OVX Female Rats Research Articles

Effect of probiotic supplementation in murines with induced osteoporosis: systematic review with meta-analysis

Postmenopausal osteoporosis results in low bone mineral density, interference in bone microarchitecture, decreased bone strength, and an increased risk of fragility fractures. This systematic review aimed to increase the level of evidence on supplementation with bacteria of the genus Bifidobacterium in induced osteoporosis in murines, rats, and ovariectomized female mice (OVX). The search was initially conducted using the PubMed, Embase, ScienceDirect, Scopus, Web of Science, and Scielo databases in 2024, using the keywords “osteoporosis”, “murine”, “ovariectomized”, “OVX”, and “Bifidobacterium”. In total, 89 full articles, abstracts, or book chapters were found and after detailed screening, 5 studies met the inclusion criteria, using 66 animals, randomly divided between control and treatment groups. The results demonstrated that oral supplementation with bacteria of the genus Bifidobacterium significantly improves parameters indicative of bone health BMD, BMC, BV/TV%, and Tb.Sp. In conclusion, this meta-analysis provides evidence that bacteria of the genus Bifidobacterium are involved in anti-osteoporotic mechanisms in female OVX rats and mice, as a preclinical model, and suggests the need for further studies in postmenopausal women as a therapeutic alternative or complementary therapy to conventional osteoporosis treatments.

Estrogens and Olfactomedins May Induce a Feedback Loop that Increases Nicotine Consumption in Females via the Brain Reward Circuitry

Estrogen is associated with enhanced nicotine use in women, with enhanced vulnerability to nicotine use disorders being associated with higher levels of circulating estrogen. In ovariectomized (OVX) female rats, estradiol (E2) treatments increased estrogen receptor β (ERβ) and not ERα in a key brain reward region, the nucleus accumbens core (NAcore). Therefore, we hypothesized that ERβ might be important in the reward circuitry for regulating nicotine use in females. To determine ERβ-specific functions that might regulate nicotine use, we analyzed large sequencing datasets of estrogen-induced genes and narrowed the list to those expressed in the brain and that have a hormone function. We found one class of genes known as the olfactomedins (OLFMs), which meet these criteria. Therefore, we wanted to determine whether ERα or ERβ regulates the expression of the OLFM isoforms. We used human uterine cells (Ishikawa) that express both ER isoforms and the OLFMs and found that two-hour E2 treatments induced the expression of OLFM1 and OLFM3. However, nicotine only suppressed the E2-induction of OLFM1 and not the others. We next performed chromatin immunoprecipitation (ChIP) assays with these cells and treatments using antibodies for ERα, ERβ, or GFP control. We found that the OLFM1 promoter was only enriched by ERβ, and no binding of ERα was observed. We next treated OVX female rats with E2, nicotine, E2+nicotine, or vehicle and found that Olfm1 and Olfm2 significantly increased in the NAcore, and all three OLFM isoforms were significantly higher in the ventral tegmental area (VTA), another key region within the mesolimbic reward pathway. Nicotine suppressed the E2-induced OLFM expression in the NAcore and VTA. These new findings suggest that ERβ-induction of the olfactomedins might serve as a feedback loop for driving nicotine addiction processes, which is suppressed when nicotine is on board. These results indicate a potential new target for therapeutic cessation of nicotine use in women. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cross-sexual Effects of Testosterone on Mesenteric Arterial Reactivity in Ovariectomized Female Rats

The number of transgender individuals is on the rise in the United States. Although there are studies on the effects of sex hormones on cardiovascular function of cisgender male and female, still much is not known about the effects of cross-sex hormone therapy (CSHT) on cardiovascular function in transgender individuals. It has been shown that the risk factors of cardiovascular diseases (CVD) are increased in transgenders after they undergo CSHT. Epidemiological studies suggest that transgender females (male to female, MtF) on estrogen therapy develop higher risk of CVD compared to transgender males (female to male, FtM) taking testosterone. This study investigates the effects of testosterone or estrogen treatment on the third branch of mesenteric arterial reactivity in ovariectomized female rats. Briefly, 8-10 weeks old female Sprague Dawley (SD) rats were ovariectomized (OVX) and subcutaneously implanted with placebo (OVX + PL) or testosterone (OVX + T, 7.5 mg) or 17β- estradiol (OVX + E2, 1.5 mg) pellets for about 35 days. Age matched intact female SD rats were also included in the experimental groups. Endothelium-dependent vasorelaxation (EDV) to acetylcholine (ACh) was measured in the precontracted mesenteric arteries with phenylephrine (PE), using wire myography. Responses to sodium nitroprusside (SNP)-induced vasorelaxation, and PE- and endothelin-1 (ET-1) induced vasocontraction were also determined. We demonstrated that the responses to ACh in rat mesenteric arteries were not altered by either ovariectomy or estrogen treatment. However, testosterone treatment of OVX female rats significantly enhanced the sensitivity to ACh-induced vasorelaxation compared with those in other experimental groups. Moreover, the mesenteric arteries of OVX + T exhibited a trend of reduced sensitivity to PE responses compared to other groups. Furthermore, the maximum tension to ET-1 was significantly reduced in mesenteric arteries of OVX + T compared to OVX+E2 and OVX+PL groups. The vasorelaxation responses to SNP were not altered in any of experimental groups studied. These data suggest that elevated sensitivity to ACh in testosterone-treated ovariectomized female rats might be, in part, due to reduced vessel responses to contractile agents, but not altered sensitivity of smooth muscle to nitric oxide (NO) in this group. Additional studies are needed to investigate the underlying mechanisms and document the consequences for the improvement of EDV observed in OVX + T rats. NIDA/NIGMS, 1SC1DA052120-01 to MF, Bridge Grant from University of the Pacific to RR. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

