The overall goal of this proposal is to develop a microchip-based cell culture model that is integrated with a separation-based analysis system to eventually 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 (iNOS), which produces NO that may enter the dopaminergic cells (such as PC 12 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. These findings may lead to a possible mechanism for NO-mediated damage to dopaminergic cells; however, a molecular level understanding of the mechanisms leading to dopaminergic degeneration is lacking. Since understanding these mechanisms at the molecular level is difficult to achieve in vivo, cell culture models are often employed. To gain insight into these mechanisms, we propose to develop a microchip-based system that incorporates PC 12 cells to study the affect of NO on dopaminergic degeneration. To accomplish this, a three-dimensional PC 12 cell culture model will be immobilized on a planar microchip. These cells will be shown to be bioresponsive by monitoring the neurotransmitters (dopamine and norepinephrine) that are released upon stimulation with calcium. To enable the quantitative identification of the neurotransmitters released from the PC 12 cells, a microchip capillary electrophoresis-based separation system will be developed. The system will utilize amperometric detection to monitor dopamine and norepinephrine released by the cells upon stimulation as well possible products from NO oxidation of dopamine and norepinephrine. Finally, a device that will allow the integration of the cell reactor with the analysis system will be developed using a on-chip valving network. While the majority of the work described in this proposal pertains to the separate parts of the integrated cell reactor/analysis system, the final device will result in a system that can be used in future work to study in detail the mechanisms leading to dopaminergic degeneration by NO.
