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Decreased blood-tissue oxygenation at high altitude (HA) increases mitochondrial oxidant production and reduces exercise capacity. 5-Hydroxymethylfurfural (5-HMF) is an antioxidant that increases hemoglobin's binding affinity for oxygen. For these reasons, we hypothesized that 5-HMF would improve muscle performance in rats exposed to a simulated HA of ~5500 m. A secondary objective was to measure mitochondrial activity and dynamic regulation of fission and fusion because they are linked processes impacted by HA. Fisher 344 rats received 5-HMF (40 mg/kg/day) or vehicle during exposure to sea level or HA for 72 h. Right ankle plantarflexor muscle function was measured pre- and post-exposure. Post-exposure measurements included arterial blood gas and complete blood count, flexor digitorum brevis myofiber superoxide production and mitochondrial membrane potential (ΔΨm), and mitochondrial dynamic regulation in the soleus muscle. HA reduced blood oxygenation, increased superoxide levels and lowered ΔΨm, responses that were accompanied by decreased peak isometric torque and force production at frequencies >75 Hz. 5-HMF increased isometric force production and lowered oxidant production at sea level. In HA exposed animals, 5-HMF prevented a decline in isometric force production at 75-125 Hz, prevented an increase in superoxide levels, further decreased ΔΨm, and increased mitochondrial fusion 2 protein expression. These results suggest that 5-HMF may prevent a decrease in hypoxic force production during submaximal isometric contractions by an antioxidant mechanism., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Objective: Carbon monoxide (CO) may counteract obesity and metabolic dysfunction in rodents consuming high-fat diets, but the skeletal effects are not understood. This study investigated whether low-dose inhaled CO (250 ppm) with or without moderate intensity aerobic exercise (3 h/wk) would limit diet-induced obesity and metabolic dysregulation and preserve bone health., Methods: Obesity-resistant (OR) rats served as controls, and obesity-prone (OP) rats were randomized to sedentary, sedentary plus CO, exercise, or CO plus exercise. For 10 weeks, OP rats consumed a high-fat, high-sucrose diet, whereas OR rats consumed a low-fat control diet. Measurements included indicators of obesity and metabolism, bone turnover markers, femoral geometry and microarchitecture, bone mechanical properties, and tibial morphometry., Results: A high-fat, high-sucrose diet led to obesity, hyperinsulinemia, and hyperleptinemia, without impacting bone. CO alone led only to a modest reduction in weight gain. Exercise attenuated weight gain and improved the metabolic profile; however, bone fragility increased. Combined CO and exercise led to body mass reduction and a metabolic state similar to control OR rats and prevented the exercise-induced increase in bone fragility., Conclusions: CO and aerobic exercise training prevent obesity and metabolic sequelae of nutrient excess while stabilizing bone physiology., (© 2020 The Obesity Society.)

Introduction: The consequences of low partial pressure of O 2 include low arterial O 2 saturations (SaO 2 ), low blood O 2 content (CaO 2 ), elevated mean pulmonary artery pressure (PAP), and decreased O 2 consumption VO 2 . 5-hydroxymethyl-2-furfural (5-HMF) binds to the N-terminal valine of hemoglobin (HgB) and increases its affinity to O 2 . We used an instrumented, sedated swine model to study the effect of 5-HMF on cardiovascular parameters during exposure to acute normobaric hypoxia (NH)., Methods: Twenty-three sedated and instrumented swine were randomly assigned to one of three treatment groups and received equal volume of normal saline (VEH), 20 mg/kg 5-HMF (5-HMF-20) or 40 mg/kg 5-HMF (5-HMF-40). Animals then breathed 10% FiO 2 for 120 min. Parameters recorded were Cardiac Output (CO), Mean Arterial Blood Pressure (MAP), Heart Rate (HR), Mean Pulmonary Artery Pressure (PAP), SaO 2 and saturation of mixed venous blood (SvO 2 ). The P 50 was measured at fixed time intervals prior to and during NH., Results: 5-HMF decreased P 50 . In the first 30 min of NH, treatment with 5-HMF-20 and 5-HMF-40 resulted in a (1) significantly smaller decrement in SaO 2 and SvO 2 , (2) significantly lower HR and CO, and (3) smaller increase in PAP compared to VEH. In the 120 min of NH there was a trend toward improved mortality with 5-HMF treatment., Conclusion: 5-HMF treatment decreased P 50 , improved SaO 2 , and mitigated increases in PAP in this swine model of NH.

