Fig 2: SDF1 complements the effects of Ocm and other treatments on optic nerve regeneration. (A and B) Exogenous SDF1 (0.3 µg per eye) stimulates axon regeneration (***P < 0.001 compared with NC only) and augments the effects of Ocm combined with 50 µM CPT-cAMP (Ocm/cAMP: **P < 0.01, SDF1 + Ocm/cAMP vs. SDF1; ***P < 0.001, SDF1 + Ocm/cAMP vs. Ocm/cAMP). A higher concentration of SDF1 (0.9 µg per eye) plus Ocm/cAMP increases regeneration to a similar extent as zymosan. Asterisks in A indicate the injury site. (Scale bar, 200 µm.) (C) SDF1 increases RGC survival. Whereas Ocm/cAMP does not alter RGC survival, SDF1, either alone or combined with Ocm/cAMP, increases RGC survival to the same extent as zymosan (**P < 0.01, ***P < 0.001). Error bars in B and C: standard error of mean; one-way ANOVA plus Bonferroni correction (B and C).

Fig 3: Infiltrative macrophages express SDF1. (A) Retinal cross-sections from normal mouse and mice D1 after optic nerve crush alone (NC) or NC plus intraocular zymosan (NC + Zymo) costained for SDF1 (red) and either the macrophage marker F4/80, neutrophil marker Gr1, or RGC marker TUJ1 (for ßIII-tubulin; green). DAPI is a nuclear marker (blue). Note the colocalization of SDF1 with F4/80+ cells lying outside or invading the GCL but not with Gr1 or TUJ1. (A, Bottom) Enlarged images of SDF1 coimmunostained with the same three cell markers 1 d after NC + Zymo. INL, inner nuclear layer; ONL, outer nuclear layer. (B) Immunostained cells extracted from the vitreous D1 or D4 after intraocular zymosan show colocalization of SDF1 with F4/80 but not Gr1. (C) Enlarged images showing SDF1 immunostaining in vesicles within a mononuclear macrophage and weak staining in a polymorphonuclear neutrophil. (D) SDF1 mRNA distribution. Whole eyes (dark gray bars), which include infiltrative cells, show large increases in SDF1 mRNA D1 and D4 after zymosan injections whereas neural retina (light gray) shows little elevation. (Scale bars, 75 µm [A], 45 µm [A, Bottom], 20 µm [B], and 10 µm [C].) (D) One-way ANOVA plus Sidak correction; error bars in D: standard error of mean; *P < 0.05, ***P < 0.001 (whole eye: NC + Zymo vs. NC alone).

Fig 4: Schematic summary of results and signaling pathways. (A) Effects of myeloid cell–derived growth factors SDF1 and Ocm on optic nerve regeneration: cellular sources and responses of RGC subtypes. (A, Top) AAV2 expressing an shRNA to suppress Pten expression (AAV2-shPten) induces axon growth primarily from aRGCs (15) (green cells); the control AAV2-shLuciferase virus (AAV2-shLuc) has no effects. (A, Middle) SDF1, either as a recombinant protein (rSDF1) or via AAV2-mediated expression of monomeric SDF1 (AAV2-SDF1), induces moderate axon growth primarily from non-aRGCs (Middle Left, orange cells). SDF1 combined with Pten deletion enables non-aRGCs to regenerate lengthy axons but simultaneously suppresses the ability of aRGCs to respond to Pten deletion (Middle Right). (A, Bottom) Zymosan, by elevating levels of neutrophil/macrophage-derived Ocm, macrophage-derived SDF1, and other factors, stimulates regeneration from both aRGCs and non-aRGCs (Bottom Left); growth from both subtypes is strongly augmented by combining zymosan with Pten deletion (Bottom Right). (B) Signaling pathways activated by SDF1, Ocm, CPT-cAMP, and Pten deletion. SDF1 binds to its cognate G protein–coupled receptor CXCR4, thereby 1) activating adenylate cyclase (AC) to elevate cAMP and consequently PKA, which phosphorylates the Ocm receptor (Ocm-R, or a chaperone protein), causing it to translocate to the cell membrane (9, 10) (green arrows); 2) activating PI3K and its downstream effectors, including an AKT-dependent pathway (AKT, mTOR/S6K, to increase phosphorylation of ribosomal subunit pS6 and other substrates) and AKT-independent pathways (90) (dashed arrow); 3) besides cAMP generated downstream of SDF1, CPT-cAMP, a membrane-permeable, nonhydrolyzable cAMP analog, also activates PKA and potentiates the effects of Ocm; and 4) SDF1 antagonizes the repellent effects of slit/robo signaling at the optic chiasm and diencephalon (43). The binding of Ocm to Ocm-R promotes axon growth via CaMKII and other kinases. Inhibiting CaMKII alone, or any combination of MAPK, JAK, and PI3K, blocks the effects of Ocm (9, 10). AAV2-shPten decreases Pten expression, thereby augmenting PIP3 phosphorylation and activating AKT, mTOR, and S6K. Pten deletion also promotes axon growth through AKT-independent pathways (e.g., focal adhesion kinase, RAS, and others; not shown) (90). Figure created with Biorender.com.

Fig 5: SDF1 activates downstream effectors of axon growth and enhances long-distance regeneration. (A and B) SDF1 increases phosphorylation of Creb and ribosomal protein S6 (pCreb, pS6; red) in RGCs (green; stained with antibody TUJ1+ for ßIII-tubulin) 1 d after intraocular injection (*P < 0.05, **P < 0.01, SDF1 vs. saline). (Scale bars, 25 µm.) (C–E) Mice received intraocular AAV2-shPten 2 wk prior to NC and Zymo/cAMP, with or without SDF1, immediately after NC and again 3 wk later. Regenerating axons were visualized by CTB (green) injected intraocularly 2 d before euthanasia. (C) Quantitation of regenerating axons at the indicated distances from the injury site (*P < 0.05 comparing shPten/Zymo/cAMP/SDF1 vs. shPten + Zymo/cAMP + PBS). (D) Regenerating axons extending into the optic tract (OPT; Left, arrows) and entering the ventral and dorsal lateral geniculate nuclei (vLGN, dLGN; Right, arrowheads) in a mouse treated with AAV2-shPten, zymosan/cAMP, and SDF1. (Scale bar, 150 µm.) (E) Longitudinal section (14-µm thickness) through the optic nerve of a mouse treated with AAV2-shPten, zymosan/cAMP, and SDF1. Note the numerous CTB+ axons regenerating the full length of the optic nerve after 6 wk. (E1) The enlarged image shows individual axons or axon bundles at the distal end of the optic nerve. The asterisk indicates the injury site. (Scale bar, 200 µm.) Error bars in A–C: standard error of mean; Student t test (A and B) and multiple t test (C).