本研究は、文部省が出資する韓国国立研究財団(NRF)を通じて基礎科学研究プログラム(NRF-2018R1D1A1A0202049346)の支援を受けています。

すべての動物研究手順は、実験動物福祉法、実験動物のケアと使用のためのガイド、および韓国カトリック大学医学部の施設動物ケアおよび使用委員会(IACUC)が提供するげっ歯類実験のガイドラインと方針に従って提供されました。
1. MSCおよび注射ゼラチンハイドロゲルの調製
<ol><li>37°Cと5%CO2で100mm培養皿で培養MSC.CSの成長が80%の合流に達したら、DPBSで皿を2回洗い、37°Cでトリプシン置換の1 mLを3分間加えます。 注:MSCは、従来の手順16に従ってマウス骨髄から単離され、10%のウシ胎児血清(FBS)および1%抗生物質抗ミコティック溶液を含むダルベッコの修飾ワシ培地(DMEM)で培養され、この研究のために7\u20129の通過間に使用された。</li>
	<li>培地と遠心分離機を500xgで9mL3分間加えます。次に、得られた上清を捨て、細胞をPBSの1mLで再懸濁し、細胞懸濁液を氷上に維持する。</li>
	<li>10 μLのトリパンブルーで10 μLの細胞懸濁液を希釈し、自動化されたセルカウンターを使用して細胞濃度を得る。</li>
	<li>1 x 107 細胞/mLの密度で1mLチューブに再中断し、MCを移します。</li>
	<li>PBSでGHコンジュゲート溶液の6.25重量%を調製し、2バイアルに分離します。次に、GH溶液をHRP(GH溶液A)の6μg/mLまたはH2O2(GH溶液B)の0.07重量%と混合します。 注:ゼラチン-ヒドロキシフェニルプロピオン酸(GH)のコンジュゲートを公開されたプロトコル12、15に従って準備します。<ol>
<li>HRP(GH溶液A)とGH共役溶液に対するGHコンジュゲート溶液の9:1体積比をH2O2(GH溶液B)にそれぞれ保持する。</li>
	</ol>
</li>
	<li>GH溶液AとMSCを混合する前に、細胞懸濁液を1,000xgで短時間遠心分離し、得られた上清を慎重に吸引する。続いて、MCsを含むペレットをGH溶液Aと混合する。</li>
</ol>2. SIU MSCローディングと3次元インビトロ培養
<ol><li>GH溶液A(MSCを含む)とGH溶液Bを二重シリンジの両側にロードする。5 x 106 細胞/mLの最終的な密度のMSCとの結合されたGHの解決の300 μLを8ウェルの部屋のスライドに置く。</li>
	<li>酵素架橋によるシトゥーヒドロゲル形成とその後のMSCカプセル化の後、10%FBSおよび1%抗生物質抗ミコティック溶液を含むDMEMの700 μLを加える。</li>
	<li>スライドを37°Cおよび5%CO2でインキュベートし、2\u20123日ごとに培地を交換します。</li>
</ol>3. GHヒドロゲル内のインビトロ増殖とMSCの生存の確認
<ol><li>GHヒドロゲル内の3D培養MCの生存率を決定するには、所定のインキュベーション時間後に生きた/死細胞染色アッセイを使用してください。</li>
	<li>3、5、7または14日間のGHヒドロゲル中のカプセル化されたMSCのインキュベーションに続き、培地を吸引し、PBSで2回洗浄した。</li>
	<li>カルセインAM 5 μL、エチジウムホモジマー-1(EthD-1)を10 mLのDPBSで含む染色液を調製します。</li>
	<li>200 μLの染色液をウェルに加え、室温で暗闇の中で30分間インキュベートします。</li>
	<li>染色液を吸引し、PBSで2回洗浄します。</li>
	<li>慎重にスライドからチャンバーを分離し、GHヒドロゲルの上に完全なカバースリップを置きます。コンフォーカル顕微鏡を使用して、カプセル化されたMSCの増殖および形態学的変化の程度を可視化する。 注:蛍光画像は200倍倍の下で取得され、カルセインの場合は470/540 nm、EthD-1では516/607nmの励起/発光波長で画像化されました。</li>
</ol>4. マウスの心筋梗塞の誘導
<ol><li>7週齢の雄C57BL/6マウス(20\u201222 g)を、ゾレチル(30mg/kg)とロンプン(10mg/kg)の混合物を生理膜内注射して生理学します。</li>
	<li>手術前に、脱毛クリームを使用してマウスの胸部を脱毛し、ヨウ素で皮膚を殺菌します。</li>
	<li>手術台の上にマウスを置き、カテーテルを気管に挿入して、機械的換気を介して補足的な酸素を提供します。</li>
