学意义。
结果
CaMKⅡ在各组的表达情况
24h和3d时间点单次惊厥组CaMKⅡ基因表达明显低于对照组(P<0.05),其余时间点无明显差异。24h、3d、7d及P60时点反复惊厥组CaMKⅡ的表达均明显低于对照组(P<0.05)；24h、3d、7d及P60时点反复惊厥组CaMKⅡ的表达均明显低于单次惊厥组(P<0.05)。
CREB在各组的表达情况
单次惊厥组p-CREB在24h、3d、7d及P60时间点的表达与对照组相比均无明显差异(P>0.05)；反复惊厥组CREB在24h、3d、7d及P60时点的表达均明显低于对照组和单次惊厥组(P<0.05)
结论
1.罗哌卡因致SD幼鼠(P21日龄)毒性单次惊厥后海马CaMKⅡ在惊厥后24h、3d表达明显降低。罗哌卡因致幼鼠反复惊厥在惊厥早期及成年后CaMKⅡ表达均明显减少。
2.罗哌卡因致SD幼鼠(P21日龄)单次毒性惊厥对海马p-CREB的表达无影响,罗哌卡因致幼鼠反复毒性惊厥海马p-CREB的表达在惊厥早期和成年均明显减少。
全文结论：
1.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥影响其突触可塑性：单次及反复惊厥大鼠均表现为海马CA1区突触数目减少、突触间隙增宽、突触后致密物变薄,反复惊厥比单次惊厥所致损害程度更为严重,且持续时间延长。
2.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥影响其学习记忆能力：单次惊厥大鼠表现为一过性的学习能力障碍,而反复惊厥大鼠学习记忆能力障碍持续至成年后。
3.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥对其学习记忆的影响可能与突触可塑性破坏、海马突触素表达下调、CamkⅡ及p-CREB表达下调有关。
Seizure induced by Ropivacaine central nervous system toxicity is common toxicant adverse reaction. Infants and children may be at increased risk form local anesthetics compared with adults because of their special anatomy and development feature. The previous study confirm that immature rats undergoing long term or recurrent seizure, it can effect the synaptic plasticity, learning and memory.
It is the key time for development of synapses, nerve cell differentiationin migration, and fast formation of dendritic synapse in infants. Many investigations describe that other factors induced recurrent or long time seizures induced defect of learing and memory, its mechanism including ion channel, excitatory amino acid (EAA) receptor, neurotransmitter,development of synapse,the p protein synthesis, gene expression, and signal transduction. It is still uncertain if seizure induced by LAs can damage the development of nervous system, learning, and memory.
It has been repoted about LAs toxicity seizure in the early1920century. Much has been done about the mechanism and prevention and cure by anesthetists. The mechanism of LAs toxicity seizure including the unbalance of EAA-IAA, NMDA-Ca2+-NOS, dopamine, acetylcholine, M-potassium channels et al. All above receptors, neurotransmitter, ion channels participant the development and maintenance of CNS, synapses, leaning, and memory.Hippocampus is the most important place of learning, and memory, and also the place which LAs toxicity seizure take place.
Learning and memory is an higher nervous activity of brain. The two nervous activity intercommunication and influence about learning and memory. Learning means human or biology can change its performance in response to its environment, and memory means the imformation storage and output by learning activity. The nerves base of learning and memory is high plasticity of CNS, its including neural network connection, the plastic relative neurotransmitter, protein, ion channels, and receptors.
Synapses is the junction point of shape structure and functional about neuron, it's the key site of maintenance about information transport. The synapse plastic means when changing of external environment, the synapse take a adaptive change to keep the relative stable condition, the changing including form structure and function changing. The studay consider after recurrent or long time seizures, the synapses form structure was destruction in hippocampal area, the changing impact function of nervous, and its destroy level are positively correlate with learning and memory performance. The studay about LAs CNS toxicity describe that LAs inhibit synapses axon growth, induce nerve impulse attenuated, and impairment the physiologic function.
Synaptophysin is a calcium binding glucoprotein which located in presynaptic membrane of axon peripheral. Synaptophysin participate in the release procedure of ACH, glutamic acid,et al, it has a close relation with synaptic plastics. Synaptophysin is a specificity protein of synaptic vesicle, its density and distribution reflect synapsis amount, and distribution. Synaptophysin has a close relation with synaptic restitution and recognition. Synaptophysin can regulate the formation, development, and mature of neurons, and also effect differentiation of neurons by regulate the cytoskeletal protein pack. Synaptophysin has a considerable meaning to axon lengthen, synaptic linkage, and synaptic maturation. The opposite research describe that it is not necessary of synaptophysin to synaptic development, learning, and memory, and it is still uncertain of its mechanism.
Ca2+-CaMK Ⅱ-CREB path is one of the main signal of formation synaptic plasticity and learning memory of CNS. CaMK Ⅱ is a protein has a the highest content in the hippocamp, it's a binding protein of Ca2+ion, has a close correlation of LTP, it can also be called"molecular switch of learning, and memory". Lidocaine. cocaine, and procaine can inhibit LTP in hippocampal by effect of calmodulince. LAs induced CNS toxicity seizure cause calcium overload, if it can effect CaMK Ⅱ, synaptic plasticity, learning, and memory.
cAMP-responsive element binding protein (CREB) is a intracellular transcicription factor, which mediates the genetic transcription and the proteinous synthesis, plays a important role in the long term memory. It effect CREB phosphorylation induced learning,memory impairment in with acute or repeat treatment of cocaine.
We determine ED95of0.5%Ropivacaine which the dose induced CNS toxicity seizure with postnatal21days(P21) mice, and preparation single and repeated seizure mice model. The learning,memory ability test by Morris water maze (MWM) at time after seizure. Synaptic ultrastructure in hippocamp after seizure transmission electron microscope Immunohistochemical staining and western blotting methods were used in the assays of the expressive levels of synaptophysin in CA1area of hippocamp. Fluorescent quantitation polymerase chain reaction(Q-t PCR) technique was used in the assays of the exprwssive levels of CaMK Ⅱ and p-CREB gene. We detect if LAs CNS toxicity seizure will effect the synaptic plasticity, learning and memory of immature mice, through test ethology; morphology,protein, and gene level, and discuss the possible mechanism about it.
Objective
We determine the ED50, ED95of ropivacaine induced seizure through up-down and unit probability regression method.
