Name: visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle
Uploaded: 2016-10-19T13:00:00.000+00:00
Duration: 6 min 10 s
Description: Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

この研究は、シンガポール国立大学の施設内動物管理使用委員会（承認されたプロトコルがない。R15-0225）によるものです。 動物モデルの1外科の準備<ol><li> 血管カニューレ挿入 <ol><li>ケタミン（37.5 mg / mlで）および腹腔内を介して、キシラジン（5 mg / mlで）カクテル（IP）注射（2ミリリットル/ kg）でグラム - （7週齢6）（203±20）のオスのSprague-Dawleyラットを麻酔。針をリキャップまたは注射後に注射器からそれを削除しないでください。 </li><li>動物は（爪先つまんで確認）、麻酔された後、37℃で、その体温を維持するために加温パッド上に置きます。そっと肩甲骨、前頸部、下腹部、内側後肢と陰嚢の毛を剃ります。ゆっくりと粘着紙テープを使用して足を拘束。 </li><li>見ながら顕微解剖ハサミと角度のついたピンセットを使用して、すべての外科的処置を実行します実体顕微鏡。手術中にけがを防ぐために、パンク防止トレイ上のすべての鋭い手術器具を配置します。 </li><li>切開を行う前に、すべての手術部位を交互にヨウ素、70％アルコールで3回スクラブ。 30 IU / mlのヘパリン - 食塩水溶液を持つすべてのカテーテルをフラッシュします。 </li><li>右頸静脈の上に手術用ハサミを使用して、肩甲骨1.5センチ正中皮膚切開 - 1を加えます。頸静脈を露出させるために鈍的切開により筋膜を分離し、これをポリエチレンチューブ（PE-50）5-0絹縫合糸を用いて、ヘパリン生理食塩水で満たされてカニューレを挿入。補足麻酔を注入する際に必要な（1/3初期投与量の1/2に、静脈内（IV））手術と実験の過程を通して。 </li><li>気道開存性を維持するために気管切開を行います。前頸部エリア1.5センチ切開 - 1を加えます。所定の位置にカテーテルを固定するために2-0絹縫合糸でポリエチレンチューブ（PE-205）を使用して、気管にカニューレを挿入。 </li><li>血圧を測定します大腿動脈にカニュレーションを通じて。後肢の左内側面で1.5センチ切開 - 1を加えます。鈍的切開によって大腿動脈を分離します。ヘパリン生理食塩水は、5-0絹縫合糸を用いて充填したポリエチレンチューブ（PE-10）と大腿動脈にカニューレを挿入。 </li></ol></li><li> 精巣挙筋の準備と流れの可視化 <ol><li>それを拡張するために陰嚢の頂部を通って5-0絹縫合糸を挿入します。陰嚢の腹側表面に沿って切開を行います。露出した筋肉に、定期的に暖かい等張液（pH 7.4、37℃）を適用します。 </li><li>慎重に周囲の結合組織を削除し、徹底的に綿棒を使用して。 </li><li>精巣挙筋の頂点を通って5-0絹縫合糸を挿入します。同じ長さの2つのピースに縫合糸をカットし、それぞれの側に結び目を作ります。 2ノットの間の筋肉をカットし、静かに縫合糸を引っ張ることにより、カスタマイズされた透明なプレキシグラスのプラットフォームにそれを伸ばします。フィックスブルータックとプラットフォームへの縫合糸の終わり。 注：周囲の結合組織の徹底的な除去は、最適な画像コントラストを得る上で重要です。 </li><li> 5〜6凝視が行われるまで、ステップ1.2.3を繰り返します。慎重に高温焼灼を使用して、精巣上体から精巣挙筋を取り除きます。組織の脱水を防ぐために露出した筋肉に温かい等張液を表面かん流します。 <ol><li>ガーゼの折り返し片と精巣挙筋を囲みます。ポリビニルフィルムで露出した筋肉をカバーしています。フィルムとガーゼ片は、水浸顕微鏡対物レンズ（ 図1A）のための温かい等張液を保持するために浅い盆地を形成しています。 </li></ol></li><li>生体顕微鏡（ 図1C）の動物ステージ上に動物を転送します。連続圧力監視（ 図1E）のための生理的データ収集システムに動脈カニュレーションを接続します。 </li><li>筋肉temperatを維持動物のプラットフォーム（ 図1B）の下に取り付けられた発熱体と35℃でURE。加熱素子（ 図1D）のパワー・コントローラへの負のフィードバックを提供するために、筋肉の横に温度プローブを配置します。 </li><li>環境と平衡に15分間のステージ上で動物を残します。 </li><li> 40X水浸対物レンズと長い作業冷却器を備えた生体顕微鏡で血液の流れを可視化します。 </li><li>血管の直径平面上に顕微鏡を集中させるためにはRBCコア、CFLと血管壁との間に明確な映像のピントやコントラストに基づいて分岐していない細動脈を（&lt;60μm）で、選択してください。垂直方向に血管壁を整列させるために、顕微鏡に取り付けたカメラを回転させます。 </li><li> 1秒間3,000 /秒のフレームレートの高速ビデオカメラを使用して血流を記録します。画像品質を維持するために、非圧縮8ビットグレースケールAVI形式として記録されたビデオを保存します。 