Hindlimb Unloading Induces Bone Microarchitectural and Transcriptomic Changes in Murine Long Bones in an Age-Dependent Manner

Age and disuse-related bone loss both result in decreases in bone mineral density, cortical thickness, and trabecular thickness and connectivity. Disuse induces physiological changes in bone like those seen with aging. Bone microarchitecture and biomechanical properties were compared between 6- and 22-month-old C57BL/6J male control mice and 6-month-old mice that were hindlimb unloaded (HLU) for 3 weeks. Epiphyseal trabecular bone was the compartment most affected by HLU and demonstrated an intermediate bone phenotype between age-matched controls and aged controls. RNA extracted from whole-bone marrow-flushed tibiae was sequenced and analyzed. Differential gene expression analysis additionally included 4-month-old male mice unloaded for 3 weeks compared to age-matched controls. Gene ontology analysis demonstrated that there were age-dependent differences in differentially expressed genes. Genes related to downregulation of cellular processes were most affected in 4-month-old mice after disuse whereas those related to mitochondrial function were most affected in 6- month-old mice. Cell-cycle transition was downregulated with aging. A publicly available dataset (GSE169292) from 3-month female C57BL/6N mice unloaded for 7 days was included in ingenuity pathway analysis with the other datasets. IPA was used to identify the leading canonical pathways and upstream regulators in each HLU age group. IPA identified "Senescence Pathway" as the second leading canonical pathway enriched in mice exposed to HLU. HLU induced activation of the senescence pathway in 3- month and 4-month-old mice but inhibited it in 6-month-old mice. In conclusion, we demonstrate that hindlimb unloading and aging initiate similar changes in bone microarchitecture and gene expression. However, aging is responsible for more significant transcriptome and tissue-level changes compared to hindlimb unloading. HighlightsO_LIEpiphyseal trabecular bone is most susceptible to hindlimb unloading. C_LIO_LIHindlimb unloaded limbs resemble an intermediate phenotype between age-matched and aged controls. C_LIO_LIHindlimb unloading induces gene expression changes that are age dependent and may lead to inflammation and/or mitochondrial dysfunction depending on context. C_LIO_LIYounger mice (3-4 months) activate the senescence pathway upon hindlimb unloading, whereas skeletally mature (6 months) mice inhibit it. C_LI