Genetic variation influences the skeletal response to hindlimb unloading in the eight founder strains of the diversity outbred mouse population.

During disuse, mechanical unloading causes extensive bone loss, decreasing bone volume and strength. Variations in bone mass and risk of osteoporosis are influenced by genetics; however, it remains unclear how genetic variation affects the skeletal response to unloading. We previously found that genetic variation affects the musculoskeletal response to 3 weeks of immobilization in the 8 Jackson Laboratory J:DO founder strains: C57Bl/6J, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Hindlimb unloading (HLU) is the best model for simulating local and systemic contributors of disuse and therefore may have a greater impact on bones than immobilization. We hypothesized that genetic variation would affect the response to HLU across the eight founder strains. Mice of each founder strain were placed in HLU for 3 weeks, and the femurs and tibias were analyzed. There were significant HLU and mouse strain interactions on body weight, femur trabecular BV/TV, and femur ultimate force. This indicates that unloading only caused significant catabolic effects in some mouse strains. C57BL/6 J mice were most affected by unloading while other strains were more protected. There were significant HLU and mouse strain interactions on gene expression of genes encoding bone metabolism genes in the tibia. This indicates that unloading only caused significant effects on bone metabolism genes in some mouse strains. Different mouse strains respond to HLU differently, and this can be explained by genetic differences. These results suggest the outbred J:DO mice will be a powerful model for examining the effects of genetics on the skeletal response to HLU.

A comparison of bone microarchitectural and transcriptomic changes in murine long bones in response to hindlimb unloading and aging.

Age- and disuse-related bone loss both result in decreases in bone mineral density, cortical thickness, and trabecular thickness and connectivity. Disuse induces changes in the balance of bone formation and bone resorption like those seen with aging. There is a need to experimentally compare these two mechanisms at a structural and transcriptomic level to better understand how they may be similar or different. Bone microarchitecture and biomechanical properties were compared between 6- and 22-month-old C57BL/6 J 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 in young adult mice. 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/6 N mice unloaded for 7 days was included in ingenuity pathway analysis (IPA) 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.

Genetic diversity modulates the physical and transcriptomic response of skeletal muscle to simulated microgravity in male mice.

Developments in long-term space exploration necessitate advancements in countermeasures against microgravity-induced skeletal muscle loss. Astronaut data shows considerable variation in muscle loss in response to microgravity. Previous experiments suggest that genetic background influences the skeletal muscle response to unloading, but no in-depth analysis of genetic expression has been performed. Here, we placed eight, male, inbred founder strains of the diversity outbred mice (129S1/SvImJ, A/J, C57BL/6J, CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, PWK/PhJ, and WSB/EiJ) in simulated microgravity (SM) via hindlimb unloading for three weeks. Body weight, muscle morphology, muscle strength, protein synthesis marker expression, and RNA expression were collected. A/J and CAST/EiJ mice were most susceptible to SM-induced muscle loss, whereas NOD/ShiLtJ mice were the most protected. In response to SM, A/J and CAST/EiJ mice experienced reductions in body weight, muscle mass, muscle volume, and muscle cross-sectional area. A/J mice had the highest number of differentially expressed genes (68) and associated gene ontologies (328). Downregulation of immunological gene ontologies and genes encoding anabolic immune factors suggest that immune dysregulation contributes to the response of A/J mice to SM. Several muscle properties showed significant interactions between SM and mouse strain and a high degree of heritability. These data imply that genetic background plays a role in the degree of muscle loss in SM and that more individualized programs should be developed for astronauts to protect their skeletal muscles against microgravity on long-term missions.

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. Highlights: Epiphyseal trabecular bone is most susceptible to hindlimb unloading.Hindlimb unloaded limbs resemble an intermediate phenotype between age-matched and aged controls.Hindlimb unloading induces gene expression changes that are age dependent and may lead to inflammation and/or mitochondrial dysfunction depending on context.Younger mice (3-4 months) activate the senescence pathway upon hindlimb unloading, whereas skeletally mature (6 months) mice inhibit it.

Connexin 43 and cell culture substrate differentially regulate OCY454 osteocytic differentiation and signaling to primary bone cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests that osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption; however, the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using three-dimensional (3D) culture substrates in OCY454 cells suggest that 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and receptor activator of nuclear factor-κB ligand (RANKL). In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds with traditional 2D tissue culture, both with [wild-type (WT)] and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures were used to determine soluble signaling to differentiate primary bone marrow cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared with that of Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion.NEW & NOTEWORTHY 3D cell culture of OCY454 cells promoted increased differentiation compared with traditional 2D culture. Although Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest that Cx43 deficiency promotes increased bone remodeling in a cell-autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Connexin 43 and Cell Culture Substrate Differentially Regulate OCY454 Osteocytic Differentiation and Signaling to Primary Bone Cells.

