氟哌利多对大鼠脑缺血海马CA1区锥体细胞持续钠通道电流的影响

背景脑缺血后离子通道通透性的异常和神经细胞内外离子平衡的紊乱是缺血性脑损伤的重要因素,钠通道激活引起的去极化是脑缺血损伤的始动环节.目的采用膜片钳技术测定氟哌利多对脑缺血海马CA1区锥体细胞持续性钠通道电流的影响,分析氟哌利多是否对脑缺血损伤产生保护?设计随机对照动物实验.单位上海交通大学附属第六人民医院麻醉科,上海交通大学附属第一人民医院麻醉科.材料实验于2002-04/2003-04在上海交通大学附属第一人民医院麻醉实验室完成.选择出生10～14 d未断乳的SD大鼠14只,每只鼠各选择2个海马CA1区细胞,共28个细胞,随机分为4组缺血对照组、氟哌利多3 μmol/L组、氟哌利多10 μmol/L组、氟哌利多30μmol/L组,7个/组. 方法酶消化法急性分离全部大鼠脑海马CA1区锥体细胞,通过低氧和无糖法制备神经元缺血模型.选择贴壁良好、呈三角形或星形、胞体较亮,折光性良好、突起明显、胞浆均匀一致、核仁明显的细胞用于实验.采用"Y-tube"系统快速给药,氟哌利多3,10,30 μmol/L组各自给予氟哌利多3,10,30μmol/L,缺血对照组不给药.全细胞膜片钳技术记录各组持续钠电流的基础值以及缺血3 min和5 min时钠通道电流的变化.主要观察指标①脑海马CA1区神经元正常持续钠电流的记录.②脑缺血时海马CA1区神经元持续性钠电流的记录.③不同浓度氟哌利多对脑缺血时持续性钠电流的影响.结果实验选用14只大鼠脑海马CA1区的28个细胞,全部进入结果分析.①脑海马CA1区神经元正常持续钠电流的记录使用钙通道阻滞剂CdCl2 0.5 mmol/L及钾通道阻滞剂TEA 20 mmol/L,在钳制电压-105 mV、刺激电压-30 mV下给予长为400 ms的方波刺激,可记录到一个较小的、激活较晚且持续时间较长的内向电流,经河豚毒素阻断证实为持续性钠电流.②脑缺血时海马CA1区神经元持续性钠电流的记录缺血对照组缺血3 min时持续钠电流增加为正常情况的(1.60±0.21)倍,缺血5 min时持续钠电流增加为正常情况的(2.87±0.45)倍,差异显著(P＜0.05).③不同浓度氟哌利多对脑缺血时持续性钠电流的影响缺血对照组、氟哌利多3,10,30 μmol/L组持续钠电流的基础值分别是(77.42±15.17)pA,(87.44±21.56)pA,(84.13±20.06)pA,(80.22±19.30)pA,组间比较差异无显著性意义.缺血5min后氟哌利多3,10,30μmol/L组持续性钠电流分别为(105.36±17.16)pA,(94.74±18.88)pA,(84.88±13.94)pA,明显低于缺血对照组(218.31±29.34)pA.结论在钳制电压-105mV、刺激电压-30mV条件下,脑缺血损伤时持续性钠电流增加,氟哌利多可能通过抑制持续性钠电流的增强而发挥神经元保护作用.

Effects of droperidol on persistent sodium channel currents of pyramidal cell in hippocampal CA1 area of rats with cerebral ischemia

BACKGROUND: Both abnormal permeability of ionic channel and disturbance of ionic balance between inside and outside nerve cell are key factors for ischemic brain injury after ischemia. Depolarization induced by activation of sodium channel is starting link for cerebral ischemic injury.OBJECTIVE: To study the effects of droperidol on persistent sodium channel currents of pyramidal cell in hippocampal CA1 area of rats with cerebral ischemia with patch clamp technique so as to analyze whether droperidol can protect cerebral ischemic injury.DESIGN: Randomized controlled animal study.SETTING: Department of Anesthesiology of the Sixth People's Hospital Affiliated to Shanghai Jiaotong University and Department of Anesthesiology of the First People's Hospital Affiliated to Shanghai Jiaotong University.MATERIALS: The experiment was carried out at the Department of Anesthesiology of the First People's Hospital Affiliated to Shanghai Jiaotong University from April 2002 to April 2003. Totally 14 SD rats, aging 10-14days, without ablactation, were selected. Two cells in hippocampal CA1area of each rat were collected, totally 28 cells were divided into 4 groups:ischemic control group, 3 μmol/L droperidol group, 10 μmol/L droperidol group and 30 μmol/L droperidol group, with 7 cells in each group.METHODS: Pyramidal cells in hippocampal CA1 area were separated with digested enzyme method, and ischemic model of neuron was established through hypoxia and no sugar method. Cells were selected with the following conclusion criteria: well adherent wall, triangle or starry shape,bright soma, well refraction, obvious apophysis, steady plasma, and transparent nucleolus. Y-tube system was used for rapid medication. 3, 10 and 30 μmol/L droperidol were given to rats in 3, 10 and 30 μmol/L droperidols respectively, but rats in ischemic control group were not given any medicine. Whole-cell patch-clamp was used to recorded basic value of persistent sodium currents and changes of sodium channel currents during 3-minute and 5-minute ischemia.MAIN OUTCOME MEASURES: ① Record of normal persistent sodium current of neuron in cerebral hippocampal CA1 area; ② Record of persistent sodium current of neuron in cerebral hippocampal CA1 area during cerebral ischemia; ③ Effect of droperidol in various concentrations on persistent sodium current of neuron in cerebral hippocampal CA1 area during cerebral ischemia.RESULTS: Totally 28 cells in cerebral hippocampal CA1 area of 14 rats were entered the final analysis. ① Record of normal persistent sodium current of neuron in cerebral hippocampal CA1 area: 0.5 mmol/L CdCl2 calcium channel blocking agent and 20 mmol/L TEA kalium channel blocking agent were used to perform 400 ms square-wave stimulation under -105 mV claw voltage and -30 mV stimulated voltage. Introversion current,slight, late activation and lasting for a long time, was recorded and deter mined as persistent sodium currents by blocking toxin of puffer fish. ② Record of persistent sodium current of neuron in cerebral hippocampal CA1 area during cerebral ischemia: After 3-minute ischemia, persistent sodium currents in ischemic control group was increased as (1.60±0.21) times as that in normal group, and was (2.87 ±0.45) times after 5-minute ischemia. The difference was significant (P ＜ 0.05). ③ Effect of droperidol at various concentrations on persistent sodium current of neuron in cerebral hippocampal CA1 area during cerebral ischemia: Basic values of persistent sodium currents were (77.42±15.17) pA, (87.44±21.56) pA, (84.13±20.06) pA and (80.22±19.30) pA in ischemic control, 3, 10 and 30 μmol/L droperidol groups respectively, and the differences among groups were not significant. After 5-minute ischemia, values of persistent sodium currents were (105.36±17.16) pA, (94.74±18.88) pA and (84.88±13.94) pA in 3, 10 and 30 μmol/L droperidol groups respectively, which were obviously lower than that in the ischemic control group (218.31±29.34) pA.CONCLUSION: Persistent sodium currents increase under -105 mV claw voltage and -30 mV stimulated voltage during cerebral ischemic injury. Droperi dol can protect neuron by inhibiting the increase of persistent sodium current.