红藻氨酸诱发大鼠复杂部分性癫痫发作的海马神经元和星形胶质细胞反应及相互关系

目的：观察大鼠癫痫发作后海鸟内神经元与星形胶质细胞反应变化的时空效应及相互关系。方法：以红藻氨酸诱发的大鼠复杂部分性癫痂发作为模型，利用免疫组织化学法，在原位显示癫痫发作后15、30、60、90、120、180min6个时间点海马神经元Fos蛋白及星形胶质细胞内胶质原纤维酸性蛋白（GFAP）的表达变化、相互关系及分布规律。结果：致痫后15min海马内GFAP表达开始增多，60min达高峰。Fos阳性神经元在癞痴诱发后30min开始出现，120min达高峰。海马内GFAP阳性细胞与Fos阳性神经元分布规律基本一致。结论：在癫痫病理状态下，海马内星形胶质细胞的反应略早于神经元，两者之间分布呈平行关系，它们之间可能存在着复杂的信息通讯，以复合体的形式其同对各种病理生理刺激作出反应。     

GFAP和Fos蛋白在戊四氮致痫大鼠前脑中的表达变化

目的 研究大鼠在戊四氮导致癫痫发作时前脑内星形胶质细胞和神经元的形态学反应及其相互关系。方法 应用免疫组织化学单标记法分别显示前脑内GFAP和Fos蛋白表达的时间规律，并用免疫组织化学双重标记显示GFAP和Fos蛋白表达的相互关系。结果 在戊四氮导致大鼠癫痫发作早期，前脑的星形胶质细胞被激活，细胞体积增大，突起粗大，GFAP表达阳性，随着存活时间的变化，星形胶质细胞的反应经历先逐渐升高后降低的过程。被激活的星形胶质细胞和神经元表达Fos蛋白阳性，也呈现逐渐升高又降低的变化；另外，GFAP阳性星形胶质细胞和Fos阳性神经元在前脑主要分布在大脑皮层、海马、杏仁核等部位，二者的分布特征基本一致。结论 星形胶质细胞可能和神经元一起参与了戊四氮所致癫痫发作的变化。     

LPS激发大鼠延髓内脏带星形胶质细胞GFAP和神经元FOS表达水平的变化

观察腹腔注射细菌内毒素（LPS）后,大鼠延髓内脏带（MVZ）星形胶质细胞胶质原纤维酸性蛋白（GFAP）及神经元FOS表达水平随时间变化的规律及其相互关系。大鼠经LPS腹腔注射后1,3,6,12h分别行固定取材制片。每个时间点的切片分为3组,分别进行抗FOS、抗GFAP免疫组织化学染色及抗FOS／GFAP／酪氨酸羟化酶（TH）三重免疫组织化学染色。结果表明：（1）FOS反应在LPS注射后3h达到高峰,阳性产物主要分布于MVZ内。（2）GFAP反应在注射后1h即达到高峰,表现为胶质细胞肥大,数量增多。其分布于FOS基本相同。（3）三重染色观察到GFAP与FOS的多种聚集方式（FOS／GFAP／TH,FOS／GFAP,GFAP／TH）,FOS阳性神经元周围GFAP免疫反应产物更密集。提示星形胶质细胞对LPS起反应,其反应高峰的出现先于神经元。     

红藻氨酸诱发大鼠复杂部分性癫癎发作的海马神经元和星形胶质细胞反应及相互关系

目的:观察大鼠癫发作后海马内神经元与星形胶质细胞反应变化的时空效应及相互关系。方法:以红藻氨酸诱发的大鼠复杂部分性癫发作为模型,利用免疫组织化学法,在原位显示癫发作后153、0、60、901、20、180 min 6个时间点海马神经元Fos蛋白及星形胶质细胞内胶质原纤维酸性蛋白(GFAP)的表达变化、相互关系及分布规律。结果:致后15 min海马内GFAP表达开始增多,60 min达高峰。Fos阳性神经元在癫诱发后30 min开始出现,120 min达高峰。海马内GFAP阳性细胞与Fos阳性神经元分布规律基本一致。结论:在癫病理状态下,海马内星形胶质细胞的反应略早于神经元,两者之间分布呈平行关系,它们之间可能存在着复杂的信息通讯,以复合体的形式共同对各种病理生理刺激作出反应。     

