A expressão da proteína Fos após injeção intracerebroventricular de adrenalina em ratos saciados

Dissertação (Mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Neurociências, Florianópolis, 2014Estudos prévios em nosso laboratório sugerem que a injeção de adrenalina (AD) no núcleo mediano da rafe (NMR) diminui a ingestão de alimentos em ratos em restrição alimentar e aumenta a ingestão em ratos saciados. A injeção de agonista a-2 adrenérgico no NMR provoca aumento da ingestão de alimento em ratos saciados. Por outro lado, a administração de agonista a-1 adrenérgico, no NMR, não provoca alteração no consumo alimentar, provavelmente devido à presença de elevada atividade adrenérgica e manutenção de um tônus inibitório persistente (possivelmente serotonérgico) sobre a ingestão de alimento em animais saciados. O presente estudo teve como objetivo avaliar a expressão da proteína Fos e sua co-localização com neurônios serotonérgicos após a injeção de AD no espaço intracerebroventricular (ICV) em ratos saciados. Foram utilizados 84 ratos Wistar com cânulas-guia cronicamente implantadas no ventrículo lateral (VL) ou 4º ventrículo (4V). Primeiramente, foram feitas curvas dose/resposta para a escolha da dose mais efetiva de AD sobre os comportamentos ingestivos. Após a injeção de AD no VL ou 4V, avaliou-se os comportamentos ingestivos e não ingestivos durante 30 minutos e a quantidade de comida e água consumida foram mensuradas. Os resultados mostraram que a dose de 20 nmol de AD injetada no VL e a dose de 60 nmol de AD injetada no 4V, foram as mais efetivas sobre os comportamentos ingestivos, aumentando a ingestão de alimentos. Após, novos grupos experimentais foram feitos com as doses escolhidas no VL e 4V, e realizaram-se reações imunoistoquímicas para proteína Fos e serotonina (5-HT) no NMR e núcleo dorsal da rafe (NDR), e para Fos em núcleo hipotalâmicos. Os resultados apontaram que, após administração de AD 20 nmol no VL, ocorreu aumento na expressão de Fos no núcleo paraventricular do hipotálamo (NPV) e hipotálamo ventromedial (HVM) e um aumento de Fos e sua co-localização com 5-HT no NDR. Nenhuma modificação estatiscamente significante no NMR foi observada. Os resultados após a administração de AD 60 nmol no 4V indicaram que houve apenas aumento de duplas marcações no NDR, não sendo observada nenhuma alteração no NMR. Os dados presentes neste trabalho mostram que a injeção de AD ICV aumenta a ingestão de alimentos em ratos saciados. A expressão de Fos e duplas marcações no NDR demonstra atividade neuronal serotonérgica e não-serotonérgica nesta região. No entanto, não obtivemos evidências de que uma modificação na neurotransmissão adrenérgica em ratos saciados modificasse a expressão de Fos no NMR e que os efeitos adrenérgicos sobre a ingestão de alimentos sejam mediados por ativação de neurônios serotonérgicos deste núcleo.Abstract: Previous studies from our laboratory have shown that adrenaline (AD) injection into the median raphe nucleus (NMR) decreased food intake in food-restricted rats and increases food intake in free-feeding rats. Injection of a-2 adrenergic agonist into the NMR produced increases food intake in satiated rats. Moreover, a-1 adrenergic agonist administration into the NMR do not change feeding behavior, may be due to the presence of a high adrenergic activity and maintenance of a persistent inhibitory tone (possibly serotonergic) on food intake in satiated animals. The aim of the present study was to evaluate the Fos protein expression and its colocalization in serotonergic neurons after intraventricular (ICV) injection of AD in free-feeding rats. A guide cannula was chronically implanted in the lateral ventricle (VL) or fourth ventricle (4V) of 84 male wistar rats. First, curves dose/response were made to choose the most effective dose of AD on ingestive behavior. After AD injection in VL or 4V, we assessed ingestive and non ingestive behaviors for 30 minutes and the amount of food and water consumed. The results showed that a dose of AD 20 nmol injected into the VL and a dose of AD 60 nmol injected into the 4V, were the most effective on changing the ingestive behavior. After, new experimental groups were made with the selected VL and 4V doses, and immunohistochemical reactions were performed to measure Fos protein and serotonin (5-HT) in NMR and NDR, and Fos expression in the hypothalamic nuclei. The results showed that administration of AD 20 nmol in VL increased Fos expression in NPV and HVM hypothalamic nuclei and increased Fos and its colocalization with 5-HT in the NDR. No statistically significant change was observed in NMR. The results after administration of AD 60 nmol in 4V showed an increase of double staining on the NDR, but no changes were observed in NMR. The data presented in this paper show that AD ICV injections increases food intake in free-feeding rats. The expression of Fos and double staning on the NDR showed serotonergic and non-serotonergic neuronal activity in this region stimulated by AD. However, we did not obtain evidence that a change in adrenergic neurotransmission in satiated rats modifies the Fos expression in NMR and that the adrenergic effects on food intake are mediated by the activation of serotonergic neurons in this nucleus

