摘要
目的:应用盐酸利多卡因致大鼠中毒惊厥模型,测定中毒惊厥过程中海马CA1区细胞外液谷氨酸(Glu)和γ-氨基丁酸(GABA)含量,并应用N-甲基-D-天门冬氨酸受体(NMDAR)拮抗剂MK-801,观察的NMDAR1活性以及Glu和GABA含量的变化,探讨MK-801在利多卡因致大鼠中毒惊厥过程中的作用机制。
方法:
1实验分组及动物模型的制备
22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3组,对照组(C组,6只)、利多卡因组(L组,8只)、MK-801+利多卡因组(MK-801+L组,8只)。大鼠异氟烷(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,右股静脉切开置管以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)。用微型颅钻在左侧海马CA1区(前囟[bregma]后方3.6 mm和中线左旁开2 mm处)打开一直径约2 mm的圆孔,小心挑开硬脑膜,将微透析针垂直刺入大脑皮层2 mm,以2μl/min的速度持续输注乳酸钠林格氏液。手术操作完成后,持续吸入1.5 %异氟烷维持麻醉。微透析针置入60 min后开始收集脑组织微透析液,每隔10 min收集一次,首先收集20 min,之后按照实验分组给与不同的处理。(1)C组:腹腔注射生理盐水0.5 mg/kg,30 min后继续收集透析液40 min,于-20℃冰箱内保存待测。(2)L组:腹腔注射生理盐水0.5 mg/kg,30 min后2%利多卡因2 mg静注,然后以4 mg·kg-1·min-1的速度经股静脉持续泵入,C组以相同速度静注或泵入乳酸钠林格氏液。观察惊厥波(EEG上出现振幅超过100μV宽大快速的棘波并持续10 s以上)出现后,停止泵入利多卡因,其他两组泵入利多卡因或乳酸钠林格氏液时间与所求得的预试验中L组输入利多卡因时间的95%可信区间相当,记录惊厥波发生及持续时间,呼吸抑制及持续时间,余操作与C组相同。(3)MK-801+L组:静脉泵入利多卡因前30 min腹腔注射MK-801 0.5 mg/kg,余操作与L组相同。各组分别在微透析针置入60分钟及L组开始出现惊厥波时(其他两组于相应时间点)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),呼吸抑制期间给与辅助呼吸,以维持PaO2和PaCO2在正常范围。
2标本的采集及检测方法
2.1 NMDAR1表达的检测
实验进行2.5小时后,大鼠深麻醉下经心脏用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,冠状切取前囟部位3～13mm的组织,浸入4%多聚甲醛溶液中保存备用。之后酒精脱水,二甲苯透明,浸蜡,包埋,连续冠状切片(片厚5μm),用抗NMDAR1多克隆抗体进行免疫组化染色(ABC法)。在右侧海马CA1区相同部位分别取4个相邻的40倍视野,以胞浆或胞核染成棕黄色作为阳性细胞,计阳性细胞数,计算出平均数和标准差,数据以x_±s表示,采用SPSS 11.5软件进行方差分析及t检验, P < 0. 05表示差异有显著性。
2.2海马CA1区谷氨酸和GABA浓度的检测
利用柱前衍生高效液相色谱-荧光法测定出各种浓度的谷氨酸和GABA标准品的峰面积,并绘制标准曲线、求得回归方程。利用该方法测出每个样品中上述两种氨基酸的峰面积,根据回归方程求得氨基酸的浓度。
结果:
1各组间体重、心率、微透析针置入并稳定60 min及L组开始出现惊厥波时(其他两组于相应时间点)两个时间点动脉血气指标比较差异无统计学意义。
2各组间惊厥波出现时间、呼吸抑制出现时间及持续时间的比较
C组未见惊厥波,后两组均见惊厥波出现,其中L组每只均见惊厥波,而MK-801组只有两只出现了惊厥波。与L组比较,MK-801+L组呼吸抑制出现较晚(P<0.05),呼吸抑制持续时间较短(P < 0.05)。
3各组间海马CA1区NMDAR1表达的比较
与C组比较,L组、MK-801+L组NMDAR1免疫阳性细胞数均显著增多(P < 0.05);与L组比较,MK-801+L组NMDAR1免疫阳性细胞数显著减少(P < 0.05)。
4各组间海马CA1区Glu和GABA浓度的比较
与C组比较,相同时间点L组Glu浓度显著升高(P < 0.05);与L组比较,相同时间点MK-801+L组Glu浓度显著降低(P < 0.05)。
各组间相同时间点与C组相应时间点比较,T2时L组GABA浓度升高(P < 0.05);余差别无统计学意义。
结论:MK-801能够抑制利多卡因中毒惊厥效应,机制与NMDA受体活性降低有关。
Objective: Using the model of seizure induced by hydrochloride lidocaine to detect the concentration of glutmate (Glu) and GABA in extracellular fluid of the brain in rats during the convulsion; and the N-methy-D-aspartate receptor (NMDAR) antagonist MK-801 was administered to observe the NMDAR’s activity and the variations of Glu and GABA’s concentration, and to investigate the effect of MK-801 on the brain against convulsion induced by lidocaine in rats and the underlying mechanism.
