长期使用抗癫药物对发育期大鼠脑的影响

目的探讨长期使用抗癫药（AEDs）对发育期大鼠脑的影响，在细胞及分子水平上观察AEDs影响认知功能的机制。方法将生后7 d的Wistar大鼠234只任意分为13组：4种AEDs［苯巴比妥（PB），丙戊酸（VPA），拉莫三嗪（LTG），托吡酯（TPM）］分别分为高、中、低剂量组和对照组，每组18只大鼠。PB 80、 40、20 mg·kg-1和VPA 200、100、50 mg·kg-1分别溶于超纯水腹腔注射每日1次共21 d，LTG 80、40、20 mg·kg-1和TPM 80、40、20 mg·kg-1分别溶于1％纤维素钠灌胃每日1次共21 d，对照组予相同体积1％纤维素钠灌胃每日1次共21 d。从第22天始每组6只大鼠断头取脑，用Annexin-V FITC/PI双标检测细胞凋亡，用实时定量PCR检测脑源性神经营养因子（BDNF）和神经营养物质-3（NT-3）mRNA的表达；余12只大鼠过量麻醉后灌注固定，分别用于BrdU染色和Timm's染色。 结果①4种AEDs均会造成幼鼠脑重减轻，其中VPA高、中、低剂量组大鼠脑重降低最明显，VPA高剂量组导致脑重降低15％。②4种AEDs均引起神经细胞凋亡的增加，其阈值分别为：PB 20 mg·kg-1, VPA 50mg·kg-1, LTG 80 mg·kg-1，TPM 40 mg·kg-1。③实时定量PCR结果显示，4种AEDs可导致海马区BDNF和NT-3 mRNA表达的减少，其阈值分别为PB 40 mg·kg-1、VPA 100 mg·kg-1、LTG 80 mg·kg-1、TPM 40 mg·kg-1。VPA和LTG低剂量组可引起海马齿状回和门区BrdU标记的细胞数增加。Timm's染色结果显示4种AEDs CA3区和颗粒细胞上层Timm评分与对照组比较差异均无统计学意义(P>0.05)。 结论长期使用AEDs会对发育期大脑造成损害，不同AEDs可引起大脑细胞凋亡增加和海马区神经营养物质表达减少，其阈值有所不同，这可能是造成认知损害的机制之一。VPA和LTG会造成神经发生增加，但不伴有明显的苔藓纤维发芽。

Effects of long-term antiepileptic treatment on the developing brain of rats

ObjectiveTo study the effects of long-term treatment with antiepileptic drugs (AEDs) on the developing brain of rats, and to explain possible mechanisms of adverse effects of AEDs at cellular and molecular levels. MethodsA total of 234 neonatal Wistar rats（P7）were divided into 13 groups (the control group, PB, VPA, LTG and TPM with high, middle and low dosage), with 18 rats in each group. After 3-weeks treatment with AEDs the treatment groups and the controls were divided into two parts. One part was sacrificed by decapitation and the brain was removed and washed with ice-cold saline. These brains were used in the study of Annexin-V FITC/PI double staining and quantitative real-time PCR detection. The other part used in the study of BrdU staining and Timm's staining received an overdose of sodium pentobarbital (60 mg·kg-1, i.p.) and was perfused with different solution. Results①Long-term treatment with AEDs caused significant reduction in brain weight, especially in VPA groups. VPA (200 mg·kg-1) resulted in 15% decrease in brain weight. ②AEDs caused apoptotic neurodegeneration, the threshold of PB, VPA, LTG and TPM was 20, 50, 80 and 40 mg·kg-1, respectively. ③ Quantitative real-time PCR showed 4 AEDs decreased the expression of BDNF and NT-3, the threshold of PB, VPA, LTG and TPM was 40, 100, 80 and 40 mg·kg-1, respectively. Neurogenesis increased in the rats treated with valproate and lamotrigine but their effect on mossy fiber sprouting was not obvious in any rats (P>0.05). ConclusionLong-term treatment with AEDs damages developing brain of rats, PB, VPA, LTG and TPM cause apoptotic neurodegeneration in the developing brain at different dose levels. Neuronal death is associated with reduced expression of BDNF and NT-3. Interestingly, VPA and LTG cause increased neurogenesis in dentate gyrus with an absence of mossy fiber sprouting. These findings presented one possible mechanism to explain that cognitive impairment was associated with exposure of humans to antiepileptic therapy.