Este trabalho foi financiado em parte por uma concessão de desenvolvimento cientista da associação americana do coração (14SDG18160021 para YD), o US NIH R01 concede HL 81753 e HL 107304 para XX e a Fundação de Mayo para XX.

Todos os procedimentos descritos aqui foram realizados de acordo com o guia para o cuidado e o uso de animais de laboratório (academias nacionais imprensa. 2011), e foram aprovados pelo Comitê de uso e Mayo Clinic institucional Cuidado Animal.
1. adultos Zebrafish preparação
<ol><li>Configure o suficiente casais reprodutores cruzando tanques para adquirir pelo menos duas vezes como muitos como o peixe total necessário para a injeção de DOX. Se comparando peixes com diferentes origens genéticas, todos os peixes se reproduzem dentro da mesma semana para assegurar controles idade.</li>
	<li>Recolher os embriões de peixe na manhã seguinte, transferi-los para pratos de Petri de 100mm e mantê-los em uma incubadora de 28,5 ° C. Manter os embriões em uma densidade baixa (< 100 embriões/placa de Petri).</li>
	<li>Atualizar o diário da água do embrião para evitar desequilíbrios de sexo e remover manualmente os ovos mortos em tempo hábil com uma pipeta de transferência.</li>
	<li>Colocar o mesmo número de embriões em cada tanque (por exemplo, 60 embriões/3 L de meio tanque inicialmente) para assegurar a densidade-controles.</li>
	<li>Comece paramécios alimentando na fertilização de pós 4 dias (dpf).</li>
	<li>Inspecione o peixe diariamente durante a fase juvenil. Ajustar o número de peixes conforme necessário para garantir o peixe similar densidade.</li>
	<li>Quando o peixe atingir 4 semanas de idade, transferi até 20 peixes para cada novo tanque médio de 3L para subsequente crescimento. Começa a alimentar o peixe com artêmias. hachuradas ao vivo.</li>
</ol>2. preparação e armazenamento da solução-mãe DOX
Nota: DOX pode ser comprado de várias empresas-bio. O composto é geralmente adquirido como um pó em recipientes de marrons escuros.
<ol><li>Dissolva completamente o pó DOX em água deionizada para assegurar que não aglomerados são visíveis, com uma concentração final de 5 mg/mL como solução-mãe. Alíquotas de 1ml de estoque DOX em cada tubo de segurança-bloqueio de 1,5 mL. Envolva os tubos de 1,5 mL com papel da folha de alumínio para proteger o DOX da exposição à luz. Nota: Execute esta etapa em uma capa de química.</li>
	<li>Manter a solução estoque de DOX a 4 ° C para armazenamento. Para armazenamento a longo prazo (> 4 semanas) de solução-mãe de DOX, executar a seção opcional 3 descrito abaixo.</li>
</ol>3. controle de qualidade da DOX utilizando embriões de peixe-zebra (opcionais)
Nota: DOX é ambos e luz-sensíveis à umidade, assim ele pode perder sua eficácia de drogas para modelagem DIC após armazenamento prolongado. Para DOX comprado de empresas diferentes, ou mesmo diferentes lotes da mesma empresa, é útil calibrar sua eficácia de drogas usando embriões de zebrafish do selvagem-tipo (WT) antes de realizar os experimentos no peixe adulto. Este método é derivado de um zebrafish relatado embrionárias DIC modelo21.
<ol><li>Colete embriões de zebrafish WT de pelo menos 2 pares de peixes. Dechorionate de embriões na pós-fertilização 24h (hpf) manualmente usando uma seringa com uma agulha de micro. Alternativamente, trate o embrião com proteinase K em concentração final de 10 µ g/mL por 10-15 min em uma incubadora de 30 ° C. Atualize a água do embrião após a dechorionation. Remover os embriões mortos e manter pelo menos 36 embriões de cada lote.</li>
	<li>Dilua a solução-estoque DOX em água fresca embrião para uma concentração final de 100 µM. O volume da solução é de 100 µ l de cada 3 embriões. Misture a solução diluída de DOX pelo vórtice. A solução diluída final deve ser uma luz, a cor vermelha.</li>
	<li>Adicione 100 µ l/poço de solução diluída de DOX de uma placa de 96 poços limpa transparente.</li>
