Figure 2.

NO treatment induces iron uptake in retinal cultures and purified RGCs. Dissociated primary pan-retinal (A,B) and immunopurified RGC (C) cultures were prepared from neonatal day 4–5 C57BL/6J mice. (A) 1 hr incubation with the NO-donor GSNO induces iron uptake, measured as the presence of ascorbate-⁵⁵Fe²⁺ (*p < 0.05). (B) 24 h treatment with NO-donor DETA-NONOate (200 μM) increases retinal cell death (***p < 0.001) compared with control cultures. Treatment with 200 µM deferiprone for 24 h significantly (^@@@p < 0.001 compared with DETA-NONOate treatment alone) attenuates retinal cell death induced by DETA-NONOate. Deferiprone treatment alone (without DETA-NONOate) does not affect retinal cell survival, with no difference in cell viability compared to control cultures, and viability remains significantly higher than in cultures treated with DETA-NONOate alone (^@@@p < 0.001). (C) Purified RGCs were incubated with GSNO, then intracellular iron levels were detected with iron-sensitive dye, Phen-GreenSK (PG SK) and 2,2′-bipyridyl (BPD). 2,2′-BPD chelates intracellular iron, increasing PG SK fluorescence. RGCs treated with GSNO show increased intracellular iron levels compared with control RGCs (*p < 0.05, N = 20 cells/treatment group). The normalized change of fluorescence (ΔF/F) was used to estimate change in cytosolic Fe²⁺ levels. All experiments were repeated 3 times.