Article Figures & Data
Figures
- Figure 1.
Optic nerve crush results in both proximal and distal axon degeneration. A–D, Optic nerves immunostained for NF-M to label axons 12 h, 24 h, 7 d, and 30 d after optic nerve crush. *Crush site. E, Naive mouse optic nerve immunostained for βIV spectrin to label nodes of Ranvier (green, arrow) and Caspr to label flanking paranodes (red). F, Flat-mount of mouse retina immunostained for βIV spectrin to label AIS (green, arrow) and NF-M to label axons (red). Scale bars: A–D, 200 μm; E, 5 μm; F, 30 μm.
- Figure 2.
Nodes of Ranvier degenerate after optic nerve crush. A–P, Representative images of optic nerves immunostained for βIV spectrin (green) and caspr (red) at the indicated time points after nerve crush and distances from the crush site. Q–T, The ratio of nodes of Ranvier per FOV in crushed optic nerve to uninjured control optic nerve in areas proximal (−325 μm), within (0 μm), and distal (+325 and +975 μm) to the crush site. Scale bars, 10 μm.
- Figure 3.
AISs of RGCs degenerate after optic nerve crush. A–F, Representative images of flat-mounts of retina immunostained for βIV spectrin reveal RGC AIS (arrow) and AII amacrine cell lobular dendritic processes (arrowhead) in control (A) and after optic nerve crush (B–F). G, H, Quantification of AIS degeneration (G) and changes in AIS length (H) after optic nerve crush. Scale bars: A–F, 20 μm. **p < 0.01 (unpaired t test with Mann–Whitney post test). ***p < 0.001 (unpaired t test with Mann–Whitney post test).
- Figure 4.
Axons regenerate after genetic deletion of PTEN coupled with intravitreal injection of zymosan and CTP-cAMP (Cre+Zymo+cAMP). A–C, Optic nerves immunostained for NF-M in control saline-injected and Cre+zymosan+cAMP-treated mice at 2 weeks (A), 6 weeks (B), and 12 weeks (C) after crush. *Crush site. Scale bar, 200 μm.
- Figure 5.
Nodes of Ranvier are reassembled in regenerating CNS axons. A–F, Representative images of optic nerves immunostained for βIV spectrin (green, arrows) and Caspr (red) proximal to the crush (A, C, E) and at the crush site (B, D, F). Optic nerves were examined at 2, 6, and 12 weeks after crush. G, H, The ratio of nodes of Ranvier per FOV in crushed optic nerve to uninjured control optic nerve at 2, 6, and 12 weeks after crush. Measurements were made proximal to the crush site (−325 μm) and within the crush site (0 μm). Treatment = Cre+zymosan+cAMP. Scale bars: A–F, 10 μm. *p < 0.05 (unpaired t test with Mann–Whitney post test).
- Figure 6.
Nodes of Ranvier are reassembled in regenerating CNS axons distal to the crush site. A–F, Representative images of optic nerves immunostained for βIV spectrin (green, arrows) and Caspr (red) distal to the crush (A, C, E: 325 μm; B, D, F: 925 μm). Optic nerves were examined at 2, 6, and 12 weeks after crush. G, H, The ratio of nodes of Ranvier per FOV in crushed optic nerve to uninjured control optic nerve at 2, 6, and 12 weeks after crush. Measurements were made distal to the crush site (+325 and +975 μm). Treatment = Cre+zymosan+cAMP. Scale bars: A–F, 10 μm. *p < 0.05 (unpaired t test with Mann–Whitney post test).
- Figure 7.
Regenerating CNS axons can be remyelinated and reassemble paranodal junctions. A, B, Proximal (A) and distal (D) regions of optic nerve immunostained for NF-M showing examples of “U-turn” axons (arrows) 12 weeks after optic nerve crush in Cre+zymosan+cAMP mice. C, D, Proximal optic nerve immunostained for Nav1.6 (C) or AnkG (D) to label nodes of Ranvier (red, arrow) and Caspr to label flanking paranodes (green) 12 weeks after optic nerve crush in Cre+zymosan+cAMP mice. E–H, Optic nerve immunostained for Caspr (green) and NF-M (red) to label paranodes and axons, respectively, in Cre+zymosan+cAMP mice 12 weeks after optic nerve crush. I, Quantification of Caspr-labeled paranodes along regenerating optic nerve in saline-treated and Cre+zymosan+cAMP-treated mice 12 weeks after optic nerve crush. J, K, Electron microscopy of control (J) and regenerating axons (K) showing the electron dense reaction product from HRP-CTB histochemistry (asterisks) in the axoplasm of myelinated axons. L, The number of regenerating CTB-HRP-labeled myelinated axons per nerve. N = 3 mice per group. ***p < 0.001 (unpaired t test). Scale bars: A, B, 5 μm; C, D, 10 μm; E, 300 μm; F–H, 5 μm; J, K, 0.5 μm.
- Figure 8.
AISs are reassembled in regenerating CNS axons. A–C, Flat-mounts of uninjured control (A), 12 week saline-treated (B), and 12 week Cre+zymosan+cAMP-treated retinas (C) immunostained for NF-M to label axons. D–K, Immunostaining of retinas using antibodies against βIV spectrin (green) and NF-M (red) to label axons in saline-treated (D, G) and Cre+zymosan+cAMP-treated (E, H–K), retinas at 6 weeks (D, E) and 12 weeks (G–K) after optic nerve crush. F, Uninjured controls. L, The number of AISs per FOV after optic nerve crush. M, AIS length after optic nerve crush. Treatment = Cre+zymosan+cAMP. Labels on x-axis of M apply to the x-axis of L. Scale bars: A–C, 300 μm; D–K, 20 μm. *p < 0.05 (unpaired t test with Mann–Whitney post test). ***p < 0.001 (unpaired t test with Mann–Whitney post test).
- Figure 9.
AnkG is not required for axon regeneration. A, GAP-43 immunostatining of optic nerve after intravitreal injection of β-1,3-glucan (Curdlan) or saline, 2 weeks after optic nerve crush. B, Quantification of GAP-43-labeled axons in mice administered either Curdlan or saline 2 weeks after optic nerve crush. C, Immunostaining of Ank3f/f mouse optic nerves after intravitreal injection of either AAV-GFP or AAV-Cre-GFP using antibodies against ankG (red), Caspr (blue), and GFP (green). Arrows indicate nodes of Ranvier. Red arrowheads indicate paranodal ankG in the AAV-Cre-GFP injected mice. D, F, Optic nerves from Ank3f/f mice treated with Curdlan and either AAV-GFP or AAV-Cre-GFP and immunostained with GAP-43 (D) or GFP (F) 2 weeks after optic nerve crush. E, G, Quantification of GAP-43 (E) or GFP (G) positive axons in regenerating axons in Ank3f/f mice administered curdlan and either AAV-GFP or AAV-Cre-GFP 2 weeks after optic nerve crush. Scale bars: A, D, 300 μm; F, 200 μm. ****p < 0.0001 (unpaired t test with Mann–Whitney post test).
