私たちは、顕微鏡分析および心臓修復、技術者や私たちの動物施設の管理に関連するプロジェクトを支援するためのMagdiヤコブ研究所（MYI）に感謝。この作品は、英国心臓財団（BHF）、プロジェクト無償PG/10/019によってサポートされています。 MPSは、MYIとBHFによってサポートされています。 TPはBHF-優良研究員である。 NRは、NH＆MRCオーストラリアフェローである。

すべての動物実験は、国際（欧州議会の指令2010/63/EU）と国家（英国内務省、アクト1986）規制に準拠して行った。本明細書に記載の手順は、英国のライセンス当局の下での作業の我々の計画の一部であり、記録の目的のために行われていない。 1。細胞の調製このプロトコルは、デモ用の特定の細胞株（ヒト胎児腎臓HEK293細胞）の調製を記載する。細胞特異的なプロトコルは、成長して、最終的には、特定の細胞系統に分化多能性または成体幹細胞を区別するために使用されなければならない。 <ol><li> DMEM培地（高グルコース、4,500ミリグラム/ L）中の細胞を、1％ピルビン酸ナトリウム、2mMグルタミン、1％の抗生物質ペニシリン/ストレプトマイシン及び10％ウシ胎児血清（FBS）を含有する成長する。一つの10cmプレートは通常、午前1時03分で分割されるべきであり、メディアは2日ごとに新鮮な補足。 </li><li> trypsで穏やかに細胞を処理1：01に記載されるようにDMEM培地で1×に再懸濁した。 </li><li>血球計数器チャンバー内の細胞をカウントし、別のチューブに3×10 6個の細胞を収集します。 </li><li> 5分間100グラムで、スイングバケットローターで遠心分離する。 </li><li>上澄み液を捨て、滅菌したリン酸緩衝液2倍で細胞ペレットを洗浄する。 </li><li>免疫除去したマウスの左心室に注入するための10 50分の6 ULの濃度でPBS 1X中に再懸濁します。 </li></ol> 2。手術前の条件<ol><li>各手術前に、温度制御されたアイソレータ（ 表1 22℃）で、滅菌食物ペレットと水で免疫除去マウス（NOD.CB17-Prkdscid/JHliHSD）を維持する。 </li><li>層流フードの下免疫除去したマウスの手術を行う。 70％エタノールで作業領域をきれいにし、手術器具（鉗子やハサミ）をオートクレーブ。 </li><li>手術と回復のための加熱パッドをオンにします。加熱パッドは重要です中に発生した体温の低下および術後を遮断する。 </li><li>リカバリー·加熱パッド上にきれいにし、オートクレーブケージを置きます。 </li><li>無菌オートクレーブ処理袋に手術室にアイソレータから元のケージでマウスを輸送する。 </li><li>マウスを秤量し、体重に従って注入皮下（sc）投与メデトミジンおよびマウスを麻酔するために0.9％滅菌生理食塩液で希釈した塩酸ケタミンを含有する溶液、ならびに（筋肉組織を緩和するメデトミジン1 mg / kgを、塩酸ケタミン75 mg / kgで）。 </li><li>完全に麻酔をかけたときに（刺激した後、1〜2分以内にごつま先ピンチ反射を）ケージからマウスを削除しない</li><li>胸の左側にある脱毛クリームとスロート面積が適用されます。適切な時間（約2〜5分）した後、水に濡れたティッシュで緩い毛を乾いた布で拭き取ってください。 </li><li>仰臥位での手術パネル上にマウスを置きます。から外科医の視点位置は、テールダウンヘッドアップ垂直である。 </li><li>後肢皮下に無菌生理食塩水（ブプレノルフィン）で希釈した0.1〜0.2μgの/ gで鎮痛ソリューションを注入し、綿棒を使用して無菌性溶液のポビドンヨードで剃毛面積をカバーしています。 </li><li>スロート面積に顕微鏡（2.5X目標を）集中する。 </li></ol> 3。外科的条件<ol><li>鈍ハサミで少し輪状軟骨の下に約0.5〜cm長さの正中線腹部皮膚切開を行います。唾液腺を視覚化するために結合組織から皮膚を分離する。 </li><li>同時に鉗子と磁気胸のリトラクターで各パーツの方に引いて、自然の中線部門での唾液腺を分割し、気管を公開します。 </li><li>喉頭を公開する側にゆっくりと舌を引っ張る。慎重に気管内に挿管チューブをスライドさせます。チューブの先端が表示されたLを通して見えるarynxと気管。チューブには、しかしはっきりと見えない場合に重要なことに、それは、食道内にある。それを削除して、管の先端の位置を変更してください。 </li><li>通風機にチューブを接続します。 200μLおよび150ストローク/分で拍出量を設定します。テープで手術パネルの通気チューブを固定します。 </li><li>鈍ハサミとピンセットで、ヘッド1に垂直尾翼を作る - 左胸部領域の上1.5センチ、長皮膚切開する。 </li><li>切開を加えずに、結合組織/筋肉層から皮膚やメジャーとマイナーの胸の筋肉を緩めます。 </li><li>以下のように第三及び第四肋骨の間開胸を行います。 <ol><li>つまんで小さくて丸みを帯びたピンセットで引っ掻いて肋間筋層を穿孔。 </li><li>肋間筋層が穿孔された後、最大限に胸腔を開くために胸のリトラクターを配置。その上層の耳介（心房）と左心室（LV）の上部及び中央部である今見える。 </li></ol></li><li>細胞を用いた高精度のシリンジを準備します。注射器は、それぞれの注射のために10のULの容積の量を送達することができるはずです。注射器の先端に30ゲージ針をキャスト。 </li><li>針の開口先端を含む露出のみ1ミリメートルを残して、テープで針上（introcan-W 20 Gから）小さなプラスチック製のカニューレを固定します。これは全体の左心室壁を穿孔し、左心室腔に細胞を注入し、針を防ぐことができます。 </li><li> 5Xの目的の助けを借りて、この倍率で左心室を視覚化し、5つの異なる場所（各注入注入106個、合計10 ULをお届けします）での左心室壁に細胞を注入します。注入が成功した場合は、白い領域と、細胞の主要な逆流がないことは、（注入が成功しなかった場合、動物は、機能分析から除外されます）表示されます。 </li><li>開創器を取り外します。胸壁Bを閉じます6-0絹縫合糸の2針を持つ2つの別々のリブを囲むY。 </li><li> PBS 1Xでびっしょり綿棒で胸筋と皮膚に潤いと静かにピンセットで元の位置に筋肉を置く。 </li><li> 6-0絹縫合糸の縫い目3-4で皮膚を閉じます。肌を閉じるには、2〜3ステッチと喉の切開を縫合。 </li><li>麻酔の効果を元に戻すアチパメゾール（1 mg / kgの最終濃度）を注入。 </li><li>とすぐにマウスが自律的に呼吸を開始すると、気管からチューブを取り出して、その右側にマウスを置く。マウスを数分以内に回復すると予想されています。 </li><li>マウスを回して歩いて、時間暖かい回復ケージ（クローズドフィルタートップと小さなIVCケージ）に入れて起動すると。 </li><li>制御加熱箱（37℃）中で一晩、動物を残す。 </li><li>手術後の最初の2日間は、回復を支援するために水とペレット食物と一緒にお皿を埋める。 </li></ol>次のように細胞と10コントロール注入されていない心を注入十心臓は組織学的に分析されています。 <ol><li> 1週間の注入（ 図1、図3）の後に移植された細胞の分析を実行します。 <ol><li>イソフルランの過剰投与により安楽死させた後、組織を固定し、4％パラホルムアルデヒド（PFA）20mlで心臓を灌流。 （百分の70から100）のエタノールの割合を増加させるのに固定された組織を脱水し、パラフィンブロックに埋め込む。 </li><li>形態素解析のためにヘマトキシリン​​およびエオシンで5ミクロンとシミにミクロトームでパラフィンセクションの心を。 </li><li>マッソントリクローム染色を使用して結合組織を可視化する。 </li><li>スライドスキャナ顕微鏡像（ 図1A、2A、3A、3B、および3C）を分析し、デジタルスライドスキャナ顕微鏡を用いて短軸像で心臓全体を視覚化する（ ふぃぎゅ解像度1Bおよび1C）。 </li></ol></li><li>以下の上記の分析の後、キシレン中に心臓組織を脱パラフィン、（100％の水）エタノールの割合を減少させるに試験片を浸漬することによって再水和すると、走査電子顕微鏡のための処理（SEM）として： <ol><li> 1時間、0.1 Mリン酸緩衝液中の1％のタンニン酸で脱パラフィンブロックをインキュベートする。 </li><li> （25から100まで%）エタノールの割合を増やすことで標本を脱水。 </li><li> HMDS（ヘキサメチルジシラザン）で乾燥したサンプル。 </li><li>アチソンシルバーダグと金 - パラジウムでコーティングしてとスタッブス上の標本をマウントします。 </li><li>分析走査型電子顕微鏡（ 図2B）のビューのサンプル。 </li></ol></li></ol>

