Muscle secreted factors enhance activation of the PI3K/Akt and ß-catenin pathways in murine osteocytes.

Skeletal muscle and bone interact at the level of mechanical loading through the application of force by muscles to the skeleton and more recently focus has been placed on molecular/biochemical coupling of these two tissues. We sought to determine if muscle and muscle-derived factors were essential to the osteocyte response to loading. Botox® induced muscle paralysis was used to investigate the role of muscle contraction during in vivo tibia compression loading. 5-6 month-old female TOPGAL mice had their right hindlimb muscles surrounding the tibia injected with either BOTOX® or saline. At four days post injections when muscle paralysis peaked, the right tibia was subjected to a single session of in vivo compression loading at ∼2600 µÎµ. At 24 h post-load we observed a 2.5-fold increase in ß-catenin signaling in osteocytes in the tibias of the saline injected mice, whereas loading of tibias from Botox® injected mice failed to active ß-catenin signaling in osteocytes. This suggests that active muscle contraction produces a factor(s) that is necessary for or conditions the osteocyte's ability to respond to load. To further investigate the role of muscle derived factors, MLO-Y4 osteocyte-like cells and a luciferase based ß-catenin reporter (TOPflash-MLO-Y4) cell line we developed were treated with conditioned media (CM) from C2C12 myoblasts (MB) and myotubes (MT) and ex vivo contracted Extensor Digitorum Longus (EDL) and Soleus (Sol) muscles under static or loading conditions using fluid flow shear stress (FFSS). 10 % C2C12 myotube CM, but not myoblast or NIH3T3 fibroblast cells CM, induced a rapid activation of the Akt signaling pathway, peaking at 15 min and returning to baseline by 1-2 h under static conditions. FFSS applied to MLO-Y4 cells for 2 h in the presence of 10 % MT-CM resulted in a 6-8 fold increase in pAkt compared to a 3-4 fold increase under control or when exposed to 10 % MB-CM. A similar response was observed in the presence of 10 % EDL-CM, but not in the presence of 10 % Sol-CM. TOPflash-MLO-Y4 cells were treated with 10 ng/ml Wnt3a in the presence or absence of MT-CM. While MT-CM resulted in a 2-fold activation and Wnt3a produced a 10-fold activation, the combination of MT-CM + Wnt3a resulted in a 25-fold activation of ß-catenin signaling, implying a synergistic effect of factors in MT-CM with Wnt3a. These data provide clear evidence that specific muscles and myotubes produce factors that alter important signaling pathways involved in the response of osteocytes to mechanical load. These data strongly suggest that beyond mechanical loading there is a molecular coupling of muscle and bone.

Rats genetically selected for low and high aerobic capacity exhibit altered soleus muscle myofilament functions.

Aerobic exercise capacity is critical to bodily health. As a model to investigate the mechanisms that determine health and disease, we employed low (LCR) and high (HCR) capacity running rat models selectively bred to concentrate the genes responsible for divergent aerobic running capacity. To investigate the skeletal muscle contribution to this innate difference in running capacity we employed an approach combining examination of the myofilament protein composition and contractile properties of the fast fiber extensor digitorum longus (EDL) and slow fiber soleus (SOL) muscles from LCR and HCR rats. Intact muscle force experiments demonstrate that SOL, but not EDL, muscles from LCR rats exhibit a three times greater decrease in fatigued force. To investigate the mechanism of this increased fatigability in the LCR SOL muscle, we determined the myofilament protein composition and functional properties. Force-Ca2+ measurements demonstrate decreased Ca2+ sensitivity of single skinned SOL muscle fibers from LCR compared with that of HCR rats. Segregating SOL fibers into fast and slow types demonstrates that the decreased Ca2+ sensitivity in LCR SOL results from a specific decrease in slow-type SOL fiber Ca2+ sensitivity such that it was similar to that of fast-type fibers. These results identify that the altered myofilament contractile properties of LCR SOL slow-type fibers result in a fast muscle type Ca2+ sensitivity and the LCR muscle phenotype. Overall our findings demonstrate alterations of the myofilament proteins could contribute to fatigability of the SOL muscle and the decreased innate aerobic running performance of LCR compared with HCR rats.

