Figure 2

Activation of NADPH oxidase in the presence of glial iNOS is synergistic in killing co-cultured neurons. Cultures were stained with the cell-impermeable dye propidium iodide (PI) to count necrotic cells and the cell-permeable dye Hoechst 33342 to count neuronal nuclei showing chromatin condensation/fragmentation (CC), 48 hours after treatment (a). PMA stimulation of NADPH oxidase did not substantially affect neuronal survival, but in the presence of LPS/IFN-γ had synergistic effects on neuronal death, which were blocked by inhibitors of iNOS (25 μM 1400W), NADPH oxidase (1 mM apocynin), or a peroxynitrite scavenger (100 μM FeTPPS) but not by a blocker of the NMDA receptor (10 μM MK-801). Nitrite and nitrate levels were not affected by the presence of PMA or apocynin but were significantly reduced by 1400W (b). Statistical differences were established using ANOVA at *p <0.05, **p < 0.01 and ***p < 0.001, the symbol # replaces * when comparing protection against LPS/IFN-γ/PMA induced neuronal death. The symbol ¶ is used to demonstrate a significant difference in comparison to PMA or LPS/IFN-γ alone. Statistical significance refers to the total death (black + white parts of the bar). Data expressed is mean ± SEM, n = 3 or more.