Immunolocalization of markers for bone formation during guided bone regeneration in osteopenic rats

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Extended periods of alcohol intake negatively affects osseointegration in rats

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Hypoxic and hypercapnic ventilatory responses in rats with polycystic ovaries

In female rats, a single injection of estradiol valerate (EV) results in effects that are similar to those observed in women with polycystic ovary syndrome (PCOS). We hypothesized that EV-induced PCOS affects breathing control based on evidence showing an influence of sex hormones on ventilation. To test this hypothesis, we studied the effects of EV treatment on the ventilation of female rats in air, in 7% CO2 and in 7% O2, at 30, 45 and 60 days after EV injection. The group examined 30 days after EV treatment showed a 61% reduction in the hypercapnic ventilatory response compared to the control group. Basal ventilation, hypoxic ventilatory response, and body temperature were not affected. These results, suggest that the hormonal changes observed in PCOS may result in a temporary inhibition of the central chemoreflex but do not influence basal ventilation or the hypoxic peripheral chemoreflex.

Reproductive physiology, and physical and sexual development of female offspring born to diabetic dams

Objectives: The objective of this study was to evaluate physical and sexual development and reproductive physiology in female rat offspring that developed in hyperglycemia conditions in utero and during lactation. Materials and methods: Maternal diabetes was induced in female rats by a single IV injection of streptozotocin before mating. Female offspring development was evaluated by means of the following parameters: physical development; age of vaginal opening and first estrus; weight and histological evaluation of uterus and ovaries; duration of the estrous cycle, sexual behavior, and fertility after natural mating. Results: In the female offspring, maternal diabetes caused delays in initial physical development; diminution in ovary weight and number of follicles; and inferior reproductive performance compared with the control group. Conclusions: The exposure to hyperglycemia in uterus and during lactation caused delays in physical and sexual development, and affected the reproductive physiology of female rats negatively.

Medial amygdaloid nucleus 5-HT2C receptors are involved in the hypophagic effect caused by zimelidine in rats

The medial amygdaloid nucleus (MeA) is a sub-region of the amygdaloid complex that has been described as participating in food intake regulation. Serotonin has been known to play an important role in appetite and food intake regulation. Moreover, serotonin 5-HT2C and 5-HT1A receptors appear to be critical in food intake regulation. We investigated the role of the serotoninergic system in the MeA on feeding behavior regulation in rats. The current study examined the effects on feeding behavior regulation of the serotonin reuptake inhibitor, zimelidine, administered directly into the MeA or given systemically, and the serotoninergic receptors mediating its effect. Our results showed that microinjection of zimelidine (0.2, 2 and 20 nmol/100 nL) into the MeA evoked dose dependent hypophagic effects in fasted rats. The selective 5-HT1A receptor antagonist WAY-100635 (18.5 nmol/100 nL) or the 5-HT1B receptor antagonist SB-216641 microinjected bilaterally into the MeA did not change the hypophagic effect evoked by local MeA zimelidine treatment. However, microinjection of the selective 5-HT2C receptor antagonist SB-242084 (10 nmol/100 nL) was able to block the hypophagic effect of zimelidine. Moreover, microinjection of the 5-HT2C receptor antagonist SB-242084 into the MeA also blocked the hypophagic effect caused by zimelidine administered systemically. These results suggest that MeA 5-HT2C receptors modulate the hypophagic effect caused by local MeA administration as well as by systemic zimelidine administration. Furthermore, 5-HT2C into the MeA could be a potential target for systemic administration of zimelidine. (C) 2012 Elsevier Ltd. All rights reserved.

