The development of an in vitro model of CNS injury to identify factors which promote repair.

Spinal cord injury results in paralysis and loss of sensation because of the interruption of communication between the brain and spinal cord below the injury. These deficits are permanent because the CNS, once damaged has little capacity for repair. The CNS is made up of nerve cells (neurons) and specialised support cells called glia which can promote the growth of nerves and provide them with an insulating protective sheath (myelin). There have been a growing number of reports from different laboratories providing evidence that transplants of glia into spinal cod injury provides a promising means of promoting return of function to the injured spinal cord. However more recently it is apparent that cell transplantation alone is not sufficient to promote functional regeneration and that combining cell transplantation with other approaches, eg. growth factors, and agents that reduce the inhibitory molecules in the spinal cord after injury may be more successful. The study of a combination of treatments would involve the use of many animals. For this reason we would like to establish an in vitro model of spinal cord injury building on preliminary data we have acquired on a culture system (ie in test tubes) that allows us to study intact myelinated axons. We wish to cut these axons and see if they will regenerate. Furthermore we wish to add cells, or reagents that could promote repair and allow us to then study combined treatments in culture without the use of large numbers of animals. Information gained in these investigations will be valuable in making progress towards the rational design of treatments for spinal cord injury.

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 damaged 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 (PCL) substrates to align the axons allowing a more organized topology prior to axotomy. The substrates contain grooves micorengineered to have widths of 5.25micron to 100micron and 4.5-5.0micron 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.