Methods
Forty healthy P21Sprague-Dawley (SD) rats. Using sodium chloride configuration0.5%Ropivacaine. Experiment dose divide to5groups, the geometric proportion coefficient is0.8, and the initial dose is24mg/kg. A rat accept intraperitoneal injection only one time. The seizure standard according to Racine V degree (righting reflex loss, generalized tonic-clonic seizures).
grade0, normal behavior,1, facial twitches; wet dog shakes, arres t;2, head nodding, chewing;3, forelimb clonus;4, rearing, falling on forelimbs;5, falling on the side or back, hindlimb clonus. The rats seizure degree achieve V bring into experiment.
Termination of seizure by means of twitch disappear and recover of self-action(righting reflex recover). Performance according to the up-down method: The first rat treatment with0.5%Ropivacaine intraperitoneal injection dose is24mg/kg, if it appearance seizure,and the next rat will accept the down regulation dose of Ropivacaine, but if the first rat did not appearance seizure, and the next rat will accept the upper regulation dose. Record positive, negative, and all cases in per dose group. Record the latency and duration of rat that undergone seizure.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). The unit probability regression method (probit) was used toanalyze ED50and ED95of seizure induced by ropivacaine toxicity.
Results
The seizure rats were characterized by free-running decreased, restlessness, run in a hurry aimless, loss of right reflex and generalized tonic-clonic convulsions. From inject time to occurrence of seizure(latency) is4.7±0.94min, and from occurrence to terminal of seizure(duration) is26.4±6.0min. According probit method calculated, equation y=-17.89±12.78(1g dose), the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg). and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).
Conclusion
According probit method calculated, the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg), and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. The learning,memory ability test by Morris water maze (MWM) at time after seizure.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=10):control group(Cgroup), single seizure group (R group), and recurrent seizure group (FR group). N group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The learning,memory ability test by Morris water maze (MWM) at time (1h～3d;5d～7d; P60～P62) after seizure in each group.
Test content①Place navigation, it used to test the learning and memory ability of the rats. The rats swim2min in the pool before the starting formal experiment. The formal test experience three days,and test1time at am and pm per day. Putting the rats into water with face toward barrel wall at different point, the software recorded the swimming image of the rats(the residence time is5s), and analyzing the swimming path, velocity, and latency. If the rats could not find the platform in120s, the operater guide the rats to the platform ang stady for5s, and recording the latency was120s.②Spatial probe:It used to test the space memory ability, spatial probe carried out at the time after thelast place navigationtest, removing the platform, putting the rats at different point into water recording the times of accrosing and the swimming time in the quadrantic which the platform was located.(4d,8d, and P62),
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). Repeat measurement F test was ued to analyze escape latency. One-Way ANOVA was used to analyze the latency at different time in different groups or in different groups at the same time. One-Way ANOVA was used to analyze the times of accrosing and the swimming time in the quadrantic which the platform was located. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
D1-3Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased (F=225.09, P<0.001; F=152.614, P<0.001; F=46.603, P<0.001). There were significant deviation of latency time in C, R and FR groups on day1-3(F=7.371, P<0.001; F=111.73, P<0.001; F=172.096, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C vs R, C vs FR groups at day1-2(P<0.05); and there was significant deviation of latency time between C vs FR, R vs FR groups at day3(P<0.05);Day4spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day4(F=43.246, P<0.001), and there was significant deviation of time accrosing in C vs R, and R vs FR groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=129.646, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)
D5～7Search platfonn latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C and R groups by train time increased (F=14.75, P<0.001:F=17.194, P<0.001),but not in FR group(F=2.777, P>0.05). There were significant deviation of latency time in C, R and FR groups on day5～7(F=169.044, P<0.001; F=235.824, P<0.001; F=285.290, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day5-7(P<0.05). Day8spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=57.474, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=124.692, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)
P60～P62Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased(F=16.718, P<0.001; F=20.580, P<0.001; F=14.214, P<0.001)There were significant deviation of latency time in C, R and FR groups on day P60-P62((F=177.218, P<0.005; F=353.348, P<0.001; F=269.125, P<0.001) The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day P60-P62(P<0.05). Day P62spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=25.327, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=120.061, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not N vs R groups (P<0.05)
Conclusion
It can effect the learning and memory ability transiently with single seizures induced by Ropivacaine, but the long-term effect by repeated seizures.
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptic morphology in the hippocampal CA1area by transmission electron microscope.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP. which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=12):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days, I/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=3) according to the24h,3d,7d, and P60after covulsion or injection respective.
According the time, perfusing the rats with paraformaldehyde and glutaric dialdehyde mixed liquor. After that taking out the hippocampus quickly and putting the glutaric dialdehyde. Using the transmission electron microscope to observe the synaptic cleft, synaptic number, and PSD.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptic cleft, synaptic number, and PSD at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
The synaptic number in R group at time24and3d, was less than C group at the same time. And the synaptic number in FR group at time24h,3d and7d, was less than C group at the same time (P<0.05). The synaptic cleft in R and FR groups were reduced than it in the C group at time24and3d (P<0.05).The PSD in R and FR groups were reduced than it in the C group at time24(P<0.05). The synaptic number, synaptic cleft and PSDin Both R and FR groups were the same as in the C at time P60.
Conclusion
Ropivacaine-induced seizures effect the synaptic morphology in the hippocampal CAI area. The synaptic number reduced, synaptic clef widen and PSD thinningz atearlier period (24h,3d, and7d)
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptophysin in the hippocampal by Immunohistochemical stain and western blotting method.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=24):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=6) according to the24h,3d,7d, and P60after covulsion or injection respective.
According the time, perfusing the rats with paraformaldehyde liquor. Another rats without perfusing to obtain western blotting sample.After that taking out the hippocampus Using t Immunohistochemical stain and western blotting method to observe the synaptophysin content at different time.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptophysin content at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
Synaptophysin content expression by Immunohistochemical stain
The synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in C group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as C group at P60. Synaptophysin content expression by western blotting
The synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in N group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as N group at P60.
Conclusion
The synaptophysin content expression was reduced transiently at time3d after single seizure induced bu Ropivacaine, and it continue reduced until7d after repeat seizures.The synaptophysin content were same as normal in both single and repeat seizures at P60.
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the CaMKⅡand p-CREB in the hippocampal by Fluorescent quantitation polymerase chain reaction technique and Western Blot..
Methods
One hundred and twenty rats healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The one hundred and twenty rats were randomized into three groups (n=40): control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=10) according to the24h,3d,7d, and P60after covulsion or injection respective.
