神经元GABA合成、分解和分泌与癫痫发病的研究

目的 研究神经元的γ－ 氨基丁酸（GABA）合成、分解和分泌与癫痫发病的关系。方法 用致痫剂马桑内酯（CL）和抗痫剂苯妥英钠（PHT）处理培养大鼠神经元,用高效液相色谱仪,紫外分光光度计分别检测培养神经元内外的GABA浓度和神经元内γ－ 氨基丁酸转氨酶（GABA－T）活性。结果 与对照组比较,CL使神经元内GABA－T活性显著增强（P＜0．05）,对神经元内外GABA浓度没有影响。与CL处理相似100μmol/L CL培养神经元24h）比较,PHT使CL致痫神经元内GABA－T活性显著减弱（P＜0．05）,基本恢复到对照组水平。但神经元内外GABA浓度仍相对稳定。结论 癫痫发病中,痫性神经元合成、分解GABA增强,分泌GABA相对稳定。     

神经元GAD和GABA—T的活性与癫痫发病的关系

目的：研究探索神经元内谷氨酸脱羧酶（GAD）和γ－氨基丁酸转氨酶（GABA－T）的活性与癫痫发病的关系。方法：用致痫剂马桑内酯（CL）和抗痫剂苯妥因钠（PHT）处理大鼠培养海马神经元,用液体闪烁计数仪,紫外分光光度计检测培养神经元内GAD和GABA－T的活性。结果：与对照组比较,CL致痫组培养神经元内GAD活性和GABA－T的活性显著增高,运用抗痫剂PHT处理致痫神经元后,与CL致痫组比较,神经元内GABA－T活性显著降低,与对照组比较差异不显著。结论：在癫痫发病中,痫性神经元GAD和GABA－T活性增高。     

戊四氮所致慢性癫痫发病过程中谷氨酸脱羧酶基因表达的研究

目的：观察谷氨酸脱羧酶（GAD）67mRNA表达在慢性癫痫演变过程的变化,并探讨其作用。方法：采用寡核苷酸探针原位组织杂交技术检测慢性癫痫演变过程中各期皮层,海马GAD67mRNA的表达水平。结果：慢性癫痫演变过程中GAD67mRNA阳性细胞数略有减少,但与对照组比较无显著性差异。而继续存活的GAD67mRNA阳性细胞平均吸光度明显增加,与对照组比较有显著性差异（P＜0．01）,结论：继续存活的GAD67m RNA阳性细胞GAD67mRNA表达水平的增高可能是机体的一种内源性抗病表现。     

癫痫患者脑脊液谷氨酸脱羧酶活性的改变

癫痫患者脑脊液谷氨酸脱羧酶活性的改变侯国庆阮旭中沈安华方思羽史庭慧谷氨酸脱羧酶（ＧＡＤ）作为中枢抑制性神经递质γ－氨基丁酸（ＧＡＢＡ）的合成限速酶，在癫痫（ＥＰ）的研究中很受重视。我们通过测定ＥＰ患者ＣＳＦ内ＧＡＤ活性，以探讨其临床意义。资料：ＥＰ组...     

癫痫状态大鼠海马HSP70mRNA及GAD67mRNA表达水平的研究

目的 研究癫痫演变过程中特定脑区选择性神经元损伤与修复机制。方法 采用分子杂交方法动态观察了马桑内酯（coriaria Lactone,CL)诱导的癫痫状态大鼠海马区早期神经元损伤标志物热休克蛋白（heat shock protein,HSP)70mRNA及谷氨酸脱羧酶（glutamin acid decarboxylase,GAD)67mRNA表达水平的变化,同时也观察了大鼠行为学及脑电图改变。结果 侧脑室注射CL后20分钟,大鼠出现连续发作的四肢抽搐伴有EEG持续性的棘慢波及尖波发放。注射CL后2小时组大鼠海马GAD67 mRNA表达水平有所降低,但HSP70mRNA变化不明显,注射CL后8小时组大鼠海马GAD67mRNA表达水平有所降低,但HSP70mRNA表达变化不明显,注射CL后8小时组大鼠海马GAD67mRNA表达水平明显低于对照组（P＜0．01）,HSP70mRNA表达水平较对照组明显增高（P＜0．01）。结论 癫痫状态期间大鼠海马区可能存在神经元损伤与修复过程,而且GABA能神经元受到不同程度的损伤。     

