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.

HINWEIS: Achten Sie stets darauf, dass IACUC oder Ethikkommission die Genehmigung ist an Ort und Stelle vor dem Beginn einer Versuchsdurchführung mit Zebrafisch sein. 1. Bereiten Sie Reagenzien und Setup <ol><li> Bereiten Sie die folgenden Lösungen. Finden Sie die Rezepte für alle diese Lösungen in Standard Zebrafisch Handbücher 10. <ul><li> 500 ml Wasser Egg </li><li> 200 ml 0,03% Tricaine in Egg Wasser </li><li> 100 ml 1x phosphatgepufferter Kochsalzlösung (PBS) </li><li> RBC Lysis Buffer (optional) </li><li> Zielpuffer der Wahl (Fixiermittel Trizol, PBS, etc.) in einem 1,5 ml Mikrozentrifugenröhrchen auf Eis HINWEIS: Fixativ und Trizol sind giftig, wenn sie in Kontakt mit der Haut kommen. Tragen Sie Handschuhe beim Umgang mit diesen Stoffen. </li></ul></li><li> Bereiten Dissektion Setup: <ol><li> Stellen Sie den Schwanenhals-Lichtquelle über das Binokular. Setzen Sie den Deckel eines 9 cm Petrischale auf dem Binokular Bühne, und gießen Sie eine dünne Schicht von 1 × PBS iner (der Deckel verwendet wird, weil die Schale selbst ist zu tief für eine einfache Zerlegung). </li><li> Legen Sie die Petrischale selbst (nicht der Deckel) auf der Arbeitsplatte in der Nähe des Binokular und gießen Sie etwa 15 ml 1x PBS darin. Verwenden Sie diese Option, um das Herz nach der Sektion zu waschen. Alternativ können Sie RBC Lysis Buffer. </li><li> Ort Rasierklinge zwei Zangenpaaren, Mikroscheren (optional) und eine Transferpipette neben dem Mikroskop. Besorgen Sie sich einen Behälter mit Eis, wenn Euthanasie durch schnelles Abkühlen gewählt wird. </li><li> Gießen 200 ml von 0,03% Tricaine in Egg Wasser in einen 250 ml Glasbecher, wenn Euthanasie durch Tricaine gewählt wird. Bereiten Sie eine kleine Biohazard Sack zur Dekontaminierung von Zebrafisch-Kadaver. </li></ol></li></ol> 2. Bereiten Sie Zebrafisch <ol><li> Für feste Fisch, bringen festen Fisch, in PBS gespült, mit dem Mikroskop-Setup. <ol><li> Euthanize die ganze erwachsenen Zebrafisch durch schnelles Abkühlen mit Eis für&gt; 10 min. </li><li> Verwenden einer Pinzette, um ein Loch in der Haut über das Bauchfell machen(Vom Herzen), um das Eindringen von Fixiermittel zu unterstützen, und sie dann in 4% Paraformaldehyd in Fix-Puffer 10 für 2 h bei RT oder O / N bei 4 ° C. </li></ol></li><li> Alternativ für frischen Fisch, statt Fisch in einem kleinen Vorratstank eingeschläfert werden und bringen auf den Mikroskopaufbau. Je nach Fisch Protokoll der individuellen Einrichtung und die nachgelagerten Anwendungen für die seziert Fischherzen, betäuben Fisch mit Tricaine oder einschläfern durch schnelles Abkühlen, bevor Enthauptung. <ol><li> Um Eis zu verwenden, nehmen Sie einen Fisch mit dem Fisch net und in Eiswasser, bis der Fisch nicht mehr bewegt, ca. 5 min. </li><li> Alternativ zu Tricaine verwenden, nehmen Sie einen Fisch mit dem Fischnetz und in das Becherglas von der Lösung, bis Kiemenbewegungen zu stoppen. </li></ol></li><li> Holen schnell auf die eingeschläfert Fisch durch seine Schwanzflosse und legen Sie sie auf die Seite, in der Petrischale Abdeckung, die mit 1x PBS gefüllt war. Verwenden Sie die Pinzette, um eine Brustflosse mit einer Hand zu heben, während der Verwendung der razor Klinge, um die Fische mit der anderen Hand zu köpfen, nur zur Befestigung der Brustflosse (1A) posterior. Führen Sie diesen Schritt, indem Sie entweder dem Mikroskop oder durch direkt Visualisierung der Fisch auf dem Mikroskoptisch. HINWEIS: frisch getöteten Fischen noch nach Enthauptung bluten. </li></ol><img alt="Figur 1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> Abbildung 1: Zebrafisch erwachsenen Herzen Dissektion nutzt Zebrafisch anatomischen Landmarken. (A), um die Fische zu enthaupten, heben Sie den Brustflosse mit einer Pinzette und mit einem scharfen Rasierklinge entlang der rot gestrichelten Linie, wie dargestellt. (B), um die Fischkopf zu stabilisieren, legen Sie die Zinken der Zange im Fischmaul während