Use of the senolytics dasatinib and quercetin for prevention of pelvic organ prolapse in a mouse animal model

Abstract

Objective: Senolytic agents have the potential to target age-related pathology associated with cellular senescence and reduce senescent cell activity in several disease processes. We utilized a mouse model of pelvic organ prolapse, Fibulin-5 knockout (Fbln-5^-/-) mice, to assess the ability of dasatinib and quercetin (D+Q) to prevent development of prolapse.

Methods: Four-week-old female Fbln-5^-/- (n=63) and wild-type (WT) mice (n=54) were assigned to control (vehicle injection) or treatment (D = 5 mg/kg, Q = 50 mg/kg) groups. Weekly oral gavage injections were administered from weeks 4-8 of life. Pelvic organ prolapse quantification system measurements were obtained weekly. Vaginal tissue was harvested at 10, 12 and 20 weeks. Tissue analysis included immunostaining for cell cycle inhibitors, multiplex cytokine analysis, senescence-associated-β-galactosidase (SA-β-Gal) and histologic analysis of extracellular matrix proteins.

Results: Perineal body length was significantly longer in Fbln-5^-/- treatment mice at 20 weeks. Expression of p16 and p53 was decreased in Fbln-5^-/- treatment mice compared to controls (4.0% vs. 26.7%, p=0.0124 and 2.9% vs. 16.8%, p=0.272) at 20 weeks. Expression of SA-β-Gal and senescence-associated cytokines did not vary significantly between groups. At 20 weeks, vaginal tissue elastin content in Fbln-5^-/- treatment mice increased compared to controls (1.04% vs. 0.84%, p=0.999).

Conclusions: D+Q injections did not result in clinically significant differences in prolapse development but did demonstrate decreased expression of cellular senescence markers in Fbln-5^-/- mice. This suggests senolytic agents may mitigate contributions of cellular senescence to tissue dysfunction associated with prolapse. Further studies are needed to confirm ideal timing, dosage, and route of senolytics in prevention of prolapse.

Keywords: animal model; cellular senescence; pelvic organ prolapse; senolytic agents.

Figure 1

Impact of serial dasatinib and quercetin (D+Q) injections on development of pelvic organ prolapse in wild type (WT) and Fibulin-5 knockout (Fbln-5^-/-) mice. D+Q or placebo injections were administered at weeks 4, 5, 6, 7 and 8 of life to treatment and control groups, respectively. Mouse pelvic organ prolapse quantification measurements were used to obtain weekly perineal body length (A) and vaginal bulge height (B) values. WT control (n=27), WT treatment (n=27), Fbln-5^-/- control (n=33), Fbln-5^-/- treatment (n=30). p <0.05 between Fbln-5^-/- treatment mice and Fbln-5^-/- control mice, and between Fbln-5^-/- treatment mice and WT treatment mice (A).

Figure 2

Impact of serial dasatinib and quercetin (D+Q) injections on p16 expression in the vaginal tissue of wild type (WT) and Fibulin-5 knockout (Fbln-5^-/-) mice at 20 weeks of life. Representative tissue sections from WT control, WT treatment, Fbln-5^-/- control and Fbln-5^-/- treatment mice. Top panels demonstrate differences in p16 expression between groups. Bottom panels display corresponding DAPI staining to label cellular nuclei for anatomical reference. Epi=epithelium, Musc=muscularis. 20x magnification.

Figure 3

Impact of serial dasatinib and quercetin (D+Q) injections on p53 expression in the vaginal tissue of wild type (WT) and Fibulin-5 knockout (Fbln-5^-/-) mice at 20 weeks of life. Representative tissue sections from WT control, WT treatment, Fbln-5^-/- control and Fbln-5^-/- treatment mice. Top panels demonstrate differences in p53 expression between groups. Bottom panels display corresponding DAPI staining to label cellular nuclei for anatomical reference. Epi=epithelium, Musc=muscularis. 20x magnification.

Figure 4

Impact of serial dasatinib and quercetin (D+Q) injections on elastin content in the vaginal tissue of wild type (WT) and Fibulin-5 knockout (Fbln-5^-/-) mice at 20 weeks of life. Representative tissue sections from 20-week-old WT and Fbln-5^-/- mice (A). Top panels correspond to WT and Fbln-5^-/- mice in the control group and the bottom panels correspond to WT and Fbln-5^-/- mice in the D+Q group. Quantification of vaginal tissue elastin content at 10, 12 and 20 weeks of life in WT control, WT treatment, Fbln-5^-/- control and Fbln-5^-/- treatment groups (B). Results are expressed as raw mean percent of total tissue ± SEM. 20x magnification.

Figure 5

Impact of serial dasatinib and quercetin (D+Q) injections on collagen content in the vaginal tissue of wild type (WT) and Fibulin-5 knockout (Fbln-5^-/-) mice at 20 weeks of life. Representative tissue sections from 20-week-old WT and Fbln-5^-/- mice (A). Top panels correspond to WT and Fbln-5^-/- mice in the control group and the bottom panels correspond to WT and Fbln-5^-/- mice in the D+Q group. Quantification of vaginal tissue collagen content at 10, 12 and 20 weeks of life in WT control, WT treatment, Fbln-5^-/- control and Fbln-5^-/- treatment groups (B). Results are expressed as raw mean percent of total tissue ± SEM. 20x magnification.