Introduction: Individuals with a known susceptibility to high altitude pulmonary edema (HAPE) demonstrate a reduced ventilation response and increased pulmonary vasoconstriction when exposed to hypoxia. It is unknown whether reduced sensitivity to hypercapnia is correlated with increased incidence and/or severity of HAPE, and while acute exercise at altitude is known to exacerbate symptoms the effect of exercise training on HAPE susceptibility is unclear. Purpose: To determine if chronic intermittent hypercapnia and exercise increases the incidence of HAPE in rats. Methods: Male Wistar rats were randomized to sedentary (sed-air), CO 2 (sed-CO 2 ,) exercise (ex-air), or exercise + CO 2 (ex-CO 2 ) groups. CO 2 (3.5%) and treadmill exercise (15 m/min, 10% grade) were conducted on a metabolic treadmill, 1 h/day for 4 weeks. Vascular reactivity to CO 2 was assessed after the training period by rheoencephalography (REG). Following the training period, animals were exposed to hypobaric hypoxia (HH) equivalent to 25,000 ft for 24 h. Pulmonary injury was assessed by wet/dry weight ratio, lung vascular permeability, bronchoalveolar lavage (BAL), and histology. Results: HH increased lung wet/dry ratio (HH 5.51 ± 0.29 vs. sham 4.80 ± 0.11, P < 0.05), lung permeability (556 ± 84 u/L vs. 192 ± 29 u/L, P < 0.001), and BAL protein (221 ± 33 μg/ml vs. 114 ± 13 μg/ml, P < 0.001), white blood cell (1.16 ± 0.26 vs. 0.66 ± 0.06, P < 0.05), and platelet (16.4 ± 2.3, vs. 6.0 ± 0.5, P < 0.001) counts in comparison to normobaric normoxia. Vascular reactivity was suppressed by exercise (-53% vs. sham, P < 0.05) and exercise+CO 2 (-71% vs. sham, P < 0.05). However, neither exercise nor intermittent hypercapnia altered HH-induced changes in lung wet/dry weight, BAL protein and cellular infiltration, or pulmonary histology. Conclusion: Exercise training attenuates vascular reactivity to CO 2 in rats but neither exercise training nor chronic intermittent hypercapnia affect HH- induced pulmonary edema.

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.

Small ruminants have been implicated in outbreaks of Escherichia coli O157:H7 at livestock exhibitions throughout the United States. Additionally, goat meat or milk may serve as a reservoir for foodborne transmission of the organism. These associations highlight the public health importance of an effective strategy to reduce E. coli O157:H7 shedding in goats. We examined the efficacy of the SRP ® vaccine in goats orally challenged with E. coli O157:H7. Mixed-breed goats (n = 14) were randomly allocated into vaccinated and unvaccinated treatments (n = 7 per treatment). Goats were housed with a vaccinated and unvaccinated animal in each pen. Feces were collected for 3 weeks, then at necropsy, gastrointestinal contents were collected to determine the concentration of E. coli O157:H7. Three isolates per positive sample were saved and evaluated by pulsed-field gel electrophoresis (PFGE) to assess strain persistence over time. The mean concentration of E. coli O157:H7 in the feces of goats was numerically reduced in the vaccinated treatment; however, it was not statistically significant. In addition, the total number of days goats were fecal positive for E. coli O157:H7 were not different between vaccinated and unvaccinated treatments. Pulsotypes of isolates revealed that goats initially shed two of the four challenge strains of E. coli O157:H7, after which there was a distinct shift to two different strains. Further work is needed to evaluate cost-effective intervention strategies that reliably reduce E. coli O157:H7 shedding in goats, particularly those that may reduce the risk of transmission at public events, including petting zoos and fairs.

Exploration missions to the Moon or Mars will expose astronauts to galactic cosmic radiation and low gravitational fields. Exposure to reduced weightbearing and radiation independently result in bone loss. However, no data exist regarding the skeletal consequences of combining low-dose, high-linear energy transfer (LET) radiation and partial weightbearing. We hypothesized that simulated galactic cosmic radiation would exacerbate bone loss in animals held at one-sixth body weight (G/6) without radiation exposure. Female BALB/cByJ four-month-old mice were randomly assigned to one of the following treatment groups: 1 gravity (1G) control; 1G with radiation; G/6 control; and G/6 with radiation. Mice were exposed to either silicon-28 or X-ray radiation. (28)Si radiation (300 MeV/nucleon) was administered at acute doses of 0 (sham), 0.17 and 0.5 Gy, or in three fractionated doses of 0.17 Gy each over seven days. X radiation (250 kV) was administered at acute doses of 0 (sham), 0.17, 0.5 and 1 Gy, or in three fractionated doses of 0.33 Gy each over 14 days. Bones were harvested 21 days after the first exposure. Acute 1 Gy X-ray irradiation during G/6, and acute or fractionated 0.5 Gy (28)Si irradiation during 1G resulted in significantly lower cancellous mass [percentage bone volume/total volume (%BV/TV), by microcomputed tomography]. In addition, G/6 significantly reduced %BV/TV compared to 1G controls. When acute X-ray irradiation was combined with G/6, distal femur %BV/TV was significantly lower compared to G/6 control. Fractionated X-ray irradiation during G/6 protected against radiation-induced losses in %BV/TV and trabecular number, while fractionated (28)Si irradiation during 1G exacerbated the effects compared to single-dose exposure. Impaired bone formation capacity, measured by percentage mineralizing surface, can partially explain the lower cortical bone thickness. Moreover, both partial weightbearing and (28)Si-ion exposure contribute to a higher proportion of sclerostin-positive osteocytes in cortical bone. Taken together, these data suggest that partial weightbearing and low-dose, high-LET radiation negatively impact maintenance of bone mass by lowering bone formation and increasing bone resorption. The impaired bone formation response is associated with sclerostin-induced suppression of Wnt signaling. Therefore, exposure to low-dose, high-LET radiation during long-duration spaceflight missions may reduce bone formation capacity, decrease cancellous bone mass and increase bone resorption. Future countermeasure strategies should aim to restore mechanical loads on bone to those experienced in one gravity. Moreover, low-doses of high-LET radiation during long-duration spaceflight should be limited or countermeasure strategies employed to mitigate bone loss.