CaMK Ⅱ and p-CREB up, down-stream primer were synthesis. According the time, to obtain hippocampus sample. extracting the sumRNA, reverse transcription, PCR amplification. Using the quantitive method analysis gene expression.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way AN OVA was used to analyze the CaMK Ⅱ and p-CREB at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
CaMK Ⅱ gene expression
The CaMK Ⅱ gene content expression in R group was less than it in C group at time24h and3d (P<0.05), and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05) p-CREB gene expression
The p-CREB gene content expression in R group was same as it in C group at time24h,3d,7d, and P60; and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05)
Conclusion
The CaMK Ⅱ gene expression content expression was reduced transiently at time3d after single seizure induced by Ropivacaine, and it continue reduced until P6o after repeated seizures.The p-CREB gene content expression were same as normal in single group at all the time, but the expression were all reduced significantly in repeated group at all the time.
婴幼儿或幼年动物神经系统发育处于突触爆发期,是神经细胞快速分化、迁徙、树突快速形成神经网络的关键时期,此期对各种伤害性刺激抵御能力差易对其中枢神经系统生理发育和功能形成产生影响。众多研究报道其它非局麻药因素致发育期反复或长程惊厥可导致动物成年认知功能和学习记忆能力下降,其机制与离子通道、氨基酸受体、神经递质、突触发育及学习i记忆相关的蛋白质合成、基因表达、信号转导有关。局麻药致发育期婴幼儿中枢神经系统毒性惊厥,是否会对其神经系统发育造成损害,对其学习记忆能力的形成是否有影响尚无研究定论。
早在上世纪20年代已有关于局麻药中枢神经毒性的相关报道。此后麻醉领域学者们展开了大量关其机制和防治的研究。局麻药中枢神经毒性以惊厥发作为主要临床表现,其机制主要与兴奋性—抑制性氨基酸学说、NMDA-Ca2+-NOS通路学说、多巴胺、乙酰胆碱、M型钾通道等有关。而以上受体、神经递质、及离子通道等均不同程度参与中枢神经系统神经突触发育及学习记忆能力的形成。海马作为与学习记忆最为相关的解剖部位,也是局麻药中枢神经毒性惊厥的发生形成关键部位。。
学习记忆是大脑的高级神经功能。学习和记忆是两个相互联系和影响的神经活动过程,学习是指人或生物依赖经验来改变自身环境的神经活动过程,而记忆则是将通过学习得到的信息储存和输出的一种神经活动。学习记忆的神经基础是中枢神经系统的高度可塑性,包括神经网络连接形态及与神经突触发育相关的各种神经递质、蛋白、离子通道、受体等多种不同层次不同阶段的可塑性。而其中突触连接是神经可塑性的关键部位,也是实现神经元之间传递信息、实现生理功能的关键部位。
突触可塑性是指在外环境改变时,神经元突触发生适应性改变从而维持其相对稳定的状态,包括形态结构和功能上的改变。研究认为在反复或长程惊厥后,海马突触形态结构遭到不同程度的破坏,从而影响学习记忆的维持和神经功能的发挥,其破坏程度与学习记忆功能的减退呈正相关。以往局麻药中枢神经毒性研究发现,局麻药抑制突触轴突生长,并减弱神经冲动传导,从而削弱了突触维持正常生理功能的能力。突触素是一种钙结合糖蛋白,特异性位于轴突末梢的突触前膜上。突触素参与谷氨酸、乙酰胆碱等神经递质的释放过程,与突触可塑性关系密切。突触素作为突触囊泡的一种特异性标志蛋白,其密度和分布可间接反应单位体积内突触数量和分布情况,与突触重建及认知过程密切相关。另一方面突触素调节神经元的发生、发育及成熟,还可通过调节细胞骨架蛋白组装而影响神经元的分化。突触素在神经轴突延伸、突触连接形成和突触成熟中具有重要的意义。
钙/钙调素依赖性蛋白激酶Ⅱ(Calcium/calmodulin dependent protein kinase Ⅱ, CaMK Ⅱ) CaMK Ⅱ是海马内含量最高的一种蛋白质,是钙离子的主要结合蛋白,且与突触长时程增强密切相关,有“学习记忆分子开关”之称。环磷酸腺苷反应单元结合蛋白(cAMP-responsive element binding protein,CREB) CREB是一种真核生物细胞核内转录转导因子,具有调节基因转录的功能,是细胞内多种信号转导通路的关键成分之一。研究发现CREB在长期记忆的维持中起关键作用。在以往对可卡因急性或反复应用研究中发现,可卡因通过影响CREB磷酸化,对学习记忆产生影响。Ca2+-CaMK Ⅱ-CREB通路是中枢神经系统突触可塑性和学习记忆能力形成与维持的主要信号通路之一。以往研究发现,利多卡因、可卡因、普鲁卡因抑制海马长时程增强,与影响钙调素有关。局麻药中枢神经毒性惊厥时引发钙超载,严重钙超载与CaMK Ⅱ、CREB之间影响如何?是否会对突触可塑性和学习记忆产生影响目前尚不明了
本课题通过测定0.5%罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥ED95值,分别制备幼鼠单次惊厥和反复惊厥实验模型。应用Morris水迷宫测定实验鼠惊厥后近期及成年后学习i记忆能力变化；采用透射电镜观察中枢神经毒性惊厥后实验鼠在不同时间海马突触超微结构变化；以免疫组织化学染色和Western Blot免疫印迹技术观察海马CA1区突触素蛋白的表达；应用Western Blot和荧光实时定量PCR技术测定CaMK Ⅱ和p-CREB蛋白和基因表达水平变化。本研究从行为学、形态学、蛋白和基因水平检测局麻药致发育鼠中枢神经毒性惊厥对其突触可塑性及学习记忆的影响,并探讨和研究其可能涉及的机制。
目的
采用序贯法(up-down method)结合单位概率回归法(probit法)测定SD幼鼠(P21日龄)清醒状态下,0.5%罗哌卡因腹腔注射致其惊厥ED50及ED95。
方法
选用健康Sprague Dawley(SD)P21日龄幼鼠40只。用生理盐水配置浓度为0.5%盐酸罗哌卡因备用。罗哌卡因腹腔注射剂量分为5组,起始剂量为24mg/kg,相邻剂量间隔等比系数为0.8。其余剂量组依次为15.4mg/kg、19.2mg/kg、30mg/kg、37.5mg/kg。
