RESUMEN
As part of the ACuteTox project aimed at the development of non-animal testing strategies for predicting human acute oral toxicity, aggregating brain cell cultures (AGGR) were examined for their capability to detect organ-specific toxicity. Previous multicenter evaluations of in vitro cytotoxicity showed that some 20% of the tested chemicals exhibited significantly lower in vitro toxicity as expected from in vivo toxicity data. This was supposed to be due to toxicity at supracellular (organ or system) levels. To examine the capability of AGGR to alert for potential organ-specific toxicants, concentration-response studies were carried out in AGGR for 86 chemicals, taking as endpoints the mRNA expression levels of four selected genes. The lowest observed effect concentration (LOEC) determined for each chemical was compared with the IC20 reported for the 3T3/NRU cytotoxicity assay. A LOEC lower than IC20 by at least a factor of 5 was taken to alert for organ-specific toxicity. The results showed that the frequency of alerts increased with the level of toxicity observed in AGGR. Among the chemicals identified as alert were many compounds known for their organ-specific toxicity. These findings suggest that AGGR are suitable for the detection of organ-specific toxicity and that they could, in conjunction with the 3T3/NRU cytotoxicity assay, improve the predictive capacity of in vitro toxicity testing.
Asunto(s)
Encéfalo/citología , Pruebas de Toxicidad Aguda , Alternativas a las Pruebas en Animales , Animales , Células Cultivadas , Hemo Oxigenasa (Desciclizante)/genética , Proteína Básica de Mielina/genética , Proteínas del Tejido Nervioso/genética , Proteínas de Neurofilamentos/genética , ARN Mensajero/metabolismo , RatasRESUMEN
Serum-free aggregating brain cell cultures are free-floating three-dimensional primary cell cultures able to reconstitute spontaneously a histotypic brain architecture to reproduce critical steps of brain development and to reach a high level of structural and functional maturity. This culture system offers, therefore, a unique model for neurotoxicity testing both during the development and at advanced cellular differentiation, and the high number of aggregates available combined with the excellent reproducibility of the cultures facilitates routine test procedures. This chapter presents a detailed description of the preparation, maintenance, and use of these cultures for neurotoxicity studies and a comparison of the developmental characteristics between cultures derived from the telencephalon and cultures derived from the whole brain. For culture preparation, mechanically dissociated embryonic brain tissue is used. The initial cell suspension, composed of neural stem cells, neural progenitor cells, immature postmitotic neurons, glioblasts, and microglial cells, is kept in a serum-free, chemically defined medium under continuous gyratory agitation. Spherical aggregates form spontaneously and are maintained in suspension culture for several weeks. Within the aggregates, the cells rearrange and mature, reproducing critical morphogenic events, such as migration, proliferation, differentiation, synaptogenesis, and myelination. For experimentation, replicate cultures are prepared by the randomization of aggregates from several original flasks. The high yield and reproducibility of the cultures enable multiparametric endpoint analyses, including "omics" approaches.
Asunto(s)
Encéfalo/citología , Técnicas de Cultivo de Célula , Animales , Agregación Celular , Células Cultivadas , Medio de Cultivo Libre de Suero , Embrión de Mamíferos/citología , Femenino , Neuronas/citología , Embarazo , RatasRESUMEN
Ochratoxin A (OTA), a fungal contaminant of basic food commodities, is known to be highly cytotoxic, but the pathways underlying adverse effects at subcytotoxic concentrations remain to be elucidated. Recent reports indicate that OTA affects cell cycle regulation. Therefore, 3D brain cell cultures were used to study OTA effects on mitotically active neural stem/progenitor cells, comparing highly differentiated cultures with their immature counterparts. Changes in the rate of DNA synthesis were related to early changes in the mRNA expression of neural stem/progenitor cell markers. OTA at 10nM, a concentration below the cytotoxic level, was ineffective in immature cultures, whereas in mature cultures it significantly decreased the rate of DNA synthesis together with the mRNA expression of key transcriptional regulators such as Sox2, Mash1, Hes5, and Gli1; the cell cycle activator cyclin D2; the phenotypic markers nestin, doublecortin, and PDGFRα. These effects were largely prevented by Sonic hedgehog (Shh) peptide (500ngml(-1)) administration, indicating that OTA impaired the Shh pathway and the Sox2 regulatory transcription factor critical for stem cell self-renewal. Similar adverse effects of OTA in vivo might perturb the regulation of stem cell proliferation in the adult brain and in other organs exhibiting homeostatic and/or regenerative cell proliferation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Homeostasis/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Ocratoxinas/toxicidad , Telencéfalo/efectos de los fármacos , Animales , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Medio de Cultivo Libre de Suero , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Proteína Doblecortina , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telencéfalo/citología , Telencéfalo/metabolismo , Factores de Transcripción/metabolismoRESUMEN