Glucose Homeostasis Is Not Affected in a Murine Model of Parkinson’s Disease Induced by 6-OHDA

There is a mutual relationship between metabolic and neurodegenerative diseases. However, the causal relationship in this crosstalk is unclear and whether Parkinson’s disease (PD) causes a posterior impact on metabolism remains unknown. Considering that, this study aimed to evaluate the appearance of possible changes in metabolic homeostasis due to 6-hydroxydopamine (6-OHDA) administration, a neurotoxin that damage dopaminergic neurons leading to motor impairments that resemble the ones observed in PD. For this, male Wistar rats received bilateral 6-OHDA administration in the dorsolateral striatum, and the motor and metabolic outcomes were assessed at 7, 21, or 35 days post-surgical procedure. Dexamethasone, a diabetogenic glucocorticoid (GC), was intraperitoneally administered in the last 6 days to challenge the metabolism and reveal possible metabolic vulnerabilities caused by 6-OHDA. Controls received only vehicles. The 6-OHDA-treated rats displayed a significant decrease in locomotor activity, exploratory behavior, and motor coordination 7 and 35 days after neurotoxin administration. These motor impairments paralleled with no significant alteration in body mass, food intake, glucose tolerance, insulin sensitivity, and biochemical parameters (plasma insulin, triacylglycerol, and total cholesterol levels) until the end of the experimental protocol on days 35–38 post-6-OHDA administration. Moreover, hepatic glycogen and fat content, as well as the endocrine pancreas mass, were not altered in rats treated with 6-OHDA at the day of euthanasia (38th day after neurotoxin administration). None of the diabetogenic effects caused by dexamethasone were exacerbated in rats previously treated with 6-OHDA. Thus, we conclude that bilateral 6-OHDA administration in the striatum causes motor deficits in rats with no impact on glucose and lipid homeostasis and does not exacerbate the adverse effects caused by excess GC. These observations indicate that neurodegeneration of dopaminergic circuits in the 6-OHDA rats does not affect the metabolic outcomes

Physics with the KLOE-2 experiment at the upgraded DAϕ\phiNE

Investigation at a $\phi$--factory can shed light on several debated issues in particle physics. We discuss: i) recent theoretical development and experimental progress in kaon physics relevant for the Standard Model tests in the flavor sector, ii) the sensitivity we can reach in probing CPT and Quantum Mechanics from time evolution of entangled kaon states, iii) the interest for improving on the present measurements of non-leptonic and radiative decays of kaons and eta/eta$^\prime$ mesons, iv) the contribution to understand the nature of light scalar mesons, and v) the opportunity to search for narrow di-lepton resonances suggested by recent models proposing a hidden dark-matter sector. We also report on the $e^+ e^-$ physics in the continuum with the measurements of (multi)hadronic cross sections and the study of gamma gamma processes.Comment: 60 pages, 41 figures; added affiliation for one of the authors; added reference to section

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Modificações na neurotransmissão adrenérgica no núcleo dorsal da rafe e seus efeitos sobre os comportamentos relacionados à ingestão de alimentos em ratos

