杏仁核点燃癫痫大鼠丘脑中线核和皮层EEG表现

癫痫研究的目的之一就是探索阻止癫痫发生或改变癫痫信号传播的过程。而丘脑中线核群在颞叶癫痫发作中的作用日益受到人们的重视。本实验通过建立杏仁核点燃癫痫模型，记录不同部位EEG表现来探讨丘脑中线核群在颞叶癫痫发作中的作用。     

低频电刺激丘脑底核对癫(癎)大鼠海马谷氨酸和γ-氨基丁酸表达的影响

目的 探讨低频电刺激(LFS)丘脑底核对癫(癎)大鼠海马谷氨酸(Glu)及γ-氨基丁酸(GABA)表达的影响.方法 用30只大鼠制备杏仁核电刺激点燃模型,同时在丘脑底核埋置刺激电极;制模成功的大鼠随机分为LFS组及对照组;两组大鼠每日点燃1次,共10 d;LFS组大鼠每次点燃前予以LFS丘脑底核.观察10 d后两组大鼠癫(癎)发作程度和持续时间,应用免疫组化法测定大鼠海马Glu、GABA阳性细胞数及灰度值.结果 与对照组比较,LFS组癫(癎)发作的Racine评分明显降低,癫(癎)发作持续时间明显缩短;海马GABA阳性细胞数明显增多、灰度值降低;Glu阳性细胞数明显减少,灰度值明显增加(均P＜0.05＝.结论 LFS丘脑底核能有效抑制大鼠杏仁核点燃发作,其作用机制可能是改变了脑内Glu、GABA的表达.     

塞来昔布对慢性颞叶癫癎大鼠海马核因子-κBp65和P-糖蛋白表达的影响

目的探讨环氧合酶-2抑制药塞来昔布对慢性颞叶癫癎大鼠海马核因子-κBp65和P-糖蛋白表达的影响,以及核因子-κBp65和P-糖蛋白与颞叶癫癎发病机制的关系,以为环氧合酶-2抑制药用于抗癫癎药物辅助治疗提供实验依据。方法采用大鼠海马CA3区微量注射海人酸的方法制备颞叶癫癎动物模型,免疫组织化学染色和Western blotting法观察塞来昔布治疗后大鼠海马核因子-κBp65和P-糖蛋白表达变化。结果与对照组相比较,颞叶癫癎大鼠海马核因子-κBp65、P-糖蛋白表达水平,以及核因子-κBp65核移位现象明显增加(均P<0.05);经塞来昔布治疗后,海马组织中核因子-κBp65、P-糖蛋白表达水平及核因子-κBp65核移位现象显著改善,与模型组比较差异有统计学意义(均P<0.05)。结论核因子-κBp65和P-糖蛋白在颞叶癫癎慢性期表达上调、核因子-κBp65核移位现象增加,有可能是难治性癫癎发生与发展的分子生物学机制之一。环氧合酶-2抑制药塞来昔布通过降低慢性颞叶癫癎大鼠海马CA3区核因子-κBp65和P-糖蛋白表达水平,抑制核因子-κBp65核移位,最终降低炎性反应,逆转多药耐药而发挥抗癫癎作用。     

颞叶癫(癎)大鼠海马中miRNA表达谱的检测和分析

目的 探讨颞叶癫(癎)大鼠海马组织中miRNA分子表达谱的差异,为进一步研究相关miRNA在颞叶癫(癎)发病机制中的作用打下基础.方法 对同一父系和母系的子代大鼠,利用氯化锂-匹罗卡品化学诱导方法制备慢性颞叶癫(癎)大鼠模型.分别提取1只正常和3只颞叶癫(癎)大鼠海马组织的miRNA,采用高通量的miRNA微阵列芯片杂交,筛选颞叶癫(癎)海马组织中差异表达的内源性miRNA.结果 在大鼠海马组织中共检测到125个miRNA基因.与正常大鼠相比,颞叶癫(癎)大鼠海马组织中差异表达的miRNA有23个,其中有5个miRNA下调,18个miRNA上调.结论 与正常大鼠相比,颞叶癫(癎)大鼠海马组织中存在差异表达的miRNA分子,差异表达的miRNA分子可能参与癫(癎)的发病过程,具有潜在的研究价值.     

