Name: a doxorubicin-induced cardiomyopathy model in adult zebrafish
Uploaded: 2018-06-07T13:30:00.000+00:00
Duration: 8 min 9 s
Description: Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

この作品は、アメリカの中心連合からの科学者の開発補助金による部分で支えられました (YD に 14SDG18160021)、米国 NIH R01 付与 HL 81753 と HL 107304 xx、XX にメイヨー財団。

ここで説明したすべての手順を行ったガイドに従ってケアおよび実験動物の使用 (国立アカデミー プレス 2011), とメイヨー クリニック機関動物ケアおよび使用委員会によって承認されました。
1. 大人のゼブラフィッシュの準備
<ol><li>DOX の注入に必要な総魚の 2 倍多くを少なくとも取得するタンクを横切る際に十分な繁殖ペアを設定します。異なる遺伝的背景を持つ魚を比較すると、同じ週に年齢をマッチさせたコントロールを確保するためにすべての魚を繁殖します。</li>
	<li>収集魚胚次の朝、100 mm のペトリ皿に転送、28.5 ° C の定温器でそれらを保ちます。低密度で胚を維持 (< 100 胚/シャーレ)。</li>
	<li>性の不均衡を避けるために胚の水を毎日をリフレッシュし、手動で転送ピペットを使用してタイムリーに死んだ卵を削除します。</li>
	<li>各タンクに胚の同じ数を入れます (たとえば、60 胚/3 L 型中戦車が最初になる) 密度をマッチさせたコントロールを確保するため。</li>
	<li>ゾウリムシの 4 日後受精 (dpf) で供給を開始します。</li>
	<li>幼若期の間に毎日魚を検査します。似たような魚を確保するため必要に応じて魚の数を調整する密度。</li>
	<li>魚が 4 週齢に達したら、さらなる成長のため各新しい 3 L 中型タンクに最大 20 の魚を転送します。ライブに孵化したブラインシュリンプを魚の餌に開始します。</li>
</ol>2. 準備中と DOX 原液の貯蔵
注: DOX は様々 なバイオ企業から購入できます。化合物は、暗い茶色の容器に粉体として通常取得されます。
<ol><li>徹底的に最終濃度 5 mg/mL の原液として、目に見える塊がないように脱イオン水中で DOX 粉を溶解します。DOX 在庫安全ロックにある各 1.5 mL のチューブに分注 1 mL。軽い露出から、DOX を保護するアルミ箔紙に 1.5 mL チューブをラップします。 注: は、化学のフードでこの手順を実行します。</li>
	<li>ストレージのための 4 ° C で DOX 原液を維持します。長期保存のため (> 4 週間) DOX 原液の以下省略可能セクションの 3 を実行します。</li>
</ol>3。 DOX ゼブラフィッシュ胚 (オプション) を使用しての品質管理
注: 拡張ストレージ後 DIC をモデル化するための薬の有効性を失うことができるので、DOX は両方湿気と光感受性、です。DOX は、さまざまな企業、または同じ会社からもさまざまなバッチから購入、成魚の実験を行う前に野生型 (WT) ゼブラフィッシュ胚を用いたその薬効を調整する便利です。このメソッドは、報告されたゼブラフィッシュ胚性 DIC モデル21から派生されます。
<ol><li>魚の少なくとも 2 つのペアから WT ゼブラフィッシュ胚を収集します。マイクロ針と注射器を使用して手動で 24 時間後受精 (hpf) で Dechorionate 胚。また、10 μ g/mL の 30 の ° C の定温器で 10-15 分のための最終的な集中にプロテイナーゼ K を持つ胚を扱います。Dechorionation 後胚水を更新します。死胚を削除し、各バッチの少なくとも 36 の胚を維持します。</li>
	<li>新鮮な胚水 100 μ M の最終的な集中に DOX 原液を希釈します。ソリューションのボリュームは、すべての 3 の胚のため 100 μ L です。渦による DOX 薄めたをミックスします。最終希釈液は、赤色光をする必要があります。</li>
	<li>100 μ L/ウェルの 96 ウェルきれいな透明板を薄めた DOX を追加します。</li>
	<li>3 dechorionated 胚転送プラスチック ピペット、ピペット チップの終わり近くに胚をようにましょう。DOX ソリューションの各ウェルにピペット チップを置きます。胚は井戸に泳ぐことができ、ソリューションに触れる先端の終わりをしましょう。 注: 手動で同様に多くの水を追加し、希薄 DOX 溶液胚を押すことを避けます。</li>
	<li>48 で DOX ソリューションを更新 hpf。この時点で、浮腫を伴う死胚 (ハートビートの停止) または胚を識別するために 10 倍の倍率での顕微鏡の下の井戸を観察します。カウントし、タイムリーなファッションは、それ以外の場合に死胚と液残りの胚がすぐに死ぬことができるだけでなく任意の死胚を削除します。</li>
