Domoic Acid-Induced Neurotoxicity Is Mainly Mediated by the AMPA/KA Receptor: Comparison between Immature and Mature Primary Cultures of Neurons and Glial Cells from Rat Cerebellum

Domoic acid (DomA) is a naturally occurring shellfish toxin that can induce brain damage in mammalians. Neonates have shown increased sensitivity to DomA-induced toxicity, and prenatal exposure has been associated with e.g. decreased brain GABA levels, and increased glutamate levels. Here, we evaluated DomA-induced toxicity in immature and mature primary cultures of neurons and glial cells from rat cerebellum by measuring the mRNA levels of selected genes. Moreover, we assessed if the induced toxicity was mediated by the activation of the AMPA/KA and/or the NMDA receptor. The expression of all studied neuronal markers was affected after DomA exposure in both immature and mature cultures. However, the mature cultures seemed to be more sensitive to the treatment, as the effects were observed at lower concentrations and at earlier time points than for the immature cultures. The DomA effects were completely prevented by the antagonist of the AMPA/KA receptor (NBQX), while the antagonist of the NMDA receptor (APV) partly blocked the DomA-induced effects. Interestingly, the DomA-induced effect was also partly prevented by the neurotransmitter GABA. DomA exposure also affected the mRNA levels of the astrocytic markers in mature cultures. These DomA-induced effects were reduced by the addition of NBQX, APV, and GABA

Metabolomics reveals metabolic alterations by intrauterine growth restriction in the fetal rabbit brain

Background: Intrauterine Growth Restriction (IUGR) due to placental insufficiency occurs in 5-10% of pregnancies and is a major risk factor for abnormal neurodevelopment. The perinatal diagnosis of IUGR related abnormal neurodevelopment represents a major challenge in fetal medicine. The development of clinical biomarkers is considered a promising approach, but requires the identification of biochemical/molecular alterations by IUGR in the fetal brain. This targeted metabolomics study in a rabbit IUGR model aimed to obtain mechanistic insight into the effects of IUGR on the fetal brain and identify metabolite candidates for biomarker development. Methodology/Principal Findings: At gestation day 25, IUGR was induced in two New Zealand rabbits by 40-50% uteroplacental vessel ligation in one horn and the contralateral horn was used as control. At day 30, fetuses were delivered by Cesarian section, weighed and brains collected for metabolomics analysis. Results showed that IUGR fetuses had a significantly lower birth and brain weight compared to controls. Metabolomics analysis using liquid chromatographyquadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and database matching identified 78 metabolites. Comparison of metabolite intensities using a t-test demonstrated that 18 metabolites were significantly different between control and IUGR brain tissue, including neurotransmitters/peptides, amino acids, fatty acids, energy metabolism intermediates and oxidative stress metabolites. Principle component and hierarchical cluster analysis showed cluster formations that clearly separated control from IUGR brain tissue samples, revealing the potential to develop predictive biomarkers. Moreover birth weight and metabolite intensity correlations indicated that the extent of alterations was dependent on the severity of IUGR. Conclusions: IUGR leads to metabolic alterations in the fetal rabbit brain, involving neuronal viability, energy metabolism, amino acid levels, fatty acid profiles and oxidative stress mechanisms. Overall findings identified aspargine, ornithine, Nacetylaspartylglutamic acid, N-acetylaspartate and palmitoleic acid as potential metabolite candidates to develop clinical biomarkers for the perinatal diagnosis of IUGR related abnormal neurodevelopment

Toward Good In Vitro Reporting Standards

A good experiment reported badly is worthless. Meaningful contributions to the body of science are made by sharing the full methodology and results so that they can be evaluated and reproduced by peers. Erroneous and incomplete reporting does not do justice to the resources spent on conducting the experiment and the time peers spend reading the article. In theory peer-review should ensure adequate reporting – in practice it does not. Many areas have developed reporting standards and checklists to support the adequate reporting of scientific efforts, but in vitro research still has no generally accepted criteria. It is characterized by a “Wild West” or “anything goes” attitude. Such a culture may undermine trust in the reproducibility of animal-free methods, and thus parallel the “reproducibility crisis” discussed for other life science fields. The increasing data retrieval needs of computational approaches (in extreme as “big data” and artificial intelligence) makes reporting quality even more important so that the scientific community can take full advantage of the results. The first priority of reporting standards is to ensure the completeness and transparency of information provided (data focus). The second tier is a quality of data display that makes information digestible and easy to grasp, compare and further analyze (information focus). This article summarizes a series of initiatives geared towards improving the quality of in vitro work and its reporting. This shall ultimately lead to Good In Vitro Reporting Standards (GIVReSt)

Developmental neurotoxicity : challenges in the 21st century and in vitro opportunities

In recent years neurodevelopmental problems in children have increased at a rate that suggests lifestyle factors and chemical exposures as likely contributors. When environmental chemicals contribute to neurodevelopmental disorders developmental neurotoxicity (DNT) becomes an enormous concern. But how can it be tackled? Current animal test-based guidelines are prohibitively expensive, at $1.4 million per substance, while their predictivity for human health effects may be limited, and mechanistic data that would help species extrapolation are not available. A broader screening for substances of concern requires a reliable testing strategy, applicable to larger numbers of substances, and sufficiently predictive to warrant further testing. This review discusses the evidence for possible contributions of environmental chemicals to DNT, limitations of the current test paradigm, emerging concepts and technologies pertinent to in vitro DNT testing and assay evaluation, as well as the prospect of a paradigm shift based on 21st century technologies

