La fibrillation atriale est l'arythmie cardiaquela plus fréquente. Les études sur les modèles animaux ont fourni beaucoup de renseignements sur les mécanismes de cette arythmie, mais, à ce jour, nous ne disposons pas de modèle animal d'arythmie atriale spontanée.Nous avons cherché à développer un modèle d'arythmie atriale spontanée chez le rat et àidentifier les mécanismes physiopathologiques de ces arythmies. Nous avons également cherché àévaluer la présence et la sévérité de l'inflammation et de la dysfonction endothéliale, impliquées dansla survenue des complications de la fibrillation atriale comme les accidents vasculaires cérébraux, chezles patients avec fibrillation atriale. Nous avons également constaté des niveauxélevés de facteur de croissance endothélial vasculaire et de facteur von Willebrand chez les patientsavec fibrillation atriale par rapport aux contrôles. Ces résultats suggèrent un profil spécifique du risquethromboembolique en fonction de la forme clinique de l'arythmie et mettent en évidence une évolutionparallèle de la fibrillation atriale et de la dysfonction endothéliale.Ce nouveau modèle animal permettra d'étudier les mécanismes physiopathologiques desarythmies atriales et d'évaluer de nouveaux agents thérapeutiques dans un cadre qui reproduitfidèlement la présentation clinique de l'arythmie / Atrial fibrillation is the most prevalent form of cardiac arrhythmia. Studies in animal modelshave provided important insights into arrhythmia mechanisms. However, to date, we do not dispose ofanimal models of spontaneous atrial arrhythmia.Thus, we aimed to develop a model of spontaneous atrial arrhythmia in rats and to assesspathophysiological mechanisms of these arrhythmias by using a multidisciplinary approach. We alsoaimed to assess the presence and the extent of inflammation and endothelial dysfunction, incriminatedin atrial fibrillation-related complications such as stroke, in atrial fibrillation patients.The animal study describes the first animal model of spontaneous atrial arrhythmias. We alsoprovide evidence that multiple mechanisms participate in arrhythmia occurrence in this model,particularly autonomic imbalance with relative vagal hyperactivity, left atrial endocardial fibrosis, anddecreased left atrial expression of the Pitx2 gene. In our clinical study, we found high levels ofvascular endothelial growth factor and von Willebrand factor in atrial fibrillation patients compared tosinus rhythm controls. These results suggest specific thromboembolic risk patterns according to theclinical form of arrhythmia and highlight a parallel evolution of atrial fibrillation and endothelialdysfunction. These results add new insights into the understanding of atrial arrhythmias. This new animalmodel could facilitate studies of pathophysiological mechanisms involved in atrial arrhythmias andallow assessment of efficacy and toxicity of therapeutic agents in a setting that faithfully reproducesthe clinical presentation of the arrhythmia

Modelos experimentais do transtorno obsessivo compulsivo (TOC) com humanos mostram que uma forma de evocar comportamentos de checagem é apresentar instruções que especificam consequências aversivas para o comportamento inefetivo na execução de tarefas. Atualmente há na área somente um estudo experimental com delineamento de sujeito único. Os presentes dois experimentos com 16 participantes verbalmente habilidosos tiveram o objetivo de testar se instruções com especificação de consequência aversiva ou apetitiva poderiam ter o efeito de produzir respostas de checagem. Em um restaurante experimental, as instruções foram apresentadas durante uma tarefa de separação de sementes misturadas. No Experimento 1, cinco de oito participantes apresentaram maiores porcentagens de checagens sob especificação de consequência aversiva. No Experimento 2, sete de oito participantes apresentaram maiores porcentagens sob especificação de consequência apetitiva. Concluiu-se que determinadas instruções alteraram a função discriminativa e/ou motivadora dos estímulos envolvidos na tarefa experimental. Sugere-se que o presente delineamento pode permitir a formulação de análises funcionais do fenômeno comportamental normalmente envolvido em alguns casos de TOC / Experimental models of obsessive compulsive disorder (OCD) with humans show that a way to evoke checking behaviors is to provide instructions that specify aversive consequences for behavior ineffective in performing tasks. Currently there is only one experimental study with a single subject design in this area. The present study presents two experiments with 16 verbally skilled participants tested whether instructions specifying the appetitive or aversive consequence could have the effect of producing checking behaviors. In an experimental restaurant, the instructions were presented during a task of separation of mixed seeds. In Experiment 1, five of eight participants showed higher percentages of checks under specification of aversive consequence. In Experiment 2, seven of eight participants had higher percentages under specification of appetitive consequence. It was concluded that certain instructions alter the discriminative and motivate function of the stimuli involved in experimental task. It is suggested that this design may allow the formulation of functional analysis of behavioral phenomenon normally involved in some cases of OCD

Recentemente, descobrimos que coelhos, Nova Zelândia, imunizados com colágeno tipo V humano mais adjuvante de Freund apresentavam fibrose e vasculite de órgãos normalmente afetados na esclerose sistêmica. Deste modo, nós estudamos o processo de fibrilogênese para identificar possíveis fatores envolvidos na alteração do remodelamento observado neste modelo de esclerodermia. Adicionalmente, fizemos uma comparação preliminar com pele humana obtida de pacientes com esclerodermia (n=3). Coelhos fêmeas, Nova Zelândia (n=14), foram imunizados subcutaneamente com duas doses de 1mg de colágeno V mais adjuvante completo de Freund, com intervalo de 30 dias, seguido de duas imunizações em adjuvante incompleto de Freund, via intramuscular, com intervalo de 15 dias. Os animais do grupo controle (n- 14) foram inoculados somente com adjuvante completo e incompleto de Freund, nas mesmas condições dos imunizados. Foram realizadas análises histológicas das peles dos animais e pacientes através da coloração com tricrômico de Masson e imunofluorescêcia, a fim de detectar fibras de colágeno e interação dos colágenos I, III e V. A análise da pele dos animais demonstrou depósito precoce de fibras de colágeno na derme após 7 dias da sensibilização, com aumento destes depósitos após 75 e 120 dias respectivamente. Depósito de colágeno na pele e atrofia de anexos foram mais intensos nos animais sacrificados em 120 dias e se correlacionaram com a quantidade aumentada de colágeno. Surpreendentemente, o colágeno V foi expresso em animais e pacientes, formando fibras densas e atípicas na derme. Sugerimos que esta expressão anômala de colágeno V, morfologicamente diferente, possa justificar o remodelamento observado na placa esclerodérmica / Recently, we discovered that New Zealand rabbits immunized with human type V collagen