OVX Animals Research Articles

A novel RANKL-targeted flavonoid glycoside prevents osteoporosis through inhibiting NFATc1 and reactive oxygen species.

Background and purposeOsteoporosis is characterized by excessive bone resorption due to enhanced osteoclast activation. Stimulation of nuclear factor of activated T cells 1 (NFATc1) and accumulation of reactive oxygen species (ROS) are important mechanisms underlying osteoclastogenesis. Robinin (Rob) is a flavonoid glycoside that has shown anti‐inflammatory and antioxidative effects in previous studies, but little is known about its effects on bone homeostasis. The purpose of our research was to investigate whether Rob could prevent bone resorption in ovariectomized (OVX) mice by suppressing osteoclast production through its underlying mechanisms.MethodsThe docking pose of Rob and RANKL was identified by protein‐ligand molecular docking. Rob was added to bone marrow macrophages (BMMs) stimulated by nuclear factor‐κB (NF‐κB) ligand (RANKL). The effects of Rob on osteoclastic activity were evaluated by positive tartrate resistant acid phosphatase (TRAcP) staining kit and hydroxyapatite resorption assay. RANKL‐induced ROS generation in osteoclasts was detected by H2DCFDA and MitoSox Red staining. The classic molecular cascades triggered by RANKL, such as NF‐κB, ROS, calcium oscillations, and NFATc1‐mediated signaling pathways, were investigated using Fluo4 staining, western blot, and quantitative real‐time polymerase chain reaction. In addition, an OVX mouse model mimicking estrogen‐deficient osteoporosis was created to evaluate the therapeutic effects of Rob in vivo.ResultsComputational docking results showed that Rob could bind specifically to RANKL's predicted binding sites. In vitro, Rob inhibited RANKL‐mediated osteoclastogenesis dose‐dependently without obvious cytotoxicity at low concentrations. We also found that Rob attenuated RANKL‐induced mitochondrial ROS production or enhanced activities of ROS‐scavenging enzymes, and ultimately reduced intracellular ROS levels. Rob abrogated the RANKL‐induced mitogen‐activated protein kinase (MAPK) and NF‐κB signaling pathways, and subsequently blocked NFATc1 signaling and TRAcP expression. In addition, Rob inhibited osteoclast proliferation by downregulating the expression of osteoclast target genes (Acp5, Cathepsin K, Atp6v0d2, Nfact1, c‐Fos, and Mmp9) and reducing Ca2+ oscillations. Our in vivo results showed that Rob reduced bone resorption in OVX animal model by repressing osteoclast activity and function.ConclusionsRob inhibits the activation of osteoclasts by targeting RANKL and is therefore a potential osteoporosis drug.

Effects of estrogen deficiency during puberty on maxillary and mandibular growth and associated gene expression \u2013 an \u03bcCT study on rats

BackgroundEstrogen is a well-known and important hormone involved in skeletal homeostasis, which regulates genes involved in bone biology. Some studies support that estrogen is important for craniofacial growth and development. Therefore this in vivo animal study aimed to investigate, whether and in which way low estrogen levels in the prepubertal period affect craniofacial development in the postpubertal stage and to quantify the gene expression of RANK, RANKL and OPG in cranial growth sites in ovariectomized estrogen-deficient rats during puberty.MethodsControl (sham-operated, n = 18) and ovariectomy (OVX, n = 18) surgeries were performed on 21-days-old female Wistar rats. Animals euthanized at an age of 45 days (pubertal stage) were used for gene expression analyses (n = 6 per group) and immunohistochemistry of RANK, RANKL and OPG. Animals euthanized at 63 days of age (post-pubertal stage) were used for craniofacial two-dimensional and three-dimensional craniofacial measurements using μCT imaging (n = 12 per group).ResultsIn the μCT analysis of the mandible and maxilla many statistically significant differences between sham-operated and OVX groups were observed, such as increased maxillary and mandibular bone length in OVX animals (p < 0.05). Condylar volume was also significantly different between groups (p < 0.05). The sham-operated group showed a higher level of RANK expression in the midpalatal suture (p = 0.036) and the RANKL:OPG ratio levels were higher in the OVX group (p = 0.015).ConclusionsOur results suggest that estrogen deficiency during the prepubertal period is associated with alterations in the maxillary and mandibular bone length and condylar growth.

