抗癫痫药物对癫痫幼鼠认知的影响及其机制的研究
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摘要
研究背景
癫痫是临床常见的慢性神经系统疾病,以生后第一年发病率最高,部分患儿还伴有认知功能的损害,不仅危害了身心健康,还降低了整个家庭的生活质量。与癫痫相关的认知障碍一直是医生及家长非常关注的问题。癫痫患儿伴发的认知障碍与发病原因、起病年龄、病灶部位、发作类型、持续时间、发作频率及抗癫痫药物等多种因素均有关,但由于抗癫痫药物是可触及、可选择因素,且为目前治疗癫痫最主要方法而受到更多的关注。传统抗癫痫药物对认知功能的影响已有大量研究报道,发现对认知的多个方面包括空间学习记忆,运动水平,注意力甚至情绪等均可产生不良影响。虽然有一些研究显示新型抗癫痫药物较传统抗癫痫药物对认知功能的影响小,但研究并不充分,对发育期大脑影响的报道较少见,对托吡酯等药物到底是利是弊也存有争议。因此本研究中的抗癫痫药物既有存在争论的托吡酯(TPM),也有近年临床普遍应用的其它新型抗癫痫药左乙拉西坦(LEV)、拉莫三嗪(LTG)和奥卡西平(OXC),还包括目前仍广泛应用的传统抗癫痫药丙戊酸钠(VPA),通过不同的测试方法评价这些药物对幼鼠空间学习记忆、工作记忆及活动水平的影响。
抗癫痫药物对认知影响的具体机制至今尚不明了,使得临床上医生对于防治抗癫痫药物对认知的副作用无的放矢。以往已经进行了一些关于抗癫痫药物对大脑功能损害的研究,大多局限于对大脑细胞凋亡的影响,但是对其他可能的机制如对神经营养物质、神经元前体细胞增殖和迁移影响的研究很少,对突触结构及功能可塑性等方面的研究更是罕见,而这些也有可能是造成生前或生后接触抗癫痫药引起认知损害的重要原因,有必要对这些机制进行进一步的研究。
目前的癫痫动物实验多使大鼠经历单次痫性发作持续状态,而临床上的癫痫患儿常有反复发作而非单次的长时程惊厥。数项研究也显示在未成熟动物中,单次的长时发作极少导致细胞丧失及发芽,也极少有学习记忆及行为缺陷,因此本实验以经历3次匹罗卡品诱发癫痫发作的新生期幼鼠为模型,并与正常大鼠进行对照研究,在模拟临床的同时观察抗癫痫药物是否对正常大鼠和癫痫大鼠有不同的影响。此前的实验多集中于成年期,对于发育期大脑的研究并不多,而小儿尤其是新生儿的神经生理及解剖与成人有很大不同,如突触产生及发育尚未完成,未成熟海马和皮层神经元对惊厥活动更易感,电惊厥传播异常,黑质惊厥抑制系统发育不足等,从而推测发育期的脑对抗癫痫药物的反应可能与成年期不同,之前也有研究显示抗癫痫药物对幼年动物认知功能的损伤大于成年动物。因此非常有必要深入研究长期应用抗癫痫药对发育期大脑认知功能的影响及其机制,对临床药物选择提供理论依据,实现有效控制癫痫发作同时降低对认知的影响。
目的
研究长期应用抗癫痫药物对癫痫幼鼠的空间学习记忆、工作记忆、活动水平以及神经营养物质、神经发生、苔藓纤维发芽、突触可塑性相关蛋白的影响,探讨抗癫痫药物对发育期大脑认知功能的影响及其机制。
方法
1.造模、分组及投药：
选用岀生后1d(P1)的雄隆Wistar大鼠,随机分为癫痫组及对照组，癫痫组大鼠分别在生后1d,4d及7d予腹腔注射匹罗卡品诱发癫痫发作。对照组大鼠腹腔注射同等量生理盐水。将生后8d的存活的癫痫组大鼠随机分为生理盐水对照组(EP+NS)、VPA治疗组(EP+VPA)、LEV(?)治疗组(EP+LEV)、OXC治疗组(EP+OXC)、TPM治疗组(EP+TPM)、LTG治疗组(EP+LTG)对照组大鼠随机分为生理盐水对照组(CON+NS)、应用VPA组(CON+VPA)、应用LEV组(CON+LEV)、应用OXC组(CON+OXC)、应用TPM组(CON+TPM)、应用LTG组(CON+LTG)。自生后8d,各药物干预组大鼠分别接受以下药物每日灌胃1次VPA200mg/kg、LEV100mg/kg、 TPM40mg/kg、OXC100mg/kg、LTG40mg/kg。生理盐水对照组每日接受生理盐水灌胃。连续用药3周。
2.研究长期应用抗癫痫药物对幼鼠认知的影响：
所有大鼠于P29-P30进行旷场测试以评价其活动水平,P31-P36时进行Morris水迷宫测试评价其视觉—空间学习和记忆能力,P37P-38进行改良的Y迷宫以评价其工作记忆。
3.研究长期应用抗癫痫药物对发育期大脑神经营养物质、神经发生、苔藓纤维发芽及突触可塑性相关蛋白的影响：
用药3周后,每组大鼠取6只断头取脑,测量脑重,用实时定量PCR检测脑源性神经营养因子(BDNF)和突触小体相关蛋白(SNAP-25) mRNA的表达,Western blot检测BDNF、SNAP-25蛋白的表达。每组剩余的大鼠过量麻醉后分别灌注固定取脑行BrdU染色与Timm染色观察海马神经发生与苔藓纤维发芽。
结果
1.长期应用抗癫痫药物对幼鼠认知的影响：
(1)体重增长：VPA处理组大鼠用药4天后出现体重增长加快(P<0.05), LTG处理大鼠用药1周后体重增张减慢(P＜0.05)。TPM、LEV及OXC处理组大鼠体重增长与相应对照组无差异(P＞0.05)。
(2)旷场实验：EP+NS组大鼠在固定时间内穿越的方格数明显低于CON+NS组大鼠(P＜O.01)。LEV处理组大鼠穿越的方格数较相应对照组增多(P<0.05)。LTG处理组大鼠穿越的方格数较相应对照组减少(P＜0.05)。VPA、TPM及OXC处理大鼠穿越的方格数与相应盐水对照组无差异(P＞.05)。
(3)Morris水迷宫：EP+NS组大鼠较CON+NS组找到水下平台的时间明显延长,穿越原平台位置次数减少,在目的象限停留时间缩短(P<0.05)。VPA处理组及CON+TPM组大鼠也较各自对照组找到平台的时间延长,穿越原平台位置的次数减少,在目的象限停留时间缩短(P<0.05); EP+LEV组大鼠搜寻平台的潜伏期缩短,穿越原平台位置的次数增加,在目的象限停留时间延长(P＜0.05)。其余各组大鼠的成绩与相应对照组无差异(P＞0.05)
