【作者】 乔玉敏；

【导师】 张山；

【作者基本信息】 河北医科大学 ， 麻醉学， 2008， 硕士

【摘要】 目的:应用利多卡因致大鼠惊厥模型,在儿茶酚胺合成限速酶酪氨酸羟化酶(tyrosine hydroxylase,TH)抑制剂α-甲基-ρ-酪氨酸(alpha-methyl-ρ-tyrosine,AMPT)以及去甲肾上腺素转运蛋白(Norepinephrine transporter,NET)抑制剂地昔帕明的作用下,观察TH免疫活性以及测定惊厥过程中纹状体部位细胞外液去甲肾上腺素(norepinephrine,NE)与多巴胺(dopamine, DA)含量,探讨去甲肾上腺素与多巴胺在利多卡因致大鼠惊厥过程中的作用。方法:1实验分组及动物模型制备24只Wistar雄性大鼠,体重250±20 g (河北医科大学实验动物中心提供)。随机分为4组,每组6只。大鼠5%异氟烷(isoflurane)麻醉后气管插管,手术过程中持续吸入3 %异氟烷维持麻醉,右股静脉置管以1ml/h的速度输入乳酸钠林格氏液。刺入电极持续监测心电图(ECG)和脑电图(EEG)。纹状体定位(A,- 1.2 cm; M,-2.8 cm; D, 3.5 mm)、钻颅孔、划破硬膜,微透析针以此坐标点垂直刺入硬膜下脑组织。操作结束1小时脑电图波形稳定后,以2μl/min的速度注入乳酸钠林格氏液,收集脑组织微透析液20分钟,之后按照实验分组给予不同处理。L组:注射生理盐水1ml,30分钟后单次泵入2%利多卡因0.1 ml,见脑电图无异常棘波继续以4mg·kg-1·min-1速度持续泵入,直到EEG上出现振幅超过100μV宽大快速的簇状棘波并持续10秒以上,停止输入利多卡因。A+L组:在泵入利多卡因前30分钟经腹腔注射α-甲基-ρ-酪氨酸( alpha-methyl-ρ-tyrosine AMPT)溶液250mg/kg,输入利多卡因的量和速度以及停止时间均与L组相同。D+L组:在泵入利多卡因前30分钟,腹腔注射地昔帕明20mg/kg,余操作同L组。C组:与L组相比仅在泵入利多卡因时泵入相同剂量的乳酸钠林格氏液,余操作均与其相同。记录惊厥波发生及持续时间、呼吸抑制及持续时间。分别在微透析针置入60分钟及L组出现惊厥波时(其他三组于相应时间点)两个时间点采集股动脉血测动脉血氧分压(PaO2)和二氧化碳分压(PaCO2),呼吸抑制期间给予辅助呼吸,维持PaO2和PaCO2在正常范围。2标本采集操作结束1小时待脑电图波形稳定后(T1)收集脑微透析液20分钟,输入利多卡因后(T2)再收集20分钟。实验后,大鼠深麻醉经左心0.9%生理盐水和4%多聚甲醛溶液各50 ml灌流、固定,完整取出脑组织,浸入4%多聚甲醛溶液中保存备用。3标本的检测方法3.1免疫组织化学染色法脑组织切片5μm、脱蜡,经抗原修复滴加试剂、抗体,经DAB染色在显微镜下观察显色,每个切片在高倍镜下取4个视野进行TH阳性细胞计数并计算平均值。3.2高效液相色谱—荧光检测法测定脑微透析液NE、DA的含量样品测定:绘制标准曲线,求得去甲肾上腺素、盐酸多巴胺的浓度与峰面积回归方程,对样品进行测定求得去甲肾上腺素、盐酸多巴胺的峰面积,检测探针回收率,按照回归方程、探针回收率及盐酸多巴胺与多巴胺的换算关系计算大鼠脑纹状体去甲肾上腺素、多巴胺的实际含量。结果: 1各组呼吸抑制持续时间比较C组和A+L组均无呼吸抑制;L组呼吸抑制持续时间为21.83±1.69分钟;D+L组呼吸抑制持续时间为14.67±1.37分钟。与L组比较,D+L组呼吸抑制持续时间明显减少(P<0.05)。2各组棘波持续时间比较C组无棘波出现,L组棘波持续时间为25.67±0.75分钟;A+L组棘波持续时间为8.33±1.13分钟;D+L组棘波持续时间为13.5±2.07分钟。与L组比较,A+L组、D+L组棘波持续时间均减少(P<0.05);与A+L组比较,D+L组棘波持续时间延长(P<0.05)。3 C组、L组、A+L组大鼠大脑黑质部位酪氨酸羟化酶阳性细胞数比较C组阳性细胞数为34.83±1.47个;L组阳性细胞数为84.33±3.78个;A+L组阳性细胞数为54.00±1.55个。与C组比较,L组阳性细胞数明显增加,A+L组阳性细胞数亦增加(P<0.05);与L组比较,A+L组阳性细胞数减少(P<0.05)。4 C组、L组、A+L组大鼠脑细胞外液T2去甲肾上腺素、多巴胺的含量比较C组去甲肾上腺素、多巴胺的含量分别为351.78±10.15 ng/ml,316.80±6.62 ng/ml,L组去甲肾上腺素、多巴胺的含量分别为524.61±17.85 ng/ml,758.42±15.46 ng/ml,A+L组420.68±5.79 ng/ml,483.61±6.24 ng/ml与C组比较,L组去甲肾上腺素、多巴胺的含量均增加(P<0.05);A+L组去甲肾上腺素、多巴胺的含量均增加(P<0.05);与L组比较,A+L组去甲肾上腺素、多巴胺的含量均降低(P<0.05)。5 TH阳性细胞数与C组、L组、A+L组棘波持续时间呈正相关,直线回归方程为Y=0.52X-18.65 R2=0.99。6三组TH阳性细胞数与T2大鼠脑细胞外液去甲肾上腺素、多巴胺的含量呈正相关关系,直线回归方程分别为Y=3.5X+230.28 R2=1.00;Y=8.87X+7.47 R2=1.00。7 T2大鼠脑细胞外液去甲肾上腺素、多巴胺的含量与棘波持续时间呈正相关关系,直线回归方程分别为Y=0.15X-52.33 R2=0.98;Y=0.06X-19.11 R2=0.99。8 L组T1与T2去甲肾上腺素、多巴胺含量的比较L组T1去甲肾上腺素含量358.9±9.12ng/ml,多巴胺319.35±6.64ng/ml, T2去甲肾上腺素、多巴胺含量与T1比较明显升高(P<0.05)9 A+L组T1与T2去甲肾上腺素、多巴胺含量的比较A+L组T1去甲肾上腺素含量357.63±10.58ng/ml,多巴胺319.83±8.58ng/ml,T2去甲肾上腺素、多巴胺含量与T1比较明显升高(P<0.05)结论:1利多卡因致大鼠惊厥过程中纹状体脑细胞外液NE、DA均升高,其原因可能是神经元TH免疫活性增强。2 DA在利多卡因致大鼠惊厥过程中起着更重要的作用3 NE对利多卡因致大鼠惊厥有抑制作用。更多还原