Combined Effects of Exercise and Phytoanabolic Extracts in Castrated Male and Female Mice.

Dry extracts from the Eurasian plants, Ajuga turkestanica, Eurycoma longifolia, and Urtica dioica have been used as anabolic supplements, despite the limited scientific data on these effects. To assess their actions on early sarcopenia signs, male and female castrated mice were supplemented with lyophilized extracts of the three plants, isolated or in association (named TLU), and submitted to resistance exercise. Ovariectomy (OVX) led to body weight increase and non-high-density cholesterol (HDL) cholesterol elevation, which had been restored by exercise plus U. dioica extract, or by exercise and TLU, respectively. Orchiectomy (ORX) caused skeletal muscle weight loss, accompanied by increased adiposity, being the latter parameter reduced by exercise plus E. longifolia or U. dioica extracts. General physical activity was improved by exercise plus herbal extracts in either OVX or ORX animals. Exercise combined with TLU improved resistance to fatigue in OVX animals, though A. turkestanica enhanced the grip strength in ORX mice. E. longifolia or TLU also reduced the ladder climbing time in ORX mice. Resistance exercise plus herbal extracts partly altered gastrocnemius fiber size frequencies in OVX or ORX mice. We provide novel data that tested ergogenic extracts, when combined with resistance exercise, improved early sarcopenia alterations in castrated male and female mice.

Effects of quercetin, vitamin E, and estrogen on Metabolic-Related factors in uterus and serum of ovariectomized rat models

Aims Estrogen (E2) deficiency has been related to uterine metabolic dysfunction, which could be accompanied by infertility in the reproductive ages. Despite having adverse effects, estrogen replacement therapy is considered the fundamental treatment strategy for this problem. The current study sought to determine the palliative effects of quercetin (Q) and vitamin E (Vit.E) on some of the uterine's metabolism-related factors in ovariectomized (OVX) rats and compare them with the effects of estrogen. Materials and methods Sixty-four rats were divided into eight groups. OVX animals were treated with Q (15 mg/kg/day), Vit.E (60 mg/kg/day), E2 (10 µg/kg/day), and Q (7.5 mg/kg/day) + Vit.E (30 mg/kg/day) for 10 weeks. Glucose and adiponectin were measured using glucose oxidase and ELISA, respectively. Furthermore, the present study investigated the alterations in the expression of AdipoR1, nesfatin1, and GluT4 genes. Results Antioxidants suppress the weight gain of OVX animals. Also, Q, Vit.E, and E2 cause a significant decline in glucose and adiponectin levels (p-value < .05). Finally, the expression of AdipoR1, nesfatin1, and GLUT4 genes was significantly increased in treated OVX rats' uterus. Conclusion The present findings suggest that the administration of Q and Vit.E could demonstrate promising characteristics in a similar approach with estradiol and thus be considered as alternatives for estrogen replacement therapy.

Potential Estrogenic Properties of 17β-Hydroxy-ethylimine Estradiol Derivative Targeted to Menopause Stage.

Hormone replacement therapy during menopause increases the risk of thromboembolic diseases and cancer, so safety alternative therapeutic strategies are needed. 17β-Aminoestrogens are a synthetic estrogens group that possess mild anticoagulant activity that contrasts with the pro-coagulant effects showed by estradiol's (E2) in rodents. Being considered an alternative to conventional hormone replacement therapy during menopause without thrombogenic risks producing. The present study aimed to determine the estrogenic profile and anxiolytic activity of 17β-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE2), a related compound unknown until now. IE2 was assessed in immature rats by uterotrophic assay administering IE2, E2, or vehicle. In ovariectomized adult Wistar rats (Ovx) to facilitating the lordotic behavior compared with E2, estradiol benzoate, or vehicle. The effect of IE2 on anxiety was estimated in Ovx animals treated with IE2, E2, or vehicle group and evaluated in the elevated plus-maze model. IE2 produced an uterotrophic effect, lordotic behavior, and anxiolytic effect in a dose-dependent manner, similar to E2. IE2 depicted estrogenicity, indicating potential clinical use as hormone replacement therapy during menopause.

