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. 2021 Jan 25;11:608294. doi: 10.3389/fimmu.2020.608294

Figure 1.

Figure 1

A lack of RGC-32 affects astrocyte density, process morphology, and intermediate filament protein expression in EAE spinal cords. Spinal cords from WT (A–F) and RGC-32 KO (G–L) mice were harvested at the peak of EAE (day 14) and stained with anti-GFAP (A, D, G, J), anti-vimentin (B, E, H, K), or anti-FABP7 antibody (C, F, I, L), and then counterstained with Harris hematoxylin. GFAP-stained astrocytes from WT spinal cords show reactive changes in areas rich in inflammation (A, D, arrows), whereas RGC-32 KO spinal cords astrocytes have a bipolar morphology reminiscent of radial glia and astrocyte progenitors (G, J, arrows). Some of these reactive astrocytes are also positive around the inflammatory infiltrate for vimentin and FABP7 in WT spinal cords (B, C, arrows and insert), but they are scarce in areas outside of the inflammation (E, F, arrows and inserts). In contrast, vimentin- and FABP7-positive astrocytes with radial glia morphology are present throughout the spinal cords in KO mice (H, I, K, L, arrows). We noticed that the inflammatory infiltrate is also staining positive for vimentin and FABP7 (B, C, arrowheads). The blood vessels were also vimentin-positive (H). Original magnification: x20. Scale bars: 40 µm. OD of the white matter GFAP-positive cells was determined from 10x-magnified spinal cord sections, and the mean OD per area was compared between WT and RGC-32 KO spinal cords as described in Materials and Methods. A significantly higher OD value for GAFP+ cells was seen in WT than in RGC-32 KO spinal cords (M). Vimentin- and FABP7-positive cells with astrocyte and radial morphology were manually counted from white matter areas of 10x-magnified spinal cord sections, and the values were compared between WT and RGC-32 KO spinal cords. A significantly higher number of vimentin- and FABP7-positive cells were observed in RGC-32 KO spinal cords (N, O). Results are expressed as mean ± SEM (M: n = 11 areas per 4 mice in WT; n = 14 areas per 5 mice in RGC-32 KO. N: n = 13 areas per 4 mice in WT; n = 17 areas per 4 mice in RGC-32 KO. O: n = 8 areas per 3 mice in WT; n = 10 areas per 4 mice in RGC-32 KO). ** = p<0.01; **** = p<0.0001.