地芬诺酯灌胃对小鼠癫痫发作及神经行为的影响

Effect of intragastric administration of diphenoxylate on epileptic seizures and neurobehavioral changes in mice

目的探究地芬诺酯灌胃对小鼠癫痫的诱发效果及小鼠神经行为学和海马神经元的变化。方法选取2~3周龄的C57雄性小鼠40只,按照随机数字表法分为对照组和地芬诺酯组,每组20只。地芬诺酯组小鼠每天给予地芬诺酯(200 mg/kg)灌胃1次,连续灌胃14 d,对照组小鼠每天给予等体积0.9%氯化钠溶液。每次灌胃后观察2 h,根据Racine评分观察小鼠癫痫发作等级。应用旷场实验、高架十字实验和Morris水迷宫实验观察小鼠神经行为活动的改变;应用数字化脑电图机监测地芬诺酯小鼠癫痫发作脑电波;应用尼氏染色观察小鼠海马神经元损伤情况。采用SPSS 25.0软件对数据进行统计分析,两两比较采用独立样本t检验。结果Racine分级结果显示,地芬诺酯组小鼠在灌胃后1 h出现2、3级发作。脑电监测结果显示,与灌胃前比较,地芬诺酯组小鼠脑电波频率增加,波幅增加。在旷场实验中,地芬诺酯组小鼠在中央区域的停留时间显著低于对照组[(12.21±3.37)s,(17.05±4.34)s,t=3.29,P<0.01]。在高架十字实验中,地芬诺酯组小鼠在开放臂的停留时间低于对照组[(17.36±5.41)s,(26.70±9.06)s,t=3.31,P<0.01]。在Morris水迷宫测试中,地芬诺酯组在平台象限的停留时间低于对照组[(22.08±6.76)s,(27.64±4.60)s,t=2.54,P<0.05],地芬诺酯组在平台区域的停留时间与停留次数均低于对照组(均P<0.05)。尼氏染色显示,地芬诺酯组小鼠CA3区海马神经元数量显著低于对照组[(135.67±4.59)个,(140.67±2.73)个,P<0.05]。结论过量地芬诺酯可诱发小鼠癫痫发作,并且小鼠出现焦虑样行为增加及空间学习记忆能力下降。

Objective To explore the effect of intragastric administration of diphenoxylate on inducing epilepsy in mice,as well as the changes in neurobehavioral and hippocampal neurons in mice.Methods Forty C57 male mice aged 2-3 weeks were selected and divided into control group and diphenoxylate group using random number table method,with 20 mice in each group.The mice in the diphenoxylate group were given diphenoxylate(200 mg/kg)by gavage once a day for 14 consecutive days,while the control group mice were given an equal volume of 0.9%sodium chloride solution every day.After each gavage,the seizure status of mice within 2 hours was observed and the mice were graded based on the Racine score.Open field test,elevated cross test,and Morris water maze test were used to observe the neurobehavioral activities of mouse.A digital electroencephalogram machine was used to monitor the epileptic seizures of mice induced by diphenoxylate.Nissl staining was used to observe the damage of hippocampal neurons in mice.SPSS 25.0 software was used for statistical analysis of the data,and independent sample t-test was used for pairwise comparison.Results The Racine grading results showed that the mice in diphenoxylate group exhibited grade 2 and 3 seizures at 1 hour after gavage.The EEG monitoring results showed that compared with before gavage,the frequency and amplitude of brain waves of mice in diphenoxylate group increased.In the open field test,the residence time in the central region of mice in diphenoxylate group was significantly lower than that of the control group((12.21±3.37)s,(17.05±4.34)s,t=3.29,P<0.01).In the elevated cross test,the residence time in the open arm of mice in diphenoxylate group was significantly lower than that of the control group((17.36±5.41)s,(26.70±9.06)s,t=3.31,P<0.01).In the Morris water maze test,the residence time in the platform quadrant of diphenoxylate group was significantly lower than that of the control group((22.08±6.76)s,(27.64±4.60)s,t=2.54,P<0.05).The residence time and the number of stays in the platform area of diphenoxylate group were both significantly lower than those of the control group(both P<0.05).Nissl staining showed that the number of hippocampal neurons in the CA3 region of mice treated with diphenoxylate was significantly lower than that in the control group((135.67±4.59),(140.67±2.73),P<0.05).Conclusion Excessive diphenoxylate can induce seizures in mice,and the mice exhibit increased anxiety-like behavior and decreased spatial learning and memory abilities.