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: Asegúrese siempre de que la aprobación del IACUC o el comité de ética está en su lugar antes de comenzar cualquier procedimiento experimental utilizando el pez cebra. 1. Preparar reactivos y configuración <ol><li> Preparar las siguientes soluciones. Encuentra las recetas de todas estas soluciones en los manuales estándar de pez cebra 10. <ul><li> 500 ml de agua del huevo </li><li> 200 ml de 0,03% tricaína en Egg Agua </li><li> 100 ml de tamponada con fosfato salino (PBS 1x) </li><li> RBC tampón de lisis (opcional) </li><li> Búfer de destino de elección (fijador, Trizol, PBS, etc.) en un tubo de microcentrífuga de 1,5 ml en hielo NOTA: Fijador y Trizol son tóxicos si entran en contacto con la piel. Use guantes para manipular estas sustancias. </li></ul></li><li> Preparar configuración disección: <ol><li> Coloque la fuente de luz de cuello de cisne sobre el microscopio de disección. Coloque la tapa de una placa de Petri de 9 cm en la platina del microscopio de disección, y se vierte una capa delgada de 1x PBS enella (la tapa se utiliza porque el plato en sí es demasiado profunda para una disección fácil). </li><li> Coloque la propia placa de Petri (no la tapa) en la encimera cerca del microscopio de disección y vierta aproximadamente 15 ml de PBS 1x en ella. Use esto para lavar el corazón después de la disección. Como alternativa, utilice tampón de lisis RBC. </li><li> Hoja de afeitar Place, dos pares de pinzas, microtijeras (opcional), y una pipeta de transferencia al lado de la microscopio. Obtener un recipiente con hielo si se elige la eutanasia por enfriamiento rápido. </li><li> Vierta 200 ml de 0,03% tricaína en Egg agua en un vaso de vidrio de 250 ml si se elige la eutanasia por tricaína. Preparar una pequeña bolsa de riesgo biológico para la eliminación de cadáveres de pez cebra. </li></ol></li></ol> 2. Prepare Pez Cebra <ol><li> Para los peces fijo, traer peces fijo, se enjuagan en PBS, a la configuración de microscopio. <ol><li> La eutanasia a todo el pez cebra adulto de un rápido enfriamiento con hielo durante&gt; 10 min. </li><li> El uso de fórceps para hacer un agujero en la piel sobre el peritoneo(Lejos del corazón) para ayudar a la penetración del fijador, y luego ponerlos en paraformaldehído al 4% en Fix Buffer 10 durante 2 horas a temperatura ambiente o O / N a 4 ° C. </li></ol></li><li> Alternativamente, para el pescado fresco, pescado lugar a ser sacrificado en un pequeño tanque de retención y traer a la configuración de microscopio. Dependiendo del protocolo de peces de la instalación individual y los usos posteriores de los corazones de peces disecados, anestesiar pescado con tricaína o la eutanasia de un enfriamiento rápido antes de la decapitación. <ol><li> Para utilizar hielo, recoger un pez con la red de pesca y el lugar en agua helada hasta que el pescado deja de moverse, aproximadamente 5 minutos. </li><li> Como alternativa, utilizar tricaína, coger un pez con la red de pesca y el lugar en el vaso de la solución hasta que los movimientos de enmalle se detienen. </li></ol></li><li> Rápidamente recoger los peces sacrificados por su aleta de la cola y lo pondré sobre su lado en la cubierta placa de Petri que estaba lleno de PBS 1x. Utilice las pinzas para levantar una aleta pectoral con una mano, mientras que con el razocuchilla r para decapitar a los peces con la otra mano, justo posterior a la fijación de la aleta pectoral (Figura 1A). Realice este paso mirando por el microscopio o simplemente visualizar directamente el pescado sobre la platina del microscopio. NOTA: Pescados recién sacrificados todavía sangran después de la decapitación. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> Figura 1. disección corazón adulto pez cebra utiliza referencias anatómicas de pez cebra. (A) para decapitar a los peces, levante la aleta pectoral con una pinza y utilizar una hoja de afeitar limpia agudo a lo largo de la línea de puntos de color rojo, como se muestra. (B) Para estabilizar la cabeza de pescado, coloque una púa de la pinza en la boca de pescado, mientras que el otro diente miente a través del ojo, y luego gire la cabeza de pescado para que la superficie ventral es y ambas puntas de la pinza son estables frente a la parte inferior de la placa de Petri. (C) Utilice el forc gratiseps para cortar el apego del opérculo (flecha). (D) de elevación de esto, la aorta dorsal es visible como una estructura blanca con una raya rosada denota sangre luminal (flecha). