The study was supported by the Swiss National Foundation [SNF 310030-149986 to MNG], the University of Fribourg, and Fribourg Hospital.

すべての動物は動物ケアに関する欧州条約に準拠した人道的なケアを受けました。外科的手順は、フリブール、スイス連邦獣医局、スイスで承認され、国家獣医局の許可を得た後、スイスの動物保護法に従って行われました。 1.ハート収穫注：すべての外科的介入は、イソフルラン麻酔下で実施しました。努力が動物の苦痛を軽減するために行われました。具体的には、全ての動物は、0.1ミリグラム/ kgのブプレノルフィン予め麻酔の皮下注射を受けました。心筋梗塞入校するための外科的プロトコルは、以前に他の場所13に記載されています。 <ol><li>麻酔下myocardially梗塞動物（挿管動物、2.5％イソフルラン、足のピンチ反射により確認し、適切な麻酔）に胸骨切開を行います。 <ol><li>皮膚を切断することにより、動物を開き、筋肉手術用ハサミで。左右のリブをカットしてから、胸を削除します。 </li><li>以前の手術（LAD結紮）の後に、残りの癒着を削除します。大動脈をカットし、心を取り除きます。 （PBS中）1 MのKClに組織を入れ、その後、PBSで洗ってください。 </li></ol></li></ol> 2.組織標本<ol><li>アクリルラット心臓マトリックス中に縦方向に梗塞心を置きます。 1時間-20℃で保管してください。 </li><li>横方向とカミソリの刃を使用して、マトリックスに直接心をカット。各スライスは2ミリメートルの厚さであることを確認してください。 ;（改造レベルに基づいて）各心臓のための7のスライド - 約5をカットこれは、体系的なサンプリングと呼ばれています。 </li><li>このステップでは、2,3,5-トリフェニルテトラゾリウムクロリド（TTC）染色を行うこと（心臓スライスのベース - 頂点の向きを保つ）は任意です。 <ol><li> 37℃で50分間、PBS中の1％TTCでインキュベートします。 1時間20分間、4％パラホルムアルデヒド（PFA）でそれをインキュベートします。 betweeセクションを入れてnは2 mmのスペーサーを有する2枚のガラス板とは15X倍率でカメラに接続された立体顕微鏡で写真を撮ります。 注意！パラホルムアルデヒドは有毒です。 </li></ol></li><li>スライド凍結（1 番目のオプション） <ol><li> （金型の底に下り区間の頂点指向側を配置）方向を維持、cryotomy用培地（最適切断温度、10月）を取り付けたプラスチック金型（10×10×5ミリメートル）で、各スライドを置きます。 15分 - 10用の液体窒素冷却下、2-メチルブタン蒸気でブロックをフリーズします。最後に、-80℃で組織を保存します。 </li></ol></li><li>パラフィン除去（2 番目のオプション） <ol><li> 24時間、4％PFAで各カセットを埋め込んでスライド（ダウンカセットの底の部分の頂点指向側を配置することによって方向を維持）とを配置します。ティッシュプロセッサマシンで一晩でそれを置きます。 <ol><li> 2時間エタノール70％でそれをインキュベートします。イーサンでそれをインキュベート2時間95％をオール。 3時間エタノール100％でそれをインキュベートします。 4時間キシロールでそれをインキュベートします。 5時間（オーブンで60℃で溶融）パラフィンでそれをインキュベートします。最後に、パラフィンの各心臓スライドを埋め込むことにより、ブロックを作ります。 </li></ol></li></ol></li></ol> 3.