der andere Zinken liegt über das Auge, und drehen Sie den Fischkopf, so dass die Bauchseite ist und die beiden Zinken der Zange sind gegen den Boden der Petrischale stabil. (C) Verwenden Sie die kostenlose forceps, um die Befestigung der Kiemendeckel (Pfeil). (D) Deinstallations diese geschnitten, als weißer Struktur mit einem rosa Streifen bezeichnet Lumen Blut (Pfeil) zu erkennen ist die dorsale Aorta. <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> 3. Präparieren Sie die Herz <ol><li> Visualisieren Sie die Fischkopf unter dem Mikroskop. Mit einer Pinzette, beruhigen die Fischkopf, indem Sie eine Zinken der Zange im Fischmaul, durch das Gehirn, während die andere Zinke ist außerhalb des Kopfes, über das Auge (Abbildung 1B). Sicherzustellen, dass beide Zinken der Zange sind stabil gegen den Boden der Petrischale. Halten der Fischkopf auf diese Weise, sollte seine Bauchseite nach oben zeigen. </li><li> Mit der anderen Hand verwendet man die zweite Pinzette zum ventralen Befestigung des Verschlusses an dem Körper (Pfeil, 1C) geschnitten. Heben Sie diese leicht mit der Pinzette, die dØrsal Aorta wird unterhalb als weißer Aufbau mit einem roten Streifen von Blut in den Aortenlumen (Pfeil, 1D) angezeigt. Schneiden Sie die dorsale Aorta mit der Zange durch Zusammendrücken der Aorta und nach oben ziehen; alternativ können Sie Mikroschere, um die dorsale Aorta geschnitten. </li><li> Verwenden Sie nun die zweite Zange, um die Brustflosse von seiner Basis zu erfassen, einschließlich des Körpers Knorpel an der Basis der Flosse, und heben Sie diese ab. Wiederholen Sie mit der anderen Brustflosse. Gelegentlich kommt das Herz mit den Brustflossen, so prüfen, diese Stücke um sicherzustellen, dass das Herz nicht vor dem Wegwerfen ihnen verbunden. HINWEIS: An dieser Stelle sollte das Herz sichtbar, noch intakt und mit dem restlichen Fischkopf verbunden sein (obwohl es gelegentlich kommt mit den Brustflossen). Das Herz kann durch seine Form, seine rosa Farbe, und sein Wesen durch pigmentierte Herzbeutel umgeben identifiziert werden. Da die Euthanasie der Fische wird schnell ausgeführt wird, können frisch getöteten Fischen Herz noch schlägt slo seinWLY. </li><li> Lassen Sie den Fischkopf mit der ersten Zange, so dass beide Hände halten Pinzette und sind kostenlos. Benutzen Sie beide Zangen, um das Herz von der Herzbeutel zu necken. Fassen Sie die Herzen durch den Rest der dorsalen Aorta während die restlichen Herzbeutel weggezogen. HINWEIS: Ob fest oder frisch, ist robust leichtes Ziehen, während das umgebende Bindegewebe ist brüchig das Herz. Daher kann jeder umgebenden Herzbeutelgewebe weg mit das Herz intakt bleibt seziert werden. </li><li> Entsorgen Sie die übrig gebliebenen Fisch Karkasse in der biohazard Tasche. </li></ol> 4. Bereiten Sie den Inneres für Downstream-Anwendungen <ol><li> Mit einer Pinzette, fassen Sie die Herzen durch den Rand der dorsalen Aorta / Bulbus arteriosus und legen Sie die Herzen in der frischen Schüssel mit 1x PBS. Alternativ können Sie einen Transferpipette. Bewegen Sie die Herzen in der PBS und zurück zu 10-fach zu wie möglich weg zu waschen, so viel Blut. </li><li> Für Anwendungen, bei denen es wichtig ist, alle possib entfernenle Blutkörperchen, Waschen in einer 9 cm Petrischale mit 15 ml RBC Lysispuffer anstelle von PBS mit derselben hin- und herbewegt. Wenn die Erhaltung der Struktur des Herzens ist nicht wichtig für die nachgeschaltete Anwendung (zB, was RNA), mit den Zangen oder Mikroscheren, um die Herzhohlraum zu öffnen und zu erleichtern Abspülen von Blutzellen. </li><li> Für Anwendungen, bei denen separate Herzkammern gewünscht werden, verwenden Pinzette oder Mikroschere, um den Bulbus arteriosus, Vorhof und Herzkammer voneinander zu sezieren. </li><li> Übertragen Sie das Herz, oder getrennte Kammern, in die Zielpuffer auf Eis. HINWEIS: Gemeinsame Ziele sind Puffer für Zelldissoziation, 4% Paraformaldehyd oder 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.

