Exercise training modifies the bone and endocrine response to graded reductions in energy availability in skeletally mature female rodents.

Introduction: Reductions in energy availability leading to weight loss can induce loss of bone and impact important endocrine regulators of bone integrity. We sought to elucidate whether endurance exercise (EX) can mitigate bone loss observed in sedentary (SED) skeletally mature rodents subjected to graded energy deficits. Methods: Female virgin rats (n=84, 5-mo-old; 12/group) were randomized to baseline controls and either sedentary (SED) or exercise (EX) conditions, and within each exercise status to adlib-fed (ADLIB), or moderate (MOD) or severe (SEV) energy restriction diets for 12 weeks. Rats assigned to EX groups performed treadmill running to increase weekly energy expenditure by 10%. MOD-ER-SED, SEV-ER-SED, MOD-ER-EX and SEV-ER-EX were fed modified AIN93M diets with 20%, 40% 10%, and 30% less energy content, respectively, with 100% of all other nutrients provided. Results: Energy availability (EA) was effectively reduced by ~14% and ~30% in the MOD-ER and SEV-ER groups, respectively. MOD-ER for 12 weeks resulted in few negative impacts on bone and, except for serum leptin in MOD-ER-SED rats, no significant changes in endocrine factors. By contrast, SEV-ER in SED rats resulted in significantly lower total body and femoral neck bone mass, and reduced serum estradiol, IGF-1 and leptin. EX rats experiencing the same reduction in energy availability as SEV-ER-SED exhibited higher total body mass, lean mass, total BMC, and higher serum IGF-1 at the end of 12 weeks. Bone mechanical properties at 3 bone sites (mid-femur, distal femur, femoral neck) were minimally impacted by ER but positively affected by EX. Discussion: These findings indicate that combining increased EX energy expenditure with smaller reductions in energy intake to achieve a targeted reduction in EA provides some protection against loss of bone mass and lean mass in skeletally mature female rats, likely due to better preservation of circulating IGF-1, and that bone mechanical integrity is not significantly degraded with either moderate or severe reduced EA.

Rapid Transition from a High-Fat, High-Fructose to a Low-Fat, Low-Fructose Diet Reverses Gains in Bone Mass and Strength.

PURPOSE: Obesity is thought to negatively impact bone quality and strength despite improving bone mineral density. We hypothesized that 1) continuous consumption of a high-fat, high-sugar (HFS) diet would impair bone quality and strength, and 2) a change from an HFS diet to a low-fat, low-sugar (LFS) would reverse HFS-induced impairments to bone quality and strength. METHODS: Six-week-old male C57Bl/6 mice ( n = 10/group) with access to a running wheel were randomized to an LFS diet or an HFS diet with simulated sugar-sweetened beverages (20% fructose in place of regular drinking water) for 13 wk. HFS mice were subsequently randomized to continuing HFS feeding (HFS/HFS) or transition to the LFS diet (HFS/LFS) for four additional weeks. RESULTS: HFS/HFS mice exhibited superior femoral cancellous microarchitecture (i.e., greater BV/TV, Tb.N, Tb.Th, and decreased Tb.Sp) and cortical bone geometry (i.e., lower Ct.CSA and pMOI) compared with all other groups. At the femoral mid-diaphysis, structural, but not material, mechanical properties were greatest in HFS/HFS mice. However, HFS/HFS exhibited greater femoral neck strength only when compared with mice assigned to diet transition (HFS/LFS). Osteoclast surface and the percentage of osteocytes staining positive for interferon-gamma were greater in HFS/LFS mice, consistent with reduced cancellous microarchitecture postdiet transition. CONCLUSIONS: HFS feeding enhanced bone anabolism and structural, but not material, mechanical properties in exercising mice. A change from an HFS to LFS diet returned the bone structure to that of continuously LFS-fed mice while compromising strength. Our results indicate rapid weight loss from obese states should be performed with caution to prevent bone fragility. A deeper analysis into the altered bone phenotype in diet-induced obesity from a metabolic standpoint is needed.

Combined effects of heavy ion exposure and simulated Lunar gravity on skeletal muscle.

