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Universidade Federal Do Espírito Santo Centro De Ciências Da Saúde UNIVERSIDADE FEDERAL DO ESPÍRITO SANTO CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS FARMACÊUTICAS IGOR SIMÕES ASSUNÇÃO FELIPPE Avaliação in vitro e in vivo dos efeitos da intoxicação aguda com o inseticida organofosforado, clorpirifós, e da eficácia do tratamento farmacológico empregado na intoxicação sobre a modulação cardiorrespiratória tônica e reflexa. VITÓRIA 2017 IGOR SIMÕES ASSUNÇÃO FELIPPE Avaliação in vitro e in vivo dos efeitos da intoxicação aguda com o inseticida organofosforado, clorpirifós, e da eficácia do tratamento farmacológico empregado na intoxicação sobre a modulação cardiorrespiratória tônica e reflexa Dissertação apresentada ao Programa de Pós- Graduação em Ciências Farmacêuticas da Universidade Federal do Espírito Santo como requisito para obtenção do título de mestre em ciências farmacêuticas. Orientadora: Profa. Dra. Karla Nívea Sampaio. Co-Orientadora: Profa. Dra. Vanessa Beijamini Harres VITÓRIA 2017 Dados Internacionais de Catalogação-na-publicação (CIP) (Biblioteca Setorial do Centro de Ciências da Saúde da Universidade Federal do Espírito Santo, ES, Brasil) Felippe, Igor Simões Assunção, 1990 - F315a Avaliação in vitro e in vivo dos efeitos da intoxicação aguda com o inseticida organofosforado, clorpirifós, e da eficácia do tratamento farmacológico empregado na intoxicação sobre a modulação cardiorrespiratória tônica e reflexa / Igor Simões Assunção Felippe – 2017. 158 f. : il. Orientador: Karla Nívea Sampaio. Coorientador: Vanessa Beijamini Harres. Dissertação (Mestrado em Ciências Farmacêuticas) – Universidade Federal do Espírito Santo, Centro de Ciências da Saúde. 1. Compostos Organofosforados. 2. Clorpirifós. 3. Barorreflexo. 4. Atropina. 5. Compostos de Pralidoxima. I. Sampaio, Karla Nívea. II. Harres, Vanessa Beijamini. III. Universidade Federal do Espírito Santo. Centro de Ciências da Saúde. IV. Título. CDU: 61 Agradecimentos Primeiramente, gostaria de agradecer a Deus, pois foi ele quem muitas vezes me guiou e me mostrou o caminho, mesmo que por algum momento não tenha sido aquele que planejei; no final se mostrou o caminho correto. À minha mãe Eliane, aos meus avós José Nicomedes e Dalva, assim como toda minha família, por todo carinho e suporte ao longo de vários anos. Aos “migos lab” dos laboratórios de “Investigação Fármaco-Toxicológica da Função Cardiovascular” e de “Psicofarmacologia”. Agradeço também a todos os funcionários da UFES por todos esses anos e ajuda. Em especial ao Rodolfo e todos os funcionários do Biotério. Aos amigos Guto, Vitor, Wanderson, Xandão, Marcelo, Rovena, Susi, Yuri, Kainá e tantos outros. Gostaria de agradecer muito à professora Vanessa Beijamini Harres, por todos esses anos de ensinamento e amizade. Sem o auxílio da senhora eu não teria conseguido terminar essa dissertação. Além disso, de ter me passado um pouquinho do gosto e de seus conhecimentos sobre análise de dados e estatística. Por fim, gostaria de agradecer em especial à minha orientadora, Karla Nívea Sampaio por todos esses anos de paciência, amizade e conhecimento. Eu devo muito da minha vida profissional e acadêmica a senhora. Cada conselho e ensinamento ao longo desses anos eu guardo de coração para o resto da vida. Você sabe o carinho e o apreço que tenho pela senhora, que com certeza não é apenas minha orientadora, para mim a senhora é minha família. Muito obrigado!! SUMÁRIO SUMÁRIO.................................................................................................................... 6 LISTA DE FIGURAS ................................................................................................... 9 LISTA DE TABELAS, QUADROS E FÓRMULAS ..................................................... 15 LISTA DE ABREVIATURAS E/ OU SIGLAS ............................................................. 18 RESUMO................................................................................................................... 22 ABSTRACT ............................................................................................................... 25 1. INTRODUÇÃO ...................................................................................................... 27 1.1. DEFINIÇÃO ........................................................................................................ 27 1.2. MERCADO MUNDIAL E BRASILEIRO .............................................................. 28 1.3. COMPOSTOS ORGANOFOSFORADOS .......................................................... 30 1.3.1. História dos inseticidas organofosforados ....................................................... 30 1.3.2. Epidemiologia das intoxicações ...................................................................... 