	<li>72 で胚を確認 hpf とそれらを数えます。DOX の治療は、「良い薬効」場合と見なされます > 胚の両方のバッチで 25% 死 (ハートビートの停止) を観察できます。</li>
</ol>4. 事前注入準備
注: 生後 6 ヶ月に 8 週間の魚は、DOX の注入に使用されます。注入する成熟した野生インド核型 (WIK) 魚の体重 (BWs) の範囲から 0.2 0.5 g。
<ol><li>高速で射出する前に 24 時間の魚。</li>
	<li>0.16 mg/mL tricaine を含む胚水で魚を麻酔します。体の両側から水を乾燥してきれいなフィルター ペーパーを使用します。規模でそれぞれの魚の帯域幅を測定します。後注入のために BW で 10% の差で魚をグループ化できます。 注: この手順で作業負荷を最小限に抑える、BW で 10% の差で魚と見なされます同じサイズしたがって、BW の平均によると 1 つの DOX 作業ソリューションを準備します。</li>
	<li>ソリューションの 5 μ L で各の成魚を注入する予定です。魚の数、BWs によると DOX 作業濃度を計算します。 注: を 6 ヶ月間慢性毒性を勉強するには、20 mg/kg の用量で DOX を使用します。DOX の急性心毒性を調査するには、50 mg/kg BW の DOX 線量を増やすことができます。</li>
	<li>1 ハンクの平衡塩溶液 (HBSS) 対応する作業濃度 x DOX 株式を希釈します。ソリューションをミックスする渦。暫時スピンダウンしてソリューションを収集します。</li>
</ol>5. 成魚で DOX 注入
<ol><li>場所きれいな 100 mm ペトリ皿を解剖顕微鏡の下で内部にスポンジは、フォーカスを調整します。魚を保持する長さ約 4 cm の空洞にスポンジをカットします。大きな魚の長い空洞を作る。</li>
	<li>10 μ L マイクロ注射器で 34 G の針を準備します。任意の泡とブロック注射器とチューブから削除する 1 x HBSS バッファーで針をすすいでください。</li>
	<li>2 分 0.16 mg/mL tricaine を含む胚水の成魚を麻酔します。 注: 長期 anesthetization DOX 注入後 5 分以上は簡単に魚を引き起こすことができます死。</li>
	<li>Tricaine、胚水でスポンジを浸し、転送を注射用スポンジ魚。</li>
	<li>
下記 2 つの方法のいずれかで IP DOX の注入を実行します。
	<ol>
<li>古典的な IP 注入29<ol>
<li>スポンジの空洞を腹部に魚を置きます。すぐに挿入針、45 ° 骨盤フィンの間の正中線に角度魚体に浸透約 1 2 ミリメートル。 はゆっくりとすべての DOX ソリューションをリリースします。針を抜く前に待機 5 s。魚の腹に見える赤い色で DOX 配信を確認してください。</li>
		</ol>
</li>
		<li>代替 IP 注入<ol>
<li>横方向を右側に前方にスポンジ魚をプレイス.優しく鈍い端鉗子を使用して、左の手で魚を安定させるし、右手でマイクロ シリンジを保持します。</li>
			<li>上向き傾斜面で骨盤鰭の上横行下針を配置します。45 ° の角度で 7 位置を指して、骨盤とアナルのフィンの間に位置する魚の空洞に 3-4 mm の針を挿入し、ゆっくりとピストンを低下させます。魚の腹に見える赤い色で DOX 配信を確認してください。</li>
		</ol>
</li>
	</ol>
</li>
	<li>すばやく回復する魚を許可するように新鮮なシステムで水で満ちているきれいな交差タンクに注入された魚を転送します。注射の間 1 x HBSS バッファーに一度針をすすいでください。</li>
</ol>6. 投与後魚管理
<ol><li>注入後、魚を循環を実行しているシステムに戻ります。可能であれば、循環を共有する別のタンク間のクロス汚染を避けるために、メイン ・ システムから個別に DOX 扱われる魚を維持します。</li>
	<li>回復のための別の 24 時間注入された魚を高速です。最初の週の間に毎日魚を観察します。他の魚への感染を避けるために適切なタイミングで死んだ魚を削除します。 注: 最初の 24 時間以内の魚の死亡は、注射による物理的な病変が原因可能性があります。</li>
	<li>さらに長期観察の DOX 重点を置かれた魚を維持します。タンクの他の魚に感染症を避けるために時間で死んだ魚を削除します。 注: 魚の数は、生存曲線を生成する記載されています。</li>
	<li>表現型心エコー法30、心機能レポーター遺伝子組換え行23、水泳チャレンジ26, と他の病理学的改造の定量化など、DOX 強調した魚に異なる実験的アッセイを使用します。マーカー23。</li>
</ol>

