FIG. 1.

Concentration- and duration-dependent neuronal injury induced by hydrogen peroxide (H₂O₂) in the presence of blockers of glutamate receptors and L-type Ca²⁺ channels. (a) Example phase-contrast images showing neurons before or 24 h after incubation with 100 μM H₂O₂ (upper panels) and summary bar graph showing concentration-dependent neuronal injury by H₂O₂ (lower panel). Cultured mouse cortical neurons grown in 24-well plates were treated with different concentration of H₂O₂ for 1 h in the presence of 10 μM (5R,10S)-(+)-5-Methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine (MK801), 20 μM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 5 μM nimodipine. Neuronal injury was assayed by measurement of the lactate dehydrogenase (LDH) released into the culture medium at 24 h after the H₂O₂ exposure. The relative LDH release was calculated by normalizing the amount of LDH released at 24 h to the maximal releasable amount of LDH induced by permeabilizing cells with 1% triton X-100. The relative LDH release for control, 10, 30, 50, 100, and 300 μM H₂O₂ was 0.11 ± 0.01, 0.11 ± 0.02, 0.45 ± 0.05, 0.5 ± 0.05, 0.55 ± 0.02, and 0.60 ± 0.05, respectively (n = 7–8). (b) Summary data demonstrating duration-dependent neuronal injury by 100 μM H₂O₂. Neurons were treated with 100 μM H₂O₂ in the presence of MK801, CNQX, and nimodipine for 10 min, 30 min, 60 min, and continuous treatment. Relative LDH release was measured at 6 or 24 h after H₂O₂ exposure. At 6 h after the exposure of H₂O₂ for 0 min, 10 min, 30 min, 60 min, and 6 h (continuous), relative LDH release was 0.07 ± 0.01, 0.40 ± 0.03, 0.38 ± 0.02, 0.49 ± 0.06, 0.35 ± 0.05, and 0.46 ± 0.06 (n = 4 wells each). At 24 h after the exposure of H₂O₂ for 0 min, 10 min, 30 min, 60 min, 120 min, and 24 h (continuous), relative LDH release was 0.017 ± 0.01, 0.62 ± 0.06, 0.57 ± 0.04, 0.65 ± 0.06, and 0.55 ± 0.07 (n = 4 wells each). No statistic significant difference in relative LDH release was detected between the treatment with H₂O₂ for 10 min, 30 min, 60 min, and 24 h. **p < 0.01 compared with the control group.