Figure 2

STAT3 depletion in RGCs compromises the IS-induced switch into a regenerative state. (a) βIII tubulin-positive RGCs of STAT3-floxed mice either exposed to vehicle (con) or CNTF (200 ng/ml) after 3 days in culture. Animals were intravitreally injected with either AAV2-GFP or AAV2-Cre 14 days prior to preparing cultures. Scale bar: 50 μm. (b) Quantification of neurite length per RGC in retinal cultures as described in a. Values represent the mean of 12 wells from three independent retinae per group. Depletion of STAT3 in RGCs significantly compromised CNTF-induced neurite growth. (c) Quantification of RGCs per well in cultures as described in a. STAT3 knockdown did not affect the survival of mature RGCs in these cultures. (d) In vivo pre-conditioned, βIII tubulin-positive RGCs after 24 h in culture. Pre-treatment: STAT3-floxed mice received intravitreal injections of either AAV2-GFP (gfp) or AAV2-Cre (cre). Two weeks later, animals were subjected either to optic nerve crush (onc) or onc+inflammatory stimulation (onc/is). Retinal cell cultures were prepared 5 days thereafter. Scale bar: 50 μm. (e) Quantification of neurite length per cultured RGC from mice treated as described in d. Values represent the mean of 16 wells from four independent retinae per group. STAT3 depletion significantly compromised IS-induced neurite growth. (f) Quantification of RGCs per well in retinal cultures as described in d. Virus-mediated STAT3 depletion did not affect RGC survival in retinal cell cultures. (g, h) Quantitative real-time PCR: retinal Gap43 (g) and Sprr1a (h) expression was quantified relative to GAPDH expression in animals as described in d. Values represent the mean of four retinae per group. ONC- and IS-induced upregulation of GAP43 and Sprr1a expression was impeded upon STAT3 depletion. (i) Western blot analysis of retinal lysates from animals treated as described in d using antibodies against GAP43, CNTF and pSTAT3. Tubulin served as loading control. Expression of GAP43 and CNTF strongly increased after ONC+IS compared with untreated AAV2-GFP control mice (gfp). IS-induced upregulation of GAP43 expression was compromised upon STAT3 depletion (cre), whereas the increase in CNTF expression was not affected. Treatment effects: n.s., non-significant; **P<0.01; ***P<0.001