卡马西平对成年癫癎大鼠海马齿状回BrdU/NeuN表达水平及其对空间记忆的影响

目的 研究卡马西平对成年癫癎大鼠海马齿状回新生神经元的影响及其与空间记忆之间的关系.方法 采用氯化锂和匹罗卡品联合诱导大鼠癫痫 间模型,利用5-溴脱氧尿苷嘧啶与神经元核性蛋白双标记观察海马齿状回内源性神经前体细胞分化为成熟神经元的情况;利用行为学分析评价大鼠的空间记忆.结果 (1)卡马西平可增加癫癎大鼠海马齿状回新生成熟神经元的数量(P＜0.05);(2)卡马西平对癫癎大鼠的空间记忆有明显改善作用(P＜0.01).结论 卡马西平增加癫癎大鼠海马齿状回新生成熟神经元形成,是其改善癫癎大鼠空间记忆的可能机制之一.     

卡马西平对匹罗卡品诱导成年癫间疒大鼠海马齿状回神经前体细胞增殖的影响

目的研究卡马西平对成年癫大鼠海马齿状回内源性神经前体细胞增殖的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫模型,将癫大鼠随机分为癫对照组和癫卡马西平组,正常大鼠随机分为正常对照组和正常卡马西平组。癫对照组和正常对照组给以蒸馏水灌胃,同时癫卡马西平组和正常卡马西平组给予卡马西平灌胃。于灌胃后第6d腹腔注射5-溴脱氧尿苷嘧啶(BrdU)标记海马齿状回的内源性神经前体细胞的增殖情况;用免疫组化方法观察各组大鼠在注射BrdU后第1d、第7d齿状回BrdU阳性细胞数量的表达。结果①注射BrdU后第1d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组减少(P<0.05);②注射BrdU后第7d,癫对照组大鼠海马齿状回BrdU阳性细胞数较正常对照组明显增加(P<0.01),癫卡马西平组大鼠海马齿状回BrdU阳性细胞数较癫对照组明显减少(P<0.05)。结论卡马西平抑制癫大鼠海马齿状回内源性神经前体细胞增殖。     

卡马西平对匹罗卡品诱导成年癫癎大鼠海马齿状回神经发生影响的研究

目的研究卡马西平对成年癫癎大鼠海马齿状回神经发生的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠癫癎模型,于干预后第6d腹腔注射5-溴脱氧尿核苷嘧啶标记海马齿状回内源性神经前体细胞的增殖情况;用免疫组织化学方法及免疫荧光双标方法观察海马齿状回新生细胞的增殖、存活、分化及迁移情况。结果 （1）卡马西平可明显抑制癫癎大鼠海马齿状回新生细胞增殖;（2）卡马西平可明显促进癫癎大鼠海马齿状回新生细胞的存活;（3）卡马西平可增加癫癎大鼠海马齿状回新生神经元的数量,但不增加新生细胞分化为成熟神经元的比例;（4）卡马西平对新生神经细胞的异位迁移无抑制作用。结论卡马西平对癫癎大鼠海马齿状回神经发生的影响是其控制癫癎临床症状的可能机制之一。     

不同程度的性发作对成年大鼠空间学习记忆影响的研究

目的研究不同程度的性发作对成年大鼠空间学习记忆的影响。方法采用氯化锂和匹罗卡品联合诱导大鼠不同程度的癫模型(轻型和重型)。于造模后第6d给所有大鼠腹腔注射5-溴脱氧尿苷嘧啶(BrdU+)标记海马齿状回增殖的内源性神经前体细胞;用免疫组化方法观察各组大鼠注射BrdU+后第1d和第28d齿状回BrdU+阳性细胞数以及第28d的BrdU+/神经元核性蛋白(NeuN+)阳性细胞数及分布情况;利用Morris水迷宫评价大鼠的学习记忆功能。结果与正常组及轻型组比较,在各个时间点重型组海马齿状回BrdU+细胞数均增加(P<0.05),28d时BrdU+/NeuN+细胞数相应增多,但其占BrdU+细胞数的比例明显下降(P<0.05)。28d时重型组大鼠的学习记忆功能较正常组及轻型组明显下降(P<0.05)。结论严重的癫发作造成大鼠对空间学习记忆功能的损害,可能与其刺激大鼠海马齿状回内源性神经前体细胞增殖水平,抑制其分化为新生的成熟神经元有关。     

