长期使用抗癫药物对发育期大鼠脑的影响

刘玉洁 石秀玉 于新颖 胡琳燕 邹丽萍

·论著·

刘玉洁1,3石秀玉1,3于新颖2胡琳燕1邹丽萍1

1 方法

1.1 动物分组 生后7 d的Wistar大鼠234只任意分为13组：4种AEDs(PB，VPA，LTG，TPM)分别分为高、中、低剂量组和对照组，每组18只。分别标记组别后母鼠与新生鼠同笼存放。

PB溶于超纯水2 mL(终浓度为10 mg·mL-1)，PB高、中、低剂量组分别予PB 80、 40、20 mg·kg-1腹腔注射；VPA溶于超纯水2 mL(终浓度为20 mg·mL-1)，VPA高、中、低剂量组分别予VPA 200、100、50 mg·kg-1腹腔注射；LTG溶于1%纤维素钠2 mL(终浓度为10 mg·mL-1)，LTG高、中、低剂量组分别予LTG 80、40、20 mg·kg-1灌胃；TPM溶于1%纤维素钠2 mL(终浓度为10 mg·mL-1)，TPM高、中、低剂量组分别予TPM 80、40、20 mg·kg-1灌胃，对照组予相同体积1%纤维素钠，灌胃。均每日1次，共用21 d。

从第22 d始，①每组大鼠取6只断头取脑，迅速用冰冷的生理盐水洗净血液，滤纸吸干立即称重，用于流式细胞术和RT-PCR检测。②每组再取6只大鼠将溶于生理盐水的Brdu(sigma公司)100 mg·kg-11次性经腹腔注射[13]。36 h后予3%戊巴比妥60 mg·kg-1过量麻醉，4%多聚甲醛左心灌注固定取脑，在海马部位做冠状连续冰冻切片，切片厚度20 μm，每隔2张取1张，组成1套8～10张，行BrdU单标免疫荧光染色。③每组余下的6只大鼠左心先后灌注硫化钠溶液和4%多聚甲醛固定取脑，在海马部位做冠状冰冻连续切片，切片厚20 μm，每隔3张取1张行Timm染色和评分。

1.2 流式细胞仪Annexin-V/PI法检测细胞凋亡 将剥离的脑组织制成每毫升1×106细胞悬液，室温1 000 r·min-1离心5 min，去除上清，PBS清洗，加入1.25 μL 200 μg·mL-1Annexin V-FITC(晶美)室温避光孵育15 min， 室温1 000 r·min-1离心5 min，去除上清，加入10 μL碘化丙啶，流式细胞仪检测。

1.3 荧光实时定量PCR法分析BDNF和NT-3的表达

1.3.1 引物设计 BDNF、NT-3和β-actin的PCR扩增引物序列由上海Invitrogen公司设计合成，β-actin sense: AAGATCCTGACCGAGCGTGG，antisense: CAGCACTGTGTT-GGCATAGAGA；BDNF sense: CGACGTCCCTGGCTGGACAC-TTTT，antisense: AGTAAGGGCCCGAACATACGATTGG； NT-3 sense: GGTCAGAATTCCAGCCGATGATTGC，antisense: CAGCGCCAGCC TACGTTTGTTGT。

1.3.2 组织总RNA的提取和逆转录 将称重后的脑组织立即分离，取出两侧海马，放入EP管中-80℃保存备用，按照硫氰酸胍-酚-氯仿抽提法提取总RNA，溶于适量RNAase 水，立即使用或保存于-70℃。随机引物法进行逆转录反应，按试剂说明书操作。

1.3.3 PCR反应 荧光实时定量PCR的反应体系为20 μL，包括SYBR Premix Ex Taq(大连宝生物)10 μL，Primer1 1.2 μL，Primer 2 1.2 μL，cDNA 2 μL，dH2O 5.6 μL。PCR反应参数：预变性95℃ 10 s, 56℃ 10 s退火、72℃ 10 s延伸，共50个循环。最后65℃ 15 s、95℃每秒改变0.1℃、40℃ 30 s进行融解曲线分析。

