Morfologia e desenvolvimento do sistema reprodutor masculino de carrapatos do gênero Amblyomma (Acari, Ixodidae): uma análise comparativa

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

Avaliação histopatológica utilizando PAS para análise de alterações hepáticas em ratos tratados com 4-NQO

O fígado é a maior glândula e o segundo maior órgão do corpo, podendo ser dividido em zona 1, próxima à região do espaço porta, zona 2 ou intermediária, e zona 3, próxima da veia centro-lobular. A zona 1 é responsável pela gliconeogenese, enquanto a zona 3 é responsável pela detoxicação. O óxido de 4-nitroquinolina (4-NQO) é um agente carcinogênico sintético capaz de aumentar o risco individual ao desenvolvimento de neoplasia maligna na língua de ratos, e sua biotransformação 4-HAQO ocorre no compartimento hepático. Neste estudo, 5 grupos de 8 ratos cada, foram submetidos à administração oral de 4-NQO na concentração de 25 ppm (massa solvente/massa soluto) através da água de beber, com a finalidade de identificar a ocorrência de alterações metabólicas hepáticas devido ao acúmulo de glicogênio nos hepatócitos dos animais, analisado pela coloração de ácido periódico de Shiff (PAS). O glicogênio é o principal polissacarídeo de reserva energética dos animais, e é fundamental para manter a homeostase do organismo, sendo seu acúmulo nos hepatócitos uma resposta fisiológica normal após a ingestão de alimentos, ou ainda, pode devido a perturbações metabólicas provocadas por tratamentos que os animais foram expostos. Neste estudo, o fígado dos animais dos diferentes grupos analisados demonstraram níveis distintos de marcação para glicogênio, mas não apresentaram diferenças estatisticamente significantes, podendo considerar que os animais foram pouco ou não foram afetados pela exposição às diferentes substâncias estudadas no projeto e que não foram tóxicas aos animais, havendo apenas um ligeiro aumento nos níveis de glicogênio hepático geral.

Sex differences in the excretion of fecal glucocorticoid metabolites in the Syrian hamster

We verified the relevance of measuring fecal glucocorticoid metabolites (FGM) to assess the stress response of the Syrian hamster. Male and female hamsters (n = 10 each) were submitted to an adrenocorticotropic hormone (ACTH) challenge test, whereas animals in the control group received 0.5 mL of sterile isotonic saline solution. All feces voided by each animal were collected at 4 h intervals from 24 h before (baseline) until 48 h after injections. FGM were quantified using an 11-oxoetiocholanolone enzyme immunoassay (EIA). Basal concentrations of FGM were almost four times higher in males than in females. Following ACTH administration, FGM levels started rising from 8 h onwards, reaching peak concentrations 20 or 28 h post injection in males and females, respectively. Despite the much higher absolute concentrations present in males, the relative increase (500%) in response to the ACTH stimulation was similar in both sexes. Sex differences in FGM levels are in accordance with results reported by others regarding the hamster adrenal physiology. The comparison of the adrenocortical response of males and females to an ACTH challenge provided new information about the amplitude and the timing of such a response and the excretion of glucocorticoids in both sexes. We demonstrated for the first time in the Syrian hamster that adrenocortical activity can be monitored in fecal samples in a noninvasive way. Our study provides a humane, practical, and noninvasive alternative to blood removal and therefore a powerful tool for stress-related studies in a species frequently used as an animal model in medical research.

Divergent Roles of the CRH Receptors in the Control of Gonadotropin Secretion Induced by Acute Restraint Stress at Proestrus

