海人酸致痫大鼠海马细胞外氨基酸类神经递质的变化

目的 分析海马细胞外氨基酸递质在癫痫发生中的作用,探讨海人酸致痫模型大鼠癫痫发生的机制.方法 应用立体定向方法建立海人酸大鼠颞叶癫痫模型,观察大鼠行为学和电生理变化,应用电镜观察大鼠海马超微结构,应用微透析获取大鼠海马细胞外液,高压液相色谱法测定透析液中的兴奋性氨基酸谷氨酸、抑制性氨基酸牛磺酸及γ-氨基丁酸的含量.结果 海人酸注射后大鼠出现典型的颞叶癫痫发作,皮层脑电显示痫性发作,电镜显示兴奋性神经递质增加,高效液相色谱分析显示海马细胞外谷氨酸、牛磺酸和γ-氨基丁酸含量明显高于对照组(P＜0.05),虽然谷氨酸、γ-氨基丁酸都升高,而谷氨酸升高的更明显.结论 兴奋性氨基酸与抑制性氨基酸的失衡在海人酸致痫大鼠模型的癫痫发生过程中发挥重要作用,是癫痫发生的原因之一.     

伽玛刀对癫痫大鼠模型的治疗作用及机制研究

目的 研究伽玛刀照射对癫痫大鼠的放射生物学作用,探讨伽玛刀治疗癫痫的作用机制.方法 制备海人酸大鼠癫痫模型,利用自行设计的伽玛刀动物定向头架,分别应用100Gy和20Gy的伽玛射线对癫痫大鼠海马组织进行照射,观察大鼠行为学、脑电图、MRI及超微结构、脑组织氨基酸含量及GABA能神经元表达变化.结果 伽玛刀照射后大鼠癫痫发作次数明显减少.100Gy组照射后2个月MRI表现为靶区高信号,20Gy组5个月MRI未见改变.伽玛刀照射后兴奋性氨基酸含量显著低于癫痫组,GABA能神经元表达低于正常,但高于癫痫组.结论 伽玛刀照射对癫痫大鼠具有治疗作用,高剂量伽玛射线(100Gy)对致痫灶有毁损作用.低剂量伽玛射线(20Gy)通过抑制兴奋性神经元释放兴奋性氨基酸使癫痫发作减少.     

癫痫患者脑脊液中GABA,Glu,Asp的变化及其临床意义

采用高效液相色谱紫外检测３７例特发性癫痫患者和２０例正常对照者脑脊液中抑制性氨基酸递质γ－氨基丁酸（ＧＡＢＡ）、兴奋性氨基酸递质谷氨酸（Ｇｌｕ）、天门冬氨酸（Ａｓｐ）水平。结果表明患者脑脊液中γ－氨基丁酸含量显著低于正常对照组，谷氨酸含量显著高于正常对照组。初步证明了特发性癫痫的发病机制与脑内兴奋性氨基酸递质与抑制性氨基酸递质平衡失调有关。     

丙戊酸对癫癎大鼠海马兴奋性氨基酸类神经递质动态释放的影响

目的:观察丙戊酸(valproate,VPA)对戊四氮(pentylenetetrazol,PTZ)点燃的自由活动大鼠海马胞外兴奋性氨基酸类神经递质动态释放过程的影响。方法:建立大鼠慢性癫癎模型及自由活动的清醒动物脑内在体微透析(microdialysis,MD)模型,用高效液相色谱测定急性腹腔注射VPA 200 mg·kg-1对大鼠海马胞外兴奋性氨基酸类神经递质释放的影响。结果:在惊厥发作期及发作间期,自由活动大鼠海马胞外谷氨酸及天冬氨酸水平显著上升,给予VPA 200 mg·kg-1后,两者浓度明显下降。结论:①VPA对海马胞外主要的兴奋性氨基酸类神经递质释放的调控为其发挥抗癎作用的重要机制之一;②在VPA对CNS兴奋性氨基酸类神经化学递质调控机制的研究中,PTZ点燃的大鼠癫癎模型较为理想。     