plus Freund`s adjuvant presented fibrosis and vasculitis of organs usually affected in systemic sclerosis. In this way, we studied the fibrillogenesis process regarding to identify any possible factor involved in altered remodeling observed in this scleroderma-like model. Additionally, we done a very preliminary comparison with human skins obtained from scleroderma patients (N=3). Female New Zealand rabbits (N=14) were immunized subcutaneously with two doses of 1mg collagen V plus complete Freunds adjuvant at a 30 days interval, followed by two additional intramuscular booster immunizations in incomplete Freunds adjuvant at a 15-day interval. Animals from control group (N=14), were only inoculated with complete and incomplete Freunds adjuvant given at same conditions of collagen type V group. Histological analysis of skins from animals and patients were done by Massons trichrome staining, and immunofluorescence method used to detect collagen fibers and interactions of types I, III and V collagen in the remodeling process. The analysis of animal skins showed precocious collagen fibril deposits in the dermis after 7 days of immunization and increase of this process in 75 and 120 days. Skin collagen deposit and atrophy of annexes were progressively more intense in late sacrificed animals and correlated with increased amount of collagen deposition. Surprisingly, type V collagen was over expressed both in animals and patients, forming dense and atypical collagen fibers in the dermis. We suggest that this anomalous expression of morphologically different type V collagen could justify the remodeling observed in scleroderma plaque

Submitted by Cássia Santos (cassia.bcufg@gmail.com) on 2017-07-17T11:35:24Z No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-07-18T10:31:42Z (GMT) No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-07-18T10:31:42Z (GMT). No. of bitstreams: 2 Tese - Daniel Silva Goulart -2017.pdf: 3553497 bytes, checksum: 6ec13ae3cfbad63a4fdfdcf4e9b0dab1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-03-02 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The genus Brachiaria has provided important forage species to the countries located in tropical regions. However, this genus of grass is associated with photosensitization and poisoning in animal production. Thus, the aim of this study was to establish the standard for collection, processing, and conservation of Brachiaria brizantha for protodioscin quantification and spore count from Pithomyces chartarum and the evaluation of guinea pig (Cavia porcellus) as an experimental model of poisoning by diosgenin. For Brachiaria brizantha evaluation, samples were collected from eight pastures, which were subdivided into young leaves, mature leaves, old leaves, and whole plant. After that, the samples were submitted to nine different drying and conservation treatments. After separation and treatment of the samples, saponins were quantified and spores were counted. In most of treatments, the young leaves presented larger amounts of protodioscin than the old leaves and the whole plant. The treatments that maintained the highest amounts of protodioscin were room temperature, oven, and oven with forced air circulation. No fungal spores were found in the pasture samples. Thus, we concluded that the concentration of protodioscin varies with the maturation stage of the leaves; drying at room temperature, in an oven, or in an oven with forced air circulation are the best methods, and freezing plants for preservation of protodioscin is not recommended. In the guinea pigs evaluation, 14 guinea pigs were divided into two groups, the treatment group (TG) and the control group (CG). The TG received 480 mg/kg of diosgenin and the CG received only vegetable oil. Both groups received the treatment for 30 days. Blood samples were collected on day 0 (M0) and day 30 (M30) for blood cell count and biochemical analyzes. In clinical biochemistry, the analyzes were performed for alanine aminotransferase, aspartate aminotransferase, gamma glutamyl trasferase, alkaline phosphatase, urea, creatinine, glucose, cholesterol, HDL, triglycerides, lactate, cholinesterase, and calcium. Hematology and biochemistry did not present alterations, which could not be related to hepatic disease caused by saponins. In histology, only one animal had bile duct hyperplasia, the other alterations observed were generally nonspecific. Therefore, we concluded that the administration of 480 mg/kg of diosgenin to guinea pigs did not produce significant hepatic lesions or hematological and biochemical alterations related to liver disease. / O gênero Brachiaria possui importantes espécies de forrageiras utilizadas em países localizados em regiões tropicais. No entanto, esse gênero de gramíneas está associado à fotossensibilização e intoxicações em animais de produção. Deste modo, objetivou-se com este estudo estabelecer o padrão para colheita, processamento e conservação de amostras de Brachiaria brizantha para quantificação de protodioscina e contagem de esporos de Pithomyces chartarum e a avaliação da Cavia porcellus (cobaias) como modelo experimental na intoxicação por diosgenina. Para a avaliação da Brachiaria brizantha, amostras foram colhidas de oito pastagens as quais foram subdivididas em folhas jovens, folhas maduras, folhas velhas e planta inteira e submetidas a nove diferentes métodos de processamento. Após separação e tratamento das amostras foi realizada a quantificação de saponinas e a contagem de esporos. Observou-se que na maioria dos tratamentos as folhas jovens apresentaram quantidades maiores de protodioscina que as folhas velhas e a planta inteira. Os tratamentos que mantiveram as maiores quantidades de protodioscina foram temperatura ambiente, estufa e estufa com ventilação forçada. Nas amostras das pastagens não foram encontrados esporos do fungo. Desta forma, conclui-se que a concentração de protodioscina é maior nas folhas jovens; a secagem em temperatura ambiente, em estufa ou em estufa de ventilação forçada são os melhores métodos para preservação de protodioscina e o congelamento de plantas para a quantificação de protodioscina não é recomendado. Para a avaliação das cobaias como modelos experimentais na intoxicação por diosgenina foram utilizadas 14 cobaias, divididas em dois grupos, o grupo tratado (GT) e o grupo controle (GC). O GT recebeu 480 mg/kg de diosgenina diluída em óleo vegetal e o GC recebeu somente o óleo vegetal, ambos por 30 dias. Para avaliação do hemograma e análises bioquímicas foram colhidas amostras sanguíneas no dia 0 (M0) e no dia 30 (M30). Na bioquímica plasmática foram realizadas análises para ALT, AST, GGT, ALP, ureia, creatinina, glicose, colesterol, HDL, triglicerídeos, lactato, colinesterase e cálcio. A hematologia e a bioquímica não apresentaram alterações relacionadas à doença hepática causada por saponinas. Na histologia apenas um animal apresentou hiperplasia de ductos biliares, as outras alterações observadas foram de forma geral inespecíficas. Conclui-se que, na dose utilizada, a administração da diosgenina a cobaias não produz lesões hepáticas expressivas nem alterações hematológicas e bioquímicas relacionadas a hepatopatia.