The present study investigated the detrimental effects of non-lethal, high-dose (whole body) γ-irradiation on bone, and the impact that radiation combined with skin trauma (i.e. combined injury) has on long-term skeletal tissue health. Recovery of bone after an acute dose of radiation (RI; 8 Gy), skin wounding (15-20% of total body skin surface), or combined injury (RI+Wound; CI) was determined 3, 7, 30, and 120 days post-irradiation in female B6D2F1 mice and compared to non-irradiated mice (SHAM) at each time-point. CI mice demonstrated long-term (day 120) elevations in serum TRAP 5b (osteoclast number) and sclerostin (bone formation inhibitor), and suppression of osteocalcin levels through 30 days as compared to SHAM (p<0.05). Radiation-induced reductions in distal femur trabecular bone volume fraction and trabecular number through 120 days post-exposure were significantly greater than non-irradiated mice (p<0.05) and were exacerbated in CI mice by day 30 (p<0.05). Negative alterations in trabecular bone microarchitecture were coupled with extended reductions in cancellous bone formation rate in both RI and CI mice as compared to Sham (p<0.05). Increased osteoclast surface in CI animals was observed for 3 days after irradiation and remained elevated through 120 days (p<0.01). These results demonstrate a long-term, exacerbated response of bone to radiation when coupled with non-lethal wound trauma. Changes in cancellous bone after combined trauma were derived from extended reductions in osteoblast-driven bone formation and increases in osteoclast activity., (Published by Elsevier Inc.)

Exposure to high-dose radiation results in detrimental effects on survival. The effects of combined trauma, such as radiation in combination with hemorrhage, the typical injury of victims exposed to a radiation blast, on survival and hematopoietic effects have yet to be understood. The purpose of this study was to evaluate the effects of radiation injury (RI) combined with hemorrhage (i.e., combined injury, CI) on survival and hematopoietic effects, and to investigate whether hemorrhage (Hemo) enhanced RI-induced mortality and hematopoietic syndrome. Male CD2F1 mice (10 weeks old) were given one single exposure of γ- radiation (60Co) at various doses (0.6 Gy/min). Within 2 hr after RI, animals under anesthesia were bled 0% (Sham) or 20% (Hemo) of total blood volume via the submandibular vein. In these mice, Hemo reduced the LD50/30 for 30-day survival from 9.1 Gy (RI) to 8.75 Gy (CI) with a DMF of 1.046. RI resulted in leukocytopenia, thrombopenia, erythropenia, and bone marrow cell depletion, but decreased the caspase-3 activation response. RI increased IL-1β, IL-6, IL-17A, and TNF-α concentrations in serum, bone marrow, ileum, spleen, and kidney. Some of these adverse alterations were magnified by CI. Erythropoietin production was increased in kidney and blood more after CI than RI. Furthermore, CI altered the global miRNAs expression in kidney and the ingenuity pathway analysis showed that miRNAs viz., let-7e, miR-30e and miR-29b that were associated with hematopoiesis and inflammation. This study provides preliminary evidence that non-lethal Hemo exacerbates RI-induced mortality and cell losses associated with high-dose γ-radiation. We identified some of the initial changes occurring due to CI which may have facilitated in worsening the injury and hampering the recovery of animals ultimately resulting in higher mortality.