低剂量伽玛刀照射对癫痫大鼠皮层及海马NMDA受体亚基表达的影响

目的观察低剂量伽玛刀照射对癫痫大鼠皮层和海马N-甲基-D-天氡氨酸(N-methyl-Daspartate,NMDA)受体亚基表达的影响。方法根据动物是否致痫及接受伽玛刀照射,将大鼠分为4组:对照组、伽玛刀组、药物致痫组、伽玛刀+药物组。腹腔连续注射戊四氮(pentylenetetrazole,PTZ)制备癫痫大鼠模型,以双侧额叶为照射靶区对大鼠进行低剂量伽玛刀照射,边缘剂量为15Gy。观察并记录各组大鼠伽玛刀照射前、后癫痫发作情况,并于伽玛刀照射后12周后留取脑组织,分别利用免疫组化及免疫蛋白印迹法对大鼠皮层及海马NMDA受体亚基NR1、NR2A和NR2B进行检测。结果对照组及伽玛刀组大鼠无痫性发作表现,与药物致痫组大鼠相比,伽玛刀+药物组大鼠经低剂量伽玛刀照射后12周,痫性发作明显减轻(P0.05)。与对照组相比,药物致痫组大鼠额叶皮层及海马CA1、CA3区NR1、NR2A和NR2B表达均明显增强(P0.05),阳性神经元数目及平均吸光度值均明显增加(P0.05);与药物致痫组比较,伽玛刀+药物组额叶皮层及海马CA1、CA3区NR1、NR2A和NR2B表达均明显降低(P0.05),阳性神经元数目及平均吸光度值明显减少(P0.05);伽玛刀组与对照组无明显差别(P0.05)。结论癫痫大鼠额叶皮层及海马NR1、NR2A及NR2B亚单位蛋白表达增强,低剂量伽玛刀照射可能引起癫痫大鼠皮层及海马NMDA受体亚基表达减少,这可能是低剂量伽玛刀抑制癫痫发作的分子机制之一。     

依达拉奉对癫痫大鼠脑内谷氨酸的影响

目的 探讨依达拉奉对戊四氮致痫大鼠脑中谷氨酸的影响.方法 30只成年SD大鼠随机分为对照组、癫痫组和治疗组,癫痫组和治疗组大鼠腹腔注射戊四氮60mg/kg,诱导癫痫发作.治疗组于注射戊四氮之前1h经腹腔注射依达拉奉30mg,并观察1h.然后处死大鼠取脑,应用柱前衍生HPLC-荧光法测定大鼠脑皮质谷氨酸(Glu)的含量.结果 治疗组痫性发作潜伏期、平均痫性发作等级及脑内谷氨酸含量与癫痫组相比差异均有显著性(P<0.01).结论 依达拉奉可以通过拮抗氧自由基并抑制谷氨酸的释放,在癫痫发作中发挥保护作用.     

经皮三叉神经慢性电刺激对慢性癫痫大鼠海马和皮层内VGLUT1表达的影响

目的观察经皮三叉神经慢性电刺激(trigeminal nerve stimulation,TNS)对匹罗卡品诱导的癫痫大鼠海马和皮层内囊泡型谷氨酸转运体-1(vesicular glutamate transporter 1,VGLUT1)表达的影响,探讨TNS治疗癫痫的可能机制。方法将达到癫痫持续状态(status epilepticus,SE)的实验大鼠随机分为模型(Pilo)组和经皮三叉神经慢性电刺激(TNS)组;正常对照组给予同剂量的生理盐水代替Pilo腹腔注射,随后对照组和Pilo组均给予为期一个月的经皮三叉神经假性电刺激(TNS)组给予为期一个月的真性电刺激。通过免疫荧光双标和Western blot分析海马和皮层内VGLUT1的表达。结果 TNS组和Pilo组海马、皮层内VGLUT1的表达均呈先升高后下降的趋势,约在72 h左右达到高峰。在24 h时,TNS组海马和皮层内VGLUT1的表达均明显高于Pilo组(P<0.01),而在以后各个时间点均低于Pilo组(P<0.05,P<0.01)。结论经皮三叉神经慢性电刺激能够减少癫痫大鼠脑内VGLUT1的表达,从而影响谷氨酸能神经元兴奋性神经递质的传递,推测TNS抗癫痫的机制可能与此有关。     

缺氧缺血性脑病时大鼠兴奋性氨基酸含量以及某些药物的影响

为了探讨药物干预对缺氧缺血性脑损伤(HIE)脑内兴奋性氨基酸(EAAs)含量的影响,采用新生大鼠HIE模型观察了脑活素、神经生长因子(NGF)及丹参对脑组织EAAs的影响.1材料和方法7日龄Wistar大鼠随机分为对照组、HIE组及药物干预组.药物干预组在造模型前30 min腹腔注药1次,剂量分别为:按体重脑活素0.05 mL/20 g,NGF 0.02 mg/20 g,丹参0.2 mL/20 g.在HIE后30 min处死分离左右皮层及海马,用HITACHI 835-50型氨基酸自动分析仪测定谷氨酸(Glu),天冬氨酸(Asp)含量.     