BACKGROUND: The limitations to prolonged spaceflight include unloading-induced atrophy of the musculoskeletal system which may be enhanced by exposure to the space radiation environment. Previous results have concluded that partial gravity, comparable to the Lunar surface, may have detrimental effects on skeletal muscle. However, little is known if these outcomes are exacerbated by exposure to low-dose rate, high-energy radiation common to the space environment. Therefore, the present study sought to determine the impact of highly charge, high-energy (HZE) radiation on skeletal muscle when combined with partial weightbearing to simulate Lunar gravity. We hypothesized that partial unloading would compromise skeletal muscle and these effects would be exacerbated by radiation exposure. METHODS: For month old female BALB/cByJ mice were -assigned to one of 2 groups; either full weight bearing (Cage Controls, CC) or partial weight bearing equal to 1/6th bodyweight (G/6). Both groups were then divided to receive either a single whole body absorbed dose of 0.5 Gy of 300 MeV 28Si ions (RAD) or a sham treatment (SHAM). Radiation exposure experiments were performed at the NASA Space Radiation Laboratory (NSRL) located at Brookhaven National Laboratory on Day 0, followed by 21 d of CC or G/6 loading. Muscles of the hind limb were used to measure protein synthesis and other histological measures. RESULTS: Twenty-one days of Lunar gravity (G/6) resulted in lower soleus, plantaris, and gastrocnemius muscle mass. Radiation exposure did not further impact muscle mass. 28Si exposure in normal ambulatory animals (RAD+CC) did not impact gastrocnemius muscle mass when compared to SHAM+CC (p>0.05), but did affect the soleus, where mass was higher following radiation compared to SHAM (p<0.05). Mixed gastrocnemius muscle protein synthesis was lower in both unloading groups. Fiber type composition transitioned towards a faster isoform with partial unloading and was not further impacted by radiation. The combined effects of partial loading and radiation partially mitigated fiber cross-sectional area when compared to partial loading alone. Radiation and G/6 reduced the total number of myonuclei per fiber while leading to elevated BrdU content of skeletal muscle. Similarly, unloading and radiation resulted in higher collagen content of muscle when compared to controls, but the effects of combined exposure were not additive. CONCLUSIONS: The results of this study confirm that partial weightbearing causes muscle atrophy, in part due to reductions of muscle protein synthesis in the soleus and gastrocnemius as well as reduced peripheral nuclei per fiber. Additionally, we present novel data illustrating 28Si exposure reduced nuclei in muscle fibers despite higher satellite cell fusion, but did not exacerbate muscle atrophy, CSA changes, or collagen content. In conclusion, both partial loading and HZE radiation can negatively impact muscle morphology.

A rodent model of human dose-equivalent progestin-only implantable contraception.

BACKGROUND: Long-acting, reversible contraceptives (LARC; progestin only) are an increasingly common hormonal contraceptive choice in reproductive aged women looking to suppress ovarian function and menstrual cyclicity. The overall objective was to develop and validate a rodent model of implanted etonogestrel (ENG) LARC, at body size equivalent doses to the average dose received by women during each of the first 3 years of ENG subdermal rod LARC use. METHODS: Intact, virgin, female Sprague-Dawley rats (16-wk-old) were randomized to 1 of 4 groups (n = 8/group) of ENG LARC (high-0.30µg/d, medium-0.17µg/d, low-0.09µg/d, placebo-0.00µg/d) via a slow-release pellet implanted subcutaneously. Animals were monitored for 21 days before and 29 days following pellet implantation using vaginal smears, ultrasound biomicroscopy (UBM), saphenous blood draws, food consumption, and body weights. Data were analyzed by chi-square, non-parametric, univariate, and repeated measures 2-way ANOVA. RESULTS: Prior to pellet implantation there was no difference in time spent in estrus cycle phases among the treatment groups (p > 0.30). Following pellet implantation there was a dose-dependent impact on the time spent in diestrus and estrus (p < 0.05), with the high dose group spending more days in diestrus and fewer days in estrus. Prior to pellet insertion there was not an association between treatment group and estrus cycle classification (p = 0.57) but following pellet implantation there was a dose-dependent association with cycle classification (p < 0.02). Measurements from the UBM (ovarian volume, follicle count, corpora lutea count) indicate an alteration of ovarian function following pellet implantation. CONCLUSION: Assessment of estrus cyclicity indicated a dose-response relationship in the shift to a larger number of acyclic rats and longer in duration spent in the diestrus phase. Therefore, each dose in this model mimics some of the changes observed in the ovaries of women using ENG LARC and provides an opportunity for investigating the impacts on non-reproductive tissues in the future.

Oral Estradiol Impact on Mitigating Unloading-Induced Bone Loss in Ovary-Intact Rats.

