丙戊酸钠对戊四氮致痫大鼠海马神经元凋亡的影响

目的 探讨丙戊酸钠(VAP)对戊四氮(PTZ)致痫大鼠海马神经元凋亡的影响.方法 将48只成年Wistar大鼠随机平均分为正常对照(NC)组、PTZ组和VAP组,PTZ组和VAP组大鼠腹腔注射阈下剂量的PTZ 35mg/(kg·d),直至达到点燃标准.点燃后,VAP组大鼠经腹腔注入VAP15mg/(kg·d),PTZ组大鼠经腹腔注入生理盐水,30min后,再腹腔注射PTZ诱发癫痫发作.应用免疫组化法检测大鼠海马神经元Fas、Caspase-3和Survivin的表达.结果 PTZ组大鼠海马神经元Fas、Caspase-3阳性细胞数和光密度明显高于VAP组和NC组(均P<0.01),Survivin阳性细胞数和光密度明显低于VAP组(P<0.01),但高于NC组(P<0.05);VAP组大鼠海马神经元Fas、Caspase-3阳性细胞数和光密度明显高于NC组(均P<0.05),Survivin阳性细胞数和光密度明显高于NC组(P<0.01).结论 癫痫发作可以导致大鼠海马神经元的凋亡,而丙戊酸钠有对抗癫痫发作导致细胞凋亡的作用,主要通过促进Survivin的表达和抑制Fas和Caspase-3的表达发挥作用.     

甘松治疗戊四氮致大鼠的实验性研究

目的观察甘松对戊四氮致大鼠的疗效。方法将50只Wistar大鼠随机分为5组,每组各10只,分别为正常对照组、模型组、甘松治疗组、丙戊酸钠治疗组和甘松合并丙戊酸钠治疗组;采用腹腔注射戊四氮制作建立癫模型;观察各组大鼠行为学及脑电图、NSE(神经元特异性烯醇化酶)浓度变化。结果与模型组比较,甘松治疗组、丙戊酸钠治疗组、甘松合并丙戊酸钠组可明显减轻大鼠样发作程度,减少发作频率和持续时间,改善大鼠皮层脑电图,降低脑脊液神经元特异性烯醇化酶浓度,其中以甘松合并丙戊酸钠组最为明显。结论甘松对戊四氮致大鼠具有一定的抗癫及脑保护作用,与丙戊酸钠联用有协同作用。     

海人酸颞叶癫痫模型及丙戊酸钠治疗作用的观察

目的 建立海人酸癫痫动物模型并给予丙戊酸钠治疗，探讨其治疗作用。方法 42只Wistar大鼠置于立体定向仪上，于右侧海马注射海人酸制备大鼠癫痫模型。根据癫痫发作病程，随机分为急性期、静止期、慢性期等实验组；另设丙戊酸钠治疗组，分别于海人酸注射后24h和自发性癫痫出现后给予丙戊酸钠治疗。在实验过程中以视频录像监测大鼠症状的改变，深部脑电图观察癫痫的病理过程及丙戊酸钠治疗前后脑电活动的改变。组织切片经Nissl和Timm染色观察海马神经元数目、苔藓状纤维发芽。结果 (1)电生理学改变：海人酸注射后数分钟大鼠即出现癫痫发作并呈现急性期、静止期和慢性期等病变过程。脑电图于急性期和慢性期均表现为典型的癫痫发作电活动及发作间期表现。(2)病理学改变：海马神经元死亡主要出现在急性期，以实验侧CA3、CA4区最为明显，齿状回无明显神经元缺失。Timm染色显示从静止期开始出现苔藓状纤维发芽并进行性增加。(3)丙戊酸钠治疗前后症状的改变：早期应用丙戊酸钠治疗可抑制癫痫发作及其症状的发展；出现自发性癫痫后丙戊酸钠的治疗效果欠佳，停药后症状再次出现。结论 海人酸模型是模拟人类颞叶癫痫较为理想的动物模型。早期丙戊酸钠治疗效果较好，否则不能有效控制癫痫。     

