头孢曲松钠抗颞叶癫痫的效果

目的研究头孢曲松钠(Cef)对颞叶癫痫模型小鼠的抗癫痫效果以及对谷氨酸转运蛋白(GLT-1)表达情况的作用。方法首先构建颞叶癫痫小鼠模型,利用同步视频脑电监测(v EEG)技术,24 h不间断记录小鼠癫痫发作情况。实验组腹腔注射Cef 200 mg/(kg·d),对照组腹腔注射0.9%氯化钠溶液,从癫痫发作频率、间期棘波及海马硬化等方面评价Cef对癫痫发作的控制情况,并用Western blot检测其对谷氨酸转运蛋白GLT-1表达情况的影响。结果单侧海马注射200 ng海人酸(KA)可模拟内侧颞叶癫痫患者反复自发性癫痫发作和海马硬化等两个疾病症状,成功构建内侧颞叶癫痫模型。Cef处理使癫痫发作次数从2.145次/d降低到1.597次/d,平均发作次数降低了31.2%(P0.05)。KA癫痫小鼠较正常小鼠GLT-1表达明显降低,但Cef处理并未明显提升GLT-1的表达。结论腹腔注射Cef部分抑制KA癫痫小鼠的慢性自发性癫痫发作,但无明显提升海马中GLT-1的表达,提示其可能并非是通过提高星形胶质细胞的谷氨酸清除能力而抑制癫痫发作。     

海人酸致癫痫大鼠海马结构中noggin表达变化

目的:研究海人酸(kainic acid,KA)侧脑室注射并诱发癫痫持续状态(status epilepticus,SE)后大鼠海马结构中noggin基因在大鼠海马的表达变化。方法:大鼠在侧脑室注射KA后1、3、7、14、30和60d等不同时间,采用RT-PCR研究noggin mRNA含量变化,采用免疫组化观察noggin蛋白表达变化。结果:在正常大鼠海马结构中noggin mRNA有少量表达。noggin阳性细胞主要位于齿状回及CA3、CA1区,数量较少。侧脑室注射KA诱发SE后,noggin表达持续升高,3d达到高峰;7d在脑内的表达开始降低;注射后2个月,noggin表达降至术前水平,仅见散在的阳性细胞。结论:侧脑室注射KA并诱发SE后,大鼠海马结构中noggin表达明显增加。     

癫痫大鼠血清和脑组织IL-1β、IL-6和TNF-α水平变化

目的:探讨白介素-1β(IL-1β)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)对癫痫的神经免疫调节作用及卡马西平(CBZ)对它的影响。方法:180只Wistar大鼠随机分为对照组、红藻氨酸(KA)组和CBZ组,后两组再按癫痫发作后的不同时点分为6个亚组(1h、4h、12h、24h、48h和72h);以KA建立癫痫模型,采用放射免疫分析(RIA)测定IL-1β、IL-6和TNF-α的水平变化。结果:癫痫发作后72h内,血清和脑匀浆液中IL-1β、IL-6和TNF-α的水平变化不同;在不同时点,KA和CBZ组的三者水平均明显高于对照组(P<0.01或P<0.05),同时,CBZ组的三者水平明显低于KA组(P<0.01)。结论:癫痫大鼠免疫系统处于活化状态,细胞因子水平的失衡可能参与了癫痫的免疫病理过程;CBZ对癫痫具有一定的免疫抑制作用。     

