ここに記載された装置および実験は、循環器科、ボストン小児病院によっておよびHaseotesファミリーからの慈善寄付によって賄われました。我々は、博士に感謝しています。フランク・マッゴーワンと華美彼のアートワークの支援のために、このモデルでは、とリンゼイ・トムソンに早期の経験を私たちに提供します。

<ol>
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The authors have nothing to disclose.

この作業心臓モデルは、心室プリロードおよび後負荷、灌流液の酸素張力、ならびに心拍数の完全な制御を持つ心室性能の評価を可能にします。他の要因の中でも、in vivoモデルを使用して可能ではない方法で後負荷と前負荷の独立変力薬の本質的な心筋の効果の評価を可能にします。このモデルは、晶質灌流液を利用するので、例えば、心筋のエネルギー状態の分光分析を単純化する、ヘモグロビンからの干渉なしに評価心筋を許可ことが可能であるが、このモデルでは、図14に示すように 、右心房は、我々の器具の一部としてカニューレを挿入されていませんそうするために。我々は意図的に心筋酸素消費量の評価のための冠状静脈洞の流れのサンプリングを容易にするためにそうしないことを選びました。それは共同ポンプとして重要なことは、しかし、右心はまだこのモデルでは、圧力と体積の作業を行い、ronary洞は、肺動脈カニューレに流れ込みます。いくつかの右心室プリロードを提供することは、心室中隔のポジショニングを改善し、左心室のパフォーマンスを向上させ、そしてこのモデルの重要な要素である。15 言及するには、いくつかの実験的な落とし穴があります。最初は、虚血の期間を最小にするために（2分未満で、 すなわち ）好都合に実行されるべき最初の逆行カニュレーションです。マスターへの最も重要なスキルは、上行大動脈の効率的な単離、調製及​​び取扱いです。大動脈断端が大動脈弁上記のカニュレーションのための不十分な余地を残して、過度に短くカットされないことが重要です。しかし、大動脈断端がカニューレの周りの大動脈のトルク締めが発生する可能性があり、長すぎるではないことも重要です。大動脈カニューレと大動脈根が適切にサイズを一致させたことも重要です。小さなカニューレ上の過大​​な大動脈こともできますカニューレ上の大動脈基部のトルク締めにつながります。右鎖骨下動脈は、典型的には、約7ミリメートル大動脈弁上に上行大動脈から離陸します。解剖時に腕頭容器（直径約1ミリメートル）を識別し、大動脈のトリミングは、横方向の大動脈切開用として重要なランドマークを果たします。ちょうど最初の腕頭動脈の離陸の下に大動脈をトリミングすることをお勧めします。トリミングされた大動脈基部で、この容器を含めることは、一般的に取り組んで心臓モードに移行する際に、大動脈根圧力のKHBの漏洩、損失につながります。 挿管のもう一つの技術的に困難な側面は、左心房のカニューレ挿入です。それは左心耳にカニューレを挿入することが可能であるが、我々は、カニューレが頻繁に心耳内で立ち往生し、左心房の体内に容易に通過しないことがわかりました。したがって、我々は、ほぼ、左心房の身体に切開を作ることを好みます房室溝に優れた2ミリメートル。カニューレを固定するとき薄肉アトリウムを引き裂く回避するために挿入する前に、適切な平面内の左心房のカニューレを配置することが重要です。 私たちは、左心房切開の理想的な大きさは約3mmであることがわかりました。切開の小さすぎ作成すると、左心房のカニューレの配置がより困難になる場合があり、および左心房の裂けにつながる可能性があります。私たちは、左心房ブロック上の酸素不透過性チューブ（内径2.9ミリメートル）のストレート、8ミリメートル、面取り部分を使用してください。我々は、傾斜エッジでこれではなく、カニューレを使用して、最も安定した心房カニューレ挿入を導き、左心房ブロックを確保する処理を容易にすることを見出しました。かかわらず使用するチューブの、上に示したように、チューブの端が（心房中隔または僧帽弁によって閉塞されていないことを確認することが重要である、我々は左心房圧のトレースがこのREGAに役立つことがわかりました心房カニューレのようであっても微妙な動きRD）は、大幅に左心室プリロードし、得られた血行動態測定値を変更することができます。同じ理由から、左心房、左心房ブロックを開いた後、次漏れないことを確実にするために重要です。それは関係なく、このシステム内のチューブは心臓への十分な酸素供給を確保するために、酸素不透過性であることを確実にするために使用されるチューブの種類の重要です。 手順の他の技術的に困難な側面は、圧力 - 容積（PV）カテーテルの配置でした。私たちは、最初は大動脈ブロックを通してカテーテルの逆行配置を支持しました。技術的に可能なものの、我々は尖穿刺を介してPVカテーテルを配置するためにはるかに簡単であり、好都合であることが判明しました。時間にカテーテル内または左心室の外に移動することができるようにケアは、実験期間を通してカテーテルの位置を監視するために注意しなければなりません。これはpressuを監視することによって行うことができます時間をかけて再とボリューム追跡。 最後に、注意がKHB溶液を各実験のために新鮮に作成されることを保証するために注意すべきです。 KHBの成分を秤量し、前もって粉末形態でコニカルチューブに保存することが可能です。実験の日に、これらは、滅菌濾過し、水、二酸化炭素/酸素との混合物に添加し、カルシウムと混合することができます。このようなTergazyme（または類似）などの酵素アクティブ粉末洗剤を使用してシステムを洗浄し、定期的に灌流液フィルタを交換することも重要です。 この実験準備のいくつかの制限が注意すべきです。まず、すべて晶質灌流ランゲンドルフ調製物と同様に、KHBおよびその他のasanguinous灌流液は血液に、容量の相対を運ぶ大幅に減少した酸素を持っています。これは部分的冠動脈血管拡張および超生理学的冠動脈の流れを介して補償されるが、準備が完全にphysiologiではありませんこのような理由のために、C。この機器で使用されるウインドケッセル室のほぼ無限のコンプライアンス、収縮期および拡張期の圧力が最小限にしか分離されているため、2番目、（ 図2Aを参照）、したがって、冠動脈灌流圧は、非生理的です。これは、後負荷ブロックにエラスタンス成分を組み込むことにより、将来のモデルで克服することができます。心筋傷害または機能不全を作成する可能性がある温虚血の - （3分2）第三に、すべての単離心臓標本のように、心が定義された期間を受けます。技術の実践を通して、この損傷を最小化することは代表的な結果に最も重要です。心がKHBで再灌流されるように、この効果はすぐに廃止されることが期待されるが、さらに、動物福祉のために必要なものの、吸入麻酔薬は、早期の再灌流過程における心筋抑制剤として機能することができます。 説明ワーキングハートシステムはphysiolの多種多様なことができます患者ケア、研究と教育に関連するogic調査。いくつかの付加的な変更を加えて、システムは、肺高血圧症および単心室生理機能を含む先天性心疾患に関連する重要な生理機能をシミュレートするために使用することができます。制限は、心臓の代わりに、より高い酸素含有血液のバッファによって灌流されていることをエクスビボ製剤であることが挙げられます。

