In vitro models to study insulin and glucocorticoids modulation of trimethyltin (TMT)-induced neuroinflammation and neurodegeneration, and in vivo validation in db/db mice.

Brain susceptibility to a neurotoxic insult may be increased in a compromised health status, such as metabolic syndrome. Both metabolic syndrome and exposure to trimethyltin (TMT) are known to promote neurodegeneration. In combination the two factors may elicit additive or compensatory/regulatory mechanisms. Combined effects of TMT exposure (0.5-1 µM) and mimicked metabolic syndrome-through modulation of insulin and glucocorticoid (GC) levels-were investigated in three models: tridimensional rat brain cell cultures for neuron-glia effects; murine microglial cell line BV-2 for a mechanistic analysis of microglial reactivity; and db/db mice as an in vivo model of metabolic syndrome. In 3D cultures, low insulin condition significantly exacerbated TMT's effect on GABAergic neurons and promoted TMT-induced neuroinflammation, with increased expression of cytokines and of the regulator of intracellular GC activity, 11ß-hydroxysteroid dehydrogenase 1 (11ß-Hsd1). Microglial reactivity increased upon TMT exposure in medium combining low insulin and high GC. These results were corroborated in BV-2 microglial cells where lack of insulin exacerbated the TMT-induced increase in 11ß-Hsd1 expression. Furthermore, TMT-induced microglial reactivity seems to depend on mineralocorticoid receptor activation. In diabetic BKS db mice, a discrete exacerbation of TMT neurotoxic effects on GABAergic neurons was observed, together with an increase of interleukin-6 (IL-6) and of basal 11ß-Hsd1 expression as compared to controls. These results suggest only minor additive effects of the two brain insults, neurotoxicant TMT exposure and metabolic syndrome conditions, where 11ß-Hsd1 appears to play a key role in the regulation of neuroinflammation and of its protective or neurodegenerative consequences.

Consensus statement on the need for innovation, transition and implementation of developmental neurotoxicity (DNT) testing for regulatory purposes.

This consensus statement voices the agreement of scientific stakeholders from regulatory agencies, academia and industry that a new framework needs adopting for assessment of chemicals with the potential to disrupt brain development. An increased prevalence of neurodevelopmental disorders in children has been observed that cannot solely be explained by genetics and recently pre- and postnatal exposure to environmental chemicals has been suspected as a causal factor. There is only very limited information on neurodevelopmental toxicity, leaving thousands of chemicals, that are present in the environment, with high uncertainty concerning their developmental neurotoxicity (DNT) potential. Closing this data gap with the current test guideline approach is not feasible, because the in vivo bioassays are far too resource-intensive concerning time, money and number of animals. A variety of in vitro methods are now available, that have the potential to close this data gap by permitting mode-of-action-based DNT testing employing human stem cells-derived neuronal/glial models. In vitro DNT data together with in silico approaches will in the future allow development of predictive models for DNT effects. The ultimate application goals of these new approach methods for DNT testing are their usage for different regulatory purposes.

An adverse outcome pathway for parkinsonian motor deficits associated with mitochondrial complex I inhibition.

Epidemiological studies have observed an association between pesticide exposure and the development of Parkinson's disease, but have not established causality. The concept of an adverse outcome pathway (AOP) has been developed as a framework for the organization of available information linking the modulation of a molecular target [molecular initiating event (MIE)], via a sequence of essential biological key events (KEs), with an adverse outcome (AO). Here, we present an AOP covering the toxicological pathways that link the binding of an inhibitor to mitochondrial complex I (i.e., the MIE) with the onset of parkinsonian motor deficits (i.e., the AO). This AOP was developed according to the Organisation for Economic Co-operation and Development guidelines and uploaded to the AOP database. The KEs linking complex I inhibition to parkinsonian motor deficits are mitochondrial dysfunction, impaired proteostasis, neuroinflammation, and the degeneration of dopaminergic neurons of the substantia nigra. These KEs, by convention, were linearly organized. However, there was also evidence of additional feed-forward connections and shortcuts between the KEs, possibly depending on the intensity of the insult and the model system applied. The present AOP demonstrates mechanistic plausibility for epidemiological observations on a relationship between pesticide exposure and an elevated risk for Parkinson's disease development.

Direct angiotensin type 2 receptor (AT2R) stimulation attenuates T-cell and microglia activation and prevents demyelination in experimental autoimmune encephalomyelitis in mice.

