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.

NOTE: Always be sure that IACUC or ethics committee approval is in place before beginning any experimental procedure using zebrafish.
1. Prepare reagents and setup
<ol>
	<li>Prepare the following solutions. Find the recipes for all of these solutions in standard zebrafish manuals10.
	<ul>
		<li>500 ml of Egg Water</li>
		<li>200 ml of 0.03% Tricaine in Egg Water</li>
		<li>100 ml of 1x Phosphate-Buffered Saline (PBS)</li>
		<li>RBC Lysis Buffer (optional)</li>
		<li>Destination buffer of choice (fixative, Trizol, PBS, etc.) in a 1.5 ml microfuge tube on ice 
		NOTE: Fixative and Trizol are toxic if they come into contact with skin. Wear gloves when handling these substances.</li>
	</ul>
	</li>
	<li>Prepare dissection setup:
	<ol>
		<li>Position the gooseneck light source over the dissecting microscope. Place the lid of a 9 cm Petri dish on the dissecting microscope stage, and pour a thin layer of 1x PBS into it (the lid is used because the dish itself is too deep for an easy dissection).</li>
		<li>Place the Petri dish itself (not the lid) on the countertop near the dissecting scope and pour approximately 15 ml of 1x PBS in it. Use this to wash the heart after dissection. Alternatively, use RBC Lysis Buffer.</li>
		<li>Place razor blade, two pairs of forceps, microscissors (optional), and a transfer pipette beside the microscope. Obtain a container of ice if euthanasia by rapid cooling is chosen.</li>
		<li>Pour 200 ml of 0.03% Tricaine in Egg Water into a 250 ml glass beaker if euthanasia by Tricaine is chosen. Prepare a small biohazard bag for disposal of zebrafish carcasses.</li>
	</ol>
	</li>
</ol>
2. Prepare Zebrafish
<ol>
	<li>For fixed fish, bring fixed fish, rinsed in PBS, to the microscope setup.
	<ol>
		<li>Euthanize the whole adult zebrafish by rapid cooling with ice for &gt;10 min.</li>
		<li>Use forceps to make a hole in the skin over the peritoneum (away from the heart) to aid penetration of fixative, and then put them in 4% paraformaldehyde in Fix Buffer10 for 2 hr at RT or O/N at 4 °C.</li>
	</ol>
	</li>
	<li>Alternatively, for fresh fish, place fish to be euthanized in a small holding tank and bring to the microscope setup. Depending on the individual facility’s fish protocol and the downstream uses for the dissected fish hearts, anesthetize fish with Tricaine or euthanize by rapid cooling before decapitation.
	<ol>
		<li>To use ice, pick up one fish with the fish net and place in ice water until the fish stops moving, approximately 5 min.</li>
		<li>Alternatively, to use Tricaine, pick up one fish with the fish net and place in the beaker of the solution until gill movements stop.</li>
	</ol>
	</li>
	<li>Quickly pick up the euthanized fish by its tail fin and lay it on its side in the Petri dish cover that was filled with 1x PBS. Use the forceps to lift a pectoral fin with one hand, while using the razor blade to decapitate the fish with the other hand, just posterior to the attachment of the pectoral fin (Figure 1A). Perform this step either by looking through the microscope or by just directly visualizing the fish on the microscope stage. 
	NOTE: Freshly euthanized fish will still bleed after decapitation.</li>
</ol>
<img alt="Fish dissection process; diagrams of sectioning and analysis of gill structure and anatomy." src="/files/ftp_upload/52248/52248fig1highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Zebrafish adult heart dissection utilizes zebrafish anatomical landmarks. (A) To decapitate the fish, lift the pectoral fin with a forceps and use a sharp clean razor blade along the red dotted line as shown. (B) To steady the fish head, place one tine of the forceps in the fish mouth while the other tine lies across the eye, and then turn the fish head so that the ventral surface is up and both tines of the forceps are stable against the bottom of the Petri dish. (C) Use the free forceps to cut the attachment of the operculum (arrow). (D) Lifting this, the dorsal aorta is visible as a white structure with a pink stripe denoting luminal blood (arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Dissect the Heart
<ol>
	<li>Visualize the fish head under the microscope. With one forceps, steady the fish head by placing one tine of the forceps in the fish mouth, through the brain, while the other tine is outside the head, across the eye (Figure 1B). Ensure both tines of the forceps are steady against the bottom of the petri dish. Holding the fish head this way, its ventral surface should be facing upwards.</li>
	<li>With the other hand, use the second forceps to cut the ventral attachment of the operculum to the body (arrow, Figure 1C). Lifting this slightly with the forceps, the dorsal aorta will appear underneath as a white structure with a red stripe of blood in the aortic lumen (arrow, Figure 1D). Cut the dorsal aorta with the forceps by pinching the aorta and pulling upwards; alternatively, use microscissors to cut the dorsal aorta.</li>
	<li>Now, use the second forceps to grasp the pectoral fin from its base, including the body cartilage at the base of the fin, and lift this off. Repeat with the other pectoral fin. Occasionally, the heart comes out with the pectoral fins, so examine these pieces to make sure the heart is not attached before discarding them. 
	NOTE: At this point, the heart should be visible, still intact and connected to the remaining fish head (although occasionally it comes off with the pectoral fins). The heart can be identified by its shape, its pink color, and its being surrounded by pigmented pericardium. Because euthanasia of the fish is performed quickly, freshly euthanized fish hearts may still be beating slowly.</li>
	<li>Let go of the fish head with the first forceps, so that both hands hold forceps and are free. Use both forceps to tease the heart away from the pericardium. Grasp the heart by the remainder of the dorsal aorta while the remaining pericardium is pulled away. 
	NOTE: Whether fixed or fresh, the heart is robust to gentle pulling while the surrounding connective tissue is more friable. Therefore, any surrounding pericardial tissue can be dissected away with the heart remaining intact.</li>
	<li>Discard the leftover fish carcass in the biohazard bag.</li>
</ol>
4. Prepare the Heart for Downstream Applications
<ol>
	<li>Using forceps, grasp the heart by the edge of the dorsal aorta/bulbus arteriosus and place the heart in the fresh dish of 1x PBS. Alternatively, use a transfer pipette. Move the heart back and forth in the PBS about 10x to wash away as much blood as possible.</li>
	<li>For applications in which it is important to remove all possible blood cells, wash in a 9 cm Petri dish with 15 ml RBC Lysis Buffer instead of PBS, using the same back-and-forth motion. If preserving the structure of the heart is not important for the downstream application (e.g., making RNA), use the forceps or microscissors to open the heart cavity and facilitate rinsing away of blood cells.</li>
	<li>For applications in which separate heart chambers are desired, use forceps or microscissors to dissect the bulbus arteriosus, atrium, and ventricle apart from each other.</li>
	<li>Transfer the heart, or separate chambers, to the destination buffer on ice. 
	NOTE: Common destinations include buffers for cell dissociation, 4% paraformaldehyde, or Trizol.</li>
</ol>