To study inflammatory reactions occurring in relation to demyelination, aggregating rat brain cell cultures were subjected to three different demyelinating insults, i.e., (i) lysophosphatidylcholine (LPC), (ii) interferon-gamma combined with lipopolysaccharide (IFN-gamma+LPS), and (iii) anti-MOG antibodies plus complement (alpha-MOG+C). Demyelination was assessed by measuring the expression of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), and the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). The accompanying inflammatory reactions were examined by the quantification of microglia-specific staining, by immunostaining for glial fibrillary acidic protein (GFAP), and by measuring the mRNA expression of a panel of inflammation-related genes. It was found that all three demyelinating insults decreased the expression of MBP and MOG, and induced microglial reactivity. LPC and alpha-MOG+C, but not IFN-gamma+LPS, decreased CNP activity; they also caused the appearance of macrophagic microglia, and increased GFAP staining indicating astrogliosis. LPC affected also the integrity of neurons and astrocytes. LPC and IFN-gamma+LPS upregulated the expression of the inflammation-related genes IL-6, TNF-alpha, Ccl5, Cxcl1, and iNOS, although to different degrees. Other inflammatory markers were upregulated by only one of the three insults, e.g., Cxcl2 by LPC; IL-1beta and IL-15 by IFN-gamma+LPS; and IFN-gamma by alpha-MOG+C. These findings indicate that each of the three demyelinating insults caused distinct patterns of demyelination and inflammatory reactivity, and that of the demyelinating agents tested only LPC exhibited general toxicity.
Asunto(s)
Encéfalo/citología , Citocinas/metabolismo , Enfermedades Desmielinizantes/metabolismo , Neuronas/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Anticuerpos/farmacología , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Citocinas/genética , Enfermedades Desmielinizantes/fisiopatología , Interacciones Farmacológicas , Embrión de Mamíferos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Factores Inmunológicos/genética , Factores Inmunológicos/metabolismo , Interferón gamma/farmacología , Lectinas/metabolismo , Lisofosfatidilcolinas/farmacología , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Asociada a Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Neuronas/efectos de los fármacos , Polisacáridos/farmacología , ARN Mensajero/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
BACKGROUND: Brain inflammation plays a central role in numerous brain pathologies, including multiple sclerosis (MS). Microglial cells and astrocytes are the effector cells of neuroinflammation. They can be activated also by agents such as interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS). Peroxisome proliferator-associated receptor (PPAR) pathways are involved in the control of the inflammatory processes, and PPAR-beta seems to play an important role in the regulation of central inflammation. In addition, PPAR-beta agonists were shown to have trophic effects on oligodendrocytes in vitro, and to confer partial protection in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In the present work, a three-dimensional brain cell culture system was used as in vitro model to study antibody-induced demyelination and inflammatory responses. GW 501516, a specific PPAR-beta agonist, was examined for its capacity to protect from antibody-mediated demyelination and to prevent inflammatory responses induced by IFN-gamma and LPS. METHODS: Aggregating brain cells cultures were prepared from embryonal rat brain, and used to study the inflammatory responses triggered by IFN-gamma and LPS and by antibody-mediated demyelination induced by antibodies directed against myelin-oligodendrocyte glycoprotein (MOG). The effects of GW 501516 on cellular responses were characterized by the quantification of the mRNA expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), inducible NO synthase (i-NOS), PPAR-beta, PPAR-gamma, glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), and high molecular weight neurofilament protein (NF-H). GFAP expression was also examined by immunocytochemistry, and microglial cells were visualized by isolectin B4 (IB4) and ED1 labeling. RESULTS: GW 501516 decreased the IFN-gamma-induced up-regulation of TNF-alpha and iNOS in accord with the proposed anti-inflammatory effects of this PPAR-beta agonist. However, it increased IL-6 m-RNA expression. In demyelinating cultures, reactivity of both microglial cells and astrocytes was observed, while the expression of the inflammatory cytokines and iNOS remained unaffected. Furthermore, GW 501516 did not protect against the demyelination-induced changes in gene expression. CONCLUSION: Although GW 501516 showed anti-inflammatory activity, it did not protect against antibody-mediated demyelination. This suggests that the protective effects of PPAR-beta agonists observed in vivo can be attributed to their anti-inflammatory properties rather than to a direct protective or trophic effect on oligodendrocytes.