マッソン - ゴールドナートリクローム染色<ol><li> （：厚さ5μm）または-18℃に設定したクライオスタットと10月のブロックから（厚さ：7μm）のマニュアルミクロトームでパラフィンブロックから組織切片を作成します。 </li><li>マッソン-ゴールドナートリクローム染色で- （スライドあたり4横断セクション3）（ 附属書を参照 ）、各ラットについて各心部からのスライドを染色します。 注：パラフィン切片について、このステップから開始します。 <ol><li>オーブンで60℃でスライドを溶かします。ヒュームフードの下で、10分間ずつ二回キシロールでそれらを脱パラフィン。 3分ごとに100％エタノール、95％エタノール、70％エタノール、蒸留水に再水和します。 注：凍結切片に対して、この手順から開始します秒。 </li><li>ブアン液に一晩でそれらを修正しました。 15分 - そして、10のために水道水でそれらを洗い流してください。蒸留水でそれらを洗い流してください。 3分間マイヤーのヘマトキシリンでそれらをインキュベートします。 </li><li>スライドを削除し、5分間蒸留水にそれらを残します。その後、5分間酸フクシン、ポンソーでそれらをインキュベートします。 1分間1％酢酸でそれらを洗い流します。 </li><li>次に、1分間リンモリブデン酸オレンジGでそれらをインキュベートします。 、1分間1％酢酸でそれらを洗い流し10分間ライトグリーン色素でそれらをインキュベートし、1分間1％酢酸でそれらをすすぎます。 </li><li> 70％エタノール（30秒）、95％エタノール（30秒）、及び100％エタノール（5分）でそれらを脱水します。 、組織切片上の樹脂封入剤の1滴を入れてカバースリップでそれらをカバーし、それらを乾燥させてください。 </li></ol></li></ol> 4.梗塞サイズ分析<ol><li>カメラに結合された実体顕微鏡（15X倍率）上の各スライドから1つの画像を取得します。同じ設定で定規を撮影秒。 </li><li> 、梗塞の中間に隔壁厚さ、左心室（LV）腔面積、梗塞領域、およびLVの組織領域を使用して瘢痕の厚さを測定するために画像解析ソフトウェアを使用します。 <ol><li>定規の画像（ 図1A）とのスケールを設定します。 <ol><li> セット変換係数を選択し測定をクリックします。キャリブレーションバー上に線を描画します。右の写真をクリックすると、エンド・キャリブレーションをクリックしてください。バー値をメモして、[OK]をクリックします。 </li></ol></li><li>染色されたハートの絵（ 図1B）からLVを選択します。 複数のセグメントのツールを使用します。 LVポイントバイポイントを選択し、右クリック コピー/どこにでも貼り付けます。 </li><li> 単一セグメントの測定ツールを使用して傷や隔壁の厚さを測定する（ 図1C </st栄&gt;）。 </li><li>自動LV腔、梗塞を検出し、LV領域はRGBモード（ 図1D）における自動面積測定ツールを使用して。 <ol><li>ツールをクリックします。アクティブにのみ罫線と RGBモードでの連続 。最後に、それを検出するために、関心領域内をクリックします。必要に応じて、精密なツールを使用しています。 </li></ol></li></ol></li><li> LV面積に対する梗塞面積の割合としての梗塞サイズを計算します。 </li><li>次のように拡張インデックスを計算します[LV腔面積/全LV面積] / [梗塞厚さ/隔壁厚さ]、LV空洞とLVの組織領域の両方を含む全LVエリアと。 </li><li>次のように最後に、それぞれの心のための「平均」を算出：[梗塞スライドから拡張インデックスの平均] * [梗塞スライド/スライドの総数の数]。 </li></ol>