1.3.4 结果分析 结果以目的基因与内参照β-actin的比值表示。每次反应时标准品(cDNA的PCR产物经纯化后做倍比稀释：1、1×10-2、1×10-4、1×10-6、1×10-8、1×10-10)，待测样本及阴性对照(以去离子水为模板)同时扩增，并做溶解曲线以检测非特异扩增。BDNF(或NT-3)/β-actin比值代表待测样本的BDNF(或NT-3)相对表达。

1.4 免疫组织化学分析神经发生和苔藓纤维发芽

1.4.1 BrdU单标免疫荧光染色 以鼠抗Brdu单克隆抗体(Sigma公司)作为一抗，FITC标记的羊抗小鼠IgG(北京中山公司)作为二抗，荧光显微镜下观察并照相，Brdu阳性者发绿色荧光。

1.4.2 Timm's 染色 将切片自然晾干，三蒸水冲洗后，放置于含有120 mL阿拉伯树胶(50%)、60 mL氢醌(5.78%)、10 mL柠檬酸(51%)、10 mL柠檬酸钠(47%)和212.5 mg硝酸银的混合溶液中，暗室显影40～60 min，流水终止染色15 min。切片晾干后，梯度乙醇脱水、透明、封片。以半定量的记分方法分析Timm染色结果，观察CA3区和颗粒细胞上层苔藓纤维发芽。

2 结果

2.1 脑重 表1显示，PB高剂量组较对照组脑重下降12%，(2.03±0.16)vs(2.32±0.24) g,P<0.05。VPA中和高剂量组脑重较对照组下降最明显，其中高剂量组脑重降低15%，(1.95±0.26)vs(2.32±0.24) g,P<0.01。LTG和TPM各剂量组脑重下降与对照组差异无显著差异。

Notes The high, middle and low dose groups of phenobarbital (PB) were given 80, 40 and 20 mg·kg-1; the corresponding doses of valproate(VPA) in three groups were 200, 100 and 50 mg·kg-1, doses of lamotrigine(LTG) were 80, 40 and 20 mg·kg-1, doses of topiramate(TPM) were 80,40 and 20 mg·kg-1. 1)vscontrol group，P<0.05

2.2 细胞凋亡 表1显示，4种AEDs(PB、VPA、LTG、TPM)分别的3个剂量组Annexin V+/PI -细胞的百分数均有高于对照组的趋向，其中PB和VPA分别的3个剂量组，LTG高剂量组，TPM中、高剂量组Annexin V+/PI -细胞的百分数与对照组的差异有统计学意义。

2.3 AEDs对神经营养因子mRNA表达的影响 实时荧光定量PCR结果分析显示PB 中、高剂量组, VPA中、高剂量组, LTG高剂量组和 TPM中、高剂量组可导致BDNF 和 NT-3 mRNA的水平较对照组显著降低(表1)。

2.4 BrdU 染色和Timm's染色

2.4.1 神经发生的影响 表2显示，VPA和LTG分别的3个剂量组均可导致海马门区、齿状回以及海马CA3区和海马外区域 BrdU-标记的细胞数较对照组显著增加,P<0.05(图1)。 PB和TPM分别的3个剂量组与对照组比较，BrdU-标记的细胞数差异均无统计学意义。

2.4.2 苔藓纤维发芽的影响 表2显示，4种AEDs分别的3个剂量组C3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)。

Notes The high, middle and low dose groups of phenobarbital (PB) were given 80, 40 and 20 mg·kg-1; the corresponding doses of valproate(VPA) in three groups were 200, 100 and 50 mg·kg-1, doses of lamotrigine(LTG) were 80, 40 and 20 mg·kg-1, doses of topiramate(TPM) were 80,40 and 20 mg·kg-1. 1)vscontrol group，P<0.05

图1 海马外神经发生(×100)

