我々は、イメージング研究のヘルプは博士ウィリアムCashdollar、およびウィスコンシン州の血液研究所のノースウェスタン·ミューチュアル財団イメージングセンターに感謝します。また、この原稿で、有用な議論の重要な読書のための博士ダリアIlatovskayaに感謝します。本研究は、（AS）に健康補助金HL108880と（AMG）はDP2-OD008396の国立研究所によってサポートされていました。

以下に記載の実験手順は、ウィスコンシン医科大学の施設内動物管理使用委員会（IACUC）によって承認され、実験動物の管理と使用に関する健康ガイドの国立研究所に応じてありました。 ラット大動脈の1の単離<ol><li>イソフルラン（5％誘導、1.5から2.5パーセントのメンテナンス）または別のIACUC承認の方法でラットを麻酔。 </li><li>仰臥位でラットを配置します。腹部の臓器を露出させるために、皮膚と皮下組織を通って腹部の小さな腹側正中切開を行います。ガーゼパッドを使用して、穏やかに、図1に示すように、大動脈と大静脈を露出するために腸を偏向させます。 </li><li>簡単に言えば、結合組織からと大静脈から大動脈を解剖鈍いです。縫合糸を使用して、腎臓にできるだけ近い大動脈を縛ります。大動脈2センチ、15ミリリットルの円錐TUに配置 - 約1を分析氷の上で：;（140のNaCl、1.2のMgCl 2、2のCaCl 2、4.5のKCl、6グルコース、10 mMのHEPESでPSS）は、通常の生理食塩液を含むこと。 </li><li>大動脈が分離されると、承認されたIACUCプロトコルに従って動物を安楽死させます。すべての非生存手続きの完了時に動物の人道的な終焉を確実にするために開胸誘導気胸により深く麻酔をかけた動物を安楽死させる。3 </li><li>解剖顕微鏡を使用して、先の細いピンセット、およびmicroscissorsは大動脈のオフ脂肪および結合組織を解剖。大動脈がきれいになったら、縦に大動脈を切断し、後で氷上PSSに配置します。 </li></ol> 2.色素添加およびインキュベーション<ol><li>アルミ箔で覆われている個々の500μlのチューブに、DMSOベースのソリューションで提供されている1 mMののFluo-4 AM色素のピペット7μlの。時間をかけて、色素の性質を維持するために-20℃の冷凍庫に保管してください。注：これらは非常に分注しlutionsは、少なくとも6ヶ月間安定であるべきです。 </li><li>使用前に、色素溶液を開く前に、室温まで昇温することができます。使用直前に作業溶液を調製します。後で使用するために希釈された試薬を保管しないでください。 </li><li>何のカルシウムを含有しないPSSの450 ULにDMSO溶液に溶解した既製の1mMのFluo-4 AM色素の7μlを添加します。アセトキシメチル（AM）エステルを分散させる助け、カルシウム色素の負荷を改善するために、ローディングカクテルに非DMSOベースの10％プルロニック酸溶液40μlを追加します。 </li><li> 1時間（2.3で説明したように）、ローディングカクテルを含む500μlのチューブに解剖した大動脈を置きます。 RTで回転シェーカー上箔と場所ですべてのチューブをカバーしています。 </li><li>血管系におけるNO産生を監視しない場合は、DAF-FMジアセテート染料を使用しています。 450μlのPSS、40μlのプルロニック酸および10mM DAF-FMジアセテートの5μLを含有する溶液中に大動脈をインキュベートします。 （2.3で説明）のカルシウムのインキュベーションプロトコルと同様に、大動脈を配置室温で次の30分間、続いて4℃で30分間回転シェーカー上に箔と場所でカバーNO染料溶液を含む500μlのチューブです。 </li><li> NO産生が測定されていない場合、 図4（b）に示すように、対照としてNO産生を遮断しないように、内皮のNO合成酵素阻害薬、L-NAMEを使用します。その手順については、（室温で）インキュベーションの最後の30分間（2.5で説明したように）カクテル100 nmのL-NAMEに追加します。 </li></ol> 3.レーザー走査2光子顕微鏡のプロトコル<ol><li>細胞外色素を除去し、クリーンPSSで3回 - 1時間のインキュベーションの後、大動脈2を洗います。 </li><li>通常のPSSまたはCa 2+を含まない緩衝液（実験プロトコルに依存します）のいずれかを含むシリコーン被覆皿に大動脈を転送します。ピンダウンμ状のピンを使用してシリコーン（皿開口部に露出され、すなわち、内皮ルーメン）と接触している外膜を有するシリコーンコーティング上に大動脈。あるいは、スライスアンカーを使用グリッドまたは接着剤は、組織を固定します。 注：ストレスと可能な機械的なアーティファクト、伝達を低減およびCa 2+フリーソリューションを大動脈を修正し、5待つに-実験開始の10分前。 </li><li>薬物の自動または手動のアプリケーションのために、または細胞外溶液を変更するためのシリコーン被覆プレートに蠕動ポンプまたはシリンジを接続します。 </li><li>直立二光子顕微鏡のノーズピースの下に皿を置き、試料の上に25×（NA 1.05と作動距離2mm）を水浸対物レンズを取り付け、位置決めします。注：この特定のレンズが使用できない場合は、通常の目的と比較して飛躍的に信号の解像度を向上させることができるので、特に2光子イメージングのために製造され対物レンズを使用しています。 </li><li>ソフトウェアを使用して、2つの光子レーザーをアクティブにします（レーザーは、ポンプとオンし始めます）。チューニング820 nmのレーザー励起。 </li><li>エピ蛍光を用いて、大動脈を探し、上の目的を集中粗調整を使用して、内皮表面。 </li><li>励起レーザは、モードロックされ、非デスキャン検出器が係合されると予想される発光スペクトルに適切な放射フィルタがインストールされていることを確認して、2光子レーザ走査顕微鏡モードに切り替えます。注：ここでは、FV10-MRL / R（540 nmの495）を使用しました。 </li><li>約5％のレーザー出力を使用して、生のスキャンを開始し、微細血管内皮細胞を可視化するために、目的の焦点を合わせます。 注：通常のPSSでインキュベートしたときに内皮細胞は強い蛍光シグナルを示すであろう。容器はカルシウムを含まない緩衝液中でインキュベートされたとき、これは著しく弱くなります。内皮細胞は、開いた大動脈の上に存在し、平滑筋細胞は、単に内皮細胞（ 図2参照 ）の下に見られます。血管はいずれも平滑であってもあるので、平滑筋細胞および内皮細胞の両方が存在している場所があります。&lt;/ LI&gt; <li>セルがフォーカスに入ると、顕微鏡ソフトウェアプログラムは、高速スキャンモードで連続画像（ラスタースキャン、1.1秒のフレームコレクションの頻度で512×512ピクセルのウィンドウ）を収集します。フルオ4 AM蛍光と並行して、血管収縮の変化を検出し、より良い実験条件を可視化するために光画像を送信し収集します。 </li><li>ベースライン信号画像のコレクションの後、外液中のCa 2+イオン濃度を変更したり、ゆっくりと撮影しながら蠕動ポンプを使用して、薬理学的な刺激剤を適用します。負荷細胞内の蛍光シグナルの増加を観察します。 注：内皮細胞は、フローの変化にこのようにして、それがのCa 2+またはNOシグナル伝達の薬理学的活性剤を塗布する前に、低い層アプリケーションや車両試験を使用することをお勧めし応答します。のFluo-4（のFluo-4 Iの解離定数がロードされた血管内nMの濃度値を、細胞内カルシウムを再計算しますS 345 nM）を、蛍光強度は、ベースライン時およびイオノマイシンとのMnCl 2を添加した後に記録することができた。4 </li></ol> 4.画像処理と計算<ol><li>フィジーの画像処理パッケージをダウンロードし、インストールします。 </li><li>フィジーの画像ファイルを開きます。プロンプトが表示されたら、ファイルをインポートすると、チャンネル（透過光と蛍光信号）を分割します。データ分析のための唯一の蛍光シグナルを使用してください。 </li><li>フィジープログラム内で、分析タブをクリックし、[ツール]オプションまでスクロールダウンします。ツールのオプションでは、ROIのマネージャーを言うタブを見つけ、それをクリックしてください。新しいウィンドウが表示されます。 </li><li>この後、分析]タブで設定された測定値をクリックしてください。関心のある特定の測定値を選択します。カルシウムトランジェント測定では、平均グレー値のオプションを使用します。 </li><li>円形トレースツールでは、関心領域（ROI）を追跡し、すべてのRのためのROIマネージャ画面で「追加」をクリックして始めますOI。一度興味のあるすべてのセルは、「より」タブの下にマルチメジャーを選択し、ROIマネージャウィンドウで、トレースされています。スプレッドシートにこれらの測定の全てを直接測定値を保存するか、コピー＆ペーストするかを覚えておいてください。 </li><li>フィジーから* .txtファイルやExcelスプレッドシートを開き、新しい原点ワークシートに番号を転送します。注：または、さらなる分析のためにシグマプロットやプリズムのようなプログラムを使用します。 </li><li>オリジンプログラム内では、すべての過渡応答の概要を観察するためにプロット→線+シンボルメニューを使用します。応答しない細胞の選択を解除します。 </li><li>時間スケール（X軸）へのトレース数を再計算し、列→列値の設定を使用し、（我々の場合1.1sで）画像収集頻度へのトレース番号の列を掛けます。 </li><li>平均（Y軸）および標準エラーのトレースを計算するために選択したすべての記録のための行の解析→統計を使用してください。細胞プロット→線+シンボルの現在のグループのための最終的なグラフをプロットします。 </li><li> MEA以下のようにn個のカルシウム過渡計算が記載されています。 <ol><li>オリジンプログラム内の総カルシウム放出のために、グラフをクリックして、次に統合→メニュー分析→微積分を使用しています。曲線下面積の全積分は、結果ログに表示されます。 </li><li>一過性の動態については、グラフ上でクリックし、メニューを使用します。分析→非線形曲線フィット→[データセット（最大値から過渡応答の最後の範囲でX軸を選択します）。完了→指数関数的減衰1→関数選択→フィッティングを開始します。 注：指数フィッティングがグラフウィンドウに表示され、ログイン結果。得られたデータ（値）をリリースし、カルシウムの総量を表し、チャンネルの動力学は、過渡応答を媒介しました。 </li></ol></li></ol>