THE IMPACT OF SEX ON METABOLIC AND FUNCTIONAL ABNORMALITIES, INDUCED BY HIGH-FRUCTOSE-DIET IN RATS

Metabolic syndrome (MetS) is a cluster of cardiometabolic risk factors, which manifestation may be different for men and women. Changes in sex hormone levels in men and women during lifespan contribute to increased cardiometabolic risk. Data of clinical researches suggest that transition from normal glucose tolerance to dysglycemia lead to more adverse differences in cardiometabolic risk factors in women than in men, suggesting an important impact of sex on the development of metabolic abnormalities. A significant increase in risk may be due to the sex differences in manifestation of some components of metabolic syndrome, but accurate reasons of these discrepancies need future investigations. The aim of this study was to determine the differences in impairment of glucose, lipid metabolism and cardiovascular systems function between male and female rats with different levels of estrogen sufficiency. Materials and Methods. MetS was modelled in male and female rats with different levels of estrogens by chronic (for 8 weeks) intake of fructose (high-fructose diet - HFD) with drinking water at a concentration 200 g/L. Estrogens deficiency was reproduced by bilateral ovariectomy. Experimental rats were divided into five groups: control males (n=6), control females (n=6), males with HFD (n=6), sham-operated females with intact ovaries and HFD (n=6), ovariectomized (OVX) females with HFD (n=6). Glucose homeostasis was assessed by basal glycemia, glycemia during the intra-abdominal glucose tolerance test and fructosamine level. Insulin sensitivity was determined using intra-abdominal insulin tolerance test. Body weight gain, visceral fats, level of total cholesterol, triglycerides, levels of estradiol and testosterone in blood serum were determined. At the end of the study, electrocardiograms were recorded in three standard leads from the limbs. Data are presented as mean ± standard error of mean (SEM). Results. It was established that HFD have not been induced metabolic abnormalities in female rats with intact ovaries. HFD, independently of sex, led to the development of insulin resistance and significant increase in fructosamine level in blood serum of rats. Ovariectomized females with HFD had greater intensity of body weight gain, significantly higher levels of relative mass of fats, more pronounced alterations of lipid metabolism with increased total cholesterol and significantly higher levels of triglycerides, than HFD-treated male rats. However, impairment of glucose tolerance, induced by HFD, was more pronounced in male than in OVX female rats. We found the decline testosterone levels and slight increase in estradiol levels in male rats with HFD. OVX females with HFD had a moderate significant increase in testosterone levels and dramatically decrease in estradiol concentration as result of bilateral ovariectomy. HFD, independently from sex, induced the development of sinus tachycardia in experimental animals. Metabolic abnormalities, induced by HFD, in OVX female rats led to more pronounced disturbances of heart depolarization and repolarization processes and development of diastolic dysfunction compare to HFD-treated males. Conclusions. We revealed that sex and estrogens levels have a great impact on the impairment of glucose and lipid metabolism and cardiovascular systems function, induced by high-fructose diet in rats. The obtained data justifies the necessity to take into account sex and estrogens sufficiency in the development of gender-specific prevention and treatment of metabolic syndrome.

Roflumilast ameliorates ovariectomy-induced depressive-like behavior in rats via activation of AMPK/mTOR/ULK1-dependent autophagy pathway

