海马苔藓纤维出芽与颞叶癫痫

海马颗粒细胞苔藓纤维出芽(MFS)导致局部环路重建,在颞叶癫痫的发生与发展过程中起关键性作用。本文综述了近年有关海马MFS的病理解剖生理学特点、出芽机理及其与颞叶癫痫关系的最新研究成果,以期为探讨颞叶癫痫的形成机制提供新的思路。     

颞叶癫痫海马突触重组及其与钙调控的关系

苔藓纤维出芽是颞叶癫痫突触重组的主要表现.出芽的苔藓纤维在颗粒细胞间形成兴奋性环路,使颗粒细胞产生同步化电活动的阈值降低,也可经海马结构内的传导通路,使海马结构在传出途径上的电活动增强,从而导致反复自发性癫痫发作的发生.钙离子作为一种信使参与多种细胞功能活动的调节,钙通道功能的改变可引起细胞内外钙离子浓度的变化,而细胞内高钙和细胞外低钙均可诱发神经电活动紊乱,因此钙稳态在癫痫发生发展及海马突触重组的形成中起重要作用.     

海马苔藓纤维发芽与颞叶癫痫的研究进展

颞叶癫痫是部分性发作癫痫的最主要类型，约有40％的人类癫痫起源于颞叶，这与海马结构独特的神经元环路及纤维联系有关。海马结构中异位突触的形成被认为与颞叶癫痫发作有着密切的联系。而在海马结构的突触重排中，研究最多而且被充分证明的就是苔藓纤维发芽（mossy fiber sprouting，MFS）。现就海马MFS的形态学特性、诱发因素、出芽机理及其与颞叶癫痫发作之间的关系作一综述。     

颞叶癫痫患者海马苔藓纤维发芽的实验研究

目的观察颞叶癫痫病人海马齿状回和CA3区苔藓纤维出芽情况。方法癫痫组样本来自12例颞叶癫痫病例的手术切除标本包含海马齿状回和CA3区的脑组织，对照组脑组织样本来自4例非癫痫病的尸检脑组织。应用Timm组织化学染色方法在光镜和电镜水平进行海马结构苔藓纤维发芽的研究。结果光镜下癫痫组可见苔藓纤维穿越海马齿状回颗粒细胞层到达内分子层．CA3区也可见明显的苔藓纤维发芽。癫痫组CA3区和齿状回内分子层苔藓纤维发芽评分高于对照组．统计学上差异有显著性意义。电子显微镜下观察显示癫痫组患者齿状回内分子层可见到银标记的突触末端，主要和树突形成突触连接，所形成的突触为非对称性突触。结论颞叶癫痫可致海马齿状回和CA3区苔藓纤维发芽增加，这可能是难治性癫痫形成的重要机制。     

癫痫大鼠海马出芽苔藓纤维突触的超微结构特征

目的:探讨匹罗卡品颞叶癫痫大鼠海马出芽苔藓纤维突触的超微结构特征及其在颞叶癫痫发病机制中的作用。方法:采用Timm组化染色标记出芽苔藓纤维突触末端,在电镜下观察新生突触的类型、比例、定位、以及突触后靶成分。结果:颞叶癫痫大鼠齿状回内分子层可见到银标记的突触末端,出芽苔藓纤维突触主要是轴棘型非对称性突触,其次是轴树型非对称性突触,偶可看到出芽轴突和颗粒细胞体形成突触联系。结论:轴棘型非对称性突触是颞叶癫痫大鼠海马出芽苔藓纤维突触的主要类型,出芽苔藓纤维突触的超微结构特性支持重组突触形成重复的兴奋性环路,而且形成的新的兴奋性环路可能在颞叶癫痫的发生与发展中起重要作用。     