	<li>Leve 3 embriões de dechorionated com uma pipeta de transferência de plástico e mantenha os embriões perto do fim da ponta da pipeta. Colocar a ponta da pipeta para cada poço com solução DOX. Deixe a extremidade da ponta toque a solução e permitir que os embriões a nadar dentro do poço. Nota: Evite manualmente empurrando os embriões, que irão adicionar mais água no poço e diluir a solução DOX.</li>
	<li>Atualizar a solução DOX em 48 hpf. Por esta altura, observe os poços sob um microscópio com ampliação de 10x para identificar embriões mortos (cessação dos batimentos cardíacos) ou embriões com edema. Conte e remover qualquer embriões mortos em tempo hábil, caso contrário os restantes embriões expostos à solução com embriões mortos podem morrer rapidamente também.</li>
	<li>Verifique os embriões em 72 hpf e contá-los. O tratamento de DOX é considerado "eficácia boa droga" se > 25% morte (cessação dos batimentos cardíacos) pode ser observado em ambos os lotes de embriões.</li>
</ol>4. pré-injeção preparação
Nota: O peixe de 8 semanas a 6 meses de idade é utilizados para injeção de DOX. Os pesos de corpo (BWs) de um peixe selvagem Indian cariótipo (WIK) amadurecido para ser injetado podem variar de 0,2-0,5 g.
<ol><li>Rapidamente o peixe para 24 h antes da injeção.</li>
	<li>Anestesia o peixe com água do embrião que contém metanosulfonato de 0,16 mg/mL. Use um papel de filtro limpo para secar a água de ambos os lados do corpo. Medir o BW de cada peixe em escala. Grupo peixe dentro de 10% de diferença em BW juntos para posterior injeção. Nota: Para minimizar a carga de trabalho a este passo, peixe dentro de 10% de diferença em BW é considerados o mesmo tamanho; Portanto, prepare uma solução de trabalho de DOX, de acordo com sua média de BW.</li>
	<li>Plano para injetar cada peixe adulto com 5 µ l de solução. Calcule a concentração de trabalho DOX de acordo com o número de peixes e BWs. Nota: Para estudar a cardiotoxicidade crônica acima de 6 meses, use DOX na dose de 20 mg/kg. Para estudar a cardiotoxicidade aguda de DOX, a dose DOX pode ser aumentada até 50 mg/kg de BW.</li>
	<li>Dilua o estoque DOX em 1 x solução salina equilibrada de Hank (HBSS) para as concentrações de trabalho correspondente. Vórtice para misturar a solução. Brevemente spin para baixo, para recolher a solução.</li>
</ol>5. DOX injeção no peixe adulto
<ol><li>Lugar uma limpa 100 mm placa de Petri com uma esponja dentro dela, sob um microscópio de dissecação, então ajuste o foco. Corte a esponja para fazer uma cavidade de cerca de 4 cm de comprimento para segurar um peixe. Fazer uma cavidade mais para um peixe maior.</li>
	<li>Prepare uma agulha 34g com um microseringa de 10 µ l. Enxague a agulha com 1 x HBSS buffer para remover quaisquer bolhas e blocos dos tubos e de seringa.</li>
	<li>Anestesia o peixe adulto na água do embrião que contém metanosulfonato de 0,16 mg/mL por 2 min. Nota: Anesthetization prolongada mais de 5 min, seguido por injeção DOX pode facilmente causar peixes morte.</li>
	<li>Mergulhe a esponja na água de embrião com metanosulfonato e transfira o peixe sobre a esponja para injeção.</li>
	<li>
Injeção de IP DOX execute qualquer um dos dois métodos descritos abaixo.
	<ol>
<li>Clássico de injeção IP29<ol>
<li>Posicione o peixe com o abdômen acima na cavidade da esponja. Inserir a agulha, com uma 45° ângulo para o corpo do peixe em relação à linha mediana entre as nadadeiras pélvicas e penetrar cerca de 1-2 mm. libere rapidamente toda solução DOX lentamente. Aguardar 5 s antes de retirar a agulha. Verificar a entrega DOX, uma cor vermelha visível na barriga do peixe.</li>
		</ol>
</li>
		<li>Injeção de IP alternativa<ol>
<li>Coloque o peixe lateralmente sobre a esponja com o anterior para a direita. Delicadamente estabilizar o peixe usando um fórceps fim brusco com a mão esquerda e segure a seringa com a mão direita.</li>
			<li>Posicione a agulha abaixo da linha lateral, acima da nadadeira pélvica, com o bisel voltado para cima. Apontando para a posição de 07:00 em um ângulo de 45 °, introduza a agulha 3 a 4 mm para a cavidade de peixe, localizada entre a pélvica e as barbatanas anais e em seguida, pressione lentamente o êmbolo. Verificar a entrega DOX, uma cor vermelha visível na barriga do peixe.</li>