分離された臓器の研究は、 生体内で可能であるものを超えた生理学的条件の制御を可能にする。 エクスビボ心臓製剤は、最初の逆行性灌流で分離されたモデルを説明したオットー・ランゲンドルフ、1によって説明されました。その後、他の人が心筋が両方の圧力と体積の作業を実行するには「働く心」モデルを、説明した。2そのような調製物は、心筋の作用メカニズム、3心筋代謝、4-6と強心薬の効果を解明に尽力してきています。7- 9 心筋の収縮性を高める薬物の使用は、重症患者では一般的です。しかし、いくつかのデータは、術後の設定での心不全の臨床徴候を有する患者のケアに有用である可能性が収縮し、心筋酸素消費量、データに対するこれらの薬剤の相対的効果を比較ご利用いただけます。10しかし、ほとんどの強心薬だけでなく、心筋だけでなく、動脈抵抗、静脈容量11、および患者の代謝率、12 ex vivoで単離された心臓モデルは上のような薬剤の効果を研究することにより、最適な手段のままに影響を与えるので、心筋適切。 私たちは、心筋機能と酸素消費量に強心薬の負荷に依存しない研究のためのex vivoモデルの使用を記載しています。 SDラットから心臓を心臓モデルを作業左心室を使用してカニューレを挿入し、変更されたクレブスヘンゼライト灌流液を用いて灌流しました。大動脈と左心房の圧力を制御しました。圧 - 容積インピーダンスカテーテルは、収縮期および拡張期機能を連続的に監視するための頂端穿刺を介して左心室に入れました。酸素消費量を連続的に左心房perfus間の酸素含有量でインデックス付きの差として測定しました。食べたと肺動脈流出液。試験される薬物は、左心房のブロックに注入し、心臓の性能および酸素代謝の変化を測定し、直前のベースラインと比較しました。

