罗哌卡因、布比卡因和利多卡因对小鼠中枢神经系统毒性作用的比较

背景有证据表明罗哌卡因导致的心脏毒性比相同剂量的布比卡因低,但布比卡因脂溶性高,且作用强度大,临床上只需较小的剂量即可.中枢神经系统明显的神经毒性作用(如惊厥)往往是心脏毒性产生的先兆,半数致死剂量和半数致惊厥剂量的比值能够反应局麻药的安全性.目的确定罗哌卡因、布比卡因、利多卡因的半数致惊厥剂量和半数致死量,比较在引起50%致惊厥反应率时3种酰胺类局部麻醉药对中枢神经系统的毒性作用.设计随机对照,3个实验各自独立.单位华中科技大学同济医学院附属同济医院麻醉科.材料实验于2002-07/12在武汉华中科技大学同济医学院附属同济医院麻醉科实验室完成.选取1月龄雄性昆明小白鼠310只,清洁级.方法①局麻药剂量与致惊厥率及致死率关系的确定选择240只小鼠,确定罗哌卡因的量效关系时采取的小鼠数量为50只,分为76.80,68.69,61.44,49.15,31.46 mg/kg 5种剂量,10只/剂量组;确定布比卡因的量效关系时采取的小鼠数量为90只,分为50.00,47.29,44.72,42.29,40.00,35.78,32.00,28.62,25.60 mg/kg 9种剂量,10只/剂量组;确定利多卡因的量效关系时采取的小鼠数量为100只,分为183.11,163.77,146.48,131.02,117.19,93.75,75.00,60.00,48.00,38.40 mg/kg 10种剂量,10只/剂量组.不同剂量的局麻药腹腔注射时,尽量使每种麻醉药致各组小鼠的惊厥率和致死率对称分布在50%左右,给药5 min后记录各组的致惊厥率和死亡率.使用Probit法将量效曲线直线化,建立3种局麻药的对数剂量一概率单位的回归直线方程,确定各自的量效关系.②不同剂量利多卡因对小鼠惊厥时间和大脑c-Fos表达的影响选择40只小鼠,随机分成4组空白对照组、30%致惊厥剂量组、60%致惊厥剂量组、90%致惊厥剂量组,10只/组.空白对照组腹腹腔注射生理盐水,利多卡因各剂量组依次腹腔注射47,57,73 mg/kg的利多卡因,观察并记录各组小鼠惊厥的持续时间.将发生惊厥的小鼠妥善标记,2 h后制作冰冻冠状切片,检测神经元核蛋白c-Fos的表达,镜下计算不同区域表达c-Fos的棕黄色颗粒数目,每个颗粒代表1个神经元.③50%致惊厥剂量的局麻药对小鼠毒性反应的测定选择30只小鼠,随机分成3组罗哌卡因组、布比卡因组、利多卡因组,10只/组.各组依次腹腔注射50%致惊厥剂量的对应局麻药后,对发生惊厥的小鼠观察其惊厥持续时间,2 h后制作冰冻冠状切片,免疫组织化学ABC法检测神经元核蛋白c-Fos的表达.主要观察指标观察小鼠对腹腔注射不同局麻药的反应,记录惊厥持续时间,使用免疫组织化学方法检测小鼠脑组织c-Fos蛋白的表达.结果实验选用310只小鼠,全部进入结果分析.①各种局麻药不同剂量与惊厥及死亡率的关系利多卡因、布比卡因、罗哌卡因的治疗指数(半数致死量/50%致惊厥剂量)分别为2.89,1.48,1.34.②利多卡因致小鼠惊厥后脑组织c-Fos蛋白的表达情况表达c-Fos蛋白的神经元主要分布在侧脑室以下,第3脑室周围的丘脑区域;正中隆起以上,第3脑室旁边的下丘脑区域;以及下侧部皮质的梨状皮质与杏仁体.③不同剂量利多卡因致小鼠惊厥持续时间和c-Fos阳性神经元数目的比较与空白对照组比较,30%,60%,90%致惊厥剂量组的惊厥持续时间呈逐渐上升趋势(P均＜0.05),杏仁体表达的c-Fos神经元数亦呈逐渐上升趋势(P均＜0.05).④各种局麻药50%致惊厥剂量对小鼠毒性反应的测定结果利多卡因组、布比卡因组引起的惊厥时间和梨状皮质及杏仁体c-Fos阳性神经元数基本相似(P＞0.05),而罗哌卡因均显著高于利多卡因组和布比卡因组(P＜0.05).结论罗哌卡因在临床麻醉中的使用剂量较少超过布比卡因,很少达到其致惊厥剂量,具有较高的安全性.但一旦发生中枢毒性反应,罗哌卡因导致的惊厥则会更加严重,可能与其脂溶性低,吸收后分布容积小,血浆浓度高有关.

