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: Accertarsi sempre che l&#39;approvazione IACUC o comitato etico è a posto prima di iniziare qualsiasi procedura sperimentale utilizzando zebrafish. 1. Preparare i reagenti e installazione <ol><li> Preparare le seguenti soluzioni. Trova le ricette per tutte queste soluzioni in manuali di zebrafish normali 10. <ul><li> 500 ml di acqua Egg </li><li> 200 ml di 0,03% Tricaine a Egg Water </li><li> 100 ml di 1x tampone fosfato salino (PBS) </li><li> RBC Lysis Buffer (optional) </li><li> Il buffer di destinazione di scelta (fissante, Trizol, PBS, etc.) in una provetta da 1,5 ml microcentrifuga sul ghiaccio NOTA: fissativo e Trizol sono tossici se entrano in contatto con la pelle. Maneggiare con guanti queste sostanze. </li></ul></li><li> Preparare l&#39;installazione dissezione: <ol><li> Posizionare la sorgente luminosa collo di cigno sul microscopio da dissezione. Mettere il coperchio di un piatto 9 centimetri Petri sul palco microscopio da dissezione, e versare un sottile strato di 1x PBS in(il coperchio è utilizzato perché il piatto stesso è troppo profonda per un facile dissezione). </li><li> Porre la capsula Petri in sé (non il coperchio) sul piano di lavoro in prossimità del campo di applicazione dissezione e versare circa 15 ml di 1x PBS in esso. Usare questo per lavare il cuore dopo la dissezione. In alternativa, utilizzare RBC Lysis Buffer. </li><li> Luogo lametta, due paia di pinze, microscissors (opzionale), e una pipetta di trasferimento accanto al microscopio. Ottenere un contenitore di ghiaccio se viene scelto eutanasia per raffreddamento rapido. </li><li> Versare 200 ml di 0,03% Tricaine a Egg acqua in un bicchiere di vetro da 250 ml se si sceglie l&#39;eutanasia per Tricaine. Preparare una piccola borsa di rischio biologico per lo smaltimento delle carcasse di zebrafish. </li></ol></li></ol> 2. Preparare Zebrafish <ol><li> Per il pesce fissa, portano pesce fisso, sciacquati in PBS, per la configurazione del microscopio. <ol><li> Euthanize tutta zebrafish adulto per un rapido raffreddamento con ghiaccio per&gt; 10 min. </li><li> Usare pinze per fare un buco nella pelle sopra il peritoneo(Dal cuore) per favorire la penetrazione di fissativo, e poi metterli in 4% paraformaldeide in Fix Buffer 10 per 2 ore a temperatura ambiente o O / N a 4 ° C. </li></ol></li><li> In alternativa, per il pesce fresco, pesce posto da eutanasia in un piccolo serbatoio e portare alla messa a punto del microscopio. A seconda del protocollo di pesce della singola struttura e gli usi a valle per i cuori dei pesci sezionati, anestetizzare pesce con Tricaine o eutanasia da un raffreddamento rapido prima decapitazione. <ol><li> Per usare il ghiaccio, prendere un pesce con la rete di pesce e posto in acqua ghiacciata fino a quando il pesce si ferma, a circa 5 min. </li><li> In alternativa, per usare Tricaine, prendere un pesce con la rete di pesce e posto nel bicchiere della soluzione fino a quando i movimenti delle branchie fermano. </li></ol></li><li> Prendere rapidamente il pesce eutanasia dal suo pinna caudale e adagiarla su un fianco nel coperchio piatto Petri che era pieno di 1x PBS. Utilizzare le pinze per sollevare un pettorale con una mano, mentre con l&#39;Razolama r per decapitare il pesce con l&#39;altra mano, appena posteriormente al fissaggio della pinna pettorale (Figura 1A). Eseguire questo passaggio sia guardando attraverso il microscopio o semplicemente visualizzare direttamente il pesce sul palco microscopio. NOTA: Il pesce appena eutanasia saranno ancora sanguinano dopo la decapitazione. </li></ol><img alt="Figura 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> Figura 1. Zebrafish dissezione cuore adulto utilizza punti di repere anatomici zebrafish. (A) di decapitare i pesci, sollevare la pinna pettorale con una pinza e utilizzare una lama di rasoio pulito affilata lungo la linea tratteggiata rossa come mostrato. (B) Per stabilizzare la testa di pesce, mettere un dente della pinza nella bocca del pesce, mentre l&#39;altro dente