The death of RGCs in certain models of glaucoma is known to be apoptotic.
3 6 70 71 72 73 In our experimental system, exposure to 70 mm Hg pressure for 48 hours led to clear morphologic changes in RGCs, including the retraction of cellular processes, a more rounded appearance, and decreased density
(Fig. 4) . These morphologic changes were accompanied by increased TUNEL staining
(Fig. 4) . Quantitatively, in the postplating period necessary to reach homeostasis, RGCs stabilized at densities of 550 to 650 cells/mm
2. We maintained cultures at ambient pressure for an additional 48 hours without significant change in density (
P = 0.60;
Fig. 5A ), and the variability for this period was small (552 ± 22 cells/mm
2;
Fig. 5A ). Exposure to either 6 or 12 hours of elevated pressure induced little or no change in survival (
P = 0.98 and
P = 0.69, respectively;
Fig. 5A ). However, after 24 hours of elevated pressure, the density of RGCs decreased by 27% compared with their ambient levels at the same time point (
P < 0.01;
Fig. 5A ). Similarly, exposure for 48 hours induced a 37% decrease in RGC density from ambient levels for the same time (
P < 0.01;
Fig. 5A ). Because the densities of RGCs did not change appreciably for 6- and 12-hour exposures, we focused additional measurements on the longer exposures. At 24 hours of elevated pressure, the fraction of TUNEL-positive RGCs increased by 65% from the fraction at ambient pressure for the same time (
P < 0.01); at 48 hours of pressure, the fraction nearly doubled from the ambient level (
P < 0.01;
Fig. 5B ). Thus, exposure to elevated pressure for 24 hours or more induces a decrease in cell density that is accompanied by a reciprocal increase in TUNEL-positive cells. The small concentration of cytosolic LDH in the medium did not change after 24 hours of elevated pressure (
P = 0.17;
Fig. 5C ), indicating no change in the degree of necrotic death.