A study of selective vulnerability to diabetes of nerves supplying the ileum using in vitro models Eleni Voukali A thesis submitted for the degree of Doctor of Philosophy At the University College London 2013 Department of Cell and Developmental Biology University College London 1 I, Eleni Voukali, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. ELENI VOUKALI 21-05-2013 2 Abstract Autonomic neuropathy is a complication of diabetes and, where the innervation of the gut is involved, results in disordered gut motility. In vivo, sympathetic nerves and subpopulations of enteric neurons supplying the ileum are differentially affected by diabetes. The overall of aim of this study was to establish whether such differential susceptibility could be reproduced in vitro using wholemount preparations of myenteric plexus and sympathetic ganglion explants from the adult rat and to use such models to examine potential mechanisms underlying the development of neuropathy and its prevention. Preparations were exposed to a range of stimuli that mimic the diabetic environment including high glucose, advanced glycation endproducts (AGEs), carbonyl stress and oxidative stress. Evidence is presented that exposure of myenteric neurons to oxidative stress in vitro mimicked the effects of diabetes as reflected by increased expression of vasoactive intestinal polypeptide (VIP), decreased expression of neuronal nitric oxide synthase (nNOS) and unaltered calbindin expression. However, the mechanism underlying oxidative stress was not uniform, increased VIP expression only occurred on exposure to high glucose and carbonyl stress whereas decreased nNOS expression was only induced by AGEs. Neurons containing calbindin were resistant to all stimuli. Potential therapeutic agents produced differing effects depending on whether they primarily acted against oxidative or carbonyl stress. Using two photon microscopy and fluorescence lifetime imaging (FLIM), the effect of high glucose on reduced nicotinamide adenine (phosphate) (NAD(P)H) metabolism was investigated over time. Comparisons were made between sympathetic neurons from superior cervical ganglia (SCG), which are unaffected in diabetes, and from superior mesenteric/coeliac ganglia (CG/SMG) which develop axonal dystrophy. High glucose temporarily increased NAD(P)H levels selectively in the CG/SMG which coincided with a significant difference between the two ganglia in the fluorescence lifetime of free NAD(P)H. These results may explain the complex pattern of change that occurs in enteric nerves in diabetes. 3 Acknowledgments This work was funded in part by a joint award from the NIDDK and Juvenile Diabetes Research Foundation International (DK58010) and the Sir Richard Stapley Educational Trust. Their support is gratefully acknowledged. I would like to thank Dr. Chris Thrasivoulou for his kind assistance and technical expertise on the expreriments with the multiphoton microscope. I would like to acknowledge my gratitude to my supervisor and outstanding scientist Dr. Jill Lincoln, from whom I have learned much. I would also like to acknowledge Dr. Hannah Shotton, Dr. Greg Campbell, Dave Blundell, Jane Pendjiky, Dr. Anthony Pullen for their invaluable assistance throughout this thesis. Thanks also, to my family and friends for their support all these years. 4 Dedicated to my father 5 Abbreviations 5HT 5-hydroxytryptamine (Serotonin) ACh Acetylcholine ACTION Chronic aminoguanidine treatment in overt diabetic neuropathy ADP/ATP Adenosine diphosphate/triphosphate AGE Advanced glycation end product AH After-hyperpolarisation ANS Autonomic nervous system B1 Thiamine B3 Nicotinamide BSA Bovine Serum Albumin CEL N6-(1-carboxylethyl)-lysine CGRP Calcitonin gene-related peptide CML N6-carboxymethyl-lysine CNS Central nervous system CG/SMG Coeliac/superior mesenteric ganglionic complex DCCT Diabetes control and complications trial DMSO Dimethylsulfoxide DNA Deoxyribonucleic acid 6 DOLD 3-deoxyglucosome-derived lysine dimers DRG Dorsal root ganglion ELAV Embryonic lethal, abnormal vision ENS Enteric nervous system FADH2 Flavin adenine dinucleotide FLIM Fluorescence lifetime imaging microscopy GABA Gamma aminobutyric acid GI tract Gastrontestinal tract GOLD Glyoxal-derived lysine dimers GSH/GSSG Glutathione HbA1c Glycated hemoglobin levels HBSS Hanks balanced salt solution ICC Interstitial cells of Cajal IGF-1 Insulin-like growth factor 1 IMG Inferior mesenteric ganglion IPANs Intrinsic Primary Afferent Neurons LDL Low density lipoproteins MAPK Mitogen associated protein kinases MOLD Methylglyoxal-derived lysine dimers NA Noradrenaline NAD(H) Nicotinamide adenine dinucleotide 7 NADP(H) Nicotinamide adenine dinucleotide phosphate NANC Non-adrenergic non-cholinergic NFκB