著者らは、開示することは何もありません。

本稿では、我々は、マウスの心の中の細胞の心筋内注入を実行する方法が示されている。この方法論の証明として、我々は、HEK293細胞を使用している。これは、HEK293細胞は任意の細胞治療研究では使用しないので、この原稿の知見は治療的アプローチへの直接変換のための適切ではないことを強調することが重要である。しかしながら、HEK293細胞は収縮性細胞ではなく、他の細胞型に分化転?...

様々な細胞送達プロトコルは、ヒト患者におけるこの実験手順の効率を翻訳し、有効性及び安全性を目的とした心血管疾患のマウスおよびラットモデルにおいて試験されている。ラット心臓順行3と逆行4冠動脈内の細胞注入でも使用することができるのに対し、小型のげっ歯類の心の中で、心筋内細胞送達は、細胞送達1,2の最も実現可能な方法である。どちらの方法も限界と利点を持っている。冠動脈内経路を介して細胞送達は、グローバルセル3普及を促進する上で直接的な筋肉内注射の理論上の利点を有するが、それはまた、冠動脈塞栓3,5を引き起こす危険性を有している。心筋内配達の制限事項は、機械的な損傷、急性炎症、および心筋障害6,7に関連している。ヒトでは、心臓の修復のための細胞は、心内膜または外科心外膜を通して心筋内注射によって送達されるアプローチまたは冠動脈内動脈ルート8による。経血管経路による注射は、急性心筋梗塞および再灌流心筋症患者に適しているが、完全閉塞や影響領土9の血管内であまり流れの場合にはできないことがあります。経心または経心外膜注入による心室壁への直接注入は、患者の健康状態に応じて技術的に可能である。実際、経心外膜注射用開胸手術が必要であり、経心ための実行可能な虚血または心筋瘢痕9の部位を区別するために必要とされる各患者の電気生理学的マッピングに近づくが、この技術は、10,11安全であることが示されている。 重要なのは、細胞治療の研究に移植するための最良のセルの選択は、調査中です。短期分析（4週間）は、心臓幹細胞の注入がcardiospheresとして定義することを示した<sアップ&gt; 12または骨髄から13サイドポピュレーション細胞瘢痕の大きさおよび細胞死を減少させることによって、心筋梗塞のマウス14及び15のモデルラットにおける心機能回復を誘導した。免疫抑制なしのラットの心筋梗塞モデルにおけるcardiospheresの同種移植は、心臓の再生を促進し、安全であることが判明し、内因性の修復機構15の刺激により心機能を改善しました。心の中Lin-/c-kit +大人の前駆細胞は、in vitroおよびin vivoで自己再生、クローン原性、および多能であることが示され、虚血性ラットの心臓に注射した場合には、障害を受けた心筋壁16の大部分を再構成していたた導電性の中間サイズの冠状動脈17を形成する能力。これらの有望なデータがフェーズを煽っIおよびヒトにおけるII臨床試験：自己および同種間葉系幹細胞（MSC）18の注入は、19を cardiospheresまたはc-Kitの陽性心臓幹細胞（CSC）虚血性心臓におけるヒト20それぞれは、長期試験において、心機能に有益な効果を示した。それにもかかわらず、大規模な長期追跡及び遡及的メタ分析は、幹細胞療法はなく、予測不可能な結果21の範囲内の他、一部の患者に有意な利益を提供することを実証した。それは、これらの制限は、各個人や各疾患のための細胞送達の特定のプロトコルの設計を必要とする可能性があります。 マウスおよびラットモデルにおいて、長期的な研究は、細胞注射は、さらに（12ヶ月）の心機能を改善しなかったことを明らかにした。実際に、ヒト胚性幹細胞由来心筋細胞（hESCの-CM）の移植片は主として線維性組織22,23の層によって宿主心筋から単離した。同様の結果は、梗塞マウス24の中心部に骨格筋芽細胞の心筋内移植後に観察されている。 FurthermoRE、梗塞中心部に機能を維持するために、同種のMSCの長期的な能力は、分化25の後に免疫特権の免疫原性状態への遷移によって制限されてきた。 考察上記で概説した課題と展望を考慮して、我々は、マウスの心筋内注射することによって細胞を送達する方法をここに表示されます。我々は心筋細胞収縮特性のない細胞を宿主の心筋に接続されており、薄い線維性バリアで凝集塊を形成していないことを確認します。いくつかのケースでは、この結果が有利であり得るが、以下の分析は、細胞移植は、同様に機能的に接続された心筋の構造を生成するために変調することができる方法を理解するのに有用であり得る。

