The poly(I:C)-induced maternal immune activation model; a systematic review and meta-analysis of cytokine levels in the offspring.

The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1ß, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment.

Is the time right for in vitro neurotoxicity testing using human iPSC-derived neurons?

Current neurotoxicity testing heavily relies on expensive, time consuming and ethically debated in vivo animal experiments that are unsuitable for screening large number of chemicals. Consequently, there is a clear need for (high-throughput) in vitro test strategies, preferably using human cells as this increases relevance and eliminates the need for interspecies translation. However, human stem cell-derived neurons used to date are not well characterised, require prolonged differentiation and are potentially subject to batch-to-batch variation, ethical concerns and country-specific legislations. Recently, a number of human induced pluripotent stem cell (iPSC)-derived neurons became commercially available that may circumvent these concerns. We therefore used immunofluorescent stainings to demonstrate that human iPSC-derived neurons from various suppliers form mixed neuronal cultures, consisting of different types of (excitatory and inhibitory) neurons. Using multi-well microelectrode array (mwMEA) recordings, we demonstrate that these human iPSC-derived cultures develop spontaneous neuronal activity over time, which can be modulated by different physiological, toxicological and pharmacological compounds. Additional single cell calcium imaging illustrates the presence of functional GABA, glutamate, and acetylcholine receptors as well as voltage-gated calcium channels. While human iPSC-derived neuronal cultures appear not yet suitable to fully replace the rat primary cortical model, our data indicate that these rapidly differentiating, commercially available human iPSC-derived neuronal cultures are already suitable for in vitro prioritisation and effect screening studies. Further characterisation and toxicological validation is now required to facilitate acceptance and large-scale implementation of these animal-free, physiologically-relevant human iPSC-based modelsfor future neurotoxicity testing.

In Vitro Developmental Neurotoxicity Following Chronic Exposure to 50 Hz Extremely Low-Frequency Electromagnetic Fields in Primary Rat Cortical Cultures.

Exposure to 50-60 Hz extremely low-frequency electromagnetic fields (ELF-EMFs) has increased considerably over the last decades. Several epidemiological studies suggested that ELF-EMF exposure is associated with adverse health effects, including neurotoxicity. However, these studies are debated as results are often contradictory and the possible underlying mechanisms are unknown. Since the developing nervous system is particularly vulnerable to insults, we investigate effects of chronic, developmental ELF-EMF exposure in vitro. Primary rat cortical neurons received 7 days developmental exposure to 50 Hz block-pulsed ELF-EMF (0-1000 µT) to assess effects on cell viability (Alamar Blue/CFDA assay), calcium homeostasis (single cell fluorescence microscopy), neurite outgrowth (ß(III)-Tubulin immunofluorescent staining), and spontaneous neuronal activity (multi-electrode arrays). Our data demonstrate that cell viability is not affected by developmental ELF-EMF (0-1000 µT) exposure. Depolarization- and glutamate-evoked increases in intracellular calcium concentration ([Ca(2+)]i) are slightly increased at 1 µT, whereas both basal and stimulation-evoked [Ca(2+)]i show a modest inhibition at 1000 µT. Subsequent morphological analysis indicated that neurite length is unaffected up to 100 µT, but increased at 1000 µT. However, neuronal activity appeared largely unaltered following chronic ELF-EMF exposure up to 1000 µT. The effects of ELF-EMF exposure were small and largely restricted to the highest field strength (1000 µT), ie, 10 000 times above background exposure and well above current residential exposure limits. Our combined data therefore indicate that chronic ELF-EMF exposure has only limited (developmental) neurotoxic potential in vitro.

Assessment of the neurotoxic potential of exposure to 50Hz extremely low frequency electromagnetic fields (ELF-EMF) in naïve and chemically stressed PC12 cells.

Increasing exposure to extremely low frequency electromagnetic fields (ELF-EMF), generated by power lines and electric appliances, raises concern about potential adverse health effects of ELF-EMF. The central nervous system is expected to be particularly vulnerable to ELF-EMF as its function strongly depends on electrical excitability. We therefore investigated effects of acute (30min) and sub-chronic (48h) exposure to 50Hz ELF-EMF on naïve and chemically stressed pheochromocytoma (PC12) cells. The latter have higher levels of iron and/or reactive oxygen species (ROS) and display increased vulnerability to environmental insults. Effects of ELF-EMF on Ca(2+)-homeostasis, ROS production and membrane integrity were assessed using Fura-2 single cell fluorescence microscopy, H2-DCFDA and CFDA assays, respectively. Our data demonstrate that acute exposure of naïve PC12 cells to 50Hz ELF-EMF up to 1000µT fails to affect basal or depolarization-evoked [Ca(2+)]i. Moreover, sub-chronic ELF-EMF exposure up to 1000µT has no consistent effects on Ca(2+)-homeostasis in naïve PC12 cells and does not affect ROS production and membrane integrity. Notably, in chemically stressed PC12 cells both acute and sub-chronic ELF-EMF exposure also failed to exert consistent effects on Ca(2+)-homeostasis, ROS production and membrane integrity. Our combined findings thus indicate that exposure to 50Hz ELF-EMF up to 1000µT, i.e. 10,000 times above background exposure, does not induce neurotoxic effects in vitro, neither in naïve nor in chemically stressed PC12 cells. Though our data require confirmation, e.g. in developing neuronal cells in vitro or (developing) animals, it appears that the neurotoxic risk of ELF-EMF exposure is limited.

