この作業は、教育および研究 (BMBF 01EO1003 および BMBF 01EO1503) ドイツの大臣によって支えられました。

雄マウスの年齢 8 ~ 12 週間ラインラント = プファルツ州 (承認番号 23 177-07/G12-1-002 と 23 177-07/G15-1-051) から動物実験に倫理委員会の承認の下で古い実験を行った。
1. マウスの麻酔や手術の準備
<ol>
	<li>手術前に良好な健康状態と動物の状態を確認します。術前に体重、食糧や水の消費量だけでなく、全身状態 (外観、姿勢、自発的行動) の 2-3 日に一度動物を監視します。</li>
	<li>腹腔内にミダゾラム (5 mg/kg 体重)、メデトミジン (0.5 mg/kg 体重)、フェンタニル (0.05 mg/kg 体重) と麻酔のマスターの組合せを準備 (i. p.) 26 G 針で 1 mL 注射器で 200 μ L/30 g マウスを注入します。</li>
	<li>浸透圧ポンプ注入と IVM の実験のための動物の準備は、プラットフォームを温暖化 39 ° C と操作フィールドで実行されます。滅菌消毒風呂のすべてのツール、地球温暖化のプラットフォームを消毒してください。</li>
	<li>手続き中に眼軟膏を動物の目を保護します。浸透圧ポンプ注入前にカミソリで毛を削除します。頸動脈と頸静脈カテーテルの注入を IVM 実験の隔離のための脱毛クリームと綿棒を使用します。2 つの皮膚の消毒剤を使用して動物の皮を消毒: 1 つの防腐剤と消毒剤のポビドン ヨードとの 1 つを含む皮膚アルコール ・ ベースのソリューションで防腐剤の含まれている octenidine。</li>
	<li>麻酔 (「antisedan ミックス」) atipamezol (0.05 mg/kg 体重) とフルマゼニル (0.01 mg/kgbody 重量) を敵に回したマスター ミックスを調製し皮下に 26 G 針で 1 mL 注射器で 200 μ L/マウスを準備 (サウスカロライナ) 注入。</li>
</ol>
2. 浸透圧ポンプの準備と移植
注: 浸透圧のポンプを使用して皮下 AngII 注入によって記述されていた詳細に Luら17
<ol>
	<li>滅菌生理食塩水の凍結乾燥粉末を再構成することによって、AngII を準備し、動物の重量とポンプの配信レートで濃度を調整します。プラスチック チューブ (ガラス管を使用していない) に再構成された AngII をしてください。</li>
	<li>7 日間の 1 mg/(kg·d) を提供する AngII ソリューションでポンプを入力します。調製後ポンプを維持、4-6 時間滅菌生理食塩水で最低 37 ° C で</li>
	<li>リア混ぜ麻酔の注射は足の反射と温かみのある操作フィールド上に配置後は、マウスの完全な麻酔をテストします。麻酔導入は 10-15 分かかります、暗い環境で動物を配置している場合、良い作品します。</li>
	<li>マウスの腰をそるし、アルコールの皮膚消毒と消毒します。</li>
	<li>はさみを使って背骨に垂直な 1 cm の切開を行います。海峡の止血を使用してポンプのためのポケットを作成し、ポンプを挿入 (まず司会の流れ)。ポケット傷口に圧力がないとポンプの自由な移動を許可する必要があります。</li>
	<li>炎症; を制限するために縫合糸、およびないクリップで傷を閉じる皮膚消毒剤で消毒します。</li>
	<li>Antisedan ミックスを 200 μ l 添加の皮下注射による麻酔の反感を買う、そのケージにマウスを戻ります。</li>
	<li>ブプレノルフィンによる術後鎮痛を行う (0.075 mg/kg 皮下) 動物の行動に応じてオンデマンド手術後 1 時間。</li>
