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>

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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.

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			<td>.024&#34; OD X .011&#34; ID&#160;</td>
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			<td>.038&#34; OD X .023&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
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			<td>.082&#34; OD X .062&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
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			<td style="width:119px;">113-S</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
			<td style="width:167px;">Deknatel</td>
			<td style="width:119px;">106-S</td>
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			<td height="21" style="height:21px;width:251px;">Water circulating heating pad</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Water bath</td>
			<td style="width:167px;">Fisher Scientific</td>
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			<td>Equipment</td>
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			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">22-415-468</td>
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			<td style="width:167px;">Fisher Scientific</td>
			<td style="width:119px;">23-400-124</td>
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			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
		</tr>
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			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14225</td>
			<td>Surgical instrument</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vessel clip</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14120</td>
			<td>Surgical instrument</td>
		</tr>
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			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

電離放射線への応答でγH2AX病巣の定量化

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

骨格筋からの前駆体の精製

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

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 ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

蛍光を用いたmRNA分子の可視化と分析<em&gt;その場で</emで&gt;ハイブリダイゼーション<em&gt;サッカロミセス·セレビシエ（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

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 ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

マウス冠動脈血管平滑筋細胞の単離

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

ミオシン重鎖免疫組織化学を使用して、ラット筋断面における筋線維人口分析のための迅速な自動化されたプロトコル

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...