Mechanisms Underlying Early Visual Dysfunctions in Rodent Models of Type 1 Diabetes Público

Abstract

Diabetic retinopathy is a devastating ocular complication of diabetes mellitus. Due to the wide prevalence of diabetes, diabetic retinopathy is the leading global cause of blindness in working-age adults. As severe vision loss is associated with microvasculopathy in the retinal vasculature, such as aneurysm, hemorrhage, and neovascularization, clinical diagnosis and treatment of diabetic retinopathy is based on the onset and severity of these irreversible structural lesions. However, diabetic patients with angiographically normal retinas have been found to suffer subtle defects in vision (e.g. scotopic vision, color vision, contrast sensitivity). The appearance of these visual deficits can potentially provide an earlier detection and therapeutic window for diabetic retinopathy. Therefore, the purpose of this thesis project is to elucidate plausible mechanisms underlying early diabetes-induced visual dysfunction. Using rodent models of Type 1 diabetes, I first delineated the temporal relationship of visual deficits with other early diabetes-evoked changes in the retina, namely retinal alterations, vascular dysfunction, and cataract formation. Specifically, I found that visual defects found in early-stage diabetic retinopathy may initially involve dysfunctions of the neural retina and retinal vasculature, and worsen with later development of cataracts. Subsequently, with pharmacologic means and genetic models, I investigated the contributions of two specific abnormalities to the visual deficits - disrupted dopamine neurotransmission and defective functional hyperemia. The results show that both retinal dopamine deficiency and reduced functional hyperemic response due to diabetes result in profound visual deficits in rodent models of diabetes, and restoration of these functions ameliorates the severity of visual defects. Collectively, these findings uncover new therapeutic targets that may yield not only visual improvement but also slowing of disease progression. Moreover, the mechanisms revealed in this thesis work can potentially provide novel biomarkers to characterize preclinical diabetic retinopathy and strengthen the importance of viewing and treating diabetic retinopathy as a neurovascular disorder.

