この作品は、アメリカ心臓協会14FTF20370058（DMA）およびNIH T32 HL007101-35（DMA）によってサポートされています。

1.コンダクタンスカテーテルの準備と圧力校正<ol><li>血行動態カテーテルモジュールにコンダクタンスカテーテルを接続します。電子的にカテーテルモジュールの設定設定圧力と体積を記録することにより、圧力と体積の測定値を校正。 0ミリメートルHgのと25ミリメートルHgの（ 図1A）のトレースを記録し、両方の圧力トレーシング（ 図1Bおよび1C）に電圧を割り当てます。同様に、5 RVUおよび25 RVU（ 図1D）の量のトレースを記録し、両方のボリュームトレーシング（1Eおよび1F）に電圧を割り当てます。 </li><li>水銀柱血圧計を使用して、手動圧力校正と電子圧力キャリブレーションを確認してください。 3方活栓で血圧計のカフポートを取り付けます。サイドポートを用いてRT水でしばしば冠動脈形成術に使用される3ポート止血弁システムを、入力します。 <ol><li>流体満たされた止血弁にコンダクタンスカテーテルの先端を置き、静かにウィットを確保ハウトのよじれカテーテル。血圧計に止血弁を接続し、200 mmHgのに膨らませると、3方活栓をロックします。測定された圧力が血圧計に膨張圧力に対応しているかどうかを調べます。 </li></ol></li><li>生理食塩水に入れカテーテルは、動作フィールドと測定圧力のレベルである37℃に加熱しました。記録された圧力がゼロになるまで圧力制御を調整します。 </li></ol> 2.麻酔/挿管<ol><li>腹腔内注射として（80〜100/10 mgのキロ-1）ケタミン/キシラジンを管理します。 注意：代替の麻酔薬を使用することができます。麻酔薬の広範なリストは、この技術11,12の以前のレビューで提供されています。適切な麻酔は穏やかテールピンチで確認することができます。 </li><li>麻酔した後、加熱されたパッドの上にバリカンと場所で首と胸を剃ります。 36.5から37.5ºCでマウスの直腸温度を維持します。低体温落ち込んで心拍数になります。乾燥を防ぐために、目に軟膏を適用します。 </li><li>首の正中切開を行い、気管を露出させ、気管の筋肉を離れて解剖。挿管を確実にするために気管を可視化しながら、口から気管内チューブを配置し、人工呼吸器に接続します。 </li><li>手順の間に人工呼吸器の上でマウスを維持します。先に記載11のように設定され、人工呼吸器の設定は、動物の体重に基づきます。一回換気量（ミリリットル）= 6.2 X（キログラム動物体重）1.01と呼吸率= 53.5 X（キログラム動物体重）-0.26。 </li></ol> LVチャンバー内のコンダクタンスカテーテルの3配置<ol><li>頸動脈アプローチ<ol><li>無菌性を確保するために、無菌の手術器具の二組は、最初の皮膚切開や開胸で動作するいずれかのused-一つです。楽器は、個々の外科セッションA中に動物の乾燥滅菌器を使用して除染する必要がありますND各外科手術日の終わりにオートクレーブ処理。 </li><li>皮膚はクロルヘキシジン+アルコールスキンスクラブ（0.5％クロルヘキシジン/ 70％アルコール溶液）の3サイクルで除染された後、胸骨に下顎から右頸動以上の切開を行います。右頸動脈を露出させるために周囲の組織を解剖し、頸動脈に隣接して実行される迷走神経を切断します。 </li><li>ネクタイとセキュア、（離れて胸から）頸動脈の遠位端の周りに滅菌6-0絹縫合糸を配置します。緩く中間の縫合糸を結ぶ、第1の縫合糸に頸動脈近位（胸に近い方）の下に二つの追加の縫合糸を配置します。そっと近位縫合糸を引き、肌にクランプして固定します。頸動脈に進む前に、両方の近位および遠位クランプされていることを確認してください。 </li><li> 、R.の頚動脈に小さな切開を行い第1の縫合糸の近位に、胸に向かって長手方向に延びます。 </li><li>コンダクタンスカテーテル先端を挿入し、以前の切開を通って容器の中に、30分間温生理食塩水に浸し、中央の縫合糸を使用してカテーテルを固定します。 </li><li>正しい配置を確実にするためにトレース圧 - 容積ループを見ながらゆっくり、頸動脈を介して左心室にカテーテルを進めます。 注：長方形に見える圧-容積ループを生じるはずであるカテーテルの最適な配置（ 図2を参照してください ）。カテーテルの通過への抵抗が発生した場合は、ゆっくりと引いて、穏やかな圧力を再び進めます。カテーテルのジェントル回転は、左心室に配置して助けることができます。コンダクタンスカテーテルを強制すると、深刻な心血管系合併症またはカテーテルの損傷につながることができます。 </li><li>レコードベースラインの圧力-体積は、カテーテル留置後〜10分をループし、定常状態（ 図2）を達成 。 </li></ol></li><li>アピカルアプローチ<ol><li>麻酔をかけ、換気マウスでは、切開FRを作ります剣状突起OMとダイアフラムが表示されるまで横方向に胸壁を切断。 </li><li>ダイアフラム関わらずカットし、心尖を視覚化します。 </li><li>近位電極はちょうど心室の内側になるまで、（25〜30 G針を使用）、針の刺し傷を介して左心室の頂点にコンダクタンスカテーテルを挿入します。 </li><li>レコードベースラインの圧力 - 体積は、カテーテル留置後〜10分をループし、定常状態を達成します。 </li></ol></li></ol>過渡大動脈閉塞を使用した4変後負荷<ol><li>一過性の大動脈閉塞を実行するには、胸の上部に小さな水平切開を作り、横大動脈を露出するために周囲の組織を解剖。 </li><li>横大動脈の下に6-0絹糸結紮を配置します。