NB-3 signaling mediates the cross-talk between post-traumatic spinal axons and scar-forming cells (2016)
Figure 9

Figure 9. Co-induction of CHL1 and NB-3 after SCI mediates downstream signaling transduction.

A Co-immunostaining of CHL1 (blue), NB-3 (green), and BDA (red) in injured spinal cords of NB-3+/+ (A1) and NB-3−/− (A5) mice. (A2–A4 and A6–A8) High-magnification images of boxed areas in (A1 and A5), respectively. Images in (A2 and A6) are presented as single-channel fluorescence images in (A3–A4 and A7–A8), respectively.

B Quantification of fluorescence intensity of CHL1 at the spinal lesion sites of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. n = 11 mice per group.

C, D Western blot analysis (C) and quantification (D) of CHL1 expression in spinal lesion areas of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 2 mice per group.

E Co-immunoprecipitation of NB-3 and CHL1 from spinal lesion areas of NB-3+/+ and NB-3−/− mice.

F Co-immunostaining of CHL1 and NB-3 in FG-labeled corticospinal neurons (arrowheads) of NB-3+/+ and NB-3−/− mice 14 days post-injury (dpi).

G Quantification of the fluorescence intensity of CHL1 in (F). *p < 0.05, and **p < 0.01; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 corticospinal neurons from 3 mice in each group were quantified.

H, I Western blot analysis (H) and quantification (I) of CHL1 expression in sensorimotor cortices of NB-3+/+ and NB-3−/− mice after SCI. *p < 0.05; one-way ANOVA followed by Bonferroni post-test. Data were analyzed from 3 independent experiments each including 3 mice per group.

J CHL1 expression in the somas and axons of NB-3+/+ and NB-3−/− pyramidal neurons when co-culturing with NB-3–expressing astrocytes (dashed lines and asterisks) or astrocytes that didn't express NB-3 (dashed lines). Co-localization of CHL1 and NB-3 in astrocytes overexpressed NB-3 (asterisks). Co-localization of CHL1 and NB-3 in the somas and axons of co-cultured NB-3+/+ pyramidal neurons (arrowheads). Left inset, high-magnification view of the soma (white arrowhead). Right inset, high-magnification view of the axon tip (green arrowhead).

K Quantification of fluorescence intensity of CHL1 in (J). *p < 0.05, **p < 0.01, and ***p < 0.001; one-way ANOVA followed by Bonferroni post-test. The intensities of more than 300 astrocytes or pyramidal neurons from 3 independent experiments in each group were quantified.

L–N Sagittal sections showing the corticospinal axons in injured spinal cords following injection of LV-nc-GFP (L) and LV-CHL1 shRNA-GFP (M) into both the sensorimotor cortex and lesion sites. High-magnification image (N) of the area in (M) (asterisk). The arrows indicate the lesion sites; the asterisk indicates the lesion epicenter.

O Quantification of intensity index of BDA-labeled axons at certain distances from the lesion border in (L and M). *p < 0.05 and **p < 0.01; two-way ANOVA followed by Fisher’s LSD. n = 9 mice in (L), and n = 10 mice in (M).

Data are represented as mean ± SEM (B, D, G, I, K, and O). Scale bars, 500 µm (A1 and A5), 40 µm (A2–A4 and A6–A8), 20 µm (F), 10 µm (J), 400 µm (L and M), and 40 µm (N).