An Investigation into Changes to Trace Metals and Metabolic Profiling in the Diabetic Retina A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2017 Sandra Callagy School of Biological Sciences Division of Evolution and Genomic Sciences 1 Contents List of Figures ........................................................................................................................ 8 List of Tables ........................................................................................................................ 10 List of Abbreviations ........................................................................................................... 11 Abstract ................................................................................................................................. 14 Declaration ............................................................................................................................ 15 Disclaimer ............................................................................................................................. 15 Copyright statement ............................................................................................................ 16 Dedication ............................................................................................................................. 17 Acknowledgements ............................................................................................................. 18 About the Author .................................................................................................................. 19 Chapter 1 | Introduction ....................................................................................................... 20 1.1 Introduction ....................................................................................................................... 21 1.2 Overview of Glucose Metabolism and Insulin Modulation ................................................ 21 1.3 Introduction to Diabetes Mellitus ...................................................................................... 23 1.3.1 Diagnosis ................................................................................................................... 25 1.3.2 Cost of Diabetes ........................................................................................................ 26 1.3.3 Global Prevalence ..................................................................................................... 26 1.3.4 Treatments for Diabetes ............................................................................................ 27 1.3.5 Diabetic Complications .............................................................................................. 27 1.4 Diabetic Retinopathy: Introduction and Pathogenesis ...................................................... 31 1.4.1 Introduction ................................................................................................................ 31 1.4.2 Introduction to Diabetic Retinopathy ......................................................................... 33 1.4.3 Pathogenesis of Diabetic Retinopathy ...................................................................... 33 1.4.4 Changes to the Retinal Vasculature during Diabetic Retinopathy ............................ 36 1.5 Diabetic Retinopathy: Metabolic and Inflammatory Changes ........................................... 37 1.5.1 The Polyol Pathway ................................................................................................... 38 1.5.2 Hexosamine pathway ................................................................................................ 38 2 1.5.3 AGEs ......................................................................................................................... 41 1.5.4 Lipid mediators .......................................................................................................... 43 1.5.5 Inflammatory Molecular Mediators of Diabetic Retinopathy ...................................... 44 1.5.6 Oxidative Stress ........................................................................................................ 45 1.5.7 Transition Metals ....................................................................................................... 46 1.6 Current treatments for Diabetic Retinopathy .................................................................... 47 1.6.1 Preventative Therapy ................................................................................................ 47 1.6.2 Treatments for Diabetic Macular Oedema ................................................................ 47 1.6.3 Treatment for Proliferative Diabetic Retinopathy ...................................................... 48 1.6.4 Prospective Therapeutics .......................................................................................... 49 1.7 Rodent models of Diabetic Retinopathy ........................................................................... 50 1.7.1 Chemically-Induced Diabetic Models ........................................................................ 50 1.7.2 Spontaneous Models of Diabetes ............................................................................. 51 1.7.3 Proliferative Diabetic Retinopathy Models ................................................................ 51 1.8 Summary and Aims .......................................................................................................... 53 Chapter 2 | Overview and validation of the in vivo Experiments used in this Study .... 54 2.1 Introduction ....................................................................................................................... 55 2.2 Methods ............................................................................................................................ 56 2.2.1 In vivo Methods ......................................................................................................... 56 2.2.2 Tissue Extraction and Analysis ................................................................................. 58 2.2.3 Statistical Analysis ..................................................................................................... 60 2.3 Results .............................................................................................................................. 61 2.3.1 Characteristics of in vivo Studies .............................................................................. 61 2.3.2 Changes in the mRNA expression of Molecular Biomarkers in 12-Week Diabetic Retinas ............................................................................................................................... 61 2.3.3 Changes in Retinal Biomarker mRNA Expression after 16 Weeks of Diabetes ....... 61 2.4 Discussion......................................................................................................................... 65 2.4.1 Altered mRNA expression of Established Biomarkers Confirmed the Molecular Pathology after 12 and 16 weeks of STZ-induced Diabetes .............................................. 65 2.4.2 Gat3 and Nppa were changed in rat retinas after 12 weeks but not 16 weeks of STZ- induced diabetes ................................................................................................................ 66 3 2.4.3 Additional biomarkers Txnip and Vegfa were also changed in rat retinas after 16 weeks of STZ-induced diabetes ......................................................................................... 67 2.4.4 Overall Conclusions .................................................................................................. 67 Chapter 3 | Characterising Trace Metals in Human and Rat Retinas and the Effects of Diabetes ................................................................................................................................ 69 3.1 Introduction ....................................................................................................................... 70 3.2 Methods ............................................................................................................................ 74 3.2.1 Donor Retinas............................................................................................................ 74 3.2.2 Dissection of Rat Retinas .......................................................................................... 74 3.2.3 Tissue Digestion ........................................................................................................ 74 3.2.4 ICP-MS ...................................................................................................................... 75 3.2.5 Statistical Analysis ..................................................................................................... 75 3.3 Results .............................................................................................................................. 77 3.3.1 Donor Retinas............................................................................................................ 77 3.3.2 Effect of Diabetes on Retinal Trace Metal Content ................................................... 77 3.3.3 The Influence of Sex on Retinal Trace Metal Concentrations ................................... 80 3.3.4 Effects of Age on Retinal Trace Metal Content ........................................................