癫痫苔藓纤维出芽的研究进展

癫痫的发病机制非常复杂．迄今尚未完全阐明。苔藓纤维出芽（mossy fiber sprouting，MFS）作为癫痫发作后最常见的病理改变之一，与癫痫的反复自发性发作密切相关。近年来，已有不少的研究从苔藓纤维出芽的角度探讨癫痫的发生、发展．并为癫痫的治疗提出新的观点。现就癫痫苔藓纤维出芽的研究进展做一综述。[第一段]     

SCG10在癫痫苔藓纤维出芽中的作用

苔藓纤维出芽(mossy fiber sprouting.MFS)是癫痫后最重要的形态学改变之一,与痫性活动的反复自发性发作密切相关.MFS和突触重塑是难治性癫痫的主要病理基础,而MFS又是突触重塑的主要形式,在难治性颞叶癫痫患者标本和癫痫动物模型中普遍存在.     

TRPC6在匹罗卡品致痫大鼠海马苔藓纤维出芽中的作用

目的 观察传统型瞬时受体电位通道6(TRPC6)蛋白在匹罗卡品致痫大鼠海马中的表达变化,探讨其在海马苔藓纤维出芽中的作用.方法 72只SD大鼠随机分为实验组(n=60)和对照组(n=12).实验组采用氯化锂-匹罗卡品腹腔注射法建立颞叶癫痫模型;对照组腹腔注射等量无菌生理盐水.实验组按癫痫持续状态(SE)后1d、7d、15d、30 d和60 d分为5个亚组,每亚组12只大鼠.以上各亚组及对照组再分为2个小组,分别进行Western blot检测TRPC6及突触重建标志蛋白Synaptophysin在海马中的表达和Timm染色观察海马苔藓纤维出芽并评分.结果 实验组TRPC6蛋白表达量在SE后1d达高峰(P＜0.01),其他时间点均显著高于对照组(P＜0.01).Synaptophysin蛋白表达量在SE后7d、15d、30 d和60 d显著增加(7 d:P＜0.05;15 d、30 d、60 d:P＜0.01),30 d达峰值(P＜0.01).实验组大鼠齿状回内分子层在SE后7d出现Timm颗粒,并呈进行性增加.结论 TRPC6可能参与了苔藓纤维出芽这一过程.     

匹罗卡品癫痫模型中突触素Ⅰ的表达及苔藓纤维出芽的动态变化

目的探讨癫痫发生过程中突触素Ⅰ(SYNⅠ)在海马和齿状回的表达及齿状回苔藓纤维出芽的动态变化。方法建立匹罗卡品癫痫持续状态模型,用图像分析系统测定海马和齿状回不同时点SYNⅠ免疫反应吸光度值．Neo-timms’染色观察齿状回苔藓出芽的演变。结果SYNⅠ在海马和齿状回的表达于癫痫状态后2d、7d出现降低,14d开始升高,30d、60d表达明显增高；齿状回内分子层于14d开始出现苔藓纤维出芽,大鼠在同期开始出现自发发作。结论在癫痫状态后2d即出现了突触可塑性的变化,14d后由于神经轴突的再生,齿状回内分子层出现苔藓纤维出芽,形成了兴奋性的环路,可能是癫痫反复自发发作的病理基础,SYNⅠ及苔藓纤维出芽较好的反应了神经可塑性的变化。     

Tau蛋白在点燃大鼠海马苔藓纤维出芽中的作用探讨

目的观察总tau蛋白及其p-ptauser202在戊四氮点燃癫模型海马中的表达变化,探讨其在苔藓纤维出芽中的作用。方法 180只雄性SD大鼠随机分为对照组(腹腔注射生理盐水)和模型组(腹腔注射戊四氮),均n=90。于不同的时间点应用Timm染色观察苔藓纤维出芽,免疫组化和Western blot检测各时间点海马总tau蛋白及p-ptauser202蛋白表达情况。结果模型组各时间点CA1、CA3、DG区苔藓纤维出芽较对照组明显增多,差异有统计学意义(P<0.05);模型组第1周CA1区、CA3区,第2周DG区的p-ptauser202蛋白表达与对照组比较明显增多,差异均有统计学意义(分别为CA1区P<0.05,CA3区P<0.01,DG区P<0.01);门区(H区)tau蛋白及p-ptauser202蛋白在各时间点的表达明显增多,差异有显著统计学意义(P<0.01);对照组无动态变化。结论 tau蛋白可能通过其磷酸化水平的增高参与癫点燃大鼠的苔藓纤维出芽,在癫的发生和发展中起重要作用。     