BACKGROUND: The impact of the spaceflight environment on endogenous estrogen production in female crewmembers and the resulting impact on other adaptations, like bone loss, is an under-investigated topic. Hence, we investigated the interaction of exogenous 17- estradiol (E2) treatment and disuse to test the hypothesis that E2 treatment would mitigate disuse-induced bone loss.METHODS: There were 40 virgin female Sprague-Dawley rats (5 mo old) randomized to placebo (PL; 0 ppm E2) or estrogen (E2; 10 ppm E2) treatments, delivered via custom-made rodent diets; half of each group was randomized to either weightbearing (WB) or hindlimb unloading (HU) for 39 d.RESULTS: We observed expected lower values after HU (615%) in volumetric BMD and cross-sectional areas at the proximal tibia metaphysis (PTM, by pQCT), 20% lower %BV/TV (nonsignificant) at the PTM, and 11% lower femoral neck maximal load; none of these HU-induced impacts were modified by E2. Impaired PTM periosteal expansion was observed in all E2-treated rats, with smaller (13 to 18%) cross-sectional areas. Midshaft tibial geometry was unaffected by E2 treatment, but large reductions (73 to 81%) in periosteal bone formation indices were observed in E2-treated rats.DISCUSSION: In summary, modest supplementation of exogenous E2 did not mitigate decrements in volumetric BMD, PTM cross-sectional geometry, or femoral neck strength observed with HU. However, numerous independent impacts of E2 treatment were observed, with significant suppression of periosteal bone formation indices. If maintained over time, this might impact negatively on cortical bone integrity during prolonged nonweightbearing.Mantri AV, Allaway HCM, Brezicha JE, Hogan HA, Bloomfield SA. Oral estradiol impact on mitigating unloading-induced bone loss in ovary-intact rats. Aerosp Med Hum Perform. 2021; 92(2):6574.

Omega-3 fatty acid modulation of serum and osteocyte tumor necrosis factor-α in adult mice exposed to ionizing radiation.

Chronic inflammation leads to bone loss and fragility. Proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) consistently promote bone resorption. Dietary modulation of proinflammatory cytokines is an accepted therapeutic approach to treat chronic inflammation, including that induced by space-relevant radiation exposure. As such, these studies were designed to determine whether an anti-inflammatory diet, high in omega-3 fatty acids, could reduce radiation-mediated bone damage via reductions in the levels of inflammatory cytokines in osteocytes and serum. Lgr5-EGFP C57BL/6 mice were randomized to receive diets containing fish oil and pectin (FOP; high in omega-3 fatty acids) or corn oil and cellulose (COC; high in omega-6 fatty acids) and then acutely exposed to 0.5-Gy 56Fe or 2.0-Gy gamma-radiation. Mice fed the FOP diet exhibited consistent reductions in serum TNF-α in the 56Fe experiment but not the gamma-experiment. The percentage osteocytes (%Ot) positive for TNF-α increased in gamma-exposed COC, but not FOP, mice. Minimal changes in %Ot positive for sclerostin were observed. FOP mice exhibited modest improvements in several measures of cancellous microarchitecture and volumetric bone mineral density (BMD) postexposure to 56Fe and gamma-radiation. Reduced serum TNF-α in FOP mice exposed to 56Fe was associated with either neutral or modestly positive changes in bone structural integrity. Collectively, these data are generally consistent with previous findings that dietary intake of omega-3 fatty acids may effectively mitigate systemic inflammation after acute radiation exposure and facilitate maintenance of BMD during spaceflight in humans.NEW & NOTEWORTHY This is the first investigation, to our knowledge, to test the impact of a diet high in omega-3 fatty acids on multiple bone structural and biological outcomes following space-relevant radiation exposure. Novel in biological outcomes is the assessment of osteocyte responses to this stressor. These data also add to the growing evidence that low-dose exposures to even high-energy ion species like 56Fe may have neutral or even small positive impacts on bone.

Hindlimb unloading causes regional loading-dependent changes in osteocyte inflammatory cytokines that are modulated by exogenous irisin treatment.

Disuse-induced bone loss is characterized by alterations in bone turnover. Accruing evidence suggests that osteocytes respond to inflammation and express and/or release pro-inflammatory cytokines; however, it remains largely unknown whether osteocyte inflammatory proteins are influenced by disuse. The goals of this project were (1) to assess osteocyte pro-inflammatory cytokines in the unloaded hindlimb and loaded forelimb of hindlimb unloaded rats, (2) to examine the impact of exogenous irisin during hindlimb unloading (HU). Male Sprague Dawley rats (8 weeks old, n = 6/group) were divided into ambulatory control, HU, and HU with irisin (HU + Ir, 3×/week). Lower cancellous bone volume, higher osteoclast surfaces (OcS), and lower bone formation rate (BFR) were present at the hindlimb and 4th lumbar vertebrae in the HU group while the proximal humerus of HU rats exhibited no differences in bone volume, but higher BFR and lower OcS vs. Con. Osteocyte tumor necrosis factor-α (TNF-α), interleukin-17 (IL-17), RANKL, and sclerostin were elevated in the cancellous bone of the distal femur of HU rats vs. Con, but lower at the proximal humerus in HU rats vs. Con. Exogenous irisin treatment increased BFR, and lowered OcS and osteocyte TNF-α, IL-17, RANKL, and sclerostin in the unloaded hindlimb of HU + Ir rats while having minimal changes in the humerus. In conclusion, there are site-specific and loading-specific alterations in osteocyte pro-inflammatory cytokines and bone turnover with the HU model of disuse bone loss, indicating a potential mechanosensory impact of osteocyte TNF-α and IL-17. Additionally, exogenous irisin significantly reduced the pro-inflammatory status of the unloaded hindlimb.