Connexin 43 (Cx43), the predominate gap junction protein in bone, is essential for intercellular communication and skeletal homeostasis. Previous work suggests osteocyte-specific deletion of Cx43 leads to increased bone formation and resorption, however the cell-autonomous role of osteocytic Cx43 in promoting increased bone remodeling is unknown. Recent studies using 3D culture substrates in OCY454 cells suggest 3D cultures may offer increased bone remodeling factor expression and secretion, such as sclerostin and RANKL. In this study, we compared culturing OCY454 osteocytes on 3D Alvetex scaffolds to traditional 2D tissue culture, both with (WT) and without Cx43 (Cx43 KO). Conditioned media from OCY454 cell cultures was used to determine soluble signaling to differentiate primary bone marrow stromal cells into osteoblasts and osteoclasts. OCY454 cells cultured on 3D portrayed a mature osteocytic phenotype, relative to cells on 2D, shown by increased osteocytic gene expression and reduced cell proliferation. In contrast, OCY454 differentiation based on these same markers was not affected by Cx43 deficiency in 3D. Interestingly, increased sclerostin secretion was found in 3D cultured WT cells compared to Cx43 KO cells. Conditioned media from Cx43 KO cells promoted increased osteoblastogenesis and increased osteoclastogenesis, with maximal effects from 3D cultured Cx43 KO cells. These results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Finally, 3D cultures appear better suited to study mechanisms from Cx43-deficient OCY454 osteocytes in vitro due to their ability to promote osteocyte differentiation, limit proliferation, and increase bone remodeling factor secretion. New and Noteworthy: 3D cell culture of OCY454 cells promoted increased differentiation compared to traditional 2D culture. While Cx43 deficiency did not affect OCY454 differentiation, it resulted in increased signaling, promoting osteoblastogenesis and osteoclastogenesis. Our results suggest Cx43 deficiency promotes increased bone remodeling in a cell autonomous manner with minimal changes in osteocyte differentiation. Also, 3D cultures appear better suited to study mechanisms in Cx43-deficient OCY454 osteocytes.

Reambulation following hindlimb unloading attenuates disuse-induced changes in murine fracture healing.

Patients with bone and muscle loss from prolonged disuse have higher risk of falls and subsequent fragility fractures. In addition, fracture patients with continued disuse and/or delayed physical rehabilitation have worse clinical outcomes compared to individuals with immediate weight-bearing activity following diaphyseal fracture. However, the effects of prior disuse followed by physical reambulation on fracture healing cellular processes and adjacent bone and skeletal muscle recovery post-injury remains poorly defined. To bridge this knowledge gap and inform future treatment and rehabilitation strategies for fractures, a preclinical model of fracture healing with a history of prior unloading with and without reambulation was employed. First, skeletally mature male and female C57BL/6J mice (18 weeks) underwent hindlimb unloading by tail suspension (HLU) for 3 weeks to induce significant bone and muscle loss modeling enhanced bone fragility. Next, mice had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Then, mice were randomly assigned to continued HLU or allowed normal weight-bearing reambulation (HLU + R). Mice given normal cage activity throughout the experiment served as healthy age-matched controls. All mice were sacrificed 4-days (DPF4) or 14-days (DPF14) following fracture to assess healing and uninjured hindlimb musculoskeletal properties (6-10 mice per treatment group/biological sex/timepoint). We found that continued disuse following fracture led to severely diminished uninjured hindlimb skeletal muscle mass (gastrocnemius and soleus) and femoral bone volume adjacent to the fracture site compared to healthy age-matched controls across mouse sexes. Furthermore, HLU led to significantly decreased periosteal expansion (DPF4) and osteochondral tissue formation by DPF14, and trends in increased osteoclastogenesis (DPF14) and decreased woven bone vascular area (DPF14). In contrast, immediate reambulation for 2 weeks after fracture, even following a period of prolonged disuse, was able to increase hindlimb skeletal tissue mass and increase osteochondral tissue formation, albeit not to healthy control levels, in both mouse sexes. Furthermore, reambulation attenuated osteoclast formation seen in woven bone tissue undergoing disuse. Our results suggest that weight-bearing skeletal loading in both sexes immediately following fracture may improve callus healing and prevent further fall risk by stimulating skeletal muscle anabolism and decreasing callus resorption compared to minimal or delayed rehabilitation regimens.