Während Verfahren zum Präparieren des erwachsenen Zebrafisch Herzen beschrieben worden sind, sind diese Methoden zeitaufwendig und häufig verursacht beim Präparieren von Schädigungen des Herzens. Experimente, wo eine große Anzahl von erwachsenen Herzen erforderlich sein, führen und / oder wenn die Vermeidung von Abbau von Herzgewebe ist wichtig für die Downstream-Anwendungen ist die Zeit unter Verwendung traditioneller Techniken Dissektion erforderlich unerschwinglich. Ebenso ist reproduzierbar erhalten unbesch?...

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

Verwendung des Zebrafisch-Modellsystem für die Untersuchung der Entwicklung, Regeneration und Krankheit wird in Richtung der Anwendung erwachsenen Herzen für Zelldissoziationslösung und Aufreinigung von RNA, DNA und Proteinen erweitert. Alle diese Anwendungen fordern die rasche Erholung der eine erhebliche Anzahl von Zebrafisch-Herz genregulatorischen, Stoffwechsel und andere Veränderungen, die nach dem Tod beginnen zu vermeiden. Erwachsenen Zebrafisch Herzen sind auch zur Untersuchung von Herzstruktur für eine Vielzahl von Mutanten und zur Untersuchung der Herzregeneration erforderlich. Jedoch ist die herkömmliche Zebrabärbling Herz Dissektion langsam und schwierig und erfordert Spezialwerkzeuge, so dass großflächige Dissektion erwachsenen Zebrafisch Herzen mühsam. Traditionelle Verfahren bergen auch das Risiko einer Beschädigung des Herzens während der Präparation. Hier beschreiben wir eine Methode zur Präparation der erwachsenen Zebrafisch Herzen, schnell, reproduzierbar ist, und bewahrt Herzen Architektur. Außerdem funktioniert diese Methode nicht verlangen Spezialwerkzeugen, ist schmerzfrei für den Zebrafisch,kann auf frischem oder feste Proben durchgeführt werden und kann an Zebrafischen im Alter von 1 Monat alt durchgeführt werden. Die beschriebene Vorgehensweise erweitert die Verwendung von erwachsenen Zebrafisch für Herz-Kreislauf-Forschung.

Unter Verwendung dieses Verfahrens kann ein Erwachsener Zebrabärbling Herz in weniger als 1 min präpariert werden, im Vergleich zu über 5 min mit traditionellen Methoden 8. Herz seziert mit dieser Methode sind zuverlässig intakt (2A), während die traditionellen Methoden 8 erfordern Schneiden blindlings in den Herzbeutel und damit häufig Beschädigung oder Verlust von Atrium oder Bulbus arteriosus (2B) verursachen. Herz präpariert behalten ihre strukturelle I...

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.

Rückgewinnung von Adult Zebrafisch-Herzen für Hochdurchsatz-Anwendungen

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 Aufrufe.  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.

Serie: 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.

Großzebrafisch Embryonale Herz Dissection für Transkriptionsanalyse

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 ...

Forschung

Entwicklungsbiologie

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.

Induktion eines Myokardinfarktes in Adult Zebrafisch Mit Cryoinjury

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

Medizin

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.

Hochfrequenz-Ultraschall-Echokardiographie zur Beurteilung der Herzfunktion von Zebrafischen

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.

Herz Dissection in Larven, juvenile und adulte Zebrafisch, 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 ...

Biologie

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.

Immunfärbung von Dissected Zebrafisch embryonale Herz

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

Analyse der Herzkontraktion von Zebrafischen mit Virtual-Reality-Interaktion

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 im Rampenlicht: Hochauflösende 4D-Lichtblatt-Bildgebung und Virtual Reality im Zebrafisch zur Einzelzellanalyse der Herzfunktion

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.

4D-Lichtblatt-Bildgebung der Herzkontraktion von Zebrafischen

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

Biotechnik

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.

Beurteilung der Schwimmausdauer und des Schwimmverhaltens bei erwachsenen Zebrafischen

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

Neurowissenschaften

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.

Rückgewinnung von Adult Zebrafisch-Herzen für Hochdurchsatz-Anwendungen

Zusammenfassung

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Immunfärbung von Dissected Zebrafisch embryonale Herz

4D-Lichtblatt-Bildgebung der Herzkontraktion von Zebrafischen

4D-Lichtblatt-Bildgebung der Herzkontraktion von Zebrafischen

4D-Lichtblatt-Bildgebung der Herzkontraktion von Zebrafischen

Beurteilung der Schwimmausdauer und des Schwimmverhaltens bei erwachsenen Zebrafischen

Intrathorakische Injektion für die Erforschung des erwachsenen Zebrafischherz