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1.  An immunohistochemical study of the antinociceptive effect of calcitonin in ovariectomized rats 
Calcitonin is used as a treatment to reduce the blood calcium concentration in hypercalcemia and to improve bone mass in osteoporosis. An analgesic effect of calcitonin has been observed and reported in clinical situations. Ovariectomaized (OVX) rats exhibit the same hormonal changes as observed in humans with osteoporosis and are an animal model of postmenopousal osteoporosis. The aim of this study to investigate antinociceptive effect of calcitonin in OVX rats using the immunohistochemical study.
We assessed the antinociceptive effects of calcitonin in an ovariectomized (OVX) rat model, which exhibit osteoporosis and hyperalgesia, using the immunohistochemical method. Fifteen rats were ovariectomized bilaterally, and ten rats were received the same surgery expected for ovariectomy as a sham model. We used five groups: the OVX-CT (n = 5), the sham-CT (n = 5), and the OVX-CT-pcpa (n = 5) groups recieved calcitonin (CT: 4 U/kg/day), while OVX-vehi (n = 5) and the sham-vehi (n = 5) groups received vehicle subcutaneously 5 times a week for 4 weeks. The OVX-CT-pcpa-group was given traperitoneal injection of p-chlorophenylalanine (pcpa; an inhibitor of serotonin biosynthesis) (100 mg/kg/day) in the last 3 days of calcitonon injection. Two hours after 5% formalin (0.05 ml) subcutaneously into the hind paw, the L5 spinal cord were removed and the number of Fos-immunoreactive (ir) neurons were evaluated using the Mann-Whitney-U test.
The numbers of Fos-ir neurons in the OVX-CT and sham-CT groups were significantly less than in the OVX-vehi and sham-vehi groups, respectively (p = 0.0090, p = 0.0090). The number of Fos-ir neurons in the OVX-CT-pcpa-group was significantly more than that of the OVX-CT-group (p = 0.0283), which means pcpa inhibits calcitonin induced reduction of c-Fos production.
The results in this study demonstrated that 1) the increase of c-Fos might be related to hyperalgesia in OVX-rats. 2) Calcitonin has an antinociceptive effect in both OVX and sham rats. 3) The central serotonergic system is involved in the antinociceptive properties of calcitonin.
PMCID: PMC2654564  PMID: 19077321
2.  Olive Oil effectively mitigates ovariectomy-induced osteoporosis in rats 
Osteoporosis, a reduction in bone mineral density, represents the most common metabolic bone disease. Postmenopausal women are particularly susceptible to osteoporosis when their production of estrogen declines. For these women, fracture is a leading cause of morbidity and mortality. This study was conducted to evaluate the protective effects of olive oil supplementation against osteoporosis in ovariectomized (OVX) rats.
We studied adult female Wistar rats aged 12-14 months, divided into three groups: sham-operated control (SHAM), ovariectomized (OVX), and ovariectomized rats supplemented with extravirgin olive oil (Olive-OVX) orally for 12 weeks; 4 weeks before ovariectomy and 8 weeks after. At the end of the experiment, blood samples were collected. Plasma levels of calcium, phosphorus, alkaline phosphatase (ALP), malondialdehyde (MDA), and nitrates were assayed. Specimens from both the tibia and the liver were processed for light microscopic examination. Histomorphometric analysis of the tibia was also performed.
The OVX-rats showed a significant decrease in plasma calcium levels, and a significant increase in plasma ALP, MDA, and nitrates levels. These changes were attenuated by olive oil supplementation in the Olive-OVX rats. Light microscopic examination of the tibia of the OVX rats revealed a significant decrease in the cortical bone thickness (CBT) and the trabecular bone thickness (TBT). In addition, there was a significant increase in the osteoclast number denoting bone resorption. In the Olive-OVX rats these parameters were markedly improved as compared to the OVX group. Examination of the liver specimens revealed mononuclear cellular infiltration in the portal areas in the OVX-rats which was not detected in the Olive-OVX rats.
Olive oil effectively mitigated ovariectomy-induced osteoporosis in rats, and is a promising candidate for the treatment of postmenopausal osteoporosis.
PMCID: PMC3045997  PMID: 21294895
3.  The combination therapy with alfacalcidol and risedronate improves the mechanical property in lumbar spine by affecting the material properties in an ovariectomized rat model of osteoporosis 
We conducted the present study to investigate the therapeutic effects of a combination treatment of alfacalcidol (ALF) and risedronate (RIS) on the bone mechanical properties of bone and calcium (Ca) metabolism using an ovariectomized (OVX) rat model of osteoporosis.
Female Wistar rats were OVX- or sham-operated at 40 weeks of age. Twelve weeks post-surgery, rats were randomized into seven groups: 1) sham + vehicle, 2) OVX + vehicle, 3) OVX + ALF 0.025 μg/kg/day, 4) OVX + ALF 0.05 μg, 5) OVX + RIS 0.3 mg, 6) OVX + RIS 3.0 mg, 7) OVX + ALF 0.025 μg + RIS 0.3 mg. Each drug was administered orally five times a week for 12 weeks. After treatment, we evaluated the mechanical properties of the lumbar vertebra and femoral midshaft. In the lumbar vertebra, structural and material analyses were performed using micro-computed tomography (micro-CT) and microbeam X-ray diffraction (micro-XRD), respectively. Biochemical markers in serum and urine were also determined.
(1) With respect to improvement in the mechanical strength of the lumbar spine and the femoral midshaft, the combination treatment of ALF and RIS at their sub-therapeutic doses was more effective than each administered as a monotherapy; (2) In the suppression of bone resorption and the amelioration of microstructural parameters, the effects of ALF and RIS were considered to be independent and additive; (3) The improvement of material properties, such as microstructural parameters and the biological apatite (Bap) c-axis orientation, contributed to the reinforcement of spinal strength; and (4) The combination treatment of ALF and RIS normalized urinary Ca excretion, suggesting that this treatment ameliorated the changes in Ca metabolism.