Exercise training prevents diastolic dysfunction induced by metabolic syndrome in rats

OBJECTIVE: High fructose consumption contributes to the incidence of metabolic syndrome and, consequently, to cardiovascular outcomes. We investigated whether exercise training prevents high fructose diet-induced metabolic and cardiac morphofunctional alterations. METHODS: Wistar rats receiving fructose overload (F) in drinking water (100 g/l) were concomitantly trained on a treadmill (FT) for 10 weeks or kept sedentary. These rats were compared with a control group (C). Obesity was evaluated by the Lee index, and glycemia and insulin tolerance tests constituted the metabolic evaluation. Blood pressure was measured directly (Windaq, 2 kHz), and echocardiography was performed to determine left ventricular morphology and function. Statistical significance was determined by one-way ANOVA, with significance set at p<0.05. RESULTS: Fructose overload induced a metabolic syndrome state, as confirmed by insulin resistance (F: 3.6 +/- 0.2 vs. C: 4.5 +/- 0.2 mg/dl/min), hypertension (mean blood pressure, F: 118 +/- 3 vs. C: 104 +/- 4 mmHg) and obesity (F: 0.31 +/- 0.001 vs. C: 0.29 +/- 0.001 g/mm). Interestingly, fructose overload rats also exhibited diastolic dysfunction. Exercise training performed during the period of high fructose intake eliminated all of these derangements. The improvements in metabolic parameters were correlated with the maintenance of diastolic function. CONCLUSION: The role of exercise training in the prevention of metabolic and hemodynamic parameter alterations is of great importance in decreasing the cardiac morbidity and mortality related to metabolic syndrome.

Glutamate-induced obesity leads to decreased sperm reserves and acceleration of transit time in the epididymis of adult male rats

Background: Given the established fact that obesity interferes with male reproductive functions, the present study aimed to evaluate sperm production in the testis and storage in the epididymis in a glutamate-induced model of obesity. Methods: Male rats were treated neonatally with monosodium glutamate (MSG) at doses of 4 mg/kg subcutaneously, or with saline solution (control group), on postnatal days 2, 4, 6, 8 and 10. On day 120, obesity was confirmed by the Lee index in all MSG-treated rats. After this, all animals from the two experimental groups were anesthetized and killed to evaluate body and reproductive organ weights, sperm parameters, plasma hormone levels (FSH, LH and testosterone), testicular and epididymal histo-morphometry and histopathology. Results: Significant reductions in absolute and relative weights of testis, epididymis, prostate and seminal vesicle were noted in MSG-treated animals. In these same animals plasma testosterone and follicle-stimulating hormone (FSH) concentrations were decreased, as well as sperm counts in the testis and epididymis and seminiferous epithelium height and tubular diameter. The sperm transit time was accelerated in obese rats. However, the number of Sertoli cells per seminiferous tubule and stereological findings on the epididymis were not markedly changed by obesity. Conclusions: Neonatal MSG-administered model of obesity lowers sperm production and leads to a reduction in sperm storage in the epididymis of adult male rats. The acceleration of sperm transit time can have implications for the sperm quality of these rats.

STIM 1/Orai 1 contributes to sex differences in vascular responses to calcium in spontaneously hypertensive rats

Sex differences in Ca2+-dependent signalling and homoeostasis in the vasculature of hypertensive rats are well characterized. However, sex-related differences in SOCE (store-operated Ca2+ entry) have been minimally investigated. We hypothesized that vascular protection in females, compared with males, reflects decreased Ca2+ mobilization due to diminished activation of Orai 1/STIM 1 (stromal interaction molecule I). In addition, we investigated whether ovariectomy in females affects the activation of the Orai 1/STIM 1 pathway. Endothelium-denuded aortic rings from male and female SHRSP (stroke-prone spontaneously hypertensive rats) and WKY (Wistar Kyoto) rats and from OVX (ovariectomized) or sham female SHRSP and WKY rats were used to functionally evaluate Ca2+ influx-induced contractions. Compared with females, aorta from male SHRSP displayed: (i) increased contraction during the Ca2+-loading period; (ii) similar transient contraction during Ca2+ release from the intracellular stores; (iii) increased activation of STIM 1 and Orai1, as shown by the blockade of STIM 1 and Orai1 with neutralizing antibodies, which reversed the sex differences in contraction during the Ca2+-loading period; and (iv) increased expression of STIM I and Orai I. Additionally, we found that aortas from OVX-SHRSP showed increased contraction during the Ca2+-loading period and increased Orai1 expression, but no changes in the SR (sarcoplasmic reticulum)-buffering capacity or STIM I expression. These findings suggest that augmented activation of STIM 1/Orai 1 in aortas from male SHRSP represents a mechanism that contributes to sex-related impaired control of intracellular Ca2+ levels. Furthermore, female sex hormones may negatively modulate the STIM/Orai 1 pathway, contributing to vascular protection observed in female rats.