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著者らは、開示することは何もありません。

実験の概要。より良い血管生理学にカルシウムの寄与を理解し、NOに、新規な方法を測定するために開発されたの[Ca 2+] iと内の平滑筋および単離された無傷の大動脈の内皮細胞NO。 1）機械的分離と準備（酵素消化ない）容器の：一緒に、このプロトコルは、これらの重要なステップで構成されています。これは最適な生理学的記録を得ることが可能な限り健康で無傷?...

カルシウムは、内皮細胞および平滑筋細胞などの血管細胞内の基本的なセカンドメッセンジャーです。これは、血管収縮のための主要な刺激であり、内皮内のNO発生を介してその効果を含め、血管拡張において重要な役割を果たしています。これにより、撮像技術の制限のために、それは、無傷の血管内カルシウム処理を観察することは事実上不可能でした。 2光子イメージングシステムと新しいカルシウムの作成またはNO標識色素の開発は、カルシウム動態及び血管系内のNO産生を理解し始めるために十分な深さと解像度でイメージすることができます。 二光子顕微鏡は、最近ので、深く低いバックグラウンド蛍光と高い信号感度で組織に浸透するその優れた能力の組織、器官、さらには全体の動物試験に適用されている。1,2イルミネータで2光子励起の狭いスペクトルイオン焦点と非デスキャン検出器の使用は、2光子顕微鏡は、従来の共焦点顕微鏡よりも優れている理由です。共焦点顕微鏡は、自動蛍光および共焦点ピンホールへのピンボケ光の散乱に起因する必要な組織深さで高品質の画像を生成することはできません。したがって、我々は、[Ca 2+] i のシグナルを測定するために2つの光子顕微鏡を使用する方法と、高解像度、低信号対雑音比を有する無傷の、個々の血管細胞におけるNO産生を開発しました。