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Por favor, haga clic aquí para ver una versión más grande de esta figura.</a> 3. Disección del Corazón <ol><li> Visualice la cabeza de pescado bajo el microscopio. Con uno fórceps, estabilizar la cabeza de pescado mediante la colocación de una púa de la pinza en la boca de pescado, a través del cerebro, mientras que el otro diente está fuera de la cabeza, a través del ojo (Figura 1B). Asegúrese de que ambas puntas de las pinzas son estables contra la parte inferior de la placa de Petri. La celebración de la cabeza de pescado de esta manera, su superficie ventral debe estar mirando hacia arriba. </li><li> Con la otra mano, utilizar los segundo fórceps para cortar el enganche ventral del opérculo al cuerpo (flecha, Figura 1C). Esta elevación ligeramente con las pinzas, la dOrsal aorta aparecerá debajo, como una estructura de color blanco con una franja roja de la sangre en la luz aórtica (flecha, Figura 1D). Cortar la aorta dorsal con las pinzas por pellizcar la aorta y tirando hacia arriba; alternativamente, utilizar microtijeras para cortar la aorta dorsal. </li><li> Ahora, usa los segundos pinzas para agarrar la aleta pectoral de su base, incluyendo el cartílago del cuerpo en la base de la aleta, y levantar esto. Repita con la otra aleta pectoral. En ocasiones, el corazón sale con las aletas pectorales, por lo que examinar estas piezas para asegurarse de que el corazón no está conectado antes de desecharlos. NOTA: En este punto, el corazón debe ser visible, sigue intacto y conectado a la cabeza de pescado restante (aunque en ocasiones se corre con las aletas pectorales). El corazón puede ser identificado por su forma, su color rosa, y su ser rodeado por el pericardio pigmentada. Debido a la eutanasia de los peces se realiza de forma rápida, corazones pescado recién sacrificados todavía puede ser vencer sloWLY. </li><li> Dejar de lado la cabeza de pescado con los primeros fórceps, de modo que ambas manos sostienen fórceps y son gratuitos. Use las dos pinzas para embromar el corazón lejos del pericardio. Agarre el corazón por el resto de la aorta dorsal, mientras que el pericardio restante se apartó. NOTA: Ya sea fijo o fresco, el corazón es robusto a suave tirando mientras el tejido conectivo que rodea es más friable. Por lo tanto, cualquier tejido pericárdico circundante puede ser disecado de distancia con el corazón intacto restante. </li><li> Deseche la canal pescado sobrante en la bolsa de riesgo biológico. </li></ol> 4. Preparar el Corazón para aplicaciones posteriores <ol><li> Con unas pinzas, agarre el corazón por el borde de la aorta dorsal / bulbo arterioso y poner el corazón en el plato fresco de 1x PBS. También puede utilizar una pipeta de transferencia. Mover el corazón de ida y vuelta en la PBS sobre 10x para lavar tanta sangre como sea posible. </li><li> Para aplicaciones en las que es importante para eliminar todo possibglóbulos le, se lavan en una placa de Petri de 9 cm con 15 ml de tampón de lisis RBC en lugar de PBS, con el mismo movimiento hacia atrás y hacia adelante. Si la preservación de la estructura del corazón no es importante para la aplicación de aguas abajo (por ejemplo, haciendo RNA), utilizar los fórceps o micro tijeras para abrir la cavidad del corazón y facilitar el aclarado de distancia de células sanguíneas. </li><li> Para aplicaciones en las que se desean cámaras del corazón separadas, el uso de fórceps o microtijeras para diseccionar la arterioso bulbus, atrio, ventrículo y separados unos de otros. </li><li> Transferir el corazón, o cámaras separadas, a la memoria intermedia de destino en hielo. NOTA: los destinos comunes incluyen tampones de disociación celular, el 4% de paraformaldehído, o 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.

