In vivo and in vitro studies on the effects of corticosteroids on retinal ganglion cells.

Ho Yi-Fong.Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.Includes bibliographical references (leaves 120-131).Abstracts in English and Chinese.Abstract --- p.iAcknowledgements --- p.ivTable of Contents --- p.vList of Figures --- p.ixList of Tables --- p.xiAbbreviations --- p.xiiChapter Chapter 1 --- Introduction --- p.1Chapter 1.1 --- Corticosteroids in ophthalmology --- p.1Chapter 1.1.1 --- History of the clinical use of corticosteroids --- p.1Chapter 1.1.2 --- Administration --- p.1Chapter 1.1.3 --- General biological effects of corticosteroids --- p.4Chapter 1.1.4 --- Application of corticosteroids in ocular diseases --- p.5Chapter 1.1.5 --- Side effects of ocular corticosteroid treatment --- p.6Chapter 1.1.6 --- General introduction to commonly used corticosteroids in ophthalmology --- p.6Chapter 1.1.6.1 --- Hydrocortisone --- p.6Chapter 1.1.6.2 --- Dexamethasone --- p.7Chapter 1.1.6.3 --- Triamcinolone --- p.7Chapter 1.1.6.4. --- Chemical structures and relative anti-inflammatory potencies --- p.8Chapter 1.1.7 --- Cytotoxicity of triamcinolone --- p.12Chapter 1.2 --- Retinal ganglion cells --- p.13Chapter 1.2.1 --- Basic structures of the eye --- p.13Chapter 1.2.2 --- Anatomical structure of retina --- p.13Chapter 1.2.3 --- Functions of retinal ganglion cells --- p.18Chapter 1.2.4 --- Culture models to study RGCs --- p.20Chapter 1.3 --- Aim of study --- p.25Chapter Chapter 2 --- Methodology --- p.26Chapter 2.1 --- Intravitreal injection of TA (IVTA) --- p.26Chapter 2.1.1 --- Materials --- p.26Chapter 2.1.1.1 --- Animals --- p.26Chapter 2.1.1.2 --- Chemicals --- p.26Chapter 2.1.1.3 --- Instruments --- p.26Chapter 2.1.2 --- Procedures --- p.26Chapter 2.2 --- Peripheral Nerve - Optic Nerve Grafting (PN-ON) Procedure --- p.27Chapter 2.3 --- Retrograde Labeling of regenerating RGCs --- p.27Chapter 2.3.1 --- Materials --- p.27Chapter 2.3.2 --- Procedures --- p.27Chapter 2.3.3 --- Statistical analysis --- p.28Chapter 2.4 --- Immunohistochemistry --- p.28Chapter 2.4.1 --- Materials --- p.28Chapter 2.4.2 --- Procedures --- p.29Chapter 2.4.3 --- Statistical analysis --- p.29Chapter 2.5 --- Histology --- p.29Chapter 2.5.1 --- Materials --- p.29Chapter 2.5.2 --- Procedures --- p.29Chapter 2.6 --- Primary rat retinal ganglion cell culture --- p.30Chapter 2.6.1 --- Materials --- p.30Chapter 2.6.1.1 --- Animals --- p.30Chapter 2.6.1.2 --- Chemicals --- p.30Chapter 2.6.1.3 --- Solutions and buffers --- p.30Chapter 2.6.1.4 --- Instruments --- p.31Chapter 2.6.2 --- Preparations --- p.31Chapter 2.6.2.1 --- Working media --- p.31Chapter 2.6.2.2 --- Plate coating --- p.32Chapter 2.6.3 --- Cell culture process --- p.32Chapter 2.6.3.1 --- Dissection of retinal tissues --- p.32Chapter 2.6.3.2 --- Purification of RGCs --- p.33Chapter 2.6.3.3 --- Culture condition and cell seeding --- p.34Chapter 2.7 --- Corticosteroid treatment --- p.34Chapter 2.7.1 --- Materials --- p.34Chapter 2.7.2 --- Preparations --- p.34Chapter 2.7.3 --- Treatment --- p.35Chapter 2.8 --- Cell viability assay and morphometric study --- p.36Chapter 2.8.1 --- Materials --- p.36Chapter 2.8.2 --- Calcein-AM staining --- p.36Chapter 2.8.3 --- Cell viability --- p.37Chapter 2.8.4 --- Morphometry study --- p.37Chapter 2.9 --- TUNEL Assay --- p.38Chapter 2.9.1 --- Materials --- p.38Chapter 2.9.2 --- Procedure --- p.38Chapter 2.9.3 --- Statistical analysis --- p.39Chapter 2.10 --- Quantitative Reverse transcription - Polymerase Chain Reaction (qRT-PCR) --- p.39Chapter 2.10.1 --- Materials --- p.39Chapter 2.10.1.1 --- "Chemicals, reagents, and kits" --- p.39Chapter 2.10.1.2 --- Primers --- p.40Chapter 2.10.1.3 --- Equipment --- p.41Chapter 2.10.1.4 --- Software --- p.41Chapter 2.10.2 --- Procedures --- p.41Chapter 2.10.2.1 --- Cell collection and RNA isolation --- p.41Chapter 2.10.2.2 --- Reverse Transcription --- p.42Chapter 2.10.2.3 --- Real-time PCR --- p.43Chapter 2.10.3 --- Statistical analysis --- p.43Chapter 2.11 --- Western blotting --- p.44Chapter 2.11.1 --- Sample preparation --- p.44Chapter 2.11.1.1 --- Materials --- p.44Chapter 2.11.1.1.1 --- Chemicals and materials --- p.44Chapter 2.11.1.1.2 --- Equipment --- p.44Chapter 2.11.1.2 --- Procedures --- p.44Chapter 2.11.2 --- Protein assay --- p.45Chapter 2.11.2.1 --- Materials --- p.45Chapter 2.11.2.1.1 --- Chemicals and materials --- p.45Chapter 2.11.2.1.2 --- Equipment and software --- p.46Chapter 2.11.2.2 --- Procedures --- p.46Chapter 2.11.3 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.46Chapter 2.11.3.1 --- Materials --- p.46Chapter 2.11.3.1.1 --- Chemicals and reagents --- p.46Chapter 2.11.3.1.2 --- Equipment --- p.46Chapter 2.11.3.1.3 --- Solutions and buffers --- p.47Chapter 2.11.3.2 --- Gel preparation --- p.48Chapter 2.11.3.3 --- Electrophoresis --- p.49Chapter 2.11.3.4 --- Transblotting (semi-dry transfer) --- p.49Chapter 2.11.3.5 --- Band visualization --- p.49Chapter 2.11.4 --- Immunostaining --- p.50Chapter 2.11.4.1 --- Materials --- p.50Chapter 2.11.4.1.1 --- Antibodies --- p.50Chapter 2.11.4.1.2 --- Chemicals and reagents --- p.50Chapter 2.11.4.1.3 --- Equipment --- p.50Chapter 2.11.4.2 --- Procedures --- p.50Chapter 2.12 --- Gas-chromatography-electron-capture negative-ion mass spectrometry (GC-NCI-MS) --- p.51Chapter 2.12.1 --- Standard and reagents --- p.51Chapter 2.12.2 --- Chromatography and mass spectrometry --- p.52Chapter 2.12.3 --- Sample collection --- p.52Chapter 2.12.3 --- Standard and sample preparation --- p.53Chapter 2.12.4 --- Validation --- p.54Chapter Chapter 3 --- ResultsChapter 3.1 --- Effect of triamcinolone on RGCs in vivo --- p.55Chapter 3.2 --- Cell viability of RGCs after IVTA plus PN-ON grafting --- p.55Chapter 3.3 --- Abnormal retinal morphology under different IVTA conditions --- p.59Chapter 3.4 --- Cell viability assay --- p.66Chapter 3.4.1 --- Effects of triamcinolone on RGC viability --- p.66Chapter 3.4.2 --- Effects of dexamethasone on RGC viability --- p.68Chapter 3.4.3 --- Effects of hydrocortisone on RGC viability --- p.70Chapter 3.4.4 --- Effects of filtered fraction of triamcinolone on RGC viability --- p.72Chapter 3.5 --- Morphometric study analysis of RGCs --- p.74Chapter 3.5.1 --- Percentage of RGCs showing neurite outgrowth --- p.74Chapter 3.5.2 --- Average neurite length --- p.77Chapter 3.5.3 --- Neurite spanning area --- p.77Chapter 3.5.4 --- Neurite count --- p.80Chapter 3.5.5 --- Neurite branching --- p.83Chapter 3.6 --- Determination of concentration of TA in culture media by GC-NCI-MS --- p.85Chapter 3.7 --- TUNEL assay --- p.90Chapter 3.8 --- Real-time quantitative Reverse transcription - Polymerase Chain Reaction --- p.93Chapter 3.9 --- Western blot --- p.99Chapter Chapter 4 --- Discussion --- p.102Chapter Chapter 5 --- References --- p.12