Figure 5

Effects of FGF-2 on cellular migration and clearance of degenerated neuronal debris by microglia. (A) Microglial migration assay; aFKN or aFGF-2 were added to lower plates and microglia set on upper Transwell inserts and cultured for 48 h, with isotype-matched IgG as control. Migrated cells in lower plates were counted by FACS. One-way ANOVA with Tukey’s post-hoc test: *, significant difference compared with untreated samples using healthy conditioned medium (***: P < 0.001); +, significant difference compared with untreated samples (n.s., not significant; +, P < 0.05). (B) Effect of FGFR blockers and antibodies on microglial migration induced by 100 ng/ml FGF-2. PD, pan-FGFR blocker 1 μM PD173074; SU, selective FGFR1 blocker 500 nM SU11652; aR2, anti-FGFR2 antibody; aR3, anti-FGFR3 antibody; aR4, anti-FGFR4 antibody; aR5, anti-FGFR5 antibody; isotype-matched IgG control. **, P < 0.01 compared with FGF-2 treatment alone (one-way ANOVA with Dunnett’s post-hoc test). (C) Effect of IWR-1-endo on microglial migration induced by FGF-2 treatment. **, P < 0.01 compared with FGF-2 treatment alone (one-way ANOVA with Dunnett’s post-hoc test). (D) Microglial phagocytosis assay. CM-DiI-labeled neurons were treated with or without glutamate, and microglia were added to the culture. These cells were treated with or without 100 ng/ml FGF-2 and with FGFR blockers or each of the anti-FGFR neutralizing antibodies of (B) for 24 h. Scale bars, 20 μm. (E) Phagocytosis index, defined as the percentage of total microglia staining (green) overlapping with DiI staining (red). Columns indicate mean with SEM from three independent experiments. Significant differences compared with sample used degenerated neuronal debris and FGF-2 treatment: **, P < 0.01; ***, P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).