		</ol>
</li>
	</ol>
</li>
	<li>Rapidamente transferi os peixes injetados para um tanque limpo cruzamento cheio de água fresca do sistema para permitir que o peixe se recuperar. Enxague a agulha uma vez com 1 buffer de x HBSS entre as injeções.</li>
</ol>6. pós-injeção peixe gestão
<ol><li>Após a injeção, retorne o peixe para o sistema com circulação em execução. Se possível, manter o peixe DOX-tratados separadamente do sistema principal para evitar a contaminação cruzada entre tanques diferentes que compartilham a circulação.</li>
	<li>Rapidamente o peixe injetado para outro 24h para recuperação. Observe os peixes diariamente durante a primeira semana. Remova os peixes mortos em tempo hábil para evitar a infecção para os outros peixes. Nota: Mortes de peixes dentro as primeiras 24 horas são provavelmente devido a lesões físicas causadas por injeção.</li>
	<li>Mais manter o peixe DOX-estressado para observações longitudinais. Remova o peixe morto no tempo para evitar infecções de outros peixes do aquário. Nota: Os números de peixes estão documentados para gerar uma curva de sobrevivência.</li>
	<li>Usar diferentes ensaios experimentais de fenótipo o peixe DOX-forçado, tais como a Ecocardiografia30, função cardíaca repórter transgénicos linha23, natação desafio26e quantificação de outros remodelamento patológico marcadores de23.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+cardiomyopathy.">Doxorubicin cardiomyopathy.</a> Cardiology. 115 (2), 155-162 (2010).</li><li>Bang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+fibroblast-derived+microRNA+passenger+strand-enriched+exosomes+mediate+cardiomyocyte+hypertrophy.">Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy.</a> J Clin Invest. 124 (5), 2136-2146 (2014).</li><li>Rassaf, T., Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+diabetic+cardiomyopathy+-+putative+role+of+the+retinoid+receptor-mediated+signaling.">Protection from diabetic cardiomyopathy - putative role of the retinoid receptor-mediated signaling.</a> J Mol Cell Cardiol. 59, 179-180 (2013).</li><li>Wahbi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dilated+cardiomyopathy+in+patients+with+mutations+in+anoctamin+5.">Dilated cardiomyopathy in patients with mutations in anoctamin 5.</a> Int J Cardiol. 168 (1), 76-79 (2013).</li><li>Zhou, M. D., Sucov, H. M., Evans, R. M., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid-dependent+pathways+suppress+myocardial+cell+hypertrophy.">Retinoid-dependent pathways suppress myocardial cell hypertrophy.</a> Proc Natl Acad Sci U S A. 92 (16), 7391-7395 (1995).</li><li>Hershman, D. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin,+cardiac+risk+factors,+and+cardiac+toxicity+in+elderly+patients+with+diffuse+B-cell+non-Hodgkin's+lymphoma.">Doxorubicin, cardiac risk factors, and cardiac toxicity in elderly patients with diffuse B-cell non-Hodgkin's lymphoma.</a> J Clin Oncol. 26 (19), 3159-3165 (2008).</li><li>Silber, J. H., Barber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+cardiotoxicity.">Doxorubicin-induced cardiotoxicity.</a> N Engl J Med. 333 (20), 1359-1360 (1995).</li><li>Von Hoff, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risk+factors+for+doxorubicin-induced+congestive+heart+failure.">Risk factors for doxorubicin-induced congestive heart failure.</a> Ann Intern Med. 91 (5), 710-717 (1979).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injection+in+adult+zebrafish.">Retro-orbital injection in adult zebrafish.</a> J Vis Exp. (34), (2009).</li><li>Zang, L., Morikane, D., Shimada, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protocol+for+the+oral+administration+of+test+chemicals+to+adult+zebrafish.">A novel protocol for the oral administration of test chemicals to adult zebrafish.</a> Zebrafish. 8 (4), 203-210 (2011).</li><li>Collymore, C., Rasmussen, S., Tolwani, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gavaging+adult+zebrafish.">Gavaging adult zebrafish.</a> J Vis Exp. (78), (2013).</li></ol>

