Neuronal injury/dysfunction is common to all acute and chronic neurodegenerative disease. Much of our understanding of neuronal injury mechanisms is derived from in vivo experiments. Extensive in vitro experimentation has also been carried out to address this question; however the neuron offers unique challenges for in vitro culture, due to the significant polarity and size of the cell. Current in vitro systems offer only limited control over different sub-cellular compartments of the neuron and can be difficult and inefficient to use. We propose to develop a simple, cost-effective, easy to use, multiple application, multi-chambered in vitro culture system for neurons. The chambers will render individual neuronal compartments, i.e. the soma/axon and growth cone, accessible to experimental manipulations, including mechanical and cell mediated injury. The devices will be amenable to repeat imaging studies over the duration of an experiment. The described in vitro system will replace much of the need for in vivo studies investigating the molecular events underpinning axon degeneration. Successful advanced in vitro culture systems will result in more efficient and effective in vitro experiments. Our advanced culture systems will aim to significantly reduce the number of animals required for studying these important aspects of neuropathology. The culture systems comprise chambers separated by precisely controlled interconnecting channels of sufficient size to allow axon penetration, but small enough to prevent rapid fluid equilibrium between the chambers. Hydrostatic pressure and flow will inhibit molecular diffusion and will allow manipulation and control of the environment within and between the chambers. Patterned stripes, on the chamber base, of a non-adhesive substrate will promote directional growth and fasciculation of small bundles of axons in the experimental chamber. Once we have established precise manufacturing conditions we will investigate a number of critical parameters before embarking on neuronal culture. We will use PC-12 and SH-SY5Y cell-lines to optimise the culture system dimensions. Microscopical imaging of cell cultures will be a mainstay of both the device optimisation and future intended experiments. The culture system will then be used to investigate the response of axons to a physical or cell mediated injury. Our proposed in vitro system will result in a reduction in animal numbers by minimising the numbers of animals required for establishment of cultures and reducing the need for whole animal experiments.
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