<ol>
	<li>Amsterdam, E. A., 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=AHA/ACC+Guideline+for+the+Management+of+Patients+with+Non-ST-Elevation+Acute+Coronary+Syndromes:+a+report+of+the+American+College+of+Cardiology/American+Heart+Association+Task+Force+on+Practice+Guidelines.">AHA/ACC Guideline for the Management of Patients with Non-ST-Elevation Acute Coronary Syndromes: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines.</a> J Am Coll Cardiol. 64, e139-e228 (2014).</li><li>Konstam, M. A., Kramer, D. G., Patel, A. R., Maron, M. S., Udelson, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Left+ventricular+remodeling+in+heart+failure:+current+concepts+in+clinical+significance+and+assessment.">Left ventricular remodeling in heart failure: current concepts in clinical significance and assessment.</a> JACC Cardiovasc Imaging. 4, 98-108 (2011).</li><li>Frobert, A., 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=Prognostic+Value+of+Troponin+I+for+Infarct+Size+to+Improve+Preclinical+Myocardial+Infarction+Small+Animal+Models.">Prognostic Value of Troponin I for Infarct Size to Improve Preclinical Myocardial Infarction Small Animal Models.</a> Front Physiol. 6, 353 (2015).</li><li>Redfors, B., Shao, Y., Omerovic, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myocardial+infarct+size+and+area+at+risk+assessment+in+mice.">Myocardial infarct size and area at risk assessment in mice.</a> Exp Clin Cardiol. 17, 268-272 (2012).</li><li>Guex, A. 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=Plasma-functionalized+electrospun+matrix+for+biograft+development+and+cardiac+function+stabilization.">Plasma-functionalized electrospun matrix for biograft development and cardiac function stabilization.</a> Acta Biomater. 10, 2996-3006 (2014).</li><li>Landa, N., 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=Effect+of+injectable+alginate+implant+on+cardiac+remodeling+and+function+after+recent+and+old+infarcts+in+rat.">Effect of injectable alginate implant on cardiac remodeling and function after recent and old infarcts in rat.</a> Circulation. 117, 1388-1396 (2008).</li><li>Valente, M., 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=Optimized+heart+sampling+and+systematic+evaluation+of+cardiac+therapies+inmouse+models+of+ischemic+injury:+Assessment+of+cardiacremodeling+and+semi-automated+quantification+of+myocardial+infarctsize.">Optimized heart sampling and systematic evaluation of cardiac therapies inmouse models of ischemic injury: Assessment of cardiacremodeling and semi-automated quantification of myocardial infarctsize.</a> Curr. Protoc. Mouse Biol. 5, 359-391 (2015).</li><li>Csonka, 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=Measurement+of+myocardial+infarct+size+in+preclinical+studies.">Measurement of myocardial infarct size in preclinical studies.</a> J. Pharmacol. Toxicol. Methods. 61, 163-170 (2010).</li><li>Zornoff, L. A., Paiva, S. A., Minicucci, M. F., Spadaro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+myocardium+infarction+in+rats:+Analysis+of+the+model.">Experimental myocardium infarction in rats: Analysis of the model.</a> Arq. Bras Cardiol. 93, 434-440 (2009).</li><li>Lichtenauer, M., 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=Myocardial+infarct+size+measurement+using+geometric+angle+calculation.">Myocardial infarct size measurement using geometric angle calculation.</a> Eur J Clin Invest. 44, 160-167 (2014).</li><li>Lutgens, 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=Chronic+myocardial+infarction+in+the+mouse:+cardiac+structural+and+functional+changes.">Chronic myocardial infarction in the mouse: cardiac structural and functional changes.</a> Cardiovasc Res. 41, 586-593 (1999).</li><li>Frobert, A., Valentin, J., Cook, S., Lopes-Vicente, L., Giraud, M. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-based+therapy+for+heart+failure+in+rat:+double+thoracotomy+for+myocardial+infarction+and+epicardial+implantation+of+cells+and+biomatrix.">Cell-based therapy for heart failure in rat: double thoracotomy for myocardial infarction and epicardial implantation of cells and biomatrix.</a> J Vis Exp. , e51390 (2014).</li><li>Takagawa, J., 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=Myocardial+infarct+size+measurement+in+the+mouse+chronic+infarction+model:+comparison+of+area-+and+length-based+approaches.">Myocardial infarct size measurement in the mouse chronic infarction model: comparison of area- and length-based approaches.</a> J Appl Physiol (1985). 102, 2104-2111 (2007).</li><li>Fishbein, M. 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=Early+phase+acute+myocardial+infarct+size+quantification:+validation+of+the+triphenyl+tetrazolium+chloride+tissue+enzyme+staining+technique.">Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique.</a> Am Heart J. 101, 593-600 (1981).</li><li>Weisman, H. F., Bush, D. E., Mannisi, J. A., Weisfeldt, M. L., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+of+myocardial+infarct+expansion.">Cellular mechanisms of myocardial infarct expansion.</a> Circulation. 78, 186-201 (1988).</li><li>Mannisi, J. A., Weisman, H. F., Bush, D. E., Dudeck, P., Healy, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Steroid+administration+after+myocardial+infarction+promotes+early+infarct+expansion.+A+study+in+the+rat.">Steroid administration after myocardial infarction promotes early infarct expansion. A study in the rat.</a> J Clin Invest. 79, 1431-1439 (1987).</li></ol>

