Date of Award

5-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Bioengineering

Committee Member

Dr. Bruce Gao, Committee Chair

Committee Member

Dr. Ken Web

Committee Member

Dr. Delphine Dean

Committee Member

Dr. Mark Kindy

Abstract

The developing central nervous system is a unique target for environmental toxicants both pre- and postnatal. Exposure to industrial chemical toxicants at various stages throughout development are known to contribute to injuries that result in autism, attention-deficit hyperactivity disorder (ADHD), dyslexia, and other cognitive impairments [81]. The damage caused by these exposures is often untreatable and frequently permanent, resulting in reduced intelligence (expressed in terms of lost IQ points) or behavioral abnormalities. It is now reported that 10-15% of all births are associated with disorders of neurobehavioral development [81], where 1 in 68 children in the United States is diagnosed with some form of an autism spectrum disorder (ASD) [7, 93, 188] and 14% of the roughly 4 million children born each year suffer from ADHD [124]. It is estimated that 3% of developmental disabilities are the direct result of environmental exposure, and that another 25% stem from interactions between environmental factors and genetic susceptibility [80, 146]. With more diagnosed cases and rising costs, the identification of the chemicals responsible for the deleterious effects on the developing nervous system has become significant topic of research. Current developmental neurotoxicity (DNT) testing relies heavily on whole animal approaches for hazard identification and dose-response evaluations. These methods are not practical for screening the over 82,000 chemicals already used in commerce with an additional 700 new chemicals introduced annually [24]. Following the first workshop on “Incorporating In Vitro Alternative Methods for Developmental Neurotoxicity (DNT) Testing into International Hazard and Risk Assessment Strategies” in 2005, it was determined that in vitro DNT testing methods should be included as part of a tiered approach to help create a reference list of potential developmentally neurotoxic chemicals and catalog the effects they have on various developmental mechanistic endpoints [40, 127]. Using directionality of pioneer-neuron axonal pathfinding as the mechanism for evaluation, we developed a biochip-based single-neuron axonal pathfinding assay to subjugate extending axons to simultaneous geometric and chemical guidance. To achieve this we devised a laser cell-micropatterning system to facilitate the placement of individual-neurons to exact locations on a PDMS substrate. The cell-culture conditions were optimized to promote single-neuron axonal extension through and beyond the confinements of a geometric guidance microchannel. Evaluation of the pathfinding direction in response to geometric guidance was compared to that of geometric and chemical stimuli. We found using our system that the addition of a chemical guidance component 1) increased the number of individual-neurons extending an axon at least 20 µm beyond the end of a guidance microchannel structure and 2) showed the potential to elicit a growth cone turning event by abruptly changing the initial pathfinding trajectory of an axon. Based on our previous study that single-neuron axonal pathfinding under geometric guidance is one order of magnitude more sensitive to a chemical toxicant, our research data demonstrate that we have created a platform that can be used to test the possible effects that low dose (nM concentrations) chemical exposures may have on pioneer-neuron axonal pathfinding.

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