新一代抗癫痫药对新生大鼠脑发育的影响

Effect of new generation antiepileptic drugs on brain development of neonatal rats

目的探讨新一代抗癫痫药一次性单药或联合苯巴比妥(PB)用药对新生大鼠脑发育的影响。方法将健康清洁级7日龄Wistar大鼠270只随机分为对照组、奥卡西平(OXC)30组、OXC60组、托吡酯(TPM)组、左乙拉西坦(LEV)组、PB组、PB+OXC30组、PB+TPM组和PB+LEV组,每组30只。OXC30组、OXC60组、TPM组、LEV组和PB组大鼠分别灌胃OXC187.5mg·kg^-1、OXC375.0mg·kg^-1、TPM62.5mg·kg^-1、LEV375.0mg·kg^-1、PB62.5mg·kg^-1;PB+OXC30组、PB+TPM组和PB+LEV组大鼠均腹腔注射PB62.5mg·kg^-1,4h后,再分别灌胃OXC187.5mg·kg^-1、TPM62.5mg·kg^-1、LEV375.0mg·kg^-1;对照组大鼠腹腔注射生理盐水0.1mL。每组取10只大鼠测给药前后体质量及给药后脑质量;给药后24h,每组取10只大鼠采用苏木精-伊红(HE)、尼氏染色观察大鼠脑组织病理学改变;给药后16d,每组取10只大鼠行Morris水迷宫试验检测大鼠学习记忆能力;水迷宫试验结束后次日处死大鼠,取脑组织观察病理学改变,并进行颞叶皮层及海马下托复合体神经细胞计数。结果用药前各组大鼠体质量比较差异无统计学意义(P>0.05);给药后24h,OXC60组、PB+OXC30组、PB+TPM组、PB+LEV组及PB组大鼠体质量均低于对照组(P<0.05);OXC30组、TPM组、LEV组与对照组大鼠体质量比较差异无统计学意义(P>0.05)。PB+OXC30组、PB+TPM组、PB+LEV组与PB组大鼠体质量比较差异均无统计学意义(P>0.05)。OXC60组、PB+OXC30组大鼠脑质量显著低于对照组(P<0.05);其余各给药组大鼠脑质量与对照组比较差异均无统计学意义(P>0.05)。PB+OXC30组、PB+TPM组、PB+LEV组大鼠脑质量与PB组比较差异均无统计学意义(P>0.05)。给药后24h,HE染色可见,各给药组大鼠脑组织颞叶皮层结构未见明显异常。OXC60组、PB组、PB+OXC30组、PB+TPM组及PB+LEV组大鼠脑组织海马下托复合体出现部分细胞核固缩、深染,核周空泡,细胞皱缩等病理学改变,且PB+OXC30组病变严重程度明显大于PB组;OXC30、TPM、LEV组大鼠脑组织海马下托均未见明显病理改变。尼氏染色显示,OXC60组、PB+OXC30组、PB+TPM组及PB+LEV组大鼠脑组织颞叶皮层少数神经细胞尼氏小体减少,部分溶解消失;OXC60组和PB组大鼠海马下托可见尼氏小体减少,部分溶解。给药后23d,PB+OXC30组大鼠脑组织颞叶皮层可见退行性红色神经细胞明显增多;PB+TPM组、PB+LEV组及OXC60组大鼠脑组织病理学改变有所恢复。OXC60组和PB+OXC30组大鼠穿越平台次数少于对照组(P<0.05)。OXC30组、TPM组、LEV组、PB组、PB+TPM组和PB+LEV组与对照组大鼠穿越平台次数比较差异无统计学意义(P>0.05);PB+OXC30组、PB+TPM组和PB+LEV组与PB组大鼠穿越平台次数比较差异无统计学意义(P>0.05)。第1、2、3、4天,各给药组与对照组大鼠寻台潜伏期比较差异均无统计学意义(P>0.05)。PB+OXC30组大鼠第5天寻台潜伏期长于对照组(P<0.05),其余各给药组与对照组大鼠第5天寻台潜伏期比较差异均无统计学意义(P>0.05)。第1、2、3、4、5天,PB+OXC30组、PB+TPM组、PB+LEV组与PB组大鼠寻台潜伏期比较差异无统计学意义(P>0.05)。各给药组与对照组大鼠颞叶皮层神经细胞计数比较差异均无统计学意义(P>0.05)。PB+OXC30组大鼠海马下托区神经细胞计数少于对照组(P<0.05),其余各组大鼠海马下托区神经细胞计数与对照组比较差异均无统计学意义(P>0.05)。PB+OXC30组、PB+TPM组、PB+LEV组与PB组大鼠海马下托区神经细胞计数比较差异无统计学意义(P>0.05)。结论一次性使用375.0mg·kg^-1OXC可导致新生健康大鼠急性期脑损伤和远期空间记忆能力稍减弱;较低剂量(187.5mg·kg^-1)的OXC不会引起发育未成熟脑出现明显损伤,但却能明显加重PB引起的脑损伤,且遗留持久性脑损伤和记忆功能障碍;抢救剂量的LEV(375.0mg·kg^-1)和TPM(62.5mg·kg^-1)不存在脑损伤危险,也不加重PB引起的损伤。