Orexin-1受体拮抗剂对慢性点燃癫大鼠学习记忆的影响及海马神经细胞的增殖变化

目的:研究orexin-1受体(OX1R)拮抗剂(SB334867,SB)对戊四氮(PTZ)慢性点燃癫大鼠空间学习记忆能力及海马齿状回神经细胞增殖的影响。方法:Wistar大鼠随机分为①对照组[腹腔和侧脑室均注射生理盐水(NS)];②PTZ组(腹腔注射PTZ+侧脑室注射NS);③PTZ+orexin-A(OXA)组(腹腔注射PTZ+侧脑室注射OXA);④PTZ+SB组(腹腔注射PTZ+侧脑室注射SB);⑤PTZ+SB+OXA组(腹腔注射PTZ+侧脑室注射SB和OXA)。观察各组大鼠的空间学习记忆能力及海马齿状回区BrdU+和BrdU+/NeuN+细胞的表达。结果:与PTZ+OXA组比较,PTZ+SB+OXA组大鼠逃避潜伏期延长、穿越平台象限的次数减少(P<0.05)。免疫荧光显示,PTZ+OXA组大鼠齿状回区BrdU+和BrdU+/NeuN+细胞表达增多(P<0.01),而PTZ+SB+OXA组大鼠齿状回区BrdU+/NeuN+细胞表达比PTZ+OXA组减少(P<0.01)。结论:OXA通过OX1R能改善癫大鼠的空间学习记忆能力,可能与OX1R介导的海马齿状回神经细胞增殖与分化作用有关。     

丰富环境对成年颞叶癫大鼠认知功能及神经发生的影响

目的对成年颞叶癫大鼠进行丰富环境干预,探讨丰富环境是否能促进认知功能以及是否对齿状回的神经发生产生影响。方法将成年Wistar大鼠随机分为4组:假手术组、丰富环境+假手术组、癫组、丰富环境+癫组。丰富环境干预30 d后,应用Morris水迷宫实验进行学习记忆功能评价,并应用免疫荧光技术观察海马齿状回新生神经细胞的情况。结果一般线性重复变量分析示逃避潜伏期[F(3,36)=9.810,P0.05)]、上台前路程[F(3,36)=11.092,P0.05)],各组间差异有显著性;而游泳速度差异无显著性[F(3,36)=0.776,P0.05)]。丰富环境+假手术组海马齿状回DCX阳性细胞(新生神经细胞标志物)[(65.00±6.10)个]显著多于假手术组[(32.43±2.76)个](P0.05),癫组DCX阳性细胞[(21.30±2.97)个]较假手术组明显减少(P0.05);丰富环境+癫组DCX阳性细胞[(51.50±3.16)个]明显多于癫组(P0.05)。结论丰富环境有利于成年颞叶癫大鼠认知功能的恢复。     

卡马西平对诱发电位的影响及意义

目的研究长期口服卡马西平治疗癫对诱发电位的影响,并讨论其意义。方法选择尚未治疗的癫病人31例作为试验组;以性别、年龄与癫组相匹配的健康正常人26例作为对照组。两组先分别做脑干听觉诱发电位(BAEP)、事件相关电位P300、视觉诱发电位(VEP)和体感诱发电位(SEP),之后癫组开始卡马西平治疗,服药一年后再作上述各项检查。结果癫组病人治疗前各项电生理学指标与正常对照组相比无显著性差异;癫组卡马西平治疗后各项电生理指标与治疗前相比BAEP各波、P300以及VEP的P100波潜伏期均显著延长;SEP的潜伏期无显著变化。结论神经电生理学检查可以早期发现长期服用卡马西平导致的亚临床毒性。     