<ol><li>Arnaout, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+model+for+human+long+QT+syndrome%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system%5BTitle%5D)+AND+%22PLoS+Biology%22%5BJournal%5D)">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/pubmed?term=((Getting+to+the+heart+of+regeneration+in+zebrafish%5BTitle%5D)+AND+%22Seminars+in+Cell+%26+Developmental+Biology%22%5BJournal%5D)">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell & Developmental Biology. 18 (1), 36-45 (2007).</li>
<li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1%2FStat3+injury+response+required+for+zebrafish+heart+regeneration%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Fluorescence-activated+cell+sorting+%28FACS%29+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">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/pubmed?term=((Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development%5BTitle%5D)+AND+%22Mechanisms+of+Development%22%5BJournal%5D)">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/pubmed?term=((RNA-Seq%3A+a+revolutionary+tool+for+transcriptomics%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">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/pubmed?term=((Dissection+of+organs+from+the+adult+zebrafish%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Heart+dissection+in+larval%2C+juvenile+and+adult+zebrafish%2C+Danio+rerio%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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://scholar.google.com/scholar?hl=en&safe=off&q=author%3aWesterfield%2C+M+%22The+zebrafish+book%3A+a+guide+for+the+laboratory+use+of+zebrafish+%28Brachydanio+rerio%22">The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio</a>. , University of Oregon Press. Eugene, OR. (1993).</li>
</ol>

The authors have no disclosures.