癫(癎)幼鼠海马和颞叶皮质区缝隙连接蛋白43和突触体素表达的变化

目的观察神经元缝隙连接蛋白43(Cx43)和突触体素(synaptophysin P38)在戊四氮(PTZ)点燃癫癎幼鼠海马及颞叶皮质区中的表达,探讨两者与癫癎的关系及其在癫形成中的作用。方法将50只21日龄Wistar大鼠分为对照组和实验组。实验组采用PTZ点燃癫癎幼鼠,按点燃进程分为Ⅰ级、Ⅱ级、Ⅲ级、Ⅳ级及Ⅴ级发作组。采用免疫组化和图像分析技术,观察海马及颞叶皮质区Cx43和P38表达的变化。结果应用PTZ点燃后,实验各组幼鼠海马及颞叶皮质区Cx43和P38的表达明显高于对照组(P<0.01),且随发作级别的增高,幼鼠海马及颞叶皮质各区Cx43和P38的表达均增加。但各组间海马区和颞叶皮质区Cx43和P38的表达情况的比较差异无统计学意义(P>0.05)。结论Cx43和P38的表达水平与癫癎的发生发展有密切关系,为研究小儿癫癎的病因及发病机制提供依据。     

大鼠海马快速点燃模型的建立及机制研究

目的：建立大鼠海马快速点燃模型并对其机制进行初步探讨。方法：制备大鼠海马快速点燃模型;观察经典抗杏仁核点燃药物对该模型的影响;观察大鼠海马快速点燃模型和大鼠杏仁核点燃模型海马CA_3区和皮层区c-fos蛋白的表达情况及蛋白抑制剂对其表达的影响。结果：大鼠海马快速点燃模型点燃成功率与刺激参数有关;苯巴比妥20,50mg·kg~1 及地西泮2,5mg·kg~(-1)对大鼠海马快速点燃ADD和Racine＇s分级有抑制作用(P＜0．05);与正常组比较,海马快速点燃大鼠和杏仁核点燃大鼠海马CA_3区、大脑皮层c-fos免疫阳性细胞增多(P＜0．05),在蛋白抑制剂作用下,海马c-fos免疫阳性细胞减少,但仍比正常组高(P＜0．05)。结论：大鼠海马快速点燃成功率与刺激参数具有相关性;传统抗点燃药物具有抗海马快速点燃作用;大鼠海马快速点燃模型c-fos蛋白在不同脑区和核团的免疫阳性表达细胞增加。     

颞叶癫大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化

目的 研究颞叶癫(癎)大鼠海马轴突导向分子Sema3F及其受体Np2表达的变化.方法 给SD大鼠腹腔注射匹罗卡品、氯化锂制作颞叶癫(癎)模型.用免疫组化法和原位杂交技术对致(癎)后不同时间点大鼠海马CA1区、CA3区、齿状回的Sema3F mRNA、Np2 mRNA和蛋白表达进行检测,并与正常对照组比较.结果 颞叶癫(癎)大鼠致(癎)后7 d、15 d,海马CA1区、CA3区Sema3F mRNA、Np2 mRNA和蛋白的表达明显低于正常对照组(P＜0.05～0.01), 致(癎)后30 d、60 d表达与正常对照组差异无统计学意义;而齿状回Sema3F mRNA、Np2 mRNA和蛋白的表达与正常对照组的差异无统计学意义.结论 颞叶癫(癎)大鼠海马CA1区、CA3区Sema3F、Np2表达在致(癎)后早期明显下调,而在慢性期恢复正常.     

