私たちは彼の技術支援のためのトレントウォーに感謝したいです。また、病気の子供や、このプロジェクトのために惜しみない資金調達のための治療先天性筋ジストロフィー（CMD）のための病院の小児科を認めます。

寒天注入プレートの調製（時間：45分） <ol><li> E3培地で2％〜3％のアガロースを煮て、その溶液をベンチにわずかに冷却します。注：準備されている注射板の数は、必要なアガロースの量を決定します。各注入プレートは、アガロース溶液の約35ミリリットルが必要です。 </li><li>煮沸後に、所望の温度に到達するまで、アガロースが冷却する（ 例えば 、45°C）射出成形金型の製造者の指示に従って。 </li><li> 100ミリメートル皿にアガロース冷却の約35ミリリットルを注ぎます。 </li><li>溶液中に好適な射出成形金型の一方の端を置き、その後、（これは気泡の発生を減らすのに役立ちます）アガロース溶液に金型の残りの部分を置きます。 </li><li>アガロース溶液を約30分間、室温または4℃のいずれかで固化することを許可します。 </li><li>固体アガロースから金型の一方の端部を分離するために、スパチュラを使用します。ゆっくりメートルの残りの部分を削除<html>古い。 </li></ol>エバンスブルー色素（EBD）注射ミックスの調製（時間：30分） <ol><li> 1Xリンゲル液中EBDの1％の株式を行います（; 5のKCl、155 mMの塩化ナトリウムを2 mMの塩化カルシウム、1mMのMgCl 2、2 mMのの Na 2 HPO 4、10mMのHEPES、10mMのグルコース、7.2のpH）、これは室温で保存することができます。 </li><li> -20℃で1×リンゲル液およびストアに25 mg / mlの時MW万キロダルトン-デキストランフルオレセインイソチオシアネート（FITC）の原液を作ります</li><li> （ すなわち 、100μlの最終作業容量のために：FITCデキストラン株式の90μlの1％EBD10μlのミックス）FITC-デキストラン株式の原液で直接0.1％にEBDを希釈することにより、注射ミックスを調製します。 </li><li>徹底的にボルテックス注射ミックス（それがグリーンに点灯します）とアルミ箔で注入ミックスチューブをラップすることによって、直接光のうちに保ちます。 </li></ol> 3. EBD注入準備（時間：約30分） ENT &quot;&gt;注：プロトコルは3-7日後に受精（DPF）から幼虫に最適です。 <ol><li> RTへの事前暖かい注入プレート。 </li><li>金属板にマイクロマニピュレーターを配置することにより、射出装置を設定し、注入のために使用されている顕微鏡の隣に立っています。空気駆動マイクロインジェクションコントローラーの電源をオンにします。注：優先噴射システムは、ラボによって変化し、分析の結果を変更しないでください。 </li><li>バックEBDミックスの約2〜4μlの注射針を埋めます。 </li><li> EBDミックスの約5 NLに注入量を調整します。注：注入量の校正が校正方法に依存するであろう。ガス圧力がインジェクタマイクロメータを使用してボリュームを介して較正されたボーラス注入量を必要とするのに対し、ピストン駆動注射、直接指定された噴射量に設定することができます。 </li><li> 1Xリンゲル液で濡れ注入プレート、ウェルから過剰を取り除きます。 </li><li> 0.04％の3-アミノ安息香酸エチルメタンサルによる前治療の幼虫噴射の開始に先立って、幼虫を固定する1Xリンゲル液中に希釈トン（トリカイン）。注意：適切な注入が任意の残留運動では困難であるとして、幼虫が完全に動けないです確保することが重要です。 </li><li>ガラスピペットを用いて寒天注入プレートのウェルに麻酔幼虫を置きます。幼虫が完全にウェル内とその側に横たわっていることを確認してください。注：ウェル当たり幼虫数は実験者に任されています。 </li><li>幼虫をウェルに入れられた後、ウェル内の幼虫の動きを最小限に抑えるために、過剰なリンガー溶液を除去します。幼虫は脱水しないように、溶液の残量を残します。 </li></ol> EBDとゼブラフィッシュ幼虫の4心膜注射（時間：幼虫注入の数に依存し、推定1-3時間） <ol><li>注射が行われる解剖スコープの幼虫を含む注入プレートを置きます。 </li><li>含む注入ピペットの針を置きEBDは、ゼブラフィッシュの幼虫の上に混ぜます。 </li><li>位置を再注射針は、幼虫の心の近くにあり、約45°の腹側に前後軸からので、それを回転させることによって注入プレート。 </li><li>静脈が最初に背側方向に旋回している卵黄（ 図1）の前部に静脈の領域に共通の主静脈（CCV）に注射針を挿入します。注意：最大40倍までの倍率がはっきりCCVを参照することが有用であり得ます。 </li><li> EBDミックスの5 NLを注入し、EBDミックスの即時漏れを最小限にするために5-8秒の位置に注射針を保ちます。注：良い注入が心室（ 図1）で見られる色素の着色を持つことになります。 EBDミックスを中心に観察されない場合には、EBDミックスさらに5 NLを注入し、色素の取り込みを誘導するのに十分であり得ます。あるいは、胚を廃棄することができます。 注意：いくつかの状況では、心臓が鼓動を停止することがあります。このOの場合ccurs、20〜40秒間の幼虫を監視し続けます。色素が循環系を通って移動するように一般的に、心臓は鼓動を再開します。 </li><li>次の幼虫と繰り返しに移動します。 </li><li> （ 図2）注射直後に血管系におけるFITC-デキストランの存在を観察することによって成功した注入された胚を特定します。 </li></ol> 5.インキュベーションおよびEBD取り込み（時間：4-6時間） <ol><li>幼虫の所望の数が注入された後、戻り値は、100mm皿にトリカインなし1Xリンゲル液に幼虫を注射しました。 </li><li>アルミホイルで包んで料理をしてください。注：キーピングが著しく生存率が増加し、信号強度の最大の一貫性を確実に暗所で幼虫を注射しました。アルミ箔でラッピングは幼虫がインキュベーターの外にある一定の期間のために特に重要です。 </li><li>幼虫は十分EBD取り込みを確実にするために4-6時間28.5℃でインキュベートすることを許可します。 </li></オール&gt; 筋肉内EBD 6.可視化（時間：幼虫注入顕微鏡の種類の数に応じて、推定0.5〜3時間） <ol><li>画像化の前に、移動を防止するために、0.04％のトリカインで幼虫を麻酔。 </li><li> EBDの取り込みが骨格筋（ 図3）で発生しているかどうかを判断するために赤色蛍光の下にあるViewの幼虫。 </li></ol>