PS激发大鼠延髓内脏带星形胶质细胞GFAP和神经元FOS表达水平的变化

观察腹腔注射细菌内毒素 (LPS)后 ,大鼠延髓内脏带 (MVZ)星形胶质细胞胶质原纤维酸性蛋白 (GFAP)及神经元FOS表达水平随时间变化的规律及其相互关系。大鼠经LPS腹腔注射后 1,3,6,12h分别行固定取材制片。每个时间点的切片分为 3组 ,分别进行抗FOS、抗GFAP免疫组织化学染色及抗FOS/GFAP/酪氨酸羟化酶 (TH)三重免疫组织化学染色。结果表明 :①FOS反应在LPS注射后 3h达到高峰 ,阳性产物主要分布于MVZ内。②GFAP反应在注射后 1h即达到高峰 ,表现为胶质细胞肥大、数量增多。其分布与FOS基本相同。③三重染色观察到GFAP与FOS的多种聚集方式 (FOS/GFAP/TH ,FOS/GFAP ,GFAP/TH) ,FOS阳性神经元周围GFAP免疫反应产物更密集。提示星形胶质细胞对LPS起反应 ,其反应高峰的出现先于神经元     

LPS激发大鼠前脑神经元Fos和小胶质细胞OX42表达改变

目的 探讨单次腹腔注射LPS后前脑神经元和小胶质细胞的可塑性变化和相互关系。方法 应用抗Fos、抗TH或抗OX42单一、以及抗Fos／抗TH／抗OX42三重免疫组化标记方法，观察大鼠单次腹腔注射LPS后，Fos阳性神经元、Fos／TH阳性神经元、OX42阳性小胶质细胞在脑内的表达分布及时程变化，以及Fos阳性神经元或Fos／TH阳性神经元与OX42阳性小胶质细胞之间的关系。结果：Fos阳性神经元分布在额、顶皮质，扣带回和梨状皮质，外侧隔核腹侧部，杏仁中央核，海马CA2区、CA3区、齿状回，下丘脑室旁核、视上核、下丘脑外侧区和第三脑室周围灰质等。Fos阳性神经元在注射后30min出现表达，注射后1～3h为表达高峰。反应阳性小胶质细胞首先于脑室周围灰质出现，注射后6h达到高峰，胞体变大，突起变粗，OX42呈阳性深染，密集分布于Fos阳性神经元的表达区域。下丘脑Fos／TH／OX42三重染色切片显示：由LPS激活的Fos／TH阳性神经元周围被OX42阳性细胞包绕并接触，表明神经元和小胶质细胞在对LPS刺激的反应中关系密切。结论 在外周免疫刺激下，下丘脑、扣带回、梨状皮质和海马内的神经元和小胶质细胞可能参与免疫调节。     

16Hz次声作用对大鼠海马TRPV4、GFAP和fos蛋白表达的影响

目的研究16Hz，90dB和130dB次声作用后，大鼠海马瞬时感受电位香草酸家族4（TRPV4）通道蛋白、胶质纤维酸性蛋白（GFAP）和fos蛋白的表达情况。方法16Hz，90dB和130dB次声作用于大鼠，2h／d，作用7d后采用免疫组织化学染色方法，观察大鼠海马中TRPV4蛋白、GFAP和fos蛋白表达的情况。结果16Hz，130dB次声作用7d后，与对照组相比较大鼠海马中显著表达TRPV4阳性神经元，GFAP阳性星形胶质细胞和fos阳性神经元（P〈0．05），三者分布一致，关系密切；90dB组大鼠的上述三种蛋白表达均较130dB组弱（P〈0．05）。结论16Hz，90dB和130dB次声作用可以引起大鼠海马TRPV4阳性细胞表达增多，且能够激活神经元和星形胶质细胞。     

