Journal: Development (Cambridge, England)

Article Title: Microglia refine developing retinal astrocytic and vascular networks through the complement C3/C3aR axis.

doi: 10.1242/dev.201047

Figure Lengend Snippet: Fig. 6. Microglia express C3aR and loss of C3aR increases astrocyte density. (A) Representative P5 C3aR-tdTomato (C3aR-tdT)-expressing retinal flatmount showing Pdgfra (astrocytes, green) and P2ry12 (microglia, blue) immunolocalization around the vascular growth front (n=3). Arrows indicate the localization of tdTomato in P2ry12-expressing microglia. (B) Representative 3D-reconstructed image from C3aR-tdTomato expressing P5 retinal flatmount immunostained for P2ry12 and Pdgfra, revealing C3aR reporter expression in microglia (P2ry12, green) that are closely interacting with astrocytes (Pdgfra, gray) (n=3). (C) Bar graph represents the quantification of C3aR-tdTomato-expressing microglia in the vascular (center) and avascular (periphery) retinal regions (n=3). (D) Retinal flatmounts from wild-type or C3aR KO groups immunostained for Sox9 and Pdgfra showing astrocyte nucleus distribution and network patterning in the vascularized central retina (n=3-5). Bar graphs showing the quantification of Sox9 and Pdgfra density in the vascularized central retina. (E) Retinal flatmounts from wild-type or C3aR KO groups immunostained for Sox9 and Pdgfra showing astrocyte nucleus distribution and network patterning in the avascular mid-peripheral retina (n=3-5). Bar graphs showing the quantification of Sox9 and Pdgfra density in the avascular mid-peripheral retina. All error bars represent s.e.m. Statistical differences between wild type and C3aR KO were calculated using an unpaired t-test (*P<0.05, **P<0.01, ****P<0.0001). Scale bars: 75 µm in A; 10 µm in B; 25 µm in D,E.

Article Snippet: Microglial cells were then treated with DiI-labeled dead astrocyte bodies in a 1:1 ratio and incubated for 2 h. For receptor neutralization assays, microglial cells were pre-incubated with 20 μg/ml of anti-C3aR antibody (MAB10417) or control IgG (R&D Systems) suspended in DMEM/F12 supplemented with 5% heatinactivated FBS, followed by treatment with DiI-labeled astrocyte dead bodies in the presence of antibodies for 2 h at 37°C.

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Microglia refine developing retinal astrocytic and vascular networks through the complement C3/C3aR axis.

doi: 10.1242/dev.201047

Figure Lengend Snippet: Fig. 7. Loss of C3aR reduces microglial phagocytosis of astrocytes and increases vascular density. (A) Z-stack images were acquired from P5 wild-type and C3aR KO retinal flatmounts immunostained for P2ry12, Gfap and CD68, and 3D reconstructed to visualize microglia (P2ry12)-engulfed astrocyte (Gfap) bodies within microglial lysosomal/ endosomal membrane protein CD68. Bar graph shows the percentage of Gfap debris localized within P2ry12 and CD68 co-labelled microglia in wild-type and C3aR KO retinas (n=3). (B) Primary microglial cultures isolated from P5 retinas were treated with DiI-labeled retinal astrocyte apoptotic bodies in the presence of control IgG or anti-C3aR IgG for 2 h, then the number of DiI-astrocyte bodies engulfed by microglia in the two treatment conditions were quantified and are shown in the bar graph (n=3). (C) Representative P5 retinal flatmounts of wild-type and C3aR KO immunostained for CD31 showing vascular outgrowth and quantification of vascularized area (n=5). (D) P5 retinal flatmounts of wild type and C3aR KO were immunostained for CD31, images were taken in the vascularized central retina, and the differences in vessel density and spatial branching (lacunarity) were measured and quantified using ‘ImageJ AngioTool’ software tool (n=5). (E) Real-time PCR data showing gene expression levels of Pax2, Lamc3, Col4a3, Col4a4, Col8a1, Col8a2, Fbn1 and Fmod in wild type and C3aR KO P5 retinas (n=6). All error bars represent s.e.m. Statistical differences were calculated using an unpaired t-test (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Scale bars: 10 µm in A; 50 µm in B,D; 500 µm in C.

Article Snippet: Microglial cells were then treated with DiI-labeled dead astrocyte bodies in a 1:1 ratio and incubated for 2 h. For receptor neutralization assays, microglial cells were pre-incubated with 20 μg/ml of anti-C3aR antibody (MAB10417) or control IgG (R&D Systems) suspended in DMEM/F12 supplemented with 5% heatinactivated FBS, followed by treatment with DiI-labeled astrocyte dead bodies in the presence of antibodies for 2 h at 37°C.

Techniques: Membrane, Isolation, Labeling, Control, Software, Real-time Polymerase Chain Reaction, Gene Expression