この刊行物は、サウスカロライナ医科大学の学術家、NIH / NCATS認可番号UL1 TR000062と、サウスカロライナ州臨床およびトランスレーショナルリサーチ（SCTR）研究所によってサポートされていました。さらにサポートがDRMにVAのメリット賞BX002327-01によって提供されました。 DJHは、NIH / NCATS認可番号TL1 TR000061によっておよびNIH認可番号T32 GM008716によってサポートされていました。 SEAは、NIHグラント番号T32 HL07260によってサポートされていました。

ここに記載されたすべての手順は、サウスカロライナ医科大学の施設内動物管理使用委員会によって承認されています。ここに記載した実験は、急性、非生存実験です。このように、そこに眼軟膏のない使用はありませんし、無菌手術室は必要ありません。安楽死は、心臓の収穫時に放血することによって達成されます。 1。実験の準備<ol><li>一定圧力または一定流量、ランゲンドルフ灌流装置4のいずれかを設定します。 </li><li> （：112のNaCl、5 KClを、1.2 硫酸マグネシウム、1 K 2 HPO 4、1.25のCaCl 2、25のNaHCO 3、11 D-グルコース、0.2オクタン酸、pH値= 7.4ミリモル）に変更されたクレブスヘンゼライト緩衝液の4 Lを準備します。 <ol><li> 10×ストックバッファを作成します。 <ol><li> 3 Lの超純水に塩化ナトリウム、塩化カリウム、 硫酸マグネシウム、及びK 2 HPO 4を溶解します。 </li><li> 500ミリリットル超純水のwiでのCaCl 2を溶解1 N HClを40mlの目。 </li><li>ゆっくり1.2.1.1で行われたバッファへのCaCl 2溶液を加えます。 4 L.合計するのに十分な超純水を追加し、0.8ミクロンフィルターを通して濾過する前に、1時間攪拌します。 4℃で保存します。 </li><li> 4 Lの超純水に炭酸水素ナトリウムを溶解します。 0.8μmのフィルターで通して濾過する前に1時間攪拌します。 4°Cで保存</li></ol></li><li> 400ミリリットル10倍クレブスヘンゼライト緩衝液を400mlの10倍のNaHCO 3緩衝液で結合します。攪拌しながら、グルコースおよびオクタン酸を溶かし4 L.合計するのに十分な超純水を追加します。 0.8μmのフィルターを通して濾過します。 </li></ol></li><li>心のためのシステムを準備します。 <ol><li> 4 Lボトルにろ過し、バッファの2 Lを追加大動脈カニューレに取り付けられたコック上記に38を置きました。これは推奨灌流圧4である心臓に70〜80 mmHgでの灌流圧をお届けします。 </li><li>ボトル内のバッファにガス分散バブラーを挿入します。 </li><li>チューブの内部から気泡を除去するようにしてくださいされ、システムを介してバッファを実行します。 </li><li>心臓の前に少なくとも30分（95％O 2/5％CO 2）ガスをオンにすると、システム上に配置されることになります。酸素飽和度を確認するために、ガス流プローブおよび血液ガス分析装置を使用します。 </li></ol></li><li>外科的供給とバルーンを準備します。 <ol><li>心の収穫については、6.75「手術用はさみ、4.5の1対」ハサミ、止血の二対、caramalt鉗子の二組、27 G針を持つ2つのシリンジ、0 - ゲージ絹縫合糸の2個（約15の1組を準備長さはセンチ）、1 1/16インチ有刺カニューレ、1気管カニューレ。 </li><li>気管カニューレを組み立てるには、Yコネクタに1/16インチ径のプラスチック管の短い断片を接着。 </li><li>バルーン装置を組み立てるには：1/16インチチューブの片に強制経口投与針を取り付け、その後、圧力変換器ハウジングにチューブを接続します。圧力TRの反対側に取り付けられた小型の注射器ansducerハウジングは、バルーンの膨張および収縮が可能になります。 <ol><li>中心部の強制経口投与針のプラスチックラップと場所先端の正方形をカットします。経管栄養針の周りにラップを包みます。 </li><li>生理食塩水でバルーンを記入し、2本の縫合糸で縛ります。漏れがないことを確認するためにバルーンを膨らませます。膨張したバルーン内の生理食塩水の量​​は、およそ200μLであるべきであるが、ラットの大きさに依存して変化し得ます。 </li><li>あるいは、プラスチックラップの代わりにラテックス指サックを使用しています。指サックの中に強制経口投与針を置き、生理食塩水で満たし、縛ります。 </li></ol></li></ol></li></ol> 2.収穫の心<ol><li> decapiconeまたは他の手動の拘束装置を使用して、スプラーグドーリーラットを抑制する。ラットを計量。 </li><li>ラットを麻酔するために腹腔内注射を介して、ケタミン/キシラジン混合物（0.85ミリグラム/ kgのケタミンおよび0.15ミリグラム/ kgのキシラジン）を管理します。麻酔薬のこの用量は、のために十分な麻酔を提供します20〜40分が、麻酔が確認された後の手順では、可能な限り迅速に完了する必要があります。 </li><li>つま先ピンチ反射をテストすることによって、麻酔の適切度を確認してください。 </li><li>気管切開<ol><li>切開を作るために毛皮やはさみを上昇させるために止血剤を使用して、顎のベースに半ば前足から毛皮を削除します。一つは毛皮に沿って切断し、それを削除することができ、基礎となる筋肉からそれを分離するために上昇した毛皮を保管してください。 </li><li>止血剤を使用して、腺組織を持ち上げ、腺組織に筋膜の横切開が悪くなります。ニックにない頸静脈を注意しながら、オープン切開部を広げるために止血剤を使用してください。 </li><li> 、止血剤を使用してピンチの筋肉の気管の上にあるし、使用してハサミだけで止血の先端下の筋肉に横切開を行います。止血鉗子を使用して、気管を明らかに離れて組織を広げます。 </li><li>静かに止血を前進および後退させることによって進行しながら、筋膜をクリアし、気管の下に止血剤を慎重に挿入します。 </li><li>止血剤が正常気管の後ろに配置されると、止血剤で0-0絹縫合糸をつまんではさみの小さな、鋭いペアで気管を二等分します。 </li><li>二等分気管に気管カニューレを挿入し、気管の後ろに縫合糸を引っ張ると、カニューレを挿入し、気管の周りに縫合糸を結ぶ、外科医の結び目とカニューレのY-鼠径部に縫合糸を固定します。 </li><li> 27 G針を使用して、頸静脈に千U / kgのヘパリンを注入し、30秒間を循環させています。 </li></ol></li><li>開胸<ol><li>止血剤でピンチとハサミで切断することにより、ミッド前足領域に半ば腹部から毛皮を削除します。 </li><li>ちょうどダイヤフラムの下に、腹部の筋肉壁に¾インチ横切開を加えます。 </li><li>胸骨に沿って切断し、腹壁と胸の壁まで縦切開を行います。 </li><li>左右のバックダイアフラムをカットし、それが普及のまま確保するために止血剤と心を明らかにするために胸郭、クランプ胸郭を広げます。 </li&gt; <li>止血剤を使用して、静かに上行大動脈を露出し、胸腺を削除します。 </li><li>進退止血剤を、ゆっくりと止血の先端を介してできるように筋膜を広げ、大動脈ループを介して止血をいじめます。 </li><li> 、上行大動脈の後ろに0-0絹縫合糸を引っ張る緩い半正方形の結び目に縫合糸を結ぶために止血剤を使用してください。 </li><li>灌流バッファ、二等分する大動脈の流れを開始し、すぐに大動脈内腔にカニューレを挿入するためにコックを回します。急速半正方形の結び目を締める、徹底的に締め、結び目を完了します。 </li><li> 、離れて大血管から心臓を解剖胸から心臓を除去し、灌流列に接続するはさみを使用してください。 </li><li>過剰な肺組織を切り取ると、心臓がバルーンの挿入前に約15分間カラムに平衡化することができます。 