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. 2016 Mar 14;5:e11903. doi: 10.7554/eLife.11903

Figure 3. Deletion or inactivation of GSK3β promotes axon regeneration.

(A) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 2 weeks after optic nerve crush from Gsk3a−/− mice, Gsk3bf/f mice injected with AAV-GFP or AAV-Cre, or AAV-GSK3β K85A injected wild-type mice. *, crush site. Scale bar, 200 µm. (B) Confocal images of retinal whole-mounts showing surviving Tuj1+ RGCs at 2 weeks after optic nerve injury. Scale bar, 50 µm. (C) Quantification of regenerating axons at different distances distal to the lesion site. Data are presented as mean ± s.d., n=5 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (D) Quantification of RGC survival at 2 weeks after injury, expressed as a percentage of total Tuj1+ RGCs in the uninjured retina. Data are presented as mean ± s.d., n=5 per group.

DOI: http://dx.doi.org/10.7554/eLife.11903.009

Figure 3.

Figure 3—figure supplement 1. Cre-mediated Gsk3b deletion in RGCs.

Figure 3—figure supplement 1.

(A) Confocal images of retinal sections showing double immunolabeling for Tuj1+ RGCs and GSK3β from AAV-GFP or AAV-Cre-injected Gsk3bf/f eyes, Scale bar, 20 µm. (B) Quantification of GSK3β immunofluorescence intensity. Data are presented as mean ± s.d., n=3 per group. *p<0.01, Student’s t test.
DOI: http://dx.doi.org/10.7554/eLife.11903.010
Figure 3—figure supplement 2. A time course study of axon regeneration in Gsk3b-deleted RGCs.

Figure 3—figure supplement 2.

(A-C) Confocal images of optic nerve sections showing regenerating axons labeled by anti-GAP43 immunohistochemistry at 1, 3, or 7 days from Gsk3bf/f mice injected with AAV-GFP or AAV-Cre. *, crush site. Scale bar, 100 µm. (D) Quantification of regenerating axons at 7 days after injury from Gsk3bf/f mice injected with AAV-GFP or AAV-Cre at different distances distal to the lesion site. Data are presented as mean ± s.d., n=4 per group. *p<0.01, Two-way ANOVA with Bonferroni post hoc test. (E) Confocal images of optic nerve sections showing double immunolabeling for GAP43+ regenerating axons and chondroitin sulfate proteoglycan (CSPG). Scale bar, 20 µm. (F) Confocal images of optic nerve sections showing double immunolabeling for GAP43+ regenerating axons and glial fibrillary acidic protein (GFAP). Scale bar, 20 µm.
DOI: http://dx.doi.org/10.7554/eLife.11903.011
Figure 3—figure supplement 3. AKT activation increases GSK3α phosphorylation.

Figure 3—figure supplement 3.

(A) Immunohistochemical detection of GSK3α phosphorylation at Ser21 in retinal sections from AAV-GFP or AAV-caAkt injected eyes, with or without optic nerve injury. Scale bar, 20 µm. (B) Quantification of RGC phospho-GSK3α immunofluorescence intensity. Data are presented as mean ± s.d., n=4 per group. *p<0.001, Two-way ANOVA with Bonferroni post hoc test.
DOI: http://dx.doi.org/10.7554/eLife.11903.012