Fig 2: Effect of anti-CCL20 antibody or CCR6 knock down on microgliosis and astrogliosis in optic nerve following rTBI. a Representative immunofluorescence images showing Iba1 (red) or GFAP (green) immunoreactivity in the optic nerve. Scale bar 100 µm. b, c Histograms showing average count of Iba1-positive cells (b) or average fluorescence intensity of GFAP immunoreactivity (c) as quantitated by ImageJ. Values are shown as mean ± SEM. *p < 0.01, **p < 0.001, ***p < 0.0001

Fig 3: rTBI induces RGC loss and CCL20 expression in the retina 7 days post-injury. a Representative low magnification, bright field image of H&E staining of cross section of the whole eye. Images, as shown in b and g, are representations of one of the boxed areas in the a. Cells were counted or integrated density was measured from both the boxed areas, and the average numbers were plotted as the histogram shown in c and h respectively. b Photomicrograph of H&E staining of retinal sections from one of the boxed areas in a under different experimental conditions. Arrows indicate RGC loss. c Histogram showing the RGC counts from the H&E-stained sections. d Retinal flat mount preparation showing immunostaining with anti-RBPMS antibody. e Representative high-magnification immunofluorescence images of the boxed area in d showing RBPMS-positive ganglionic cells under different experimental conditions. f Histogram showing RBPMS positive cell counts under different experimental conditions. g Photomicrograph showing immunoperoxidase staining with anti-CCL20 antibody of the retinal sections from one of the boxed areas shown in a. Scale bar 100 µm, inset, high-magnification image of the boxed area of the ganglionic cell layer, scale bar, 20 µm. h ImageJ quantitation of CCL20 immunoreactivity in the retina. i CCL20 protein expression in the retina as measured by Quantikine ELISA. *p < 0.01, **p < 0.001, ***p < 0.0001

Fig 4: Anti-CCL20 antibody significantly reduces RGC loss, microgliosis, Müller cell activation and CCL20 expression in the retina. a Representative immunofluorescence images showing RGC (H&E), Iba1 (red), GFAP (green), or CCL20 (bright field) immunoreactivity in the retina with or without anti-CCL20 antibody or isotype treatment 7 days after rTBI. Scale bar 100 µm. Inset high-magnification images showing CCL20 expression in the retinal ganglionic cells, scale bar 20 µm. Blue fluorescence shows DAPI. b–e Histograms showing average count of RGC (b), Iba1-positive cells (c), average fluorescence intensity of GFAP immunoreactivity (d), or CCL20 immunoreactivity (e) as quantitated by ImageJ. f CCL20 protein expression in the retina as estimated by Quantikine ELISA. *p < 0.01, **p < 0.001, ***p < 0.0001