CRH has been implicated as a mediator of stress-induced effects on the hypothalamus-pituitary-gonad axis, acting via CRH receptors in various brain regions. We investigated whether the effects of restraint stress on the secretion of gonadotropins on the morning of proestrus are mediated by the CRH-R1 or CRH-R2 receptors in the oval subdivision of the anterolateral BST, the central amygdala, the locus coeruleus (LC), or the A1 and A2 neuron groups in the medulla. At proestrus morning, rats were injected with antalarmin (a CRH-R1 antagonist), asstressin2-B (a CRH-R2 antagonist) or vehicles. Thirty minutes after the injection, the animals were placed into restraints for 30 min, and blood was sampled for 2 h. At the end of the experiment, the brains were removed for immunofluorescence analyses. Restraint stress increased the levels of FSH and LH. Antalarmin blocked the stress-induced increases in FSH and LH secretion, but astressin2-B only blocked the increase in FSH secretion. LC showed intense stress-induced neuronal activity. FOS/tyrosine-hydroxylase coexpression in LC was reduced by antalarmin, but not astressin2-B. The CRH-R1 receptor, more than CRH-R2 receptor, appears to be essential for the stimulation of the hypothalamus-pituitary-gonad axis by acute stress; this response is likely mediated in part by noradrenergic neurons in the LC. We postulate that the stress-induced facilitation of reproductive function is mediated, at least in part, by CRH action through CRH-R1 on noradrenaline neurons residing in the LC that trigger GnRH discharge and gonadotropin secretion. (Endocrinology 153: 4838-4848, 2012)

Expressão do gene da leptina e seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras

Objetivou-se com este trabalho avaliar os efeitos de uma dieta de alto nível de energia e proteína combinada com a aplicação de bST no perfil de expressão dos genes da leptina e de seu receptor Ob-Rb no parênquima mamário de novilhas leiteiras. Foram utilizadas amostras de parênquima mamário de 32 novilhas holandesas distribuídas aleatoriamente em quatro tratamentos (n=8): dieta com alto ou baixo teor de energia e proteína combinada ou não com a aplicação de bST. O delineamento utilizado foi em blocos casualizados com arranjo de tratamentos em esquema fatorial 2 × 2. A extração do RNA total das amostras de tecido foi feita e o nível de expressão gênica foi analisado por qRT-PCR utilizando-se o gene da glicuronidase β como controle, pelo método 2-ΔΔCt. Animais que receberam a dieta com alto conteúdo de energia e proteína apresentaram maior expressão de mRNA de leptina, com aumento de 56%, e menor expressão de mRNA do receptor Ob-Rb, com redução de 18%. Por outro lado, a aplicação de bST resultou em diminuição da expressão do mRNA de leptina e do receptor Ob-Rb em 74% e 23%, respectivamente. Não houve interação entre dieta e aplicação de bST. O aumento na expressão de leptina pode explicar, ao menos em parte, os efeitos negativos da dieta de alta energia e proteína, oferecida no período pré-púbere, sobre a produção de leite de novilhas leiteiras.

Impact of Glucocorticoid Receptor Gene Polymorphisms on the Metabolic Profile of Adult Patients with the Classical Form of 21-Hydroxylase Deficiency

Background: CAH patients have an increased risk of cardiovascular disease, and it remains unknown if lifelong glucocorticoid (GC) treatment is a contributing factor. In the general population, glucocorticoid receptor gene (NR3C1) polymorphisms are associated with an adverse metabolic profile. Our aim was to analyze the association between the NR3C1 polymorphisms and the metabolic profile of CAH patients. Methodology: Sixty-eight adult patients (34SV/34SW) with a mean age of 28.4 +/- 9 years received dexamethasone (mean 0.27 +/- 0.11 mg/day) to obtain normal androgen levels. SW patients also received fludrocortisone (50 mu g/day). Metabolic syndrome (MetS) was defined by the NCEP ATPIII criteria and obesity by BMI >= 30 kg/m(2). NR3C1 alleles were genotyped, and association analyses with phenotype were carried out with Chi-square, t-test and regression analysis. Results: Obesity and MetS were observed in 23.5% and 7.3% of patients, respectively, and were not correlated with GC doses and treatment duration. BMI was positively correlated with blood pressure (BP), triglycerides (TG), LDL-c levels and HOMA-IR and inversely correlated with HDL-c levels. BclI and A3669G variants were found in 26.4% and 9.6% of alleles, respectively. Heterozygotes for the BclI polymorphism presented with higher BMI (29 kg/m(2) +/- 5.3 vs. 26 kg/m(2) +/- 5.3, respectively) and waist circumference (89 cm +/- 12.7 vs. 81 cm +/- 13, respectively) compared to wild-type subjects. Hypertension was found in 12% of patients and heterozygotes for the BclI polymorphism presented higher systolic BP than wild type subjects. Low HDL-c and high TG levels were identified in 30% and 10% of patients, respectively, and were not associated with the NR3C1 polymorphisms. A3669G carriers and non-carriers did not differ. Conclusion: In addition to GC therapy, the BclI GR variant might play an important role in obesity susceptibility in CAH patients. Genotyping of GR polymorphisms could result in the identification of a subgroup at risk patients, allowing for the establishment of personalized treatment and the avoidance of long-term adverse consequences.