腺苷对癫痫大鼠海马缝隙连接蛋白43表达的影响

目的:探讨腺苷对癫痫大鼠发作行为、脑电图(EEG)及海马缝隙连接蛋白43(CX43)的影响。方法:雄性Wistar大鼠随机分成对照组、红藻氨酸(KA)组及腺苷组。采用视频监视观察2周和5周大鼠癫痫发作情况以及EEG,应用免疫印迹法检测CX43的表达。结果:2周时腺苷组大鼠行为学发作级别、EEG与KA组无明显差异,5周时腺苷组行为学发作级别明显降低,EEG癫痫放电频率降低。对照组无发作,EEG无癫痫波。KA和腺苷组大鼠在2周和5周时CX43表达均明显增加;5周时腺苷组CX43表达水平显著低与KA组。结论:CX43在癫痫大鼠海马组织中的表达变化与癫痫发作相关;腺苷可以明显减轻大鼠癫痫发作级别,降低癫痫放电频率。腺苷可能通过抑制KA诱导的大鼠癫痫发作时CX43的表达而发挥抗癫痫作用。     

左乙拉西坦对红藻氨酸致发育期癫痫幼鼠海马主穹窿蛋白表达的影响

目的:建立发育期慢性癫痫大鼠模型,观察海马主穹窿蛋白(MVP)的表达及左乙拉西坦(LEV)对其表达的影响。方法:腹腔注射红藻氨酸(KA)1 mg/kg(浓度0.5 mg/ml),建立大鼠慢性癫痫模型,注射后连续观察8 h,癫痫发作达5级以上并出现癫痫持续状态的大鼠,且两周后出现自发性反复惊厥发作者视为模型成功。将造模成功后的40只大鼠随机分为未治疗组(KA组)20只,左乙拉西坦治疗组(KA+LEV组)20只,另取40只大鼠腹腔注射生理盐水,并分为NS组20只、NS+LEV组20只。用药组均于癫痫自发性反复发作后开始用药,疗程为6周,然后将所有大鼠断头取脑,用免疫组化及RT-PCR法测定大鼠海马内MVP及其mRNA的表达。结果:(1)大鼠海马MVP的阳性细胞计数与MVP的mRNA表达趋势相一致。(2)NS组、NS+LEV组大鼠海马有少量MVP阳性细胞及mRNA表达,NS+LEV组MVP阳性细胞数及mRNA的含量与NS组相比差异无统计学意义(P>0.05);KA组MVP阳性细胞计数及mRNA的含量与NS组相比显著增高(P<0.05);KA+LEV组与KA组相比,MVP的阳性细胞及mRNA含量减少(P<0.05)。结论:MVP可能参与慢性癫痫耐药的发生,LEV可以控制大鼠痫性发作,并下调MVP的表达。     

癫痫发作敏感大鼠齿状回苔状纤维侧枝发芽

目的　探讨海马齿状回苔状纤维侧枝发芽与癫痫发作敏感性形成之间的关系。方法　在颈部皮下注射惊厥剂量的海人酸 (KA ,10mg/kg)诱发大鼠出现癫痫发作后 ,采用Timm’s染色法 ,分别在注射KA后3d、7d和 1个月 3个时间点观察致痫大鼠海马齿状回内苔状纤维发芽的情况。结果　Timm’s染色发现 ,注射KA后 7d ,海马齿状回分子层内带和颗粒细胞上层出现苔状纤维的异常发芽 ,注射KA后 1个月海马齿状回内Timm’s染色颗粒颜色加深 ,范围增大。提示海马苔状纤维发芽形成的时间过程与癫痫发作敏感性形成的时间过程一致。结论　海马齿状回分子层内带和颗粒细胞上层出现异常的苔状纤维发芽可能与癫痫发作敏感性形成有关。     