31 1.3.3. Química e toxicologia dos inseticidas organofosforados ................................. 32 1.3.4. Quadro clínico das intoxicações por organofosforados ................................... 34 1.4. ORGANOFOSFORADOS E SISTEMA CARDIOVASCULAR ............................ 35 1.5. REFLEXOS NEURAIS E O SISTEMA CARDIORESPIRATÓRIO ...................... 36 1.6. ORGANOSFOSFORADOS E OS REFLEXOS CARDIOVASCULARES ............................................................................................ 43 1.7. TRATAMENTO PADRÃO EM INTOXICAÇÕES POR ORGANOFOSFORADOS ......................................................................................... 44 1.8. CLORPIRIFÓS ................................................................................................... 46 2. OBJETIVOS .......................................................................................................... 50 3. MÉTODOS ............................................................................................................ 52 3.1. ANIMAIS ............................................................................................................. 52 3.2. PRODUTO QUÍMICO ......................................................................................... 52 3.3. DELINEAMENTO DO PROTOCOLO IN VITRO. ............................................... 53 3.4. PREPARAÇÃO TRONCO CEREBRAL-CORAÇÃO ISOLADO (WHBP) ..................................................................................................................... 54 3.4.1. Cirurgia e isolamento dos nervos .................................................................... 54 3.4.2. Protocolo experimental na WHBP ................................................................... 57 3.4.3. Atividades dos nervos frênico, laríngeo recorrente, simpático torácico e acoplamento simpato-respiratório ............................................................. 58 3.4.4. Quimiorreflexo ................................................................................................. 59 3.4.5. Barorreflexo ..................................................................................................... 60 3.4.6. Modulação da atividade simpática e parassimpática ...................................... 60 3.5. DELINEAMENTO DO MODELO ANIMAL IN VIVO ............................................ 62 3.5.1. Grupos de tratamento e doses adotadas: ....................................................... 62 3.6. ENSAIOS CARDIORESPIRATÓRIOS: .............................................................. 63 3.6.1. Canulação da artéria e veia femoral ................................................................ 63 3.6.2. Registro da pressão arterial e da frequência cardíaca .................................... 65 3.6.3. Avaliação do quimiorreflexo ............................................................................ 65 3.6.4. Análise da frequência respiratória (fR) ............................................................. 67 3.6.5. Análise do volume corrente e do volume minuto nos animais ......................... 67 3.7. DOSAGENS ENZIMÁTICAS .............................................................................. 69 3.7.1. Obtenção e processamento das amostras de plasma para dosagem da BuChE .................................................................................................. 69 3.7.2. Determinação da atividade da colinesterase plasmática (BuChE) .................. 69 3.7.3. Obtenção e processamento das amostras do tronco encefálico para dosagem da AChE cerebral .............................................................................. 70 3.7.5. Determinação da atividade da acetilcolinesterase cerebral (AChE) ...................................................................................................................... 72 3.7.3. Obtenção e processamento dos punchs de cortes do tronco encefálico contendo as amostras dos núcleos ambíguo, NTS e RVLM/preBötC. ......................................................................................................... 73 3.8. ANÁLISE DOS DADOS ...................................................................................... 76 3.8.1. Análise da atividade basal dos nervos frênico, laríngeo recorrente, acomplamento simpato-respiratório, atividade do barorreflexo, modulação simpática e parassimpática e atividade das enzimas BuChE e AChE na WHBP. .......................................................................... 76 3.8.2. Análise da atividade do quimiorreflexo na WHBP ........................................... 76 3.8.5. Análise dos parâmetros de avaliação da toxicidade aguda no modelo in vivo ........................................................................................................... 77 3.8.6. Análise dos valores das pressões arteriais médias
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