The overall goal of this proposal is to develop a microchip-based system that integrates a cell culture model with a separation-based analysis system to study the affect of nitric oxide (NO) on dopaminergic degeneration. Degeneration of dopaminergic neurons is a characteristic of Parkinson's disease and studies have shown that inflammation of microglia results in the activation of inducible nitric oxide synthase, which produces NO that may enter the dopaminergic cells and oxidize dopamine found in storage vesicles. This NO-mediated oxidation may render dopamine stores depleted, thus decreasing amounts of exocytotic dopamine, a known trait of Parkinson's patients. Given all that is known about intracellular neuronal changes that may be related to the genesis of Parkinson's disease, a detailed molecular level understanding of the mechanisms leading to dopaminergic degeneration by NO is still lacking. An optimal situation to study these mechanisms is to employ an in vitro cell culture system that mimics an in vivo system and is easily coupled to an analysis system that can discretely analyze the products formed when NO depletes dopamine stores. In this proposal, we describe the development of such a system using microchip technology and PC 12 cells. Great strides towards the development of this system were made in the currently funded grant. The proposed studies will continue the development of a microchip-based cell culture system/analysis system that incorporates PC 12 cell immobilization, on-chip valving, electrophoresis-based separations, and electrochemical detection. To accomplish this, we first will integrate PC 12 cell immobilization and amperometric detection with the continuous flow reactor/microchip electrophoresis device developed in the current studies. Variables such as immobilization conditions and chip dimensions will be optimized so that the cells become nearly confluent in rounded microchannels. We then propose to use this device to study the effect of NO on dopaminergic degeneration by monitoring changes in dopamine and norepinephrine release as the cells are incubated with varying concentrations of NO. We also propose to determine the different products that result from the interaction of NO with these neurotransmitters. Finally, we propose to develop a more realistic model of Parkinson's onset by culturing both PC 12 cells and microglia cells in a microchip device, with the long term goal of using the microglia as the NO source in these studies. This will involve optimization of immobilization conditions for the microglia as well as studies to show that the microglia cells are bioresponsive. While this proposal is focused on PC 12 cells and neurotransmitter analysis, the final device will be a general analytical tool that is amenable to immobilization of a variety of cell lines and analysis of released analytes by electrophoretic means. ? The overall goal of this proposal is to develop a microchip device that can be used to study the role nitric oxide plays in dopaminergic degeneration, which is a characteristic of Parkinson's disease. This final device will utilize micron-sized valves to integrate a neuron cell model to an analysis system that can differentiate between the neurotransmitters released from the cells. This device will allow neuroscientists to study the role that microglia cells play in the onset of Parkinson's disease. ? ? ?
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