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.

注：常に動物実験委員会または倫理委員会の承認は、ゼブラフィッシュを使用して、任意の実験手順を開始する前に、所定の位置にあることを確認してください。 1.試薬およびセットアップを準備<ol><li>以下のソリューションを準備します。標準のゼブラフィッシュマニュアル10におけるこれらのソリューションのすべてのレシピを検索します。 <ul><li>卵水500mlの</li><li>卵、水で0.03％トリカインの200ミリリットル</li><li> 1×リン酸緩衝生理食塩水（PBS）100ml中</li><li> RBC溶解バッファー（オプション） </li><li>氷上の1.5mlマイクロチューブ中の選択肢の宛先バッファ（固定液、トリゾール、PBS など ） 注：彼らは皮膚に接触した場合に固定液とトリゾールは有毒である。これらの物質を取り扱う際は手袋を着用してください。 </li></ul></li><li>解剖のセットアップを準備します。 <ol><li>解剖顕微鏡上グースネック光源を配置します。解剖顕微鏡のステージ上で9センチメートルシャーレの蓋を置き、中に1×PBSの薄い層を注ぐそれ（ディッシュ自体は簡単に解剖のために深すぎるので、蓋が使用されている）。 </li><li>解剖スコープの近くにカウンター上のペトリ皿自体（いない蓋）を配置し、その中に1×PBSの約15ミリリットルを注ぐ。解剖後に心臓を洗浄するために使用します。また、RBC溶解バッファーを使用しています。 </li><li>置きかみそりの刃、顕微鏡の横に鉗子、microscissors（オプション）、および転送ピペットの2組。急速冷却による安楽死を選択した場合の氷のコンテナを取得します。 </li><li>トリカインによって安楽死を選択した場合250mlのガラスビーカーに卵水中の0.03％トリカインを200ml注ぐ。ゼブラフィッシュ死骸の処分のための小さなバイオハザードバッグを準備します。 </li></ol></li></ol> 2.ゼブラフィッシュを準備<ol><li>固定魚の場合は、顕微鏡のセットアップに、PBSですすぎ、固定された魚を、持って来る。 <ol><li> &gt; 10分間氷で急冷することによって全体の大人のゼブラフィッシュを安楽死させる。 </li><li>腹膜上の皮膚に穴を作るために鉗子を使用して、（心臓から）固定液の浸透を補助するため、その後、4℃でRTまたはO / Nで2時間修正バッファ10に4％パラホルムアルデヒドでそれらを置くために。 </li></ol></li><li>また、新鮮な魚のために、小さな保持タンクに安楽死させ、顕微鏡のセットアップにもたらすことが魚を置く。個々の施設の魚のプロトコルと解剖魚の心のために下流の用途に応じて、トリカインで魚を麻酔または断頭前に急速に冷却することにより安楽死させる。 <ol><li>魚は、約5分の移動を停止するまで、氷を使用するには、氷水で魚網と場所で1魚を拾う。 </li><li>鰓の動きが停止するまでまたは、トリカインを使用するために、溶液のビーカー内の魚網と場所で1魚を拾う。 </li></ol></li><li>すぐにそのテールフィンにより安楽死させた魚をピックアップし、1×PBSで満たしたペトリ皿のカバーでその側にそれを置く。 RAZOを使用しながら、片手で胸びれを持ち上げるために鉗子を使用して、Rもう一方の手で魚を斬首するブレード、ちょうど胸びれ（ 図1A）の結合を後方。顕微鏡を通して見ることによって、または単に直接顕微鏡ステージ上の魚を視覚化することによってどちらか、この手順を実行します。 注：新鮮な安楽死魚はまだ断頭後に出血します。 </li></ol><img alt="図1" src="/files/ftp_upload/52248/52248fig1highres.jpg" /> 図1.ゼブラフィッシュの大人の心臓解剖は、ゼブラフィッシュ、解剖学的ランドマークを利用しています。 （A）は 、魚を斬首ピンセットで胸びれを持ち上げ、図のように赤の点線に沿ってシャープなきれいなカミソリの刃を使用します。（B）、魚の頭を安定魚の口のしばらく鉗子の1歯を配置するには他の歯は、目全体に位置し、腹面がアップしていると鉗子の両方の歯は、ペトリ皿の底に対して安定しているように、その後魚の頭を回す。（C）無料FORCを使用してくださいEPSは蓋（矢印）。（D）は 、このリフティングの添付ファイルをカットする、背側大動脈を管腔血液（矢印）を示すピンクのストライプと白の構造として表示されている。 <a href="https://www.jove.com/files/ftp_upload/52248/52248fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 3.ハートを解剖<ol><li>顕微鏡下で魚​​の頭を可視化。他の歯は、目（ 図1B）全体で、頭部の外にある間に1鉗子で、脳を通して、魚の口の中で鉗子の1歯を配置することにより、魚の頭を安定。鉗子の両方の歯は、ペトリ皿の底に対して安定していることを確認してください。魚の頭をこのように保持し、その腹面を上向きにする必要があります。 </li><li>もう一方の手で、体（矢印、 図1C）に蓋の腹側添付ファイルをカットする第二鉗子を使用しています。鉗子で少しこのリフティング、Dorsalの大動脈、大動脈内腔（矢印、 図1D）の血液の赤のストライプと白の構造体としての下に表示されます。大動脈をつまんで上向きに引いて、ピンセットで背側大動脈をカット。代わりに、背側大動脈をカットするmicroscissorsを使用しています。 </li><li>さて、フィンのベースのボディ軟骨を含む、そのベースから胸びれを把握することが第二の鉗子を使用し、これをオフに持ち上げます。他の胸鰭を繰り返します。時折、心臓は胸びれで出てくるので、心がそれらを廃棄する前に接続されていないことを確認するこれらの作品を調べます。 注：この時点で、心臓は（時にはそれが胸びれで外れが）まだそのまま、残りの魚の頭に接続され、表示されるはずです。心臓は、その形状、そのピンク色、および色素性心膜に囲まれた人間によって同定することができる。魚の安楽死を迅速に実行されているので、新鮮に安楽死魚の心はまだSLOを破っできるWLY。 </li><li>両手が鉗子を保持し、自由であるように、最初の鉗子で魚の頭を手放す。離れて心膜から心をいじめるために両方の鉗子を使用してください。残りの心膜が引き離されている間背部大動脈の残りの部分によって心をつかみます。 注：固定または新鮮かどうか、心が周囲の結合組織はよりもろい状態で引っ張って穏やかに堅牢である。そのため、周囲の心膜組織は、心臓が無傷で残っていると離れて解剖することができます。 </li><li>バイオハザードバッグ内の残りの魚の死体を捨てる。 </li></ol> 4.ダウンストリームアプリケーションのハートを準備<ol><li>鉗子を使用して、背側大動脈/動脈球の縁によって心をつかみ、1×PBSの新鮮な皿に心を置く。また、転送ピペットを使用しています。約10倍はできるだけ多くの血を洗い流すために、PBS中で前後に心を移動します。 </li><li>それはすべてpossibを除去することが重要である用途のためにルの血液細胞は、同一の往復運動を使用して、代わりにPBSを15mlのRBC溶解バッファーと9センチメートルシャーレに洗う。心臓の構造を維持すること（ 例えば、RNAする）、心臓腔を開いて離れ、血液細胞のすすぎ容易にするために、鉗子またはmicroscissorsを使用する下流側のアプリケーションのために重要でない場合。 </li><li>心腔を分離する用途では、所望の動脈球を分析するために鉗子またはmicroscissorsを使用し、心房、および互いに離間心室される。 </li><li>氷の上の宛先バッファに、心臓、または別々のチャンバを転送します。 注：一般的な目的地は、細胞解離、4％パラホルムアルデヒド、またはトリゾールための緩衝液を含む。 </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.