	<li>手術用ハサミを使って皮膚をそっと切り抜け、肋間筋をマイクロハサミで貫通する。5-0シルク縫合糸を使用して2番目と3番目の左肋骨を分離し、開いた胸腔を維持します。</li>
	<li>8-0の針ホルダーを使用して、左前前下降(LAD)冠状動脈を慎重にリゲートするポリプロピレン縫合糸を電気焼き液を用いて縫合糸を切断する。</li>
	<li>左心室前壁の色の変化を観察します。</li>
</ol>5. MSC負荷GHヒドロゲルの心筋内移植
<ol><li>LADライゲーションにより心筋梗塞を誘発した後、26G針を装着したデュアルシリンジを用いて、梗塞境界領域(合計:2 x 105 MSC/20 μL)の2つの異なる点に10μLのMSC負荷GH溶液を注入します。<ol>
<li>ステップ1で説明したのと同じ手順に従って、MSC-LOADING GH溶液を二重シリンジに準備し、移します。 注:梗塞領域内のMSC負荷GHヒドロゲルの生着を評価するために、MSCおよびGHコンジュゲートは、それぞれPHK26およびフルオレセインイソチオシアネート(FITC)で事前に標識しました。</li>
	</ol>
</li>
	<li>開いた胸腔を復元し、5-0縫合を使用して筋肉と皮膚を閉じます。 注意:胸を閉める前に、カテーテルの注射器を使用して空気を取り除きます。</li>
	<li>気管チューブを取り外し、回復中に赤外線ランプの下のケージにマウスを置きます。</li>
	<li>術後鎮痛の場合、皮下ケトプロフェン注射剤(1日5mg/kg)を最低72時間投与する。すべてのマウスは、外科的処置と適切な疼痛治療の後に適切な回復を確実にするために適切な時間を注意深く監視する必要があります。</li>
</ol>6. 心エコー検査
<ol><li>移植後4週間、最初に5%のイオブルランでマウスを麻酔し、イオブルラン濃度を1%に調整する。</li>
	<li>脱毛クリームを使用して胸を脱退し、加熱パッドの上にマウスを置きます。胸部に超音波トランスデューサーゲルを適用します。</li>
	<li>2次元の準船骨短軸ビューを取得し、乳頭筋のレベルでMモードトレースを記録します。 注: 左寄尾線に線形配列トランスデューサ(7\u201215 MHz)を配置し、解剖構造を表示します。</li>
	<li>LVAW、LVID、LVPWの対応するラインを測定して、心臓壁の厚さ、チャンバー寸法、および分数短縮を得ます。 注:同じ解剖学的位置で適切な評価を確実にするために、乳頭筋のレベルで、排出分率(EF)、分数短縮(FS)、および末収縮期容積(ESV)を含む心機能を比較してください。</li>
</ol>7. 組織学的評価
<ol><li>MSC負荷GHヒドロゲルを梗塞した心臓に移植した後の所定の時間に、マウスをCO2チャンバーに安楽死させ、組織学的分析用の心臓を集める。</li>
	<li>ヘマトキシリンとエオシン(H&E)とマッソンのトリクローム(MT)染色の場合、解剖した心臓組織を4%パラホルムアルデヒド(PFA)に固定し、パラフィンに埋め込みます。次に、パラフィン埋め込み心臓ブロックをミクロトームを用いて4μmシリアルセクションに切断し、標準プロトコル17に従ってMT染色でセクションを染色する。</li>
	<li>20倍の倍率でスライドスキャナ上の画像を取得し、治療群の梗塞サイズを計算します。 梗塞サイズ(%)=全梗塞周周/総LV周回×100</li>
	<li>正中長の測定で両方の円周を計算します。LV中線周周では、心内膜面と心外膜表面の中心線の長さを測定します。中線梗塞の円周の場合、心筋18の全厚さの50%以上を含む梗塞の長さを測定する。 注:すべての画像解析は、ImageJ ソフトウェアを使用して実行されました。</li>
	<li>乳頭筋レベルでの瘢痕の壁の厚さを測定します。</li>
	<li>コラーゲン領域の分数を計算します。 コラーゲン領域(%)=間質線維化/筋細胞領域の総面積 x 100</li>
</ol>

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注射可能なGHヒドロゲルは、多様な治療薬をその一体的にその中に組み込む能力のために、インビボアプリケーションに大きな可能性を秘めています。さらに、それらの物理的および生化学的特性は、疾患に依存する要件に基づいて容易に操作することができる。この点で、注射ヒドロゲルは、負傷した心臓19,20における生存不良および細胞保持(す...