Past and recent radiation events have involved a high incidence of radiation combined injury where victims often succumb to serious infections as a consequence of bacterial translocation and subsequent sepsis. The risk of infection is exacerbated in radiation combined skin-burn injury (RCI), which increase vulnerability. Furthermore, no suitable countermeasures for radiation combined skin-burn injury have been established. In this study, we evaluated captopril as a potential countermeasure to radiation combined skin-burn injury. Captopril is an FDA-approved angiotensin-converting enzyme inhibitor that was previously reported to stimulate hematopoietic recovery after exposure to ionizing radiation. Female B6D2F1/J mice were whole-body bilateral (60)Co gamma-photon irradiated (dose rate of 0.4 Gy/min) with 9.5 Gy (LD70/30 for RCI), followed by nonlethal dorsal skin-burn injury under anesthesia (approximately 15% total-body surface-area burn). Mice were provided with acidified drinking water with or without dissolved captopril (0.55 g/l) for 30 days immediately after injury and were administered topical gentamicin (0.1% cream; day 1-10) and oral levofloxacin (90-100 mg/kg; day 3-16). Surviving mice were euthanized on day 30 after analyses of water consumption, body weight and survival. Our data demonstrate that, while treatment with captopril did mitigate mortality induced by radiation injury (RI) alone (55% captopril vs. 80% vehicle; n = 20, P < 0.05), it also resulted in decreased survival after radiation combined skin-burn injury (22% captopril vs. 41% vehicle; n = 22, P < 0.05). Moreover, captopril administration via drinking water produced an uneven dosage pattern among the different injury groups ranging from 74 ± 5.4 to 115 ± 2.2 mg/kg/day. Captopril treatment also did not counteract the negative alterations in hematology, splenocytes or bone marrow cellularity after either radiation injury or radiation combined skin-burn injury. These data suggest that captopril may exert its actions differently between the two injury models (RI vs. RCI) and that captopril dosing, when combined with topical and systemic antibiotic treatments, may not be a suitable countermeasure for RCI.

Background: Exposure to high dose radiation in combination with physical injuries such as burn or wound trauma can produce a more harmful set of medical complications requiring specialist interventions. Currently these interventions are unavailable as are the precise biomarkers needed to help both accurately assess and treat such conditions. In the present study, we tried to identify and explore the possible role of serum exosome microRNA (miRNA) signatures as potential biomarkers for radiation combined burn injury (RCBI)., Methodology: Female B6D2F1/J mice were assigned to four experimental groups (n = 6): sham control (SHAM), burn injury (BURN), radiation injury (RI) and combined radiation skin burn injury (CI). We performed serum multiplex cytokine analysis and serum exosome miRNA expression profiling to determine novel miRNA signatures and important biological pathways associated with radiation combined skin-burn trauma., Principal Findings: Serum cytokines, IL-5 and MCP-1, were significantly induced only in CI mice (p<0.05). From 890 differentially expressed miRNAs identified, microarray analysis showed 47 distinct miRNA seed sequences significantly associated with CI mice compared to SHAM control mice (fold change ≥ 1.2, p<0.05). Furthermore, only two major miRNA seed sequences (miR-690 and miR-223) were validated to be differentially expressed for CI mice specifically (fold change ≥ 1.5, p<0.05)., Conclusions: Serum exosome miRNA signature data of adult mice, following RCBI, provides new insights into the molecular and biochemical pathways associated with radiation combined skin-burn trauma in vivo.

Ionizing radiation exposure combined with wound injury increases animal mortalities than ionizing radiation exposure alone. Ciprofloxacin (CIP) is in the fluroquinolone family of synthetic antibiotic that are available from the strategic national stockpile for emergency use and is known to inhibit bacterial sepsis. The purpose of this study was to evaluate the efficacy of ciprofloxacin as a countermeasure to combined injury mortality and determine the signaling proteins involved in energy machinery. B6D2F1/J female mice were randomly assigned to receive either 9.75 Gy irradiation with Co-60 gamma rays followed by skin wounding (combined injury; CI) or sham procedure (sham). Either ciprofloxacin (90 mg/kg/day) or vehicle (VEH) (water) was administered orally to these mice 2 h after wounding and thereafter daily for 10 days. Determination of tissue adenosine triphosphate (ATP) was conducted, and immunoblotting for signaling proteins involved in ATP machinery was performed. Combined injury resulted in 60% survival after 10 days compared to 100% survival in the sham group. Furthermore, combined injury caused significant reductions of ATP concentrations in ileum, pancreas, brain, spleen, kidney and lung (-25% to -95%) compared to the sham group. Ciprofloxacin administration after combined injury resulted in 100% survival and inhibited reductions in ileum and kidney ATP production. Ileum protein levels of heat-shock protein 70 kDa (HSP-70, a chaperone protein involved in ATP synthesis) and pyruvate dehydrogenase (PDH, an enzyme complex crucial to conversion of pyruvate to acetyl CoA for entrance into TCA cycle) were significantly lower in the CI group (vs. sham group). Using immunoprecipitation and immunoblotting, HSP-70-PDH complex was found to be present in the ileum tissue of CI mice treated with ciprofloxacin. Furthermore, phosphorylation of serine residues of PDH resulting in inactivating PDH enzymatic activity, which occurred after combined injury, was inhibited with ciprofloxacin treatment, thus enabling PDH to increase ATP production. Increased ileum levels of pyruvate dehydrogenase kinase 1 protein (PDK1, an enzyme responsible for PDH phosphorylation) after combined injury were also prevented by ciprofloxacin treatment. Taken together, these data suggest that ciprofloxacin oral administration after combined injury had a role in sustained ileum ATP levels, and may have acted through preservation of PDH by HSP-70 and inhibition of PDK1. These molecular changes in the ileum are simply one of a host of mechanisms working in concert with one another by which ciprofloxacin treatment mitigates body weight loss and drastically enhances subsequent survival after combined injury. To this end, our findings indicate that oral treatment of ciprofloxacin is a valuable therapeutic treatment after irradiation with combined injury and warrants further analyses to elucidate the precise mechanisms involved.