10〜20ミリリットル/分の中心部を介してバッファの通常の流量を使用してください。 </li></ol></li></ol> 3.ランゲンドルフ灌流と虚血再灌流障害<ol><li> LV前の配置ssureバルーン<ol><li>円錐状に回転させながらバルーンを収縮し、バルーンが収縮したまま確保するためにコックを回します。 </li><li>消費税​​は、僧帽弁へのアクセスを露出するようにはさみの小さなペアで心房を残しました。 </li><li>左心房と左心室に僧帽弁を介してバルーンを挿入します。 </li><li>圧力監視ソフトウェアを起動します（LabChartプロ）。 </li><li>コックを開け、ゆっくりとマイクロマノメーターから拡張期圧の読みがゼロを超えるまでバルーンに生理食塩水の量​​を増加させることにより、バルーンを膨らませます。 </li><li>ゆっくりと収縮、長さ張力応答を確認75 mmHgでの拡張期圧にゆっくりバルーンを膨張させるために、一回繰り返します。 </li><li>バルーンに生理食塩水の量​​を調節することによって、10 mmHgでの拡張期圧を設定します。インフレのこのレベルでバルーンをシールし、LVのバルーンと圧力変換器との間の閉鎖圧力システムを維持するためにコックを回します。 </li><li>プラスチックと加熱室とカバー室内への低い心。 </li></ol></li><li>広告虚血再灌流障害の奉仕<ol><li>加熱室の底部を接続し、チャンバが流出し、バッファを充填することを可能にします。 </li><li>バッファが心を沈めたときに、全体的虚血を誘導する、心臓にバッファの流れを止めるためにストップコックを回します。虚血時間は、実験の目的に依存して変化し得ます。 </li><li>虚血の30分後、再灌流を開始し、心臓にバッファの流れを回復するためにコックを回します。 </li><li>実験を終了する前に1時間、心臓の再灌流を可能にします。実験の目的に応じて、変数の再灌流時間を使用してください。 </li></ol></li><li>実験の終了および組織の収穫<ol><li>栓の側面ポートを使用して、心臓を停止させるために10ml /分の速度で心臓に4℃でリン酸緩衝生理食塩水を10mlを投与。 </li><li>残りの心房組織を切り取る、潅流カラムから心臓を取り出します。 </li><li>ペンシルベニア州で覆い、ステンレス鋼の組織スライスマトリックスに心を置きます8分間、または心臓まで-80℃でrafilm、および場所は、マシュマロのような一貫性を持っています。 </li><li>冷凍庫からブロックを削除し、2mmのスライスに心をスライスする行列をスライスにrazorbladesを挿入します。組織ブロックは2ミリメートル以外のスライスサイズで購入することができます。 </li><li>スライスを一度に1つを削除して、生化学的研究のための凍結をフラッシュする液体窒素の中にドロップします。梗塞染色のための頂点から第三脳室のスライスを保持します。 </li><li>生化学実験に使用するまで-80℃で液体窒素とストアから組織を削除します。 </li></ol></li><li>梗塞染色<ol><li> 37℃で15分間/ Vの塩化トリフェニルテトラゾリウム（TTC）の1％w 10mlに第心室切片をインキュベートします。 注：TTCでのインキュベーションは、有効15変化なしで15または30分間行うことができます。 </li><li> 10％緩衝ホルマリンにTTCから組織切片を移し、室温で一晩インキュベートすることができます。最適な結果を得るために、聞きますTSは、ホルマリンから取り出し、24時間以内に4画像化されるべきです。 </li><li>写真はスライスを染色し、製造業者のプロトコルに従って梗塞の大きさを推定するために、このようなImageJのようなソフトウェアを使用しています。色素が時間をかけてフェードインしますように、固定後できるだけ早く写真を撮ります。 </li></ol></li></ol>