These results demonstrate that the combination treatment of ALF and RIS at their sub-therapeutic doses can improve the mechanical properties of the spine as well as the femur and ameliorate changes in Ca metabolism in an animal model of osteoporosis, suggesting that the combination treatment of ALF and RIS has a therapeutic advantage over each monotherapy for the treatment of osteoporosis.
PMCID: PMC2706792  PMID: 19527501
4.  Nigella Sativa reverses osteoporosis in ovariectomized rats 
Osteoporosis poses a significant public health issue. It is a skeletal disorder characterized by compromised bone strength that predisposes to increased risk of fracture. There is a direct relationship between the lack of estrogen after menopause and the development of osteoporosis. About 33% of women over 50 will experience bone fractures as a result of osteoporosis. Nigella Sativa (NS) has been shown to have beneficial effects on bone and joint diseases. The present study was conducted to elucidate the protective effect of Nigella Sativa on osteoporosis produced by ovariectomy in rats.
Female Wistar rats aged 12–14 months were divided into three groups: sham-operated control (SHAM), ovariectomized (OVX), and ovariectomized supplemented with nigella sativa (OVX-NS) orally for 12 weeks; 4 weeks before ovariectomy and 8 weeks after. After 12 weeks, plasma levels of calcium (Ca+2), phosphorous (Pi), alkaline phosphatase (ALP), amino terminal collagen type 1 telopeptide, malondialdehyde (MDA), nitrates, nitric oxide surrogate, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were measured. Histological examination of the liver and the tibia was conducted. Histomorphometric analysis of the tibia was also performed.
OVX rats showed significant decrease in plasma Ca+2, accompanied by a significant increase in plasma ALP, amino terminal collagen type 1 telopeptide, MDA, nitrates, TNF-α and IL-6. These changes were reversed by NS supplementation in OVX-NS group to be near SHAM levels. Histological examination of the tibias revealed discontinuous eroded bone trabeculae with widened bone marrow spaces in OVX rats accompanied by a significant decrease in both cortical and trabecular bone thickness compared to Sham rats. These parameters were markedly reversed in OVX-NS rats. Histological examination of the liver showed mononuclear cellular infiltration and congestion of blood vessels at the portal area in OVX rats which were not found in OVX-NS rats.
Nigella sativa reverses osteoporosis in ovariectomized rats, which could be attributed to its high content of unsaturated fatty acids as well as its antioxidant and anti-inflammatory properties.
PMCID: PMC3898005  PMID: 24422662
Osteoporosis; Nigella sativa; Ovariectomy; Rats
5.  Establishing a rapid animal model of osteoporosis with ovariectomy plus low calcium diet in rats 
The objective of this study was to rapidly develop osteoporotic model animals by combining ovariectomy with a low calcium diet in rats. Thirty, eight-week-old, female, Sprague-Dawley rats were either sham-operated (Sham) or ovariectomized (Ovx) and divided into three groups: Sham, Ovx, and Ovx + low calcium diet. Rats in the Sham and Ovx groups were fed a standard diet containing 1.1% w/w calcium while rats in the Ovx + low calcium diet group were fed a diet containing 0.1% w/w calcium. Serum osteocalcin and bone mineral density (BMD) of the lumbar vertebrae were measured 4 and 8 weeks after surgery. The rats were euthanized 12 weeks after surgery, and the BMD of the right femur and histomorphometry of the femoral neck were assessed at that time. The Ovx + low-calcium diet group had a significantly lower mean BMD of the lumbar vertebra and higher mean serum osteocalcin concentration than the Sham and Ovx groups. Twelve weeks after surgery, rats in the Ovx + low calcium diet group had a significantly lower BMD, smaller Tb.Th and Tb.N, and larger Tb.Sp of the right femoral neck than did rats in the Sham and Ovx groups. These data indicate that a low calcium diet can significantly accelerate bone loss in ovariectomized rats. Combining ovariectomy and a low calcium diet can save considerable time in the creation of osteoporotic model animals.
PMCID: PMC4152076  PMID: 25197386
Animal model; osteoporosis; ovariectomy; low calcium diet
6.  Effect of dietary legumes on bone-specific gene expression in ovariectomized rats 
Nutrition Research and Practice  2013;7(3):185-191.
In previous studies, we found that the consumption of legumes decreased bone turnover in ovariectomized rats. The purpose of the present study is to determine whether the protective effects on bone mineral density (BMD) and the microarchitecture of a diet containing legumes are comparable. In addition, we aim to determine their protective actions in bones by studying bone specific gene expression. Forty-two Sprague-Dawley rats are being divided into six groups during the 12 week study: 1) rats that underwent sham operations (Sham), 2) ovariectomized rats fed an AIN-93M diet (OVX), 3) ovariectomized rats fed an AIN-93M diet with soybeans (OVX-S), 4) ovariectomized rats fed an AIN-93M diet with mung beans (OVX-M), 5) ovariectomized rats fed an AIN-93M diet with cowpeas (OVX-C), and 6) ovariectomized rats fed an AIN-93M diet with azuki beans (OVX-A). Consumption of legumes significantly increased BMD of the spine and femur and bone volume of the femur compared to the OVX. Serum calcium and phosphate ratio, osteocalcin, expression of osteoprotegerin (OPG), and the receptor activator of nuclear factor κB ligand (RANKL) ratio increased significantly, while urinary excretion of calcium and deoxypyridinoline and expression of TNF-α and IL-6 were significantly reduced in OVX rats fed legumes, compared to OVX rats that were not fed legumes. This study demonstrates that consumption of legumes has a beneficial effect on bone through modulation of OPG and RANKL expression in ovariectomized rats and that legume consumption can help compensate for an estrogen-deficiency by preventing bone loss induced by ovarian hormone deficiency.
PMCID: PMC3679327  PMID: 23766879
Bone mass density; bone-specific gene expression; cytokines; legumes; ovariectomized rats
7.  Effects of green tea or Sasa quelpaertensis bamboo leaves on plasma and liver lipids, erythrocyte Na efflux, and platelet aggregation in ovariectomized rats 
Nutrition Research and Practice  2012;6(2):106-112.
