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.
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.
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.
Planned Impact
Beneficiaries of this research and how they will benefit:
Pharmaceutical and industrial base in the UK
This project will provide the pharmaceutical industry with a high throughput screen using a complex culture system that recapitulates many aspects of the central nervous system in the living animal. Specifically in the first instance, it will enable screening of molecular libraries to identify potential treatments to enhance myelination in patients with disease of myelin.
Health care professionals
The methodology developed in the course of this project will provide a robust and reliable assay to identify patients with neurological diseases who will benefit from treatments that target antibody-dependent effector mechanisms
Members of the academic community
While it is expected that many University departments will not have access to automated liquid handling platforms, we will make this methodology available to our academic colleagues in the UK, thus providing broadened collaborative opportunities.
Patients with MS and other myelin-related diseases, and their carers
Patients will ultimately benefit through improved treatment and diagnosis of their disorders, but in the shorter term publication of our results will provide important psychological support by demonstrating continued interest and progress in developing treatments for these devastating disorders.
Members of the public with an interest in animal welfare
We are aware that there is tremendous public interest in minimising the use of experimental animals. Open communication of our progress will demonstrate the academic and industrial communities take these concerns seriously and are acting to reduce animal usage whilst ensuring research output is maximised. This will help to achieve public and user confidence in medical research practice involving experimental animals.
Pharmaceutical and industrial base in the UK
This project will provide the pharmaceutical industry with a high throughput screen using a complex culture system that recapitulates many aspects of the central nervous system in the living animal. Specifically in the first instance, it will enable screening of molecular libraries to identify potential treatments to enhance myelination in patients with disease of myelin.
Health care professionals
The methodology developed in the course of this project will provide a robust and reliable assay to identify patients with neurological diseases who will benefit from treatments that target antibody-dependent effector mechanisms
Members of the academic community
While it is expected that many University departments will not have access to automated liquid handling platforms, we will make this methodology available to our academic colleagues in the UK, thus providing broadened collaborative opportunities.
Patients with MS and other myelin-related diseases, and their carers
Patients will ultimately benefit through improved treatment and diagnosis of their disorders, but in the shorter term publication of our results will provide important psychological support by demonstrating continued interest and progress in developing treatments for these devastating disorders.
Members of the public with an interest in animal welfare
We are aware that there is tremendous public interest in minimising the use of experimental animals. Open communication of our progress will demonstrate the academic and industrial communities take these concerns seriously and are acting to reduce animal usage whilst ensuring research output is maximised. This will help to achieve public and user confidence in medical research practice involving experimental animals.