Exposure to high-dose radiation results in deleterious effects on skeletal tissue. However, the effects of combined trauma such as radiation and hemorrhage on skeletal properties have yet to be elucidated. The purpose of this study was to evaluate the effects of radiation injury combined with hemorrhage on trabecular bone properties and biomarkers of bone metabolism, and to determine whether hemorrhage enhances radiation-associated bone loss. Male CD2F1 mice (10 weeks old) were exposed to one single dose of gamma radiation ((60)Co): 0 or 7.25 Gy. Two hours after irradiation, animals were bled 0% (n = 8) or 20% (n = 8) of total blood volume via the submandibular vein. Mice were euthanized 30 days after irradiation, and distal femora were analyzed using standard histomorphometry to determine changes in trabecular bone volume (BV/TV), thickness (Tb.Th), spacing (Tb.Sp), number (Tb.N) and marrow adipocyte density. Femurs from mice euthanized 1, 7 and 15 days post injury were flushed and total bone marrow cells were counted. Radiation exposure resulted in deleterious effects on distal femur BV/TV (-63%), Tb.Th (-34%), Tb.N (-45%), Tb.Sp (+125%) and adipocyte density (+286%) compared with the sham-irradiated mice (0 Gy; P < 0.05). Hemorrhage after irradiation resulted in greater deleterious effects on the distal femur with BV/TV (-13%), Tb.Th (-44%), Tb.N (-26%), Tb.Sp (+29%) and marrow adipocyte density (+33%) compared with radiation exposure only (P < 0.05). Analysis of the biomarkers of bone metabolism in serum from irradiated and hemorrhaged mice revealed significantly lower levels of osteocalcin (-60%) and procollagen type 1 amino-terminal propeptide (-36%; P1NP, biomarkers of bone formation activity), as well as elevations in sclerostin (+56%; SOST, an inhibitor of bone formation) compared with serum from irradiated only mice (P < 0.05). Additionally, the onset of bone marrow cell depletion in irradiated and hemorrhaged mice occurred earlier and to a greater extent compared to that in irradiated only mice. This study provides definitive, preliminary evidence that hemorrhage further exacerbates trabecular bone loss associated with nonlethal high-dose gamma radiation.

Estrogen receptor-α (ER-α) is an important mediator of the bone response to mechanical loading. We sought to determine whether restricting dietary energy intake by 40% limits the bone formation rate (BFR) response to mechanical loading (LOAD) by downregulating ER-α-expressing osteocytes, or osteoblasts, or both. Female rats (n = 48, 7 mo old) were randomized to ADLIB-SHAM and ADLIB-LOAD groups fed AIN-93M purified diet ad libitum or to ER40-SHAM and ER40-LOAD groups fed modified AIN-93M with 40% less energy (100% of all other nutrients). After 12 wk, LOAD rats were subjected to a muscle contraction protocol three times every third day. ER40 produced lower proximal tibia bone volume (-22%), trabecular thickness (-14%), and higher trabecular separation (+127%) in SHAM but not LOAD rats. ER40 rats exhibited reductions in mineral apposition rate, but not percent mineralizing surface or BFR. LOAD induced similar relative increases in these kinetic measures of osteoblast activity/recruitment in both diet groups., but absolute values for ER40 LOAD rats were lower vs. ADLIB-LOAD. There were fourfold and eightfold increases in proportion of estrogen receptor-α protein-positive osteoblast and osteocytes, respectively, in LOAD vs. SHAM rats, with no effect of ER40. These data suggest that a brief period of mechanical loading significantly affects estrogen receptor-α in cancellous bone osteoblasts and osteocytes. Chronic energy restriction does result in lower absolute values in indices of osteoblast activity after mechanical loading, but not by a smaller increment relative to unloaded bones; this change is not explained by an associated downregulation of ER-α in osteoblasts or osteocytes.

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.