Oxytocin interference in the effects induced by inhalation of 7.5% CO2 in healthy volunteers

Objective The objective of this study was to assess the acute effect of intranasally administered oxytocin (OT) on subjective states, cardiovascular, and endocrine parameters in healthy volunteers who inhaled 7.5% CO2. Methods Forty-five subjects were allocated into three matched groups of subjects who received 24?international units (IU) of OT, 2?mg of lorazepam (LZP), or placebo (PL). The challenge consisted of medical air inhalation for 20?min, 10?min of rest, and CO2 7.5% inhalation for 20?min. Subjective effects were evaluated by self-assessment scales; heart rate, blood pressure, skin conductance, and salivary cortisol were also measured. Assessments were performed at four time points: (i) baseline (-15?min); (ii) post-air inhalation (90?min); (iii) post-CO2 inhalation (120?min), and (iv) post-test (160?min). Results CO2 inhalation significantly increased the anxiety score in the PL group compared with the post-air measurement but not in the OT or LZP groups. The LZP reduced anxiety after medical air inhalation. Other parameters evaluated were not affected by OT. Conclusion OT, as well as LZP, prevented CO2-induced anxiety, suggesting that this hormone has anxiolytic properties. Copyright (C) 2012 John Wiley & Sons, Ltd.

Characterization of a Novel Oral Glucocorticoid System and Its Possible Role in Disease

Synthetic corticosteroids are used widely for the treatment of a variety of diseases of the mouth. However, little is known as to whether the oral mucosa is able to modulate the local concentration of active corticosteroids or to produce steroids de novo. This has important clinical implications, because tissue-specific regulation of glucocorticoids is a key determinant of the clinical efficacy of these drugs. In the present study, we show that oral fibroblasts and keratinocytes expressed ACTH receptor (MC2R), glucocorticoid receptor (GR), and 11 beta-hydroxysteroid dehydrogenases (11 beta-HSDs). Unlike keratinocytes, fibroblasts lacked 11 beta-HSD2 and could not effectively deactivate exogenously administered cortisol. However, both cell types were able not only to activate cortisone into the active form cortisol, but also to synthesize cortisol de novo following stimulation with ACTH. 11 beta-HSD2, the enzyme controlling cortisol deactivation, exhibited different patterns of expression in normal (squamous epithelium and salivary glands) and diseased oral mucosa (squamous cell carcinoma and mucoepidermoid carcinoma). Blocking of endogenous cortisol catabolism in keratinocytes with the 11 beta-HSD2 inhibitor 18 beta-glycyrrhetinic acid mimicked the effect of exogenous administration of hydrocortisone and partially prevented the detrimental effects induced by pemphigus vulgaris sera. Analysis of the data demonstrates that a novel, non-adrenal glucocorticoid system is present in the oral mucosa that may play an important role in disease.

Laparoscopic adrenalectomy in children

Purpose: Reporting on the laparoscopic technique for adrenal disease in children and adolescents has been limited. We review here our experience with laparoscopic adrenal surgery in children. Patients and methods: 19 laparoscopic unilateral adrenalectomies were performed in 10 girls and 7 boys (mean age 3.9 years) during 1998-2011. The clinical diagnosis before surgery was virilizing tumor (n = 8), pheochromocytoma (n = 3), nonfunctioning solid adrenal tumor (n = 3), mixed adrenocortical tumor (n = 2), cystic adrenal mass (n = 1). Unilateral adrenal lesions were 20-65 mm at the longest axis on computerized tomography (12 right side, 7 left side). Results: The final clinicopathological diagnosis was cortical adenoma (n = 9), pheochromocytoma (n = 3, bilateral in two), neuroblastoma (n = 1), ganglioneuroblastoma (n = 1), ganglioneuroma (n = 1), adrenocortical carcinoma (n = 1), benign adrenal tissue (n = 1). Average operative time was 138.5 min (range 95-270). Blood transfusion was required in one case (5%). No conversion to open surgery was required and no deaths or postoperative complications occurred. Average hospital stay was 3.5 days (range 2-15). Average postoperative follow-up was 81 months (range 2-144). Two contralateral metachronic pheochromocytomas associated with von Hippel-Lindau syndrome occurred, treated with partial laparoscopic adrenalectomy (one without postoperative need of cortisone replacement therapy). Conclusions: Laparoscopic adrenalectomy is a feasible procedure that produces good results. It can be used safely to treat suspected benign and malignant adrenal masses in children with minimal morbidity and short hospital stay. (C) 2011 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.

