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Studies on the effects of hydrostatic pressure on rat retinal ganglion cell line RGC5.
Abstract/OtherAbstract :
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.
Authors :
Li, Shaojuan, Shaojuan.Li@SESIAHS.HEALTH.NSW.GOV.AU
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Publisher :  University of New South Wales. School of Medical Sciences     Type :  -     Format :  -    
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Copyright Information :, Copyright Shaojuan Li
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Languages :  en    
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