Cervical cancer screening and chinese women: insights from focus groups.

OBJECTIVE: Despite extensive efforts to raise awareness, Papanicolaou (Pap) testing rates among Chinese women living in North America remain low compared with Euro-American women. Although the lower Pap testing rate and ensuing health repercussions among Chinese women are well characterized, mechanisms underlying such health disparities are not. The aim of this study was to use a qualitative approach to delineate such mechanisms. Qualitative approaches to understand constructs within the domain of sexual and reproductive health have been shown to be particularly appropriate, and offer a nuanced view of sexuality that is not afforded by traditional quantitative methods. METHOD: We carried out two focus groups aimed at exploring how Mandarin-speaking and English-speaking Chinese women experience Pap testing (N = 12). The women were invited to partake in the focus groups from having participated in a large-scale quantitative study. Participants were all first-generation immigrants and their average age was 53-years-old. We used content analyses to analyze transcripts and extract themes. RESULTS AND DISCUSSION: The women heavily endorsed traditional Chinese medicine philosophy, conceptualizing physical health holistically, and valuing preventative measures over screening and interceptive measures. Pap testing was described as qualitatively different from other screening procedures, such that women assigned a sexually charged meaning to Pap testing, often discussing it in relation to sexual activity and promiscuity. Women expressed their preference for the compulsory and depersonalized manner that Pap tests are performed in their home country of China, as this lessens the embarrassment associated with undergoing Pap testing. CONCLUSION: Three mechanisms may contribute to lower Pap testing among middle-aged first-generation Chinese immigrants: preference for Chinese medicine philosophy, perceived sexualization of Pap testing, and the institutionalization of medical care. Implications for improving the reproductive health of Chinese women are discussed.

Skeletal muscle secreted factors prevent glucocorticoid-induced osteocyte apoptosis through activation of ß-catenin.

It is a widely held belief that the sole effect of muscle on bone is through mechanical loading. However, as the two tissues are intimately associated, we hypothesized that muscle myokines may have positive effects on bone. We found that factors produced by muscle will protect osteocytes from undergoing cell death induced by dexamethasone (dex), a glucocorticoid known to induce osteocyte apoptosis thereby compromising their capacity to regulate bone remodeling. Both the trypan blue exclusion assay for cell death and nuclear fragmentation assay for apoptosis were used. MLO-Y4 osteocytes, primary osteocytes, and MC3T3 osteoblastic cells were protected against dex-induced apoptosis by C2C12 myotube conditioned media (MT-CM) or by CM from ex vivo electrically stimulated, intact extensor digitorum longus (EDL) or soleus muscle derived from 4 month-old mice. C2C12 MT-CM, but not undifferentiated myoblast CM prevented dex-induced cell apoptosis and was potent down to 0.1 % CM. The CM from EDL muscle electrically stimulated tetanically at 80 Hz was more potent (10 fold) in prevention of dex-induced osteocyte death than CM from soleus muscle stimulated at the same frequency or CM from EDL stimulated at 1 Hz. This suggests that electrical stimulation increases production of factors that preserve osteocyte viability and that type II fibers are greater producers than type I fibers. The muscle factor(s) appears to protect osteocytes from cell death through activation of the Wnt/ß-catenin pathway, as MT-CM induces ß-catenin nuclear translocation and ß-catenin siRNA abrogated the positive effects of MT-CM on dex-induced apoptosis. We conclude that muscle cells naturally secrete factor(s) that preserve osteocyte viability.

Advanced ovarian cancer: phase III randomized study of sequential cisplatin-topotecan and carboplatin-paclitaxel vs carboplatin-paclitaxel.

