戊四氮点燃癫痫大鼠空间学习记忆障碍及海马突触素表达减少的研究

目的:探讨戊四氮点燃癫痫对大鼠空间学习记忆的影响及可能的分子机制。方法:戊四氮(pentylenetet-razol,PTZ)点燃建立慢性癫痫(chronic epileptic,CEP)模型,Morris水迷宫进行行为学检测,western blot方法观察大鼠海马突触素(synaptophysin,P38)蛋白的表达变化。结果:水迷宫试验检测癫痫组大鼠空间学习记忆能力受损(P<0.05,P<0.01);western blot结果表明海马P38蛋白表达较对照组明显减少(P<0.05)。结论:戊四氮点燃癫痫大鼠伴有学习记忆功能减退,其海马P38蛋白的表达减少可能参与了空间学习记忆受损。     

边缘癫痫实验模型海马内突触体素表达

目的探讨癫痫时突触体素(P^38)在海马表达的时间变化及意义。方法建立匹罗卡品边缘癫痫模型，用图像分析系统测定海马不同时间点P^38免疫反应吸光度值。结果P^38免疫反应性在海马呈现两次高峰：致痫后3～6h在海马门区及CA3区P^38短期升高，30～60dCA3区呈现第2次高峰。在内分子层，从第7天开始直至第60天P^38呈进行性增多，且与Neo—Timm染色结果相平行。结论P^38在海马第2次表达增高，平行于苔藓纤维出芽，与自发性发作形成有关。急性期表达增高则与癫痫持续状态的产生与维持有关。     

戊四氮点燃过程中大鼠海马胶质细胞增生和突触重建的研究

目的探讨戊四氮点燃过程中海马胶质细胞增生及突触重建与慢性癫痫发病机制的关系。方法大鼠随机分为对照组、非药物干预组(戊四氮35mg/kg,腹腔注射,每日一次)和药物干预组(苯巴比妥30mg/kg,戊四氮35mg/kg,均为腹腔注射,每日一次)。采用免疫组织化学方法观察胶质原纤维酸性蛋白(GFAP)和神经细胞粘附分子(NCAM)表达水平。结果非药物干预组大鼠注射戊四氮后在行为学未出现惊厥,脑电图未出现痫性放电的点燃前潜伏期内,出现突触重建和胶质细胞增生,以海马CA3区、门区明显,与对照组比较,有显著性差异(P<0.05);与药物干预组对应时间点比较,亦有显著性差异(P<0.05)。结论大鼠注射戊四氮后引起反应性胶质细胞增生和神经元可塑性改变,可能与形成异常神经元放电环路,最终诱发癫痫发作有关,苯巴比妥可抑制异常神经网络的建立,预防癫痫发生。     

戊四氮点燃大鼠中海马谷氨酸转运体的作用研究

目的 研究点燃形成过程中和点燃后谷氨酸转运体的变化,进一步探讨慢性癫痫的点燃机制。方法 将78只雄性成年Wistar 大鼠随机分为对照组（I组）和戊四氮（PTZ）组（Ⅱ组）。Ⅱ组腹腔注射阈下剂量的PTZ（35mg/kg),每日1次,直至达到点燃标准;Ⅰ组腹腔注射等量生理盐水。采用逆转录聚合酶链式反应（RT－PCR）方法检测海马区谷氨酸转运体－1（GLT－1）mRNA和兴奋性氨基酸载体－1（EAAC1）mRNA的表达。结果 GLT－1mRNA的表达在0h、48h时显著升高,随后下降;EAAC1mRNA的表达呈上升趋势。点燃后第60d时,基本恢复至对照组水平。结论 海马区GLT－1的下降和EAAC1的升高可能与癫痫敏感性的形成与维持有关。     