</ol>
3. 頸静脈カテーテル注入と頸動脈の準備
<ol>
	<li>麻酔の注射と後部食品反射を混ぜて位麻酔動物背 recumbence 39 ° C 手術板上軟膏をつけ、温度を維持するために直腸プローブを持つ後マウスの完全な麻酔をテストします。プロシージャの間にそれらを保護するために目。</li>
	<li>首毛、脱毛クリームを使用してを削除します。普及し、毛が脱落する開始まで 2 分間クリームをこする綿棒を使用します。さらに 2 分後ヘラでクリームと毛を削除し、アルコールの皮膚消毒で皮膚を消毒します。</li>
	<li>顕微鏡下で手術を行います。頸部、気管の横に 1 cm の縦皮膚で 1 〜 1.5 cm の切開を行います。最初の切開の両方の下肢を垂直に他の 2 つの切開を行います。</li>
	<li>慎重に皮膚の横にある組織、左内頚静脈と気管を覆っている皮膚の部分を取り外します。</li>
	<li>慎重に分離; 耳下腺2 カーブタイプ鉗子と利子のゾーンから舌下腺と顎下腺。カットにまたは損傷腺、頸静脈、頸動脈の最高アクセスを許可するようにそれらを配置しないでください。</li>
	<li>頸静脈を分離するには、細い鉗子を使用して、ゆっくりと開き、クローズして周囲の組織から血管を優しく解放します。静脈が完全にきれいになったら容器の下に鉗子を配置し、それの下で 10 cm の 2 つの 7-0 縫合糸を配置します。</li>
	<li>2 つの結び目の頭部に近位の縫合を閉じます。その他の縫合の 1 つの結び目を準備が終了しません。</li>
	<li>37 ° C; 片手で鉗子を滅菌生理食塩水で満たされたカテーテル (0.61 mm 外径、内径 0.28 mm) を保持します。他の手で、薄いはさみや湾曲した 26 G 針で容器に小切開を確認します。その後、頚静脈にカテーテルを挿入し、2 回結び目を閉じます。完全な固定を確保するために、カテーテルの上頭の近位の縫合を閉じます。</li>
	<li>特定し頚動脈を準備するには、区域を薄いカーブタイプ鉗子を使用して気管を解剖します。</li>
	<li>頸動脈が周囲の組織から解放されたら、船から (それは頸動脈に隣接する白い線のように見える) 迷走神経を削除 (しかしそれをカットされません)。細い鉗子をクローズし、ゆっくりと優しく神経を動員して開くことができます。</li>
	<li>両方の動脈が完全にクリーンな最初の頚動脈下鉗子を置き、船下小黒 3 mm 幅 x 2.5 cm の長さのプラスチック片を配置します。伸び過ぎて血管が血流が容器ホルダーが配置される存在であることを確認する非常に重要です。容器ホルダーに 2 つ目の動脈を置きます。顕微鏡下で直接マウスを挿入しない場合は気管上腺を戻すし、37 ° C ゾーンの乾燥を避けるために滅菌生理食塩水を適用します。</li>
</ol>
4. ローリングと白血球の付着の評価する LysM + セル IVM の/
<ol>
	<li>アクリジン オレンジ 2 mg/mL 原液-20 ° C で保存からを準備し、それを 1 mL の注射器の滅菌生理食塩水 (最終濃度が 0.5 mg/mL) で 1:4 希釈します。</li></ol>注射器を光から保護し、マウスを一回あたり 200 μ L を使用します。<ol>
		<li>さまざまな条件をテスト: アクリジン オレンジ頸静脈カテーテル; (1) 注射((この状態で内頸静脈カテーテルが注入なし); 尾外側静脈に直接アクリジン オレンジの注入 2)(3) アクリジン オレンジ蛍光 LysMCre+IRG+を用いたマウス (ここは再び頸静脈カテーテルが注入なし) することがなく測定。</li>
	</ol>
	