Os autores declaram não há conflitos de interesses.

Para modelar uma DIC progressiva, a dose de 20 mg/kg DOX foi determinada experimentalmente como a dose mais elevada que não causa morte de peixes significativo durante 1 wpi mas ainda resultados na morte de peixes e a redução da função cardíaca após 4 wpi (Figura 3 e Figura 4). Esta dose é comparável aos frequentemente usados em modelos de roedores DIC (15-25 mg/kg) e para a dose cumulativa de limite em humanos (550 mg/m2, que é equivalente...

Doxorrubicina (DOX), também chamada Adriamicina, foi desenvolvida como uma droga anti-neoplásicos desde a década de 1960,1,2. Agora é ainda ativamente utilizado como uma droga de quimioterápico importante para um amplo espectro de tumores. No entanto, aplicação clínica da DOX foi prejudicada por sua toxicidade dose-dependente, especialmente a cardiotoxicidade caracterizada por sintomas variáveis, variando de alterações eletrocardiográficas assintomáticas a pericardite e descompensada cardiomiopatia 1 , 2. até à data, pelo menos, três grandes hipóteses têm sido levantadas para explicar a DIC, incluindo registrados reativas de oxigênio (ROS) de espécies1,3,4,5, inibição da topoisomerase II-β ( TOP2β)6,7e modulação de cálcio intracelular release1,8,9. Acumular evidência também sugere predisposição genética como fator de risco importante para DIC10,11,12,13. Identidades de gene relacionado com estas predisposições DIC, no entanto, permanecem em grande parte desconhecidos. Dexrazoxane é o único agente adjuvante aprovado pelo Food and Drug Administration (FDA) para tratar o DIC, mas com aplicação limitada14,15,16, ressaltando a necessidade de identificar adicionais estratégias terapêuticas. Modelos animais de DIC, portanto, são explorados para estes fins. Devido à sua acessibilidade e simplicidade, mecanicistas estudos sobre modelos DIC potencialmente poderiam ter impactos mais amplos em outros tipos de cardiomiopatias: patogênese comum pode ser compartilhado entre cardiomiopatias de diferentes etiologias, especialmente à tarde estágios patológicos17,18,19,20.
Além de modelos de roedores de DIC, modelos DIC zebrafish com uma taxa de transferência foram desenvolvidos para facilitar a descoberta de novos fatores genéticos e terapêutica. Um modelo DIC embrionário foi estabelecido nos embriões de peixe-zebra transparente para a seleção de compostos terapêuticos21. Dado que as miocardiopatias são doenças de início adulto com uma patogênese progressiva, cardiomiopatia zebrafish adulto modelos têm sido desenvolvidos22,23,24,25,26. Geramos o primeiro modelo adquirido para cardiomiopatia resultante da anemia crônica24, seguido por DIC como o segundo modelo de cardiomiopatia adquirida no zebrafish adultos23. Nós achamos que a injeção de um único bolus de DOX em zebrafish adulto induz cardiotoxicidade que consiste de uma fase aguda, mais ou menos dentro de injeção de pós de 1 semana (wpi), seguida por uma fase crônica de cardiomiopatia de injeção após 6 meses. Enquanto haploinsuficiência do alvo da rapamicina mecanicista(mtor) melhora a cardiomiopatia na fase crônica, ele exagera peixe mortalidade na fase aguda, ressaltando o valor do modelo adulto DIC para discernir a fase dependente mecanismos de23. Mais demonstrámos que o modelo DIC adulto pode ser usado para salientar uma coleção de zebrafish insercional cardíaca (ZIC) mutantes que estão sendo gerados através de uma abordagem de mutagênese insercional baseados em transposon27. Uma tela piloto identificado 3 genes de cardiomiopatia conhecida bem como DnaJ (Hsp40) homólogo, subfamília B e membro 6b (dnajb6b) como novo DIC genes de suscetibilidade28. Portanto, a geração do modelo adulto DIC no zebrafish levou a uma nova metodologia que permite a identificação de modificadores genéticos para DIC, que complementa o estudo de associação de genoma-largo existente (GWAS) e locus traço quantitativos (QTL sistematicamente ) análise.
Durante a geração e implementação do modelo adulto zebrafish DIC, notamos variações significativas entre os diferentes pesquisadores e/ou mesmo entre diferentes injecções realizadas pelo mesmo investigador. A natureza longitudinal do modelo impõe desafios para registrar os resultados de diferentes investigadores e ao processo de solução de problemas sequencial. Para facilitar o uso desse método simples stress induzindo cardiomiopatia pela comunidade de pesquisa, descrevemos nosso protocolo detalhadamente, presentes dois tipos de injeção do IP e discutir considerações para reduzir as variações entre diferentes pesquisadores.