Nuclear factor κB NGF Nerve growth factor NO Nitric oxide NOD Non obese diabetic nNOS Neuronal nitric oxide synthase NPY Neuropeptide Y PARP Poly(ADP ribose)polymerase PBS Phophate-buffered saline solution PFA Paraformaldehyde PI3K Phosophoinositol-3-kinase PKC Protein kinase C RAGE Receptor for AGE RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species S Synaptic SCG Superior cervical ganglia SOD Superoxide dismutase STZ Streptozotocin 8 TCA Tricarboxylic Acid TCSPC Time correlated single photon counting TH Tyrosine hydroxylase TUNEL Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling VIP Vasoactive intestinal polypeptide 9 Contents Abstract ............................................................................................................................. 3 Acknowledgments ............................................................................................................. 4 Abbreviations .................................................................................................................... 6 Contents .......................................................................................................................... 10 List of Figures ................................................................................................................ 15 List of tables .................................................................................................................... 18 Chapter 1: General introduction ................................................................................... 19 1.1 Diabetes and gastrointestinal problems ................................................. 19 1.2 Diabetes and peripheral neuropathy ...................................................... 20 1.3 Innervation of the GI tract ..................................................................... 22 1.3.1 Extrinsic Innervation ...................................................................... 23 1.3.2 Extrinsic primary afferent/sensory innervation of the GI tract ...... 25 1.3.3 Intrinsic innervation ....................................................................... 26 1.3.3.1 Coordination of enteric neurons .............................................. 28 1.3.3.2 Classification of enteric neurons ............................................. 29 1.4 Changes in intrinsic and extrinsic nerves due to diabetes mellitus ....... 34 1.4.1 Clinical diabetes ............................................................................. 35 1.4.2 Animal models of diabetes ............................................................. 36 1.4.3 Experimental studies ...................................................................... 38 1.4.3.1 Enteric neurons........................................................................ 38 1.4.3.2 Sympathetic neurons ............................................................... 41 10 1.5.1 Metabolism of nutrients in neurons and cell respiration ................ 43 1.5.2 Oxidative stress, anti-oxidants and diabetic neuropathy ................ 44 1.5.3 Altered NADPH and NADH metabolism ...................................... 46 1.5.4 Carbonyl Stress and AGEs ............................................................. 48 1.5.5 Neurotrophic support deficiency and alterations in gene expression ................................................................................................................. 50 Chapter 2: Establishment of the in vitro models ........................................................... 52 2.1 Introduction ........................................................................................... 52 2.1.1 AGEs & methylglyoxal .............................................................. 53 2.1.1.1 Functional effects of AGEs on neurons .................................. 55 2.1.1.2 Correlation of AGEs with HbA1c levels ................................ 56 2.1.2 Oxidative stress and accelerated ageing ......................................... 56 2.1.3 Culture models for the study of diabetic neuropathy ..................... 58 2.1.4 Heterogeneity of enteric neuronal responses in diabetes ............... 62 2.2 Materials and methods .......................................................................... 64 2.2.1 Culture preparations ....................................................................... 64 2.2.2 Immunofluorescence procedures: double staining for VIP, nNOS, or calbindin with HuC/D. ........................................................................ 65 2.2.3 Analysis and statistics .................................................................... 66 2.3 Results ..................................................................................................