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David C. Broadway, Andrew Osborne, Jeremy Rhodes, Julie Sanderson; Does Elevated Hydrostatic Pressure Cause Loss Of Retinal Ganglion Cell Viability In Human Organotypic Retinal Cultures?. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6692.
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© ARVO (1962-2015); The Authors (2016-present)
Elevated IOP, the major risk factor in glaucoma, has been proposed to have a direct effect on retinal ganglion cells (RGCs). The effect of "pure" hydrostatic pressure on stress pathway signalling and RGC survival in human organotypic retinal cultures (HORCs) was investigated.
A novel culture chamber was made to expose cells to increased hydrostatic pressure (constant or fluctuating) maintaining constancy of other parameters. Accurate pressure control (0-100mmHg) was achieved using mass flow controllers. HORCs from human donors (<24h PM) were cultured in serum-free DMEM/HamF12 medium. RGC-5 cells were used for comparison, since these have been used previously in similar published studies. To simulate ischaemia, HORCs were cultured for 3h in glucose-free DMEM in a modular incubator gassed with 95%N2/5%CO2, followed by control conditions for 21h. Cell viability was measured by MTS assay; cell death and apoptosis by LDH and TUNEL assays; RGC marker expression by QRTPCR (THY-1) and immunohistochemistry (NeuN). Activated p38 and JNK levels were detected by Western blot.
Exposure of HORCs to constant (60mmHg) or fluctuating (0-100mmHg; 1 cycle/min) pressure for 24 or 48h caused no loss of structural integrity or retinal architecture compared with controls (n=4). No significant change in RGC marker (THY-1 or NeuN) levels or LDH release was observed (n=4; p>0.05) with pressure elevation. In RGC-5 cells exposed to elevated pressure, no change in cell viability (n=4), LDH release (n=3) or apoptosis (24 or 72h) was observed (n=3; p>0.05). Simulated ischaemia reduced RGC number by 40.3±2.3% at 24h in HORCs (n=9; p<0.05) and caused an 82.8±11.7% increase in LDH release (n=10; p<0.05). p38 and JNK phosphorylation remained unchanged in HORCs exposed to fluctuating pressure (15, 30, 60 and 90min)(n=3; p>0.05), whereas ischaemia for 3h followed by culture for 0-90min increased activation of p38 and JNK in HORCs (2.8-fold & 2.5-fold respectively at t=60min; n=3; p<0.05).
Use of a sophisticated pressure control system enabled the effect of "pure" pressure changes to be investigated in a human retina model system. Increased pressure did not affect RGC survival. Simulated ischaemia however was sufficient to activate stress pathways and reduce RGC number. It is suggested that direct hydrostatic pressure alone is not responsible for degeneration of RGCs in glaucoma.
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