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

A confocal scanning laser ophthalmoscope (CSLO) was used to image the axonal and dendritic aborizations of RGCs in the Thy-1 YFP mice. With quantitative analysis of cell body area, axon diameter, dendritic field, number of terminal branches, total dendritic branch length, branching complexity, symmetry and distance from the optic disc, the morphologies of RGCs and the patterns of axonal and dendritic degeneration were analyzed. After optic nerve crush, RGC damage was observed prospectively to begin with progressive dendritic shrinkage, followed by loss of the axon and the cell body. Similar pattern of RGC degeneration was observed after 90 minutes of retinal ischemia although no morphological changes were detected when the duration of ischemia was shortened to 30 minutes. The rate of dendritic shrinkage was variable and estimated on average 2.0% per day and 11.7% per day with linear mixed modeling, after optic nerve crush and retinal ischemic injury, respectively. RGCs with a larger dendritic field had a slower rate of dendritic shrinkage. / In summary, we demonstrated that dendritic shrinkage could be evident even before axonal degeneration after optic nerve crush and retinal ischemic injury. We have established a methodology for in vivo and direct visualization of RGCs and retinal microglia, which could provide reliable and early markers for neuronal damage. Measuring the rate of dendritic shrinkage and tracking the longitudinal activation of microglia would provide new paradigms to study the mechanism of neurodegenerative diseases and offer new insights in testing novel therapies for neuroprotection. / Progressive neuronal cell death and microglial activation are the key pathological features in most neurodegenerative diseases. While investigating the longitudinal profiles of neuronal degeneration and microglial activation is pertinent to understanding disease mechanism and developing treatment, analyzing progressive changes has been obfuscated by the lack of a non-invasive approach that allows long term, serial monitoring of individual neuronal and microglial cells. Because of the clear optical media in the eye, direct visualization of the retinal ganglion cells (RGCs) and microglia is possible with high resolution in vivo imaging technique. In this study, we developed experimental models to visualize and characterize the cellular morphology of RGCs and retinal microglia in vivo in the Thy-1 YFP and the CX3CR1 +/GFP transgenic mice, described the patterns of axonal and dendritic shrinkage of RGCs, discerned the dynamic profile of microglial activation and investigated the relationship between RGC survival and microglial activation after optic nerve crush and retinal ischemic injury induced by acute elevation of intraocular pressure. / The longitudinal profile of microglial activation was investigated by imaging the CX3CR1GFP/+ transgenic mice with the CSLO. Activation of retinal microglia was characterized with an increase in cell number reaching a peak at a week after optic nerve crush and retinal ischemic injury, which was followed by a gradual decline falling near to the baseline at the 4 th week. The activation of retinal microglia was proportional to the severity of injury. The number of RGCs survival at 4 weeks post-injury was significantly associated with the number of activated retinal microglia. / Li, Zhiwei. / Adviser: Leung Kai Shun. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 50-66). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Chiasm cells, which include glia and neurons, are generated early before any retinal axon arrives at the midline of the mouse ventral diencephalon. These cells have been shown to affect retinal axon growth and patterning in the optic chiasm. In this study, we used EdU (5-ethyny1-2'-deoxyuridine) for birthdating these chiasm cells, aiming to find out when these cells are generated; then we tried to trace their fates at later stages of development. EdU injection at embryonic day (E) 9.5 to El 1 labeled a number of chiasmatic neurons and radial glial cells at E13, which were immunoreactive for SSEA-1 and RC2, respectively. After colocalization studies, we found that most of these neurons were born as early as E9.5, while a large number of radial glial cells were born as from El 1. Both E9.5-born chiasmatic neurons and Ell-born radial glia decreased by E14-E16; the radial glia even disappeared finally from the midline. Furthermore, we found that some chiasmatic neurons underwent apoptotic cell death as from El 4, and that the radial glia likely differentiated into other cell types after finishing their retinal axon guidance mission at the midline. So it is reasonable that some of the earliest born chiasm cells disappear during development. / During development, retinal ganglion cell axons grow from the eye to the ventral diencephalon, where axons from the two eyes converge and segregate into crossed and uncrossed projections, forming the optic chiasm. This pattern is critical for binocular vision. Although significant progress has been obtained over the past decades, how retinal axon growth and guidance are regulated at the chiasm is largely unknown. Our research will focus on those problems. / In the last part of this thesis, we investigated the retinal axon pathway in the ventral diencephalon of the Sox10Dom mutant embryos and gamma-crystallin mutant embryos. Our findings indicate that Sox10 may not contribute to axon guidance in the developing optic pathway whereas gammaA-crystallin may only play a role in the later uncrossed axons. / N-methyl-D-aspartate (NMDA) receptor is one of the ionotropic glutamate receptors, which are important in synaptic plasticity, apart from implications in dendritic spine remodeling, neurite outgrowth, elongation and branching and glutamate neurotoxicity. There are several subtypes of NMDA receptor channel subunits, NR1, NR2A-D, NR3A&B. The functional diversity of NMDA receptor resides in the different assembly of subunits. In this study, we used RT-PCR to analyze the mRNA expression of all the NMDA receptor subunits in mouse embryos. After that we chose the NR1, NR2B and NR3A antibodies to investigate NMDA receptor subunit expression in the optic pathway during mouse optic pathway development. Using immunohistochemistry, we found that NR1, NR2B and NR3A were expressed in the mouse retina and optic pathway as from E13 when the optic chiasm is forming. Expression of the NMDA receptor subunits were found in the inner cell layers and along retinal axons. Colocalization studies showed that NR1, NR2B and NR3A were localized on the ganglion cells and their axons. In the ventral diencephalon, these subunits were expressed extensively, but NR1 and NR3A were particularly strong along the optic nerve and optic tract. Furthermore, to identify the function of NMDA receptor during optic chiasm development, we cultured E14 retinal explants on laminin and poly-D-ornithine in the presence of the NMDA receptor antagonists MK-801 or Dextrorphan-D-tartrate. These two antagonists can significantly inhibit the retinal axon outgrowth, suggesting that the NMDA receptor promotes retinal axon outgrowth in the retinofugal pathway during optic chiasm development. / Li, Jia. / Adviser: Chan Sun On. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 145-158). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Wai-Chi Kong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 83-92). / Acknowledgements --- p.i / Abstract --- p.ii / Abbreviations Frequently Used --- p.v / Chapter Chapter1 --- General Introduction --- p.1 / Chapter Chapter2 --- Long term culture of whole-mount retinal explant --- p.16 / Chapter Chapter3 --- Responses of retinal ganglion cells after peripheral nerve transplantation in vivo and in vitro --- p.46 / Chapter Chapter4 --- Effect of optic nerve or peripheral nerve explants on cultured whole-mount retinal explants --- p.62 / Chapter Chapter5 --- General Discussion --- p.78 / References --- p.83 / Tables --- p.93

