published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Glaucoma is a devastating blinding disease, caused by retinal ganglion cell (RGC) loss via apoptosis and clinically associated with raised intraocular pressure (IOP). Mainstream theories of glaucoma's pathogenesis detail loss of RGCs via indirect links to lOP; including mechanodistortion of optic nerve axons, trophic factor deficiency, ischaemia or excitotoxicity. A novel concept in the pathogenesis of glaucoma is that pressure alone could be a direct stimulus for RGC loss. Currently available glaucoma treatments are solely aimed at lowering lOP. Reduction ofIOP has been shown to reduce glaucomatous progression however RGC losses continue. Neuroprotection is an emerging field of research offering hope to neurodegenerative diseases, including glaucoma. This thesis investigated known glaucoma therapeutics with suggested neuroprotective activity in an in vitro glaucoma model using the RGC-5 cell line. To evaluate therapies, a suitable in vitro model was initially evaluated. The RGC-5 cell line was immunochemically demonstrated to possess NSE, a neuronal marker and Thy-1, an RGC marker. Glutamate excitotoxicity was investigated however excessive concentrations were required to cause significant in vitro RGC-5 cytotoxicity. Using a modified hydrostatic pressure model, reproducible pressure-induced RGC-5 apoptosis was demonstrated. Apoptosis was detected using cell morphology and confirmed with both early (caspase-3 and annexin V) and late (TUNEL) apoptotic markers. Despite the advantages of rapid, objective quantification, results indicated that flow cytometry of RGC-5 cells was not technically possible. I defined a modified laser scanning cytometry protocol, allowing for objective apoptosis quantification of TUNEL stained cells. Brimonidine is postulated to mediate receptor mediated RGC protection. Experiments conducted for this thesis, were the first to immunochemically demonstrate alpha2 adrenergic receptor expression on human RGCs and the RGC-5 cell line, suggesting direct cell mediated protection on the target neuron of glaucoma is possible and that the RGC-5 line is a useful in vitro target for experimentation. Both brimonidine and betaxolol were shown to confer protection to the RGC-5 cell line from pressure-induced apoptosis, under defined conditions. These results suggest that these drugs may confer direct cell-mediated protection, rather than indirect protection conferred via other retinal or glial cells, the anterior segment, the vasculature or some other means.

Thesis (Ph. D.)--University of Hong Kong, 1994. / Includes list of author's publication (leaves viii-xiv). Includes bibliographical references (leaves 253-281).

No description available.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Glaucoma is characterized by retinal ganglion cell apoptosis leading to a corresponding loss of the visual field. Elevated intraocular pressure is the principal clinical association of this disease and its reduction remains the mainstay of current therapy. This research established an in-vitro glaucoma model and investigated the direct effects of increased hydrostatic pressure on retinal ganglion cell survival as well as the cellular response to changes in pressure. In the first part of this thesis (chapter 3) the direct effects of pressure on retinal ganglion cell survival was established. The differentiated RGC5 cell line was subjected to elevated pressure 100 mmHg for a period of two hours in a pressure chamber. Cell apoptosis was then detected by TdT-mediated dITP Nick-End Labelling (TUNEL). Quantitative analysis of the percentage of apoptotic cells between the control and pressure groups by Laser Scanning Cytometry (LSC) revealed that pressure alone induced significant apoptosis. Furthermore, caspase-3 cleavage was detected in the pressure treated cells by Western blot analysis. The next three chapters investigated how the applied pressure may be mediated through cellular mechno-sensitive structures. TWIK Related Arachiodonic Acid stimulated K+ channel (TRAAK) is a mechano-gated neuronal potassium channel, which can be opened by pressure and arachidonic acid. In chapter 4, TRAAK was identified as expressed on the rat RGC5 cell line. This was determined by both immunostaining and RT-PCR. Opening this channel by arachidonic acid induced significant apoptosis in RGC5 neurons; elevated extracellular K+ concentration and blockage of TRAAK by gadolinium inhibited both arachidonic acid and pressure-induced apoptosis. These results indicated that elevated pressure resulted in opening of the outward potassium channel-TRAAK and consequently potassium ion efflux and apoptotic volume decrease (AVD). Data from chapter 5 revealed that pressure also caused actin reorganization with both F- and G-actin shifts. At the early stage (following 2 hours pressure treatment), actin polymerization led to G-actin pool decrease and disinhibition of DNase1 in the cytoplasm. This has been suggested to lead to DNase1 nuclear translocation and contribution to DNA fragmentation associated with apoptosis. The preliminary microarray results of chapter 6 revealed pressure effects on gene expression Included in the many up- and down-regulated genes was; down-regulation of antiapoptotic gene- BcL-x and up- regulation of Damage-Induced Neuronal Endopeptidase (DINE) after pressure treatment. This study showed that elevated pressure induced RGC5 apoptosis and affected multi cellular mechanosnesitive structures. These results may indicate new mechanisms of RGC neuron apoptosis and further therapeutic strategies.

Thesis (Ph.D.)--Texas Christian University, 2007. / Title from dissertation title page (viewed Apr. 25, 2007). Includes abstract. Includes bibliographical references.

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Glaucoma is characterized by retinal ganglion cell apoptosis leading to a corresponding loss of the visual field. Elevated intraocular pressure is the principal clinical association of this disease and its reduction remains the mainstay of current therapy. This research established an in-vitro glaucoma model and investigated the direct effects of increased hydrostatic pressure on retinal ganglion cell survival as well as the cellular response to changes in pressure. In the first part of this thesis (chapter 3) the direct effects of pressure on retinal ganglion cell survival was established. The differentiated RGC5 cell line was subjected to elevated pressure 100 mmHg for a period of two hours in a pressure chamber. Cell apoptosis was then detected by TdT-mediated dITP Nick-End Labelling (TUNEL). Quantitative analysis of the percentage of apoptotic cells between the control and pressure groups by Laser Scanning Cytometry (LSC) revealed that pressure alone induced significant apoptosis. Furthermore, caspase-3 cleavage was detected in the pressure treated cells by Western blot analysis. The next three chapters investigated how the applied pressure may be mediated through cellular mechno-sensitive structures. TWIK Related Arachiodonic Acid stimulated K+ channel (TRAAK) is a mechano-gated neuronal potassium channel, which can be opened by pressure and arachidonic acid. In chapter 4, TRAAK was identified as expressed on the rat RGC5 cell line. This was determined by both immunostaining and RT-PCR. Opening this channel by arachidonic acid induced significant apoptosis in RGC5 neurons; elevated extracellular K+ concentration and blockage of TRAAK by gadolinium inhibited both arachidonic acid and pressure-induced apoptosis. These results indicated that elevated pressure resulted in opening of the outward potassium channel-TRAAK and consequently potassium ion efflux and apoptotic volume decrease (AVD). Data from chapter 5 revealed that pressure also caused actin reorganization with both F- and G-actin shifts. At the early stage (following 2 hours pressure treatment), actin polymerization led to G-actin pool decrease and disinhibition of DNase1 in the cytoplasm. This has been suggested to lead to DNase1 nuclear translocation and contribution to DNA fragmentation associated with apoptosis. The preliminary microarray results of chapter 6 revealed pressure effects on gene expression Included in the many up- and down-regulated genes was; down-regulation of antiapoptotic gene- BcL-x and up- regulation of Damage-Induced Neuronal Endopeptidase (DINE) after pressure treatment. This study showed that elevated pressure induced RGC5 apoptosis and affected multi cellular mechanosnesitive structures. These results may indicate new mechanisms of RGC neuron apoptosis and further therapeutic strategies.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.