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D., Eames, S. C., Philipson, L. H., Prince, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+injection+into+adult+zebrafish.">Intraperitoneal injection into adult zebrafish.</a> J Vis Exp. (42), (2010).</li><li>Wang, L. W., 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=Standardized+echocardiographic+assessment+of+cardiac+function+in+normal+adult+zebrafish+and+heart+disease+models.">Standardized echocardiographic assessment of cardiac function in normal adult zebrafish and heart disease models.</a> Dis Model Mech. 10 (1), 63-76 (2017).</li><li>Desai, V. G., 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=Development+of+doxorubicin-induced+chronic+cardiotoxicity+in+the+B6C3F1+mouse+model.">Development of doxorubicin-induced chronic cardiotoxicity in the B6C3F1 mouse model.</a> Toxicol Appl Pharmacol. 266 (1), 109-121 (2013).</li><li>Zhu, W., Shou, W., Payne, R. M., Caldwell, R., Field, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+juvenile+doxorubicin-induced+cardiac+dysfunction.">A mouse model for juvenile doxorubicin-induced cardiac dysfunction.</a> Pediatr Res. 64 (5), 488-494 (2008).</li><li>Chatterjee, K., Zhang, J., Honbo, N., Karliner, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin+cardiomyopathy.">Doxorubicin cardiomyopathy.</a> Cardiology. 115 (2), 155-162 (2010).</li><li>Bang, 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=Cardiac+fibroblast-derived+microRNA+passenger+strand-enriched+exosomes+mediate+cardiomyocyte+hypertrophy.">Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy.</a> J Clin Invest. 124 (5), 2136-2146 (2014).</li><li>Rassaf, T., Kelm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protection+from+diabetic+cardiomyopathy+-+putative+role+of+the+retinoid+receptor-mediated+signaling.">Protection from diabetic cardiomyopathy - putative role of the retinoid receptor-mediated signaling.</a> J Mol Cell Cardiol. 59, 179-180 (2013).</li><li>Wahbi, K., 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=Dilated+cardiomyopathy+in+patients+with+mutations+in+anoctamin+5.">Dilated cardiomyopathy in patients with mutations in anoctamin 5.</a> Int J Cardiol. 168 (1), 76-79 (2013).</li><li>Zhou, M. D., Sucov, H. M., Evans, R. M., Chien, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoid-dependent+pathways+suppress+myocardial+cell+hypertrophy.">Retinoid-dependent pathways suppress myocardial cell hypertrophy.</a> Proc Natl Acad Sci U S A. 92 (16), 7391-7395 (1995).</li><li>Hershman, D. L., 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=Doxorubicin,+cardiac+risk+factors,+and+cardiac+toxicity+in+elderly+patients+with+diffuse+B-cell+non-Hodgkin's+lymphoma.">Doxorubicin, cardiac risk factors, and cardiac toxicity in elderly patients with diffuse B-cell non-Hodgkin's lymphoma.</a> J Clin Oncol. 26 (19), 3159-3165 (2008).</li><li>Silber, J. H., Barber, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+cardiotoxicity.">Doxorubicin-induced cardiotoxicity.</a> N Engl J Med. 333 (20), 1359-1360 (1995).</li><li>Von Hoff, D. D., 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=Risk+factors+for+doxorubicin-induced+congestive+heart+failure.">Risk factors for doxorubicin-induced congestive heart failure.</a> Ann Intern Med. 91 (5), 710-717 (1979).</li><li>Pugach, E. K., Li, P., White, R., Zon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injection+in+adult+zebrafish.">Retro-orbital injection in adult zebrafish.</a> J Vis Exp. (34), (2009).</li><li>Zang, L., Morikane, D., Shimada, Y., Tanaka, T., Nishimura, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protocol+for+the+oral+administration+of+test+chemicals+to+adult+zebrafish.">A novel protocol for the oral administration of test chemicals to adult zebrafish.</a> Zebrafish. 8 (4), 203-210 (2011).</li><li>Collymore, C., Rasmussen, S., Tolwani, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gavaging+adult+zebrafish.">Gavaging adult zebrafish.</a> J Vis Exp. (78), (2013).</li></ol>

