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。     

红藻氨酸致痫大鼠海马Fos和GFAP的共同表达

目的 研究红藻氨酸（kainic acid,KA)诱导大鼠癫痫发作后海马（hippocampus,HI)内神经元和星形胶质细胞的时空效应性反应变化。方法 大鼠侧脑室内注射KA，用抗即刻早期基因Fos蛋白和抗胶质原纤维酸性蛋白（GFAP）的双重免疫荧光组织化学方法结合激光共聚焦显微镜技术，显示痫性发作后HI同一部位内反应性神经元与星形胶质细胞的分布。结果 KA诱导大鼠癫痫发作，HI内的Fos阳性神经元和GFAP阳性星形胶质细胞明显增多。两分布范围基本一致，且癫痫诱发30min后GFAP开始增多，1h达高峰；1h后Fos阳性产物开始增多；2h达高峰；部分Fos阳性神经元周围有GFAP免疫反应产物包绕，显示反应性神经元（Fos阳性）与反应性星形胶质细胞（GFAP阳性）之间关系密切。结论 HI内的神经元和星形胶质细胞与癫痫发作直接相关且存在相互关系。可能共同参与癫痫的发生及其调节。     

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。     

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.     

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。     

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。     

胍丁胺对戊四氮诱导慢性癫癎大鼠的保护作用和海马星形胶质细胞表达的影响

目的:探讨不同剂量胍丁胺对戊四氮诱导的慢性癫癎大鼠模型的保护作用及对海马区星形胶质细胞表达的影响。方法:连续28 d腹腔注射戊四氮35 mg.kg-1建立大鼠慢性癫癎模型。不同剂量胍丁胺(20、40、80 mg.kg-1)进行干预。观察大鼠癫癎发作行为学及海马的形态学变化,检测海马星形胶质细胞的表达。结果:胍丁胺40、80 mg.kg-1可降低癫癎发作的日均等级评分,减少海马神经元丢失及星形胶质细胞增生。结论:胍丁胺40、80 mg.kg-1可抑制慢性癫癎大鼠发作,降低惊厥发作后海马星形胶质细胞的异常增生及神经元损伤。     

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。     

Long‐lasting pro‐ictogenic effects induced in vivo by rat brain exposure to serum albumin in the absence of concomitant pathology

Purpose: Dysfunction of the blood–brain barrier (BBB) is a common finding during seizures or following epileptogenic brain injuries, and experimentally induced BBB opening promotes seizures both in naive and epileptic animals. Brain albumin extravasation was reported to promote hyperexcitability by inducing astrocytes dysfunction. To provide in vivo evidence for a direct role of extravasated serum albumin in seizures independently on the pathologic context, we did the following: (1) quantified the amount of serum albumin extravasated in the rat brain parenchyma during status epilepticus (SE); (2) reproduced a similar concentration in the hippocampus by intracerebroventricular (i.c.v.) albumin injection in naive rats; (3) measured electroencephalography (EEG) activity in these rats, their susceptibility to kainic acid (KA)–induced seizures, and their hippocampal afterdischarge threshold (ADT). Methods: Brain albumin concentration was measured in the rat hippocampus and other forebrain regions 2 and 24 h after SE by western blot analysis. Brain distribution of serum albumin or fluorescein isothiocyanate (FITC)‐albumin was studied by immunohistochemistry and immunofluorescence, respectively. Naive rats were injected with rat albumin or FITC‐albumin, i.c.v., to mimic the brain concentration attained after SE, or with dextran used as control. Inflammation was evaluated by immunohistochemistry by measuring glial induction of interleukin (IL)‐1β. Western blot analysis was used to measure inward rectifying potassium channel subunit Kir4.1 protein levels in the hippocampus. Seizures were induced in rats by intrahippocampal injection of 80 ng KA and quantified by EEG analysis, 2 or 24 h after rat albumin or dextran administration. ADT was measured by electrical stimulation of the hippocampus 3 months after albumin injection. In these rats, EEG was continuously monitored for 2 weeks to search for spontaneous seizures. Key Findings: The hippocampal serum albumin concentration 24 h post‐SE was 0.76 ± 0.21 μm . Similar concentrations were measured in other forebrain regions, whereas no changes were found in cerebellum. The hippocampal albumin concentration was similarly reproduced in naive rats by i.c.v. administration of 500 μg/4 μl rat albumin: albumin was predominantly detected extracellularly 2 h after injection, whereas at 24 h it was visible inside pyramidal neurons and in only a few scattered chondroitin sulphate proteoglycan (NG2)‐positive cells, but not in glial fibrillary acidic protein (GFAP)‐positive astrocytes or CR‐3 complement receptor (OX‐42)‐positive microglia. The presence of albumin in naive rat hippocampus was associated with induced IL‐1β in GFAP‐positive astrocytes and a concomitant tissue down‐regulation of Kir4.1. Spiking activity was evoked by albumin in the hippocampus lasting for 2 h. When KA was intrahippocampally applied either 2 or 24 h after albumin injection, the number of total interictal spikes in 3 h EEG recording was significantly increased by twofold on average. Three months after albumin injection, neither albumin nor inflammation was detected in brain tissue; at this time, the ADT was reduced by 50% but no spontaneous seizures were observed. Significance: Transient hippocampal exposure to albumin levels similar to those attained after prominent BBB breakdown resulted in increased seizure susceptibility and long‐term reduction in seizure threshold, but it did not evoke spontaneous seizures. These effects may be mediated by albumin‐induced astrocytes dysfunction and the associated induction of proinflammatory molecules.     