The authors have nothing to disclose.

プロトコル内の重要なステップ 線維性組織は正確に採取し、心臓や画像の系統的なサンプリングを使用して、慢性MIラットモデルで評価することができる頂点にベースから得た三色染色した組織切片の分析。 2つのステップが成功したプロトコル実装のために特に重要です。まず、心臓の収穫のためのKClを使用することは、心臓の筋肉が弛緩した状態に維持?...

心筋梗塞（MI）は、世界中の死亡および障害の主要な原因です。冠状動脈性心臓病は、主な原因です。 MIは、閉塞などの冠動脈イベントの連続虚血に起因します。再灌流の最初の6時間以内に行われていない場合、虚血は、不可逆的心筋壊死を誘発します。患者では、MIの特徴付けは、臨床徴候、心電図、バイオマーカー、心エコー検査、MRIイメージングの血漿レベルの評価を含む様々な診断ツールに依存し、そして組織1を解析します 。急性および慢性MIは、冠動脈閉塞の時間に対する心筋壊死のタイミングに応じてけがの二つの異なる相として分類されています。最初の7日の間に起こる急性期は、心筋細胞の損失、大規模な炎症、および線維芽細胞の動員と関連しています。心臓組織の治癒および瘢痕の形成を特徴と亜急性期、発生し、1〜4から6週間。梗塞の拡大、心室壁の菲薄化、および心室の拡張は、慢性期を特徴づけます。左心室の大規模な改造が次第に重症心不全2になります。 永久左前下行枝（LAD）結紮によって誘導される心筋梗塞、慢性心筋梗塞の標準的なげっ歯類モデルを表します。冠動脈結紮を冠動脈閉塞を模倣します。梗塞の大きさは、合字のサイトに依存します。げっ歯類モデルにおける心筋虚血損傷の特徴付けは、古典的に、このようなトロポニンIおよびT 3、心エコー検査、MRI、および組織学4,5などのバイオマーカーの血漿レベルを用いて行われます。バイオマーカーのレベルは、心筋細胞死の程度と相関しています。心エコー検査は、局所壁運動異常に起因する左心室機能障害を評価します。例えばMRIや高解像度などの他に、非侵襲的イメージング技術、心エコー検査、壁運動の減少、減少した灌流および生存心筋と瘢痕領域の容量、および減肉の評価を可能にします。 LV寸法は梗塞サイズの正確な評価を可能にします。最後に、生存可能であり、死んだ心筋の定量化は、収穫の心の組織切片の特定の汚れを使用して、死後を行い、梗塞サイズ（IS）の検証を可能にすることができます。別の重要な特徴は、梗塞拡大指数（EI）6の評価です。 EIは、最初の3日以内に貫梗塞と開始と関連しています。 EIは、壁の厚さの漸進的な減少、LV腔サイズの増大、およびLV形状の結果として変化することを特徴とします。 新規の治療の治療効果を評価するために - 特に、再生戦略細胞、マトリックスに基づいて、および齧歯類におけるMIの遺伝子送達の正確な評価は極めて重要です。乳頭筋のレベルで得られた単一の断面で測定した場合、ISサイズは、LAD結紮後により梗塞発達に存在する大きな変動にバイアスされてもよいです。頂点梗塞は、その後、掩蔽されることがあります。重要なことは、決定MIサイズに、より正確な方法は、マウス7-9またはラット10について記載されています。それにもかかわらず、IS正確LVリモデリングやリモデリングの治療誘発される減少（または予防策）を定量化するには不十分です。実際、一般的に心臓の断面で評価総LV容積のパーセンテージとして表されています。この方法は、急性心筋梗塞のために有効ですが、改造中に発生したLV壁の菲薄化は、11,12評価の下で、残っています。梗塞サイズ、構造的な変更の完全な形態学的定量は、心内膜および心外膜の長さや直径、ならびに梗塞や健康分野などのいくつかのパラメータを、定量化する必要があります。私たちは、方法論的APPRを記述します正確慢性ラットモデルにおいてMIおよびリモデリングを評価するoach。

