Name: recovery of adult zebrafish hearts for high-throughput applications
Uploaded: 2014-12-12T15:00:00.000+00:00
Duration: 6 min 51 s
Description: Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.

NOTA: Certifique-se sempre de que a aprovação IACUC ou comitê de ética está no lugar antes de iniciar qualquer procedimento experimental usando peixe-zebra. 1. Prepare os reagentes e configuração <ol><li> Prepare as seguintes soluções. Encontrar as receitas para todas estas soluções em manuais de peixe-zebra padrão de 10. <ul><li> 500 ml de água ovo </li><li> 200 ml de 0,03% em tricaina ovo Água </li><li> 1x 100 ml de solução salina tamponada com fosfato (PBS) </li><li> Tampão de lise de RBC (opcional) </li><li> Destino tampão de escolha (fixador, Trizol, PBS, etc.) num tubo de microcentrífuga de 1,5 ml em gelo NOTA: fixador e Trizol são tóxicos se entrarem em contacto com a pele. Usar luvas ao manusear estas substâncias. </li></ul></li><li> Prepare configuração dissecção: <ol><li> Posicione a fonte de luz gooseneck sobre o microscópio de dissecação. Colocar a tampa de uma placa de Petri de 9 centímetros na fase de microscópio de dissecação, e verter uma fina camada de fosfato em 1x-lo (a tampa é usada porque o prato em si é muito profunda para a dissecção fácil). </li><li> Coloque a própria placa de Petri (não a tampa) na bancada perto do escopo de dissecação e despeje cerca de 15 ml de PBS 1x nele. Use-o para lavar o coração após a dissecação. Em alternativa, utilizar um tampão de lise de RBC. </li><li> Lâmina de barbear lugar, dois pares de pinças, microtesouras (opcional), e uma pipeta de transferência ao lado do microscópio. Obter um recipiente de gelo, se a eutanásia de um arrefecimento rápido é escolhido. </li><li> Despeje 200 ml de 0,03% tricaina em Egg água em um copo de vidro de 250 ml, se a eutanásia por tricaina é escolhido. Prepare um pequeno saco de risco biológico para eliminação de carcaças de peixe-zebra. </li></ol></li></ol> 2. Prepare Zebrafish <ol><li> Para peixes fixo, trazer peixe fixo, lavadas em PBS, para a configuração do microscópio. <ol><li> Eutanásia todo o peixe-zebra adulto de um arrefecimento rápido com gelo durante&gt; 10 min. </li><li> Use uma pinça para fazer um furo na pele sobre o peritoneu(Longe do coração) para auxiliar a penetração do fixador, e, em seguida, colocá-los em 4% de paraformaldeído em tampão de Fix 10 durante 2 h à TA ou O / N a 4 ° C. </li></ol></li><li> Alternativamente, para o peixe fresco, peixe local para ser sacrificado em um pequeno tanque de retenção e trazer para a configuração do microscópio. Dependendo do protocolo de peixes da instalação individual e os usos a jusante para os corações de peixes dissecados, anestesiar peixe com tricaina ou eutanásia de um arrefecimento rápido antes de decapitação. <ol><li> Para usar o gelo, pegar um peixe com a rede de pesca e coloque em água gelada até que o peixe pára de se mover, cerca de 5 min. </li><li> Como alternativa, para usar tricaina, pegar um peixe com a rede de pesca e coloque no copo da solução até movimentos de emalhar parar. </li></ol></li><li> Rapidamente pegar os peixes mortos por sua barbatana caudal e colocá-lo em seu lado na tampa placa de Petri que foi preenchido com 1x PBS. Use a pinça para levantar uma nadadeira peitoral com uma mão, enquanto estiver usando o razolâmina r para decapitar o peixe com o outro lado, apenas posterior à fixação da barbatana peitoral (Figura 1A). Execute esta etapa ou olhando através do microscópio ou apenas visualizar diretamente os peixes no palco microscópio. NOTA: Peixes recentemente sacrificados ainda vai sangrar após a decapitação. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> Figura 1. Zebrafish dissecção coração adulto utiliza marcos anatômicos de peixe-zebra. (A) para decapitar o peixe, levante a nadadeira peitoral com uma pinça e usar uma lâmina limpa afiada ao longo da linha pontilhada vermelha, como mostrado. (B) Para firmar a cabeça de peixe, coloque um dente do fórceps na boca dos peixes enquanto o outro tine fica do outro lado do olho, e, em seguida, virar a cabeça de peixe de modo que a superfície ventral é para cima e as duas pontas da pinça são estáveis ​​contra a parte inferior da placa de Petri. (C) Use a forc livreeps para cortar a ligação do opérculo (seta). (D) elevação isso, a aorta dorsal é visível como uma estrutura branca com uma listra rosa denotando sangue luminal (seta). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Por favor, clique aqui para ver uma versão maior desta figura.</a> 3. Dissecar o Coração <ol><li> Visualize a cabeça de peixe sob o microscópio. Com uma pinça, estabilizar a cabeça de peixe, colocando um dente da pinça na boca de peixe, através do cérebro, enquanto que o outro dente é fora da cabeça, através do olho (Figura 1B). Certifique-se de ambas as garras de pinça são estáveis ​​contra o fundo do prato de petri. Segurando a cabeça de peixe desta forma, a sua superfície ventral deve estar voltado para cima. </li><li> Com a outra mão, utilizar a segunda pinça para cortar a ligação ventral do opérculo ao corpo (seta, Figura 1C). Levantar esta ligeiramente com a pinça, o dØrsal aorta aparece por baixo como uma estrutura branca com uma listra vermelha de sangue na luz aórtica (seta, Figura 1D). Corte as aorta dorsal, com a pinça por beliscar a aorta e puxando para cima; em alternativa, usar microtesouras para cortar a aorta dorsal. </li><li> Agora, use a segunda pinça para agarrar a barbatana peitoral de sua base, incluindo a cartilagem corpo, na base da nadadeira, e levante esta off. Repita com a outra barbatana peitoral. Ocasionalmente, o coração sai com as nadadeiras peitorais, então examinar estas peças para certificar-se o coração não está conectado antes de descartá-los. NOTA: Neste ponto, o coração deve estar visível, ainda intacto e conectado à cabeça de peixe restante (embora ocasionalmente ele sai com as nadadeiras peitorais). O coração pode ser identificado pela sua forma, a sua cor-de-rosa, e os seus estar rodeado por pericárdio pigmentada. Porque a eutanásia dos peixes é realizada rapidamente, corações peixe acabado de eutanásia pode ainda estar batendo slowly. </li><li> Deixe de lado a cabeça de peixe com os primeiros fórceps, para que ambas as mãos segurando uma pinça e são gratuitas. Use as duas pinças de provocar o coração longe do pericárdio. Agarrar o coração pelo restante da aorta dorsal, enquanto o restante pericárdio é puxado para fora. NOTA: Se fixa ou fresco, o coração é robusto a suave puxando enquanto o tecido conjuntivo circundante é mais friável. Portanto, qualquer tecido de pericárdio circundante pode ser dissecado com o coração restante intacto. </li><li> Descarte a carcaça de peixe sobra no saco de risco biológico. </li></ol> 4. Prepare o coração para aplicações a jusante <ol><li> Utilizando uma pinça, segure o coração pela borda da aorta dorsal / arterial bulbus e colocar o coração no prato fresco de 1x PBS. Em alternativa, utilizar uma pipeta de transferência. Mova o coração e para trás na PBS cerca de 10x para lavar o máximo de sangue possível. </li><li> Para aplicações em que é importante para remover todos possibiliOs glóbulos le, lavar em uma placa de Petri de 9 centímetros, com 15 ml de tampão de lise de RBC em vez de PBS, usando o mesmo movimento de vai-e-vem. Se preservar a estrutura do coração não é importante para a aplicação a jusante (por exemplo, tornando o RNA), usar os fórceps ou microtesouras para abrir a cavidade do coração e facilitar enxaguando de células sanguíneas. </li><li> Para aplicações em que são desejados diferentes câmaras cardíacas, utilize uma pinça ou microtesouras para dissecar o bulbo arterial, átrio, e ventrículo afastados uns dos outros. </li><li> Transferir o coração, ou câmaras separadas, para o buffer de destino em gelo. NOTA: destinos comuns incluem buffers para dissociação celular, paraformaldeído 4%, ou Trizol. </li></ol>

<ol>
	<li>Arnaout, R., 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=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. 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=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., 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=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., 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=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

The authors have no disclosures.

