Figure 6.

Reactive oxygen species (ROS) and JNK phosphorylation are required for programmed necrosis of microglia. (a) Abrogation of LPS/zVAD-induced microglial cell death by SP600125 (10 μM) and BHA (150 μM). Pure rat microglia were treated as indicated and cell survival was determined by counting Calcein AM⁺/PI^- cells 20–24 h later. Vitamin K1 (1 μM) and K2 (1 μM, Menaquinone-4) had a moderate effect but not statistically significant. AA861 (10 μM) had no protective effect. Data represent mean±S.E.M. of three to five independent experiments. ***P<0.001; NS, not significant as compared with LPS/zVAD-treated cells. (b) Increased JNK phosphorylation in LPS/zVAD-treated microglia and complete inhibition by Nec-1. Rat microglia were pre-treated with vehicle, Nec-1 (20 μM), BHA (150 μM), or SP600125 (10 μM) for 1 h followed by stimulation with or without LPS (0.1 ng/ml) and zVAD (10 μM) for 3 h. Microglial lysates were then prepared and subjected to western blot analyses as described. Data represent mean±S.E.M. of densitometry analyses of three independent experiments. *P<0.05; **P<0.01; ***P<0.001; two-tailed Student's t test