Co-variation of free amino acids in human epileptogenic cortex

The concentration of free amino acids was measured in 41 surgically removed samples of human epileptogenic brain and in 7 specimens of non-epileptic brain tissue, removed during surgery for meningiomas, etc. The material was subdivided according to the neuropathological diagnosis: mild cortical dysplasia (MCD), gliosis astrocytoma infiltration and a histologically heterogeneous group. The non-tumoral epileptogenic samples had five times higher than normal concentration of ethanolamine and 50% elevated concentration of glycine. The concentration of other neurotransmitter amino acids did not differ markedly between epileptogenic and non-epileptic samples. The concentration of neurotransmitter amino acids showed a strong correlation with the enzyme neuron specific enolase (NSE) and were low in most samples with astrocytoma infiltration. On the other hand, tyrosine and leucine had higher concentrations in samples with lower NSE concentration. Factor analysis of the amino acids revealed four groups of covarying compounds in the brain samples, first, a neurotransmitter group, including aspartate, glutamate, GABA and phosphoethanolamine. Another group contained ethanolamine, glutamine, glycine and taurine. Factor analysis on corresponding extracellular amino acids showed two groups, the first being a neurotransmitter group, containing serine, taurine phosphoethanolamine and ethanolamine in addition to aspartate and glutamate. The other group consisted of asparagine, glycine, alanine, tyrosine, valine, phenylalanine, isoleucine and leucine.Special issue dedicated to Dr. Claude Baxter.     

Immunohistochemical localization of S-100 protein,glial fibrillary acidic protein,and neuron-specific enolase in the pars distalis of quail,rat, and human hypophyses

Summary In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Immunohistochemical localization of S-100 protein, glial fibrillary acidic protein, and neuron-specific enolase in the pars distalis of quail, rat, and human hypophyses

In the present study, we have localized immunohistochemically S-100 protein, glial fibrillary acidic (GFA) protein, and neuron-specific enolase (NSE) by the unlabelled antibody peroxidase-antiperoxidase technique. Special attention was paid to the influence of fixation and of pretreatment of sections with proteolytic enzymes. It appeared that the final immunostaining of a given antigen largely depends on the fixative and on the species used. Moreover, pepsin pretreatment proved to be necessary to unmask S-100 protein in quail and GFA protein in rat. S-100 protein (rat, human) and GFA protein (human) immunoreactivities were detected in the folliculo-stellate (FS) cells. In quail, S-100 protein was also found in cells, which were not arranged around a follicular lumen and, in rat, the endothelial cells were immunostained for GFA protein. Clusters of granular cells were weakly immunostained for NSE in all species. An exclusive relationship between FS cells and S-100 protein could not be ascertained from this study.     

Quantitative immunochemistry on neuronal loss, reactive gliosis and BBB damage in cortex/striatum and hippocampus/amygdala after systemic kainic acid administration

Cell specific markers were quantified in the hippocampus, the amygdala/pyriform cortex, the frontal cerebral cortex and the striatum of the rat brain after systemic administration of kainic acid. Neuron specific enolase (NSE) reflects loss of neurons, glial fibrillary acidic protein (GFAP) reflects reactive gliosis, and brain levels of serum proteins measures blood-brain-barrier permeability. While the concentration of NSE remained unaffected in the frontal cerebral cortex and the striatum, their GFAP content increased during the first three days. In the hippocampus and amygdala, NSE levels decreased significantly. GFAP levels in the hippocampus were unaffected after one day and decreased in the amygdala/pyriform cortex. After that, GFAP increased strikingly until day 9 or, in the case of amygdala/pyriform cortex, even longer. This biphasic time course for GFAP was accompanied by a decrease of S-100 during days 1-9 followed by a significant increase at day 27 above the initial level. The regional differences in GFAP and S-100 could result from the degree of neuronal degeneration, the astrocytic receptor set-up and/or effects on the blood-brain barrier.     