圧 - 容積ループがベースラインに戻った後、静かに、ゆっくりと、針クランプで縫合糸の両端を留める1-2秒かけて縫合糸を上げ、ゆっくりと緊張を解放します。 </LI&gt; <li> 3つの別々の最適な録音が同じ動物から作られるまで、この手順を繰り返します。 注：最適な録音は、少なくとも5圧力容積ループサイクルおよび縫合糸（ 図3Aおよび3C）の張力印加時の収縮末期圧の安定した上昇を持っている必要があります。 </li></ol> 5.変プリロード一時下大静脈閉塞を使用して（IVC） <ol><li>一過性下大静脈閉塞を実行するには、IVCを露出するように、振動板の下に、剣状突起下の水平方向の切開を行います。 </li><li> IVCの下に6-0絹糸結紮を配置します。圧 - 容積ループがベースラインに戻った後、針クランプ付き縫合糸の両端を握ります。静かに、ゆっくりと1〜2秒かけて縫合糸を上げ、ゆっくりと緊張を解放します。 IVCの閉塞も、綿棒をひっくり返した綿を使用して圧力を穏やかに適用することによって行うことができます。 </li><li> 3つの別々の最適な録音がMになるまで、この手順を繰り返し同じ動物からADE。 注：最適な録音は、少なくとも5圧力容積ループサイクルと縫合糸の張力印加時の左心室拡張末期圧（ 図4Aおよび4B）は着実に低下している必要があります。 </li></ol> 6.生理食塩水校正<ol><li>研究の終了時に、並列コンダクタンス（V p）の値は、頸静脈（ 図5（a）及び（b））を介して動物に高張食塩水（15％）の10μlのボーラスを注入することによって得ることができます。 注：このボーラスは、圧力の変化がないとの体積の明らかな増加の原因となります。ボリュームでこの明らかな変化は、ボリューム内の実際の増加に血液プールコンダクタンスではなく、変化の結果です。高張食塩水は、陰性変力作用13を有するもののdP / dtの最大の過渡的減少を観察することができます。計算されたV pは、に沿った圧力容積ループの分析ソフトウェアに入力することができますキュベット校正パラメータとし、マイクロリットル中のRVUへ変換します。 </li></ol> 7.キュベット校正<ol><li>キュベットのキャリブレーションを実行するために、加熱パッドまたは水浴に製造業者によって提供既知の直径のウェルにキュベットを配置37ºCに温め。血行動態の評価を受けたマウスからの新鮮な温かいヘパリン化血液の最初の4-5の穴を埋めます。 注意：キュベット校正は、マウスの血液を用いて、左心室の血液プールの正確な評価を可能にし、マイクロリットルのRVUからボリュームデータの変換を可能にします。 </li><li>すべての電極が水没するまで、最初のウェル内にコンダクタンスカテーテルを挿入します。ゆっくりのRVUを変える生成された、ウェル内にカテーテルを移動します。 </li><li> RVUでボリュームチャネルコンダクタンスの変化を記録します。キャリブレーションのための最高のRVUを選択します。 注意：ウェルの容積は、どちらvolumの式を使用して、1）により算出することができます半径がキュベットウェルと長さのものであるシリンダーのeは内側の2つの検出電極間の長さに基づいて、または2）を製造者の指示でチェックしています。コンダクタンスの出力は、マイクロリットルの11へのRVUからのデータを変換するキャリブレーション式を開発することが知られているボリュームと相関させることができます。 </li></ol> 8.安楽死<ol><li>プロトコルの最後に、マウスを麻酔下ながら頸椎脱臼を使用して安楽死させます。 </li><li>死を確実にするために、経子宮頸部脱臼を有するマウスは、安楽死の第二の方法を受けなければなりません。私たちは、麻酔下の実験のための心臓組織の収穫、または両側開胸で、麻酔下で放血を使用しています。 </li></ol>圧力ボリュームループ解析ソフトウェアを使用して9.データ解析<ol><li>生理食塩水のキャリブレーションから、V pを計算します<ol><li>選択圧とvolu私は、高張食塩水の噴射の間に得 ​​られたループ（ 図5Aおよび5B） </li><li>エクスポートは、圧力ボリューム解析ソフトウェアにループします。生理食塩水の校正のためのオプションを選択します（ 図5C） </li><li>計算されたV p値（ 図5D）を記録します。 注：V p値は、1）生理食塩水校正から収縮末期容積対拡張末期容積、および2）拡張末期容量=収縮末期容積線の交点を識別することによって計算されます。これらの線の交点は、圧力容量ループ解析ソフトにより算出されるV pを、提供します。 </li></ol></li><li>ボリュームへのコンダクタンスを入力し、ボリュームチャネルオプションで（RVU）の関係（ 図1F） </li><li>ベースライン圧 - 容積ループ関係を測定します<ol><li>定常状態が達成された後、圧力と体積のチャンネル8から10の心周期を選択します（ 図2A）とソフトの解析へのエクスポートウェア。 5-6呼気終末ループ（ 図2B）を特定 </li><li>並列コンダクタンスを補正するために、V p値を使用してください。 「定常状態」オプションを選択し、血行動態の要約表を生成する（ 図2C） </li></ol></li><li>大動脈狭窄時の測定圧 - 容積ループの関係<ol><li>拡張末期圧（ 図3A）および解析ソフトウェアへの輸出の増加に先立って大動脈狭窄に対応した圧力と体積のチャンネル8から10の心周期を選択します。 5-6呼気終末ループ（ 図3C）を特定 </li><li>収縮末期圧容積関係を計算します選択し、「収縮」の分析（ 図3B）、（ESPVR） </li></ol></li><li> IVC収縮時の測定圧 - 容積ループの関係<ol><li>くびれをIVCに対応した圧力と体積のチャンネルから8-10心周期を選択します（FIGUREの4A）および解析ソフトウェアへのエクスポート。 