Addressing Challenges of Opportunistic Computed Tomography Bone Mineral Density Analysis.

Computed tomography (CT) offers advanced biomedical imaging of the body and is broadly utilized for clinical diagnosis. Traditionally, clinical CT scans have not been used for volumetric bone mineral density (vBMD) assessment; however, computational advances can now leverage clinically obtained CT data for the secondary analysis of bone, known as opportunistic CT analysis. Initial applications focused on using clinically acquired CT scans for secondary osteoporosis screening, but opportunistic CT analysis can also be applied to answer research questions related to vBMD changes in response to various disease states. There are several considerations for opportunistic CT analysis, including scan acquisition, contrast enhancement, the internal calibration technique, and bone segmentation, but there remains no consensus on applying these methods. These factors may influence vBMD measures and therefore the robustness of the opportunistic CT analysis. Further research and standardization efforts are needed to establish a consensus and optimize the application of opportunistic CT analysis for accurate and reliable assessment of vBMD in clinical and research settings. This review summarizes the current state of opportunistic CT analysis, highlighting its potential and addressing the associated challenges.

Lipopolysaccharide-Induced Bone Loss in Rodent Models: A Systematic Review and Meta-Analysis.

Osteoporosis has traditionally been characterized by underlying endocrine mechanisms, though evidence indicates a role of inflammation in its pathophysiology. Lipopolysaccharide (LPS), a component of gram-negative bacteria that reside in the intestines, can be released into circulation and stimulate the immune system, upregulating bone resorption. Exogenous LPS is used in rodent models to study the effect of systemic inflammation on bone, and to date a variety of different doses, routes, and durations of LPS administration have been used. The study objective was to determine whether systemic administration of LPS induced inflammatory bone loss in rodent models. A systematic search of Medline and four other databases resulted in a total of 110 studies that met the inclusion criteria. Pooled standardized mean differences (SMDs) and corresponding 95% confidence intervals (CI) with a random-effects meta-analyses were used for bone volume fraction (BV/TV) and volumetric bone mineral density (vBMD). Heterogeneity was quantified using the I2 statistic. Shorter-term (<2 weeks) and longer-term (>2 weeks) LPS interventions were analyzed separately because of intractable study design differences. BV/TV was significantly reduced in both shorter-term (SMD = -3.79%, 95% CI [-4.20, -3.38], I2 62%; p < 0.01) and longer-term (SMD = -1.50%, 95% CI [-2.00, -1.00], I2 78%; p < 0.01) studies. vBMD was also reduced in both shorter-term (SMD = -3.11%, 95% CI [-3.78, -2.44]; I2 72%; p < 0.01) and longer-term (SMD = -3.49%, 95% CI [-4.94, -2.04], I2 82%; p < 0.01) studies. In both groups, regardless of duration, LPS negatively impacted trabecular bone structure but not cortical bone structure, and an upregulation in bone resorption demonstrated by bone cell staining and serum biomarkers was reported. This suggests systemically delivered exogenous LPS in rodents is a viable model for studying inflammatory bone loss, particularly in trabecular bone. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Internal calibration for opportunistic computed tomography muscle density analysis.

INTRODUCTION: Muscle weakness can lead to reduced physical function and quality of life. Computed tomography (CT) can be used to assess muscle health through measures of muscle cross-sectional area and density loss associated with fat infiltration. However, there are limited opportunities to measure muscle density in clinically acquired CT scans because a density calibration phantom, allowing for the conversion of CT Hounsfield units into density, is typically not included within the field-of-view. For bone density analysis, internal density calibration methods use regions of interest within the scan field-of-view to derive the relationship between Hounsfield units and bone density, but these methods have yet to be adapted for muscle density analysis. The objective of this study was to design and validate a CT internal calibration method for muscle density analysis. METHODOLOGY: We CT scanned 10 bovine muscle samples using two scan protocols and five scan positions within the scanner bore. The scans were calibrated using internal calibration and a reference phantom. We tested combinations of internal calibration regions of interest (e.g., air, blood, bone, muscle, adipose). RESULTS: We found that the internal calibration method using two regions of interest, air and adipose or blood, yielded accurate muscle density values (< 1% error) when compared with the reference phantom. The muscle density values derived from the internal and reference phantom calibration methods were highly correlated (R2 > 0.99). The coefficient of variation for muscle density across two scan protocols and five scan positions was significantly lower for internal calibration (mean = 0.33%) than for Hounsfield units (mean = 6.52%). There was no difference between coefficient of variation for the internal calibration and reference phantom methods. CONCLUSIONS: We have developed an internal calibration method to produce accurate and reliable muscle density measures from opportunistic computed tomography images without the need for calibration phantoms.

Trabecular and cortical bone are unaltered in response to chronic lipopolysaccharide exposure via osmotic pumps in male and female CD-1 mice.