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.     

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.     

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。     

Changes of hippocampal Cu/Zn-superoxide dismutase after kainate treatment in the rat

In order to evaluate the putative role of Cu,Zn-superoxide dismutase (SOD-1) in the antioxidant defense mechanism during the neurodegenerative process, we examined the level of mRNA, the specific activity and immunocytochemical distribution for SOD-1 in the rat hippocampus after systemic injection of kainic acid (KA). Hippocampal SOD-1 mRNA levels were significantly increased by the seizure intensity 3 and 7 days after KA. These enhanced mRNA levels for SOD-1 were consistent with the increased specific activities for SOD-1, suggesting that the superoxide radical generated in neurotoxic lesion, induced SOD-1 mRNA. The CA1 and CA3 neurons lost their SOD-1-like immunoreactivity, whereas SOD-1-positive glia-like cells mainly proliferated throughout the CA1 sector and had an intense immunoreactivity at 3 and 7 days after KA. This immunocytochemical distribution for SOD-1-positive non-neuronal elements was similar to that for glial fibrillary acidic protein (GFAP)-positive cells. Each immunoreactivity for SOD-1-positive non-neuronal cell or GFAP in the layers of CA1 and CA3 disappeared 3 and 7 days after a maximal stage 5 seizure. On the other hand, activated microglial cells as selectively marked with the lectin occurred in the areas affected by KA-induced lesion. Double-labeling immunocytochemical analysis demonstrated the co-localization of SOD-1-positive glia-like cells and reactive astrocytes as labeled by GFAP or S-100 protein immunoreactivity. This finding suggested that the mobilization of astroglial cells for the synthesis of SOD-1 protein is a response to the KA insult designed to decrease the neurotoxicity induced by oxygen-derived free radicals. Therefore, these alterations might reflect the regulatory role of SOD-1 against oxygen-derived free radical-induced neuronal degeneration after systemic KA administration.     

Acute hyperosmotic stimulus‐induced Fos expression in neurons depends on activation of astrocytes in the supraoptic nucleus of rats

Acute hyperosmolarity induced a time‐dependent expression of Fos protein in both neurons and astrocytes of the rat supraoptic nucleus, with peak Fos expresion occurring at 45 min in astrocytes and at 90 min in neurons after hypertonic stimulation in vivo. To determine whether the two cell types were activated separately or in an integrated manner, animals were pretreated with fluorocitrate, a glial metabolic blocker or carbenoxolone, a gap junction blocker followed by an acute hypertonic stimulation similar to that of the controls. Antibodies against glial fibrillary acidic protein, connexin 43, vasopressin, and oxytocin were used in serial sections to identify the cellular elements of the supraoptic nucleus. It was found that interruption of astrocyte metabolism with fluorocitrate significantly reduced Fos protein expression in both astrocytes and neurons, whereas blockage of gap junctions with carbenoxolone clearly reduced Fos protein expression in neurons, but not in astrocytes. These results indicate that both neurons and astrocytes in the rat supraoptic nucleus are involved in regulating osmolarity. Astrocytes are activated first, whereas connexin 43 functional hemichannels in SON astrocytes are required for the subsequent activation of the neurons. © 2009 Wiley‐Liss, Inc.