AG490对氯化锂-匹罗卡品致癫癎大鼠星形胶质细胞活化及癫癎的影响

目的 探讨匹罗卡品致癫癎大鼠海马肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达、星形胶质细胞活化及JAK/STAT信号转导通路在颞叶癫癎发作中的作用.方法 应用腹腔注射氯化锂-匹罗卡品(PILO)的方法建立颞叶癫痫 间模型,应用JAK/STAT信号转导通路特异性抑制剂AG490进行腹腔注射建立干预模型组;用Western blotting方法测定大鼠海马匀浆后TNF-α表达水平的变化,用免疫荧光观察AG-490阻滞JAK/STAT通路后对大鼠海马TNF-α表达水平与星形胶质细胞活化及癫痫 间发作的影响.结果 (1)PILO致癫痫 间模型组有80.00%(32/40)的大鼠达Racine分级Ⅳ级以上发作;10.00%(4/40)的雄性大鼠死亡;10.00%(4/40)的雄性大鼠未达Racine分级Ⅳ级发作,模型成功率为80.00%;1月后有自发发作者8只(25.00% 8/32).AG490干预组雄性大鼠Ⅳ级以上发作的发生率为75.00%(30/40);病死率为7.50%(3/40);17.50%(7/40)的雄性大鼠未达Ⅳ 级发作,模型成功率为75%;1月后有自发发作者1只(3.3% 1/30),AG490组Ⅳ级及以上发作及自发发作数与PILO致癫痫 间模型组比较,有显著性差异(P<0.05);(2)PILO致癫癎模型组TNF-α表达水平较对照组开始增高,AG490干预组TNF-α表达水平均较 PILO致癫癎模型组明显降低(P<0.05);(3)PILO癫痫 间模型组可见阳性颗粒在星形胶质细胞内的空间分布,AG490腹腔注射建立干预后阳性颗粒的星形胶质细胞明显减少(P<0.05).结论 癫痫 间发作后TNF-α表达水平明显增高,提示星形胶质细胞增生.AG490可阻断JAK/STAT信号转导通路进而抑制星形胶质细胞活化,同时也可以影响癫痫 间大鼠的行为学变化,提示AG490对癫癎的发作可能有一定的抑制作用,其机制可能与其抑制星形胶质细胞增生有关.     

STAT3在匹罗卡品致大鼠星形胶质细胞增生中的作用

目的探讨致状态下大鼠海马内信号转导与转录激活因子3(STAT3)与星形胶质细胞增生的关系。方法匹罗卡品(PILO)腹腔注射建立大鼠颞叶癫模型,免疫组织化学方法观察阻滞JAK/STAT通路前后大鼠海马p-STAT3与胶质纤维酸性蛋白(GFAP)阳性细胞的表达规律,双重免疫荧光方法观察p-STAT3与GFAP阳性细胞的关系。结果癫发作3h(SE3h)时即出现STAT3在海马内被激活,SE3d时达高峰,之后渐降低,至SE30d时仍维持在较正常时略高的水平上;GFAP阳性细胞数的变化规律与之类似。预先用AG490阻断STAT3通路后,海马区p-STAT3及GFAP阳性细胞数均明显减少。双重免疫荧光结果发现p-STAT3阳性胞核位于GFAP阳性细胞胞浆中。结论匹罗卡品导致的癫伴有大鼠海马星形胶质细胞内STAT3的激活,STAT3的活化可能促进星形胶质细胞的反应性增生。     

STAT3在匹罗卡品致(癎)大鼠星形胶质细胞增生中的作用

目的 探讨致(癎)状态下大鼠海马内信号转导与转录激活因子3(STA3)与星形胶质细胞增生的关系.方法 匹罗卡品(PILO)腹腔注射建立大鼠颞叶癫(癎)模型,免疫组织化学方法观察阻滞JAK/STAT通路前后大鼠海马p-STAT3与胶质纤维酸性蛋白(GFAP)阳性细胞的表达规律,双重免疫荧光方法观察p-STAT3与GFAP阳性细胞的关系.结果 癫(癎)发作3 h(SE 3 h)时即出现STAT3在海马内被激活,SE 3 d时达高峰,之后渐降低,至SE 30 d时仍维持在较正常时略高的水平上;GFAP阳性细胞数的变化规律与之类似.预先用AG490阻断STAT3通路后,海马区p-STAT3乃及GFAP阳性细胞数均明显减少.双重免疫荧光结果发现p-STAT3阳性胞核位于GFAP阳性细胞胞浆中.结论 匹罗卡品导致的癫(癎)伴有大鼠海马星形胶质细胞内STAT3的激活,STAT3的活化可能促进星形胶质细胞的反应性增生.     