GAD在颞叶癫痫大鼠海马内源性促痫机制中的作用

目的:探讨GAD65、GAD67在颞叶癫痫发生后海马内源性促痫机制中的作用。方法:112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),实验组大鼠选用海人酸腹腔注射法建立颞叶癫痫模型,对照组大鼠腹腔注射无菌生理盐水。选取腹腔注射后3小时、6小时、12小时、24小时、48小时、7天、30天为研究的时间点,颞叶海马的CA1区、CA3区、齿状回为研究部位。腹腔给药后每天观察大鼠的行为学变化,大鼠处死前进行EEG描记。用原位杂交方法检测不同时间点海马不同区域GAD65、GAD67mRNA的表达,免疫组织化学法检测GAD65、GAD67蛋白的表达。结果:实验组大鼠海马GAD65 mRNA及其蛋白的表达随时间呈逐渐增高趋势,致痫后48小时～30天,GAD65 mRNA及其蛋白表达较对照组增高(48小时P<0.05;7～30天P<0.01);海人酸致痫后6小时、24小时实验组大鼠海马的GAD67mRNA及其蛋白表达较对照组增高(分别为P<0.01、P<0.05)。结论:颞叶癫痫急性期海马GAD67表达的增高及慢性期海马GAD65表达的增高是癫痫发生后机体的内源性抗痫机制。     

癫痫动物模型脑中神经肽Y的动态含量变化及意义

目的:探讨匹罗卡品(PILO)诱发的癫痫大鼠模型脑组织中神经肽Y(Neuropeptide Y,NPY)含量的动态变化及意义,进一步明确NPY与癫痫的关系,为抗癫痫治疗,研制抗癫痫药物提供新途径。方法:健康成年雄性SD大鼠120只,随机分为两组:单纯腹腔注射匹罗卡品组(癫痫模型组);单纯腹腔注射生理盐水组(对照组);注射后根据Racine制定的标准判定是否有癫痫发作,并行脑电图检查,观察有无癫痫样波(棘波,尖波,棘慢波,尖慢波)发放。两组大鼠分别于给药后1h,3h,6h,24h,3d,7d,15d,30d,60d将大鼠麻醉,取出脑组织,对脑组织中的NPY含量进行测定。结果:癫痫模型组60只大鼠中,2只死于癫痫持续状态,其余大鼠可观察到边缘发作行为表现,脑电图有典型的癫痫样波发放,对照组无癫痫发作及癫痫样波发放。癫痫模型组脑NPY含量与对照组相比,差异有显著性P<0.05;癫痫模型组急性期(1h-7d)与慢性期(15d-60d)比较差异有显著性P<0.05,对照组差异无显著性;癫痫模型组与对照组脑中的NPY含量在12h,24h,15d,30d,60d.差异有显著性P<0.05或P<0.01,癫痫模型组脑中的NPY含量各组(各时间段)比较有差异有显著性P<0.05,对照组差异无显著性,癫痫模型组大鼠Ⅳ-Ⅴ级发作与Ⅱ-Ⅲ级发作,脑NPY含量比较,差异有显著性P<0.05。结论:1.神经肽Y与癫痫密切相关,癫痫发作后?     

苯妥英钠和丙戊酸钠预防术后癫痫的对照研究

目的　对比苯妥英钠和丙戊酸钠预防术后癫痫的作用、毒副反应以及和血药浓度的关系。方法　采用随机前瞻对照性研究 ,选择幕上开颅手术病人 ,苯妥英纳组 72例 ,丙戊酸钠组 80例 ,术前术后分别规则服用苯妥英钠和丙戊酸钠 ,监测抗癫痫药物的血药浓度和术后癫痫及毒副反应发生情况。结果　两组共 15例术后发生早期癫痫 ,10例 (10 /15 )达到有效血浓度 ;其中 ,苯妥英钠组 6例 (8 3 % )发生早期癫痫 ,2例达到有效血浓度 ,丙戊酸钠组 9例 (11 3 % )发生早期癫痫 ,8例达到有效血浓度 ;远期癫痫发作 7例中丙戊酸钠组 5例 ,苯妥英钠组 2例。苯妥英钠组 11例 (15 3 % )及丙戊酸钠组2例 (2 5 % )出现了毒副反应。经χ2 检验 ,两组术后癫痫发生率无显著性差异 ,术后未发癫痫组和癫痫发作组间药物血浓度无显著性差异 (P >0 0 5 ) ,苯妥英钠组毒副反应发生率高于丙戊酸钠组。结论　苯妥英钠和丙戊酸钠预防控制术后癫痫无差别 ,苯妥英钠毒副反应较丙戊酸钠严重。     