Aunque se han descrito métodos para la disección de la corazón del pez cebra adultos, estos métodos eran mucho tiempo y comúnmente causaron daños al corazón durante la disección. Para llevar a cabo experimentos en los que puede ser necesario un gran número de corazones adultos, y / o al evitar la degradación del tejido del corazón es importante para aplicaciones posteriores, el tiempo requerido usando técnicas de disección tradicionales es prohibitivo. Del mismo modo, la obtención reproducible en buen esta...

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

El uso del sistema de modelo de pez cebra para estudiar el desarrollo, la regeneración, y la enfermedad se está expandiendo hacia el uso de los corazones adultos para disociación celular y purificación de ARN, ADN y proteínas. Todas estas aplicaciones exigen la rápida recuperación de un número significativo de los corazones de pez cebra para evitar gen regulador, metabólicos y otros cambios que comienzan después de la muerte. También se requieren adultos corazones de pez cebra para el estudio de la estructura del corazón para una variedad de mutantes y para el estudio de la regeneración del corazón. Sin embargo, la disección corazón del pez cebra tradicional es lenta y difícil y requiere herramientas especializadas, haciendo la disección a gran escala de corazones de pez cebra adultos tedioso. Los métodos tradicionales también albergan el riesgo de dañar el corazón durante la disección. Aquí se describe un método para la disección de corazones adultos de pez cebra que es rápido, reproducible, y preserva la arquitectura del corazón. Además, este método no requiere herramientas especializadas, es indoloro para el pez cebra,se pueden realizar en muestras frescas o fijos, y se puede realizar en el pez cebra de tan sólo un mes de edad. El enfoque descrito se expande el uso de adultos de pez cebra para la investigación cardiovascular.

Usando este método, un corazón del pez cebra adulto puede ser diseccionado en menos de 1 min, en comparación con más de 5 min usando métodos tradicionales 8. Corazones disecados utilizando este método son de forma fiable intacta (Figura 2A), mientras que los métodos tradicionales requieren el corte de 8 a ciegas en el pericardio y por lo tanto comúnmente causar daños o pérdida de la aurícula o bulbus arterioso (Figura 2B). Corazones disecados mantienen s...

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.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

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

12.3K vistas.  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.

Video: 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.

A gran escala de pez cebra embrionario del corazón Disección de Análisis transcripcional

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 ...

Investigación

Biología del Desarrollo

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.

La inducción de infarto de miocardio en adultos de pez cebra Uso de 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.

Ecocardiografía por ultrasonido de alta frecuencia para evaluar la función cardíaca del pez cebra

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.

La disección del corazón en las larvas, juveniles y adultos de pez cebra, 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 ...

Biología

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.

Inmunotinción de Disección del corazón embrionario de pez cebra

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

Análisis de la contracción cardíaca del pez cebra con interacción de realidad 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 destacado: Imágenes de lámina de luz 4D de alta resolución y realidad virtual en pez cebra para el análisis de la función cardíaca en una sola célula

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.

Imágenes de lámina de luz 4D de la contracción cardíaca del pez cebra

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

Bioingeniería

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.

Evaluación de la resistencia a la natación y el comportamiento de natación en peces cebra adultos

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

Neurociencias

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.

Recuperación de adultos de pez cebra Corazones para aplicaciones de alto rendimiento

Resumen

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