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Article
Makarand Risbud
Thomas Jefferson University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3913-0649
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This work is licensed under a CC BY 4.0 License. Read Full License
Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts showed lower incidences of degeneration, and the treatment resulted in a significant decrease in senescent markers p16INK4a, p19ARF, and SASP molecules IL-1β, and IL-6. Treated animals also showed preserved cell viability, phenotype, and matrix content. Although transcriptomic analysis showed disc compartment-specific effects of the treatment, cell death and cytokine response pathways were commonly modulated across tissue types. Results suggest that senolytics may provide a novel approach to mitigating age-dependent disc degeneration.
Keywords
Aging, Senescence, intervertebral disc, transcriptome, SASP, Mouse model
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There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigures.pdf
Supplemental Figure 1: (A, B) Representative histology of Veh and D+Q treated mice from (A) 6-23M and (B) 18-23M cohorts. (A) Representative low (5X) and high (20X) magnification views of a whole disc and NP, AF, and CEP compartments are shown. 6-23M animals showed preservation of tissue architecture and cell morphology in the NP, AF, and CEP of D+Q-treated mice. (B) Representative histology of Veh and D+Q animals from the 18-23M cohort showing whole disc and high magnification views of the NP, AF, and EP compartments. The 18-23M cohort showed comparable aging and deterioration of tissue architecture and cell morphology in the NP, AF, and CEP across both groups. (C-D) Level-by-level analysis of Modified Thompson Grading averages of NP and AF compartments from L3-6 lumbar discs of 6-23M, 14-23M, and 18-23M cohorts. 6-23M Veh (n=13), D+Q (n=15); 18-23M Veh (n=11), D+Q (n=9); 3 lumbar levels per group were analyzed. Low magnification scale bar = 200 µm (first column: A, B); high magnification scale bar = 50 µm. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate. Supplemental Figure 2: (A) Representative percentage of upregulated DEGs in the AF and NP comparison from 23-month-old mice from the 14-23M Veh cohort. (B-C) Schematic summarizing the DEGs between AF and NP of 23-month-old mice related to focal adhesion and endochondral bone ossification pathways. Supplemental Figure 3: Representative GO processes and select DEGs that change between D+Q and Veh groups in the AF and NP. (A) Representative percentage of up- and downregulated DEGs between D+Q and. Veh AF tissues from the 14-23M cohort, p ≤0.05 (B) Hierarchical clustering analysis of DEGs between D+Q and Veh AF tissues. (C) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. tissues (D) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. AF tissues (E) Representative percentage of up- and downregulated DEGs between D+Q and Veh NP tissues, p ≤0.05. (F) Hierarchical clustering analysis of DEGs between D+Q and Veh. NP tissues (G) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. NP tissues. GO process enrichment analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. (H) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. NP tissues (I) Representative Top 50 upregulated DEGs between D+Q and Veh NP tissues, p ≤0.05 Supplemental Figure 4: Multiplex analysis of the (A-A’) proinflammatory molecules, (B-B’) cytokines, and (C-C’) Th17-related proteins in serum from 6-23M and 18-23M Veh and D+Q cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 5: (A) Survival curve from 6-23M Veh and D+Q cohorts. (B) Weight progression in males from 6-23M, Veh and D+Q cohorts. (C) Weight progression in females from 6-23M, Veh and D+Q cohorts. (D) Survival curve from 18-23M, Veh and D+Q cohorts. (E) Weight progression in males from 18-23M, Veh and. D+Q cohorts. (F) Weight progression in females from 18-23M, Veh and D+Q cohorts. (G) Grip test comparison of 6-23M Veh and D+Q cohorts. Supplemental Figure 6: (A-C) Disc height, vertebral height, and disc height index (DHI) comparisons between Veh