研究は、KPにNIH HL75503によってサポートされていました。

テネシー大学健康科学センターの施設内動物管理使用委員会によって承認された動物で行われ、すべての実験であった。 灌流はラット肺の準備のための1。チューブ<ol><li> 図1に示すように、圧力変換器（P23XL）を接続します。血液灌流用タイゴンチューブ（＃18）と配管システムを準備し、顕微鏡ステージ上にチューブを置きます。 </li><li> 37℃の水浴温度を設定する</li><li>肺の入口および出口はタイゴンチューブを一時的に接続する。 </li></ol><img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51552/51552fig1highres.jpg" width="500" /> 図1。血液灌流チューブ 。回路図は、孤立した肺の準備を通して血液を循環させるために使用される管のセットアップを示しています。また、示されたが、部品を通る配管I関連しているSがルーティングされた。心臓と肺の概略管および肺動脈（PA）、および左心房（LA）、カニューレ（青色破線）との接続部位を示すために含まれている。 ラット摘出肺の2。準備<ol><li>ケタミン（80〜100 mg / kg）を持つとキシラジン（5月10日mg / kg）を持つ、ラット（250〜300グラムのオスのSDラット）を麻酔。麻酔の外科的平面性を確保した後、仰臥位で動物を配置します。 </li><li>気管切開を行い、気管カニューレ（PE-90）を挿入し、外科用縫合糸で固定します。 </li><li>心臓穿刺（21のG翼状針）で心にヘパリン（100〜200 U）を注入、60秒、放血させる血液（〜12〜15ミリリットルの血液）を待つ。 </li><li> 2カニューレ（;長さ4cm、タイゴンチューブ、直径3mm一端にフレア）を準備し、生理食塩水で満たします。 </li><li>開胸を行います。右心室に切開（3ミリメートル）を作成し、肺に向かって切開部にカニューレのフレア端部をスライドさせ動脈。手術用縫合糸で肺動脈にカニューレを固定します。 </li><li> （3ミリメートル）左心室の頂点を切開し、切開部に第二のカニューレのフレア端部をスライドさせます。左心房にカニューレをガイド。臍テープ（2ミリメートル幅）を左心室にカニューレを固定します。 </li><li>任意の結合組織を解剖し、付属のカニューレと一緒に肺と心臓を取り出します。ペトリ皿に、肺や心臓を配置します。横隔膜面が上になるように、肺の位置を変更。 3カニューレは、同じ方向を向くようにします。 </li><li> XY方向に操作することができ、ステージ上の肺の準備を置く。顕微鏡に付属しているいずれかの段階では、チューブや、必要に応じて、構築されたカスタムメイドの段階を保持するように変更することができます。 </li></ol>ラット摘出肺の3。血液灌流<ol><li>同体積のアルブミン（5％）溶液を用いて放血血液を混ぜてリザーバに追加します。蠕動ポンプを起動し、フローRを設定14ミリリットル/分に食べました。血液がチューブを埋めることができます。 </li><li>シャント開いて、一時的な肺の出入口コネクタを外します。それぞれ、肺の入口と出口に、肺動脈と左心房のカニューレを取り付けます。配管内に気泡がないことを確認してください。 </li><li>第3の圧力トランスデューサー（P23XL）に気管カニューレを接続します。気管カニューレを介して30％の酸素で肺を膨張させ5cmのH 2 Oの圧力を維持する</li><li>肺灌流を開始するクランプでシャントを閉じます。 〜10で、肺動脈と左心房の圧力を維持し、3センチメートルH 2 Oであった。 </li></ol> 4。微小血管注入のための肺の準備<ol><li>肺で安全のレベルを超えてカテーテル用延長チューブの後端を持ち上げます。 </li><li>約30センチPE90チューブに長さ40cmのPE10チューブを約30cmを挿入して注入カテーテルを準備します。針（30 G）のATにPE10チューブの露出端を接続注射器（1 CC）に取り付けられ。 </li><li>延長チューブの隆起後端にカテーテルの組み合わせを挿入します。それは抵抗を受けるまで、左心房を通って肺にカテーテルの組み合わせを導く。それは抵抗を受けるまで、肺の中にさらに単独のPE10カテーテルを押してください。カテーテルを挿入しながら、無理な力を適用することは、肺を傷つけることに注意してください。 </li><li>リンゲル液で1ccの注射器を満たし、シリンジポンプに取り付けます。 10μL/分に注入速度を設定します。 </li><li>注入部位は、肺の他の部分に比べて薄いとなります。 </li><li>生理食塩水で肺を湿らし、明らかに注入部位を残すのに十分な大きさの穴をラップで肺をカバーしています。 </li><li> Oリング（オーダーメイド）の下にいくつかのストップコックグリースを綿棒＆Oリングの底にガラスカバースリップ（＃1.5、直径22mm）を配置します。優しく注入部位でのカバーガラスでOリングを配置。標準試験管HOLにOリングを固定しますデア。最近、15を報告したようにカバースリップ/ Oリングの組み合わせは、その下歯槽構造を歪曲していません。 </li><li>位置Oリング、上記蛍光顕微鏡の対物レンズ（20X）および肺表面に焦点を当てる。 </li></ol>微小血管スルー5。イメージング蛍光デキストラントランジット<ol><li> FITCの蛍光を画像化するための顕微鏡を準備します。 </li><li>撮像すべき領域を選択し、画像取得ソフトウェアを用いて1/minuteの速度で画像を取得する。 </li><li>シリンジポンプを使用してFITC-デキストラン20kDの（0.5 mg / ml）での注入を開始する。血管内の蛍光が徐々に増加し、最大値に達する。 60分間注入を続ける。 </li><li>その後、管腔の蛍光を洗い流しリンゲル輸液に切り替える。 10分かけて点滴用を維持する。洗い流し時の限界回転で画像収集を続ける。 </li></ol> 6。画像解析<ol><li>画像取得ファイルを開きます。 </li><li>場所領域画像フレーム内の微小血管の上の関心の。 </li><li>イメージファイルフレームごとに再生すると、すべてのフレームのための関心の各領域での蛍光強度を記録。 </li><li>時間の関数としての関心領域毎の蛍光強度の変化をプロットする。 </li><li>ウォッシュオフの10分後、最大蛍光強度と残留蛍光強度を定量化する。 </li><li>関心のその領域に関連する微小血管の透過性の指標を得るために、関心の各領域での残留蛍光強度を最大の比率を計算します。 </li></ol>