<table><tbody><tr><td>Acrylic rat heart matrix 2 mm</td><td>72-5015</td><td>Harvard Appartus</td><td></td></tr><tr><td>Inspira advanced volume controlled ventilator</td><td>Harvard Apparatus</td><td>557058</td><td></td></tr><tr><td>Catheter Insyte 14 G</td><td>BD</td><td>381267</td><td></td></tr><tr><td>O.C.T</td><td>BDHA361603E</td><td>VWR</td><td></td></tr><tr><td>TTC</td><td>T8877-10G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Mayer hematoxylin</td><td>MHS32-1L</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Acid Fuchsin&lt;br/&gt; CI 42685</td><td>F8129-50G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Ponceau Xylidin&lt;br/&gt; CI 16150</td><td>P2395-25G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Orange G&lt;br/&gt; CI 16230</td><td>O3756-100G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Light green&lt;br/&gt; CI 42095</td><td>L5382-25G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>KCl</td><td>P9333-500G</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Xylol</td><td>10315083</td><td>HoneyWell</td><td></td></tr><tr><td>Ethanol absolute</td><td>10303990</td><td>HoneyWell</td><td></td></tr><tr><td>2-methylbutane</td><td>M32631-1L</td><td>Sigma Aldrich</td><td></td></tr><tr><td>Stereogical microscope</td><td>SM2800</td><td>Nikon</td><td></td></tr><tr><td>Formaldehyde</td><td>99340</td><td>Reactolab</td><td></td></tr><tr><td>Embedding cassette</td><td>K113.1</td><td>Carl Roth</td><td></td></tr><tr><td>Bersoft Image measurement Software</td><td></td><td>Bersoft.com</td><td>Licensed software</td></tr></tbody></table>

histological quantification of chronic myocardial infarct in rats

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ischemic injury. Following the initial acute phase, chronic remodeling of the left ventricle (LV) modifies the structure and function of the heart. Permanent coronary ligation in small animals has been widely used as a reference model for a chronic model of MI. Thinning of the infarcted wall progressively develops to transmural fibrosis. Histological assessment of infarct size is commonly performed; nevertheless, a standardization of the methods for quantification is missing. Indeed, important methodological aspects, such as the number of sections analyzed and the sampling and quantification methods, are usually not described and therefore preclude comparison across investigations. Too often, quantification is performed on a single section obtained at the level of the papillary muscles. Because novel strategies aimed at reducing infarct expansion and remodeling are under investigation, there is an important need for the standardization of accurate heart sampling protocols. We describe an accurate method to quantify the infarct size using a systematic sampling of harvested rat heart and image analyses of trichromatic stained histological sections obtained from base to apex. We also provide evidence that calculating the expansion index (EI) allowed for infarct size assessment, taking into account changes of the left ventricle throughout the remodeling.

6週間後のLAD結紮、心臓をLewisラットから採取しました。 2mmの組織切片は、頂部から基部に得られました。 TTC染色手順は、白で表示され、梗塞面積、および赤で表示され、健康な心筋（ 図2）を可視化するために行きました。 LADの結紮の部位に応じて、梗塞サイズが異なります。大MIについて、貫梗塞は、ベース（ 図2A）に頂点から観察され?...

Watch this Scientific Journal Video about Histological Quantification of Chronic Myocardial Infarct in Rats at JoVE.com

Histological Quantification of Chronic Myocardial Infarct in Rats

The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections.

ラットにおける慢性心筋梗塞の組織学的定量

Myocardial infarction is defined as cardiomyocyte death due to prolonged ischemia; an inflammatory response and scar formation (fibrosis) follow the ...

histological-quantification-of-chronic-myocardial-infarct-in-rats

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

14.4K ビュー.  University of Fribourg. The post-mortem assessment of myocardial infarction (MI) in rodents is based on quantification of the infarct on stained heart sections. We describe an accurate method to quantify the infarct size using systematic sampling of harvested rat hearts from base to apex and image analyses of trichrome-stained histological sections. 

ビデオ: Histological Quantification of Chronic Myocardial Infarct in Rats

Coronary Artery Ligation and Intramyocardial Injection in a Murine Model of Infarction