【Abstract】 Objective: In order to determine the contention of NE and DA in extracellular fluid in striatum of rats with convulsion,we make a model of lidocaine-induced convulsion. TH immunoactivity and change of concentration of NE and DA are observed, AMPT and desipramine were injected to investigate different role of norepinephrine and dopamine in the process of lidocaine-induced convulsion in striatum in rats.Methods: Twenty-four male wistar rats weighing 250±20mg were randomly divided into 4 groups(n=6 . After anesthetized by isoflurane, a rat was placed in the stereotaxic frame and a microdialysis probe was placed into the striatum (A–1.2cm, M–2.8cm, D 3.5mm, from bregma and dural surface). Microdialysis samples were collected per 20 min after EEG waves were stable. (1) group L: After saline was injected i.p, lidocaine was single pumped into femoral vein for 30 min, and pumped at a rate of 4mg·kg-1·min-1 if multiple high-voltage spikes could not be seen. (2) group C: The process was same with group L except lactated Ringer Solution was pumped into femoral vein. (3) group A+L: The process was same with group L except AMPT was pumped into femoral vein. (4) group D+L: The process was same with group L except desipramine was pumped into femoral vein. After collecting microdialysis samples,rats were perfused and fixed through cardia. Tissue sections of brain tissue were underwent antigen recovering and droping agent and DAB, TH positive immunoactive neurons were observed under high power field of optical microscope. The concentrations of extracellular norepinephrine and dopamine in striatum and the recolleting rate of microdialysis probe were detected by the method of HPLC-fluorescence, according to recolleting rate and reforming relation of dopamine hydrochloride with dopamine to calculate concentrations of NE and DA in striatum in rats.Results:1 Results of respiratory depression and spikes Group D+L lasting time of respiratory depression was significantly shortencompared with group L (P<0.05). Group A+L and group D+L lasting time of multiple high-voltage spikes were significantly shorten compared with group L (P<0.05 ).2 Results of TH immunoactivity in substantia nigraTH positive immunoactive neurons in group A+L were significantly decreased compared with group L (P<0.05). TH positive immunoactive neurons in group A+L were significantly increased compared with group C (P<0.05).3 Concentration of NE and DA in T2Concentrations of NE and DA in group A+L were significantly decreased compared with group L (P<0.05)., Concentrations of NE and DA in group A+L were significantly increased compared with group C (P<0.05 each). There were significant differences among them (P<0.05).4 Relation of each indexTH positive immunoactive neurons were directly correlated with spikes lasting time, Y=0.52X-18.65 R2=0.99. TH positive immunoactive neurons were directly correlated with concentrations of NE and DA in T2, Y=3.5X+230.28 R2=1.00 Y=8.87X+7.47 R2=1.00. Spikes lasting time were directly correlated with concentrations of NE and DA in T2, Y=0.15X-52.33 R2=0.99; Y=0.06X-19.11 R2=0.99.5 Concentrations of NE and DA in T2 compared with T1Concentrations of NE and DA in T2 were significantly increased compared with that in T1 in group L(P<0.05). Concentrations of NE and DA in T2 were significantly increased compared with that in T1 in group A+L (P<0.05 each).Conclusion:1 Concentrations of NE and DA in extracellular fluid in striatum in rats were increased during lidocain-induced convulsion. The increase of NE and DA may be attribute to activation of TH .2 DA play a importent role in convulsion induced by lidocaine.3 NE may play a depressant role in convulsion induced by lidocaine.更多还原