电针对癫痫模型大鼠自发性反复发作及海马CA3和DG区谷氨酸脱羧酶67表达的影响

背景由于对药物疗法不良反应的担忧,近几年人们对使用针灸治疗癫痫越来越感兴趣。尽管现已报道了不少针灸抗癫痫作用的临床证据,但其确切机制仍不清楚。目的 研究电针(EA)对癫痫大鼠自发性复发性癫痫(SRS),以及海马CA3和齿状回(DG)区中谷氨酸脱羧酶67(GAD67)表达的影响。方法 将50只Sprague-Dawley(SD)大鼠随机分为对照组、癫痫组、假刺激组、非穴位电针组和穴位电针组,每组10只。除对照组外,其余4组均制备氯化锂—匹罗卡品颞叶癫痫大鼠模型。造模成功的大鼠采用针刺或电针穴位治疗,8周后观察自发性复发性癫痫发作的次数。分别取5组大鼠海马组织,采用荧光定量PCR和Western blotting分别检测各组大鼠海马组织中GAD67 mRNA水平和蛋白水平表达变化;采用免疫组化法检测各组大鼠海马CA3和DG区GAD67蛋白水平表达变化。结果 治疗8周后,穴位电针组与癫痫组、假刺激组、非穴位电针组比较,减少了自发性复发性癫痫发作的次数,差异有统计学意义(P<0.05)。qRT-PCR结果显示：与对照组相比,癫痫组、假刺激组、非穴位电针组和穴位电针组大鼠海马组织中GAD6...     

癫痫大鼠海马CA3区钙超载与癫痫发病机制的相关性研究

目的:探讨癫痫的发病机制,海马CA3区钙超载是否在癫痫的发病机制中起主要作用。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。并把点燃成功的大鼠麻醉后做海马脑片,看海马的组织病理学改变。结果:癫痫大鼠海马CA1、CA3区钙超载高于对照组,CA3区钙超载高于CA1区,在尼莫地平的作用下CA3区钙超载明显降低。组织病理学改变亦显示CA3区是神经元变性、坏死是海马区最明显的部位。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。     

实验性癫痫发作后GABA在海马结构中变化的研究进展

各类癫痫的发生几乎都与脑内γ-氨基丁酸(GABA)的功能变化有关。海马结构在癫痫的发生发展中起重 要作用。本文着重介绍海马结构中GABA、对GABA数量和功能有影响的谷氨酸脱羧酶(GAD)、GABA转运体(GAT) 及GABA受体在实验性癫痫发作后发生的重要变化,以了解其在癫痫中发生发展机理,认为EP发作后海马结构 GABA能抑制作用增强,可防止EP灶过度兴奋的扩散,此可能属机体对神经网络过度兴奋的代偿或适应性反应。     

癫痫持续发作对大鼠海马神经元的影响

目的 研究实验性癫痫大鼠在癫痫发作持续不同时间对海马神经元的影响。方法24只SD大鼠随机分成4组：诱发火鼠瘢痫持续状态（status cpilepticus，SE）〈10、10～30、〉30min组及正常对照组。于电镜下观察各组海马神经元超微结构变化；采用免疫组化方法检测凋亡相关基因bcl-2、bax的蛋白表达及通过流式细胞技术检测细胞凋亡情况。结果SE〈10min组海马神经元所受影响不大．SE10～30min组海马神经元具有明显凋亡特征．SE〉30min组多数海马神经元呈坏死性改变。结论大鼠SE对海马神经元损伤有凋亡和坏死两种不同形式．这与癫痫发作的持续时间密切相关。     

褪黑素对癫痫状态大鼠海马GAD、GABA-T活性和GABA、Glu含量的影响

目的从神经递质方面研究褪黑素(melatonin,Mel)抗癫痫作用的生化机制。方法采用匹罗卡品(pilocarpine,PILO)诱发大鼠癫痫持续状态(status epilepticus,SE)模型,观察大鼠SE后4个时相点即6、48、72h和7 d海马γ-氨基丁酸、谷氨酸含量和谷氨酸脱羧酶、γ-氨基丁酸转氨酶活性的变化,以及Mel对其变化的影响。结果PILO诱导大鼠SE后6 h~7 d海马谷氨酸含量显著升高,γ-氨基丁酸含量和谷氨酸脱羧酶活性显著降低,与对照组比较差异有统计学意义(P<0.05),γ-氨基丁酸转氨酶活性在SE后72 h才显著下降(P<0.05);给予Mel的大鼠在SE后6 h~7 d,海马γ-氨基丁酸含量和谷氨酸脱羧酶活性均显著高于仅予PILO处理的大鼠(P<0.05),而谷氨酸含量和γ-氨基丁酸转氨酶活性与仅予PILO处理的大鼠比较差异无统计学意义(P>0.05)。结论Mel通过增强SE大鼠海马谷氨酸脱羧酶活性及γ-氨基丁酸合成发挥抗癫痫作用。     