(4)改良的Y迷宫：EP+NS组大鼠每天第一轮与第二轮测试中,每轮的后10次尝试,与前10次相比,错误次数无明显变化(P＞0.05),无论是训练阶段还是保持阶段,总错误次数均高于CON+NS组大鼠(P<0.05)。EP+LEV组大鼠成绩优于EP+NS组,每日两轮测试中,每轮的后10次尝试与前10次相比错误次数明显减少(P＜0.05)。CON+TPM组大鼠的成绩落后于CON+NS组,每日每轮测试后10次尝试中的错误次数与前10次相比无统计学意义(P＞0.05)。其余各组大鼠的表现与相应对照无明显差异(P＞0.05)。
2.长期应用抗癫痫药对BDNF、神经发生、苔藓纤维发芽及SNAP-25的影响。
(1)脑重：EP+NS组大鼠脑重较CON+NS组降低约15%(P＜0.01)。VPA及TPM处理组大鼠脑重均较相应对照组降低(P<0.05)。EP+LEV组大鼠脑重较EP+NS组增加(P<0.01)。OXC、LTG处理组及CON+LEV组大鼠脑重与相应对照组无差异(P>0.05)。
(2)BDNF:EP+NS组大鼠海马中BDNF mRNA及蛋白表达均高于CON+NS组(P<0.01).VPA及TPM处理组大鼠海马中BDNF mRNA与蛋白的表达均较相应对照组降低(P<0.05)。LEV及LTG处理组大鼠海马中BDNF mRNA与蛋白的表达较相应对照组增加(P<0.05)、OXC处理组大鼠的表达与相应对照组无差异(P＞0.05)。
(3)神经发生：EP+NS组大鼠海马齿状回、CA3区、门区和海马外区域中BrdU标记的细胞数较CON+NS组显著增加(P<0.01)。EP+LEV组、EP+TPM组、EP+OXC组、EP+LTG组大鼠齿状回BrdU阳性细胞数均少于EP+NS组(P＜0.05);EP+VPA组大鼠齿状回BrdU阳性细胞数则较EP+NS组增多(P＜0.05)。对照组中所有用药组大鼠BrdU阳性细胞数与CON+NS组相比均无显著性差异(P＞0.05)。
(4)苔藓纤维发芽:Timm染色结果显示EP+NS组大鼠海马CA3区及颗粒上层的Timm记分均高于CON+Ns组(P<0.05)。但所有药物处理组大鼠CA3区及颗粒上层的Timm记分与相应生理盐水对照组相比均无统计学意义(P＞0.05)。
(5)SNAP-25:EP+NS组大鼠海马中·SNAP-25mRNA及蛋白的表达低CON+NS组(P＜0.01)。VPA处理组大鼠SNAP-25mRNA及蛋白的表达也较相应对照组降低(P<0.05)。LEV、TPM、OXC及LTG处理组与相应对照组的NAP-25mRNA及蛋白表达无差异。(P＞0.05)
结论
1.不同的抗癫痫约物对认知的影响不同
(1)TPM可损害正常幼鼠的空间学习记忆能力；LEV能改善癫痫幼鼠的空间学习记忆；VPA对癫痫幼鼠及正常幼鼠的空间学习记忆能力均有损害;LTG及OXC对癫痫幼鼠及正常幼鼠的空间学习记忆均无不良影响。
(2)TPM损害正常幼鼠的工作记忆;LEV改善了癫痫幼鼠的工作记忆；VPA、 LTG及OXC对癫痫幼鼠及正常幼鼠的工作记忆无不良影响。
(3) TPM、OXC、VPA不影响幼鼠的活动水平；LEV可增加活动水平;LTG可致活动水平降低及体重增长减慢；VPA致体重增长加快；TPM、OXC、LEV对体重无影响。
2.不同抗癫痫药物对认知影响的机制不同。
(1)VPA、TPM、OXC、LEV、LTG均不影响正常幼鼠的神经发生及所有大鼠的苔藓纤维,但VPA增强了癫痫幼鼠的神经发生；TPM、OXC、LEV、LTG降低了癫痫幼鼠神经发生。
(2)VPA可致海马中SNAP-25的表达减少；TPM、OXC、LEV、LTG对SNAP-25的表达无影响。
(3) VPA、TPM可致癫痫幼鼠及正常幼鼠海马中BDNF的表达减少且脑重降低；LTG、LEV可使癫痫幼鼠及正常幼鼠海马中BDNF的表达增高,且LEV可致癫痫幼鼠脑重增加,LTG则对脑重无影响；OXC对脑重及BDNF表达均无影响。
Background
Epilepsy is a common chronic disease of nervous system, wiht high incidence childhood involving cognitive impairement. Multiple factors led to this problem, including the seizure type, frequency and duration, etiology, local ization of the epileptic focus, age at onset of epilepsy, antiepi1optic drugs and so on. Anti-epileptic drugs are the most widely used medications for the treatment of epilepsy. However, many clinical and animal experiments have found that traditional anti-epileptic drugs affect cognition in various aspects, including spatial learning and memory, locomot ion, at tention and even emotion. In the recent years, new anti epileptic drugs such as sodium valproate, levetiracet am, lamotrigine topiramate, oxcarbazopine were widely used in pediatric practice. Although solid evidences are scarcely available, the general belief is that newer anti epileptic drugs may have mild effects on cognition than traditional anti-epileptic drugs. To support, this belief, the cognitive influence of newer anti epileptic drugs should be thoroughly studied.