This study was conducted to investigate the effects of Sasa quelpaertensis bamboo and green tea on plasma and liver lipids, platelet aggregation, and erythrocyte membrane Na channels in ovariectomized (OVX) rats. Thirty female rats were OVX, and ten female rats were sham-operated at the age of 6 weeks. The rats were divided into four groups at the age of 10 weeks and fed the experiment diets: sham-control, OVX-control, OVX-bamboo leaves (10%), or OVX-green tea leaves (10%) for four weeks. Final body weight increased significantly in the OVX groups compared with that in the sham-control, whereas body weight in the OVX-green tea group decreased significantly compared with that in the OVX-control (P < 0.01). High density lipoprotein (HDL)-cholesterol level decreased in all OVX groups compared with that in the sham-control rats (P < 0.05) but without a difference in plasma total cholesterol. Plasma triglycerides in the OVX-green tea group were significantly lower than those in the sham-control or OVX-control group (P < 0.05). Liver triglycerides increased significantly in the OVX-control compared with those in the sham-control (P < 0.01) but decreased significantly in the OVX-green tea group compared with those in the OVX-control or OVX-bamboo group (P < 0.01). Platelet aggregation in both maximum and initial slope tended to be lower in all OVX rats compared with that in the sham-control rats but was not significantly different. Na-K ATPase tended to increase and Na-K cotransport tended to decrease following ovariectomy. Na-K ATPase decreased significantly in the OVX-green tea group compared with that in the OVX-control group (P < 0.01), and Na-K cotransport increased significantly in the OVX-bamboo and OVX-green tea groups compared with that in the OVX-control (P < 0.05). Femoral bone mineral density tended to be lower in OVX rats than that in the sham-control, whereas the green tea and bamboo leaves groups recovered bone density to some extent. The results show that ovariectomy caused an increase in body weight and liver triglycerides, and that green tea was effective for lowering body weight and triglycerides in OVX rats. Ovariectomy induced an increase in Na efflux via Na-K ATPase and a decrease in Na efflux via Na-K cotransport. Furthermore, consumption of green tea and bamboo leaves affected Na efflux channels, controlling electrolyte and body water balance.
PMCID: PMC3349031  PMID: 22586498
Green tea; Sasa quelpaertensis bamboo; Na efflux channel; platelet aggregation; ovariectomized rats
8.  Genistein, a phytoestrogen, improves total cholesterol and Synergy®, a prebiotic, improved calcium utilization but there were no synergistic effects 
Menopause (New York, N.Y.)  2011;18(8):923-931.
Prebiotics and phytoestrogens have sparked great interest because evidence indicates that consumption of these dietary constituents leads to lower cholesterol levels and inhibition of postmenopausal bone loss. The aim of this study was to determine the effect of both a prebiotic (Synergy®) and phytoestrogen (genistein) on bone and blood lipid levels in an animal model of postmenopausal women.
A four week feeding study was conducted in 5 mo old ovariectomized (OVX) Sprague Dawley rats to examine the effect of genistein, Synergy® (prebiotic) and genistein + Synergy® on bone density and strength, calcium metabolism and lipid biomarkers. There were six treatment groups: SHAM Control, OVX Control, OVX rats receiving daily estradiol injections, and OVX rats receiving an AIN 93M diet supplement with either 200 ppm genistein, 5% Synergy®, or 200 ppm genistein + 5% Synergy®.
Rats receiving genistein had significantly lower total serum cholesterol concentration than OVX Control (17%), OVX rats receiving daily estradiol injections (14%), and OVX rats on the 5% Synergy® diet (19%). Synergy® improved calcium absorption efficiency (41%) compared to OVX Control. SHAM Control rats had significantly higher femoral bone density, as determined by underwater weighing, than all OVX groups. Genistein consumption restored total and trabecular BMD at the distal femur similar to levels of SHAM rats.
Genistein supplementation imparts modest heart health benefits and improves bone geometry at the distal femur, and prebiotic consumption (Synergy®) results in improved calcium utilization strength in ovariectomized rats but the combination produced no synergistic effects.
PMCID: PMC3181048  PMID: 21659907
genistein; prebiotics; cholesterol; bone rodents
9.  Fermented soybeans by Rhizopus oligosporus reduce femoral bone loss in ovariectomized rats 
Nutrition Research and Practice  2014;8(5):539-543.
Soy isoflavones are structurally similar to estrogen and bind to estrogen receptors, suggesting that they exhibit estrogenic activities; therefore, they are referred to as phytoestrogens. Fermentation may affect the bioavailability of isoflavones altering soy isoflavone glycosides in the form of aglycones. Thus, this study investigated the effects of fermented soybeans by Rhizopus oligosporus on bone metabolism in both young rats as a pilot test and in ovariectomized (ovx) old rats as a model of menopause.
In the pilot test, a total of 24 seven-week-old female Sprague-Dawley (SD) rats were fed one of three diets for a period of four weeks: casein, unfermented soybean product, or fermented soybean product by R. oligosporus. In the ovx rat model, 20-week-old SD rats weighing 260-290 g underwent either sham-operation (n = 10) or bilateral ovariectomy (n = 30) and were then fed the AIN-93M diet for one week. Thereafter, rats were fed sham-casein, ovx-casein, ovx-soybean, or ovx-fermented soybean diet for five weeks. After decapitation, femoral bones were isolated and preserved in 9% formalin for assessment of bone mineral density (BMD), bone mineral content (BMC), and bone-breaking strength (BBS).
Ovx rats showed significantly increased weight gain and decreased uterine wet weight. Of particular interest, ovx rats fed fermented soybeans showed increased uterine wet weights compared to control rats. Fermented soybean diet caused a significant increase in plasma 17-β estradiol concentrations in young rats, and 17-β estradiol levels were enhanced in ovx rats to match those of sham-operated ones. Significantly lower femoral BMD and BMC were observed in ovx rats compared to sham-operated controls, whereas bone areas did not differ statistically among the groups. In addition, BBS tended to be increased in ovx rats fed soybeans and fermented soybeans.