注意：<html> 3000フレーム/秒の最小記録フレームレートがCFLの測定は、生理的細動脈の流れの条件の下で少なくとも一回RBCあたりに実行できることを確認することをお勧めします。 </li><li>赤血球と血漿との間のコントラストを高めるために、510 nmの - 310で394 nmおよびスペクトル帯域の波長でピーク透過と青色フィルタを使用してください。 注：光スペクトルは、微小光源（100 Wハロゲンランプ）からの青色フィルタを通過したことを確認し、任意の潜在的な組織の損傷を防止するために、低光強度です。 </li><li>実験の最後に、ペントバルビタールナトリウムの過剰投与により動物を安楽死させます。 </li></ol></li></ol> 2.画像解析 <ol><li> CFL幅測定のための前処理 <ol><li> MATLABを開き、「CFL_pre.m &#39;ファイルを実行します。 （これと他のMATLABファイルはで見つけることができますIP &quot;&gt;補足MATLABアーカイブ。） </li><li>分析するために、ビデオファイルを選択するには、「ファイルを開く」をクリックしてください。 </li><li>垂直方向に血管壁を整列させるために「回転」スライダを調整します。 注：ユーザーは「グリッドオン]ラジオボタンを選択することで、血管の位置合わせのための補助グリッド線を表示し、「ズーム」スライダーをスライドさせることにより、画像のズームレベルを調整することができます。 </li><li>血管の位置合わせを確認する「確認して編集」ボタンをクリックします。 </li><li>関心領域（ROI）を定義する」に設定したROI作物へ」ボタンをクリックします。配置画像がポップアップウィンドウに表示されます。画像上に長方形の目標を調整し、二重のROIをクリックして確認します。画像のトリミングが必要とされない場合は、このステップをスキップします。 注：のみ容器からCFL幅を分析するためのROI内の単一の容器が含まれます。必要に応じて、元の形にイメージを復元するには、「リセットイメージ」ボタンをクリックします。 </li><li>クリック9; &#39;（BMP&#39;形式の連続したビットマップ画像の8ビットグレースケール）に、すべての編集されたビデオフレームを抽出するためにボタン &#39;イメージを抽出します。抽出された画像は、選択したビデオファイルと同じ名前のフォルダに格納されています。 </li></ol></li><li> CFL幅の測定 <ol><li> MATLABを開き、「CFL_measure.m &#39;ファイルを実行します。 </li><li>抽出された画像を含むフォルダを選択するには、「[フォルダの選択]をクリックします。 </li><li>画像を含むフォルダをクリックし、「フォルダの選択」をクリックします。フォルダ内の最初の画像フレームがロードされ、「画像のヒストグラム」パネルで、その灰色の強度ヒストグラムと一緒に、「グレースケール画像」パネルに表示されます。 </li><li>分析を実行するために、リストボックスから所望の画像フレームを選択し、そうでなければ第1の画像フレームが選択されます。 </li><li>位置で決定された画像で血管内壁を識別するために、「血管壁を探す」をクリックします暗いから2ピクセル以上の光に光強度プロファイルのピーク遷移します。 </li><li> 「塩、コショウ」ノイズを低減する画像にメディアンフィルタを適用するには、「メジアンフィルタ」を確認してください。 </li><li>画像のコントラストを向上させるために、デジタル画像強度を調整する「自動コントラスト」を確認してください。 </li><li> τ（RBCコア）以下のグレーレベルでτ上記のグレーレベル（CFL）と白画素と黒画素 - 自動的に2つのクラスにグレイレベルを分割しきい値値（τ）を決定するリストボックスのしきい値アルゴリズムを選択します。 注：自動化された閾値処理アルゴリズムのいずれも適切な画像閾値化を提供しない場合は別の方法としては、手動のしきい値を使用します。 「マニュアル」のラジオボタンをクリックして、手動のしきい値の値を定義するには、スライダを調整します。 </li><li> 、CFL幅の空間的変化を測定する「ピクセル解像度」ボックス内のピクセル解像度（解像度を入力するにはこの実験で0.42ミクロン/ピクセル）でした。 </li><li> CFL幅の空間的な変化を得るために「計算」ボタンをクリックします。集計形式でCFL幅データをエクスポートするには、「書き出した.csv]をクリックします。 </li><li> 、血管に沿った特定の分析線でCFL幅の時間変化を測定する「時間変化]ラジオボタンをクリックして、フレームレート情報を入力します（この実験で使用されるフレームレートは3000フレーム/秒でした）。 </li><li>それぞれ、「フレームを起動 &#39;と&#39;最後のフレーム」ボックスに最初のフレームおよび分析のための画像の最後のフレームを入力します。 </li><li> 「分析ライン」のスライドバーをスライドさせることにより、血管に沿って分析線の位置を選択します。 「グレースケール画像」と「バイナリイメージ」の両方に示されている分析線の位置を確認してください。 </li><li> CFL幅の時間変化を取得するために「計算」をクリックしてください。 CLICKを表形式でCFL幅データをエクスポートするには、「エクスポートは.csvファイル」。 </li></ol></li></ol>