著者は利益相反行為を宣言しません。

進歩的な DIC をモデルに DOX の 20 の mg/kg の線量だった魚の死の 1 の wpi がまだ結果の間に重要な魚の死と 4 wpi (図 3および後心機能の低下が発生しない最高の線量として実験的に決定図 4)。この線量はこれらの齧歯動物の DIC モデル (15 〜 25 mg/kg) で頻繁に使用して人間 (550 mg/m2、15 mg/kg に相当) の限界累積投与量に匹敵する4...

1960 年代1,2以来抗腫瘍薬として開発されたドキソルビシン (DOX)、アドリアマイシンとも呼ばれます。それが今はまだ腫瘍の広範なスペクトルの重要な化学療法薬として積極的に使用します。しかし、DOX の臨床応用は、その用量毒性によって妨げられた、心毒性特に無症候性の心電図変化から心膜炎に至るまで可変の症状によって特徴づけられる、心筋症の時1,2活性酸素種 (ROS)1,3,4、5、トポイソメラーゼ II β (の抑制を含む DIC を説明するまでに、少なくとも 3 つの主要な仮説を提起されている。TOP2β)6、7、および細胞内カルシウムの変調は、1,8,9をリリースします。また DIC10、11,12,13の極めて重要なリスク因子として遺伝的素因を示唆証拠の蓄積します。しかしこれらの DIC の素因に関連する遺伝子のアイデンティティ、主不明のまま。Dexrazoxane は追加を識別する必要性を強調、唯一のアジュバント エージェント、DIC の治療に米国食品医薬品局 (FDA) が限定的に実施14,15,16, 承認治療戦略。DIC の動物モデルはこれらの目的のため検討されます。そのアクセシビリティとシンプルさに、DIC モデルの解明いる可能性のある広範な影響心筋症の他の種類の: 一般的な病因共有可能性があります別の病因による心筋症の中で特に後病理学的ステージ17,18,19,20。
DIC の齧歯動物モデルに加えてより高いスループットのゼブラフィッシュ DIC モデルは新しい遺伝的要因と治療法の探索を容易にするために発達しました。21治療化合物をスクリーニングするための透明の zebrafish の胚の胚の DIC モデルを設置します。心筋症には、進歩的な病因と成人発症疾患がされますが、モデルがされている大人のゼブラフィッシュ心筋症は22,23,24,25,26を開発しました。我々 は、心筋症による慢性貧血24、続いて DIC アダルト ゼブラフィッシュ232 番目の取得した心筋症モデルとしての最初の獲得モデルを生成されます。アダルト ゼブラフィッシュに DOX の単一ボーラス注入が続いて心筋症の慢性期 6 ヶ月投与後に投与後 1 週間 (wpi) 内ほぼ急性期から成る心毒性を誘導することがわかった。ラパマイシンの機構のターゲットのハプロ不全により中(mtor) 心筋症の改善も慢性の段階でそれは誇張魚死亡率急性期でステージ依存を識別する大人の DIC モデルの値を強調します。機構23。我々 はさらに大人の DIC モデルを挿入変異のトランスポゾン ベース アプローチ27経由で生成されているゼブラフィッシュ挿入の心臓 (ジック) 変異のコレクションを強調する使用ことができることを実証しました。パイロット画面識別 DnaJ (Hsp40) の相同物、亜科 B、およびメンバーの 6 b と同様に、3 の知られている心筋症遺伝子 (dnajb6b) として新しい DIC 感受性遺伝子28。したがって、ゼブラフィッシュの大人の DIC モデルの生成は、体系的に DIC は、既存のゲノム広い連合の調査 (GWAS) と量的形質遺伝子座 (QTL を補完の遺伝的修飾因子の同定を可能にする新しい方法論につながった) 分析。
