Fluoro-Jade C揭示匹罗卡品模型中梨状皮质神经元变性

目的应用Fluoro-Jade C(FJC)染色方法在小鼠匹罗卡品癫痫模型中检测梨状皮质结构中神经元的变性情况,以了解梨状皮质结构在慢性颞叶癫痫发生中的病理变化和癫痫反复发作的神经基础。方法雄性昆明小鼠10只(对照组5只,匹罗卡品处理组5只)。处理组在癫痫持续状态后3d处死处理组小鼠。在梨状皮质水平切制冠状切片,行FJC染色,在荧光显微镜下观察FJC阳性细胞的形态和在梨状皮质中的整体分布情况。结果在处理组,FJC染色的脑切片上可以很清楚地看到呈亮黄绿色荧光的FJC阳性细胞,呈神经元形态,胞体和突起均清晰显示。在梨状皮质和梨状内核内出现大量FJC阳性细胞,而对照组未见。结论在小鼠匹罗卡品癫痫模型中运用FJC染色技术显示梨状皮质内发生了大量神经元变性,此研究有利于更好地理解颞叶癫痫中中枢神经系统所发生的长期病理变化和自发反复发作的癫痫机制。     

Fluoro-Jade C染色在小鼠匹罗卡品癫痫模型中的应用

目的应用Fluoro-Jade C(FJC)染色技术在小鼠匹罗卡品癫痫模型中探测神经元变性,以了解FJC应用价值及其细胞死亡模式。方法雄性昆明小鼠6只:对照组3只;匹罗卡品处理组3只。处理组小鼠在癫痫持续状态后12 h处死,在海马水平切制冠状切片,先行FJC染色,用荧光显微镜观察;之后在切片上行FJC与Hoechst双标。结果处理组,FJC阳性细胞清晰显示,呈神经元形态,对照组未见。双标资料显示FJC(100%)与Hoechst33342双标记。结论小鼠匹罗卡品癫痫模型成功应用FJC染色技术探测了神经元变性,证实此技术在探测神经元变性方面敏感、可靠且简单,并显示此模型中FJC阳性细胞也许主要是凋亡性的。     

拉莫三嗪及丙戊酸钠对锂-匹罗卡品致(癎)大鼠的神经保护作用及其对癫(癎)的治疗作用

目的:观察拉莫三嗪(LTG)及丙戊酸钠(VPA)对锂-匹罗卡品癫(癎)持续状态(SE)大鼠海马锥体细胞、齿状回门区神经元的保护作用及对癫(癎)的治疗作用.方法:用大鼠制作锂-匹罗卡品SE动物模型,分三组:SE对照组,LTG治疗组和VPA治疗组.此三组在SE后2h给予安定阻断(癎)性发作,再分别给予适量生理盐水(NS)、...     

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.     

活化素A对致痫小鼠行为、脑电及海马神经元损伤的影响

目的观察重组人活化素A(rhACT)对匹鲁卡品(PC)致癎小鼠行为、脑电及海马神经元损伤的影响.方法利用脑立体定位手段将rhACT注入侧脑室,1 h后腹腔注射PC.采用发作程度评分、脑电记录观察rhACT对癫癎发作与放电的影响;利用尼氏染色及组织原位凋亡检测观察rhACT对注射PC后24及48h海马神经元损伤的影响.结果预先脑室注射rhACT后PC诱导的癫癎发作显著减轻,癎样放电明显受抑制;24及48h海马神经元未见明显损害.结论rhACT可能有抗K诱导的癫癎发作及保护海马神经元的作用.     

普瑞巴林对慢性颞叶癫癎大鼠海马凋亡调控基因的影响

目的通过观察普瑞巴林对匹罗卡品慢性癫癎大鼠海马区Bcl-2和Bax表达的影响,探讨普瑞巴林治疗癫癎的药理学机制及对大鼠海马神经元的抗凋亡作用。方法采用氯化锂-匹罗卡品化学诱导方法建立慢性颞叶癫癎模型。经腹腔注射普瑞巴林40mg(/kg·d)连续治疗3周,免疫组织化学染色和Western blotting法检测不同处理组大鼠海马区Bcl-2和Bax表达变化。结果与生理盐水对照组比较,模型组大鼠海马区Bcl-2和Bax表达水平显著升高(均P=0.000);与模型组比较,普瑞巴林治疗组大鼠海马区Bcl-2表达水平升高、Bax表达水平降低,组间差异具有统计学意义(均P=0.000)。结论新型抗癫癎药物普瑞巴林可通过降低慢性颞叶癫癎大鼠海马区Bax表达、上调Bcl-2表达而抑制细胞凋亡,发挥神经元保护作用。     