Le chat est la seule espèce animale qui développe spontanément une entité clinique similaire à bien des égards à lasthme allergique humain. Communément appelée « asthme félin » par homologie à la maladie humaine, cette entité pathologique est le résultat dune inflammation persistante des voies respiratoires, associée à des phases aiguës de bronchospasme, une hyperréactivité bronchique à des stimuli divers et dans les stades avancés à des remaniements tissulaires de la paroi bronchique (e.g., érosions épithéliales, hypertrophie de la musculature lisse, hyperplasie glandulaire). Des modèles dasthme félin, obtenus par sensibilisation expérimentale à un allergène, reproduisent la majorité des caractéristiques cliniques, fonctionnelles et lésionnelles de la maladie naturelle et sont au premier plan de la découverte de nouvelles stratégies thérapeutiques (i.e., cyclosporine A, corticostéroïdes oraux et inhalés, immunothérapie spécifique). De la pathogénie de lasthme, ressort toute limportance de dominer lobstruction récurrente des voies respiratoires. Même si les bronchodilatateurs inhalés sont recommandés dans la prise en charge des crises dasthme chez le chat, leur efficacité clinique reste incertaine et peu étayée. Ainsi, les principes actifs, les posologies et les protocoles thérapeutiques actuellement préconisés sont dans une large mesure empiriques et/ou extrapolés de la médecine humaine, et nécessitent dêtre validés dans des conditions expérimentales contrôlées. Nos travaux sinscrivent donc dans cette optique et visent à définir une approche thérapeutique tant préventive que symptomatique des crises de bronchospasme chez le chat, ainsi quune utilisation rationnelle des bronchodilatateurs à longue durée daction pour un contrôle optimal de linflammation asthmatique. Lors dune première étude, nous avons investigué lactivité in vitro, vis-à-vis du muscle lisse bronchique félin, de différentes agents bronchodilatateurs parmi lesquels des agonistes β2-adrénergiques (isoprotérénol, salbutamol [SAL], fénotérol, formotérol, salmétérol [SLM]), un anticholinergique (ipratropium bromide [IB]) et une méthylxanthine (théophylline). Tous ont produit une relaxation dose-dépendante de la musculature lisse bronchique, avec des spécificités propres en termes de puissance, defficacité ou dactivité intrinsèque. Sur une base comparative, le formotérol est le ß2-mimétique le plus puissant, et lisoprotérénol le plus efficace avec le fénotérol. Le fénotérol et le formotérol sont des agonistes complets des récepteurs ß2-adrénergiques, le SAL et le SLM des agonistes partiels. Tenant compte des résultats des essais in vitro et de la disponibilité de formulations administrables par aérosols dans lespèce féline, les effets bronchoprotecteurs (intensité, durée daction) de six médications inhalées ont été caractérisés chez le chat sain, en préambule à leur utilisation dans des conditions pathologiques. Un modèle de bronchoconstriction induite par le carbachol a été retenu dans le cadre de cette deuxième étude. Il ressort de cette étude que le SLM en aérosol-doseur (25µg) présente un effet bronchoprotecteur soutenu persistant 24 heures mais est aussi la médication la moins efficace. Le SAL et lIB (nébulisation ou aérosol-doseur) sont des bronchodilatateurs à courte durée daction (48 heures) dont lutilisation combinée en aérosol-doseur (SAL IB : 100µg/20µg) met en exergue une synergie daction. Les conditions naturelles étant souvent incompatibles avec une approche anticipative de la crise dasthme, les traitements instaurés sont davantage à visée curative que prophylactique. Dans une troisième étude, nous avons exploré les effets bronchorelaxants du SAL (100µg) et de lIB (20µg), administrés seuls ou en traitement combiné, sur une bronchoconstriction induite par un aéroallergène (Ascaris suum) chez des chats rendus expérimentalement asthmatiques. La technique dinhalation par aérosol-doseur a été privilégiée vu les effets synergiques de la combinaison « SAL IB » et lintérêt de cette voie dadministration dans la gestion à domicile des crises dasthme. Aux doses testées, les bronchodilatateurs inhalés nont exercé aucun effet notable, naffectant en rien la résolution du bronchospasme induit. Chez lhomme, les crises et les épisodes dexacerbation de lasthme sont fréquents dans le cours évolutif de la maladie. Aucune étude chez le chat asthmatique en crise aiguë (induite ou spontanée) natteste de lefficacité des corticostéroïdes inhalés ni ne mentionne dans ce contexte lintérêt scientifique dune association aux ß2-mimétiques à longue durée daction. Nous avons donc comparé les effets de la prednisolone orale (1mg/kg q12h) à ceux dune dose élevée de fluticasone inhalée, seule (500µg q12h aérosol-doseur) ou en traitement combiné avec du SLM (500µg/50µg q12h aérosol-doseur), sur la fonction et linflammation pulmonaires dans un modèle félin dasthme aigu. Cette quatrième étude a montré quune courte cure orale de prednisolone diminue significativement linflammation bronchique à éosinophiles. De fortes doses de fluticasone inhalée se sont avérées bénéfiques dans le contrôle de lhyperréactivité bronchique non spécifique, sans toutefois exercer des effets marqués sur linflammation bronchique sous-jacente. Combiner cette corticothérapie inhalée au SLM a permis dobtenir des effets anti-éosinophiliques accrus et comparables à ceux de la prednisolone orale. Aucune des médications testées na modifié significativement la réponse clinique et fonctionnelle consécutive à linhalation de lallergène. Létude précédente permet de supposer que le SLM potentialise lactivité anti-inflammatoire des corticostéroïdes inhalés et/ou est doté de propriétés anti-inflammatoires intrinsèques. Dans une cinquième étude, nous avons donc investigué les effets du SLM en monothérapie (50µg q12h aérosol-doseur) sur la fonction et linflammation pulmonaires. Il ressort de cette étude, conduite sur un modèle félin dasthme aigu, que le SLM en monothérapie nexerce pas deffet protecteur sur la survenue dun bronchospasme allergique et est dépourvu de tout effet anti-inflammatoire propre. Les résultats obtenus, dans les conditions expérimentales de nos essais, conduisent aux conclusions suivantes : 1) les bronchodilatateurs inhalés (SAL, IB, SLM) sont dune efficacité thérapeutique limitée dans la prévention et le traitement symptomatique du bronchospasme allergique chez le chat sensibilisé à Ascaris suum 2) en revanche, recourir au SLM comme thérapie adjuvante aux corticostéroïdes inhalés ouvre de nouvelles perspectives thérapeutiques dans le contrôle de linflammation et de lhyperréactivité bronchiques chez le chat asthmatique./The cat is the only animal species that spontaneously develops a clinical entity closely similar to human allergic asthma and commonly referred to as feline asthma. Feline asthma is a chronic inflammatory disease of the lower airways characterised by intermittent respiratory distress due to bronchoconstriction, non-specific bronchial hyperresponsiveness and airway remodeling at latter stages (e.g., epithelial erosions, smooth muscle hypertrophy, glandular hyperplasia). Based on experimental sensitisation to allergens, models of feline asthma mimic many clinical, functional and lesional features of the naturally developing condition. Moreover, development and implementation of feline asthma models have greatly facilitated the search for novel therapies (i.e., cyclosporin A, oral and inhaled corticosteroids, specific immunotherapy). In considering the pathogenesis of asthma, it is of major importance to control recurrent airway obstruction. Although inhaled bronchodilators are recommended for the management of acute asthmatic exacerbations in cats, their clinical efficacy remains uncertain and poorly documented. Most currently recommended drugs, dosages and therapeutic schemes are largely empirical and extrapolated from human medicine, and need to be further validated under controlled experimental conditions. Hence, our work was aimed at determining the potential of inhaled bronchodilators for preventive and curative treatment of acute bronchospasms in asthmatic cats as well as at exploring the benefit of using long-acting bronchodilators to optimally control the airway inflammation. In the first study, we investigated in vitro effects on isolated feline bronchi of different bronchodilating agents including β2-adrenergic agonists (isoproterenol, salbutamol [SAL], fenoterol, formoterol, salmeterol [SLM]), an anticholinergic (ipratropium bromide [IB]) and a methylxanthin derivative (theophylline). All compounds caused a dose-related relaxation of bronchial smooth muscle, each exhibiting specificities in terms of potency, efficacy and intrinsic activity. On a comparative basis, isoproterenol and fenoterol are the most efficacious β2-mimetics while formoterol is the most potent one. Fenoterol and formoterol act as full agonists of β2-adrenoceptors, SAL and SLM as partial agonists. Taking into account results from in vitro testing procedures and availability of aerosol formulations suitable for lung delivery in cats, we compared the antispasmodic effects (magnitude, duration of action) of six inhaled medications against carbachol-induced bronchoconstriction in healthy cats. This second study showed that SLM by metered-dose inhaler (25µg) has a sustained activity for as long as 24 hours but is also the least efficacious medication. SAL and IB (nebulisation or metered-dose inhaler) are short-acting bronchodilators (4-8 hours) whose combination delivered with a metered-dose inhaler (SAL IB: 100µg/20µg) exhibits a synergistic antispasmodic effect. In the natural disease, asthmatic crisis are difficult to predict in terms of occurrence. Therefore, therapeutic interventions are more curative than preventive. In a third study, we explored the bronchodilating effects of SAL (100µg) and IB (20µg), delivered either alone or as a combined therapy, on allergen-induced bronchospasms in Ascaris suum-sensitised cats. The inhalation technique using a metered-dose inhaler was retained given the synergistic effects of the combination SAL IB with this method and its therapeutic interest for at-home management of asthmatic crisis. At the tested doses, these bronchodilators failed to reverse allergen-induced bronchospasms in cats with experimental asthma. In human beings, crisis and exacerbations of asthma are frequent events in the natural course of the disease. Efficacy of inhaled corticosteroids in cats with acute asthmatic exacerbations (induced or spontaneous) has not yet been determined, nor the potential benefit of adding long-acting β2-agonists in this context. Thus, the fourth study was aimed at comparing the effects of oral prednisolone (1mg/kg q12h) with those of inhaled fluticasone at high doses, alone (500µg q12h metered-dose inhaler) or combined with salmeterol (500µg/50µg q12h metered-dose inhaler), on lung function and airway inflammation in a feline model of acute asthma. This study showed that a short course of oral prednisolone significantly reduced allergen-induced bronchial eosinophilic inflammation. High doses of inhaled fluticasone proved to be efficient for decreasing non specific airway hyperresponsiveness but failed to markedly reduce the underlying airway inflammation. Adding salmeterol to inhaled fluticasone led to anti-eosinophilic effects of the same magnitude as those found for oral prednisolone. None of these treatments improved clinical and functional responses to allergen exposure. According to the previous study, it may be that SLM has an anti-inflammatory effect on its own and/or functions as a steroid-potentiating agent. In a fifth study, we investigated the effects of salmeterol as monotherapy (50 µg q12h metered-dose inhaler) on lung function and airway inflammation in our feline model of acute asthma. This inhaled medication did not prevent occurrence of allergic bronchospasm in Ascaris suum-sensitised and challenged cats, nor did it possess intrinsic anti-inflammatory activity. Under our experimental conditions, the main results achieved led to the following conclusions: 1) the inhaled bronchodilators (SAL, IB, SLM) are of limited efficacy for the prevention and symptomatic treatment of allergic bronchospasm in cats sensitised to Ascaris suum; 2) in contrast, using salmeterol as adjuvant therapy to inhaled corticosteroids opens up new perspectives for the treatment of bronchial inflammation and hyperresponsiveness in asthmatic cats.