<table><tbody><tr><td>Sodium bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td>8.401 g/4 L</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6758</td><td>0.744 g/4 L</td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333</td><td>1.580 g/4 L</td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506</td><td>0.578 g/4 L</td></tr><tr><td>Sodium pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td>0.220 g/ 4 L</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S3014</td><td>27.584 g/4 L</td></tr><tr><td>Dextrose</td><td>Sigma-Aldrich</td><td>D9434</td><td>7.208 g/4 L</td></tr><tr><td>Calcium chloride dihydrate</td><td>Sigma-Aldrich</td><td>C7902</td><td>1.470 g/4 L</td></tr><tr><td>Biventricular working heart model</td><td>Harvard Apparatus</td><td>IH-51</td><td></td></tr><tr><td>Pressure volume catheter</td><td>Millar, Inc</td><td>SPR-944-1</td><td>6 mm spacing catheter used</td></tr><tr><td>LabChart Pro 8</td><td>AD Instruments</td><td>Version 8.1</td><td></td></tr></tbody></table>

rodent working heart model for the study of myocardial performance and oxygen consumption

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways that cannot be accomplished in vivo. For example, inotropic medications commonly also affect preload and afterload, precluding load-independent assessments of their myocardial effects in vivo. Additionally, this model allows for sampling of coronary sinus effluent without contamination from systemic venous return, permitting assessment of myocardial oxygen consumption. Further, the advent of miniaturized pressure-volume catheters has allowed for the precise quantification of markers of both systolic and diastolic performance. We describe a model in which the left ventricle can be studied while performing both volume and pressure work under controlled conditions. 
In this technique, the heart and lungs of a Sprague-Dawley rat (weight 300-500 g) are removed en bloc under general anesthesia. The aorta is dissected free and cannulated for retrograde perfusion with oxygenated Krebs buffer. The pulmonary arteries and veins are ligated and the lungs removed from the preparation. The left atrium is then incised and cannulated using a separate venous cannula, attached to a preload block. Once this is determined to be leak-free, the left heart is loaded and retrograde perfusion stopped, creating the working heart model. The pulmonary artery is incised and cannulated for collection of coronary effluent and determination of myocardial oxygen consumption. A pressure-volume catheter is placed into the left ventricle either retrograde or through apical puncture. If desired, atrial pacing wires can be placed for more precise control of heart rate. This model allows for precise control of preload (using a left atrial pressure block), afterload (using an afterload block), heart rate (using pacing wires) and oxygen tension (using oxygen mixtures within the perfusate).