Methods: Twenty-two male Wistar rats weighing 200±50g were randomly divided into 3 groups : controling group/ group C (n=6); Lidocaine group / group L (n=8); MK-801+lidocaine group/group MK-801+L (n=8). The model of seizure was induced by lidocaine and confirmed by EEG (five electrodes were implanted under the epicranium of rats respectively) on which multiple sharp waves were seen. Anesthesia was induced with 5% isoflurane in oxygen in a container. The trachea was cannulated via endotracheal intubation. Needle electrodes were placed for recording lead II of the electrocardiogram (ECG) and frontooccipital electroencephalogram (EEG). During surgical preparation, anesthesia was maintained with 3% isoflurane in oxygen. A cannula was placed through the right femoral vein into the vena cava for local anesthetic and sodium lactated Ringer’s solution infusion. EEG, and ECG were continuously recorded.
A constant infusion of sodium lactated Ringer’s solution was started at the rate of 1 ml·h-1. A microdialysis probe was pricked 2 mm depth into the pallium of the left hippocampal CA1 field of a rat, and the sodium lactated Ringer’s solution exchanged was drived at the flow rate of 2μl·min-1 . After surgical preparation, anesthesia was maintained with 1.5% isoflurane in oxygen. 60 mins later, the dialysate was collected every 10 mins, after the first 20 mins’collection , the rats were dealt differrently according to subgroups. (1) group C: Normal saline was injected into abdominal cavity at a dose of 0.5mg·kg-1, 30 mins later, dialysate was collected for about 40 mins continiously. (2) group L: Normal saline was injected into abdominal cavity at a dose of 0.5ml·kg-1, 30 mins later, lidocaine hydrochloride with the concentration of 2% was quickly infused about 2 mg ,then it was continuously infused at a rate of 4 mg·kg-1·min-1 until the multiple sharp waves were seen on a relatively calm backgroud on EEG (confirmed by two independent persons). Sodium lactated Ringer’s solution was infused in group C at the same time. Then, the infusion of lidocaine was stopped and the sodium lactated Ringer’s solution was infused at the formal rate. At the same time the emergency time of convulsion and the emergency time and duration of respiratory depression were recorded. (3) group MK-801+L: All of the steps were same with group L except that MK-801 was injected via peritoneal injection at a dose of 0.5mg·kg-1 30 mins before lidocaine’s infusion. Arterial blood was collected when the microdialysis probe was implanted into the pallium of the left hippocampal CA1 field of a rat and when the high spike wave appeared on a relatively calm backgroud in EEG in group L to detect PaO2 and PaCO2.
After the experiments, all rats were fixed with 4% PFA perfused via ascending aorta. Then, the rats’brain were carefully sampled and stored under 4℃. What’s more, specimans of the brain were used in immunohistochemical test to observe the expression of N-methy-D-aspartate receptor 1 (NMDAR1). The dialysate was stored under -20℃and would be used to determine the concentration of Glu and GABA by high-performance liquid chromatography (HPLC).
Result
1 There are no differences of the rats’weight、HR、PaO2 and PaCO2 in the three groups.
2 Comparision of the emergency time of convulsion wave and the emergency time and duration of respiratory depresion. No high spike waves appeared on the EEG in group C.
Each of the rats in group L was found of high spike waves appeared in EEG. But only two of the rats in group MK-801+L was found high spike waves appeared in EEG. Compared with group L, resperatory depresion occurred later in group MK-801 (P<0.05), and the duration of resperatory depresion lasted shorter in group MK-801 (P<0.05).
3 The expression of NMDAR1 level in hippocampal CA1 field
Compared with group C , the expression of NMDAR1 level in hippocampal CA1 field was significantly higher in both group L and group MK-801+L (P<0.05) ; Compared with group L , the expression of NMDAR1 level in hippocampal CA1 field was significantly lower in group MK-801+L (P<0.05).
4 The concentration of Glu in dialysate
The average of the Glu’s concentration of the first two points was definited as a control value .
Compared with group C, the concentrations of the corresponding time points are significant higher in group L (P<0.05); Compared with group L, the concentrations of the corresponding time points are significant lower in group MK-801+L (P<0.05).
5 The concentration of GABA in dialysate
The average of the concentration GABA of the first two points was definited as a control value .
Compared with the concentrations of the corresponding time point in group C, only the concentration of 40 min is higher in group L (P<0.05), and there are no statistical significance during every other time points of the three groups (P>0.05).
Conclusion
MK-801 play a positive role in the convulsion induced by lidocaine in the hippocampal CA1 field of the rats’brain, the decrease of the activity of NMDAR is involved in the mechanism.
引文
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