β-Amyloid Formation, Memory, and Learning Decline Following Long-term Ovariectomy and Its Inhibition by Systemic Administration of Apigenin and β-Estradiol.

Introduction:The increasing cases of Alzheimer Disease (AD) has caused numerous problems. The risk of developing AD increases in menopausal women, too. Apigenin and β-estradiol are effective antioxidant and neuroprotective agents. We conducted the present study to explore their combined effects on β-amyloid plaque formation, memory, and learning in ovariectomized rats.Methods:Forty-two Wistar rats were randomly assigned into 6 groups: 1) ovariectomized (OVX), 2) OVX + apigenin, 3) OVX + β-estradiol, 4) OVX + apigenin + β-estradiol, 5 &6) vehicle shams for E2 and API, and 7) surgical sham. Treatment was done with apigenin and β-estradiol. Then, we studied the formation of β-amyloid plaques, neuronal density in the hippocampus area, apoptosis, memory, and learning.Results:Findings showed the significant formation of β-amyloid plaques in the hippocampus of OVX animals and their memory impairment. Apigenin and β-estradiol significantly reduced the number of β-amyloid plaques, as well as the symptoms of memory impairment and learning, and decreased the expression of caspase-3 in treated animals.Conclusion:Accordingly, β-estradiol and apigenin could have more potent therapeutic effects on AD.

Impact of low bone mass and antiresorptive therapy on antibiotic efficacy in a rat model of orthopedic device-related infection.

A significant proportion of orthopedic devices are implanted in osteoporotic patients, but it is currently unclear how estrogen deficiency and/or exposure to antiresorptive bisphosphonates (BPs) influence orthopedic device-related infection (ODRI), or response to therapy. The aim of this study is to characterize the bone changes resulting from Staphylococcus epidermidis infection in a rodent ODRI model and to determine if ovariectomy (OVX) or BP treatment influences the infection or the success of antibiotic therapy. A sterile or S. epidermidis-contaminated screw was implanted into the proximal tibia of skeletally mature female Wistar rats (n = 6-9 per group). Bone changes were monitored over 28 days using in vivo micro-computed tomography scanning. OVX was performed 12 weeks before screw implantation. The BP zoledronic acid (ZOL) was administered 4 days before screw insertion. A combination antibiotic regimen (rifampin plus cefazolin) was administered from Days 7-21. In skeletally healthy animals, S. epidermidis induced marked changes in bone, with peak osteolysis occurring at Day 9 and woven bone deposition and periosteal mineralization from Day 14 onwards. Antibiotic therapy cleared the infection in the majority of animals (2/9 infected) but did not affect bone responses. OVX did not affect the pattern of infection-induced changes in bone, nor bacterial load, but reduced antibiotic efficacy (5/9 infected). ZOL treatment did not protect from osteolysis in OVX animals, or further affect antibiotic efficacy (5/9 infected) but did significantly increase the bacterial load. This study suggests that both BPs and OVX can influence host responses to bone infections involving S. epidermidis.

Kisspeptin Neurons in the Infundibular Nucleus of Ovariectomized Cats and Dogs Exhibit Unique Anatomical and Neurochemical Characteristics.

Neurons co-synthesizing kisspeptin (KP), neurokinin B (NKB), and dynorphin (“KNDy neurons”) in the hypothalamic arcuate/infundibular nucleus (INF) form a crucial component of the gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) “pulse generator.” The goal of our study was to characterize KP neuron distribution, neuropeptide phenotype and connectivity to GnRH cells in ovariectomized (OVX) dogs and cats with immunohistochemistry on formalin-fixed hypothalamic tissue sections. In both species, KP and NKB neurons occurred in the INF and the two cell populations overlapped substantially. Dynorphin was detected in large subsets of canine KP (56%) and NKB (37%) cells and feline KP (64%) and NKB (57%) cells; triple-labeled (“KNDy”) somata formed ∼25% of all immunolabeled neurons. Substance P (SP) was present in 20% of KP and 29% of NKB neurons in OVX cats but not dogs, although 26% of KP and 24% of NKB neurons in a gonadally intact male dog also contained SP signal. Only in cats, cocaine- and amphetamine regulated transcript was also colocalized with KP (23%) and NKB (7%). In contrast with reports from mice, KP neurons did not express galanin in either carnivore. KP neurons innervated virtually all GnRH neurons in both species. Results of this anatomical study on OVX animals reveal species-specific features of canine and feline mediobasal hypothalamic KP neurons. Anatomical and neurochemical similarities to and differences from the homologous KP cells of more extensively studied rodent, domestic and primate species will enhance our understanding of obligate and facultative players in the molecular mechanisms underlying pulsatile GnRH/LH secretion.