锂-匹罗卡品致痫大鼠海马胶质纤维酸性蛋白的表达及意义

目的观察其海马经HE染色后组织病理学、胶质纤维酸性蛋白免疫反应阳性表达细胞在LPS中各观察时间点海马CA1、CA3、齿状回的表达,探讨其致机制。方法锂-匹罗卡品急性诱导SD癫痫持续状态模型鼠形成后,采用免疫组化和图像分析方法观察海马HE染色组织病理学、胶质纤维酸性蛋白免疫反应阳性表达细胞。结果模型组各时间点海马细胞形态出现病理性改变,部分细胞脱失,胞浆浓缩,胞核固缩深染;胶质纤维酸性蛋白免疫反应阳性表达细胞亦显著上调（P〈0.05）。结论Pilo诱导SD大鼠癫痫发作后存在显著的海马神经元结构和胶质细胞的损伤,以胶质细胞损伤更显著,胶质纤维酸性蛋白持续高表达可能是这种功能异常的胶质细胞增生的重要原因,也可能是锂-匹罗卡品致癫痫发作的重要因素之一。     

氯化锂-匹罗卡品致痫大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致     

匹罗卡品癫幼鼠模型B1与B2激肽受体表达研究

目的检测B1及B2激肽受体在匹罗卡品癫幼鼠模型中表达的变化并探讨其作用机制。方法健康、雄性幼年(3周)SD大鼠35只,随机分为空白对照组5只;癫模型组15只,采用匹罗卡品法制作癫模型,分为急性期组、静止期组、慢性期组3亚组,每亚组5只;生理盐水对照组(盐水组)15只,与匹罗卡品实验组大鼠相同时间点给予腹腔注射盐水,分为与实验癫组各时间点相对应的盐水6h组、盐水5d组、盐水60d组3个亚组,每亚组5只。各组于相应时间点处死动物取海马标本,应用逆转录聚合酶链反应(RT-PCR)方法检测脑组织海马区的B1及B2激肽受体的表达变化,并相互比较。结果与盐水6h组、盐水5d组比较,急性期组、静止期组的B1激肽受体mRNA表达显著上调,差异具有统计学意义(P<0.05),慢性期与生理盐水60d组间比较差异无统计学意义(P>0.05);与盐水60d组比较,在实验癫模型各亚组B2激肽受体mRNA表达均显著上调,差异具有统计学意义(P<0.05);盐水各亚组与空白对照组相比,B1、B2激肽受体mRNA表达差异无统计学意义(P>0.05)。结论B1与B2激肽受体mRNA表达失衡在癫的发生、发展过程中起重要作用。     

杏仁核电点燃癫(痫)模型与氯化锂-匹罗卡品大鼠癫(痫)模型的对比研究

目的:探讨不同类型癫(痫)大鼠在急性期和慢性期反复自发性发作时、发作前后脑电图(EEG)相应指标改变以及行为学改变.方法:选取雄性SD大鼠40只,随机分为A、B、C、D4组.A组10只为杏仁核电点燃癫(痫)模型(不作电刺激)对照组;B组:10只,为制作杏仁核电刺激点燃癫(痫)模型组;C组:10只,氯化锂-匹罗卡品癫(痫)模型(只注生理盐水)对照组;D组:10只,制作氯化锂-匹罗卡品癫(痫)模型组.结果:D组慢性期发作时EEG频率与急性期频率相比差异有统计学意义(P＜0.05),与B组各时期相比差异有统计学意义(P＜0.05);D组慢性期发作后3 min发作频率与B组急性期相比差异有统计学意义(P＜0.05);A组与C组在频率、波幅方面比较差异无统计学意义(P＞0.05).结论:B、D两种不同类型的癫(痫)大鼠不同时期EEG和行为学都会发生相应的改变.     