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著者は全く競合する金融利害や関心のある他の競合を持っていません。

ゼブラフィッシュは、神経筋疾患2,29の研究のための強力なツールとして浮上しています。現在までに、ゼブラフィッシュ系は、新たな筋疾患を引き起こす突然変異16,17,30を検証する新規な発症機序を解明18、及び潜在的に新しい治療薬12,24を識別するために使用されています。これらの集団的努力は、人間の神経筋疾患をモデル化するためにゼブラフィッシュの有用性を確立しています。しかし、ゼブラフィッシュおよび哺乳動物モデルで作られた進歩にもかかわらず、神経筋条件の広いスペクトル内の患者のための限られた治療の選択肢があります。そのため、高い需要が壊滅的な疾患のこのグループの治療法の開発のために存在します。治療のためのこの要求を並列接続すると、新しい動物モデルおよび推定治療戦略を検証するための継続的な実験技術革新だけでなく、厳密な分析のための対応が必要です。 EBDの分析は、一般的にマウスモデルで使用されています組織や脳、心臓、骨格筋27,31における細胞損傷を研究。最も顕著なのは、EBDは、筋膜の不安定性の深刻度を示し、8を損傷するために様々な筋ジストロフィーのサブタイプのマウスモデルにおいて広く使用されています。筋膜損傷を明らかにするEBDの使用は、ヒトの疾患状態9に動物モデルの類似性を確立支援するパラメータです。マウスではEBDの電力は、開発し、神経筋疾患のゼブラフィッシュのモデルにEBDを適用するために、私たち自身を含むいくつかの研究室が、リードしてきました。原因EBD分析の適用に、この技術は積極的にヒトの疾患状態11,15,22,24,32にゼブラフィッシュモデルを裏付けるために実施されています。損傷した筋肉の膜との幼虫は、筋線維内EBD取り込み、したがって、赤色の蛍光を持つことになります。蛍光は、繊維間の空間内に観察されたが、個々の筋線維内でTにおける基底膜から脱着繊維の有益かもしれません膜損傷の彼が不在、有用な診断の詳細を提供します。 EBD分析は、動物モデルの検証を超えた潜在的な用途を有しています。私たちの研究室からの努力は、最近EBD分析は、潜在的に新規の治療薬24を検証するのに有益であることを実証しました。潜在的な治療処置が軽減または関連する治療作用8を意味することができます神経筋疾患モデルにおけるEBD取り込みを廃止するかどうかの確認。このタイプの分析は、治療薬の機構（単数または複数）を確立する助けとEBD解析の適用を拡大することができます。 多くの技術と同様に、EBD解析は、実験計画と実践の間に観察するには、いくつかの注意点を持っています。例えば、それは、経年組織の肥厚にCCVを識別するために挑戦することができます。加えて、実験的な数を減少させ、幼虫の多数プリパレーションする必要性を増大させる、心膜注入前との間に調製物中の幼虫を損傷することは容易です。損傷した筋肉はEBDを取ることができますようにまた、取り扱いおよび注入中の幼虫に行っ物理的な損傷は、偽陽性になる可能性があります。これらの障害のいくつかを克服するために、我々は、注入の前、その後の分析に成功した直後に、色素注入と幼虫の簡単かつ信頼性のある同定を可能にするこのビデオ資料に共射出戦略を記載しています。前の筋線維によるその取り込みを血管系内のEBDの確認を可能にすることによって成功した注射用の共射出コントロールFITCデキストラン。筋線維に回収されていない場合EBD蛍光は、数時間後に幼虫に非常にびまん性になるように、これは特に有用である可能性があります。このように、検出するのが困難であることができます。また、CCVが欠落し、卵黄または体腔にEBDを注入して、インキュベーション後、胚を制御するために同様の拡散赤色蛍光で、まだ損傷した筋線維による取り込み可能性の減少をもたらすことができます。まとめると、これらの洞窟ATSはEBD注入が一貫して信頼性の高い結果を得るためには忍耐と練習が必要示唆しています。 すべてでは、我々は、ゼブラフィッシュの幼虫にEBD分析を実行するための実用的かつ簡単な方法を説明します。現在までに、モデル系としてゼブラフィッシュの使用は、特にヒト疾患モデルとして、急速に拡大しています。この拡張は、ゼブラフィッシュシステムの現在の利点を改良実験技術の継続的な開発と変更に部分的に起因しています。 EBD注入技術は、ゼブラフィッシュ筋疾患モデルの検証と研究のための研究者の兵器庫に追加し、強力なツールを提供します。この技術の継続的な実施と修正は、新規治療戦略だけでなく、疾患を引き起こすメカニズムを解明するのに役立つ可能性を秘めています。