Neural (N-) cadherin,a synaptic adhesion molecule,is induced in hippocampal mossy fiber axonal sprouts by seizure

Aberrant mossy fiber sprouting and synaptic reorganization are plastic responses in human temporal lobe epilepsy, and in pilocarpine-induced epilepsy in rodents. Although the morphological features of the hippocampal epileptic reaction have been well documented, the molecular mechanisms underlying these structural changes are not understood. The classic cadherins, calcium-dependent cell adhesion molecules, are known to function in development in neurite outgrowth, synapse formation, and stabilization. In pilocarpine-induced status epilepticus, the expression of N-cadherin mRNA was sharply upregulated and reached a maximum level (1- to 2.5-fold) at 1- to 4 weeks postseizure in the granule cell layer and the pyramidal cell layer of CA3. N-cadherin protein was correspondingly increased and became concentrated in the inner molecular layer of the dentate gyrus, consistent with the position of mossy fiber axonal sprouts. Moreover, N-cadherin labeling was punctate; colocalized with definitive synaptic markers, and partially localized on polysialated forms of neural cell adhesion molecule (PSA-NCAM)-positive dendrites of granule cells in the inner molecular layer. Our findings show that N-cadherin is likely to be a key factor in responsive synaptogenesis following status epilepticus, where it functions as a mediator of de novo synapse formation.     

The organization and development of the hippocampal mossy fiber system

The anatomical organization and development of the hippocampal mossy fiber system has been reviewed with special reference to its organization in the common laboratory rat.The mossy fibers originate from the granule cells of the dentate granular layer and the few granule cells found scattered in the dentate molecular layer and hilus. Via a complex system of collaterals the mossy fibers terminate on several types of neurons in the hilus, e.g. the basket cells and the mossy cells. Upon leaving the hilus to pass into Ammon's horn, the mossy fibers converge to form a distinct band of fibers that terminates on the proximal part of the apical and basal dendrites of the pyramidal and basket cells of the regio inferior. In some mammalian species the mossy fibers may continue into the adjacent part of the regio superior. Despite differences in the number of granule cells and pyramidal cells at different septotemporal levels this organization is relatively uniform along the septotemporal extent of the hippocampus.During development the mossy fibers grow out in a sequential manner that matches the pattern of neurogenesis and the aggregation of the cells of origin. From the level at which they originate, the fibers diverge along the septotemporal axis in such a way that the oldest granule cells have the most extensive projections. The adult topographic organization, which is already apparent at the earliest developmental stages, is thus formed in a stepwise fashion. It is concluded that the organization of the hippocampal mossy fibers indicates that neuronal specificity should not be explained by cellular recognition alone, but rather as the cumulated product of the preceding sequence of developmental events that include neurogenesis, migration, aggregation and directed axonal outgrowth.     

