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. 2023 Mar 31;18(11):2526–2534. doi: 10.4103/1673-5374.371372

Figure 1.

Figure 1

AAV-mediated transduction of caRheb increases p-S6 levels in RGCs and protects RGCs following ONC by increasing the survival of non-αRGCs.

(A) AAV-caRheb was administrated intravitreally to mice 2 weeks before ONC, and eyes were harvested on days 14, 21, and 42 after ONC. (B, C) caRheb increased the expression of p-S6. Immunofluorescence of retinal sections before and after injury in eyes transfected with AAV-caRheb or AAV-EGFP; staining was performed using p-S6 (red) or Tuj1 (green) antibodies. (D) Quantification of p-S6+ RGCs in EGFP and caRheb groups with and without ONC. Data are presented as the mean ratios of p-S6+ Tuj1+ cells among total Tuj1+ cells of each retina. Cell counts were performed in at least three nonconsecutive sections (mean ± SD, n = 3–4 per group). ****P < 0.0001, unpaired t-test. (E) Expression of caRheb increased the survival of RGCs. Immunofluorescence of flat-mount retinas from sham operation, AAV-EGFP, and AAV-caRheb groups on 14, 21, and 42 days after ONC showing surviving Tuj1+ RGCs (green). (F) Quantification of surviving Tuj1+ RGCs (mean ± SD, n = 6–8). **P < 0.01, ***P < 0.001, ****P < 0.0001, one-way analysis of variance with Bonferroni’s post hoc test. (G) Quantification of the size of surviving RGCs from AAV-EGFP and AAV-caRheb groups (mean ± SD, n = 6–8). ****P < 0.0001, one-way analysis of variance with Bonferroni’s post hoc test. (H) Rheb-mediated increase of RGC survival was not through increasing the survival of α-RGCs. Immunofluorescence of flat-mount retinas for Tuj1 (red) (pan-RGCs) and SMI-32 (green) (α-RGCs) showed the surviving pan-RGCs and α-RGCs in AAV-EGFP and AAV-caRheb groups on day 14 after ONC. Scale bars: 40 µm (B, C, H), 60 µm (E). (I) Quantification of surviving non-α-RGCs. Data are presented as mean numbers of SMI-32– Tuj1+ cells of each retina, n = 4 for each group, ***P < 0.001, unpaired t-test. (J) Quantification of surviving α-RGCs. Data are presented as mean numbers of SMI-32+ Tuj1+ cells of each retina (mean ± SD, n = 4 for each group, unpaired t-test). AAV: Adeno-associated virus; caRheb: constitutively active Ras homolog enriched in brain; dpc: days post crush; DAPI: 4′,6-diamidino-2′-phenylindole; EGFP: enhanced green fluorescent protein; ONC: optic nerve crush; p-S6: phospho-S6 ribosomal protein; RGC: retinal ganglion cell; Tuj1: β3-tubulin; SMI-32: neurofilament H.