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Equine laminitis pain and modulatory mechanisms at a potential analgesic target, the TRPM8 ion channel Ignacio Viñuela-Fernández Thesis presented for the degree of Doctor of Philosophy The College of Medicine and Veterinary Medicine The University of Edinburgh 2011 DECLARATION I hereby declare that the composition of this thesis and the work presented are my own, with the exception of the ATF-3 and NPY immunohistochemistry which was carried out by Emma Jones. The contribution of others is also appropriately credited. Some of the data included in this thesis have been published and are included in the Appendix. Ignacio Viñuela-Fernández i ACKNOWLEDGEMENTS This work was supported by a Scholarship from the Royal (Dick) Veterinary School at Edinburgh University. I would like to thank Professor Sue Fleetwood- Walker and Dr Rory Mitchell for their supervision, support and guidance throughout my PhD. I would like to thank Professor Vince Molony for his continuing encouragement, support and generous advice. I would also like to thank the members of the Fleetwood-Walker and Mitchell’s lab, including Heather Anderson, Pam Holland and Dr Louise Dickson. Especial thanks to Bobbie Rosie for her patience and generosity in sharing her academic skills and experience, and for her personal support. And Emma Jones for her crucial role in the laminitis study. I am also very grateful to Dr Ada Delaney and Andrew Allchorne who helped and advised me throughout my PhD. And to Dr John Curtis, who oversaw the calcium imaging study. I am also very grateful to Iain McKendrick and Darren Shaw for their statistical advice. Finally I would like to thank two great friends I made during my time in the lab, Dr Hayley Gooding and Dr Anisha Patel, who helped me with the science and are the best company for travelling. On a personal level, I would like to thank Ever Dundas, Sergio Mori and Paul Wilson, for their friendship, which is one of the most wondrous gifts life has granted me. Peter Landsborough, showed me that friendship is a conversation, and what a kind and joyful one his has been. David Wingrove, shared with me amazing books and movies and made life more bearable. I also must thank Judas, the soul of my home and the surest comfort through bad times. Finally, I would like to thank my parents, for their support and their love. And Ryan McQuade, who makes everything better with his honesty, kindness and affection. This thesis is dedicated to the memory of my grandfather, Luciano Fernandez, who always inspired me with his curiosity and respect for humans and animals. ii CONTENTS DECLARATION.......................................................................................................i ACKNOWLEDGEMENTS.....................................................................................ii CONTENTS.............................................................................................................iii LIST OF FIGURES AND TABLES......................................................................ix ABSTRACT............................................................................................................xii ABBREVIATIONS ...............................................................................................xiv Chapter 1: INTRODUCTION ................................................................................1 1.1 Pain definition ...............................................................................................1 1.2 Types of pain..................................................................................................2 1.3 Pain plasticity ................................................................................................2 1.4 Neuropathic pain...........................................................................................4 1.5 Laboratory models of neuropathic pain .....................................................4 1.6 Chronic and neuropathic pain in domestic animals ..................................6 1.7 Neural circuits of pain ..................................................................................7 1.7.1 Primary afferent neurons and nociceptors ......................................................7 1.7.2 Nerve injury-related alterations in the properties of primary afferent neurons..........................................................................................................10 1.7.3 Organization of the dorsal horn of the spinal cord........................................11 1.7.4 Effect of nerve injury on the behaviour of spinal cord neurons....................13 1.7.5 Ascending pathways .....................................................................................14 1.7.6 Descending modulation of pain ....................................................................14 1.8 Molecular mechanisms of nociception ......................................................16 1.8.1 Molecular transduction of noxious stimuli ...................................................17 1.8.2 Voltage-gated ion channels in nociceptive conduction.................................24 1.8.3 Peripheral molecular mechanisms of neuropathic pain ................................25 1.8.4 Role of the sympathetic nervous system in neuropathic pain.......................29 1.9 Spinal molecular mediators of nociception and chronic pain.................30 1.9.1 Glutamate neurotransmission in the dorsal horn...........................................30 iii 1.9.2 GABA and glycine influences in the dorsal horn .........................................31 1.9.3 Central sensitisation in chronic pain states ...................................................32 1.9.4 Spinal disinhibition in chronic pain states ....................................................33 1.10 Mechanisms of cooling-induced analgesia ..............................................34 1.10.1 Neural circuits mediating cold temperature sensation ..................................35 1.10.2 TRPM8-mediated cooling-induced analgesia...............................................36 1.11 Pain assessment in animals.......................................................................38 1.11.1 Physiological measures of pain.....................................................................38 1.11.2 Quantitative sensory testing (QST)...............................................................39 1.11.3 Behavioural quantification............................................................................41 1.12 Overview of experimental work described in this thesis .......................42 Chapter 2: MATERIALS AND METHODS.......................................................43 2.1 Animals ........................................................................................................43 2.1.1 Horses (laminitic and control) ......................................................................43 2.1.2 Rats (chronic constriction injury (CCI) model of neuropathic pain) ............43 2.2 Quantitative sensory testing.......................................................................44 2.2.1 Hoof testing...................................................................................................44 2.2.2 Mechanical allodynia (von Frey filaments test)............................................46 2.2.3 Thermal sensitivity (Hargreaves’ test)..........................................................47 2.3 Drug administration in laboratory rats ....................................................47 2.3.1 Intrathecal application of drugs ....................................................................47 2.4 Electron microcrographic analysis of nerve morphology .......................48 2.5 Immunohistochemical analysis of protein localisation............................49 2.5.1 Tissue processing for immunohistochemistry ..............................................49 2.5.2 Immunohistochemistry procedure................................................................49 2.6 Western blot analysis of proteins...............................................................51 2.6.1 Tissue removal and processing for Western blot..........................................51 2.6.2 Western blot procedure.................................................................................51 iv 2.7 Cell culture...................................................................................................54 2.7.1 Dorsal root ganglion neuron culture .............................................................54 2.7.2 Stable TRPM8 HEK293 culture ...................................................................55 2.7.3 Transient cell transfection.............................................................................55