Figure 1

Calpain-mediated spectrin proteolysis during acute axonal degeneration after optic nerve crush in vivo.

(a,b) Representative Western blots of spectrin proximal (a) and distal (b) to the crush site in the optic nerves at indicated time points after crush and uncrushed optic nerves (0 min after crush). Below, the quantification of the 145 kDa cleaved spectrin band is depicted, which represents the breakdown product (BDP) of spectrin specifically attributed to calpain cleavage, normalized to GAPDH. 3–6 optic nerves are included at each time point. Error bars represent the standard error of the mean (SEM). Differences are considered significant with *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA and Dunnett’s test. (c) Overview images (20×) of a native uncrushed optic nerve (control) and an optic nerve at 1 h after crush immunostained against cleaved spectrin. The antibody against cleaved spectrin selectively detects the calpain-specific BDP of spectrin. Below, quantification of the staining intensity along the longitudinal sections of native optic nerves and optic nerves at 1 h after crush. (d) Higher magnification micrographs of the representative areas immunostained against cleaved spectrin and the axonal marker smi31 in a native optic nerve and within 300 μm of the crush site in an optic nerve at 1 h after crush. Below, quantification of the intensity of intra-axonal cleaved spectrin up to 300 μm proximal and distal to the crush site. 3 optic nerves per group. *P < 0.05 by independent samples t-test.