序贯法操作：第一只实验鼠腹腔注射剂量为24mg/kg,如出现惊厥则下一只实验鼠腹腔注射剂量下调至较低相邻剂量,即19.2mg/kg,如未出现惊厥则下一只实验鼠腹腔注射剂量上调至较高相邻剂量,即30mg/kg,依次类推,直至所有动物均接受一次注射。统计每个剂量组阳性例数、阴性例数及总例数。记录发生惊厥实验鼠惊厥潜伏时间及持续时间。
实验鼠行为依照Racine分级法进行分级,0级：无任何反应,呈常态表现；Ⅰ级：点头样抖动、面部肌肉痉挛表现如节律性眨眼、咀嚼、胡须抖动等；Ⅱ级：痉挛性表现,点头或甩尾；Ⅲ级：一侧前肢体阵挛性抖动；Ⅳ级：双侧前肢体阵挛,但仍可站立；V级：全身阵挛性发作,失去平衡,无法站立,翻正不能。
纳入标准：鼠发生惊厥以Racine分类V级(翻正不能、直至全身强直-阵挛性发作)为标准。惊厥结束以抽搐停止,恢复自主行动(翻正恢复)为标准。统计学处理
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。用单位概率回归法(probit法)测定罗哌卡因腹腔注射ED50及ED95。
结果
发生惊厥实验鼠表现为自由活动减少,逐渐转变为躁动、无目的性奔跑、肢体挛缩倒地、翻正不能、直至全身强直-阵挛性发作。腹腔注射罗哌卡因后从注射到出现惊厥时间(潜伏期)为4.7±0.9min,自出现惊厥到惊厥结束(持续时间)为26.4±6.0mmin。经probit法计算得出方程式为：y=-17.89+12.75(1g剂量),得出0.5%罗哌卡因P21日龄SD幼鼠腹腔注射致惊厥剂量ED50为25.12mg/kg(95%可信区间为22.38-27.54mg/kg)。ED95为33.79mg/kg(95%可信区间为29.99-48.38mg/kg)。
结论
采用序贯法结合单位概率回归法(probit法)测定清醒状态下,SD大鼠(P21日龄)腹腔注射0.5%罗哌卡因致惊厥ED50及ED95值分别为25.12mg/kg和33.79mg/kg。
目的
采用第一章测得的0.5%罗哌卡因致P21日龄SD幼鼠惊厥ED95值,制备中枢神经单次和反复毒性惊厥模型。用Morris水迷宫测定惊厥后实验鼠的近期和成年期学习记忆能力变化。
方法
选用21日龄SD幼鼠,采用第一章实验测得的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,纳入实验鼠30只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=10)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=10)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=10)：腹腔注射注射等量的生理盐水。各组分别注射后1h～第3d、第5d～第7d、第60日龄(P60)～第62日龄进行行为学测试。
测试内容：①定位航行实验：测试经历3天,每天上、下午各测试1次。实验鼠由不同落水点面朝桶壁入水,软件自动记录大鼠自入水至找到平台(在平台停留时间为5s)的图像,并分析其路径、游泳速度和所用时间(逃避潜伏期)。如实验鼠在120s内仍未寻找到平台,则由操作者引导至平台并停留5s,记录其潜伏期为120s。②空间探索实验：最后一次定位航行实验结束后(训练第4d、8d、P62),撤除水池中的平台。将实验鼠由3个落水点面壁放入水中,记录大鼠在120s内穿越原平台所在象限的次数及在此象限内的游泳时间。③观察寻找平台潜伏期探索策略。统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。大鼠寻找平台逃避潜伏期采用重复测量的方差分析进行统计；同一时点不同组间,组内不同时间点间均采用One-Way ANOVA方差分析。穿越平台次数、在平台所在象限游泳时均采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05差异有统计学意义。
结果
定位航行实验寻找平台潜伏期
第1-3d定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时间点寻找平台潜伏期有显著差异(F=288.96,P<0.001)；对照组、单次组及反复组均随训练时间的增加,潜伏期逐渐缩短(F=225.094,P<0.001；F=152.614,P<0.001；F=46.603,P<0.001)；第1天、第2天和第3天3组间训练潜伏期均有显著差异(F=7.371,P<0.005；F=111.73,P<0.001；F=172.096,P<0.001)进一步用LSD两两比较分析各组之间的差异：第1、2天潜伏期时间,对照组分别与单次组、反复组比较组间均有显著差异(P<0.05),单次组和反复组潜伏期时间明显长于对照组；而单次组和反复组间无统计学差异(P>0.05)；第3天潜伏期时间分析,对照组与单次组间潜伏期时间无统计学差异(P>0.05),对照组、单次组分别与反复组比较,均有显著性差异(P<0.05),反复组潜伏期时间明显长于对照组和单次组。
第5-7d定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时点寻找平台潜伏期有显著差异(F=19.361,P<0.001)；对照组和单次惊厥组均随训练时间增加潜伏期逐渐缩短(F=14.75,P<0.001；F=17.194,P<0.001)反复惊厥组随训练时间延长寻找平台潜伏期无明显变化(F=2.777,P>0.05)第5、6、7天,3组间寻找平台潜伏期有显著差异(F=169.044,P<0.001：F=235.824,P<0.001； F=285.290,P<0.001),进一步用LSD两两比较分析各组间的差异：第5、6、7天寻找平台潜伏期,对照组、单次惊厥组与反复惊厥组比较,组间均有显著差异(P<0.05),反复组潜伏期时间明显长于对照组；而对照组和单次惊厥组间无统计学差异(P>0.05)
P60-P62天定位航行实验寻找平台潜伏期：经重复测量方差分析,3组间不同时点寻找平台潜伏期有显著差异(F=80.667,P<0.001)；3组寻找平台潜伏期均随训练天数增加逐渐缩短(F=16.718,P<0.001；F=20.580,P<0.001；F=14.214,P<0.001)；寻找平台潜伏期相同时点组间存在显著差异(F=177.218,P<0.005；F=353.348,P<0.001；F=269.125,P<0.001),进一步用LSD两两比较分析各组之间的差异：在P60-P62天,反复惊厥组与对照组、单次惊厥组比较均有显著差异(P<0.05),反复惊厥组潜伏期时间明显长于对照组；而对照组和单次惊厥组无统计学差异(P>0.05)
空间探索实验
第4天空间探索实验：①穿越原平台所在象限次数：3组在第4天穿越原平台所在象限次数有显著差异(F=43.246,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。②在原平台所在象限游泳时间：3组在原平台所在象限的游泳时间有显著差异(F=129.646,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