Repression of Ppargc1a Gene in Liver of Hyperglycemic Rats Induced with High Fat Diet Combined with Streptozotocin

Type 2 diabetes mellitus implies deregulation of multiple metabolic processes, being the maintenance of glycemia one of the most important. Many genes are involved in the deregulation of this particular process. Therefore, the aim of this study was to evaluate gene expression of genes related to type 2 diabetes mellitus, in the liver and pancreas of rats with hyperglycemia induced by high fat diet along with a low single dose of streptozotocin. Ahsg and Ppargc1a genes were studied in liver, whereas Kcnj11 and Slc2a2 genes were analyzed in pancreas. For this purpose, 210-240 g female rats were fed a high fat diet or a control diet for three weeks. At day 14, animals fed with high fat diet were injected with a single low dose of streptozotocin (35 mg/kg) and the control group rats were injected only with the vehicle. Plasmatic glucose, triglycerides and total cholesterol levels were measured at the beginning, day 14 and end of treatment. Body weight was also measured. Once the treatment was complete, rats were appropriately euthanized and then, pancreas and liver were surgically removed and frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent, treated with DNase land reversely transcribed to cDNA. Gene expression analysis was performed using SYBR Green - Real time PCR and comparative Cq method, using three reference genes. Rats fed with high fat diet and treated with streptozotocin showed higher values of plasmatic glucose (17.09 +/- 0.43 vs. 5.91 +/- 0.29 mmol/L, p < 0.01) and a minor expression of Ppargc1a versus the control group (2-fold less expressed, p < 0.05) in liver. We conclude that repression of Ppargc1a gene may be an important process in the establishment of chronic hyperglycemia, probably through deregulation of hepatic gluconeogenesis. However, further studies need to be performed in order to clarify the role of Ppargc1a deregulation in liver glucose homeostasis.

Bioelectrical impedance analysis and anthropometry for the determination of body composition in rats: effects of high-fat and high-sucrose diets

Objective The aim of the present study was to determine the impedance of Wistar rats treated with high-fat and high-sucrose diets and correlate their biochemical and anthropometric parameters with chemical analysis of the carcass. Methods Twenty-four male Wistar rats were fed a standard (AIN-93), high-fat (50% fat) or high-sucrose (59% of sucrose) diet for 4 weeks. Abdominal and thoracic circumference and body length were measured. Bioelectrical impedance analysis was used to determine resistance and reactance. Final body composition was determined by chemical analysis. Results Higher fat intake led to a high percentage of liver fat and cholesterol and low total body water in the High-Fat group, but these changes in the biochemical profile were not reflected by the anthropometric measurements or bioelectrical impedance analysis variables. Anthropometric and bioelectrical impedance analysis changes were not observed in the High-Sucrose group. However, a positive association was found between body fat and three anthropometric variables: body mass index, Lee index and abdominal circumference. Conclusion Bioelectrical impedance analysis did not prove to be sensitive for detecting changes in body composition, but body mass index, Lee index and abdominal circumference can be used for estimating the body composition of rats.

Impact of maternal mild hyperglycemia on maternal care and offspring development and behavior of Wistar rats

The aim of the present study was to evaluate the effect of maternal mild hyperglycemia on maternal behavior, as well as the development, behavior, reproductive function, and glucose tolerance of the offspring. At birth, litters were assigned either to Control (subcutaneous (sc)-citrate buffer) or STZ groups (streptozotocin (STZ)-100 mg/kg-sc.). On PND 90 both STZ-treated and Control female rats were mated. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) were performed during pregnancy. Pregnancy duration, litter size and sex ratio were assessed. Newborns were classified according to birth weight as small (SPA), adequate (APA), or large for pregnancy age (LPA). Maternal behavior was analyzed on PND 5 and 10. Offspring body weight, length, and anogenital distance were measured and general activity was assessed in the open field. Sexual behavior was tested in both male and female offspring. Levels of reproductive hormones and estrous cycle duration were evaluated in female offspring. Female offspring were mated and both a GTT and ITT performed during pregnancy. Neonatal STZ administration caused mild hyperglycemia during pregnancy and changed some aspects of maternal care. The hyperglycemic intrauterine milieu impaired physical development and increased immobility in the open field in the offspring although the latter effect appeared at different ages for males (adulthood) and females (infancy). There was no impairment in the sexual behavior of either male or female offspring. As adults, female offspring of STZ-treated mothers did not show glucose intolerance during pregnancy. Thus, offspring of female rats that show mild hyperglycemia in pregnancy have fewer behavioral and developmental impairments than previously reported in the offspring of severely diabetic dams suggesting that the degree of impairment is directly related to the mother glycemic intensity. (C) 2012 Elsevier Inc. All rights reserved.

