Combination Of Ovariectomy Research Articles

EXPERIMENTAL JUSTIFICATION OF THE PREVENTION OF PERIODONTAL BONE TISSUE DESTRUCTION IN OVARIOECTOMIZED RATS WITH CALCIUM-CONTAINING ADDITIVES

Purpose. Study of periodontoprotective properties of preventive complexes based on calcium, quercetin, vitamins, macro- and microelements in ovariectomized rats with alimentary deficiency of protein and calcium. Materials and methods. The study was conducted on 32 female rats in which pathology of periodontal bone tissue was simulated due to hipoestrogenicity; as well as the prevention of the said pathology with biologically active complexes based on calcium citrate from the shells of Black Sea oysters and "Minerol". Hypoestrogenicity was caused by bilateral ovariectomy, the negative effect of which on bone tissue was further complicated by a diet with a low content of calcium and protein. After 5 months, the animals were removed from the experiment; the degree of atrophy of the alveolar process was determined on the drugs; the content of acid and alkaline phosphatases, elastase, catalase and MDA was determined in the homogenate of the alveolar process. Results. The combination of ovariectomy and an inferior diet caused characteristic inflammatory-dystrophic changes in the bone tissue of the jaws, which are expressed in an increase in the activity of elastase, catalase, and MDA content in the alveolar bone homogenate. Also, under the condition of pathology, an increase in the degree of atrophy of the alveolar process, an increase in the activity of acid and alkaline phosphatases was observed, which indicates a violation of mineralization processes. Prophylactic administration of a complex based on calcium citrate from the shells of Black Sea oysters and a complex based on the biologically active additive "Minerol" showed a pronounced osteoprotective effect, which consisted in the decrease of the degree of the alveolar process atrophy and the reduction of the activity of markers of bone resorption and peroxidation lipids.

Intervertebral Disc Degeneration Induced by Needle Puncture and Ovariectomy: A Rat Coccygeal Model.

Objectives To establish a novel animal model of intervertebral disc degeneration (IVDD) in rats and to investigate the effect of 17β-estradiol (E2) intervention in this model. Methods This study was divided into two parts: animal model (four groups: Sham, ovariectomy (OVX), Puncture, and OVX+Puncture; three-time points: 4, 8, and 12 weeks; three female rats/group/time point) and drug intervention (Sham, OVX+Puncture+corn oil, and OVX+Puncture+E2; three female rats/group). The rats were analyzed by micromagnetic resonance imaging (MRI), hematoxylin and eosin (HE) staining, and safranin-O staining. Results MRI and histological scores significantly differed among the four groups at the three-time points (all P < 0.05). IVDD progressed with time in the OVX, Puncture, and OVX+Puncture groups (all P < 0.05). The changes were the most obvious in the OVX+Puncture group. In the E2 intervention part, the Veh group had the worst MRI signals and histological scores (P < 0.05). The MRI scores in the E2 group were less obvious compared to the Sham group (P > 0.05). Also, the histological scores were significantly different between the Sham and E2 groups (P < 0.05). Conclusions The combination of ovariectomy and needle puncture can synergically induce IVDD in rat coccygeal discs. Estrogen treatment can effectively ameliorate IVDD progression in the newly established IVDD models.

Chronic High Fat Diet Intake Impairs Hepatic Metabolic Parameters in Ovariectomized Sirt3 KO Mice.

High fat diet (HFD) is an important factor in the development of metabolic diseases, with liver as metabolic center being highly exposed to its influence. However, the effect of HFD-induced metabolic stress with respect to ovary hormone depletion and sirtuin 3 (Sirt3) is not clear. Here we investigated the effect of Sirt3 in liver of ovariectomized and sham female mice upon 10 weeks of feeding with standard-fat diet (SFD) or HFD. Liver was examined by Folch, gas chromatography and lipid hydroperoxide analysis, histology and oil red staining, RT-PCR, Western blot, antioxidative enzyme and oxygen consumption analyses. In SFD-fed WT mice, ovariectomy increased Sirt3 and fatty acids synthesis, maintained mitochondrial function, and decreased levels of lipid hydroperoxides. Combination of ovariectomy and Sirt3 depletion reduced pparα, Scd-1 ratio, MUFA proportions, CII-driven respiration, and increased lipid damage. HFD compromised CII-driven respiration and activated peroxisomal ROS scavenging enzyme catalase in sham mice, whereas in combination with ovariectomy and Sirt3 depletion, increased body weight gain, expression of NAFLD- and oxidative stress-inducing genes, and impaired response of antioxidative system. Overall, this study provides evidence that protection against harmful effects of HFD in female mice is attributed to the combined effect of female sex hormones and Sirt3, thus contributing to preclinical research on possible sex-related therapeutic agents for metabolic syndrome and associated diseases.