AimsRoflumilast, a well-known phosphodiesterase-4 (PDE-4) inhibitor, possess an anti-inflammatory activity with approved indications in chronic obstructive pulmonary disease. This study aimed to evaluate the neuroprotective role of roflumilast in ovariectomy (OVX)-induced depressive-like behavior in female rats and to shed light on a potential autophagy enhancing effect. Main methodsRats were randomly divided into four groups: sham, OVX, OVX + roflumilast (1 mg/kg, p.o), and OVX + roflumilast + chloroquine (CQ) (50 mg/kg, i.p). Drugs were administered for 4 weeks starting 2 weeks after OVX. Key findingsRoflumilast improved the depressive-like behaviors observed in OVX rats as evidenced by decreasing both forced swimming and open field immobility times while, increasing % sucrose preference and number of open field crossed squares. Histopathological analysis provides further evidence of roflumilast's beneficial effects, demonstrating that roflumilast ameliorated the neuronal damage caused by OVX. Roflumilast antidepressant potential was mediated via restoring hippocampal cAMP and BDNF levels as well as down-regulating PDE4 expression. Moreover, roflumilast revealed anti-inflammatory and anti-apoptotic effects via hindering TNF-α level and diminishing Bax/Bcl2 ratio. Roflumilast restored the autophagic function via up-regulation of p-AMPK, p-ULK1, Beclin-1 and LC3II/I expression, along with downregulation of P62 level and p-mTOR protein expression. The autophagy inhibitor CQ was used to demonstrate the suggested pathway. SignificanceThe present study revealed that roflumilast showed an anti-depressant activity in OVX female rats via turning on AMPK/mTOR/ULK1-dependent autophagy pathway; and neurotrophic, anti-inflammatory, and anti-apoptotic activities. Roflumilast could offer a more secure alternative to hormone replacement therapy for postmenopausal depression treatment.

Hypoxic ventilatory response and respiratory chemosensitivity in differing rat models of female sex hormone loss

Respiratory function is modulated by circulating sex steroids. Specifically, concentrations of circulating 17β-estradiol and progesterone regulate the responsiveness of the respiratory system in adult female rats. Progesterone was first noted to influence breathing following the observation that pregnancy impacted blood gasses. 17β-estradiol is the most abundant and neuroactive form of estrogen, and female respiratory neuroplasticity is dependent on its presence. Circulating sex hormone levels fluctuate across the normal estrus cycle and gradually decline as part of the natural aging process. The specific influences of sex hormones on hypoxic ventilatory response (HVR) and chemosensitivity are slowly emerging in the literature. A developing pattern is that short-term hormone fluctuations, such as estrus cycle, do not appear to significantly change basic respiratory responsiveness. However, less is known about larger hormone shifts, such as those following the loss of gonads or with natural aging. We hypothesized that the loss of sex hormones would differentially impact HVR and chemosensitivity in an ovariectomy (OVX) model versus a naturally aged female rat. Specifically, we theorized that aged rats would demonstrate reduced sensitivity to hypoxia and reduced dynamic range within ventilatory output, as compared to OVX and intact female rats. Three experimental groups of female rats were used: young rats (3 mos old; n=4), young OVX rats (n=5), and aged rats (>24 mos old; n=5). Chemosensitivity was measured during progressive hypoxic challenges at 15%, 12%, and 9% oxygen (O 2 ) using unrestrained barometric plethysmography. Serum samples were taken for measurement of estrogen (E 2 ) and progesterone (P) using ELISA assays. At the highest level of hypoxia (9% O 2 ), all groups exhibited intact chemosensitivity as demonstrated by significant changes in minute ventilation (V E ; < 0.05), V E controlling for metabolism (V E / VCO 2 ; < 0.05), and respiratory neural drive (calculated as the ratio of tidal volume (T V ) to inspiratory time (Ti), controlling for metabolism; T V /T i /VCO 2 ; < 0.05). Further analysis showed that young and OVX groups demonstrated greater sensitivity to hypoxia as compared to the aged group. Using analyses targeting rate of change, the young and OVX groups had a faster rate of change in V E , V E / VCO 2 , and T V /T i /VCO 2 (< 0.05) as the hypoxia intensity increased. ELISA analyses suggest that E 2 , but not P, concentration is significantly correlated with these results (< 0.05). Collectively, these data indicate that aging impacts respiratory function in complex and unique ways that differ from OVX in female rats. This may be significant since OVX is commonly used as a model of age-related physiological changes. University of Minnesota BIRCWH/Women's Health Research Program Seed Grant, National Heart Lungs Brain Institute R01HL146477, Rehabilitation Science Graduate Program, UMN Medical School This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