In the present study, we evaluated stimulation of the angiotensin type 2 receptor (AT2R) by the selective non-peptide agonist Compound 21 (C21) as a novel therapeutic concept for the treatment of multiple sclerosis using the model of experimental autoimmune encephalomyelitis (EAE) in mice. C57BL-6 mice were immunized with myelin-oligodendrocyte peptide and treated for 4 weeks with C21 (0.3 mg/kg/day i.p.). Potential effects on myelination, microglia and T-cell composition were estimated by immunostaining and FACS analyses of lumbar spinal cords. The in vivo study was complemented by experiments in aggregating brain cell cultures and microglia in vitro. In the EAE model, treatment with C21 ameliorated microglia activation and decreased the number of total T-cells and CD4+ T-cells in the spinal cord. Fluorescent myelin staining of spinal cords further revealed a significant reduction in EAE-induced demyelinated areas in lumbar spinal cord tissue after AT2R stimulation. C21-treated mice had a significantly better neurological score than vehicle-treated controls. In aggregating brain cell cultures challenged with lipopolysaccharide (LPS) plus interferon-γ (IFNγ), AT2R stimulation prevented demyelination, accelerated re-myelination and reduced the number of microglia. Cytokine synthesis and nitric oxide production by microglia in vitro were significantly reduced after C21 treatment. These results suggest that AT2R stimulation protects the myelin sheaths in autoimmune central nervous system inflammation by inhibiting the T-cell response and microglia activation. Our findings identify the AT2R as a potential new pharmacological target for demyelinating diseases such as multiple sclerosis.

Putative adverse outcome pathways relevant to neurotoxicity.

The Adverse Outcome Pathway (AOP) framework provides a template that facilitates understanding of complex biological systems and the pathways of toxicity that result in adverse outcomes (AOs). The AOP starts with an molecular initiating event (MIE) in which a chemical interacts with a biological target(s), followed by a sequential series of KEs, which are cellular, anatomical, and/or functional changes in biological processes, that ultimately result in an AO manifest in individual organisms and populations. It has been developed as a tool for a knowledge-based safety assessment that relies on understanding mechanisms of toxicity, rather than simply observing its adverse outcome. A large number of cellular and molecular processes are known to be crucial to proper development and function of the central (CNS) and peripheral nervous systems (PNS). However, there are relatively few examples of well-documented pathways that include causally linked MIEs and KEs that result in adverse outcomes in the CNS or PNS. As a first step in applying the AOP framework to adverse health outcomes associated with exposure to exogenous neurotoxic substances, the EU Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) organized a workshop (March 2013, Ispra, Italy) to identify potential AOPs relevant to neurotoxic and developmental neurotoxic outcomes. Although the AOPs outlined during the workshop are not fully described, they could serve as a basis for further, more detailed AOP development and evaluation that could be useful to support human health risk assessment in a variety of ways.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

Effects of the PPAR-beta agonist GW501516 in an in vitro model of brain inflammation and antibody-induced demyelination.

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.

Protein pathway analysis to study development-dependent effects of acute and repeated trimethyltin (TMT) treatments in 3D rat brain cell cultures.

Trimethyltin is an organometallic compound, described to be neurotoxic and to trigger neuroinflammation and oxidative stress. Previous studies associated TMT with the perturbation of mitochondrial function, or neurotransmission. However, the mechanisms of toxicity may differ depending on the duration of exposure and on the stage of maturation of brain cells. This study aim at elucidating whether the toxicity pathways triggered by a known neurotoxicant (TMT) differs depending on cell maturation stage or duration of exposure. To this end omics profiling of immature and differentiated 3D rat brain cell cultures exposed for 24 h or 10 days (10-d) to 0.5 and 1 µM of TMT was performed to better understand the underlying mechanisms of TMT associated toxicity. Proteomics identified 55 and 17 proteins affected by acute TMT treatment in immature and differentiated cultures respectively, while 10-day treatment altered 96 proteins in immature cultures versus 353 in differentiated. The results suggest different sensitivity to TMT depending on treatment duration and cell maturation. In accordance with known TMT mechanisms oxidative stress and neuroinflammation was observed after 10-d treatment at both maturation stages, whereas the neuroinflammatory process was more prominent in differentiated cultures than in the immature, no development-dependent difference could be detected for oxidative stress or synaptic neurodegeneration. Pathway analysis revealed that both vesicular trafficking and the synaptic machinery were strongly affected by 10-d TMT treatment in both maturation stages, as was GABAergic and glutamatergic neurotransmission. This study shows that omics approaches combined with pathway analysis constitutes an improved tool-set in elucidating toxicity mechanisms.

