High content in vitro testing strategy to reduce animals in neurotoxicity evaluation

Summary | Background | Current state of the art | What could your solution be used for? Collaboration | 3Rs impact


Summary

We are seeking agrochemical, pharmaceutical and chemical company collaborators to help validate our integrated approach for assessing neurotoxicity in vitro. Our model uses rat primary cortical neurons to establish the toxicity profile and the dose-response relationship of compounds.


Background

Current in vitro test systems developed for regulatory neurotoxicity assessment are often based on general cytotoxicity assays and do not sufficiently represent the endpoints specific for mechanisms of neuronal and glial toxicity.

In vitro neuronal networks are simplified models, coupled to microelectrode arrays (MEAs). These approaches have already demonstrated their suitability to measure the neuronal specific endpoint, spontaneous electrical activity, and its perturbation due to an acute or chronic exposure of toxicants (Novellino et al., 2011; Defranchi et al., 2011, Hogdberg et al., 2011). Computing the Mean Firing Rate (MFR) of the neuronal network is the most common approach used to characterize its overall electrical activity, but this only provides on/off information, rather than describing changes in firing pattern. We have recently developed a new integrated analysis of specific parameters related to the bursting behavior of neurons. This is widely considered the most important property for analyzing the dynamics of electrical activity during the development of neuronal networks (Van Pelt J et al., 2004; Chen CP et al., 2006, Li X et al., 2006), and provides a more detailed temporal view of global network signaling. It also provides a tool for identifying the mechanism of action or unknown off-target effects of the chemical.


Current state of the art

Detecting neurotoxicity induced by chemicals represents a major challenge due to the physiological and morphological complexity of the central (CNS) and peripheral nervous system (PNS). Predicting neurotoxic effects of a new chemical or drug is a key feature of many of the test guidelines accepted by regulatory authorities (OECD 2008; US EPA 2008). Currently only in vivo test methods are accepted for regulatory purposes. Four OECD Test Guidelines (TGs) describe in vivo neurotoxicity studies.

  • Delayed Neurotoxicity of Organophosphorus Substances Following Acute Exposure (TG 418) involves a single oral dose to hens, which are then observed for 21 days. Primary observations include the hen’s behavior, weight, and gross and microscopic pathology.
  • Delayed Neurotoxicity of Organophosphorus Substances: 28-day Repeated Dose Study (TG 419) involves daily oral dosing of hens with an organophosphorous pesticide for 28 days followed by biochemical and histopathological assessments.
  • Neurotoxicity Study in Rodents (TG 424) involves daily oral dosing of rats for acute, subchronic, or chronic assessments (28 days, 90 days, or one year or longer). Primary observations include behavioral assessments and evaluation of nervous system histopathology.
  • Developmental Neurotoxicity (DNT) Study (TG 426) evaluates in utero and early postnatal effects by daily dosing of at least 60 pregnant rats from implantation through lactation. Offspring are evaluated for neurologic and behavioral abnormalities, and brain weights and neuropathology are assessed at different times through adulthood.

Although to date these methods are the best available for assessing the potential for neurotoxicity and DNT in human health risk assessment, they have several limitations: they provide limited mechanistic information regarding the toxic effect (Worth and Balls, 2002; Coecke et al., 2005; Harry and Tiffany-Castiglioni, 2005), even though neurotoxicants may act on a number of targets within the nervous system and by a variety of mechanisms. Since no single test is capable of thoroughly assessing the neurotoxic potential of all substances, an integrated in vitro and in vivo strategy could help to reveal the risks by elucidating toxicity mechanisms and identifying the molecular target of neurotoxicants, while minimizing the cost, time and number of animals used.

Moreover, existing test methods for neurotoxicity assessment are no longer suitable for the demands of 21st century large scale chemical risk assessment (e.g. REACH, and the High Production Volume Programme (HPVP)). There is increasing pressure to develop alternative test strategies based on the latest technological advances which are more predictive, faster and cheaper, and which reduce the use of animals. On the basis of feasibility studies conducted to date, our MEA-based high-throughput method is a valuable tool to perform sensitive, quick and low cost neurotoxicological screening. In this approach, rat and mouse neurons have served as the gold standard, but more recently human adult stem cells and induced pluripotent stem (iPS) cells offer the opportunity for the development of predictive in vitro models without using animals or embryonic stem cells.


What could your Solution be used for?

Our primary neuronal networks derived from embryonic rat cortex and cultured on MEA neurochips provide a testing platform to evaluate effectively the neurotoxic potential of chemicals, agrochemicals and drugs in order to predict safeguards for human health. Our multi-parameter approach can rapidly and accurately extract relevant features of the spontaneous electrical network activity to elucidate the alteration caused by chemical exposure and to identify the mechanism of action or its unknown off- target effects.

Furthermore, the MEA neurochip technology can be used to study the influence of chemicals during early neuronal network electrical activity maturation and the long-term functioning of the active networks over many months. We are also developing standard operating procedures for the commitment of human adult stem cells and engineered cell lines into a neural lineage, to create a readily available human-based predictive cell model that can be used as part of an integrated strategy.


Need for collaboration

We are seeking collaborations with pharmaceutical, agrochemical and chemical industry partners able to provide reagents or compounds with known or unknown neurotoxicity to test either alone or in combination, to validate our system in identifying the neurotoxicological profile of compounds. This is necessary to generate a robust data base to validate the system.

In addition, we would like to collaborate with research groups and/or companies developing human neuronal cell models or tissues. We would be keen to use our MEA system to evaluate the ability of these models to produce spontaneous electrical activity. The validation of an alternative human cell-based method is of high interest for the pharmaceutical, agrochemical and chemical industry. For example, generating data to comply with REACH will require many millions of vertebrate animals and cost about €10 billion over the next 10 years (Hartung and Rovida, 2009; Schoeters, G., 2010).


3Rs impact assessment

Currently there are no alternative methods for neurotoxicity assessment. Our Solution integrates the latest in MEA technology and in vitro cultured neuronal networks to create a platform to characterise and assess chemical-induced neurotoxic effects, while reducing the number of animals used. The OECD neurotoxicity study in rodents (TG 424) requires at least three dose groups and a control group, with at least 20 animals (10 females and 10 males) in each dose group. By using MEA-based method, it is possible to prepare about thirty MEAs from only one embryonic rat cortex. This is enough for testing six concentrations of ten substances in triplicate.

Furthermore, the number of fetuses (average n.8-10) of each dam is high and exceeds the experimental need. We have optimized a tissue cryopreservation protocol by which we can preserve fetuses which are surplus to requirement and use them later, minimizing the number of pairs required.


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ETT S.p.A
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