In vivo studies using animal models have suggested that the astrocytes of the ONH region acquire a reactive phenotype in glaucoma, such that they no longer support retinal ganglion cell survival. This has motivated the study of the effects of elevated hydrostatic pressure on astrocytes. Several research groups have applied elevated hydrostatic pressure to optic nerve head astrocytes and retinal ganglion cells cultured on a rigid substrate as an in vitro model for glaucoma. These studies have generally shown significant biological effects and this hydrostatic pressure model is now becoming generally accepted in the ophthalmic community. However, since the applied pressures were modest (<100 mm Hg), the finding of significant biological effects due to pressure alone is surprising. We hypothesized that the application of hydrostatic pressure as described in these studies also altered gas tensions (including oxygen tension) in the culture media. Our goal was to design equipment and carry out experiments to separate the biologic effects of hydrostatic pressure from those of gas tension (including hypoxia) on cultured astrocytes. To this end, we designed equipment and carried out experiments to subject astrocyte cultures to the four combinations of two levels of hydrostatic pressure and two levels of oxygen concentration. Using DITNC1 cells, a transformed astrocyte cell line from rat brain, we measured the morphology of astrocytes and the rate at which the cells in the culture populated a cell-free area. No significant change was observed in either migration rate or morphology as a result of elevated hydrostatic pressure or hypoxia. We conclude that the levels of elevated pressure and hypoxia used in these experiments were not sufficient to trigger measurable changes in the morphology or migration rates of DITNC1 cells.