Article Figures & Data
Figures
- Figure 1.
Time course of RGC survival in culture. A, B, Dissociated retinal cells from adult B6.Cg(Thy1-CFP)23Jrs/j mice were cultured in the presence or absence of 3.3 nm Otx2 for 4 h to 6 d. CFP+ (A) or NF+ (B) cells were counted. A significant survival effect of Otx2 was observed starting at 24 h. (*p < 0.005 and **p < 0.001 compared to control, 2-tailed t test). Six coverslips were analyzed for each condition.
- Figure 2.
Otx2 promotes survival of adult RGCs in culture in a dose-dependent manner. A, Otx2 significantly increased the number of surviving NF+ RGCs after 7 days in culture in a dose-dependent manner (ANOVA, F = 21.712, p < 0.0001; *p < 0.05, **p < 0.005 compared to control (Cont), post hoc test, Fisher's PLSD). These results are representative of 20 independent experiments. B, Otx2 stimulates the survival of immunopurified adult rat RGCs 6 days in culture, whereas an anti-Otx2 abrogates the survival effect (ANOVA, F = 7.014, p = 0.001; **p < 0.005 compared to control, post hoc test, Fisher's PLSD). These results are representative of six independent experiments. Four to six coverslips were analyzed for each condition in A and B.
- Figure 3.
NMDA excitotoxicity of RGCs. A, Four days after intraocular injection of NMDA (2 mm final) in B6.Cg(Thy1-CFP)23Jrs/j mice, the number of CFP-positive RGC profiles appeared reduced throughout the retina compared to the uninjected (Uninj) retina (top). When Otx2 (30 ng) was injected at the time of NMDA, fewer CFP+ cells appeared lost (bottom). Scale bar, 500 μm. B, Counting CFP-positive RGCs in the experiment depicted in A showed a significant reduction of about 33% compared to the uninjected left eye (2-tailed t test). Thirty nanograms of Otx2 fully protected fully against the NMDA-induced loss of CFP+ RGCs. C, NMDA (2 mm final) significantly reduced Brn3a mRNA, while mRNAs specific for other cell types were little or not affected. (*p < 0.005, one sample t test against null hypothesis that the right eye to left eye ratio = 1.0). The numbers at the bottom of each bar represent the number of mice in each condition.
- Figure 4.
Otx2 protects adult RGCs against NMDA excitotoxicity. A, Otx2 injected into the eye does not alter Brn3a mRNA levels. NMDA significantly reduces Brn3a levels compared to the uninjected eye (*p < 0.005, one sample t test against null hypothesis = 1.0). In eyes injected with NMDA and 30 ng of Otx2, the levels of Brn3a in the injected eye are indistinguishable from those in the uninjected eye. Three nanograms provided 50% protection. B, In a separate experiment, 144 ng of Otx2 provided the same full protection as 36 ng. C, Preincubation of Otx2 with anti-Otx2 blocked the ability of Otx2 to protect against excitotoxic decrease in Brn3a levels (**p < 0.05, one sample t test against null hypothesis = 1.0). Injection of the antibody alone did not alter Brn3a mRNA levels (data not shown). D, A single injection of Otx2 at the time of NMDA (2 mm final) protected against NMDA-induced decrease in Brn3a expression for up to 21 d. R/L ratio, Right/left ratio.
- Figure 5.
Otx2 protects RGCs against the loss of visual acuity. A, Mice were separated into two groups and were tested on the optomotor with a square wave grating of 0.375 cpd before intraocular injection. Four days after intraocular injection of NMDA, the number of head turns was significantly reduced compared to day 0 (group A, t test). In mice injected with Otx2 and NMDA (group B), the number of head turns was significantly greater than mice injected with NMDA alone and was not significantly different from the number of head turns before eye injection. B, The protection by Otx2 against visual acuity loss due to NMDA is associated with significantly greater Brn3a mRNA.
- Figure 6.
Otx2YL does not promote RGC survival in vitro or in vivo. A Otx2wt or Otx2YL were produced with a myc tag and were detected using an anti-myc antibody. Anti-myc signal is detected in the GCL and INL and to a lesser extent in the outer ONL of mice injected with Otx2wt, but not in mice injected with Otx2YL. Scale bar, 100 μm. B, Cellular localization of Otx2wt-FITC injected into the eye. Bi, Six hours after injection of 156 ng of Otx2, exogenous protein detected by anti-myc was detected in all cell body layers of the retina. Bii, Confocal image of a single focal plane of the FITC signal revealed Otx2 in cells in the GCL. Biii, TO-PRO-3 fluorescence delimited the cell nuclei and nucleoli, and the merged image (Biv) shows Otx2 in GCL nuclei. Bv, DAB precipitate of exogenous Otx2 detected by the myc-tag in and around the nucleus of a cell in the GCL. Scale bars: Bi, 100 μm; Bii, Bv, 10 μm. C, Otx2wt promotes the survival of adult mouse RGCs 6 d in vitro. [ANOVA, F = 27.538, p < 0.0001; **p < 0.0005 compared to control (Cont), Fisher's PLSD]. Otx2YL did not promote the survival of adult mouse RGCs. D, Brn3a mRNA was significantly decreased 4 d after intraocular injection of NMDA compared to mice injected with Otx2wt and NMDA. Brn3a mRNA in mice injected with Otx2YL was significantly reduced compared to mice injected with Otx2wt and NMDA (ANOVA, F = 21.287, p < 0.0001; **p < 0.005, Fisher's PLSD). Three to eight coverslips were analyzed for each condition.
- Figure 7.
RGCs in mixed culture are lost via apoptosis. Dissociated cells from adult C56BL/6 mice were cultured for 24 h with 3.3 nm Otx2, 20 μm ZVAD, or both and NF+ RGCs were counted. Otx2 promoted the survival of RGCs as did ZVAD. There was no additive effect of Otx2 + ZVAD (ANOVA, F = 18.674, p < 0.0001; *p < 0.005, Fisher's PLSD). Six coverslips were analyzed for each condition.