Objective To investigate the effect of new generation of antiepileptic drugs alone or combined with phenobarbital(PB)on the brain development of newborn rats.Methods Two hundred and seventy healthy cleaning seven days old Wistar rats were randomly divided into control group,oxcarbazepine(OXC)30 group,OXC60 group,topiramate(TPM)group,levetiracetam(LEV)group,PB group,PB+OXC30 group,PB+TPM group and PB+LEV group,with 30 rats in each group.The rats in OXC30 group,OXC60 group,TPM group,LEV group and PB group were given OXC 187.5 mg·kg^-1,OXC 375.0 mg·kg^-1,TPM 62.5 mg·kg^-1,LEV 375.0 mg·kg^-1,PB 62.5 mg·kg^-1 by intragastric administration;the rats in PB+OXC30 group,PB+TPM group and PB+LEV group were given PB 62.5 mg·kg^-1 by intraperitoneal injection,after 4 hours,the rats were given OXC 187.5 mg·kg^-1,TPM 62.5 mg·kg^-1 and LEV 375.0 mg·kg^-1 respectively;the rats in the control group were intraperitoneally injected with 0.1 mL saline.The body weight and brain weight of rats in all groups(n=10)were measured before and after 24 hours administration;the histopathological changes of rats in each group(n=10)were observed by hematoxylin-eosin(HE)and Nissl staining at 24 hours after administration.The learning and memory ability of rats in each group(n=10)were tested by Morris water maze test on the 16 th day after administration;the rats were sacrificed on the next day after the water maze test and the brain tissues were taken to observe the pathological changes,and the hippocampal neurons in temporal cortex and subhippocampal complex were counted.Results There was no significant difference in body weight of rats in each group before administration(P>0.05);at the 24 hours after administration,the body weight of rats in OXC60 group,PB+OXC30 group,PB+TPM group,PB+LEV group and PB group was lower than that in the control group(P<0.05);there was no significant difference in the body weight of rats between OXC30 group,TPM group LEV group and control group(P>0.05).There was no significant difference in body weight between PB+OXC30 group,PB+TPM group,PB+LEV group and PB group(P>0.05).The brain weight of rats in OXC60 group and PB+OXC30 group was significantly lower than that in the control group(P<0.05),while there was no significant difference in the brain weight between other intervention groups and control group(P>0.05).There was no significant difference in the brain weight of rats between PB+OXC30 group,PB+TPM group,PB+LEV group and control group(P>0.05).At 24 hours after administration,HE staining showed that there was no significant abnormality in temporal cortex structure of brain tissue in all intervention groups.Some pathological changes such as nuclear pyknosis,hyperchromatism,perinuclear vacuoles and cell shrinkage were observed in the hippocampal substratum complex of rats in OXC60 group,PB group,PB+OXC30 group,PB+TPM group and PB+LEV group;and the severity of the lesions of rats in PB+OXC30 group was significantly higher than that in PB group.There were no obvious pathological changes in the subhippocampal substratum of rats in OXC30 group,TPM group and LEV group.Nissl staining showed that the Nissl bodies decreased in a small number of neurons in the temporal cortex of brain tissue of rats in OXC60 group,PB+OXC30 group,PB+TPM group and PB+LEV group and partial Nissl bodies dissolved;the Nissl bodies decreased in the hippocampal substratum of brain tissue of rats in OXC60 group and PB group and partial Nissl bodies dissolved.At 23 days after administration,the number of degenerative red neurons in temporal lobe cortex of brain tissue of rats increased in PB+OXC30 group,and the pathological changes of brain tissue of rats in PB+TPM group,PB+LEV group and OXC60 group recovered.The number of crossing the platform of rats in OXC60 group and PB+OXC30 group was less than that in the control group(P<0.05);there was no statistic difference in the number of crossing the platform of rats between OXC30 group,TPM group,LEV group,PB group,PB+TPM group,PB+LEV group and control group(P>0.05).There was no statistic difference in the number of crossing the platform of rats between the PB+OXC30 group,PB+TPM group,PB+LEV group and PB group(P>0.05).There was no significant difference in the latency of seeking platform between all intervention groups and control group on the 1 st,2 nd,3 rd and 4 th day of Morris water maze test(P>0.05).The latency of seeking platform of rats in PB+OXC30 group was longer than that in the control group on the 5 th day of Morris water maze test(P<0.05);there was no significant difference in the latency of seeking platform between the other intervintion groups and the control group(P>0.05).There was no significant difference in the latency of seeking platform between PB+OXC30 group,PB+TPM group,PB+LEV group and PB group on the 1 st,2 nd,3 rd,4 th and 5 th day of Morris water maze test(P>0.05).Conclusion One-time use of rescue dose(375.0 mg·kg^-1)of OXC can cause acute stage brain injury and weak long-term spatial memory ability of neonatal rats.The low doses(187.5 mg·kg^-1)of OXC dose not cause significant acute phase damage in immature brain,but it can aggravated the brain damage caused by PB,and leave persistent brain damage and memory dysfunction.The rescue dose of LEV(375.0 mg·kg^-1)and TPM(52.5 mg·kg^-1)do not present the risk of brain injury and do not aggravate the damage caused by PB.