杏仁核点燃癫痫大鼠P-糖蛋白及苔藓纤维出芽变化的研究

目的 观察杏仁核点燃癫痫大鼠海马区P-糖蛋白(P-gp)表达及苔藓纤维出芽(MFS)的动态变化. 方法 90只大鼠采用随机数字表法分为假手术对照组(10只)、癫痫组(40只)和治疗组(40只),假手术对照组只安装电极,不予刺激;癫痫组和治疗组制作杏仁核点燃模型,治疗组加用左乙拉西坦灌胃治疗[100 mg/(kg·d),2次/d)].采用Timm银染组织化学方法观察海马区MFS,免疫组化法检测P-gp的表达. 结果 (1)成功制造癫痫模型后,在海马CA3区透明层出现异常MFS,其中S1亚组大鼠MFS评分最低,与假手术对照组比较差异无统计学意义(P＞0.05);S2亚组大鼠评分开始增高,S4亚组大鼠明显增高,S8亚组大鼠达到高峰,与假手术对照组比较差异均有统计学意义(P＜0.05).而治疗组大鼠MFS评分各时间点与假手术对照组比较差异均无统计学意义(P＞0.05).(2)癫痫发作后癫痫组大鼠P-gp表达量呈现出逐渐降低的趋势,S1、S2、S4亚组与假手术对照组差异有统计学意义(P＜0.05);S8亚组接近正常水平,与假手术对照组比较差异无统计学意义(P＞0.05).治疗组大鼠除Y1亚组外,余各亚组P-gp表达量与假手术对照组比较差异均无统计学意义(P＞0.0S). 结论 MFS是慢性癫痫形成的重要机制,P-gp是癫痫发生的产物,是癫痫药物耐药的主要原因.     

戊四氮点燃模型大鼠Cdk5活性与苔藓纤维出芽相关性

目的研究戊四氮(PTZ)点燃模型大鼠细胞周期素依赖性激酶5(Cdk5)活性改变与苔藓纤维出芽(MFS)动态变化的相关性。方法将120只雄性成年大鼠随机分为PTZ组和对照组,PTZ组连续每天腹腔注射PTZ30mg/kg,对照组同时注射等体积生理盐水。按戊四氮第1次给药后3d、1、2、4和6周各随机分为5个亚组。每个亚组再随机分为2个小组:一组用于液体闪烁计数仪测定各时间点大鼠海马的Cdk5活性;另一组用于Timm染色检测苔藓纤维出芽情况。结果 PTZ组MFS评分3d时增加,2周达高峰并维持至6周;海马Cdk5活性3d时增加,2周达高峰,4周下降,6周恢复对照组水平;较对照组有显著差异。额区Cdk5活性各时间点之间及与对照组之间均无显著性差异。结论 Cdk5可能通过活性增高参与苔藓纤维出芽,从而促使癫发生。     

法舒地尔对戊四氮点燃大鼠海马丝切蛋白及苔藓纤维出芽的影响

目的 探讨法舒地尔对戊四氮(PTZ)点燃大鼠海马组织中丝切蛋白(cofilin,非磷酸化形式)表达与苔藓纤维出芽程度关系的影响.方法 210只SD雄性大鼠分成戊四氮组、法舒地尔干预组和生理盐水对照组,采用PTZ慢性点燃癫癎模型,应用SABC法检测cofilin表达,用Timm染色检测苔鲜纤维出芽情况.结果 PTZ组大鼠点燃率、病死率与法舒地尔组比较差异无统计学意义.PTZ组和法舒地尔组CA3区苔藓纤维出芽评分差异无统计学意义,与对照组相比差异均有统计学意义(P＜0.05).PTZ组和法舒地尔组海马非磷酸化cofilin表达差异无统计学意义.结论 丝切蛋白可能通过苔藓纤维出芽与癫癎的发生相关.     

Seizure, neuron loss, and mossy fiber sprouting in herpes simplex virus type 1-infected organotypic hippocampal cultures

