Development of a in vitro screening system to minimise animal use in the search for factors that modulate (re)myelination

Lead Research Organisation: University of Glasgow
Department Name: College of Medical, Veterinary &Life Sci

Abstract

Disorders of the myelin sheath, the insulating material that surrounds nerve fibres, are amongst the most common and disabling in neurology. Multiple sclerosis (MS), which has a prevalence of 1 in 500 in the United Kingdom, is the best known disorder of myelin. However, there are also rarer, genetically determined disorders of myelin that have devastating consequences for the families affected. This is particularly true for those disorders, such as childhood forms of X-linked adrenoleukodystrophy, that manifest early and reduce life expectancy to less than two decades.

Currently, there are no effective treatments available for either these genetically determined disorders of myelin, or for progressive forms of MS. However, there is now considerable evidence that treatments aimed at enhancing myelin formation, maintenance or restoration (replacement of damaged myelin) represent a promising therapeutic strategy that will restore function and prevent further neurological decline. Several potential molecular targets have been identified but further progress has been severly limited because identifying specific treatments is time-consuming, expensive, labour intensive and furthermore, requires large numbers of experimental animals. A further problem being that many current cell culture models fail to capture the complexity of the living brain.

We have contributed to the development of a cell culture system that overcomes this last problem and allows us to study myelination and demyelination at the laboratory bench. We have already used this system to identify a small number of factors that either enhance or inhibit myelination. Our major goal is now to adapt this system to allow us to screen thousands of potential therapeutic agents in the shortest possible time, while using an absolute minimum number of experiemental animals. Our preliminary experiments show this is feasible, and that animal numbers can be reduced by at least a factor of 10, compared to current usage. However, before the system can be put into practical use, further modifcations, refinements and validations have to be carried out. Only then will scientists have confidence that the system is sufficiently sensitive and reliable to be used to identify therapeutic agents.

Technical Summary

The aim is to develop a high throughput screen, that uses a minimal number of animals, to identify therapeutic agents that will enhance remyelination in the context of a complex cellular environment mimicing that of the CNS in vivo.

The objectives are:
(i) to adapt an established myelinating cell culture system, from embryonic murine spinal cord, to a multi-well (96 or 384) format, that will minimise animal usage by a factor of at least 10.
(ii) automate handling and analysis for high throughput
(iii) automate data acquisition and analysis for high throughput and to eliminate user bias
(iv) validate the methodology, using factors known to modulate myelination in vitro, to confirm it provides the sensitivity and specificity required for high throughput screen
(v) to achieve i-iv using minimum numbers of experimental animals

Methodology
(i) adapt existing myelinating cell culture techniques into a multi-well format to minimise animal usage
(ii) establish and validate the use of a robotic integrated liquid handling platform to automate all dispensing, feeding and processing steps
(iii) accelerate objective data acquisition using an IN CELL Analyzer 2000 high content screening microscope (GE Healthcare)
(iv) develop template analysis protocols using currint IN CELL level 3 software to quantify effects on myelination

Scientific and medical opportunities
The proposed project will provide ultimately a high throughput screening protocol that will have diverse scientific/medical applications. In the first instance it will provide, for the first time, the opportunity to screen molecular libraries for compounds that stimulate myelination by endogenous progenitor cells; an approach predicted to provide substantial clinical benefits in multiple sclerosis and other de- or dysmyelinating disorders of the central nervous system. It will also facilitate studies of autoantibody mediated effects in neurological diseases.

Publications

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