Advances in 3D neuronal microphysiological systems : towards a functional nervous system on a chip

Microphysiological systems (MPS) designed to study the complexities of the peripheral and central nervous systems have made marked improvements over the years and have allowed researchers to assess in two and three dimensions the functional interconnectivity of neuronal tissues. The recent generation of brain organoids has further propelled the field into the nascent recapitulation of structural, functional, and effective connectivities which are found within the native human nervous system. Herein, we will review advances in culture methodologies, focused especially on those of human tissues, which seek to bridge the gap from 2D cultures to hierarchical and defined 3D MPS with the end goal of developing a robust nervous system-on-a-chip platform. These advances have far-reaching implications within basic science, pharmaceutical development, and translational medicine disciplines.publishe

COVID-19 : prime time for microphysiological systems, as illustrated for the brain

The development of therapies for and preventions against infectious diseases depends on the availability of disease models. Bioengineering of human organoids and organs-on-chips is one extremely promising avenue of research. These miniature, laboratory-grown organ systems have been broadly used during the ongoing, unprecedented coronavirus 2019 (COVID-19) pandemic to show the many effects of the etiologic agent, severe acute respiratory coronavirus 2 (SARS-CoV-2) on human organs. In contrast, exposure of most animals either did not result in infection or caused mild clinical signs - not the severe course of the infection suffered by many humans. This article illuminates the opportunities of microphysiological systems (MPS) to study COVID-19 in vitro, with a focus on brain cell infection and its translational rel-evance to COVID-19 effects on the human brain. Neurovirulence of SARS-CoV-2 has been reproduced in different types of human brain organoids by 10 groups, consistently showing infection of a small portion of brain cells accompanied by limited viral replication. This mirrors increasingly recognized neurological manifestations in COVID-19 patients (evidence of virus infection and brain-specific antibody formation in brain tissue and cerebrospinal fluid). The pathogenesis of neuro-logical signs, their long-term consequences, and possible interventions remain unclear, but future MPS technologies offer prospects to address these open questions.publishe

Human IPSC-Derived Model to Study Myelin Disruption

Myelin is of vital importance to the central nervous system and its disruption is related to a large number of both neurodevelopmental and neurodegenerative diseases. The differences observed between human and rodent oligodendrocytes make animals inadequate for modeling these diseases. Although developing human in vitro models for oligodendrocytes and myelinated axons has been a great challenge, 3D cell cultures derived from iPSC are now available and able to partially reproduce the myelination process. We have previously developed a human iPSC-derived 3D brain organoid model (also called BrainSpheres) that contains a high percentage of myelinated axons and is highly reproducible. Here, we have further refined this technology by applying multiple readouts to study myelination disruption. Myelin was assessed by quantifying immunostaining/confocal microscopy of co-localized myelin basic protein (MBP) with neurofilament proteins as well as proteolipid protein 1 (PLP1). Levels of PLP1 were also assessed by Western blot. We identified compounds capable of inducing developmental neurotoxicity by disrupting myelin in a systematic review to evaluate the relevance of our BrainSphere model for the study of the myelination/demyelination processes. Results demonstrated that the positive reference compound (cuprizone) and two of the three potential myelin disruptors tested (Bisphenol A, Tris(1,3-dichloro-2-propyl) phosphate, but not methyl mercury) decreased myelination, while ibuprofen (negative control) had no effect. Here, we define a methodology that allows quantification of myelin disruption and provides reference compounds for chemical-induced myelin disruption.publishe

A LUHMES 3D dopaminergic neuronal model for neurotoxicity testing allowing long-term exposure and cellular resilience analysis

Several shortcomings of current Parkinson's disease (PD) models limit progress in identification of environmental contributions to disease pathogenesis. The conditionally immortalized cell line LUHMES promises to make human dopaminergic neuronal cultures more easily available, but these cells are difficult to culture for extended periods of time. We overcame this problem by culturing them in 3D with minor medium modifications. The 3D neuronal aggregates allowed penetration by small molecules and sufficient oxygen and nutrient supply for survival of the innermost cells. Using confocal microscopy, gene expression, and flow cytometry, we characterized the 3D model and observed a highly reproducible differentiation process. Visualization and quantification of neurites in aggregates was achieved by adding 2 % red fluorescent protein-transfected LUHMES cells. The mitochondrial toxicants and established experimental PD agents, rotenone and MPP(+), perturbed genes involved in one-carbon metabolism and transsulfuration pathways (ASS1, CTH, and SHTM2) as in 2D cultures. We showed, for the first time in LUHMES, down-regulation of mir-7, a miRNA known to target alpha-synuclein and to be involved in PD. This was observed as early as 12 h after rotenone exposure, when pro-apoptotic mir-16 and rotenone-sensitive mir-210 were not yet significantly perturbed. Finally, washout experiments demonstrated that withdrawal of rotenone led to counter-regulation of mir-7 and ASS1, CTH, and SHTM2 genes. This suggests a possible role of these genes in direct cellular response to the toxicant, and the model appears to be suitable to address the processes of resilience and recovery in neurotoxicology and Parkinson's disease in future studies.publishe