<table>
	<tbody>
		<tr>
			<td>Crossing tank</td>
			<td>Aquaneering</td>
			<td>ZHCT100</td>
			<td>Fish breeding</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Maintaining embryo</td>
		</tr>
		<tr>
			<td>3 L medium tank</td>
			<td>Aquaneering</td>
			<td>ZT280</td>
			<td>Maintaining fish</td>
		</tr>
		<tr>
			<td>Paramecia</td>
			<td>Carolina</td>
			<td>131560</td>
			<td>Food for juvenile fish</td>
		</tr>
		<tr>
			<td>Live hatched brine shrimp</td>
			<td>in house</td>
			<td></td>
			<td>Food for adult fish</td>
		</tr>
		<tr>
			<td>Doxorubicin hydrochloride</td>
			<td>Sigma</td>
			<td>D1515-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml safe-lock tube</td>
			<td>Eppendorf</td>
			<td>No. 022363204</td>
			<td>For drug storage</td>
		</tr>
		<tr>
			<td>Aluminum foil paper</td>
			<td>Fisher</td>
			<td>1213104</td>
			<td>For preventing light exposure</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Roche</td>
			<td>No. 03115887001</td>
			<td>For dechorionating embryo</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBBS)</td>
			<td>ThermoFisher</td>
			<td>14025076</td>
			<td>Vehicle for DOX</td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>431741</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L NanoFil micro-syringe</td>
			<td>WPI</td>
			<td>NANOFIL</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>34 gauge needle&#160;</td>
			<td>WPI</td>
			<td>NF34BV-2</td>
			<td>For injection</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Argent</td>
			<td>MS-222</td>
			<td>Anesthetizing fish</td>
		</tr>
		<tr>
			<td>96 well plate</td>
			<td>Costar</td>
			<td>3539</td>
			<td>For embryo drug treatment</td>
		</tr>
		<tr>
			<td>Transfer pipette</td>
			<td>Bel-art product</td>
			<td>F37898</td>
			<td>For transfering embryo</td>
		</tr>
	</tbody>
</table>