心臓幹細胞療法は、心筋修復および再生1,2の潜在的アプローチとして出現した。動物モデルおよび臨床試験における最近の肯定的な結果にもかかわらず、心筋修復のための幹細胞ベース療法の適用は、梗塞した心臓組織3、4における注入された細胞の低い保持および生存不良のために制限される。その結果、細胞系組織工学の使用は、注射可能な生体材料5、心臓パッチ6、および細胞シート7を含む、宿主心筋内の細胞の保持および統合を改善するために集中的に研究されてきた。
生体工学的心臓組織修復に対する様々な潜在的アプローチの中で、間葉系幹細胞(MSC)、胚性幹細胞(ESC)、および人工多能性幹細胞(iPSC)などの適切な細胞タイプと組み合わせた注射可能なヒドロゲルは、心筋領域8,9に細胞を効果的に送達するための魅力的な選択肢である。ゼラチンは、よく知られている天然ポリマーであり、生物医学用途で使用される幅広い生体材料と比較して、その優れた生体適合性、かなりの生分解性、および免疫原性の低下により、注射可能なマトリックスとして使用することができます。ゼラチンベースの注入プラットフォームは大きな可能性を秘めていますが、生体内での適用性は、低い機械的剛性と生理学的環境における容易な劣化性に基づいて制限されたままです。
これらの制限を克服するために、インビボ用途に対してヒドロキシフェニルプロピオン酸からなるゼラチン系ヒドロゲルの新規かつシンプルな設計が提案されている。ゼラチンヒドロキシフェニルプロピオン酸(GH)コンジュゲートは、酵素、西洋ワサビペルオキシダーゼ(HRP)の存在下でその時に架橋することができ、その後、様々な薬物、生体分子、またはヒドロゲル内の細胞を封入し、組織工学アプリケーション10、11、12、13、14に大きな可能性を示唆している。さらに、我々は最近、カプセル化されたMSCを含むGHヒドロゲルの治療効果を調査し、マウスモデル15におけるMI後の心臓修復および再生に成功した心臓修復および再生におけるそれらの使用を実証した。本プロトコルでは、GHヒドロゲル内のMCのカプセル化およびインビトロ3次元(3D)増殖に関する簡単な手法について述べた。また、梗塞した心臓へのMSC負荷GHヒドロゲルの冠動脈結紮および筋膜内移植を介してマウスMIモデルを生成するように設計された外科的処置を導入する。

<table>
	<tbody>
		<tr>
			<td>4 % paraformaldehyde (PFA)</td>
			<td>Intron</td>
			<td>IBS-BP031-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 silk suture</td>
			<td>AILEE</td>
			<td>SK534</td>
			<td></td>
		</tr>
		<tr>
			<td>8-0 polypropylene suture</td>
			<td>ETHICON</td>
			<td>M8732H</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Nunc LAB-TEK</td>
			<td>154534</td>
			<td></td>
		</tr>
		<tr>
			<td>Angiocath Plus (22GA) catheter</td>
			<td>BD Angiocath Plus</td>
			<td>REF382423</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-antimyocotic</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>GYROGEN</td>
			<td>1582MGR</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slipe</td>
			<td>MARIENFELD</td>
			<td>101242</td>
			<td></td>
		</tr>
		<tr>
			<td>Deluxe High Temperature Cautery kit</td>
			<td>Bovie</td>
			<td>QTY1</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual-syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EOSIN</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT110116</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>EMSURE</td>
			<td>K49350783 739</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16000-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Fechtner conjunctiva forceps titanium</td>
			<td>WORLD PRECISISON INSTRUMENTS</td>
			<td>WP1820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate isomer I (FITC)</td>
			<td>SIGMA-ALDRICH</td>
			<td>F7250</td>
			<td></td>
		</tr>
		<tr>
			<td>Forcep</td>
			<td>HEBU</td>
			<td>HB0458</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Ildong Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Stoelting</td>
			<td>50300</td>
			<td>Homeothermic Blanket System</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>50301</td>
			<td>Replacement Heating Pad for 50300 (10 X 12.5cm)</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>SIGMA-ALDRICH</td>
			<td>HHS80</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxide (HRP; 250-330 U/mg)</td>
			<td>SIGMA-ALDRICH</td>
			<td>P8375</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide (H2O2; 30 wt % in H2O)</td>
			<td>SIGMA-ALDRICH</td>
			<td>216763</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td>Green Pharmaceutical</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD cell staining kit</td>
			<td>Thermo Fisher</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical ventilator</td>
			<td>Harvard Apparatus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge</td>
			<td>HANIL</td>
			<td>Micro 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro needle holder</td>
			<td>KASCO</td>
			<td>37-1452</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro scissor</td>
			<td>HEBU</td>
			<td>HB7381</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>OLYMPUS</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>MT staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>HT1079-1SET</td>
			<td>Weigert&#8217;s iron hematoxylin solution</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>HT15-1KT</td>
			<td>Trichrome Stain (Masson) Kit</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>LK LABKOREA</td>
			<td>H06-660-107</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS buffer</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>PHK26 staining kit</td>
			<td>SIGMA-ALDRICH</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>HEBU</td>
			<td>HB7454</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M micopore</td>
			<td>1530-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue cassette</td>
			<td>Scilab Korea</td>
			<td>Cas3003</td>
			<td></td>
		</tr>
		<tr>
			<td>Transducer gel</td>
			<td>SUNGHEUNG</td>
			<td>SH102</td>
			<td></td>
		</tr>
		<tr>
			<td>Trout-Barraquer needle holder curved</td>
			<td>KASCO</td>
			<td>50-3710c</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound system</td>
			<td>Philips</td>
			<td>Affiniti 50</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>JUNSEI</td>
			<td>25175-0430</td>
			<td></td>
		</tr>
	</tbody>
</table>