逆行性灌流（ 図1A）で、左心室の作業心臓（ 図1B）で完全に計装心の概略図。典型的な拡張末期圧は約3であるD - - 。典型的な大動脈、左心房と左心室圧と容積トレーシングは 、 図2Aに示されているこのモデルでは5 mmHgで、ピーク収縮期圧は約100 mmHgである。図2Eは、変化を示しています。 LAカニューレは、カニューレの配置および位置決め中に離れて心房中隔から移動したときに、左心房でトレース。これらの実験では、大動脈圧が90 mmHgのに設定し、LA圧が10 mmHgのに設定しました。 カテコールアミンの効果をテストするには、（圧力 - 容積カテーテルおよび関連ソフトウェアから主に由来する）各生理学的パラメータは、直前と比較しました<html>ベースライン期間。図示の例では、ドーパミンは、左心房のブロックには15μg/ kg /分で注入しました。拡張末期圧（このモデルでは、固定心房圧与えられた）二つの条件が同一であるが、左心室拡張末期容積は2.5％減少し、左心室収縮末期容積が増加し、ストローク量を得、4.9％減少します（ 図3A）。プラセボ点滴、左心室ストロークワーク、圧力-容積曲線内の領域として同定され、ドーパミンでの処理中に32％増加した（ 図3B、P &lt;0.001、t検定、群当たりn = 10）と比較。これは、プラセボ注入に対する心筋酸素消費量（ 図3C）において大きな増加と関連していました。このようにして、別の強心薬や用量の相対的な効力およびエネルギーコストが負荷条件に対するそれらの効果を互いに独立に比較することができます。 コンテンツ &quot;FO：キープtogether.withinページ=&quot; 1 &quot;&gt; <img alt="図1" src="/files/ftp_upload/54149/54149fig1.jpg" /> 図1： 逆行性灌流およびワーキングハートモードで完全計装心臓内の流れの図 （ パネルA：ランゲンドルフモード; パネルB：。。作業心臓モード逆行モードでは、KHBは大動脈ルートに設定された灌流圧で注入されます。このモードは、虚血時間や計測中に次の心筋を回復するために利用される。心臓の作業モードでは、灌流液は、冠循環を灌流する前に、左心を通って流れる。このモードでは、心筋が独自の灌流圧を生成する必要があります。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig1large.jpg" target="_blank">ご覧になるにはこちらをクリックしてください。この図の拡大版。</a> <img alt="図2" src="/files/ftp_upload/54149/54149fig2.jpg" /> Figure 2： ベースライン測定時に得られた代表的な圧力とボリュームトレーシング。 （A）大動脈根圧力、（B）左心房圧、（C）、左心室の圧力及び（D）は、ベースライン測定時心室容積の追跡を左に表示されています。一回拍出量、脳卒中作業、心拍出量、タウ、および他のパラメータを自動的に計算し、ソフトウェアによりリアルタイムで表示することができます。ワーキング心モードの貧しい心臓出力に関連鈍化左心房トレース（E）は、カニューレが左心房に位置異常されていることを手がかりとすることができます。うまく配置された左心房圧トレースの著名なV波が原因で完全にインストルメント動物で減少した左心房のコンプライアンスに、共通の可能性があることに注意してください。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 図 3： 圧力-容積曲線に対するドパミンの効果が増加行程容積を含むPV曲線（A）中左シフト、内ドーパミン輸液結果は、ベースライン測定と比較して、収縮期容積を終了減少しました。これらのPV曲線のいくつかの構成要素の形状が原因で動脈と静脈のエラスタンスの不在に（ 図4を参照）は、典型的には、 生体内で測定されたものとは異なること。（B）直前のベースラインと比較して、ストロークの作業は注入の間に有意に多くの増加に注意してくださいプラセボよりもドーパミンの（**、P = 0.0017、t検定）、心筋酸素消費をしたとして（*、P = 0.013、t検定、C）。このモデルを使用して、ベースラインでの平均心筋酸素消費量は、推定dissolvを用いて、0.22±0.02ミリモルO 2 /グラム組織/分40℃の生理食塩水で165マイクロモル/ LのEDの酸素含有量このような測定は、様々な薬の心筋酸素消費量を比較するために使用することができます。 <a href="https://www.jove.com/files/ftp_upload/54149/54149fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/54149/54149fig4.jpg" /> 図4： 圧力ボリュームループの解析。示さ理論的圧-容積ループは、正常な心臓サイクルを記述し （1）、isovolemic収縮が発生した大動脈弁（AV）の閉鎖（1から2）に続いて。心室圧として心房圧未満に減少します。この段階の持続時間は、タウによって表されます。僧帽弁（MV）を心室（ - 2,3）を充填し、心房収縮と同時に開きます。収縮期はその後isovolemic contracti始まります（3 - 4）に心室圧までAVが開く時の拡張期動脈圧を超えます。 2と3 - -ストロークボリュームはライン1との間の差である。2 - - 3 - 4ストローク作業が1内の領域である。4、曲線<a href="https://www.jove.com/files/ftp_upload/54149/54149fig4large.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

