Article Figures & Data
Figures
- Figure 1.
AAV-mediated transfection of RGCs. A-C, Flat-mounted rat retina 3 weeks after intravitreal injection with AAV-NgRWT-IGFP, double-stained for GFP to detect transfected cells (A) and βIII tubulin, a selective marker for RGCs in the retina (B). Note staining of cell bodies and axon fascicles (arrows) throughout the retina. C, Merged image. D-F, Retinal cross section, double labeled as above. GFP-expressing cells (D) are located within the innermost retina and show positive staining for βIII tubulin (E). F, Merged image. Scale bars: A-C, 100 μm; D-F, 20 μm.
- Figure 2.
Overexpression of NgR in transfected RGCs. A-C, Controls transfected with AAV-GFP and immunostained for GFP (A) and NgR (B). C, Merged image. D-F, Animals transfected with AAV-NgRWT-IGFP and immunostained for GFP (D) and NgR (E). F, Merged image. Scale is the same as in Figure 1D-F.
- Figure 3.
Role of the Nogo receptor in axon regeneration. A-F, Longitudinal sections through the optic nerve showing GAP-43-positive axons distal to the injury site (asterisk) 2 weeks after surgery with or without lens injury. A-C, Axon regeneration after transfection of growth-activated RGCs with AAV expressing GFP alone (A), NgRWT (B), or NgRDN (C). D, NgRDN expression fails to increase regeneration when RGCs have not been sensitized to grow. E, F, Distal optic nerve from the NgRDN-expressing, growth-sensitized case shown in C. Many of the longest axons coexpress GAP-43 (E) and GFP (F) (arrows). Scale bars: A-D, 200 μm; E, F, 100 μm.
- Figure 4.
Quantitation of axon regeneration and RGC survival. A, Total number of axons at 0.5 mm (light bars) and 1 mm (dark bars) distal to the injury site. B, Cell survival (βIII tubulin-positive RGCs per section). ††p < 0.01, decrease relative to GFP-transfected controls; **p < 0.01, increase relative to GFP-transfected controls.
- Figure 5.
Overexpression of NgRWT causes axon retraction. GFP-labeled axons in the optic nerve proximal to the injury site 2 weeks after nerve surgery and lens injury. A, C, Growth-sensitized control transfected with AAV-GFP. B, D, Growth-sensitized case transfected with AAV-NgRWT-IGFP. Note reduced number of axons proximal to the injury site (B, asterisk) and axons turning away from the long axis of the nerve (D, arrows). A, B, Arrowheads show the head of the myelinated portion of the nerve. In all cases, the lesion site (not shown) is to the right of the area depicted. Scale bar, 100 μm.
- Figure 6.
Axon regeneration on permissive and nonpermissive substrates. A-F, Retinal explants grown on a permissive PLL substrate. A, Control retina not exposed to macrophage-derived factors in vivo (i.e., no lens injury). B-D, Retinas transfected with AAV-NgRWT-IGFP and exposed to macrophage-derived factors in vivo. Axons (visualized by phase-contrast microscopy in B) arise from transfected RGCs, as shown by positive immunostaining for βIII tubulin (C) and GFP (D). E, Axons arising from growth-activated retina transfected with AAV-NgRDN-IGFP show positive immunostaining for the epitope tag expressed on the NgR-HA fusion protein. F, Quantitation of axon growth. G, Growth of transfected retinal explants (exposed to macrophage-derived factors in vivo) on myelin (percentage of axons arising from explants that extend ≥500 μm). †††p < 0.001, decrease relative to controls; ***p < 0.001, increase relative to controls. Scale bar, 100 μm.