戊四唑致痫大鼠脑内缝隙连接的作用

目的: 研究缝隙连接(gap junction, GJ)在癫痫发病中的作用及机制。方法: 以戊四唑 (pentylene-tetrazol, PTZ)致痫大鼠模型为研究对象,采用免疫组织化学和实时定量RT-PCR技术,分别检测缝隙连接蛋白Cx32和Cx43在癫痫发作后不同时点皮层和海马神经元的表达。加用卡马西平(carbamazepine, CBZ)和甘珀酸(carbenoxolone, CBX)干预,观察二者对Cx32/43表达以及大鼠癫痫发作的影响。结果: 免疫组织化学染色显示PTZ致痫2 h后大鼠脑内Cx32/43阳性细胞开始增多,8 h后增多更为明显。实时定量RT-PCR示致痫2 h Cx32 mRNA迅速升高,5 h达高峰。Cx43 mRNA表达水平较低,但明显高于对照组。CBX显著抑制了Cx32/43的表达,CBZ对Cx32和Cx43的表达无明显影响。二者均抑制了大鼠的痫样发作。结论: GJ参与癫痫的发病过程,具有促进癫痫发作的作用。CBZ不影响Cx32/43的表达,表明其抗癫痫作用机制与阻断GJ 无关。     

合成红藻氨酸诱发大鼠癫痫作用的研究

目的: 观察合成红藻氨酸(SKA) 诱发大鼠癫痫的作用及其作用特点。方法: Wistar大鼠40只,随机分为正常对照组、SKA 12 mg/kg、SKA 10 mg/kg 和 SKA 5 mg/kg 剂量组及红藻氨酸 (KA)10 mg/kg阳性对照组。腹腔注射给药,连续8 h观察大鼠癫痫发作的行为学变化及连续3.5 h记录其脑电图变化。结果: 合成红藻氨酸5、10、12 mg/kg 腹腔注射,可诱发大鼠癫痫发作,其行为学及脑电改变与KA对照组无明显差异。但合成红藻氨酸诱发动物癫痫呈现规律、稳定及阶段性明显的特征,且大鼠的死亡率较天然红藻氨酸低。结论: 合成红藻氨酸腹腔注射可诱发大鼠癫痫发作,以10 mg/kg为较合适剂量。     

KA诱发癫痫反复发作损伤大鼠空间学习记忆及中脑多巴胺能神经元

目的探讨颞叶癫痫反复发作(Spontaneous recurrent se izure,SRS)对大鼠空间学习记忆影响及中脑内多巴胺能神经元变化。方法以红藻氨酸(kain ic ac id,KA)制备颞叶癫痫大鼠模型,以是否出现SRS为标准将KA大鼠分为伴有反复发作和不伴有反复发作组,盐水为对照组。分别进行水迷宫行为测试,评价其学习记忆能力;并用酪氨酸羟化酶(Tyrosine hydroxylase,TH)免疫组化方法来观察各组大鼠中脑内多巴胺能神经元变化。结果KA处理后,按照Rac ine描述标准,KA组动物发作全部达到4~5级。KA后3周大鼠19只出现SRS,16只未见SRS;Morris水迷宫发现,在5 d的空间学习记忆测试中,反复发作KA大鼠的寻找潜伏期明显长于不伴有SRS的KA大鼠和盐水对照组(P<0.01),而不伴有SRS组与盐水对照组没有明显差别;伴有SRS的KA组大鼠总共穿过平台次数显著少于不伴有SRS的KA组大鼠和盐水对照组(P<0.01)。TH免疫组织化学结果发现与不伴有SRS的KA大鼠和盐水对照组比较,伴有SRS的KA大鼠在腹侧被盖的多巴胺能神经元大量脱失(P<0.01)。结论KA大鼠癫痫反复发作可能与空间学习记忆障碍和在腹侧被盖多巴胺能神经元大量脱失相关。     