intramyocardial transplantation of msc-loading injectable hydrogels after myocardial infarction in a murine model

梗塞後の心不全を予防するための現在の心臓幹細胞療法が直面している主要な問題の1つは、負傷した心筋内の移植細胞の低い保持率と生存率であり、治療効果を制限する。近年、スキャフォールディング生体材料の使用は、幹細胞治療の改善と最大化に注目されています。このプロトコルの目的は、注射用ヒドロキシフェニルプロピオン酸(GH)ヒドロゲルを使用して骨髄由来間葉系幹細胞(MCC)を移植するための簡単で簡単な技術を導入することです。ヒドロゲルは、その場で架橋され、高い生体適合性を持つ能力のために、心臓組織工学アプリケーション用の細胞送達プラットフォームとして好ましい。MSC負荷GHヒドロゲル(MSC/ヒドロゲル)を製造し、その生存と増殖を3次元(3D)インビトロ培養で評価する簡単な方法を紹介します。また、マウスにおけるMSC/ヒドロゲルの心筋内移植の手法を示し、左前下降(LAD)冠動脈結紮術とその後のMSC/ヒドロゲル移植を介して心筋梗塞(MI)を誘導する外科的処置を説明する。

梗塞心筋にMCを効果的に送達するために、 図1 に記載されたシンチクロスリンク可能なヒドロゲル中のMSC負荷をこのプロトコルで使用した。インビボ移植の前に、GHヒドロゲル中のMCの増殖および生存は、3Dインビトロ生きている/死細胞染色アッセイ(生:緑;死んだ:赤)によって確認された。 図2に示すように、代表的な画像は十分なMSC増殖を示し、GH?...

Watch this Scientific Journal Video about Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model at JoVE.com

Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

幹細胞ベースの治療は、心筋梗塞後に負傷した心臓組織を修復するための効率的な戦略として浮上している。酵素的に架橋できるゼラチンハイドロゲルを用いた幹細胞移植に最適なインビボアプリケーションを提供します。

マウスモデルにおける心筋梗塞後のMSC負荷注入性ヒドロゲルの筋内移植

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of ...

intramyocardial-transplantation-msc-loading-injectable-hydrogels

Research

JoVE Journal

Medicine

4.3K ビュー.  The Catholic University of Korea. 幹細胞ベースの治療は、心筋梗塞後に負傷した心臓組織を修復するための効率的な戦略として浮上している。酵素的に架橋できるゼラチンハイドロゲルを用いた幹細胞移植に最適なインビボアプリケーションを提供します。

ビデオ: Intramyocardial Transplantation of MSC-Loading Injectable Hydrogels after Myocardial Infarction in a Murine Model