Table of Contents

TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ..................................................................................... 1 1.1 Visual Processing by the Retina.......................................................................... 1 1.2 Overview of Diabetic Retinopathy.......................................................................... 6 1.3 Signs of Neuronal Dysfunctions in Preclinical Stages of Diabetic Retinopathy... 12 1.3.1 Alterations in Retinal Function..................................................................... 12 1.3.2 Early Diabetes-associated Visual Dysfunction............................................. 17 1.4 Potential Underlying Pathogenic Mediator(s) for Retinal & Visual Deficit.......... 18 1.4.1 Neurodegeneration....................................................................................... 18 1.4.2 Abnormalities in Visual Processing.............................................................. 20 1.4.3 Abnormal Hemodynamic Responses & Resulting Retinal Hypoperfusion... 27 1.5 Thesis Overview............................................................................................... 34 CHAPTER 2: Associations between Early Retinal Alterations & Visual Deficits in Streptozotocin-induced Diabetic Long Evans Rats............................................................................................................ 38 2.1 Abstract............................................................................................................. 38 2.2 Introduction...................................................................................................... 39 2.3 Materials & Methods........................................................................................ 41 2.3.1 Animals & Experimental Design.................................................................. 41 2.3.2 Visual Function Test..................................................................................... 42 2.3.3 Cataract Examination & Scoring................................................................. 43 2.3.4 Retinal Function Test with ERG................................................................... 44 2.3.5 Statistical Analysis........................................................................................ 44 2.4 Results.................................................................................................................... 45 2.4.1 Diabetes Resulted in Profound Visual Deficits............................................. 47 2.4.2 Cataract Formation Impaired Visual Function in Diabetic Animals........... 49 2.4.3 Retinal Dysfunction may be a Potential Contributor to Early Visual Deficits ....................................................................................................................... 53 2.5 Discussion.............................................................................................................. 57 2.5.1 Cataract & Visual Dysfunction in Early-stage DR....................................... 59 2.5.2 Current OKT Findings on Animal Models of Diabetes................................ 59 2.5.3 Retinal Origins of Visual Defects in Diabetes.............................................. 60 2.5.4 Future Implications....................................................................................... 62 CHAPTER 3: DOPAMINE DEFICIENCY CONTRIBUTES TO EARLY VISUAL DYSFUNCTION IN A RODENT MODEL OF TYPE 1 DIABETES.................. 64 3.1 Abstract.............................................................................................................. 64 3.2 Introduction...................................................................................................... 65 3.3 Materials & Methods....................................................................................... 67 3.3.1 Animals & Experimental Design.................................................................. 67 3.3.2 Visual Psychophysical Test........................................................................... 70 3.3.3 Retinal Function Test.................................................................................... 71 3.3.4 Dopamine & DOPAC Analysis..................................................................... 72 3.3.5 RT-PCR Analysis.......................................................................................... 72 3.3.6 Statistical Analysis........................................................................................ 75 3.4 Results.................................................................................................................... 75 3.4.1 Diabetes Significantly Reduced Retinal Dopamine Level............................ 75 3.4.2 Restoring Dopamine Content Delayed Diabetes-induced Visual Dysfunction ...................................................................................................................... 80 3.4.3 Retinal Dysfunction Underlies the Dopamine-mediated Visual Deficits in Diabetes........................................................................................................ 82 3.4.4 Retinal Transcript Levels of Key Dopamine Proteins Unchanged with Diabetes........................................................................................................ 90 3.4.5 Selective Improvement in Visual Function with Dopamine Receptor Agonists......................................................................................................... 93 3.5 Discussion.............................................................................................................. 95 3.5.1 Dopamine Deficiency in Diabetes................................................................ 95 3.5.2 Dopamine Deficiency & Visual Deficits....................................................... 97 3.5.3 Diabetes-induced Dopamine Deficiency & Clinical Relevance................... 99 CHAPTER 4: DEFECTIVE FUNCTIONAL HYPEREMIA MAY CONTRIBUTE TO EARLY DIABETES-INDUCED VISUAL DEFICITS IN STREPTOZOTOCIN-INDUCED DIABETIC ANIMALS................................. 101 4.1 Abstract................................................................................................................ 101 4.2 Introduction.......................................................................................................... 103 4.3 Materials & Methods........................................................................................... 105 4.3.1 Animals & Experimental Design................................................................ 105 4.3.2 Assessing Flicker-induced Vasodilation..................................................... 107 4.3.3 Optokinetic Tracking Assessements............................................................ 111 4.3.4 Slit Lamp Cataract Exam................................................................... 111 4.3.5 Statistical Analysis...................................................................................... 111 4.4 Results.................................................................................................................. 112 4.4.1 Diabetic Rats Exhibited Diminished Hyperemic Response Prior to Reduced Visual Function.... ......................................................................................112 4.4.2 HET0016 Treatment Ameliorated Diabetes-induced Visual Deficits......... 115 4.4.3 AUDA Treatment Improved Visual Function of Diabetic Animals............ 115 4.4.4 Comparison of the Visual Benefits of HET0016 & AUDA Treatments................................................................................................... 118 4.4.5 HET0016 or AUDA Treatment Did Not Alter the General Health & Lens Opacity of the Treated Diabetic Animals.................................................... 120 4.5 Discussion............................................................................................................ 124 4.5.1 Detecting Functional Hyperemia in Early-stage DR.................................. 124 4.5.2 Underlying Causes for Abnormal Functional Hyperemia...................... 125 4.5.3 Role of 20-HETE & EETs in Early-stage Diabetic Retinopathy................ 126 4.5.4 Regulating Levels of Arachidonic Acid Metabolites & the Effects on Visual Function...................................................................................................... 128 4.5.5 Therapeutic Efficacies of HET0016 & AUDA............................................ 130 4.5.6 Conclusions.............. ................................... .............................................. 131 CHAPTER 5: CONCLUDING REMARKS ............................................................... 132 5.1 Summary of Findings...................................................................................... 132 5.2 Technical Implications for Experimental Study of Diabetic Retinopathy........... 134 5.3 Insights to the Pathogenesis of Early-stage Diabetic Retinopathy...................... 136 5.4 Potential Clinical Applications............................................................................ 141 5.5 Limitations, Future Direction, & Final Words..................................................... 142 REFERENCES............................................................................................................... 147