To date, the mechanism of the impact of antiepileptic drugs on cognition is not well understood. This has caused confusion amongst clinicians as to how to prevent side effects of anti-epileptic drugs. Previous experiments have shown that anti-epileptic drugs in newborn rodents may cause neurotoxicity, of which the mechanism is believed to the enhancement of neurodegeneration due to increased apoptosis. However, it is not certain whether other common mechanisms also contribute to human neurological deficits caused by perinatal exposure to anti-epileptic drugs, these mechanisms including impairment of proliferation or migration of neuronal progenitors and disturbance of synaptogenesis. Thus, the present study investigated the effects of anti-epileptic drugs on neurotrophins, neurogenesis, mossy fiber sprouting and synaptic plasticity, in an attempt to elucidate the possible mechanisms of anti-epileptic drugs associated cognitive impairment.
Currently, rat models usually experience a single status epilepticus. However, children with the clinical condition often have recurring seizures. Several studies have also shown that in immature animals, a single long seizure rarely results in cell loss and germination, and has little effect on learning, memory and behavior. Therefore, in the experiments presented here, we have used neonatal rats that have experienced three pilocarpine-induced seizures as a model to observe the effect of long-term application of anti-epileptic drugs on cognition in the developing brain.
in addition, since the neonatal brain is immature due to underdeveloped synaptogenesis, immature brain is different from the mature one both in physiology and anatomy, immature brain is easier to occur seizure. On the other hand, children with epilepsy often receive anti-epileptic drugs for longer time, effets of anti-epileptic drugs on immature brain may be different from the mature one. Therefore it is necessary to explore the effects and mechanism of anti-epileptic drugs on cognition to pave the way for safe and efficacious clinical practice for neonate and infants.
Objectives
The aim of is to elucidate the effects and mechanisms of long term anti-epileptic drugs treatment on immature brain cognition. From the aspects of spatial learning and working memory, locomotor activity, neurotrophins expression, neurogenesis, mossy fiber sprouting, synaptic plasticity
Methods
1. The male Wistar rats (P1) were randomly divided into two groups, group epilepy and group control. Rats in group epilepsy were given intraperitoneal injections of pilocarpine on P1, P4and P7after birth. Rats in Group control were injected with the same volume of saline in the same way. In epilepy group, all surviving rats were randomly divided into six groups on P8:EP+NS, EP+LEV, EP+VPA, EP+OXC, EP+TPM, EP+LTG. In control group, rats were also randomly divided into six groups:CON+NS, CON+LEV, CON+VPA, CON+OXC, CON+TPM, CON+LTG. From the P8, rats in the drug treatment group were administered VPA, LEV, TPM, OXC, LTG respectively; the remaining rats were treated with saline.
2. After3-weeks treatment with anti-epileptic drugs, locomotor activity were checked by using open field tests on P29, spatial learning ware checked by using Morris water maze on P31and working memory were checked by using Y maze on P37.
3. After above tost, all rats in the epilepsy groups and the controls were sacrificed and randomly subgouped for, western blot and real time PCR detection, BrdU staining or Timm's staining, respectively.
Results
1. VPA treated rats gain weight and LTG treated rats lost weight compared with the control rats (P<0.05). In open field test EP+NS group has lower locomotor activity than CON+NS group (P<0.05); LEV treatment promoted rat locomotor activity while LTG treatment suppress rats activity. Inmorris water maze, rats in EP+NS group, VPA group and CON+TPM group need longer time to find the platform but shorter time in the target quadrant than CON+NS group (P<0.05). Rats in EP+LEV group need shorter time to find the platform and longer time in the target quadrant.(P<0.05); Rats in OXC group, LTG group, EP+TPM group and CON+LEV group had no differences with corresponding control group in the test (P>0.05); In Y maze test, all control rats(except CON+TPM group)and EP+LEV group rats have a good grasp of the left-right alternative memory model. rats in CON+TPM group and all epileptic rats(except EP+LEV group) are difficult in working memory, total errors of the initial ten trials were similar to the latter ten trials for these rats.