慢性癫痫大鼠海马细胞外液卡马西平浓度的研究

研究表明癫痫灶附近多种药物转运体(m u ltidrug trans-porter,m dt)的过度表达可能降低靶点细胞外液抗癫痫药(AED)浓度而产生癫痫耐药现象。关于慢性癫痫脑细胞外AED浓度的研究国内未见报道。因此我们建立大鼠慢性癫痫模型,采用微透析活体取样的方法,检测大鼠海马脑细胞外液卡马西平浓度。1材料与方法健康雄性Sprague-D aw ley大鼠,20只,体重160~200g,随机分成正常对照组、慢性癫痫组。慢性癫痫模型制作:大鼠每日腹腔注射戊四氮(35m g/kg),观察注射后1h内癫痫发作强度,按R ac ing分级评分:0级,无抽搐发作;1级,面肌抽搐;2级,面肌抽搐与…     

戊四氮致痫大鼠不同脑区神经肽Y的表达及意义

目的研究神经肽Y(neuropeptideY,NPY)在戊四氮(pentylenetetrazol,PTZ)致痫大鼠不同脑区中的变化,以探讨NPY与癫痫的关系。方法清洁级近交系雄性SD大鼠50只,分为对照组(A组,n=10)及实验组(40只)。实验组按体重50mg/kg经腹腔注射PTZ1次,对照组腹腔注射同等容量的生理盐水。根据癫痫发作分级,0~1级7只为B组,立即取脑;2级以上发作33只,随机于癫痫发作后0(C组,n=10)、6(D组,n=11)、24(E组,n=10)、72h(F组,n=2)取脑。采用放射免疫方法动态观察脑组织中海马、中脑、纹状体和额叶中NPY的改变;SP免疫组化染色法染色,观察各组大鼠海马CA1区NPY的表达。结果B组中脑、纹状体及额叶NPY含量较A组升高(P<005),而两组间海马NPY含量差异无显著性;癫痫发作组(C组、D组、E组)各部位NPY含量均明显高于A组。动态观察结果显示,癫痫发作后在中脑、海马、纹状体和额叶的NPY含量明显增高随后呈逐步下降,在癫痫发作后24h其含量与未发生惊厥的B组NPY含量差异无显著性(P>005);免疫组化染色显示癫痫大发作后海马NPY的表达明显增加,但随着时间的推移而降低。结论NPY与大鼠癫痫的发生、发展有密切关系。     

匹罗卡品诱发成年大鼠普遍癫痫与难治性癫痫海马神经发生的变化

背景：神经发生包括细胞增殖、迁移、分化和存活等,是人和多数哺乳动物部分脑区产生新生神经元的过程,主要分布在侧脑室下区和海马齿状回。癫痫可导致海马齿状回的神经发生改变,BrdU是目前公认最理想检测未成熟细胞增殖的标记物之一。 目的：观察匹罗卡品诱发成年大鼠癫痫后神经发生的特点,以及普通癫痫与难治性癫痫神经发生的差异。 设计、时间及地点：随机分组,细胞分子生物学实验,于2006-08/2007-06在华中科技大学同济医学院中心实验室及附属协和医院中心实验室完成。 材料：选用健康Sprague-Dawley雄性大鼠100只,随机分为2组,对照组8只,实验组92只,分为普通癫痫组,自发发作组,难治性癫痫组和非耐药组。匹罗卡品为武汉晶美公司产品,兔抗鼠BrdU抗体为美国sigma公司产品,辣根过氧化物酶标记的羊抗兔IgG为武汉博士德公司产品。 方法：对照组大鼠腹腔注射生理盐水;实验组腹腔注射匹罗卡品15mg/kg,最多注射4次,出现癫痫持续状态的大鼠注射水合氯醛终止发作。致癫大鼠癫痫发作终止后6h腹腔注射BrdU,观察自发发作情况。以脑电图、自发发作频率持续时间,无自发发作为普通癫痫组,有自发发作为自发发作组。行卡马西平灌胃2周并记录发作频率,对卡马西平治疗无效,发作频率减少<50%的为难治性癫痫组,治疗有效即发作频率减少>50%为非耐药组。普通癫痫组分别于注射后第1,2,3,7,14,21和28d取鼠脑海马部做冠状连续切片。非耐药组和难治性癫痫组的大鼠,再次行腹腔注射BrdU,每次为50mg/kg,连续注射4次,每次间隔2h,48h后取脑海马部制作石蜡切片。 主要观察指标：脑海马部切片行免疫组化染色,镜下观察不同时间点BrdU阳性细胞的分布、形态和数量及注射匹罗卡品后大鼠癫痫发作状况。 结果：匹罗卡品第1次注射后所有大鼠无癫痫发作,第2次注射发作16只,第3次注射发作42只,第4次注射后发作11只,14只大鼠4次注射仍无癫痫发作,9只大鼠癫痫持续状态后死亡,77只进入结果分析。神经发生主要位于海马颗粒细胞层和齿状回。与对照组比较,普通癫痫组BrdU阳性细胞明显增多（P<0.01）,难治性癫痫组新生细胞较普通癫痫组明显减少（P<0.01）,神经发生减少。癫痫后第2天BrdU 阳性细胞数目开始增加,14-15d达到高峰,1个月后回到初始水平。 结论：与普通癫痫相比,难治性癫痫可导致神经发生减少。     