	<li>カテーテルは、場所と 2 つの頸動脈が分離、顕微鏡下でマウスを置きます。長距離コンデンサーと 10 X を使用して高速ワイド フィールド蛍光顕微鏡を用いた測定を行う (NA 0.3) 分光器、ビームスプリッター、電荷結合素子カメラと水浸対物レンズです。画像の獲得およびリアルタイム イメージング システムの分析を実行します。</li>
	<li>血管内皮細胞表面で頸動脈の真ん中に対物レンズの焦点を合わせます。アクリジン オレンジの 50 μ L (0.5 mg/mL) (最初の注入用カテーテルのデッド ボリュームを考慮に入れなさい) をゆっくりと注入します。画像ごとに 120 ミリ秒の露光時間で 100 の画像を記録するソフトウェアを設定します。</li>
	<li>各ビデオの動脈上の異なる場所で内頚動脈 (左と右) あたりの 4 つのビデオを作る。蛍光信号が小さくなる場合は、アクリジン オレンジの別の 50 μ L を挿入します。</li>
	<li>すべてのビデオを記録した後頚部転位によって、動物を安楽死させます。</li>
	<li>毎秒 10 枚で動画の速度を設定して各ビデオの容器の中央に置かれた 200 μ x 250 μ m 四角形ビューでローリングと付着性細胞を定量化します。ローリングと付着性のセルを数える;付着性のセルは、セルを移動しない、または 10 の s ビデオ内血管内皮細胞からデタッチとして定義されます。定量化を簡素化し、赤い蛍光を用いたチャネルを削除し、ローリングと付着細胞をカウントする緑の蛍光性を使用します。</li>
	<li>アクリジン オレンジを用いた実験では、血管内皮細胞によって作られたバック グラウンドを除去するために蛍光性の手動によるしきい値を確認します。</li>

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著者がある何も開示するには

高血圧5の開発に関与する LysM+単球は以前示した。LysM+の免疫細胞がロールし、AngII 注入に応えて内皮層に準拠を示します。この発見は、LysMCre+IRG+を使用して得られたアクリジン オレンジ6,10の注入と白血球の可視化による生体内で高血圧症における免疫細胞の役割を探る私たちの以前の研究からマウス。したがって、内皮に付着した細胞の種類の識別は不可能だった。
IVM は血管の検査や、血管に直接細胞の生体内観察の非常に便利なツールが、高血圧症の炎症性のコンテキストで外科的に挿入されたカテーテルの助けを借りて、染料を注入することの影響は不明のまま。カテーテルとアクリジン オレンジの潜在的な効果を評価する射出我々 は IRG+マウス LysMCre+の利点を取った。AngII 注入増加 LysM+細胞を血管内皮細胞に圧延の。頸静脈へのカテーテルの挿入増幅効果とカテーテル インプラントなしでマウスと比較されたカテーテルと計測 AngII 注入マウスよりローリングと付着した白血球が検出されました。これはその注入のコンテキストにおける全身性炎症反応は創傷治癒のオーバーレイされる高血圧18に見られる免疫反応を頸動脈カテーテル原因のことを示します。また、頸動脈、頸静脈の近接のため頚静脈に影響を与える追加免疫活性化を発生している可能性があります。この効果が直接に尾静脈注射が行われたとき存在していないので効果が期限であると仮定ことができます染料ではなく、カテーテルの挿入のプロシージャに。我々 は実験中に生体内のプロセスに干渉されていない蛍光蛋白質を表現する遺伝子改変レポーター マウスの重要性を示します。さらに、蛍光トレーサーの尾静脈注射頸静脈カテーテル注射に可能な選択肢があります。
プロシージャの 1 つの重要なステップは、AngII 注入です。ポンプによって血圧の尾カフ評価 AngII 配信の有効性を制御するために可能です。血圧は、注入の 2−3 日後増やす必要があります。炎症 LysM+細胞活性化に影響を及ぼす可能性を制限するには、ポンプが注入されて切開の閉鎖は、クリップではなく縫合すべきであります。1 つの制限は、尾静脈注射に関する注意されなければならない: 最高の可能なデータ収集をするためには、4 つのビデオ、通常各頸動脈の撮影します。蛍光信号が小さくなる、アクリジン オレンジの 50 μ L を注入が、尾の注入といくつかの注射をして、頸動脈を対物レンズの下で安定させるためにより困難です。頸静脈カテーテルの存在の期間かもしれない免疫細胞活性化を調節するもそれがカテーテル注入19後 4 h を作製した多血小板血漿の凝固活性化に影響を与えるので。カテーテル注入のこの側面はさらに調べる必要があります。
最後に、頸静脈カテーテル注入 AngII 注入後 LysM+細胞の活性化の影響を感謝して、たとえ完全には推定不可能で準備、皮膚の除去、および潜在的な LysM+セルに頸動脈の分離の役割活性化。ような蛍光レポーター マウスの使用を終えて LysMCre+IRG+マウス生体内イメージングのため動物の準備時に容器の完全性の混乱を避けるためにお勧めします。