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo. With the advent of genetic modifications to the whole organism or the myocardium and an array of biological and/or synthetic materials, there is great potential for any combination of these to assuage the extent of acute ischemic injury and impede the onset of heart failure pursuant to myocardial remodeling. 
Here we present the methods and materials used to reliably perform this microsurgery and the modifications involved for temporary (with reperfusion) or permanent coronary artery occlusion studies as well as intramyocardial injections. The effects on the heart that can be seen during the procedure and at the termination of the experiment in addition to histological evaluation will verify efficacy. 
Briefly, surgical preparation involves anesthetizing the mice, removing the fur on the chest, and then disinfecting the surgical area. Intratracheal intubation is achieved by transesophageal illumination using a fiber optic light. The tubing is then connected to a ventilator. An incision made on the chest exposes the pectoral muscles which will be cut to view the ribs. For ischemia/reperfusion studies, a 1 cm piece of PE tubing placed over the heart is used to tie the ligature to so that occlusion/reperfusion can be customized. For intramyocardial injections, a Hamilton syringe with sterile 30gauge beveled needle is used. When the myocardial manipulations are complete, the rib cage, the pectoral muscles, and the skin are closed sequentially. Line block analgesia is effected by 0.25% marcaine in sterile saline which is applied to muscle layer prior to closure of the skin. The mice are given a subcutaneous injection of saline and placed in a warming chamber until they are sternally recumbent. They are then returned to the vivarium and housed under standard conditions until the time of tissue collection. At the time of sacrifice, the mice are anesthetized, the heart is arrested in diastole with KCl or BDM, rinsed with saline, and immersed in fixative. Subsequently, routine procedures for processing, embedding, sectioning, and histological staining are performed. 
Nonsurgical intubation of a mouse and the microsurgical manipulations described make this a technically challenging model to learn and achieve reproducibility. These procedures, combined with the difficulty in performing consistent manipulations of the ligature for timed occlusion(s) and reperfusion or intramyocardial injections, can also affect the survival rate so optimization and consistency are critical.

Numerous genetic manipulations and/or intramyocardial injections of genes, proteins, cells, and/or biomaterials are superimposed upon the dimension of time in studies of acute ischemia/ reperfusion injury and chronic remodeling in mice. This video illustrates the microsurgical procedures for ischemia/reperfusion, permanent coronary artery ligation, and intramyocardial injection studies.

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

Mouse models are a valuable tool for studying acute injury and chronic remodeling of the myocardium in vivo.  With the advent of genetic modifications to ...

医学

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that MicroCT provides quantitative high-resolution three-dimensional (3D) anatomical data non-destructively and longitudinally. Most importantly, with the development of a novel preclinical iodinated contrast agent called eXIA160, functional and metabolic assessment of the heart became possible. However, prior to the advent of commercial MicroCT scanners equipped with X-ray flat-panel detector technology and easy-to-use cardio-respiratory gating, preclinical studies of cardiovascular disease (CVD) in small animals required a MicroCT technologist with advanced skills, and thus were impractical for widespread implementation. The goal of this work is to provide a practical guide to the use of the high-speed Quantum FX MicroCT system for comprehensive determination of myocardial global and regional function along with assessment of myocardial perfusion, metabolism and viability in healthy mice and in a cardiac ischemia mouse model induced by permanent occlusion of the left anterior descending coronary artery (LAD).

In Vivo Quantitative Assessment of Myocardial Structure, Function, Perfusion and Viability Using Cardiac Micro-computed Tomography

This method outlines the use of Quantum Micro-Computed Tomography (MicroCT) to assess cardiac morphology, function, perfusion, metabolism and viability with iodinated contrast agent in mice with experimentally-induced myocardial ischemia. The technique can be applied for non-destructive high-throughput longitudinal in vivo imaging of various animal models of human heart disease.

心筋の構造、機能の in vivo定量的評価では 、灌流および生存率は、心臓マイクロコンピュータ断層撮影法を使用しました

The use of Micro-Computed Tomography (MicroCT) for in vivo studies of small animals as models of human disease has risen tremendously due to the fact that ...

生物工学

Minimal Invasive Surgical Procedure of Inducing Myocardial Infarction in Mice

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last decades. For clarification of the underlying mechanisms and the development of new therapeutic strategies, the availability of valid animal models are mandatory. Since we need new insights into pathomechanisms of cardiovascular diseases under in vivo conditions to combat myocardial infarction, the validity of the animal model is a crucial aspect. However, protection of animals are highly relevant in this context. Therefore, we establish a minimally invasive and simple model of myocardial infarction in mice, which assures a high reproducibility and survival rate of animals. Thus, this models fulfils the requirements of the 3R principle (Replacement, Refinement and Reduction) for animal experiments and assure the scientific information needed for further developing of therapeutical strategies for cardiovascular diseases.

A highly reproducible model for myocardial infarction in mice with minimal invasive manipulations is described. The model can be easily performed, resulting in a high reproducibility and survival rate. Thus, the described model will reduce the number of required animals as requested by the 3R principle (Replacement, Refinement and Reduction).