托吡酯对癫痫大鼠海马细胞外液氨基酸和神经元凋亡的影响

目的研究托吡酯对癫癎大鼠海马区细胞外液氨基酸和神经元凋亡的影响.方法采用戊四氮(PTZ)致癎模型,大鼠癫癎发作后连续给予托吡酯(TPM)80 mg·kg-1·d-1和卡马西平40 mg·kg-1·d-1,共14 d.以TUNEL方法标记DNA片段,原位检测海马凋亡的神经细胞.脑内微透析技术采集大鼠海马细胞外液,反相高效液相色谱技术测定氨基酸类神经递质的含量.结果TPM组、卡马西平组与对照组比较,凋亡细胞数存在显著差异(P＜0.001),TPM组与卡马西平组相比无显著差异(P＞0.05).TPM可明显升高海马细胞外液γ-氨基丁酸(GABA)水平,并降低谷氨酸(Glu)浓度.结论TPM可减轻大鼠癫癎发作后的神经元损伤,这种作用可能是氨基酸变化的结果;但在我们的实验中,没有发现TPM对癫癎后脑损伤比卡马西平有更明显的神经保护作用.     

奥卡西平与卡马西平单药治疗儿童部分性癫的对照研究

目的比较奥卡西平与卡马西平单药治疗儿童部分性癫的疗效。方法71例新诊断的儿童部分性癫患者按单双号顺序分为奥卡西平组(35例)和卡马西平组(36例),并给予相应的药物治疗;6个月后进行疗效评价,观察不良反应。结果奥卡西平组和卡马西平组分别有25例及29例完成6个月治疗;脑电图改善率分别为44.0%及44.8%;显效率分别为92.0%及86.2%;不良反应发生率分别为22.2%及41.2%,两组间比较差异无统计学意义(均P>0.05);卡马西平组2例出现严重不良反应。结论奥卡西平与卡马西平单药治疗新诊断的儿童部分性癫均有很好的疗效,不良反应及耐受性相似,但奥卡西平组未见严重的不良反应。     

丰富环境促进颞叶癫(痫)大鼠神经发生及其机制的研究

目的 探讨丰富环境对颞叶癫(痫)大鼠齿状回新生细胞分化和存活的影响及其相关分子机制.方法 成年Wistar 大鼠随机分为4组:假手术组、丰富环境+假手术组、癫(痫)组、丰富环境+癫(痫)组,各组均n=15.大鼠侧脑室注射海人酸制作颞叶癫(痫)模型.丰富环境干预30 d后,应用免疫荧光技术观察大鼠海马齿状回的新生细胞分化和存活情况,用Western blot方法检测各组海马脑源性神经营养因子(BDNF)、cAMP应答元件结合蛋白(pCREB)、蛋白激酶A(PKA)表达水平.结果 丰富环境+假手术组、丰富环境+癫(痫)组齿状回新生细胞标记物(BrdU)和新生成熟神经细胞标记物(BrdU/NeuN)阳性细胞数分别多于假手术组、癫(痫)组(P＜0.05),而新生星形胶质细胞BrdU/GFAP阳性细胞数无统计学意义,并且丰富环境+假手术组、丰富环境+癫(痫)组海马BDNF和pCREB蛋白表达水平分别高于假手术组、癫(痫)组(P＜o.05),而PKA蛋白表达水平无增高.结论 丰富环境可能通过增强pCREB/BDNF通路促进成年颞叶癫(痫)大鼠海马齿状回的神经发生.     

Roles of astrocytes and microglia in seizure‐induced aberrant neurogenesis in the hippocampus of adult rats