Asunto(s)
Antiinflamatorios/farmacología , Anticuerpos/inmunología , Enfermedades Desmielinizantes , Encefalitis , PPAR-beta/agonistas , Tiazoles/farmacología , Animales , Antiinflamatorios/uso terapéutico , Astrocitos/citología , Astrocitos/metabolismo , Encéfalo/citología , Células Cultivadas , Enfermedades Desmielinizantes/tratamiento farmacológico , Enfermedades Desmielinizantes/inmunología , Enfermedades Desmielinizantes/patología , Encefalitis/tratamiento farmacológico , Encefalitis/inmunología , Encefalitis/patología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Interferón gamma/inmunología , Interferón gamma/farmacología , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Microglía/citología , Microglía/metabolismo , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Proteínas de Neurofilamentos/genética , Proteínas de Neurofilamentos/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , PPAR-beta/genética , Ratas , Tiazoles/uso terapéutico , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Hyperammonemic disorders in pediatric patients lead to poorly understood irreversible effects on the developing brain that may be life-threatening. We showed previously that some of these NH4+-induced irreversible effects might be due to impairment of axonal growth that can be protected under ammonium exposure by creatine co-treatment. The aim of the present work was thus to analyse how the genes of arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), allowing creatine synthesis, as well as of the creatine transporter SLC6A8, allowing creatine uptake into cells, are regulated in rat brain cells under NH4+ exposure. Reaggregated brain cell three-dimensional cultures exposed to NH4Cl were used as an experimental model of hyperammonemia in the developing central nervous system (CNS). We show here that NH4+ exposure differentially alters AGAT, GAMT and SLC6A8 regulation, in terms of both gene expression and protein activity, in a cell type-specific manner. In particular, we demonstrate that NH4+ exposure decreases both creatine and its synthesis intermediate, guanidinoacetate, in brain cells, probably through the inhibition of AGAT enzymatic activity. Our work also suggests that oligodendrocytes are major actors in the brain in terms of creatine synthesis, trafficking and uptake, which might be affected by hyperammonemia. Finally, we show that NH4+ exposure induces SLC6A8 in astrocytes. This suggests that hyperammonemia increases blood-brain barrier permeability for creatine. This is normally limited due to the absence of SLC6A8 from the astrocyte feet lining microcapillary endothelial cells, and thus creatine supplementation may protect the developing CNS of hyperammonemic patients.