Embora os métodos para dissecar o coração de adulto peixe-zebra foram descritos, estes métodos foram demorada e normalmente causado dano ao coração durante a dissecção. Para realizar experiências onde pode ser necessário um grande número de coração do adulto, e / ou ao evitar a degradação do tecido do coração é importante para aplicações a jusante, o tempo necessário utilizando técnicas tradicionais de dissecção é proibitivo. Da mesma forma, a obtenção de reprodutível, corações intactos nã...

Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.

<table><tbody><tr><td>Small tank for transporting fish</td><td>Aquaneering</td><td>ZHCT100</td><td></td></tr><tr><td>Fish net</td><td>Petsmart</td><td>36-16731</td><td></td></tr><tr><td>250 ml glass beaker</td><td>Kimble</td><td>14005-250</td><td></td></tr><tr><td>9 cm polystyrene Petri dish</td><td>Nunc</td><td>172958</td><td></td></tr><tr><td>Razor blade</td><td>Personna American Safety Razor Company</td><td>94-120-71</td><td></td></tr><tr><td>2 Dumont #5SF forceps</td><td>Fine Science Tools</td><td>11252-00</td><td></td></tr><tr><td>Dissecting microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A-5040</td><td></td></tr><tr><td>Plastic transfer pipette</td><td>Thermo Scientific</td><td>202-20S</td><td></td></tr><tr><td>Gooseneck light source</td><td>Dolan-Jenner Industries, Inc</td><td>Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td><td></td></tr><tr><td>Fluorescent light source</td><td>Lumen Dynamics</td><td>X-Cite 120Q</td><td>optional</td></tr><tr><td>Micro-scissors</td><td>Biomedical Research Instruments, Inc</td><td>11-1000</td><td>optional</td></tr><tr><td>RBC lysis buffer</td><td>eBioscience</td><td>00-4333-57</td><td>optional</td></tr></tbody></table>

recovery of adult zebrafish hearts for high-throughput applications

A utilização do sistema modelo para estudar o desenvolvimento do peixe-zebra, a regeneração, e a doença está a expandir para utilização de coração do adulto para dissociação de células e purificação de RNA, DNA e proteínas. Todas estas aplicações exigem a rápida recuperação de um número significativo de corações de peixe-zebra para evitar gene regulador, metabólicas e outras mudanças que começam após a morte. Corações de peixe-zebra adultos são também necessários para estudar a estrutura do coração para uma variedade de mutantes e para estudar a regeneração cardíaca. No entanto, a dissecção do coração do peixe-zebra tradicional é lento e difícil e requer ferramentas especializadas, tornando dissecção em larga escala de corações de peixe-zebra adultos tedioso. Os métodos tradicionais também abrigam o risco de danificar o coração durante a dissecção. Aqui, descrevemos um método para dissecção de corações peixe-zebra adulto que é rápido, reprodutível, e preserva a arquitetura coração. Além disso, este método não requer ferramentas especializadas, é indolor para o peixe-zebra,pode ser realizada em amostras frescas ou fixos, e pode ser realizado em peixes-zebra tão jovens como um mês de idade. A abordagem descrita amplia o uso de peixe-zebra adulto para a investigação cardiovascular.

Usando este método, um peixe-zebra coração adulto pode ser dissecado em menos de 1 minuto, em comparação com mais de 5 min, utilizando métodos tradicionais 8. Corações dissecados utilizando este método são seguramente intacta (Figura 2A), enquanto que os métodos tradicionais requerem 8 cortar cegamente no pericárdio e, portanto, comumente causar dano ou perda do átrio ou bulbus arterial (Figura 2B). Corações dissecados manter a sua integridade estrut...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

recovery-of-adult-zebrafish-hearts-for-high-throughput-applications

Research

JoVE Journal

Biology

12.2K visualizações.  University of California San Francisco. Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Vídeo: Recovery of Adult Zebrafish Hearts for High-throughput Applications

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Em larga escala Zebrafish Embryonic Coração de dissecação para a análise transcricional

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Biologia do Desenvolvimento

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

High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

We describe a protocol to assess heart morphology and function in adult zebrafish using high-frequency echocardiography. The method allows visualization of the heart and subsequent quantification of functional parameters, such as heart rate (HR), cardiac output (CO), fractional area change (FAC), ejection fraction (EF), and blood inflow and outflow velocities.

