Date of Award

Summer 8-16-2018

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Engineering

First Advisor

Mark Decoster

Abstract

Astrocytes and microglia (glial cells) are active elements of the brain maintaining numerous homeostatic functions. Disturbances result in worsening of neuro-inflammation, traumatic brain injury, and various stages of brain tumors. Glial cells contribute to homeostasis for dynamic second messengers in the CNS, including intracellular calcium concentration ([Ca2+] i). Calcium is a central secondary messenger which signals for example, through the N-methyl-D-aspartate (NMDA) glutamate receptor on the neuronal membrane. A large, dynamic Ca2+ influx ensues after glutamate binds to the NMDA receptor. This influx initiates several molecular mechanisms within the cell. Disturbances in calcium homeostasis can lead to neurological diseases such as epilepsy and major depressive disorder.

In this project, we set out to gain a better understanding of the effect of glial density on neural signalling. This was done by accomplishing four main objectives:

1. Develop neural micro-environments with quantifiable variations in glial cell densities.

2. Use calcium imaging methods to analyze the calcium information processing capacity of the various neural micro-environment developed.

3. Develop mathematical tools for testing calcium dynamics iv

4. Study short term and interactions of novel biomaterial (CuHARS) used for tissue engineering in brain cell micro-environments (Ca+2 signaling as an indicator of cell “health”)

To do so, tissue engineered microenvironments were constructed to test the effects of the glial cell density have on calcium information processing. We investigated the response of glia rich, mildly glia depleted, partially depleted, and severely depleted neuronal cultures to sub-maximal (nM to µM) glutamate concentrations using calcium imaging. This was used to assist in predicting and interpreting chaotic neural networks experimentally. Anti-mitotic agents, cytosine arabinoside (AraC), or 2-deoxy-5- fluorouridine (FdU) were used to inhibit proliferating glia and develop the three classes of glia density. Imaging was done with Fluo 3/AM, nine to fourteen days after plating. Neuronal cultures severely depleted (greater than sixty percent depletion) of glia responded to increasing glutamate additions with large, slightly unsynchronized responses with the greatest area under the curve (AUC) observed which returned to baseline the slowest of the three micro-environments developed. Cultures partially depleted (thirty to sixty percent depletion) of glia, responded to increasing glutamate addition with mid-sized, synchronized responses with lower AUC than cultures with severely depleted glial cells. Mildly depleted cultures behaved similarly to glia rich cultures. The difference between their AUC was not statistically significant. Studying how the brain behaves in altered systems, such as in glia depleted micro-environments will help us explore cell loss in the brain and develop more targeted protective strategies.

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