Browsing by Author "Bhadouria, Neharika"

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    Raloxifene Stimulates Estrogen Signaling to Protect Against Age- and Sex-Related Intervertebral Disc Degeneration in Mice
    (Frontiers Media, 2022-08-11) Bhadouria, Neharika; Berman, Alycia G.; Wallace, Joseph M.; Holguin, Nilsson; Mechanical and Energy Engineering, Purdue School of Engineering and Technology
    Estrogen agonist raloxifene is an FDA-approved treatment of osteoporosis in postmenopausal women, which may also be a promising prophylactic for painful intervertebral disc (IVD) degeneration. Here, we hypothesized that 1) aging and biological sex contribute to IVD degeneration by reducing estrogen signaling and that 2) raloxifene stimulates estrogen signaling to protect against age- and sex-related IVD degeneration in mice. 2.5-month-old (male and female) and 22.5-month-old (female) C57Bl/6J mice were subcutaneously injected with raloxifene hydrochloride 5x/week for 6 weeks (n = 7–9/grp). Next, female mice were ovariectomized (OVX) or sham operated at 4 months of age and tissues harvested at 6 months (n = 5–6/grp). Advanced aging and OVX increased IVD degeneration score, weakened IVD strength, reduced estrogen receptor-α (ER-α) protein expression, and increased neurotransmitter substance P (SP) expression. Similar to aging and compared with male IVDs, female IVDs were more degenerated, mechanically less viscoelastic, and expressed less ER-α protein, but unlike the effect induced by aging or OVX, IVD mechanical force was greater in females than in males. Therapeutically, systemic injection of raloxifene promoted ER-α protein to quell these dysregulations by enlarging IVD height, alleviating IVD degeneration score, increasing the strength and viscoelastic properties of the IVD, and reducing IVD cell expression of SP in young-adult and old female mice. Transcriptionally, injection of raloxifene upregulated the gene expression of ER-α and extracellular matrix-related anabolism in young-adult and old IVD. In vertebra, advanced aging and OVX reduced trabecular BV/TV, whereas injection of raloxifene increased trabecular BV/TV in young-adult and old female mice, but not in young-adult male mice. In vertebra, advanced aging, OVX, and biological sex (females > males) increased the number of SP-expressing osteocytes, whereas injection of raloxifene reduced the number of SP-expressing osteocytes in young-adult female and male mice and old female mice. Overall, injection of estrogen agonist raloxifene in mice normalized dysregulation of IVD structure, IVD mechanics, and pain-related SP expression in IVD cells and osteocytes induced by aging and biological sex. These data suggest that, in addition to bone loss, raloxifene may relieve painful IVD degeneration in postmenopausal women induced by advanced age, biological sex, and estrogen depletion.
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    Suppression of sost/sclerostin and dikkopf-1 augment intervertebral disc structure in mice
    (Wiley, 2022) Kroon, Tori; Bhadouria, Neharika; Niziolek, Paul; Edwards, Daniel; Clinkenbeard, Erica L.; Robling, Alexander; Holguin, Nilsson; Biomedical Engineering, School of Engineering and Technology
    Intervertebral disc (IVD) degeneration is a leading cause of low back pain, characterized by accelerated extracellular matrix breakdown and IVD height loss but there is no approved pharmacological therapeutic. Deletion of Wnt ligand competitor Lrp5 induces IVD degeneration, suggesting that Wnt signaling is essential for IVD homeostasis. Therefore, the IVD may respond to neutralization of Wnt ligand competitors sost(gene)/sclerostin(protein) and/or dikkopf-1 (dkk1). Anti-sclerostin antibody (scl-Ab) is an FDA-approved bone therapeutic that activates Wnt signaling. We (1) determined if pharmacological neutralization of sclerostin, dkk1 or their combination would stimulate Wnt signaling and augment IVD structure and (2) determined the prolonged adaptation of the IVD to global, persistent deletion of sost. Nine-week-old C57Bl/6J female mice (n = 6-7/grp) were subcutaneously injected 2x/wk for 5.5 wk with scl-Ab (25 mg/kg), dkk1-Ab (25 mg/kg), 3:1 scl-Ab/dkk1-Ab (18.75:6.25 mg/kg) or vehicle (veh). Separately, IVD of sost KO and wildtype (WT) mice (n = 8/grp) were harvested at 16 weeks of age. First, compared to vehicle, injection of scl-Ab, dkk1-Ab and 3:1 scl-Ab/dkk1-Ab similarly increased lumbar IVD height and β-catenin gene expression. Despite these similarities, only injection of scl-Ab alone strengthened IVD mechanical properties and decreased heat shock protein gene expressions. Genetically and compared to WT, sost KO enlarged IVD height, increased proteoglycan staining and imbibed IVD hydration. Notably, persistent deletion of sost was compensated by upregulation of dkk1, which consequently reduced the cell nuclear fraction for Wnt signaling co-transcription factor β-catenin in the IVD. Lastly, RNA-sequencing pathway analysis confirmed the compensatory suppression of Wnt signaling and revealed a reduction of cellular stress-related pathways. Together, suppression of sost/sclerostin or dkk1 each augmented IVD structure by stimulating Wnt signaling, but scl-Ab outperformed dkk1-Ab in strengthening the IVD. Ultimately, postmenopausal women prescribed scl-Ab injections to prevent vertebral fracture may also benefit from a restoration of IVD height and health.