<table border="0" cellpadding="0" cellspacing="0" width="560">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Isolator</td>
			<td style="width:115px;">Pfi systems</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Quotation needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Heating Pad</td>
			<td style="width:115px;">Vet Tech Solutions</td>
			<td style="width:88px;">HE006</td>
			<td style="width:255px;">For small animals</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">medetomidine</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">ketamine hydrochloride</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">atipamezole</td>
			<td style="width:115px;">National veterinary Service</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Hair removal cream</td>
			<td style="width:115px;">Commercial shops</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">buprenorphine</td>
			<td style="width:115px;">NVS</td>
			<td style="width:88px;">-</td>
			<td style="width:255px;">Veterinary prescription is necessary</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Leica MZFLIII microscope</td>
			<td style="width:115px;">Leica</td>
			<td style="width:88px;">Model S6E</td>
			<td style="width:255px;">With swing arm stand TS0</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:103px;">Hamamatsu Nanozoomer digital slide scanner</td>
			<td style="width:115px;">Hamamatsu</td>
			<td style="width:88px;">RS series</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">Scanning Electron Microscope</td>
			<td style="width:115px;">Jeol</td>
			<td style="width:88px;">JSM-6610</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Blunt scissors</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">14084-09</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Minivent</td>
			<td style="width:115px;">Harvard apparatus</td>
			<td style="width:88px;">73-0043</td>
			<td style="width:255px;">Including small Y adapter (73-0027) and intubation cannula (73-2844)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">Forceps</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">11052-10</td>
			<td style="width:255px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">Retraction system</td>
			<td style="width:115px;">FST</td>
			<td style="width:88px;">18200-20</td>
			<td style="width:255px;">Kit for animals up to 200grams</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:103px;">30G 12mm; &#189; inch</td>
			<td style="width:115px;">BBraun</td>
			<td style="width:88px;">A210</td>
			<td style="width:255px;">Fine yellow</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:103px;">microliter syringe</td>
			<td style="width:115px;">ESSLAB</td>
			<td style="width:88px;">81201</td>
			<td style="width:255px;">Also include a Hamilton repeating dispenser PB 600-1 Catalogue number 83700</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:103px;">6-0 silk suture</td>
			<td style="width:115px;">Ethicon</td>
			<td style="width:88px;">W1614T</td>
			<td style="width:255px;"></td>
		</tr>
	</tbody>
</table>

intramyocardial cell delivery: observations in murine hearts

以前の研究は、細胞送達は、サイトカインおよび心臓組織の血管再生および細胞生存を増加させる因子の放出によって心機能改善を促進することが示された。また、さらなる観察は、例えば、心臓幹細胞、間葉系幹細胞およびcardiospheresなどの特定の幹細胞は、心筋細胞、平滑筋細胞および内皮細胞に分化することによって周囲の心筋層内に統合する能力を有していることが明らかになった。 ここでは、確実に免疫枯渇マウ​​スの左心室壁への非収縮細胞を送達するための材料および方法を提示する。この顕微手順の顕著なステップは、麻酔および鎮痛注射、気管内挿管、胸を開いて、無菌の30ゲージの針、精密マイクロリットル注射器で細胞の心と配信を露出させるために、切開を伴う。 心臓収穫、embeddiからなる組織処理NG、切片および組織学的染色は、心筋内細胞注入は、小さな心外地域の被害だけでなく、心室壁にを生成することを示した。非収縮細胞は、免疫不全マウスの心筋壁に保持され、心臓の圧力と機械的負荷から保護する可能性線維性組織の層に囲まれた。

我々は、細長い心筋細胞（ 図1A）と比較して、玉石状に、それらの異なる形態（ 図1）により心臓の細胞から区別可能であるHEK293細胞を注射した。 HEK293細胞は、それらの増加、核コンテンツ（ 図1A）の可能性が高い心筋細胞（ピンク色）と比較して、ヘマトキシリン色素（青色）に、より反応性であった。さらに、宿主組織から注入された細胞を区別す?...