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fluorescein isothiocyanate-dextran MW 10,000</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">FD10S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Evan&#39;s Blue Dye</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm Petri dish</td>
			<td style="width:173px;">Fischerbrand</td>
			<td style="width:140px;">FB0875712</td>
			<td>Injection mold base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Thin wall glass capillaries</td>
			<td style="width:173px;">World Precision Instruments</td>
			<td style="width:140px;">TW100F-4</td>
			<td>For Injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Agarose</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">AGA001</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microinjection mold</td>
			<td style="width:173px;">Adaptive Science Tools</td>
			<td style="width:140px;">TU-1</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">SOD001</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Potassium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">POC888</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Magnessium chloride hexahydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">M2670</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium phosphate monobasic monohydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">S9638</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">H4034</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glucose</td>
			<td style="width:173px;">BioBasic</td>
			<td style="width:140px;">GB0219</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Calcium chloride</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">C1061</td>
			<td>Ringer&#39;s solution</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

EBD注入ミックスは3 DPFでsapjeホモ接合変異体および野生型同胞のCCVに注入しました。 （ 図1B）は、心臓チャンバを充填注射は、次に緑色蛍光（ 図2）の下に血管系にFITCデキストランを可視化することによって成功した注射のために分析しました。 4時間のインキュベーション期間の後、EBD取り込みは蛍光顕微鏡を用いて体節のレベルで調べました。 sapjeホモ接合変異体は筋肉膜15（ 図3B）への損傷を示す、EBD取り込みを示した野生型の兄弟は、目に見える筋線維（ 図3A）内にはEBD蛍光を示さありませんでした。 <img alt="図1" src="/files/ftp_upload/53183/53183fig1.jpg" /> 図1.ジェクトEBD注入ミックスゼブラフィッシュエンブリーの総主静脈（CCV）へO。 （A）非注入胚。矢印は、CCV注入のための理想的な場所を示します。 （B）CCVに成功した注射。染料は、心腔（矢印）に入り、血管系を通って圧送され始めます。失敗したCCV注入は胚（矢印）の卵黄嚢に入る色素の一部または全てになります（C）。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53183/53183fig1large.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> <img alt="図2" src="/files/ftp_upload/53183/53183fig2.jpg" /> 図2.胚は、直ちに注入前EBD取り込みに以下の血管系全体に緑色蛍光下のFITC-デキストランの分布を観察することによって成功した注射用ソートすることができます。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。 <img alt="図3" src="/files/ftp_upload/53183/53183fig3.jpg" /> 図3：EBDは、損傷した膜を有する繊維によって取り込まれる（A）野生型兄弟が筋線維にはEBD蛍光を示さありません。 （B）は、複数の筋線維（矢印）内EBD蛍光とSapje ​​ホモ接合変異体。すべての幼虫がEBD注入ミックスを注射し、3 DPFで4時間のインキュベーション期間後に分析しました。兄弟および変異体は、CCVの注入前に筋線維脱離することにより選別した。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/53183/53183fig3large.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>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

エバンスブルー色素でゼブラフィッシュ幼生骨格筋の整合性の分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

analysis-zebrafish-larvae-skeletal-muscle-integrity-with-evans-blue

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

9.0K ビュー.  The Hospital for Sick Children. In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

ビデオ: Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

タイムラプス明実体顕微鏡でゼブラフィッシュ幼虫における組織修復をキャプチャ

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

発生生物学

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

心臓毒注入による急性骨格筋再生の誘導

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

テストの子孫によるゼブラフィッシュ致死の骨格変異の浸透度をシフト

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

最適化されたマイクロインジェクションおよびゼブラフィッシュ幼虫の画像処理用微細構造デバイス

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性的な刺激の収縮活動に伴うラット骨格筋の表現型適応を研究への応用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 とラミニン抗体抗体法による骨格筋衛星細胞の同定

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

筋肉疾患の構造的基礎を研究するためのツールとしての無傷のマウス骨格筋のX線回折

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

骨格筋生検手順からのヒト筋前駆細胞の単離、培養、特徴付け、分化

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

免疫不全マウスにおけるヒト骨格筋異種移植片の実行

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

マウス骨格筋移植からの原発性筋芽生の単離と分化

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...