成体ゼブラフィッシュの心臓を切開するための方法が記載されているが、これらの方法は時間がかかりました、一般に切開の間に心臓への損傷を引き起こした。成人の心臓の多数必要になることが実験を実行するために、および/または心臓組織の劣化を回避することが下流の適用のために重要である場合、従来の切開技術を使用してかなりの時間が必要である。同様に、再現可能に損傷を受...

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

発達、再生、および疾患を研究するためのゼブラフィッシュモデル系の使用はRNA、DNA、およびタンパク質の細胞分離および精製のための大人の心の使用に向けて拡大している。これらのアプリケーションはすべて、調節遺伝子、代謝、および死後に始まる他の変更を避けるために、ゼブラフィッシュの心のかなりの数の急速な回復を要求する。大人のゼブラフィッシュの心も、突然変異体のさまざまな心臓の構造を研究するため、心臓の再生を研究するために必要とされる。しかし、従来のゼブラフィッシュの心臓解剖が遅いとは困難であり、退屈な大人のゼブラフィッシュの心の大規模な解剖をする、特殊なツールが必要です。伝統的な方法はまた、切開中に心臓を損傷する危険性を保有する。ここでは、高速で再現性があり、心臓のアーキテクチャを保持成体ゼブラフィッシュの心臓の解剖するための方法を記載する。さらに、この方法は、特殊なツールを必要としない、ゼブラフィッシュのために無痛である、新鮮なまたは固定された試料で行うことができ、生後1ヶ月の幼いゼブラフィッシュで行うことができる。記載されたアプローチは、心血管研究のための大人のゼブラフィッシュの使用を拡大する。

この方法を用いて、成体ゼブラフィッシュの心臓は、伝統的な方法8を用いて5分間かけて比較して、1分未満で解剖することができる。ハーツは、この方法を使用して解剖し、従来の方法8は心膜に盲目的に切断が必要なので、一般的にアトリウムまたは球部動脈（ 図2B）の損傷または損失の原因となりながら、確実に（ 図2A）は無傷である。切開した?...

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.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

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 ビュー.  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.

ビデオ: 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.

転写分析のための大規模ゼブラフィッシュ胚心臓解剖

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 ...

発生生物学

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.

Cryoinjuryを使用した大人のゼブラフィッシュにおける心筋梗塞の誘発

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

医学

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.

ゼブラフィッシュ心臓機能を評価する高周波超音波心エコー検査

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.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

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

生物学

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.

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

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

バーチャルリアリティインタラクションによるゼブラフィッシュ心収縮の解析

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.

著者スポットライト:Zebrafishの高分解能4Dライトシートイメージングとバーチャルリアリティによる心機能のシングルセル解析

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.

ゼブラフィッシュの心臓収縮の4Dライトシートイメージング

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

生物工学

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.

成体ゼブラフィッシュの水泳持久力と水泳行動の評価

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

神経科学

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.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

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転写分析のための大規模ゼブラフィッシュ胚心臓解剖

Cryoinjuryを使用した大人のゼブラフィッシュにおける心筋梗塞の誘発

ゼブラフィッシュ心臓機能を評価する高周波超音波心エコー検査

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

ゼブラフィッシュの心臓収縮の4Dライトシートイメージング

ゼブラフィッシュの心臓収縮の4Dライトシートイメージング

ゼブラフィッシュの心臓収縮の4Dライトシートイメージング

成体ゼブラフィッシュの水泳持久力と水泳行動の評価

成人ゼブラフィッシュハートの研究のための胸腔内圧注射