痛刺激后大鼠三叉神经尾侧亚核星形细胞和神经元相互关系的电镜观察

目的：观察大鼠上唇皮下注射福尔马林后，三叉神经尾侧亚核（Sp5C)内反应性星形细胞和神经元之间相互关系的超微结构。方法：用DAB染色的抗胶质原纤维酸性蛋白（GFAP）、抗connexin43(Cx43）和金颗粒标记抗Cx32双标记免疫电镜方法。结果：电镜下观察到Sp5C内反应性星形细胞和神经元之间存在4种联系结构：第一种是突触样结构；第二种是三种成分的突触复合体，第三种是缝隙连接；第四种是由Cx32和Cx43构成的异源性缝隙连接（HGJ）。HGJ表现为两侧膜增厚，Cx43阳性物质和Cx32阳性金颗粒分别位于星形细胞和神经元一侧，痛刺激后HGJ数明明显增加。结论：神经元和星形细胞之间有多种信息通道，HGJ可能是一种快速，适应性信息通道。Sp5C星形细胞可能通过HGJ调节神经元的活动，共同参与中枢神经系统对刺激反应的调节。     

脑发育异常性癫痫脑组织的MDR-1、MRP和GFAP表达一例并文献复习

目的：观察灰质异位性难治性癫痫中多药耐药基因蛋白（MDR-1）、耐药基因相关蛋白（MRP）及胶质纤维酸性蛋白（GFAP）在发育异常性脑组织中的表达情况。方法：对确诊为脑灰质异位性症状性癫痫患者手术切除下病灶组织行免疫组化染色，观察脑组织中MDR-1、MRP及GFAP的表达情况。结果：在手术切除的病灶组织中，除一些增生性的星形胶质细胞同时具上述三种蛋白的阳性标记外，在一些异形的神经元内同时还存在有MDR-1和MRP的阳性表达。结论：灰质异位性难治性癫痫中MDR-1、MRP及GFAP不仅可在一些反应性的星形胶质细胞中表达，而且还可在一些发育异常的异形神经元中表达。     

星形胶质细胞在实验性癫痫中的作用及意义探讨

目的 探讨星形胶质细胞在实验性癫痫中的作用及意义。方法 建立大鼠杏仁核点燃癫痫模型．应用免疫组化方法检测大鼠点燃癫痫后海马及颞叶GFAP免疫反应阳性细胞。并测定其细胞数、面积、周长、积分光密度。结果 实验组海马回和颞叶皮层GFAP免疫反应阳性的胶质细胞与对照组和手术对照组相比，数量明显增多、胞体截面积增大、突起及其分支增多、GFAP免疫反应性增强。结论 星形胶质细胞的增多是癫痫后脑损伤的代偿结果，另一方面．又可以反过来成为影响癫痫病程发展的原因。     

Interactions between limbic, thalamo-striatal-cortical, and central autonomic pathways during epileptic seizure progression.

We used immunocytochemistry to determine the regional and temporal distribution of Fos protein expression in awake and unrestrained rats after a unilateral stereotaxic microinjection of a cholinergic agonist, carbachol, in the thalamic ventroposterolateral and reticular nuclei, previously shown to cause limbic and generalized convulsive seizures. The microinjection of carbachol elicits behavioral alterations including immobilization, staring, facial and jaw clonus, rearing, and falling, followed by recurrent generalized convulsive seizures, and a pattern of c-fos expression throughout the brain. In addition to the hypothalamic paraventricular and supraoptic nuclei, the initial induction of c-fos expression was observed as early as 15 minutes after the carbachol microinjection, in the piriform and entorhinal cortices, the thalamic paraventricular, the supramammilary, the lateral parabrachial nuclei, and the central gray. From 30 minutes to 2 hours, corresponding to the occurrence of motor expression of limbic and recurrent generalized convulsive seizures, Fos immunoreactivity was seen in a number of functionally related brain regions including the hippocampus, the amygdala, and the anterior thalamic nucleus (limbic system); the thalamus, the basal ganglia, and the cortex (thalamo-striatal-cortical system); and the hypothalamus, the central nucleus of the amygdala, the pons, and the medulla (central autonomic system). On the basis of the present results showing regional and temporal c-fos expression and well known neuroanatomical connections, we have constructed a neural network relating the limbic, thalamo-striatal-cortical, and central autonomic systems. This analysis provides, for the first time, neuronal circuits and pathways relating epilepsy-elicited behavioral expression of convulsive seizures and adaptive homeostatic responses and could serve as a basis for studying central autonomic regulation during epileptic disorders.     