<ol>
	<li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+variations+of+cell-free+layer+width+in+arterioles.">Temporal and spatial variations of cell-free layer width in arterioles.</a> Am J Physiol Heart Circ Physiol. 293 (3), H1526-H1535 (2007).</li><li>Ong, P. K., Namgung, B., Johnson, P. C., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+erythrocyte+aggregation+and+flow+rate+on+cell-free+layer+formation+in+arterioles.">Effect of erythrocyte aggregation and flow rate on cell-free layer formation in arterioles.</a> Am J Physiol Heart Circ Physiol. 298 (6), H1870-H1878 (2010).</li><li>Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+uneven+red+cell+influx+on+formation+of+cell-free+layer+in+small+venules.">Effect of uneven red cell influx on formation of cell-free layer in small venules.</a> Microvasc Res. 92, 19-24 (2014).</li><li>Goldsmith, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Microcirculatory+Society.">The Microcirculatory Society.</a> Eugene M. Landis Award Lecture. The Microrheology of Human-Blood. Microvasc Res. 31 (2), 121-142 (1986).</li><li>Buerk, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+We+Model+Nitric+Oxide+Biotransport?+A+Survey+of+Mathematical+Models+for+a+Simple+Diatomic+Molecule+with+Surprisingly+Complex+Biological+Activities.">Can We Model Nitric Oxide Biotransport? A Survey of Mathematical Models for a Simple Diatomic Molecule with Surprisingly Complex Biological Activities.</a> Annu Rev Biomed Eng. 3 (1), 109-143 (2001).</li><li>Tateishi, N., Suzuki, Y., Soutani, M., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+dynamics+of+erythrocytes+in+microvessels+of+isolated+rabbit+mesentery:+cell-free+layer+and+flow+resistance.">Flow dynamics of erythrocytes in microvessels of isolated rabbit mesentery: cell-free layer and flow resistance.</a> J Biomech. 27 (9), 1119-1125 (1994).</li><li>Ong, P. K., Cho, S., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cell-free+layer+formation+on+NO/O2+bioavailability+in+small+arterioles.">Effects of cell-free layer formation on NO/O2 bioavailability in small arterioles.</a> Microvasc Res. 83 (2), 168-177 (2012).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+NO+bioavailability+by+temporal+variation+of+the+cell-free+layer+width+in+small+arterioles.">Modulation of NO bioavailability by temporal variation of the cell-free layer width in small arterioles.</a> Ann Biomed Eng. 39 (3), 1012-1023 (2011).</li><li>Park, S. W., Intaglietta, M., Tartakovsky, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+stochastic+fluctuations+in+the+cell+free+layer+on+nitric+oxide+bioavailability.">Impact of stochastic fluctuations in the cell free layer on nitric oxide bioavailability.</a> Front Comput Neurosci. 9, 131 (2015).</li><li>Ng, Y. C., Namgung, B., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+transient+model+for+prediction+of+arteriolar+NO/O2+modulation+by+spatiotemporal+variations+in+cell-free+layer+width.">Two-dimensional transient model for prediction of arteriolar NO/O2 modulation by spatiotemporal variations in cell-free layer width.</a> Microvasc Res. 97, 88-97 (2015).</li><li>Sriram, 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=The+effect+of+small+changes+in+hematocrit+on+nitric+oxide+transport+in+arterioles.">The effect of small changes in hematocrit on nitric oxide transport in arterioles.</a> Antioxid Redox Sign. 14 (2), 175-185 (2011).</li><li>Hightower, C. 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=Integration+of+cardiovascular+regulation+by+the+blood/endothelium+cell-free+layer.">Integration of cardiovascular regulation by the blood/endothelium cell-free layer.</a> Wiley Interdiscip Rev Syst Biol Med. 3 (4), 458-470 (2011).