BACKGROUND: Topotecan has single-agent activity in recurrent ovarian cancer. It was evaluated in a novel combination compared with standard frontline therapy. METHODS: Women aged 75 years or younger with newly diagnosed stage IIB or greater ovarian cancer, Eastern Cooperative Oncology Group Performance Status of 1 or less, were stratified by type of primary surgery and residual disease, treatment center, and age; then randomly assigned to one of the two 21-day intravenous regimens. Patients in arm 1 (n = 409) were administered four cycles of cisplatin 50 mg/m(2) on day 1 and topotecan 0.75 mg/m(2) on days 1-5, then four cycles of paclitaxel 175 mg/m(2) over 3 hours on day 1 followed by carboplatin (area under the curve = 5) on day 1. Patients in arm 2 (n = 410) were given paclitaxel plus carboplatin as in arm 1 for eight cycles. We compared progression-free survival (PFS), overall survival, and cancer antigen-125 normalization rates in the two treatment arms. A stratified log-rank test was used to assess the primary endpoint, PFS. All statistical tests were two-sided. RESULTS: A total of 819 patients were randomly assigned. At baseline, the median age of the patients was 57 years (range = 28-78); 81% had received debulking surgery, and of these, 55% had less than 1 cm residual disease; 66% of patients were stage III and 388 (47.4%) patients had measurable disease. After a median follow-up of 43 months, 650 patients had disease progression or died without documented progression and 406 had died. Patients in arm 1 had more hematological toxicity and hospitalizations than patients in arm 2; PFS was 14.6 months in arm 1 vs 16.2 months in arm 2 (hazard ratio = 1.10, 95% confidence interval = 0.94 to 1.28, P = .25). Among patients with elevated baseline cancer antigen-125, fewer in arm 1 than in arm 2 had levels return to normal by 3 months after random assignment (51.6% vs 63.3%, P = .007) CONCLUSIONS: Topotecan and cisplatin, followed by carboplatin and paclitaxel, were more toxic than carboplatin and paclitaxel alone, but without improved efficacy. Carboplatin plus paclitaxel remains the standard of care for advanced epithelial ovarian cancer.

Psychological profiles among women with vulvar vestibulitis syndrome: a chart review.

The purpose of this study was to assess the prevalence and type of psychological distress in women with vulvar vestibulitis syndrome (VVS). A retrospective chart review was conducted of all women receiving a diagnosis of VVS referred to a tertiary care facility during a two-year period. Brief psychological questionnaires, including the Personality Assessment Screener, Fear of Negative Evaluation Scale, Golombok-Rust Inventory of Sexual Satisfaction, and the Phobia Rating Scale were administered. Fifty-consecutive cases were reviewed along with 12-15 month follow-up data for 41 cases. Phobic anxiety to vaginal touch or entry was significantly higher in women with VVS than normative data. Fear of Negative Evaluation was a strong associated feature, and for 30% approached clinically significant levels. Twenty-six percent showed a moderate, while another 26% showed a mild clinically distressed profile. Negative affect and social withdrawal were among the most frequently endorsed variables. Improvement in allodynia and intercourse were both related to these psychological variables, and a multiple regression analysis supported the use of psychological instruments in addition to standard medical assessment. A subgroup of women with VVS display clinically significant broad based psychological distress that warrants additional assessment. The use of psychological questionnaires in addition to medical assessment of women with VVS may provide valuable information predictive of treatment needs and response.

Definitions of women's sexual dysfunction reconsidered: advocating expansion and revision.

In light of various shortcomings of the traditional nosology of women's sexual disorders for both clinical practice and research, an international multi-disciplinary group has reviewed the evidence for traditional assumptions about women's sexual response. It is apparent that fullfilment of sexual desire is an uncommon reason/incentive for sexual activity for many women and, in fact, sexual desire is frequently experienced only after sexual stimuli have elicited subjective sexual arousal. The latter is often poorly correlated with genital vasocongestion. Complaints of lack of subjective arousal despite apparently normal genital vasocongestion are common. Based on the review of existing evidence-based research, many modifications to the definitions of women's sexual dysfunctions are recommended. There is a new definition of sexual interest/desire disorder, sexual arousal disorders are separated into genital and subjective subtypes and the recently recognized condition of persistent sexual arousal is included. The definition of dyspareunia reflects the possibility of the pain precluding intercourse. The anticipation and fear of pain characteristic of vaginismus is noted while the assumed muscular spasm is omitted given the lack of evidence. Finally, a recommendation is made that all diagnoses be accompanied by descriptors relating to associated contextual factors and to the degree of distress.

