早期惊厥样放电对发育中大鼠皮层神经元细胞内游离钙的远期影响

Long-term effects of early seizure-like discharge on [Ca^(2+)]_i in developing cortical neurons of rats

目的探讨早期惊厥样放电对发育中大鼠皮层神经元功能及NMDA激发后细胞内[Ca2+]i的远期影响。方法以原代培养的大鼠大脑皮层神经元无镁诱导的反复惊厥样放电为模型。根据对培养6d皮层神经元的不同处理分为三组正常DMEM培养液组(CONT1)、正常细胞外液组(CONT2)和无镁细胞外液组(MGF)。神经元分别在上述三种液体中孵育3h,然后恢复正常DMEM培养基继续培养,在培养7,12及17d时测定皮层神经元噻唑蓝(MTT)代谢率及荧光数码扫描显微镜测定[Ca2+]i信号的变化,100μmol/LNMDA刺激神经元。[Ca2+]i反应通过静息钙、峰值和上升时间表示。结果与CONT2和CONT1组相比,MGF组MTT代谢率在培养7和12d皮层神经元均明显降低(P<0.05);不同培养时间各组静息钙和峰值钙水平均升高(P<0.05);上升时间在培养7和17d较短(P<0.05),而12d较长(P<0.05)。CONT2组与CONT1组相比无统计学差异(P>0.05)。结论发育中皮层神经元早期无镁诱导惊厥样放电对细胞内钙具有远期影响,提供了神经元早期惊厥样放电引起远期功能损伤的可能机制。

Objective To explore the long-term effects of early seizure-like discharge on neuronal function and the changes of intracellular calcium concentration and calcium responses to NMDA in developing rat cortical neurons. Methods The seizure model was recurrent epileptiform discharges induced by transient magnesium-free media treatment in the primary cultured embryo cortical neurons of rat. According to different treatment to cortical neurons at cultured day 6, three groups were divided: regular DMEM medium (CONT1)、regular extracellular solution media (CONT2) and magnesium-free extracellular solution media (MGF). The neurons were exposed to above three solutions for 3 h in cultured day 6, and then cultured in regular DMEM medium again. At day 7, 12, and 17 in vitro, intracellular calcium concentration was determined for individual cells using microscopic fluorescent digital imaging. Neurons were stimulated with micropressure application of NMDA, intracellular calcium responses were quantified by measurement of the peak amplitude, resting calcium levels, and risetime. MTT metabolic rate were detected at different culture day (7,12,17). Results Compared with group CONT1 and CONT2 respectively, in group MGF MTT metabolic rate decreased at day 7 and day 12, the peak calcium amplitudes were enhanced, the resting calcium levels were increased (all P<0.05), the rise time was shorter at culture day 7 and 17, longer at culture day 12(all P<0.05). There were not statistic significant differences of MTT metabolic rat at culture day 17 among three groups (P>0.05). Compared with CONT1, there were not statistic significant differences in group CONT2. Conclusion The change of neuronal function and NMDA evoked [Ca^2+ ]_i may be resulted by long-term effects of early seizure-like discharge in developing cortical neurons of rat.