Effect of neurotoxicity of ropivacaine, bupivacaine and lidocaine on central nervous system of mice

BACKGROUND: It is demonstrated that ropivacaine has less toxicity than bupivacaine, but bupivacaine has higher liposolubility and efficacy, so a less dose of bupivacaine is needed in clinical comparing with ropivacaine. Serious convulsion is usually followed by cardiotoxicity induced by local anesthetics. The ratio of medial lethal dose (CD50) and median convulsant doses (LD50) is usually used to assess the comparative safety of local anesthetics.OBJECTIVE: To establish CD50 and LD50 of 2% lidocaine, 0.75% bupivacaine and 0.75% ropivacaine for Kunming mice and select proper indicator for neurotoxicity, then to compare neurotoxicity of the three local anesthetics on central nervous system.DESIGN: Randomized and controlled study.SETTING: Department of Anesthesiology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: This study was carried out from July to December in 2002 in the Laboratory of Anesthesiology, Tongji Hospital, Tonji Medical College, Huazhong University of Science and Technology. Totally 310Kunming mice aged of 1-month with clean grade were enrolled in this study.METHODS: ① To determine the relation of dose of local anesthetics with conclusion rate and death rate in mice. Todetermine the dose-effect relationship for ropivacaine, 50 mice were selected and divided into 5 groups with 10rates in each group who received dose of 76.80, 68.69, 61.44,49.15, 31.46 mg/kg respectively. For bupivacaine, 90 mice were divided into 9 groups, with 10 rates in each group who received intraperitoneal dose of 50.00, 47.29, 44.72, 42.29, 40.00, 35.78, 32.00, 28.62, 25.60 mg/kg respectively. For lidocaine, 100 mice were divided into 10 groups,with10rates in each group who received dose of 183.11, 163.77, 146.48,131.02, 117.19, 93.75, 75.00, 60.00, 48.00, 38.40 mg/kg respectively. For each local anes thetic, the rates of convulsion or death were tried to distribute on both sides of 50% symmetrically. On the dose-response curve, 4or 5 well-spaced points were obtained for probit analysis to determine CD50 and LD50 of each agent. ② The effect of different dose of lidocaine on conclusion duration and c-fos expression in brain with different doses.Forty mice were divided into 4 groups with 10 rates in each group who received 0, 30%, 60% and 90% convulsant doses of lidocaine intraperitoneally. The duration of convulsion were recorded carefully for the convulsant mice that should be marked correctly for next procedure. Two hours later, the convulsant mice were anesthetized deeply and fixed by transcardiac perfusion for Immunohistochemistry to detect c-Fos expression. ③ Comparison of neurotoxicity induced by CD50 of three agents.Thirty mice were randomly divided into 3 groups with 10 rates in each group who received intraperitoneally CD50 of lidocaine, bupivacaine and ropivacaine respectively. The duration of convulsion and the number of neurons expressed with c-Fos in mice brain were compared among these three groups.MAIN OUTCOME MEASURES: The response of mice to intraperitoneal local anesthestics, duration of convusion and c-Fos expression using immunohistochemistry methods.RESULTS: Date of totally 310 mice was entered into final results analysis. ① The relation of dose of local anesthetics and conclusion rate or death rate in mice. The therapeutic index (LD50/CD50) of 2% lidocaine,0.75% bupivacaine and 0.75% ropivacaine were 2.89, 1.48 and 1.34, respectively. ② c-Fos expression induced by lidocaine in mice brain: The cFos expression in mice brain was mainly distributed in three zones-thalamencephal, hypothalamus, amydyla and pyriform cortex. ③ Compare of the duration of convulsion and number of neurons with c-Fos expression induced by different dose oflidocaine. Compared with control group, the duration of convulsion and number of neurons with c-Fos expression in amydyla and pyriform cortex all increased significantly in CD30, CD60and CD90 group (P ＜ 0.05). ④ Neurotoxicity induced by CD50 of lidoacaine, bupivacaine and ropivacasine The duration of convulsion and expression of c-fos in amydyla and pyriform cortex were significantly increased in ropivacaine group compared to bupivacaine or lidocaine group intraperitoneally (P ＜ 0.05). There were no significant differences in the duration of convulsion and expression of c-Fos between lidocaine and bupivacaine group (P ＞ 0.05).CONCLUSION: Compared with bupivacaine, ropivacaine produced less toxicity when identical dose was used in clinic. It is indicated that if an accidental convulsion induced by ropivacaine, it may be more severe than that induced by correspondent either lidocaine or bupivacaine. It may be the reason that ropivacaine have less lipid solubility, absorbed easily from this tissue compartment, and to get a high concentration in blood.