胞二磷胆碱、氯脂醒、神经节苷脂(GM1)对青霉素致痫大鼠脑内某些内源性物质的影响

目的 探讨胞二磷胆碱、氯脂醒、神经节苷脂（GM1）对青霉素致痫大鼠脑内某些内源性物质的影响，为其临床应用提供依据。方法 将35只成年健康SD大鼠随机分为5组：对照组、模型组、胞二磷胆碱组、氯脂醒组、GM1组。制作癫痫模型，制模后1h处死大鼠，取额顶叶脑皮质测定其中超氧化物岐化酶（SOD）、丙二醛（MDA）、谷氨酸（Glu）、γ-氨基丁酸（GABA）含量。结果 ①模型大鼠均制模成功，腹腔注射胞二磷胆碱（500mg／kg）、氯脂醒（100mg／kg）、GM1（30mg／kg）预处理均不能阻止青霉素诱导的大鼠癫痫发作。②与对照组相比，模型组大鼠脑皮质内SOD活性明显降低，MDA、Glu、Glu／GABA值含量明显升高（P〈O．05或尸〈0．01）。③与模型组相比，胞二磷胆碱组、氯脂醒组、GM1组大鼠脑皮质内SOD活性明显升高，MDA、Glu／GABA值含量明显降低（P〈0．05），GM1组Glu含量亦明显降低（P〈0．05）。结论 胞二磷胆碱、氯脂醒、GM1虽不能预防青霉素诱导的大鼠癫痫发作，但可提高其脑组织清除自由基、增强抗氧化以及调节神经递质紊乱、减轻兴奋性氨基酸的毒性等作用，从而起到保护作用。     

伽玛刀低剂量照射对致疒间大鼠海马中氨基酸递质的影响

目的观察伽玛刀低剂量照射对致疒间大鼠海马中谷氨酸(Glu)、γ-氨基丁酸(GABA)的影响,探讨伽玛刀治疗癫疒间的作用机制。方法将50只锂-匹罗卡品致疒间大鼠随机分为照射组和非照射组,各25只,另取正常大鼠25只(腹腔注射生理盐水)作为对照组。照射组大鼠进行伽玛刀低剂量照射(周边剂量20Gy),对照组和非照射组大鼠不进行伽玛刀照射。用高效液相色谱-质谱-质谱(HPLC-MS-MS)分析法检测各组大鼠海马Glu、GABA的含量。结果伽玛刀照射2周后,照射组和非照射组大鼠海马中Glu含量均高于对照组,照射组GLu含量低于非照射组,差异具有统计学意义(P0.05);照射组和非照射组GABA含量均低于对照组,照射组GABA含量高于非照射组,差异具有统计学意义(P0.05)。结论伽玛刀低剂量照射通过调节海马中Glu与GABA的平衡,以达到抗癫疒间的作用。     

血清神经元特异性烯醇化酶在癫癎患者中的应用价值

目的 探讨癫痫患者发作后不同时间神经元特异性烯醇化酶（NSE）的动态变化。方法 采用放射免疫法测定20例癫痫患者发作后48h内、2-4d、5-7d血清中NSE水平，并与22例正常健康体检者进行对照。结果 癫痫患者发作48h内及2～4d血清NSE水平与对照组比较有显著性差异（t=4．31，2．57；P〈0．01，0．05）。结论 癫痫发作后血清NSE水平升高可能与脑神经元损害有关。     

慢性癫痫大鼠脑组织神经生长因子阳性神经元的变化

应用免疫组织化学的方法观察了戊四氮诱导的慢性癫痫大鼠脑组织神经生长因子(NGF)阳性神经元的变化。结果发现慢性癫痫大鼠海马回、齿状回NGF阳性神经元数目明显增多。在所选取的时间点,即末次抽搐发作后24h、72h、7d均较对照组明显升高。结果提示:NGF参与了癫痫的发病过程,可能通过介导突触的重建面具有促痫作用。也可能作为一种保护因子而防止癫痫后脑损害。     