Longitudinal Changes of Circulating miRNAs During Bisphosphonate and Teriparatide Treatment in an Animal Model of Postmenopausal Osteoporosis.

ABSTRACTMicroRNAs regulate bone homeostasis, and circulating microRNAs have been proposed as novel bone biomarkers. The effect of anti‐osteoporotic treatment on circulating microRNAs has not been described in detail. Therefore, we performed a comprehensive analysis of microRNA serum levels in ovariectomized (OVX) and sham‐operated (SHAM) rats over 12 weeks of antiresorptive or osteoanabolic treatment. Forty‐two Sprague Dawley rats underwent SHAM surgery (n = 10) or ovariectomy (n = 32). After 8 weeks, OVX rats were randomized to antiresorptive treatment with zoledronate (n = 11), osteoanabolic treatment with teriparatide (n = 11), or vehicle treatment (n = 10). Serum samples were collected at weeks 8, 12, 16, and 20 after surgery. A total of 91 microRNAs were analyzed by RT‐qPCR in serum samples collected at week 20. Based on the results, 29 microRNAs were selected for longitudinal analysis at all four study time points. Changes in bone mineral density and microstructure were followed up by in vivo micro‐CT and ex vivo nano‐CT. Ovariectomy resulted in the loss of trabecular bone, which was reversed by osteoanabolic and antiresorptive treatment. Differential expression analysis identified 11 circulating miRNAs that were significantly regulated after treatment. For example, miR‐107 and miR‐31‐5p increased in vehicle‐treated OVX animals, whereas they decreased during teriparatide treatment. Additional miRNAs were identified that showed significant correlations to bone microstructure or bone miRNA expression, including miR‐203a‐3p, which exhibited a significant negative correlation to vertebral and tibial trabecular bone volume fraction (%). Longitudinal analysis confirmed eight microRNAs with significant changes in serum over time that were prevented by teriparatide and zoledronate treatment (miR‐34a‐5p, miR‐31‐5p, miR‐30d‐3p, miR‐378a‐5p) or teriparatide treatment only (miR‐375‐3p, miR‐183‐5p, miR‐203a‐3p, miR‐203b‐3p). Gene target network analysis identified WNT and Notch signaling as the main signaling pathways controlled by these miRNAs. Thus, ovariectomy results in time‐dependent deregulation of circulating miRNAs compared with SHAM animals. Anti‐osteoporotic treatments can rescue this effect, showing that bone‐related miRNAs might act as novel biomarkers for treatment monitoring. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Hormonal and biochemical changes in female Proechimys guyannensis, an animal model of resistance to pilocarpine-induced status epilepticus

The Amazon rodent Proechimys guyannensis is widely studied for hosting various pathogens, though rarely getting sick. Previous studies on male Proechimys have revealed an endogenous resistance to epilepsy. Here, we assess in female Proechimys, whether sex hormones and biochemical aspects can interfere with the induction of status epilepticus (SE). The lithium-pilocarpine ramp-up protocol was used to induce SE, and blood sera were collected at 30 and 90 min after SE, alongside brains, for biochemical, western blot and immunohistochemical analyses. Results from non-ovariectomised (NOVX) Proechimys were compared to ovariectomised (OVX) animals. Data from female Wistars were used as a positive control of SE inductions. SE latency was similar in NOVX, OVX, and female Wistars groups. However, the pilocarpine dose required to induce SE in Proechimys was higher (25- to 50-folds more). Despite a higher dose, Proechimys did not show strong SE like Wistars; they only reached stage 2 of the Racine scale. These data suggest that female Proechimys are resistant to SE induction. Glucose and progesterone levels increased at 30 min and returned to normal at 90 min after SE. A relevant fact because in humans and rodents, SE leads to hypoglycaemia after 30 min of SE and does not return to normal levels in a short time, a typical adverse effect of SE. In OVX animals, a decrease in GABAergic receptors within 90 min of SE may suggest that ovariectomy produces changes in the hippocampus, including a certain vulnerability to seizures. We speculate that progesterone and glucose increases form part of the compensatory mechanisms that provide resistance in Proechimys against SE induction.