不同化学致癇剂对大鼠癫癇发作和神经元凋亡的影响

目的观察几种不同癫癇持续状态模型发作的特点和海马区的组织病理学改变.方法采用匹罗卡品、锂-匹罗卡品和戊四氮腹腔注射制成大鼠癫持续状态模型,以TUNEL方法标记DNA片段,原位检测海马CA1和CA3区的凋亡神经细胞.结果海马CA1和CA3区神经细胞凋亡数,PILO组多于Li-PILO组和PTZ组,差异有显著性(P《0.05).结论匹罗卡品、锂-匹罗卡品和戊四氮均可诱发大鼠癫持续状态,并导致海马神经元损伤.     

匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素和微清蛋白mRNA表达研究

目的 观察匹罗卡品致(癎)大鼠海马γ-氨基丁酸能中间神经元生长抑素(SS)mRNA和微清蛋白(PV)mRNA表达水平变化,拟从基因水平探讨其表达阳性γ-氮基丁酸能中间神经元在颞叶癫(癎)发生发展中的作用.方法 建立匹罗卡品致(癎)大鼠模型,采用原位杂交法检测各观察时间点海马SSmRNA和PVmRNA表达阳性神经元数目.结果 模型组大鼠海马各区γ-氨基丁酸能中间神经元SSmRNA表达水平均于出现癫(癎)持续状态后3d降低最为显著(均P=0.000),随后逐渐升高;至发病后60d,海马CA3区SSmRNA表达水平高于对照组(t=1.021,P=0.005),海马门区(t=3.211,P=0.009)和CA1区(t=1.902,P=0.048)则仍低于对照组.模型组大鼠海马门区γ-氨基丁酸能中间神经元PVmRNA表达水平于出现癫(癎)持续状态后6h开始降低,至发病后60d降低最为显著(均P=0.000);海马CA1区PVmRNA表达水平于发病后3d降低最为显著(均P=0.000),随后逐渐升高但仍低于对照组(江2.216,P=0.048);癫(癎)持续状态早期,海马CA3区PVmRNA表达水平无明显变化,至发病后7d逐渐升高且高于对照组(t=1.021,P=0.005).结论 γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的下调可能在颞叶癫(癎)的发生中起重要作用,至慢性期γ-氨基丁酸能中间神经元SSmRNA和PVmRNA表达水平的恢复或上调可能与颞叶癫(癎)的发展或修复有关.γ-氨基丁酸能中间神经元数目的 变化,部分是由于其标志物mRNA表达水平的调节所致,并非神经元数目变化的唯一因素.     

锂-匹罗卡品致癎大鼠认知功能与海马苔藓纤维发芽和5-HT表达的变化

目的探讨癫癎发作后海马结构和海马5-羟色胺(5-HT)水平的变化,以及与认知功能改变的关系。方法采用锂-匹罗卡品复制大鼠癫癎模型,观察大鼠癫癎发作后大鼠海马组织结构、海马组织中5-HT水平的变化,并用Morris水迷宫观察大鼠认知功能的改变。结果 (1)大鼠癫癎发作后海马Timms染色显示苔藓纤维发芽(MFS)明显,半定量评分显示评分明显增高;(2)癫癎发作后海马5-HT免疫组织化学染色显示海马组织中5-HT神经元数量以及5-HT水平均明显减少;(3)癫癎发作后大鼠认知功能明显受到影响,大鼠寻找目标的潜伏期明显延长、游泳轨迹发生明显变化,以及规定时间内穿越平台次数减少。结论大鼠癫癎发作后认知功能明显下降,其原因可能与海马5-HT神经元数量以及5-HT水平的减少有关;癫癎的发作可能与海马苔藓纤维发芽有关。     

大鼠杏仁核点燃癫痫模型丘脑中线核群中c－fos蛋白的表达

目的探讨丘脑中线核群与颞叶癫癎的病理生理联系.方法采用免疫组化方法,在杏仁核点燃的大鼠癫癎模型上,观察丘脑有关核群c-fos蛋白表达细胞分布情况,用c-fos阳性细胞密度作定量研究指标.结果在杏仁核点燃的大鼠,癫癎发作达到4～5级时,丘脑中线核群的室旁核、菱形核、连结核以及丘脑背内侧核均有显著的c-fos蛋白表达.结论丘脑中线核群参与颞叶癫癎的传播过程.     