Cardioprotective action of chia (Salvia hispanica L.) in ovariectomized rats fed a high fat diet.

The reduction in estrogen levels is associated with the increased risk factors for cardiovascular disease development. The present study aimed to evaluate the effect of chia consumption in a standard diet (SD) or high fat diet (HFD) on ovariectomized (OVX) and non-ovariectomized (SHAM) rats, in relation to biometric measurements, oxidative stress, mineral content and ATPase enzymes in the heart. The study was conducted with 80 female Wistar rats, which received a SD or HFD for 18 weeks. During the first 7 weeks, the animals received the SD or HFD. Then, 40 rats were ovariectomized and 40 rats were SHAM operated. After recovery from surgery, the animals were allocated to 8 groups (n = 10) and they received one of the following diets for 8 weeks: SD, SD + chia, HFD and HFD + chia. In the OVX group, HFD increased weight gain, adiposity, cardiac hypertrophy, and nitric oxide (NO) and K concentration and decreased the Na+/K+ATPase activity. In combination with HFD, ovariectomy decreased the catalase activity, Mg, Cu and Zn concentration, total ATPase activity, and Na+/K+ATPase and Mg2 + ATPase activities; this group also presented higher NO, Ca, K, Fe and Mn concentration in the heart. The SHAM group fed chia presented a lower fat content in the heart. In the OVX group fed HFD, chia increased the activity of superoxide dismutase, decreased NO and maintained the content of minerals and ATPase enzymes. Thus, chia improved the biometric parameters of the heart, the antioxidant activity and maintained the content of minerals and ATPase enzymes, showing a cardioprotective action, but without reversing the deleterious effects of ovariectomy.

The effect of estrogen deficiency and its combination with chronic stress on the condition of periodontal in old rats

The aim of the study was to investigate the effect of ovariectomy and its combination with stress (femoral fracture) on the periodontal condition in old rats. Materials and methods. A total of 24 white female rats were used in the experiment. The first group consisted of intact rats. Rats of the second group were subjected to an ovariectomy (OE) at the age of 2 months. Rats of the third group underwent a fracture of the one femur 1 month before they were euthanized. The animals were sacrificed at 15 months of age. Results. The level of LPO was increased in the liver, salivary glands and alveolar bone process after experimental ovariectomy; the recession defects on molars were significantly increased. Stress (femoral fracture) aggravated the intensification of peroxidation processes in the salivary glands and liver of rats. The levels of nucleic and higher polyene fatty acids in the liver lipids were decreased in the combination of ovariectomy and stress. The level of LPO in the alveolar bone process was not changed significantly. Histomorphometric study of periodontium in rats revealed a decrease in the upper margin of epithelial attachment. Conclusions. Experimental estrogen deficiency caused the most significant pathogenic effect on the periodontium in old rats. The obtained data demonstrate that female sex hormone deficiency is one of the mechanisms in periodontitis pathogenesis.

