Fig 2: CCL2 expression is reduced in the spinal cords of ASK1GFAP KO EAE mice at d30, but not d17. (A) qPCR analysis of CCL2 expressions in the spinal cords. Two-tailed Student’s t test was used. n = 4 mice per group. *P < 0.05. (B) Schematic representation of the experimental procedure for mRNA purification of astrocytes from the spinal cords of Rpl22HA:GFAP EAE mice on d17 and d30. Immunoprecipitation using mouse monoclonal anti-HA antibody (IP-HA) was performed before mRNA purification. (C) qPCR analysis of CCL2 expressions in astrocytes. Two-tailed Student’s t test was used. n = 4 mice per group. *P < 0.05. (D) Triple immunohistochemical staining of CCL2, iba1, and GFAP in the spinal cords of WT and ASK1GFAP KO EAE mice on d17 and d30. (Scale bar: 50 µm.) (E) Quantification of the percentage of CCL2-, CCL2/iba1-, and CCL2/GFAP-positive area in the gray matter of the spinal cord. The one-way ANOVA with Tukey–Kramer post hoc test was used. n = 4 to 5 mice per group. ***P < 0.001; **P < 0.01; *P < 0.05.

Fig 3: GFAP immunoreactivity in the midbrain of sham and BLG-sensitized mice. GFAP-positive astrocytes were identified by immunohistochemically staining brain sections (40 μm). Representative midbrain sections from sham (A,a',a”) and BLG-sensitized (B,b',b”) male mice are shown. The white rectangles in (A,B) indicate where the respective higher magnification images a'–b” were taken. The open arrowheads indicate dense GFAP-immunoreactive astrocyte end-feet along the blood vessel. The low-magnification (A,B) and high-magnification (a',a”, b',b”) images taken with a 10X and 40X objectives, respectively. Scale bars: 100 μm for (A) and (B); 25 μm for a'–b”.

Fig 4: PGM aggrandized glial cell activation in the hippocampal regions of APP/PS1 mice. (A) Fluorescent GFAP (red)/DAPI (blue) colocalization in the DG regions of the hippocampus in APP/PS1 mice. Scale bar = 100 μm. (B) Fluorescent GFAP (red)/DAPI (blue) colocalization in the DG regions of the hippocampus in WT mice. Scale bar = 100 μm. (C) Fluorescent Iba1 (red)/DAPI (blue) colocalization in the DG regions of the hippocampus in APP/PS1 mice. Scale bar = 100 μm. (D) Fluorescent Iba1 (red)/DAPI (blue) colocalization in the DG regions of the hippocampus in WT mice. Scale bar = 100 μm. (E) Quantification of GFAP-positive cells in each APP/PS1 mice group. n = 5–8 per group, three sections per animal. (F) Quantification of GFAP-positive cells in each WT mice group. n = 5–8 per group, three sections per animal. (G) Quantification of Iba1-positive cells in each APP/PS1 mice group. n = 5–8 per group, three sections per animal. (H) Quantification of Iba1-positive cells in each WT mice group, n = 5–8 per group, three sections per animal. *P < 0.05 vs. APP/PS1-saline, **P < 0.01 vs. APP/PS1-saline.

Fig 5: Effects of macrophage-derived soluble factors on spinal cord organotypic cultures. a Whole-slice MTT shows increased cellular respiration following incubation with M1-conditioned, but not M2-conditioned medium. b Cord cultures incubated for 72 h with conditioned medium of M1, but not M2, macrophages show increased numbers of dividing cells, as shown by EdU nucleotide incorporation. c The proportion of EdU incorporating cells that also express GFAP is not changed in experimental conditions. d–o Immunohistochemistry on cryosectioned slice cultures shows a significant fraction of dividing cells are GFAP+ astrocytes