DSS-induced colitis produces inflammation-induced bone loss while irisin treatment mitigates the inflammatory state in both gut and bone.

Chronic pediatric inflammatory bowel disease (IBD) leads to lack of bone accrual, bone loss, and increased fractures. Presently there is no cure, and many IBD treatments incur negative side effects. We previously discovered treatment with exogenous irisin resolved inflammatory changes in the colon, gut lymphatics, and bone in a mild IBD rodent model. Here we assess irisin treatment in severe IBD induced via dextran sodium sulfate (DSS). Male Sprague Dawley rats (2-mo-old) were untreated (Con) or given 2% DSS in drinking water. In week two, half of each group (Con + Ir and DSS + Ir) received injections of recombinant irisin (i.p., 2x/wk). After 4 weeks, gut inflammation was associated with declines in bone mineral density and cancellous bone volume. Furthermore, elevated osteocyte TNF-α, interleukin-6, RANKL, OPG, and sclerostin corresponded with higher osteoclast surfaces and lower bone formation rate in DSS animals as well as lower ultimate load. While irisin treatment improved colon inflammation, there were no improvements in bone density or bone mechanical properties; however, irisin elevated bone formation rate, decreased osteoclast surfaces, and reduced osteocyte pro-inflammatory factors. These data highlight the negative impact of chronic gut inflammation on bone as well as the therapeutic potential of irisin as an anti-inflammatory treatment.

Positive impact of low-dose, high-energy radiation on bone in partial- and/or full-weightbearing mice.

Astronauts traveling beyond low Earth orbit will be exposed to galactic cosmic radiation (GCR); understanding how high energy ionizing radiation modifies the bone response to mechanical unloading is important to assuring crew health. To investigate this, we exposed 4-mo-old female Balb/cBYJ mice to an acute space-relevant dose of 0.5 Gy 56Fe or sham (n = ~8/group); 4 days later, half of the mice were also subjected to a ground-based analog for 1/6 g (partial weightbearing) (G/6) for 21 days. Microcomputed tomography (µ-CT) of the distal femur reveals that 56Fe exposure resulted in 65-78% greater volume and improved microarchitecture of cancellous bone after 21 d compared to sham controls. Radiation also leads to significant increases in three measures of energy absorption at the mid-shaft femur and an increase in stiffness of the L4 vertebra. No significant effects of radiation on bone formation indices are detected; however, G/6 leads to reduced % mineralizing surface on the inner mid-tibial bone surface. In separate groups allowed 21 days of weightbearing recovery from G/6 and/or 56Fe exposure, radiation-exposed mice still exhibit greater bone mass and improved microarchitecture vs. sham control. However, femoral bone energy absorption values are no longer higher in the 56Fe-exposed WB mice vs. sham controls. We provide evidence for persistent positive impacts of high-LET radiation exposure preceding a period of full or partial weightbearing on bone mass and microarchitecture in the distal femur and, for full weightbearing mice only and more transiently, cortical bone energy absorption values.

A moderately elevated soy protein diet mitigates inflammatory changes in gut and in bone turnover during chronic TNBS-induced inflammatory bowel disease.

Inflammatory bowel disease is a condition that leads to gut pathologies such as abnormal lymphatic architecture, as well as to systemic comorbidities such as bone loss. Furthermore, current therapies are limited to low efficacy and incur side effects. Dietary interventions have been explored minimally, but may provide a treatment for improving gut outcomes and comorbidities. Indeed, plant-based soy protein has been shown to exert anti-inflammatory effects. Here, we tested the impact of a moderately elevated soy protein diet in a chronic, 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis model on gut and bone inflammatory-mediated pathophysiological adaptations. Colitis was induced by intrarectal administration of TNBS. Gut histopathology was scored, and lymphatic structural changes and the local inflammatory state were assessed via immunofluorescence. In addition, the effects of gut inflammation on bone turnover and osteocyte proteins were determined via histomorphometry and immunohistochemistry, respectively. The moderately elevated soy protein diet produced improvements in both colonic and bone tissues. In TNBS animals given the soy protein intervention, colon histological scores were reduced and the abnormal lymphatic architecture resolved. There were also improvements in bone formation and reduced bone resorption. In addition, TNBS increased inflammatory cytokines such as tumor necrosis factor-α and receptor activator of nuclear factor κ-B ligand in the gut and bone, but this was resolved in both tissues with the dietary soy protein intervention. The moderately elevated soy protein diet mitigated gut and bone inflammation in a chronic, TNBS-induced colitis model, demonstrating the potential for soy protein as a potential anti-inflammatory dietary intervention for inflammatory bowel disease.