SALIVARY CORTISOL AND IMMUNOGLOBULIN A RESPONSES TO SIMULATED AND OFFICIAL JIU-JITSU MATCHES

Moreira, A, Franchini, E, Freitas, CG, Arruda, AFS, Moura, NR, Costa, EC, and Aoki, MS. Salivary cortisol and immunoglobulin A responses to simulated and official Jiu-Jitsu matches. J Strength Cond Res 26(8): 2185-2191, 2012-The aim of this study was to compare the salivary cortisol (sC) and the salivary immunoglobulin A (sIgA) responses to simulated and official Brazilian Jiu-Jitsu (BJJ) matches. Saliva samples were collected from 9 male BJJ athletes before (pre) and after (post) 2 simulated matches (SMs) and 2 official matches (OMs) performed during 2 different competitions. Salivary cortisol and sIgA concentrations (absolute concentration of sIgA [sIgA(abs)] and the secretion rate of sIgA [sIgA(rate)]) were measured by an enzyme-linked immunosorbent assay. For sC, there was an effect of condition (SM vs. OM) (p < 0.05) and a time effect (pre and post) (p < 0.05). The sC was lower during SMs as compared with that during OMs and lower at premeasurement when compared with postmeasurement. No changes were observed for sIgA measurements. In summary, both SMs and official BJJ matches can increase sC levels. Moreover, the higher sC resting levels, observed before OMs, suggest that psychological factors associated with high physical-physiological demands from official BJJ competitions maximize stress hormone responses. In addition, the present findings suggest that the acute effect of BJJ matches on mucosal immunity is minimal, and it seems unlikely that changes in cortisol play a major role in the alterations in sIgA levels in response to BJJ matches. The findings of this study suggest that the use of sC can provide valuable information for coaches regarding athletes' responses to competition. In addition, psychological strategies should be implemented before events, to improve the manner in which BJJ athletes cope with the stress inherent to official matches.

Influence of N-methyl-D-aspartate receptors on ouabain activation of nuclear factor-kappa B in the rat hippocampus

It has been shown that ouabain (OUA) can activate the Na,K-ATPase complex and mediate intracellular signaling in the central nervous system (CNS). Inflammatory stimulus increases glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptors, which are usually coupled to the activation of nitric oxide synthase (NOS). Nuclear factor-kappa B (NF-kappa B) activation modulates the expression of genes involved in development, plasticity, and inflammation. The present work investigated the effects of OUA on NF-kappa B binding activity in rat hippocampus and the influence of this OUA-Na,K-ATPase signaling cascade in NMDA-mediated NF-kappa B activation. The findings presented here are the first report indicating that intrahippocampal administration of OUA, in a concentration that did not alter Na,K-ATPase or NOS activity, induced an activation of NF-kappa B, leading to increases in brain-derived neurotrophic factor (Bdnf), inducible NOS (iNos), tumor necrosis factor-alpha (Tnf-alpha), and B-cell leukemia/lymphoma 2 (Bcl2) mRNA levels. This response was not linked to any significant signs of neurodegeneration as showed via Fluoro-Jade B and Nissl stain. Intrahippocampal administration of NMDA induced NF alpha B activation and increased NOS and alpha 2/3-Na,K-ATPase activities. NMDA treatment further increased OUA-induced NF-kappa B activation, which was partially blocked by MK-801, an antagonist of NMDA receptor. These results suggest that OUA-induced NF-kappa B activation is at least in part dependent on Na,K-ATPase modulatory action of NMDA receptor in hippocampus. The interaction of these signaling pathways could be associated with biological mechanisms that may underlie the basal homeostatic state linked to the inflammatory signaling cascade in the brain. (c) 2011 Wiley Periodicals, Inc.