2. The brain weight of EP+NS group rats was lower than CON+NS group (P<0.05), Long-term treatment with VPA and TPM reduced brain weight while LEV increased the brain weight of EP+LEV group rats(P<0.05);VPA and TPM suppressed the expression of BDNF in the developing brain, LEV and LTG increased the expression of BDNF (P<0.05);Compared with those in the control+NS group, rats in the EP+NS group showed an increased number of BrdU-labeled cells in the dentate gyrus, hilus, CA3region and outer area of the hippocampus (P<0.01).Neurogenesis increased in the rats treated with VPA but reduced in the rats treated with LEV, TPM, OXC, LTG; EP+NS group had significantly more Timm particles than CON+NS group In the hippocampal CA3area and supragranular layer (P<0.05),the effect of anti-epileptic drug on mossy fiber sprouting was not significant(P>0.05); VPA suppressed the expression of SNAP-25in the developing brain while other anti-epileptic drugs in this study have no effect on it.
Conclusions
1. Anti-epileptic drugs exhibit different effects on cognition.
(1) VPA cause damage to the spatial learning and memory ability but have no adverse effect on working memory and locomotor activity.
(2)LEV improve the spatial learning and memory ability, working memory of epileptic rats but have no positive effects on normal rats, LEV increase locomotor activity.
(3)TPM impair spatial learning and working memory of normal rats but have no adverse effect on epileptic rats. TPM have on effect on locomotor activity.
(4)LTG have no adverse effect on spatial learning and working memory but depress locomotor activity.
(5) Oxc have no adverse effect on spatial learning and memory ability, working memory and locomotor activity.
2. Different anti-epileptic drugs affect cognition through different mechanisms
(1) VPA cause reduction in brain weight, suppresse the expression of BDNF and SNAP-25in brain, increase neurogenesis, but have no effect on mossy fiber sprouting in young rats, with or without epilepsy
(2)LHV increase the expression of BDNF and the brain weight of epileptic rats, reduce HP-induced neurogenesis, but have no effect on the expression of SNAP-25, mossy fiber sprouting and the brain weight, of normal rats.
(3)TPM cause reduction in brain weight, suppresse the expression of BDNF in brain, reduce EP-induced neurogenesis, but have no effect on the expression of SNAP-25and mossy fiber sprouting
(4) LTG increase the expression of BDNF and the brain weight of epileptic rats, reduce EP-induced neurogenesis, but have no effect on the expression of SNAP-25, mossy fiber sprouting and the brain weight.
(5) OXC reduce EP-induced neurogenesis, have no effect on the expression of BDNF and SNAP-25, mossy fiber sprouting and the brain weight.