A new animal model for menopausal transition: combination of ovariectomy and empty bottle stimulation

Previous small animal models for menopausal transition are mainly performed to mimic the biochemical changes of patients with menopausal transition. The psychological symptoms in animal models are often overlooked. To mimic clinical situation, we developed a new mouse model of menopausal transition: the combination of bilateral ovariectomy (OVX) and empty bottle stimulation (EBS). After OVX and 21 days of EBS, behavioral test and pentobarbital-induced sleep test were carried out. Then all the animals were euthanized for further investigations. Compared with the control group, the combination of OVX and EBS group (OVX + EBS) showed higher bodyweight, lower organ index of uteri, and lower concentrations of serum estradiol. In addition, behavioral manifestations, sleep quality, and morphologic features in OVX + EBS group showed significant differences compared with the control group. OVX alone or EBS alone failed to include all the changes of OVX + EBS group. These findings indicate ovariectomy or EBS alone could not well mimic the symptoms of menopausal transition. The combination of bilateral ovariectomy and EBS offers an easy and repeatable method for building comprehensive menopausal transition model in mice.

Combined effects of ovariectomy and streptozotocin-induced diabetes in the articular cartilage of rats

Aim: To evaluate the combined effects of streptozotocin-induced diabetes (Di) and ovariectomy in the articular cartilage of rats.Methods: Forty adult female Wistar rats were ovariectomized (OVX) or sham-operated. After recovery from surgery, the animals were assigned randomly into four groups: OVX control (OVX-C); OVX treated with 10 µg/kg/day of 17β-estradiol (OVX-E); sham-operated subjected to Di (Sham-Di); and OVX subjected to Di (OVX-Di). After 60 days of treatment, the animals were euthanized and the distal femurs with articular cartilage were processed for paraffin-embedding. Sections were stained with hematoxylin and eosin for histomorphometry, Picro-Sirius Red for collagen, or Alcian Blue for glycosaminoglycan (GAG) content. To detect apoptosis, sections were stained with an antibody to cleaved caspase-3 (casp-3).Results: Articular cartilage thickness and GAG content were significantly lower (p < 0.05) in the OVX-Di group, which also showed a higher number of casp-3-positive chondrocytes than the other groups. Interestingly, the higher percentage (p < 0.05) of mature collagen fibers was seen in the OVX-Di group, may be as a result of a reduced extracellular matrix remodeling of the articular cartilage.Conclusion: Our results indicate that the combination of ovariectomy and streptozotocin-induced diabetes produces more deleterious effects in articular cartilage of rats than either condition alone.

Estrogen promotes the brain metastatic colonization of triple negative breast cancer cells via an astrocyte-mediated paracrine mechanism.

Brain metastases (BM) are a devastating consequence of breast cancer. BM occur more frequently in patients with estrogen receptor-negative (ER−) breast cancer subtypes; HER2 overexpressing (HER2+) tumors and triple-negative (TN) (ER−, progesterone receptor-negative (PR−) and normal HER2) tumors. Young age is an independent risk factor for development of BM, thus we speculated that higher circulating estrogens in young, pre-menopausal women could exert paracrine effects through the highly estrogen-responsive brain microenvironment. Using a TN experimental metastases model, we demonstrate that ovariectomy decreased the frequency of MRI detectable lesions by 56% as compared to estrogen supplementation, and that the combination of ovariectomy and letrozole further reduced the frequency of large lesions to 14.4% of the estrogen control. Human BM expressed 4.2-48.4% ER+ stromal area, particularly ER+ astrocytes. In vitro, E2-treated astrocytes increased proliferation, migration and invasion of 231BR-EGFP cells in an ER-dependent manner. E2 upregulated EGFR ligands Egf, Ereg, and Tgfa mRNA and protein levels in astrocytes, and activated EGFR in brain metastatic cells. Co-culture of 231BR-EGFP cells with E2-treated astrocytes led to upregulation of the metastatic mediator S100 Calcium-binding protein A4 (S100A4) (1.78-fold, P<0.05). Exogenous EGF increased S100A4 mRNA levels in 231BR-EGFP cells (1.40±0.02 fold, P<0.01 compared to vehicle-control) and an EGFR/HER2 inhibitor blocked this effect, suggesting that S100A4 is a downstream effector of EGFR activation. ShRNA-mediated S100A4 silencing in 231BR-EGFP cells decreased their migration and invasion in response to E2-CM, abolished their increased proliferation in co-cultures with E2-treated astrocytes, and decreased brain metastatic colonization. Thus, S100A4 is one effector of the paracrine action of E2 in brain metastatic cells. These studies provide a novel mechanism by which estrogens, acting through ER+ astrocytes in the brain microenvironment, can promote BM of TN breast cancers, and suggests existing endocrine agents may provide some clinical benefit towards reducing and managing BM.