Progression to Adrenocortical Tumorigenesis in Mice and Humans through Insulin-Like Growth Factor 2 and beta-Catenin

Dysregulation of the WNT and insulin-like growth factor 2 (IGF2) signaling pathways has been implicated in sporadic and syndromic forms of adrenocortical carcinoma (ACC). Abnormal beta-catenin staining and CTNNB1 mutations are reported to be common in both adrenocortical adenoma and ACC, whereas elevated IGF2 expression is associated primarily with ACC. To better understand the contribution of these pathways in the tumorigenesis of ACC, we examined clinicopathological and molecular data and used mouse models. Evaluation of adrenal tumors from 118 adult patients demonstrated an increase in CTNNB1 mutations and abnormal beta-catenin accumulation in both adrenocortical adenoma and ACC. In ACC, these features were adversely associated with survival. Mice with stabilized beta-catenin exhibited a temporal progression of increased adrenocortical hyperplasia, with subsequent microscopic and macroscopic adenoma formation. Elevated Igf2 expression alone did not cause hyperplasia. With the combination of stabilized beta-catenin and elevated Igf2 expression, adrenal glands were larger, displayed earlier onset of hyperplasia, and developed more frequent macroscopic adenomas (as well as one carcinoma). Our results are consistent with a model in which dysregulation of one pathway may result in adrenal hyperplasia, but accumulation of a second or multiple alterations is necessary for tumorigenesis. (Ant J Pathol 2012, 181:1017-1033; http://dx.doi.org/10.1016/j.ajpath.2012.05.026)

Plasma corticosterone levels in mouse models of pain

Background: Pain markedly activates the hypothalamic-pituitary-adrenal (HPA) axis and increases plasma corticosterone release interfering significantly with nociceptive behaviour as well as the mechanism of action of analgesic drugs. Aims/Methods: In the present study, we monitored the time course of circulating corticosterone in two mouse strains (C57Bl/6 and Balb/C) under different pain models. In addition, the stress response was investigated following animal handling, intrathecal (i.t.) manipulation and habituation to environmental conditions commonly used in nociceptive experimental assays. We also examined the influence of within-cage order of testing on plasma corticosterone. Results: Subcutaneous injection of capsaicin precipitated a prompt stress response whereas carrageenan and complete Freund's adjuvant induced an increased corticosterone release around the third hour post-injection. However, carrageenan induced a longer increased corticosterone in C57Bl/6 mice. In partial sciatic nerve ligation, neuropathic pain model corticosterone increased only in the first days whereas mechanical hypersensitivity remained much longer. Animal handling also represents an important stressor whereas the i.t. injection per se does not exacerbate the handling-induced stress response. Moreover, the order of testing animals from the same cage does not interfere with plasma corticosterone levels in the intrathecal procedure. Animal habituation to the testing apparatus also does not reduce the immediate corticosterone increase as compared with non-habituated mice. Conclusion: Our data indicate that HPA axis activation in acute and chronic pain models is time dependent and may be dissociated from evoked hyperalgesia. Therefore, HPA-axis activation represents an important variable to be considered when designing experimental assays of persistent pain as well as for interpretation of data.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Risk assessment behaviors associated with corticosterone trigger the defense reaction to social isolation in rats: Role of the anterior cingulate cortex

The extent to which the hypothalamic-pituitary-adrenal axis is activated by short-term and long-term consequences of stress is still open to investigation. This study aimed to determine (i) the correlation between plasma corticosterone and exploratory behavior exhibited by rats subjected to the elevated plus maze (EPM) following different periods of social isolation, (ii) the effects of the corticosterone synthesis blocker, metyrapone, on the behavioral consequences of isolation, and (iii) whether corticosterone produces its effects through an action on the anterior cingulate cortex, area 1 (Cg1). Rats were subjected to 30-min, 2-h, 24-h, or 7-day isolation periods before EPM exposure and plasma corticosterone assessments. Isolation for longer periods of time produced greater anxiogenic-like effects on the EPM. However, stretched attend posture (SAP) and plasma corticosterone concentrations were increased significantly after 30 min of isolation. Among all of the behavioral categories measured in the EPM, only SAP positively correlated with plasma corticosterone. Metyrapone injected prior to the 24 h isolation period reversed the anxiogenic effects of isolation. Moreover, corticosterone injected into the Cg1 produced a selective increase in SAP. These findings indicate that risk assessment behavior induced by the action of corticosterone on Cg1 neurons initiates a cascade of defensive responses during exposure to stressors.