Chaotic electrical stimulation of the subthalamic nucleus-mossy fiber sprouting, epileptic seizures, and brain electrical activity in pentylenetetrazol-kindled rats

BACKGROUND: Previous studies have demonstrated that appropriate interventions can alter brain electrical activity of epileptic patients prior to and during a seizure, leading to maintenance of a highly chaotic state, thereby inhibiting abnormal epileptic discharges, and eventually controlling epileptic seizure. OBJECTIVE: This study was designed to observe the effects of chaotic electrical stimulation to the subthalamic nucleus on mossy fiber sprouting, epileptic seizures, and electrical discharges, and to summarize the most suitable intervention. DESIGN, TIME AND SETTING: This randomized grouping, neuroelectrophysiological study was performed at the Laboratory of Neurology, Union Hospital Affiliated to Fujian Medical University in September 2007.MATERIALS: Fifty-five healthy, male, Sprague Dawley rats were subjected to an epileptic model by an intraperitoneal injection of pentylenetetrazol. The YC-2 programmed electrical stimulator was provided by Chengdu Instrument Factory, China; the video electroencephalographic system (KT-88-2400) and 24-hour active electroencephalographic system were products of Contec Medical System Co., Ltd., China; pentylenetetrazol was purchased from Sigma, USA.METHODS: The present interventional method consisted of electrical stimulation to the subthalamic nucleus with an intensity of 500 μ A, pulse width 0.05 ms, frequency 30 Hz, and a duration of 20 minutes for 14 successive days. Fifty-five rats were divided into 6 groups: (1) pre-stimulation (n = 10), pentylenetetrazol was administered and 30 minutes later, chaotic electrical stimulation was performed; (2) synchronous stimulation (n = 10), rats received pentylenetetrazol and chaotic electrical stimulation concurrently; (3) post-administration stimulation (n = 10), after pentylenetetrazol administration, chaotic electrical stimulation was performed immediately after cessation of a seizure; (4) sham-stimulation (n = 10), following pentylenetetrazol administration, an electrode was connected to the stimulator, but electrical stimulation was not performed; (5) control (n = 10), pentylenetetrazol administration, but no electrode was implanted; (6) blank control (n = 50), administration of the same amount of physiological saline and chaotic electrical stimulation.MAIN OUTCOME MEASURES: Timm-stained granule change was scored. Simultaneously, electroencephalography was performed to acquire seizure counts and time course of epileptic discharge within 24 hours.RESULTS: Timm scores were lower in the electrically stimulated rats than in the non-stimulated rats (P < 0.01). Timm scores were lowest in the synchronous stimulation group. When the rats suffered from tonic clonic seizure, the electroencephalogram primarily showed a persistent spike-slow wave and sharp wave. For the electrically stimulated rats, the mean values of seizure counts and time course of epileptic discharge during each hour were noticeably decreased compared with the non-stimulated rats. The synchronous stimulation group, however, had the lowest seizure counts and the shortest time course, followed by the pre-stimulation group, and lastly the post-administration stimulation group. Significant differences existed among the groups (P < 0.01). Compared with the pre-stimulation group and the post-administration stimulation group, the latent period of grades Ⅰ and Ⅳ epileptic seizures was significantly prolonged, and the time course of tonic clonic seizure, as well as total time course, were significantly shortened in the synchronous stimulation group (P < 0.01). CONCLUSION: Simultaneous administration of pentylenetetrazol together with chaotic electrical stimulation produced the greatest inhibitory effects on epileptic seizures. This is possibly related to inhibition of abnormal mossy fiber spouting in the hippocampus.     

电压门控钾通道Kv1.1在戊四唑致痫大鼠中的表达

目的通过检测戊四唑(PTZ)致痫大鼠钾通道Kv1.1蛋白表达,探讨钾通道Kv1.1与癫痫发病的相关性。方法40只SD大鼠分成实验组30只和正常对照组10只。实验组30只大鼠通过腹腔注射PTZ建立全身强直-阵挛发作大鼠癫痫模型,取成功致痫鼠24只均分成3组,分别于致痫后3个时间段(1h、24h、48h)取脑组织。用免疫组化法和Westernblot法检测大鼠钾通道Kv1.1蛋白。结果实验组大鼠海马区钾通道Kv1.1蛋白表达水平在致痫后3个时间段(1h、24h、48h)均明显低于对照组(P0.05),但3个时间段之间钾通道Kv1.1蛋白表达水平差异无统计学意义(P0.05)。结论大鼠海马区钾通道Kv1.1表达的减少与全身强直阵挛发作大鼠癫痫发病密切相关。     