Alteration of the oral microbiota may be a responsible factor, along with estrogen deficiency, by the development of larger periapical lesions.

To answer the questions: (1) Does reducing estrogen levels influence the microbial composition of the oral cavity? (2) Does the presence of periapical lesion (PL) cause changes in the oral microbiota? (3) Since estrogen deficiency alters the oral microbiota, can this be one of the factors that contribute to the increase of the PL? Thirty-six rats were divided into four groups: sham (control), ovariectomy (OVX), control with PL (Sham + PL), and OVX + PL. After 9 weeks of OVX, the lower first molars were submitted to PL induction. After 21 days, the microbiological collection of the oral cavity was performed, and the animals were euthanized. The contents were evaluated by the checkerboard DNA-DNA hybridization method, to verify the prevalence of 40 bacterial species (divided into 7 microbial complexes). The blocks containing the lower first molars were submitted to histotechnical processing and staining with hematoxylin and eosin (HE), for the measurement of the periapical lesion area. The results were submitted to ANOVA and Kruskal-Wallis tests and Tukey and Dunn post-tests, with a significance level of 5%. In conditions of estrogen deficiency, there was alteration of the oral microbiota. The OVX groups had a higher amount of bacteria compared to the SHAM group in most of the microbial complexes (p < 0.001). The animals in the control group (with or without lesion) did not present a statistically significant difference (p > 0.001) in any of the microbial complexes. The PLs in OVX animals were significantly higher compared to SHAM animals (p < 0.001). Hypoestrogenicity conditions interfere in the oral microbiota by increasing the amount of bacteria in the saliva and influencing the progression of periapical lesions. This inedited study shows that deficiency of estrogen leads to alteration of the oral microbiota.

Impact of Single or Combined Drug Therapy on Bone Regeneration in Healthy and Osteoporotic Rats.

Complications in bone regeneration in patients with systemic impaired bone metabolism (e.g., osteoporosis) represent a rapidly increasing clinical challenge. Alendronate and simvastatin are drugs commonly used to promote bone metabolism in osteoporotic conditions. The aim of this study was to evaluate initial bone regeneration within osseous defects grafted with beta-tricalcium phosphate (β-TCP) in adjunction with systemic coadministrations of alendronate and simvastatin (i.e., daily subcutaneous injection for 3 weeks) in healthy and osteoporotic rats. Eighty Wistar female rats were ovariectomized (OVX; n = 40) or sham operated (n = 40). Six weeks later, osseous defects (a 3-mm critical-sized defect) were created in the left femoral condyles and then grafted with β-TCP. From the day following graft installation, OVX and sham animals received for 3 weeks a daily subcutaneous injection of alendronate (50 μg/kg of body weight) and simvastatin (5 mg/kg of body weight), alone or in combination. A control group was included, which received subcutaneous saline administration. At the end of the 3 weeks, rats were euthanized and specimens (femoral condyles) were retrieved for histological evaluation and histomorphometric measurements, that is, bone area (BA%) and remaining bone graft (RBG%). In osteoporotic rats, 3 weeks of daily subcutaneous injection of combined therapy (alendronate plus simvastatin) led to a significant (p < 0.05) increase in BA% and a significant decrease in RBG% compared to healthy controls in osseous defects grafted with β-TCP (BA%: 28.6 ± 12.0 vs. 18.2 ± 7.6, RBG% 61.3 ± 11.1 vs. 70.7 ± 7.3). No significant differences in BA% and RBG% were found in the OVX rats for single treatments. Furthermore, healthy controls showed similar BA% and RBG% upon single or combined therapy compared to nontreated control rats. Daily coinjections (for 3 weeks) of alendronate plus simvastatin result in a significant enhancement of bone regeneration within osseous defects grafted with β-TCP in osteoporotic rats. Despite the expected effects on osteoporotic bone, our study did not confirm the hypothesized benefit of alendronate and simvastatin on bone regeneration in osseous defects in healthy conditions. The efficacy of the combination drug therapy on bone regeneration demands further investigation to elucidate molecular and cellular aspects underlying this therapy.