Global cannabinoid receptor 1 deficiency affects disuse-induced bone loss in a site-specific and sex-dependent manner.

Bone loss during mechanical unloading increases fracture risk and is a major concern for the general population and astronauts during spaceflight. The endocannabinoid system (ECS) plays an important role in bone metabolism. One of the main ECS receptors, cannabinoid receptor 1 (CB1), has been studied in regards to basic bone metabolism; however, little is known as to how CB1 and the ECS affect bone in different mechanical environments. In this study, we analyzed the influence of global CB1 deficiency and sex on mice during disuse caused by single limb immobilization. Female mice were more sensitive to disuse-induced BV/TV loss than males in both the femoral metaphysis and tibial epiphysis. Genotype also affected bone loss in a sex-dependent manner, with male mice deficient in CB1 receptors (CB1KO) and female wildtype (WT) mice experiencing increased bone loss in both the tibial metaphysis and femoral epiphysis. Genotype affected the response to disuse as CB1KO mice displayed greater changes in femoral ultimate force, along with lower tibial ultimate stress, compared to WT mice. Female mice had a significantly higher femoral, and lower tibial ultimate force compared to male mice. These results reveal that disuse-induced bone loss due to CB1 deficiency is sex-dependent. CB1 deficiency in male mice exacerbated bone loss, while in females CB1 deficiency appeared to protect against disuse-induced bone loss. Regardless of genotype, female mice were more sensitive than males to disuse. These results suggest that CB1 receptors may represent a potential therapeutic target for mitigation of disuse-induced bone loss.

Calcium and phosphorus supplemented diet increases bone volume after thirty days of high speed treadmill exercise in adult mice.

Weight-bearing exercise increases bone mass and strength. Increasing bone loading frequency during exercise can strengthen bone. Combining exercise with a calcium- and phosphorus-supplemented diet increases cortical area more than exercise alone in mice. Thus, we hypothesized that combining high-speed treadmill exercise while feeding mice a mineral-supplemented diet would lead to greater cortical area than high-speed exercise on a standard diet and low-speed exercise on a supplemented diet. Fifteen-week old male C57BL/6 mice were assigned to seven groups-(1) baseline, (2) non-exercise fed a control diet, (3) non-exercise fed a supplemented diet, (4) low-speed exercise fed a control diet, (5) low-speed exercise fed a supplemented diet, (6) high-speed exercise fed a control diet, and (7) high-speed exercise fed a supplemented diet. Mice exercised thirty days for 20 min/day at 12 m/min or 20 m/min. Tibiae were assessed by micro-CT and 4-point bending. Cortical area fraction and trabecular bone volume fraction (BV/TV) were significantly increased by the supplemented diet. High-speed exercised mice had significantly lower body weight, with no detrimental effects to bone health. Increasing running speed can decrease body weight while maintaining the benefits of exercise and nutrition on bone health. Running can lower body weight without harming bone health.

Similarities Between Disuse and Age-Induced Bone Loss.

Disuse and aging are known risk factors associated with low bone mass and quality deterioration, resulting in increased fracture risk. Indeed, current and emerging evidence implicate a large number of shared skeletal manifestations between disuse and aging scenarios. This review provides a detailed overview of current preclinical models of musculoskeletal disuse and the clinical scenarios they seek to recapitulate. We also explore and summarize the major similarities between bone loss after extreme disuse and advanced aging at multiple length scales, including at the organ/tissue, cellular, and molecular level. Specifically, shared structural and material alterations of bone loss are presented between disuse and aging, including preferential loss of bone at cancellous sites, cortical thinning, and loss of bone strength due to enhanced fragility. At the cellular level bone loss is accompanied, during disuse and aging, by increased bone resorption, decreased formation, and enhanced adipogenesis due to altered gap junction intercellular communication, WNT/ß-catenin and RANKL/OPG signaling. Major differences between extreme short-term disuse and aging are discussed, including anatomical specificity, differences in bone turnover rates, periosteal modeling, and the influence of subject sex and genetic variability. The examination also identifies potential shared mechanisms underlying bone loss in aging and disuse that warrant further study such as collagen cross-linking, advanced glycation end products/receptor for advanced glycation end products (AGE-RAGE) signaling, reactive oxygen species (ROS) and nuclear factor κB (NF-κB) signaling, cellular senescence, and altered lacunar-canalicular connectivity (mechanosensation). Understanding the shared structural alterations, changes in bone cell function, and molecular mechanisms common to both extreme disuse and aging are paramount to discovering therapies to combat both age-related and disuse-induced osteoporosis. © 2022 American Society for Bone and Mineral Research (ASBMR).