動脈高血圧症は、心血管疾患と死亡のリスクを増加させるし、アテローム性動脈硬化、冠動脈疾患、動脈または静脈の最も1の開発を促進します。高血圧の開発は、環境、遺伝、内分泌、および血行力学的要因の相互作用に依存します。現在、高血圧2の病因に重要な役割を果たすには免疫と関連炎症も歓迎です。
T リンパ球、単球、マクロファージは免疫細胞の中で AngII 誘発血管炎症と高血圧、活性酸素種3をトリガーする能力に関連する部分で作因的に関与する見つかりました。大食細胞のコロニー刺激因子欠損のマウスは、血圧上昇や血管炎症4に関する AngII に還元反応を示した。前作、AngII 血圧5LysM + 単球の血管機能不全や炎症ドライブを見せてください。最近では、血小板とトロンビン依存性血管炎症6を誘発する血管壁が凝固第 XI 因子が協力して経路について述べる。高血圧症における免疫系の役割について現在のナレッジ最近要約されロドリゲス Iturbeらによって審査します。7
高血圧の開発に免疫細胞の関与が明らかになったのでモデルと容器と免疫細胞の相互作用を研究する手法が必要となった。血管の落射蛍光 IVM は循環血液細胞と内皮細胞8,9,10の生体内での相互作用を観察する役に立つツールです。この手法により、dna (などアクリジン オレンジ) 間をインターカ レート染料の注入は、有核細胞 (循環と同様、内皮から) を視覚化できます。分離された血小板が前のヴィヴォローダミン 6 G のステンド グラスやジクロルフルオレッセン (DCF) を注入して、動脈または静脈の損傷モデルにおける血小板血栓を視覚化することができます。
頸静脈カテーテルのトレーサーを注入するために使用またはマークの通常、血小板。単球の活性化に効果があると、内皮の変化と凝固カスケードの後続活性化両方知られています。血管内皮細胞障害はすぐにその後単球の魅力と活性化11組織をシールする内皮下マトリックス分子を介して血小板の活性化に します。それどころか、そのまま血管内皮細胞が抗凝固特性 (例えば、組織因子経路阻害剤またはトロンボモジュリン経由)12と単球などの分泌を直接抑制効果を持っている知られています。マイクロ Rna の13を含む細胞小胞。単球は組織因子、凝固カスケードのエクスプレス プロテアーゼ活性化受容体 (PARs) トロンビンによってアクティブにすることができ、単球活性化14,15 に参加する外因性活性剤を生成する知られています。.したがって、任意の活性化血小板または血管損傷による血液凝固カスケードの可能性があります予期しない単球活性化に対する影響とみられる現象を妨げる。IRG トランスジェニック LysM Cre トランスジェニック マウス、二重蛍光 Cre レポーター マウス (LysMCre+IRG+) の助けを借りて、提案する LysM+のカテーテルでの代替方法で注射の効果の詳細に検討するには動脈高血圧症16マウス モデルにおける骨髄単球性細胞。