第8天空间探索实验：①穿越原平台所在象限次数：第8天3组穿越原平台所在象限次数有显著差异(F=57.474,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。②在原平台所在象限游泳时间：3组在前3天测试结束后,在原平台所在象限的游泳时间有显著差异(F=124.692,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
P62空间探索实验：①穿越原平台所在象限次数：3组在P62时间穿越原平台所在象限次数有显著差异(F=25.327,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组穿越次数明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)。
②在原平台所在象限游泳时间：3组在前3天测试结束后,在原平台所在象限的游泳时间有显著差异(F=120.061,P<0.001)；进一步LSD两两比较发现,反复惊厥组与对照组、单次惊厥组均有显著差异(P<0.05),反复惊厥组游泳时间明显少于对照组和单次组；而单次惊厥组与对照组无显著差异(P>0.05)
平台潜伏期探索策略
3组大鼠寻找平台潜伏期探索策略：对照组大鼠多采用直线和趋向式策略；单次组大鼠1～3d多采用边缘式和趋向式,5～d和P60～62多以直线式和趋向式为主；反复组多以边缘式和随机式为主。
结论
1.罗哌卡因致SD幼鼠(P21日龄)单次中枢神经毒性惊厥后学习记忆能力有一过性减弱,至惊厥后3天即恢复至正常水平,对其成年后学习记忆能力无明显损害。
2.罗哌卡因致SD幼鼠(P21日龄)中枢神经反复毒性惊厥对其近期及成年后学习记忆能力均有影响,表现为学习记忆能力减弱。
目的
本章节通过制备惊厥模型,观察发生惊厥SD幼鼠海马突触发育结构变化,以明确罗哌卡因致幼鼠中枢神经毒性惊厥对其突触发育是否会造成影响,以及与学习记忆能力的改变有何联系。
方法
选用21日龄SD幼鼠,采用第一章实验得出0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,共有实验鼠36只纳入实验。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=12)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=12)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=12)：腹腔注射注入等量生理盐水。每组根据处死时间点不同(24h、3d、7d、P60)分为4个亚组(n=3)。
各亚组取大鼠3只,多聚甲醛和戊二醛混合液灌注。灌注结束后快速于冰上剥出脑组织,取出双侧海马组织。按照大鼠脑定位图谱,在解剖显微镜下切取大小约1mm3的海马CA1区组织块,放入2.5%的戊二醛电镜液中固定。每只实验鼠选取2张铜网进行观察测量,观察内容包括神经元超微结构变化。在每张铜网上随机选取10个结构清楚的突触,运用电镜标尺对突触前后膜间隙和突触后致密物厚度进行测量。在透射电镜放大46000倍数下对突触数目进行测量分析。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。突触后致密物厚度、突触间隙宽度、突触数目均采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05有统计学意义。
结果
海马CA1区神经毡一般形态变化
对照组各时间点海马神经元形态规则完整,结构清楚。细胞结构清晰完整,细胞核呈圆形,染色质颗粒分布均匀；胞内线粒体丰富,呈圆形或椭圆形,线粒体内嵴清晰,规则排列：内质网和高尔基小体发达。神经毡树突中线粒体数目较多,轴突内可见神经微管。反复惊厥组在24h、3d、7d时间点神经元数目明显减少；神经元胞浆固缩、浓染,星形胶质细胞高度水肿、液化、核固缩。视野内可见大量细胞核肿胀,形态不规则,核膜肿胀,结构不清晰细胞核内染色质颗粒分布不均；线粒体出现肿胀,可见空泡、固缩现象。在P60时间点电镜下海马神经元、线粒体结构形态基本恢复正常。单次惊厥组电镜下观察,仅在惊厥后24h、3d时间点神经元出现水肿,核膜尚完整,细胞核形态基本正常,少量线粒体出现水肿,偶见线粒体嵴断裂和空泡现象；在7d、P60时间点基本恢复正常。
突触超微结构变化
对照组各时间点突触数目丰富,散在。突触结构清晰,突触前膜、突触间隙及突触后膜致密物清晰可见。在突触前膜靠近突触间隙一侧可见较多形态规则、大小均匀的突触小泡。突触间隙清晰可见,突触后致密结构较厚,散在均匀致密颗粒。反复惊厥组,除P60时间点外,其余各时间点突触数目均较对照组显著减少,尤其在3d、7d时间点。突触结构模糊不清,突触间隙明显增宽,突触小泡散在,形态大小不均,突触后致密物变薄。单次惊厥组在24h、3d时间点,突触数目稍减少,突触结构尚清晰,间隙稍增宽,突触后致密物厚度无明显变化。在7d和P60时间点,突触结构基本正常。
海马CA1区突触数、突触间隙和突触后致密物厚度比较
单次惊厥早期(24h、3d),突触数目较对照组减少,突触间隙较对照组增宽(P<0.05),在惊厥后7d及实验鼠成年期(P60)和对照组比较无统计学差异(P>0.05)；单次惊厥组突触后致密物在惊厥后24h时间点与对照组比较明显变薄,其余时间点与对照组比较无统计学意义(P<0.05)；反复惊厥组在惊厥后24h、3d、7d时间点,突触数目较对照组和单次惊厥组均明显减少,突触间隙明显增宽,突触后致密物明显变薄(P<0.05),成年期(P60)与对照组及单次组比较三者均无统计学差异(P<0.05)。
结论
罗哌卡因致SD幼鼠(P21日龄)中枢神经毒性惊厥影响海马CA1区突触形态结构,表现为线粒体破坏,突触数目减少,突触间隙增宽,突触后致密物变薄。单次及反复惊厥对其成年后海马突触发育无明显影响。
目的
本章节内容采用免疫组化及Western Blot方法检测惊厥大鼠海马不同时点突触素表达,观测局麻药致大鼠惊厥是否影响其表达,以及与突触发育及学习记忆的联系。
方法
选用21日龄SD幼鼠,采用第一章实验得出的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者予以剔除,共选用实验鼠108只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=36)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=36)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=36)：腹腔注射注射等量生理盐水。每组根据处死时间点不同(24h、3d、7d、P60)分为4个亚组(n=9)。
各亚组取6只大鼠制备免疫组织化标本,灌注后迅速取脑切成薄片置入多聚甲醛固定。进入脑片包埋、切片、染色过程。在光学显微镜下观察切片并拍片,每只动物取6张切片,用IPP图像处理软件,测量海马CA1区免疫反应产物的光密度值。另外每组各时间点再各取3只,给予3%水合氯醛腹腔注射麻醉。迅速断头于冰上取脑,分离出双侧海马组织,用Western Blot免疫印迹法测定大鼠海马突触素蛋白含量。扫描X胶片,采用Quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为突触素蛋白的表达水平。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。不同时间点间各组突触素表达采用One-Way ANOVA方差分析；两两分析采用LSD法,P<0.05为有统计学意义。