Supplementation of fermented soybeans could have preventive and therapeutic effects against osteoporosis in postmenopausal women.
PMCID: PMC4198967  PMID: 25324934
Fermented soybean; 17-β estradiol; bone mineral density (BMD); bone-breaking strength
10.  The influence of long-term treadmill exercise on bone mass and articular cartilage in ovariectomized rats 
Loss of bone quality and deterioration of articular cartilage are commonly seen after menopause. While exercise may protect against tissue degeneration, a clear link has yet to be established. The aim of the present study is to investigate the influence of long-term treadmill exercise on changes in bone mass and articular cartilage in ovariectomized rats.
Sixty female Sprague-Dawley rats were randomly assigned to 4 groups: ovariectomized (OVX), ovariectomized plus treadmill exercise (OVX-RUN), treadmill exercise alone (RUN), and control (CON) groups. After 36 weeks, the following variables were compared among the 4 groups. Bone mass was evaluated by trabecular bone volume and bone mineral density (BMD). Articular cartilage in the knee joints was evaluated by histology analysis and a modified Mankin score.
Rats in the ovariectomized groups (OVX and OVX-RUN) had significantly lower BMD and bone mass than the non-ovariectomized rats (CON and RUN), indicating that exercise did little to preserve bone mass. However, the sedentary OVX group had a significantly worse modified Mankin score (7.7 ± 1.4) than the OVX-RUN group (4.8 ± 1.0), whose scores did not differ significantly from the other 2 non-operated groups. The articular cartilage in the sedentary OVX rats was relatively thinner, hypocellular, and had more clefts than in the other 3 groups.
This study suggests that long-term exercise protects articular cartilage in OVX rats but does not retard the loss of bone mass seen in after menopause.
PMCID: PMC2936345  PMID: 20727126
11.  Preventive Effects of Collagen Peptide from Deer Sinew on Bone Loss in Ovariectomized Rats 
Deer sinew (DS) has been used traditionally for various illnesses, and the major active constituent is collagen. In this study, we assessed the effects of collagen peptide from DS on bone loss in the ovariectomized rats. Wister female rats were randomly divided into six groups as follows: sham-operated (SHAM), ovariectomized control (OVX), OVX given 1.0 mg/kg/week nylestriol (OVX + N), OVX given 0.4 g/kg/day collagen peptide (OVX + H), OVX given 0.2 g/kg/day collagen peptide (OXV + M), and OVX given 0.1 g/kg/day collagen peptide (OXV + L), respectively. After 13 weeks of treatment, the rats were euthanized, and the effects of collagen peptide on body weight, uterine weight, bone mineral density (BMD), serum biochemical indicators, bone histomorphometry, and bone mechanics were observed. The data showed that BMD and concentration of serum hydroxyproline were significantly increased and the levels of serum calcium, phosphorus, and alkaline phosphatase were decreased. Besides, histomorphometric parameters and mechanical indicators were improved. However, collagen peptide of DS has no effect on estradiol level, body weight, and uterine weight. Therefore, these results suggest that the collagen peptide supplementation may also prevent and treat bone loss.
PMCID: PMC4102020  PMID: 25101135
12.  Tibia bone properties at different time course of ovariectomized rats 
The model of bilaterally ovariectomized rats mimics the accelerated bone loss observed in postmenopausal women due to estrogen deficiency. Although calcium is main mineral in bone, previous study in human showed there is hypermineralization and higher calcium level in hydroxyapatite crystal structure from osteoporosis patients. This study was aimed to investigate the effect of time course ovariectomized on tibia bone turn over markers, mineral elements, hydroxyapatite crystale, mesostructure, and histomorphometry.
A total of 30 Wistar female rats were randomly assigned into three groups (n = 10 each): control group, ovariectomy group follow up for one month and two month. All animals procedures was according to Animal Ethics Guidelines and approval by ethic committee of the Medical Faculty, Lambung Mangkurat University which obtained prior the study. Expression of osteocalcin (OC) and C-telopeptyde collagen type I (CTX) was analyzed by ELISA method. Tibia bone mineral element was measured using X-Ray Fluorescence. Hydroxyapatite crystale structure was analyzed using X-Ray Diffracttion. Mesostructure was determined using Scanning Electron Microscope. Histomorphometry was analyzed using BoneJ software analyzer. ANOVA test was used to analyze the different level of serum bone turnover markers and bone mineral elements.
Serum OC and CTX were significantly decrease in one month and two month after ovariectomized groups compared to sham-operated group (P < 0.05). The levels Ca, P, Fe, Cu, Zn, Ni, Ca/P, and Cu/Zn were not significantly different in all groups (P > 0.05). The structure of hydroxyapatite crystal in one month and two month after ovariectomized groups were different compared with sham-operated control group. Mesostructure of tibia bone after one and two month ovariectomized procedure significantly different than that in sham-operated rats. The level of trabecular volume were lower significantly on OVX-1 and OVX-2 groups compared with sham group (P < 0.05). The trabecular thickness and spacing were increase significantly on OVX-1 and OVX-2 groups compared with sham group (P < 0.05). The trabecular number were significantly decrease OVX-1 and OVX-2 groups than that sham group (P < 0.05).
We found that two month after ovariectomized decrease serum osteocalcin but not change bone mineral elements in rats. Also, we found the difference of lattice parameter of hydroxyapatite crystale structure and trabecular properties which determined bone mesostructure.