癫痫是临床常见的慢性神经系统疾病,以生后第一年发病率最高,部分患儿还伴有认知功能的损害,不仅危害了身心健康,还降低了整个家庭的生活质量。与癫痫相关的认知障碍一直是医生及家长非常关注的问题。癫痫患儿伴发的认知障碍与发病原因、起病年龄、病灶部位、发作类型、持续时间、发作频率及抗癫痫药物等多种因素均有关,但由于抗癫痫药物是可触及、可选择因素,且为目前治疗癫痫最主要方法而受到更多的关注。传统抗癫痫药物对认知功能的影响已有大量研究报道,发现对认知的多个方面包括空间学习记忆,运动水平,注意力甚至情绪等均可产生不良影响。虽然有一些研究显示新型抗癫痫药物较传统抗癫痫药物对认知功能的影响小,但研究并不充分,对发育期大脑影响的报道较少见,对托吡酯等药物到底是利是弊也存有争议。因此本研究中的抗癫痫药物既有存在争论的托吡酯(TPM),也有近年临床普遍应用的其它新型抗癫痫药左乙拉西坦(LEV)、拉莫三嗪(LTG)和奥卡西平(OXC),还包括目前仍广泛应用的传统抗癫痫药丙戊酸钠(VPA),通过不同的测试方法评价这些药物对幼鼠空间学习记忆、工作记忆及活动水平的影响。
抗癫痫药物对认知影响的具体机制至今尚不明了,使得临床上医生对于防治抗癫痫药物对认知的副作用无的放矢。以往已经进行了一些关于抗癫痫药物对大脑功能损害的研究,大多局限于对大脑细胞凋亡的影响,但是对其他可能的机制如对神经营养物质、神经元前体细胞增殖和迁移影响的研究很少,对突触结构及功能可塑性等方面的研究更是罕见,而这些也有可能是造成生前或生后接触抗癫痫药引起认知损害的重要原因,有必要对这些机制进行进一步的研究。
目前的癫痫动物实验多使大鼠经历单次痫性发作持续状态,而临床上的癫痫患儿常有反复发作而非单次的长时程惊厥。数项研究也显示在未成熟动物中,单次的长时发作极少导致细胞丧失及发芽,也极少有学习记忆及行为缺陷,因此本实验以经历3次匹罗卡品诱发癫痫发作的新生期幼鼠为模型,并与正常大鼠进行对照研究,在模拟临床的同时观察抗癫痫药物是否对正常大鼠和癫痫大鼠有不同的影响。此前的实验多集中于成年期,对于发育期大脑的研究并不多,而小儿尤其是新生儿的神经生理及解剖与成人有很大不同,如突触产生及发育尚未完成,未成熟海马和皮层神经元对惊厥活动更易感,电惊厥传播异常,黑质惊厥抑制系统发育不足等,从而推测发育期的脑对抗癫痫药物的反应可能与成年期不同,之前也有研究显示抗癫痫药物对幼年动物认知功能的损伤大于成年动物。因此非常有必要深入研究长期应用抗癫痫药对发育期大脑认知功能的影响及其机制,对临床药物选择提供理论依据,实现有效控制癫痫发作同时降低对认知的影响。
目的
研究长期应用抗癫痫药物对癫痫幼鼠的空间学习记忆、工作记忆、活动水平以及神经营养物质、神经发生、苔藓纤维发芽、突触可塑性相关蛋白的影响,探讨抗癫痫药物对发育期大脑认知功能的影响及其机制。
方法
1.造模、分组及投药：
选用岀生后1d(P1)的雄隆Wistar大鼠,随机分为癫痫组及对照组，癫痫组大鼠分别在生后1d,4d及7d予腹腔注射匹罗卡品诱发癫痫发作。对照组大鼠腹腔注射同等量生理盐水。将生后8d的存活的癫痫组大鼠随机分为生理盐水对照组(EP+NS)、VPA治疗组(EP+VPA)、LEV(?)治疗组(EP+LEV)、OXC治疗组(EP+OXC)、TPM治疗组(EP+TPM)、LTG治疗组(EP+LTG)对照组大鼠随机分为生理盐水对照组(CON+NS)、应用VPA组(CON+VPA)、应用LEV组(CON+LEV)、应用OXC组(CON+OXC)、应用TPM组(CON+TPM)、应用LTG组(CON+LTG)。自生后8d,各药物干预组大鼠分别接受以下药物每日灌胃1次VPA200mg/kg、LEV100mg/kg、 TPM40mg/kg、OXC100mg/kg、LTG40mg/kg。生理盐水对照组每日接受生理盐水灌胃。连续用药3周。
2.研究长期应用抗癫痫药物对幼鼠认知的影响：
所有大鼠于P29-P30进行旷场测试以评价其活动水平,P31-P36时进行Morris水迷宫测试评价其视觉—空间学习和记忆能力,P37P-38进行改良的Y迷宫以评价其工作记忆。
3.研究长期应用抗癫痫药物对发育期大脑神经营养物质、神经发生、苔藓纤维发芽及突触可塑性相关蛋白的影响：
用药3周后,每组大鼠取6只断头取脑,测量脑重,用实时定量PCR检测脑源性神经营养因子(BDNF)和突触小体相关蛋白(SNAP-25) mRNA的表达,Western blot检测BDNF、SNAP-25蛋白的表达。每组剩余的大鼠过量麻醉后分别灌注固定取脑行BrdU染色与Timm染色观察海马神经发生与苔藓纤维发芽。
结果
1.长期应用抗癫痫药物对幼鼠认知的影响：
(1)体重增长：VPA处理组大鼠用药4天后出现体重增长加快(P<0.05), LTG处理大鼠用药1周后体重增张减慢(P＜0.05)。TPM、LEV及OXC处理组大鼠体重增长与相应对照组无差异(P＞0.05)。
(2)旷场实验：EP+NS组大鼠在固定时间内穿越的方格数明显低于CON+NS组大鼠(P＜O.01)。LEV处理组大鼠穿越的方格数较相应对照组增多(P<0.05)。LTG处理组大鼠穿越的方格数较相应对照组减少(P＜0.05)。VPA、TPM及OXC处理大鼠穿越的方格数与相应盐水对照组无差异(P＞.05)。
(3)Morris水迷宫：EP+NS组大鼠较CON+NS组找到水下平台的时间明显延长,穿越原平台位置次数减少,在目的象限停留时间缩短(P<0.05)。VPA处理组及CON+TPM组大鼠也较各自对照组找到平台的时间延长,穿越原平台位置的次数减少,在目的象限停留时间缩短(P<0.05); EP+LEV组大鼠搜寻平台的潜伏期缩短,穿越原平台位置的次数增加,在目的象限停留时间延长(P＜0.05)。其余各组大鼠的成绩与相应对照组无差异(P＞0.05)
(4)改良的Y迷宫：EP+NS组大鼠每天第一轮与第二轮测试中,每轮的后10次尝试,与前10次相比,错误次数无明显变化(P＞0.05),无论是训练阶段还是保持阶段,总错误次数均高于CON+NS组大鼠(P<0.05)。EP+LEV组大鼠成绩优于EP+NS组,每日两轮测试中,每轮的后10次尝试与前10次相比错误次数明显减少(P＜0.05)。CON+TPM组大鼠的成绩落后于CON+NS组,每日每轮测试后10次尝试中的错误次数与前10次相比无统计学意义(P＞0.05)。其余各组大鼠的表现与相应对照无明显差异(P＞0.05)。
2.长期应用抗癫痫药对BDNF、神经发生、苔藓纤维发芽及SNAP-25的影响。
(1)脑重：EP+NS组大鼠脑重较CON+NS组降低约15%(P＜0.01)。VPA及TPM处理组大鼠脑重均较相应对照组降低(P<0.05)。EP+LEV组大鼠脑重较EP+NS组增加(P<0.01)。OXC、LTG处理组及CON+LEV组大鼠脑重与相应对照组无差异(P>0.05)。
(2)BDNF:EP+NS组大鼠海马中BDNF mRNA及蛋白表达均高于CON+NS组(P<0.01).VPA及TPM处理组大鼠海马中BDNF mRNA与蛋白的表达均较相应对照组降低(P<0.05)。LEV及LTG处理组大鼠海马中BDNF mRNA与蛋白的表达较相应对照组增加(P<0.05)、OXC处理组大鼠的表达与相应对照组无差异(P＞0.05)。
(3)神经发生：EP+NS组大鼠海马齿状回、CA3区、门区和海马外区域中BrdU标记的细胞数较CON+NS组显著增加(P<0.01)。EP+LEV组、EP+TPM组、EP+OXC组、EP+LTG组大鼠齿状回BrdU阳性细胞数均少于EP+NS组(P＜0.05);EP+VPA组大鼠齿状回BrdU阳性细胞数则较EP+NS组增多(P＜0.05)。对照组中所有用药组大鼠BrdU阳性细胞数与CON+NS组相比均无显著性差异(P＞0.05)。