Osteocytes reflect a pro-inflammatory state following spinal cord injury in a rodent model.

Profound bone loss occurs following spinal cord injury (SCI) resulting in a high incidence of fractures. While likely caused in part by loss of weight-bearing, there is greater bone loss following SCI when compared to that observed in other disuse animal models. Patients with SCI have a protracted inflammatory response, with elevated circulating levels of pro-inflammatory markers. This chronic inflammation could compound the bone loss attributed to disuse and the loss of neural signaling. To assess this, we examined inflammatory markers and bone turnover regulators in osteocytes from rats with a moderate spinal contusion injury (SCI) and intact controls (CON). We counted osteocytes positive for cytokines [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-17 (IL-17), and interleukin-10 (IL-10)], osteoclastogenesis regulators RANKL and OPG, and the bone formation inhibitor sclerostin, 32 days after the spinal contusion. By day 9 post-injury, the majority of SCI rats had recovered significant locomotor function and were bearing weight on their hindlimbs. However, despite weight-bearing, peripheral QCT scans demonstrated lower bone mass due to SCI in the proximal tibia metaphysis compared to CON. SCI animals also had lower cancellous bone volume, lower bone formation rate (BFR), lower osteoid surface (OS), and higher osteoclast surface (Oc.S). Tibial mid-shaft periosteal BFR was also lower after SCI. Immunohistochemical staining of the distal femur bone revealed cancellous osteocytes positive for TNF-α, IL-6, IL-17, and IL-10 were elevated in SCI animals relative to intact controls. Protein expression of RANKL+, OPG+, and sclerostin+ osteocytes was also higher in SCI rats. At the cortical midshaft, osteocyte TNF-α, IL-6, and sclerostin were statistically higher in SCI vs. CON. With regression analysis, inflammatory factors were associated with changes in bone turnover. In conclusion, inflammatory factors as well as altered mechanical loading influence bone turnover following a moderate SCI. Treatments aimed at minimizing fracture risk after SCI may need to target both the chronically altered inflammatory state as well as disuse-induced bone loss.

Fat and Lean Mass Predict Bone Mass During Energy Restriction in Sedentary and Exercising Rodents.

Energy restriction (ER) causes bone loss, but the impact of exercise during ER is less understood. In this study, we examined the impact of metabolic hormones and body composition on both total body bone mineral content (BMC) and local (proximal tibia) volumetric bone mineral density (vBMD) during short- (4 weeks) and long-term (12 weeks) ER with and without exercise in adult female rats. Our first goal was to balance energy between sedentary and exercising groups to determine the impact of exercise during ER. Second, we aimed to determine the strongest predictors of bone outcomes during ER with energy-matched exercising groups. Methods: Female Sprague-Dawley rats were divided into three sedentary groups (ad libitum, -20% ER, and -40% ER) and three exercising groups (ad libitum, -10% ER, and -30% ER). Approximately a 10% increase in energy expenditure was achieved via moderate treadmill running (∼60-100 min 4 days/week) in EX groups. n per group = 25-35. Data were analyzed as a 2 × 3 ANOVA with multiple linear regression to predict bone mass outcomes. Results: At 4 weeks, fat and lean mass and serum insulin-like growth factor-I (IGF-I) predicted total body BMC (R 2 = 0.538). Fat mass decreased with ER at all levels, while lean mass was not altered. Serum IGF-I declined in the most severe ER groups (-40 and -30%). At 12 weeks, only fat and lean mass predicted total body BMC (R 2 = 0.718). Fat mass declined with ER level regardless of exercise status and lean mass increased due to exercise (+5.6-6.7% vs. energy-matched sedentary groups). At the same time point, BMC declined with ER, but increased with exercise (+7.0-12.5% vs. energy-matched sedentary groups). None of our models predicted vBMD at the proximal tibia at either time point. Conclusion: Both fat and lean mass statistically predicted total body BMC during both short- and long-term ER. Fat and lean mass decreased with ER, while lean mass increased with EX at each energy level. Measures that predicted total body skeletal changes did not predict site-specific changes. These data highlight the importance of maintaining lean mass through exercise during periods of ER.

Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic disease with gastrointestinal dysfunction as well as comorbidities such as inflammation-induced bone loss and impaired immune response. Current treatments for IBD all have negative, potentially severe side effects. We aimed to test whether exogenous treatment with irisin, a novel immunomodulatory adipomyokine, could ameliorate IBD-induced lymphatic and bone alterations. Irisin treatment improved both gut and bone outcomes by mitigating inflammation and restoring structure. In the gut, IBD caused colonic lymphatic hyperproliferation into the mucosal and submucosal compartments. This proliferation in the rodent model is akin to what is observed in IBD patient case studies. In bone, IBD increased osteoclast surface and decreased bone formation. Both gut and osteocytes in bone exhibited elevated levels of TNF-α and receptor activator of NF-κB ligand (RANKL) protein expression. Exogenous irisin treatment restored normal colonic lymphatic architecture and increased bone formation rate concurrent with decreased osteoclast surfaces. After irisin treatment, gut and osteocyte TNF-α and RANKL protein expression levels were no different from vehicle controls. Our data indicate that the systemic immunologic changes that occur in IBD are initiated by damage in the gut and likely linked through the lymphatic system. Additionally, irisin is a potential novel intervention mitigating both local inflammatory changes in the gut and distant changes in bone.-Narayanan, S. A., Metzger, C. E., Bloomfield, S. A., Zawieja, D. C. Inflammation-induced lymphatic architecture and bone turnover changes are ameliorated by irisin treatment in chronic inflammatory bowel disease.

Exposure to Low-Dose X-Ray Radiation Alters Bone Progenitor Cells and Bone Microarchitecture.

Exposure to high-dose ionizing radiation during medical treatment exerts well-documented deleterious effects on bone health, reducing bone density and contributing to bone growth retardation in young patients and spontaneous fracture in postmenopausal women. However, the majority of human radiation exposures occur in a much lower dose range than that used in the radiation oncology clinic. Furthermore, very few studies have examined the effects of low-dose ionizing radiation on bone integrity and results have been inconsistent. In this study, mice were irradiated with a total-body dose of 0.17, 0.5 or 1 Gy to quantify the early (day 3 postirradiation) and delayed (day 21 postirradiation) effects of radiation on bone microarchitecture and bone marrow stromal cells (BMSCs). Female BALBc mice (4 months old) were divided into four groups: irradiated (0.17, 0.5 and 1 Gy) and sham-irradiated controls (0 Gy). Micro-computed tomography analysis of distal femur trabecular bone from animals at day 21 after exposure to 1 Gy of X-ray radiation revealed a 21% smaller bone volume (BV/TV), 22% decrease in trabecular numbers (Tb.N) and 9% greater trabecular separation (Tb.Sp) compared to sham-irradiated controls (P < 0.05). We evaluated the differentiation capacity of bone marrow stromal cells harvested at days 3 and 21 postirradiation into osteoblast and adipocyte cells. Osteoblast and adipocyte differentiation was decreased when cells were harvested at day 3 postirradiation but enhanced in cells isolated at day 21 postirradiation, suggesting a compensatory recovery process. Osteoclast differentiation was increased in 1 Gy irradiated BMSCs harvested at day 3 postirradiation, but not in those harvested at day 21 postirradiation, compared to controls. This study provides evidence of an early, radiation-induced decrease in osteoblast activity and numbers, as well as a later recovery effect after exposure to 1 Gy of X-rays, whereas osteoclastogenesis was enhanced. A better understanding of the effects of radiation on osteoprogenitor cell populations could lead to more effective therapeutic interventions that protect bone integrity for individuals exposed to low-dose ionizing radiation.

Differential responses of mechanosensitive osteocyte proteins in fore- and hindlimbs of hindlimb-unloaded rats.