结果海马CA1区突触素表达
单次惊厥组与对照组相比,海马CA1区突触素蛋白水平表达仅在惊厥后24h时间点低于对照组(P<0.05),其余各时间点均无显著差异；反复惊厥组与对照组比较,海马CA1区突触素蛋白表达除P60时间点无统计学差异外,其余各时间点均显著低于对照组(P<0.05)；反复惊厥组与单次惊厥组比较,鼠海马CA1区突触素表达水平除P60时间点无统计学差异外,其余时间点均显著低于单次惊厥组(P<0.05)。
Western Blot检测大鼠海马突触素表达
单次惊厥组与对照组相比,海马突触索蛋白水平表达仅在惊厥后24h时间点显著低于对照组(P<0.05),其余各时间点均无显著差异；反复惊厥组与对照组比较,海马突触素蛋白表达除P60时间点无统计学差异外,其余各时间点均显著低于对照组(P<0.05)；反复惊厥组与单次惊厥组比较,鼠海马CA1区突触素表达水平除P60时间点无统计学差异外,其余时间点均显著低于单次惊厥组(P<0.05)。
结论
1.罗哌卡因致SD幼鼠(P21日龄)单次惊厥对海马突触素表达有一过性影响,在惊厥后第3天基本恢复至正常,对成年期海马突触素表达无影响。
2.SD幼鼠(P21日龄)经历反复惊厥后,海马突触素表达减少至惊厥后第7天,对其成年后表达无影响。
目的
本章通过制备罗哌卡因惊厥模型,运用Western Blot和QT-PCR技术对海马CaMK Ⅱ和p-CREB蛋白和基因表达水平进行检测,观察CaMK Ⅱ和p-CREB在局麻药毒性惊厥对突触发育及学习记忆的影响。
方法
选选用21日龄SD幼鼠,采用第一章实验得出的0.5%罗哌卡因致幼鼠惊厥ED95值(33.8mg/kg)腹腔注射,达到惊厥标准者入选实验,死亡或未发生惊厥者剔除实验,共选用实验鼠120只。
纳入标准：同第一章。
实验分组：单次惊厥组(R,n=40)：单次腹腔注射0.5%罗哌卡因33.8mg/kg；反复惊厥组(FR,n=40)：腹腔注射0.5%罗哌卡因33.8mg/kg,1次/d,连续5d；对照组(C,n=40)：腹腔注射注射等量生理盐水。每组根据处死时点不同(24h、3d、7d、P60)分为4个亚组(n=10)。
合成CaMKⅡ和CREB上下游引物。每亚组取5只大鼠提取总RNA、逆转录、PCR扩增,反应条件为：93。C3min,然后93℃45s,55℃1min,共循环40次,对整个升温过程进行全程荧光信号收集,绘制融解曲线并分析。定量的方法以2-AA Ct(Ct代表循环闽值)来表达基因的表达量,计算公式为A△Ct=[Ct(待测组目标基因)—Ct(内参)]—[Ct(对照组目标基因)-Ct(内参)]。
另外每组各时间点再各取5只,给予3%水合氯醛腹腔注射麻醉。迅速断头于冰上取脑,分离出双侧海马组织,用Western Blot免疫印迹法测定大鼠海马CaMKⅡ和p-CREB含量。扫描X胶片,采用Quantity one图像分析软件分析目标条带的吸光度值,以β-actin条带吸光度值为参照,两者比值为蛋白的表达水平。
统计学分析
采用SPSS13.0软件进行数据统计分析,计量资料以均数±标准差(x±s)表示。各时间点不同组间CaMKⅡ和p-CREB蛋白和基因表达水平比较采用One-Way ANOVA；两两比较采用LSD法,P<0.05为有统计学意义。
结果
CaMKⅡ在各组的表达情况
24h和3d时间点单次惊厥组CaMKⅡ基因表达明显低于对照组(P<0.05),其余时间点无明显差异。24h、3d、7d及P60时点反复惊厥组CaMKⅡ的表达均明显低于对照组(P<0.05)；24h、3d、7d及P60时点反复惊厥组CaMKⅡ的表达均明显低于单次惊厥组(P<0.05)。
CREB在各组的表达情况
单次惊厥组p-CREB在24h、3d、7d及P60时间点的表达与对照组相比均无明显差异(P>0.05)；反复惊厥组CREB在24h、3d、7d及P60时点的表达均明显低于对照组和单次惊厥组(P<0.05)
结论
1.罗哌卡因致SD幼鼠(P21日龄)毒性单次惊厥后海马CaMKⅡ在惊厥后24h、3d表达明显降低。罗哌卡因致幼鼠反复惊厥在惊厥早期及成年后CaMKⅡ表达均明显减少。
2.罗哌卡因致SD幼鼠(P21日龄)单次毒性惊厥对海马p-CREB的表达无影响,罗哌卡因致幼鼠反复毒性惊厥海马p-CREB的表达在惊厥早期和成年均明显减少。
全文结论：
1.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥影响其突触可塑性：单次及反复惊厥大鼠均表现为海马CA1区突触数目减少、突触间隙增宽、突触后致密物变薄,反复惊厥比单次惊厥所致损害程度更为严重,且持续时间延长。
2.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥影响其学习记忆能力：单次惊厥大鼠表现为一过性的学习能力障碍,而反复惊厥大鼠学习记忆能力障碍持续至成年后。
3.罗哌卡因致幼鼠(P21日龄)中枢神经毒性惊厥对其学习记忆的影响可能与突触可塑性破坏、海马突触素表达下调、CamkⅡ及p-CREB表达下调有关。
Seizure induced by Ropivacaine central nervous system toxicity is common toxicant adverse reaction. Infants and children may be at increased risk form local anesthetics compared with adults because of their special anatomy and development feature. The previous study confirm that immature rats undergoing long term or recurrent seizure, it can effect the synaptic plasticity, learning and memory.
It is the key time for development of synapses, nerve cell differentiationin migration, and fast formation of dendritic synapse in infants. Many investigations describe that other factors induced recurrent or long time seizures induced defect of learing and memory, its mechanism including ion channel, excitatory amino acid (EAA) receptor, neurotransmitter,development of synapse,the p protein synthesis, gene expression, and signal transduction. It is still uncertain if seizure induced by LAs can damage the development of nervous system, learning, and memory.