癫痫幼鼠海马和颞叶皮质区缝隙连接蛋白43和突触体素表达的变化

目的观察神经元缝隙连接蛋白43（Cx43）和突触体素（synaptophysin P38）在戊四氮（PTZ）点燃癫痫幼鼠海马及颞叶皮质区中的表达，探讨两者与癫痫的关系及其在癫痫形成中的作用。方法将50只21日龄Wistar大鼠分为对照组和实验组。实验组采用PTZ点燃癫痫幼鼠，按点燃进程分为Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级及Ⅳ级发作组。采用免疫组化和图像分析技术，观察海马及颞叶皮质区Cx43和P38表达的变化。结果应用PTZ点燃后，实验各组幼鼠海马及颞叶皮质区Cx43和P38的表达明显高于对照组（P〈0．01），且随发作级别的增高，幼鼠海马及颞叶皮质各区Cx43和P38的表达均增加。但各组间海马区和颞叶皮质区Cx43和P38的表达情况的比较差异无统计学意义（P〉0．05）。结论Cx43和P38的表达水平与癫痫的发生发展有密切关系，为研究小儿癫痫的病因及发病机制提供依据。     

海马神经细胞活化在戊四氮点燃大鼠癫痫形成过程中的作用

目的 以核因子κB/P65(nuclear factor κB,NF-κB/P65)的核转位作为神经细胞活化的标志,观察在戊四氮(pentylenetetrazol,PTZ)点燃大鼠出现惊厥之前,即点燃过程中海马神经细胞NF-κB活化在癫痫形成过程中的作用.方法 将大鼠随机分为对照组、非药物干预组、药物干预组(苯巴比妥30 mg/kg, 腹腔注射,每日一次).除对照组外均以低于急性致惊剂量的PTZ(40mg/kg,腹腔注射,每日一次)点燃大鼠,用行为学观察和脑电图确定癫痫存在,免疫组织化学方法 检测大鼠癫痫形成过程中不同时间点海马CA各区和齿状回神经细胞NF-κB活化,用图像分析系统分析对照组、非药物干预组和药物干预组三大组间海马神经细胞NF-κB活化的差异.结果 非药物干预组大鼠均于17d～22d点燃,而药物干预组PTZ点燃大鼠所需时间明显延长(于30d～35d点燃),且行为学惊厥程度和脑电痫样放电明显轻于非药物干预组.非药物干预组大鼠在行为学未出现惊厥,脑电图未出现痫样放电的点燃前潜伏期内,其海马CA各区、齿状回NF-κB活化的阳性神经细胞数明显增加,与对照组相比两者有显著性差异(P<0.05);药物干预组在与非药物干预组相应时间点的海马CA各区、齿状回NF-κB活化的阳性神经细胞明显减少,两者有显著性差异(P<0.05).结论 海马神经细胞活化是PTZ点燃大鼠癫痫形成的重要机制之一,苯巴比妥可通过抑制神经细胞活化,预防癫痫发生.     

去势对戊四氮点燃大鼠癫痫模型的行为学表现

目的 研究去势对戊四氮点燃大鼠癫痫模型行为学表现的影响.方法 采用戊四氮腹腔注射制作癫痫大鼠模型,对照研究去势大鼠同正常大鼠的潜伏期及持续时间等行为学表现.结果 大鼠癫痫模型全部点燃,去势组平均潜伏期(8.09±0.89) min((-x)±SD)长于非去势组的(3.94±0.65) min((-x)±SD).发作时间也有所缩短,去势组(19.16 ±3.06) min((-x)±SD)略短于非去势组的(26.37±2.90) min((-x)±SD) (P ＜0.05).非去势组点燃时间明显短于去势组,非去势组平均点燃时间(20.83±6.15) d((-x)±s),而去势组平均点燃时间(24.6±5.64) d((-x)±SD).结论 戊四氮点燃大鼠致痫模型是一种成熟的、较为安全的癫痫模型.去势后,SD雄鼠较正常非去势SD雄鼠相比致痫潜伏期延长、持续时间缩短、发作频率及程度减轻,点燃时间也明显长于正常SD雄鼠.     