地塞米松对海人酸致（癇）大鼠脑P-糖蛋白表达的影响

目的：探讨地塞米松对海人酸致痫大鼠脑P-糖蛋白（P-gp）表达的影响.方法：将SD大鼠随机分为假手术组（Sham组,n=8）、癫痫组（EP组,n=12）、地塞米松干预癫痫组（DEX组,n=12）.后两组采用海马注射海人酸方法制作癫痫模型,DEX组癫痫造模前30 min给予腹腔注射地塞米松0.4 mg/kg.分别记录各组大鼠达到Ⅲ级和Ⅴ级发作时所需的时间（潜伏期）,初次至第6次≥Ⅳ级发作的间隔时间作为评价癫痫发作严重程度的指标;大鼠术后24 h处死,使用HE染色和免疫组织化学方法,比较各组海马CA3区、齿状回、杏仁核复合体区P-gp表达及脑损伤情况.结果：①Sham 组未见癫痫发作;DEX组与EP组达到Ⅲ级发作的潜伏期分别为（87.92±45.80）min和（67.50±22.91）min,达到Ⅴ级发作的潜伏期分别为（103.33±51.27 ）min和（75.60±22.10）min,差异均无统计学意义（P〉0.05）;与EP组相比,DEX组样发作严重程度降低（P=0.004）;②与EP组相比,DEX组于所观察的脑区损伤均减轻,以海马CA3区和杏仁核复合体区较为显著;③与Sham组比较,EP组各观察脑区P-gp表达均明显升高（P〈0.01）;与EP组相比,DEX组海马CA3区和杏仁核复合体区P-gp表达显著减少（P〈0.05）,而在齿状回表达量差异无统计学意义（P=0.078）.结论：地塞米松可降低海人酸致痫大鼠发作严重程度和脑损伤,抑制P-gp表达上调,其中以海马CA3区和杏仁核区较为显著.     

Long-term effects of neonatal seizures on subsequent N-methyl-D-aspartate receptor-1 and gamma-aminobutyric acid receptor A-alpha 1 receptor expression in hippocampus of the Wistar rat

To evaluate the pathophysiological mechanism of subsequent reduced seizure threshold following neonatal seizures, single or recurrent prolonged seizures were induced to neonatal rats by the inhalant flurothyl. The expression of N-methyl-d-aspartate receptor 1 (NR1) and gamma-aminobutyric acid receptor A-alpha 1 (GABA-A-alpha 1) immunoreactivity in hippocampus were examined by Western blotting analysis at the day 7 (P7) and day 75 (P75) after the last seizure. Whereas there were no significant changes in single seizure group and recurrent seizure group of P75, NR1 expression enhanced significantly in P7 rats of recurrent seizure group. Meanwhile, polypeptide levels of GABA-A-alpha 1 receptor subunit decreased significantly in both single and recurrent seizure-treated P7 and P75 rats. Our results suggest that recurrent or single prolonged seizures during the neonatal period may have long-term effects on the balance between excitatory NMDA system and inhibitory GABA system in hippocampus of rats.     

CCK与海人酸诱发癫痫敏感性形成的关系

用海人酸 (Kainic acid,KA) 10 mg/ kg给 Sprague- Dawley (SD)大鼠颈部皮下注射 ,诱发急性癫痫发作 ,在急性癫痫发作后一周 ,用阈下剂量 (5 mg/ kg)的 KA检测癫痫敏感性。同时分别用原位杂交和免疫组化技术检测并发现癫痫敏感性形成大鼠额叶皮层胆囊收缩素原 (PCCK) m RNA明显增加 ;海马结构中 ,靠近海马齿状回颗粒细胞 (DGCs)基底部锥体样胆囊收缩素 (Cholecystokinin,CCK)免疫反应阳性神经元染色明显增强 ,但海马门尖部 CCK免疫反应阳性神经元明显减少。而癫痫敏感性未形成大鼠未见上述变化 ,提示上述变化与癫痫敏感性形成有关     

