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目的探讨临床常用的新生儿惊厥治疗方案中单用尤其联合使用传统抗惊厥药物对新生鼠脑发育的影响。方法以临床剂量换算大鼠用药剂量,新生7d(P7)大鼠120只分为苯巴比妥(phenobarbital,PB)、地西泮(diazepam,DZP)、苯妥英(phenytoin,PHT)、苯巴比妥联合地西泮(PB+DZP)、苯巴比妥联合苯妥英(PB+PHT)和正常对照(给予等量生理盐水)共6组,每组各20只。分别进行行为学(每组10只)及组织学观测(每组10只)。给药后24h(P8)检测急性期脑损伤:ELISA法测定血清神经元特异性烯醇化酶(neuron-specific enolase,NSE)含量,免疫组化检测Bax、Bcl-2蛋白表达;原位末端标记法(TUNEL)检测凋亡细胞。P24行Morris水迷宫实验,HE、尼氏染色观察病理改变。结果 PB、PHT、PB+DZP、PB+PHT组血清NSE浓度较正常对照组显著增高,分别高出52.48%、67.02%、152.48%和175.53%(P<0.01);PB+DZP和PB+PHT组分别高出PB单药65.58%和80.20%(P<0.01)。PB+PHT组Bcl-2蛋白表达较正常对照组显著降低(P<0.05),各用药组Bax蛋白的表达显著强于正常对照组(P<0.05);PB、DZP、PHT组海马下托TUNEL染色阳性细胞数较正常对照组(14.86±5.40)分别增加4.74倍、3.27倍、5.44倍(P<0.01);PB+DZP和PB+PHT组分别高出PB组1.31倍和1.47倍(P<0.01)。Morris水迷宫实验,单药组PB、DZP组和正常对照组间差异无统计学差异(P>0.05),PHT组仅穿越平台的次数较正常对照组少,但其寻台潜伏期与对照组差异无显著性;联合用药组平均寻台潜伏期较正常对照组显著延长(P<0.01),且穿越平台次数较正常对照组减少,差异有统计学意义(P<0.01)。P30幼鼠尼氏体染色400倍光镜下海马下托部神经元计数,与正常对照组(82.80±14.44)比较,PHT组、PB+DZP组、PB+PHT组的神经细胞数量分别减少22.6%、23.8%、31.3%(P<0.01)。结论单用PB、DZP、PHT能引起不同程度的急性期脑损伤,其脑损伤随3周生长发育得到恢复;PB+DZP、PB+PHT联合用药,其脑损伤更为严重,对发育脑造成长期的学习记忆损害。
Objective To investigate the effect of single antiepileptic drug combined with traditional anticonvulsant therapy on neonatal rat brain development in the treatment of neonatal seizures. Methods 120 rats were randomly divided into two groups: phenobarbital (PB), diazepam (DZP), phenytoin (PHT), phenobarbital A total of 6 groups were treated with PB + DZP, PBT plus PHT, and normal control (with an equal volume of saline), 20 in each group. Behavioral studies (10 in each group) and histological observations (10 in each group) were performed. Serum levels of neuron-specific enolase (NSE) were measured by ELISA. Expression of Bax and Bcl-2 protein was detected by immunohistochemistry. Apoptotic cells were detected by TUNEL. P24 line Morris water maze test, HE, Nissl staining pathological changes. Results The levels of serum NSE in PB, PHT, PB + DZP and PB + PHT groups were significantly higher than those in control group (52.48%, 67.02%, 152.48% and 175.53% Group were 65.58% and 80.20% higher than that of PB alone (P <0.01). The expression of Bcl-2 protein in PB + PHT group was significantly lower than that in normal control group (P <0.05), and the expression of Bax protein in each group was significantly higher than that in normal control group (P <0.05) (P <0.01). PB + DZP and PB + PHT groups were 1.31 times and 1.47 times higher than that of PB group (P <0.01), respectively, compared with the control group (14.86 ± 5.40) 0.01). Morris water maze test, single drug group PB, DZP group and the normal control group no significant difference (P> 0.05), PHT group only cross the platform less than the normal control group, but its search platform latency and control group differences (P <0.01), and the number of crossing platform decreased compared with the control group, the difference was statistically significant (P <0.01). Compared with the normal control group (82.80 ± 14.44), the number of neurons in PHT group, PB + DZP group and PB + PHT group decreased by 22.6% %, 23.8%, 31.3% (P <0.01). Conclusions PB, DZP and PHT can induce different degrees of acute brain injury, and their brain injury recovered with the growth of 3 weeks. Combined with PB + DZP and PB + PHT, their brain injury was more serious, Long-term learning and memory impairment.