电针水沟穴对脑缺血再灌注大鼠海马CA1区L-Ca2+通道的影响

Effect of Electroacupuncture at Shuigou Acupoint on L-type Voltage-gated Calcium Channels in hippocampal CA1 region of MCAO rats

目的:通过观察电针水沟穴对脑缺血再灌注大鼠神经功能恢复及海马CA1区L-Ca^2+通道神经电生理学特性的影响,探讨电针水沟穴治疗缺血性脑卒中的脑保护机制。方法:选用30只健康雄性成年Wistar大鼠,采用大脑中动脉线栓法建立大脑中动脉缺血(MCAO)模型。大鼠苏醒后按照Zausinger六分法进行神经功能缺损评分,选择1~3分大鼠入组,按照随机数字表法分为水沟穴组、非穴组和对照组,每组10只。水沟穴组大鼠在清醒1h后接受电针水沟穴治疗,治疗参数为连续波方波,波宽0.2ms,电流强度1mA,频率2Hz,刺激时间10min,每日1次,共治疗7d。非穴组大鼠选取水沟穴左侧旁开0.5cm进行针刺,与水沟穴组实施同等条件的电针干预。对照组造模成功后实施同等条件的抓取与固定,不实施任何干预。治疗结束后采用神经功能缺损评分评估大鼠的神经功能,随后采用酶急性分离技术获取脑缺血再灌注大鼠海马CA1区椎体神经元细胞,单细胞膜片钳技术检测海马CA1区L-Ca^2+通道的电流幅度和平均开放时间常数。结果:①神经功能缺损评分方面,干预前3组神经功能缺损评分差异无统计学意义(P>0.05),说明3组造模大鼠针刺干预前神经功能缺损评分基线一致,具有可比性。干预后,水沟穴组神经功能缺损评分明显优于干预前(P<0.05);而非穴组和对照组神经功能缺损评分与干预前比较均无统计学差异(P>0.05);水沟穴组干预后的神经功能缺损评分明显优于对照组和非穴组,差异均有统计学意义(P<0.05);非穴组干预后的神经功能缺损评分与对照组比较,差异无统计学意义(P>0.05)。②水沟穴组与对照组大鼠L-Ca^2+电流幅度比较差异有统计学意义(P<0.05);水沟穴组与非穴组L-Ca^2+电流幅度比较,差异具有统计学意义(P<0.05),水沟穴组均值小于非穴组;非穴组与对照组大鼠L-Ca^2+电流幅度比较,差异无统计学意义(P>0.05)。③水沟穴组L-Ca^2+通道的平均开放时间常数明显小于对照组和非穴组,差异均有统计学意义(P<0.05);非穴组与对照组比较,差异无统计学意义(P>0.05)。结论:电针水沟穴可改善脑缺血再灌注大鼠的神经功能,与电针非穴比较具有经穴效应的特异性。电针水沟穴可能通过降低脑缺血再灌注大鼠海马CA1区L-Ca^2+通道的电流幅度,减少L-Ca^2+通道开放时间,抑制神经细胞内钙超载的作用,促进脑缺血再灌注大鼠神经功能的恢复,起到脑保护作用。

Objective: To observe the effect of electroacupuncture at Shuigou acupoint on the recovery of neurological function to explore the mechanism of the L-type voltage-gated calcium channels in hippocampal CA1 region of middle cerebral artery occlusion(MCAO) model mice, and to investigate the brain protection mechanism of electroacupuncture at Shuigou acupoint on cerebral ischemia. Methods: A total of thirty health male adult Wistar mice were choosed. The MCAO was established by Zea Longa middle cerebral artery occlusion. After the mice waking up, Zausinger six acupoint scale was used to the neurological function score of the mice, and the mice which scored 1 to 3 points were selected for the experiment. They were randomly divided into Shuigou group, control group and non-acupiont group according the random number method, with 10 mice in each group. Mice in the Shuigou group were treated with electroacupuncture at Shuigou acupoint by continuous square wave(2 Hz, 1 m A) for 10 minutes, once a day for 7 days.The mice in the non-acupoint group accepted acupuncture at the acupoint 0.5 cm on the left of Shuigou acupoint, and the electroacupuncture intervention was performed under the same conditions as that in the Shuigou group. The mice of the control group were grasped and fixed on the operating plank as the other two groups without any intervention. After treatment, the neurological function of the mice was evaluated by neurological function defect score. Then vertebral neurons in the hippocampus CA1 area of the MCAO model mice were obtained by acute enzyme isolation technology. In addition, the current amplitude and average open time coustant of the L-type voltage-gated calcium channels in hippocampal CA1 region were recorded by the single cell patch clamp technology. Results:(1) In terms of neurological function score, there were no statistically significant differences among the control group, Shuigou group and non-acupoint group before intervention(P>0.05), indicating that the baseline score of neurological function defect before acupuncture treatment in the three MCAO groups were consistent and comparable. After intervention, the neurological function score of the Shuigou group was significantly smaller than the prior(P<0.05). There was no statistically significant difference in neurological function score of the non-acupoint group than the prior(P>0.05). There was no statistically significant difference in neurological function score of the control group than the prior(P>0.05). Furthermore, the Shuigou group was significantly superior to the control group and the non-acupoint group respectively with statistically significant differences(P<0.05). However, there was no statistical difference between the non-acupoint group and the control group(P>0.05).(2) The difference in current amplitude of the L-type voltage-gated calcium channels between the Shuigou group and the control group was statistically significant(P<0.05). There was a statistically significant difference in current amplitude of the L-type voltage-gated calcium channels between the Shuigou group and the non-acupoint group(P<0.05), and the L-type voltage-gated calcium channels of the Shuigou group was smaller than the non-acupoint group.There was no statistically significant difference in current amplitude of the L-type voltage-gated calcium channels between the non-acupoint group and the control group(P>0.05).(3) The mean opening time constant of the L-type voltage-gated calcium channels in the Shuigou group was significantly shorter than that in the control group and the non-acupoint group respectively( P<0.05). There was no statistically significant difference between the non-acupoint group and the control group(P>0.05). Conclusion: This study suggested that electroacupuncture at Shuigou acupoint could enhance the recovery of the neurological function of MCAO model mice on account of the acupoint specificity effect. Electroacupuncture at Shuigou acupoint may significantly reduce the current amplitude of the L-type voltage-gated calcium channels in the hippocampus CA1 region of mice after cerebral ischemia reperfusion, and reduce the open time of the L-type voltage-gated calcium channels in hippocampal CA1 region. Electroacupuncture at Shuigou acupoint may play an importment role in inhibiting calcium overload in nerve cells, promoting the recovery of nerve function in mice after cerebral ischemia reperfusion and playing a role in brain protection.