<table><tbody><tr><td>DAF-FM</td><td>Life Technologies</td><td>D-23842</td><td></td></tr><tr><td>Fluo-4 AM</td><td>Life Technologies</td><td>F14217</td><td>500 &amp;micro;l in DMSO</td></tr><tr><td>Pluronic F-68 solution</td><td>Sigma-Aldrich</td><td>P5556</td><td></td></tr><tr><td>L-NAME</td><td>Tocris</td><td>0665</td><td></td></tr><tr><td>Olympus upright microscope</td><td>Olympus</td><td>Fluoview FV1000</td><td></td></tr><tr><td>MaiTai HP DeepSee-OL&amp;nbsp;</td><td>Spectra Physics</td><td></td><td>Ti:sapphire laser 690nm &amp;mdash; 1040nm</td></tr><tr><td>Filters</td><td>Olympus</td><td>FV10-MRL/R&amp;nbsp;</td><td>495 to 540 nm&amp;nbsp;</td></tr><tr><td>25&amp;times; water-immersion objective lens</td><td>Olympus</td><td>XLPL25XWMP</td><td>N.A. 1.05 and working distance 2 mm</td></tr><tr><td>Slice Anchor grid&amp;nbsp;</td><td>Warner Instrument&amp;nbsp;</td><td>SHD-27LH</td><td></td></tr><tr><td>Other basic reagents&amp;nbsp;</td><td>Sigma-Aldrich</td><td></td><td></td></tr></tbody></table>

two-photon imaging of intracellular ca2+ handling and nitric oxide production in endothelial and smooth muscle cells of an isolated rat aorta

カルシウムは、血管組織の多くの生理学的プロセスの非常に重要な調節因子です。ほとんどの内皮および平滑筋の機能は非常に細胞内カルシウム（[Ca 2+] i） の一酸化窒素（NO）の変化に依存します。の[Ca 2+] i の 、NOおよび下流分子は血管収縮薬および血管拡張薬に反応して血管によってどのように処理されるかを理解するために、我々はカルシウム標識（またはNO標識）を適用する新規な技術を開発した2光子顕微鏡を有する色素単離された血管におけるカルシウム処理（またはNO産生）を測定しました。ここで説明した内皮細胞または平滑筋細胞内にラット、ラベルカルシウムまたはNOから大動脈を分離する方法を示す詳細なステップバイステップの手順であり、画像のカルシウムトランジェント（またはNO産生）生理次の2光子顕微鏡を用いて、または薬理学的刺激。この方法を使用する利点は、複数倍である：1）それは、同時にすることが可能ですLY異なる刺激に応答して、両方の内皮細胞および平滑筋細胞におけるカルシウムトランジェントを測定します。 2）それは、画像の内皮細胞とそれらの天然の環境における平滑筋細胞のいずれかを可能にします。 3）この方法は、細胞内カルシウムまたはNO変化に非常に敏感であり、正確な測定のための高解像度の画像を生成します。 4）記載の手法は、活性酸素種のような他の分子の測定に適用することができます。要するに、2光子レーザ発光顕微鏡の応用カルシウムトランジェントを監視し、単離された血管の内皮細胞および平滑筋細胞におけるNO産生は、高品質の定量的データを提供しておらず、血管機能を調節するメカニズムの理解を推進してきました。

正確に血管の生理学（血管拡張および血管収縮）のカルシウムの寄与を評価するために、プロトコルは、単離された無傷の大動脈の両方の内皮細胞および平滑筋細胞にカルシウム色素をロードするように設計されました。 図1に示されている設定の一般的な実験では、単離およびイメージングの前に容器の製造のための基本戦略を示しています。簡単に言うと、ラッ?...