G蛋白偶联内向整流钾通道亚基2 mRNA在颞叶癫疒间大鼠海马内的表达

目的探讨G蛋白偶联内向整流钾通道亚基2(GIRK 2)在颞叶癫癎大鼠海马内的表达变化.方法应用腹腔注射海人酸致癎大鼠,采用原位杂交法检测大鼠海马GIRK 2 mRNA的表达.结果 GIRK 2 mRNA在癫癎大鼠海马齿状回表达增加,与正常对照组相比差异有显著性( P<0.01).结论癫癎大鼠海马内GIRK2增高是机体对神经元网络过度兴奋的代偿反应.     

GIRK1在颞叶癫痫大鼠海马齿状回中的表达及其意义

目的探讨GIRK1在颞叶癫癎大鼠海马齿状回的表达及其意义.方法112只雄性SD大鼠随机分为实验组(n=70)与对照组(n=42),同时建立海人藻酸(KA)颞叶癫模型.选取KA腹腔注射后3、6、12、24、48 h,7、30 d为研究的时间点.用原位杂交法及免疫组织化学法检测海马齿状回GIRK1 mRNA及蛋白的表达.结果实验组大鼠海马齿状回GIRK1 mRNA表达在致癎后6 h较对照组减少,而在致癎后至7～30 d较对照组增高.结论在颞叶癫癎的不同时期海马齿状回GIRK1表达的变化反映出颞叶癫癎的复杂性.     

应用Fluoro-Jade C评价匹罗卡品模型丘脑神经变性

目的 应用Fluoro-Jade C(FJC)染色方法 观察小鼠匹罗卡品癫(癎)模型丘脑神经元变性情况,以了解丘脑结构在慢性颞叶癫痫发生中的病理变化和癫(癎)反复发作的神经学基础.方法 以盐酸匹罗卡品腹腔注射(220 mg/kg)制备小鼠癫痼持续状态模型.脑组织切片经FJC染色后.荧光显微镜下观察FJC阳性细胞形态和在丘脑的整体分布情况.结果 匹罗卡品模型组小鼠FJC阳性细胞呈神经元形态,丘脑结构损害呈连续性.功能不同的丘脑核其细胞损害程度有所不同.结论 采用FJC染色技术观察匹罗卡品癫(癎)持续状态小鼠模型丘脑神经元变性情况,有利于更好地理解颞叶癫(癎)的中枢神经系统长期病理变化和自发性反复发作机制.     

失神性癫痫持续发作诱导丘脑C-fos基因表达的实验研究

目的　探讨失神性癫痫持续发作的病理生理机制。　　方法　采用C -fos基因探针原位杂交法在γ -羟丁酸诱发的大鼠失神性癫痫持续发作模型上 ,观察癫痫持续发作后不同时间丘脑各核团中C -fos基因表达的分布状况 ,用C -fos基因阳性细胞密度作定量研究指标。　　结果　失神性癫痫发作后 1 0min ,双侧丘脑室旁核内可见到低密度C -fos阳性细胞 ;3 0min时 ,高密度C -fos阳性细胞广泛分布于外侧缰核、室旁核、菱形核和丘脑板内核群。　　结论　外侧缰核及丘脑中线和板内核群极可能涉及失神性癫痫发作的病理生理机制。     

γ—羟基丁酸诱发大鼠失神型癫痫持续状态时丘脑c—fos基因的表达

探讨失神性癫痫持续状态的病理生理机制。方法采用ｃ－ｆｏｓ基因探针原位杂交法，在γ－羟基丁酸诱发的大鼠失神性癫痫持续状态模型上，观察癫痫持续状态后不同时间丘脑各核团中ｃ－ｆｏｓ基因表达的分布状况，用ｃ－ｆｏｓ基因阳性细胞密度作定量研究指标。结果失神性癫痫发作后１０分钟，双侧丘脑室旁核内可见到低密度ｃ－ｆｏｓ阳性细胞；３０分钟时，高密度及中等密度ｃ－ｆｏｓ阳性细胞广泛分布于外侧缰核、室旁核、菱形核和丘脑板内核群。结论外侧缰核及丘脑中线和板内核群极可能涉及失神性癫痫发作的病理生理机制。     