Estrogen increases the severity of anaphylaxis in female mice through enhanced endothelial nitric oxide synthase expression and nitric oxide production

Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.

Ovarektomie und Kalzium-/Vitamin D2/D3-arme Diät als Osteoporosemodell an der Wirbelsäule von Sprague-Dawley-Ratten

Osteoporosis is a widespread disease characterised by low bone mass and structural deterioration of bone resulting in an increased susceptibility to fractures. Osteoporosis affects women more frequently than men; every second woman older than 50 years suffers from an osteoporotic fracture, frequently a vertebral fracture. The aim of this study was to induce osteoporosis in rats to establish an osteoporotic small-animal model that simulates the human pathology particularly in the spine. Therefore, bone density parameters, which are routinely determined in the spine of osteoporotic patients, were investigated by Dual-Energy X-ray Absorptiometry (DEXA). Fourteen-week-old female Sprague-Dawley rats (n = 50) were either sham-operated (control group: sham) or ovariectomised (experimental group). Ovariectomised rats were further divided into two groups; one received calcium/vitamin D2/D3 deficient diet (OVX + diet), and the other received subcutaneous steroid injections (dexamethasone 0.3 mg/kg body weight) twice a month (OVX + steroid). Rats were scanned by DEXA at three time points (Month = M, 0 M, 1 M and 3 M). DEXA measurement of the spine delivered T-value, Z-value, bone mineral content (BMC), and the scanned area. Fifteen female patients at an age of 57-72 years were scanned in 8-10 regions of the spine (150 measurements). T-values and Z-values were pre-calculated based on patient databases. Statistical analysis was performed using two-way ANOVA followed by Bonferroni correction, with significance considered at p < 0.05. T-value and Z-value of both rat groups were compared with the patient data as well as with each others. Both treated rat groups revealed significantly lower T- and Z-values than controls. Despite the significant difference, the reference line (-2.5 for T-value and -1.5 for Z-value) was only reached by the OVX + diet group. On the other hand, the sham group showed an increase in BMC over time, while no change was seen in OVX + diet or OVX + steroid. Bone area demonstrated a significant increase up to M3. However, the increase in bone area within the OVX + diet group was notably higher than in both sham and OVX + steroid groups. Patients showed significantly lower T- and Z-values than sham and OVX + steroid but insignificant ones when compared with OVX + diet. A reproducible vertebral osteoporosis can be generated in a rat model by combination of ovariectomy with administration of a calcium/vitamin D3 deficient diet. T- and Z-values of this experimental group mimicked values obtained from osteoporotic patients, reflecting a simulation of their pathology. Interestingly, the increase in bone area over time with the steady BMC results in lower mineral density (BMD) of the OVX + diet group. Therefore, this rat model presents a reliable experimental set-up that may serve as a tool to better understand and treat osteoporosis.

Combination of chronic stress and ovariectomy causes conditioned fear memory deficits and hippocampal cholinergic neuronal loss in mice

We have recently found that the combination of ovariectomy (OVX) and chronic restraint stress (CS) causes hippocampal pyramidal cell loss and cognitive dysfunction in female rats and that estrogen replacement prevents the OVX/CS-induced morphological and behavioral changes. In this study, to clarify the mechanisms underlying the OVX/CS-mediated memory impairment further, we examined the roles of cholinergic systems in the OVX/CS-induced memory impairment in mice. Female Slc:ICR strain mice were randomly divided into two groups: OVX and sham-operated groups. Two weeks after the operation, the mice of each group were further assigned to CS (6 h/day) or non-stress group. Following the 3-week-stress period, all mice were subjected to contextual fear conditioning, and context- and tone-dependent memory tests were conducted 1 or 24 h after the conditioning. Overburden with 3 weeks of CS from 2 weeks after OVX impaired context- and tone-dependent freezing and the OVX/CS caused significant Nissl-stained neuron-like cell loss in the hippocampal CA3 region, although OVX and CS alone did not cause such behavioral and histological changes. Replacement of 17β-estradiol for 5 weeks after OVX suppressed OVX/CS-induced memory impairment and hippocampal Nissl-positive cell loss. Furthermore, the OVX/CS mice exhibited a significant decrease in choline acetyltransferase in the hippocampus compared with other groups. The cholinesterase inhibitors donepezil and galantamine ameliorated OVX/CS-induced memory impairment. These data suggest that cholinergic dysfunction may be involved in the OVX/CS-induced conditioned fear memory impairment. Overall, our findings suggest that the OVX/CS mouse model is useful to study the mechanisms underlying estrogen loss-induced memory deficits.

