A novel tool that embeds 3Rs principles in multiple sclerosis research

Why did we fund this project?

This award aims to replace the use of mice in studies of multiple sclerosis (MS) by developing an in vitro model using embryo-derived neurons from murine spinal cords.

MS is modelled in rodents by immunising against myelin, causing the immune system to target the myelin that insulates axons. This induces experimental autoimmune encephalomyelitis (EAE), which has similar myelin damage to that seen in MS. EAE results in weakness, fore- and hindlimb paralysis and incontinence, although the degree of severity varies between mice. The studies are associated with a high level of suffering and the need for specialist husbandry and care. Current in vitro models do not contain all cell types present in the central nervous system and use neuronal cells that are not electrically active or myelinated, limiting their use as an alternative to the mouse EAE model. Dr Julia Edgar and colleagues have developed a model using murine neurons derived from embryonic spinal cords, which recapitulate myelination events, both cellular and molecular, in vitro.

The student will expand the utility of these cells by combining them with various supplements, such as myelin-reactive T cells, to form an in vitro model of MS. They will optimise the model by determining concentrations and durations of exposure that most accurately mimic MS lesions and evaluate the suitability for a high-throughput drug efficacy screen. The student will develop skills in time-lapse fluorescent microscopy, phase contrast microscopy and quantitative PCR to evaluate gene expression changes.

Multiple sclerosis (MS) is the most common cause of nontraumatic neurological disability in young adults in Europe and North America. This debilitating disease is modelled in animals by inducing central nervous system (CNS) autoimmunity in which the animal's defence system against bacteria and viruses, instead attacks cells in the animal's CNS. This results in experimental autoimmune encephalomyelitis (EAE); a condition characterised by paralysis in the experimental animal. Thus, EAE is classed as severe under the Animals (Scientific Procedures) Act 1986. Although EAE has helped us understand aspects of MS and informed drug discovery and testing, there is an urgent need to find an alternative that eliminates or reduces its use. Here we will develop and verify a cell culture (brain in a dish) model of EAE to replace experiments in living animals.

With our combined expertise in EAE, myelinating cell cultures, axon degeneration and electrophysiology, we will provide a validated cell culture tool to:

• Model EAE/MS using embryonic mice, that (i) reduces the use of EAE by the research and pharmaceutical communities (ii) will be used to generate new concepts, ideas, toxicity screening and target validation and (iii) to investigate how axons are injured and can be rescued in the context of progressive MS.
• Publicise this model to maximise its uptake
• Provide training in the 3Rs principles in general, and in the context of understanding, investigating and identifying therapies for neurodegenerative disorders.
• Instil a long-term appreciation and adoption of the 3Rs principles
• Elucidate the mechanisms and molecules responsible for axon injury, providing rationale targets for therapeutic intervention.
• Provide mentoring and training in many skills, including adoption of rigorous and unbiased approaches to addressing research questions.

The model will be amenable to:

• Electrophysiological analysis which replace the EAE disability score for testing the benefits/toxicity of putative therapeutics.
• Screening up to 180 molecules simultaneously for axon protection properties, using immunocytochemistry and a 96-well format
• Single cell transcriptomic analysis, immunohistochemistry, western blotting, qRT-PCR, ELISA and other assays to provide mechanistic insights in basic research applications.
• The model cannot replace all in vivo work, but it will be particularly useful in testing therapies for progressive forms on MS in which CNS-intrinsic processes are principal contributors to disability progression. More details on 3Rs benefits are detailed in the supporting information.

The PhD student will receive practical and theoretical training in 3Rs principles throughout the duration of the studentship from Biological Services staff including qualified veterinarians, animal care staff and the Dr Edgar, who has almost 20 years hands-on experience in a wide range of procedures, training and reporting.

We will provide ‘hands-on’ training as required and also detailed written instructions and videos if hands-on training is not feasible, making adoption straight forward. The model cannot fully replicate the in vivo situation involving the gut microbiome, peripheral and/or adaptive immune system, vasculature and endocrine systems, but conversely, this limitation allows uncoupling of CNS-intrinsic molecules and mechanism to address important questions, such as what drives progression in progressive forms of MS.