Short-term effects induced by nicotinamide in ovariectomized females.

Nicotinamide adenine dinucleotide (NAD)+ precursors such as nicotinamide activate sirtuins and enhance energy metabolism. The aim of this study was to evaluate the metabolic effects of nicotinamide in ovariectomized (OVX) female rats to establish molecular targets against obesity, which support the safe therapeutic application of nicotinamide. The OVX animals were divided into groups: SHAM (simulated surgery), SHAMn (two weeks of 35mg·kg-1 nicotinamide per day, by gavage), OVX, and OVXn (two weeks of 35mg·kg-1 nicotinamide per day, by gavage). The results indicated that nicotinamide favored lipolysis, as evidenced by an increase in free fatty acid and hepatic triglyceride levels, which were not fully normalized during the treatment period. The lipolysis appeared to be due to increased SIRT1 and mitochondrial oxidative phosphorylation in muscle and adipose tissue. There were decreases in muscle and fat nicotinamide N-methyltransferase (NNMT), which were associated with decreases in mass and triglyceride, low-density lipoprotein cholesterol (LDLc), and total cholesterol content. Nicotinamide appeared to be beneficial for the glycemic profile, with normal hepatic glycogen storage and a tendency towards insulin sensitivity in the OVXn. In the SHAMn group, nicotinamide led to glucose intolerance, together with reduced muscle expressions of nicotinamide phosphoribosyltransferase (NAMPT) and SIRT3, suggesting that there were no short-term benefits. Supplementation with nicotinamide led to tissue-specific adaptive lipid and molecular changes in OVX rats.

Inflammatory response in postmenopausal osteoporotic fracture healing.

A balanced inflammatory response is important for successful fracture healing. The response of osteoporotic fracture healing is deranged and an altered inflammatory response can be one underlying cause. The objectives of this review were to compare the inflammatory responses between normal and osteoporotic fractures and to examine the potential effects on different healing outcomes. A systematic literature search was conducted with relevant keywords in PubMed, Embase, and Web of Science independently. Original preclinical studies and clinical studies involving the investigation of inflammatory response in fracture healing in ovariectomized (OVX) animals or osteoporotic/elderly patients with available full text and written in English were included. In total, 14 articles were selected. Various inflammatory factors were reported; of those tumour necrosis factor-α (TNF-α) and interleukin (IL)-6 are two commonly studied markers. Preclinical studies showed that OVX animals generally demonstrated higher systemic inflammatory response and poorer healing outcomes compared to normal controls (SHAM). However, it is inconclusive if the local inflammatory response is higher or lower in OVX animals. As for clinical studies, they mainly examine the temporal changes of the inflammatory stage or perform comparison between osteoporotic/fragility fracture patients and normal subjects without fracture. Our review of these studies emphasizes the lack of understanding that inflammation plays in the altered fracture healing response of osteoporotic/elderly patients. Taken together, it is clear that additional studies, preclinical and clinical, are required to dissect the regulatory role of inflammatory response in osteoporotic fracture healing.Cite this article: Bone Joint Res 2020;9(7):368–385.

Biological Effect of Single or Combined Pharmacological Therapy Using Alendronate and Simvastatin on Implant Osseointegration: An In Vivo Study in Healthy and Osteoporotic Rat Models