Placental Extract Improves Hippocampal Neuronal Loss and Fear Memory Impairment Resulting From Chronic Restraint Stress in Ovariectomized Mice

We have recently found that combination of ovariectomy (OVX) and chronic restraint stress causes cognitive dysfunction and reduces hippocampal CA3 neurons in female rats and mice and that estrogen replacement and chronic treatment with Ginkgo biloba extract EGb 761 suppress the OVX/stress-induced behavioral and morphological changes. In this study, we examined the effect of placental extract on the memory impairment and neuromorphological change in OVX/stress-subjected mice. Female Slc:ICR strain mice were randomly divided into four groups: vehicle-treated OVX, porcine placental extract (120 and 2160 mg/kg)-treated OVX, and sham-operated control groups. Two weeks after surgical operation, OVX mice underwent restraint stress for 21 days (6 h/day), and all animals were then subjected to a contextual fear conditioning test followed by morphological examination by Nissl staining. Placental extract was orally administered once daily until the behavioral analysis was carried out. Chronic treatment with both doses of placental extract improved the OVX/stress-induced fear memory impairment and Nissl-positive cell loss of the hippocampal CA3 region, although it did not affect the loss of bone mineral density and increase in body weight after OVX. These results have important implications for the neuroprotective and cognition-enhancing effects of placental extract in postmenopausal women.

Characterization of a rabbit osteoporosis model induced by ovariectomy and glucocorticoid

Background and purpose Experimental models of osteoporosis in rabbits are useful to investigate anabolic agents because rabbits have an active Haversian remodeling and achieve skeletal maturiaty quickly. In this study, an experimental model of osteoporosis in rabbits induced by a combination of ovariectomy and glucocorticoid was characterized to provide a useful model for prevention and therapy of osteoporosis.Methods 32 skeletally mature female rabbits were divided randomly into 4 groups: sham control, bilateral ovariectomy (OVX), 1mg/kg/day methylprednisolone (MP) for 8 weeks, and OVX in combination with MP. All rabbits were killed 10 weeks after surgery. Bone mineral density (BMD) of lumbar spine was measured by dual-energy X-ray absorptiometry at baseline, and at 6 and 10 weeks postoperatively. Bone microarchitecture and mechanical properties of lumbar vertebrae were investigated by high-resolution micro-computed tomography and compression test, respectively, after killing.Results Mean BMD of the OVX+MP group at 10 weeks was reduced by about 36% from baseline (p < 0.001). Bone microarchitecture of lumbar vertebrae in the OVX+MP group indicated osteoporosis-associated deterioration. There was a statistically significant reduction in maximum load, stiffness, and energy absorption capacity of lumbar vertebrae in the OVX+MP group, but not in the OVX group, compared to the sham control. In the MP group, BMD and some microarchitecture parameters such as trabecular thickness and bone volume fraction were reduced. The mechanical properties were not statistically significantly different from those in the sham control group, however, although a negative trend was observed.Interpretation Osteopenia can be induced experimentally in rabbits through a combination of OVX and MP, and can be evaluated by BMD, bone microstructure, and mechanical parameters.