Fig 1 Neurogenesis outside of the hippocampus (×100)

Notes A, B represented neurogenesis in the entorhinal cortex outside of the hippocampus in VPA 200mg·kg-1group and LTG 80 mg·kg-1group, respectively. The arrows pointed to newborn neurons

3 讨论

既往研究[7，9，14]认为AEDs对新生啮齿类动物可能造成神经毒性损害，不同AEDs对发育期大脑所造成的神经损害差异很大。Bittigau等[7]研究发现苯妥英、VPA、氨己烯酸、地西泮和氯硝西泮可增加神经细胞凋亡，而其对应的AEDs浓度与人类控制惊厥发作的血药浓度接近。Glier等[8]发现治疗剂量的TPM对发育期大脑没有毒性。本研究发现4种AEDs均会造成脑重下降，但VPA的不同剂量组脑重降低最明显，LTG不同剂量组虽脑重也呈降低趋势，但与对照组差异无统计学意义。AEDs可引起神经细胞凋亡增加，但不同AEDs引起凋亡增加的阈值不同，PB为20 mg·kg-1, VPA为50 mg·kg-1, LTG为80 mg·kg-1，TPM为40mg·kg-1。神经细胞凋亡增加的同时也伴有神经营养因子BDNF和NT-3 mRNA表达的降低。

神经细胞凋亡在大脑发育过程中起着非常重要的作用，任何影响这一过程的物质都会导致神经元凋亡的改变[15]，影响正常的脑发育，从而影响认知。AEDs导致发育期大脑神经细胞凋亡增加可能有不同的机制，其中之一为内源性神经营养物质系统如BDNF和NT-3表达的降低[16]，BDNF对中枢胆碱能神经元有刺激生长作用，可促进背根神经节神经元突起向中枢生长，也可延长离体培养胚胎大鼠脑中隔胆碱能神经元细胞的存活时间，并增加乙酰胆碱酶和胆碱乙酰转移酶的酶活性。NT-3结构上与BDNF相似，可促进由神经基板发生的神经元突起的生长，也可促进离体培养的背根神经节神经细胞突起的生长。目前认为脑的发育、老化等改变，以及病理性损害等均与BDNF和NT-3的作用有关。其表达的降低可导致神经细胞凋亡增加影响脑发育，还可直接影响了脑的正常功能，从而导致认知功能的损害。这可能是本研究观察到的AEDs导致脑重下降的原因之一。

本研究中AEDs导致BDNF和NT-3 mRNA表达下降的剂量比既往研究报道的低，Bittigau等[7]研究中发现PB和VPA引起BDNF和NT-3 mRNA表达降低的阈值分别为50和200 mg·kg-1。结果的不一致可能与发育阶段不同有关，本研究大鼠在生后7 d开始应用AEDs直至生后28 d，有研究显示AEDs对大鼠的神经毒性呈年龄依赖性，多集中在生后21 d内，与脑生长高峰符合[17]。此外，本研究连续应用AEDs 21 d，相当于人类整个婴幼儿时期，而Bittigau等[7]研究用药时间仅1 d，提示AEDs治疗时间越长，导致神经营养物质表达降低的阈值呈降低趋势。

[1] Perrine K, Kiolbasa T. Cognitive deficits in epilepsy and contribution to psychopathology. Neurology, 1999, 53(5S2):39-48

[2] Meador KJ. Current discoveries on the cognitive effects of antiepileptic drugs. Pharmacotherapy, 2000, 20(8 Pt 2):85-90

[3] Kwan P, Brodie MJ. Neuropsychological effects of epilepsy and antiepileptic drugs . Lancet, 2001, 20,357(9251):216-222

[4] Wu Y, Wang L. The effect of antiepileptic drugs on spatial learning and hippocampal protein kinase C γ in immature rats. Brain Dev, 2002, 24(2):82-87

