Age-Associated Changes in Knee Osteoarthritis, Pain-Related Behaviors, and Dorsal Root Ganglia Immunophenotyping of Male and Female Mice

Abstract

Objective: Osteoarthritis (OA) is a leading cause of chronic pain, yet OA pain management remains poor. Age is the strongest predictor of OA development, and mechanisms driving OA pain are unclear. We undertook this study to characterize age-associated changes in knee OA, pain-related behaviors, and dorsal root ganglion (DRG) molecular phenotypes in mice of both sexes.

Methods: Male or female C57BL/6 mice 6 or 20 months of age were evaluated for histopathologic knee OA, pain-related behaviors, and L3-L5 DRG immune characterization via flow cytometry. DRG gene expression in older mice and humans was also examined.

Results: Male mice at 20 months of age had worse cartilage degeneration than 6-month-old mice. Older female mouse knees showed increased cartilage degeneration but to a lesser degree than those of male mice. Older mice of both sexes had worse mechanical allodynia, knee hyperalgesia, and grip strength compared to younger mice. For both sexes, DRGs from older mice showed decreased CD45+ cells and a significant increase in F4/80+ macrophages and CD11c+ dendritic cells. Older male mouse DRGs showed increased expression of Ccl2 and Ccl5, and older female mouse DRGs showed increased Cxcr4 and Ccl3 expression compared to 6-month-old mouse DRGs, among other differentially expressed genes. Human DRG analysis from 6 individuals >80 years of age revealed elevated CCL2 in men compared to women, whereas CCL3 was higher in DRGs from women.

Conclusion: We found that aging in male and female mice is accompanied by mild knee OA, mechanical sensitization, and changes to immune cell populations in the DRG, suggesting novel avenues for development of OA therapies.

Fig. 1.

Aging is associated with mild knee osteoarthritis in mice. (A) Medial cartilage degeneration scores for male mice right knees aged 6 months (n=10; Mean ± SEM = 0.600 ± 0.267) or 20 months (n=12; Mean ± SEM = 11.5 ± 2.39) old as determined by OARSI scoring methods; (B) lateral cartilage degeneration for males aged 6 months (n=10; Mean ± SEM = 1.9 ± 0.3786) or 20 months (n=12; Mean ± SEM = 5.58 ± 0.656) ; (C) Total cartilage degeneration score for males aged 6 months (n=10; Mean ± SEM = 2.5 ± 0.543 or 20 months (n=12; Mean ± SEM = 17.13 ± 2.49) plotted as sum of medial and lateral compartments; (D) Osteophyte width for males plotted in micron size found in either medial or lateral compartments of the knee; (E) Synovitis hyperplasia score for males showing an average of two blinded scores (maximum score is 12); (F) Synovitis cellularity score for males showing an average of two blinded scores (maximum score is 12); (G) Synovitis fibrosis score for males showing an average of two blinded scores (maximum score is 4); (H – N) Same as in (A – G) but shown for female mice aged 6 months (n=6; Medial Mean ± SEM = 0.1667 ± 0.1667; Lateral Mean ± SEM = 0.0 ± 0.0; Total Mean ± SEM = 0.1667 ± 0.1667) or 20 months old (n=6; Medial Mean ± SEM = 3.667 ± 1.116; Lateral Mean ± SEM = 1.884 ± 0.4773; Total Mean ± SEM = 5.50 ± 1.586). Statistical analysis by Mann-Whitney test. Significant if p < 0.05. Error bars show Mean ± SEM.

Fig. 2.

Aging is associated with development of pain-related behaviors. (A) Mechanical allodynia by von Frey fiber testing; (B) Knee hyperalgesia by pressure application measurement testing; (C) Grip strength by square grid grip testing of all four paws observed in 10-week (n=10), 6-month (n=10), or 20-month (n=12) old male mice. (D - F) Same as in (A - C) but for 10-week (n=10), 6-month (n=6), or 20-month (n=6) old females. Statistical analysis by Ordinary One-Way ANOVA test with Tukey post test. Mechanical allodynia values were first log transformed before running statistical analysis as the von Frey fibers are on a log scale. Significant if p < 0.05. Error bars show mean±SEM.

Fig. 3.

Immune cell populations in dorsal root ganglia of aged male mice. (A) Frequency of CD45+ leukocytes; (B) F4/80+ macrophages; (C) CD11c+ Dendritic cells; (D) CD3+ T cells; (E) CD11b+ myeloid cells; (F) Ly6Chi monocytes; (G) Ly6G+ granulocytes; (H) Ly6C-antigen presenting precursor cells; (I) CCR2+ cells in 6-month (n=5), or 20-month (n=6) old male mice. Flow cytometry gating strategy in Suppl. Fig. 2. Statistical analysis by Mann-Whitney test. Significant if p < 0.05. Error bars show mean±SEM.

Fig. 4.

Immune cell populations in dorsal root ganglia of aged female mice. (A) Frequency of CD45+ leukocytes; (B) F4/80+ macrophages; (C) CD11c+ Dendritic cells; (D) CD3+ T cells; (E) CD11b+ myeloid cells; (F) Ly6Chi monocytes; (G) Ly6G+ granulocytes; (H) Ly6C-antigen presenting precursor cells; (I) CCR2+ cells in 6-month (n=6), or 20-month (n=6) old female mice. Flow cytometry gating strategy in Suppl. Fig. 2. Statistical analysis by Mann-Whitney test. Significant if p < 0.05. Error bars show mean±SEM.

Fig. 5.

Macrophage phenotypes in male and female aged DRGs. (A) Frequency of F4/80+MHCII+ pro-inflammatory macrophages; (B) F4/80+CX3CR1+ pro-inflammatory macrophages; (C) F4/80+CD206+ anti-inflammatory macrophages; (D) F4/80+CD163+ anti-inflammatory macrophages from 6-month (n=5) or 20-month old (n=6) male mice DRGs. (E - H) Same as in (A - D) but for 6-month or 20-month old female mice (n=6) DRGs. Flow cytometry gating strategy in Suppl. Fig. 3. Statistical analysis by Mann-Whitney test. Significant if p < 0.05. Error bars show mean±SEM.

Fig. 6.

Expression of CCL2 and CCL3 in male and female human dorsal root ganglia. (A-C) RNAscope used to quantify the expression of CCL2 or (D-F) CCL3 in serial sections of human dorsal root ganglia (n=6 donors, three male, three female); Mean±SEM. (A-B, D-E) Representative sections of (A, D) male and (B, E) female human DRG. 60X magnification (scale bar = 25 μm). White arrows indicate examples of positive signal. (C, F) RNAscope signal was quantified using RS-FISH in 5-6 60X images and averaged per donor. Significant if, p < 0.05, by unpaired two-tailed t-test.