<ol>
	<li>Ware, L. B., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. 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=The+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Bhattacharya, J., Matthay, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+repair+of+the+alveolar-capillary+barrier+in+acute+lung+injury.">Regulation and repair of the alveolar-capillary barrier in acute lung injury.</a> Annu Rev Physiol. 75, 593-615 (2013).</li><li>Parthasarathi, K., 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=Connexin+43+mediates+spread+of+Ca2+-dependent+proinflammatory+responses+in+lung+capillaries.">Connexin 43 mediates spread of Ca2+-dependent proinflammatory responses in lung capillaries.</a> J Clin Invest. 116, 2193-2200 (2006).</li><li>Parthasarathi, K., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+Acid+instillation+by+a+wedged-catheter+method+reveals+a+role+for+vascular+gap+junctions+in+spatial+expansion+of+Acid+injury.">Localized Acid instillation by a wedged-catheter method reveals a role for vascular gap junctions in spatial expansion of Acid injury.</a> Anat Rec (Hoboken). 294, 1585-1591 (2011).</li><li>Gorin, A. B., Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+permeability+of+endothelial+and+epithelial+barriers+to+albumin+flux.">Differential permeability of endothelial and epithelial barriers to albumin flux.</a> J Appl Physiol Respir Environ Exerc Physiol. 47, 1315-1324 (1979).</li><li>Boutoille, 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=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp Lung Res. 35, 263-271 (2009).</li><li>Thorball, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-dextran+tracers+in+microcirculatory+and+permeability+studies+using+combined+fluorescence+stereo+microscopy,+fluorescence+light+microscopy+and+electron+microscopy.">FITC-dextran tracers in microcirculatory and permeability studies using combined fluorescence stereo microscopy, fluorescence light microscopy and electron microscopy.</a> Histochemistry. 71, 209-233 (1981).</li><li>Stevens, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+heterogeneity+of+pulmonary+endothelial+cells.">Functional and molecular heterogeneity of pulmonary endothelial cells.</a> Proc Am Thorac Soc. 8, 453-457 (2011).</li><li>Ofori-Acquah, S. F., 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=Heterogeneity+of+barrier+function+in+the+lung+reflects+diversity+in+endothelial+cell+junctions.">Heterogeneity of barrier function in the lung reflects diversity in endothelial cell junctions.</a> Microvasc Res. 75, 391-402 (2008).</li><li>Kandasamy, K., 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=Real-time+imaging+reveals+endothelium-mediated+leukocyte+retention+in+LPS-treated+lung+microvessels.">Real-time imaging reveals endothelium-mediated leukocyte retention in LPS-treated lung microvessels.</a> Microvasc Res. 83, 323-331 (2012).</li><li>Kandasamy, K., 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=Lipopolysaccharide+induces+endoplasmic+store+Ca2+-dependent+inflammatory+responses+in+lung+microvessels.">Lipopolysaccharide induces endoplasmic store Ca2+-dependent inflammatory responses in lung microvessels.</a> PloS One. 8, (2013).</li><li>Qiao, R. L., Bhattacharya, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segmental+barrier+properties+of+the+pulmonary+microvascular+bed.">Segmental barrier properties of the pulmonary microvascular bed.</a> J Appl Physiol. 71, 2152-2159 (1991).</li><li>Parthasarathi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+connexin43+mediates+acid-induced+increases+in+pulmonary+microvascular+permeability.">Endothelial connexin43 mediates acid-induced increases in pulmonary microvascular permeability.</a> Am J Physiol Lung Cell Mol Physiol. 303, (2012).</li><li>Wu, Y., Perlman, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+methods+for+assessing+alveolar+mechanics.">In situ methods for assessing alveolar mechanics.</a> J Appl Physiol 1985. 112, 519-526 (2012).</li><li>Kuebler, W. M., 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=A+novel+signaling+mechanism+between+gas+and+blood+compartments+of+the+lung.">A novel signaling mechanism between gas and blood compartments of the lung.</a> Journal Clin Invest. 105, 905-913 (2000).</li></ol>