Asunto(s)
Encéfalo/metabolismo , Creatina/biosíntesis , Creatina/deficiencia , Proteínas de Transporte de Membrana/metabolismo , Compuestos de Amonio Cuaternario/toxicidad , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Encéfalo/citología , Encéfalo/embriología , Técnicas de Cultivo de Célula/métodos , Células Cultivadas , Creatina/genética , Femenino , Proteínas de Transporte de Membrana/biosíntesis , Proteínas de Transporte de Membrana/genética , Embarazo , RatasRESUMEN
There is a need for more efficient methods giving insight into the complex mechanisms of neurotoxicity. Testing strategies including in vitro methods have been proposed to comply with this requirement. With the present study we aimed to develop a novel in vitro approach which mimics in vivo complexity, detects neurotoxicity comprehensively, and provides mechanistic insight. For this purpose we combined rat primary re-aggregating brain cell cultures with a mass spectrometry (MS)-based metabolomics approach. For the proof of principle we treated developing re-aggregating brain cell cultures for 48 h with the neurotoxicant methyl mercury chloride (0.1-100 microM) and the brain stimulant caffeine (1-100 microM) and acquired cellular metabolic profiles. To detect toxicant-induced metabolic alterations the profiles were analysed using commercial software which revealed patterns in the multi-parametric dataset by principal component analyses (PCA), and recognised the most significantly altered metabolites. PCA revealed concentration-dependent cluster formations for methyl mercury chloride (0.1-1 microM), and treatment-dependent cluster formations for caffeine (1-100 microM) at sub-cytotoxic concentrations. Four relevant metabolites responsible for the concentration-dependent alterations following methyl mercury chloride treatment could be identified using MS-MS fragmentation analysis. These were gamma-aminobutyric acid, choline, glutamine, creatine and spermine. Their respective mass ion intensities demonstrated metabolic alterations in line with the literature and suggest that the metabolites could be biomarkers for mechanisms of neurotoxicity or neuroprotection. In addition, we evaluated whether the approach could identify neurotoxic potential by testing eight compounds which have target organ toxicity in the liver, kidney or brain at sub-cytotoxic concentrations. PCA revealed cluster formations largely dependent on target organ toxicity indicating possible potential for the development of a neurotoxicity prediction model. With such results it could be useful to perform a validation study to determine the reliability, relevance and applicability of this approach to neurotoxicity screening. Thus, for the first time we show the benefits and utility of in vitro metabolomics to comprehensively detect neurotoxicity and to discover new biomarkers.
Asunto(s)
Cafeína/toxicidad , Estimulantes del Sistema Nervioso Central/toxicidad , Cloruro de Mercurio/toxicidad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotoxinas/toxicidad , Animales , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina/metabolismo , Creatina/metabolismo , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Glutamina/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Análisis de Componente Principal , Ratas , Espermina/metabolismo , Espectrometría de Masas en Tándem/métodos , Telencéfalo/citología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Despite the widespread use of Cannabis as recreational drug or as medicine, little is known about its toxicity. The accumulation, metabolism and toxicity of THC were analyzed 10 days after a single treatment, and after repeated exposures during 10 days. Mixed-cell aggregate cultures of fetal rat telencephalon were used as in vitro model, as well as aggregates enriched either in neurons or in glial cells. It was found that THC accumulated preferentially in neurons, and that glia-neuron interactions decreased THC accumulation. The quantification of 11-OH-THC and of THC-COOH showed that brain aggregates were capable of THC metabolism. No cell-type difference was found for the metabolite 11-OH-THC, whereas the THC-COOH content was higher in mixed-cell cultures. No cell death was found at THC concentrations of 2 microM in single treatment and of 1 microM and 2 microM in repeated treatments. Neurons, and particularly GABAergic neurons, were most sensitive to THC. Only the GABAergic marker was affected after the single treatment, whereas the GABAergic, cholinergic and astrocytic markers were decreased after the repeated treatments. JWH 015, a CB2 receptor agonist, showed effects similar to THC, whereas ACEA, a CB1 receptor agonist, had no effect. The expression of the cytokine IL-6 was upregulated 48 h after the single treatment with 5 microM of THC or JWH 015, whereas the expression of TNF-alpha remained unchanged. These results suggest that the adverse effects of THC were related either to THC accumulation or to cannabinoid receptor activation and associated with IL-6 upregulation.
Asunto(s)
Encéfalo/patología , Dronabinol/metabolismo , Dronabinol/toxicidad , Animales , Encéfalo/citología , Cannabinoides/metabolismo , Agregación Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Cromatografía Líquida de Alta Presión , Dronabinol/análisis , Femenino , GTP Fosfohidrolasas/metabolismo , Glutamato Descarboxilasa/metabolismo , Interleucina-6/biosíntesis , L-Lactato Deshidrogenasa/metabolismo , Espectrometría de Masas , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Embarazo , Ratas , Receptor Cannabinoide CB1/agonistas , Receptor Cannabinoide CB2/agonistas , Estándares de Referencia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solventes , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
This is the report of the first workshop on Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies, held in Ispra, Italy, on 19-21 April 2005. The workshop was hosted by the European Centre for the Validation of Alternative Methods (ECVAM) and jointly organized by ECVAM, the European Chemical Industry Council, and the Johns Hopkins University Center for Alternatives to Animal Testing. The primary aim of the workshop was to identify and catalog potential methods that could be used to assess how data from in vitro alternative methods could help to predict and identify DNT hazards. Working groups focused on two different aspects: a) details on the science available in the field of DNT, including discussions on the models available to capture the critical DNT mechanisms and processes, and b) policy and strategy aspects to assess the integration of alternative methods in a regulatory framework. This report summarizes these discussions and details the recommendations and priorities for future work.
