Figure 3.
Efficient neurite growth of mature retinal ganglion cells (RGCs) upon hIL-6 stimulation. (a) Representative pictures of dissociated adult mouse RGCs cultured for 4 days either untreated (-) or in the presence of IL-6, CNTF or hIL-6 at 104 pmol/l and visualized by βIII-tubulin staining. Scale bar = 50 µm. (b) Quantification of RGC neurite length in retinal cultures treated with control-supernatant from empty plasmid-transfected HEK293 cells (GFP), various concentrations of hIL-6 supernatant, recombinant IL-6 or CNTF as depicted in a). Whereas control-supernatant showed no effect compared to untreated cultures (-), incubation with hIL-6 increased neurite growth concentration-dependently. In comparison to IL-6 and CNTF, hIL-6 was more efficacious (###P ≤ 0.001), as ~3.5 increased neurite growth was observed with 10 nmol/l hIL-6 compared to twofold increase with 10 nmol/l IL-6 and CNTF, respectively. Data were normalized to untreated controls (-) with an average axon length of 10.5 µm/neuron and represent means ± SEM of three independent experiments. Treatment effects compared to untreated controls: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, n.s.=non-significant. (c) Quantification of neurite length of dissociated RGCs treated with 10 nmol/l hIL-6 either alone or in combination with 5 µmol/l PI3K inhibitor LY294002 (ly), 7 µmol/l JAK/STAT3 inhibitor AG490 (ag) or 10 nmol/l mTOR inhibitor rapamycin (rap). All signaling inhibitors significantly impaired neurite growth of hIL-6 treated RGCs, indicating that hIL-6 induced neurite growth depended on PI3K/AKT/mTOR and JAK/STAT3 signaling. Data were normalized to untreated controls with an average axon length of 8.6 µm/neuron and represent means ± SEM of three independent experiments. Treatment effects: **P ≤ 0.01, ***P ≤ 0.001. (d) Quantification of neurite length of mouse RGCs cultured either on laminin or myelin in the presence of 10 nmol/l CNTF and hIL-6, respectively. The ROCK inhibitor Y27632 (y27) was used as positive control for disinhibition on myelin. Neurite growth on myelin was reduced compared to laminin in untreated control (-) and CNTF-treated cultures, but not after hIL-6 incubation, indicating a disinhibitory effect of hIL-6. Data were normalized to untreated controls on laminin with an average axon length of 9 µm/neuron and represent means ± SEM of three independent experiments. Treatment effects: **P ≤ 0.01, ***P ≤ 0.001, n.s., = nonsignificant.