The enteric nervous system (ENS) is commonly referred to as the ‘second brain’ due to its complex networks of neuronal cells. The abnormality of these neurons and/or their absence has been shown to play a fundamental role in diseases of both the ENS and the central nervous system. Accordingly, electrophysiological studies of the ENS and general understanding of how enteric neurons behave in the gastrointestinal tract are critical in the characterization of the pathophysiology of enteric and neurodevelopmental diseases. To date, studies on these aspects have been limited by the difficulty of culturing enteric neurons in-vitro, as well as by their poor adhesion properties. The primary objectives of this thesis are to develop strategies to investigate electrodynamics processes of enteric neurons and close in on their interactions with polymeric substrates, aiming at optimizing conventional experimental approaches and expanding the current knowledge and critical understanding of this elusive cell type.
By capitalizing on a rapid and efficient culturing method developed by our group, different polymers were tested in order to assess their ability to promote adhesion of enteric neurons, as confirmed by immunofluorescence analysis. The most effective polymer resulting from this initial screening was then applied as a coating onto the glass surface of multichannel electrode arrays (MEAs) allowing for the analysis of neuron dynamics. While Matrigel® was the most effective at promoting both neuron adhesion and neurite outgrowth, it acted as an insulating material which prevented the MEA electrodes from picking up electrical signals. Therefore, we opted instead for laminin protein and poly-d-lysine immobilized on glass by polydopamine, to study the electrophysiology of the neurons. Of note, polydopamine was found to be critical in enhancing the stability of the protein coating and ensuring cellular viability.
The same protein coating was also used to functionalize the surface of blends of poly(styrene) and poly(methyl methacrylate), which segregate when mixed to give rise to varying topographical features. These surfaces aimed at elucidating fundamental processes that dictate how neurons interact with surfaces when compared to smooth rigid surfaces (i.e. glass).
Finally, the most effective surface for neuron adhesion was applied to study how chemotaxis influences neurite elongation and directionality. Enteric neurons were cultured onto both a linear concentration gradient of protein created using a microfluidic system and a uniform concentration profile to compare their response to chemical signals. In general, their motion was random and lacked directionality on the uniform protein surface. The neuronal response to the chemical gradient could not be evaluated to completion; however, this analysis still provided meaningful insight as a starting point for future studies.
The results presented in this thesis serve as a significant stepping-stone for the improvement of the in-vitro study of the ENS and will be used to gain a deeper understanding of enteric diseases, ultimately contributing to the development of novel polymeric scaffolds for tissue-engineering applications.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/37895 |
| Date | 17 July 2018 |
| Creators | Jakupovic, Dilara |
| Contributors | Variola, Fabio |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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