Watch this Scientific Journal Video about Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta at JoVE.com

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

細胞内カルシウムの二光子イメージング<sup&gt; 2+</sup&gt;取扱い及び内皮における一酸化窒素産生および単離されたラット大動脈の平滑筋細胞

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

two-photon-imaging-intracellular-ca2-handling-nitric-oxide-production

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

11.2K ビュー.  Medical College of Wisconsin. Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta. 

ビデオ: Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcification of Vascular Smooth Muscle Cells and Imaging of Aortic Calcification and Inflammation

Cardiovascular disease is the leading cause of morbidity and mortality in the world. Atherosclerotic plaques, consisting of lipid-laden macrophages and calcification, develop in the coronary arteries, aortic valve, aorta, and peripheral conduit arteries and are the hallmark of cardiovascular disease. In humans, imaging with computed tomography allows for the quantification of vascular calcification; the presence of vascular calcification is a strong predictor of future cardiovascular events. Development of novel therapies in cardiovascular disease relies critically on improving our understanding of the underlying molecular mechanisms of atherosclerosis. Advancing our knowledge of atherosclerotic mechanisms relies on murine and cell-based models. Here, a method for imaging aortic calcification and macrophage infiltration using two spectrally distinct near-infrared fluorescent imaging probes is detailed. Near-infrared fluorescent imaging allows for the ex vivo quantification of calcification and macrophage accumulation in the entire aorta and can be used to further our understanding of the mechanistic relationship between inflammation and calcification in atherosclerosis. Additionally, a method for isolating and culturing animal aortic vascular smooth muscle cells and a protocol for inducing calcification in cultured smooth muscle cells from either murine aortas or from human coronary arteries is described. This in vitro method of modeling vascular calcification can be used to identify and characterize the signaling pathways likely important for the development of vascular disease, in the hopes of discovering novel targets for therapy.

Vascular calcification is an important predictor of and contributor to human cardiovascular disease. This protocol describes methods for inducing calcification of cultured primary vascular smooth muscle cells and for quantifying calcification and macrophage burden in animal aortas using near-infrared fluorescence imaging.

血管平滑筋細胞と大動脈石灰化と炎症のイメージングの石灰化

Cardiovascular disease is the leading cause of morbidity and mortality in the world.  Atherosclerotic plaques, consisting of lipid-laden macrophages and ...

医学

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 ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

マウス抵抗動脈から毛細血管内皮の管の隔離

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

生物学

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to correlate this with local hemodynamics. This protocol uses transgenic mice that have been prepared with a chronic cranial window and express the genetically encoded calcium indicator, RCaMP1.07, under the &#945;-smooth muscle actin promoter to specifically label mural cells, such as vascular smooth muscle cells and ensheathing pericytes. Steps are outlined on how to prepare a tail vein catheter for intravenous injection of fluorescent dyes to trace blood flow, as well as how to measure brain pericyte calcium and local blood vessel hemodynamics (diameter, red blood cell velocity, etc.) by two photon microscopy in vivo through the cranial window in ketamine/xylazine anesthetized mice. Finally, details are provided for the analysis of calcium fluctuations and blood flow movies via the image processing algorithms developed by Barrett et al. 2018, with an emphasis on how these processes can be adapted to other cellular imaging data.

Brain Pericyte Calcium and Hemodynamic Imaging in Transgenic Mice In Vivo

This protocol presents steps to acquire and analyze fluorescent calcium images from brain ensheathing pericytes and blood flow data from nearby blood vessels in anesthetized mice. These techniques are useful for studies of mural cell physiology and can be adapted to investigate calcium transients in any cell type.

生体内のトランスジェニックマウスにおける脳ペリサイトカルシウムと血行力学イメージング

Recent advances in protein biology and mouse genetics have made it possible to measure intracellular calcium fluctuations of brain cells in vivo and to ...

