Su, Nan: Effects of subretinal injection of human lipofuscin on murine retina as a novel animal model of age-related macular degeneration. 2022
Inhalt
- Table of Contents
- List of Figures
- List of Tables
- List of Abbreviations
- 1. Introduction
- 1.1 Synopsis
- 1.2 Introduction of LF
- 1.2.1 The Composition and Distribution of Lipofuscin
- 1.2.2 Mechanisms of LF Accumulation
- 1.2.3 Lipid Metabolism
- 1.2.4 Relationship between Mitochondria and Lipofuscinogenesis
- 1.2.5 AMD and Lysosome
- 1.2.6 Relationship between AMD and Lipofuscinogenesis
- 1.2.7 Closing Words on Lipofuscin
- 1.3 Glia Cells in the Mammalian Retina
- 1.3.1 Retinal Müller Cells
- 1.3.2 The Microglial Cells
- 1.3.2.1 The Way of Maintenance and Origin of Microglia in the Retina
- 1.3.2.2 The Function of Microglia in Healthy Retina
- 1.3.2.3 The Behaviour of Microglia in the Diseased Retina
- 1.3.2.3.1 Microglial Cells and AMD
- 1.3.2.3.2 Microglial Cells and RPE
- 1.3.2.3.3 Accumulation of LF (A2E) in Retinal Microglial Cells
- 1.3.2.3.4 Microglial Cells and Ageing
- 1.3.2.3.5 Microglial Cells and Diabetic Retinopathy
- 1.3.2.4 Frequently Used Markers of Microglial Cells
- 1.4 RPE, Bruch’s Membrane and Choriocapillaris
- 1.4.1 The RPE in AMD
- 1.4.2 Bruch’s Membrane
- 1.4.3 The Choriocapillaris (CC)
- 1.4.4 The Relationship between the RPE, BM and CC
- 1.5 Purpose of this Study
- 2 Methods and Materials
- 2.1 Devices
- 2.2 Chemicals
- 2.3 Buffers and solutions
- 2.4 Laboratory equipment and consumptive materials
- 2.5 Isolation of Lipofuscin
- 2.6 Animals
- 2.7 Subretinal Injection of LF Suspensions
- 2.8 The Inhibitors of Microglia
- 2.9 Electroretinography (ERG)
- 2.10 In vivo Imaging by OCT
- 2.11 Tissue Processing and Fluorescent Immunohistochemistry
- 2.12 Counting the Cells in Histological Sections
- Fig. 2.5: Example for Counting Cells in Mouse Retina.
- 2.13 Measurement of Thicknesses of RPE and Retina
- 2.14 Investigation of Gene Expression by Quantitative PCR
- 2.15 Electron Microscopy
- 2.16 Statistics
- 3 Results
- 3.1 Synopsis
- 3.2 In vivo Imaging OCT (Optical Coherence Tomography)
- 3.2.1 The Detachment of the Retina after Injection
- 3.2.2 The Auto-Fluorescence of LF in Retina
- 3.2.3 The Difference between the LF and PBS Injection
- 3.2.4 The Difference among the Sham, PBS and LF Injection is Visible in Multicolour Images and OCT
- 3.2.5 The Thickness of RPE and Retina Measured in OCT Images
- 3.2.6 The Difference among the Sham, PBS, and LF Injection Detected by OCTA
- 3.3 Investigation of the Function of the Retina Measured by ERG
- 3.4 Histology
- 3.4.1 Distribution of LF Histology
- 3.4.2 The Behaviour of Microglia
- 3.4.3 Microglia Inhibitors and Immumohistochemical Staining
- 3.4.4 Double Staining of Microglia and some Pro-inflammatory Cytokines
- 3.4.4.1 Double Staining of Microglia and IL-6
- 3.4.4.2 Double Staining of Microglia and TNF-α
- 3.4.4.3 Double Staining of Microglia and CXCL2
- 3.4.4.4 Double Staining of Microglia and HIF-1-α
- 3.4.4.5 Double Staining of Microglia and P2X4
- 3.4.4.6 Double Staining of Microglia and VEGF-R1
- 3.4.4.7 Double Staining of Müller Cells and VEGF-R2
- 3.4.4.8 Double Staining of Microglia and VEGF
- 3.4.4.9 Double Staining of Ganglion Cells and IL-6 or TNF-α
- 3.4.4.10 The Cell Number of Microglial Cells in Subretinal Space and the Inner Retina
- 3.5 Analysis of Retinal Structure in Histological Sections
- 3.6 The Thickness of the RPE
- 3.7 Examination of mRNA Levels Using Quantitative PCR
- 3.8 Ultrastructural Evaluation
- 4 Discussion
- 4.1 Synopsis
- 4.2 Feasibility of LF Isolation from Human RPE
- 4.4 Epiretinal Membrane Imaged by Multicolour Imaging
- 4.5 ERG and AMD
- 4.6 Effects of Increased Pro-inflammatory Cytokines and Chemokines on Retinal and Neuronal Degeneration
- 4.6.1 TNF-α
- 4.6.2 IL-6
- 4.6.3 TNF-α and IL-6
- 4.6.4 The Effect of HIF-1α and Its Target Genes in AMD
- 4.6.5 Pro-inflammation and AMD
- 4.6.6 FGF-2 and Ocular Angiogenesis
- 4.7 Microglial Inhibitors
- 4.8 The Effects of Injected LF Studied by Electron Microscopy
- 4.9 The Features of AMD in OCTA
- 4.10 About Immunostaining
- 5. Conclusions
- Reference
- List of Animals Used in this Study
- Acknowledgements