Epilepsy due to malformations of cortical development--correlation of clinical, MRI and Tc-99mECD SPECT findings

Malformations of cortical development (MCD) have been increasingly recognized as an important cause of intractable epilepsy. The aim of our study was to define epileptogenicity of MCDs by correlating MRI, EEG and semiology of epileptic attacks, and to determine the effect of MCD on drug resistant epilepsy. We also intended to reveal the utility of interictal single photo emission computed tomography (SPECT) in verification of MCD lesions and relative prevalence of different MCDs. Based on interictal EEG finding, semiology of the epileptic attacks and brain magnetic resonance imaging (MRI) "electroclinical epileptogenicity" of MCD was defined. Brain MRI revealed cortical dysplasia (CD) in nine patients, polymicrogyria in four patients, lissencephaly and schizencephaly in one patient each. Three patients had a combination of malformations. The localization of SPECT hypoperfusion corresponded to MCD lesion in ten (66.67%) patients. Electroclinically confirmed epileptogenicity of MCD overlapped with MR and interictal SPECT findings in fourteen (93.3%) and nine (60.0%) patients, respectively. Our study results demonstrated the MCD lesions to be highly epileptogenic and a frequent cause of intractability.     

Unchanged glutamine synthetase activity and increased NMDA receptor density in epileptic human neocortex: implications for the pathophysiology of epilepsy

We investigated whether alterations in glutamate metabolising glutamine synthetase activity occur in human epileptic neocortex, as shown previously for human epileptic hippocampus [Eid, T., Thomas, M.J., Spencer, D.D., Rundén-Pran, E., Lai, J.C.K., Malthankar, G.V., Kim, J.H., Danbolt, N.C., Ottersen, O.P., de Lanerolle, N.C., 2004. Loss of glutamine synthetase in the human epileptic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy. Lancet 363, 28-37]. Glutamine synthetase activity was equivalent in both non-epileptic and epileptic human neocortex. Epileptic tissue, however, was characterised by a 37% increase in the density of synaptosomal NMDA receptor sites compared to non-epileptic tissue, as revealed by a radioligand binding assay (B max(non-epileptic) 1.45 pmol/mg protein and B max(epileptic) 1.99 pmol/mg protein, P < 0.05). Our findings shed some doubts on a role of glutamine synthetase in the pathophysiology of epilepsy in the neocortex. However, the detection of a significantly reduced enzymatic activity in the epileptic amygdala supports the assumption that the enzyme defect is localized to the epileptic mesial temporal lobe of corresponding patients.     

Masking of epitopes in tissue sections

Summary Antisera to chicken brain antigen (CBA) isolated by hydroxyapatite chromatography from 8 M urea extracts following repeated extractions with phosphate buffer selectively decorate neurofilaments (NF) in neuronal perikarya, dendrites and axons. The antisera also reacted with GFA protein, the astrocyte-specific intermediate filament protein, as indicated by the adsorption of NF immunoreactivity following passage of the antisera through columns prepared with purified GFA protein. Moreover, the antisera stained the polypeptides of the NF triplet (70 kd, 150 kd, 200 kd) and GFA protein by the immunoblotting procedure. Monoclonal antibodies selectively decorating NF in tissue sections were isolated from a fusion of mouse myeloma cells with spleen cells of mice immunized with CBA. By the immunoblotting procedure the antibodies decorated the 150 kd NF polypeptide and GFA protein. No staining of glial filaments or any other structure on tissue sections was also observed with antibodies derived from another fusion strongly reacting with GFA protein on immunoblots. All antibodies (monoclonal and polyclonal) appeared to react with the same region of the GFA polypeptide as indicated by immunoblots of cleavage products.     

Rats with Malformations of Cortical Development Exhibit Decreased Length of AIS and Hypersensitivity to Pilocarpine-Induced Status Epilepticus

Malformations of cortical development (MCD) are critical brain development disorders associated with varied abnormalities in both anatomic structures and neural functioning. It is also a very common etiology to the epilepsy, in which the alteration on excitability of cortical neurons is hypothesized as one of important causes to the epileptic seizures. Due to the key role in regulating neuron firing properties, the plasticity of axon initial segment (AIS) was investigated in present study to further determine the relation between MCD and epilepsy. Our results showed a prolonged decrease in the length of AIS occurred in MCD animal models. Besides, the AIS was also found greatly shortened in MCD models during the acute, but not chronic phase of status epileptics compared with intact controls. Our findings of identification of AIS plasticity in MCD animal models and its hypersensitivity to status epilepsy are significant in furthering our understanding of the pathophysiological mechanisms involved in this disorder.     

Neurospecific proteins in human malignant brain tumors

The content of neurospecific proteins S-100, GFA and D2 was measured in malignant cerebral tumors by electrophoresis with the use of monospecific antisera. Concomitant measurement of proteins S-100 and GFA is a more reliable diagnostic criterion as to the tumor histogenesis than study of each protein alone. D2 protein appeared to be the most stable specific marker.     