著者らは、開示することは何もありません。

リアルタイムイメージングと相まって孤立血液潅流肺標本は、単一の肺の微小血管内の透過性の変化を測定するためのシンプルなツールを提供しています。我々は、LPSの注入に応じて透過性の変化を定義するには、この方法を適用した。我々のデータは明らかに、LPS注入は、微小血管透過性の増加を引き起こしたことを示唆している。また、データも、LPSによって誘導された透過性の変化は?...

肺における微小血管透過性の増加は、浮腫および肺胞の妥協ガス交換の開発につながり、急性肺損傷（ALI）1-3の主要な特徴である。従って、血管透過性の推定値は、肺損傷および提案された治療的介入の有効性の範囲を定義する上で重要である。このような血中遊離肺のウェット対ドライ比と微小血管ろ過係数として重量分析は、広く浸透性4,5を推定するための方法を使用します。他の方法は、肺組織中の6-8放射性または蛍光プローブの保持を定量することが挙げられる。しかしながら、上記の方法は、透過性データを解明に向けて肺組織サンプルのpostexperiment処理を必要とする。一匹の動物は、単一の治療プロトコルに使用することができるので、大型動物番号は完全な研究のために必要とされ得る。上記の方法の共通の特徴は、彼らが平均血管透過性を決定するということです組織サンプル内の全ての血管。しかし、周知の肺ミクロおよびマクロ血管が表現型的に異なっている9ことが確立されている。このため、透過性の応答は、同様に9,10の様々な血管セグメントの中で異質かもしれません。このように、組織サンプル中のすべての肺血管の平均透過性を定量化することは十分にこの不均一性を反映しない場合があります。 単離された血液灌流肺製剤において、肺表面上の血管が正立顕微鏡4,11,12によって可視化することができる。これは、単一の容器内の応答を特徴づけるため、応答13内の任意の不均一性に対処することができます。また、微小血管の蛍光イメージングを利用して、蛍光ベースのアッセイは、組み込むことができる。また、左心房のマイクロカテーテルは、血管11,14内剤および蛍光プローブを送達するために使用することができる。マイクロカテーテルは、小さな肺領域への配信を制限し、このようにEX注入された薬剤と蛍光団にリージョン内でのみ血管を装った。これは、同じ肺内の複数の小領域は、研究に必要な動物の全体的な減少をもたらす、別々の実験のために使用することができる。 リアルタイムイメージングは​​、孤立した肺の準備の単一微小血管の血管および血管外蛍光の動的な変更の取得を可能にします。したがって、画像フィールド内の各微小血管のために、フルオロフォアおよびウォッシュオフの注入の間蛍光の変化を記録し、オフライン14を定量することができる。最大残留血管の蛍光値を用いて、撮像視野内の各微小血管の透過性指数を決定することができる。炎症性または有害な薬剤に応じて、透過性の変化を決定するために、所望の薬剤を最初に投与することができ、その後の透過性指数を決定した。また、画像フィールドには、注入によって肺の領域内のどこにでも設定することができるマイクロカテーテル、所望の血管網を選択する際の高度な柔軟性を可能にする。このように、リアルタイムで画像化と並行して孤立した血液灌流肺の準備は、単一の肺の微小血管内の透過性を定量化するための魅力的な実験モデルを提供しています。

<table><tbody><tr><td>Tygon Tubing</td><td>Fisher Scientific</td><td></td><td>#18</td></tr><tr><td>Pressure Transducer</td><td>Data Sciences International</td><td>P23XL</td><td>Need quantity 3</td></tr><tr><td>Butterfly Needle</td><td>Greiner Bio-One</td><td>450081</td><td>21 G</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer</td><td>Masterflex L/S</td><td></td></tr><tr><td>PE-90 tubing</td><td>Becton Dickinson</td><td>427421</td><td>30 cm needed</td></tr><tr><td>PE-10 tubing</td><td>Becton Dickinson</td><td>427401</td><td>40 cm needed</td></tr><tr><td>Syringe Pump</td><td>Braintree Scientific</td><td>BS8000</td><td></td></tr><tr><td>O-ring</td><td></td><td></td><td>Custom made with a 20 mm diamter hole and a handle to secure O-ring to holder</td></tr><tr><td>Upright fluorescence microscope</td><td>Olympus America</td><td>BX61WI</td><td></td></tr><tr><td>Image Acquisition Software</td><td>Molecular Devices</td><td>Metamorph</td><td></td></tr><tr><td>FITC Dextran 20KD</td><td>Sigma Aldrich</td><td></td><td>0.5 mg/ml (A dextran of different molecular size can be selected, if trial experiments indicate its suitability based on the calculated permeability index values)&amp;nbsp;</td></tr><tr><td>Lipopolysaccharide</td><td>Sigma Aldrich</td><td></td><td>Serotype 0111:B4</td></tr></tbody></table>

quantifying single microvessel permeability in isolated blood-perfused rat lung preparation

単離された血液灌流肺製剤が広く可視化し、単一の微小血管内シグナリングを定義するために使用される。リアルタイムイメージングにより、この調製物を結合することによって、個々の肺微小血管透過性における変化を決定することが可能となる。ここに我々は、ラットの肺を隔離し、自己血でそれらを灌流する手順について説明します。その後、我々は小さな肺領域にマイクロカテーテルを経由してフルオロフォアまたは薬剤を注入するための手順の概要を説明します。記載され、これらの手順を使用して、我々は、細菌のリポ多糖の注入を受けてラット肺微小血管内の透過性の増加を決定した。データは、リポ多糖の両方細静脈や毛細血管の微小血管セグメントにわたって流体の漏れを増加させたことを明らかにした。したがって、この方法は、血管透過性のセグメント間の応答を比較することが可能となり、従って、応答内の任意の不均一性を規定する。肺の透過性を定義するために一般的に使用される方法は、肺組織サンプルの後処理を必要としながら、リアルタイムイメージングの使用は、本方法から明らかなように、この要件を回避する。このように、リアルタイムで画像化と組み合わせた孤立性肺準備が肺微小血管透過性を決定するために、従来の方法に比べていくつかの利点を提供しています、まだ開発し、実装するための簡単​​な方法です。