<table>
	<tbody>
		<tr>
			<td>Midazolam</td>
			<td>Ratiopharm GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Pfizer Deutschland GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Fentanyl</td>
			<td>Janssen-Cilag GmbH</td>
			<td></td>
			<td>Anesthesia mix</td>
		</tr>
		<tr>
			<td>Alzane</td>
			<td>Zoetis</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Flumazenil</td>
			<td>Hikma pharma</td>
			<td></td>
			<td>Antisedan mix</td>
		</tr>
		<tr>
			<td>Braunol</td>
			<td>B Braun</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Octeniderm</td>
			<td>Sch&#252;lke</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Osmotic pumps</td>
			<td>Alzet</td>
			<td>1007D</td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Angiotensin II</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>7-0 prolene suture</td>
			<td>Ethicon</td>
			<td></td>
			<td>Osmotic pump implantation</td>
		</tr>
		<tr>
			<td>Acridine orange</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Smiths Medical Deutschland GmbH</td>
			<td></td>
			<td>Inside diameter, 0.28 mm; outer diameter, 0.61 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>BX51WI fluorescence microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Beam Splitter</td>
			<td>Photometrics</td>
			<td>CMR-DV2-SYS - DualView2 MicroImager</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Olympus</td>
			<td>UMPLFLN10X</td>
			<td>Water immersion objective with a monochromator</td>
		</tr>
		<tr>
			<td>Camera</td>
			<td>Hamamatsu Photonics</td>
			<td>ORCA-R2</td>
			<td></td>
		</tr>
		<tr>
			<td>Image acquisition and analysis software</td>
			<td>Olympus</td>
			<td>Realtime Imaging System eXcellence RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 008705</td>
			<td>B6.Cg-Tg(CAG-DsRed,-EGFP)5Gae/J</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>The Jackson Laboratory</td>
			<td>Jax 004781</td>
			<td>LysM Cre (B6.129P2-Lyz2tm1(cre)Ifo/J )</td>
		</tr>
	</tbody>
</table>

visualizing leukocyte rolling and adhesion in angiotensin ii-infused mice: techniques and pitfalls

落射蛍光生体ビデオ顕微鏡 (IVM) 血管は、免疫細胞の活性化と役割する能力を評価し、内皮層に付着する確立された方法です。循環し、蛍光色素や蛍光体結合抗体の注射によって細胞の可視化、使用されます。また、蛍光レポーター マウスを使用できます。特定リゾチーム M における白血球の相互作用+ (LysM+) 単球は、血管壁に血管機能不全や動脈高血圧を促進する上で極めて重要な役割を再生します。ここで視覚化し、白血球ローリングとアンジオテンシン II (AngII) 頚動脈で密着性を定量化する手法を提案する-マウス IVM の血圧。
カテーテルの注入は、血管壁を損傷、変更された血液細胞の反応に 。別の射出成形技術と LysMCre+IRG+で白血球を視覚化する投与経路を比較したマウスの赤色けい光たんぱく質の広範な表現とする LysM で緑の蛍光蛋白質の条件式+細胞。AngII 注入マウスを用いて LysM+細胞の活性化を研究、圧延および内皮に白血球の付着が増加します。我々 はどちらかアクリジン オレンジ、頚を使用してカテーテルを注入または直接尾静脈し、の量を比較しても圧延付着細胞と。わかった頸静脈カテーテル注入自体圧延の数が増加に付着する LysM+細胞偽注入 LysMCre+IRG+マウス コントロールと比較して.この活性化は、AngII 注入マウスの拡張だった。興味深いことに、アクリジン オレンジ直接尾静脈増加しなかった LysM+細胞接着を注入または偽注入マウスでローリングします。それによって、実験中に生体内のプロセスに干渉されていない蛍光蛋白質を表現する遺伝子改変レポーター マウスの重要性を示しました。さらに、蛍光トレーサーの尾静脈注射頸静脈カテーテル注射に可能な選択肢があります。