マウスにおける心筋梗塞を誘発する低侵襲手術手順

Myocardial infarction still remains the main cause of death in western countries, despite considerable progress in the stent development area in the last ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

急性心筋梗塞の豚モデルにおける主要アウトカム評価

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Murine Myocardial Infarction Model using Permanent Ligation of Left Anterior Descending Coronary Artery

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine models of MI with permanent ligation of left-anterior descending (LAD) coronary artery closely mimics MI in humans. Murine models benefit from the extensive genetic engineering available nowadays. Here we propose a reproducible murine surgical model of myocardial infarction by permanent LAD coronary ligation. Our technique comprises anesthesia with ketamine/xylazine that can be rapidly reversed by administration of an antagonist, intubation without tracheotomy for mechanical-assisted ventilation, ventilation with application of extrinsic positive end-expiratory pressure (PEEP) to avoid alveolar collapse, a thoracotomy method limiting to the minimum surgical lesions made to skeletal muscles, and lung inflation without thoracentesis. This method is sparsely invasive, reproducible and reduces post-surgery mortality and complications.

Herein we describe a surgical procedure showing how to achieve permanent ligation of the left-anterior descending coronary artery in mice. This model is of high relevance to investigate the pathophysiology of myocardial infarction and the concomitant biological processes.

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

Myocardial infarction (MI) and acute coronary diseases are among the most prominent causes of death in population with western lifestyle. The murine ...

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial infarction (MI). However, the disadvantages of this model include high mortality rate after ligation and larger variations both in the infarct size and the degree of impaired cardiac function. In addition, a ventilator or exteriorization of the heart is indispensable for the previous models, which complicates the procedure during the ligation. In this study, we developed a reliable and reproducible model without the ventilator or exteriorization of the heart by ligating the LAD coronary artery. Four weeks after the procedure, we found that the serum concentrations of CK-MB, NT-proBNP, and Renin, which were used to assist diagnoses of MI and HF, were significantly higher in the MI group compared to the sham group. In contrast, the value of left ventricle ejection fraction (LVEF) in the MI group was obviously less than in the sham group. Furthermore, the infarct size and cardiac fibrosis area were individually confirmed and quantitatively analyzed by TTC staining and Masson's trichrome staining. Smaller variations were found in either infarct size or fibrosis area in the MI group, which helped to develop a reliable and reproducible model of HF post-MI. This new model of HF post-MI in the rat is vital for studying the potential mechanisms of MI and HF. This new method can be used to develop the new drug for treatment of MI and HF in rats by using pharmacological strategies.

We successfully developed a reliable and reproducible model of heart failure post-myocardial infarction in the rat without ventilation or exteriorization of the heart. This simplifies the procedure and benefits the further studies about the potential mechanisms behind the heart failure.

Ligation of the left anterior descending (LAD) coronary artery has been widely used to establish the rat model of heart failure (HF) post myocardial ...

An Intact Pericardium Ischemic Rodent Model

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to induce myocardial injury). The majority of pre-clinical myocardial infarction models require the disruption of pericardial integrity with loss of the homeostatic cellular milieu. However, recently a methodology has been developed by us to induce myocardial infarction, which minimizes pericardial damage and retains the heart&#39;s resident immune cell population. An improved cardiac functional recovery in mice with an intact pericardial space following coronary ligation has been observed. This method provides an opportunity to study inflammatory responses in the pericardial space following myocardial infarction. Further development of the labeling techniques can be combined with this model to understand the fate and function of pericardial immune cells in regulating the inflammatory mechanisms that drive remodeling in the heart, including fibrosis.

This protocol outlines the steps for inducing myocardial infarction in mice while preserving the pericardium and its contents.

無傷心膜虚血性げっ歯類モデル

This protocol has shown that the pericardium and its contents play an essential anti-fibrotic role in the ischemic rodent model (coronary ligation to ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

マウスにおける右冠動脈の永久結紮による右心室心筋梗塞の生成と特徴付け

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Ultrasound-Guided Induced Pluripotent Stem Cell-Derived Cardiomyocyte Implantation in Myocardial Infarcted Mice

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising cell source for cardiac repair after ischemic damage. However, a low grafted rate is a significant obstacle for effective cardiac tissue regeneration after transplantation. This protocol shows that multiple hiPSC-CM ultrasound-guided percutaneous injections into an MI area effectively increase cell transplantation rates. The study also describes the entire hiPSC-CM culture process, pretreatment, and ultrasound-guided percutaneous delivery methods. In addition, the use of human mitochondrial DNA help detect the absence of hiPSC-CMs in other mouse organs. Lastly, this paper describes the changes in cardiac function, angiogenesis, cell size, and apoptosis at the infarcted border zone in mice 4 weeks after cell delivery. It can be concluded that echocardiography-guided percutaneous injection of the left ventricular myocardium is a feasible, relatively invasive, satisfactory, repeatable, and effective cellular therapy.