Asunto(s)
Alternativas a las Pruebas en Animales/normas , Modelos Biológicos , Sistema Nervioso/efectos de los fármacos , Pruebas de Toxicidad/métodos , Xenobióticos/toxicidad , Alternativas a las Pruebas en Animales/legislación & jurisprudencia , Animales , Humanos , Técnicas In Vitro , Sistema Nervioso/embriología , Sistema Nervioso/crecimiento & desarrollo , Estudios de Validación como AsuntoRESUMEN
Aggregating brain cell cultures are three-dimensional primary cell cultures derived from embryonal rat brain cells. Within 3-4 weeks in culture under continuous agitation, they exhibit organotypic structures and functions. The transient arrest of agitation induced adverse effects comparable with those occurring in the ischemic brain in vivo. This culture system therefore offers a suitable in vitro model for the elucidation of ischemia-related pathogenic processes in the brain.
Asunto(s)
Encéfalo/patología , Accidente Cerebrovascular/patología , Alternativas a las Pruebas en Animales , Animales , Encéfalo/embriología , Agregación Celular , Técnicas de Cultivo de Célula/métodos , Muerte Celular , Humanos , Ratones , Persona de Mediana Edad , Ratas , Factores de Riesgo , Accidente Cerebrovascular/epidemiología , Células Tumorales Cultivadas/patologíaRESUMEN
The incidence of neurodegenerative disease like Parkinson's disease and Alzheimer's disease (AD) increases dramatically with age; only a small percentage is directly related to familial forms. The etiology of the most abundant, sporadic forms is complex and multifactorial, involving both genetic and environmental factors. Several environmental pollutants have been associated with neurodegenerative disorders. The present article focuses on results obtained in experimental neurotoxicology studies that indicate a potential pathogenic role of lead and mercury in the development of neurodegenerative diseases. Both heavy metals have been shown to interfere with a multitude of intracellular targets, thereby contributing to several pathogenic processes typical of neurodegenerative disorders, including mitochondrial dysfunction, oxidative stress, deregulation of protein turnover, and brain inflammation. Exposure to heavy metals early in development can precondition the brain for developing a neurodegenerative disease later in life. Alternatively, heavy metals can exert their adverse effects through acute neurotoxicity or through slow accumulation during prolonged periods of life. The pro-oxidant effects of heavy metals can exacerbate the age-related increase in oxidative stress that is related to the decline of the antioxidant defense systems. Brain inflammatory reactions also generate oxidative stress. Chronic inflammation can contribute to the formation of the senile plaques that are typical for AD. In accord with this view, nonsteroidal anti-inflammatory drugs and antioxidants suppress early pathogenic processes leading to Alzheimer's disease, thus decreasing the risk of developing the disease. The effects of lead and mercury were also tested in aggregating brain-cell cultures of fetal rat telencephalon, a three-dimensional brain-cell culture system. The continuous application for 10 to 50 days of non-cytotoxic concentrations of heavy metals resulted in their accumulation in brain cells and the occurrence of delayed toxic effects. When applied at non-toxic concentrations, methylmercury, the most common environmental form of mercury, becomes neurotoxic under pro-oxidant conditions. Furthermore, lead and mercury induce glial cell reactivity, a hallmark of brain inflammation. Both mercury and lead increase the expression of the amyloid precursor protein; mercury also stimulates the formation of insoluble beta-amyloid, which plays a crucial role in the pathogenesis of AD and causes oxidative stress and neurotoxicity in vitro. Taken together, a considerable body of evidence suggests that the heavy metals lead and mercury contribute to the etiology of neurodegenerative diseases and emphasizes the importance of taking preventive measures in this regard.
Asunto(s)
Exposición a Riesgos Ambientales/efectos adversos , Plomo/toxicidad , Mercurio/toxicidad , Enfermedades Neurodegenerativas/inducido químicamente , Encéfalo/patología , Humanos , Enfermedades Neurodegenerativas/patologíaRESUMEN
By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.