世代と成人ゼブラフィッシュ DIC モデルの実装は、我々 は別の研究者の間でおよび/または同じ調査官によって実行される別の注射の中でも大きな変化に気づいた。モデルの縦の性質は、別の捜査官からの結果を登録して、連続するトラブルシューティング プロセスに課題を課しています。この単純な心筋症を誘発するストレスによる研究コミュニティの使用を容易にするには、我々 のプロトコルの詳細、IP 注入の存在の 2 つのタイプと異なる研究者のばらつきを減らすための考慮事項について説明.

<table><tbody><tr><td>Crossing tank</td><td>Aquaneering</td><td>ZHCT100</td><td>Fish breeding</td></tr><tr><td>Incubator</td><td>ThermoFisher</td><td></td><td>Maintaining embryo</td></tr><tr><td>3 L medium tank</td><td>Aquaneering</td><td>ZT280</td><td>Maintaining fish</td></tr><tr><td>Paramecia</td><td>Carolina</td><td>131560</td><td>Food for juvenile fish</td></tr><tr><td>Live hatched brine shrimp</td><td>in house</td><td></td><td>Food for adult fish</td></tr><tr><td>Doxorubicin hydrochloride</td><td>Sigma</td><td>D1515-10MG</td><td></td></tr><tr><td>1.5 ml safe-lock tube</td><td>Eppendorf</td><td>No. 022363204</td><td>For drug storage</td></tr><tr><td>Aluminum foil paper</td><td>Fisher</td><td>1213104</td><td>For preventing light exposure</td></tr><tr><td>Proteinase K</td><td>Roche</td><td>No. 03115887001</td><td>For dechorionating embryo</td></tr><tr><td>Hank&#039;s balanced salt solution (HBBS)</td><td>ThermoFisher</td><td>14025076</td><td>Vehicle for DOX</td></tr><tr><td>100 mm petri dish</td><td>Falcon</td><td>431741</td><td></td></tr><tr><td>10 &amp;mu;L NanoFil micro-syringe</td><td>WPI</td><td>NANOFIL</td><td>For injection</td></tr><tr><td>34 gauge needle</td><td>WPI</td><td>NF34BV-2</td><td>For injection</td></tr><tr><td>Tricaine</td><td>Argent</td><td>MS-222</td><td>Anesthetizing fish</td></tr><tr><td>96 well plate</td><td>Costar</td><td>3539</td><td>For embryo drug treatment</td></tr><tr><td>Transfer pipette</td><td>Bel-art product</td><td>F37898</td><td>For transfering embryo</td></tr></tbody></table>