While methods for dissecting the adult zebrafish heart have been described, these methods were time-consuming and commonly caused damage to the heart during dissection. To perform experiments where a large number of adult hearts may be needed, and/or when avoiding degradation of heart tissue is important for downstream applications, the time required using traditional dissection techniques is prohibitive. Similarly, reproducibly obtaining undamaged, intact hearts is important for study of heart structure and for immunohistochemical and microscopy methods. We found that even in the hands of beginners, 100% of hearts dissected were recovered structurally intact. Overall, the method for adult heart dissection presented here presents an advance in technique that expands the use of adult zebrafish for a variety of current and future applications, including use in heart injury and regeneration experiments.
There are several critical steps within this protocol. First, depending on the experiment for which the zebrafish hearts are required, it is important to rinse hearts as much as possible from blood cells. Examples of experiments that would benefit from removing blood cells include recovery of hearts for RNA-Seq, where presence of blood will alter gene expression profiles, or for FACS sorting, where blood cells may be difficult to sort from myocardial cells. After the fish is euthanized with Tricaine or ice, it is important to decapitate the fish quickly and allow bleeding to occur to help rid the heart of excess blood. If an intact heart is not required for the planned experiment, it can be helpful to cut the atrium and ventricle open when in PBS to further rinse out the blood. RBC lysis buffer can also be used instead of PBS.
Second, as mentioned above, occasionally the heart comes off with one of the pectoral fins rather than staying attached to the head. Therefore, pull the pectoral fins off under the microscope so that the heart can be identified. Finally, when dissecting the heart away from surrounding pericardium, try not to grasp the heart itself; instead, grasp two pieces of pericardium and pull them both away from the heart, which has more structural integrity than the friable pericardium. In this way, even minimal damage to the heart can be avoided.
The heart should be easily identifiable by its shape and contractions. As an additional aid in finding the heart, one can dissect a heart expressing a fluorescent protein under a fluorescence dissecting microscope. This is usually not needed but may be useful for beginners and/or for dissections of very small hearts.
One limitation of this technique is that dissecting hearts from small fish, i.e., younger than 3 weeks old, is difficult; other techniques have been designed for larval dissections9. Also, while it is easy to dissect the bulbus arteriosus, atrium, and ventricle from each other, it is not possible to dissect the atrioventricular valve on its own. The valve remains attached to the ventricle in most cases.

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

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Using this method, an adult zebrafish heart can be dissected in less than 1 min, compared to over 5 min using traditional methods8. Hearts dissected using this method are reliably intact (Figure 2A), while traditional methods8 require cutting blindly into the pericardium and therefore commonly cause damage or loss of the atrium or bulbus arteriosus (Figure 2B). Hearts dissected maintain their structural integrity and are suitable for histology (Figure 2C), electron microscopy (Figure 2D), as well as for applications where the heart is dissociated or pulverized. In our hands, from a dissection of 50 hearts over the course of a week’s experiments, 50 hearts were obtained structurally intact. We also taught this technique to a colleague in the laboratory who had no experience with zebrafish dissection. 9 out of 9 hearts dissected by the beginner colleague were also structurally intact. This dissection method is suitable for juveniles older than 1 month old, and for adults of all ages.
<img alt="Embryonic heart tissue microscopy; histological sections; structural analysis; scale bar 0.5mm, 1µm." src="/files/ftp_upload/52248/52248fig2highres.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Dissected hearts are intact and maintain architectural integrity. (A) In less than 1 min, an adult zebrafish heart with an intact atrium (A), ventricle (V), and bulbus arteriosus (BA) can be dissected from a euthanized fish. (B) Traditional adult zebrafish heart dissections take longer and even when care is taken, run the risk of damage. Here, only the ventricle is intact. (C) A heart dissected from a Tg(myl7dsRed) adult fish maintained its structural integrity for use in histology. The atrium (A), ventricle (V), and bulbus arteriosus (BA) are seen, and even smaller structures like the atrioventricular valve (AV, arrow) and the outflow tract (OFT, arrow) are intact. (D) Electron microscopy of a dissected wild-type adult heart shows that sarcomeres remain undamaged by the dissection process. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52248/52248fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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.

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.3K 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.

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 ...

Developmental Biology

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.

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

Medicine

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.

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.

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

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.

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

Analysis of Zebrafish Cardiac Contraction with Virtual Reality Interaction

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.

Author Spotlight: High-Resolution 4D Light-Sheet Imaging and Virtual Reality in Zebrafish for Single-Cell Analysis of Heart Function

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.

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

Bioengineering

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.

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

Neuroscience

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.

The adult zebrafish heart provides a powerful model in cardiac regeneration research. Although the strength of this system is based on transgenic ...

In Vivo Surface Electrocardiography for Adult Zebrafish

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of zebrafish not only as a research model for human cardiac electrophysiology and myopathies but also as a surrogate model in high throughput pharmaceutical screening for potential cardiotoxicities to humans, such as QT prolongation. As such, in vivo electrocardiography for adult zebrafish is an electrical phenotyping tool that is necessary, if not indispensable, for cross-sectional or longitudinal in vivo electrophysiological characterizations. However, too often, the lack of a reliable, practical, and cost-effective recording method remains a major challenge preventing this in vivo diagnostic tool from becoming more readily accessible. Here, we describe a practical, straightforward approach to in vivo electrocardiography for adult zebrafish using a low-maintenance, cost-effective, and comprehensive system that yields consistent, reliable recordings. We illustrate our protocol using healthy adult male zebrafish of 12-18 months of age. We also introduce a rapid real-time interpretation strategy for quality validation to ensure data accuracy and robustness early in the electrocardiogram recording process.

Here, we present a reliable, minimally invasive, and cost-effective method to record and interpret electrocardiograms in live anesthetized adult zebrafish.

The electrocardiogram waveforms of adult zebrafish and those of humans are remarkably similar. These electrocardiogram similarities enhance the value of ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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