Fos expression in GHB-induced generalized absence epilepsy in the thalamus of the rat

Using the model of gamma-hydroxybutyrate (GHB)-induced generalized absence epilepsy, the present work investigated the distribution of fos oncoprotein expression in the rat thalamus with fos antibody immunohistochemistry. Thirty minutes after absence-like seizures, some fos-immunoreactive cell nuclei were found in bilateral thalamic paraventricular nuclei (PV). After a further 30 min, a massive bilateral induction of fos was observed in the lateral habenular nucleus (LHb), the PV, the rhomboid thalamic nucleus, and the intralaminar nuclei of the thalamus. These results suggest that the LHb and the midline and intralaminar thalamic nuclei may very likely be involved in the pathophysiology of absence seizures.     

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.     

Evidence for a critical role of GABAergic transmission within the thalamus in the genesis and control of absence seizures in the rat.

The involvement of GABAergic transmission within the thalamus in the generation and control of spike and wave discharges (SWD) in generalized non-convulsive or absence epilepsy was studied in rats with spontaneous SWD and in non-epileptic rats. In epileptic rats, bilateral injections of gamma-vinyl GABA (GVG, 10 micrograms/side) or muscimol (10 ng/side) into the medial part of the ventral lateral thalamus, i.e. the specific relay nuclei, significantly increased spontaneous cortical SWD whereas similar injections into the most lateral part of the thalamus, i.e. the area of the reticular nuclei, significantly suppressed these seizures. Injections of GVG (20 micrograms) or muscimol (20 ng) into the midline thalamus had no direct effect on the spontaneous SWD. In non-epileptic rats, injections of GVG (25 micrograms/side) or muscimol (100 ng/side) into the thalamic relay nuclei produced short SWD on the cortical EEG. These results suggest that GABAergic neurons in the reticular nuclei and their projections to the specific relay nuclei of the thalamus are involved in the elicitation and control of generalized non-convulsive seizures.     

Role of the thalamus in the physiopathology of epilepsy

Early hypotheses on the origin of primary generalized epilepsy suggested a determinant role for the "centrencephalon" in the triggering of discharges of generalized spike-waves (GSW) and tonic-clonic crises (TCC). It was demonstrated in this respect that bilateral cortical spike-wave discharges at 3 c/sec were obtained by electrical stimulation of intralaminar nuclei in the rat. The role of the thalamus in the genesis of GSW and TCC was subsequently demonstrated in several experimental models. However, the thalamus is apparently not involved in the genesis of EEG manifestations of generalized epilepsy and most authors agree that the latter are of cortical origin since a) with or without cardiazol, generalized seizures may occur after ablation of cat thalamus; b) in the cat diffuse cortical application of dilute penicillin can reproduce signs of generalized epilepsy observed after systemic injection of penicillin to this animal; c) finally paroxysmal discharges which appear in subcortical structures in the photosensitive baboon are always preceded by frontal cortical spike-waves; similarly intermittent light stimulation-provoked TCC always arise from the frontal cortex and furthermore hemispheric synchronization of attacks disappears after callosotomy. These findings indicate that the origin of GSW and TCC, is not in the thalamus but that the latter nevertheless plays a role in their elaboration.     

Expression of cholecystokinin and somatostatin genes in the human thalamus.

The cholecystokinin (CCK) gene is expressed in thalamocortical and thalamo-striatal neurons of the rat. In the cat, this peptide is found in some intralaminar and midline nuclei, whereas somatostatin (SRIF) is expressed in the reticular nucleus of the cat but not in rat. Since the putative neurotransmitters used by thalamic neurons are still incompletely known, especially in humans, we investigated the expression of the CCK and SRIF genes in the human thalamus by using hybridization histochemistry. CCK mRNA was found in many neurons, located in several nuclei of the dorsal thalamus. They were especially numerous and widespread in the nuclei associated with the internal thalamic lamina. They formed a continuum in the basal medial thalamus, from the central-medial nucleus, through the centre median/parafascicular complex to the limitans and suprageniculate nuclei. In addition, neurons with CCK mRNA were found medially and laterally to the mediodorsal nucleus, in the midline and intralaminar nuclei. Only rare neurons with CCK mRNA were found in other nuclei (e.g., in the ventral group of nuclei). SRIF mRNA was found in many neurons of the reticular nucleus, but not in the dorsal thalamus. Neurochemical features of the human thalamus, for the genes studied here, resemble those found in the cat. SRIF may play a role in modulating dorsal thalamic impulses, which may be conveyed through CCK innervation to the striatum and, partly, to the cortex.     