5-6呼気終末ループ（ 図4B）を特定 </li><li> 「収縮」分析プリロード補充可能なストローク作業を計算します（ 図3B）、（PRSW）（図4C）を選択し 、EDV（ 図4D）、ならびにESPVRと拡張末期圧容積関係対DT最大DP /（ 図4E） </li></ol></li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+contractile+state+on+curvilinearity+of+in+situ+end-systolic+pressure-volume+relations.">Influence of contractile state on curvilinearity of in situ end-systolic pressure-volume relations.</a> Circulation. 79 (1), 167-178 (1989).</li><li>Little, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+left+ventricular+dP/dtmax-end-diastolic+volume+relation+in+closed-chest+dogs.">The left ventricular dP/dtmax-end-diastolic volume relation in closed-chest dogs.</a> Circ Res. 56 (6), 808-815 (1985).</li><li>Glower, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearity+of+the+Frank-Starling+relationship+in+the+intact+heart:+the+concept+of+preload+recruitable+stroke+work.">Linearity of the Frank-Starling relationship in the intact heart: the concept of preload recruitable stroke work.</a> Circulation. 71 (5), 994-1009 (1985).</li><li>Sharir, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventricular+systolic+assessment+in+patients+with+dilated+cardiomyopathy+by+preload-adjusted+maximal+power.+Validation+and+noninvasive+application.">Ventricular systolic assessment in patients with dilated cardiomyopathy by preload-adjusted maximal power. Validation and noninvasive application.</a> Circulation. 89 (5), 2045-2053 (1994).</li><li>Baan, J., Van der Velde, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitivity+of+left+ventricular+end-systolic+pressure-volume+relation+to+type+of+loading+intervention+in+dogs.">Sensitivity of left ventricular end-systolic pressure-volume relation to type of loading intervention in dogs.</a> Circ Res. 62 (6), 1247-1258 (1988).</li><li>Wei, C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volume+catheter+parallel+conductance+varies+between+end-systole+and+end-diastole.">Volume catheter parallel conductance varies between end-systole and end-diastole.</a> IEEE Trans Biomed Eng. 54 (8), 1480-1489 (2007).</li><li>Porterfield, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+correction+for+parallel+conductance,+GP,+and+gain+factor,+alpha,+in+invasive+murine+left+ventricular+volume+measurements.">Dynamic correction for parallel conductance, GP, and gain factor, alpha, in invasive murine left ventricular volume measurements.</a> J Appl Physiol (1985). 107 (6), 1693-1703 (2009).</li></ol>