</li><li>Ng, Y. C., Namgung, B., Leo, H. L., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+aggregation+may+promote+uneven+spatial+distribution+of+NO/O+in+the+downstream+vessel+of+arteriolar+bifurcations.">Erythrocyte aggregation may promote uneven spatial distribution of NO/O in the downstream vessel of arteriolar bifurcations.</a> J Biomech. , (2015).</li><li>Ong, P. K., Jain, S., Kim, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+variations+of+the+cell-free+layer+width+may+enhance+NO+bioavailability+in+small+arterioles:+Effects+of+erythrocyte+aggregation.">Temporal variations of the cell-free layer width may enhance NO bioavailability in small arterioles: Effects of erythrocyte aggregation.</a> Microvasc Res. 81 (3), 303-312 (2011).</li><li>Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocyte+rheology+in+microcirculation.">Erythrocyte rheology in microcirculation.</a> Jpn J Physiol. 46 (1), 1-14 (1996).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels+-+Influence+of+Erythrocyte+Aggregation.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels - Influence of Erythrocyte Aggregation.</a> Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Kim, S., Kong, R. L., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+computer-based+method+for+determination+of+the+cell-free+layer+width+in+microcirculation.">A computer-based method for determination of the cell-free layer width in microcirculation.</a> Microcirculation. 13 (3), 199-207 (2006).</li><li>Soutani, M., Suzuki, Y., Tateishi, N., Maeda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Evaluation+of+Flow+Dynamics+of+Erythrocytes+in+Microvessels.">Quantitative Evaluation of Flow Dynamics of Erythrocytes in Microvessels.</a> Influence of Erythrocyte Aggregation. Am J Physiol Heart Circ Physiol. 268 (5), H1959-H1965 (1995).</li><li>Namgung, B., 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+comparative+study+of+histogram-based+thresholding+methods+for+the+determination+of+cell-free+layer+width+in+small+blood+vessels.">A comparative study of histogram-based thresholding methods for the determination of cell-free layer width in small blood vessels.</a> Physiol Meas. 31 (9), N61-N70 (2010).</li><li>Ong, P. 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=An+automated+method+for+cell-free+layer+width+determination+in+small+arterioles.">An automated method for cell-free layer width determination in small arterioles.</a> Physiol Meas. 32 (3), N1-N12 (2011).</li><li>Otsu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Threshold+Selection+Method+from+Gray-Level+Histograms.">A Threshold Selection Method from Gray-Level Histograms.</a> IEEE Trans. Syst., Man, Cybern. 9 (1), 62-66 (1979).</li><li>Prewitt, J. M., Mendelsohn, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+cell+images.">The analysis of cell images.</a> Ann N Y Acad Sci. 128 (3), 1035-1053 (1966).</li><li>Ridler, T. W., Calvard, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picture+Thresholding+Using+an+Iterative+Selection+Method.">Picture Thresholding Using an Iterative Selection Method.</a> IEEE Trans. Syst., Man, Cybern. 8 (8), 630-632 (1978).</li><li>Shanbhag, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+Information+Measure+as+a+Means+of+Image+Thresholding.">Utilization of Information Measure as a Means of Image Thresholding.</a> Cvgip-Graph Model Im. 56 (5), 414-419 (1994).</li><li>Bishop, J. J., Popel, A. S., Intaglietta, M., Johnson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+erythrocyte+aggregation+and+venous+network+geometry+on+red+blood+cell+axial+migration.">Effects of erythrocyte aggregation and venous network geometry on red blood cell axial migration.</a> Am J Physiol Heart Circ Physiol. 281 (2), H939-H950 (2001).</li><li>Yamaguchi, S., Yamakawa, T., Niimi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+plasma+layer+in+cerebral+microvessels.">Cell-free plasma layer in cerebral microvessels.</a> Biorheology. 29 (2-3), 251-260 (1992).</li></ol>