Different patterns of neuronal activation and neurodegeneration in the thalamus and cortex of epilepsy‐resistant Proechimys rats versus Wistar rats after pilocarpine‐induced protracted seizures

Purpose: To analyze cellular mechanisms of limbic‐seizure suppression, the response to pilocarpine‐induced seizures was investigated in cortex and thalamus, comparing epilepsy‐resistant rats Proechimys guyannensis with Wistar rats. Methods: Fos immunoreactivity revealing neuronal activation, and degenerating neurons labeled by Fluoro‐Jade B (FJB) histochemistry were analyzed on the first day after onset of seizures lasting 3 h. Subpopulations of γ‐aminobutyric acid (GABA)ergic cells were characterized with double Fos‐parvalbumin immunohistochemistry. Results: In both cortex and thalamus, degenerating neurons were much fewer in Proechimys than Wistar rats. Fos persisted at high levels at 24 h only in the Proechimys thalamus and cortex, especially in layer VI where corticothalamic neurons reside. In the parietal cortex, about 50% of parvalbumin‐containing interneurons at 8 h, and 10–20% at 24 h, were Fos‐positive in Wistar rats, but in Proechimys, Fos was expressed in almost all parvalbumin‐containing interneurons at 8 h and dropped at 24 h. Fos positivity in cingulate cortex interneurons was similar in both species. In the Wistar rat thalamus, Fos was induced in medial and midline nuclei up to 8 h, when <30% of reticular nucleus cells were Fos‐positive, and then decreased, with no relationship with cell loss, evaluated in Nissl‐stained sections. In Proechimys, almost all reticular nucleus neurons were Fos‐positive at 24 h. Discussion: At variance with laboratory rats, pilocarpine‐induced protracted seizures elicit in Proechimys limited neuronal death, and marked and long‐lasting Fos induction in excitatory and inhibitory cortical and thalamic cell subsets. The findings implicate intrathalamic and intracortical regulation, and circuits linking thalamus and cortex in limbic seizure suppression leading to epilepsy resistance.     

Seizures produced by pilocarpine in mice: A behavioral, electroencephalographic and morphological analysis

Increasing doses of pilocarpine, 100-400 mg/kg, were given intraperitoneally to mice and the resulting behavioral, electroencephalographic and neuropathological alterations were studied. No behavioral phenomena were observed in mice treated with the lowest dose of pilocarpine. Occasional tremor and myoclonus of hindlimbs were found in animals which received pilocarpine in a dose of 200 mg/kg. At doses of 300, 325 and 350 mg/kg, pilocarpine produced a sequence of behavioral alterations including staring spells, limbic gustatory automatisms and motor limbic seizures that developed over 15-30 min and built up progressively into a limbic status epilepticus lasting for several hours. The highest dose of pilocarpine, 400 mg/kg, was generally lethal to mice. Pilocarpine produced both interictal and ictal epileptiform activity in the electroencephalogram (EEG). The earliest EEG alterations appeared in the hippocampus and then spread to cortical areas. EEG seizures started 10-15 min after injection of large doses of pilocarpine, 300-350 mg/kg. Ictal periods lasted for 1-2 min, recurred every 5-10 min and were followed by periods of depression of the EEG activity. By 30-45 min paroxysmal activity resulted in a status epilepticus. Examination of frontal forebrain sections with light microscopy revealed a widespread damage to several brain regions including the hippocampus, amygdala, thalamus, olfactory cortex, neocortex and substantia nigra. Scopolamine, 10 mg/kg, and diazepam, 10 mg/kg, prevented the development of convulsive activity and brain damage produced by pilocarpine. The results emphasize that excessive and sustained stimulation of cholinergic receptors can lead to seizures and seizure-related brain damage in mice. It is proposed that systemic pilocarpine in mice provides a useful animal model for studying mechanisms of and therapeutic approaches to temporal lobe epilepsy.     

Malnutrition in infancy as a susceptibility factor for temporal lobe epilepsy in adulthood induced by the pilocarpine experimental model

Malnutrition during the earliest stages of life may result in innumerable brain problems. Moreover, this condition could increase the chances of developing neurological diseases, such as epilepsy. We analyzed the effects of early-life malnutrition on susceptibility to epileptic seizures induced by the pilocarpine model of epilepsy. Wistar rat pups were kept on a starvation regimen from day 1 to day 21 after birth. At day 60, 16 animals (8 = well-nourished; 8 = malnourished) were exposed to the pilocarpine experimental model of epilepsy. Age-matched well-nourished (n = 8) and malnourished (n = 8) rats were used as controls. Animals were video-monitored over 9 weeks. The following behavioral parameters were evaluated: first seizure threshold (acute period of the pilocarpine model); status epilepticus (SE) latency; first spontaneous seizure latency (silent period), and spontaneous seizure frequency during the chronic phase. The cell and mossy fiber sprouting (MFS) density were evaluated in the hippocampal formation. Our results showed that the malnourished animals required a lower pilocarpine dose in order to develop SE (200 mg/kg), lower latency to reach SE, less time for the first spontaneous seizure and higher seizure frequency, when compared to well-nourished pilocarpine rats. Histopathological findings revealed a significant cell density reduction in the CA1 region and intense MFS among the malnourished animals. Our data indicate that early malnutrition greatly influences susceptibility to seizures and behavioral manifestations in adult life. These findings suggest that malnutrition in infancy reduces the threshold for epilepsy and promotes alterations in the brain that persist into adult life.     