and D+Q mice from (A) 6-23M, (B) 14-23M, and (C) 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 7: (A-B) 3D reconstruction showing a transverse section through representative male and female lumbar vertebrae from 6-23M and 18-23M Veh and D+Q cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of trabecular bone parameters (C-C’) BV/TV (D-D’) trabecular thickness (Tb.Th), (E-E’) trabecular number (Tb.N), and (F-F’) trabecular space (Tb.Sp) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 8: (A-B) 3D reconstruction showing surface view and hemisection through representative male and female lumbar motion segment from 6-23M and 18-23M cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of cortical bone parameters (C-C’) Bone volume (BV) (D-D’) bone area (B.Ar) (E-E’) cortical bone thickness (Cs.Th), and (F-F’) eccentricity cross-sectional thickness (Ecc) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 9: (A-D) Bone mineral density of aged male and female mouse lumbar spines for Veh- and D+Q-treated mice from 6-23M, 14-23M, and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 10: (A) OARSI histological score (summed across 4 quadrants, 0-6 score for each) in Veh and D+Q animals from the 6-23M and 18-23M cohorts. (B) OARSI grading in males and females in the Veh and DQ groups across all treatment durations analyzed in the study (Veh: 12 male, 7 female; DQ: 14 male, 5 female)). (C-E’’) Staining and abundance analysis of key markers of senescence in the knee: P16INK4a, P19ARFand P21. M: Meniscus, 5 knees per group were analyzed. t-test or Mann-Whitney used as appropriate.
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This preprint is under consideration at a Nature Portfolio Journal. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Nature journals have partnered with Research Square to offer In Review, a journal-integrated preprint deposition service.
Article
Makarand Risbud
Thomas Jefferson University
Corresponding Author
ORCiD: https://orcid.org/0000-0003-3913-0649
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 21 Jan, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Intervertebral disc degeneration is highly prevalent within the elderly population and is a leading cause of chronic back pain and disability. Due to the link between disc degeneration and senescence, we explored the ability of the Dasatinib and Quercetin drug combination (D + Q) to prevent an age-dependent progression of disc degeneration in mice. We treated C57BL/6 mice beginning at 6, 14, and 18 months of age, and analyzed them at 23 months of age. Interestingly, 6- and 14-month D + Q cohorts showed lower incidences of degeneration, and the treatment resulted in a significant decrease in senescent markers p16INK4a, p19ARF, and SASP molecules IL-1β, and IL-6. Treated animals also showed preserved cell viability, phenotype, and matrix content. Although transcriptomic analysis showed disc compartment-specific effects of the treatment, cell death and cytokine response pathways were commonly modulated across tissue types. Results suggest that senolytics may provide a novel approach to mitigating age-dependent disc degeneration.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
There is NO Competing Interest.
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryFigures.pdf
Supplemental Figure 1: (A, B) Representative histology of Veh and D+Q treated mice from (A) 6-23M and (B) 18-23M cohorts. (A) Representative low (5X) and high (20X) magnification views of a whole disc and NP, AF, and CEP compartments are shown. 6-23M animals showed preservation of tissue architecture and cell morphology in the NP, AF, and CEP of D+Q-treated mice. (B) Representative histology of Veh and D+Q animals from the 18-23M cohort showing whole disc and high magnification views of the NP, AF, and EP compartments. The 18-23M cohort showed comparable aging and deterioration of tissue architecture and cell morphology in the NP, AF, and CEP across both groups. (C-D) Level-by-level analysis of Modified Thompson Grading averages of NP and AF compartments from L3-6 lumbar discs of 6-23M, 14-23M, and 18-23M cohorts. 