氯化锂-匹罗卡品致(疒间)大鼠脑髓鞘转录因子的表达及其意义

目的探讨氯化锂-匹罗卡品致疒间大鼠脑髓鞘转录因子1(MyT1)的表达及其意义.方法给SD大鼠先后腹腔注射氯化锂、匹罗卡品,制成癫疒间动物模型;用免疫荧光组化法检测癫疒间大鼠癫疒间发作后不同时间大脑皮质和海马CA1区MyT1阳性细胞数.结果与对照组相比,癫疒间后1 d组大鼠海马CA1区MyT1阳性细胞数显著减少(P＜0.05),癫疒间后其他各时间组大鼠脑皮质和海马CA1区MyT1阳性细胞数均有明显的增加,其中癫疒间后7 d组MyT1阳性细胞数最多(P＜0.01,P＜0.05).结论氯化锂-匹罗卡品致疒间大鼠早期大脑MyT1表达增加,并有时程性变化.     

氯化锂-匹罗卡品致痫大鼠海马EphA5及ephrinA3的表达及意义

目的探讨颞叶癫痫发作后海马EphA5及ephrinA3基因的表达变化和轴突出芽的关系。方法建立氯化锂-匹罗卡品颞叶癫痫大鼠模型,利用原位杂交方法检测致痫后12h、24h、7d、15d、30d、60d海马CA3区、CA1区EphA5及ephrinA3 mRNA的表达,快速Golgi染色观察CA1区的轴突出芽。结果致痫后,EphA5 mRNA在CA3区表达下调,ephrinA3 mRNA在CA1区表达下调,均在7d降至最低点,与对照组相比差异有显著意义(P<0.01),此后逐渐回升,但15d时仍低于对照组(P<0.05),在30d和60d与对照组相比差异无统计学意义(P>0.05)。快速Golgi染色显示,对照组大鼠CA1区轴突走行正常,匹罗卡品致大鼠SE后7dCA1区锥体细胞层出现显著增多的轴突染色。结论CA3区的EphA5和CA1区的ephrinA3的表达下调可能与CA1区的轴突出芽、突触重建有关。     

锂-匹罗卡品致癫大鼠早期大脑皮质MyT1 mRNA表达变化

目的 探讨氟化锂-匹罗卡品致癫大鼠脑髓鞘转录因子1基因表达变化及其意义。方法 以氯化锂、匹罗卡品对雄性成年SD大鼠先后腹腔注射,制成癫痫持续状态动物模型;采用5′末端标记地高辛的寡核苷酸探针荧光原位核酸分子杂交检测癫性发作后早期大鼠大脑皮质MyT1 mRNA阳性细胞数量。结果 与对照组相比,癫痫后1d组大鼠脑皮质MyT1 mRNA阳性细胞数减少（P＜0.05）;其他各组大鼠脑皮质MyT1 mRNA阳性细胞数都有明显的增加,其中癫痫后7d和14d组MyT1mRNA阳性细胞数都有非常显著的增加（P＜0.05,P＜0.01）。结论 氯化锂-匹罗卡品致癫大鼠早期大脑MyT1 mRNA表达增加,并有时程性变化,提示与早期脑损伤修复有关。     

改良锂-匹罗卡品大鼠癫痫模型的制作及性别特点

目的 建立稳定可靠的颞叶癫痫动物模型，摸索并证实建模规律。方法 选用56只健康SD大鼠，雌雄近半，随机分配为试验组48只，对照组8只。氯化锂3mmol／kg腹腔注射预处理：18-20h后首剂注射匹罗卡品30mg／kg，此后每隔30分钟注射10mg／kg追加剂量，极量为60mg／kg，直至出现癫痫持续状态（SE）；SE1h后地西泮10mg／kg止痉，2h后注射生理盐水5mL／只，2／d．连续2d；SE后次日起灌胃鸡蛋牛奶糊7mL／只，2／d，连续6d；观察大鼠行为学改变。对照组大鼠除用等量生理盐水代替匹罗卡品外，其余处理措施均相同。结果大鼠成功点燃44只，病死10只．造模总成功只数为34。雌雄大鼠点燃只数相近．雄鼠病死只数低于雌鼠。结论中等剂量首剂．不超过3次小剂量累加腹腔注射匹罗卡品造模简易有效，可操作性强，效应匀齐。改良护理方法可明显降低病死率．雄鼠较雌鼠更适于造模。     