TLR9、MyD88在颞叶癫痫大鼠海马组织中表达的动态变化

目的观察氯化锂-匹罗卡品致痫大鼠各期海马中Toll-样受体9(TLR9)、髓样分化因子(MyD88)表达的变化,探讨其是否与颞叶癫痫发生有关。方法 SD雄性大鼠120只,随机分为对照组(30只)和模型组(90只),腹腔注射氯化锂。18 h~20 h后模型组腹腔注射匹罗卡品诱导癫痫持续状态(SE);对照组予等量生理盐水取代匹罗卡品腹腔注射。对照组和造模成功的模型组依据腹腔注射后时间随机分为10个亚组:急性模型组(SE后3 h、6 h、9 h、12 h、1 d、3 d、7 d);潜伏模型组(SE后14 d、28 d);慢自发发作组(SE后56 d)。每亚组动物模型组9只,对照组3只。免疫组化、蛋白印迹、RT-PCR技术测定各亚组癫痫大鼠海马内TLR9、MyD88的表达。结果TLR9、MyD88在模型组海马内表达明显增多,与对照组相比,差异有显著性(P0.05)。模型亚组内,TLR9、MyD88在急性期和慢性期表达明显增高,而潜伏期无明显表达变化。其中急性期内的增高多集中在癫痫发作后6 h;3组比较差异有显著性(P0.05)。结论大鼠海马内TLR9、MyD88表达增多可能与颞叶癫痫发病有关,探讨其机制可能为颞叶癫痫的治疗提供新的靶点。     

托吡酯对慢性癫痫幼鼠神经元保护作用的实验研究

目的 研究托吡酯(TPM)单药对慢性癫痫幼鼠损伤神经元的影响.方法 将3～4周龄雄性Wistar大鼠72只采用随机数字表法分为6组,每组各12只,A组:阴性对照组;B组:阳性对照组;C组:TPM低剂量组(20 mg/kg);D组:TPM中等剂量组(40 mg/kg);E组:TPM高剂量组(80 mg/kg);F组:合药组(TPM 40 mg/kg+丙戊酸钠200 mg/kg),其中B～F组为慢性癫痫组,于给药前腹腔注射戊四氮.连续用药2个月,观察幼鼠体重、行为学表现、血清神经元特异性烯醇化酶(NSE)及病理改变.结果 (1)E组对幼鼠体重的影响比C组、F组显著.(2)D组、E组戊四氮诱发痫性发作的时间延长,痫性发作的程度减轻.(3)TPM各剂量组NSE水平显著低于B组,F组与TPM单药组无差异.(4)E组海马组织神经元的退行性变和胶质细胞的增生程度减轻明显.结论 TPM可减轻幼鼠痫性发作后的神经损伤程度,且存在剂量效应,但与丙戊酸钠联合可削弱其神经保护作用.     