PS-B02-10: MTOR INHIBITOR IMPROVES TESTOSTERONE-INDUCED MYOCARDIAL HYPERTROPHY IN HYPERTENSIVE RATS

Compelling evidence have described the incidence of hypertension and left ventricular hypertrophy in postmenopausal women is significantly increased worldwide. Our team previous research identified that androgen was an underlying factor contributing to increased blood pressure and LVH in postmenopausal women. However, little is known about how androgens affect LVH in postmenopausal hypertensive women. The purpose of this study was to evaluate the role of mTOR signaling pathway in myocardial hypertrophy in androgen-induced postmenopausal hypertension and whether mTOR inhibitors can protect the myocardium from androgen-induced interference to prevent and treat cardiac hypertrophy. For that, ovariectomized spontaneously hypertensive rats aged 12 weeks were used to study the effects of testosterone T 2.85 mg/kg/weekly im on blood pressure and myocardial tissue. On the basis of antihypertensive therapy chlorthalidone 8 mg/kg/day ig, the improvement of blood pressure and myocardial hypertrophy in rats treated with different dose gradients of rapamycin 0.8 mg/kg/day Vs 1.5 mg/kg/day Vs 2 mg/kg/day ip in OVX+ estrogens E 9.6 mg/Kg/day, ig+T group was further evaluated. After T intervention, the OVX female rats exhibited significant increments in the heart weight / tibial length, cross-sectional area of cardiomyocytes and the mRNA expressions of atrial natriuretic peptide, myosin heavy chain and matrix metalloproteinase 9 accompanied by a significant reduction in the uterine weight/TL and issue inhibitor of metalloproteinase 1. Mammalian rapamycin receptor mTOR, ribosomal protein S6 kinase S6K1, 4E-bindiong protein 1 4EBP1 and eukaryotic translation initiation factor 4E in myocardial tissue of OVX+E+T group were expressed at higher levels than those of the other four groups. On the other hand, rapamycin abolished the effects of T-induced cardiac hypertrophy, decreased the systolic and diastolic blood pressure of SHR, and inhibited the activation of mTOR/ S6K1/4EBP1 signaling pathway in a concentration-dependent manner. Collectively, these data suggest that the mTOR/S6K1/4EBP1 pathway is an important therapeutic target for the prevention of LVH in postmenopausal hypertensive women with high T levels. Our findings also support the standpoint that the mTOR inhibitor, rapamycin, can eliminate T-induced cardiomyocyte hypertrophy.

Androgens Enhance Recognition Memory and Dendritic Spine Density in the Hippocampus and Prefrontal Cortex of Ovariectomized Female Rats

Estrogen replacement has been repeatedly shown to enhance memory and increase dendritic spine density in the hippocampus and prefrontal cortex of ovariectomized (OVX) female rats. Given the potential deleterious effects of chronic estrogen administration, the present study assessed cognitive function using recognition memory tasks and measured dendritic spine density in the CA1 region of the hippocampus and medial prefrontal cortex after subchronic androgen replacement to adult OVX female rats. All androgens enhanced recognition memory in OVX rats, but object placement (OP) and object recognition (OR) results differed. Only testosterone enhanced OR. Testosterone had no effect on OP while dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT) and androstenedione (AD) enhanced OP. Dendritic spine density was increased by both TP and DHEA in both brain areas (DHT and AD were not tested). Lastly, we used the aromatase inhibitor, letrozole, to discriminate between potential androgenic and estrogenic effects of androgens on behavior. Letrozole alone did not alter recognition memory in OVX rats and did not block the effects of either TP or DHEA on recognition memory suggesting that effects were mediated via androgenic mechanisms. The present results expand previous information on gonadal hormone actions and show that, in addition to estrogens, androgens also improve memory and increase spine density in brains of OVX female rats. While requiring further investigation, these observations provide a basis for therapeutic interventions in the treatment of menopausal, age or disease related memory loss.

MTOR INHIBITOR IMPROVES TESTOSTERONE-INDUCED MYOCARDIAL HYPERTROPHY IN HYPERTENSIVE RATS

Objective: Compelling evidence have described the incidence of hypertension and left ventricular hypertrophy (LVH) in postmenopausal women is significantly increased worldwide. Our team’s previous research identified that androgen was an underlying factor contributing to increased blood pressure and LVH in postmenopausal women. However, little is known about how androgens affect LVH in postmenopausal hypertensive women. The purpose of this study was to evaluate the role of mTOR signaling pathway in myocardial hypertrophy in androgen-induced postmenopausal hypertension and whether mTOR inhibitors can protect the myocardium from androgen-induced interference to prevent and treat cardiac hypertrophy. Design and method: For that, ovariectomized (OVX) spontaneously hypertensive rats (SHR) aged 12 weeks were used to study the effects of testosterone (T 2.85 mg/kg/weekly im) on blood pressure and myocardial tissue. On the basis of antihypertensive therapy (chlorthalidone 8 mg/kg/day ig), the improvement of blood pressure and myocardial hypertrophy in rats treated with different dose gradients of rapamycin (0.8 mg/kg/day Vs 1.5 mg/kg/day Vs 2 mg/kg/day ip) in OVX+ estrogens (E 9.6 mg/Kg/day, ig)+T group was further evaluated. Results: After T intervention, the OVX female rats exhibited significant increments in the heart weight / tibial length (TL), cross-sectional area of cardiomyocytes and the mRNA expressions of atrial natriuretic peptide, B- myosin heavy chain and matrix metalloproteinase 9 accompanied by a significant reduction in the uterine weight/TL and issue inhibitor of metalloproteinase 1. Mammalian rapamycin receptor (mTOR), ribosomal protein S6 kinase (S6K1), 4E-bindiong protein 1(4EBP1) and eukaryotic translation initiation factor 4E in myocardial tissue of OVX+E+T group were expressed at higher levels than those of the other four groups. On the other hand, rapamycin abolished the effects of T-induced cardiac hypertrophy, decreased the systolic and diastolic blood pressure of SHR, and inhibited the activation of mTOR/ S6K1/4EBP1 signaling pathway in a concentration-dependent manner. Conclusions: Collectively, these data suggest that the mTOR/S6K1/4EBP1 pathway is an important therapeutic target for the prevention of LVH in postmenopausal hypertensive women with high T levels. Our findings also support the standpoint that the mTOR inhibitor, rapamycin, can eliminate T-induced cardiomyocyte hypertrophy.