The midline thalamus: alterations and a potential role in limbic epilepsy

PURPOSE: In limbic or mesial temporal lobe epilepsy, much attention has been given to specific regions or cell populations (e.g., the hippocampus or dentate granule cells). Epileptic seizures may involve broader changes in neural circuits, and evidence suggests that subcortical regions may play a role. In this study we examined the midline thalamic regions for involvement in limbic seizures, changes in anatomy and physiology, and the potential role for this region in limbic seizures and epilepsy. METHODS: Using two rat models for limbic epilepsy (hippocampal kindled and chronic spontaneous limbic epilepsy) we examined the midline thalamus for evidence of involvement in seizure activity, alterations in structure, changes in the basic in vitro physiology of the thalamic neurons. We also explored how this region may influence limbic seizures. RESULTS: The midline thalamus was consistently involved with seizure activity from the onset, and there was significant neuronal loss in the medial dorsal and reuniens/rhomboid nuclei. In addition, thalamic neurons had changes in synaptically mediated and voltage-gated responses. Infusion of lidocaine into the midline thalamus significantly shortened afterdischarge duration. CONCLUSIONS: These observations suggest that this thalamic region is part of the neural circuitry of limbic epilepsy and may play a significant role in seizure modulation. Local neuronal changes can enhance the excitability of the thalamolimbic circuits.     

The contribution of the lateral posterior and anteroventral thalamic nuclei on spontaneous recurrent seizures in the pilocarpine model of epilepsy

The pilocarpine model of epilepsy in rats is characterised by the occurrence of spontaneous seizures (SRSs) during the chronic period that recur 2-3 times per week during the whole animal life. In a previous study on brain metabolism during the chronic period of the pilocarpine model it was possible to observe that, among several brain structures, the lateral posterior thalamic nuclei (LP) showed a strikingly increased metabolism. Some evidences suggest that the LP can participate in an inhibitory control system involved in the propagation of the seizures. The aim of the present study was to verify the role of LP in the expression and frequency of spontaneous seizures observed in the pilocarpine model. Ten adult male rats presenting SRSs were monitored for behavioural events by video system one month before and one month after LP ibotenic acid lesion. Another group of chronic epileptic rats (n=10) had the anteroventral thalamic nuclei (AV) lesioned by ibotenic acid. After the surgical procedure, the animals were sacrified and the brains were processed for histological analysis by the Nissl method. The LP group seizure frequency was 3.1+/-1.9 before ibotenic acid injection and showed an increase (16.3+/-7.2 per week) after LP lesion. No changes in SRSs frequency were observed in the AV group after ibotenic lesion in these nuclei. These results seem to suggest that LP play a role in the seizure circuitry inhibiting the expression of spontaneous seizures in the pilocarpine model.     

Rapamycin has paradoxical effects on S6 phosphorylation in rats with and without seizures

Purpose: Accumulating data have demonstrated that seizures induced by kainate (KA) or pilocarpine activate the mammalian target of rapamycin (mTOR) pathway and that mTOR inhibitor rapamycin can inhibit mTOR activation, which subsequently has potential antiepileptic effects. However, a preliminary study showed a paradoxical exacerbation of increased mTOR pathway activity reflected by S6 phosphorylation when rapamycin was administrated within a short period before KA injection. In the present study, we examined this paradoxical effect of rapamycin in more detail, both in normal rats and KA‐injected animals. Methods: Normal rats or KA‐treated rats pretreated with rapamycin at different time intervals were sacrificed at various time points (1, 3, 6, 10, 15, and 24 h) after rapamycin administration or seizure onset for western blotting analysis. Phosphorylation of mTOR signaling target of Akt, mTOR, Rictor, Raptor, S6K, and S6 were analyzed. Seizure activity was monitored behaviorally and graded according to a modified Racine scale (n = 6 for each time point). Neuronal cell death was detected by Fluoro‐Jade B staining. Key Findings: In normal rats, we found that rapamycin showed the expected dose‐dependent inhibition of S6 phosphorylation 3–24 h after injection, whereas a paradoxical elevation of S6 phosphorylation was observed 1 h after rapamycin. Similarly, pretreatment with rapamycin over 10 h before KA inhibited the KA seizure–induced mTOR activation. In contrast, rapamycin administered 1–6 h before KA caused a paradoxical increase in the KA seizure–induced mTOR activation. Rats pretreated with rapamycin 1 h prior to KA exhibited an increase in severity and duration of seizures and more neuronal cell death as compared to vehicle‐treated groups. In contrast, rapamycin pretreated 10 h prior to KA had no effect on the seizures and decreased neuronal cell death. The paradoxical effect of rapamycin on S6 phosphorylation was correlated with upstream mTOR signaling and was reversed by pretreatment of perifosine, an Akt inhibitor. Significance: These data indicate the complexity of S6 regulation and its effect on epilepsy. Paradoxical effects of rapamycin need to be considered in clinical applications, such as for potential treatment for epilepsy and other neurologic disorders.     