Structural and functional analysis of the human cone-rod homeobox transcription factor.

The cone-rod homeobox (CRX) protein is a critical K50 homeodomain transcription factor responsible for the differentiation and maintenance of photoreceptor neurons in the vertebrate retina. Mutant alleles in the human gene encoding CRX result in a variety of distinct blinding retinopathies, including retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis. Despite the success of using in vitro biochemistry, animal models, and genomics approaches to study this clinically relevant transcription factor over the past 25 years since its initial characterization, there are no high-resolution structures in the published literature for the CRX protein. In this study, we use bioinformatic approaches and small-angle X-ray scattering (SAXS) structural analysis to further understand the biochemical complexity of the human CRX homeodomain (CRX-HD). We find that the CRX-HD is a compact, globular monomer in solution that can specifically bind functional cis-regulatory elements encoded upstream of retina-specific genes. This study presents the first structural analysis of CRX, paving the way for a new approach to studying the biochemistry of this protein and its disease-causing mutant protein variants.

Genetic variability affects the skeletal response to immobilization in founder strains of the diversity outbred mouse population.

Mechanical unloading decreases bone volume and strength. In humans and mice, bone mineral density is highly heritable, and in mice the response to changes in loading varies with genetic background. Thus, genetic variability may affect the response of bone to unloading. As a first step to identify genes involved in bones' response to unloading, we evaluated the effects of unloading in eight inbred mouse strains: C57BL/6J, PWK/PhJ, WSB/EiJ, A/J, 129S1/SvImJ, NOD/ShiLtJ, NZO/HlLtJ, and CAST/EiJ. C57BL/6J and NOD/ShiLtJ mice had the greatest unloading-induced loss of diaphyseal cortical bone volume and strength. NZO/HlLtJ mice had the greatest metaphyseal trabecular bone loss, and C57BL/6J, WSB/EiJ, NOD/ShiLtJ, and CAST/EiJ mice had the greatest epiphyseal trabecular bone loss. Bone loss in the epiphyses displayed the highest heritability. With immobilization, mineral:matrix was reduced, and carbonate:phosphate and crystallinity were increased. A/J mice displayed the greatest unloading-induced loss of mineral:matrix. Changes in gene expression in response to unloading were greatest in NOD/ShiLtJ and CAST/EiJ mice. The most upregulated genes in response to unloading were associated with increased collagen synthesis and extracellular matrix formation. Our results demonstrate a strong differential response to unloading as a function of strain. Diversity outbred (DO) mice are a high-resolution mapping population derived from these eight inbred founder strains. These results suggest DO mice will be highly suited for examining the genetic basis of the skeletal response to unloading.

Genetic variability affects the response of skeletal muscle to disuse.

OBJECTIVE: To examine whether genetic variability plays a role in skeletal muscle response to disuse. METHODS: We examined skeletal muscle response to disuse in five different strains of mice: CAST/EiJ, NOD/ShiLtJ, NZO/HILtJ, 129S1/SvImJ and A/J. Mice had one limb immobilized by a cast for three weeks. RESULTS: Response to immobilization was dependent on the strain of mice. Skeletal muscle mass/body weight was decreased by immobilization in all strains except 1291/SvImJ. Immobilization decreased absolute skeletal muscle mass in quadriceps and gastrocnemius in NOD/ShiltJ and NZO/HILtJ mice. Three weeks of immobilization resulted in an increase in quadriceps levels of atrogenes in CAST/EiJ. Immobilization resulted in an increase in quadriceps and gastrocnemius levels of Myh4 in CAST/EiJ. A similar trend was observed for Myh7 in gastrocnemius muscle. Immobilization resulted in a decrease of the p-p70S6K1/total p706SK1 ratio in quadriceps of NOD/ShiLtJ mice and the gastrocnemius of A/J mice. Immobilization did not affect the p-4EBP1/total 4EBP1 ratio in quadriceps of any of the strains examined. However, the p-4EBP1/total 4EBP1 ratio in gastrocnemius was greater in immobilized, relative to control, limbs in CAST/EiJ mice. CONCLUSION: Genetic variability affects the response of skeletal muscle to disuse.