Watch this Scientific Journal Video about Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption at JoVE.com

Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

心筋パフォーマンスと酸素消費量の研究のための齧歯類のワーキングハートモデル

Isolated working heart models have been used to understand the effects of loading conditions, heart rate and medications on myocardial performance in ways ...

rodent-working-heart-model-for-study-myocardial-performance-oxygen

Research

JoVE Journal

Biology

20.1K ビュー.  Boston Children’s Hospital. Isolated working heart models can be used to measure the effect of loading conditions, heart rate, and medications on myocardial performance and oxygen consumption. We describe methods for preparation of a rodent left heart working model that permits study of systolic and diastolic performance and oxygen consumption under various conditions.

ビデオ: Rodent Working Heart Model for the Study of Myocardial Performance and Oxygen Consumption

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of cardiovascular disease. Although molecular biology is necessary in identifying key changes in the signaling pathway, it is not a surrogate for functional significance. While physiology can provide answers to the question of function, combining physiology with biochemical assessment of metabolites in the intact, beating heart allows for a complete picture of cardiac function and energetics. For years, our laboratory has utilized isolated heart perfusions combined with nuclear magnetic resonance (NMR) spectroscopy to accomplish this task. Left ventricular function is assessed by Langendorff-mode isolated heart perfusions while cardiac energetics is measured by performing 31P magnetic resonance spectroscopy of the perfused hearts. With these techniques, indices of cardiac function in combination with levels of phosphocreatine and ATP can be measured simultaneously in beating hearts. Furthermore, these parameters can be monitored while physiologic or pathologic stressors are instituted. For example, ischemia/reperfusion or high workload challenge protocols can be adopted. The use of aortic banding or other models of cardiac pathology are apt as well. Regardless of the variants within the protocol, the functional and energetic significance of molecular modifications of transgenic mouse models can be adequately described, leading to new insights into the associated enzymatic and metabolic pathways. Therefore, 31P NMR spectroscopy in the isolated perfused heart is a valuable research technique in animal models of cardiovascular disease.

Assessment of Cardiac Function and Energetics in Isolated Mouse Hearts Using 31P NMR Spectroscopy

Langendorff-mode isolated heart perfusion, in conjunction with 31P NMR spectroscopy, combines the fields of biochemistry and physiology into one experiment. The protocol allows for the dynamic measurement of high energy phosphate content and turnover in the heart while concurrently monitoring physiologic function. When performed correctly, this is a valuable technique in the assessment of cardiac energetics.

使用する絶縁マウスハーツの心機能およびエネルギー論の評価 31 P NMR分光法

Bioengineered mouse models have become powerful research tools in determining causal relationships between molecular alterations and models of ...