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa Multicêntrico de Pós-Graduação em Ciências Fisiológicas, Florianópolis, 2018.O presente estudo teve como objetivo avaliar os efeitos da injeção de agonistas a-adrenérgicos no núcleo dorsal da rafe (DRN) sobre os comportamentos relacionados à ingestão de alimentos em ratos saciados e submetidos o jejum noturno. Ratos wistar, machos e adultos, com cânulas- guia implantadas cronicamente no DRN foram injetados com adrenalina (AD) (doses de 6, 20 e 60 nmol) ou com noradrenalina (NA) nas mesmas doses , além de agonista a-1 adrenérgico Fenilefrina (FEN) e agonista a-2 adrenérgico Clonidina (CLO) seguida pela avaliação de parâmetros comportamentais ingestivos. O comportamento foi monitorado durante 60 minutos e a quantidade de alimento e água ingerida foi avaliada. Os tratamentos com AD e NA na dose de 60 nmol e com CLO na dose de 20 nmol aumentaram a ingestão de alimento em ratos saciados, aumentando também a duração e a frequência e diminuindo a latência para iniciar o consumo. Em ratos submetidos ao jejum noturno, a dose de 60 nmol de AD e NA e a dose de 20 nmol de FEN diminuíram a ingestão alimentar quando comparado ao grupo controle. Este efeito veio acompanhado também de queda na duração do comportamento ingestivo. A ingestão hídrica foi alterada apenas nos animais submetidos ao jejum noturno, ocorrendo uma diminuição na quantidade de água consumida. As análises imunistoquímicas revelaram que houve um aumento de atividade neuronal no núcleo paraventricular do hipotálamo (PVN) e um aumento de atividade de neurônios a-MSH+ no núcleo arqueado (ARC) após a injeção de CLO 20 nmol no DRN de ratos saciados. Em contrapartida, houve uma diminuição da atividade neuronal no PVN e uma diminuição da atividade de neurônios a-MSH+ no ARC após a injeção de FEN 20 nmol no DRN de ratos submetidos ao jejum noturno. Em conclusão, os resultados indicam uma ativação diferencial dos receptores a adrenérgicos do DRN, que parece ser dependente da disponibilidade do alimento. Além disso, as modificações na atividade neuronal encontradas no PVN e em neurônios POMC do ARC após as modificações na neurotransmissão adrenérgica no DRN sugere que este núcleo atue através de mecanismos homeostáticos no controle da ingestão alimentar.Abstract : The present study aimed to evaluate the effects of the injection of a- adrenergic agonists into the dorsal raphe nucleus (DRN) on behaviors related to food intake in free-feeding rats and and fasted. Adult male wistar rats with chronically implanted cannulae in the DRN were injected with adrenaline (AD) or noradrenaline (NA) (both at doses of 6, 20 and 60 nmol), or a-1 adrenergic agonist Phenylephrine (FEN) or a-2 adrenergic agonist Clonidine (CLO) (both at doses of 6 and 20 nmol). The injection were followed by the evaluation of ingestive behavioral parameters. Food and water intake was evaluated for 60 minutes. Administration of AD and NA at 60 nmol and CLO at 20 nmol increased food intake and decreased latency to start consumption in free-feeding rats. AD, NA (both at dose of 60 nmol) and FEN (20 nmol) decreased food intake in fasted rats when compared to the controle group. This effect was followed by a decrease in the duration of ingestive behavior. Water intake was decreased only in fasted animals. Immunohistochemical analyzes revealed an increase in Fos positive neurons in the paraventricular nucleus of the hypothalamus (PVN) and an increase of a-MSH + neurons activity in the arcuate nucleus (ARC) after injection of CLO 20 nmol in the DRN of free-feeding rats. In contrast, there was a decrease in neuronal activity in the PVN and a decrease in a- MSH + neurons activity in the ARC after the injection of FEN 20 nmol in the DRN of fasted. In conclusion, these results highlight the functional role of DRN adrenergic receptors in the control of food intake and indicate a differential activation of the DRN adrenergic receptors, which appears to be dependent on food availability. In addition, the modifications in neuronal activity found in PVN and in POMC neurons of the ARC after the modifications in adrenergic neurotransmission in DRN suggest that this nucleus controls food intake through homeostatic mechanisms

Feeding behaviour after injection of α-adrenergic receptor agonists into the median raphe nucleus of food-deprived rats