Asunto(s)
Anticuerpos/efectos adversos , Cristalinas/metabolismo , Enfermedades Desmielinizantes/metabolismo , Oligodendroglía/efectos de los fármacos , Telencéfalo/citología , Factor de Necrosis Tumoral alfa/metabolismo , 2',3'-Nucleótido Cíclico Fosfodiesterasas/metabolismo , Animales , Apoptosis/fisiología , Western Blotting/métodos , Células Cultivadas , Proteínas del Sistema Complemento/efectos adversos , Enfermedades Desmielinizantes/inducido químicamente , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Embrión de Mamíferos , Proteína Ácida Fibrilar de la Glía/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Hemo-Oxigenasa 1 , Inmunohistoquímica/métodos , Hibridación in Situ/métodos , Proteína Básica de Mielina/genética , Proteína Básica de Mielina/metabolismo , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Necrosis/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Regulación hacia ArribaRESUMEN
The abnormal vascular system of brain cancers inappropriately expresses membrane proteins, including proteolytic enzymes, ultimately resulting in blood extravasation. The production of inflammatory mediators, such as cytokines and nitric oxide, and tumor hypoxia have been implicated in these effects. We have previously shown that the activity of aminopeptidase A is increased in the abnormal vascular system of human and rat brain tumors. To study the mechanisms regulating the activities of peptidases in cerebral vasculature in brain tumors, we have developed a three-dimensional model of differentiated rat brain cells in aggregate cultures in which rat brain microvessels were incorporated. The secretion of interleukin-6 (IL-6) in the culture medium of aggregates was used as an indicator of inflammatory activation. Addition to these aggregates of C6 glioma cell medium (C6-CM) conditioned under hypoxic or normoxic conditions or serum mimicked tumor-dependent hypoxia or conditions of dysfunction of brain tumor vasculature. Hypoxic and normoxic C6-CM, but not serum, regulated peptidase activity in aggregates, and in particular it increased the activity of aminopeptidase A determined using histoenzymography. Serum, but not C6-CM, increased IL-6 production, but did not increase aminopeptidase A activity in aggregates. Thus soluble glioma-derived factors, but not serum-derived factors, induce dysfunctions of cerebral vasculature by directly regulating the activity of peptidases, not involving inflammatory activation. Tumor hypoxia is not necessary to modulate peptidase activity.
Asunto(s)
Aminopeptidasas/metabolismo , Neoplasias Encefálicas , Glioblastoma , Neovascularización Patológica/enzimología , Animales , Agregación Celular , Diferenciación Celular , Hipoxia de la Célula , Medios de Cultivo Condicionados , Femenino , Glutamil Aminopeptidasa , Técnicas In Vitro , Embarazo , Ratas , Solubilidad , Células Tumorales Cultivadas/enzimologíaRESUMEN
When freshly dissociated embryonic tissues are kept under gyratory agitation, the cells aggregate to form three-dimensional spheroids in which the cells can migrate and organize themselves, attaining maximal cellular differentiation after weeks of culture. The three-dimensional architecture of the aggregates permits direct cell-to-cell interactions and the formation of a natural cell matrix, which is fundamental to the acquisition of the histotypic properties of the aggregates. This unit describes protocols for preparing forebrain cells from embryonic rodents for aggregating cultures and maintaining these cultures to the differentiated state.
Asunto(s)
Neuronas/citología , Prosencéfalo/embriología , Animales , Técnicas de Cultivo de Célula , Femenino , Ratones , Embarazo , Prosencéfalo/citología , Ratas , Ratas Sprague-DawleyRESUMEN
Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.