a doxorubicin-induced cardiomyopathy model in adult zebrafish

遺伝的にアクセスできるアダルト ゼブラフィッシュ (動脈分布) は、心筋症などの疾患を理解するため脊椎動物モデルとしてますます使用されています。利便性、こうしてハイスループット遺伝子操作のため取得した心筋症モデルの生成アダルト ゼブラフィッシュのドキソルビシン誘発心筋症 (DIC) モデルなどは扉を開いて、新しい研究手段に前方遺伝学的スクリーニングを介して心筋症修飾子の発見を含みます。ゼブラフィッシュ胚の DIC モデルとは異なり、急性初期と後で慢性期心筋症決定できますアダルト ゼブラフィッシュ DIC モデルでは、ステージ依存性シグナル伝達機構と治療戦略の研究を有効にします。ただし、現在のモデルでは、経験豊富な研究者の手にも変数の結果を取得できます。DIC モデルの将来の実装を容易にするには、大人のゼブラフィッシュのこの DIC モデルを生成および腹腔内 (IP) 投与の 2 つの代替方法について説明する方法で詳細なプロトコルを紹介します。我々 はさらに信頼性の高い結果を取得し、結果を適切に解釈する方法に関する提案を提供する変化を少なくための方法について説明します。

ここでは、大人のゼブラフィッシュ モデル DIC への IP の注入を実行する 2 つの方法が掲載されています。古典的な確立された IP 注入法29を使用している間注入 DOX ソリューション (赤い色) が針が浸透した場所から時々 にじみでていることが指摘されました。代替の IP 注入は腹膜から 3-4 mm、DOX のリリース、針の浸透のため別の場所で使用 (<strong clas...

Watch this Scientific Journal Video about A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish at JoVE.com

A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

大人のゼブラフィッシュのドキソルビシン誘発心筋症モデル

アダルト ゼブラフィッシュ (動脈分布) のドキソルビシン誘発心筋症モデルを生成する方法を次に紹介します。腹腔内投与の 2 つの方法を提示し、さまざまな実験グループのばらつきを減らすための条件について説明。

The genetically accessible adult zebrafish (Danio rerio) has been increasingly used as a vertebrate model for understanding human diseases such as ...

a-doxorubicin-induced-cardiomyopathy-model-in-adult-zebrafish

Research

JoVE Journal

Medicine

9.7K ビュー.  Mayo Clinic Graduate School of Biomedical Sciences. アダルト ゼブラフィッシュ (動脈分布) のドキソルビシン誘発心筋症モデルを生成する方法を次に紹介します。腹腔内投与の 2 つの方法を提示し、さまざまな実験グループのばらつきを減らすための条件について説明。

