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.
| Identifer | oai:union.ndltd.org:ADTP/258309 |
| Date | January 2007 |
| Creators | Kalapesi, Freny B., Medical Sciences, Faculty of Medicine, UNSW |
| Publisher | Publisher:University of New South Wales. Medical Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://unsworks.unsw.edu.au/copyright, http://unsworks.unsw.edu.au/copyright |
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