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The authors declare that they have no competing financial interests.

単離された灌流ラット心臓が正常虚血再灌流傷害9心臓プレコンディショニングにおける薬理学的介入の効果を研究するために使用することができます。しかし、再現可能な結果を​​保証するために標準化されなければならない手順をいくつかの重要なステップがあります。システム内で37.4℃の温度を維持することはあっても軽度の低体温と高体温が心臓プレコンディショニング<su...

虚血および再灌流の両方に対する心臓応答の基礎となる事象の解明は、心筋梗塞1および大動脈クロスクランプ2を必要とする心臓の外科手術の治療の向上に不可欠です。虚血再灌流障害のin vivoモデルは、非常に有用なエンドポイント解析を可能にするが、それらはリアルタイムで急性虚血再灌流障害の機能的効果を研究するように有効ではありません。さらに、 インビボでの虚血再灌流モデルは、一般的に梗塞サイズで有意な変動を生成し、再灌流時の心臓への薬剤の直接送達は、困難です。虚血再灌流障害を研究するためのランゲンドルフ単離心臓システムの利用は薬理学的治療、梗塞組織の均一な領域と、直接心筋への薬物の瞬間デリバリーのリアルタイム機能評価を可能にします。 最初のBを記載1895年3中のyオスカーランゲンドルフ、ランゲンドルフ単離された心臓は、過去40年間4,5のために、虚血再灌流研究に用いられている、虚血再灌流障害を研究するための堅牢なモデルです。ここでは、いくつかの変更は、機能解析のために、単離された心臓を最適化するために行われる。大動脈のその場挿管で心臓が鼓動を心臓が虚血再灌流試験6の結果を変化させる虚血プレコンディショニングを、経験していないことが保証されています。これを容易にするために、気管切開は、換気を可能にし、手術中、ラットの生理学的安定性を確保することが行われます。心臓は、その後、クレブスヘンゼライト緩衝液を大動脈に直接逆行性灌流を介して配信され、それを通してガラスウォータージャケットスパイラルカラムに取り付けられています。生理食塩水で満たされたバルーンを左心室に挿入し、心室AN内からの圧力の実時間測定を可能にする圧力トランスデューサーに取り付けられています。複数の機能パラメータのd計算。実験の終了時に、心臓が収縮し、DNA、RNAおよびタンパク質レベルのダウンストリーム解析を可能にするために、液体窒素中で急速冷凍を逮捕するために、冷生理食塩水でフラッシュされます。このように変更され、ランゲンドルフ灌流心臓は虚血再灌流障害の間に急性任意の時点での薬理学的介入の生理的効果を直接監視するための効果的なシステムとして機能します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td align="right">203726</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>Sigma Aldrich</td>
			<td>RES20765-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride Dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C8106</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr>
			<td>Octanoic Acid</td>
			<td>Sigma Aldrich</td>
			<td>C2875</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride</td>
			<td>Sigma Aldrich</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical Pressure Transducer</td>
			<td>MEMSCAP</td>
			<td>SP844</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Peristaltic Pump</td>
			<td>Cole Parmer</td>
			<td>EW-07521-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex Easy Load Pump Head</td>
			<td>Cole Parmer</td>
			<td>EW-07518-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated circulating water bath</td>
			<td>Lauda</td>
			<td>M3</td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing Flow Module</td>
			<td>Transonic</td>
			<td>TS410</td>
			<td></td>
		</tr>
		<tr>
			<td>Modular Research Console</td>
			<td>Transonic</td>
			<td>T402</td>
			<td></td>
		</tr>
		<tr>
			<td>Inline flow sensor</td>
			<td>Transonic</td>
			<td>ME2PXN</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLab Data Acquisition Device</td>
			<td>AD Instruments</td>
			<td>PL3508</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart data acquisition software</td>
			<td>AD Instruments</td>
			<td>MLU60/8</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

左心室のバルーン装置は、左心室収縮（ 図1）によって開発された圧力のリアルタイムモニタリングを可能にします。先に7に説明したように、この圧力トレースは、心室機能のパラメータの多くを計算することができます。これらの計算は、ベースライン期と同様に再灌流段階で行うことができ、各グループ内で複数のトレース上で平均し、我々は以前に9を行っ...

Watch this Scientific Journal Video about Induction and Assessment of Ischemia-reperfusion Injury in Langendorff-perfused Rat Hearts at JoVE.com

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

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 ...

induction-assessment-ischemia-reperfusion-injury-langendorff-perfused

Research

JoVE Journal

Medicine

22.6K ビュー.  Medical University of South Carolina. 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.