灌流チューブおよび関連機器に接続された単離された血液灌流肺製剤が図2に示されている。本明細書に記載された手順は、任意のネズミ種を用いることができるが説明のために、我々は、スプラーグドーリーラットを使用した。左心房マイクロカテーテルを通して注入は、肺のわずかな領域に到達する。注入された領域は注入によって誘発される変色（ 図3）によっ...

Watch this Scientific Journal Video about Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation at JoVE.com

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

孤立した血液灌流肺準備が肺表面上の微小血管ネットワークを可視化することが実現可能になります。ここでは、リアルタイムの蛍光画像を用いて単離された肺における単一微小血管の透過性を定量化するためのアプローチを記載する。

単離された血液灌流ラットの肺の準備にシングル微小血管透過性を定量化する

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

quantifying-single-microvessel-permeability-isolated-blood-perfused

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.4K ビュー.  The University of Tennessee Health Science Center. 孤立した血液灌流肺準備が肺表面上の微小血管ネットワークを可視化することが実現可能になります。ここでは、リアルタイムの蛍光画像を用いて単離された肺における単一微小血管の透過性を定量化するためのアプローチを記載する。 

ビデオ: Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

アン<em生体内で&gt;</em血管透過性をテストする&gt;アッセイ

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

医学

Angiogenesis in the Ischemic Rat Lung

The adult lung is perfused by both the systemic bronchial artery and the entire venous return flowing through the pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that responds to a need for enhanced lung perfusion and shows robust neovascularization. Pulmonary vascular ischemia induced by pulmonary artery obstruction has been shown to result in rapid systemic arterial angiogenesis in man as well as in several animal models. Although the histologic assessment of the time course of bronchial artery proliferation in rats was carefully described by Weibel 1, mechanisms responsible for this organized growth of new vessels are not clear. We provide surgical details of inducing left pulmonary artery ischemia in the rat that leads to bronchial neovascularization. Quantification of the extent of angiogenesis presents an additional challenge due to the presence of the two vascular beds within the lung. Methods to determine functional angiogenesis based on labeled microsphere injections are provided.

The lung is perfused by both the systemic bronchial artery and pulmonary arteries. In most lung pathologies, it is the smaller systemic vasculature that shows robust neovascularization. Cessation of pulmonary blood flow promotes brisk bronchial angiogenesis. We provide surgical details of inducing left pulmonary artery ischemia that promotes bronchial neovascularization.

虚血ラットの肺の血管新生

The  adult lung is perfused by both the systemic bronchial artery and the entire  venous return flowing through the pulmonary arteries. In most lung ...

Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 &#181;m in diameter3.
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 &#181;m inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar arteries with inner diameter between 20-40 &#181;m which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar arteries with inner diameters between 40-100 &#181;m which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2 and the response fades after 30-40 min at hypoxia.

Hypoxic pulmonary vasoconstriction (HPV) is an important physiological phenomenon by which at alveolar hypoxia lung perfusion is matched to ventilation. The major vascular segment contributing to HPV is the intra-acinar artery. Here, we describe our protocol for the analysis of HPV of murine pulmonary vessels with diameters of 20-100 &mu;m.