Chronic low-grade inflammation has been identified as an underlying cause of many diseases including osteoporosis. Lipopolysaccharide (LPS) is a potent inducer of the inflammatory response that can negatively affect bone outcomes by upregulating bone resorption and inhibiting bone formation. The objective of this study was to assess the longitudinal response of trabecular and cortical bone structure and bone mineral density to LPS continuously administered for 12 weeks in male and female CD-1 mice. Mice were assigned to one of four LPS groups at 8-weeks of age: placebo (0.0 µg/d), low (0.9 µg/d), mid (3.6 µg/d) and high (14.4 µg/d) dose. Trabecular and cortical bone outcomes were measured at 8, 12, 16, and 20 weeks of age using in vivo micro-computed tomography. The anticipated serum LPS dose-dependent response was not observed. Therefore, the low, mid, and high LPS groups were combined for analysis. Compared to the placebo group, endpoint serum LPS was elevated in both males (p < 0.05) and females (p < 0.05) when all LPS treatment groups were combined. However, there was no significant change in trabecular or cortical bone outcomes in the combined LPS groups compared to the placebo following the 12-week LPS intervention for either sex. This suggests that although serum LPS was elevated following the 12-week LPS intervention, the dosages administered using the osmotic pumps was not sufficient to negatively impact trabecular or cortical bone outcomes in either male or female CD-1 mice.

Sex-specific responses in trabecular and cortical microstructure of tibia due to repeated irradiation from micro-computed tomography in adult CD-1 mice.

While repeated in vivo micro-computed tomography (µCT) allows for longitudinal measurement of bone outcomes in rodent models, it is important to determine that the resulting irradiation - dependent on the frequency and number of scans - does not exceed the effects of the intervention. The objective of this study was to determine whether repeated irradiation exposure from µCT scans at 1-month intervals for a total of four scans would alter trabecular or cortical bone structure outcomes and/or bone mineral density in tibias from both male and female CD-1 mice. The right tibia of male (n = 12) and female (n = 11) CD-1 mice were scanned using µCT at 2, 3, 4, and 5 months of age, while the contralateral left tibia served as a control and was scanned only at 5 months of age. All scans were performed at a resolution of 9 µm using a radiation dose of 460 mGy per scan. Some outcomes of trabecular bone structure were affected by repeated irradiation in both males and females. The bone volume fraction was lower in the irradiated right tibia compared to the non-irradiated left tibia in both males (p < 0.05) and females (p < 0.01) as a result of decreased trabecular number (males p < 0.05; females p < 0.05) and increased trabecular separation (males p < 0.05; females p < 0.01). Some cortical measures were also affected in females but not in males, including lower cortical bone periosteal perimeter (p < 0.05), lower total area (p < 0.01) and lower marrow area (p < 0.05) with repeated irradiation. Exposure to repeated radiation at intervals of 1 month, for a total of four scans, altered trabecular bone in both male and female CD-1 mice while outcomes of cortical bone structure were altered only in females.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Low dose lithium supplementation activates Wnt/ß-catenin signalling and increases bone OPG/RANKL ratio in mice.

Lithium, a well-known inhibitor of glycogen synthase kinase-3ß (GSK3ß), can improve bone formation by activating the Wnt/ß-catenin signalling pathway. However, most studies have used higher doses of lithium, which potentially have adverse effects. Herein, we report that low dose lithium supplementation (10 mg/kg/d for 6 weeks) in mice results in a serum lithium concentration of 0.02 mM significantly inhibiting GSK3ß while activating Wnt/ß-catenin in bone. In turn, we observed a significant increase in the expression of osteoprotegerin (OPG), with unaltered expression of nuclear-factor kß ligand (RANKL), ultimately leading to a significant increase in the OPG/RANKL ratio. Altogether, our findings provide initial evidence that low dose lithium supplementation can promote the signalling pathways associated with bone formation.

Evaluation of neuropathological effects of a high-fat high-sucrose diet in middle-aged male C57BL6/J mice.

Metabolic dysfunction related to diet-induced obesity has recently been linked to the pathogenesis of sporadic Alzheimer's disease (AD). However, the underlying mechanisms linking obesity and AD remain unclear. The purpose of this study was to examine early alterations in brain insulin signaling, inflammatory/stress markers, and energetic stress in a model of diet-induced obesity during middle age. Male C57BL/6J mice were randomized to either a control diet (AGE n = 12) or high-fat and sucrose diet (AGE-HFS n = 12) for 13-weeks from 20-weeks of age. Prefrontal cortex and hippocampal samples were collected at 20-weeks of age (BSL n = 11) and at 33-weeks of age (AGE and AGE-HFS). The HFS diet resulted in increased body weight (30%; P = 0.0001), increased %fat mass (28%; P = 0.0001), and decreased %lean mass (33%; P = 0.0001) compared to aged controls. In the prefrontal cortex, AGE-HFS resulted in increased 5' adenosine monophosphate - activated protein kinase (AMPK) phosphorylation (P = 0.045). In the hippocampus, AGE-HFS resulted in increased extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) phosphorylation and protein kinase B (Akt) serine473 and glycogen synthase kinase (GSK) phosphorylation (P < 0.05). Results from this study demonstrate that aging combined with a HFS diet results in increased inflammation (pERK and pJNK) and energetic stress (pAMPK) in the hippocampus and prefrontal cortex, respectively. Together these novel results provide important information for future targets in early AD pathogenesis.

