Figure 2. OPN Sensitizes the Responsiveness to IGF1 in Adult CSNs.

(A) Schematic diagram of the experimental timeline. Wild type mice received intraspinal injection (at the spinal cord T12-L4 segments) with HiRet-GFP at P12–P14, T10 spinal cord lateral hemisection at P60, bilateral cortical injection of saline, soluble human recombinant IGF1, OPN or OPN/IGF1 at P74, and were sacrificed at P77 for histological analysis.

(B) Representative images showing intact and axotomized CSNs (green: retrogradely labeled by GFP) co-stained with IGFR, pIGFR, and pS6 in saline (top lanes), IGF1 (middle lanes), and OPN & IGF1 protein (bottom lanes) injected animals. Arrowheads indicate the co-localization of GFP with IGFR, pIGFR, and pS6 immunofluorescence. Scale bar: 20 μm.

(C,D,E) Quantification of immunofluorescence intensities of IGFR, pIGFRβ and pS6 in various conditions. All images were taken using identical optical parameters and scan settings. In each case, the intensities were normalized to that in intact CSNs with saline injection. ** and n.s., p < 0.01 and no statistical significance. One way-ANOVA, followed by post hoc Bonferroni correction. For IGFR quantification, n = 122, 107, 123, 126, 94, 93, 121, 113 for the intact and axotomized CSNs in saline (n = 3), IGF1 (n = 3), OPN (n=3) and OPN/IGF1 (n = 3) injected animals, respectively. For pIGFRβ quantification, n = 104, 119, 107, 122, 104, 96, 126, 118 for the intact and axotomized CSNs in saline (n = 3), IGF1 (n = 3), OPN (n=3) and OPN/IGF1 (n = 3) injected animals, respectively. For pS6 quantification, n = 130, 131, 130, 134, 119, 108, 133, 139 for the intact and axotomized CSNs in saline (n = 3), IGF1 (n = 3), OPN (n=3) and OPN/IGF1 (n = 3) injected animals, respectively. Note that the images of OPN protein injected group were present in Figure S4A.