si trova attraverso l&#39;occhio, e poi girare la testa di pesce in modo che la superficie ventrale è alto e due denti della pinza sono stabili contro il fondo della scatola di Petri. (C) Utilizzare la forc gratuitoeps per tagliare l&#39;attaccamento del opercolo (freccia). (D) Sollevamento questo, l&#39;aorta dorsale è visibile come una struttura bianca con una striscia rosa denota sangue luminale (freccia). <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> 3. Sezionare il Cuore <ol><li> Visualizza la testa di pesce sotto il microscopio. Con una pinza, stabilizzare la testa di pesce mettendo una tine della pinza nella bocca di pesce, attraverso il cervello, mentre l&#39;altro dente è al di fuori della testa, attraverso l&#39;occhio (Figura 1B). Garantire entrambi denti della pinza sono costanti contro il fondo della capsula di Petri. Tenendo la testa di pesce in questo modo, la sua superficie ventrale deve essere rivolto verso l&#39;alto. </li><li> Con l&#39;altra mano, utilizzare la seconda pinza per tagliare l&#39;attacco ventrale dell&#39;opercolo al corpo (freccia, Figura 1C). Sollevamento questo un po &#39;con le pinze, il dOrsal aorta apparirà sotto una struttura bianca con una striscia rossa di sangue nel lume aortico (freccia, Figura 1D). Tagliare le aorta dorsale con la pinza pizzicando l&#39;aorta e tirando verso l&#39;alto; in alternativa, utilizzare microscissors per tagliare l&#39;aorta dorsale. </li><li> Ora, utilizzare la seconda pinza per afferrare la pinna pettorale dalla sua base, compresa la cartilagine corpo alla base della pinna, e sollevare fuori questo. Ripetere con l&#39;altra pinna pettorale. Di tanto in tanto, il cuore esce con le pinne pettorali, quindi esaminare questi pezzi per assicurarsi che il cuore non è collegato prima di scartare. NOTA: A questo punto, il cuore deve essere visibile, ancora intatta e collegato alla restante testa di pesce (anche se a volte si stacca con le pinne pettorali). Il cuore può essere identificato dalla sua forma, il suo colore rosa, e il suo essere circondato da pericardio pigmentata. Perché l&#39;eutanasia del pesce viene effettuata rapidamente, cuori di pesce fresco eutanasia possono essere ancora battendo sloWLY. </li><li> Lasciate andare la testa di pesce con le prime pinze, in modo che entrambe le mani tengono pinze e sono gratuiti. Usare entrambe le pinze per prendere in giro il cuore lontano dal pericardio. Afferrare cuore dal resto dei dell&#39;aorta dorsali mentre il pericardio rimanente viene tirato via. NOTA: Sia fisso o fresco, il cuore è robusta a leggera trazione mentre il tessuto connettivo circostante è più friabile. Pertanto, qualsiasi tessuto pericardico circostante può essere sezionato via con il cuore rimasta intatta. </li><li> Eliminare la carcassa di pesce rimasto nel sacchetto. </li></ol> 4. Preparare il Cuore per applicazioni a valle <ol><li> Utilizzando pinze, afferrare il cuore dal bordo della aorta dorsale / bulbo arterioso e mettere il cuore nel piatto fresco di 1x PBS. In alternativa, utilizzare una pipetta di trasferimento. Spostare il cuore avanti e indietro nel PBS circa 10x per lavare via il più sangue possibile. </li><li> Per applicazioni in cui è importante rimuovere tutto possibLe cellule del sangue, lavare in un piatto 9 centimetri Petri con 15 ml RBC Lysis Buffer invece di PBS, utilizzando lo stesso movimento di va e vieni. Se mantenendo la struttura del cuore non è importante per l&#39;applicazione a valle (ad esempio, facendo RNA), utilizzare le pinze o microscissors per aprire la cavità cardiaca e facilitare risciacquo distanza di cellule del sangue. </li><li> Per le applicazioni in cui sono determinanti: camere cardiache separati, utilizzare forcipe o microscissors per sezionare il bulbo arterioso, atrio e il ventricolo uno dall&#39;altro. </li><li> Trasferire il cuore o camere separate, al buffer di destinazione su ghiaccio. NOTA: destinazioni più comuni includono buffer per dissociazione cellulare, 4% paraformaldeide, 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.

