Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture

Rockliffe, Alice (2021) Modelling changes in excitability of the peripheral nervous system using compartmentalised microfluidic culture. PhD thesis, University of Nottingham.

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This thesis describes the use of compartmentalised microfluidic devices to investigate changes in neuronal excitability. All studies carried out in this work were completed in line with principles of the NC3Rs (reduction, replacement and refinement). Particular interest was given to the study of the excitability of dorsal root ganglion neurons (DRGs) in the context of pain-based signalling. This also included the in vitro culture and characterisation of non-neuronal cells involved in inflammation and nociception.

Current methods for In vitro modelling of pain pathways often fails to replicate the unique morphology of the DRG neurons. These pseudo-unipolar neurons detect nociceptive stimuli at the peripheral terminals, and transduce long range action-potentials to higher processing centres in the central nervous system. Unlike in vivo modelling of pain behaviours, in vitro models of nociception provide the capacity to monitor changes in neuronal function at a cellular and molecular level. However, until the development of technology such as microfluidics, the standard methods of culture failed to isolate the axons from the soma.

The primary aim of this project was to develop a model capable of replicating the complex microenvironment that terminals of the DRG neurons encounter during the development and onset of pain. This involved the optimisation of cell culture methods for inflammatory cells used to induce changes in neuronal excitability, both from the context of the peripheral terminals, or from the CNS if desired. At a molecular level, the microfluidic model was also used to investigate the role of small non-coding RNA (microRNAs) on regulating DRG excitability in the context of nociception. This Thesis hypothesises that voltage-gated potassium channels form an interesting target for a microRNA of interest. However, it is widely acknowledged that microRNAs regulate the expression of multiple mRNAs.

The use of functional studies using the microfluidic model have shown here that there are differences in the way in which a neuron responds to a stimulus, dependent on whether it is applied locally to the axon or the soma. Live cell imaging was used to measure evoked changes in Ca2+ transients as a proxy for cell excitability. As well as significant differences in the response to depolarising agents such as potassium chloride (KCL), the use of biologically relevant stimuli to the study of nociception was also developed. The culture of inflammatory cells such as bone marrow derived macrophages led to the development of cytokine-rich media which was used to evoke changes in neuronal excitability. By exploiting the microfluidic nature of the device, subsequent investigations to the role of microRNA 138-5p in regulating neuronal excitability were undertaken. The use of cell permeable microRNA inhibition showed a reduction in cell excitability if applied locally to the axons. Bioinformatics led to the development of Kv1.2 as a potential target for miR-138-5p in vivo, which could explain the effects of miR-138-5p in modulating excitability of the DRGs.

The findings in this work have demonstrated the potential for development of more biologically relevant in vitro models using microfluidic compartmentalised cell culture. For example, fluidic isolation has characterised the role of miR-138-5p in regulating DRG excitability at the axons.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Dajas-Bailador, Federico
Hathway, Gareth
Bellamy, Tomas
Keywords: neuroscience, in vitro modelling, microRNA, excitability, pain
Subjects: Q Science > QP Physiology > QP351 Neurophysiology and neuropsychology
Faculties/Schools: UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID: 65702
Depositing User: Rockliffe, Alice
Date Deposited: 04 Aug 2021 04:43
Last Modified: 04 Aug 2021 04:43

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