雌激素对癫癎大鼠中枢神经系统Fos蛋白表达的影响及其作用部位的研究

目的 :了解雌激素致作用的部位。方法 :利用红藻氨酸和 6 氟二乙酯致的两种大鼠癫模型 ,应用免疫组化法检测单纯致及给予雌激素后再致大鼠海马 ,大脑皮质 ,纹状体的Fos表达。结果 :两种模型单纯致组海马、皮质、纹状体Fos表达较正常组显著增高 (P <0 0 1)。给予雌二醇 (E2 )后再致 ,红藻氨酸模型中海马 ,皮质Fos表达较单纯致组增加 (P <0 0 1,P <0 0 5 ) ;而 6 氟二乙酯模型中无变化。结论 :E2 促进癫发作的敏感性不是普遍增加全脑神经元兴奋的结果 ,而是与海马有关     

Nestin expression in hippocampal astrocytes after injury depends on the age of the hippocampus

Analysis of the expression of nestin in reactive astrocytes facilitates quantification of the extent of activation of astrocytes after injury in the mature CNS. We hypothesize that the capability of astrocytes for re-expressing nestin in response to CNS injury diminishes as a function of age. We quantified astrocytes positive for S-100beta protein, glial fibrillary acidic protein (GFAP) and nestin in the hippocampus of young adult, middle-aged, and aged Fischer 344 rats after an intracerebroventricular kainic acid (KA) administration. In all age groups, KA administration induced degeneration of CA3 pyramidal neurons, which led to a significant deafferentation in the CA1 region. The KA-induced neurodegeneration and deafferentation resulted in an increased population of astrocytes positive for S-100beta and glial fibrillary acidic protein (GFAP) in all age groups. Interestingly, these increases were highly comparable across the three age groups. However, in areas of both neurodegeneration and deafferentation, the overall numerical density of nestin-positive reactive astrocytes varied depending on the age at the time of injury with noticeably decreased numerical density in the injured middle-aged and aged hippocampus. In contrast, nestin-immunoreactive radial glia framework after lesion is not impaired with aging in the ependymal lining of the CA3 region.     

FOS induction in brain associated with seizure and sustained cortical vasodilation in anesthetized rat.

PURPOSE: By estimating the anatomical distribution of neurons expressing c-fos protein, we sought to establish whether the intrinsic neural systems known to be implicated in the cerebrovascular regulation were activated during the increase in cortical blood flow associated with epileptic seizures. METHODS: A single unilateral microinjection of the cholinergic agonist, carbachol, in the thalamic generalized convulsive seizure area was used in anesthetized rats to elicit recurrent episodes of electrocortical epileptiform activity and an increase in cortical blood flow. Neuronal expression of Fos protein was analyzed to identify activated brain regions. RESULTS: We identified two cortical vasodilatory responses: a sustained cortical vasodilatory response associated with the continuous low-frequency, high-amplitude spiking and a transient cortical vasodilatory response invariably related to the recurrent spike-burst activity. The sustained cortical blood flow began to increase at 55-65 min, remaining significantly (p < 0.05) increased and reaching at the end of the experiment < or =182+/-17% of the prestimulated control. The electrocortical epileptic activity and the cerebral cortical vasodilation were associated with a marked increase in Fos immunoreactivity in the entorhinal and piriform cortices, the dentate gyrus, the hippocampus, and the amygdala. Fos-positive neurons also were found in specific thalamic nuclei, the cerebral cortex, the caudate-putamen, the hypothalamus, the pontine parabrachial nuclei, the dorsal raphe, and the rostral ventrolateral medulla. CONCLUSIONS: These results provide evidence that convulsive seizures elicited by cholinergic stimulation of the thalamus, in addition to limbic and somatic motor systems, activate central autonomic nuclei and their pathways, including those implicated in cerebrovascular regulation.     