Variations in maternal care alter corticosterone and 17beta-estradiol levels, estrous cycle and folliculogenesis and stimulate the expression of estrogen receptors alpha and beta in the ovaries of UCh rats

Background: Variations in maternal care are associated with neonatal stress, hormonal disturbances and reproductive injuries during adulthood. However, the effects of these variations on sex hormones and steroid receptors during ovary development remain undetermined. This study aimed to investigate whether variations in maternal care are able to influence the hormonal profile, follicular dynamics and expression of AR, ER-alpha and ER-beta in the ovaries of UCh rat offspring. Methods: Twenty-four adult UCh rats, aged 120 days, were randomly divided into two groups (UChA and UChB) and mated. Maternal care was assessed from birth (day 0) to the 10th postnatal day (PND). In adulthood, twenty adult female rats (UChA and UChB offspring; n = 10/group), aged 120 days, were euthanized by decapitation during the morning estrus. Results: UChA females (providing high maternal care) more frequently displayed the behaviors of carrying pups, as well as licking/grooming and arched back nursing cares. Also, mothers providing high care had elevated corticosterone levels. Additionally, offspring receiving low maternal care showed the highest estrous cycle duration, increased corticosterone and 17beta-estradiol levels, overexpression of receptors ER-alpha and ER-beta, increased numbers of primordial, antral and mature follicles and accentuated granulosa cell proliferation. Conclusions: Our study suggests that low maternal care alters corticosterone and 17beta-estradiol levels, disrupting the estrous cycle and folliculogenesis and differentially regulating the expression of ER-alpha and ER-beta in the ovaries of adult rats.

Validation of a new method to assess the long-term Hypothalamic-Pituitary-Adrenal Activity using hair glucocorticoids as biomarkers. - Studies on pigs and laboratory Sprague-Dawley Rats

The evaluation of chronic activity of the hypothalamic-pituitary-adrenal (HPA) axis is critical for determining the impact of chronic stressful situations. The potential use of hair glucocorticoids as a non-invasive, retrospective, biomarker of long term HPA activity is of great interest, and it is gaining acceptance in humans and animals. However, there are still no studies in literature examining hair cortisol concentration in pigs and corticosterone concentration in laboratory rodents. Therefore, we developed and validated, for the first time, a method for measuring hair glucocorticoids concentration in commercial sows and in Sprague-Dawley rats. Our preliminary data demonstrated: 1) a validated and specific washing protocol and extraction assay method with a good sensitivity in both species; 2) the effect of the reproductive phase, housing conditions and seasonality on hair cortisol concentration in sows; 3) similar hair corticosterone concentration in male and female rats; 4) elevated hair corticosterone concentration in response to chronic stress manipulations and chronic ACTH administration, demonstrating that hair provides a good direct index of HPA activity over long periods than other indirect parameters, such adrenal or thymus weight. From these results we believe that this new non-invasive tool needs to be applied to better characterize the overall impact in livestock animals and in laboratory rodents of chronic stressful situations that negatively affect animals welfare. Nevertheless, further studies are needed to improve this methodology and maybe to develop animal models for chronic stress of high interest and translational value in human medicine.