(4)苔藓纤维发芽:Timm染色结果显示EP+NS组大鼠海马CA3区及颗粒上层的Timm记分均高于CON+Ns组(P<0.05)。但所有药物处理组大鼠CA3区及颗粒上层的Timm记分与相应生理盐水对照组相比均无统计学意义(P＞0.05)。
(5)SNAP-25:EP+NS组大鼠海马中·SNAP-25mRNA及蛋白的表达低CON+NS组(P＜0.01)。VPA处理组大鼠SNAP-25mRNA及蛋白的表达也较相应对照组降低(P<0.05)。LEV、TPM、OXC及LTG处理组与相应对照组的NAP-25mRNA及蛋白表达无差异。(P＞0.05)
结论
1.不同的抗癫痫约物对认知的影响不同
(1)TPM可损害正常幼鼠的空间学习记忆能力；LEV能改善癫痫幼鼠的空间学习记忆；VPA对癫痫幼鼠及正常幼鼠的空间学习记忆能力均有损害;LTG及OXC对癫痫幼鼠及正常幼鼠的空间学习记忆均无不良影响。
(2)TPM损害正常幼鼠的工作记忆;LEV改善了癫痫幼鼠的工作记忆；VPA、 LTG及OXC对癫痫幼鼠及正常幼鼠的工作记忆无不良影响。
(3) TPM、OXC、VPA不影响幼鼠的活动水平；LEV可增加活动水平;LTG可致活动水平降低及体重增长减慢；VPA致体重增长加快；TPM、OXC、LEV对体重无影响。
2.不同抗癫痫药物对认知影响的机制不同。
(1)VPA、TPM、OXC、LEV、LTG均不影响正常幼鼠的神经发生及所有大鼠的苔藓纤维,但VPA增强了癫痫幼鼠的神经发生；TPM、OXC、LEV、LTG降低了癫痫幼鼠神经发生。
(2)VPA可致海马中SNAP-25的表达减少；TPM、OXC、LEV、LTG对SNAP-25的表达无影响。
(3) VPA、TPM可致癫痫幼鼠及正常幼鼠海马中BDNF的表达减少且脑重降低；LTG、LEV可使癫痫幼鼠及正常幼鼠海马中BDNF的表达增高,且LEV可致癫痫幼鼠脑重增加,LTG则对脑重无影响；OXC对脑重及BDNF表达均无影响。
Background
Epilepsy is a common chronic disease of nervous system, wiht high incidence childhood involving cognitive impairement. Multiple factors led to this problem, including the seizure type, frequency and duration, etiology, local ization of the epileptic focus, age at onset of epilepsy, antiepi1optic drugs and so on. Anti-epileptic drugs are the most widely used medications for the treatment of epilepsy. However, many clinical and animal experiments have found that traditional anti-epileptic drugs affect cognition in various aspects, including spatial learning and memory, locomot ion, at tention and even emotion. In the recent years, new anti epileptic drugs such as sodium valproate, levetiracet am, lamotrigine topiramate, oxcarbazopine were widely used in pediatric practice. Although solid evidences are scarcely available, the general belief is that newer anti epileptic drugs may have mild effects on cognition than traditional anti-epileptic drugs. To support, this belief, the cognitive influence of newer anti epileptic drugs should be thoroughly studied.
To date, the mechanism of the impact of antiepileptic drugs on cognition is not well understood. This has caused confusion amongst clinicians as to how to prevent side effects of anti-epileptic drugs. Previous experiments have shown that anti-epileptic drugs in newborn rodents may cause neurotoxicity, of which the mechanism is believed to the enhancement of neurodegeneration due to increased apoptosis. However, it is not certain whether other common mechanisms also contribute to human neurological deficits caused by perinatal exposure to anti-epileptic drugs, these mechanisms including impairment of proliferation or migration of neuronal progenitors and disturbance of synaptogenesis. Thus, the present study investigated the effects of anti-epileptic drugs on neurotrophins, neurogenesis, mossy fiber sprouting and synaptic plasticity, in an attempt to elucidate the possible mechanisms of anti-epileptic drugs associated cognitive impairment.