Aortic Relaxation in Castrated Male Rats and Responses to Estrogen Treatment

Previous studies suggest that risk factors for cardiovascular diseases (CVD) have increased in the transgender population after they undergo cross‐sex hormone therapy (CSHT). Several studies, including ours in animal models as well as in humans, have shown that estrogen exerts direct beneficial effects on the vessel wall in healthy females. A growing body of evidence also suggests that androgen exhibits protective actions on the cardiovascular system. However, the effects of CSHT in cardiovascular physiology in males or females are less understood.The current study was, therefore, undertaken to evaluate the effects of estrogen treatment on aortic reactivity in castrated (CAS) male rats. Specifically, we investigated the effects of in vivo treatment with 17β‐estradiol (E2) on the aortic responses of CAS male as well as ovariectomized (OVX) female rats.Age‐matched CAS male and OVX female rats were implanted subcutaneously with either 17β‐estradiol (E2,1.5 mg) or a placebo‐containing pellet for 30 days. Endothelium‐dependent vasorelaxation (EDV) in response to acetylcholine (ACh, 10‐8 to 10‐5 M) in aorta precontracted with phenylephrine (PE, 2 µM) was measured before and after pretreatment with indomethacin (10 µM, cyclooxygenase inhibitor), followed by N‐nitro‐ L‐arginine methyl ester [L‐NAME, 200 µM, a nonselective nitric oxide synthase (NOS) inhibitor]. Endothelium‐independent vasorelaxation was assessed by measuring sodium nitroprusside (SNP)‐induced relaxation response in endothelium‐denuded rings in addition to the contractile response curves to PE before and after pretreatment with L‐NAME in aortic rings.EDV to ACh was significantly impaired in aortic rings from E2‐treated CAS rats compared to their placebo‐treated counterparts and E2‐treated OVX rats. The impairment of EDV to ACh in E2‐treated CAS rats was accompanied by a significant decrease in nitric oxide (NO) contribution to ACh‐induced relaxation in this group. The smooth muscle sensitivity to NO as measured by SNP relaxation response curve was not altered, whereas the responsiveness to PE was slightly enhanced in the aortas of E2‐treated CAS group compared to those in other experimental groups. Endothelium‐derived NO release during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was not different in the aortas of experimental groups.Overall, our data demonstrate that aortic function was altered in E2‐treated CAS male rats. Here, we provide the first evidence of impairment in aortic relaxation in E2‐treated CAS male rats, possibly via a significant decrease in the relative contribution of NO to the regulation of vascular relaxation response. Furthermore, the comparison of current data in the CAS male rats with our recently published data on the aortic reactivity in the intact males appears to rule out any effect of testosterone deprivation on the stimulated release of NO in male rat aortas. Additional studies will be needed to document the direction and magnitude of CSHT interaction with vasculatures.

Mechanism of Soy Isoflavone Daidzein-Induced Female-Specific Anorectic Effect.

Epidemiological studies suggest that regular intake of soy isoflavone exerts a preventive effect on postmenopausal obesity and other forms of dysmetabolism. Estrogens inhibit eating behavior. Soy isoflavones may act as estrogen agonist in estrogen-depleted conditions, whereas they may either act as an estrogen antagonist or be ineffective in estrogen-repleted conditions. We investigated the effects of dietary soy isoflavone on food intake under various estrogen conditions using male, ovariectomized (OVX), and non-OVX female rats, and compared the effects with those of estradiol. We found that soy isoflavones reduced food intake in females specifically, regardless of whether ovariectomy had been performed, whereas subcutaneous implantation of estradiol pellet did not reduce food intake in intact female rats, but did so in OVX female and male rats. Contrary to this hypothesis, the reduction in food intake may not be caused by the estrogenic properties of soy isoflavones. It is of great interest to understand the mechanisms underlying the anorectic effects of soy isoflavones. In this non-systematic review, we summarize our recent studies that have investigated the bioactive substances of anorectic action, pharmacokinetic properties of soy isoflavones, and the modification of central and peripheral signals regulating appetite by soy isoflavones, and selected studies that were identified via database mining.

