Senolytic treatment for low back pain

Abstract

Senescent cells (SnCs) accumulate because of aging and external cellular stress throughout the body. They adopt a senescence-associated secretory phenotype (SASP) and release inflammatory and degenerative factors that actively contribute to age-related diseases, such as low back pain (LBP). The senolytics, o-vanillin and RG-7112, remove SnCs in human intervertebral discs (IVDs) and reduce SASP release, but it is unknown whether they can treat LBP. sparc^-/- mice, with LBP, were treated orally with o-vanillin and RG-7112 as single or combination treatments. Treatment reduced LBP and SASP factor release and removed SnCs from the IVD and spinal cord. Treatment also lowered degeneration scores in the IVDs, improved vertebral bone quality, and reduced the expression of pain markers in the spinal cord. Together, our data suggest RG-7112 and o-vanillin as potential disease-modifying drugs for LBP and other painful disorders linked to cell senescence.

Fig. 1.. Validation of senescence in 9-month-old sparc^−/− IVDs.

(A) Representative images of multichromatic FAST (Fast Green, Alcian Blue, Safranin-O, and tartrazine)–stained sections sparc^−/− and wild-type spines. (B) Quantification of the histological degeneration grade in the upper (T13-L3) and lower (L3-S1) lumbar IVDs (n = 10 animals per strain). (C) Photomicrographs showing p16^Ink4a-positive nucleus pulposus (NP) and annulus fibrosis (AF) cells with 4′,6-diamidino-2-phenylindole (DAPI) counterstain. The red arrows point to p16^Ink4a-positive cells, and the green arrows indicate non-SnCs. (D) Quantifying of p16^Ink4a-positive IVD cells. (E) Scatter plot showing the correlation between IVD degeneration and p16^Ink4a-positive cells (n = 6 to 8 WT and 9 sparc^−/− animals per strain). (F) Principal components analysis (PCA) analysis showing the transcriptomic signatures in upper IVDs. (G) Venn diagram of differentially expressed genes (DEGs). PC, principal component. (H) Heatmap showing the expression of 37 senescence and SASP-associated genes. Data shown are relative to the calculated z scores across the samples and ranked by significance adjusted to P < 0.05. Red represents relatively high expression, and blue represents relatively low expression (n = 9 animals per strain). (I) Multiplex gene set enrichment analysis (GSEA) of the SenMayo, Reactome (cellular senescence, SASP, stress-induced senescence, and G₂-M checkpoints), and inflammatory response (MSigDB) gene sets upper (T13-L3) IVDs. Nominal P value, calculated as a two-sided t test, with no adjustment since only one gene set was tested (n = 9 animals per strain). (J to S) Luminex evaluation of circulating SASP factors (n = 18 animals per strain). Values are presented as means ± SD; significance was evaluated by a two-tailed unpaired t test in (B) and repeated-measures one-way analysis of variance (ANOVA) in (D) and (J) to (S). Significance is indicated as *P < 0.05, ***P < 0.001, and ****P < 0.0001. n.s., not significant.

Fig. 2.. Targeting SnCs with senolytic drugs improves axial discomfort, cold, and mechanical sensitivity in sparc^−/− mice.

(A) Schematic of the experimental setup. Animals received vehicle or senolytic treatment weekly through oral gavage [as described in the treatment groups (box)]. Pain assessment tests were performed every 2 weeks (grip strength, acetone-evoked behavior, and von Frey) and every 4 weeks for tail suspension. mo, months; μCT, micro–computed tomography; IF, immunofluorescence; IHC, immunohistochemistry. Axial pain was assessed by (B) grip strength and (D) tail suspension tests. Radiating pain was assessed by (F) acetone-evoked behavior (cold sensitivity) and (H) von Frey (mechanical sensitivity) tests. The area under the curve (AUC) between baseline and week 4 and between week 4 and week 8 was calculated using the trapezoid method [(T2 − T1) × (B1 + B2)/2], where T is time and B is the behavioral score for (C) grip strength, (E) tail suspension, (G) acetone-evoked behavior, and (I) von Frey assay. n = 14 to 20 animals per group (7 to 10 males and 7 to 10 females). Data are presented as means ± SEM and analyzed by one-way ANOVA followed by Tukey’s post hoc test (C, E, G, and I). */#P < 0.05, **/##P < 0.01, ***/###P < 0.001, and ****/#### P < 0.0001. * indicates a significant difference compared with sparc^−/−, and # indicates a significant difference compared with single-drug treatment.