Melatonin protects against the effects of chronic stress on sexual behaviour in male rats.

The effects of chronic mild stress (CMS) on both sexual behaviour and wet dog shakes (WDS), a serotonergic type 2A (5-HT2A) receptor-mediated behaviour, were explored in the male rat. In addition, the possible attenuation of these effects by chronic treatment with melatonin, a putative 5-HT2A antagonist, was examined. The CMS procedure resulted in a significant increase in WDS and an overall decrease in all aspects of sexual behaviour. Concurrent melatonin administration attenuated the CMS-induced effects on sexual behaviour, but not the effects on either spontaneous WDS or WDS in response to the 5-HT2A agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, suggesting a mechanism of action other than exclusive 5-HT2A antagonism. These results are the first to demonstrate that melatonin significantly protects against the detrimental effects of a chronic stressor on sexual behaviour.

Paradoxical effects of chronic corticosterone on forced swim behaviours in aged male and female rats.

The effects of chronically administered corticosterone on forced swim test and open field test behaviours were explored in aged male and female rats. Though corticosterone has typically been associated with depressive behaviours, recent data have suggested a putative antidepressive effect of corticosterone. The current study used the forced swim test as a model of antidepressant efficacy in order to explore this. Aged male and female rats received either corticosterone (20 mg/kg) or the vehicle for 10 days before testing in the forced swim test, then for an additional 3 days before testing in the open field test. On day 11, each animal was individually tested on the duration of swimming, immobile, and struggling behaviours, and on day 14, for the display of rearing and line crossing behaviours. Results revealed that corticosterone significantly increased swimming and decreased immobility behaviour in females, but failed to do so in males. Additionally, there was a main effect of corticosterone on struggling behaviour such that it decreased it in males. There were no effects of corticosterone or sex on open field test behaviours, suggesting that the present findings are not accounted for by a general effect of corticosterone on motor behaviour. Overall, the data suggest that chronically administered corticosterone possesses effects that are sex-specific, and that it may exert mildly antidepressive effects in females, but the opposite effects in males. These data are consistent with emerging evidence that corticosterone may play a paradoxical antidepressive effect.

Hypoxia/fatigue-induced degradation of troponin I and troponin C: new insights into physiologic muscle fatigue.

Conditions such as respiratory failure and cardiopulmonary arrest can expose the diaphragm to hypoxemia. In skeletal muscles, fatiguing stimulation renders muscles hypoxic, which has long been known to dramatically reduce muscle function. We have previously demonstrated that fatiguing stimulation under hypoxic conditions disrupts both the excitation-contraction coupling (ECC) process and the isometric contractile properties (ICP) in intact diaphragm muscle strips and the contractile properties of skinned fibers isolated from these muscles. Here we have analyzed the effects of intermittent fatiguing stimulation on specific muscle proteins in muscle strips from mouse diaphragms that have been exposed to hypoxia. We report for the first time that the effects of hypoxia-fatigue, namely to decrease maximal tetanic force, maximal calcium-activated force and calcium sensitivity of the mouse diaphragm muscle, are associated with the degradation of troponins TnI and TnC (Western blot analysis). The concentrations of TnT and actin did not change under these same conditions. Because troponins are integrally involved in regulating the interaction between actin and myosin during the cross-bridge cycle, the degradation of TnI and TnC may explain the effects of hypoxia-fatigue on the ICP. This interpretation is supported by the observations that extraction of troponins from control skinned fibers mimics the effects of hypoxia-fatigue on contractile function and that incorporation of native troponins into fibers isolated from hypoxic-fatigued muscles partially restores function.

Chronic corticosterone enhances the rewarding effect of hypothalamic self-stimulation in rats.

Excessive levels of glucocorticoids have been implicated in the etiology of affective disorders in humans, and in a range of behavioral deficits in animals. In the present study, we used an established regimen of corticosterone administration (40 mg/kg, for 21 days) to determine its effects upon responding for rewarding brain stimulation in rats. After chronic treatment, subjects exhibited an unexpected but significantly increased sensitivity to the rewarding effects of brain stimulation. These results suggest that chronic, high levels of corticosterone are unlikely to cause anhedonia in rodents.