医学

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

隔離された両心室ワーキングウサギ心臓のNADH蛍光イメージング

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

An Isolated Working Heart System for Large Animal Models

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart model, have been invaluable tools for studying cardiovascular function and disease1-15. Although the Langendorff heart preparation can be used for any mammalian heart, most studies involving this apparatus use small animal models (e.g., mouse, rat, and rabbit) due to the increased complexity of systems for larger mammals1,3,11. One major difficulty is ensuring a constant coronary perfusion pressure over a range of different heart sizes &#8211; a key component of any experiment utilizing this device1,11. By replacing the classic hydrostatic afterload column with a centrifugal pump, the Langendorff working heart apparatus described below allows for easy adjustment and tight regulation of perfusion pressures, meaning the same set-up can be used for various species or heart sizes. Furthermore, this configuration can also seamlessly switch between constant pressure or constant flow during reperfusion, depending on the user&#8217;s preferences. The open nature of this setup, despite making temperature regulation more difficult than other designs, allows for easy collection of effluent and ventricular pressure-volume data.

Most studies involving the Langendorff apparatus use small animal models due to the increased complexity of systems for larger mammals. We describe a Langendorff system for large animal models that allows for use across a range of species, including humans, and relatively easy data acquisition.

大型動物モデルの絶縁型ワーキングハートシステム

Since its introduction in the late 19th century, the Langendorff isolated heart perfusion apparatus, and the subsequent development of the working heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

循環死後の心臓寄付の前臨床モデル

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...

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 ...

Semi-Minimal Invasive Method to Induce Myocardial Infarction in Rats and the Assessment of Cardiac Function by an Isolated Working Heart System

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily and highly reproducible MI induction procedure is required to obtain further insight and better understanding of the underlying pathological changes. This procedure can also be used to evaluate the effects or potency of new and promising treatments (as drugs or interventions) in acute MI, subsequent remodeling and heart failure (HF). After intubation and pre-operative preparation of the animal, an anesthetic protocol with isoflurane was performed, and the surgical procedure was conducted&#160;quickly. Using a minimally invasive approach, the left anterior descending artery (LAD) was located and occluded by a ligature. The occlusion can be performed acutely for subsequent reperfusion (ischemia/reperfusion injury). Alternatively, the vessel can be ligated permanently to investigate the development of chronic MI, remodeling or HF. Despite common pitfalls, the drop-out rates are minimal. Various treatments such as remote ischemic conditioning can be examined for their cardioprotective potential pre-, peri- and post-operatively. The post-operative recovery was quick as the anesthesia was precisely controlled and the duration of the operation was short. Post-operative analgesia was administered for three days. The minimally invasive procedure reduces the risk of infection and inflammation. Furthermore, it facilitates rapid recovery. The &#8220;working heart&#8221; measurements were performed ex vivo and enabled precise control of preload, afterload and flow. This procedure requires specific equipment and training for adequate performance. This manuscript provides a detailed step-by-step introduction for conducting these measurements.

This article presents an efficient method to perform myocardial ischemia and subsequent chronic reperfusion in rats using a minimally invasive approach. In addition, left ventricular hemodynamic function of rats is assessed by echocardiography and isolated working heart methods.&#160;

ラットの心筋梗塞を誘導する半最小侵襲法と、単離した働く心臓システムによる心機能の評価

Myocardial infarction (MI) remains the main contributor to morbidity and mortality worldwide. Therefore, research on this topic is mandatory. An easily ...

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

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

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

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

Improved Rodent Model of Myocardial Ischemia and Reperfusion Injury

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence suggests that thrombolytic therapy or primary percutaneous coronary intervention (PPCI) does not prevent reperfusion injury. There is still no ideal animal model for MIRI. This study aims to improve the MIRI model in rats to make surgery easier and more feasible. A unique method for establishing MIRI is developed by using a soft tube during a key step of the ischemic period. To explore this method, thirty rats were randomly divided into three groups: sham group (n = 10); experimental model group (n = 10); and existing model group (n = 10). Findings of triphenyltetrazolium chloride staining, electrocardiography, and percent survival are compared to determine the accuracies and survival rates of the operations. Based on the study results, it has been concluded that the improved surgery method is associated with a higher survival rate, elevated ST-T segment, and larger infarct size, which is expected to mimic the pathology of MIRI better.