养血清脑颗粒和丙戊酸联用对戊四氮点燃癫痫大鼠的影响

目的 探讨养血清脑颗粒(YXQNG)联用丙戊酸(VPA)对戊四氮(PTZ)慢性点燃模型大鼠癫痫发作、EEG、认知功能及颞叶、海马T型Ca2+通道蛋白(Cav3.2)表达的影响. 方法 成年雄性SD大鼠40只按随机数字表法分为PTZ组、VPA组、VPA+YXQNG组、NS组,每组10只.前3组大鼠腹腔注射PTZ溶液制作慢性点燃模型,VPA组大鼠在注射PTZ前1 h给予VPA灌胃;VPA+YXQNG组除给予VPA外,注射PTZ前1.5 h给予YXQNG灌胃;NS组腹腔注射生理盐水,每天一次.8周后观察各组大鼠的行为学变化、Y型电迷宫检查大鼠的正确反应率、捕记EEG并应用免疫组化染色检测颞叶和海马Cav3.2的表达. 结果 给药8周后PTZ组大鼠全部达到完全点燃(连续3 d出现Ⅳ级发作或达到Ⅴ级发作),VPA组和VPA+YXQNG组大鼠仅出现0～Ⅱ级发作;Y型电迷宫检查结果显示VPA+YXQNG组大鼠正确反应率高于PTZ组,差异有统计学意义(P＜0.05);EEG结果显示PTZ组大鼠癫痫发作时EEG有明显异常放电,总功率高于用药前,差异有统计学意义(P＜0.05).VPA组、VPA+YXQNG组大鼠用药前后EEG总功率的差值均高于PTZ组,差异有统计学意义(P＜0.05);免疫组化染色结果显示VPA组、VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于PTZ组,VPA+YXQNG组大鼠颞叶和海马Cav3.2表达低于VPA组,差异均有统计学意义(P＜0.05). 结论 YXQNG和VPA联用能降低癫痫大鼠发作级别、改善认知功能、减少脑部异常放电并降低脑组织Cav3.2水平,有抗癫痫和脑保护作用.

Abstract：

Objective To explore the effet of Yangxue Qingnao granule (YXQNG) on seizures and cognition function of pentylenetetrazole (PTZ)-kindled chronic epileptic rats models, expression of Cav3.2 in the hippocampus and the temporal lobe of these rats, and EEG features of the rats. Methods Forty healthy adult male SD rats were equally divided into 4 groups at random: PTZ group, VPA treatment group, VPA+YXQNG treatment group, normal saline (NS)-control group (n=10). PTZ solution was intraperitoneally injected for 8 weeks to induce the kindling model in the above 3 groups except the NS-control group. VPA by intragastric administration was given to the rats in the VPA treatment group 1 h before PTZ injection; YXQNG and VPA by intragastric administration were given to the rats in the VPA+YXQNG treatment group 1.5 h before PTZ injection. Behavioral changes of the rats were observed 8 weeks after PTZ injection; accuracy rate of response of the rats were examined by electric maze test;EEG was performed; and the expression ofT-type Ca2+ channel protein (Cav3.2) in the temporal lobe and hippocampus was detected by immunohistochemical staining. Results Rats in the PTZ group appeared grade Ⅳ or Ⅴ seizures for 3 consecutive d, and rats in the VPA treatment group, VPA+YXQNG treatment group appeared grade 0-Ⅱ seizures. The accuracy rate of response of the rats in the VPA+YXQNG treatment group was significantly higher than that in the PTZ group (P＜0.05). EEG indicated that paradoxical discharge was noted in rats of PTZ group when seizures appeared, and the total power at the time was obviously higher than that before PTZ injection (P＜0.05). The D-value of total power of EEG in rats of the VPA treatment group and VPA+YXQNG treatment group before and after treatment was significantly higher than that in the PTZ group (P＜0.05). And the level of Cav3.2 in the temporal and hippocampus in rats of the VPA treatment group and VPA+YXQNG treatment group was significantly lower than that in the PTZ group (P＜0.05); as compared with that in the VPA treatment group, the expression of Cav3.2 in the temporal and hippocampus in rats of the VPA+YXQNG treatment group was significantly reduced (P＜0.05). Conclusion The combination use of YXQNG and VPA can decrease the seizure stage, the paradoxical discharge of the brain and the level of Cav3.2 in brain tissue,and improve the cognitive function of the PTZ-kindled rats, indicating that using VAP and YXQNG simultaneously can treat epileptic seizure and protect the neurons.     