PMCID: PMC4195878  PMID: 25317398
Trabecular; Osteoblast; Osteoclast; Turn over; Osteoporosis
13.  Differential Maintenance of Cortical and Cancellous Bone Strength Following Discontinuation of Bone-Active Agents 
Osteoporotic patients treated with antiresorptive or anabolic agents experience an increase in bone mass and a reduction in incident fractures. However, the effects of these medications on bone quality and strength after a prolonged discontinuation of treatment are not known. We evaluated these effects in an osteoporotic rat model. Six-month-old ovariectomized (OVX) rats were treated with placebo, alendronate (ALN, 2 µg/kg), parathyroid hormone [PTH(1–34); 20 µg/kg], or raloxifene (RAL, 2 mg/kg) three times a week for 4 months and withdrawn from the treatments for 8 months. Treatment with ALN, PTH, and RAL increased the vertebral trabecular bone volume (BV/TV) by 47%, 53%, and 31%, with corresponding increases in vertebral compression load by 27%, 51%, and 31%, respectively (p < .001). The resulting bone strength was similar to that of the sham-OVX control group with ALN and RAL and higher (p < .001) with PTH treatment. After 4 months of withdrawal, bone turnover (BFR/BS) remained suppressed in the ALN group versus the OVX controls (p < .001). The vertebral strength was higher than in the OVX group only in ALN-treated group (p < .05), whereas only the PTH-treated animals showed a higher maximum load in tibial bending versus the OVX controls (p < .05). The vertebral BV/TV returned to the OVX group level in both the PTH and RAL groups 4 months after withdrawal but remained 25% higher than the OVX controls up to 8 months after withdrawal of ALN (p < .05). Interestingly, cortical bone mineral density increased only with PTH treatment (p < .05) but was not different among the experimental groups after withdrawal. At 8 months after treatment withdrawal, none of the treatment groups was different from the OVX control group for cortical or cancellous bone strength. In summary, both ALN and PTH maintained bone strength (maximum load) 4 months after discontinuation of treatment despite changes in bone mass and bone turnover; however, PTH maintained cortical bone strength, whereas ALN maintained cancellous bone strength. Additional studies on the long-term effects on bone strength after discontinuation and with combination of osteoporosis medications are needed to improve our treatment of osteoporosis. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179292  PMID: 20839286
14.  Organic Nitrate Maintains Bone Marrow Blood Perfusion in Ovariectomized Female Rats: A Dynamic, Contrast-Enhanced Magnetic Resonance Imaging (MRI) Study 
Pharmaceutics  2012;5(1):23-35.
This study investigated the effects of nitrate on bone mineral density (BMD) and bone marrow perfusion in ovariectomized (OVX) female rats, and also the effects of nitrate on in vitro osteoblastic activity and osteoclastic differentiation of murine monocyte/macrophage RAW 264.7 cells. Female Sprague–Dawley rats were divided into OVX + nitrate group (isosorbide-5-mononitrate, ISM, 150 mg/kg/ day b.i.d), OVX + vehicle group, and control group. Lumbar spine CT bone densitometry and perfusion MRI were performed on the rats at baseline and week 8 post-OVX. The OVX rats’ BMD decreased by 22.5% ± 5.7% at week 8 (p < 0.001); while the OVX + ISM rats’ BMD decreased by 13.1% ± 2.7% (p < 0.001). The BMD loss difference between the two groups of rats was significant (p = 0.018). The OVX rats’ lumbar vertebral perfusion MRI maximum enhancement (Emax) decreased by 10.3% ± 5.0% at week 8 (p < 0.005), while in OVX + ISM rats, the Emax increased by 5.5% ± 6.9% (p > 0.05). The proliferation of osteoblast-like UMR-106 cells increased significantly with ISM treatment at 0.78 µM to 50 μM. Treatment of UMR-106 cells with ISM also stimulated the BrdU uptake. After the RAW 264.7 cells were co-treated with osteoclastogenesis inducer RANKL and 6.25 μM ~ 100 μM of ISM for 3 days, a trend of dose-dependent increase of osteoclast number was noted.
PMCID: PMC3834938  PMID: 24300395
nitrates; osteoporosis; DCE MRI; bone mineral density; osteoblast; osteoclast
15.  Efficacy of Static Magnetic Field for Locomotor Activity of Experimental Osteopenia 
In order to examine the effectiveness of applying a static magnetic field (SMF) for increasing bone mineral density (BMD), we assessed the degree of osteopenia by dual-energy X-ray absorptiometry (DEXA), the metabolism measuring system, and histological examination of bone tissue in an ovariectomized (OVX) rat model. Thirty-six female Wistar rats (8 weeks old, 160–180 g) were divided into three groups. The rats in the OVX-M group were exposed to SMF for 12 weeks after ovariectomy. The ovariectomized rats in the OVX-D group were not exposed to SMF as a control. The rats in the normal group received neither ovariectomy nor exposure to SMF. Twelve-week exposure to SMF in the OVX-M group inhibited the reduction in BMD that was observed in the OVX-D group. Moreover, in the OVX rats, before exposure to SMF, there was no clear difference in the level of locomotor activity between the active and resting phases, and the pattern of locomotor activity was irregular. After exposure of OVX rats to SMF, the pattern of locomotor activity became diphasic with clear active and resting phases, as was observed in the normal group. In the OVX-M group, the continuity of the trabecular bone was maintained more favorably and bone mass was higher than the respective parameters in the OVX-D group. These results demonstrate that exposure to SMF increased the level of locomotor activity in OVX rats, thereby increasing BMD.
PMCID: PMC1810356  PMID: 17342247
locomotor activity; ovariectomized (OVX) rat; static magnetic fields (SMF); thermography
16.  Distributional Variations in Trabecular Architecture of the Mandibular Bone: An In Vivo Micro-CT Analysis in Rats 
PLoS ONE  2015;10(1):e0116194.
To evaluate the effect of trabecular thickness and trabecular separation on modulating the trabecular architecture of the mandibular bone in ovariectomized rats.
Materials and Methods
Fourteen 12-week-old adult female Wistar rats were divided into an ovariectomy group (OVX) and a sham-ovariectomy group (sham). Five months after the surgery, the mandibles from 14 rats (seven OVX and seven sham) were analyzed by micro-CT. Images of inter-radicular alveolar bone of the mandibular first molars underwent three-dimensional reconstruction and were analyzed.