Musculoskeletal structure and function in response to the combined effect of an obesogenic diet and age in male C57BL/6J mice.

SCOPE: The effects of a long-term high fat and sucrose diet (HFS) superimposed with aging on bone and muscle structure and/or function. METHODS AND RESULTS: Male C57BL/6J mice (20 weeks of age) were randomized to 1 of 3 groups: baseline (BSL, n = 12), or assigned to a control (AGE, n = 12) or HFS (HFS-AGE, n = 11) diet for 13 weeks. Trabecular bone structure, volumetric bone mineral density (vBMD), and body composition, were measured longitudinally at 20, 24, and 32 weeks of age. In vitro contractile measures were performed on isolated soleus and extensor digitorum longus (EDL) muscles for each group. Both AGE and HFS-AGE had similar declines in trabecular bone structure, while HFS-AGE resulted in increased soleus cross-sectional area (CSA) compared to AGE, but this did not translate to greater twitch or tetanic peak force. The ratio of outcomes of bone to muscle declined in both AGE and HFS-AGE compared to BSL as a result of greater declines in trabecular bone structure than muscle function. CONCLUSION: Consumption of a 13-week HFS diet at 20 weeks of age did not exacerbate age-related declines in bone or muscle, but these tissues do not decline in a coordinate manner with greater declines in bone than muscle.

Skeletal site-specific effects of endurance running on structure and strength of tibia, lumbar vertebrae, and mandible in male Sprague-Dawley rats.

Bone microarchitecture, bone mineral density (BMD), and bone strength are affected positively by impact activities such as running; however, there are discrepancies in the magnitude of these effects. These inconsistencies are mainly a result of varying training protocols, analysis techniques, and whether or not the skeletal sites measured are weight bearing. This study's purpose was to determine the effects of endurance running on sites that experience different weight bearing and load. Eight-week-old male Sprague-Dawley rats (n = 20) were randomly assigned to either a group with a progressive treadmill running protocol (25 m/min for 1 h, incline of 10%) or a nontrained control group for 8 weeks. The trabecular structure of the tibia, lumbar vertebra (L3), and mandible and the cortical structure at the tibia midpoint were measured using microcomputed tomography to quantify bone volume fraction (i.e., bone volume divided by total volume (BV/TV)), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and cortical thickness. BMD at the proximal tibia, lumbar vertebrae (L1-L3), and mandible was measured using dual energy X-ray absorptiometry. The tibia midpoint strength was measured by 3-point bending using a materials testing system. Endurance running resulted in superior bone structure at the proximal tibia (12% greater BV/TV (p = 0.03), 14% greater Tb.N (p = 0.01), and 19% lower Tb.Sp (p = 0.05)) but not at other sites. Contrary to our hypothesis, mandible bone structure was altered after endurance training (8% lower BV/TV (p < 0.01) and 15% lower Tb.Th (p < 0.01)), which may be explained by a lower food intake, resulting in less mechanical loading from chewing. These results highlight the site-specific effects of loading on the skeleton.

Higher PLIN5 but not PLIN3 content in isolated skeletal muscle mitochondria following acute in vivo contraction in rat hindlimb.

Contraction-mediated lipolysis increases the association of lipid droplets and mitochondria, indicating an important role in the passage of fatty acids from lipid droplets to mitochondria in skeletal muscle. PLIN3 and PLIN5 are of particular interest to the lipid droplet-mitochondria interaction because PLIN3 is able to move about within cells and PLIN5 associates with skeletal muscle mitochondria. This study primarily investigated: 1) if PLIN3 is detected in skeletal muscle mitochondrial fraction; and 2) if mitochondrial protein content of PLIN3 and/or PLIN5 changes following stimulated contraction. A secondary aim was to determine if PLIN3 and PLIN5 associate and whether this changes following contraction. Male Long Evans rats (n = 21; age, 52 days; weight = 317 ± 6 g) underwent 30 min of hindlimb stimulation (10 msec impulses, 100 Hz/3 sec at 10-20 V; train duration 100 msec). Contraction induced a ~50% reduction in intramuscular lipid content measured by oil red-O staining of red gastrocnemius muscle. Mitochondria were isolated from red gastrocnemius muscle by differential centrifugation and proteins were detected by western blotting. Mitochondrial PLIN5 content was ~1.6-fold higher following 30 min of contraction and PLIN3 content was detected in the mitochondrial fraction, and unchanged following contraction. An association between PLIN3 and PLIN5 was observed and remained unaltered following contraction. PLIN5 may play a role in mitochondria during lipolysis, which is consistent with a role in facilitating/regulating mitochondrial fatty acid oxidation. PLIN3 and PLIN5 may be working together on the lipid droplet and mitochondria during contraction-induced lipolysis.