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

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

ラットの精巣挙筋の虚血再灌流障害における白血球 - 内皮相互作用のリアルタイムデジタルイメージング（IRI）

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

医学

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite tissue. Ischemia reperfusion injury (IRI) is a known cause of partial free flap failure and has no effective treatment. Establishing a laboratory model of this injury can prove costly both financially as larger mammals are conventionally used and in the expertise required by the technical difficulty of these procedures typically requires employing an experienced microsurgeon. This publication and video demonstrate the effective use of a model of IRI in rats which does not require microsurgical expertise. This procedure is an in situ model of a transverse abdominis myocutaneous (TRAM) flap where atraumatic clamps are utilized to reproduce the ischemia-reperfusion injury associated with this surgery. A laser Doppler Imaging (LDI) scanner is employed to assess flap perfusion and the image processing software, Image J to assess percentage area skin survival as a primary outcome measure of injury.

In situ Transverse Rectus Abdominis Myocutaneous Flap: A Rat Model of Myocutaneous Ischemia Reperfusion Injury

Free tissue transfer is widely employed in reconstructive surgery to restore form and function following oncological resection and trauma. Preconditioning this tissue prior to surgery may improve outcome. This article describes an in situ transverse rectus abdominis myocutaneous flap (TRAM) in rats as a means for testing preconditioning strategies.

筋皮虚血再灌流傷害のラットモデル： 現場横腹直筋皮フラップで

Free tissue transfer is the gold standard of reconstructive surgery to repair complex defects not amenable to local options or those requiring composite ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

マウスにおける急性腎障害とポスト傷害線維の虚血再灌流モデル

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Evaluation of Coronary Flow Reserve After Myocardial Ischemia Reperfusion in Rats

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via recanalization of the coronary artery is the most effective strategy for reducing the size of ischemic myocardium. The coronary microvasculature cannot be visualized and imaged&#160;in vivo, but there are several invasive and noninvasive techniques that can be used to assess parameters which depend directly on coronary microvascular function. The endothelial function after ischemia reperfusion can be assessed also at the level of the coronary circulation via the coronary flow reserve (CFR).&#160;In this study, peak velocity of left anterior descending (LAD) coronary arteries was measured in rats in vivo via Transthoracic Doppler Echocardiography during resting and stress challenge (induced by Dobutamine). A normal heart can increase its coronary blood flow up to four times above the resting values during stress induction. Following ischemia reperfusion, we found a significantly diminished CFR, which can be used as a marker of coronary microvascular dysfunction. CFR has opened a window on the importance of microvascular dysfunction and has been shown to predict cardiovascular risk independent of whether the severe obstructive disease is present.

Coronary flow reserve (CFR), is defined as the ratio of maximal coronary blood flow to the resting coronary blood flow. We present a protocol for evaluating CFR in rats via ultrasound, which offers the opportunity to predict cardiovascular risk factors in the absence of obstructive coronary disease.

ラットにおける心筋虚血再灌流後の冠動脈流量の評価

Coronary artery disease is the leading cause of death worldwide. After an acute myocardial infarction, early and successful myocardial intervention via ...

A Pre-clinical Rat Model for the Study of Ischemia-reperfusion Injury in Reconstructive Microsurgery

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many areas of biomedical research due to its cost-effectiveness and its translation to humans. This protocol describes a method to create a preclinical free skin flap model in rats with ischemia-reperfusion injury. The described 3 cm x 6 cm rat free skin flap model is easily obtained after the placement of several vascular ligatures and the section of the vascular pedicle. Then, 8 h after the ischemic insult and completion of the microsurgical anastomosis, the free skin flap develops the tissue damage. These ischemia-reperfusion injury-related damages can be studied in this model, making it a suitable model for evaluating therapeutic agents to address this pathophysiological process. Furthermore, two main monitoring techniques are described in the protocol for the assessment of this animal model: transit-time ultrasound technology and laser speckle contrast analysis.

Here, we describe a pre-clinical animal model for studying the pathophysiology of ischemia-reperfusion injury in reconstructive microsurgery. This free skin flap model based on the superficial caudal epigastric vessels in the rat may also allow for the evaluation of different therapies and compounds to counteract ischemia-reperfusion injury-related damage.