Hypoxia and fatigue-induced modification of function and proteins in intact and skinned murine diaphragm muscle.

Fatigue studies of isolated, intact muscles typically utilize solutions saturated with O2. However, under in vivo fatiguing conditions, less oxygen is delivered to the muscles and they actually experience hypoxia. No studies to date have correlated the effects of acute hypoxia on the isometric contractile properties of intact muscles, skinned fibers isolated from the same muscles, and the cellular content of specific muscle proteins. Therefore, we have studied the effects of in vitro acute hypoxia on the fatigability of intact diaphragm muscle strips and on the isometric contractile properties of single Triton-skinned fibers isolated from control and hypoxic diaphragm muscles. We found that hypoxia and fatiguing stimulation per se affect the tetanic force of intact muscle strips without exhibiting any significant deleterious effects on the calcium-activated force of skinned muscle fibers dissected from the intact muscles. In contrast, fatiguing stimulation under hypoxic conditions decreased both the tetanic force of muscle strips and the calcium-activated force of skinned muscle fibers. Gel electrophoresis of muscles subjected to hypoxia and hypoxic-fatigue revealed that there is a significant reduction in three protein bands when compared to control muscles. Protein modification may be the underlying mechanism of muscle fatigue under physiologic conditions.

Sex differences in forced-swim and open-field test behaviours after chronic administration of melatonin.

The effects of melatonin administered chronically on forced-swim test and open-field test behaviours were examined in male and female rats. The forced-swim test has been shown to be sensitive to all major classes of antidepressants and evidence indicates that melatonin possesses putative antidepressive properties. Male and female Long-Evans rats received either a regimen of chronic administration of melatonin or the control condition for 14 days via the drinking water. On day 15, each animal was individually introduced into a swim chamber, and was scored for 15 min on the duration of swimming, struggling, and immobility. After 24 h, each animal was again tested in the forced-swim test for 10 min. On day 18, all animals were tested in the open-field test apparatus for 5 min. Results revealed that females consistently showed higher activity levels than males in the forced-swim and open-field tests. Melatonin significantly increased struggling in males on day 15, but failed to do so in females. Also, whereas melatonin-treated females showed higher levels of behavioural immobility during their first exposure to the forced-swim test, this effect was prevented upon a second exposure. In both males and females, melatonin decreased swimming in the forced-swim test while increasing open-field ambulatory behaviour. Therefore, it is unlikely that melatonin's mechanism of action is a general inhibitory effect on motor activity. Taken together, the results suggest that the effects of melatonin treatment on forced-swim test behaviours are sex- and test-dependent.

Melatonin enhances sexual behavior in the male rat.

Anecdotal reports suggest that melatonin enhances libido in men. However, controlled trials remain to be published for any species. Accordingly, adult male rats were chronically treated for 12 weeks with melatonin via the drinking water. On the 13th week, all males were tested in the presence of sexually receptive females on measures of sexual behavior. Moreover, because of the established inverse relationship between male sexual behavior and serotonergic type 2A (5-HT(2A)) receptor activity, "wet-dog shakes" (WDS), a 5-HT(2A) receptor mediated behavior, were measured concurrently. All aspects of sexual activity were significantly facilitated in males treated with melatonin. In addition, there was a consistent, progressive reduction in the frequency of WDS, suggestive of a temporal decrement in serotonergic receptor activity and supportive of previous indications that melatonin possesses 5-HT(2A) antagonistic properties. These results provide the first empirical evidence for a facilitatory role of melatonin in sexual behavior, and suggest that its mechanism of action may involve the 5-HT(2A) receptor.

Nefazodone attenuates the stress-induced facilitation of wet dog shaking behaviour but not the facilitation of sexual behaviour in female rats.