PURPOSE: Epileptic seizures are frequently seen after viral encephalitis. Herpes simplex virus type 1 (HSV-1) encephalitis is the most common cause of acquired epilepsy in humans. However, little information is available about the neuropathogenesis of HSV-1-associated seizures. We have developed an in vitro HSV-1-infected organotypic hippocampal slice culture to elucidate the underlying mechanisms of HSV-1-associated acute seizure activity. METHODS: Hippocampal slice cultures were prepared from postnatal day 10 to 12 rat pups. Wild-type HSV-1 strain RE (1 x 10(5) PFU) was applied to cultures at 14 days in vitro. The excitability of CA3 pyramidal cells and hippocampal network properties were measured with electrophysiological recordings. Hematoxylin-eosin (H&E) and Timm stains were used. RESULTS: HSV-1 infection induces epileptiform activity, neuron loss, and subsequently a dramatic increase of mossy fiber sprouting in the supragranular area. With intracellular recordings, surviving CA3 pyramidal cells exhibited a more depolarizing resting membrane potential concomitant with an increase in membrane input resistance and had a lower threshold to generate synchronized bursts and a decrease in the amplitude of afterhyperpolarization than did controls. When the antiherpes agent acyclovir was applied with a delay of 1 or 24 h after HSV-1 infection, a dramatic inhibition of HSV-1 replication and protection of the neuron loss were observed. CONCLUSIONS: These results suggest that a direct change in the excitability of the hippocampal CA3 neuronal network and HSV-1-induced neuron loss resulting in subsequent mossy fiber reorganization may play an important role in the generation of epileptiform activity.     

皮质发育障碍大鼠小脑回形态学和海马苔藓纤维发芽的研究

目的 探讨液氮损伤诱导局灶性皮质发育障碍大鼠海马形态学及苔藓纤维发芽的情况。方法 实验随机分为正常对照组、假手术组和液氮损伤组，建立局灶性皮质发育障碍动物模型，察看其行为改变；采用常规HE染色、Nissl染色和Timm’s硫化银组织化学方法染色，肉眼和光镜下观察大鼠脑皮质形态变化，光镜下评估海马苔藓纤维发芽情况，各组数据取苔藓纤维发芽评分，采用非参数秩和Kruskal—Wallis H检验，组间两两比较用Nemenyi法。结果 液氮损伤组大鼠行为轻微改变，鼠脑嘴尾方向形成了一小的脑回，同侧海马CA3区有苔藓纤维发芽．而正常对照组和假手术组却没有。结论 幼鼠早期液氮损伤可导致小脑回形成及海马CA3区苔藓纤维发芽。小脑回周围异常兴奋性突触环路和海马CA3区苔藓纤维发芽形成推测是局灶性皮质发育障碍导致癫痫发生的重要机制。     

Increased densities of AMPA GluR1 subunit proteins and presynaptic mossy fiber sprouting in the fascia dentata of human hippocampal epilepsy

In human hippocampal epilepsy, there is a consistent pathology of cell loss and reactive synaptic reorganization of ‘excitatory' mossy fibers (MF) into the inner molecular layer (IML) of the fascia dentata (FD). In this study, neo-Timm's histochemistry of MFs and immunocytochemistry of GluR1 were used to determine, in patients with or without hippocampal sclerosis (HS), if there was a correlation between aberrant supragranular (IML) mossy fiber sprouting and increased densities of AMPA GluR1 subunit proteins in the IML of the FD. Computerized quantified densitometric grey values of Timm and GluR1 densities were corrected for the densities of granule cell losses using cell counts. In the IML of the HS group, despite the losses of granule cells, mossy fiber sprouting was significantly greater (P<0.000001) and GluR1 protein densities were significantly higher (P<0.0005) than those of the non-HS group. Unlike supragranular mossy fiber sprouting, which was limited to the IML, the increased GluR1 stainings were distributed throughout the whole molecular layer. For all cases, MF synaptic reorganization in the supragranular ML was correlated with GluR1 subunit protein densities in the IML (R=0.784, P<0.0093). These data demonstrate that in the human epileptic fascia dentata, there are significantly increased AMPA GluR1 subunit proteins associated with aberrant MF synaptic reorganizations. This suggests that the hyperexcitability of sclerotic hippocampus occurs, at least in part, from the associated changes of both presynaptic mossy fiber glutamatergic neoinnervation and increased GluR1 subunit proteins in the dendritic domains of the FD.