Figure 1
AAV2-mediated conditional STAT3 depletion in RGCs. (a) Retinal flat-mount from a STAT3-floxed mouse 2 weeks after intravitreal application of AAV2-Cre. HA-tagged Cre recombinase (cre, red) was detected in the nuclei of ∼90% of βIII tubulin-positive RGCs (green) using an anti-HA-antibody. Scale bar: 50 μm. (b) Quantification of βIII tubulin-positive RGCs per mm2 in flat-mounted retinae 3 weeks after intravitreal injection of either AAV2-GFP (control) or AAV2-Cre. Virus-mediated knockdown of STAT3 (cre) did not affect the survival of uninjured RGCs compared with AAV2-GFP-treated control retinae (gfp). Values represent the mean of three retinae per group. (c) Immunohistochemical staining of cross sections of untreated control retinae (con) and retinae 5 days after optic nerve crush (onc) or onc+inflammatory stimulation (onc/is) from either AAV2-GFP- (gfp) or AAV2-Cre- (cre) injected mice with antibodies against phosphorylated STAT3 (pSTAT3, red) and βIII tubulin (tubulin, green). Virus-mediated STAT3 knockdown reduced IS-induced activation of STAT3 specifically in RGCs, but not in cells of the fiber and inner nuclear layer. Scale bar: 50 μm; GCL, ganglion cell layer; INL, inner nuclear layer. (d) Western blot analysis of retinal lysates from animals treated as described in c with an antibody against phosphorylated STAT3 (pSTAT3). Tubulin served as loading control. Activation of STAT3 strongly increased after ONC+IS compared with untreated (con) and optic nerve crushed (ONC) AAV2-GFP control mice (gfp). Virus-mediated STAT3 knockdown (cre) strongly diminished STAT3 phosphorylation upon ONC+IS. (e) Photometric quantification of western blots as in d. Virus-mediated STAT3 knockdown (cre) reduced STAT3 activation by 78%. Values represent the mean of four independent experiments. Treatment effects: ***P<0.001