戊四氮癫痫幼鼠海马内NF-κB与N-Cadherin定位与苔藓纤维发芽现象

目的:探讨单次癫痫发作后脑内NF-κB和N-Cadherin表达与海马结构中苔藓纤维发芽现象之间的关系与作用。方法:利用腹腔注射戊四氮(PTZ)制作发育期SD大鼠(14 d、28 d)单次惊厥发作模型,各日龄组随机分为生理盐水(NS)组、PTZ组、吡咯二硫氨基甲酸酯(PDTC)组和PDTC+PTZ组,采用免疫组化法检测NF-κB和N-Cadherin的表达,利用Timm染色法观察海马苔藓纤维发芽现象。结果:NS组在海马CA3区可见极少Timm染色颗粒;致惊后1周偶见Timm染色颗粒、3周可见Timm染色颗粒沿海马CA3区呈条带样分布(P<0.01);PDTC预处理组Timm染色颗粒较PTZ致惊组明显减少(P<0.05)。NS组大鼠海马CA1、CA3区无NF-κB p65核转位细胞,致惊后24 h各日龄组幼鼠海马CA1、CA3区NF-κB p65核转位细胞较NS组明显增加(P<0.01);PDTC预处理后NF-κB p65的核转位细胞较致惊组明显减少(P<0.05)。NS组海马CA1、CA3区可见少量N-Cadherin阳性细胞;致惊组海马CA3和齿状回门区的N-Cadherin的阳性细胞与NS组相比明显增多(P<0.01),PDTC预处理后相同区域内N-Cadherin阳性细胞较PTZ致惊组明显减少(P<0.05)。NF-κB p65、N-Cadherin及Timm染色颗粒的表达结果与惊厥鼠的日龄并无关联性。结论:幼鼠单次惊厥发作可以引起海马不同程度的苔藓纤维发芽,而NF-κB p65、N-Cadherin的分布位置与变化时相与苔藓纤维发芽相吻合,表明NF-κB p65、N-Cadherin可能参与或伴随着苔藓纤维的发芽。     

Increased afterdischarge threshold during kindling in epileptic rats

The effects of daily electrical kindling stimulation of the perforant pathway were investigated in an excitotoxic rat model of epilepsy with chronic seizures in order to learn whether the preexisting epileptic condition would facilitate or retard kindling. Sprague-Dawley rats with recurrent spontaneous seizures 4-8 months after unilateral intrahippocampal kainic acid (KA) injection were implanted with recording electrodes in the hippocampus and stimulating electrodes in the perforant path. Daily stimulation for 10 s at 5 Hz was given for 15 days. The afterdischarge (AD) threshold and the AD duration of kindled KA rats were compared before and during kindling with those of a kindled control group. In the control group, as expected, mean AD thresholds decreased ( P<0.01), while AD duration progressively increased. Although AD threshold was the same in KA and control groups at the start of kindling, in the KA group a significant increase in threshold occurred from the beginning to the end of kindling ( P<0.01). Behaviorally, KA rats showed stage 4 or 5 seizures on the first stimulation, and stage 3-5 seizures during the remainder of kindling. Paired pulse testing showed facilitation of late components of the dentate gyrus field potential at the beginning of kindling, and suppression of late components at the end, in the KA rats. A significant decrease in the rate of spontaneous seizures in KA rats was noted during the period of kindling ( P=0.04). These results suggest that electrical stimulation of the perforant path may strengthen homeostatic seizure suppressing mechanisms, and may provide insights into novel approaches to the treatment of clinical seizures in temporal lobe epilepsy.     

经皮三叉神经电刺激减轻戊四氮诱导的大鼠癫痫发作并抑制海马内IL-1β和TNF-α的表达

 目的:探讨经皮三叉神经电刺激预处理对戊四氮（PTZ）诱发的急性癫痫大鼠的行为学及海马致痫细胞因子白细胞介素1β(IL-1β)及肿瘤坏死因子α(TNF-α)的影响。方法:动物随机分为对照组、致痫组（PTZ组）和经皮三叉神经电刺激组,分别给予7 d、14 d和28 d的假刺激和三叉神经电刺激预处理后,腹腔注射PTZ 建立急性癫痫动物模型,观察给药后大鼠癫痫行为学表现,并分别用免疫组织化学方法及ELISA方法对海马IL-1β、TNF-α进行检测。结果:经皮三叉神经电刺激可以明显减轻大鼠的痫性发作级别,减少癫痫发作持续的时间（P<0.05）, 且海马细胞因子IL-1β及TNF-α的表达明显少于PTZ组（P<0.05或P<0.01）。结论:经皮三叉神经电刺激预处理在PTZ 急性点燃癫痫大鼠模型中不仅具有抗惊厥作用, 还可以减少海马致痫细胞因子IL-1β及TNF-α的表达,可能为癫痫的防治带来新的策略。       