Mentre sono stati descritti i metodi per sezionare cuore zebrafish adulto, questi metodi sono stati in termini di tempo e di solito causato danni al cuore durante la dissezione. Per eseguire esperimenti in cui può essere necessario un gran numero di cuori adulti, e / o quando evitare il degrado del tessuto cardiaco è importante per le applicazioni a valle, il tempo necessario utilizzando tecniche di dissezione tradizionali è proibitivo. Allo stesso modo, ottenendo riproducibile intatti, cuori intatti è importante pe...

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

L&#39;uso del sistema modello per lo studio zebrafish sviluppo, rigenerazione e malattia è espansione verso l&#39;uso di cuori adulti per dissociazione cellulare e purificazione del DNA, RNA e proteine. Tutte queste applicazioni richiedono la rapida ripresa di un numero significativo di cuori zebrafish per evitare gene regolatore, metaboliche, e altri cambiamenti che iniziano dopo la morte. Cuori zebrafish adulti sono necessari anche per lo studio la struttura del cuore per una varietà di mutanti e per studiare la rigenerazione del cuore. Tuttavia, il tradizionale dissezione cuore zebrafish è lento e difficile e richiede strumenti specializzati, facendo su larga scala dissezione dei cuori di zebrafish adulto noioso. I metodi tradizionali porto anche il rischio di danneggiare il cuore durante la dissezione. Qui, si descrive un metodo per la dissezione di cuori di zebrafish adulto che è veloce, riproducibile, e conserva l&#39;architettura del cuore. Inoltre, questo metodo non richiede strumenti specializzati, è indolore per il zebrafish,possono essere eseguite su campioni freschi o fissi, e possono essere eseguite sul pesce zebra giovane come un mese. L&#39;approccio descritto espande l&#39;uso di zebrafish adulto per la ricerca cardiovascolare.

Usando questo metodo, un cuore zebrafish adulto può essere sezionato in meno di 1 minuto, a fronte di oltre 5 min con metodi tradizionali 8. Hearts sezionati con questo metodo sono affidabile intatto (figura 2A), mentre i metodi tradizionali richiedono 8 tagliando alla cieca in pericardio e quindi comunemente causare danni o la perdita dell&#39;atrio o bulbo arterioso (Figura 2B). Cuori sezionato mantengono la loro integrità strutturale e sono adatti per l&#39;is...

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.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

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 Views.  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.

Su larga scala Zebrafish embrionali Cuore dissezione per l&#39;analisi trascrizionale

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 ...

Ricerca

Biologia dello sviluppo

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.

L&#39;induzione di infarto miocardico in Zebrafish adulti 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.

Ecocardia ad ultrasuoni ad alta frequenza per valutare la funzione cardiaca del pesce zebra

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.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, 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.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

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

Analisi della contrazione cardiaca del pesce zebra con l'interazione della realtà virtuale

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.

Autore in primo piano: Imaging 4D Light-Sheet ad alta risoluzione e realtà virtuale in zebrafish per l'analisi di singole cellule della funzione cardiaca

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.

Imaging a foglio luminoso 4D della contrazione cardiaca del pesce zebra

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

Bioingegneria

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.

Valutazione della resistenza al nuoto e del comportamento di nuoto nel pesce zebra adulto

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

Neuroscienze

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.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

Riepilogo

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Capitoli in questo video

Su larga scala Zebrafish embrionali Cuore dissezione per l'analisi trascrizionale

L'induzione di infarto miocardico in Zebrafish adulti usando Cryoinjury

Ecocardia ad ultrasuoni ad alta frequenza per valutare la funzione cardiaca del pesce zebra

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio

Immunocolorazione di Dissected Cuore Zebrafish embrionali

Imaging a foglio luminoso 4D della contrazione cardiaca del pesce zebra

Imaging a foglio luminoso 4D della contrazione cardiaca del pesce zebra

Imaging a foglio luminoso 4D della contrazione cardiaca del pesce zebra

Valutazione della resistenza al nuoto e del comportamento di nuoto nel pesce zebra adulto

Iniezione intrathoracica per lo studio del cuore di zebrafish adulto