Enhanced activity of hippocampal BACE1 in a mouse model of postmenopausal memory deficits

Ovarian hormone decline after menopause may influence cognitive performance and increase the risk for Alzheimer's disease (AD) in women. We have recently demonstrated that a combination of ovariectomy and chronic stress (OVX/stress) causes hippocampus-associated cognitive dysfunction in mice. In this study, we examined whether OVX/stress could affect the levels of AD-related molecules in the mouse hippocampus. Female ICR mice were ovariectomized or sham-operated, and then randomly divided into a daily restraint stress (21 days, 6 h/day) or non-stress group. Although OVX or stress alone did not affect beta-site amyloid precursor protein (APP)-cleaving enzyme-1 (BACE1) activity, OVX/stress increased activity in hippocampal CA1 and CA3 regions, compared with other groups. In contrast, OVX/stress did not affect gamma-secretase activity, Abeta(1-40), and phosphorylated-tau levels in the hippocampus. These findings suggest that a stressful life after menopause can influence the levels of AD-related molecules and that BACE1 is the most sensitive molecule for such a situation.

Ginkgo biloba extract EGb 761 attenuates hippocampal neuronal loss and cognitive dysfunction resulting from chronic restraint stress in ovariectomized rats

We have recently found that a combination of ovariectomy (OVX) and chronic restraint stress causes cognitive dysfunction and reduces hippocampal CA3 neurons in female rats and that estrogen replacement suppresses the OVX/stress-induced behavioral and morphological changes. In this study, we examined the effect of Ginkgo biloba extract (EGb 761), a popular herbal supplement, on the cognitive dysfunction and neuromorphological change in OVX/stress-subjected rats. Female Fisher 344 rats were randomly divided into three groups: vehicle-treated OVX, EGb 761 (50 mg/kg) -treated OVX and vehicle-treated sham-operated control groups. Two months after ovariectomy, all animals received restraint stress for 21 days (6 h/day), and were then subjected to a novel object recognition test followed by morphological examination by Nissl staining. EGb 761 was orally administered once daily until the behavioral analysis was done. Treatment with EGb 761 improved memory impairment and neuronal loss of hippocampus in the OVX/stress-subjected group in the same ways as 17β-estradiol. On the other hand, EGb 761 did not affect the loss of bone mineral density and increase in body weight after OVX, although 17β-estradiol attenuated them. These results have important implications for neuroprotective and cognition enhancing effects of EGb 761 in postmenopausal women and suggest that the effects are mediated by a different mechanism from estrogen.

Glucocorticoid-treated sheep as a model for osteopenic trabecular bone in biomaterials research.

The purpose of this study was to determine the alterations in ovine trabecular bone induced by a combination of ovariectomy and steroid treatment. Twenty-four female skeletally mature Merino sheep were randomly assigned to ovariectomy alone (OVX), ovariectomy combined with glucocorticoid treatment for 6 months (OVX + GC), or no treatment (control). Biopsies of trabecular bone were harvested 6 and 12 months after the beginning of the study from the proximal tibia. The biopsies were scanned for apparent bone mineral density by quantitative computed tomography and were mechanically tested. Three-dimensional bone reconstructions were obtained by micro-computed tomography. Trabecular bone from the OVX + GC animals had a markedly reduced apparent bone mineral density (27% less than control), bone volume (34%), and elastic modulus (36%) at 6 months. At 12 months, the reductions in apparent bone mineral density (33%), bone volume (37%), and elastic modulus (62%) appeared to be even more pronounced. Ovariectomy alone did not result in a perceptible reduction in any parameter. The combination of ovariectomy and glucocorticoid treatment in sheep resulted in a successful induction of substantial loss of trabecular bone and thus may serve as a large-animal model for osteopenic trabecular bone for the development and testing of orthopedic implants and techniques under osteoporotic conditions.

Bone changes due to glucocorticoid application in an ovariectomized animal model for fracture treatment in osteoporosis.