再建微小外科における虚血再灌流損傷の研究のための前臨床ラットモデル

Ischemia-reperfusion injury is the main cause of flap failure in reconstructive microsurgery. The rat is the preferred preclinical animal model in many ...

Optocardiography and Electrophysiology Studies of Ex Vivo Langendorff-perfused Hearts

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to larger animals. Yet, larger mammals are better suited for translational research questions related to normal cardiac physiology, pathophysiology, and preclinical testing of therapeutic agents. To overcome the technical barriers associated with employing a larger animal model in cardiac research, we describe an approach to measure physiological parameters in an isolated, Langendorff-perfused piglet heart. This approach combines two powerful experimental tools to evaluate the state of the heart: electrophysiology (EP) study and simultaneous optical mapping of transmembrane voltage and intracellular calcium using parameter sensitive dyes (RH237, Rhod2-AM). The described methodologies are well suited for translational studies investigating the cardiac conduction system, alterations in action potential morphology, calcium handling, excitation-contraction coupling and the incidence of cardiac alternans or arrhythmias.

The objective of this study was to establish a method for investigating cardiac dynamics using a translational animal model. The described experimental approach incorporates dual-emission optocardiography in conjunction with an electrophysiological study to assess electrical activity in an isolated, intact porcine heart model.

元ヴィヴォ・ランゲンドルフ・パーフュード・ハーツの眼電図と電気生理学研究

Small animal models are most commonly used in cardiovascular research due to the availability of genetically modified species and lower cost compared to ...

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, ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

ラット肝移植モデルにおける血球力学的変化をシミュレートすることによる臓器虚血再灌流損傷

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

Real-Time Assessment of Spinal Cord Microperfusion in a Porcine Model of Ischemia/Reperfusion

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its incidence is still considerably high and therefore, influences patient outcome. Microcirculation plays a key role in tissue perfusion and oxygen supply and is often dissociated from macrohemodynamics. Thus, direct evaluation of spinal cord microcirculation is essential for the development of microcirculation-targeted therapies and the evaluation of existing approaches in regard to spinal cord microcirculation. However, most of the methods do not provide real-time assessment of spinal cord microcirculation. The aim of this study is to describe a standardized protocol for real-time spinal cord microcirculatory evaluation using laser-Doppler needle probes directly inserted in the spinal cord. We used a porcine model of ischemia/reperfusion to induce deterioration of the spinal cord microcirculation. In addition, a fluorescent microsphere injection technique was used. Initially, animals were anesthetized and mechanically ventilated. Thereafter, laser-Doppler needle probe insertion was performed, followed by the placement of cerebrospinal fluid drainage. A median sternotomy was performed for exposure of the descending aorta to perform aortic cross-clamping. Ischemia/reperfusion was induced by supra-celiac aortic cross-clamping for a total of 48 min, followed by reperfusion and hemodynamic stabilization. Laser-Doppler Flux was performed in parallel with macrohemodynamic evaluation. In addition, automated cerebrospinal fluid drainage was used to maintain a stable cerebrospinal pressure. After completion of the protocol, animals were sacrificed, and the spinal cord was harvested for histopathological and microsphere analysis. The protocol reveals the feasibility of spinal cord microperfusion measurements using laser-Doppler probes and shows a marked decrease during ischemia as well as recovery after reperfusion. Results showed comparable behavior to fluorescent microsphere evaluation. In conclusion, this new protocol might provide a useful large animal model for future studies using real-time spinal cord microperfusion assessment in ischemia/reperfusion conditions.

Spinal cord microcirculation plays a pivotal role in spinal cord injury. Most methods do not allow real-time assessment of spinal cord microcirculation, which is essential for the development of microcirculation-targeted therapies. Here, we propose a protocol using Laser-Doppler-Flow Needle probes in a large animal model of ischemia/reperfusion.

イシュ血症/再灌流の豚モデルにおける脊髄微小灌流のリアルタイム評価

Spinal cord injury is a devastating complication of aortic repair. Despite developments for the prevention and treatment of spinal cord injury, its ...