Osteocytes are believed to be the primary mechanosensors of bone tissue, signaling to osteoblasts and osteoclasts by releasing specific proteins. Sclerostin, interleukin-6 (IL-6), and insulin-like growth factor-I (IGF-I) are osteocyte proteins that signal to osteoblasts. The primary objective of this study was to determine if osteocyte protein response to mechanical unloading is restricted to the unloaded bone using the hindlimb unloading (HU) rodent model. We also examined tumor necrosis factor-α (TNF-α) due to its interactions with all three osteocyte proteins. We hypothesized that unloaded hindlimb cancellous bone would have an altered osteocyte protein (sclerostin, IL-6, and IGF-I) response compared to controls, while the response in the weight-bearing forelimb would not differ from ambulating controls. Male Sprague Dawley rats (7-mo old) experienced either HU (n=7) or normal cage activity (CON; n=7) for 28days. The unloaded distal femur and the weight-bearing proximal humerus were compared in HU vs CON. Metaphyseal bone density was reduced in the HU rats' hindlimb, but not in the proximal humerus, compared to CON values. Osteocyte density was 30% lower in the HU distal femur, but not different from CON in the proximal humerus. %Sclerostin+osteocytes in the distal femur were higher in HU compared to CON, but lower in the proximal humerus. Both %IGF-I+ and %IL-6+ osteocytes were lower in the distal femur for HU, but higher in the proximal humerus for HU. Osterix surface, a marker of osteoblasts, was lower in HU in the distal femur; however, the proximal humerus had more %osterix+surface in HU. In HU %Cathepsin K+ surface, a marker of osteoclasts, was higher in the distal femur and lower in the proximal humerus. %TNF-α+osteocytes were no different from CON in either bone site. HU proximal humerus osteocyte protein responses of sclerostin, IL-6, and IGF-I changed in the opposite direction as observed in the distal femur within the same animal. The opposite response of osteocyte proteins and osteoblast surface in hind- and forelimb bones within the same animal suggests that, while osteocytes in the unloaded hindlimb sense a lack of mechanical strain, osteocytes in the weight-bearing forelimb in HU animals sense some increase in local strain and generate molecular signaling to osteoblasts.

Inflammatory Bowel Disease in a Rodent Model Alters Osteocyte Protein Levels Controlling Bone Turnover.

Bone loss is a common comorbidity of inflammatory bowel disease (IBD), leading to elevated fracture risk in these patients. Inflammatory factors associated with IBD cause increased bone resorption and decreased bone formation with multiple factors implicated as instigators of these alterations. In this project, we examined the influence of IBD on osteocyte proteins in male rats (2 months old) divided into two groups: induced gut inflammation via 2,4,6-trinitrobenzenesulfonic acid (TNBS) enema, and vehicle control. We examined the prevalence of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), an anti-inflammatory cytokine, interleukin-10 (IL-10), the anabolic factor insulin-like growth factor-I (IGF-I), osteoclastogenesis regulators RANKL and OPG, and the bone formation inhibitor sclerostin in osteocytes in three bone compartments 4 weeks after initiation of gut inflammation. Histomorphometry of the proximal tibia and fourth lumbar vertebra revealed lower bone volume, lower bone formation rate (BFR), lower osteoid surface (OS), and higher osteoclast surface (Oc.S) with TNBS. Tibial mid-shaft periosteal BFR was also lower with TNBS. Immunohistochemical staining of the distal femur demonstrated that %TNF-α+ , %IL-6+ , %RANKL+ , and %OPG+ osteocytes were elevated in cancellous bone in TNBS animals compared to vehicle. These changes were coincident with increased bone resorption. With regression analysis, %RANKL+ osteocytes statistically predicted the increase in cancellous Oc.S (R2 = 0.565). Increased %sclerostin+ osteocytes observed in the TNBS treatment predicted declines in cancellous OS (R2 = 0.581) as well as BFR in cancellous and cortical bone (R2 = 0.674, R2 = 0.908, respectively). Contrary to our hypothesis, %IGF-I+ osteocytes increased in TNBS animals. In conclusion, the IBD model produced a systemic inflammation that altered the regulatory protein profile in osteocytes that control bone resorption and bone formation, likely contributing to IBD-induced bone loss. These data highlight a potential mechanistic role of osteocytes in inflammatory bone loss associated with IBD and systemic inflammation. © 2017 American Society for Bone and Mineral Research.

Microgravity Stress: Bone and Connective Tissue.

The major alterations in bone and the dense connective tissues in humans and animals exposed to microgravity illustrate the dependency of these tissues' function on normal gravitational loading. Whether these alterations depend solely on the reduced mechanical loading of zero g or are compounded by fluid shifts, altered tissue blood flow, radiation exposure, and altered nutritional status is not yet well defined. Changes in the dense connective tissues and intervertebral disks are generally smaller in magnitude but occur more rapidly than those in mineralized bone with transitions to 0 g and during recovery once back to the loading provided by 1 g conditions. However, joint injuries are projected to occur much more often than the more catastrophic bone fracture during exploration class missions, so protecting the integrity of both tissues is important. This review focuses on the research performed over the last 20 years in humans and animals exposed to actual spaceflight, as well as on knowledge gained from pertinent ground-based models such as bed rest in humans and hindlimb unloading in rodents. Significant progress has been made in our understanding of the mechanisms for alterations in bone and connective tissues with exposure to microgravity, but intriguing questions remain to be solved, particularly with reference to biomedical risks associated with prolonged exploration missions.

Space Environmental Factor Impacts upon Murine Colon Microbiota and Mucosal Homeostasis.