Atrial fibrillation is the most prevalent form of cardiac arrhythmia. Studies in animal modelshave provided important insights into arrhythmia mechanisms. However, to date, we do not dispose ofanimal models of spontaneous atrial arrhythmia.Thus, we aimed to develop a model of spontaneous atrial arrhythmia in rats and to assesspathophysiological mechanisms of these arrhythmias by using a multidisciplinary approach. We alsoaimed to assess the presence and the extent of inflammation and endothelial dysfunction, incriminatedin atrial fibrillation-related complications such as stroke, in atrial fibrillation patients.The animal study describes the first animal model of spontaneous atrial arrhythmias. We alsoprovide evidence that multiple mechanisms participate in arrhythmia occurrence in this model,particularly autonomic imbalance with relative vagal hyperactivity, left atrial endocardial fibrosis, anddecreased left atrial expression of the Pitx2 gene. In our clinical study, we found high levels ofvascular endothelial growth factor and von Willebrand factor in atrial fibrillation patients compared tosinus rhythm controls. These results suggest specific thromboembolic risk patterns according to theclinical form of arrhythmia and highlight a parallel evolution of atrial fibrillation and endothelialdysfunction. These results add new insights into the understanding of atrial arrhythmias. This new animalmodel could facilitate studies of pathophysiological mechanisms involved in atrial arrhythmias andallow assessment of efficacy and toxicity of therapeutic agents in a setting that faithfully reproducesthe clinical presentation of the arrhythmia

Introdução: Apesar dos avanços significativos no tratamento endovascular das doenças arteriais coronarianas e periféricas, a reestenose intra-stent continua sendo o principal limitante a médio prazo desses procedimentos. O mecanismo da reestenose intra-stent é principalmente a hiperplasia intimal, já que o stent impede a retração elástica aguda e resiste ao remodelamento geométrico negativo tardio. A hiperplasia intimal ocorre basicamente em resposta à formação de trombo local, à inflamação e às dissecções intimais e mediais secundárias à injúria causada pelo stent, sendo o grau de resposta intimal a base dos efeitos a longo prazo. O uso de stents com hastes menores e revestidos com drogas ou polímeros tem sido considerado uma nova alternativa para a prevenção da reestenose intra-stent. Objetivo: Analisar a resposta arterial ao implante de stent de cromo-cobalto sem e com revestimento de polímero Camouflage® em artérias carótidas de suínos, utilizando os seguintes parâmetros histológicos: grau de endotelização, conteúdo de células musculares lisas, grau de angiogênese, conteúdo de fibrina, grau de inflamação e injúria; além da análise histomorfométrica. Método: Stents balões-expansíveis de cromo-cobalto ( 8 stents CC Flex e 5 stents CC Flex Proactive) de 4 x 16 mm foram implantados em artérias carótidas comuns de oito suínos jovens, sendo um stent liberado em cada artéria. Após 30 dias, as artérias contendo os stents foram removidas, fixadas e coradas pelos métodos de hematoxilina/eosina e Verhoeff/Van Giesson. O segmento arterial contendo o stent foi dividido em 3 blocos distintos: proximal, médio e distal. Os cortes histológicos foram obtidos utilizando-se micrótomo de impacto (Polycut S, Leica, Alemanha) equipado com navalha de tungstênio de 16 cm, tipo D (Leica, Alemanha), com 5 ^m de espessura. A navalha de tungstênio mantém as hastes dos stents intactas nas secções transversas, minimizando os artefatos potenciais causados pela retirada dos stents. A avaliação foi realizada através de critérios histológicos e histomorfométricos. Resultados: Todos os stents foram implantados com sucesso e sem dificuldades técnicas. A análise histológica em 30 dias evidenciou alto grau de endotelização em todos os segmentos avaliados e leve à moderada infiltração de células musculares na íntima. Observou-se baixo grau de angiogênese em cerca de 50% dos segmentos avaliados e ausência completa de deposição de fibrina em pelo menos 80%, com distribuição semelhante entre os grupos. A resposta inflamatória e o grau de injúria causadas pelas hastes dos stents também foram discretas e similares entre os grupos e não houve correlação entre resposta inflamatória e injúria e desses parâmetros com a área de neoíntima. O grau de obstrução neo-intimal identificada neste período foi pequeno (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive) e estatisticamente não significativo entre os grupos (p=0,785). Conclusão: Os achados deste estudo experimental sugerem que o uso de stents de cromo-cobalto revestidos com polímero Camouflage® em artérias carótidas de suínos parece estar associado, pelo menos no curto prazo, a uma resposta histológica semelhante àquela encontrada após o implante de stents de cromo-cobalto não revestidos. Neste período não se observou uma menor hiperplasia intimal em virtude do revestimento de polímero. / Introduction: Despite all the advances in the endovascular treatment of coronary and peripheral artery diseases, in-stent restenosis is still the main limiting factor of these procedures in the medium and long-term. The mechanism of in-stent restenosis is mainly the intimal hyperplasia, as the stent prevents acute elastic recoil and later negative geometric arterial remodeling. Intimal hyperplasia occurs basically in response to the formation of local thrombus, inflammation and intimal and medial dissections secondary to the injury caused by the stent, with the degree of intimal response being the cause of long-term effects. Coating drug-eluting stents with polymers and drugs with thinner struts have been considered a new alternative for in-stent restenosis prevention. Objective: Analyse the arterial response to the cobalt-chromium stent implant with and without polymer coating Camouflage® in carotid arteries of pigs, using the following histological parameters: degree of endothelialization, smooth muscle cells (SMC) content, degree of angiogenesis, intimal fibrin content, degree of inflammation and injury; plus histomorphometric analysis. Method: Cobaltchromium balloon-expandable stents (8 CC Flex stents and 5 CC Flex Proactive), 4 x 16 mm, were deployed in