Compared to the sham group, trabecular thickness in OVX alveolar bone decreased by 27% (P = 0.012), but trabecular separation in OVX alveolar bone increased by 59% (P = 0.005). A thickness and separation map showed that trabeculae of less than 100μm increased by 46%, whereas trabeculae of more than 200μm decreased by more than 40% in the OVX group compared to those in the sham group. Furthermore, the OVX separation of those trabecular of more than 200μm was 65% higher compared to the sham group. Bone mineral density (P = 0.028) and bone volume fraction (p = 0.001) were also significantly decreased in the OVX group compared to the sham group.
Ovariectomy-induced bone loss in mandibular bone may be related to the distributional variations in trabecular thickness and separation which profoundly impact the modulation of the trabecular architecture.
PMCID: PMC4307973  PMID: 25625431
17.  Semen astragali complanati- and rhizoma cibotii-enhanced bone formation in osteoporosis rats 
Growing evidence shows that herb medicines have some anti-osteoporotic effects, the mechanism underlying is unknown. This study aims to investigate the therapeutic effect of Chinese herb supplements on rats that had osteoporosis-like symptom induced by ovariectomy (OVX).
OVX or sham operations were performed on virgin Wistar rats at three-month old, which were randomly divided into eight groups: sham (sham); OVX control group (OVX); OVX rats with treatments [either diethylstilbestrol (DES) or Semen Astragali Complanati decoction (SACD) or Rhizoma Cibotii decoction (RCD) or Herba Cistanches decoction (HCD) or Semen Allii Tuberosi decoction (SATD)]. Non-surgical rats were served as a normal control (NC). The treatments began 4 weeks after surgery, and lasted for 12 weeks. Bone mass and its turnover were analyzed by histomorphometry. Levels of protein and mRNA of osteoprotegerin (OPG) and receptor activator of nuclear factor κB ligand (RANKL) in osteoblasts (OB) and bone marrow stromal cells (bMSC) were evaluated by immunohistochemistry and in situ hybridization.
Compared to OVX control, TBV% in both SACD and RCD groups was increased significantly, while TRS%, TFS%, MAR, and mAR were decreased remarkably in the SACD group, only TRS% decreased dramatically in the RCD group. No significant changes in bone formation were observed in either HCD or SATD groups. OPG levels in both protein and mRNA were reduced consistantly in OB and bMSC from OVX control rats, in contrast, RANKL levels in both protein and mRNA were increased significantly. These effects were substantially reversed by treatments with either DES or SACD or RCD. No significant changes in both OPG and RANKL expression were observed in OB and bMSC from OVX rats treated with SATD and HCD.
Our study showed that SACD and RCD increased bone formation by stimulating OPG expression and downregulating RANKL expression in OB and bMSC. This suggests that SACD and RCD may be developed as alternative anti-osteoporotic agents for therapy of postmenopausal osteoporosis.
PMCID: PMC3699375  PMID: 23782721
18.  Targeted Delivery of Lovastatin and Tocotrienol to Fracture Site Promotes Fracture Healing in Osteoporosis Model: Micro-Computed Tomography and Biomechanical Evaluation 
PLoS ONE  2014;9(12):e115595.
Osteoporosis is becoming a major health problem that is associated with increased fracture risk. Previous studies have shown that osteoporosis could delay fracture healing. Although there are potential agents available to promote fracture healing of osteoporotic bone such as statins and tocotrienol, studies on direct delivery of these agents to the fracture site are limited. This study was designed to investigate the effects of two potential agents, lovastatin and tocotrienol using targeted drug delivery system on fracture healing of postmenopausal osteoporosis rats. The fracture healing was evaluated using micro CT and biomechanical parameters. Forty-eight Sprague-Dawley female rats were divided into 6 groups. The first group was sham-operated (SO), while the others were ovariectomized (OVx). After two months, the right tibiae of all rats were fractured at metaphysis region using pulsed ultrasound and were fixed with plates and screws. The SO and OVxC groups were given two single injections of lovastatin and tocotrienol carriers. The estrogen group (OVx+EST) was given daily oral gavages of Premarin (64.5 µg/kg). The Lovastatin treatment group (OVx+Lov) was given a single injection of 750 µg/kg lovastatin particles. The tocotrienol group (OVx+TT) was given a single injection of 60 mg/kg tocotrienol particles. The combination treatment group (OVx+Lov+TT) was given two single injections of 750 µg/kg lovastatin particles and 60 mg/kg tocotrienol particles. After 4 weeks of treatment, the fractured tibiae were dissected out for micro-CT and biomechanical assessments. The combined treatment group (OVx+Lov+TT) showed significantly higher callus volume and callus strength than the OVxC group (p<0.05). Both the OVx+Lov and OVx+TT groups showed significantly higher callus strength than the OVxC group (p<0.05), but not for callus volume. In conclusion, combined lovastatin and tocotrienol may promote better fracture healing of osteoporotic bone.
PMCID: PMC4272265  PMID: 25526611
19.  Effects of Multi-Deficiencies-Diet on Bone Parameters of Peripheral Bone in Ovariectomized Mature Rat 
PLoS ONE  2013;8(8):e71665.