The authors declare no competing financial interests.

CFL幅の測定は、微小循環における血行動態のより良い理解のために不可欠です。具体的には、CFL幅の測定は腸間膜6で行われた、24および脳25微小循環をspinotrapezius。 in vivoでの CFL幅の従来の測定は、記録されたビデオフレームの手動検査によって推定に制限されていました。マニュアル測定は、視覚的にRBCコアと血管壁15,16の境界を特定する前に、いくつかの連続するビデオフレームの平均化を必要としました。別の研究では、フルオレセインイソチオシアネート（FITC）は、赤血球及びローダミンBイソチオシアネート（RITC）はプラズマがネコ脳微小血管25の平均CFL幅を決定するために使用された標識-標識。これらの以前の測定方法は非常に時間がかかり、CFLのwiの空間分解能と時間分解能を制限する蛍光標識のための追加の手順が必要ですDTH測定。対照的に、効果的な画像セグメント化及び分析に高速度カメラの記録を結合することによって、技術はここで実証RBCの大きさよりも小さいオーダーの空間分解能（0.42ミクロン）でCFLの時空間変動の定量化を可能にし1/3000秒の時間分解能。 精巣挙筋の適切な外科的な準備がCFL幅測定の精度を決定する上で非常に重要です。具体的には、隣接する結合組織の徹底的な除去は、精巣挙筋における細動脈の良好なフォーカスを確保することが不可欠です。また、測定の時間と空間分解能は、顕微鏡とカメラの仕様に依存しています。高倍率の対物レンズは、空間分解能を向上させることができるが、それは順番にCFL幅の空間的変化を定量化するために得られる容器の長さを制限する視野を減少させます。したがって、microscopiCの構成は、技術の特定の用途に応じて変更することができます。 画像分割は、CFL幅測定の精度のための別の重要な要因です。開発した様々な技術の中でも、グレーレベルのヒストグラムに基づいて画像しきい値は、画像分割および分析のためのシンプルで効果的なアプローチを提供します。したがって、前景オブジェクトは、それらのグレーレベルの差に基づいて背景から抽出されます。理想的なケースでは、画像のヒストグラムが双峰性になり、谷の底部の閾値は自明です。しかし、in vivoでの実験画像は常に、このような階調プロファイルを示しません。我々の結果は、画像品質及びコントラスト画像分割プロセスに影響を与えることができる方法を示しています。光学青色フィルタの使用が大幅に赤血球および細動脈における血漿（ 図3）との間のコントラストを高め、そして時applyinことが不可欠であると思われます関係なく、アルゴリズムのCFL幅測定のためのグラムヒストグラムベースの閾値処理（ 図4）。これは、1つは、効果的に閾値を識別することを可能にする明確な二峰性、画像ヒストグラムをもたらします。しかし、それも、in vivoでの画像から得られた二峰性のヒストグラムを用いて、二つのピーク（極大値）の非常に不等分散ヒストグラムの広い谷（極小）が依然として閾値選択（ 表1に影響を与えることができることに留意すべきです）。したがって、適切な閾値化アルゴリズムの選択は、画像品質とユーザーに基づいて検討する必要があるCFL幅を定量化することで最高の適合性について、それぞれ閾値処理アルゴリズムの限界を考慮しなければなりません。 CFL幅がフロー条件に大きく依存しているように、実験の過程を通して連続的な動脈圧の測定が不可欠です。局所的な流れの状況を決定するために、血流のpseudoshearレートは血管5の平均流速を測定することによって計算することができます。 要約すると、ここで説明したラット精巣挙筋および定量的画像解析の外科的調製のためのプロトコールは、in vivoでの CFL幅の動的変化に関する定量的情報を取得するために利用されています。 CFL幅測定の精度を確保する上で主要な課題は、上記の対処されているどちらも筋肉や画像セグメンテーション、の適切な外科的な準備が含まれています。この技術は、容易に様々な生理学的および病理学的条件の血液レオロジー及び血行力学的異常を調査するために、他の微小循環の研究に適用することができます。したがって、これらの知見は、微小血管の治療アプローチと臨床的介入の将来の発展に貢献しています。