Watch this Scientific Journal Video about Intramyocardial Cell Delivery: Observations in Murine Hearts at JoVE.com

Intramyocardial Cell Delivery: Observations in Murine Hearts

例えば、高血圧または心筋梗塞などの心血管疾患のマウスモデルにおける心筋細胞送達は、回生広く研究において、異なる細胞型の治療可能性を試験するために使用される。そのため、詳細な説明およびこの外科処置の明確な可視化は、小型げっ歯類の心血管細胞治療の分析の限界と利点を定義するのに役立ちます。

心筋内細胞送達：マウスハーツの観測

Previous studies showed that cell delivery promotes cardiac function amelioration by release of cytokines and factors that increase cardiac tissue ...

intramyocardial-cell-delivery-observations-in-murine-hearts

Research

JoVE Journal

Medicine

13.4K ビュー.  Imperial College London. 例えば、高血圧または心筋梗塞などの心血管疾患のマウスモデルにおける心筋細胞送達は、回生広く研究において、異なる細胞型の治療可能性を試験するために使用される。そのため、詳細な説明およびこの外科処置の明確な可視化は、小型げっ歯類の心血管細胞治療の分析の限界と利点を定義するのに役立ちます。 

ビデオ: Intramyocardial Cell Delivery: Observations in Murine Hearts

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

Reduction in Left Ventricular Wall Stress and Improvement in Function in Failing Hearts using Algisyl-LVR

Injection of Algisyl-LVR, a treatment under clinical development, is intended to treat patients with dilated cardiomyopathy. This treatment was recently used for the first time in patients who had symptomatic heart failure. In all patients, cardiac function of the left ventricle (LV) improved significantly, as manifested by consistent reduction of the LV volume and wall stress. Here we describe this novel treatment procedure and the methods used to quantify its effects on LV wall stress and function.
Algisyl-LVR is a biopolymer gel consisting of Na+-Alginate and Ca2+-Alginate. The treatment procedure was carried out by mixing these two components and then combining them into one syringe for intramyocardial injections. This mixture was injected at 10 to 19 locations mid-way between the base and apex of the LV free wall in patients.
Magnetic resonance imaging (MRI), together with mathematical modeling, was used to quantify the effects of this treatment in patients before treatment and at various time points during recovery. The epicardial and endocardial surfaces were first digitized from the MR images to reconstruct the LV geometry at end-systole and at end-diastole. Left ventricular cavity volumes were then measured from these reconstructed surfaces.
Mathematical models of the LV were created from these MRI-reconstructed surfaces to calculate regional myofiber stress. Each LV model was constructed so that 1) it deforms according to a previously validated stress-strain relationship of the myocardium, and 2) the predicted LV cavity volume from these models matches the corresponding MRI-measured volume at end-diastole and end-systole. Diastolic filling was simulated by loading the LV endocardial surface with a prescribed end-diastolic pressure. Systolic contraction was simulated by concurrently loading the endocardial surface with a prescribed end-systolic pressure and adding active contraction in the myofiber direction. Regional myofiber stress at end-diastole and end-systole was computed from the deformed LV based on the stress-strain relationship.

This article describes procedures for implanting a novel hydrogel in failing hearts and quantifying its effect on left ventricular wall stress and function. These procedures have been successfully applied in dogs and humans.

Algisyl-LVRを使っハーツ失敗関数内で左心室壁応力と改善の削減

Injection of  Algisyl-LVR, a treatment under clinical development, is intended to treat  patients with dilated cardiomyopathy. This treatment was recently ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