[5] Shannon HE, Love PL. Effects of antiepileptic drugs on attention as assessed by a five-choice serial reaction time task in rats. Epilepsy Behav, 2005, 7(4):620-628

[6] Shannon HE, Love PL. Effects of antiepileptic drugs on working memory as assessed by spatial alternation performance in rats. Epilepsy Behav, 2004, 5(6):857-865

[7] Bittigau P, Sifringer M, Ikonomidou C. Antiepileptic drugs and apoptosis in the developing brain. Ann N Y Acad Sci, 2003, 993:103-114

[8] Glier C, Dzietko M, Bittigau P,et al. Therapeutic doses of topiramate are not toxic to the developing brain. Exp Neurol, 2004, 187(2):403-409

[9] Manthey D, Asiniadou S, Stefovska V, et al. Sulthiame but not levetiracetam exerts neurotoxic effect in the developing rat brain. Exp Neurol, 2005, 193(2):497-503

[10] Sfaello I, Baud O, Arzimanoglou A, Gressens P. Topiramate prevents excitotoxic damage in the newborn rodent brain. Neurobiol Dis, 2005, 20(3):837-848

[11] Shi XY, Sun RP, Wang JW. Consequences of Pilocarpine-induced Recurrent Seizures in Neonatal Rats. Brain Dev, 2007, 29(3):157-163

[12] Shi XY, Wang JW, Lei GF, Sun RP. Long-term effects of recurrent seizures on learning, behavior and anxiety: an experimental study in rats. World J Pediatr, 2007, 3(1):61-66

[13] McCabe BK, Silveira DC, Cilio MR, et al. Reduced neurogenesis after neonatal seizures. J Neurosci, 2001, 21(6):2094-2103

[14] Bittigau P, Sifringer M, Genz K, et al. Antiepileptic drugs and apoptotic neurodegeneration in the developing brain. Proc Natl Acad Sci USA, 2002, 99(23):15089- 15094

[15] Webb SJ, Monk CS and Nelson CA. Mechanisms of postnatal neurobiological development: implications for human development. Dev Neuropsychol, 2001,19(2): 147-171

[16] Huang EJ, Reichardt L. Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci, 2001, 24: 677-736

[17] Dobbing J, Sands J. Comparative aspects of the brain growth spurt. Early Hum Dev, 1979, 3(1):79-83

[18] Hao YL, Creson T, Zhang L, et al. Mood Stabilizer Valproate Promotes ERK Pathway-Dependent Cortical Neuronal Growth and Neurogenesis. J Neurosci, 2004, 24(29):6590-6599

[19] Wong WT, Wong RO. Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat Neurosci, 2001, 4(4): 351-352

[20] Luthi A, Schwyzer L, Mateos JM, et al. NMDA receptor activation limits the number of synaptic connections during hippocampal development. Nat Neurosci, 2001, 4(11): 1102-1107

[21] Ogura H, Yasuda M, Nakamura S,et al. Neurotoxic damage of granule cells in the dentate gyrus and the cerebellum and cognitive deficit following neonatal administration of phenytoin in mice. J Neuropathol Exp Neurol, 2002, 61(11): 956-967

(本文编辑：丁俊杰)

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

LIU Yu-jie1,3, SHI Xiu-yu1,3, YU Xin-ying2, HU Lin-yan1, ZOU Li-ping1

(1 Department of Pediatrics, Chinese PLA General Hospital, Beijing 100853; 2 Department of Pediatrics, Gaobeidian City Hospital, Gaobeidian 074000, China; 3 Co-first author)

ZOU Li-ping，E-mail:zouliping21@hotmail.com

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

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.

Antiepileptic drugs; Development ; Apoptosis; Neurotrophins; Neurogenesis

1 中国人民解放军总医院儿科 北京，100853；2 河北省高碑店市医院儿科 高碑店，074000；3 共同第一作者

邹丽萍，E-mail:zouliping21@hotmail.com

10.3969/j.issn.1673-5501.2013.05.012

2013-09-24

2013-12-12)