Many postmenopausal women have vitamin D and calcium deficiency. Therefore, vitamin D and calcium supplementation is recommended for all patients with osteopenia and osteoporosis. We used an experimental rat model to test the hypothesis that induction of osteoporosis is more efficiently achieved in peripheral bone through combining ovariectomy with a unique multi-deficiencies diet (vitamin D depletion and deficient calcium, vitamin K and phosphorus). 14-week-old Sprague-Dawley rats served as controls to examine the initial bone status. 11 rats were bilaterally ovariectomized (OVX) and fed with multi-deficiencies diet. Three months later the treated group and the Sham group (n = 8) were euthanized. Bone biomechanical competence of the diaphyseal bone was examined on both, tibia and femur. Image analysis was performed on tibia via µCT, and on femur via histological analysis. Lower torsional stiffness indicated inferior mechanical competence of the tibia in 3 month OVX+Diet. Proximal metaphyseal region of the tibia showed a diminished bone tissue portion to total tissue in the µCT despite the increased total area as evaluated in both µCT and histology. Cortical bone showed higher porosity and smaller cross sectional thickness of the tibial diaphysis in the OVX+Diet rats. A lower ALP positive area and elevated serum level of RANKL exhibited the unbalanced cellular interaction in bone remodeling in the OVX+Diet rat after 3 month of treatment. Interestingly, more adipose tissue area in bone marrow indicated an effect of bone loss similar to that observed in osteoporotic patients. Nonetheless, the presence of osteoid and elevated serum level of PTH, BGP and Opn suggest the development of osteomalacia rather than an osteoporosis. As the treatment and fracture management of both osteoporotic and osteomalacia patients are clinically overlapping, this study provides a preclinical animal model to be utilized in local supplementation of minerals, drugs and growth factors in future fracture healing studies.
PMCID: PMC3745426  PMID: 23977109
20.  Therapeutic effects of radix dipsaci, pyrola herb, and cynomorium songaricum on bone metabolism of ovariectomized rats 
The objective of this study was to evaluate the effects of herbal medicines, such as Radix Dipsaci (RDD), Pyrola Herb (PHD), and Cynomorium songaricum decoction (CSD), on osteoporotic rats induced by ovariectomy (OVX).
OVX or sham operations were performed on 69 virgin Wistar rats that were divided into six groups: sham (sham, n = 12), OVX control group (OVX, n = 12), and OVX rats with treatments (diethylstilbestrol, E2, n = 12; RDD, n = 11, PHD, n = 11, and CSD, n = 11). Non-surgical rats served as normal control (NC, n = 12). The treatments began four weeks after surgery and lasted for 12 weeks. Bone mass and bone turnover were analyzed by histomorphometry. Levels of protein expression and mRNA of OPG and RANKL in osteoblasts (OB) and bone marrow stromal cells (bMSC) were evaluated by immunohistochemistry and in situ hybridization.
Compared to NC and sham rats, trabecular bone formation was significantly reduced in OVX rats, but restored in E2-treated rats. Treatment with either RDD or PHD enhanced trabecular bone formation remarkably. No significant change of bone formation was observed in CSD-treated rats. OPG expression of protein and mRNA was reduced significantly in OB and bMSC of OVX control rats. RANKL expression of protein and mRNA was increased significantly in OB and bMSC of OVX control rats. These effects were substantially reversed (increased in OPG and decreased in RANKL) by treatment with E2, RDD, or PHD in OB and bMSC of OVX rats. No significant changes in either OPG or RANKL expression were observed in OB and bMSC of OVX rats treated with CSD.
Our study showed that RDD and PHD increased bone formation by stimulating overexpression of OPG and downregulation of RANKL in OB and bMSC. This suggests that RDD and PHD may be used as alternative therapeutic agents for postmenopausal osteoporosis.
PMCID: PMC3585854  PMID: 22639966
21.  Wnt-Signaling-Mediated Antiosteoporotic Activity of Porcine Placenta Hydrolysates in Ovariectomized Rats 
Anti-osteoporotic effects of two types of porcine placenta hydrolysates (PPH) were evaluated in ovariectomized (OVX) rats orally administered PPH without (WPPH) or with (NPPH) ovarian hormones (1 g/kg bw/day). PPH groups were compared with OVX rats with estrogen replacement (0.1 mg/kg bw conjugated estrogen; EST), or dextrose (placebo; OVX-control) All rats received high-fat/calcium-deficient diets for 12 weeks. NPPH contained less estrogen and progesterone, but more essential amino acids, whereas the opposite was true for WPPH. NPPH decreased body weight and peri-uterine fat pads, and maintained uterus weight. NPPH rats had higher femur and lumbar spine bone mass density compared to controls; but less than those of EST rats. Serum phosphorus and urinary calcium and phosphorus levels were reduced in NPPH rats compared to OVX-controls. Serum bone-specific alkaline phosphatase, osteocalcin, and bone turnover marker levels were reduced NPPH rats compared to OVX-controls. WPPH produced results similar to those of NPPH, but less significant. Both NPPH and estrogen upregulated low-density lipoprotein receptor-related protein 5 and β-catenin in OVX rats, while the expression of dickkopf-related protein 1 was suppressed. In conclusion, NPPH exerted anti-osteoporotic effects by activating osteogenesis and stimulating Wnt signaling, possibly mediated by the various amino acids and not ovarian hormones.
PMCID: PMC3522380  PMID: 23258987
22.  Greater Skeletal Gains in Ovary Intact Rats at Maturity Are Achieved by Supplementing a Standardized Extract of Butea monosperma Stem Bark that Confers Better Bone Conserving Effect following Ovariectomy and Concurrent Treatment Withdrawal 
With a longitudinally designed study, we tested whether an acetone soluble fraction (ASF) from the stem bark of Butea monosperma resulted in maximizing bone gain in rats during growth and maturation and thus protected against osteopenia following ovariectomy (OVx) with concomitant treatment withdrawal. Female rats at weaning were given ASF (100 mg/kg/d) or vehicle for 12 weeks, and baseline skeletal parameters (micro-CT) and total plasma antioxidant status (TAS) were measured. At this stage, one group was OVx and the other group was sham operated. Vehicle group (untreated) after OVx was given E2 or continued with vehicle (OVx control). ASF group after OVx was given vehicle (ASF withdrawn, ASFW). After another 12 weeks, all groups were killed and various skeletal parameters were determined. ASF resulted in substantially better skeletal parameters and higher plasma TAS over control at maturity. Rats treated with ASF before OVx had reduced rates of bone loss compared to OVx control. Twelve weeks after OVx, the ASFW group exhibited better trabecular microarchitectural preservation, bone turnover profiles, increased cortical deposition, and biomechanical strength over the OVx control, and the effects were comparable to OVx + E2 group. ASF supplementation during skeletal growth could maximize bone accrual and could confer increased resistance to post-OVx osteopenia despite treatment withdrawal.