Purpose: This study sought to elucidate the effects of a low- and high-load jump resistance exercise (RE) training protocol on cortical bone of the tibia and femur mid-diaphyses., Methods: Sprague-Dawley rats (male, 6 months old) were randomly assigned to high-load RE (HRE; n = 16), low-load RE (LRE; n = 15), or cage control (CC; n = 11) groups. Animals in the HRE and LRE groups performed 15 sessions of jump RE for 5 wk. Load in the HRE group was progressively increased from 80 g added to a weighted vest (50 repetitions) to 410 g (16 repetitions). The LRE rats completed the same protocol as the HRE group (same number of repetitions), with only a 30-g vest applied., Results: Low- and high-load jump RE resulted in 6%-11% higher cortical bone mineral content and cortical bone area compared with controls, as determined by in vivo peripheral quantitative computed tomography measurements. In the femur, however, only LRE demonstrated improvements in cortical volumetric bone mineral density (+11%) and cross-sectional moment of inertia (+20%) versus the CC group. The three-point bending to failure revealed a marked increase in tibial maximum force (25%-29%), stiffness (19%-22%), and energy to maximum force (35%-55%) and a reduction in elastic modulus (-11% to 14%) in both LRE and HRE compared with controls. Dynamic histomorphometry assessed at the tibia mid-diaphysis determined that both LRE and HRE resulted in 20%-30% higher periosteal mineralizing surface versus the CC group. Mineral apposition rate and bone formation rate were significantly greater in animals in the LRE group (27%, 39%) than those in the HRE group., Conclusion: These data demonstrate that jump training with minimal loading is equally, and sometimes more, effective at augmenting cortical bone integrity compared with overload training in skeletally mature rats.

Purpose: The purpose of this study was to investigate whether partial weight-bearing activity, at either one-sixth or one-third of body mass, blunts the deleterious effects of simulated microgravity (0G) after 21 d on muscle mass and quantitative/qualitative measures of bone., Methods: Using a novel, previously validated partial weight-bearing suspension device, mice were subjected to 16% (G/3, i.e., simulated lunar gravity) or 33% (G/6, i.e., simulated Martian gravity) weight bearing for 21 d. One gravity control (1G, i.e., Earth gravity) and tail-suspended mice (0G, i.e., simulated microgravity) served as controls to compare the effects of simulated lunar and Martian gravity to both Earth and microgravity., Results: Simulated microgravity (0G) resulted in an 8% reduction in body mass and a 28% lower total plantarflexor muscle mass (for both, P < 0.01) as compared with 1G controls, but one-sixth and one-third partial weight-bearing activity attenuated losses. Relative to 1G controls, trabecular bone volume fraction (-9% to -13%) and trabecular thickness (-10% to -14%) were significantly lower in all groups (P < 0.01). In addition, cancellous and cortical bone formation rates (BFR) were lower in all reduced weight-bearing groups compared with 1G controls (-46% to -57%, trabecular BFR; -73% to -85%, cortical BFR; P < 0.001). Animals experiencing one-third but not one-sixth weight bearing exhibited attenuated deficits in femoral neck mechanical strength associated with 0G., Conclusion: These results suggest that partial weight bearing (up to 33% of body mass) is not sufficient to protect against bone loss observed with simulated 0 g but does mitigate reductions in soleus mass in skeletally mature female mice.

Introduction: Recent data indicate a direct relationship between the sympathetic nervous system and bone metabolism. The purpose of this study was to evaluate the effects of a beta-1 adrenergic (Adrb1) agonist, dobutamine (DOB), on disuse-induced changes in bone integrity during 28 d of hindlimb unloading (HU)., Methods: Male Sprague-Dawley rats, age 6 months, were assigned to either a normal cage activity (CC) or HU (n = 24/group). Animals were given one daily bolus dose (4 mg·kg body weight a day) of DOB (n = 12) or an equal volume of saline (VEH, n = 12)., Results: In vivo peripheral quantitative computed tomography scans revealed a 9% loss in proximal tibia metaphysis (PTM) volumetric bone mineral density (vBMD) over 28 d of disuse. DOB administration during HU significantly attenuated reductions in PTM vBMD and inhibited reductions in mid-diaphysis tibia cross-sectional moment of inertia. A significant decline in PTM bone formation rate in the HU-VEH group (-56% vs CC-VEH) was completely abolished in the HU-DOB group. Significant reductions in strength of the femoral shaft and neck in the HU-VEH group (14% and 15%, respectively) were prevented with DOB treatment., Conclusion: In conclusion, DOB administration during HU effectively attenuates significant declines in total vBMD at PTM by mitigating associated decrements in bone formation rate. Positive effects of DOB were observed only in unloaded animals, with no effects observed in normal weight-bearing rats. These data provide evidence for the importance of Adrb1 signaling in maintaining osteoblast function during periods of mechanical unloading.