頸動脈 LysMCre+IRG+の AngII で注入されたマウスは、IVM を用いて観察しました。アクリジン オレンジは、頸静脈カテーテルを用いた注入しました。(これは容器との相互作用細胞型の差別に開いている残ったので船との対話可能性がありますもすべての有核循環細胞と比較して血管壁との接触する LysM+細胞の割合を見てを目指しました私たちの以前の作品からの質問)。ベースラインで頸静脈カテーテルの存在する LysM+細胞 (図 2 aD) の付着が発生します。同じ細胞が蛍光、アクリジン オレンジ注射後がまた内皮細胞が蛍光 (図 2 b, E)。内皮を制限するバック グラウンドの低減は、蛍光関連後、データ示す核付着細胞はすべている LysM+ (図 2f)。私たちの以前の結果を確認して、LysMCre+IRG+が AngII の LysM+細胞の活性化に及ぼす影響を観察するためのよいモデルであることを示す AngII 注入後は、これらの結果が当てはまります。
アクリジン オレンジ LysMCre+IRG+で AngII 注入後 LysM+細胞の活性化での投与経路の役割を評価しました。AngII 輸液、白血球の血管内皮細胞の相互作用の 1 週間後 LysMCre+IRG+の頚動脈でマウスがイメージし、IVM アクリジン オレンジ インジェクション尾静脈、頸静脈カテーテルを介しての有無で可視化しました。カテーテルや注射、なし LysM+細胞の圧延有意に増加した後 AngII 注入未処理マウスと接着に比べて増加して (図 3)。アクリジン オレンジ増加頸静脈カテーテルの接着と AngII で圧延の注入は、カテーテル (図 3) なしのマウスと比較して大きい程度にマウスを扱われます。アクリジン オレンジの尾静脈内の注入とアクリジン オレンジ (図 3) の注入を受信しなかったマウスと比較する転がりと同様の接着になります。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56948/56948fig1.jpg"> 
図 1。アンジオテンシン II 注入する LysM+の評価方式セル圧延とマウスで接着。LysMCre+IRG+マウス 7 日わいて AngII とアクリジン オレンジの尾静脈、頸静脈カテーテルを通じて注入の有無 LysM+をローリングし同様に付着、頸動脈の細胞の定量を行ったインジェクション。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56948/56948fig2.jpg"> 
図 2。LysM+細胞接着、圧延 LysMCre+の頚動脈の IRG+マウスの査定。LysM 接着+細胞 LysMCre+の IRG+マウス (A, D) 前に、頸静脈カテーテル (B, E) アクリジン注入後。赤と緑の蛍光を記録した、(緑) の LysM+細胞と平滑筋細胞 (赤) で表示されています。アクリジン オレンジの注入後、内皮細胞でも緑色で表示されますが、背景の蛍光性を削除することによりセルの視覚化 (C, F) を核だけを循環します。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56948/56948fig3.jpg"> 
図 3。アンジオテンシン II の蛍光色素の投与経路の評価は、IRG+マウス LysMCre+を注入しました。ローリング (A) と (B) LysM+を付着の定量細胞偽運営または AngII 注入動物と頸静脈カテーテルを用いたアクリジン オレンジの尾静脈注射による注入の有無。さまざまな条件 (C) の代表的な写真。結果は、平均の平均 ± 標準誤差です。2 ウェイ ANOVA を行った、ボンフェローニのホックを投稿テスト、n = 3-12/グループ;p &#60; 0.05。<a href="//ecsource.jove.com/files/ftp_upload/56948/56948fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls at JoVE.com

Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

この原稿は、トランスジェニック レポーター マウスの使用は、アンジオテンシン II による高血圧は血管の生体顕微鏡を使用して免疫細胞の活性化を評価する蛍光色素の異なる投与経路について説明しますと、ロールし、内皮に付着する機能。

