A greater number of SnOCs are associated with endplate degeneration and spinal hypersensitivity in the LSI and aged mouse models.

(a-d) Spontaneous activity, including active time (a), distance traveled (b), mean speed (c) and maximum speed (d) on the wheel within 48 hours in the sham, LSI-injury and aged mice. (e) Time in seconds spent on a hot plate in the three groups of mice. (f, g) The frequency of hind paw withdrawal (PWF) in response to mechanical stimulation (von Frey test, 0.07g (f) and 0.4g (g)) in the sham, LSI-injury and aged mice. (h) μCT images of coronal caudal endplate sections of L4-5 from 3-month-old sham and LSI- and 24-month-old aged mice. (i) Immunofluorescent (IF) staining of p16 (green), TRAP (red), and DAPI (blue) of the endplates of sham, LSI-, and aged mice. (j and k) IF staining of TRAP (red) and DAPI (blue) (j) and SA-β-gal (blue) staining (k) of endplate serial sections of sham, LSI-surgery, and aged mice. (l and m) μCT quantitative analysis of the porosity percentage (l) and trabecular separation (Tb. Sp) (m) of the endplates in the indicated groups. (n) Number of SnOCs (p16-positive and TRAP-positive cells) per mm2 in the indicated groups. (o) Number of SA-β-gal-(blue) positive cells per mm2 in the endplates in the indicated groups. (p) Number of TRAP (red) positive cells per mm2 in the endplates in the indicated groups. n ≥ 4 per group. Scale bar, 1 mm (h) and 20 μm (i, j, k). Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.

ABT263 effectively depletes endplate SnOCs in the LSI and aging mouse models.

(a-c) Immunofluorescent staining of p16 (green), TRAP (red) and nuclei (DAPI; blue) of the endplates in aged (a) and LSI-mice (b) injected with PBS (control) or ABT263 and the quantitative analysis of SnOCs based on dual staining for p16 and TRAP (c). n ≥ 4 per group. Scale bar, 20 μm. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.

ABT263 treatment improves the symptomatic spinal pain behavior in the aged and LSI mouse models.

(a, b) The PWF in response to mechanical stimulation (von Frey test, 0. 07g (a) and 0.4g (b) in aged mice treated with PBS or ABT263 compared to young adult mice. (c-e) Time (in seconds) spent on a hot plate (c), as well as spontaneous activity, including distance traveled (d) and active time (e), on the wheel within 48 hours in aged mice treated with PBS or ABT263 compared to young adult mice. (f, g) The PWF in response to mechanical stimulation (von Frey test, 0. 07g (f) and 0.4g (g)) in the LSI mouse model treated with PBS or ABT263 compared to sham-operated mice. (h-j) Time (in seconds) spent on a hot plate (h), as well as spontaneous activity analysis, including distance traveled (i) and active time (j) on the wheel within 48 hours in the sham and LSI-mice treated with PBS or ABT263. n ≥ 4 per group. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.

Depletion of SnOCs reduces spinal degeneration and sustains endplate microarchitecture in aged mice.

(a) μCT images of the aged mouse caudal endplates of L4-L5 injected with PBS or ABT263. Scale bar, 1 mm. (b) Representative images of safranin O and fast green staining of coronal sections of the caudal endplates of L4-5 in aged mice caudal endplates of L4-L5 injected with PBS or ABT263, respectively. Lower panners are zoomed in images from upper white boxes. Scale bar, 1 mm (upper panels) and 100 μm (lower panels). (c) Representative images of spine degeneration marker MMP13 (red) and nuclei (DAPI; blue) staining in aged mouse caudal endplates of L4-L5 injected with PBS or ABT263. Scale bar, 100 μm. (d) Representative images of spine degeneration marker ColX (red) and nuclei (DAPI; blue) staining in aged mouse caudal endplates of L4-L5 injected with PBS or ABT263. Scale bar, 100 μm. (e) Representative images of TRAP (magenta) staining of coronal sections of the caudal endplates of L4-5 in aged mice caudal endplates of L4-L5 injected with PBS or ABT263, respectively. Lower panners are zoomed-in images from upper white boxes. Scale bar, 100 μm. (f) The quantitative analysis of the porosity percentage. (g) The quantitative analysis of the trabecular separation. (h) The endplate score based on the safranin O and fast green staining. (i) Quantitative analysis of the intensity mean value of MMP13 in endplates per mm2. (j) Quantitative analysis of the intensity mean value of ColX in endplates per mm2. (k) The quantitative analysis of the number of TRAP-positive cells in the endplate per mm2. n ≥ 3 per group. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.