Microdialysis Study of the Neuropharmacokinetics of Phenytoin in Rat Hippocampus and Frontal Cortex

Summary: Acute administration of phenytoin (PHT) is used in the treatment of status epilepticus, yet little is known about the neuropharmacokinetics of PHT in brain extracellular fluid (ECF), the pharmacodynamically relevant compartment. To characterize the neuropharmacokinetics of brain ECF PHT we implanted microdialysis probes in rat hippocampus and frontal cortex and placed a catheter in the internal jugular vein. PHT (50 or 100 mag intraperitoneally, i.p.) was then administered, and microdialysate and serum samples were collected. PHT was rapidly absorbed, with a time to maximum concentration (T_max) of -20 min for serum concentrations. PHT rapidly entered the brain ECF compartment, with T _max values similar to those of serum. In brain ECF, PHT concentrations then plateaued for 40–60 min despite decreasing serum concentrations. The area under the brain ECF concentration-time curve (AUC) was higher in hippocampus than frontal cortex. The possible mechanisms for these observations include entry of PHT into specific brain areas both across capillaries and through the cerebrospinal fluid (CSF), extensive binding of PHT in brain white matter, and differing blood flow in different brain regions.     

Simultaneous and Continuous Measurement of Free Concentration of Valproate in Blood and Extracellular Space of Rat Cerebral Cortex

Summary: Free concentration of valproate (VPA) was measured simultaneously and continuously in blood and in the extracellular space of cerebral cortex of rats by VPA-selective microelectrodes. Constant amounts of VPA were injected into the femoral vein with differing duration of injection. Immediately after drug application, the concentration of free VPA in blood and brain increased to a peak value, the degree of which increased with the speed of injection. Ten to 15 min after VPA injection, a plateau value was reached. This plateau value was equal in the extracellular space of cortex and in blood. The data indicate that VPA can "freely" cross the blood-brain barrier (BBB).     

Valproate suppresses status epilepticus induced by 4-aminopyridine in CA1 hippocampus region

PURPOSE: We investigated the effects of valproate (VPA) on an in vivo model of status epilepticus (SE) induced by intrahippocampal application of 4-aminopyridine (4-AP). METHODS: To induce continuous epileptiform activity without a clinical component, 4-AP (100 mM) was slowly injected in the hippocampus of adult rats. Extracellular field potential from the CA1 region of the rat hippocampus was recorded to assess abnormal epileptiform activity. Once the SE seizures were induced by 4-AP, the test drug was injected. In some experiments to test the ability of a drug to prevent the induction of SE, the drug was administered before 4-AP injection. RESULTS: Intrahippocampal injection of 4-AP induced continuous epileptic activity without a clinical component that lasted >60 min. The intravenous injection of 400-600 mg/kg VPA rapidly (approximately 100 s) abolished the SE, and this effect persisted for >/=4 h in our experimental model. The intravenous injection of 100-300 mg/kg VPA did not abolish previously induced SE, but prevented the appearance of SE when applied before the induction of SE. The intravenous injection of 80 mg/kg phenytoin or carbamazepine did not abolish or prevent SE. CONCLUSIONS: We conclude that 4-AP-induced SE was suppressed by VPA at 400-600 mg/kg, whereas minor doses (100-300 mg/kg) only prevent the 4-AP-induced SE. Present results suggest the revisiting of VPA as a useful drug for the treatment of SE.     

The pharmacokinetic inter-relationship of tiagabine in blood, cerebrospinal fluid and brain extracellular fluid (frontal cortex and hippocampus)

PURPOSE: Tiagabine is a unique antiepileptic drug with a novel mechanism of action. Whilst some limited data are available as to the peripheral blood pharmacokinetics of tiagabine, data regarding the kinetics of tiagabine in the central brain compartment are very limited. We therefore sought to investigate serum, cerebrospinal fluid (CSF) and frontal cortex and hippocampal extracellular fluid (ECF) kinetic inter-relationship of tiagabine in a freely moving rat model. METHODS: Adult male rats were implanted with either a jugular vein catheter and a cisterna magna catheter for blood and CSF sampling, respectively, or a blood catheter and a microdialysis probe in the hippocampus and frontal cortex (for ECF sampling). Tiagabine was administered intraperitoneal (i.p.) at 20 or 40 mg/kg and blood, CSF and ECF were collected at timed intervals for the measurement of tiagabine concentrations by high performance liquid chromatography. RESULTS: Tiagabine concentrations in blood and CSF rose linearly and dose-dependently and time to maximum concentration (Tmax) was 15 and 29 min, respectively. Mean CSF/serum tiagabine concentration ratios (range, 0.008-0.01) were much smaller than the mean free/total tiagabine concentration ratios in serum (0.045 +/- 0.003). Entry of tiagabine into brain ECF (frontal cortex and hippocampus) was rapid with Tmax values of 31-46 min. Distribution of tiagabine in brain was not brain region specific with values in the frontal cortex and hippocampus being indistinguishable. Whilst elimination from CSF was comparable to that of serum, half-life (t(1/2)) values in ECF were three times longer. CONCLUSIONS: Tiagabine is associated with linear kinetic characteristics and with rapid brain penetration. However, CSF concentrations are not reflective of free non-protein-bound concentrations in serum. The observation that tiagabine elimination from the brain is threefold slower than that seen in blood, may explain as to the relatively long duration of action of tiagabine.     

