Article Figures & Data
Figures
- Figure 1.
Experimental design to investigate the neuroprotective role of BDNF after ONC. A, Timeline of experiments. B, C, Retinal BDNF levels were quantified by Western blot analysis. Left eyes received ONC, and relative protein levels were compared to the level of nonoperated right eyes (with right eyes of control mice designated as 1). BDNF_OE compensated for the down-regulation of retinal BDNF level induced by ONC. n = 4–7 mice in each group. N.S.: not significant; **p < 0.01 by Student’s t-test. D, In vivo imaging to identify a small group of large-soma RGCs. Left, a representative Thy-1-CreERT2 mouse retina imaged by a Micron III fundus scope. Middle, the image was processed by Matlab to identify large-soma RGCs (marked by red circles). The area marked by the orange square was highlighted on the right.
- Figure 2.
Identification of one type of RGCs with large somas. A, GFP-positive cells with large somas expressing Thy-1-CreERT2 transgene were coimmunolabeled with RGC markers: Brn-3a and Brn-3b (arrows). B, Small-soma, but not large-soma, GFP-positive cells were coimmunolabeled with amacrine cell markers: BETA3 and ChAT (arrowheads). C, GFP-positive cells with large somas were coimmunolabeled with RGC subtype markers: SMI-32 (arrows), but not with CART. D, Percentages of large-soma RGCs expressing Thy-1-CreERT2 transgene coimmunolabeled with different RGC markers. E, Confocal image of one large-soma RGC double-labeled by SMI-32 (red) and GFP (green). Orthogonal view of the same cell is shown at the bottom. Most SMI-32 and GFP cohorts in large RGCs (20 of 28) had their dendrites laminated in ON sublamina of IPL. Scale bars: 50 µm. INL, inner nuclear layer.
- Figure 3.
The loss of large-soma RGCs was delayed by BDNF overexpression. A, In vivo and Matlab-processed images show the progressive loss of large-soma RGCs after ONC. Number of surviving RGCs is shown at the bottom right of each image. Some examples of the large-soma cells are circled in solid red lines, and the same locations the next week are circled in dashed lines, demonstrating the disappearance of these large somas. Scale bar: 20 µm. B, More RGC somas survived in BDNF_OE than control mice. Control, n = 8 mice; BDNF_OE, n = 11 mice. N.S.: not significant; *p < 0.05 by Student’s t-test. Data are presented as mean ± SEM.
- Figure 4.
Axonal survival was also extended in BDNF_OE mice. A, Confocal images of flat-mounted BDNF_OE retinas before and after ONC injury (left). Axons were immune-stained with anti-GFP antibody and counted with Matlab. Each red cross marks one axon by Matlab; the number was double-checked manually. B, RGC survival was significantly higher in BNDF_OE mice at 2 weeks after ONC. n = 4–5 mice. N.S.: not significant; **p < 0.01 by two-way ANOVA, Sidak’s multiple comparison posttest. Data are presented as mean ± SEM.
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