PMCID: PMC3655608  PMID: 23710224
23.  Anti-resorptive Agents Reduce the Size of Resorption Cavities: A Three-Dimensional Dynamic Bone Histomorphometry Study 
Bone  2013;57(1):10.1016/j.bone.2013.08.018.
Alterations in resorption cavities and bone remodeling events during anti-resorptive treatment are believed to contribute to reductions in fracture risk. Here, we examine changes in the size of individual remodeling events associated with treatment with a selective estrogen receptor modulator (raloxifene) or a bisphosphonate (risedronate). Adult female rats (6 months of age) were submitted to ovariectomy (n = 17) or sham surgery (SHAM, n = 5). One month after surgery, the ovariectomized animals were separated into three groups: untreated (OVX, n = 5), raloxifene treated (OVX+Ral, n = 6) and risedronate treated (OVX+Ris, n = 6). At 10 months of age, the lumbar vertebrae were submitted to three-dimensional dynamic bone histomorphometry to examine the size (depth, breadth, volume) of individual resorption cavities and formation events. Maximum resorption cavity depth did not differ between the SHAM (23.66 ± 1.87 µm, mean ± SD) and OVX (22.88 ± 3.69 µm) groups but was smaller in the OVX+Ral (14.96 ± 2.30 µm) and OVX+Ris (14.94 ± 2.70 µm) groups (p < 0.01). Anti-resorptive treatment was associated with reductions in the surface area of resorption cavities and the volume occupied by each resorption cavity (p < 0.01 each). The surface area and volume of individual formation events (double-labeled events) in the OVX+Ris group were reduced as compared to other groups (p < 0.02). Raloxifene treated animals showed similar amounts of bone remodeling (ES/BS and dLS/BS) compared to sham-operated controls but smaller cavity size (depth, breadth and volume). The current study shows that anti-resorptive agents influence the size of resorption cavities and individual remodeling events and that the effect of anti-resorptives on individual remodeling events may not always be directly related to the degree of suppression of bone remodeling.
PMCID: PMC3818704  PMID: 23988275
Bone Remodeling; Bone Histomorphometry; Ovariectomy; Osteoporosis; Bisphosphonates; SERMS
24.  Effects of Sigma Anti-bonding Molecule Calcium Carbonate on bone turnover and calcium balance in ovariectomized rats 
Laboratory Animal Research  2011;27(4):301-307.
This study was conducted to evaluate the effect of Sigma Anti-bonding Molecule Calcium Carbonate (SAC) as therapy for ovariectomy-induced osteoporosis in rats. Three weeks after surgery, fifteen ovariectomized Sprague-Dawley rats were divided randomly into 3 groups: sham-operated group (sham), ovariectomized group (OVX) and SAC-treatment group (OVX+SAC). The OVX+SAC group was given drinking water containing 0.0012% SAC for 12 weeks. Bone breaking force and mineralization as well as blood parameters related to the bone metabolism were analyzed. In OVX animals, blood concentration of 17β-estradiol decreased significantly, while osteocalcin and type I collagen C-terminal telopeptides (CTx) increased. Breaking force, bone mineral density (BMD), calcium and phosphorus in femurs, as well as uterine and vaginal weights, decreased significantly following OVX. However, SAC treatment (0.0012% in drinking water) not only remarkably restored the decreased 17β-estradiol and increased osteocalcin and CTx concentrations, but also recovered decreased femoral breaking force, BMD, calcium and phosphorus, although it did not reversed reproductive organ weights. It is suggested that SAC effectively improve bone density by preventing bone turnover mediated osteocalcin, CTx and minerals, and that it could be a potential candidate for therapy or prevention of postmenopausal osteoporosis.
PMCID: PMC3251760  PMID: 22232638
Osteoporosis; bone mineral density; Sigma Anti-bonding Molecule Calcium Carbonate; 17β-estradiol; osteocalcin; type I collagen C-terminal telopeptides
25.  Chronic treatment with the nitric oxide synthase inhibitor, L-NAME, attenuates estradiol-mediated improvement of learning and memory in ovariectomized rats 
Clinics  2011;66(4):673-679.
The role of ovarian hormones and nitric oxide in learning and memory has been widely investigated.
The present study was carried out to evaluate the effect of the nitric oxide synthase (NOS) inhibitor, N (G)-nitro-L-arginine methyl ester (L-NAME), on the ability of estradiol to improve learning in OVX rats using the Morris water maze.
Forty rats were divided into five groups: (1) ovariectomized (OVX), (2) ovariectomized-estradiol (OVX-Est), (3) ovariectomized-L-NAME 10 (OVX-LN 10), (4) ovariectomized-L-NAME 50 (OVX-LN 50) and (5) ovariectomized-estradiol-L-NAME 50 (OVX-Est-LN 50). The animals in the OVX-Est group were treated with a weekly injection of estradiol valerate (2 mg/kg; i.m.). The OVX-LN 10 and OVX-LN 50 groups were treated with daily injections of 10 and 50 mg/kg L-NAME (i.p.), respectively. The animals in the OVX-Est-LN 50 group received a weekly injection of estradiol valerate and a daily injection of 50 mg/kg L-NAME. After 8 weeks, all animals were tested in the Morris water maze.
The animals in the OVX-Est group had a significantly lower latency in the maze than the OVX group (p<0.001). There was no significant difference in latency between the OVX-LN 10 and OVX-LN 50 groups in comparison with the OVX group. The latency in the OVX-Est-LN 50 group was significantly higher than that in the OVX-Est group (p<0.001).
These results show that L-NAME treatment attenuated estradiol-mediated enhancement of spatial learning and memory in OVX rats, but it had no significant effect in OVX rats without estrogen, suggesting an interaction of nitric oxide and estradiol in these specific brain functions.
PMCID: PMC3093799  PMID: 21655764
Estradiol; L-NAME; Morris water maze; Learning; Memory

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