Long‐lasting pro‐ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology

Purpose: Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT). Methods: Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)‐albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC‐albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)‐1β. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures. Key Findings: The hippocampal serum albumin concentration 24 h post‐SE was 0.76 ± 0.21 μm . Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 μg/4 μl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)‐positive cells, but not in glial fibrillary acidic protein (GFAP)‐positive astrocytes or CR‐3 complement receptor (OX‐42)‐positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL‐1β in GFAP‐positive astrocytes and a concomitant tissue down‐regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed. Significance: Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long‐term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin‐induced astrocytes dysfunction and the associated induction of proinflammatory molecules.     

丙戊酸钠对慢性酒中毒模型大鼠学习记忆损害的影响

目的探讨慢性酒中毒对大鼠学习记忆的影响及丙戊酸钠(VPA)的干预效应及其可能机制。方法将56只SD大鼠随机分为酒中毒模型组、VPA干预组、VPA对照组和正常对照组。以乙醇浓度梯度递增式的方式灌胃8周制作慢性酒中毒模型,而第5周始酒中毒模型组腹腔注射生理盐水,VPA干预组于第5～8周腹腔注射VPA,VPA对照组灌注等体积的生理盐水4周后第5～8周给予VPA。8周后每组随机选取7只采用Morris水迷宫和Y迷宫检测大鼠的学习记忆功能,其余7只用Westernblot检测海马脑源性神经营养因子(BDNF)蛋白的表达含量。结果与正常对照组相比,酒中毒模型组大鼠水迷宫的逃避潜伏期显著延长(P0.05),空间探索次数显著减少(P0.05);Y迷宫中2d的错误次数显著增加(P0.01);海马BDNF含量下降(P0.05)。与酒中毒模型组相比,VPA干预组大鼠的行为学成绩均得到改善(P0.01),海马BDNF含量显著增加(P0.01),与正常对照组的差异无统计学意义(P0.05)。VPA对照组与正常对照组各项指标的差异均无统计学意义(P0.05)。结论慢性酒中毒可以导致大鼠学习记忆障碍,而VPA对酒精诱导的学习记忆损害有干预作用,海马BDNF表达增加可能是其作用机制之一。     

多药转运蛋白对癫痫大鼠脑内奥卡西平浓度的影响

目的 观察多药转运蛋白家族的成员P-糖蛋白(P-glycoprotein,PGP)和多药耐药蛋白(multi-drug re-sistance associated protein,MRP)对匹罗卡品慢性癫痫大鼠模型海马内神经元细胞外液中奥卡西平浓度的影响,证明奥卡西平是否为PGP和MRP的底物,探讨PGP和MRP参与难治疗性癫痫耐药的机制.方法 建立匹罗卡品慢性癫痫动物模型,将32只大鼠分为对照组、模型组、维拉帕米干预组、丙磺舒干预组(每组8只),于腹腔注射奥卡西平(80 mg/kg)后30、60、90、120、150 min,通过微透析及高效液相色谱技术,检测大鼠海马神经元细胞外液中的药物浓度.结果 维拉帕米干预后,癫痫大鼠海马细胞外液中奥卡丙平的浓度于给药后90～120 min(1.26±0.09、0.93±0.10)明显高于模型组(0.87±0.06、0.66 4±0.04),两组比较有统计学差异(P<0.05);丙磺舒干预后60～150 min,大鼠海马内细胞外液中奥卡两平的浓度(1.07±0.11、1.32±0.13、1.02±0.10、0.87±0.08)显著高于模型组(0.81±0.08、0.87±0.06、0.66±0.04、0.58±0.06)(P<0.05).结论 奥卡西平是PGP和MRP的底物,PGP和MRP能够选择性的将奥卡西平泵出血脑屏障外,降低癫痫病灶内的药物浓度,上述机制可能参与了难治性癫痫患者对奥卡西平产生耐药.