白血球ローリングとアンジオテンシン II に注入されたマウスにおける付着の可視化: 技術と落とし穴

Epifluorescence intravital video microscopy (IVM) of blood vessels is an established method to evaluate the activation of immune cells and their ability ...

visualizing-leukocyte-rolling-adhesion-angiotensin-ii-infused-mice

Research

JoVE Journal

Immunology and Infection

8.6K ビュー.  University Medical Center Mainz. この原稿は、トランスジェニック レポーター マウスの使用は、アンジオテンシン II による高血圧は血管の生体顕微鏡を使用して免疫細胞の活性化を評価する蛍光色素の異なる投与経路について説明しますと、ロールし、内皮に付着する機能。

ビデオ: Visualizing Leukocyte Rolling and Adhesion in Angiotensin II-Infused Mice: Techniques and Pitfalls

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

白血球ホーミングと移行の非侵襲的イメージング in vivoで</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

免疫・感染学

Alphavirus Transducing System:  Tools for Visualizing Infection in Mosquito Vectors

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection. ATSs are derived from cDNA clones of mosquito-borne RNA viruses (genus Alphavirus; family Togaviridae). The Alphavirus genus contains about 30 different mosquito-borne virus species. Alphaviruses are enveloped viruses and contain single-stranded RNA genomes (~11.7 Kb). Alphaviruses transcribe a subgenomic mRNA that encodes the structural proteins of the virus required for encapsidation of the genome and maturation of the virus. Alphaviruses are usually highly lytic in vertebrate cells, but persistently infect susceptible mosquito cells with minimal cytopathology. These attributes make them excellent tools for gene expression in mosquito vectors. The most common ATSs in use are derived from Sindbis virus (SINV). The broad species tropism of SINV allows for infection of insect, avian, and mammalian cells8. However, ATSs have been derived from other alphaviruses as well9,10,20. Foreign gene expression is made possible by the insertion of an additional viral subgenomic RNA initiation site or promoter. ATSs in which an exogenous gene sequence is positioned 5' to the viral structural genes is used for stable protein expression in insects. ATSs, in which a gene sequence is positioned 3' to the structural genes, is used to trigger RNAi and silence expression of that gene in the insect.
ATSs have proven to be valuable tools for understanding vector-pathogen interactions, molecular details of viral replication and maintenance infectious cycles3,4,11,19,21. In particular, the expression of fluorescent and bioluminescent reporters has been instrumental tracking the viral infection in the vector and virus transmission5,14-16,18. Additionally, the vector immune response has been described using two strains of SINV engineered to express GFP2,9.
Here, we present a method for the production of SINV containing a fluorescent reporter (GFP) from the cDNA infectious clone. Infectious, full-length RNA is transcribed from the linearized cDNA clone. Infectious RNA is introduced into permissive target cells by electroporation. Transfected cells generate infectious virus particles expressing the GOI. Harvested virus is used to infect mosquitoes, as described here, or other host species (not shown herein). Vector competence is assessed by detecting fluorescence outside the midgut or by monitoring virus transmission7. Use of a fluorescent reporter as the GOI allows for convenient estimation of virus spread throughout a cell culture, for determination of rate of infection, dissemination in exposed mosquitoes, virus transmission from the mosquito and provides a rapid gauge of vector competence.

Methods for using alphavirus transducing systems to express fluorescent reporters in vitro and in adult mosquitoes are described. This technique may be adapted to express any protein of interest in lieu of or in addition to a reporter.

アルファウイルス形質導入システム：蚊で感染を可視化するためのツール

Alphavirus transducing systems (ATSs) are important tools for expressing genes of interest (GOI) during infection.  ATSs are derived from cDNA clones of ...