Fig. 3.. SASP factor release is reduced in treated 9-month-old sparc^−/− mice.

IVDs (L3-S1) from sparc^−/− mice treated with senolytics or vehicle were isolated, and the release of 15 SASP factors was evaluated. The release of chemokines (A to D, J, and K), cytokines (E to G, I, and M), and growth factors (H and L) was assessed using a multiplex assay. The data in (A) to (M) are presented as means ± SD; two-way ANOVA and post hoc comparison Tukey’s were used to measure significant differences between the groups. */#P < 0.05, **/##P < 0.01, ***P < 0.001, and ****/####P < 0.0001. * indicates a significant difference compared with sparc^−/−, and # indicates a significant difference compared with single-drug treatment. Four discs represent one measure per animal. n = 10 animals (5 males and 5 females) in each group.

Fig. 4.. Senolytics removed SnCs and improved IVD health.

sparc^−/−spines (L3-S1) from animals treated with senolytics or vehicle (n = 7 to 9 animals, 3 to 5 males and 4 to 5 females per group) were collected and processed for histological evaluation. Immunofluorescence staining measures the percentage of p16^Ink4a-positive cells; DAPI was used as a counterstain (A) NP and (B) AF. (C) Representative images of lumbar IVDs (L3-S1) from sparc^−/− mice treated with senolytics or vehicle. (D) FAST staining revealed histological improvement in the ventral region (clear distinction between AF and NP and increase in disc height) of treated discs. (E) Histological degeneration grade of the IVDs was independently evaluated and the average grade per animal is presented for a total of 8 to 10 animals (4 to 5 males and females) per group. Statistical comparisons were calculated using an ordinary two-way ANOVA, with a Tukey’s post hoc analysis. Data are presented as means ± SD. ###P < 0.001 and ****/####P < 0.0001. * indicates a significant difference compared with sparc^−/−, and # indicates a significant difference compared with single-drug treatment.

Fig. 5.. Senolytics reduced SnCs and pain-related neuroplastic changes in the spinal cord of sparc^−/−mice.

(A) Spinal cords from perfused animals treated with senolytic drugs or vehicle were collected and processed for histological evaluation. Representative images showing higher p16^Ink4a immunoreactivity in the dorsal horn (delineated in dashed line) in sparc^−/− compared to that in wild-type mice at 9 months. (B) Quantification of the increased immunoreactivity (percentage area) (% Area IR). (C) p16^Ink4a immunoreactivity was reduced in treated sparc^−/− animals. The spinal cords were also assessed for pain-related neuroplastic changes. (D) Representative images of calcitonin gene-related peptide (CGRP), glial fibrillary acidic protein (GFAP), and CD11b immunoreactivity in the dorsal horn of sparc^−/− mice (delineated in a dashed line). The immunoreactivity (percentage area) was quantified for (E) CGRP, (F) GFAP, and (G) CD11b. The % area with immunoreactivity above a set threshold was calculated from the total area of the dorsal horn. n = 7 to 8 animals per group (3 to 4 males and 4 females). DAPI was used as a counterstain. The average of three separate images was calculated for each animal and used to calculate the mean of the treatment group. Scale bars (A and D), 100 μm. Data are presented as means ± SD and were analyzed by a two-tailed t test or an ordinary one-way ANOVA followed by Tukey’s post hoc test. */#P < 0.05, **P < 0.01, ***/###P < 0.001, and ****P < 0.0001. * indicates a significant difference compared with sparc^−/−, and # indicates a significant difference compared with single-drug treatment.

Fig. 6.. Treatment with senolytics resulted in increased IVD volume and improved bone quality.

(A) Representative images showing the difference in disc volume (delineated in red) between 9-month-old sparc^−/− and wild-type mice. (B) Quantification of disc volume showed a lower volume in sparc^−/− compared to that in WT mice. (C) Disc volume of sparc^−/− IVDs from animals treated with senolytics or vehicle. Representative micro-CT images showing (D) vertebrae and (E and F) trabecular and cortical bone of wild-type and sparc^−/− mice (vehicle and treated). Quantification of trabecular (G to L) and cortical (M to P) bone parameters. Scale bars (A, E, and F), 500 μm. Statistical comparisons were calculated using a two-tailed t test or an ordinary one-way ANOVA, with a Dunnett’s post hoc analysis as appropriate. Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. * indicates a significant difference compared with sparc^−/−. n = 10 to 12 animals (3 to 6 males and 5 to 8 females) per group and 3 levels per animal (L4-S1).