common carotid arteries of 8 young pigs, with one stent being deployed in each artery. After 30 days, the arteries containing the stents were removed and underwent fixation and staining using the hematoxilin/eosin and Verhoeff /Van Giesson methods. The arterial segment containing the stent was divided into 3 distinct portions: proximal, middle and distal. The histological sections were obtained using impact microtome (Polycut S, Leica, Germany), equipped with a 16 cm, type D, 5 ^m thick tungsten knife (Leica, Germany). The tungsten knife maintains the stent shaft intact in cross sections, minimizing the potential artifacts caused by stent removal. The evaluation was carried out using histological and histomorfometric criteria. Results: All the stents were deployed with success and with no technical difficulties. The histological analysis performed after 30 days showed a high level of endothelialization in all the evaluated portions and mild to moderate infiltration of the SMC in the intima layer. A low level of angiogenesis of about 50% of the evaluated portions was observed and a complete absence of fibrin deposition in at least 80% of the portions, with similar distribution among the groups. The inflammatory response and the level of injury caused by the struts of the stents were also minimum and this was similar among the groups. There was no correlation between inflammatory response and injury and between the two latter parameters and the neo-intima area. The level of neo-intimal obstruction identified in this period was small (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive ) and no statistical significance between the groups (p=0,785). Conclusion: The findings of this experimental study suggest the use of balloonexpandable cobalt-chromium stents coated with polymer Camouflage® in carotid arteries of pigs seems to be associated, at least in the short-term, with a similar histological response to that found in the implantation of non-coated cobalt-chromium stents. In this period, a lower intimal hyperplasia was not observed with polymer coating stents.

Introdução: Apesar dos avanços significativos no tratamento endovascular das doenças arteriais coronarianas e periféricas, a reestenose intra-stent continua sendo o principal limitante a médio prazo desses procedimentos. O mecanismo da reestenose intra-stent é principalmente a hiperplasia intimal, já que o stent impede a retração elástica aguda e resiste ao remodelamento geométrico negativo tardio. A hiperplasia intimal ocorre basicamente em resposta à formação de trombo local, à inflamação e às dissecções intimais e mediais secundárias à injúria causada pelo stent, sendo o grau de resposta intimal a base dos efeitos a longo prazo. O uso de stents com hastes menores e revestidos com drogas ou polímeros tem sido considerado uma nova alternativa para a prevenção da reestenose intra-stent. Objetivo: Analisar a resposta arterial ao implante de stent de cromo-cobalto sem e com revestimento de polímero Camouflage® em artérias carótidas de suínos, utilizando os seguintes parâmetros histológicos: grau de endotelização, conteúdo de células musculares lisas, grau de angiogênese, conteúdo de fibrina, grau de inflamação e injúria; além da análise histomorfométrica. Método: Stents balões-expansíveis de cromo-cobalto ( 8 stents CC Flex e 5 stents CC Flex Proactive) de 4 x 16 mm foram implantados em artérias carótidas comuns de oito suínos jovens, sendo um stent liberado em cada artéria. Após 30 dias, as artérias contendo os stents foram removidas, fixadas e coradas pelos métodos de hematoxilina/eosina e Verhoeff/Van Giesson. O segmento arterial contendo o stent foi dividido em 3 blocos distintos: proximal, médio e distal. Os cortes histológicos foram obtidos utilizando-se micrótomo de impacto (Polycut S, Leica, Alemanha) equipado com navalha de tungstênio de 16 cm, tipo D (Leica, Alemanha), com 5 ^m de espessura. A navalha de tungstênio mantém as hastes dos stents intactas nas secções transversas, minimizando os artefatos potenciais causados pela retirada dos stents. A avaliação foi realizada através de critérios histológicos e histomorfométricos. Resultados: Todos os stents foram implantados com sucesso e sem dificuldades técnicas. A análise histológica em 30 dias evidenciou alto grau de endotelização em todos os segmentos avaliados e leve à moderada infiltração de células musculares na íntima. Observou-se baixo grau de angiogênese em cerca de 50% dos segmentos avaliados e ausência completa de deposição de fibrina em pelo menos 80%, com distribuição semelhante entre os grupos. A resposta inflamatória e o grau de injúria causadas pelas hastes dos stents também foram discretas e similares entre os grupos e não houve correlação entre resposta inflamatória e injúria e desses parâmetros com a área de neoíntima. O grau de obstrução neo-intimal identificada neste período foi pequeno (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive) e estatisticamente não significativo entre os grupos (p=0,785). Conclusão: Os achados deste estudo experimental sugerem que o uso de stents de cromo-cobalto revestidos com polímero Camouflage® em artérias carótidas de suínos parece estar associado, pelo menos no curto prazo, a uma resposta histológica semelhante àquela encontrada após o implante de stents de cromo-cobalto não revestidos. Neste período não se observou uma menor hiperplasia intimal em virtude do revestimento de polímero. / Introduction: Despite all the advances in the endovascular treatment of coronary and peripheral artery diseases, in-stent restenosis is still the main limiting factor of these procedures in the medium and long-term. The mechanism of in-stent restenosis is mainly the intimal hyperplasia, as the stent prevents acute elastic recoil and later negative geometric arterial remodeling. Intimal hyperplasia occurs basically in response to the formation of local thrombus, inflammation and intimal and medial dissections secondary to the injury caused by the stent, with the degree of intimal response being the cause of long-term effects. Coating drug-eluting stents with polymers and drugs with thinner struts have been considered a new alternative for in-stent restenosis prevention. Objective: Analyse the arterial response to the cobalt-chromium