著者らは、開示することは何もありません。

我々は、心臓の収縮と弛緩の両方の総合的な分析を導出するために、マウスではコンダクタンスカテーテルを使用して、圧 - 容積ループ解析をperfomingする方法について説明します。菅、佐川らは、圧力-体積は、心臓の収縮の対策、特にESPVRの傾き、または収縮末期エラスタンス（E エス ）、およびE maxを定義するためにループを利用しました。ボリューム（P / vの）への圧力?...

心臓の圧力容積ループ解析は、心機能の詳細な情報を提供し、機能評価1のゴールドスタンダードです。そのような心エコー検査や心臓MRIなどのイメージング技術が機能的対策を提供しているが、これらの措置は、負荷条件に大きく依存しています。心臓の収縮と弛緩の負荷に依存しない措置がプリロードと後負荷の範囲にわたって、心室の圧力と体積の関係の動的測定を必要とします。圧力-容積関係のこの理解は佐川や同僚2,3の画期的な作品から生じます。彼らは、圧力-容積ループ由来の収縮対策が荷重条件4とは無関係であったことをex vivoでの灌流犬の心の中で明らかにしました。 これらの分析の生体内のアプリケーションでは、1980年代にコンダクタンスカテーテルの開発を可能になりました。カー許可この技術的進歩SSらは、ヒトの5,6内の圧力-容積ループ解析を実行します。 1990年代後半7におけるコンダクタンスカテーテルおよび外科技術の向上の小型化を実行する遺伝的および薬理学的研究のためにできるように、げっ歯類心臓機能の解析が可能なりました。この進歩は、圧力 - 容積ループ解析の普及にリードするのであり、哺乳動物の心臓の生理機能への洞察力の多くを生成しています。 コンダクタンスカテーテルを使用し、そこから得られたデータの解釈の重要な概念は、ボリュームとコンダクタンスとの関係です。コンダクタンスは逆に近位に配置された電極、通常LV頂点8で、大動脈弁の下に配置され、遠位カテーテルを使用して測定された電圧に関連しています。電圧またはコンダクタンスの変化は、近位から遠位電極に流れる電流の変化によって測定されます。血液プールが寄与しているが心室壁の寄与は、コンダクタンスに大きく、並列コンダクタンス（V pを）と呼ばれるよ、測定されたコンダクタンスの絶対LV容積測定値を得るために差し引かなければなりません。 食塩較正と呼ばれるこの補正を行うための方法は、以下のプロトコールに記載されています。バーンらによって記載さコンダクタンスとボリュームの間の数学的関係は、そのボリューム= 1 /αです。 （ρL 2）（GGのP）、α=一様な場補正係数、電極間のρ=血液抵抗率、L =距離、G =コンダクタンスおよび G p =非血液コンダクタンス9。注目すべきは、マウスでの均一な場補正係数が小さいため、チャンバ容積10に1.0に近づきます。圧力変換器に結合され、コンダクタンスカテーテルは、リアルタイムの同時の圧力および容積データを提供します。 心臓pressuそれらは負荷条件の心室機能の独立および心拍数の測定を可能にするように再量分析は、心機能の他の尺度上に特定の利点を提供します。収縮末期圧容積関係（ESPVR）、DのP / D の T max の末端-拡張期容積の関係、最大エラスタンス（E max）が及び補充可能なストローク作業（PRSW）をプリロード：収縮の具体的な負荷に依存しない心臓のインデックスが含まれています。拡張機能の負荷に依存しない尺度は、拡張末期圧の体積関係（EDPVR）11です。以下のプロトコルは、頸動脈と頂端のアプローチの両方を使用して、心臓の圧力容積ループ分析の実施について説明しています。これらの研究を行うための方法が詳細以前8,11に記載されているが、我々は、生理食塩水およびキュベットのキャリブレーション補正の両方を含む、正確な圧力-体積の測定値を得るための重要なステップを確認し、THESの視覚的なデモンストレーションを提供しますE手順。この研究のために行った動物との研究が承認されたプロトコルとデューク大学医療センターの施設内動物管理使用委員会の動物福祉の規制に従って処理しました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
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			<td>AnaSed (xylazine)&#160;</td>
			<td>Lloyd Laboratories</td>
			<td>NADA no. 139-236</td>
			<td>Anesthetic</td>
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			<td>Pfizer</td>
			<td>440842</td>
			<td>Anesthetic</td>
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			<td>VIP3000</td>
			<td>Matrx Medical Inc.</td>
			<td></td>
			<td>Anesthesia machine</td>
		</tr>
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			<td>Ventilator</td>
			<td>Harvard Apparatus</td>
			<td>Model 683</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Tubing kit</td>
			<td>Harvard Apparatus</td>
			<td>72-1049</td>
			<td>Surgical Equipment</td>
		</tr>
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			<td>Homeothermic Blanket&#160;</td>
			<td>Kaz Inc.</td>
			<td>5628</td>
			<td>Surgical Equipment</td>
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			<td>Stereo microscope</td>
			<td>Carl Zeiss Optical Inc.</td>
			<td>Stemi 2000</td>
			<td>Surgical Equipment</td>
		</tr>
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			<td>Illuminator</td>
			<td>Cole&#8211;Parmer</td>
			<td>41720</td>
			<td>Surgical Equipment</td>
		</tr>
		<tr>
			<td>Dumont no. 55 Dumostar Forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11295-51</td>
			<td>Surgical Instruments</td>
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			<td>Graefe forceps, curved&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11052-10</td>
			<td>Surgical Instruments</td>
