Figure 4

oAβ(1-42) inhibits fAβ(1-42)-stimulated microglial phagocytosis. BV-2 cells or primary microglia induced by oAβ(1-42) (1.0 μM), fAβ(1-42) (5.0 μM) alone or pretreatment of oAβ(1-42) for 6 h or 12 h followed by fAβ(1-42) treatment for 30 min. Fluorescent microspheres (red) were then added for 30 min. Cells were fixed with 4% PFA and stained with alexa488-conjugated phalloidin to visualize F-actin (green). The nucleus was visualized with DAPI (blue). (A) Fluorescent images of the distinct microglial phagocytosis. Scale bar = 40 μm. (B, C) Quantification of Aβ-mediated microglial phagocytosis. Data are presented as mean ± S.E. ***P < 0.001, compared with the control. ^##P < 0.01, ^###P < 0.001, compared with fAβ(1-42) alone. (D-F) Fluorescent labeled fAβ(1-42) internalization in microglia. Cells were pre-stimulated without or with oAβ(1-42) (1.0 μM) for 3 h or for 12 h, then incubated with Hilyte-488 labeled fAβ(1-42) (5.0 μM) for 30 min, and treated cells were fixed with 4% PFA and scanned under confocal microscope. Confocal images of microglial cells ingesting green Hilyte-488 labeled fAβ were shown in the x-y plane (D left). Overlays of phase-contrast and confocal images were shown in D right. Scale bars = 25 μm. (E) z-axis scans of cuts through the cells. (F) Quantification of internalized fluorescent fAβ in microglial cells. Values were expressed as mean fluorescence intensity (arbitrary units) of internalized fAβ per cell. Data are presented as mean ± S.E. ***P < 0.001, compared with the unstimulated control group. ^#P < 0.05, between 3 h and 12 h of oAβ stimulation.