Date of Award

Winter 2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Mark DeCoster

Abstract

Tissue engineered microenvironments were constructed to test the effects glial cells have on calcium information processing, and to mimic conditions in vivo for tumor invasion and residual cancer after resection of tumor. Submaximal, nM, glutamate (GLU) stimuli were applied to the engineered environments, and the resulting calcium dynamic behavior of neuronal cells was measured to help predict and interpret chaotic systems in the experimental realm. Calcium is a key signaling ion which signals through the N-methyl-D-aspartate (NMDA) glutamate receptor on the neuronal membrane. GLU binding to the NMDA receptor (NMDAR) causes a large and dynamic increase in neuronal intracellular calcium. Perturbations in calcium homeostasis by means of the NMDAR have been linked to several neurodegenerative diseases, such as Alzheimer's, Parkinson's and Huntington's disease. Primary rat cortical cells were used in both co-culture (neurons and glia) and in cultures treated with Cytosine Arabinoside (AraC) to deplete glia. Rat glioma cells were added to the cultured cells to mimic residual cancer cells. In addition, the glioma cells were formed into novel spheroids that modeled tumor invasion. The calcium response was monitored after exogenous glutamate was added in three concentrations (250, 500 and 750 nM), in all (3!) sequences. Calcium was imaged with Fluo 3/AM, 8 to 9 days after plating. The co-culture system responded to increasing submaximal additions of glutamate with calcium spikes, as previously demonstrated in this system. Neuronal cultures depleted of glia responded to increasing nM additions of GLU with large synchronized broad transient responses which returned to baseline more slowly, leading to a greater area under the fluorescence intensity-time curve (AUC) that we believe is an indicator of excitotoxicity, as well as, normal calcium signaling. Cancer environments did not have excitotoxic calcium area under the curve AUC to glutamate stimulus; however, the residual environment did display excitotoxic conditions due to rapid glutamate induced calcium oscillatory behavior from glioma expressing system Xc-. Determining how neurons will respond and behave in altered systems, such as, in the presence of brain tumor glia may help our understanding of cell loss in the brain, and may provide better protective strategies.

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