AbstractThis study investigated the participation of median raphe nucleus (MnR) α1-adrenergic receptors in the control of feeding behaviour. The α1-adrenergic agonist phenylephrine (PHE) and α2-adrenergic agonist clonidine (CLON) (at equimolar doses of 0, 6 and 20nmol) were injected into the MnR of: a) rats submitted to overnight fasting (18h); or b) rats maintained with 15g of lab chow/day for 7days. Immediately after the drug injections, the animals were placed in the feeding chamber and feeding and non-ingestive behaviours such as grooming, rearing, resting, sniffing and locomotion were recorded for 30min. The results showed that both doses of PHE injected into the MnR of overnight fasted animals decreased food intake accompanied by an increase in the latency to start feeding. A reduction in feeding duration was observed only after treatment of the MnR with the 20nmol dose of PHE. Both locomotion duration and sniffing frequency increased after injection with the highest dose PHE into the MnR. Feeding frequency and the other non-ingestive behaviours remained unchanged after PHE treatment in the MnR. Both doses of PHE injected into the MnR of food-restricted rats decreased food intake. This hypophagic response was accompanied by a decrease in feeding duration only after treatment of the MnR with the highest dose of PHE. The latency to start feeding and feeding frequency were not affected by injection of either dose of PHE into the MnR. While both doses of PHE increased sniffing duration, the highest dose of PHE increased resting duration and resting frequency. Treatment with CLON into the MnR did not affect feeding behaviour in either of the food deprivation conditions. The present results indicate the inhibitory functional role of α1-adrenergic receptors within the MnR on feeding behaviour

Physics with the KLOE-2 experiment at the upgraded DAFNE

Investigation at a φ-factory can shed light on several debated issues in particle physics. We discuss: (i) recent theoretical development and experimental progress in kaon physics relevant for the Standard Model tests in the flavor sector, (ii) the sensitivity we can reach in probing CPT and Quantum Mechanics from time evolution of entangled-kaon states, (iii) the interest for improving on the present measurements of non-leptonic and radiative decays of kaons and η/ η' mesons, (iv) the contribution to understand the nature of light scalar mesons, and (v) the opportunity to search for narrow di-lepton resonances suggested by recent models proposing a hidden dark-matter sector. We also report on the e + e - physics in the continuum with the measurements of (multi)hadronic cross sections and the study of γ γ processes

Apolipoprotein B, Residual Cardiovascular Risk After Acute Coronary Syndrome, and Effects of Alirocumab.

Background: Apolipoprotein B (apoB) provides an integrated measure of atherogenic risk. Whether apoB levels and apoB lowering hold incremental predictive information on residual risk after acute coronary syndrome beyond that provided by low-density lipoprotein cholesterol is uncertain. Methods: The ODYSSEY OUTCOMES trial (Evaluation of Cardiovascular Outcomes After an Acute Coronary Syndrome During Treatment With Alirocumab) compared the proprotein convertase subtilisin/kexin type 9 inhibitor alirocumab with placebo in 18 924 patients with recent acute coronary syndrome and elevated atherogenic lipoproteins despite optimized statin therapy. Primary outcome was major adverse cardiovascular events (MACE; coronary heart disease death, nonfatal myocardial infarction, fatal/nonfatal ischemic stroke, hospitalization for unstable angina). Associations between baseline apoB or apoB at 4 months and MACE were assessed in adjusted Cox proportional hazards and propensity score–matched models. Results: Median follow-up was 2.8 years. In proportional hazards analysis in the placebo group, MACE incidence increased across increasing baseline apoB strata (3.2 [95% CI, 2.9–3.6], 4.0 [95% CI, 3.6–4.5], and 5.5 [95% CI, 5.0–6.1] events per 100 patient-years in strata 35–<50, and ≤35 mg/dL, respectively). Compared with propensity score–matched patients from the placebo group, treatment hazard ratios for alirocumab also decreased monotonically across achieved apoB strata. Achieved apoB was predictive of MACE after adjustment for achieved low-density lipoprotein cholesterol or non–high-density lipoprotein cholesterol but not vice versa. Conclusions: In patients with recent acute coronary syndrome and elevated atherogenic lipoproteins, MACE increased across baseline apoB strata. Alirocumab reduced MACE across all strata of baseline apoB, with larger absolute reductions in patients with higher baseline levels. Lower achieved apoB was associated with lower risk of MACE, even after accounting for achieved low-density lipoprotein cholesterol or non–high-density lipoprotein cholesterol, indicating that apoB provides incremental information. Achievement of apoB levels as low as ≤35 mg/dL may reduce lipoprotein-attributable residual risk after acute coronary syndrome. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT01663402.gov; Unique identifier: NCT01663402.URL: https://www