Fig. 1. EDN1 caused caspase 3 activation in RGCs and RGC loss.

a Caspase 3 activation in RBPMS+ RGCs after intravitreal PBS or EDN1 injection. Caspase 3 was activated (cleaved, cCASP3, red) in RBPMS+ RGCs (green) post-EDN1 injection, which peaked after 5 days. Caspase 3 was rarely activated in RGCs at any timepoint post-PBS injection (cCASP3+ RBPMS+ cells/mm² ± SEM 3, 5, 10, 14 days post injection; PBS: 0.8 ± 0.2, 0.8 ± 0.2, 1.0 ± 0.3, 0.1 ± 0.1; EDN1: 3.2 ± 1.3, 31.0 ± 11.5, 5.4 ± 2.1, 5.4 ± 2.1, 1.6 ± 0.7; n ≥ 5 per condition per timepoint, *P < 0.001 compared to respective PBS, two-way ANOVA, Holm-Sidak’s post hoc). Note: bottom row images depict RBPMS immunofluorescence (left image, green) and RBPMS/cCASP3 merged immunofluorescence (right image, green and red respectively) from the boxed area of the above image. Arrowheads indicate an RBPMS+ cCASP3+ cell. b EDN1-injured retinas had significantly fewer RBPMS+ RGCs (green) compared to PBS controls 28 days post injection (%RBPMS+ cell survival ± SEM; PBS: 100.0 ± 2.6; EDN1: 73.9 ± 3.3; n ≥ 7 per condition, *P < 0.001, unpaired two-tailed t test). Note: in some EDN1-injured retinas, caspase activation and RGC death were localized to portions of retina and were not necessarily diffuse throughout the entire retina. Images represent the average cell count for each condition. Error bars, SEM. Scale bars, 50 μm.