An Integrative Multi-Omics Workflow to Address Multifactorial Toxicology Experiments.

Toxicology studies can take advantage of omics approaches to better understand the phenomena underlying the phenotypic alterations induced by different types of exposure to certain toxicants. Nevertheless, in order to analyse the data generated from multifactorial omics studies, dedicated data analysis tools are needed. In this work, we propose a new workflow comprising both factor deconvolution and data integration from multiple analytical platforms. As a case study, 3D neural cell cultures were exposed to trimethyltin (TMT) and the relevance of the culture maturation state, the exposure duration, as well as the TMT concentration were simultaneously studied using a metabolomic approach combining four complementary analytical techniques (reversed-phase LC and hydrophilic interaction LC, hyphenated to mass spectrometry in positive and negative ionization modes). The ANOVA multiblock OPLS (AMOPLS) method allowed us to decompose and quantify the contribution of the different experimental factors on the outcome of the TMT exposure. Results showed that the most important contribution to the overall metabolic variability came from the maturation state and treatment duration. Even though the contribution of TMT effects represented the smallest observed modulation among the three factors, it was highly statistically significant. The MetaCore™ pathway analysis tool revealed TMT-induced alterations in biosynthetic pathways and in neuronal differentiation and signaling processes, with a predominant deleterious effect on GABAergic and glutamatergic neurons. This was confirmed by combining proteomic data, increasing the confidence on the mechanistic understanding of such a toxicant exposure.

Corrigendum to Recommendation on test readiness criteria for new approach methods in toxicology: exemplified for developmental neurotoxicity.

In this manuscript, which appeared in ALTEX 35 , 306-352 ( doi:10.14573/altex.1712081 ), the Acknowledgements should read: This work was supported by the Doerenkamp-Zbinden Foundation, EFSA, the BMBF, JPI-NutriCog-Selenius, and it has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No. 681002 (EU-ToxRisk).

Ammonium alters creatine transport and synthesis in a 3D culture of developing brain cells, resulting in secondary cerebral creatine deficiency.

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.

Recommendation on test readiness criteria for new approach methods in toxicology: Exemplified for developmental neurotoxicity.

Multiple non-animal-based test methods have never been formally validated. In order to use such new approach methods (NAMs) in a regulatory context, criteria to define their readiness are necessary. The field of developmental neurotoxicity (DNT) testing is used to exemplify the application of readiness criteria. The costs and number of untested chemicals are overwhelming for in vivo DNT testing. Thus, there is a need for inexpensive, high-throughput NAMs, to obtain initial information on potential hazards, and to allow prioritization for further testing. A background on the regulatory and scientific status of DNT testing is provided showing different types of test readiness levels, depending on the intended use of data from NAMs. Readiness criteria, compiled during a stakeholder workshop, uniting scientists from academia, industry and regulatory authorities are presented. An important step beyond the listing of criteria, was the suggestion for a preliminary scoring scheme. On this basis a (semi)-quantitative analysis process was assembled on test readiness of 17 NAMs with respect to various uses (e.g. prioritization/screening, risk assessment). The scoring results suggest that several assays are currently at high readiness levels. Therefore, suggestions are made on how DNT NAMs may be assembled into an integrated approach to testing and assessment (IATA). In parallel, the testing state in these assays was compiled for more than 1000 compounds. Finally, a vision is presented on how further NAM development may be guided by knowledge of signaling pathways necessary for brain development, DNT pathophysiology, and relevant adverse outcome pathways (AOP).

Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies.

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.

Expression of the monocarboxylate transporter MCT1 in the adult human brain cortex.

Distribution of the monocarboxylate transporter MCT1 has been investigated in the cortex of normal adult human brain. Similarly to the glucose transporter GLUT1 55 kDa isoform, MCT1 was found to be strongly expressed on blood vessels in all cortical layers. In addition, laminar analysis revealed intense MCT1 expression in the neuropil of layer IV in primary auditory (AI) and visual (VI) areas, while this expression was more homogeneous in the non-primary auditory area STA. The cellular distribution shows that MCT1 is strongly expressed by glial cells often associated with blood vessels that were identified as astrocytes. The observed distribution of MCT1 supports the concept that, under certain circumstances, monocarboxylates could be provided as energy substrates to the adult human brain. Moreover, the distinct laminar pattern of MCT1 expression between primary and non-primary cortical areas may reflect different types of neuronal activity requiring adequate supply of specific energy substrates.