a doxorubicin-induced cardiomyopathy model in adult zebrafish

O geneticamente acessível adulto peixe-zebra (Danio rerio) tem sido cada vez mais usado como um modelo de vertebrados para a compreensão de doenças humanas, tais como cardiomiopatia. Devido à sua conveniência e acessibilidade para manipulações genéticas de alta produtividade, a geração de modelos de cardiomiopatia adquirida, como o modelo de cardiomiopatia induzida por doxorrubicina (DIC) no zebrafish adulto, está abrindo as portas para novos caminhos de pesquisa, inclusive descobrindo modificadores de cardiomiopatia através de rastreamento genético para a frente. Diferente do modelo embrionário zebrafish DIC, tanto inicial aguda e posteriormente crônicas fases de cardiomiopatia podem ser determinadas no modelo adulto zebrafish DIC, permitindo o estudo dos mecanismos de sinalização dependente de palco e estratégias terapêuticas. No entanto, a variáveis resultados podem ser obtidos com o modelo atual, mesmo nas mãos de investigadores experientes. Para facilitar a futura implementação do modelo de DIC, apresentamos um protocolo detalhado sobre como gerar este modelo DIC no zebrafish adulto e descrever duas maneiras alternativas de injeção intraperitoneal (IP). Vamos voltar a discutir opções sobre como reduzir variações para obter resultados fiáveis e fornece sugestões sobre como interpretar adequadamente os resultados.

Aqui, apresentam-se dois métodos para executar a injeção de IP para modelo DIC no zebrafish adulto. Enquanto estiver usando o clássico, estabeleceu IP injeção método29, verificou-se que a solução DOX injetada (cor vermelha) às vezes pode escorrer para fora do local onde a agulha penetrou. A injeção de IP alternativa usa um local diferente para a penetração da agulha é de 3-4 mm longe o peritônio onde o DOX é liberado (figura 1A...

Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

Um modelo de cardiomiopatia induzida por doxorrubicina no Zebrafish adulto

Um método para gerar um modelo de cardiomiopatia induzida pela doxorrubicina em adulto peixe-zebra (Danio rerio) é descrito aqui. Apresentam-se duas formas alternativas de injeção intraperitoneal e condições para reduzir as variações entre diferentes grupos experimentais são discutidas.

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

a-doxorubicin-induced-cardiomyopathy-model-in-adult-zebrafish

Research

JoVE Journal

Medicine

9.7K visualizações.  Mayo Clinic Graduate School of Biomedical Sciences. Um método para gerar um modelo de cardiomiopatia induzida pela doxorrubicina em adulto peixe-zebra (Danio rerio) é descrito aqui. Apresentam-se duas formas alternativas de injeção intraperitoneal e condições para reduzir as variações entre diferentes grupos experimentais são discutidas.

Vídeo: A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and teleost fish retain a remarkable capacity for cardiac regeneration with little or no scarring throughout life2,3. It is not known why only some non-mammalian vertebrates can recreate a complete organ from remnant tissues4,5. To understand the molecular and cellular differences between regenerative responses in different species, we need to use similar approaches for inducing acute injuries.
In mammals, the most frequently used model to study cardiac repair has been acute ischemia after a ligation of the coronary artery or tissue destruction after cryoinjury6,7. The cardiac regeneration in newts and zebrafish has been predominantly studied after a partial resection of the ventricular apex2,3. Recently, several groups have established the cryoinjury technique in adult zebrafish8-10. This method has a great potential because it allows a comparative discussion of the results obtained from the mammalian and non-mammalian species.
Here, we present a method to induce a reproducible disc-shaped infarct of the zebrafish ventricle by cryoinjury. This injury model is based on rapid freezing-thawing tissue, which results in massive cell death of about 20% of cardiomyocytes of the ventricular wall. First, a small incision was made through the chest with iridectomy scissors to access the heart. The ventricular wall was directly frozen by applying for 23-25 seconds a stainless steel cryoprobe precooled in liquid nitrogen. To stop the freezing of the heart, fish water at room temperature was dropped on the tip of the cryoprobe. The procedure is well tolerated by animals, with a survival rate of 95%.
To characterize the regenerative process, the hearts were collected and fixed at different days after cryoinjury. Subsequently, the specimen were embedded for cryosectioning. The slides with sections were processed for histological analysis, in situ hybridization and immunofluorescence. This undertaking enhances our understanding of the factors that are required for the regenerative plasticity in the zebrafish, and provide new insights into the machinery of cardiac regeneration. A conceptual and molecular understanding of heart regeneration in zebrafish will impact both developmental biology and regenerative medicine.

Zebrafish represents a valuable model to study the mechanisms of heart regeneration in vertebrates. Here, we present a protocol for induction of a heart infarct in adult zebrafish using cryoinjury. This method results in massive cell death within 20% of the ventricular wall, similar to that observed in mammalian infarcts.