ビデオ: A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish

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

Displacement Analysis of Myocardial Mechanical Deformation (DIAMOND) Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to reliably detect segmental mechanics and are not readily feasible in zebrafish. Here we present a semiautomated, open-source method for the quantitative assessment of four-dimensional (4D) segmental cardiac function: displacement analysis of myocardial mechanical deformation (DIAMOND). Transgenic embryonic zebrafish were imaged in vivo using a light-sheet fluorescence microscopy system with 4D cardiac motion synchronization. Acquired 3D digital hearts were reconstructed at end-systole and end-diastole, and the ventricle was manually segmented into binary datasets. Then, the heart was reoriented and isotropically resampled along the true short axis, and the ventricle was evenly divided into eight portions (I&#8211;VIII) along the short axis. Due to the different resampling planes and matrices at end-systole and end-diastole, a transformation matrix was applied for image registration to restore the original spatial relationship between the resampled systolic and diastolic image matrices. After image registration, the displacement vector of each segment from end-systole to end-diastole was calculated based on the displacement of mass centroids in three dimensions (3D). DIAMOND shows that basal myocardial segments adjacent to the atrioventricular canal undergo the highest mechanical deformation and are the most susceptible to doxorubicin-induced cardiac injury. Overall, DIAMOND provides novel insights into segmental cardiac mechanics in zebrafish embryos beyond traditional ejection fraction (EF) under both physiological and pathological conditions.

Displacement Analysis of Myocardial Mechanical Deformation DIAMOND Reveals Segmental Heterogeneity of Cardiac Function in Embryonic Zebrafish

The goal of this protocol is to detail a novel method for the assessment of segmental cardiac function in embryonic zebrafish under both physiological and pathological conditions.

心筋機械変形(DIAMOND)の変位解析は、胚性ゼブラフィッシュにおける心機能のセグメント的不均一性を明らかにする

Zebrafish are increasingly utilized as a model organism for cardiomyopathies and regeneration. Current methods evaluating cardiac function fail to ...

発生生物学

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 ...

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.

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 ...

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.

成体ゼブラフィッシュの生体内心電図検査

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

成体ゼブラフィッシュにおける化合物送達のための最適化された腹腔内注射

An Intraperitoneal Injection Technique in Adult Zebrafish that Minimizes Body Damage and Associated Mortality

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as cardiomyopathy. Intraperitoneal (IP) injection is an important method that delivers compounds to the body for either testing therapeutic effects or generating disease models such as doxorubicin-induced cardiomyopathy (DIC). Currently, there are two methods of IP injection. Both&#160;methods have limitations when handling toxic compounds such as doxorubicin, which result in side effects manifesting as severe damage to the body shape and fish death. While these shortcomings could be overcome by extensive investigator training, a new IP injection method that has minimal side effects is desirable. Here, a unique IP injection method that is able to handle toxic compounds is reported. Consistently reduced cardiac function can result without incurring significant fish death. The technique can be easily mastered by researchers who have minimal experience with adult zebrafish.

著者スポットライト:ゼブラフィッシュ心毒性モデルのための新しい腹腔内注射法

A new intraperitoneal (IP) injection method in adult zebrafish is described. When handling toxic compounds such as doxorubicin, this procedure is more effective than the two previously reported IP methods. The technique is designed to be easily adopted by researchers with limited experience in the zebrafish model.

成体ゼブラフィッシュにおける腹腔内注射技術により、体の損傷とそれに伴う死亡を最小限に抑える

The adult zebrafish (Danio rerio), which is genetically accessible, is being employed as a valuable vertebrate model to study human disorders such as ...

大人のゼブラフィッシュのドキソルビシン誘発心筋症モデル

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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

心筋機械変形(DIAMOND)の変位解析は、胚性ゼブラフィッシュにおける心機能のセグメント的不均一性を明らかにする

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

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

成体ゼブラフィッシュの生体内心電図検査

成体ゼブラフィッシュにおける腹腔内注射技術により、体の損傷とそれに伴う死亡を最小限に抑える

成体ゼブラフィッシュにおける腹腔内注射技術により、体の損傷とそれに伴う死亡を最小限に抑える

成体ゼブラフィッシュにおける腹腔内注射技術により、体の損傷とそれに伴う死亡を最小限に抑える