神経科学

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular physiology. Dysfunctional eNOS such as uncoupling of eNOS leads to decrease in NO bioavailability and increase in superoxide anion (O2.&#8722;) production, and in turn promotes cardiovascular diseases. Therefore, appropriate measurement of NO and O2.&#8722; levels in the endothelial cells are pivotal for research on cardiovascular diseases and complications. Because of the extremely labile nature of NO and O2.&#8722;, it is difficult to measure NO and O2.&#8722; directly in a blood vessel. Numerous methods have been developed to measure NO and O2.&#8722; production. It is, however, either insensitive, or non-specific, or technically demanding and requires special equipment. Here we describe an adaption of the fluorescence dye method for en face simultaneous detection and visualization of intracellular NO and O2.&#8722; using the cell permeable diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE), respectively, in intact aortas of an obesity mouse model induced by high-fat-diet feeding. We could demonstrate decreased intracellular NO and enhanced O2.&#8722; levels in the freshly isolated intact aortas of obesity mouse as compared to the control lean mouse. We demonstrate that this method is an easy technique for direct detection and visualization of NO and O2.&#8722; in the intact blood vessels and can be widely applied for investigation of endothelial (dys)function under (physio)pathological conditions.

En Face Detection of Nitric Oxide and Superoxide in Endothelial Layer of Intact Arteries

In this article we describe an adapted relatively easy method using the fluorescence dye diaminofluorescein-2 diacetate (DAF-2DA) and dihydroethidium (DHE) for en face simultaneous detection and visualization of intracellular nitric oxide (NO) and superoxide anion (O2.&#8722;) respectively, in freshly isolated intact aortas of an obesity mouse model.

無傷の動脈の内皮層における一酸化窒素とスーパーオキシドのエン顔検出

Endothelium-derived nitric oxide (NO) produced from endothelial NO-synthase (eNOS) is one of the most important vasoprotective molecules in cardiovascular ...

Measurement of Calcium Fluctuations Within the Sarcoplasmic Reticulum of Cultured Smooth Muscle Cells Using FRET-based Confocal Imaging

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall health. A significant portion of intracellular Ca2+ content is found within the SR stores in VSMCs. As the only intracellular organelle with a close association to the surrounding extracellular space through plasma membrane-SR junctions, the SR can be considered to constitute the first line of response to any irregularity in Ca2+ transients, or stress experienced by the cell. Among its many functions, one of the most important is its role in the transmission of Ca2+-regulated signals throughout the cell to induce further cell-wide reactions downstream. The more common use of cytoplasmic Ca2+ indicators in this regard is overall insufficient for research into the highly dynamic changes to the intraluminal SR Ca2+ store that have yet to be fully characterized. Here, we provide a detailed protocol for the direct and clear measurement of luminal SR Ca2+. This tool is useful for investigation into the nuanced changes in SR Ca2+ that have significant subsequent effects on the normal function and health of the cell. Fluctuations in SR Ca2+ content specifically can provide us with a significant amount of information pertaining to cellular responses to disease or stress conditions experienced by the cell. In this method, a modified version of a SR-targeted Ca2+ indicator, D1SR, is used to detect Ca2+ fluctuations in response to the introduction of agents to cultured rat aortic smooth muscle cells (SMCs). Following incubation with the D1SR indicator, confocal fluorescence microscopy and fluorescence resonance energy transfer (FRET)-based imaging are used to directly observe changes to intraluminal SR Ca2+ levels under control conditions and with the addition of agonist agents that function to induce intracellular Ca2+ movement.

Currently, most available calcium indicators are used to quantify cytoplasmic calcium transients as indirect measures of calcium released from the sarcoplasmic reticulum in cultured smooth muscle cells. This protocol describes the use of a specific FRET-based indicator that allows direct measurement of calcium signals within the sarcoplasmic reticulum lumen.