Evaluation des effets de l’extrait aqueux des fleurs de Nymphaea lotus Linn. (Nymphéacées) sur la fonction de reproduction des rats normoglycémique et diabétique de type 1

In traditional medicine of Cameroon, Nymphaea lotus Linn. (Nympheaceae) is used for treatment of male sexual disorders. The aim of this study was to evaluate the effects of the N. lotus flowers aqueous extracts on general mating behavior, fertility and some androgenic parameters of normoglycemic and diabetic adult male rats. Mating behavior assessment was carried out with primiparous and with oestrus female rats. Male rats were divided into 5 groups and subjected once in a day to the following treatment: distilled water (10 mL/kg), Sildenafil citrate (5mg/kg), Testosterone enanthate (5mg/kg) and plant extract (75 mg/kg and 150 mg/kg). Treatment lasted for 8 and 55 days before sacrifice. Organ weight, epididymal sperm counts, motility, viability, histomorphometric analysis, muscular strength, seminal fructose, cholesterol level, epididymal and serum proteins testicular were determined. Results showed a dose- dependant influence of the treatment in sperm count and motility which significantly increases compared to the distilled water-administered control. An increase in some sexual performance parameters (mount and intromission frequencies) was observed in both diabetic and normal rats compared to respective controls. The latencies of mount and intromission were significantly reduced. Computed frequencies of penile licking, index of libido and sexual motivation were higher in normal and diabetic extract-treated animals; which suggest an enhancement of motivational parameters. The treatment also caused significant increases in the weight of the testis, prostate, epididymis, in serum cholesterol and epididymal protein level in normal rats compared to the distilled water-administered control. These results indicate an androgenic pro-sexual potential of N. lotus in male rats and justify the empirical use of N. lotus in the management of erectile dysfunction and fertility disorders in males. Key-words: Nymphaea lotus, prosexual, androgenic, fertility, diabetic, male rat. //Selon les tradipraticiens de l’Ouest-Cameroun, Nymphaea lotus Linn. (N. lotus), de la famille des Nymphéacées, est utilisé pour le traitement des dysfonctionnements sexuels chez les hommes. Dans cette étude, l’effet de l’extrait aqueux des fleurs de N. lotus sur la fonction de reproduction des rats mâles adultes normaux et diabétiques a été évalué. Dans le but d’évaluer les effets de l’extrait aqueux des fleurs de N. lotus sur les paramètres généraux de copulation, d’androgénicité et de fertilité, les rats normoglycémiques et diabétiques ont été divisé en 3 groupes contrôle [Groupe I- recevant de l’eau distillée, Groupe II et III recevant respectivement du citrate de sildénafil (5 mg/kg) et l’énanthate de testostérone (5 mg/kg)] et deux groupes expérimentaux traités à l’extrait aux doses 75 mg/kg (Groupe IV) et 150 mg/kg (Groupe V). L’eau distillée, l’extrait et la substance de référence était administré per os une fois par jour. Pour analyser le comportement sexuel et la fertilité, des femelles primipares et en oestrus étaient utilisées. Le traitement a duré 8 et 55 jours avant le sacrifice. Le poids relatif des organes, le nombre de spermatozoïde, la motilité, les analyses morphométriques, la force musculaire, le taux de cholestérol, le taux de protéines sériques et épididymaires étaient déterminé. Le temps de latence de monte et d’intromission a diminué significativement alors que la fréquence d’éjaculation a augmenté. L’index de libido, la fréquence de monte, d’intromission, d’éjaculation, d’orientation des mâles vis-à-vis d’eux même et l’index de motivation sexuelle a augmenté chez les animaux traités l’extrait comparés à ceux ayant reçu de l’eau distillée aussi bien chez les normaux que chez le diabétiques qui n’enregistrent d’ailleurs aucune éjaculation. Le traitement a augmenté significativement (P < 0,01) le poids des testicules, de la prostate et de l’épididyme. Il est observé une augmentation dose-dépendante du nombre et de la motilité des spermatozoïdes (P < 0,05), ainsi qu’une augmentation significative (P < 0,001) temps-dépendant du taux de cholestérol sérique et de protéines épididymaires. Ces résultats indiquent un potentiel androgénique pro-érectile de N. lotus chez les rats mâles et justifient l’utilisation empirique des fleurs de N. lotus dans le traitement des dysfonctions érectiles et des problèmes de fertilité chez les hommes. Mots-clés: Nymphaea lotus, pro-érectile, androgénique, fertilité, diabétiques, rat male

Ultrastructural changes in otoconia of osteoporotic rats

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.