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published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

The general aim of the thesis was to deduce whether selected naturally occurring flavonoids (genistein, epicatechin gallate (EC), epigallocatechin gallate (EGCG), baicalin) attenuate various secondary insults that may cause death of ganglion cells in primary open angle glaucoma (POAG). An ischemic insult to the rat retina significantly causes the inner retina to degenerate indexed by changes of various antigens, proteins and mRNAs located to amacrine and ganglion cells. These changes are blunted in animals treated with genistein as has been shown for ECGC. Studies conducted on cells (RGC-5 cells) in culture showed that hydrogen peroxide, L-buthionine sulfoximine (BSO)/glutamate and serum deprivation (mimicking oxidative stress), rotenone, sodium azide (affecting mitochondria function in specific ways) and light (where the mitochondria are generally affected) all generated reactive oxygen species and caused death of RGC-5 cells. EGCG was able to attenuate cell death caused by hydrogen peroxide, sodium azide and rotenone. Only EC was able to attenuate BSO/glutamate-induced cell death, in addition to cell death caused by hydrogen peroxide and rotenone. Genistein had no positive effect on cell death in experiments carried out on RGC-5 cells. Exposure of RGC-5 cells to flavonoids showed that EC and EGCG increased the mRNA expression of endogenous antioxidants such as HO-l (heme oxygenase 1) and Nrf-2 (nuclear erythroid factor-z-related factor 2). Light insult, rotenone and sodium azide activate the p38 (protein kinase 38) pathway, while only light and rotenone activate the JNK (c-Jun amino-terminal kinase) pathway. Serum deprivation affects mitochondrial apoptotic proteins causing an increase in the ratio of Bax/Bcl2 (Bax: Bcl-2-associated X protein; Bcl-2: B-cell lymphoma 2). An insult of light to RGC-5 cells, unlike that induced by sodium azide, is inhibited by necrostatin-I and causes an activation of AlF (apoptosis-inducing factor) with alpha-fodrin being unaffected. These studies suggest that ganglion cell death caused by insults as may occur in POAG involves various cellular signaling pathways. The selected flavonoids have diverse actions in increasing cellular defense mechanisms, and in negating the effects of ischemia and specific types of oxidative stress. The results argue for the possible use of flavonoids in the treatment of POAG to slow down ganglion cell death.