Asunto(s)
Hipoglucemiantes/farmacología , Neuroglía/efectos de los fármacos , Oxigenasas , Receptores Citoplasmáticos y Nucleares/agonistas , Tiazoles/farmacología , Tiazolidinedionas , Factores de Transcripción/agonistas , Análisis de Varianza , Animales , Anticuerpos Monoclonales/farmacología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Proteínas del Sistema Complemento/inmunología , Cristalinas/efectos de los fármacos , Cristalinas/metabolismo , Enfermedades Desmielinizantes/inducido químicamente , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Embrión de Mamíferos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Proteínas de Choque Térmico/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Hemo Oxigenasa (Desciclizante) , Inmunoglobulina G/farmacología , Técnicas In Vitro , Mediadores de Inflamación/fisiología , Proteínas de la Mielina , Glicoproteína Asociada a Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito , Neuroglía/fisiología , Pioglitazona , ARN Mensajero/biosíntesis , Ratas , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia ArribaRESUMEN
Hyperammonemia in neonates and infants affects brain development and causes mental retardation. We report that ammonium impaired cholinergic axonal growth and altered localization and phosphorylation of intermediate neurofilament protein in rat reaggregated brain cell primary cultures. This effect was restricted to the phase of early maturation but did not occur after synaptogenesis. Exposure to NH4Cl decreased intracellular creatine, phosphocreatine, and ADP. We demonstrate that creatine cotreatment protected axons from ammonium toxic effects, although this did not restore high-energy phosphates. The protection by creatine was glial cell-dependent. Our findings suggest that the means to efficiently sustain CNS creatine concentration in hyperammonemic neonates and infants should be assessed to prevent impairment of axonogenesis and irreversible brain damage.
Asunto(s)
Cloruro de Amonio/toxicidad , Creatina/farmacología , Neuroglía/metabolismo , Neuronas/efectos de los fármacos , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Axones/fisiología , Diferenciación Celular/fisiología , División Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/biosíntesis , Técnicas de Cocultivo , Creatina/metabolismo , Relación Dosis-Respuesta a Droga , Proteína GAP-43/biosíntesis , Glucosa/farmacocinética , Inmunohistoquímica , Líquido Intracelular/metabolismo , Ácido Láctico/metabolismo , Proteínas de Neurofilamentos/biosíntesis , Neuroglía/citología , Neuronas/citología , Neuronas/metabolismo , Fosfocreatina/metabolismo , Compuestos de Amonio Cuaternario/farmacocinética , Ratas , Telencéfalo/citología , Telencéfalo/embriologíaRESUMEN
Despite a wealth of data on the neurotoxic effects of lead at the cellular and molecular levels, the reasons for its development-dependent neurotoxicity are still unclear. Here, the maturation-dependent effects of lead acetate were analyzed in immature and differentiated brain cells cultured in aggregates. Markers of general cytotoxicity as well as cell-type-specific markers of glial and neuronal cells showed that immature brain cells were more sensitive to lead than the differentiated counterparts, demonstrating that the development-dependent neurotoxicity of lead can be reproduced in aggregating brain cell cultures. After 10 days of treatment, astrocytes were found to be more affected by lead acetate than neurons in immature cultures, and microglial cells were strongly activated. Eleven days after cessation of the treatment, lead acetate caused a partial loss of astrocytes and an intense reactivity of the remaining ones. Furthermore, microglial cells expressed a macrophagic phenotype, and the loss of activity of neuron-specific enzymes was aggravated. In differentiated cultures, no reactive gliosis was found. It is hypothetized that the intense glial reactions (microgliosis and astrogliosis) observed in immature cultures contribute to the development-dependent neurotoxicity of lead.
Asunto(s)
Neuroglía/efectos de los fármacos , Compuestos Organometálicos/toxicidad , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Isótopos de Carbono , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Colina O-Acetiltransferasa/metabolismo , Feto , Proteína Ácida Fibrilar de la Glía/metabolismo , Glutamato Descarboxilasa/metabolismo , Glutamato Sintasa/metabolismo , Inmunohistoquímica , Lectinas/química , Neuroglía/citología , Neuroglía/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Telencéfalo , Timidina/metabolismoRESUMEN
The neuronal effects of glucose deficiency on amino acid metabolism was studied on three-dimensional cultures of rat telencephalon neurones. Transient (6 h) exposure of differentiated cultures to low glucose (0.25 mm instead of 25 mm) caused irreversible damage, as judged by the marked decrease in the activities of two neurone-specific enzymes and lactate dehydrogenase, 1 week after the hypoglycemic insult. Quantification of amino acids and ammonia in the culture media supernatants indicated increased amino acid utilization and ammonia production during glucose-deficiency. Measurement of intracellular amino acids showed decreased levels of alanine, glutamine, glutamate and GABA, while aspartate was increased. Added lactate (11 mm) during glucose deficiency largely prevented the changes in amino acid metabolism and ammonia production, and attenuated irreversible damage. Higher media levels of glutamine (4 mm instead of 0.25 mm) during glucose deprivation prevented the decrease of intracellular glutamate and GABA, while it further increased intracellular aspartate, ammonia production and neuronal damage. Both lactate and glutamine were readily oxidized in these neuronal cultures. The present results suggest that in neurones, glucose deficiency enhances amino acid deamination at the expense of transamination reactions. This results in increased ammonia production and neuronal damage.
