A compartmentalised chamber for the in vitro study and manipulation of axon degeneration.

Diseases of the nervous system account for nearly 10% of the global burden of disease. An enormous amount of research has been undertaken to try and understand how and why nerve cells die in different conditions such as stroke, traumatic brain injury or Alzheimer?s disease. Researchers have used animals, most commonly rats and mice, to mimic aspects of these conditions to try and understand how nerve cells might be protected from degeneration and death. Much work has focussed on the nerve cell body and much less attention has been paid to other parts of the nerve cell: the axon, the fine process by which one nerve connects one region of the brain to another, or the synapse which makes contact with other nerve cells. It is clear that it is important to protect not only the nerve cell body but also the synapse and the axon since in some diseases these parts of the neuron may be the first to be injured with subsequent death of the nerve cell body. We could carry out more experiments in whole animals but it is clearly preferable to do experiments in culture and to reduce and replace the need for whole animal experiments.

Although we know little about the molecular pathways that are involved in axon or synapse degeneration we do know that it involves active biochemical pathways that can be inhibited. Understanding these pathways may offer novel routes to therapeutic interventions. We will build a device that enables us to grow nerve cells in a culture chamber so that the nerve cell bodies are in one compartment and the axons will grow through very small holes into another compartment. The axons will only grow on sticky stripes printed on the bottom of the culture chamber so that they form bundles in the same way as they do in animals brains. Because the holes between the two compartments are so small, the fluid environment on each side can be changed independently, again mimicking another aspect of the brain environment. Once we have perfected this chamber system for growing neurons we will carry out an experiment using drugs to manipulate the axon biology and test if we can protect them from injury. We think these experiments will demonstrate the advantages of the system and will encourage other scientists to use this device in their research and reduce their use of animals.

Nervous system injury and disease are significant causes of mortality and morbidity. For many years research into ways of rescuing or protecting neurons from degeneration and death have focussed on the neuronal cell body. It is now recognised that degeneration of synapses or axons may also contribute to the outcome and progression of an acute brain injury or neurodegenerative disease. The discovery of the Wlds mouse showed that degeneration of axons and synapses is an active self-destructive biochemical process. The degeneration of the soma, axon and synapses occurs by different mechanisms and appears to be compartmentalised. Large numbers of animals, mostly mice and rats, have been used to model aspect of human neurological disease. These approaches have helped our understanding of the selective vulnerability of axons and synapses. Many of these models are complex, invasive and may result in significant disability of the animal. If we are to reduce or replace the numbers of animals being used to elucidate the molecular basis of axon degeneration we need better in vitro protocols.

Axons and their cell somas reside in different microenvironments. Experimental protocols where the axon and its environment may be manipulated independently of the cell soma are needed. We have developed a system that allows proteins to be precisely stamped onto a surface to provide a substrate for axons to grow in oriented fascicles. We now propose to develop a compartmentalised chamber system using soft lithography and microfabrication techniques, using polymer-based materials that will permit the cell soma to be isolated from the axon and for the axon to grow in directed fascicles in parallel stripes across the culture system. The system will be optimised for fluid control between the two compartments and for the organisation of the axon fascicles to enable them to be individually damaged or manipulated.

To encourage the neuroscience community to adopt this approach to reduce and replace in vivo experiments we need to demonstrate its utility. We will take advantage of our recent findings demonstrating that early changes in actin treadmilling are involved in axon degeneration and that polymerisation of axonal actin may be protective. Using a pharmacological approach we will modify actin polymerisation in axons independently of the cell soma and show that this is neuroprotective.

Compartmentalised chambers will permit researchers to analyse the degeneration of distinct compartments of the neuron and reduce and replace in vivo animal experimentation.