Update on the effects of microgravity on the musculoskeletal system.

With the reignited push for manned spaceflight and the development of companies focused on commercializing spaceflight, increased human ventures into space are inevitable. However, this venture would not be without risk. The lower gravitational force, known as microgravity, that would be experienced during spaceflight significantly disrupts many physiological systems. One of the most notably affected systems is the musculoskeletal system, where exposure to microgravity causes both bone and skeletal muscle loss, both of which have significant clinical implications. In this review, we focus on recent advancements in our understanding of how exposure to microgravity affects the musculoskeletal system. We will focus on the catabolic effects microgravity exposure has on both bone and skeletal muscle cells, as well as their respective progenitor stem cells. Additionally, we report on the mechanisms that underlie bone and muscle tissue loss resulting from exposure to microgravity and then discuss current countermeasures being evaluated. We reveal the gaps in the current knowledge and expound upon how current research is filling these gaps while also identifying new avenues of study as we continue to pursue manned spaceflight.

Protective Effects of Controlled Mechanical Loading of Bone in C57BL6/J Mice Subject to Disuse.

Prolonged reduction in weightbearing causes bone loss. Disuse of bone is associated with recovery from common musculoskeletal injury and trauma, bed rest resulting from various medical conditions, and spaceflight. The hindlimb-suspension rodent model is popular for simulating unloading and disuse. We hypothesized that controlled mechanical loading of the tibia would protect against bone loss occurring from concurrent disuse. Additionally, we hypothesized that areas of high mechanical peak strains (midshaft) would provide more protection than areas of lower strain (distal shaft). Adult C57BL6/J mice were suspended for 3 weeks, with one limb subjected to tibial compression four times per week. µCT imaging was completed at days 0, 11, and 21, in addition to serum analysis. Significant bone loss caused by hindlimb suspension was detected in trabecular bone by day 11 and worsened by day 21 (p < 0.05). Bone loss was also detected in cortical thickness and area fraction by day 21. However, four short bouts per week of compressive loading protected the loaded limb from much of this bone loss. At day 21, we observed a 50% loss in trabecular bone volume/total volume and a 6% loss in midshaft cortical thickness in unloaded limbs, but only 15% and 2% corresponding losses in contralateral loaded limbs (p = 0.001 and p = 0.02). Many bone geometry parameters of the loaded limbs of suspended animals did not significantly differ from non-suspended control limbs. Conversely, this protective effect of loading was not detected in cortical bone at the lower-strained distal shaft. Analysis of bone metabolism markers suggested that the benefits of loading occurred through increased formation instead of decreased resorption. This study uniquely isolates the role of externally applied mechanical loading of the mouse tibia, in the absence of muscle stimulation, in protecting bone from concurrent disuse-related loss, and demonstrates that limited bouts of loading may be highly effective during prolonged disuse. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Single limb immobilization model for bone loss from unloading.

Hindlimb suspension is the most used model for inducing bone loss from unloading but requires a separate ground control group. This control group cannot be used for genetic studies involving outbred mice. In this study, we evaluated a single limb immobilization (SLI) model for inducing bone loss from unloading, with the contralateral limb from the same animal used as a control. Male 10-week old C57Bl/6J mice had one limb immobilized for one, two, or three weeks. Subsequently, an additional group of male 16-week old C57Bl/6J mice had one limb immobilized for three weeks. SLI resulted in decreased tibial trabecular BV/TV, Tb Th, and Tb N compared to contralateral limbs in young mice. Femoral trabecular BV/TV, Tb Th, Tb N, and femoral cortical area fraction were also decreased. Mechanical properties were not affected after three weeks. In adult mice, femoral trabecular BV/TV, Tb Th, and Tb N were decreased. Femoral stiffness, ultimate stress, and Young's modulus were decreased. Bone properties decreased by SLI were also decreased by hindlimb suspension previously. The results suggest SLI can be an effective model for inducing bone loss in growing and adult mice after three weeks of immobilization.

Combined mineral-supplemented diet and exercise increases bone mass and strength after eight weeks and maintains increases after eight weeks detraining in adult mice.