マウス精密切り出肺スライスを用いた低酸素肺血管収縮のビデオモルフォメトリクス解析

Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

設定アップ<em&gt;体外</emラット血液脳関門の&gt;モデル（BBB）：BBB不透過性および受容体媒介輸送に注力

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Automated Measurement of Microcirculatory Blood Flow Velocity in Pulmonary Metastases of Rats

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. However, very few methods exist to date to investigate lung metastases in a dynamic fashion at the microcirculatory level, due to the difficulty to access the lung with a microscope. Here, an intravital microscopy method is presented to functionally image and quantify the microcirculation of superficial pulmonary metastases in rats, using a closed-chest pulmonary window and automated analysis of blood flow velocity and direction. The utility of this method is demonstrated to measure increases in blood flow velocity in response to pharmacological intervention, and to image the well-known tortuous vasculature of solid tumors. This is the first demonstration of intravital microscopy on pulmonary metastases in a closed-chest model. Because of its minimized invasiveness, as well as due to its relative ease and practicality, this technology has the potential to experience widespread use in laboratories that specialize on pulmonary tumor research.

A method is presented to measure microcirculatory blood flow velocity in pulmonary cancer metastases of the pleural surface in rats in an automated fashion, using closed-chest pulmonary intravital microscopy. This model has potential to be used as a widespread tool to perform physiologic research on pulmonary metastases in rodents.

ラットの肺転移における微小循環血流速度の自動測定

Because the lung is a major target organ of metastatic disease, animal models to study the physiology of pulmonary metastases are of great importance. ...

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

絶縁型、ベンチレーテッド、および計装のマウス肺の特性評価は、拍動流を灌流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

生物学

Shunt Surgery, Right Heart Catheterization, and Vascular Morphometry in a Rat Model for Flow-induced Pulmonary Arterial Hypertension

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.

This protocol describes a surgical procedure to create a model for flow-induced pulmonary arterial hypertension (PAH) in rats and the procedures to analyze the principle hemodynamic and histological end-points in this model.

流れ誘導性の肺動脈性肺高血圧症のためのラットモデルにおけるシャント手術、右心カテーテル、および血管形態計測

In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt ...

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered &#34;designer&#34; rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size.

This manuscript describes in vitro video microscopy protocols for evaluating vascular function in rat cerebral resistance arteries. The manuscript also describes techniques for evaluating microvessel density with fluorescently labeled lectin and tissue perfusion using Laser Doppler Flowmetry.

分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

A Fluorescence Assay for Evaluating the Permeability of a Cardiac Microvascular Endothelial Barrier in a Rat Model of Ischemia/reperfusion

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the best strategy for improving the clinical prognosis of patients with acute coronary syndrome (ACS). Nonetheless, myocardial reperfusion following effective revascularization can trigger significant cardiomyocyte death and coronary endothelial collapse, known as myocardial ischemia/reperfusion injury (MIRI). Usually, endothelial cells and their intercellular tight junctions cooperatively maintain the microvascular endothelial barrier and its relatively low permeability but fail in reperfusion areas. 
Microvascular endothelial hyperpermeability induced by ischemia/reperfusion (IR) contributes to myocardial edema, increased infiltration of pro-inflammatory cells, and aggravated intramyocardial hemorrhage, which may worsen the prognosis of ACS. The tracer used in this study-70,000 Da FITC-dextran, a branched glucose molecule labeled by fluorescein isothiocyanate (FITC)-appears too large to infiltrate the cardiac microvascular endothelium in normal conditions. However, it is capable of infiltrating a broken barrier after MIRI. Thus, the higher the endothelial permeability is, the more FITC-dextran accumulates in the extravascular intercellular space. Thus, the intensity of fluorescence from FITC can indicate the permeability of the microvascular endothelial barrier. This protocol takes advantage of FITC-dextran to evaluate the cardiac microvascular endothelial barrier functionally, which is detected by an automated quantitative pathology imaging system.

Here, we describe a method to functionally assess the endothelial barrier of cardiac microvessels after ischemia/reperfusion injury via the measurement of the mean fluorescence intensity of extravasated 70,000 Da FITC-dextran in comparison with Evans Blue.

Revascularization therapies for culprit arteries, regardless of percutaneous coronary intervention and coronary artery bypass grafting, are considered the ...