Acute Myocardial Infarction in Rats

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular diseases. Animal models that mimic human cardiac disease, such as myocardial infarction (MI) and ischemia-reperfusion (IR) that induces heart failure as well as pressure-overload (transverse aortic constriction) that induces cardiac hypertrophy and heart failure (Goldman and Tarnavski), are useful models to study cardiovascular disease. In particular, myocardial ischemia (MI) is a leading cause for cardiovascular morbidity and mortality despite controlling certain risk factors such as arteriosclerosis and treatments via surgical intervention (Thygesen). Furthermore, an acute loss of the myocardium following myocardial ischemia (MI) results in increased loading conditions that induces ventricular remodeling of the infarcted border zone and the remote non-infarcted myocardium. Myocyte apoptosis, necrosis and the resultant increased hemodynamic load activate multiple biochemical intracellular signaling that initiates LV dilatation, hypertrophy, ventricular shape distortion, and collagen scar formation. This pathological remodeling and failure to normalize the increased wall stresses results in progressive dilatation, recruitment of the border zone myocardium into the scar, and eventually deterioration in myocardial contractile function (i.e. heart failure). The progression of LV dysfunction and heart failure in rats is similar to that observed in patients who sustain a large myocardial infarction, survive and subsequently develops heart failure (Goldman). The acute myocardial infarction (AMI) model in rats has been used to mimic human cardiovascular disease; specifically used to study cardiac signaling mechanisms associated with heart failure as well as to assess the contribution of therapeutic strategies for the treatment of heart failure. The method described in this report is the rat model of acute myocardial infarction (AMI). This model is also referred to as an acute ischemic cardiomyopathy or ischemia followed by reperfusion (IR); which is induced by an acute 30-minute period of ischemia by ligation of the left anterior descending artery (LAD) followed by reperfusion of the tissue by releasing the LAD ligation (Vasilyev and McConnell). This protocol will focus on assessment of the infarct size and the area-at-risk (AAR) by Evan's blue dye and triphenyl tetrazolium chloride (TTC) following 4-hours of reperfusion; additional comments toward the evaluation of cardiac function and remodeling by modifying the duration of reperfusion, is also presented. Overall, this AMI rat animal model is useful for studying the consequence of a myocardial infarction on cardiac pathophysiological and physiological function.

The rat model of acute myocardial infarction (AMI) is useful to study the consequence of a MI on cardiac pathophysiological and physiological function.

ラットにおける急性心筋梗塞

With heart failure leading the cause of death in the USA (Hunt), biomedical research is fundamental to advance medical treatments for cardiovascular ...

医学

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 ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

A Hydrogel Construct and Fibrin-based Glue Approach to Deliver Therapeutics in a Murine Myocardial Infarction Model.

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of treatments in vivo1. The traditional, established protocol has been further fine-tuned to minimize the damage to the animal. Notably, the pectoral muscle layers are teased away rather than simply cut, and the thoracotomy is approached intercostally as opposed to breaking the ribs in a sternotomy, preserving the integrity of the ribcage. With these changes, the overall stress on the animal is decreased. 
Stem cell therapies aimed to alleviate the damage caused by MIs have shown promise over the years for their pro-angiogenic and anti-apoptotic benefits. Current approaches of delivering cells to the heart surface typically involve the injection of the cells either near the damaged site, within a coronary artery, or into the peripheral blood stream2-4. While the cells have proven to home to the damaged myocardium, functionality is limited by their poor engraftment at the site of injury, resulting in diffusion into the blood stream5. This manuscript highlights a procedure that overcomes this obstacle with the use of a cell-encapsulated hydrogel patch. The patch is fabricated prior to the surgical procedure and is placed on the injured myocardium immediately following the occlusion of the left coronary artery. To adhere the patch in place, biocompatible external fibrin glue is placed directly on top of the patch, allowing for it to dry to both the patch and the heart surface. This approach provides a novel adhesion method for the application of a delicate cell-encapsulating therapeutic construct.

This protocol aims to alleviate the limitation of poor cell engraftment for stem cell treatment of myocardial infarctions through the use of a hydrogel system and a fibrin-based glue. With this approach, cell-to-tissue contact post-infarction can be maintained, increasing the therapeutic potential of beneficial agents at the site of injury.

ヒドロゲルは、構築し、マウス心筋梗塞モデルにおける治療薬を送達するためのフィブリンベースの接着剤のアプローチ。

The murine MI model is widely recognized in the field of cardiovascular disease, and has consistently been used as a first step to test the efficacy of ...