Differential immunocytochemical staining for glial fibrillary acidic (GFA) protein,S-100 protein and glutamine synthetase in the rat subcommissural organ,nonspecialized ventricular ependyma and adjacent neuropil

Summary Antibodies raised against glial fibrillary acidic protein (GFA), S-100 protein (S100) and glutamine synthetase (GS) are currently used as glial markers. The distribution of GFA, S100 and GS in the ependyma of the rat subcommissural organ (SCO), as well as in the adjacent nonspecialized ventricular ependyma and neuropil of the periaqueductal grey matter, was studied by use of the immunocytochemical peroxidase-antiperoxidase technique. In the neuropil, GFA, S100 and GS were found in glial elements, i.e., in fibrous (GFA, S100) and protoplasmic astrocytes (S100, GS). The presence of S100 in the majority of the ventricular ependymal cells and tanycytes, and the presence of GFA in a limited number of ventricular ependymal cells and tanycytes confirm the glial nature of these cells. The absence of S100, GFA and GS from the ependymocytes of the SCO, which are considered to be modified ependymal cells, suggests either a non-astrocytic lineage of these cells or an extreme specialization of the SCO-cells as glycoprotein-synthesizing and secreting elements, a process that may have led to the disappearance of the glial markers.     

Changes in Functional Integration with the Non-Epileptic Temporal Lobe of Patients with Unilateral Mesiotemporal Epilepsy

Purpose

To investigate epilepsy-induced changes in effective connectivity between the non-epileptic amygdalo-hippocampal complex (AHC) and the rest of the brain in patients with unilateral mesiotemporal lobe epilepsy (MTLE) associated with hippocampal sclerosis (HS).

Methods

Thirty-three patients with unilateral MTLE associated with HS (20 females, mean age: 36 years, 19 left HS) and 33 adult controls matched for age and gender underwent ¹⁸F-Fluorodeoxyglucose positron emission tomography (FDG-PET). Right-HS patients'' FDG-PET data were flipped to obtain a left–epileptic–focus–lateralized group of patients. Voxels of interest (VOI) were selected within the cytoarchitectonic probabilistic maps of the non-epileptic AHC (probability level  = 100%, SPM8 Anatomy toolbox v1.7). Patients and controls were compared using VOI metabolic activity as covariate of interest to search for epilepsy-induced changes in the contribution of the non-epileptic AHC to the level of metabolic activity in other brain areas. Age, gender, duration of epilepsy, seizure type and frequency were used as covariates of no-interest for connectivity analyses.

Key findings

Significant decrease in effective connectivity was found between the non-epileptic AHC and ventral prefrontal cortical areas bilaterally, as well as with the temporal pole and the posterior cingulate cortex contralateral to HS. Significant increase in connectivity was found between the non-epileptic AHC and midline structures, such as the anterior cingulate and dorsal medial prefrontal cortices, as well as the temporo-parietal junction bilaterally. Connectivity analyses also revealed a preserved positive connectivity between the non-epileptic and the epileptic AHC in the patients'' group.

Significance

This study evidences epilepsy-induced changes in connectivity between the non-epileptic AHC and some limbic and default mode network areas. These changes in connectivity probably account for emotional, cognitive and decision-making impairments frequently observed in MTLE patients. The preserved neurometabolic connectivity between the non-epileptic and the epileptic AHC in MTLE patients is pivotal to explain the epilepsy-induced changes found in this study.     

Immunohistochemical demonstration of neuronal and astrocytic markers and oncofoetal antigens in retinoblastomas.

General opinion is that retinoblastomas, though not everyone agrees with that view. Some authors suggest that retinoblastomas are derived from a primitive retinal cell able to differentiate into both neuronal and glial cell lines. The aim of the present work was to study immunohistochemically the expression of neuronal and astrocytic markers in retinoblastomas and at the same time the presence of the oncofoetal antigens carcinoembryonic antigen (CEA) and alpha Foeto Protein (AFP), since patients with retinoblastomas often show high oncofoetal antigen in serum levels. For this purpose we employed the streptavidin-biotin immunoperoxidase technique in 13 cases of retinoblastoma to evaluate the presence and distribution of neuron-specific enolase (NSE), neurofilament protein (NF), glial fibrillary acidic protein (GFAP), S-100 protein, CEA and AFP. All 13 tumours studied stained for NSE. Seven of them showed GFAP- and S-100 positive perivascular glial cells as well as cells distributed randomly in the tumour that were interpreted as non tumour cells. All 13 retinoblastomas lacked detectable NF, CEA, and AFP. These results support the idea that retinoblastomas are neuronal tumours, although retinal glial cells may become incorporated in the tumour and proliferate in response to the tumour.     