The effects of quercetin on the gene expression of the GABA<Subscript>A</Subscript> receptor α5 subunit gene in a mouse model of kainic acid-induced seizure

The flavonoid quercetin has recently been reported to have neuroprotective effects, and the role of the gamma-aminobutyric acid A alpha 5 subunit (GABA_A α5) receptor has been determined in some nervous system disorders. The aim of this study was to identify the molecular mechanism of the effect of quercetin administered at anticonvulsive doses on the expression of the GABA_A α5 receptor gene in kainic acid (KA)-induced seizures in mice. The experimental animals were divided into four groups: control, KA, and KA + quercetin at 50 or 100 mg/kg, respectively. The results showed a dose-dependent reduction in the behavioral seizure score with quercetin pre-treatment in the KA mouse model. Two hours after the end of the 7-day treatment regimen, expression of the GABA_A α5 receptor gene in the hippocampus was found to be increased in the KA group, but this increase was reduced in the KA + quercetin 50 or 100 mg/kg treatment groups. These results suggest that expression of the GABA_A α5 receptor could be a mechanism for reducing seizure severity or may be a marker of seizure severity. Further studies are necessary to clarify quercetin’s mechanism of action and the relation of GABA_A α5 receptor gene expression to seizure severity.     

Myo-inositol treatment prevents biochemical changes triggered by kainate-induced status epilepticus

Identification of the compounds preventing the biochemical changes underlying the epileptogenesis process is of great importance. We have previously shown that myo-inositol (MI) administration reduces kainic acid (KA) induced seizure scores. MI treatment effects on biochemical changes triggered by KA induced status epilepticus (SE) were investigated in the present study. After SE one group of rats was treated with saline, whereas the second group with MI. Control groups received either saline or MI administration. Changes in the amounts of following proteins were studied in the hippocampus and neocortex of rats: GLUR1 subunit of glutamate receptors, calcium/calmodulin-dependent protein kinase II (CaMKII), and heat shock protein 90. No changes were found 28–30 h after experiments. However on 28th day of experiment the amounts of GLUR1 and CaMKII were strongly reduced in the hippocampus of KA treated animals but MI significantly halted this reduction. Obtained results indicate anti-epileptogenic features of MI on biochemical level.     

托吡酯对癫痫持续状态大鼠海马神经元损伤的保护作用

为探讨托吡酯(topiramate,TPM)对癫痫持续状态(status epilepticus,SE)大鼠海马神经元损伤的保护作用,将大鼠随机分为正常对照组、海人酸(kanic acid,KA)组和TPM预处理组,观察海马神经元超微结构和bcl-2表达的变化。先将TPM组大鼠用TPM预处理,然后采用KA(10mg/kg)腹膜腔注射制作SE模型,在痫性发作终止后6、24和48h取海马进行研究。结果显示:KA组神经元呈凋亡特征;TPM组神经元结构大致正常,但出现核仁边聚和细胞器增多现象,亦观察到少量凋亡神经元。KA组于SE后6h观察到bcl-2表达增高,与对照组相比差异显著,(P<0.05);24h时开始减弱,48h仅有微弱表达;TPM组在24h时bcl-2呈强表达(P<0.001),并持续至48h。以上结果提示:托吡酯预处理能减轻癫痫大鼠神经元的损伤,其机制可能与上调bcl-2的表达有关。