It has been repoted about LAs toxicity seizure in the early1920century. Much has been done about the mechanism and prevention and cure by anesthetists. The mechanism of LAs toxicity seizure including the unbalance of EAA-IAA, NMDA-Ca2+-NOS, dopamine, acetylcholine, M-potassium channels et al. All above receptors, neurotransmitter, ion channels participant the development and maintenance of CNS, synapses, leaning, and memory.Hippocampus is the most important place of learning, and memory, and also the place which LAs toxicity seizure take place.
Learning and memory is an higher nervous activity of brain. The two nervous activity intercommunication and influence about learning and memory. Learning means human or biology can change its performance in response to its environment, and memory means the imformation storage and output by learning activity. The nerves base of learning and memory is high plasticity of CNS, its including neural network connection, the plastic relative neurotransmitter, protein, ion channels, and receptors.
Synapses is the junction point of shape structure and functional about neuron, it's the key site of maintenance about information transport. The synapse plastic means when changing of external environment, the synapse take a adaptive change to keep the relative stable condition, the changing including form structure and function changing. The studay consider after recurrent or long time seizures, the synapses form structure was destruction in hippocampal area, the changing impact function of nervous, and its destroy level are positively correlate with learning and memory performance. The studay about LAs CNS toxicity describe that LAs inhibit synapses axon growth, induce nerve impulse attenuated, and impairment the physiologic function.
Synaptophysin is a calcium binding glucoprotein which located in presynaptic membrane of axon peripheral. Synaptophysin participate in the release procedure of ACH, glutamic acid,et al, it has a close relation with synaptic plastics. Synaptophysin is a specificity protein of synaptic vesicle, its density and distribution reflect synapsis amount, and distribution. Synaptophysin has a close relation with synaptic restitution and recognition. Synaptophysin can regulate the formation, development, and mature of neurons, and also effect differentiation of neurons by regulate the cytoskeletal protein pack. Synaptophysin has a considerable meaning to axon lengthen, synaptic linkage, and synaptic maturation. The opposite research describe that it is not necessary of synaptophysin to synaptic development, learning, and memory, and it is still uncertain of its mechanism.
Ca2+-CaMK Ⅱ-CREB path is one of the main signal of formation synaptic plasticity and learning memory of CNS. CaMK Ⅱ is a protein has a the highest content in the hippocamp, it's a binding protein of Ca2+ion, has a close correlation of LTP, it can also be called"molecular switch of learning, and memory". Lidocaine. cocaine, and procaine can inhibit LTP in hippocampal by effect of calmodulince. LAs induced CNS toxicity seizure cause calcium overload, if it can effect CaMK Ⅱ, synaptic plasticity, learning, and memory.
cAMP-responsive element binding protein (CREB) is a intracellular transcicription factor, which mediates the genetic transcription and the proteinous synthesis, plays a important role in the long term memory. It effect CREB phosphorylation induced learning,memory impairment in with acute or repeat treatment of cocaine.
We determine ED95of0.5%Ropivacaine which the dose induced CNS toxicity seizure with postnatal21days(P21) mice, and preparation single and repeated seizure mice model. The learning,memory ability test by Morris water maze (MWM) at time after seizure. Synaptic ultrastructure in hippocamp after seizure transmission electron microscope Immunohistochemical staining and western blotting methods were used in the assays of the expressive levels of synaptophysin in CA1area of hippocamp. Fluorescent quantitation polymerase chain reaction(Q-t PCR) technique was used in the assays of the exprwssive levels of CaMK Ⅱ and p-CREB gene. We detect if LAs CNS toxicity seizure will effect the synaptic plasticity, learning and memory of immature mice, through test ethology; morphology,protein, and gene level, and discuss the possible mechanism about it.
Objective
We determine the ED50, ED95of ropivacaine induced seizure through up-down and unit probability regression method.
Methods
Forty healthy P21Sprague-Dawley (SD) rats. Using sodium chloride configuration0.5%Ropivacaine. Experiment dose divide to5groups, the geometric proportion coefficient is0.8, and the initial dose is24mg/kg. A rat accept intraperitoneal injection only one time. The seizure standard according to Racine V degree (righting reflex loss, generalized tonic-clonic seizures).
grade0, normal behavior,1, facial twitches; wet dog shakes, arres t;2, head nodding, chewing;3, forelimb clonus;4, rearing, falling on forelimbs;5, falling on the side or back, hindlimb clonus. The rats seizure degree achieve V bring into experiment.
Termination of seizure by means of twitch disappear and recover of self-action(righting reflex recover). Performance according to the up-down method: The first rat treatment with0.5%Ropivacaine intraperitoneal injection dose is24mg/kg, if it appearance seizure,and the next rat will accept the down regulation dose of Ropivacaine, but if the first rat did not appearance seizure, and the next rat will accept the upper regulation dose. Record positive, negative, and all cases in per dose group. Record the latency and duration of rat that undergone seizure.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). The unit probability regression method (probit) was used toanalyze ED50and ED95of seizure induced by ropivacaine toxicity.
Results
The seizure rats were characterized by free-running decreased, restlessness, run in a hurry aimless, loss of right reflex and generalized tonic-clonic convulsions. From inject time to occurrence of seizure(latency) is4.7±0.94min, and from occurrence to terminal of seizure(duration) is26.4±6.0min. According probit method calculated, equation y=-17.89±12.78(1g dose), the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg). and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).
Conclusion
According probit method calculated, the ED50dose is25.12mg/kg(95%confidence interval is22.38-27.54mg/kg), and the ED95dose is33.79mg/kg (95%confidence interval is29.99-48.38mg/kg).
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. The learning,memory ability test by Morris water maze (MWM) at time after seizure.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=10):control group(Cgroup), single seizure group (R group), and recurrent seizure group (FR group). N group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The learning,memory ability test by Morris water maze (MWM) at time (1h～3d;5d～7d; P60～P62) after seizure in each group.