Recent evidence showed that epileptic seizures increase hippocampal neurogenesis in the adult rat, but prolonged seizures result in the aberrant hippocampal neurogenesis that often leads to a recurrent excitatory circuitry and thus contributes to epileptogenesis. However, the mechanism underlying the aberrant neurogenesis after prolonged seizures remains largely unclear. In this study, we examined the role of activated astrocytes and microglia in the aberrant hippocampal neurogenesis induced by status epilepticus. Using a lithium‐pilocarpine model to mimic human temporal lobe epilepsy, we found that status epilepticus induced a prominent activation of astrocytes and microglia in the dentate gyrus 3, 7, 14, and 20 days after the initial seizures. Then, we injected fluorocitrate stereotaxicly into the dentate hilus to inhibit astrocytic metabolism and found that fluorocitrate failed to prevent the seizure‐induced formation of ectopic hilar basal dendrites but instead promoted the degeneration of dentate granule cells after seizures. In contrast, a selective inhibitor of microglia activation, minocycline, inhibited the aberrant migration of newborn neurons at 14 days after status epilepticus. Furthermore, with stereotaxic injection of lipopolysaccharide into the intact dentate hilus to activate local microglia, we found that lipopolysaccharide promoted the development of ectopic hilar basal dendrites in the hippocampus. These results indicate that the activated microglia in the epileptic hilus may guide the aberrant migration of newborn neurons and that minocycline could be a potential drug to impede seizure‐induced aberrant migration of newborn neurons. © 2009 Wiley‐Liss, Inc.     

Effects of drugs on the initiation and maintenance of status epilepticus induced by administration of pilocarpine to lithium-pretreated rats

The ability of various drugs to prevent the onset of status epilepticus induced by administration of the muscarinic agonist, pilocarpine, to lithium-pretreated rats was determined. Motor limbic seizures and status epilepticus occurred in 100% of rats administered pilocarpine (30 mg/kg, s.c.) 20 h after pretreatment with lithium (3 meq/kg, i.p.). The latency to spike activity and to status epilepticus was 20 +/- 1 min and 24 +/- 1 min, respectively. Atropine, diazepam, phenytoin, carbamazepine, phenobarbital, paraldehyde, and L-phenylisopropyladenosine (L-PIA) prevented all phases of seizure activity induced by lithium/pilocarpine treatment. The initiation of status epilepticus was significantly prolonged by pretreatment with sodium valproate. These findings indicate that the seizures induced by administration of lithium and pilocarpine accurately model generalized tonic-clonic epilepsy. The anticonvulsant activity of L-PIA was prevented by prior treatment with the adenosine antagonist, theophylline. The latency to spike and seizure activity was decreased by theophylline, indicating that endogenous adenosine may have a tonic inhibitory influence on cholinergic neurons. Atropine, diazepam, phenobarbital, phenytoin, sodium valproate, L-PIA, and carbamazepine did not interrupt seizure activity when administered 60 min after pilocarpine (approximately 35 min after initiation of status epilepticus). When rats were administered paraldehyde at this time, status epilepticus was rapidly terminated and all rats survived. Thus, status epilepticus induced by lithium and pilocarpine provides a seizure model that is not responsive to conventional anticonvulsants.     

Neurogenesis in the dentate gyrus of the rat following electroconvulsive shock seizures

Electroconvulsive shock (ECS) seizures provide an animal model of electroconvulsive therapy (ECT) in humans. Recent evidence indicates that repeated ECS seizures can induce long-term structural and functional changes in the brain, similar to those found in other seizure models. We have examined the effects of ECS on neurogenesis in the dentate gyrus of the adult rat using bromodeoxyuridine (BrdU) immunohistochemistry, which identifies newly generated cells. Cells have also been labeled for neuronal nuclear protein (NeuN) to identify neurons. One month following eight ECS seizures, ECS-treated rats had approximately twice as many BrdU-positive cells as sham-treated controls. Eighty-eight percent of newly generated cells colabeled with NeuN in ECS-treated subjects, compared to 83% in sham-treated controls. These data suggest that there is a net increase in neurogenesis within the hippocampal dentate gyrus following ECS treatment. Similar increases have been reported following kindling and kainic acid- or pilocarpine-induced status epilepticus. Increased neurogenesis appears to be a general response to seizure activity and may play a role in the therapeutic effects of ECT.     