戊四氮点燃大鼠海马CA3区钙超载与亚低温对点燃大鼠脑保护作用的研究

目的 海马CA1区钙超载是否在癫痫的发病机制中起主要作用及亚低温对癫痫是否有治疗意义。方法用荧光倒置显微镜来测定癫痫状态下海马CA1、CA3区钙超载的情况，并用钙离子拮抗剂尼莫地平作用于海马脑片看钙超载的变化。然后将大鼠分为头皮温度为41℃、37℃、32℃、26℃的4组，通过对4组点燃大鼠行为学和海马组织病理学的观察来看亚低温对癫痫的脑保护作用。结果癫痫大鼠海马CA1，CA3区钙超载高于对照组．CA1区钙超载高于CA1区。在尼莫地平的作用下CA1区钙超载明显降低。组织病理学改变显示CA1区是海马区神经元变性、坏死最明显的部位。而亚低温下癫痫的发作程度和神经元变性、坏死最轻。结论钙超载参与了癫痫的发病机制，其中海马CA3区的钙超载在癫痫发病机制中起主要作用。而亚低温对脑有保护作用。     

戊四氮点燃致大鼠癫痫状态后脑内HSP70的分布研究

目的：了解热休克蛋白70（HSP70）在癫痫状态大鼠脑内的分布。方法：应用免疫细胞化学方法观察戊四氮（PTZ）点燃致大鼠癫痫状态后脑内不同区域HSP70的表达。结果：PTZ点燃后大鼠脑CA3区、梨状皮层、杏仁核、丘脑、内嗅皮层、CA1区、顶叶皮层HSP70表达依次减少。结论：HSP70在脑内的表达分布与癫痫脑神经元对兴奋性损伤的耐受性密切相关,可能是癫痫脑神经元选择性脱失的内在机制之一。     

突触素与癫痫

癫痫（epilepsy）是最常见的神经系统疾病之一,其发病机制复杂,目前认为癫痫发病是由于中枢神经系统兴奋与抑制性不平衡导致大脑神经元异常放电所致。近年研究表明,这种兴奋与抑制间的不平衡主要与离子通道、突触传递及神经胶质细胞的改变有关。癫痫发生时大脑海马部位出现的苔藓纤维异常出芽及伴随的突触重建被认为是其细胞机制之一。近年来研究的热点之一是海马结构的可塑性重建,这种可塑性主要表现在突触上。     

Neuroprotective effect of nimesulide, a preferential COX-2 inhibitor, against pentylenetetrazol (PTZ)-induced chemical kindling and associated biochemical parameters in mice

Brain cyclooxygenases (COX), the rate-limiting enzyme in prostaglandin synthesis, is rapidly and transiently induced by convulsions in hippocampal and cortical neurons. Previous studies have explored the protective effect of naproxen (non-selective COX-inhibitor) or rofecoxib (selective COX-2 inhibitor) against chemical kindling in mice. With this background, the present study was designed to explore the possible effect of nimesulide (a preferential COX-2 inhibitor) against pentylenetetrazol (PTZ)-induced kindling epilepsy in mice. To induce kindling, PTZ was injected in a subconvulsive dose (40 mg/kg, i.p.) every other day for 15 days. Nimesulide (2.5 or 5 mg/kg, p.o.) was administered each day 45 min before either PTZ or vehicle challenge. The intensity of kindling was assessed immediately after PTZ administration according to a prevalidated scoring scale. On 16th day i.e. 24 h after the last dose of PTZ, animals were sacrificed and various biochemical parameters were assessed in the whole brain. Compared with normal control group, PTZ-kindled mice had significantly higher levels of malondialdehyde, nitrite, myeloperoxidase but had lower levels of reduced glutathione in the whole brain homogenate. Chronic treatment with nimesulide (2.5 or 5 mg/kg, p.o.) for 15 days showed significant decrease in kindling score and could play a role in controlling the accompanying biochemical alterations due to PTZ. These results suggested that nimesulide, a preferential COX-2 inhibitor offered neuroprotection against PTZ-induced kindling in mice.     

Reversal of a selective decrease in hippocampal acetylcholine release, but not of the persistence of kindling, after discontinuation of long-term pentylenetetrazol administration in rats