Ultrasound-guided cell delivery around the site of myocardial infarction in mice is a safe, effective, and convenient way of cell transplantation.

心筋梗塞マウスにおける超音波ガイド下多能性幹細胞由来心筋細胞移植術

The key objective of cell therapy after myocardial infarction (MI) is to effectively enhance the cell grafted rate, and human induced pluripotent stem ...

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. This paper describes a protocol for studying the role of galectin 3 (Gal-3) in the temporal evolution of cardiac healing and remodeling in an experimental animal model of MI. 
The procedures described include an experimental model of MI with a permanent coronary ligature in male C57BL/6J (control) and Gal-3 knockout (KO) mice, an echocardiography procedure to study cardiac remodeling and systolic function in vivo, a histological evaluation of interstitial myocardial fibrosis with picrosirius red-stained and rhodamine-conjugated lectin-stained sections for studying myocyte hypertrophy by the cross-sectional area (MCSA), and the quantification of infarct size and cardiac remodeling (scar thinning, septum thickness, and expansion index) by planimetry in slices stained with Masson's trichrome and triphenyl tetrazolium chloride. Gal-3 KO mice with MI showed disrupted cardiac remodeling and an increase in the scar thinning ratio and the expansion index. At the onset of MI, myocardial function and cardiac remodeling were also severely affected. At 4 weeks post MI, the natural evolution of fibrosis in infarcted Gal-3 KO mice was also affected. 
In summary, the experimental model of MI is a suitable model for studying the temporal evolution of cardiac repair and remodeling in mice with the genetic deletion of Gal-3 and other animal models. The lack of Gal-3 affects the dynamics of cardiac repair and disrupts the evolution of cardiac remodeling and function after MI.

Here, we describe an experimental model of myocardial infarction, an echocardiography procedure to study cardiac remodeling and function, and procedures for quantifying fibrosis and hypertrophy in picrosirius red-stained and rhodamine-stained sections, as well as the infarct size and expansion index in slices stained with Masson's trichrome.

Cardiovascular disease is the most prevalent cause of death in Western countries, with acute myocardial infarction (MI) being the most prevalent form. ...

ラットにおける慢性心筋梗塞の組織学的定量

要約

さらに動画を探す

この動画の章

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

心筋の構造、機能の in vivo定量的評価では、灌流および生存率は、心臓マイクロコンピュータ断層撮影法を使用しました

マウスにおける心筋梗塞を誘発する低侵襲手術手順

急性心筋梗塞の豚モデルにおける主要アウトカム評価

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

無傷心膜虚血性げっ歯類モデル

マウスにおける右冠動脈の永久結紮による右心室心筋梗塞の生成と特徴付け

心筋梗塞マウスにおける超音波ガイド下多能性幹細胞由来心筋細胞移植術

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

ラットにおける慢性心筋梗塞の組織学的定量

要約

さらに動画を探す

この動画の章

心筋梗塞のマウスモデルにおける冠動脈結紮および心筋内注入

心筋の構造、機能の in vivo定量的評価では 、灌流および生存率は、心臓マイクロコンピュータ断層撮影法を使用しました

マウスにおける心筋梗塞を誘発する低侵襲手術手順

急性心筋梗塞の豚モデルにおける主要アウトカム評価

左前皮下冠動脈の永久的なライゲーションを用いたマウス心筋梗塞モデル

A New Model of Heart Failure Post-Myocardial Infarction in the Rat

無傷心膜虚血性げっ歯類モデル

マウスにおける右冠動脈の永久結紮による右心室心筋梗塞の生成と特徴付け

心筋梗塞マウスにおける超音波ガイド下多能性幹細胞由来心筋細胞移植術

An Experimental Model of Myocardial Infarction for Studying Cardiac Repair and Remodeling in Knockout Mice

心筋の構造、機能の in vivo定量的評価では、灌流および生存率は、心臓マイクロコンピュータ断層撮影法を使用しました