Depletion of SnOCs reduces spinal degeneration and sustains endplate microarchitecture in LSI mice.

(a) μCT images of adult sham mice and 3-month-old LSI model mice caudal endplates of L4-L5 injected with PBS or ABT263. Scale bar, 1 mm. (b) Representative images of safranin O and fast green staining in different groups. Lower panners are zoomed-in images from upper white boxes. Scale bar, 1 mm (upper panels) and 100 μm (lower panels). (c) Representative images of immunofluorescent staining of spine degeneration marker MMP13 (red) and nuclei (DAPI; blue). Scale bar, 100 μm. (d) Representative images of immunofluorescent staining of spine degeneration marker ColX (red) and nuclei (DAPI; blue). Scale bar, 100 μm. (e) Representative images of TRAP (magenta) staining in different groups. Lower panners are zoomed-in images from upper white boxes. Scale bar, 100 μm. (f) The quantitative analysis of the porosity percentage of the mouse caudal endplates of L4-5 measured by the μCT. (g) The quantitative analysis of the trabecular separation (Tb.Sp) of the mouse caudal endplates of L4-5 measured by the μCT. (h) The endplate score based on the safranin O and fast green staining. (i) Quantitative analysis of the intensity mean value of MMP13 in endplates per mm2. (j) Quantitative analysis of the intensity mean value of ColX in endplates per mm2. (k) The quantitative analysis of the number of TRAP-positive cells in the endplate per mm2. n ≥ 3 per group. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.

Depletion of SnOCs abrogates sensory innervation and pain in aged and LSI mouse models.

(a) Representative images of immunofluorescent analysis of CGRP (green), PGP9.5 (red) and nuclei (DAPI; blue) of adult sham, LSI and aged mice injected with PBS or ABT263. (b) Quantitative analysis of the intensity mean value of PGP9.5 per mm2 in aged mice. (c) Quantitative analysis of the intensity mean value of CGRP per mm2 in aged mice. (d) Quantitative analysis of the intensity mean value of PGP9.5 per mm2 in the LSI mouse model. (e) Quantitative analysis of the intensity mean value of CGRP per mm2 in the LSI mouse model. (f,g) Relative fold expression of Ntn and Ngf in aged mice (f) or LSI mice (g) with or without ABT263 treatment. Statistical significance in panels b, c, and f are analyzed using t-tests, while panels d, e, and g are subjected to one-way ANOVA. All data are shown as means ± standard deviations.

(a) Representative images of immunofluorescent analysis of CD31, an angiogenesis marker (green), Emcn, an endothelial cell marker (red) and nuclei (DAPI; blue) of adult sham, LSI and aged mice injected with PBS or ABT263. (b) Quantitative analysis of the intensity mean value of CD31 per mm2 in sham, LSI mice treated with PBS or ABT263. (c) Quantitative analysis of the intensity mean value of CD31 per mm2 in aged mice treated with PBS or ABT263. (d) Quantitative analysis of the intensity mean value of Emcn per mm2 in sham, LSI mice treated with PBS or ABT263. (e) Quantitative analysis of the intensity mean value of Emcn per mm2 in aged mice treated with PBS or ABT263. n ≥ 4 per group. Statistical significance was determined by one-way ANOVA, and all data are shown as means ± standard deviations.