In a pilot experiment comparing four different modalities for inducing osteoporosis in the sheep, a combination of ovariectomy, calcium/vitamin D-restricted diet and steroid administration was found to generate the highest decrease in bone mineral density (BMD). The aim of the present study was to quantify the outcome of this triple treatment in an animal model of osteoporosis in terms of alteration in bone mass, bone structure and bone mechanics. A total of 32 sheep were divided into two equal groups. Group 1 (age 3-5 years) was used as a normal control. Group 2 (age 7-9 years) was ovariectomized, fed a calcium/vitamin D-restricted diet and injected with methylprednisolone (MP) over 7 months (22 weeks MP solution, 6 weeks MP suspension). The BMD at the distal radius and tibia was determined preoperatively and at repeated intervals bilaterally using quantitative computed tomography. Steroid blood levels were determined 4 and 24 h after selected injections. BMD was measured at L3 and L4 after 7 months. Biopsies were taken from iliac crests, vertebral bodies and femoral heads, and bone structure parameters investigated by three-dimensional micro-CT. Compressive mechanical properties of cancellous bone were determined from biopsies of vertebral bodies and femoral heads. After 7 months of osteoporosis induction the BMD of cancellous bone decreased 36 +/- 3% in the radius and 39 +/- 4% in the tibia. Steroid blood levels 24 h after injection of MP suspension were significantly higher than after injection of MP solution. Changes in structural parameters of cancellous bone from the iliac crest, lumbar spine and femoral head in group 2 indicated osteoporosis-associated changes. In group 2 there was a significant reduction in BMD of the lumbar spine and a significant reduction in stiffness and failure load in compression testing of biopsies of lumbar vertebrae. In sheep, changes in the structural parameters of bone such as trabecular number and separation during osteoporosis induction are comparable to the human situation. The sheep model presented seems to meet the criteria for an osteoporosis model for fracture treatment with respect to mechanical and morphometric bone properties.

Effects of 16 weeks of treatment with tibolone on bone mass and bone mechanical and histomorphometric indices in mature ovariectomized rats with established osteopenia on a low-calcium diet

Tibolone is a synthetic steroid with tissue-specific estrogenic, progestogenic, and androgenic properties. The therapeutic effects of tibolone on bone mass and strength, bone metabolic markers, and indices of histomorphometry were investigated in ovariectomized (ovx) rats on a low (0.1%)-calcium diet in comparison with 17α-ethynylestradiol (EE) or 1α-hydroxyvitamin D 3 [1α(OH)D 3]. Tibolone (0.1–3 mg/kg/day), EE (0.1 mg/kg/day), or 1α(OH)D 3 (0.5 μg/kg/day) was administered orally once a day for 16 weeks, starting 12 weeks after ovariectomy, when the bone mineral density (BMD) of lumbar vertebrae (L4–5) and femur (global, proximal, and distal regions) had already been decreased by the combination of ovariectomy and low dietary calcium. The BMD of the lumbar vertebrae and the femur were higher in the groups treated with tibolone, EE, or 1α(OH)D 3 than in the ovx control group. The BMD of the mid-diaphysial regions of femur and tibia, which consist mainly of cortical bone, were decreased 28 weeks after ovariectomy in the ovx control group. The BMD of the mid-diaphysial femur was higher in the groups treated with 1α(OH)D 3, and the BMD of mid-diaphysial tibia was higher in the groups treated with tibolone or 1α(OH)D 3 than in the ovx control group. Like BMD, the compressive strength of the vertebral body of L2, corrected for the volume of each individual vertebra tested, was higher in the groups treated with tibolone, EE, or 1α(OH)D 3 than in the ovx control group. Trabecular bone volume and trabecular number were reduced 12 and 28 weeks after ovariectomy but there was no change in trabecular thickness. These reduced indices were increased in the groups treated with tibolone, EE, or 1α(OH)D 3 when compared with the ovx control group. Tibolone or EE decreased serum levels of osteocalcin and bone alkaline phosphatase and urinary levels of deoxypyridinoline and pyridinoline compared with the ovx control group. Furthermore, tibolone or EE decreased the mineralizing surface and bone formation rate as well as the osteoclast surface and osteoclast numbers. 1α(OH)D 3, however, did not affect these serum and urinary parameters. These data suggest that tibolone suppresses the accelerated bone turnover induced by a combination of ovariectomy and low dietary calcium, and indicate that tibolone may be a potentially useful drug for the treatment of postmenopausal osteoporosis.