We sought to elucidate the effects of restricting calcium, energy, or food on the skeletal integrity of exercising female rats. Female Sprague-Dawley rats (4 mo old) were randomly assigned to 5 groups (n = 10/group): ad libitum intake of an AIN-93M diet (Research Diets D10012M, Research Diets, Inc.) with no exercise (AL-S) or with exercise (AL-EX) or to 1 of 3 exercising restriction groups [40% restriction of calcium only (CAR-EX), energy only (ER-EX), or food (FR-EX)]. All EX rats were treadmill trained 3 d/wk, 45 min/d for 12 wk at ~60% maximal oxygen consumption. After 12 wk, total body bone mineral content (by DXA) and body mass, but not lean mass, were lower in ER-EX (-17%) and FR-EX rats (-13%) compared with the AL-EX group. CAR-EX had few negative effects on bone geometry (by peripheral quantitative computed tomography) or histomorphometry. However, declines in total volumetric bone mineral density at the proximal tibia metaphysic (PTM) were observed in ER-EX (-6%) and FR-EX (-8%) groups; only FR-EX rats exhibited increased osteoclast surface and decreased mineral apposition rate in PTM cancellous bone. Decrements in serum estradiol, uterine weights, or both in these 2 groups implicate altered estrogen status as contributory. Urine pH declined significantly by 12 wk in all restricted groups, but net acid excretion increased only in CAR-EX rats. These findings, when compared with published data on sedentary rats, suggest that treadmill running exercise may mitigate some, but not all, deleterious effects on bone after chronic energy or food restriction but is more protective during calcium restriction.

Mechanical loading modulates the osteocyte-derived protein sclerostin, a potent inhibitor of bone formation. We hypothesized that simulated resistance training (SRT), combined with alendronate (ALEN) treatment, during hindlimb unloading (HU) would most effectively mitigate disuse-induced decrements in cortical bone geometry and formation rate (BFR). Sixty male, Sprague-Dawley rats (6-mo-old) were randomly assigned to either cage control (CC), HU, HU plus either ALEN (HU+ALEN), or SRT (HU+SRT), or combined ALEN and SRT (HU+SRT/ALEN) for 28 days. Computed tomography scans on days -1 and 28 were taken at the middiaphyseal tibia. HU+SRT and HU+SRT/ALEN rats were subjected to muscle contractions once every 3 days during HU (4 sets of 5 repetitions; 1,000 ms isometric + 1,000 ms eccentric). The HU+ALEN and HU+SRT/ALEN rats received 10 μg/kg ALEN 3 times/wk. Compared with the CC animals, HU suppressed the normal slow growth-induced increases of cortical bone mineral content, cortical bone area, and polar cross-sectional moment of inertia; however, SRT during HU restored cortical bone growth. HU suppressed middiaphyseal tibia periosteal BFR by 56% vs. CC (P < 0.05). However, SRT during HU restored BFR at both periosteal (to 2.6-fold higher than CC) and endocortical (14-fold higher than CC) surfaces (P < 0.01). ALEN attenuated the SRT-induced BFR gains during HU. The proportion of sclerostin-positive osteocytes in cortical bone was significantly higher (+121% vs. CC) in the HU group; SRT during HU effectively suppressed the higher proportion of sclerostin-positive osteocytes. In conclusion, a minimum number of high-intensity muscle contractions, performed during disuse, restores cortical BFR and suppress unloading-induced increases in sclerostin-positive osteocytes.

The purpose of this study was to assess the effectiveness of simulated resistance training (SRT) exercise combined with alendronate (ALEN) in mitigating or preventing disuse-associated losses in cancellous bone microarchitecture and formation. Sixty male Sprague-Dawley rats (6 months old) were randomly assigned to either cage control (CC), hind limb unloading (HU), HU plus either ALEN (HU + ALEN), SRT (HU + SRT), or a combination of ALEN and SRT (HU + SRT/ALEN) for 28 days. HU + SRT and HU + SRT/ALEN rats were anesthetized and subjected to muscle contractions once every 3 days during HU (four sets of five repetitions, 1000 ms isometric + 1000 ms eccentric). Additionally, HU + ALEN and HU + SRT/ALEN rats received 10 µg/kg of body weight of ALEN three times per week. HU reduced cancellous bone-formation rate (BFR) by 80%, with no effect of ALEN treatment (-85% versus CC). SRT during HU significantly increased cancellous BFR by 123% versus CC, whereas HU + SRT/ALEN inhibited the anabolic effect of SRT (-70% versus HU + SRT). SRT increased bone volume and trabecular thickness by 19% and 9%, respectively, compared with CC. Additionally, osteoid surface (OS/BS) was significantly greater in HU + SRT rats versus CC (+32%). Adding ALEN to SRT during HU reduced Oc.S/BS (-75%), Ob.S/BS (-72%), OS/BS (-61%), and serum TRACP5b (-36%) versus CC. SRT and ALEN each independently suppressed a nearly twofold increase in adipocyte number evidenced with HU and inhibited increases in osteocyte apoptosis. These results demonstrate the anabolic effect of a low volume of high-intensity muscle contractions during disuse and suggest that both bone resorption and bone formation are suppressed when SRT is combined with bisphosphonate treatment., (Copyright © 2011 American Society for Bone and Mineral Research.)