Motor map expansion in the pilocarpine model of temporal lobe epilepsy is dependent on seizure severity and rat strain

Functional alterations in movement representations (motor maps) have been observed in some people with epilepsy and, under experimental control, electrically-kindled seizures in rats also result in persistently larger motor maps. To determine if a single event of status epilepticus and its latent consequences can affect motor map expression, we assessed forelimb motor maps in rats using the pilocarpine model of temporal lobe epilepsy. We examined both pilocarpine-induced seizures, and status epilepticus (SE) in two strains that differ in their propensity for epileptogenesis; Wistar and Long-Evans. Pilocarpine was administered intraperitoneally at dosages that resulted in equivalent proportions of seizures, SE, and survival in both strains. Rats from both strains were given saline injections as a control. Diazepam was administered to all rats to attenuate seizure activity and promote survival. All rats had high-resolution movement representations derived using standard intracortical microstimulation methodologies at 48 h, 1 week, or 3 weeks following treatment. Pilocarpine-induced seizures only gave rise to motor map enlargement in Wistar rats, which also showed interictal spiking, and only at 3 weeks post-treatment indicating altered motor map expression in this strain following a latent or maturational period. Pilocarpine-induced SE yielded larger motor maps at all time points in Wistar rats but only a transient (48 h) map expansion in Long-Evans rats. Our results demonstrate that seizures and SE induced by a convulsant agent alter the functional expression of motor maps that is dependent on seizure severity and a genetic (strain) predisposition to develop epileptiform events.     

Resting and reactive astrocytes express adrenergic receptors in the adult rat brain

Adrenergic receptor subtypes were localized in situ and in cells isolated from the trigeminal motor nucleus and several other brain regions. To study receptor expression in reactive astrocytes, motor neuron degeneration and a glial reaction were induced in the trigeminal motor nucleus by the injection of the toxic lectin Ricin communis into the trigeminal motor root. Autoradiography following incubation of tissue sections in the alpha 1-ligand 125IBE 2254 (HEAT) or the beta-ligand 125Iodocyanopindolol (ICYP) showed a decrease in alpha 1- and an increase in beta-adrenergic receptor binding in the region of neuronal degeneration and gliosis. Glial hypertrophy, rather than hyperplasia, appears to be mainly responsible for the increased beta-binding, since inhibition of mitosis with cytosine arabinofuranoside only partially blocked elevations of beta-adrenergic receptor binding and GFAP immunolabelling in reactive astrocytes. More direct evidence for the expression of adrenergic receptors in normal and reactive astrocytes was obtained by combined autoradiography and immunohistochemistry of cells dissociated from the cerebral cortex, striatum, cerebellum, and trigeminal motor nucleus of adult rats. More than 88% of GFAP-positive astrocytes showed varying densities of beta-adrenergic receptor binding. In each region, the beta 2-subtype was proportionally greater than the beta 1-subtype. Astrocytes also expressed a significant density of alpha 1-receptors. Trigeminal motor neurons did not show beta-receptor binding, but had a density of alpha 1-receptors tenfold greater than astrocytes. A model for the role of astrocytes in adrenergic receptor-mediated modulation of trigeminal motor neuron excitability is discussed.     