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

<table><tbody><tr><td>Intravital microscope</td><td>Olympus</td><td>BX51WI</td><td>Equipment</td></tr><tr><td>High speed camera</td><td>Photron</td><td>1024PCI</td><td>Equipment</td></tr><tr><td>Blue filter</td><td>HOYA</td><td>B390</td><td>Equipment</td></tr><tr><td>Pressure sensor &amp;amp; biopac system</td><td>Biopac system</td><td>TSD104A, MP100</td><td>Equipment</td></tr><tr><td>Temperature controller</td><td>Shimaden</td><td>SR 1</td><td>Equipment</td></tr><tr><td>Plasma Lyte A</td><td>Baxter</td><td>NDC:0338-0221</td><td>Warm in 37 &amp;deg;C water bath before use</td></tr><tr><td>Saline 0.9%</td><td>Braun</td><td></td><td></td></tr><tr><td>Heparin (5,000 IU/ml)</td><td>LEO</td><td></td><td></td></tr><tr><td>PE-10 polyethylene tube</td><td>Becton Dickinson</td><td>427400</td><td>.024&quot; OD x .011&quot; ID&amp;nbsp;</td></tr><tr><td>PE-50 polyethene tube</td><td>Becton Dickinson</td><td>427411</td><td>.038&quot; OD x .023&quot; ID</td></tr><tr><td>PE-205 polyethene tube</td><td>Becton Dickinson</td><td>427446</td><td>.082&quot; OD x .062&quot; ID</td></tr><tr><td>2-0 non-absorbable silk suture</td><td>Deknatel</td><td>113-S</td><td></td></tr><tr><td>5-0 non-absorbable silk suture</td><td>Deknatel</td><td>106-S</td><td></td></tr><tr><td>Water circulating heating pad</td><td>Gaymar</td><td></td><td></td></tr><tr><td>Water bath</td><td>Fisher Scientific</td><td>Isotemp 205</td><td>Equipment</td></tr><tr><td>Sterile Cotton Gauze&amp;nbsp;</td><td>Fisher Scientific</td><td>22-415-468</td><td></td></tr><tr><td>Cotton-tipped applicators</td><td>Fisher Scientific</td><td>23-400-124</td><td></td></tr><tr><td>Dumont Forceps</td><td>Kent Scientific</td><td>INS14188</td><td>Surgical instrument</td></tr><tr><td>Micro Dissecting forceps</td><td>Kent Scientific</td><td>INS15915</td><td>Surgical instrument</td></tr><tr><td>Iris forceps 1 x 2 teeth</td><td>Kent Scientific</td><td>INS15917</td><td>Surgical instrument</td></tr><tr><td>Vessel cannulation forceps</td><td>Kent Scientific</td><td>INS500377</td><td>Surgical instrument</td></tr><tr><td>Micro scissor</td><td>Kent Scientific</td><td>INS14177</td><td>Surgical instrument</td></tr><tr><td>Iris scissor</td><td>Kent Scientific</td><td>INS14225</td><td>Surgical instrument</td></tr><tr><td>Vessel clip</td><td>Kent Scientific</td><td>INS14120</td><td>Surgical instrument</td></tr><tr><td>Gemini cautery system</td><td>Braintree Scientific</td><td>GEM 5917</td><td>Surgical instrument</td></tr></tbody></table>

visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

in vivoでの CFLの可視化は、動物の外科的な準備に大きく依存しています。過度の失血または拡張手術時間は収差を衝撃や血流ために動物を供することができます。手術と実験中加熱パッドだけでなく、カスタマイズされたプラットフォームを使用して、組織の温度の維持はまた、ラットの生理学的条件を維持するために重要です。顕微鏡システム100 Wのハロゲンランプを使用して、識別可能な組織の損傷があっても、実験の終了時に観察されませんでした。 図2Aは、CFLがRBCコアと内側容器壁（ 図2C）との間で観察することができるラット精巣挙筋における非分枝動脈を介して典型的なRBCの流れを示しています。実験中のこれらの構成要素間の良好なコントラストがCFL幅測定の精度を確保するために重要です。画像解析の初期位相を含みます内側の血管壁を検出します。容器に垂直な分析線に沿った光強度分布を取得することにより、場所が暗いから遷移が2ピクセル（ 図2B）の上に点灯することをピークに近似されます。 赤血球及びCFLが異なる光透過率を有するように、グレーレベルの差は、2つのクラス（二値画像）に細分することができます。しかし、画像のヒストグラムの二つのピーク間の正確な閾値の同定は悪い画質とコントラスト（ 図3A）によって制限されてもよいです。赤血球とCFLの間のコントラストを向上させるために、青色フィルタ（ 図3B）を用いることができます。これは、RBCコアの境界をより正確に青色フィルタを用いて同定することができ、 図4にも明らかです。また、閾値アルゴリズム20〜23の選択は、CFLの幅（ 図の測定に影響を与えることができます4）。これは、異なる閾値処理アルゴリズムが順番に誤ったCFLの測定につながる可能性が識別された異なるRBCコア境界をもたらしたことを図4Aには明らかです。より良い図4BにおけるCFL幅測定の閾値アルゴリズムの影響を説明するために、異なる閾値アルゴリズムを使用して得られたCFL幅の空間プロファイルは、図5に示し、 表1にまとめます。 <img alt="図1" src="/files/ftp_upload/54550/54550fig1.jpg" /> 。 図1： 生体内顕微鏡システムと精巣挙筋の準備 A：外科的に体外ラットの精巣挙筋B：精巣挙筋を配置し、35℃Cで、その温度を維持するための加熱要素を備えたカスタマイズプラットフォーム：お客さまとの顕微鏡システム微小循環血液の可視化のためtomized動物段と高速度カメラは、精巣挙筋を流れるD：ネガティブフィードバック温度制御装置及び電源E：連続的な圧力監視のための生理学的データ収集システム。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig1large.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2" src="/files/ftp_upload/54550/54550fig2.jpg" /> 図2：細動脈におけるRBCの流れの典型的なグレースケール画像（血管径= 52μm）とB：分析線に沿った光強度プロファイル（パネルAの実線） 船舶壁の位置とCFL幅 A の決意のための画像処理 。C：血管に沿っCFL測定の代表的な結果。ザ実線及び破線の矢印は、RBCのコアの内側の血管壁と外側の縁を示します。 （LWB＆RWB：LCB＆RCB、血管壁の境界を左右：左右のRBCコア境界） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig2large.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3" src="/files/ftp_upload/54550/54550fig3.jpg" /> 図3：光ブルーフィルタによる画像のコントラスト強調 （A）がなく、青色フィルタ（B）で得られたグレースケール画像の画像ヒストグラム。。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig3large.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/54550/54550fig4.jpg" /> FigurE 4：RBCコア幅は、5つの異なるしきい値処理アルゴリズムを用いて決定図3のグレースケール画像に重畳されたRBCのコアと血管壁の境界を。。（トップの行（A）：青フィルタなし、下段（B）：ブルーフィルター付き）を使用して、大津の方法、最小法、インター法、反復選択方法（ISODATA）とファジィエントロピーしきい値（Shanbhag）（左から右へ）。固体と破線はそれぞれ、血管内壁とRBCコアの外縁を示す。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig4large.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5" src="/files/ftp_upload/54550/54550fig5.jpg" /> 図5：CFL幅の空間変動 CFL幅は左（A）に沿って図4Bを対応します。そして、右（B）血管壁、それぞれ。 （D：血管径の距離） <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54550/54550fig5large.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="表1" src="/files/ftp_upload/54550/54550table1.jpg" /> 表1：図5. *のP &lt;0.001 でのしきい値のとCFL幅データ ：大津の方法から有意な差。 †P &lt;0.001：左から有意な差。統計分析は、両側不対t検定を用いて行きました。