The time course of the effect of pentylenetetrazol (PTZ)-induced kindling on acetylcholine release in the hippocampus of freely moving rats was investigated with the transversal microdialysis technique. The basal extracellular concentration of acetylcholine in the hippocampus was reduced significantly (−29%, P<0.05) after 3 weeks, and the effect was maximal (−52%, P<0.01) after 4 weeks and remained essentially unchanged during the remaining 4 weeks of PTZ treatment (30 mg/kg, i.p., 3 times/week), relative to vehicle-treated rats. The basal release of acetylcholine in the prefrontal cortex and in the striatum of kindled rats was unchanged compared with that of vehicle-treated rats. The specific binding of [³H]quinuclidinyl benzilate, a non-selective ligand of muscarinic receptors, was significantly increased (+29%, P<0.01) in hippocampal membranes, but not in membranes prepared from the prefrontal cortex or striatum, of PTZ-kindled rats. Thirty days after discontinuation of PTZ treatment, both hippocampal acetylcholine output and the density of muscarinic receptors had returned to values characteristic of vehicle-treated rats, whereas seizure susceptibility did not differ significantly from that apparent 4 days after PTZ administration. These results suggest that the selective and transient decrease in acetylcholine output and the parallel increase in the density of postsynaptic muscarinic receptors in the hippocampus may play a role in facilitating the development of kindling rather than in the maintenance of the kindled state. © 1997 Elsevier Science B.V. All rights reserved.     

戊四氮点燃过程中大鼠海马 BMP4表达与神经增殖关系

目的探讨戊四氮(PTZ)点燃过程中大鼠海马骨形成蛋白4(bone morphogenetic protein-4,BMP4)的表达变化与神经增殖的关系。方法将成年大鼠分为对照组与模型组,模型组大鼠又根据在点燃中的不同时相点分为9组。用免疫组织化学与原位杂交的方法检测海马齿状回BMP4mRNA与BrdU阳性细胞数的变化。结果正常成年大鼠BMP4阳性细胞主要分布于齿状回的门区、颗粒下层、CA3、CA1区。BrdU阳性细胞主要分布在齿状回颗粒下层。BMP4阳性细胞与BrdU阳性细胞在点燃过程中均明显增加,呈明显正相关,点燃后2月降至基线水平。结论BMP4可能通过影响成年大鼠海马神经发生在PTZ点燃过程中起重要作用。     

Amygdala kindling in Proechimys guyannensis rat: an animal model of resistance to epilepsy

PURPOSE: This study examined the effect of amygdala kindling development in Proechimys guayannensis rat, a common rodent of the Amazon basin. METHODS: Adult male P. guayannensis animals (n = 43) and adult male Wistar rats (n = 14) were submitted to electrical amygdala kindling. RESULTS: From 43 Proechimys rats submitted to the kindling process, only three animals reached stage 5 of kindling. During the kindling development (stages 4-5), these animals had behavioral alterations different from those observed in Wistar rats. A longer time spent in stages 1-3 and 5 and longer afterdischarge duration in stages 1-4 was observed in the Proechimys group compared with the Wistar group. The number of wet-dog shakes also was reduced in the Proechimys group during the kindling process. CONCLUSIONS: These findings suggest natural endogenous inhibitory mechanisms in this animal species.     

Astrocytic and synaptic response to kindling in hippocampal subfield CA1. II. Synaptogenesis and astrocytic process increases to in vivo kindling

Astrocytic glia are important for maintaining synaptic function during physiological activity. Recent hypotheses concerning epilepsy suggest a role for astrocytes in the control of neuronal excitability and in pathogenesis. This report provides morphological evidence that the periodic electrical stimulation used in the kindling model of epilepsy induces astrocytic hypertrophy and an increase in shaft synapse density in the CA1 region of the hippocampus. The Schaffer collateral pathway in the stratum radiatum of CA1 of five pairs of rats was kindled in vivo. Control animals received the same number of stimulations at a lower intensity and frequency. The animals were killed 24–48 h after reaching the criterion of five generalized seizures, and the brains were examined by electron microscopy. Kindling produced a 37% and 33% increase in the volume fraction of astrocytic processes in the middle and distal portions, respectively, of the s. radiatum in CA1. In the same tissue, the number (areal density) of shaft synapses was increased 25% in the s. radiatum of animals exhibiting generalized seizures. On the other hand, the areal density of degenerating synapses in both kindled and control animals was low and not significantly different. These results suggest that both synaptogenesis and hypertrophy of astrocytes contribute to an early stage of epileptogenesis when degenerative changes of the sort that might induce gliosis were not prominent in the tissue under study.     

