Multiple roles for the extracelllular matrix protein Tenascin-X in nerve gut function Rubina Aktar A thesis submitted in partial fulfilment of the requirements of the Degree of Doctor of Philosophy St Bartholomew’s and the Royal London School of Medicine and Dentistry. Queen Mary University of London June 2016 1 Statement of Originality I, Rubina Aktar, confirm that the research included within this thesis is my own work or that it has been carried out in collaboration with, or supported by others, that this is duly acknowledged below and my contribution indicated. All the studies were performed and analysed by myself except as stated. Mr Max Walmsley obtained IHC images for the KO mouse under my supervision. Images obtained from the DRG and spinal cord were assisted by Dr Eduardo Araujo De Almeida. Dr Jean-Marie Delalande helped obtain IHC images of rectal prolapse. Gastric emptying breath samples were run by Dr Simon Eaton and obtained and analysed by myself. Dr Madusha Peiris wrote the ethics for the gastric emptying tests and assisted with the gastric emptying experiments and analysis. I attest that I have exercised reasonable care to ensure that the work is original, and does not to the best of my knowledge break any UK law, infringe any third party’s copyright or other Intellectual Property Right, or contain any confidential material. I accept that the College has the right to use plagiarism detection software to check the electronic version of the thesis. I confirm that this thesis has not been previously submitted for the award of a degree by this or any other university. The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without the prior written consent of the author. Signature: Date: 30th June 2016 2 Acknowledgements I would like to thank my supervisors, Professor Ashley Blackshaw and Professor Qasim Aziz for allowing me this opportunity. I would like to add a special thanks to my primary supervisor, Professor Ashley Blackshaw, who made me excited for science through many scientific discussions and who always listened to and answered my infinite questions. I am especially grateful to my friend Dr Madusha Peiris who continuously supported me throughout my studies, patiently listened to me, made me laugh and motivated me to carry on. I would also like to thank the following people: Professor Amanda Page, Dr Stephen Kentish and Dr Stamatiki Kritas for teaching me the gastric emptying test. Dr Simon Eaton for measuring the gastric breath samples. Professor Helen Cox and Dr Iain Tough for teaching me how to do the secretion experiments. Dr Mark Scott and Dr Sahar Mohammed for their help in the colonic motility experiments and Professor Eduardo Araujo De Almeida, Dr Jean-Marie Delalande and Max Walmsley for helping obtain IHC data. Mr Tony Price for looking after the mice colonies, Dr Sue Surguy for management and Debbie Hampson for running the laboratory I am very grateful to my parents and my family who always believed in me and encouraged me to achieve my fullest potential. My sister Hasina who knew before I did that this was the right path for me and my nephews, Ayaan and Yasir who always make me smile. Finally to Bowel and Cancer Research for funding me. 3 Abstract Tenascin X (TNX) is a matricellular protein involved in regulating cellular functions by interacting with other extracellular matrix (ECM) proteins within the cell matrix and has anti-adhesive properties evidenced in tumours and wound healing. TNX is the only member of the tenascin family that is lost in Joint Hypermobility Syndrome (JHS) and exerts a crucial architectural function. Of importance, TNX deficient and JHS patients have gastrointestinal (GI) dysfunction. Despite this association no study has described the role of TNX in the GI tract. Thus, the aim of this thesis was to characterise the expression of TNX in the stomach and colon in mouse and human tissue. Second, we aimed to elucidate the functional role of TNX using TNX knockout (TNX KO) mice. Expression studies revealed TNX in vagal afferent endings in the mouse, and myenteric cell bodies in human stomach. In colon, TNX strongly associated with cholinergic submucous and myenteric neurons in both species, however, was not found in CGRP positive fibres. Cell bodies in nodose ganglia, dorsal root ganglia, ventral and dorsal horn were also TNX positive. Functional studies in stomach, using single fibre electrophysiology showed TNX KO mice had increased vagal afferent mechanoreceptor sensitivity. Octanoic acid breath test revealed rapid gastric emptying in TNX KO. Colonic manometry showed the amplitude and frequency of colonic contractions were reduced in TNX KO mice, particularly in the distal colon. Ussing chamber studies measuring changes in ion flux (indirect measure of secretion) showed no major difference between TNX KO and wild type (WT) mice. The specific localisation of TNX with neuronal structures in the gut is shown here for the first time suggesting that TNX is more than just an architectural protein. Indeed, its role in specific GI functions supports this observation and provides a mechanism for GI symptoms in JHS. 4 Table of Contents Table of Content .................................................................................................................... 5 List of Figures ...................................................................................................................... 10 1 Literature Review ......................................................................................................... 17 1.1 Introduction ...................................................................................................................... 17 1.2 Anatomy of the stomach and colon ................................................................................... 18 1.3 The enteric nervous system ............................................................................................... 20 1.4 Neurons defined by function and neurochemistry .............................................................. 21 1.5 Smooth muscle innervation ............................................................................................... 21 1.6 Inhibitory motor neurons to the circular muscle ................................................................. 22 1.7 Excitatory motor neurons to the circular muscle ................................................................ 22 1.8 Motor neurons innervating the longitudinal muscle ........................................................... 23 1.9 Intrinsic sensory neurons ................................................................................................... 23 1.10 Interneurons ..................................................................................................................... 25 1.11 Secretomotor/vasodilator neurons .................................................................................... 25 1.12 Inhibitory/excitatory effects of secretomotor neuron ........................................................ 26 1.13 Intestinofugal neurons ...................................................................................................... 27 1.14 AH-type neurons ............................................................................................................... 28 1.15 S-type neurons .................................................................................................................. 29 1.16 Sensory mechanoreceptors ............................................................................................... 31 1.17 Synaptic transmission ........................................................................................................ 31 1.18 Afferent innervation .......................................................................................................... 32 1.18.1 Vagal afferents .................................................................................................................... 32 1.18.2 Spinal afferents ................................................................................................................... 34 1.19 Structure and functional roles of afferent endings .............................................................. 35 1.19.1 Mucosal afferent endings ................................................................................................... 35 1.19.2 Tension receptors ............................................................................................................... 36 1.19.3 Muscular afferents .............................................................................................................. 37 1.19.4 Serosal and mesenteric afferents ....................................................................................... 38 1.20 Neural control: stomach .................................................................................................... 38 1.21 Neural control: colon ......................................................................................................... 42 1.22 Motility disorders of the stomach ...................................................................................... 44 1.22.1 Delayed gastric emptying (Gastroparesis) .......................................................................... 44 1.22.2 Rapid gastric emptying (Dumping syndrome) .................................................................... 45 1.22.3 Functional Dyspepsia .........................................................................................................