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

ラット精巣挙筋の細動脈における無細胞層の可視化と定量化

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Universidad San Sebastian

Research

JoVE Journal

Biology

6.3K ビュー.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

ビデオ: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

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

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

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

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

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

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

医学

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

免疫・感染学

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 ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

微小循環の生体内イメージングのためのマウス精巣挙筋の準備

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

生物学

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

デジタル免疫蛍光顕微鏡を用いたラット軟膜細動脈におけるエストロゲン受容体-αのイメージング

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

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 ...

Leukocyte Infiltration of Cremaster Muscle in Mice Assessed by Intravital Microscopy

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The current protocol represents a practical and reproducible method to visualize the leukocyte endothelium interaction leading to leukocyte recruitment in skeletal muscle derived tissue within the intact organism of the mouse. The model is applicable to all fields of research that focus on granulocyte activation and their role in disease.
We provide a step by step protocol to guide through the method and to highlight potential pitfalls and technical difficulties. The protocol covers the following aspects: experimental settings and required material, anesthesia of the mouse, dissection of the cremaster muscle as well as tracheal and carotid cannulation, IVM recordings and offline analysis. Data formats like adherent leukocytes, rolling flux (RF) and rolling flux fraction (RFF) are explained in detail and appropriate applications are discussed. Representative results from dystrophin deficient mdx mice are provided in the results section.
IVM is a powerful tool to assess leukocyte recruitment in an in vivo setting; however, delineating for example endothelial and leukocyte function may require a combination with ex vivo setups like flow chamber experiments. Furthermore, the genetic background of animals of interest may greatly influence baseline recruitment, requiring individual fine tuning of the protocol provided. Despite its limitations, IVM may serve as a platform to readily translate in vitro findings into a living vertebrate organism.

Here, we show how to perform intravital microscopy on post-capillary venules of the mouse cremaster muscle. Commonly applied to different models of inflammation and sepsis, particularly those induced by chemokines and cytokines, we highlight its relevance in the study of muscolopathies involving exaggerated muscular leukocyte infiltration.

インビタル顕微鏡で評価されたマウスにおけるクレマスター筋の白血球浸潤

Intravital microscopy (IVM) is widely used to monitor physiological and pathophysiological processes within the leukocyte recruitment cascade in vivo. The ...

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 ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

マウス心臓における毛細血管、細動脈、および周囲細胞の動的測定とイメージング

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.