Assessment of the seizure susceptibility of Wistar Audiogenic rat to electroshock,pentyleneterazole and pilocarpine

This work evaluates the seizure susceptibility of naïve female Wistar Audiogenic rats (WARs), a genetic model of reflex epilepsy in which seizures are induced by high-intensity sound stimulation (120 dB SPL), to other pro-convulsive stimuli: transauricular electroshock (ES), pentylenetetrazole (PTZ) and pilocarpine (PILO). Normal Wistar rats from the main breeding stock of the Institute of Biological Sciences, UFMG were taken as controls. Electroshock seizures were induced through a pair of ear-clip electrodes (10 mA, at a frequency of 60 Hz, applied for 1 s). In order to test WAR susceptibility to chemically induced seizures, animals were treated either with PTZ (37.5 mg/kg i.p.) or PILO (200, 270 and 300 mg/kg i.p.). Seizure severity was evaluated by appropriate behavioral severity index scales (SI) specific to each epilepsy model and tested for statistical significance using the non-parametric Mann–Whitney Rank Sum test. Results show a significantly greater susceptibility of WARs for ES (SI_WAR=3±3/3, SI_Wistar=1±1/1; median±interquartile range 25%/75%, P<0.01) and PTZ (SI_WAR=4±4/4, SI_Wistar=1±1/4; median±interquartile range 25%/75%, P<0.02), as indicated by significantly higher SI scores and shorter latencies for seizure onset (T_WAR=71±7 s, T_Wistar=94±8 s; P<0.05 Student t-test, mean±S.E.M.). Although PILO also caused higher SI scores in WARs (WAR_{200 mg}=1±1/1, Wistar_{200 mg}=1±1/1; WAR_{270 mg}=1.5±1/2, Wistar_{270 mg}=1±1/1.25; WAR_{300 mg}=9±1/9, Wistar_{300 mg}=4±1.5/7.5; median±interquartile range 25%/75%), statistically significant differences were not observed. In conclusion, our results show that WARs have an inherited broader predisposition for seizures.     

Cyclicity of spontaneous recurrent seizures in pilocarpine model of temporal lobe epilepsy in rat

Pilocarpine administration to rats results in status epilepticus (SE) and after a latency period to the occurrence of spontaneous seizures. The model is commonly used to investigate mechanisms of epileptogenesis as well as the antiepileptic effects of novel compounds. Surprisingly, there have been no video-EEG studies determining the duration of latency period from SE to the appearance of the first spontaneous seizures or the type and frequency of spontaneous seizures at early phase of pilocarpine-induced epilepsy even though such information is critical for design of such studies. To address these questions, we induced SE with pilocarpine in 29 adult male Wistar rats with cortical electrodes. Rats were continuously video-EEG monitored during SE and up to 23 days thereafter. The first spontaneous seizures occurred 7.2+/-3.6 days after SE. During the follow-up, the mean daily seizure frequency was 2.6+/-1.9, the mean seizure duration 47+/-7 s, and the mean behavioral seizure score 3.2+/-0.9. Typically first seizures were partial (score 1-2). Interestingly, spontaneous seizures occurred in clusters with cyclicity, peaking every 5 to 8 days. These data show that in the pilocarpine model of temporal lobe epilepsy the latency period is short. Because many of the early seizures are partial and the seizures occur in clusters, the true phenotype of epilepsy triggered by pilocarpine-induced SE may be difficult to characterize without continuous long-term video-EEG monitoring. Finally, our data suggest that the model can be used for studies aiming at identifying the mechanisms of seizure clustering.     

炎性介质阻断剂对大鼠脑缺血再灌注损伤后神经功能缺损、细胞凋亡及半胱氨酸蛋白酶-3表达的影响

目的探讨炎性介质阻断剂AG490对大鼠局灶性脑缺血再灌注损伤后神经功能缺损、细胞凋亡及半胱氨酸蛋白酶-3(caspase-3)表达的影响。方法雄性SD大鼠被随机分为假手术组、缺血再灌注组、生理盐水组、AG490组;采用大脑中动脉线栓法制作大鼠局灶性脑缺血再灌注模型;AG490组于脑缺血即刻及再灌注后12 h分别腹腔注射AG490 1 mg/kg。再灌注24 h后对各组大鼠进行神经功能缺损评分;利用原位缺口末端标记法(TUNEL法)检测神经细胞凋亡数;应用Western Blot法检测各组脑组织磷酸化酪氨酸蛋白激酶(P-JAK2)、磷酸化信号转导和转录激活因子(P-STAT3)、caspase-3表达。结果与缺血再灌注组及生理盐水组比较,AG490组大鼠神经功能缺损评分明显减低(均P<0.05);凋亡细胞数及P-JAK2、P-STAT3、caspase-3表达明显减少(均P<0.01)。结论AG490可阻断JAK2/STAT3细胞因子信号转导通路,有效抑制caspase-3表达,减轻缺血灌注损伤后神经细胞凋亡,改善神经功能缺损症状。     