Currently, rat models usually experience a single status epilepticus. However, children with the clinical condition often have recurring seizures. Several studies have also shown that in immature animals, a single long seizure rarely results in cell loss and germination, and has little effect on learning, memory and behavior. Therefore, in the experiments presented here, we have used neonatal rats that have experienced three pilocarpine-induced seizures as a model to observe the effect of long-term application of anti-epileptic drugs on cognition in the developing brain.
in addition, since the neonatal brain is immature due to underdeveloped synaptogenesis, immature brain is different from the mature one both in physiology and anatomy, immature brain is easier to occur seizure. On the other hand, children with epilepsy often receive anti-epileptic drugs for longer time, effets of anti-epileptic drugs on immature brain may be different from the mature one. Therefore it is necessary to explore the effects and mechanism of anti-epileptic drugs on cognition to pave the way for safe and efficacious clinical practice for neonate and infants.
Objectives
The aim of is to elucidate the effects and mechanisms of long term anti-epileptic drugs treatment on immature brain cognition. From the aspects of spatial learning and working memory, locomotor activity, neurotrophins expression, neurogenesis, mossy fiber sprouting, synaptic plasticity
Methods
1. The male Wistar rats (P1) were randomly divided into two groups, group epilepy and group control. Rats in group epilepsy were given intraperitoneal injections of pilocarpine on P1, P4and P7after birth. Rats in Group control were injected with the same volume of saline in the same way. In epilepy group, all surviving rats were randomly divided into six groups on P8:EP+NS, EP+LEV, EP+VPA, EP+OXC, EP+TPM, EP+LTG. In control group, rats were also randomly divided into six groups:CON+NS, CON+LEV, CON+VPA, CON+OXC, CON+TPM, CON+LTG. From the P8, rats in the drug treatment group were administered VPA, LEV, TPM, OXC, LTG respectively; the remaining rats were treated with saline.
2. After3-weeks treatment with anti-epileptic drugs, locomotor activity were checked by using open field tests on P29, spatial learning ware checked by using Morris water maze on P31and working memory were checked by using Y maze on P37.
3. After above tost, all rats in the epilepsy groups and the controls were sacrificed and randomly subgouped for, western blot and real time PCR detection, BrdU staining or Timm's staining, respectively.
Results
1. VPA treated rats gain weight and LTG treated rats lost weight compared with the control rats (P<0.05). In open field test EP+NS group has lower locomotor activity than CON+NS group (P<0.05); LEV treatment promoted rat locomotor activity while LTG treatment suppress rats activity. Inmorris water maze, rats in EP+NS group, VPA group and CON+TPM group need longer time to find the platform but shorter time in the target quadrant than CON+NS group (P<0.05). Rats in EP+LEV group need shorter time to find the platform and longer time in the target quadrant.(P<0.05); Rats in OXC group, LTG group, EP+TPM group and CON+LEV group had no differences with corresponding control group in the test (P>0.05); In Y maze test, all control rats(except CON+TPM group)and EP+LEV group rats have a good grasp of the left-right alternative memory model. rats in CON+TPM group and all epileptic rats(except EP+LEV group) are difficult in working memory, total errors of the initial ten trials were similar to the latter ten trials for these rats.
2. The brain weight of EP+NS group rats was lower than CON+NS group (P<0.05), Long-term treatment with VPA and TPM reduced brain weight while LEV increased the brain weight of EP+LEV group rats(P<0.05);VPA and TPM suppressed the expression of BDNF in the developing brain, LEV and LTG increased the expression of BDNF (P<0.05);Compared with those in the control+NS group, rats in the EP+NS group showed an increased number of BrdU-labeled cells in the dentate gyrus, hilus, CA3region and outer area of the hippocampus (P<0.01).Neurogenesis increased in the rats treated with VPA but reduced in the rats treated with LEV, TPM, OXC, LTG; EP+NS group had significantly more Timm particles than CON+NS group In the hippocampal CA3area and supragranular layer (P<0.05),the effect of anti-epileptic drug on mossy fiber sprouting was not significant(P>0.05); VPA suppressed the expression of SNAP-25in the developing brain while other anti-epileptic drugs in this study have no effect on it.
Conclusions
1. Anti-epileptic drugs exhibit different effects on cognition.
(1) VPA cause damage to the spatial learning and memory ability but have no adverse effect on working memory and locomotor activity.
(2)LEV improve the spatial learning and memory ability, working memory of epileptic rats but have no positive effects on normal rats, LEV increase locomotor activity.
(3)TPM impair spatial learning and working memory of normal rats but have no adverse effect on epileptic rats. TPM have on effect on locomotor activity.
(4)LTG have no adverse effect on spatial learning and working memory but depress locomotor activity.
(5) Oxc have no adverse effect on spatial learning and memory ability, working memory and locomotor activity.
2. Different anti-epileptic drugs affect cognition through different mechanisms
(1) VPA cause reduction in brain weight, suppresse the expression of BDNF and SNAP-25in brain, increase neurogenesis, but have no effect on mossy fiber sprouting in young rats, with or without epilepsy
(2)LHV increase the expression of BDNF and the brain weight of epileptic rats, reduce HP-induced neurogenesis, but have no effect on the expression of SNAP-25, mossy fiber sprouting and the brain weight, of normal rats.
(3)TPM cause reduction in brain weight, suppresse the expression of BDNF in brain, reduce EP-induced neurogenesis, but have no effect on the expression of SNAP-25and mossy fiber sprouting
(4) LTG increase the expression of BDNF and the brain weight of epileptic rats, reduce EP-induced neurogenesis, but have no effect on the expression of SNAP-25, mossy fiber sprouting and the brain weight.