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			<td>Moria MC31 forceps&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>11370-31</td>
			<td>Surgical Instruments</td>
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			<td>Mayo scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14512-15</td>
			<td>Surgical Instruments</td>
		</tr>
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			<td>Iris scissors&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>14041-10</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Halsey needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12501-13</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>Olsen&#8211;Hegar needle holder&#160;</td>
			<td>Fine Science Tools Inc</td>
			<td>12002-12</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>spring scissors</td>
			<td>Fine Science Tools Inc</td>
			<td>15610-08</td>
			<td>Surgical Instruments</td>
		</tr>
		<tr>
			<td>disposable underpads</td>
			<td>Kendall/Tyco Healthcare</td>
			<td>1038</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Sterile gauze sponges, sterile&#160;</td>
			<td>Dukal</td>
			<td>62208</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicators, sterile&#160;</td>
			<td>Solon</td>
			<td>368</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Surgical suture,&#160; silk, 6-0&#160;</td>
			<td>DemeTECH</td>
			<td>FT-639-1</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>1 cc Insulin syringes&#160;</td>
			<td>Becton Dickenson</td>
			<td>329412</td>
			<td>Surgical Supplies</td>
		</tr>
		<tr>
			<td>Access-9&#8482; Hemostasis Valve</td>
			<td>Merit Medical&#160;</td>
			<td>MAP111</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Sphygmomanometer</td>
			<td>Baumanometer</td>
			<td>320</td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>Millar PV system MPVS-300/400 or MPVS Ultra (includes calibration cuvette)</td>
			<td>ADInstruments Inc</td>
			<td></td>
			<td>Hemodynamic equipment</td>
		</tr>
		<tr>
			<td>1.4F conductance catheter&#160;</td>
			<td>ADInstruments Inc</td>
			<td>SPR-839</td>
			<td>Hemodynamic equipment</td>
		</tr>
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			<td>PowerLab 4/30 with Chart Pro</td>
			<td>ADInstruments Inc.</td>
			<td>ML866/P</td>
			<td>Hemodynamic software</td>
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			<td>animal clipper</td>
			<td>Wahl</td>
			<td>8787-450A</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-10 (Becton Dickinson, cat. no. )</td>
			<td>Becton Dickenson</td>
			<td>427401</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Intradermic tubing PE-50 (Becton Dickinson, cat. no.)</td>
			<td>Becton Dickenson</td>
			<td>427411</td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Needle assortment (18, 25 and 30 gauge; Thomas Scientific)</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>0.9% (wt/vol) sodium chloride injection, USP , cat. no. )</td>
			<td>Hospira</td>
			<td>NDC no. 0409-4888-50</td>
			<td>Miscellaneous</td>
		</tr>
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			<td>Surgical tape</td>
			<td></td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
		<tr>
			<td>Alconox (Alconox Inc.) for catheter cleaning</td>
			<td>ADInstruments Inc.</td>
			<td></td>
			<td>Miscellaneous</td>
		</tr>
	</tbody>
</table>