Effects of change in dietary folic acid content on pentylenetetrazol seizure threshold in rat

Pentylenetetrazol (PT) eeg thresholds were measured in rats on a folic acid (FA) deficient diet, normal diet, and a diet supplemented with large amounts of folic acid. The intraperitoneal dose per kilogram body weight of PT is significantly higher in the FA deficient group and significantly lower in the FA supplemented group than in rats on a normal diet. This indicates an effect of FA metabolism on PT epilepsy. The Lactobacillus casei minus Streptococcus faecalis activity of the brain is decreased in the FA deficient and increased in the FA supplemented group. The relation between brain folate metabolism and epilepsy is discussed.     

神经生长因子受体trkA在EAE模型中的表达及PG490的作用

目的 通过测定神经生长因子受体trkA在实验性变态反应性脑脊髓炎(EAE)模型中的含量变化以及探讨PG490在其中的作用和作用途径.方法 48只新西兰兔在已建立EAE模型的基础上按随机数字表法分为3组,即模型组、PG490 干预组1(首次发病期给予干预)及PG490干预组2(复发期给予干预).采用ELISA法检测各组动物脑组织中trkA蛋白含量变化,并观察和比较各组临床学评分变化.结果 EAE模型发病是呈缓解复发的过程,干预组其平均临床评分均较模型组明显降低,差异有统计学意义(P<0.05).PG490干预组1 trkA蛋白含量与模型组相比较差异无统计学意义(P>0.05);PG490干预组2 trkA蛋白含量与模型组相比较差异有统计学意义(P<0.05).结论 PG490能够通过调节神经生长因子受体trkA含量减轻EAE模型的临床症状.     

Regional distribution and time-course of calpain activation following kainate-induced seizure activity in adult rat brain

Systemic injection of kainic acid (KA) in adult rat elicits a pattern of neuronal pathology which exhibits several features of human temporal lobe epilepsy. KA-induced seizure activity is accompanied by the activation of the calcium-dependent protease calpain in limbic structures. In the present study, we evaluated the spatio-temporal activation of calpain after the onset of seizure activity by immunohistochemistry using an antibody for the spectrin breakdown product (sbdp) generated by calpain-mediated spectrin proteolysis. In addition, we compared the changes in sbdp immunoreactivity with those in immunoreactivity to subunits of the Glu/AMPA receptors (GluR₁ andGluR₂₃). One hour after seizure onset, sbdp accumulation was observed in selected interneurons in stratum oriens and in the hilus of the dentate gyrus. By 4 h, sbdp immunoreactivity was prominent in dendritic fields of the hippocampus as well as in neurons in thalamus and piriform cortex. By 8 h, sbdp immunoreactivity had disappeared from interneurons but was localized in pyramidal cell bodies in hippocampus. Intense labeling of cell bodies and dendritic fields persisted until 5 days following KA treatment. Changes in GluR subunit immunoreactivity were mirror images of those seen for sbdp. In general, increased sbdp immunoreactivity in dendritic fields was associated with decreased (GluR₁ immunoreactivity. However, increased sbdp immunoreactivity in neuronal perikarya was also associated with increased GluR immunoreactivity. These results indicate that calpain activation following seizure onset exhibits a specific spatio-temporal pattern, with activation in restricted interneurons preceding widespread activation in pyramidal neurons. Calpain activation also precedes neuronal pathology and could thus represent an initial trigger for neuronal pathology. Finally, the results suggest that calpain activation produces rapid alterations in GluR subunit properties which could be involved in the hyperexcitability observed following seizure activity.