Effect of (Asu 1,7)-eel calcitonin on the prevention of osteoporosis induced by combination of immobilization and ovariectomy in the rat

Experimental osteoporosis was induced in the rat by a combination of ovariectomy and immobilization by hemicordotomy. (Asu 1,7)-eel calcitonin (CT) was administered at doses of 0.1, 0.5, 1.0, 2.5, and 5.0 MRC unit/kg, and its preventive effect on bone loss was examined. Significant bone loss was observed in the untreated control group by photo-densitometry and measurement of bone mass. CT at doses of above 1.0 MRC unit/kg significantly increased femur scores and at doses of above 2.5 MRC unit/kg resulted in significantly higher photodensity. Dry weight and ash weight were significantly higher in the 2.5 and 5.0 MRC unit/kg CT-treated groups. Administration of CT had a nearly dose-dependent effect on bone mass. Histomorphometry revealed significantly lower fractional formation surface and fractional resorption surface in the 5.0 MRC unit/kg CT-treated group. Bone mineralization was studied by the quantitative analysis of tetracycline incorporation. Tetracycline uptake was significantly lower in the groups treated with CT at doses above 1.0 MRC unit/kg. The kinetic bone formation activity studied by 45Ca and 14C-proline tracer examination, in the 5.0 MRC unit/kg CT-treated group was significantly lower than that in the control group, but not lower than that in the sham-operated group.

Analysis of estradiol-independent and -dependent endogenous opioid peptide suppression of pulsatile LH release between the mornings of diestrus 2 and proestrus in the rat estrous cycle

The aim of this study was to analyze possible estradiol (E 2)-independent and -dependent endogenous opioid peptide (EOP) suppression of pulsatile LH release between the mornings of diestrus 2 (D2) and proestrus by examining the LH response to naloxone infusions in the presence or absence of proestrous levels of E 2. Pulsatile LH secretion remained unchanged between D2 and proestrus but mean blood LH levels, pulse amplitude and frequency increased within 24 hr following ovariectomy on D2. This increase was due in large part to the loss of E 2 negative feedback, since restoration of physiological proestrous E 2 levels returned LH pulse frequency to proestrous a.m. levels and greatly reduced pulse amplitude. In ovariectomized rats lacking E 2 negative feedback, continuous infusion of the EOP receptor antagonist naloxone (0.5 and 2 mg/kg/hr) caused a further increase in pulse amplitude and frequency. This naloxone-induced increment in pulsatile LH release was exerted via centrally located EOP receptors since naloxone did not alter pituitary responsiveness to LHRH, and its stimulatory action on pulsatile release was diminished by simultaneous infusion with morphine. Naloxone also increased pulsatile LH release in E 2-treated animals. The naloxone-induced increments in LH pulse amplitude were the same in the presence or absence of E 2 negative feedback. Moreover, the increments in amplitude produced by naloxone in E 2-treated rats were significantly less than those resulting from the combination of ovariectomy plus naloxone infusion in empty capsule-implanted rats. These data indicated that naloxone infusion in E 2-implanted animals blocked an E 2-independent EOP suppression of this parameter of pulsatile release. In contrast, with both naloxone doses tested, there was a trend for naloxone-induced increments in pulse frequency to be larger in ovariectomized rats previously implanted with E 2 rather than empty capsules. Although these differences were not statistically significant, the naloxone-induced increments in pulse frequency in E 2-treated rats (unlike those in pulse amplitude) were large enough so that they were also significantly different from the overall increases in pulse frequency caused by the combination of ovarian steroid removal coupled with blockade of EOP receptors during naloxone infusion in empty capsule-implanted animals. This raises the possibility that naloxone infusions in E 2-treated rats compromised both an E 2-independent EOP suppression of pulse frequency and a small, E 2-dependent component as well. Overall, the data demonstrate that between the mornings of D2 and proestrus, EOPs act independent of E 2 to suppress both LH pulse amplitude and frequency, and the negative feedback of E 2 on LH pulse amplitude is not mediated by EOPs whose receptors are antagonized by naloxone. The data also suggest the possibility that E 2 suppression of pulse frequency may be mediated, at least in part, by EOPs.