Figure 3.
Nitric oxide production is not necessary for LPS-induced preOL death in mixed glial cultures. A–C, LPS induced robust iNOS expression and NO production in mixed glial cultures. Mixed glia were treated with or without LPS (1 μg/ml) for 24 h and analyzed by immunocytochemistry (A) and Western blot (B) for iNOS expression (arrowheads). Dying preOLs with retracted processes were evident during LPS challenge (arrows). Double immunostaining for iNOS and activated microglial marker CD68 showed that iNOS was upregulated in activated microglia (bottom). C, D, The NOS inhibitor l-NMMA (1 mm) blocked NO production but had no effect on preOL survival. **p < 0.001 when compared with controls; ns, not significant. E, LPS-induced death of preOLs in mixed glial cultures was not dependent on production of NO and peroxynitrite, oxidative stress, or activation of the AMPA/kainate receptor. Mixed glial cultures were treated as indicated with or without 1 μg/ml LPS for 48 h in the presence of NOS inhibitor (1 mm NMMA), iNOS-specific inhibitor (10 μm 1400W), peroxynitrite decomposition catalyst (5 μm FeTMPyP), AMPA/kainate receptor antagonist (100 μm NBQX), or antioxidants (10 μm vitamin E or 10 μm vitamin K2). PreOL survival was quantified by counting O4+ cells. Data are representative of three separate experiments. F, Representative photomicrograph of double labeling of dying preOLs with O4 and TUNEL in preOLs plus microglia cocultures exposed to LPS for 24 h. Arrows indicate TUNEL+ preOLs.