Neuronal and glial pathological changes during epileptogenesis in the mouse pilocarpine model

The rodent pilocarpine model of epilepsy exhibits hippocampal sclerosis and spontaneous seizures and thus resembles human temporal lobe epilepsy. Use of the many available mouse mutants to study this epilepsy model would benefit from a detailed neuropathology study. To identify new features of epileptogenesis, we characterized glial and neuronal pathologies after pilocarpine-induced status epilepticus (SE) in CF1 and C57BL/6 mice focusing on the hippocampus. All CF1 mice showed spontaneous seizures by 17-27 days after SE. By 6 h there was virtually complete loss of hilar neurons, but the extent of pyramidal cell death varied considerably among mice. In the mossy fiber pathway, neuropeptide Y (NPY) was persistently upregulated beginning 1 day after SE; NPY immunoreactivity in the supragranular layer after 31 days indicated mossy fiber sprouting. beta2 microglobulin-positive activated microglia, normally absent in brains without SE, became abundant over 3-31 days in regions of neuronal loss, including the hippocampus and the amygdala. Astrogliosis developed after 10 days in damaged areas. Amyloid precursor protein immunoreactivity in the thalamus at 10 days suggested delayed axonal degeneration. The mortality after pilocarpine injection was very high in C57BL/6 mice from Jackson Laboratories but not those from Charles River, suggesting that mutant mice in the C57BL/6(JAX) strain will be difficult to study in the pilocarpine model, although their neuropathology was similar to CF1 mice. Major neuropathological changes not previously studied in the rodent pilocarpine model include widespread microglial activation, delayed thalamic axonal death, and persistent NPY upregulation in mossy fibers, together revealing extensive and persistent glial as well as neuronal pathology.     

Increased thalamus levels of glutamate and glutamine (Glx) in patients with idiopathic generalised epilepsy

OBJECTIVE: Abnormal thalamo-cortical oscillations underlie idiopathic generalised epilepsy (IGE). Although thalamic involvement has long been indicated by electrophysiological data, it has only recently become feasible to test this with independent methods. In this magnetic resonance (MR) study, we investigated the metabolic and structural integrity of the thalamus. Possible changes in glutamine and glutamate concentrations and signs of neuronal damage were of particular interest. METHOD: Forty three IGE patients and 38 age and sex matched healthy controls were investigated. Quantitative single volume MR spectroscopy (MRS, 1.5 T) was used to measure concentrations of glutamate and glutamine (Glx) and N-acetyl aspartate (NAA) in thalamus and occipital cortex. We also measured thalamic volumes on high resolution gradient-echo images and estimated fractions of thalamic grey and white matter with voxel based morphometry (VBM). RESULTS: IGE patients showed elevated Glx and reduced NAA concentrations in the thalamus compared to controls (12.2+/-2.6 v 8.9+/-4.1 mM, p = 0.0022 for Glx, and 9.9+/-1.0 v 10.7+/-0.9 mM, p = 0.017 for NAA). Thalamic grey matter fraction was reduced in IGE patients, and white matter fraction was increased with the greatest increase in the dorso-medial thalamus. Mean thalamic volume was reduced in patients (6.7+/-0.7 v 7.2+/-0.6 ml in controls, p = 0.0001), as was mean cerebral volume (1163+/-128 v 1250+/-102 ml, p = 0.0003). Patients' thalamus/whole brain ratios were normal. CONCLUSION: Quantitative MRS and VBM provide further evidence for involvement of the thalamus in IGE. The observed elevation of Glx levels together with reductions in NAA levels and grey matter fractions are consistent with epilepsy related excitoxicity as a possible underlying mechanism.