Transfer of an avian genetic reflex epilepsy by embryonic brain graft: a tissue autonomous process?

Electroencephalographic characteristics and clinical symptoms of an avian genetic reflex epilepsy have been transferred from Fayoumi epileptic (Fepi) chickens to non-epileptic chickens by embryonic homotopic grafts of brain neuroepithelium. Transplanted tissues belonging to the prosencephalic vesicle transferred epileptic electrical features while tissues from the mesencephalic vesicle were responsible for seizure motor manifestations of the disease. Thus each of these tissues can express their own specificity when grafted separately in a normal host, but they co-operate to produce the complete epileptic phenotype when grafted together.     

Immunohistochemical identification of supportive cell types in the enteric nervous system of the rat colon and rectum

Summary The non-neuronal, supportive cells of the enteric nerve plexus were investigated in the colon and rectum of adult and developing rats by means of immunohistochemistry, utilizing antisera against GFA protein and S-100 protein. Immunoreactivity to GFA protein was almost exclusively found in cells associated with the myenteric plexus and a small number of cells within the submucous ganglia. On the other hand, the use of S-100 protein antiserum resulted in the visualization of all supportive elements in the enteric nervous system. However, two types of supportive cells could be tentatively differentiated in the enteric nerve plexus during the second week of postnatal development, using GFA protein and S-100 protein antisera; GFA protein-positive cells were clearly discernible from S-100 protein-positive cells in terms of both the morphological profiles and immunohistochemical properties. It was assumed that at least two different types of supportive cells are contained in the enteric nerve plexus. We suggest that in the enteric nervous system the terms glial cells and Schwann cells should be employed to designate the supportive cells containing GFA and S-100 proteins, and cells containing S-100 protein, respectively. We discuss the possibility that glial cells are associated with the parasympathetic preganglionic fibres directly derived from the central nervous system, while Schwann cells originate from the neural crest.     

脑脊液PCT、VEGF、S-100B、NSE、MMP及CGRP水平在病毒性脑膜炎患儿中的临床研究

目的:探讨脑脊液降钙素原(PCT)、血管内皮生长因子(VEGF)、S100B蛋白(S-100B)、神经元特异性烯醇化酶(NSE)、基质金属蛋白酶(MMP)及降钙素基因相关肽(CGRP)水平联合检测在病毒性脑炎患儿中的诊断价值和意义。方法:选取2012年6月至2014年6月收治的病毒性脑炎患儿106例作为研究组,选取同时期来我院进行健康体检的60例儿童作为对照组,比较两组脑脊液PCT、VEGF、S-100B、NSE、MMP及CGRP水平,并分析研究组依照不同层次分组后以上各指标的差异。结果:研究组脑脊液中PCT、VEGF、S-100B、NSE、MMP及CGRP水平明显高于对照组,差异具有统计学意义(P0.05);研究组重症患儿高于轻症患者,急性期患者高于非急性期患儿,差异均具有统计学意义(P0.05)。以上各指标中任意二种、三种、四种及五种联合检测,其诊断效能均在0.923-0.967之间,六种指标联合检测的效能最高为0.975。结论:病毒性脑膜炎患儿血清及脑脊液PCT、VEGF、S-100B、NSE、MMP及CGRP水平均呈现升高的趋势,且不同严重程度及分期患儿的以上指标均有一定的差异,六种指标联合检测诊断病毒性脑炎患儿的效能最高。     

MicroRNA-134/CREB/pCREB通路在癫痫中的表达及意义

目的探讨miR-134、CREB、pCREB在癫痫大鼠海马及难治性癫痫患者颞叶脑组织中的表达及意义。方法难治性癫痫患者及非癫痫对照组颞叶组织、氯化锂-匹罗卡品癫痫大鼠及空白对照组海马组织中,应用实时荧光定量PCR技术检测microRNA-134（miR-134）的表达,用Western blot方法检测CREB及p CREB的表达,用免疫组织化学方法检测人脑颞叶皮质及大鼠海马区CREB、p CREB的表达。结果与对照组相比miR-134表达在难治性癫痫患者中明显降低（P〈0.05）,在癫痫模型组中点燃后3、7、14、60 d明显降低（P〈0.05）,1 d与30 d表达降低较对照组差异无显著性（P〉0.05）;癫痫模型组CREB在3、7、14、60 d时间点明显升高（P〈0.05）、pCREB各时间点表达均高于空白对照组（P〈0.05）。结论难治性癫痫患者颞叶皮质及癫痫动物海马中miR-134表达下降,CREB、pCREB表达升高,提示其可能在癫痫发生发展机制中起重要作用。     

Neuron-specific enolase,neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

Summary Immunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.     