Ecocardiografia de ultrassom de alta frequência para avaliar a função cardíaca de zebrafish

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Biologia

Immunostaining of Dissected Zebrafish Embryonic Heart 

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Imunomarcação de Dissecada Coração Zebrafish embrionárias

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

Análise da Contração Cardíaca do Peixe-Zebra com Interação de Realidade Virtual

4D Light-sheet Imaging of Zebrafish Cardiac Contraction

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for gaining insights into structural and functional changes in response to injuries. However, obtaining high-resolution and high-speed 4-dimensional (4D, 3D spatial + 1D temporal) images of the zebrafish heart to assess cardiac architecture and contractility remains challenging. In this context, an in-house light-sheet microscope (LSM) and customized computational analysis are&#160;used to overcome these technical limitations. This strategy, involving LSM system construction, retrospective synchronization, single cell tracking, and user-directed analysis, enables one to investigate the micro-structure and contractile function across the entire heart at the single-cell resolution in the transgenic Tg(myl7:nucGFP) zebrafish larvae. Additionally, we are able to further incorporate microinjection of small molecule compounds to induce cardiac injury in a precise and controlled manner. Overall, this framework allows one to track physiological and pathophysiological changes, as well as the regional mechanics at the single-cell level during cardiac morphogenesis and regeneration.

Autor em destaque: Imagens de folha de luz 4D de alta resolução e realidade virtual em peixe-zebra para análise de célula única da função cardíaca

This protocol utilizes light-sheet imaging to investigate cardiac contractile function in zebrafish larvae and gain insights into cardiac mechanics through cell tracking and interactive analysis.

Imagem de folha de luz 4D da contração cardíaca do peixe-zebra

Zebrafish is an intriguing model organism known for its remarkable cardiac regeneration capacity. Studying the contracting heart in vivo is essential for ...

Bioengenharia

Assessment of Swim Endurance and Swim Behavior in Adult Zebrafish

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection of their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain their swim capacities within 8 weeks of injury. Recovery of swim function is thus a central readout for functional spinal cord repair. Here, we describe a set of behavioral assays to quantify zebrafish motor capacity inside an enclosed swim tunnel. The goal of these methods is to provide quantifiable measurements of swim endurance and swim behavior in adult zebrafish. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. For swim behavior assessment, zebrafish are subjected to low current velocities and swim videos are captured with a dorsal view of the fish. Percent activity, burst frequency, and time spent against the water current provide quantifiable readouts of swim behavior. We quantified swim endurance and swim behavior in wild-type zebrafish before injury and after spinal cord transection. We found that zebrafish lose swim function after spinal cord transection and gradually regain that capacity between 2 and 6 weeks post-injury. The methods described in this study could be applied to neurobehavioral, musculoskeletal, skeletal muscle regeneration, and neural regeneration studies in adult zebrafish.

Capable of functional recovery after spinal cord injury, adult zebrafish is a premier model system to elucidate innate mechanisms of neural regeneration. Here, we describe swim endurance and swim behavior assays as functional readouts of spinal cord regeneration.

Avaliação da resistência ao natação e comportamento de natação em zebrafish adulto

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. ...

Neurociência

Intrathoracic Injection for the Study of Adult Zebrafish Heart

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic approaches, a rapid delivery of exogenous factors provides a complementary technique in functional studies. Here, we present a method that relies on administration of a few microliters of solution into the pericardial cavity without causing myocardial damage. Intrathoracic (IT) injections can efficiently deliver proteins and chemical compounds directly onto the heart surface. The injected substances diffuse through the epicardium into the underlying cardiac tissues. Compared to intraperitoneal (IP) injections, the main advantage of intrathoracic injections is the focal administration of the tested factors on the target organ. The delivery of molecules directly into the pericardium is a suitable strategy for studies of cardiac preconditioning and regeneration in adult zebrafish.

This method relies on the injection of 0.5&#8722;3 &#956;L of solution into the thorax of adult zebrafish. The procedure efficiently delivers proteins and chemical compounds into the proximity of the zebrafish heart without damaging the organ. The approach is suitable for testing effects of exogenous factors on various tissues of the heart.

Recuperação de Zebrafish Adulto Corações para aplicações de alto rendimento

Resumo

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