Estrogen Status and Trigeminal Ganglion Responses to Jaw Movement

Chronic temporomandibular joint disorders (TMDs) present with pain in the temporomandibular joint (TMJ) and muscles of mastication. Risk factors for TMD include localized joint/muscle inflammation and estrogen status. This study determined whether mild tissue inflammation and estrogen status influenced the responses of trigeminal ganglion neurons to jaw palpation or jaw movement, 2 key diagnostic features of clinical TMD, in adult rats. Neuronal activity was recorded from male rats, ovariectomized (OvX) female rats, and OvX female rats injected with 17β-estradiol 24 h prior to testing (OvXE). Neurons were tested for responses to deep press over the TMJ region and jaw movement in 3 directions (open, protrusion, lateral) 10 d after intra-TMJ injection of a low dose of complete Freund’s adjuvant (CFA) or vehicle (sham). Deep press evoked similar responses in all treatment groups. The response magnitude to jaw opening and protrusion was significantly greater for neurons recorded from OvXE CFA-treated rats than from OvX CFA-treated or OvXE sham rats. The responses to lateral movement of the jaw were similar across all treatment groups. Most neurons (70% to 90%) displayed a static response pattern to jaw movement independent of direction. Estradiol treatment also increased the proportion of neurons that were excited by jaw movement in >1 direction as compared with untreated OvX females or males. These results suggest that mild localized inflammation in the TMJ region during periods of elevated estrogen were sufficient to increase the peripheral driving force for jaw movement–evoked hyperalgesia.

Protective effect of propolis on myocardial ischemia/reperfusion injury in males and ovariectomized females but not in intact females.

The aim of this study is to investigate the effect of propolis, which may have estrogenic effects, on myocardial ischemia/reperfusion (mI/R) injury not only in male rats but also in intact and ovariectomized (ovx) female rats. Six groups were formed: untreated males (n=8), treated males (n=9), untreated intact females (n=9), treated intact females (n=10), untreated ovx females (n=10), and treated ovx females (n=8). An alcoholic extract of a single dose of propolis (200mg/kg) was administered orally daily for 14days. Thirty minutes of ischemia and 120min of reperfusion were performed. Blood pressure, heart rate, arrhythmias (ventricular premature contraction [VPC], ventricular tachycardia [VT], ventricular fibrillation [VF]), and myocardial infarct size were evaluated. Total antioxidant status (TAS), total oxidant status (TOS), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and 17 beta-estradiol (E2) were measured. The untreated females showed more resistance to mI/R injury than the untreated males, as evidenced by lower duration, incidence, and score of arrhythmias, and smaller infarct size (p<.05). After ovx, this resistance disappeared. Propolis improved these values in treated males and treated ovx females (p<.05). Propolis increased TAS in treated males and decreased TOS in treated ovx females as well as elevated SOD in all treated groups (p<.05). Propolis decreased E2 level in treated intact females; however, it increased E2 level in treated ovx females (p<.05). The results revealed that propolis could protect the heart against mI/R injury in males and ovx females. PRACTICAL APPLICATIONS: It is known that the female heart has an increased sensitivity to myocardial ischemia/reperfusion (mI/R) injury due to estrogen deficiency and/or estrogen deprivation following menopause or surgical removal of the ovaries. Propolis has the potential to mimic estrogen under physiological and pathophysiological conditions, as well as its antioxidant property. The results indicated that propolis decreased myocardial infarct size, arrhythmia score, arrhythmia duration, and incidence in ovariectomized female rats and male rats. In addition, the present results demonstrated that an alcoholic extract of propolis as a natural product can effectively maintain the resistance of female heart to mI/R injury after estrogen deficiency.

MTOR inhibitor improves testosterone-induced myocardial hypertrophy in hypertensive rats.

Compelling evidence has described that the incidence of hypertension and left ventricular hypertrophy (LVH) in postmenopausal women is significantly increased worldwide. Our team’s previous research identified that androgen was an underlying factor contributing to increased blood pressure and LVH in postmenopausal women. However, little is known about how androgens affect LVH in postmenopausal hypertensive women. The purpose of this study was to evaluate the role of mammalian rapamycin receptor (mTOR) signaling pathway in myocardial hypertrophy in androgen-induced postmenopausal hypertension and whether mTOR inhibitors can protect the myocardium from androgen-induced interference to prevent and treat cardiac hypertrophy. For that, ovariectomized (OVX) spontaneously hypertensive rats (SHR) aged 12 weeks were used to study the effects of testosterone (T 2.85 mg/kg/weekly i.m.) on blood pressure and myocardial tissue. On the basis of antihypertensive therapy (chlorthalidone 8 mg/kg/day ig), the improvement of blood pressure and myocardial hypertrophy in rats treated with different dose gradients of rapamycin (0.8 mg/kg/day vs 1.5 mg/kg/day vs 2 mg/kg/day i.p.) in OVX + estrogen (E 9.6 mg/kg/day, ig) + testosterone group was further evaluated. After testosterone intervention, the OVX female rats exhibited significant increments in the heart weight/tibial length (TL), area of cardiomyocytes and the mRNA expressions of ANP, β-myosin heavy chain and matrix metalloproteinase 9 accompanied by a significant reduction in the uterine weight/TL and tissue inhibitor of metalloproteinase 1. mTOR, ribosomal protein S6 kinase (S6K1), 4E-binding protein 1 (4EBP1) and eukaryotic translation initiation factor 4E in myocardial tissue of OVX + estrogen + testosterone group were expressed at higher levels than those of the other four groups. On the other hand, rapamycin abolished the effects of testosterone-induced cardiac hypertrophy, decreased the systolic and diastolic blood pressure of SHR, and inhibited the activation of mTOR/S6K1/4EBP1 signaling pathway in a concentration-dependent manner. Collectively, these data suggest that the mTOR/S6K1/4EBP1 pathway is an important therapeutic target for the prevention of LVH in postmenopausal hypertensive female rats with high testosterone levels. Our findings also support the standpoint that the mTOR inhibitor, rapamycin, can eliminate testosterone-induced cardiomyocyte hypertrophy.