Volumetric measurements of subcortical nuclei in patients with temporal lobe epilepsy.

OBJECTIVE: To determine the volumes of subcortical nuclei in patients with chronic epilepsy. BACKGROUND: Animal and human data suggest a crucial role for subcortical structures in the modulation of seizure activity, mostly as seizure-suppressing relays. Although cortical epileptogenic foci can vary in localization and extent, it nevertheless appears that these structures subsequently influence seizure propagation in a universal fashion. There is, however, little knowledge about the size of implicated subcortical structures in patients with epilepsy. METHOD: Using high-resolution MRI, the volumes of selected subcortical nuclei, such as the thalamus, caudate nucleus, putamen, and pallidum, were measured in both hemispheres of 27 patients with temporal lobe epilepsy. Fourteen healthy volunteers served as controls. Statistical analysis was done for both normalized volumes (by total brain volume) and unnormalized volumes. RESULTS: Overall, the patient group had smaller thalamic and striatal volumes in both hemispheres, mostly ipsilateral to the epileptic focus. No significant correlations were noted between volume measurements and age, age at onset, duration of epilepsy, or total seizure frequency, including frequency of generalized seizures. The putamen and thalamus seemed to be affected predominantly in patients with a history of febrile convulsions, whereas patients without febrile convulsions had smaller caudate nuclei bilaterally. CONCLUSIONS: Volumetric measurements of subcortical nuclei reveal atrophy of distinct subcortical nuclei in the patient group, predominantly ipsilateral to the focus. This finding probably reflects persistent abnormalities and not secondary change. In addition, the structural differences between patients with and patients without previous febrile convulsions suggest that these conditions may have different causes.     

Projections from the five divisions of the orbital cortex to the thalamus in the rat

The orbital cortex (ORB) of the rat consists of five divisions: the medial (MO), ventral (VO), ventrolateral (VLO), lateral (LO), and dorsolateral (DLO) orbital cortices. No previous report has comprehensively examined and compared projections from each division of the ORB to the thalamus. Using the anterograde anatomical tracer, Phaseolus vulgaris leucoagglutinin, we describe the efferent projections from the five divisions of the ORB to the thalamus in the rat. We demonstrated that, with some overlap, each division of the ORB distributed in a distinct (and unique) manner to nuclei of the thalamus. Overall, ORB projected to a relatively restricted number of sites in the thalamus, and strikingly distributed entirely to structures of the medial/midline thalamus, while completely avoiding lateral regions or principal nuclei of the thalamus. The main termination sites in the thalamus were the paratenial nucleus (PT) and nucleus reuniens (RE) of the midline thalamus, the medial (MDm) and central (MDc) divisions of the mediodorsal nucleus, the intermediodorsal nucleus, the central lateral, paracentral, and central medial nuclei of the rostral intralaminar complex and the submedial nucleus (SM). With some exceptions, medial divisions of the ORB (MO, VO) mainly targeted “limbic-associated” nuclei such as PT, RE, and MDm, whereas lateral division (VLO, LO, DLO) primarily distributed to “sensorimotor-associated” nuclei including MDc, SM, and the rostral intralaminar complex. As discussed herein, the medial/midline thalamus may represent an important link (or bridge) between the orbital cortex and the hippocampus and between the ORB and medial prefrontal cortex. In summary, the present results demonstrate that each division of the orbital cortex projects in a distinct manner to nuclei of the thalamus which suggests unique functions for each division of the orbital cortex.