To evaluate peri-implant bone formation following single or combined systemic administration of alendronate and simvastatin in healthy and osteoporotic rats, eighty female Wistar rats were ovariectomized (n = 40) or sham-operated (n = 40). At six weeks, implants were placed in femoral condyles. Then, ovariectomized (OVX) and sham-operated (SHAM) animals received daily subcutaneous alendronate (50 µg/kg), simvastatin (5 mg/kg), or both, for three weeks. Control animals received subcutaneous saline. Thereafter, specimens were retrieved for biomechanical testing, histological evaluation, and bone area (BA%) and bone-to-implant contact (BIC%). In healthy and osteoporotic rats, similar (p &gt; 0.05) push-out values were observed for all groups. For BA% analysis, control rats showed similar results for OVX (9.2% ± 2.4%) and SHAM (11.1% ± 3.5%) animals. In contrast, single or combined drug therapy significantly increased BA% compared to controls in both healthy and osteoporotic conditions (p &lt; 0.05). In osteoporotic conditions, alendronate alone showed a superior effect on BA% compared to simvastatin alone, or their combination. Systemic alendronate, simvastatin, or both showed a similar BIC% compared to controls (p &gt; 0.05). The present study demonstrates that single or combined systemic alendronate and simvastatin increases bone formation around implants (i.e., distance osteogenesis) in healthy and osteoporotic bone conditions. However, these drugs showed no beneficial effect on direct bone-to-implant contact or implant fixation.

An animal trial to study damage and repair in ovariectomized rabbits

Microdamage accumulates in bone matrix and is repaired through bone remodeling. Conditions such as osteoporosis and treatment with antiresorptive bisphosphonates can influence this remodeling process. In order to study microdamage accrual and repair in the context of osteoporosis and osteon structures, we set out to modify the rabbit forelimb fatigue model. New Zealand White rabbits (N = 43, 10 months old) received either ovariectomy (OVX) or sham surgeries and were used for forelimb fatigue loading. OVX increased fluorochrome labeling of intracortical and periosteal bone of the ulna, without changes in bone mass. Monotonic and cyclic loading of the forelimb did not reveal any statistical differences between stiffness, ultimate force, or displacement to failure between sham and OVX rabbits. Two levels of fatigue loading, chosen to represent “low” and “moderate” fatigue (25% and 40% of total displacement to failure, respectively), were used on OVX forelimbs to examine microdamage creation. However, neither group showed increased damage burden as compared to non-loaded controls. Following fatigue loading rabbit ulnae had increased intracortical remodeling and periosteal lamellar bone formation in “moderate” fatigue limbs, although no basic multicellular units or microdamage-targeted remodeling was observed. In summary, we adapted the rabbit forelimb fatigue model to accommodate OVX animals. However, loading parameters that could induce repeatable microdamage burden were not identified. Thus, while increased intracortical remodeling and periosteal bone formation were induced by our fatigue loading regimen, this preliminary study did not establish conditions to allow future study of the interactions between microdamage accrual and repair.

Quercetin alleviates cognitive decline in ovariectomized mice by potentially modulating histone acetylation homeostasis

The synergism between estrogen and histone tail acetylation-mediated memory formation is not clearly understood. Here, we attempt to study the altered histone acetylation homeostasis mediated changes in cognition following ovariectomy and evaluate the protective effect of quercetin. A significant reduction in estradiol levels with subsequent depletion in spatial memory and learning functions assessed by Morris water maze, novel object recognition test and elevated plus maze was observed in ovariectomized (OVX) mice. Correspondingly, a significant decline in neuroplasticity markers like brain-derived neurotrophic factor (BDNF), synaptophysin (SYP) and postsynaptic density-95 (PSD-95) was observed in cortex and hippocampus of OVX animals. Notably, histone acetyltransferase (HAT)/histone deacetylase (HDAC) balance was significantly disrupted in cortex and hippocampus of OVX mice. Lowered extracellular signal regulated kinases (ERK) and cAMP response element binding protein activation observed in OVX brain regions might account for HAT/HDAC imbalance. Altered HAT/HDAC homeostasis results in lowered histone 3 acetylation in OVX brain that suppressed transcriptional activation of neuroplasticity-related genes. Quercetin supplementation to OVX mice for 4 weeks was able to ameliorate cognitive impairment by restoring HAT/HDAC homeostasis through ERK activation and reversing alterations in neuroplasticity markers in cortex and hippocampus of OVX mice. Taken together, our results suggest that quercetin alleviates ovariectomy-induced cognitive decline by modulating histone acetylation homeostasis.