N-cadherin在颞叶癫痫海马苔藓纤维出芽和突触重组中的作用

目的 探讨神经性钙粘附分子(N-cadherin)在癫痫状态后海马苔藓纤维出芽和突触重组中的作用。方法取锂一匹罗卡品诱导大鼠癫痫持续状态及慢性自发性颞叶癫痫发作期的大鼠脑片，用Timm染色和免疫组化的方法分别检测苔藓纤维出芽和N-cadherin在大鼠海马组织中的表达。结果癫痫状态后第2周和第4周的实验组大鼠可见到苔藓纤维出芽，穿越齿状回颗粒细胞层到达内分子层，并在此形成一条致密的层状带(Timm染色)。免疫组化染色发现实验组大鼠在第2周和第4周，海马齿状回内分子层可以看到强染色，并形成一条致密带，与Timm染色时观察到的条带一致。结论癫痫状态后在海马齿状回内分子层N-cadherin的表达上调．N-cadherin可能参与了癫痫后苔藓纤维出芽和突触重组过程。     

Resistance of immature hippocampus to morphologic and physiologic alterations following status epilepticus or kindling.

Seizures in adult rats result in long-term deficits in learning and memory, as well as an enhanced susceptibility to further seizures. In contrast, fewer lasting changes have been found following seizures in rats younger than 20 days old. This age-dependency could be due to differing amounts of hippocampal neuronal damage produced by seizures at different ages. To determine if there is an early developmental resistance to seizure-induced hippocampal damage, we compared the effects of kainic acid (KA)-induced status epilepticus and amygdala kindling on hippocampal dentate gyrus anatomy and electrophysiology, in immature (16 day old) and adult rats. In adult rats, KA status epilepticus resulted in numerous silver-stained degenerating dentate hilar neurons, pyramidal cells in fields CA1 and CA3, and marked numerical reductions in CA3c pyramidal neuron counts (-57%) in separate rats. Two weeks following the last kindled seizure, some, but significantly less, CA3c pyramidal cell loss was observed (-26%). Both KA status epilepticus and kindling in duced mossy-fiber sprouting, as evidenced by ectopic Timm staining in supragranular layers of the dentate gyrus. In hippocampal slices from adult rats, paired-pulse stimulation of perforant path axons revealed a persistent enhancement of dentate granule-cell inhibition following KA status epilepticus or kindling. While seizures induced by KA or kindling in 16-day-old rats were typically more severe than in adults, the immature hippocampus exhibited markedly less KA-induced cell loss (-22%), no kindling-induced loss, no detectable synaptic rearrangement, and no change in dentate inhibition. These results demonstrate that, in immature rats, neither severe KA-induced seizures nor repeated kindled seizures produce the kind of hippocampal damage and changes associated with even less severe seizures in adults. The lesser magnitude of seizure-induced hippocampal alterations in immature rats may explain their greater resistance to long-term effects of seizures on neuronal function, as well as future seizure susceptibility. Conversely, hippocampal neuron loss and altered synaptic physiology in adults may contribute to increased sensitivity to epileptogenic stimuli, spontaneous seizures, and behavioral deficits.     

Seizure severity‐dependent selective vulnerability of the granule cell layer and aberrant neurogenesis in the rat hippocampus

The pilocarpine‐induced status epilepticus rodent model has been commonly used to analyze the mechanisms of human temporal lobe epilepsy. Recent studies using this model have demonstrated that epileptic seizures lead to increased adult neurogenesis of the dentate granule cells, and cause abnormal cellular organization in dentate neuronal circuits. In this study, we examined these structural changes in rats with seizures of varying severity. In rats with frequent severe seizures, we found a clear loss of Prox1 and NeuN expression in the dentate granule cell layer (GCL), which was confined mainly to the suprapyramidal blade of the GCL at the septal and middle regions of the septotemporal axis of the hippocampus. In the damaged suprapyramidal region, the number of immature neurons in the subgranular zone was markedly reduced. In contrast, in rats with less frequent severe seizures, there was almost no loss of Prox1 and NeuN expression, and the number of immature neurons was increased. In rats with no or slight loss of Prox1 expression in the GCL, ectopic immature neurons were detected in the molecular layer of the suprapyramidal blade in addition to the hilus, and formed chainlike aggregated structures along the blood vessels up to the hippocampal fissure, suggesting that newly generated neurons migrate at least partially along blood vessels to the hippocampal fissure. These results suggest that seizures of different severity cause different effects on GCL damage, neurogenesis, and the migration of new neurons, and that these structural changes are selective to subdivisions of the GCL and the septotemporal axis of the hippocampus.