Indução de Infarto do Miocárdio em Zebrafish adultos usando Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

Medicina

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Avaliar as alterações teratogênicos em um modelo de Zebrafish de exposição fetal ao álcool

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Triagem para Modificadores Melanoma usando um modelo de tumor Zebrafish autóctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

A Zebrafish Model of Diabetes Mellitus and Metabolic Memory

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined.
We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and discuss the advantages of this model.

Metabolic memory is the phenomenon by which diabetic complications persist and progress unimpeded even after euglycemia is achieved pharmaceutically. Here we describe a diabetes mellitus zebrafish model which is unique in that it allows for the examination of the mitotically transmissible epigenetic components of metabolic memory in vivo.

Um modelo Zebrafish de Diabetes Mellitus e Memória Metabólica

Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory ...

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Um Modelo Possível Zebrafish da doença renal policística: Knockdown de<em&gt; Wnt5a</em&gt; Faz com cistos no peixe-zebra Rins

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

Tachycardia-Induced Cardiomyopathy As a Chronic Heart Failure Model in Swine

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment methods. Here, we present such a model by tachycardia-induced cardiomyopathy, which can be produced by rapid cardiac pacing in swine.
A single pacing lead is introduced transvenously into fully anaesthetized healthy swine, to the apex of the right ventricle, and fixated. Its other end is then tunneled dorsally to the paravertebral region. There, it is connected to an in-house modified heart pacemaker unit that is then implanted in a subcutaneous pocket. 
After 4 - 8 weeks of rapid ventricular pacing at rates of 200 - 240 beats/min, physical examination revealed signs of severe heart failure - tachypnea, spontaneous sinus tachycardia, and fatigue. Echocardiography and X-ray showed dilation of all heart chambers, effusions, and severe systolic dysfunction. These findings correspond well to decompensated dilated cardiomyopathy and are also preserved after the cessation of pacing.
This model of tachycardia-induced cardiomyopathy can be used for studying the pathophysiology of progressive chronic heart failure, especially hemodynamic changes caused by new treatment modalities like mechanical circulatory supports. This methodology is easy to perform and the results are robust and reproducible.

Here, we present a protocol to produce tachycardia-induced cardiomyopathy in swine. This model represents a potent way to study the hemodynamics of progressive chronic heart failure and the effects of applied treatment.

Cardiomiopatia induzida por taquicardia como um modelo de insuficiência cardíaca crônica em suínos

A stable and reliable model of chronic heart failure is required for many experiments to understand hemodynamics or to test effects of new treatment ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Isolation of Atrial Myocytes from Adult Mice

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents responsible for the action potential can cause pro-arrhythmic substrates that underlie arrhythmias, such as atrial fibrillation, which are highly prevalent in many conditions and disease states. Isolating adult mouse atrial cardiomyocytes for use in patch-clamp experiments has greatly advanced our knowledge and understanding of the cellular electrophysiology in the healthy atrial myocardium and in the setting of atrial pathophysiology. In addition, studies using genetic mouse models have elucidated the role of a vast array of proteins in regulating atrial electrophysiology. Here we provide a detailed protocol for the isolation of cardiomyocytes from the atrial appendages of adult mice using a combination of enzymatic digestion and mechanical dissociation of these tissues. This approach consistently and reliably yields isolated atrial cardiomyocytes that can then be used to characterize cellular electrophysiology by measuring action potentials and ionic currents in patch-clamp experiments under a number of experimental conditions.

This protocol is used to isolate single atrial cardiomyocytes from the adult mouse heart using a chunk digestion approach. This approach is used to isolate right or left atrial myocytes that can be used to characterize atrial myocyte electrophysiology in patch-clamp studies.

Isolamento de miócitos atriais de camundongos adultos

The electrophysiological properties of atrial myocytes importantly affect overall cardiac function. Alterations in the underlying ionic currents ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

Modelo de infarto do miocárdio murino usando ligadura permanente da artéria coronária descendente anterior esquerda

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

Um modelo de murina induzida por doxorubicina de cardiomiopatia dilatada em Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...