Astronaut intestinal health may be impacted by microgravity, radiation, and diet. The aim of this study was to characterize how high and low linear energy transfer (LET) radiation, microgravity, and elevated dietary iron affect colon microbiota (determined by 16S rDNA pyrosequencing) and colon function. Three independent experiments were conducted to achieve these goals: 1) fractionated low LET γ radiation (137Cs, 3 Gy, RAD), high Fe diet (IRON) (650 mg/kg diet), and a combination of low LET γ radiation and high Fe diet (IRON+RAD) in male Sprague-Dawley rats; 2) high LET 38Si particle exposure (0.050 Gy), 1/6 G partial weight bearing (PWB), and a combination of high LET38Si particle exposure and PWB in female BalbC/ByJ mice; and 3) 13 d spaceflight in female C57BL/6 mice. Low LET radiation, IRON and spaceflight increased Bacteroidetes and decreased Firmicutes. RAD and IRON+RAD increased Lactobacillales and lowered Clostridiales compared to the control (CON) and IRON treatments. Low LET radiation, IRON, and spaceflight did not significantly affect diversity or richness, or elevate pathogenic genera. Spaceflight increased Clostridiales and decreased Lactobacillales, and similar trends were observed in the experiment using a ground-based model of microgravity, suggesting altered gravity may affect colonic microbiota. Although we noted no differences in colon epithelial injury or inflammation, spaceflight elevated TGFß gene expression. Microbiota and mucosal characterization in these models is a first step in understanding the impact of the space environment on intestinal health.

Biomechanical aspects of the muscle-bone interaction.

There is growing interest in the interaction between skeletal muscle and bone, particularly at the genetic and molecular levels. However, the genetic and molecular linkages between muscle and bone are achieved only within the context of the essential mechanical coupling of the tissues. This biomechanical and physiological linkage is readily evident as muscles attach to bone and induce exposure to varied mechanical stimuli via functional activity. The responsiveness of bone cells to mechanical stimuli, or their absence, is well established. However, questions remain regarding how muscle forces applied to bone serve to modulate bone homeostasis and adaptation. Similarly, the contributions of varied, but unique, stimuli generated by muscle to bone (such as low-magnitude, high-frequency stimuli) remains to be established. The current article focuses upon the mechanical relationship between muscle and bone. In doing so, we explore the stimuli that muscle imparts upon bone, models that enable investigation of this relationship, and recent data generated by these models.

Beta-1 adrenergic agonist treatment mitigates negative changes in cancellous bone microarchitecture and inhibits osteocyte apoptosis during disuse.

The sympathetic nervous system (SNS) plays an important role in mediating bone remodeling. However, the exact role that beta-1 adrenergic receptors (beta1AR) have in this process has not been elucidated. We have previously demonstrated the ability of dobutamine (DOB), primarily a beta1AR agonist, to inhibit reductions in cancellous bone formation and mitigate disuse-induced loss of bone mass. The purpose of this study was to characterize the independent and combined effects of DOB and hindlimb unloading (HU) on cancellous bone microarchitecture, tissue-level bone cell activity, and osteocyte apoptosis. Male Sprague-Dawley rats, aged 6-mos, were assigned to either normal cage activity (CC) or HU (n = 18/group) for 28 days. Animals were administered either daily DOB (4 mg/kg BW/d) or an equal volume of saline (VEH) (n = 9/gp). Unloading resulted in significantly lower distal femur cancellous BV/TV (-33%), Tb.Th (-11%), and Tb.N (-25%) compared to ambulatory controls (CC-VEH). DOB treatment during HU attenuated these changes in cancellous bone microarchitecture, resulting in greater BV/TV (+29%), Tb.Th (+7%), and Tb.N (+21%) vs. HU-VEH. Distal femur cancellous vBMD (+11%) and total BMC (+8%) were significantly greater in DOB- vs. VEH-treated unloaded rats. Administration of DOB during HU resulted in significantly greater osteoid surface (+158%) and osteoblast surface (+110%) vs. HU-VEH group. Furthermore, Oc.S/BS was significantly greater in HU-DOB (+55%) vs. CC-DOB group. DOB treatment during unloading fully restored bone formation, resulting in significantly greater bone formation rate (+200%) than in HU-VEH rats. HU resulted in an increased percentage of apoptotic cancellous osteocytes (+85%), reduced osteocyte number (-16%), lower percentage of occupied osteocytic lacunae (-30%) as compared to CC-VEH, these parameters were all normalized with DOB treatment. Altogether, these data indicate that beta1AR agonist treatment during disuse mitigates negative changes in cancellous bone microarchitecture and inhibits increases in osteocyte apoptosis.