cardiac pressure-volume loop analysis using conductance catheters in mice

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular systolic and diastolic function. Measures of ventricular contractility and compliance are obtained through examination of cardiac response to changes in afterload and preload. These techniques were originally developed nearly three decades ago to measure cardiac function in large mammals and humans. The application of these analyses to small mammals, such as mice, has been accomplished through the optimization of microsurgical techniques and creation of conductance catheters. Conductance catheters allow for estimation of the blood pool by exploiting the relationship between electrical conductance and volume. When properly performed, these techniques allow for testing of cardiac function in genetic mutant mouse models or in drug treatment studies. The accuracy and precision of these studies are dependent on careful attention to the calibration of instruments, systematic conduct of hemodynamic measurements and data analyses. We will review the methods of conducting pressure-volume loop experiments using a conductance catheter in mice.

圧-容積ループの分析は、薬物研究16を受けて遺伝的に改変されたマウスを14,15またはマウスで心機能を測定するために用いることができます。代表的圧力容積ループは、β-アレスチン偏ったAT1Rリガンド、TRV120023の影響を調査する以前に発表された作品16から提供されています。 TRV120023 がインビボで心機能に影響を与えるかどうかを試験するために、圧力...

Watch this Scientific Journal Video about Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice at JoVE.com

Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters.

マウスにおけるコンダクタンスカテーテルを用いて心臓圧力 - 体積ループ解析

Cardiac pressure-volume loop analysis is the &#8220;gold-standard&#8221; in the assessment of load-dependent and load-independent measures of ventricular ...

cardiac-pressure-volume-loop-analysis-using-conductance-catheters

Research

JoVE Journal

Biology

19.3K ビュー.  Duke University Medical Center. Cardiac pressure-volume loop analysis is the most comprehensive way to measure cardiac function in the intact heart. We describe a technique to perform and analyze cardiac pressure volume loops, using conductance catheters. 

ビデオ: Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

生物学

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

免疫不全マウスにおける生体工学ヒト微小血管ネットワーク

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

で嚢胞性線維症膜コンダクタンス制御因子タンパク質の発現および精製サッカロマイセスセレビシエ

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method (EFM)