Differential expression of S100beta and glial fibrillary acidic protein in the hippocampus after kainic acid-induced lesions and mossy fiber sprouting in adult rat

The expression of S100beta and glial fibrillary acidic protein (GFAP) was analyzed following bilateral injection of kainic acid (KA), a glutamate derivative, into the CA3 region of the adult rat hippocampus. This treatment produces a progressive degeneration of the pyramidal neurons of the hippocampus while sparing the granule cells of the dentate gyrus which undergo sprouting of their axons in the supragranular layer. Messenger RNA and protein levels were measured, by Northern blot and ELISA, in the hippocampus of lesioned and sham-operated rats 1, 7, and 30 days after KA injection. A significant increase of GFAP and its mRNA was demonstrated at each time point, whereas S100beta mRNA levels were significantly enhanced only 30 days after the KA injection and the levels of S100beta protein remained unchanged at all time points. However, when analyzed by immunohistochemistry the S100beta showed clear changes in its expression and distribution depending on the region considered. One month after KA injection, S100beta immunoreactivity was considerably reduced in the stratum radiatum of CA3 region, but there was increased S100beta immunoreactivity in the stratum moleculare. In particular, a notable band of S100beta positive, hypertrophic astrocytes appeared in the supragranular layer of the dentate gyrus where the sprouting of mossy fiber collaterals was detected by Timm's staining. These data show for the first time that an increase in S100beta expression in subpopulations of reactive astrocytes may be involved in the structural reorganization of the hippocampus following KA-induced neurodegeneration.     

Kainic acid-induced convulsions cause prolonged changes in the chondroitin sulfate proteoglycans neurocan and phosphacan in the limbic structures

Systemic administration of kainic acid induces repeated convulsive seizures (KA convulsions) that result in neuropathological changes similar to temporal lobe epilepsy and the appearance of spontaneous recurrent seizures (SRS). The appearance of SRS is considered a result of the remodeling of neuronal networks following neuronal degeneration. We investigated the changes in chondroitin sulfate proteoglycans (CSPGs) in the limbic structures after KA convulsions in the rat using monoclonal antibodies 1G2, which recognizes full-length neurocan and the C-terminal half of neurocan, neurocan C, and 6B4, which recognize phosphacan and protein tyrosine phosphatase zeta. After KA convulsions, full-length neurocan appeared by 24 h and reached a peak by 48 to 72 h, whereas phosphacan decreased within 24 h in the hippocampus. In immunohistochemistry, neurocan increased in the limbic structures coincident with the appearance of reactive astrocytes. Phosphacan decreased coincident with pyramidal cell loss in the hippocampus, and the number of phosphacan-positive perineuronal nets around parvalbumin neurons decreased, whereas parvalbumin neurons were relatively conserved. In contrast, phosphacan increased in the entorhinal and piriform cortices in correlation with the severity of neuronal loss. Both neurocan and phosphacan recovered to the control level by 8 weeks after KA convulsions in some rats, but the changes in neurocan and phosphacan described above still persisted in more than half the rats. The results indicate that KA convulsions induce prolonged changes in neurocan and phosphacan similar to those in the developing rat brain and suggest a role of these CSPGs in the remodeling of neuronal networks related to the establishment or enhancement of epileptogenesis.     

大鼠延髓内脏带与下丘脑室旁核、视上核往返投射通路参与高渗调节反应的研究

目的探讨延髓内脏带(MVZ)与下丘脑室旁核(PVN)和视上核(SON)之间是否存在往返渗透压投射通路。方法通过给予大鼠饮用3%氯化钠的方法制作高渗刺激模型,并用WGA-HRP逆行追踪、抗Fos、抗酪氨酸羟化酶(TH)或加压素(VP)及胶质纤维酸性蛋白(GFAP)免疫组织化学相结合的四重标记方法,观察MVZ、PVN和SON中WGA-HRP、Fos、TH、VP和GFAP阳性分布及表达状况。结果高渗刺激后MVZ、PVN和SON内Fos阳性细胞明显增多;GFAP阳性结构也明显增多,其分布与Fos阳性细胞分布基本一致,表现为胞体肥大、突起粗长。星形胶质细胞(AST)紧密包绕在神经元周围形成神经元-AST复合体(N-ASC)。结论神经元和AST以N-ASC的形式共同参与渗透压调节反应,体内存在MVZ和SON或PVN之间往返的渗透压调节通路。