海藻氨酸致痫大鼠海马谷氨酸转运体功能的变化

目的研究腹腔注射海藻氨酸致癫痫发作后海马谷氨酸转运体功能的动态变化,以探讨谷氨酸转运体在癫痫发生中的作用机制。方法60只健康成年雄性Wistar大鼠,随机分为海藻氨酸组和对照组。海藻氨酸组30只大鼠均腹腔注射海藻氨酸10mg/kg,分别于注射后4h、24h、48h、5d和7d依据Racine制定的行为学标准观察大鼠的行为学改变。同时还分别测定不同时间点海马突触膜颗粒和海马组织切片对3氢-左旋-谷氨酸(3H-L-Glu)的摄取量,以反映谷氨酸转运体于点燃后不同时间点的活性。结果与对照组相比,海藻氨酸组大鼠海马突触膜颗粒谷氨酸转运体功能于点燃后4h减弱,对3氢-左旋-谷氨酸的摄取量减少(P<0.05),并持续至注射后第5～7天(P<0.01);海马组织切片检查显示谷氨酸转运体功能在点燃后4～48h增强,于注射后第5～7天减弱(P<0.05)。结论谷氨酸转运体功能的变化与海藻氨酸致痫大鼠模型癫痫的发生及易感性有关。     

胶质细胞谷氨酸转运体在大鼠点燃效应中的作用研究

目的 研究点燃形成过程中和点燃后大鼠海马中氨酸天门氨酸转运体（GLAST）和谷氨酸转运体1（GLT－1）的变化,进一步探讨癫痫的形成机制。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（335mg/kg）,每日1次,直到达到点燃标准;I组腹腔注射等量生理盐水。采有RT－PCR方法检测海马区GLAST和GLT－1mRNA的表达。结果 PTZ组点燃后,GLASTmRNA的表达下降,60天时恢复至对照组;与对照组比较,PTZ组GLT－1mRNA的表达,在给药后15天时开始上升,点燃后0小时和48小时时显著升高,此后呈下降趋势。60天时,两组比较无明显差异。结论 海马区胶质细胞谷氨酸转运体的下降可能与癫痫敏感性的形成有关。     

Depolarization-dependent actions of dihydropyridines on synaptic transmission in the in vitro rat hippocampus

Field potential and intracellular recordings were obtained in the in vitro hippocampal slice to study the effects on synaptic transmission of dihydropyridine (DHP) derivatives. Nimodipine or nifedipine by itself had little effect upon the postsynaptic response as determined by field potential analysis. However, facilitation became evident when DHP application was coupled with manipulations which induced a moderate degree of membrane depolarization. In accordance with the hydrophobic nature of these compounds, extensive washing in normal Krebs' solution failed to reverse the facilitation indicating that the DHP effects outlasted the induced depolarization. Nifedipine is photolabile and its actions were reversed when intense light was applied to the slice. Application of the DHP Bay K 8644, resulted in a similar depolarization-dependent increase in neuronal excitability which, upon washout and exposure to light, was at first attenuated and then reversed, resulting in a long-lasting depression of the EPSP that was sensitive to caffeine. This depressant action of Bay K 8644 appeared to be mediated at a site presynaptic to the pyramidal cell because the postsynaptic component of the field potential response to pulsed applications of glutamate was not altered. Intracellular recording from CA1 neurons supports a presynaptic locus for the depressant actions of Bay K 8644; spike threshold for synaptically evoked responses was greatly increased while spike threshold to direct depolarization of the soma was unchanged. These results indicate that DHPs can exert effects on synaptic transmission in hippocampal brain slice under conditions of moderate membrane depolarization.     

Postnatal development of glutamate metabolizing enzymes in hippocampus from mice

The specific activity profiles of the glutamate synthesizing enzymes, phosphate activated glutaminase (EC 3.5.1.2), aspartate aminotransferase (EC 2.6.1.1), glutamate dehydrogenase (EC 1.4.1.2) and ornithine aminotransferase (EC 2.6.1.13) have been followed postnatally for 28 days in mouse hippocampus and compared to corresponding profiles in cerebellum and cerebral cortex (cf. refs 10 and 18). Phosphate activated glutaminase and glutamate dehydrogenase showed activity patterns similar to those found for cerebellum and glutamatergic granula cells cultured from cerebellum, whereas the aspartate aminotransferase activity pattern was found to be more similar to that previously observed for cerebral cortex as well as cultured cerebral interneurons which are likely to be GABAergic. The specific activity of ornithine aminotransferase was essentially unaltered during postnatal development, which is similar to what has been found for cerebellum and cerebral cortex.     