(5) OXC reduce EP-induced neurogenesis, have no effect on the expression of BDNF and SNAP-25, mossy fiber sprouting and the brain weight.
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28. Zhou Bo, Zhang Qin, Tian Linyu,et al. Effects of levetiracetam as an add-on therapy on cognitive function and quality of life in patients with refractory partial seizures. Epilepsy&Behavior,2008,12:305-310.
29. Birnstiel S, Wulfert E,Beck SG. Levetiracetam(ucb L059)affects in vitro models of epilepsy in CA3 pyramidal neurons without altering normalsynaptic transmission.Arch Pharmacol 1997,356:611-618.
30. Margineanu DG, Wulfert E. Ucb L059, a novel anticonvulsant, reduces bicuculline-induced hyperexcitability in rat hippocampal CA3 in vivo. Eur J Pharmacol,1995,286:321-325.
31. Birnstiel S, Wulfert E, Beck SG. Levetiracetam (L059) affects in vit-ro models of epilepsy in CA3 pyramidal neurons without altering normal synaptic transmission. Naunyn Schmiedebergs Arch Pharmacol,1997, 356:611-618.
32. Lamberty Y,Margineanu DG, Klitgaard H. Absence of Negative Impact of Levetiracetam on Cognitive Function and Memory in Normal and Amygdala-Kindled Rats.Epilepsy & Behav,2000,1:333-342.
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34. Zona C, Ciotti MT, Avoli M, et al. Topiramate attenuates voltagegated sodium currents in rat cerebellar granule cells. Neurosci Lett, 1997,231: 123-126.
35. Privitera MD. Topiramate: a new antiepileptic drug. Ann Pharmacother, 1997,31:1164-1173.
36. Perucca E. A pharmacological and clinical review on topiramate, a new antiepileptic drug. Pharmacol Res, 1997,35:241-256.
37. Sfaello I, Baud O, Arzimanoglou A, et al. Topiramate prevents exci-totoxic damage in the newborn rodent brain.Neurobiology of disease, 2005, 20:837-848.
38. Stables JP, Bertram E,Dudek FE, et al. Therapy discovery for pharma-coresistant epilepsy and for disease-modi fying therapeutics: summary of the NIH/NINDS/AES models Ⅱ workshop.Epilepsia, 2003, 44:1472-1478.
39. Aldenkamp AP, Baker G,Mulder OG, et al. A multiccnter randomized clinical study to evaluate the effect on cognitive function of topiramate compared wi th valproate as add-on therapy to carbamazepine in patients with partial-onset, seizures. Epilepsia 2000,41:1167-1178.
40.朱乐亭,赵志刚.曲莱.中国新药杂志,2004,13(8)：754-755.
41. Donati F, Gobbi G, Campistol J, et al. Effects of oxcarbazepine on cognitive function in cllildren and adolescents with partial seizures.Neurology, 2006, 67:679-682.
42. Tzitiridou M, Panou T, Ramantani G, et al. Oxcarbazepine monotherapy in benign chi Idhood epilepsy wi th centroternporal spikes:-a clinical and cognitive evaluation. Epilepsy Behav, 2005, 7:458-467.
43. Calabresi P, Picconi B,Sau1le E,et al.ls pharmacological neuro protection dependent on reduced glutamate release?Stroke, 2000,31: 766-772.
44. Crumrine RC, Bergst rand K, Cooper AT,et al. Lamotrigine protects hippocampal CA1 neurons f rom ischemic damage after cardiac arrest. Stroke,1997,28:2230-2237.
45. Gillham R,Kane K,Bryant-Comstock L,et al.A double-blind comparison of LTG and carbamazepine in newly diagnosed epilepsy with health-related quality of life as an outcome measure. Seizure,2000,9:375-379.
46. Blum D, Meador K, Biton V, et al. Cognitive effects of lamotrigine compared with topiramate in patients with epilepsy. Neurology, 2006,67:400-406.
47. Pressler RM, Binnie CD, Coleshill SG,et al. Effect of lamotrigine on cognition in children with epilepsy. Neurology,2006,66:1495-1499.
1. Wu Y, Wang L. The effect of antiepileptic drgus on spatial learning and hippocampal protein kinase Cγ in immature rats. Brain Dev, 2002, 24:82-87.
2. Shannon HE, Love PL. Effects of antiepiloptic drugs on attention as assessed by a five-choice serial reaction time task in rats. Epilepsy & Behav, 2005, 7:620-628.
3. Meador KJ. Current discoveries on the cognitive effects of antiepileptic drugs. Pharmacotherapy, 2000, 20:185S-190S.
4. Kwan P, Brodie MJ. Neuropsychological effects of epilepsy and anti epi1eptic drugs. Lancet, 2001,357:216 222.
5. Glier C, Dzietko M, Bittigau P, et al. Therapeutic doses of topiramate are not toxic to the developing brain.Exp Neurol,2004,187:403 409.
6. Manyhey D, Asi niadou S, Stefovska V, el al.Sulthiame but not levetir acetam exerts neurotoxie effect in the developing rat brain. Exp Neurol,2005, 193:497-503.
7. Pi shman RH B, Ornoy A, Yanai J. Ultrastructural evidence of long lasting cerebellar degeneration after early exposure to phenobarbital in mice.Exp Neurol,1983,79:212-222.
8. Bergman A, Feigenbaum JJ, Yanai J.Neuronal losses in mice following both prenatal and neonatal exposure to Phenobarbital. Ada Anat, 1982, 114: 185-192.
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