The effects of low-intensity exercise on cardiac glycogenesis and glycolysis in male and ovariectomized female rats on a fructose-rich diet.

We previously reported that low-intensity exercise prevented cardiac insulin resistance induced by a fructose-rich diet (FRD). To examine whether low-intensity exercise could prevent the disturbances of key molecules of cardiac glucose metabolism induced by FRD in male and ovariectomized (ovx) female rats, animals were exposed to 10% fructose solution (SF) or underwent both fructose diet and exercise (EF). Exercise prevented a decrease in cardiac GSK-3β phosphorylation induced by FRD in males (p<.001 vs. SF). It also prevented a decrease in PFK-2 phosphorylation in ovx females (p<.001 vs. SF) and increased the expression of PFK-2 in males (p<.05 vs. control). Exercise did not prevent a decrease in plasma membrane GLUT1 and GLUT4 levels in ovx females on FRD. The only effect of exercise on glucose transporters that could be indicated as beneficial is an augmented GLUT4 protein expression in males (p<.05 vs. control). Obtained results suggest that low-intensity exercise prevents harmful effects of FRD towards cardiac glycogenesis in males and glycolysis in ovx females. PRACTICAL APPLICATIONS: Low-intensity exercise, equivalent to brisk walking, was able to prevent disturbances in cardiac glycolysis regulation in ovx female and the glycogen synthesis pathway in male rats. In terms of human health, although molecular mechanisms of beneficial effects of exercise on cardiac glucose metabolism vary between genders, low-intensity running may be a useful non-pharmacological approach in the prevention of cardiac metabolic disorders in both men and postmenopausal women.

Ovariectomy and high salt increase blood pressure and alter sodium transport proteins in peripheral blood mononuclear cells of adult Wistar rats.

What is the central question of this study? In a model of salt-sensitive hypertension in ovariectomized (oVx) adult Wistar rats, what is the expression of proteins related to sodium transport in peripheral blood mononuclear cells (PBMCs), and how does the response of proteins to high sodium intake compare with changes in blood pressure in intact female rats? What is the main finding and its importance? Sodium transport proteins in PBMCs react to high sodium and blood pressure markedly differently in oVx versus intact female rats. Protein expression shows sodium and pressure sensitivity. Renal immune cells increase in oVx under high salt. Hypertension is a worldwide public health problem. High sodium consumption is associated with hypertension, and hypertensive mechanisms involve immunity cells. Peripheral blood mononuclear cells (PBMCs) are endowed with proteins related to sodium transport. We studied their abundance in PBMCs from intact (IF) or ovariectomized (oVx) adult Wistar rats under normal (NS) or high (HS) salt intake. Ovariectomy was performed at 60days of life. At 145days, one group of IF and oVx rats received NS or HS intake for 5days. Another group of IF HS and oVx HS rats received hydralazine (HDZ) to reduce blood pressure (BP). Sodium balance and BP were recorded. Expression of Na+ ,K+ -ATPase (NKA), Na+ -K+ -2Cl- cotransporter 1 (NKCC1), serum/glucocorticoid-regulated kinase 1 (SGK1), dopamine D1 like receptor (D1DR), CD4+ and CD8+ were determined in PBMCs and CD45+ leukocytes in renal tissue. IF HS rats showed increased natriuresis and normal BP. NKA and CD4+ expression diminished in IF HS. Instead, oVx HS rats had sodium retention and high BP and increased the expression of NKA, NKCC1, D1DR, CD4+ and CD8+ in PBMCs. Renal CD45+ leukocytes increased in oVx HS rats. HDZ decreased BP in all rats. Upon HDZ treatment, NKA did not change, NKCC1 decreased in oVx HS rats, while SGK1 increased in both IF HS and oVx HS rats. Hormonal background determines BP response and the expression of proteins related to sodium transport in PBMCs and renal immune cells at HS intake. The analysis of NKA, NKCC1 and SGK1 expression in PBMCs differentiated salt-sensitivity from BP variations.

Evaluating the effects of carbon monoxide releasing molecule-2 against myocardial ischemia-reperfusion injury in ovariectomized female rats.

