Fig. 7

Representative images showing intracytoplasmic OPN puncta in reactive astrocytes forming the astroglial scar. a, b Triple labeling for OPN, GFAP, and S100β at 28 days post-lesion, showing that GFAP is localized in intermediate filaments, whereas S100β is additionally expressed in the cytoplasm of soma and processes. c, d Orthogonal views showing that OPN puncta are completely enwrapped by S100β immunoreactivity (arrowheads in d), but not by GFAP-immunoreactivity (arrowheads in d). e–g The three-dimensional views from confocal z-stack images of GFAP single-labeled (e) and GFAP/S100β double-labeled cells (f). g An image subtracting the OPN puncta outside astrocytes from f, showing that OPN puncta are closely associated with, or completely enwrapped by, reactive astrocytes. Note that GFAP occupies only a small part of the S100β-positive soma and processes in reactive astrocytes. h The three-dimensional rendering of the above images showing the intracytoplasmic OPN puncta (arrows) internalized completely by the S100β-positive reactive astrocytes with the hypertrophic cytoplasm. The yellow arrow in h indicates the intracytoplasmic OPN puncta shown in c, d, and g. i, j Quantitative analysis of the time-dependent number of OPN puncta internalized by activated microglia/macrophages (i) or astrocytes (j) showing that intracytoplasmic OPN puncta in both glial cells significantly increases at days 14 and 28 post-lesion compared with the day 7 data (n = 11, 17 fields from 6 rats per time point, ^*P < 0.05 versus the day 7 one-way ANOVA (P = 0.0091 for j and P = 0.0108 for i) with Tukey’s multiple comparison test. Cell nuclei appear blue after DAPI staining. Scale bars = 20 μm for a–d; 10 μm for e–h