Myocardial ischemia-reperfusion model of rat heart is improved by using a self-made retractor, polyvinyl chloride tube, and a unique knotting method. Electrocardiogram, triphenyltetrazolium chloride and histological staining, and percent survival analysis results showed that the improved model group has higher success and survival rates than the already existing model group.

心筋虚血および再灌流傷害の改善されたげっ歯類モデル

Myocardial ischemia and reperfusion injury (MIRI), induced by coronary heart disease (CHD), causes damage to the cardiomyocytes. Furthermore, evidence ...

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of myocardial metabolism have been linked to the development of contractile dysfunction and heart failure. Therefore, unraveling the link between metabolism and contraction should shed light on some of the mechanisms governing cardiac adaptation or maladaptation in disease states. The isolated working rat heart preparation can be used to follow, simultaneously and in real time, cardiac contractile function and flux of energy providing substrates into oxidative metabolic pathways. The present protocol aims to provide a detailed description of the methods used in the preparation and utilization of buffers for the quantitative measurement of the rates of oxidation for glucose and fatty acids, the main energy providing substrates of the heart. The methods used for sample analysis and data interpretation are also discussed. In brief, the technique is based on the supply of 14C- radiolabeled glucose and a 3H- radiolabeled long-chain fatty acid to an ex vivo beating heart via normothermic crystalloid perfusion. 14CO2 and 3H2O, end byproducts of the enzymatic reactions involved in the utilization of these energy providing substrates, are then quantitatively recovered from the coronary effluent. With knowledge of the specific activity of the radiolabeled substrates used, it is then possible to individually quantitate the flux of glucose and fatty acid in the oxidation pathways. Contractile function of the isolated heart can be determined in parallel with the appropriate recording equipment and directly correlated to metabolic flux values. The technique is extremely useful to study the metabolism/contraction relationship in response to various stress conditions such as alterations in pre and after load and ischemia, a drug or a circulating factor, or following the alteration in the expression of a gene product.

The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.

単離されたワーキングラット心臓におけるグルコースおよび脂肪酸酸化の宿泊料金を決意するための方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

生物学

<meta charset="utf8"></head><body>作業ラット心臓灌流モデルを用いたドナー心臓保存の最適化

Using an Isolated Working Rat Heart Model to Test Donor Heart Preservation Strategies

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, transportation, and transplantation. Previous work from our laboratory showed that the addition of glyceryl trinitrate and erythropoietin to donor heart preservation solutions could significantly improve cardiac functional recovery after prolonged cold storage and in donation after circulatory death hearts. This supplementation protocol has been implemented in clinical use in transplant units around Australia, Belgium, and the United Kingdom. Here, we outline a protocol for testing supplementation strategies using an ex vivo isolated working rat heart (IWRH) perfusion circuit. Using this methodology, supplementation strategies can be tested in the context of prolonged cold static storage, donation after brain death, and donation after circulatory death donor heart preservation. Cardiac functional recovery, measured by aortic flow, coronary flow, cardiac output, pulse pressure, and heart rate, can be used to determine whether a particular preservation strategy can minimize ischemia-reperfusion injury of the donor heart.

<meta charset="utf8"></head><body>著者スポットライト:孤立したラット心臓灌流研究によるドナーの心臓保存の強化

We describe an ex vivo isolated working rat heart protocol to test donor heart preservation strategies. This paper describes the protocol for use in static cold storage of rodent donor hearts; however, the protocol can also be used for donor hearts obtained after donation after circulatory death and brain death.

単離された作業用ラット心臓モデルを使用して、ドナーの心臓保存戦略をテストする

Optimization of donor heart preservation solutions has played a key role in reducing ischemia-reperfusion injury in donor hearts during organ retrieval, ...