Cardioprotective effect of carbon monoxide, a gasotransmitter against myocardial ischemia-reperfusion injury (I/R), is well established in preclinical studies with male rats. However, its ischemic tolerance in post-menopausal animals has not been examined due to functional perturbations at the cellular level. The protective role of carbon monoxide releasing molecule-2 (CORM-2) on myocardial I/R was studied in female Wistar rats using the Langendorff apparatus. The animals were randomly divided into normal and ovariectomized (Ovx) female rats and were maintained 2months post-surgery. Each group was further divided into 4 subgroups (n = 6/subgroup): normal, I/R, CORM-2-control (20μmol/L), and CORM-2-I/R. The cardiac injury was estimated via myocardial infarct size, lactate dehydrogenase, and creatine kinase levels in coronary effluent and cardiac hemodynamic indices. Mitochondrial functional activity was assessed by measuring mitochondrial electron transport chain enzyme activities, swelling behavior, mitochondrial membrane potential, and oxidative stress. Hemodynamic indices were significantly lower in ovariectomized rat hearts than in normal rat hearts. Sixty minutes of reperfusion of ischemic heart exhibited deteriorated cardiac physiological recovery in both ovariectomized and normal groups, where prominent decline was observed in ovariectomized rat. However, preconditioning the isolated heart with CORM-2 improved hemodynamics parameters significantly in both ovariectomized and normal rat hearts challenged with I/R, but with a limited degree of protection in ovariectomized rat hearts. The protective effect of CORM-2 was further confirmed via a reduction in cardiac injury, preservation of mitochondrial enzymes, and reduction in oxidative stress in all groups. CORM-2 administration significantly attenuated myocardial I/R injury in ovariectomized rat hearts by attenuating I/R-associated mitochondrial perturbations and reducing oxidative stress.

Estrogen-dependent KCa1.1 modulation is essential for retaining neuroexcitation of female-specific subpopulation of myelinated Ah-type baroreceptor neurons in rats.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 μM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.

Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats

The present work was aimed to evaluate the effect of different administration modes of sodium valproate (VPA) on bone strength, bone mass and bone mineral density in ovariectomized (OVX) rats and further investigation of the possible mechanism. 60 female SD rats were randomly divided into 4 groups: Sham group (Sham, n = 15), OVX group (OVX, n = 15), OVX rats received intermittent VPA treatment group (IVPA, n = 15) and OVX rats received daily VPA treatment group (EVPA, n = 15). After 12 weeks of treatment, the rats were sacrificed, and serum and femur samples were harvested. DEXA, Micro-CT, history, biomechanical testing, biochemical index and western blot analysis were used to observe the therapeutic effect and explore the possible mechanism. Micro-CT and DEXA analysis of bones revealed better BMD and higher BV/TV, Tb. Th, Tb. N, Conn. D and lower Tb. Sp at femoral metaphysis evaluated in IVPA when compared with OVX and EVPA group (P < 0.05). Histological, fluorescent analysis and biological strength revealed more trabecular bone and higher relative mineral apposition rate, maximal load, elastic modulus and energy at break with evaluated in IVPA when compared with OVX and EVPA group (P < 0.05). The levels of P1NP, estrogen, CTX, TRAP-5b and RANKL of the IVPA group showed a significant increase when compared with the OVX and EVPA group (P < 0.05). We confirm adverse effects on protein expressions including Notch1, Jagged1, HEY1, Wnt 1, β-catenin and RUNX2 following daily VPA treatment in OVX female rats. Our current study demonstrated that intermittent administration of sodium valproate has a protective effect on bone health in OVX rats and these effects may be achieved by activating Notch/Wnt/β-catenin/RUNX2 signal axis.

Change in prostaglandin signaling during sickness syndrome hyperalgesia after ovariectomy in female rats

The present study investigated hyperalgesia during sickness syndrome in female rats. Hyperalgesia was induced by an intraperitoneal injection of lipopolysaccharide (LPS) or an intracerebroventricular injection of prostaglandin E2 (PGE2). No differences were found in basal mechanical and thermal thresholds or in LPS-induced hyperalgesia in sham-operated animals in the diestrus or proestrus phase or in ovariectomized (OVX) animals. However, higher levels of PGE2 where found in the cerebrospinal fluid of OVX animals compared to sham-operated females. Intracerebroventricular injection of PGE2 produced rapid mechanical hyperalgesia in sham-operated rats while these responses were observed at later times in OVX animals. The protein kinase A (PKA) inhibitor H-89 reduced mechanical PGE2-induced hyperalgesia in OVX female rats, whereas no effect was observed in sham-operated animals. In contrast, the exchange protein activated by cyclic adenosine monophosphate (cAMP; Epac) inhibitor ESI-09 reduced mechanical PGE2-induced hyperalgesia, whereas no effect was observed in OVX animals. PGE2 also induced thermal hyperalgesia in sham-operated and OVX female rats and a similar effect of ESI-09 was observed. These results suggest that PGE2-induced hyperalgesia that is observed during sickness syndrome has different signaling mechanisms in cycling and OVX female rats involving the activation of the cAMP-Epac or cAMP-PKA pathways, respectively.