Chemically-induced oxidative stress increases the vulnerability of PC12 cells to rotenone-induced toxicity.

In vitro models, including the widely used PC12 cell line, can increase insight into cellular and molecular mechanisms underlying neurodegenerative processes. An important determinant for the vulnerability of cells for chemical insults may be the endogenous level of oxidative stress. To test this hypothesis, we induced different degrees of cellular stress in PC12 cells by altering their ROS production using dexamethasone, l-dihydroxyphenylalanine (l-DOPA) and iron. These different PC12 models were subsequently used to investigate whether the degree of cellular stress could increase their susceptibility to environmental pollutants. The characteristics of these stressed PC12 cell subtypes and their vulnerability to the reference pesticide rotenone were investigated using a combination of biochemical (oxidative stress, cell viability, and α-synuclein expression) and functional (fluorescent calcium imaging) assays. Our combined data demonstrate that chemically-induced stress in PC12 cells increases the production of reactive oxygen species (ROS) and alters calcium homeostasis and α-synuclein expression. Moreover, l-DOPA and FeSO4 pre-treated PC12 cells show increased vulnerability to rotenone-induced cytotoxicity. These chemically-stressed cell models may therefore prove valuable to investigate how increased cellular stress influences neurotoxic outcome, for example in case of mixture toxicity.

Characterization of calcium responses and electrical activity in differentiating mouse neural progenitor cells in vitro.

In vitro methods for developmental neurotoxicity (DNT) testing have the potential to reduce animal use and increase insight into cellular and molecular mechanisms underlying chemical-induced alterations in the development of functional neuronal networks. Mouse neural progenitor cells (mNPCs) differentiate into nervous system-specific cell types and have proven valuable to detect DNT using biochemical and morphological techniques. We therefore investigated a number of functional neuronal parameters in primary mNPCs to explore their applicability for neurophysiological in vitro DNT testing. Immunocytochemistry confirmed that mNPCs express neuronal, glial, and progenitor markers at various differentiation durations (1, 7, 14, and 21 days). Because intracellular calcium ([Ca(2+)]i) plays an essential role in neuronal development and function, we measured stimulus-evoked changes in [Ca(2+)]i at these differentiation durations using the Ca(2+)-responsive dye Fura-2. Increases in [Ca(2+)]i (averages ranging from 65 to 226 nM) were evoked by depolarization, ATP, l-glutamic acid, acetylcholine, and dopamine (up to 87%, 57%, 93%, 28%, and 37% responding cells, respectively) and to a lesser extent by serotonin and gamma-aminobutyric acid (both up to 10% responding cells). Notably, the changes in percentage of responsive cells and their response amplitudes over time indicate changes in the expression and functionality of the respective neurotransmitter receptors and related calcium signaling pathways during in vitro differentiation. The development of functional intercellular signaling pathways was confirmed using multielectrode arrays, demonstrating that mNPCs develop electrical activity within 1-2 weeks of differentiation (55% active wells at 14 days of differentiation; mean spike rate of 1.16 spikes/s/electrode). The combined data demonstrate that mNPCs develop functional neuronal characteristics in vitro, making it a promising model to study chemical-induced effects on the development of neuronal function.

Don't judge a neuron only by its cover: neuronal function in in vitro developmental neurotoxicity testing.

Classical cases of developmental neurotoxicity (DNT) in humans and advances in risk assessment methods did not prevent the emergence of new chemicals with (suspected) DNT potential. Exposure to these chemicals may be related to the increased worldwide incidence of learning and neurodevelopmental disorders in children. DNT is often investigated in a traditional manner (in vivo using large numbers of experimental animals), whereas development of in vitro methods for DNT reduces animal use and increases insight into cellular and molecular mechanisms of DNT. Several essential neurodevelopmental processes, including proliferation, migration, differentiation, formation of axons and dendrites, synaptogenesis, and apoptosis, are already being evaluated in vitro using biochemical and morphological endpoints. Yet, investigation of chemical-induced effects on the development of functional neuronal networks, including network formation, inter- and intracellular signaling and neuronal network function, is underrepresented in DNT testing. This view therefore focuses on in vitro models and innovative experimental approaches for functional DNT testing, ranging from optical and electrophysiological measurements of intra- and intercellular signaling in neural stem/progenitor cells to measurements of network activity in neuronal networks using multielectrode arrays. The development of functional DNT assays will strongly support the decision-making process for measures to prevent potential chemical-induced adverse effects on neurodevelopment and cognition in humans. We therefore argue that for risk assessment, biochemical and morphological approaches should be complemented with investigations of neuronal (network) functionality.