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to achieve a high stomatal conductance (gs) to capture CO2 for photosynthesis, water is lost by transpiration1,2. Water evaporating from the airspaces is replaced from cell walls, in turn drawing water from the xylem of leaf veins, in turn drawing from xylem in the stems and roots. As water is pulled through the system, it experiences hydraulic resistance, creating tension throughout the system and a low leaf water potential (&Psi;leaf). The leaf itself is a critical bottleneck in the whole plant system, accounting for on average 30% of the plant hydraulic resistance3. Leaf hydraulic conductance (Kleaf = 1/ leaf hydraulic resistance) is the ratio of the water flow rate to the water potential gradient across the leaf, and summarizes the behavior of a complex system: water moves through the petiole and through several orders of veins, exits into the bundle sheath and passes through or around mesophyll cells before evaporating into the airspace and being transpired from the stomata. Kleaf is of strong interest as an important physiological trait to compare species, quantifying the effectiveness of the leaf structure and physiology for water transport, and a key variable to investigate for its relationship to variation in structure (e.g., in leaf venation architecture) and its impacts on photosynthetic gas exchange. Further, Kleaf responds strongly to the internal and external leaf environment3. Kleaf can increase dramatically with irradiance apparently due to changes in the expression and activation of aquaporins, the proteins involved in water transport through membranes4, and Kleaf declines strongly during drought, due to cavitation and/or collapse of xylem conduits, and/or loss of permeability in the extra-xylem tissues due to mesophyll and bundle sheath cell shrinkage or aquaporin deactivation5-10. Because Kleaf can constrain gs and photosynthetic rate across species in well watered conditions and during drought, and thus limit whole-plant performance they may possibly determine species distributions especially as droughts increase in frequency and severity11-14.
We present a simple method for simultaneous determination of Kleaf and gs on excised leaves. A transpiring leaf is connected by its petiole to tubing running to a water source on a balance. The loss of water from the balance is recorded to calculate the flow rate through the leaf. When steady state transpiration (E, mmol &bull; m-2 &bull; s-1) is reached, gs is determined by dividing by vapor pressure deficit, and Kleaf by dividing by the water potential driving force determined using a pressure chamber (Kleaf= E /- &Delta;&psi;leaf, MPa)15.
This method can be used to assess Kleaf responses to different irradiances and the vulnerability of Kleaf to dehydration14,16,17.

Measurement of Leaf Hydraulic Conductance and Stomatal Conductance and Their Responses to Irradiance and Dehydration Using the Evaporative Flux Method EFM

We describe a relatively rapid (30 min) and realistic method for simultaneously measurement of leaf hydraulic conductance (Kleaf) and stomatal conductance (gs) for transpiring excised leaves. The method can be modified to measure the light and dehydration responses of Kleaf and gs.

蒸発フラックス法（EFM）を使用した照度と脱水に葉の通水コンダクタンスと気孔コンダクタンスとその応答の測定

Water is a key resource, and the plant water transport system sets limits on maximum growth and drought tolerance. When plants open their stomata to ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

末梢血幹細胞移動の解析のためのマウス精巣挙筋で微小循環の生体顕微鏡

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

で表現する嚢胞性線維症膜コンダクタンス制御因子タンパク質の精製<em&gt;サッカロマイセス·セレビシエ</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

グレースケール解析を用いたマウスの抜け毛の定量分析のための新たな手法

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

Relating Stomatal Conductance to Leaf Functional Traits

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, morphological leaf traits have the potential to summarize plants strategies in terms of water use efficiency, growth pattern and nutrient use. The leaf economics spectrum (LES) is a recognized framework in functional plant ecology and reflects a gradient of increasing specific leaf area (SLA), leaf nitrogen, phosphorus and cation content, and decreasing leaf dry matter content (LDMC) and carbon nitrogen ratio (CN). The LES describes different strategies ranging from that of short-lived leaves with high photosynthetic capacity per leaf mass to long-lived leaves with low mass-based carbon assimilation rates. However, traits that are not included in the LES might provide additional information on the species&#39; physiology, such as those related to stomatal control. Protocols are presented for a wide range of leaf functional traits, including traits of the LES, but also traits that are independent of the LES. In particular, a new method is introduced that relates the plants&#8217; regulatory behavior in stomatal conductance to vapor pressure deficit. The resulting parameters of stomatal regulation can then be compared to the LES and other plant functional traits. The results show that functional leaf traits of the LES were also valid predictors for the parameters of stomatal regulation. For example, leaf carbon concentration was positively related to the vapor pressure deficit (vpd) at the point of inflection and the maximum of the conductance-vpd curve. However, traits that are not included in the LES added information in explaining parameters of stomatal control: the vpd at the point of inflection of the conductance-vpd curve was lower for species with higher stomatal density and higher stomatal index. Overall, stomata and vein traits were more powerful predictors for explaining stomatal regulation than traits used in the LES.

Unraveling how physiology and morphology are linked allows a deeper understanding of mechanistic functioning of plant leaves. We present both a procedure to derive parameters of stomatal regulation from stomatal conductance measurements and correlations with traditional functional leaf traits.

葉の機能上の特徴に気孔コンダクタンスの関連付け

Leaf functional traits are important because they reflect physiological functions, such as transpiration and carbon assimilation. In particular, ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

マウスの心臓変力作用の調​​査のために分離された乳頭筋の収縮の測定

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

マウスで圧力ボリュームループを測定します

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...