マウスにおける超音波ガイド下経胸壁心筋内注射

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for myocardial infarction and cardiac complications of thoracic surgery. The ability of certain drugs to precondition the myocardium against ischemia reperfusion injury has led to multiple clinical trials, with little success. The isolated heart model allows acute observation of the functional effects of ischemia reperfusion injury in real time, including the effects of various pharmacological interventions administered at any time-point before or within the ischemia-reperfusion injury window. Since brief periods of ischemia can precondition the heart against ischemic injury, in situ aortic cannulation is performed to allow for functional assessment of non-preconditioned myocardium. A saline filled balloon is placed into the left ventricle to allow for real-time measurement of pressure generation. Ischemic injury is simulated by the cessation of perfusion buffer flow, followed by reperfusion. The duration of both ischemia and reperfusion can be modulated to examine biochemical events at any given time-point. Although the Langendorff isolated heart model does not allow for the consideration of systemic events affecting ischemia and reperfusion, it is an excellent model for the examination of acute functional and biochemical events within the window of ischemia reperfusion injury as well as the effect of pharmacological intervention on cardiac pre- and postconditioning. The goal of this protocol is to demonstrate how to perform in situ aortic cannulation and heart excision followed by ischemia/reperfusion injury in the Langendorff model.

The isolated rat heart is an enduring model for ischemia reperfusion injury. Here, we describe the process of harvesting the beating heart from a rat via in situ aortic cannulation, Langendorff perfusion of the heart, simulated ischemia-reperfusion injury, and infarct staining to confirm the extent of ischemic insult.

ランゲンドルフ灌流ラット心臓における虚血再灌流傷害の誘導と評価

The biochemical events surrounding ischemia reperfusion injury in the acute setting are of great importance to furthering novel treatment options for ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection fraction (HFrEF). Before they can be considered for use, and implemented in humans, extensive preclinical studies are required in large animal models to evaluate the safety, efficacy, and fate of the injectate (e.g., stem cells) once delivered into the myocardium. Small rodent models offer advantages (e.g., cost effectiveness, amenability for genetic manipulation); however, given inherent limitations of these models, the findings in these rarely translate into the clinic. Conversely, large animal models such as rabbits, have advantages (e.g., similar cardiac electrophysiology compared to humans and other large animals), whilst retaining a good cost-effective balance. Here, we demonstrate how to perform a percutaneous contrast echocardiography-guided intramyocardial injection (IMI) technique, which is minimally invasive, safe, well tolerated, and very effective in the targeted delivery of injectates, including cells, into several locations within the myocardium of a rabbit model. For the implementation of this technique, we also have taken advantage of a widely available clinical echocardiography system. After putting in practice the protocol described here, a researcher with basic ultrasound knowledge will become competent in the performance of this versatile and minimally invasive technique for routine use in experiments, aimed at hypothesis testing of the capabilities of cardiac regenerative therapeutics in the rabbit model. Once competency is achieved, the whole procedure can be performed within 25 min after anaesthetizing the rabbit.

Novel therapeutic strategies in cardiac regenerative medicine require extensive and detailed studies in large preclinical animal models before they can be considered for use in humans. Here, we demonstrate a percutaneous contrast echocardiography-guided intramyocardial injection technique in rabbits, which is valuable for hypothesis testing the efficacy of such novel therapies.

経皮的造影エコー ガイド心筋と大規模な前臨床モデルで携帯配信

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

過期治療アプリケーションを用いてラット頸動脈圧制御区分バルーン損傷

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

生理学的条件下での豚の心臓のスライスと培養

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

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, ...

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

One of the major issues facing current cardiac stem cell therapies for preventing postinfarct heart failure is the low retention and survival rates of transplanted cells within the injured myocardium, limiting their therapeutic efficacy. Recently, the use of scaffolding biomaterials has gained attention for improving and maximizing stem cell therapy. The objective of this protocol is to introduce a simple and straightforward technique to transplant bone marrow-derived mesenchymal stem cells (MSCs) using injectable hydroxyphenyl propionic acid (GH) hydrogels; the hydrogels are favorable as a cell delivery platform for cardiac tissue engineering applications due to their ability to be cross-linked in situ and high biocompatibility. We present a simple method to fabricate MSC-loading GH hydrogels (MSC/hydrogels) and evaluate their survival and proliferation in three-dimensional (3D) in vitro culture. In addition, we demonstrate a technique for intramyocardial transplantation of MSC/hydrogels in mice, describing a surgical procedure to induce myocardial infarction (MI) via left anterior descending (LAD) coronary artery ligation and subsequent MSC/hydrogels transplantation.

Stem cell-based therapy has emerged as an efficient strategy to repair injured cardiac tissues after myocardial infarction. We provide an optimal in vivo application for stem cell transplantation using gelatin hydrogels that are able to be enzymatically cross-linked.

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 ...