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A Agar, S Li, M Hill, N Agarwal, M Coroneo; In-Vitro Pressure Induced Apoptosis in Retinal Ganglion Cell Line Shows Graded Response to Simulated Acute, Chronic Glaucoma and Normal Pressures . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4060.
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Retinal Ganglion Cell (RGC) death associated with raised intra-ocular pressures (IOP) is characteristic of glaucoma. Using an in-vitro model for hydrostatic pressure elevation we have previously shown pressure induced apoptosis in an RGC line. Purpose: To compare the effect on RGC-5 neurons of ambient hydrostatic pressures simulating 'acute' (100 mmHg), 'chronic' (30) glaucoma and 'normal' IOP (15). Methods: RGC-5 cell line derived by vector transformation of primary rat mixed retinal cultures and characterised by phenotype and morphology. Neuronal cell cultures placed in specially designed pressure chambers were incubated in a 5% CO2 & air mixture pressurised for 2h to 100, 30 & 15 mmHg (over and above atmospheric) respectively. Negative control cultures were treated identically except for the application of pressure. Positive apoptosis controls generated by 5% Ethanol treatment for 2h. Apoptosis detected by morphology and assay for specific markers including TUNEL. Quantitative automated analysis of fluorescent apoptosis markers performed by Laser Scanning Cytometry (LSC-Compucyte, MA). Results: Apoptotic morphology was confirmed by fluorescent microscopy with no significant necrosis seen. TUNEL marker and cell population data was quantified by LSC and normalised against positive controls. The proportion of apoptotic neurons was significantly higher in RGC-5 neurons subjected to 100 mmHg pressure (44% cells apoptotic) and 30 mmHg (29%) compared to controls (14%), n=8-12. Cultures exposed to 15 mmHg showed increased levels of apoptosis but this was not statistically significant. Conclusion: RGC-5 neurons showed increased apoptosis proportionate to the degree of pressure elevation; max @ 'acute', med @ 'chronic' & insignificant @ 'normal' pressure simulations. This graded cell death induced by clinically relevant levels of hydrostatic pressure alone may suggest a novel relationship linking pressure & apoptosis perhaps involving more direct mechanisms of glaucoma pathogenesis.
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