Fig. 2

Effects of microglial activation state on expression of phagocytosis-related molecules, phagocytosis, and ROS production. a Phagocytosis-related genes. Microglia were unstimulated (control, CTL) or stimulated for 24 h with 20 ng/mL IFN-γ + 50 ng/mL TNF-α (M1 activation state) or 20 ng/mL IL-4 (M2a state), with or without a subsequent 6-h exposure to 25 μg/mL myelin (plus or minus sign indicates presence/absence of myelin), as in Fig. 1a. Values are expressed as normalized mRNA counts (described in the “Methods” section), mean ± SEM (six different cell cultures), and were analyzed by two-way ANOVA with Bonferroni’s post hoc test. b Phagocytosis of myelin fragments. Microglia were exposed for 6 h to 25 μg/mL DiI-labeled myelin, and the amount of internalized myelin was determined in unstimulated (CTL), M1 (I + T), M2a (IL-4), and M2c (20 ng/mL IL-10)-stimulated microglia. Results were normalized to control microglia (dashed line) to determine activation state-dependent changes. Data are expressed as mean ± SEM (20 individual cultures) and were analyzed by one-way ANOVA with Dunnett’s post hoc test. c Production of reactive oxygen species (ROS). Intracellular ROS was monitored with the general ROS probe, dichlorofluorescein (DCF), and normalized to DCF levels in unstimulated (CTL) microglia without myelin (dashed line). Data were analyzed by two-way ANOVA with Bonferroni’s post hoc test (n = 19 individual cultures). The comparisons are as follows. Asterisk Between unstimulated microglia (CTL) and cells treated with I + T, IL-4, or IL-10. Dagger sign CTL versus different activation states in the presence of myelin. Number sign Effects of myelin within a particular activation state. One symbol p < 0.05, two symbols p < 0.01, three symbols p < 0.001