Fig 2: GFAP expression and synthesis in the ON and ONH in a murine glaucoma model. (A) GFAP immunoreactivity in the glial lamina of 1-month (green, upper panel) and 2 months (green, middle and lower panel) old ßB1-CTGF1 mice compared to WT controls. Immunoreactivity of GFAP was not altered in 1-month old TG and WT animals (green, upper panel). 2 month old ßB1-CTGF1 mice showed an increased GFAP immunoreactivity compared to WT control (green, middle panel). Lower panel shows a magnified detail of the middle panel. Nuclei are stained with Dapi (blue). (B) Schmatic illustration of the optic nerve. A depicts the region in the glial lamina where cross sections were obtained. ONH (unmyelinated part) and ON (myelinated part) were used for molecular analysis. (C) Quantification of immunohistochemical staining of GFAP in the glial lamina is not altered in 1 month old TG and WT animals (WT: n = 5; TG: n = 4). GFAP immunoreactivity is increased in 2 month old TG animals compared to WT littermates (WT: n = 10, TG: n = 13; *p = 0.039; two-tailed t-test compared to theoretical mean of 1 (normalized control)). Mean value of WT animals (control) was set at 1. (D) RT-PCR analyses revealed no alteration in the Gfap mRNA expression in the ON of 1-month (WT: n = 7; TG: n = 5) and 2 month old ßB1-CTGF1 mice (WT: n = 15; TG: n = 16) compared to WT controls. In the ONH the Gfap mRNA expression is increased in the 2 month old TG compared to WT animals (WT: n = 6, TG: n = 5; *p = 0.04). The mRNA expression of Gfap is not altered in 1 month old animals (WT: n = 6, TG: n = 5). Mean value of WT animals (control) was set at 1. RPL32 was used to normalize mRNA expression. Data represented as mean ± SEM. PRL, Prelaminar region; PSL, Postlaminar region; ON, optic nerve; ONH, optic nerve head.

Fig 3: Analysis of astrocyte reactivity, the cytoskeleton and migration rate following treatment with TGF-ß2 and CCN2/CTGF. (A) GFAP immunoreactivity (green) is increased in murine ON astrocytes following TGF-ß2 and CCN2/CTGF treatment. Nuclei are stained with Dapi (blue) (B) Western Blot experiments analyses of a-actinin protein synthesis in murine ON astrocytes after the treatment with 1 ng/ml TGF-ß2, 50 ng/ml CCN2/CTGF and 100 ng/ml CCN2/CTGF for 24 h. Densitometric analyses of Western Blot analyses shows the significant increase in a-actinin protein synthesis. Mean value of untreated cells (control) was set at 1. GAPDH was used to normalize protein intensity (n = 4; 50 ng CCN2/CTGF: *p = 0.029; 100 ng CCN2/CTGF: *p = 0.015; unpaired two-tailed t-test). (C) Scratch assay analyses show a significant increased migration rate for murine ON astrocytes after the treatment with ng/ml TGF-ß2, 50 ng/ml CCN2/CTGF and 100 ng/ml CCN2/CTGF for 12 h. Mean value of migration rate of control cells was set to 100% (control: n = 15; TGF-ß2: n = 12, **p = 0.008; 50 ng/ml CCN2/CTGF: n = 10, *p = 0.001; 100 ng/ml CCN2/CTGF: n = 4, **p = 0.0001). Data represented as mean ± SD.

Fig 4: Gliosis in the retina of pre-symptomatic APP/PS1 mice. (A) Representative images of Iba1 and GFAP immunolabelling in the retina of TG APP/PS1 and WT mice aged 3 (upper panels) and 12 (lower panels) months. Scale bar = 50 µm. (B) qPCR analysis of mRNA expression of iba1 and gfap in the retina of TG APP/PS1 and WT mice at 3 months. Results expressed as mean +/- SEM. ** p < 0.01. qPCR quantification of both GFAP and Iba1: n = 6:3M + 3F for WT mice and n = 4:2M + 2F for TG mice at 3 months.