Immunophenotypic and Ultrastructural Validation of a New Human Glioblastoma Cell Line

1. A human glioma cell line, NG97, was established by Grippo et al. in 2001 from tissue obtained from a grade III astrocytoma (WHO, 2000). In this first study, the cell line grew as two morphologically distinct subpopulations: dendritic/spindle cells and small round cells. The injection of NG97 cells into nude mice induced an aggressive tumor characterized by: severe cytological atypia, vascular proliferation and pseudopalisading necrosis (glioblastoma multiforme features). 2. The purpose of the present study was to characterize the immunophenotype and ultrastructural aspects of this cell line, using the parental tumor, cultured cells and the xenotransplant, in order to assess its glial nature and possible divergent differentiation. 3. NG97 cells and xenotransplant expressed the main neuroglial markers (GFAP, S-100 protein, NSE and Leu-7) and showed no aberrant expression of other histogenetic markers. GFAP was similarly expressed in the parental tumor and in the cells in culture, but decreased in the xenotransplant. NSE expression was reduced in NG97 cells, but substantially recovered in the xenotransplant. This variability in expression of GFAP and NSE was interpreted as either a phenomenon of dedifferentiation or to microenvironmental selection of specific subclones. S-100 was equally expressed in the three contexts. The xenotransplant's ultrastructural features were those of a highly undifferentiated tumor. No significant immunophenotypic or ultrastructural differences between the two morphologically distinct populations were found. 4. Thus, our data demonstrate that NG97 cells constitute a pure glial-committed cell line, which may prove useful as a malignant glioma model in studies addressing pathophysiological, diagnostic and therapeutic issues.     

Enhancement of asynchronous release from fast-spiking interneuron in human and rat epileptic neocortex

Down-regulation of GABAergic inhibition may result in the generation of epileptiform activities. Besides spike-triggered synchronous GABA release, changes in asynchronous release (AR) following high-frequency discharges may further regulate epileptiform activities. In brain slices obtained from surgically removed human neocortical tissues of patients with intractable epilepsy and brain tumor, we found that AR occurred at GABAergic output synapses of fast-spiking (FS) neurons and its strength depended on the type of connections, with FS autapses showing the strongest AR. In addition, we found that AR depended on residual Ca²⁺ at presynaptic terminals but was independent of postsynaptic firing. Furthermore, AR at FS autapses was markedly elevated in human epileptic tissue as compared to non-epileptic tissue. In a rat model of epilepsy, we found similar elevation of AR at both FS autapses and synapses onto excitatory neurons. Further experiments and analysis showed that AR elevation in epileptic tissue may result from an increase in action potential amplitude in the FS neurons and elevation of residual Ca²⁺ concentration. Together, these results revealed that GABAergic AR occurred at both human and rat neocortex, and its elevation in epileptic tissue may contribute to the regulation of epileptiform activities.     

突触体素、S-100蛋白、NSE免疫反应神经纤维在人淋巴结的分布

探讨突触体素、S－100蛋白、NSE免疫反应神经纤维在人淋巴结的分布,为淋巴结的神经免疫相互作用提供形态学资料。应用免疫组织化学ABC法观察人类腹股沟、腋窝、肠系膜、肺等淋巴结40例,10％福尔马林固定,石蜡包埋组织切片。结果显示：突触体素、S－100蛋白、NSE免疫反应神经纤维呈细丝状沿被膜和门部结缔组织小梁及血管进入皮质后主要分布于副皮质区,环境淋巴小结,进一步分支到达髓质。同时在淋巴小结发生中心及副皮质区有S－100蛋白免疫反应阳性细胞。在髓质髓窦内有NSE免疫反应阳性细胞。结论;淋巴结内有突触体素、S－100蛋白、NSE免疫反应神经纤维的支配、并有S－100蛋白、NSE免疫反应阳性细胞,为淋巴结的神经免疫相互作用提供形态学资料。