6-23M Veh (n=13), D+Q (n=15); 18-23M Veh (n=11), D+Q (n=9); 3 lumbar levels per group were analyzed. Low magnification scale bar = 200 µm (first column: A, B); high magnification scale bar = 50 µm. NP: nucleus pulposus; AF: annulus fibrosus; EP: endplate. Supplemental Figure 2: (A) Representative percentage of upregulated DEGs in the AF and NP comparison from 23-month-old mice from the 14-23M Veh cohort. (B-C) Schematic summarizing the DEGs between AF and NP of 23-month-old mice related to focal adhesion and endochondral bone ossification pathways. Supplemental Figure 3: Representative GO processes and select DEGs that change between D+Q and Veh groups in the AF and NP. (A) Representative percentage of up- and downregulated DEGs between D+Q and. Veh AF tissues from the 14-23M cohort, p ≤0.05 (B) Hierarchical clustering analysis of DEGs between D+Q and Veh AF tissues. (C) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. tissues (D) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. AF tissues (E) Representative percentage of up- and downregulated DEGs between D+Q and Veh NP tissues, p ≤0.05. (F) Hierarchical clustering analysis of DEGs between D+Q and Veh. NP tissues (G) Representative DEGs from selected GO processes from downregulated DEGs between D+Q and Veh. NP tissues. GO process enrichment analysis was performed using the PANTHER Overrepresentation Test with GO Ontology database annotations and a binomial statistical test with FDR ≤ 0.05. (H) Representative DEGs from selected GO processes from upregulated DEGs between D+Q and Veh. NP tissues (I) Representative Top 50 upregulated DEGs between D+Q and Veh NP tissues, p ≤0.05 Supplemental Figure 4: Multiplex analysis of the (A-A’) proinflammatory molecules, (B-B’) cytokines, and (C-C’) Th17-related proteins in serum from 6-23M and 18-23M Veh and D+Q cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 5: (A) Survival curve from 6-23M Veh and D+Q cohorts. (B) Weight progression in males from 6-23M, Veh and D+Q cohorts. (C) Weight progression in females from 6-23M, Veh and D+Q cohorts. (D) Survival curve from 18-23M, Veh and D+Q cohorts. (E) Weight progression in males from 18-23M, Veh and. D+Q cohorts. (F) Weight progression in females from 18-23M, Veh and D+Q cohorts. (G) Grip test comparison of 6-23M Veh and D+Q cohorts. Supplemental Figure 6: (A-C) Disc height, vertebral height, and disc height index (DHI) comparisons between Veh and D+Q mice from (A) 6-23M, (B) 14-23M, and (C) 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 7: (A-B) 3D reconstruction showing a transverse section through representative male and female lumbar vertebrae from 6-23M and 18-23M Veh and D+Q cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of trabecular bone parameters (C-C’) BV/TV (D-D’) trabecular thickness (Tb.Th), (E-E’) trabecular number (Tb.N), and (F-F’) trabecular space (Tb.Sp) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 8: (A-B) 3D reconstruction showing surface view and hemisection through representative male and female lumbar motion segment from 6-23M and 18-23M cohorts. Scale bar E-F = 500 µm. (C-F) Analysis of cortical bone parameters (C-C’) Bone volume (BV) (D-D’) bone area (B.Ar) (E-E’) cortical bone thickness (Cs.Th), and (F-F’) eccentricity cross-sectional thickness (Ecc) between Veh and D+Q males and females during aging and in the 6-23M and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 9: (A-D) Bone mineral density of aged male and female mouse lumbar spines for Veh- and D+Q-treated mice from 6-23M, 14-23M, and 18-23M cohorts. t-test or Mann-Whitney test was used as appropriate. Supplemental Figure 10: (A) OARSI histological score (summed across 4 quadrants, 0-6 score for each) in Veh and D+Q animals from the 6-23M and 18-23M cohorts. (B) OARSI grading in males and females in the Veh and DQ groups across all treatment durations analyzed in the study (Veh: 12 male, 7 female; DQ: 14 male, 5 female)). (C-E’’) Staining and abundance analysis of key markers of senescence in the knee: P16INK4a, P19ARFand P21. M: Meniscus, 5 knees per group were analyzed. t-test or Mann-Whitney used as appropriate.
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