琥珀酸对惊厥幼鼠小脑浦肯野细胞的保护作用

Protective effect of succinic acid on cerebellar Purkinje cells of neonatal rats with convulsion

目的探讨琥珀酸(SA)对惊厥幼鼠小脑浦肯野细胞(PC)的保护作用。方法将健康新生7 d Sprague-Dawley(SD)幼鼠120只随机分为新生期组和发育期组,两组再随机分为正常对照组、惊厥模型组、小剂量苯巴比妥(PB)组(30 mg/kg)、大剂量PB组(120 mg/kg)、小剂量琥珀酸(SA)组(30 mg/kg)、大剂量SA组(120 mg/kg)。利用腹腔注射戊四氮制备幼鼠惊厥模型,正常对照组应用生理盐水替代。新生期各组大鼠分别在注射PB或SA或生理盐水后30 min处死取小脑,发育期各组大鼠分别在注射PB或SA或生理盐水后养至30 d时处死取小脑。采用全细胞膜片钳技术,在各组幼鼠小脑脑片上记录PC动作电位(AP);采用低频刺激平行纤维(PF)诱发兴奋性突触后电流(EPSC),观察SA对各组大鼠PC长时程抑制(LTD)的影响。结果与对照组相比,新生期和发育期惊厥幼鼠PC AP频率均明显增高(P<0.05),发育期惊厥幼鼠PC AP阈刺激明显降低(P<0.01),且PC EPSC的幅值抑制程度明显增强(P<0.05);与对照组相比,新生期和发育期大剂量PB组惊厥幼鼠PC AP阈刺激明显降低(P<0.01),PC AP频率明显增高(P<0.05),PC EPSC抑制程度明显增强(P<0.05);新生期和发育期大剂量SA组惊厥幼鼠PC AP频率与惊厥组相比均明显降低(P<0.05);发育期两种剂量SA组AP产生的阈值与惊厥组相比均明显增高(P<0.05)。结论 SD幼鼠新生期惊厥导致的小脑PC兴奋性增高和PF-PC突触可塑性异常可持续至发育期,PB可能加重这种异常,而SA能降低惊厥幼鼠小脑PC的兴奋性,并对惊厥造成的PC LTD的近期和远期异常有明显的修复作用。

Objective To investigate the protective effect of succinic acid（SA） on the cerebellar Purkinje cells（PCs） of neonatal rats with convulsion. Methods A total of 120 healthy neonatal Sprague-Dawley rats aged 7 days were randomly divided into a neonatal period group and a developmental period group. Each of the two groups were further divided into 6 sub-groups： normal control, convulsion model, low-dose phenobarbital（PB）（30 mg/kg）, high-dose PB（120 mg/kg）, low-dose SA（30 mg/kg）, and high-dose SA（120 mg/kg）. Intraperitoneal injection of pentylenetetrazole was performed to establish the convulsion model. The normal control group was treated with normal saline instead. The rats in the neonatal group were sacrificed at 30 minutes after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Those in the developmental group were sacrificed 30 days after the injection of PB, SA, or normal saline, and the cerebellum was obtained. Whole cell patch clamp technique was used to record the action potential（AP） of PCs in the cerebellar slices of neonatal rats; the parallel fibers（PF） were stimulated at a low frequency to induce excitatory postsynaptic current（EPSC）. The effect of SA on long-term depression（LTD） of PCs was observed. Results Compared with the normal control groups, the neonatal and developmental rats with convulsion had a significantly higher AP frequency of PCs（P0.05）, and the developmental rats with convulsion had a significantly decreased threshold stimulus（P0.01） and a significantly greater inhibition of the amplitude of EPSC in PCs（P0.05）. Compared with the normal control groups, the neonatal and developmental rats with convulsion in the high-dose PB groups had a significantly decreased threshold stimulus（P0.01）, a significantly higher AP frequency of PCs（P0.05）, and a significantly greater inhibition of EPSC in PCs（P0.05）. Compared with the neonatal and developmental rats in the convulsion model groups, those in the high-dose SA groups had a significantly decreased AP frequency of PCs（P0.05）. The developmental rats in the low- and high-dose SA groups had a significantly higher AP threshold than those in the convulsion model group（P0.05）. Conclusions The high excitability of PCs and the abnormal PF-PC synaptic plasticity caused by convulsion in neonatal rats may last to the developmental period, which can be aggravated by PB, while SA can reduce the excitability of PCs in neonatal rats with convulsion and repair the short- and long-term abnormalities of LTD of PCs caused by convulsion.