LIST OF FIGURES CHAPTER 1: Figure 1-1. Basic overview of the eye and the retina ................................................4 Figure 1-2. A fundus image of a human eye ...................................................................5 Figure 1-3. Illustrations of clinically relevant vascular lesions and the stages of Diabetic Retinopathy .............................................................................10 Figure 1-4. Optical Coherence Tomography images comparing an eye with normal macula and one with diabetic macular edema .....................................11 Figure 1-5. Sample full-field ERG waveform (top) and filtered oscillatory potentials (bottom) from an adult rat in response to a -0.6 log cd sec/m² flash ...........................................................................................................................15 Figure 1-6. Demonstration of how stimuli are delivered in multifocal ERG and the retinal response that it elicits .......................................................................16 Figure 1-7. Representation of the molecular steps in photoactivation and deactivation of the phototransduction cascade ..............................................22 Figure 1-8. Schematics of the potential mechanisms underlying functional hyperemia ...................................................................................................................31 Figure 1-9. Synthesis pathways for the pertinent vasoactive arachidonic acid metabolites that are involved in functional hyperemia ..................................32 Figure 1-10. Proposed mechanism for arachidonic acid metabolites-mediated neurovascular coupling in the retina and how diabetes may disrupt its functioning ...........................................................................................................33 Figure 1-11. Outlines of the proposed studies and the corresponding hypotheses tested for the thesis project ......................................................................36 Figure 1-12. Configuration of the virtual optomotor system to assess optokinetic response ................................................................................................37 CHAPTER 2: Figure 2-1. Reduced visual function due to hyperglycemia over the 12-week study........................................................................................................................... 48 Figure 2-2. Cataract scores for CTRL and DM groups over time......................... 51 Figure 2-3. Stratification of visual acuity (A) and contrast sensitivity (B) of DM animals based on the presence or absence of cataract............................. 52 Figure 2-4. Retinal dysfunction due to diabetes...................................................... 55 Figure 2-5. Scatter plots of visual acuity value against corresponding (A) cataract score, (B) dim-flash OP4 latency, and (C) bright-flash OP4 latency for CTRL (n=7-8) and DM (n=5-8) rats at 4 weeks, 8 weeks, and 12 weeks post-injection................................................................................................. 56 Figure 2-6. Chronological summary of the functional deficits found in our study.................................................................................................................... 58 CHAPTER 3: Figure 3-1. Diabetes reduced retinal DA contents in STZ-induced DM rats...... 78 Figure 3-2. Diabetes lowered retinal DA levels in STZ-induced DM mice .......... 79 Figure 3-3. Chronic L-DOPA treatment delayed early diabetes-induced visual dysfunction ........................................................................................................81 Figure 3-4. Genetic model of retinal DA deficiency (rTHKO) replicated early diabetes-induced visual dysfunction and could be rescued with L-DOPA treatment.................................................................................................................. 83 Figure 3-5. Changes in DA levels due to diabetes affected light-adapted retinal function....................................................................................................... 88 Figure 3-6. Changes in DA levels due to diabetes affected dark-adapted retinal function....................................................................................................... 89 Figure 3-7. mRNA levels of the examined dopaminergic system related genes....................................................................................................................... 92 Figure 3-8. Distinct improvement in OKT responses of 8-week DM WT mice (n=7) after treatments with selective dopamine receptor agonists........................ 94 CHAPTER 4: Figure 4-1. Schematic illustrating the imaging setup........................................... 109 Figure 4-2. Images detailing the process of analyzing flicker-induced vasodilatory response.......................................................................................... 110 Figure 4-3. STZ-induced DM rats exhibited significant reductions in flicker-induce vasodilation............................................................................................... 114 Figure 4-4. Inhibition of 20-HETE synthesis with HET0016 preserved visual functions of DM rats............................................................................................. 116 Figure 4-5. Inhibition of EETs degradation with AUDA improved visual functions of DM rats............................................................................................. 117 Figure 4-6. Efficacy of HET0016 and AUDA treatments differed in maintenance of visual acuity but was similar in preservation of contrast sensitivity .................................................................................................................................... 119 Figure 4-7. Severity of cataract formation in DM animals did not differ due to HET0016 or AUDA treatment.............................................................................. 123 CHAPTER 5: Figure 5-1. Schematic illustrating how dopamine deficiency may fit in the pathogenesis of early diabetes-induced retinal and visual dysfunctions .................................................................................................................................... 139 Figure 5-2. Diagram showing the potential interactions between dopamine deficiency and defective functional hyperemia in diabetes.......................... 140 Figure 5-3. Representative HPLC chromatograms illustrating the separation of fluorescently labeled P-450 metabolites of arachidonic acid (A) in a mixture of standards and (B) in a labeled sample of rat retina................... 146
LIST OF TABLES CHAPTER 2: Table 2-1. Average weight and blood glucose levels of the two treatment groups (± SEM) for the two sets of experiments................................................. 46 CHAPTER 3: Table 3-1. Sequences of the primers used in RT-PCR reactions for genes of interest....................................................................................................................... 74 Table 3-2. Average weight and blood glucose levels of the experimental groups (± SEM) used for this project.................................................................... 77 Table 3-3. Daily L-DOPA injections restored retinal dopamine levels............... 85 Table 3-4. Fold changes in mRNA levels of genes of interest for each experimental group compared to CTRL WT group............................................. 91 CHAPTER 4: Table 4-1. Average weight and blood glucose (BG) levels of the experimental groups (± SEM) used for this project.................................................................. 122

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Neuroscience
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Biology, Neuroscience Health Sciences, Ophthalmology
Palabra Clave	rodent model visual dysfunction diabetic retinopathy dopamine deficiency functional hyperemia
Committee Chair / Thesis Advisor	Pardue, Machelle, Emory University
Committee Members	Sutliff, Roy, Emory University Hartzell Jr., Criss, Emory University Iuvone, P Michael, Emory University Thule, Peter Martin, Emory University Ressler, Kerry, Emory University

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