The development of an in vitro model of central nervous system injury to identify factors which promote repair

Aims

This project aimed to develop and assess an in vitro model of spinal cord injury as a screening tool for therapeutic agents, replacing the use of large numbers of animals.

Background

Spinal cord injury (SCI) is a major cause of permanent disability resulting in paralysis and loss of sensation. These deficits are permanent because the central nervous system (CNS), once damaged, has little capacity for repair. Laboratory models of SCI generally involve the use of large number of animals, and require technically demanding and time-consuming operations that result in substantial disability in the animals. Current opinion is that repair will require a combination of therapies which will require many animals for testing for confirmation of their efficacy.

Research details and methods

Mixed neural cells are obtained from rat or mouse embryonic spinal cords and are plated on a layer of astrocytes which over a period of weeks form a carpet of spinal cord axons. The axons are ensheathed by myelin, interspersed with nodes of Ranvier, produced by the surrounding oligodendrocytes. Many of the key axonal, glial and nodal proteins found in vivo are expressed.

Depending on the substrate the axons become arranged in long tracts which can be cut using a scalpel. A cell-free area develops and the lesioned nerve fibres respond, as in vivo, with myelin loss, changes in the expression of specific neuronal proteins, markers of scarring and cell death. No neurite growth occurs across the cell-free area. Importantly, however, neurite outgrowth and myelination have been shown to be stimulated by reagents which have been shown to promote nerve growth in animal models validating the model.

Key impacts and findings

The system has the potential to avoid the use of animals for some studies of SCI. Ten rats or mice are used per treatment or time point in a typical animal model of spinal cord injury, plus controls animals resulting in large cohorts of animals being used. With the new in vitro model, one rat provides enough cells for an experiment which if carried out in vivo would use at least 30 animals.

Related content

Laboratory models of spinal cord injury (SCI) generally involve the use of large number of animals. These experiments require technically demanding and time consuming operations which are followed by substantial disability in the animals and long-term post-operative care. In this application we aim to further develop our CNS myelination cultures as a model of CNS injury and thereby replace, refine and reduce the number of animals necessary to test potential therapeutic agents for SCI. Not only will this reduce the number of animals necessary to study SCI, but there will also be a reduction in any animal suffering, as we will only be using the animals for schedule 1 procedures (for cell preparations). Since animals will no longer be participating in any surgical intervention associated with models of spinal cord injury, they will not have to endure any post-operative pain and distress. We have established dissociated CNS cells in vitro which allow us to mimic the intact CNS. These cultures are comprised of glia and CNS axons which interact to produce many internodes of myelin interspaced with nodes of Ranvier. These cultures can be lesioned, and areas of damage detected even after 36 days in culture. These preliminary experiments illustrate the feasibility of making a lesion without damaging the viability of the entire culture and maintaining them for many weeks. We have further modified these cultures by growing them on micro-engineered polycaprolactone substrates to align the axons, allowing a more organized topology prior to axotomy. The substrates contain grooves micro-engineered to have widths of 5.25 micron to 100 micron and 4.5-5.0 micron depths. We have found that axons align along the smaller groves and the glia can interact with the axons forming mature myelin. These cultures lend themselves to the study of glia‒axonal interactions in a reproducible manner. In this proposal we intend to develop these assays further to examine in detail the changes that occur in the cultures post-lesioning. We then wish to use these cultures to investigate the array of potential combined therapies for the repair of CNS injury.

Boomkamp SD, McGrath MA, Houslay MD, Barnett SC (2014) Epac and the high affinity rolipram binding conformer of PDE4 modulate neurite outgrowth and myelination using an in vitro spinal cord injury model. British Journal of Pharmacology 171(9): 2385-98 doi:10.1111/bph.12588

Donoghue PS, Lamond R, Boomkamp SD, Sun T, Gadegaard N, Riehle MO, Barnett SC (2013) The development of a ε-polycaprolactone scaffold for central nervous system repair.Tissue Eng Part A 19(3-4):497-507  doi: 10.1089/ten.TEA.2012.0382.

Boomkamp SD, Riehle MO, Wood J, Olson MF, Barnett SC (2012) The development of a rat in vitro model of spinal cord injury demonstrating the additive effects of Rho and ROCK inhibitors on neurite outgrowth and myelination. Glia 60: 441-56 doi:10.1002/glia.22278

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Project grant

Status:

Closed

Principal investigator

Professor Susan Barnett

Institution

Unversity of Glasgow

Co-Investigator

Dr Mathis Riehle

Grant reference number

G0800572/1

Award date:

Jan 2009 - Nov 2012

Grant amount

£294,388