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2019 Gaurilcikaite Egle 14231 This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Investigating mechanisms of odontogenic pain the role of odontoblasts, trigeminal ganglion neurons, and their interaction Gaurilcikaite, Egle Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 09. Oct. 2021 Investigating mechanisms of odontogenic pain: the role of odontoblasts, trigeminal ganglion neurons, and their interaction Thesis submitted for the degree of Doctor of Philosophy Egle Gaurilcikaite Wolfson Centre for Age-Related Diseases Institute of Psychiatry, Psychology, and Neuroscience King’s College London 2014-2018 1 Abstract Despite the prevalence of pain originating from the teeth (dental, or odontogenic, pain) and its impact on patients’ lives, the cellular and molecular mechanisms of odontogenic pain are still not completely understood. The teeth are unique structures, innervated by sensory neurons that originate in the trigeminal ganglia. Factors that increase the sensitivity of these neurons, such as inflammatory cytokines, may contribute to odontogenic pain associated with dental pulp inflammation. To address this, a calcium imaging-based mouse trigeminal ganglion (TG) neuron sensitisation assay was established as part of this project. Short-term exposure to tumour necrosis factor alpha (TNFα), but not to interleukin 1 beta (IL-1β), was found to sensitise the transient receptor potential (TRP) ion channels TRPA1 and TRPV1 on TG neurons. This assay provides the opportunity to test other molecules relevant to odontogenic pain for their ability to sensitise TG neurons. Previous research suggests that initiation of dentine hypersensitivity, a type of odontogenic pain evoked by thermal, osmotic, chemical, evaporative, or mechanical stimuli to the exposed dentine, might involve both neurons and odontoblasts, the specialised mineralising cells in the teeth. In this project, mouse odontoblast-like (OB) cells were used as a cellular model to study the sensory function of odontoblasts in vitro. OB cells were demonstrated to express functional TRPV4 ion channels, known to be involved in thermal, osmotic, and mechanosensation. TRPV4 activation was also found to stimulate ATP release from OB cells, highlighting a potential means for odontoblast communication with dental afferent fibres. 2 In addition, this study was the first to demonstrate the odontoblast ability to detect biologically relevant acids. Both an acid-induced increase in OB cell intracellular calcium concentrations and subsequent ATP release were detected. These processes were found to be independent of TRPV4 activity. Finally, to address the ability of odontoblasts to transmit sensory information to adjacent neurons, a co-culture approach was used to directly study intercellular communication between mouse OB cells and TG neurons. Pharmacological activation of TRPV4 was chosen as an odontoblast-selective stimulus, based on the functional expression of TRPV4 ion channels in OB cells, but not in mouse TG neurons. Blocking purinergic signalling disrupted the interaction between stimulated OB cells and TG neurons, demonstrating the importance of ATP as a primary mediator. Taken together, the findings described in this thesis support the ability of odontoblasts to detect physiologically-relevant stimuli and respond with a release of ATP to modulate the activity of adjacent sensory neurons. Thus, this interaction may play an important role in the initiation of odontogenic pain in vivo. 3 Acknowledgements First and foremost, I would like to thank my primary supervisor, Dr Andy Grant, for his scientific expertise as well as guidance and support throughout these four years. His willingness to give his time so generously has been very much appreciated. To my second supervisor, Professor Stephen McMahon, for providing both feedback on my PhD work and insight into the wider area of pain research. I would also like to thank the members of his lab who kindly offered their help whenever I needed it. In addition, I would like to thank Professor Peter McNaughton and his lab members for the friendly research environment and advice. Also, to BSc project students Navneet Dhaliwal and Arauthy Gnanasampanthan (Gina) who have helped out along the way. A special thanks to my parents and my brother Vaidas for their continued encouragement. Lastly, I would like to thank Audrius, for his patience and all-round support, especially during the final stages of this journey. 4 Table of contents Abstract 2 Acknowledgements 4 Table of contents 5 Table of figures 10 Table of tables 13 Abbreviations 14 Chapter 1 General introduction 17 1.1 Orofacial pain .............................................................................................................. 17 1.2 Trigeminal sensory system ...................................................................................... 18 1.2.1 Primary sensory trigeminal neurons and peripheral sensitisation ............... 21 1.2.2 Second-order sensory neurons and central modulation of pain .................... 24 1.3 Structure and innervation of the teeth.................................................................. 25 1.3.1 Odontoblasts ............................................................................................................... 27 1.3.2 Dental primary afferents ......................................................................................... 31 1.3.3 Relationship between odontoblasts and dental primary afferent neurons . 34 1.4 Odontogenic pain ....................................................................................................... 36 1.4.1 Pulpitis pain ................................................................................................................ 36 1.4.2 Dentine hypersensitivity ......................................................................................... 40 1.5 Hypothesis and general aims .................................................................................. 45 Chapter 2 General materials and methods 46 2.1 Solutions ....................................................................................................................... 46 2.2 Reagents ....................................................................................................................... 47 5 2.3 Cell culture .................................................................................................................. 47 2.3.1 Mouse 17IA4/OB cells .............................................................................................. 47 2.3.2 Primary mouse trigeminal ganglion neurons ..................................................... 48 2.4 Reverse transcription polymerase chain reaction (RT-PCR) ........................... 48 2.4.1 RNA extraction and purification ........................................................................... 48 2.4.2 First-strand cDNA synthesis ................................................................................... 49 2.4.3 Amplification of cDNA by PCR ............................................................................. 49 2.4.4 Agarose gel electrophoresis .................................................................................... 51 2.5 Measurement of intracellular Ca2+ concentration .............................................. 51 2.5.1 Microplate-based calcium flux assay .................................................................... 52 2.5.2 Microscope-based single-cell calcium imaging .................................................. 53 2.6 Measurement of extracellular ATP concentration ............................................. 54 2.6.1 ATP standard curves ................................................................................................. 54 2.7 Statistical analysis ...................................................................................................... 55 Chapter 3 Characterisation of mouse odontoblast-like cells and their sensory function 56 3.1 Introduction ................................................................................................................. 56 3.1.1 Current models for studying odontoblasts.......................................................... 56 3.1.2 Sensory ion channels in odontoblasts .................................................................
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