Age and Menopause Effects on Ocular Compliance and Aqueous Outflow

PurposeGlaucoma is the second leading cause of blindness worldwide. Recent work suggests that estrogen and the timing of menopause play a role in modulating the risk of developing glaucoma. Menopause is known to cause modest changes in intraocular pressure; yet, whether this change is mediated through the outflow pathway remains unknown. Menopause also affects tissue biomechanical properties throughout the body; however, the impact of menopause on ocular biomechanical properties is not well characterized.MethodsHere, we simultaneously assessed the impact of menopause on aqueous outflow facility and ocular compliance, as a measure of corneoscleral shell biomechanics. We used young (3–4 months old) and middle-aged (9–10 months old) Brown Norway rats. Menopause was induced by ovariectomy (OVX), and control animals underwent sham surgery, resulting in the following groups: young sham (n = 5), young OVX (n = 6), middle-aged sham (n = 5), and middle-aged OVX (n = 5). Eight weeks postoperatively, we measured outflow facility and ocular compliance.ResultsMenopause resulted in a 34% decrease in outflow facility and a 19% increase in ocular compliance (P = 0.011) in OVX animals compared with sham controls (P = 0.019).ConclusionsThese observations reveal that menopause affects several key physiological factors known to be associated with glaucoma, suggesting that menopause may contribute to an increased risk of glaucoma in women.

Oral administration of bovine lactoferrin accelerates the healing of fracture in ovariectomized rats.

Lactoferrin has recently been reported for its potent bone growth effects. However, the effects of lactoferrin on the healing process of fragility fracture have not yet been studied, so the purpose of this study is to investigate whether oral administration of lactoferrin can promote the fracture healing in an OVX animal model. Three months after bilateral ovariectomy, all rats underwent unilateral tibial osteotomy and were then randomly divided into control group and bovine lactoferrin (bLF) group. At 4 and 8 weeks post-fracture, animals were sacrificed, and the fractured tibiae and serum samples were collected for evaluation. Our results showed that bLF treatment not only accelerated the bone growth at an early stage of OPF healing but also shortened the remolding process of OPF healing. When compared to control group, bLF treatment induced a significant rise in callus BMD (by 35.0% at 4 weeks and by 39.7% at 8 weeks; both p < 0.05) consistent with enhanced biomechanical strength of the callus, with ultimate force increased by 3.39-fold at 4 weeks (p < 0.05) and 1.95-fold at 8 weeks (p < 0.05). Besides, bLF administration resulted in a substantial increase in serum levels of BALP and a significant decrease in serum levels of TRAP 5b and TNF-α. Moreover, both the RANKL/OPG mRNA ratio and the expression of TNF-α in the callus of bLF-treated group were markedly lower than those in the control group. At a dose of 85mg/kg/day orally administrated bLF potently promoted the bone healing following tibial fracture in OVX rats.

Ovariectomy alters lengthening contraction induced heat shock protein expression.

Estrogen appears to play a role in minimizing skeletal muscle damage as well as regulating the expression of the protective heat shock proteins (HSPs). To clarify the relationship between estrogen, muscle HSP content, and muscle damage, tibialis anterior (TA) muscles from ovary-intact (OVI; n = 12) and ovariectomized (OVX; 3 weeks, n = 12) female Sprague-Dawley rats were subjected to either 20 or 40 lengthening contractions (LCs). Twenty-four hours after stimulation, TA muscles were removed, processed, and assessed for HSP25 and HSP72 content as well as muscle (damage) morphology. No differences in muscle contractile properties were observed in TA muscles between OVI and OVX animals for peak torque during the LCs. In unstressed TA muscles, the basal expression of HSP72 expression was decreased in OVX animals (P < 0.05) while HSP25 content remained unchanged. Following 20 LCs, HSP25 content was elevated (P < 0.05) in TA muscles from OVX animals but unchanged in muscles from OVI animals. Following 40 LCs, HSP25 content was elevated (P < 0.01) in TA muscles from both OVI and OVX animals while HSP72 content was elevated only in TA muscles from OVI animals (P < 0.05). Taken together, these data suggest the loss of ovarian hormones, such as estrogen, may impair the skeletal muscle cellular stress response thereby rendering muscles more susceptible to certain types of contraction induced damage. Novelty Ovariectomy alters muscle HSP72 content. Muscle contractile measures are maintained following ovariectomy.