stent implant with and without polymer coating Camouflage® in carotid arteries of pigs, using the following histological parameters: degree of endothelialization, smooth muscle cells (SMC) content, degree of angiogenesis, intimal fibrin content, degree of inflammation and injury; plus histomorphometric analysis. Method: Cobaltchromium balloon-expandable stents (8 CC Flex stents and 5 CC Flex Proactive), 4 x 16 mm, were deployed in common carotid arteries of 8 young pigs, with one stent being deployed in each artery. After 30 days, the arteries containing the stents were removed and underwent fixation and staining using the hematoxilin/eosin and Verhoeff /Van Giesson methods. The arterial segment containing the stent was divided into 3 distinct portions: proximal, middle and distal. The histological sections were obtained using impact microtome (Polycut S, Leica, Germany), equipped with a 16 cm, type D, 5 ^m thick tungsten knife (Leica, Germany). The tungsten knife maintains the stent shaft intact in cross sections, minimizing the potential artifacts caused by stent removal. The evaluation was carried out using histological and histomorfometric criteria. Results: All the stents were deployed with success and with no technical difficulties. The histological analysis performed after 30 days showed a high level of endothelialization in all the evaluated portions and mild to moderate infiltration of the SMC in the intima layer. A low level of angiogenesis of about 50% of the evaluated portions was observed and a complete absence of fibrin deposition in at least 80% of the portions, with similar distribution among the groups. The inflammatory response and the level of injury caused by the struts of the stents were also minimum and this was similar among the groups. There was no correlation between inflammatory response and injury and between the two latter parameters and the neo-intima area. The level of neo-intimal obstruction identified in this period was small (15,1% +/- 8,38 CC Flex x 15,5%+/- 5,39 CC Flex ProActive ) and no statistical significance between the groups (p=0,785). Conclusion: The findings of this experimental study suggest the use of balloonexpandable cobalt-chromium stents coated with polymer Camouflage® in carotid arteries of pigs seems to be associated, at least in the short-term, with a similar histological response to that found in the implantation of non-coated cobalt-chromium stents. In this period, a lower intimal hyperplasia was not observed with polymer coating stents.

Orientador: Lourenço Sbragia Neto / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-12T11:31:54Z (GMT). No. of bitstreams: 1 Nassr_AzizeCristinaCapelli_D.pdf: 6642584 bytes, checksum: 60fb43046c34664559a19ebb364b282b (MD5) Previous issue date: 2008 / Resumo: A Hérnia Diafragmática Congênita (HDC) é um defeito da formação do músculo diafragma que incide em aproximadamente 1:2500 nascidos vivos e apresenta altos índices de mortalidade fetal e neonatal decorrentes da hipoplasia e da hipertensão pulmonares. Este defeito pode ser induzido experimentalmente em ratas grávidas administrando o herbicida nitrofen que causa HDC em 24% dos fetos. A análise microscópica do pulmão da HDC demonstra a presença de hipolasia pulmonar além de alveolização e vascularização alterada. Um dos fatores de crescimento envolvidos no desenvolvimento vascular é o VEGF (vascular endothelial growth factor) e seus receptores, no entanto ainda não se conhece como a expressão desta glicoproteína e de seus receptores varia ao longo do desenvolvimento pulmonar fetal nesta doença. Utilizando o modelo experimental de HDC induzido pelo nitrofen (2,4-dicloro-4'nitrodifenil éter) investigamos o grau de hipoplasia pulmonar e por meio de análise imunoistoquímica, comparamos a expressão dos receptores para o VEGF em três fases do desenvolvimento pulmonar, pseudoglandular, canalicular e sacular de fetos de ratos normais e com HDC. Dividimos o experimento em ratas da raça Sprague-Dawley em três grupos: controle externo (CE), exposto ao óleo de oliva (OO) e expostas ao nitrofen com e sem HDC. Estudamos quatro grupos de 20 fetos cada em cinco dias gestacionais (DG) diferentes 17,5, 18,5, 19,5, 20,5 e 21,5. As variáveis morfológicas estudadas foram: peso corporal (PC), peso pulmonar total (PPT), peso do pulmão esquerdo (PPE), relação PPT/PC, volume pulmonar total (VPT) e volume do pulmão esquerdo (VPE). As variáveis histométricas estudadas foram: parênquima pulmonar (Par), espaço aéreo (EA), densidade do parênquima (DAP) e volume do parênquima do pulmão esquerdo. A avaliação imumohistoquímica foi realizada por meio da contagem de pontos de receptor de VEGFR-1 e 2. Obtivemos 37 % (100/270) de HDC nas ratas expostas ao nitrofen, todas variáveis morfológicas e histométricas indicam diminuição dos resultados no grupo nitrofen com e sem HDC em relação aos demais, mas que se acentuam mais ainda no grupo HDC. Essas alterações são mais evidentes a partir dos DG 18,5 e 19,5. A imunomarcação para os receptores VEGFR-1 aumentou nos grupos nitrofen e foram progressivamente maiores no grupo nitrofen com HDC (p<0,005) que os fetos dos grupo CE e OO a partir do dia gestacional 17,5, fase pseudoglandular com pico máximo no dia gestacional 19,5. O mesmo ocorreu com os receptores de VEGFR-2 a partir do dia gestacional 17,5, fase pseudoglandular até o dia 21,5 fase sacular do desenvolvimento pulmonar. Concluímos que o modelo é valido e que os fetos expostos ao nitrofen com e sem HDC apresentam hipoplasia pulmonar primária sendo mais acentuada nos fetos portadores de HDC. O mesmo resultado ocorre com imunomarcação para os receptores de VEGFR-1 e 2 que foram maiores na HDC. / Abstract: The Congenital Diaphragmatic Hernia (CDH) is a defect in the embryogenesis of the diaphragm with an incidence of 1:2500 liveborns and high fetal and neonatal mortality due to pulmonary hypoplasia and hypertension. This defect can be experimentally induced in fetuses of pregnant rats by the administration of Nitrofen, an herbicide that causes CDH in 24% of the fetuses. The histology of lungs in CDH shows pulmonary hipoplasia and not only the alveolarization but also the vascularization are affected. These changes lead to a high neonatal mortality because of the thickening of the