电针对脓毒症相关性脑病小鼠海马神经元钙稳态的影响

Effect of electroacupuncture on calcium homeostasis in hippocampal neurons of mice with sepsis-associated encephalopathy

目的探讨电针对脓毒症相关性脑病(SAE)小鼠海马神经元钙稳态的影响。方法健康成年雄性C57BL/6J小鼠24只,体质量18~22 g,采用随机数字表法分为4组(n=6):假手术组(Sham组)、SAE组、SAE+电针组(SAE+EA组)和SAE+假电针组(SAE+SEA组)。小鼠海马CA1区注射携带Ca^(2+)荧光探针的病毒和植入光纤,以记录Ca^(2+)荧光信号。3周后采用盲肠结扎穿孔法建立SAE模型。术前3 d时开始,SAE+EA组选取百会、双侧曲池和双侧足三里穴进行30 min/d连续7 d的电针刺激。SAE+SEA组在相应穴位旁开2 mm处针刺,但不进行电刺激。术后5 d时行旷场实验,记录站立次数和站立时海马CA1区神经元Ca^(2+)信号变化。术后6~7 d时行新物体识别实验,记录探索时间和探索新事物时海马CA1区神经元Ca^(2+)信号变化。术后7 d行为学实验结束后,处死小鼠将光纤植入侧脑组织,荧光显微镜下观察并测定海马CA1区神经元Ca^(2+)荧光强度。结果与Sham组比较,SAE组和SAE+SEA组站立次数减少,站立时海马CA1区神经元Ca^(2+)信号幅值降低(P<0.05),SAE+EA组上述指标差异无统计学意义(P>0.05),SAE组、SAE+EA组和SAE+SEA组探索指数和探索新事物时海马CA1区神经元Ca^(2+)信号幅值降低,神经元Ca^(2+)荧光强度增强(P<0.05);与SAE组和SAE+SEA组比较,SAE+EA组站立次数增加,站立时海马CA1区神经元Ca^(2+)信号幅值升高,探索指数和探索新事物时海马CA1区神经元Ca^(2+)信号幅值升高,神经元Ca^(2+)荧光强度减弱(P<0.05);SAE组和SAE+SEA组各指标比较差异无统计学意义(P>0.05)。结论电针减轻小鼠SAE的机制可能与调节海马神经元Ca^(2+)稳态有关。

Objective To investigate the effect of electroacupuncture on calcium homeostasis in hippocampal neurons of mice with sepsis-associated encephalopathy(SAE).Methods Twenty-four healthy male C57BL/6J mice,weighing 18-22 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(Sham group),SAE group,SAE plus electroacupuncture group(SAE+EA group),and SAE plus sham electroacupuncture group(SAE+SEA group).The virus carrying calcium ion(Ca^(2+))fluorescent probes was injected and then an optical fiber was implanted into the hippocampal CA1 area to record the fluorescence signals of Ca^(2+).SAE was induced by cecal ligation and puncture in anesthetized mice at 3 weeks after administration.Starting from 3 days before surgery,Baihui and bilateral Quchi and bilateral Zusanli acupoints were stimulated for 30 min per day for 7 consecutive days in SAE+EA group.In SAE+SEA group,electroacupuncture was performed at the points 0.2 mm lateral to the corresponding acupoints without electrical stimulation.Open field tests were conducted at 5 days after surgery to record the number of rearing and changes in related Ca^(2+)signals in hippocampal CA1 neurons.Novel object recognition tests were conducted at 6-7 days after surgery to record the recognition time and changes in related Ca^(2+)signals in hippocampal CA1 neurons.Mice were sacrificed after the end of behavioral testing on 7 days after surgery,and brain tissues ipsilateral to the optical fiber implant were obtained and the fluorescence intensity of Ca^(2+)in the hippocampal CA1 neurons was acquired using a fluorescent microscope.Results Compared with Sham group,the number of rearing and amplitudes of related Ca^(2+)signals in hippocampal CA1 neurons while rearing were significantly decreased in SAE group and SAE+SEA group(P<0.05),and no statistically significant changes were found in the parameters mentioned above in SAE+EA group(P>0.05),and the recognition index and amplitudes of related Ca^(2+)signals while recognizing were significantly deceased,and the fluorescence intensity of Ca^(2+)in hippocampal CA1 neurons was increased in SAE,SAE+EA and SAE+SEA groups(P<0.05).Compared with SAE group and SAE+SEA group,the number of rearing and amplitudes of related Ca^(2+)signals in hippocampal CA1 neurons while rearing were significantly increased,the recognition index and amplitudes of related Ca^(2+)signals in hippocampal CA1 neurons while recognizing were increased,and the fluorescence intensity of Ca^(2+)in hippocampal CA1 neurons was decreased in SAE+EA group(P<0.05).There were no statistically significant differences in the parameters mentioned above between SAE group and SAE+SEA group(P>0.05).Conclusions The mechanism by which electroacupuncture alleviates SAE may be related to regulation of Ca^(2+)homeostasis in hippocampal neurons of mice.