Neu3 sialidase remains inactivated after retinal axonal injury; however, exogenous sialidase can promote retinal axon regeneration. I, Schematic of timeline for retinal axon experiments. II, A–D, Fluorescent photomicrographs of adult retinal axons 15 min after axotomy. Axons are double-labeled for GD1a (A, C) and GM1 gangliosides (CTB; B, D) under normal conditions (control 15 min; A, B) or after exposure to Clostridium sialidase (sialidase 15 min; C, D). E, Fluorescent photomicrograph showing Neu3 sialidase enzyme on an adult retinal axon. The ratio of GM1/GD1a ganglioside is plotted in F (30–40 axons/chamber, n ≥ 6). G–I, Fluorescent confocal photomicrographs (longitudinal) of optic nerve uncrushed (G,G′), 6 h (H,H′), and 12 h after crush (I, I′), triple-labeled for βIII-tubulin (green), GD1a (blue), and GM1 gangliosides (CTB, red). High-magnification images (′) from the dotted squares in the corresponding pictures denote representative areas used for GD1a and GM1 quantification. Arrows indicate regions parallel to axonal staining used for measuring intensities of GM1 and GD1a gangliosides. The ratio of GM1/GD1a gangliosides (± SEM) parallel to axonal staining before and after optic nerve injury and normalized against background intensity levels is plotted in J (n = 3). p values by one-way ANOVA. K–N, Phase-contrast photomicrographs showing retinal axons at time of axotomy (0 min; K, M) and 1 h after axotomy (1 h; L, N), retracting under normal conditions (control; K, L), or regenerating after exposure to Clostridium sialidase applied 30 min before axotomy (sialidase; M, N). Plots of mean distance of axon tip retraction from site of injury measured 1 h after injury in the presence or absence of exogenous sialidase (n ≥ 6) (O) and percentage of axons that regenerated past retraction position in retinal axons 1 h after injury (n ≥ 6) (P) ± SEM. ***p < 0.001; *p < 0.05 (Student's t test, two-tailed). Scale bars: A–D, 100 μm; E, G′–I′, 25 μm; G–I, K–N, 50 μm.