Microdevices to develop ex vivo brain slice models of neurodegenerative diseases: enabling longitudinal, high-resolution, live-tissue imaging

Background: Neurodegenerative diseases such as Alzheimer's and Parkinson's Diseases are increasingly prevalent in our increasingly aging population. Current treatment options merely delay disease progression and there is a pressing need to identify new drugs and strategies targeting these devastating disorders. The intact brain is extremely complex, making a clear understanding of the primary alterations associated with neurodegeneration difficult to determine. This project will develop new methods of maintaining intact slices of brain tissue ex-vivo, in as physiologically-normal conditions as possible. Micropatterned substrates, with tailored mechanical and surface chemistry properties, will be developed to support tissue for high-resolution, subcellular imaging, integrating microfluidic channels for controlled solution exchange and localised drug delivery. These slice microsystems, in which the tissue will be bathed in media on both sides, will then be used to recapitulate chronic neurodegeneration. The microsystems will provide a unique ability to observe and quantify primary cellular events during disease onset and characterise the efficacy of potential therapeutics to prevent, treat and reverse disease-associated dysfunction and degeneration.
Parkinson's Disease (PD) and Alzheimer's Disease (AD) develop due to extensive neuronal damage and protein aggregation leading to aberrant neural network activity. A common factor in PD, AD and many other neurodegenerative diseases is the dysfunction of mitochondria. These organelles are vital as an ATP-producing metabolic hub and they influence signalling cascades, redox control and the processing of reactive oxygen species. Mitochondria are highly-motile, being trafficked along the cytoskeleton by motor proteins, and they undergo fusion and fission to maintain mitochondrial quality. The complex architectures of metabolically-demanding brain cells necessitate co-ordinated long-distance delivery of organelles, energy and substrates. As efficient calcium buffers, mitochondria are vital for appropriate physiological calcium signals and protection during pathological calcium overload. Fascinatingly, mitochondrial transport proteins disengage at sites of high calcium - delivering the organelles to sites of need for calcium buffering and ATP production. Because mitochondria are involved in these "front-line" events, they are also susceptible to damage which, if it exceeds the cell's capacity to repair, can cause a cascade of deleterious effects: spreading calcium overload that leads to a loss of ATP production, increased ROS and release of apoptosis-inducing factors. These alterations are often tightly inter-related however and further work is needed to ascertain which come first.
Research Aims: This project will exploit microengineering approaches to adapt organotypic brain slice techniques to study chronic neurodegenerative diseases. Two parallel strategies will be explored, each independently informative, and then combined to provide powerful insights into disease development, progression and therapies.
Objective 1: maintain brain slices in organotypic culture on transparent micropillar supports or ultra-thin porous membranes within a microfluidic system to allow live-cell fluorescence imaging from below using an inverted microscope to optimise resolution.
Objective 2: develop long-term chronic organotypic brain slice models of PD and AD.
Objective 3: high-resolution imaging of mitochondrial function, motility and structure, combined with calcium imaging, in various cell types and sub-cellular locations, at multiple time-points during slice degeneration.