Involvement of environmental mercury and lead in the etiology of neurodegenerative diseases.

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.

FXYD7, the first brain- and isoform-specific regulator of Na,K-ATPase: biosynthesis and function of its posttranslational modifications.

The FXYD protein family has recently been defined as a result of the search for homologues of the Na,K-ATPase gamma subunit, CHIF, and phospholemman in EST and gene data banks. FXYD7 has been seen to have a role as a brain- and isozyme-specific regulator of Na/K-ATPase. In this study, the biosynthesis, membrane topology, nature, and role of the processing of FXYD7 are investigated.

Repeated exposure to Ochratoxin A generates a neuroinflammatory response, characterized by neurodegenerative M1 microglial phenotype.

Neurotoxic effects of the environmentally abundant mycotoxin Ochratoxin A (OTA) were studied in histotypic 3D rat brain cell cultures, comprising all brain cell types. Cultures were exposed to nanomolar OTA concentrations and samples were collected 48h after a single exposure, or after 10 days of repeated administration. OTA-induced changes in gene- and protein expression, as well as alterations in cell morphology were assessed. Forty-eight-hour OTA exposure resulted in a disruption of the neuronal cytoskeleton and reduced expression of several oligodendrocyte-specific markers indicative of demyelination. Astrocyte disturbances were revealed by a decrease in two astrocytic proteins involved in regulation of inflammatory responses, metallothioneins I and II. Repeated OTA administration induced a neuroinflammatory response, as visualized by an increase of isolectin B4 labelled cells, increased expression of pro-inflammatory cytokines, and detection of macrophagic ED1/CD68 positive cells, as well as an upregulation of neurodegenerative M1 microglial phenotype markers. Partial recovery from OTA-induced deleterious effects on oligodendrocytes and astrocytes was achieved by co-treatment with sonic hedgehog (SHH). In addition, metallothionein I and II co-treatment partially restored OTA-induced effects on oligodendrocytes after 48h, and modulated microglial reactivity after 10 days. These results suggest that OTA-exposure affects Shh-signalling, which in turn may influence both oligodendrocytes and astrocytes. Furthermore, the primarily astrocytic proteins MTI/MTII may affect microglial activation. Thus the neuroinflammatory response appears to be downstream of OTA-induced effects on demyelination, axonal instabilities and astrocytes disturbances. In conclusion, repeated OTA-exposure induced a secondary neuroinflammatory response characterized by neurodegenerative M1 microglial activation and pro-inflammatory response that could exacerbate the neurodegenerative process.

Immediate and delayed effects of subchronic Paraquat exposure during an early differentiation stage in 3D-rat brain cell cultures.

Xenobiotic exposure is a risk factor in the etiology of neurodegenerative disease. It was recently hypothesized that restricted exposure during brain development could predispose for a neurodegenerative disease later in life. As neuroinflammation contributes to progressive neurodegeneration, it is suspected that neurodevelopmental xenobiotic exposure could elicit a neuroinflammatory process, which over time may assume a detrimental character. We investigated the neurotoxic effects of paraquat (PQ) in three-dimensional whole rat brain cell cultures, exposed during an early differentiation stage, comparing immediate effects-directly post exposure-with long-term effects, 20 days after interrupted PQ-administration. Adverse effects and neuroinflammatory responses were assessed by measuring changes in gene- and protein-expression as well as by determining cell morphology changes. Differentiating neural cultures were highly susceptible to PQ and showed neuronal damage and strong astrogliosis. After the 20-day washout period, neurons partially recovered, whereas astrogliosis persisted, and was accompanied by microglial activation of a neurodegenerative phenotype. Our data shows that immediate and long-term effects of subchronic PQ-exposure differ. Also, PQ-exposure during this window of extensive neuronal differentiation led to a delayed microglial activation, of a character that could promote further pro-inflammatory signals that enable prolonged inflammation, thereby fueling further neurodegeneration.

Methods to assess neuroinflammation.

Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three-dimensional cell culture system provides optimal cell-to-cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT-PCR to measure the mRNA expression of cytokines (TNF-α, IL-1ß, IL-6, IL-1ra, TGF-ß, IL-15, IFN-γ), chemokines (ccl5, cxcl1, cxcl2), and i-NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases).

Preparation, maintenance, and use of serum-free aggregating brain cell cultures.

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.