middle layer of the arterioles causing pulmonary hypertension. One of the factors involved in the growth of the arterioles is VEGF (vascular endothelial growth factor) and its receptors; however, it is not known how the expression of this glycoprotein and its receptors change during lung development in this disease. In Brazil, the experimental model has never been tested. So, we tested the model and verified the degree of pulmonary hipoplasia and, using imunohystochemistry, we compared the expression of the receptor of VEGF in three different stages of lung development, pseudoglandular, canalicular and saccular, of normal rat fetuses and fetuses with CDH. Female Sprague-Dawley rats were divided in three groups: external control (EC), exposed to olive oil (OO) and exposed to nitrofen (N). We studied four groups - EC, OO, N with CDH and N without CDH - with 20 fetuses in each five different gestational days (GD) 17,5, 18,5, 19,5, 20,5, 21,5. The morphologic variables studied were: body weight (BW), total lung weight (TLW), left lung weight (LLW), relationship TLW/BW, total lung volume (TLV) and left lung volume (LLV). The hystometric variables studied were: lung parenchyma (LP), air space (AS), left lung parenchyma density (PD) and left lung parenchyma volume (PV). The immunohystochemistry variables were: points positive and negative for the receptor for VEGF 1 and 2. We had 37% (100/270) of CDH frequency in the fetuses exposed to nitrofen. All the morphological and hystometrical variables show a reduction in the nitrofen group with and without CDH, which were more pronounced in the group of fetuses with CDH. These changes are more evident from the GD 18,5 and 19,5 on. The receptors VEGFR-1 e 2 are increased in the nitrofen groups with and without CDH, but this increase is higher in the fetuses with CDH. We conclude that the model is valid and that the fetuses exposed to nitrofen with and without CDH show primary pulmonary hypoplasia that is more pronounced in CDH, the same is also observed in the receptors of VEGFR-1 and 2. / Doutorado / Pesquisa Experimental / Doutor em Cirurgia

Os Galea spixii são roedores que tem despertado o interesse de pesquisadores devido ao seu peculiar dimorfismo sexual, uma vez que, as fêmeas possuem a genitália externa com características masculinizadas. O objetivo da presente pesquisa foi estabelecer os parâmetros morfológicos do desenvolvimento e diferenciação sexual de machos e fêmeas durante o período intrauterino e as possíveis fontes de produção de hormônios andrógenos e estrógenos durante a gestação. Foram utilizados conceptos provenientes de 30 fêmeas gestantes. Foi descrita a concentração hormonal das gestantes. Em seguida, os órgãos genitais dos conceptos foram analisados macroscopicamente e microscopicamente. E, para detectar possíveis fontes de andrógenos e estrógenos, as placentas, ovários e testículos foram submetidos a técnicas imunológicas de detecção de enzimas esteroidogênicas. Aos 25 dias de gestação (DG) se inicia o processo de diferenciação sexual das gônadas (para formar os ovários ou os testículos) e da genitália externa. O tubérculo genital sofre a canalização da uretra aos 30 DG para formar o pênis nos machos e aos 40 DG para formar o clitóris das fêmeas. Nos machos, a partir dos ductos mesonéfricos, se diferenciam os ductos epididimários e os ductos deferentes. Nas fêmeas, os ductos paramesonéfricos formam as tubas e cornos uterinos, o útero parcialmente duplo e a porção cranial da vagina. A porção caudal se origina do seio urogenital e a membrana de oclusão vaginal, da membrana urogenital. Também ficou demonstrado que, a concentração de testosterona sofre grande aumento, dos 25 DG até o final da gestação e que o mesmo não ocorre com o estradiol. Os resultados para detecção de enzimas esteroidogênicas sugerem que, a placenta pode ser o órgão que atua na produção de hormônios andrógenos e pode não realizar a conversão desses hormônios em estrógenos, devido à ausência da enzima responsável por este processo. Por fim, os testículos e ovários também podem contribuir com a produção dos principais andrógenos e o ovário também possui a enzima necessária para a produção de estrógenos. / Galea spixii are rodents that have aroused the interest of researchers because of their peculiar sexual dimorphism, since females have the external genitalia with masculinized characteristics. The aim of the present research was to establish the morphological parameters of the development and sexual differentiation of males and females during the intrauterine period and the possible sources of androgen and estrogen hormones production during pregnancy. Concepts from 30 pregnant females were used. The hormonal concentrations of pregnant were described. Then, the genital organs of the concepts were analyzed macroscopically and microscopically. And, to detect possible sources of androgens and estrogens, placentas, ovaries and testes were subjected to immunological techniques for the detection of steroidogenic enzymes. At 25 days of gestation (DG) the process of sexual differentiation of the gonads (to form the ovaries or testicles) and the external genitalia begins. The genital tubercle undergoes channeling of the urethra at 30 DG to form the penis in males and at 40 DG to form the clitoris of females. In males, the epididymal ducts and the vas deferens differentiate from the mesonephric ducts. In females, the paramesonephric ducts form the uterine tubes and horns, the partially double uterus and the cranial portion of the vagina. The caudal portion originates from the urogenital sinus and the vaginal closure membrane originates from the urogenital membrane. It has also been shown that the testosterone concentration is greatly increased from 25 DG until the end of gestation and that the same does not occur with estradiol. The results for the detection of steroidogenic enzymes suggest that the placenta may be the organ that acts in the production of androgen hormones and may not perform the conversion of these hormones into estrogens due to the absence of the enzyme responsible for this process. Finally, the testicles and ovaries can also contribute to the production of the main androgens and the ovary also has the enzyme necessary for the production of estrogens.