This study sought to elucidate the effects of a low- and high-load jump resistance exercise (RE) training protocol on cancellous bone of the proximal tibia metaphysis (PTM) and femoral neck (FN). Sprague-Dawley rats (male, 6 mo old) were randomly assigned to high-load RE (HRE; n = 16), low-load RE (LRE; n = 15), or sedentary cage control (CC; n = 11) groups. Animals in the HRE and LRE groups performed 15 sessions of jump RE during 5 wk of training. PTM cancellous volumetric bone mineral density (vBMD), assessed by in vivo peripheral quantitative computed tomography scans, significantly increased in both exercise groups (+9%; P < 0.001), resulting in part from 130% (HRE; P = 0.003) and 213% (LRE; P < 0.0001) greater bone formation (measured by standard histomorphometry) vs. CC. Additionally, mineralizing surface (%MS/BS) and mineral apposition rate were higher (50-90%) in HRE and LRE animals compared with controls. PTM bone microarchitecture was enhanced with LRE, resulting in greater trabecular thickness (P = 0.03) and bone volume fraction (BV/TV; P = 0.04) vs. CC. Resorption surface was reduced by nearly 50% in both exercise paradigms. Increased PTM bone mass in the LRE group translated into a 161% greater elastic modulus (P = 0.04) vs. CC. LRE and HRE increased FN vBMD (10%; P < 0.0001) and bone mineral content (∼ 20%; P < 0.0001) and resulted in significantly greater FN strength vs. CC. For the vast majority of variables, there was no difference in the cancellous bone response between the two exercise groups, although LRE resulted in significantly greater body mass accrual and bone formation response. These results suggest that jumping at minimal resistance provides a similar anabolic stimulus to cancellous bone as jumping at loads exceeding body mass.

This study was designed to determine the effectiveness of simulated resistance training (SRT) without weight bearing in attenuating bone and muscle loss during 28 day hindlimb unloading (HU) in mature male rats. An ambulatory control group (CC) and four groups of HU rats were used: HU, HU + anesthesia (ANHU), HU + eccentric muscle contractions (HU + ECC), and HU + isometric and eccentric muscle contractions (HU + ISO/ECC). Animals in the two SRT groups were trained once every other day at 100% daily peak isometric torque (P(0)). HU resulted in significantly lower plantarflexor muscle mass (-33% versus CC) and reduced isometric strength (-10%), which reductions were partially attenuated in both training groups. Significantly reduced total and cancellous volumetric bone mineral density (vBMD) and total bone mineral content (BMC) at the proximal tibia metaphysis (PTM) also was evidenced in HU and ANHU groups compared with both SRT groups (p < .05). Training resulted in greater increases in cortical bone mass and area compared with all other groups (p < .05). Fourfold higher material properties of PTM cancellous bone were demonstrated in SRT animals versus HU or CC animals. A significant reduction in midshaft periosteal bone formation rate (BFR) in the HU group (-99% versus CC) was completely abolished in HU + ECC (+656% versus CC). These results demonstrate that high-intensity muscle contractions, independent of weight-bearing forces, can effectively mitigate losses in muscle strength and provide a potent stimulus to bone during prolonged disuse., (Copyright 2010 American Society for Bone and Mineral Research.)

Osteopenia and an enhanced risk of fracture often accompany type 1 diabetes. However, the association between type 2 diabetes and bone mass has been ambiguous with reports of enhanced, reduced, or similar bone mineral densities (BMDs) when compared with healthy individuals. Recently, studies have also associated type 2 diabetes with increased fracture risk even in the presence of higher BMDs. To determine the temporal relationship between type 2 diabetes and bone remodeling structural and mechanical properties at various bone sites were analyzed during pre-diabetes (7 weeks), short-term (13 weeks), and long-term (20 weeks) type 2 diabetes. BMDs and bone strength were measured in the femora and tibiae of Zucker diabetic fatty rats, a model of human type 2 diabetes. Increased BMDs (9-10%) were observed in the distal femora, proximal tibiae, and tibial mid- shafts in the pre-diabetic condition that corresponded with higher plasma insulin levels. During short- and long-term type 2 diabetes, various parameters of bone strength and BMDs were lower (9-26%) in the femoral neck, distal femora, proximal tibiae, and femoral and tibial mid-shafts. Correspondingly, blood glucose levels increased by 125% and 153% during short- and long-term diabetes respectively. These data indicate that alterations in BMDs and bone mechanical properties are closely associated with the onset of hyperinsulinemia and hyperglycemia, which may have direct adverse effects on skeletal tissue. Consequently, disparities in the human literature regarding the effects of type 2 diabetes on skeletal properties may be associated with the bone sites studied and the severity or duration of the disease in the patient population studied.