Exercise has long-lasting benefits to bone mass and structural strength even after cessation. Combining exercise with a calcium- and phosphorus-supplemented diet increases cortical bone mineral content (BMC), area, and yield force more than exercise alone in adult mice. These increases could also be maintained after stopping exercise if the modified diet is maintained. It was hypothesized that combining exercise with a mineral-supplemented diet would lead to greater cortical BMC, area, and yield force immediately after a lengthy exercise program and after an equally long period of non-exercise (detraining) in adult mice. Male, 16-week old C57Bl/6 mice were assigned to 9 weight-matched groups-a baseline group, exercise and non-exercise groups fed a control or mineral-supplemented diet for 8 weeks, exercise + detraining and non-exercise groups fed a control or mineral-supplemented diet for 16 weeks. Exercise + detraining consisted of 8 weeks of exercise followed by 8 weeks without exercise. The daily exercise program consisted of running on a treadmill at 12 m/min, 30 min/day. After 8 weeks, mice fed the supplemented diet had greater tibial cortical BMC and area, trabecular bone volume/tissue volume (BV/TV), bone mineral density (vBMD), yield force, and ultimate force than mice fed the control diet. Exercise increased cortical BMC and area only when coupled with the supplemented diet. After 16 weeks, both exercised and non-exercised mice fed the supplemented diet maintained greater tibial cortical BMC and area, trabecular BV/TV, vBMD, yield force, and ultimate force than mice fed the control diet. Combining exercise with a mineral-supplemented diet leads to greater bone mass and structural strength than exercise alone. These benefits remain after an equally long period of detraining. Long-term use of dietary mineral supplements may help increase and maintain bone mass with aging in adult mice.

A clinically useful self-report measure of psychiatric patients' satisfaction with the initial evaluation.

Patient satisfaction is one component of the quality of care. Studies of satisfaction in samples of established patients are biased because dissatisfied patients are more likely to have dropped out of treatment. We, therefore, sought to develop a new instrument assessing patients' satisfaction with the initial psychiatric evaluation. In the present report from the Rhode Island Methods to Improve Diagnostic Assessment and Services (MIDAS) project we describe the development, reliability, and validity of the Clinically Useful Patient Satisfaction Scale (CUPSS). The CUPSS is a brief, self-administered questionnaire covering 3 areas: clinician's attitude and behavior, office environment and staff, and overall satisfaction. A sample of psychiatric outpatients (n=412) and partial hospital patients (n=500) completed the measure immediately after their initial meeting with the psychiatrist. The scale had high internal consistency, and all item-scale correlations were significant. All items were significantly correlated with each of the indicators of global satisfaction. There was sufficient variability in satisfaction ratings to detect differences amongst clinicians. The results of the present study of psychiatric outpatients and partial hospital patients indicate that the CUPSS was minimally to not at all burdensome to complete, it had good psychometric properties, and it can discriminate amongst clinicians.

Calcium- and Phosphorus-Supplemented Diet Increases Bone Mass after Short-Term Exercise and Increases Bone Mass and Structural Strength after Long-Term Exercise in Adult Mice.

Exercise has long-lasting benefits to bone health that may help prevent fractures by increasing bone mass, bone strength, and tissue quality. Long-term exercise of 6-12 weeks in rodents increases bone mass and bone strength. However, in growing mice, a short-term exercise program of 3 weeks can limit increases in bone mass and structural strength, compared to non-exercised controls. Short-term exercise can, however, increase tissue strength, suggesting that exercise may create competition for minerals that favors initially improving tissue-level properties over structural-level properties. It was therefore hypothesized that adding calcium and phosphorus supplements to the diet may prevent decreases in bone mass and structural strength during a short-term exercise program, while leading to greater bone mass and structural strength than exercise alone after a long-term exercise program. A short-term exercise experiment was done for 3 weeks, and a long-term exercise experiment was done for 8 weeks. For each experiment, male 16-week old C57BL/6 mice were assigned to 4 weight-matched groups-exercise and non-exercise groups fed a control or mineral-supplemented diet. Exercise consisted of treadmill running at 12 m/min, 30 min/day for 7 days/week. After 3 weeks, exercised mice fed the supplemented diet had significantly increased tibial tissue mineral content (TMC) and cross-sectional area over exercised mice fed the control diet. After 8 weeks, tibial TMC, cross-sectional area, yield force, and ultimate force were greater from the combined treatments than from either exercise or supplemented diet alone. Serum markers of bone formation (PINP) and resorption (CTX) were both decreased by exercise on day 2. In exercised mice, day 2 PINP was significantly positively correlated with day 2 serum Ca, a correlation that was weaker and negative in non-exercised mice. Increasing dietary mineral consumption during an exercise program increases bone mass after 3 weeks and increases structural strength after 8 weeks, making bones best able to resist fracture.