Test content①Place navigation, it used to test the learning and memory ability of the rats. The rats swim2min in the pool before the starting formal experiment. The formal test experience three days,and test1time at am and pm per day. Putting the rats into water with face toward barrel wall at different point, the software recorded the swimming image of the rats(the residence time is5s), and analyzing the swimming path, velocity, and latency. If the rats could not find the platform in120s, the operater guide the rats to the platform ang stady for5s, and recording the latency was120s.②Spatial probe:It used to test the space memory ability, spatial probe carried out at the time after thelast place navigationtest, removing the platform, putting the rats at different point into water recording the times of accrosing and the swimming time in the quadrantic which the platform was located.(4d,8d, and P62),
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). Repeat measurement F test was ued to analyze escape latency. One-Way ANOVA was used to analyze the latency at different time in different groups or in different groups at the same time. One-Way ANOVA was used to analyze the times of accrosing and the swimming time in the quadrantic which the platform was located. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
D1-3Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased (F=225.09, P<0.001; F=152.614, P<0.001; F=46.603, P<0.001). There were significant deviation of latency time in C, R and FR groups on day1-3(F=7.371, P<0.001; F=111.73, P<0.001; F=172.096, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C vs R, C vs FR groups at day1-2(P<0.05); and there was significant deviation of latency time between C vs FR, R vs FR groups at day3(P<0.05);Day4spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day4(F=43.246, P<0.001), and there was significant deviation of time accrosing in C vs R, and R vs FR groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=129.646, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)
D5～7Search platfonn latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C and R groups by train time increased (F=14.75, P<0.001:F=17.194, P<0.001),but not in FR group(F=2.777, P>0.05). There were significant deviation of latency time in C, R and FR groups on day5～7(F=169.044, P<0.001; F=235.824, P<0.001; F=285.290, P<0.001). The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day5-7(P<0.05). Day8spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=57.474, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=124.692, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not C vs R groups (P<0.05)
P60～P62Search platform latency in place navigation test:Ropivacaine induced CNS toxicity seizure effect the Search platform latency. The latency time was to shorten gradually in C, R and FR groups by train time increased(F=16.718, P<0.001; F=20.580, P<0.001; F=14.214, P<0.001)There were significant deviation of latency time in C, R and FR groups on day P60-P62((F=177.218, P<0.005; F=353.348, P<0.001; F=269.125, P<0.001) The two-two comparisons among the means were done by LSD method, there was significant deviation of latency time between C, R vs FR, but not C vs R groups at day P60-P62(P<0.05). Day P62spatial probe:①Times of accrosing in the quadrantic which the platform was located. There was significant deviation of times of accrosing in three groups on day8(F=25.327, P<0.001), and there was significant deviation of time accrosing in C and R vs FR, but not C vs R groups (P<0.05).②There was significant deviation of swimming time in the quadrantic which the platform was located in the three groups (F=120.061, P<0.001), and there was significant deviation of time accrosing in C vs FR, but not N vs R groups (P<0.05)
Conclusion
It can effect the learning and memory ability transiently with single seizures induced by Ropivacaine, but the long-term effect by repeated seizures.
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptic morphology in the hippocampal CA1area by transmission electron microscope.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP. which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=12):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days, I/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=3) according to the24h,3d,7d, and P60after covulsion or injection respective.
According the time, perfusing the rats with paraformaldehyde and glutaric dialdehyde mixed liquor. After that taking out the hippocampus quickly and putting the glutaric dialdehyde. Using the transmission electron microscope to observe the synaptic cleft, synaptic number, and PSD.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptic cleft, synaptic number, and PSD at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
The synaptic number in R group at time24and3d, was less than C group at the same time. And the synaptic number in FR group at time24h,3d and7d, was less than C group at the same time (P<0.05). The synaptic cleft in R and FR groups were reduced than it in the C group at time24and3d (P<0.05).The PSD in R and FR groups were reduced than it in the C group at time24(P<0.05). The synaptic number, synaptic cleft and PSDin Both R and FR groups were the same as in the C at time P60.
Conclusion
Ropivacaine-induced seizures effect the synaptic morphology in the hippocampal CAI area. The synaptic number reduced, synaptic clef widen and PSD thinningz atearlier period (24h,3d, and7d)
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the synaptophysin in the hippocampal by Immunohistochemical stain and western blotting method.
Methods
Thirty healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The thirty rats were randomized into three groups (n=24):control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=6) according to the24h,3d,7d, and P60after covulsion or injection respective.
According the time, perfusing the rats with paraformaldehyde liquor. Another rats without perfusing to obtain western blotting sample.After that taking out the hippocampus Using t Immunohistochemical stain and western blotting method to observe the synaptophysin content at different time.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way ANOVA was used to analyze the synaptophysin content at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
Synaptophysin content expression by Immunohistochemical stain
The synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in C group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as C group at P60. Synaptophysin content expression by western blotting
The synaptophysin content expression in R group was less than it in C group at time24h (P<0.05), and it expression in FR group was less than it in N group at time24h,3d, and7d (P<0.05). Both it in R and FR were same as N group at P60.
Conclusion
The synaptophysin content expression was reduced transiently at time3d after single seizure induced bu Ropivacaine, and it continue reduced until7d after repeat seizures.The synaptophysin content were same as normal in both single and repeat seizures at P60.
Objective
Using the ED95dose of Ropivacaine induced-seizure, preparation the single and repeated seizure rats model. Tset the CaMKⅡand p-CREB in the hippocampal by Fluorescent quantitation polymerase chain reaction technique and Western Blot..
Methods
One hundred and twenty rats healthy P21Sprague-Dawley (SD) rats, using the ED95dose of Ropivacaine induced-seizure IP, which achieve the seizure standard were chosen in the experiment, died or without seizure rats knock-out experiment.
The one hundred and twenty rats were randomized into three groups (n=40): control group(C group), single seizure group (R group), and recurrent seizure group (FR group). C group:the rats accept aequales Sodium Chloride; R:the rats accept single ED95Ropivacaine dose; FR group:the rats accept five days,1/day, ip ED95Ropivacaine dose; The three groups subdivided into4subgroups(n=10) according to the24h,3d,7d, and P60after covulsion or injection respective.
CaMK Ⅱ and p-CREB up, down-stream primer were synthesis. According the time, to obtain hippocampus sample. extracting the sumRNA, reverse transcription, PCR amplification. Using the quantitive method analysis gene expression.
statistics analysis
All the data were analyzed by the software SPSS13.0statistically, measurement data were measured by (x±s). One-Way AN OVA was used to analyze the CaMK Ⅱ and p-CREB at defferent time. After the equal check of variance the two-two comparisons among the means were done by LSD method.
Results
CaMK Ⅱ gene expression
The CaMK Ⅱ gene content expression in R group was less than it in C group at time24h and3d (P<0.05), and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05) p-CREB gene expression
The p-CREB gene content expression in R group was same as it in C group at time24h,3d,7d, and P60; and it expression in FR group was less than it in C group at time24h,3d,7d, and P60.(P<0.05)
Conclusion
The CaMK Ⅱ gene expression content expression was reduced transiently at time3d after single seizure induced by Ropivacaine, and it continue reduced until P6o after repeated seizures.The p-CREB gene content expression were same as normal in single group at all the time, but the expression were all reduced significantly in repeated group at all the time.
引文
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