FRETベースの共焦点イメージングを使用した培養平滑筋細胞の小胞体内カルシウム変動の測定

Maintenance of steady-state calcium (Ca2+) levels in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (VSMCs) is vital to their overall ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

の開発と特性評価<em&gt;インビトロ</em&gt;微小血管ネットワークおよび内皮の定量的測定の[Ca<sup&gt; 2+</sup&gt;]<sub&gt;私</sub&gt;と一酸化窒素の生産

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Genetically Encoded Fluorescent Nitric Oxide (NO&#8226;) Probes, the geNOps, for Real-time Imaging of NO&#8226; Signals in Single Cells

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence of a large number of methods for detecting NO&#8226; in vivo and in vitro, the real-time monitoring of NO&#8226; at the single-cell level is very challenging. The physiological or pathological effects of NO&#8226; are determined by the actual concentration and dwell time of this radical. Accordingly, methods that allow the single-cell detection of NO&#8226; are highly desirable. Recently, we expanded the pallet of NO&#8226; indicators by introducing single fluorescent protein-based genetically encoded nitric oxide (NO&#8226;) probes (geNOps) that directly respond to cellular NO&#8226; fluctuations and, hence, addresses this need. Here we demonstrate the usage of geNOps to assess intracellular NO&#8226; signals in response to two different chemical NO&#8226;-liberating molecules. Our results also confirm that freshly prepared 3-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) has a much higher potential to evoke change in intracellular NO&#8226; levels as compared with the inorganic NO&#8226; donor sodium nitroprusside (SNP). Furthermore, dual-color live-cell imaging using the green geNOps (G-geNOp) and the chemical Ca2+ indicator fura-2 was performed to visualize the tight regulation of Ca2+-dependent NO&#8226; formation in single endothelial cells. These representative experiments demonstrate that geNOps are suitable tools to investigate the real-time generation and degradation of single-cell NO&#8226; signals in diverse experimental setups.

This manuscript presents protocols for the application of novel genetically encoded nitric oxide (NO&#8226;) probes (geNOps) to monitor single cell NO&#8226; fluctuations in real-time using fluorescence microscopy. The Ca2+-triggered NO&#8226; formation on the level of individual endothelial cells was visualized by combining geNOps with a chemical Ca2+ sensor.

遺伝的にコードされた蛍光一酸化窒素の応用（•NO）シングル細胞におけるNO•信号のリアルタイムイメージングのためのプローブ、geNOps、

Nitric Oxide (NO&#8226;) is a small radical, which mediates multiple important cellular functions in mammals, bacteria and plants. Despite the existence ...

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.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Ex Vivo細胞生理学研究のため網膜細動脈の隔離

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

Measurement of Endothelium-Dependent Vasorelaxation in the Mouse Thoracic Aorta Using Tensometric Small Volume Chamber Myography

Small volume chamber tensometric myography is a commonly used technique to evaluate the vascular contractility of small and large blood vessels in laboratory animals and small arteries isolated from human tissue. The technique allows researchers to maintain isolated blood vessels in a tightly controlled and standardized (near-physiological) setting, with the option of adjusting to various environmental factors, while challenging the isolated vessels with different pharmacological agents that can induce vasoconstriction or vasodilation. The myograph chamber also provides a platform to measure vascular reactivity in response to various hormones, inhibitors, and agonists that may impact the function of smooth muscle and endothelial layers separately or simultaneously. The blood vessel wall is a complex structure consisting of three different layers: the intima (endothelial layer), media (smooth muscle and elastin fibers), and adventitia (collagen and other connective tissue). To gain a clear understanding of the functional properties of each layer, it is critical to have access to an experimental platform and system that would allow for a combinational approach to study all three layers simultaneously. Such an approach demands access to a semi-physiological condition that would mimic the in vivo environment in an ex vivo setting. Small volume chamber tensometric myography has provided an ideal environment to evaluate the impact of environmental cues, experimental variables, or pharmacological agonists and antagonists on vascular properties. For many years, scientists have used the tensometric myograph technique to measure endothelial function and smooth muscle contractility in response to different agents. In this report, a small volume chamber tensometric myograph system is used to measure endothelial function in the isolated mouse aorta. This report focuses on how small volume chamber tensometric myography can be used to evaluate the functional integrity of the endothelium in small segments of a large artery such as the thoracic aorta.

The present protocol describes the concepts and technical application of the tensometric myograph technique using a multi-chamber myograph system in the experimental ex vivo assessment of mouse aortic endothelial function.

細胞内カルシウムの二光子イメージング

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