Extracellular amino acid levels measured with intracerebral microdialysis in the model of posttraumatic epilepsy induced by intracortical iron injection

An iron induced model of posttraumatic chronic focal epilepsy in rats was studied with respect to extracellular amino acids, electrophysiology, and morphology, approx. 6 months after intracortical injection of ferrous chloride. Twenty-six of the twenty-eight (93%) rats developed spontaneous epileptiform EEG-activity and electrical cortical stimulation done in eight animals evoked seizure activity in five animals (62.5%). Epileptic brain tissue displayed significantly higher extracellular interictal levels of aspartate (ASP), compared to normal brain, measured with intracerebral microdialysis. The interictal levels of serine (SER) were significantly higher at the lesion side compared to the contralateral cortex in epileptic animals. Spontaneous elevations of ASP and glutamate (GLU) levels up to 8 times the basal level were found in 4/5 (80%). There was no consistent amino acid pattern following the electrically induced seizures, but in association with more intense seizure activity ASP and GLU were elevated. Histopathologically, the necrotic lesions in the cortex contained small vessels and iron pigment loaded astrocytes. Scattered eosinophilic neurons were found in the hippocampus, bilaterally in 37% of the animals. The results show that a focal epileptiform activity developed in a high percentage of animals that received an intracortical iron injection. The observed amino acid changes in epileptic animals may be involved in the development of seizures in this model of posttraumatic epilepsy.     

Reduction of seizure frequency by clonazepam during cobalt experimental epilepsy

Adult female Sprague-Dawley rats rendered epileptic by bilateral cerebral implantation of cobalt wire were simultaneously prepared with permanent cortical and temporalis muscle electrodes for continuous recording of electroencephalographic (EEG) and electromyographic (EMG) activities. Clonazepam (4, 10 or 40 mg/kg) dissolved in gum acacia was administered once daily intraperitoneally for 5 days beginning 9 days after cobalt implantation. The 40 mg/kg dose completely suppressed generalized seizure activity. Although no tolerance to this effect developed by the fifth day of treatment, generalized seizure activity two days after the last injection was significantly greater in epileptic rats than in control animals. These results suggest that the cobalt model of epilepsy may be useful in the study of mechanisms underlying both anticonvulsant effectiveness and rebound excitability after anticonvulsant drug withdrawal.     

Antisense GAD67ODN对戊四氮所致慢性癫痫大鼠的作用

目的 :探讨谷氨酸脱羧酶 (GAD)的抗癫疒间作用。方法 :采用戊四氮制备大鼠慢性癫疒间 模型 ,利用反义寡脱氧核苷酸技术 ,通过选择性地抑制海马GAD基因的表达来观察其对慢性癫疒间大鼠行为、发作阈值及脑电图的影响。结果 :给予AntisenseGAD67ODN处理后 ,GADmRNA表达下降、GABA浓度下降、疒间 性发作潜伏期缩短、发作程度加重及脑电图疒间 波频率增加。结论 :从反面进一步说明GAD基因可能是抗疒间基因 ,为GAD基因治疗癫疒间提供实验依据     

戊四氮致猫急性癫发作相关脑区锰离子增强功能MRI成像的研究

目的探讨锰离子(Mn2 )增强功能MRI成像对确定癫发作相关脑区的价值。并进一步确定癫与钙超载的相关性,从而对癫的发病机制和定位进行研究。方法给成年猫肌肉注射戊四氮(PTZ)制作癫疒间模型,观察猫的行为学和脑电图改变;在癫疒间急性发作时和发作后24h进行Mn2 功能MRI成像检查;对信号明显增强的脑区做病理学检查并与对照组比较。结果PTZ致疒间猫脑电图呈阵发性高波幅棘-慢波。癫发作组猫Mn2 功能MRI成像显示大脑皮质明显弥漫性增强,其中额、顶、枕叶脑皮质增强率达34.6%,颞叶皮质增强率达约22.9%,与对照组相比差异有极显著性(均P<0.01)。癫疒间发作后24h组Mn2 功能MRI成像仍显示额、顶叶明显强化。强化区的神经元有明显变性、坏死。结论额、顶叶为PTZ致疒间猫癫疒间发作形成的相关脑区,Mn2 增强功能MRI成像能显示癫疒间发作的部位,并可进一步揭示癫疒间发病机制。