a. Experimental scheme. Tamoxifen injection was started at 3 days before SCI and continued every other day until 5 weeks after SCI.
b. BMS scores during the 5 week recovery after T8 contusion injury demonstrate impaired functional recovery in Plxnb2 cKO mice as compared to littermate controls with the same tamoxifen regimen. n=12 animals per group, two-way ANOVA with Bonferroni post hoc correction for repeated measures, ***p<0.0001, ###p<0.001 for the entire recovery course, F(DFn, DFd): F(1,374)=945.8 for column factor, F(16, 374)=52.11 for row factor. Data represent mean ± SEM.
c. Motosensory behavioral assays by rotarod and ladder walking (regular and irregularly spaced rungs) at 35 dpi demonstrate impaired functional recovery in Plxnb2 cKO mice as compared to littermate controls. Rotarod tests: n=12 animals per group, unpaired two-tailed Student’s t-test, **p=0.011. Ladder walking tests: n=15 animals per group, two-way ANOVA with Bonferroni post hoc correction, ***p<0.0001. For regular, F(DFn, DFd): F(1,140)=312 for column factor, F(4, 140)=106 for row factor. For irregular, F(DFn, DFd): F(1, 140)=369 for column factor, F(4, 140)=315 for row factor. Von Frey filament test was conducted at baseline and at 35 dpi, n=4 animals per group, left and right hindpaws were measured separately, Mann Whitney test, n.s. p=0.99, ***p=0.0005. Data represent mean ± SEM.
d. IHC of sagittal sections of thoracic spinal cords at 35 dpi show larger lesion volume revealed by CSPG and fewer axon fibers at the dorsal column and the lesion center (NH-F) in Plxnb2 cKO mice than littermate controls. n=3 animals per group, unpaired two-tailed Student’s t-test. For lesion volume by CSPG, **p=0.0042. For NF-H intensity, *p=0.027 for lesion core, *p=0.014 for dorsal column. Data represent mean ± SEM.
e. IHC of sagittal sections of thoracic spinal cords at 35 dpi show fewer serotonergic fibers (5HT) at the lesion site in Plxnb2 cKO mice than in littermate controls (white arrows). n=3 for control and n=4 for cKO, unpaired two-tailed Student’s t-test. *p=0.014. Data represent mean ± SEM. R-rostral; C-caudal; D-dorsal; V-ventral; CC-central canal.