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。     

Glutamate-glutamine cycling in the epileptic human hippocampus

PURPOSE: Several findings suggest that energy metabolism and the glutamate-glutamine cycle may be impaired in epilepsy. Positron emission tomography often shows interictal hypometabolism of the epileptogenic hippocampus. In vivo microdialysis studies show that seizure-associated glutamate release is doubled, and clearance is slowed. We hypothesized that the glutamate-glutamine cycle between neurons and glia may be decreased in the epileptic human hippocampus. METHODS: A 20% solution of 2-13C-glucose was infused before resection of the epileptogenic hippocampus. Blood glucose isotopic fractions were measured every 30 min. Blood and brain specimens were frozen quickly; perchloric acid extracts of the small metabolites were prepared and analyzed by proton and carbon magnetic resonance spectroscopy (MRS) at 11.75 Tesla. RESULTS: Standard histology showed 12 with hippocampal sclerosis and five with minimal neuron loss. The relative rates of glutamate-glutamine cycling with respect to glutamate synthesis were decreased in biopsies affected by hippocampal sclerosis (mean, 0.08; 95% confidence interval, 0.04-0.12) compared with those with minimal neuron loss (0.52; 95% CI, 0.30-0.75). Mean cellular glutamate concentrations were higher in minimal neuron loss (8.9 mM; 95% CI, 7.4-10.4) than hippocampal sclerosis (7.3 mM; 95% CI, 5.9-8.7). Cellular glutamine concentrations (mean, 2.8 mM; 95% CI, 2.4-3.2; n = 17) were the same in all groups. CONCLUSIONS: The epileptogenic, gliotic human hippocampus appears to be characterized metabolically by slow rates of glutamate-glutamine cycling, decreased glutamine content, and a relative increase in glutamate content. We hypothesize that the low rate of glutamate-glutamine cycling that results from a failure of glial glutamate detoxification could account for slow glutamate clearance from synapses and continuing low-grade excitotoxicity.     

Orthogonal wave propagation of epileptiform activity in the planar mouse hippocampus in vitro

Purpose: In vitro brain preparations have been used extensively to study the generation and propagation of epileptiform activity. Transverse and longitudinal slices of the rodent hippocampus have revealed various patterns of propagation. Yet intact connections between the transverse and longitudinal pathways should generate orthogonal (both transverse and longitudinal) propagation of seizures involving the entire hippocampus. This study utilizes the planar unfolded mouse hippocampus preparation to reveal simultaneous orthogonal epileptiform propagation and to test a method of arresting propagation. Methods: This study utilized an unfolded mouse hippocampus preparation. It was chosen due to its preservation of longitudinal neuronal processes, which are thought to play an important role in epileptiform hyperexcitability. 4‐Aminopyridine (4‐AP), microelectrodes, and voltage‐sensitive dye imaging were employed to investigate tissue excitability. Key Findings: In 50‐μm 4‐AP, stimulation of the stratum radiatum induced transverse activation of CA3 cells but also induced a longitudinal wave of activity propagating along the CA3 region at a speed of 0.09 m/s. Without stimulation, a wave originated at the temporal CA3 and propagated in a temporal–septal direction could be suppressed with glutamatergic receptor antagonists. Orthogonal propagation traveled longitudinally along the CA3 pathway, secondarily invading the CA1 region at a velocity of 0.22 ± 0.024 m/s. Moreover, a local lesion restricted to the CA3 region could arrest wave propagation. Significance: These results reveal a complex two‐dimensional epileptiform wave propagation pattern in the hippocampus that is generated by a combination of synaptic transmission and axonal propagation in the CA3 recurrent network. Epileptiform propagation block via a transverse selective CA3 lesion suggests a potential surgical technique for the treatment of temporal lobe epilepsy.     

Balloon cells and dysmorphic neurons in the hippocampus associated with epileptic amnesic syndrome: a case report

Recently, we encountered a 39-year-old woman with typical epileptic amnesic syndrome. The patient underwent right anterior temporal lobectomy, which removed the right hippocampus. The patient's resected hippocampus showed typical histological features of Ammon's horn sclerosis (AHS) with dysmorphic neurons. In addition, the prominent balloon cells, admixed with dysmorphic neurons, were noted in the hippocampus. To our knowledge, this is the first reported case showing AHS with balloon cells. The presence of balloon cells reinforces the hypothesis that AHS itself might be a maldevelopment disorder.     

Relationship of seizure frequency to hippocampus volume and metabolism in temporal lobe epilepsy

PURPOSE: To examine the relationship between frequency of complex partial (CPS) and secondarily generalized tonic-clonic seizures (sGTCS) on hippocampal volume (HV) and temporal lobe metabolism. METHODS: We performed volumetric magnetic resonance imaging (MRI) and positron emission tomography with 18fluorodeoxyglucose (18FDG-PET) in 32 patients with epilepsy. Temporal lobe foci were localized by ictal video-EEG. RESULTS: We did not find any association between CPS frequency or lifetime number of sGTCS and HV or metabolism ipsilateral to electroencephalographic focus. CONCLUSION: The progress of metabolic or pathologic abnormalities of temporal lobe epilepsy may not be altered by adequate seizure control. The presence of an epileptic focus might be associated with progressive neuronal injury even in clinically well-controlled patients.