Neuronal inputs to hippocampal formation in Alzheimer's disease and in parkinsonism-dementia complex on Guam

Summary This study concerns the expression of synaptophysin in the hippocampal formation of normal controls, of patients with Alzheimer's disease (AD) and of patients with parkinsonism-dementia complex on Guam (P-D complex). A monoclonal antibody was used to visualize synaptophysin, an integral component of presynaptic vesicle membranes. In the normal controls, a strong synaptophysin immunoreactivity was seen in the stratum pyramidale, stratum radiatum and stratum lacunosum-moleculare of the hippocampus proper, in the subiculum and in the molecular layer of the dentate gyrus. In the dentate gyrus molecular layer, the reaction product was distributed in a laminar fashion. By contrast, in AD and in P-D complex a significant decrease in immunoreactivity was observed in all hippocampal strata, and especially of the hippocampal subfield CA1 and the subiculum. In both diseases, synaptophysin expression was also diminished in the molecular layer of the dentate gyrus of all patients examined, with the inner portion exhibiting almost normal and the outer portion a strikingly reduced synaptophysin immunoreactivity.     

Development of NMDA-induced theta rhythm in hippocampal formation slices

During the past 20 years experimental evidence has accumulated demonstrating that the appearance of theta rhythm requires a certain level of excitation of local neuronal networks. In this study we extended our earlier in vitro observations concerning the involvement of cholinergic and GABAergic neurotransmission in hippocampal theta production. Specifically, we investigated whether the hippocampal neuronal network is capable of generating theta oscillations in the presence of N-methyl-d-aspartic acid (NMDA) in a brain slice preparation. To answer this question, the effect of different concentrations of NMDA (Experiment I) and the effect of interaction between NMDA and GABA_A/_B agonists and antagonists on field potentials recorded in the CA3c region of hippocampal formation (HPC) slice preparations (Experiment II) was examined. We demonstrated for the first time that apart from the epileptiform activity recorded in almost all series of Experiments I and II, only the perfusion of HPC slices with NMDA in doses of 30 and 50 μM, as well as the perfusion of HPC slices with NMDA and GABA_B agonist baclofen (50 μM NMDA + 50 μM BACL), resulted in the appearance of individual theta epochs. The best synchronized theta oscillations obtained after administration of 50 μM NMDA + 50 μM BACL resembled theta activity induced by a bath perfusion of 50 μM carbachol. In light of the obtained results we conclude that besides the cholinergic and GABAergic input, NMDA glutamatergic drive is also important for the appearance of theta oscillations in HPC in vitro.     

Role of hippocampal sodium channel Nav1.6 in kindling epileptogenesis

Purpose:   Central nervous system plasticity is essential for normal function, but can also reinforce abnormal network behavior, leading to epilepsy and other disorders. The role of altered ion channel expression in abnormal plasticity has not been thoroughly investigated. Nav1.6 is the most abundantly expressed sodium channel in the nervous system. Because of its distribution in the cell body and axon initial segment, Nav1.6 is crucial for action potential generation. The goal of the present study was to investigate the possible role of changes in Nav1.6 expression in abnormal, activity-dependent plasticity of hippocampal circuits.
Methods:   We studied kindling, a form of abnormal activity-dependent facilitation. We investigated: (1) sodium channel protein expression by immunocytochemistry and sodium channel messenger RNA (mRNA) by in situ hybridization, (2) sodium current by patch clamp recordings, and (3) rate of kindling by analysis of seizure behavior. The initiation, development, and expression of kindling in wild-type mice were compared to Nav1.6 +/− med ^tg mice, which have reduced expression of Nav1.6.
Results:   We found that kindling was associated with increased expression of Nav1.6 protein and mRNA, which occurred selectively in hippocampal CA3 neurons. Hippocampal CA3 neurons also showed increased persistent sodium current in kindled animals compared to sham-kindled controls. Conversely, Nav1.6 +/− med ^tg mice resisted the initiation and development of kindling.
Discussion:   These findings suggest an important mechanism for enhanced excitability, in which Nav1.6 may participate in a self-reinforcing cycle of activity-dependent facilitation in the hippocampus. This mechanism could contribute to both normal hippocampal function and to epilepsy and other common nervous system disorders.