The effects of chronic stress both alone and in combination with the antidepressant, nefazodone, which possesses antagonistic activity at the 5-HT2A receptor, were examined on the 5-HT2A receptor-mediated behaviour, wet dog shaking and sexual behaviour. Ovariectomized female rats received either a chronic stressor or no stress for 30 days, and half of each group received concurrent nefazodone treatment (100 mg/kg/day). Following treatment with either estrogen, or estrogen combined with progesterone, sexual behaviour and wet dog shaking were recorded. Chronic stress alone was found to facilitate sexual behaviour and increase wet dog shaking, while nefazodone administration alone was without effect. Furthermore, nefazodone completely attenuated the stress-induced facilitation of wet dog shaking, but not sexual behaviour.

Corticosterone regulation of 5-HT2A receptor-mediated behaviors: attenuation by melatonin.

The effects of chronic corticosterone treatment on sexual behavior and on wet-dog shakes (WDS), a serotonergic type 2A (5-HT2A) receptor-mediated behavior, were explored in the male rat. In addition, the effects of acute melatonin treatment, both alone and in combination with corticosterone, were investigated. Chronic injections of corticosterone resulted in an overall decrease in consummatory measures of sexual behavior, and an increase in WDS. Furthermore, although an acute injection of melatonin alone had no effect on any recorded behavior, it attenuated the effects of corticosterone on sexual behavior and WDS. The data suggest that in the context of 5-HT2A receptor-mediated behaviors, melatonin has possible implications as a 5-HT2A antagonist.

Chronic stress effects on sexual behavior in male and female rats: mediation by 5-HT2A receptors.

The effects of chronic psychosocial stress on sexual behavior and on the serotonergic type 2A (5-HT2A) receptor-mediated behavior "wet dog shakes" (WDS) were investigated in male and female rats. In Experiment 1, both bilaterally adrenalectomized and sham-adrenalectomized female rats were assigned to either a psychosocial stress condition or a control condition for 62 days. On the 63rd day, estrogen-primed females were compared on measures of sexual behavior and WDS. Immediately after the behavioral tests, the same rats were primed with a subthreshold level of progesterone. Three hours after the administration of progesterone, rats were again scored for sexual behavior and WDS. Psychosocial stress was found to facilitate sexual behavior and increase WDS in sham-adrenalectomized female rats providing they were primed with both estrogen and progesterone. In Experiment 2, intact male rats were assigned to either the psychosocial stress condition or the control condition for 30 days. On the 31st day, males were compared on measures of sexual behavior and WDS. No significant differences were revealed on the spontaneous expression of sexual behavior and WDS. Subsequently, males were retested following the administration of the 5-HT2A agonist, DOI. Psychosocial stress resulted in a significant decrease in male sexual behavior and a concurrent increase in WDS, following the administration of DOI. Taken together, these results suggest that chronic psychosocial stress facilitates female sexual behavior and inhibits male sexual behavior, and that the effects of stress on sexual behavior may be mediated by 5-HT2A receptor activity.

The antidepressant, nefazodone, attenuates corticosterone-induced increases in 5-HT2A receptor-mediated behaviors in the female rat.

The effects of chronic corticosterone administration on sexual behavior and on wet-dog shakes, a 5-HT2A mediated behavior, were investigated in the female rat. In addition, effects of the antidepressant nefazodone, a selective 5-HT2A receptor antagonist, both alone and in combination with corticosterone were examined. Testing was conducted in ovariectomized animals primed with estrogen and progesterone. Corticosterone was found to significantly increase sexual receptivity, sexual proceptivity and wet dog shakes. While nefazodone alone had no significant effects, it completely attenuated the corticosterone-induced increases in both sexual behavior and wet dog shakes. This suggests that corticosterone influences sexual behavior and wet dog shakes via a 5-HT2A receptor mechanism.

Effects of housing conditions and 5-HT2A activation on male rat sexual behavior.

Adult male rats were housed individually or in groups for a period of 39 days. In Experiment 1, the effects of housing conditions on sexual behavior and concurrent spontaneous "wet dog shaking" (WDS) were investigated. Individual housing significantly impaired male sexual behavior and resulted in a trend toward increased WDS. In Experiment 2, the effects of housing conditions were examined following administration of the serotonergic type 2A (5-HT2A) agonist DOI. Individual housing significantly increased DOI-induced WDS. The implications of these findings are discussed in the context of stress-induced corticosterone secretion and the possible regulatory effect on 5-HT2A receptors.