Fig 1.
Distribution of Brn3a+ RGCs in the E18 retina.
Flat-mounted E18 retina immunostained for Brn3a RGCs. (A) Reconstruction of an entire retina with tiled 100X fluorescence micrographs indicating the retinal quadrants. Reconstruction was used for the automated RGC-counting and the total number of Brn3a+ RGCs is indicated. Yellow arrow indicates the pecten oculi. (B) Isodensity map of Brn3a+ RGCs in retina shown in A. The map is a filled contour plot generated by assigning to each individual frame a color code according to its RGC density, within a scale ranging from 0 to 2,000 (purple) to 15,000 or more (red) RGCs/mm2 (bottom left). (C) Representative micrographs from the four quadrants of the retina shown in A with Brn3a+ RGCs. White and red rectangles in (A) indicate the location of the depicted areas (a-d, a’-d’). E; embryonic day, D; dorsal, T; temporal, N; nasal, V; ventral.
Fig 2.
The fraction of Brn3a+ cells in the ganglion cell layer of E18 retina.
Representative fluorescence micrograph image sample of the ganglion cell layer (GCL) from E18 chicken retina with (A) Brn3a immunoreactivity and (B) DAPI nuclear staining. Red arrows indicate Brn3a, DAPI double positive cells. Yellow arrows indicate DAPI+, Brn3a negative cells. (C) Fraction of Brn3a+ cells of DAPI+ cells in the GCL. The number of Brn3a+ cells and DAPI+ cells was counted manually in 18 evenly distributed sample areas of an E18 flat-mount retina. Cell counts are listed in S1 Table in supporting file.
Fig 3.
Brn3a+ RGCs in normal embryonic and post-hatch control retinas.
Number and density of Brn3a+ RGCs in normal embryonic (E8—E20) and post-hatch (P4, P11) retinas. Brn3a+ RGCs were counted using automated cell counting in four retinal quadrants of flat-mount retina. (A) Retinal quadrants, (B) retinal halves (dorsal and ventral) and (A) whole retina from the different ages were compared. (C) Schematic diagram of flat-mount right (RR) and left (LR) retina with quadrants depicted. (D) The total area of flat-mounted retina plotted against the Brn3a+ RGC density (total Brn3a+ RGCs/retinal area) for each age. (E) Representative fluorescence micrographs of Brn3a+ cells in central and peripheral E10, E14, P4 and P11 retinas. One way ANOVA and Tukey’s post-hoc test, * = p<0.05, ** = p< 0.01, *** = p<0.001; £ = p<0.05; ££ = p< 0.01 and £££ = p<0.001, for retinal mean density. Cell counts are listed in Table 1. D-T; dorso-temporal, D-N; dorso-nasal, V-N; ventro-nasal, V-T; ventro-temporal.
Fig 4.
Topographic distribution of Brn3a+ RGCs in normal embryonic and post-natal control retinas.
Isodensity maps showing the topographic distribution of Brn3a+ RGCs in normal E8—E20 and P4, P11 control retinas. A representative right (RR) and left (LR) retinas is shown for each age. The total number of Brn3a+RGCs in each retina is indicated. D; dorsal, T; temporal, N; nasal, V; ventral.
Table 1.
Number, retinal area and Brn3a+ RGC density in embryonic and post-natal control retinas.
Fig 5.
Dose-dependent excitotoxic injury of Brn3a+ RGCs.
Effect of intraocular injection of different amounts of NMDA on Brn3a+ RGC survival. (A) Fluorescence micrographs of cross sections of P11 retina 14 days after injection of saline (a’-c’), 10 μg of NMDA (d’-f’), 50 μg of NMDA (g’-i’) and 100 μg of NMDA (j’-l’). Brn3a (green), rhodopsin immunoreactivity (red) and DAPI (blue). (B) Bar graph showing survival of Brn3a+ RGCs at 7 and 14 days post-lesion. 100 μg NMDA was only analyzed 14 dpl. Cell-counts were on cross-sections and are shown as percentage of Brn3a+ cells in normal control retina (100%, not shown). One way ANOVA, Tukey test post-hoc test, n = 4, ** = p<0.01, *** = p< 0.001. GCL; ganglion cell layer, INL; inner nuclear layer.
Fig 6.
Effect of brimonidine pretreatment on excitotoxically injured Brn3a+ RGCs.
Bar graphs showing survival of Brn3a+ RGC in retinas after NMDA injury pretreated with saline or brimonidine (BMD). Brn3a+ RGCs were counted using the automated cell counting in four retinal quadrants of flat-mount retina and survival is shown as percentage of the number of Brn3a+ cells in normal control retina (100%). Analysis of Brn3a+ cell survival at (A) 7 days and (B) 14 days post-lesion for each retinal quadrant and for the total retina. Cell counts are listed in Tables 2 and 3. One way ANOVA, Tukey’s post-hoc test, n = 4, * = p<0.05, ** = p< 0.01. D-T; dorso-temporal, D-N; dorso-nasal, V-N; ventro-nasal, V-T; ventro-temporal.
Table 2.
Number, retinal area and Brn3a+RGC density in experimental flat-mount retinas 7 days post-lesion.
Table 3.
Number, retinal area and Brn3a+RGC density in experimental flat-mount retinas 14 days post-lesion.
Fig 7.
Topographic distribution of Brn3a+ RGCs in excitotoxically injured retina after brimonidine pretreatment.
Isodensity maps showing the topographic distribution of Brn3a+ RGCs in a representative retina after NMDA injury pretreated with saline or brimonidine (BMD) at 7 days (A,C,E,G,I) and 14 days (B,D,F,H,J) post lesion. Experimental groups were: (A, B) Injections of saline + saline, (C, D) saline + 5 μg NMDA, (E, F) brimonidine + 5 μg of NMDA, (G, H) saline + 10 μg of NMDA, and (I, J) brimonidine + 10 μg of NMDA. The total number of Brn3a+RGCs of each retina is indicated for each map. BMD; brimonidine, D; dorsal, N; nasal, V; ventral, T; temporal.