Retinal ganglion cell line apoptosis induced by hydrostatic pressure

Abstract

Cellular responses to changes in pressure are implicated in numerous disease processes. In glaucoma apoptosis of retinal ganglion cells (RGCs) is associated with elevated intra-ocular pressure, however, the exact cellular mechanisms remain unclear. We have previously shown that pressure can induce apoptosis in B35 and PC12 neuronal cell lines, using an in vitro model for pressure elevation. A novel RGC line allows us to study the effects of pressure on retinal neurons. 'RGC-5' cultures were subjected to elevated ambient hydrostatic pressure conditions in our model. Experimental pressure conditions were 100 mm Hg and 30 mm Hg, representing acute (high) and chronic (lower-pressure) glaucoma, and 15 mm Hg for normal intra-ocular pressure, set above atmospheric pressure for 2 h. Negative controls were treated identically except for the application of pressure, while positive controls were generated by treatment with a known apoptotic stimulus. Apoptosis was determined by a combination of cell morphology and specific TUNEL and Annexin V fluorescent markers. These were assessed simultaneously by laser scanning cytometry (LSC), which also enabled quantitative marker analysis. RGC-5 neurons showed a significantly increased proportion of apoptotic cells compared with controls; maximal at 100 mm Hg, moderate at 30 mm Hg and not statistically significant at 15 mm Hg. This graded response, proportionate to the level of pressure elevation, is representative of the severity of analogous clinical settings (acute, chronic glaucoma and normal). These results complement earlier findings of pressure-induced apoptosis in other neuronal cultures. They suggest the possibility of novel mechanisms of pressure-related mechanotransduction and cell death, relevant to the pathogenesis of diseases such as glaucoma.