Asunto(s)
Aminoácidos/metabolismo , Glucosa/deficiencia , Neuronas/fisiología , Animales , Agregación Celular , Células Cultivadas , Relación Dosis-Respuesta a Droga , Metabolismo Energético , Espacio Extracelular/metabolismo , Glucosa/administración & dosificación , Glucosa/farmacología , Glutamina/administración & dosificación , Glutamina/farmacología , Líquido Intracelular/metabolismo , Ácido Láctico/farmacología , Neuronas/efectos de los fármacos , Oxidación-Reducción , Compuestos de Amonio Cuaternario/metabolismo , Ratas , Ratas Sprague-Dawley , Telencéfalo/citologíaRESUMEN
Microglial cells react early to a neurotoxic insult. However, the bioactive factors and the cell-cell interactions leading to microglial activation and finally to a neuroprotective or neurodegenerative outcome remain to be elucidated. Therefore, we analyzed the microglial reaction induced by methylmercury (MeHgCl) using cell cultures of different complexity. Isolated microglia were found to be directly activated by MeHgCl (10(-10) to 10(-6) M), as indicated by process retraction, enhanced lectin staining, and cluster formation. An association of MeHgCl-induced microglial clusters with astrocytes and neurons was observed in three-dimensional cultures. Close proximity was found between the clusters of lectin-stained microglia and astrocytes immunostained for glial fibrillary acidic protein (GFAP), which may facilitate interactions between astrocytes and reactive microglia. In contrast, immunoreactivity for microtubule-associated protein (MAP-2), a neuronal marker, was absent in the vicinity of the microglial clusters. Interactions between astrocytes and microglia were studied in cocultures treated for 10 days with MeHgCl. Interleukin-6 release was increased at 10(-7) M of MeHgCl, whereas it was decreased when each of these two cell types was cultured separately. Moreover, addition of IL-6 to three-dimensional brain cell cultures treated with 3 x 10(-7) M of MeHgCl prevented the decrease in immunostaining of the neuronal markers MAP-2 and neurofilament-M. IL-6 administered to three-dimensional cultures in the absence of MeHgCl caused astrogliosis, as indicated by increased GFAP immunoreactivity. Altogether, these results show that microglial cells are directly activated by MeHgCl and that the interaction between activated microglia and astrocytes can increase local IL-6 release, which may cause astrocyte reactivity and neuroprotection.
Asunto(s)
Astrocitos/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Gliosis/inducido químicamente , Interleucina-6/metabolismo , Intoxicación del Sistema Nervioso por Mercurio/metabolismo , Microglía/efectos de los fármacos , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Comunicación Celular/efectos de los fármacos , Comunicación Celular/fisiología , Supervivencia Celular/fisiología , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/fisiopatología , Técnicas de Cocultivo , Gliosis/metabolismo , Gliosis/fisiopatología , Inmunohistoquímica , Interleucina-6/farmacología , Intoxicación del Sistema Nervioso por Mercurio/patología , Intoxicación del Sistema Nervioso por Mercurio/fisiopatología , Microglía/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , RatasRESUMEN
Aggregate cultures are primary cell cultures prepared from dissociated fetal cells. In rotation-mediated culture under rigorously controlled conditions, the isolated cells are able to reaggregate spontaneously, and to form a large number of practically identical spheres. The three-dimensional cell structure in each aggregate provides a maximum of cell-cell interactions, and thus enables the cells to rearrange and to develop in an organotypic fashion. Relatively simple techniques are now available which permit the preparation of aggregate cultures from fetal brain and liver cells. Since they can be grown in a chemically defined medium, and because they mimic several morphogenetic events occurring in vivo, these cultures offer a unique model for developmental studies. Moreover, they may be used as well for routine testing, for example for screening purposes in toxicology, and thus contribute to the reduction of animal experiments.