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 ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

新生児マウスの心筋梗塞、心臓再生のモデル

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

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 Cryoinjury Model to Study Myocardial Infarction in the Mouse

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we demonstrate a murine cryoinjury infarct model that generates precise infarct sizes with high reproducibility and replicability. In brief, after intubation and sternotomy of the animal, the heart is lifted from the thorax. The probe of a handheld liquid nitrogen delivery system is applied onto the myocardial wall to induce cryoinjury. Impaired ventricular function and electrical conduction can be monitored with echocardiography or optical mapping. Transmural myocardial remodeling of the infarcted area is characterized by collagen deposition and loss of cardiomyocytes. Compared to other models (e.g., LAD-ligation), this model utilizes a handheld liquid nitrogen delivery system to generate more uniform infarct sizes.

This article demonstrates a model to study cardiac remodeling after myocardial cryoinjury in mice.

マウスの心筋梗塞を研究する凍結傷害モデル

The use of animal models is essential for developing new therapeutic strategies for acute coronary syndrome and its complications. In this article, we ...

Delayed Intramyocardial Delivery of Stem Cells after Ischemia Reperfusion Injury in a Murine Model

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, cardiac stem cell therapy is studied by delivering SCs concurrently with the induction of myocardial injury. However, this approach presents two significant limitations: the early hostile pro-inflammatory ischemic environment may affect the survival of transplanted SCs, and it does not represent the subacute infarction scenario where SCs will likely be used. Here we describe a two-part series of surgical procedures for the induction of ischemia-reperfusion injury and delivery of mesenchymal stem cells (MSCs). This method of stem cell administration may allow for the longer viability and retention around damaged tissue by circumventing the initial immune response. A model of ischemia reperfusion injury was induced in mice accompanied by the delivery of mesenchymal stem cells (3.0 x 105), stably expressing the reporter gene firefly luciferase under the constitutively expressed CMV promoter, intramyocardially 7 days later. The animals were imaged via ultrasound and bioluminescent imaging for confirmation of injury and injection of cells, respectively. Importantly, there was no added complication rate when performing this two-procedure approach for SC delivery. This method of stem cell administration, collectively with the utilization of state-of-the-art reporter genes, may allow for the in vivo study of viability and retention of transplanted SCs in a situation of chronic ischemia commonly seen clinically, while also circumventing the initial pro-inflammatory response. In summary, we established a protocol for the delayed delivery of stem cells into the myocardium, which can be used as a potential new approach in promoting regeneration of the damaged tissue.

Stems cells are continuously investigated as potential treatments for individuals with myocardial damage, however, their decreased viability and retention within injured tissue can impact their long-term efficacy. In this manuscript we describe an alternative method for stem cell delivery in a murine model of ischemia reperfusion injury.

マウスモデルにおける虚血再灌流損傷後の幹細胞の筋内内出産の遅延

There is significant interest in the use of stem cells (SCs) for the recovery of cardiac function in individuals with myocardial injuries. Most commonly, ...

A Modified Simple Method for Induction of Myocardial Infarction in Mice

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI remodeling and evaluation of novel therapeutics. Different methods (e.g., isoproterenol treatment, cryoinjury, coronary artery ligation, etc.) have been used to induce MI. Compared with isoproterenol treatment and cryoinjury, coronary artery ligation may better reflect the ischemic response and chronic remodeling after MI. However, traditional methods for coronary ligation in mice are technically challenging. The current study describes a simple and efficient process for induction of MI in mice with readily available materials. The mouse chest skin was cut open under stable anesthesia. The heart was immediately externalized through the intercostal space after blunt separation of the pectoralis major and pectoralis minor. The left anterior descending branch (LAD) was ligated with a 6-0 suture 3 mm from its origin. Following LAD ligation, staining with 2,3,5-Triphenyltetrazolium chloride (TTC) indicated successful induction of MI and temporal changes of post-MI scar size. Meanwhile, survival analysis results showed overt mortality within 7 days after MI, mainly due to cardiac rupture. Moreover, post-MI echocardiographic assessment demonstrated successful induction of contractile dysfunction and ventricular remodeling. Once mastered, an MI model can be established in mice within 2-3 min with readily available materials.

Under adequate anesthesia, the mouse heart was externalized through the intercostal space, and myocardial infarction was successfully induced by ligating the left anterior descending artery (LAD) using materials readily available in most laboratories.

マウスにおける心筋梗塞の誘導のための改良簡便法

Myocardial infarction (MI) represents one of the leading causes of death. MI models are widely used for investigating the pathomechanisms of post-MI ...