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host cells1. These adhesins rely on interactions with host cell surface receptors or soluble proteins acting as a bridge between bacteria and host. Adhesion is a critical first step prior to invasion and/or secretion of toxins, thus it is a key event to be studied in bacterial pathogenesis. Furthermore, adhered bacteria often induce exquisitely fine-tuned cellular responses, the studies of which have given birth to the field of 'cellular microbiology'2. Robust assays for bacterial adhesion on host cells and their invasion therefore play key roles in bacterial pathogenesis studies and have long been used in many pioneer laboratories3,4. These assays are now practiced by most laboratories working on bacterial pathogenesis.
Here, we describe a standard adherence assay illustrating the contribution of a specific adhesin. We use the Escherichia coli strain 27875, a human pathogenic strain expressing the autotransporter Adhesin Involved in Diffuse Adherence (AIDA). As a control, we use a mutant strain lacking the aidA gene, 2787&Delta;aidA (F. Berthiaume and M. Mourez, unpublished), and a commercial laboratory strain of E. coli, C600 (New England Biolabs). The bacteria are left to adhere to the cells from the commonly used HEp-2 human epithelial cell line. This assay has been less extensively described before6.

In Vitro Assay of Bacterial Adhesion onto Mammalian Epithelial Cells

This protocol is a simple bacterial adhesion assay consisting in counting the numbers of bacterial colony forming units that are adhered onto cultured cells. The assay is robust, independent of the adhesin studied, and numerous variations are used in most laboratories working on bacterial pathogenesis.

哺乳類上皮細胞上に細菌付着のインビトロアッセイ

To cause infections, bacteria must colonize their host. Bacterial pathogens express various molecules or structures able to promote attachment to host ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

のAlexa Fluor ®標識を通じてデング熱ウイルスの可視化

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

This is a method to visualise leukocyte adhesion to the endothelium in harvested pressurised vessels. The technique enables studying vascular adhesion under shear flow with differing intraluminal pressures up to 200 mmHg thus mimic-ing the pathophysiological conditions of high blood pressure.

高腔内圧で血管内皮へのイメージングの白血球接着

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

細菌付着の研究ではせん断応力を導入する

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

Quantitative In vitro Assay to Measure Neutrophil Adhesion to Activated Primary Human Microvascular Endothelial Cells under Static Conditions

Neutrophil adherence to the activated endothelium at sites of infection is an integral component of the host's inflammatory response. Described in this report is a neutrophil binding assay that allows for the in vitro quantitation of primary human neutrophil binding to endothelial cells activated by inflammatory mediators under static conditions.

定量的な<em&gt;インビトロ</em静的な条件の下で活性化された初代ヒト微小血管内皮細胞への好中球の接着を測定するために&gt;アッセイ

The vascular  endothelium plays an integral part in the inflammatory response. During the  acute phase of inflammation, endothelial cells (ECs) are ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

マウスの腸間膜静脈内の生体顕微鏡白血球 - 内皮及び血小板 - 白血球の相互作用

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Tリンパ球接着分子の相互作用の研究のための接着性の下でせん断応力の測定

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

Fast and Specific Assessment of the Halogenating Peroxidase Activity in Leukocyte-enriched Blood Samples

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on the hypotonic lysis of erythrocytes and can be applied to human samples as well as to blood of non-human origin. The small initial sample volume of about 50 to 100 &#181;l makes this method applicable to recurrent blood sampling from small laboratory animals. Moreover, leukocyte enrichment is achieved within minutes and with low material efforts regarding chemicals and instrumentation, making this method applicable in multiple laboratory environments.
Standardized purification of leukocytes is combined with a highly selective staining method to evaluate halogenating peroxidase activity of the heme peroxidases, myeloperoxidase (MPO) and eosinophil peroxidase (EPO), i.e., the formation of hypochlorous and hypobromous acid (HOCl and HOBr). While MPO is strongly expressed in neutrophils, the most abundant immune cell type in human blood as well as in monocytes, the related enzyme EPO is exclusively expressed in eosinophils. The halogenating activity of these enzymes is addressed by using the almost HOCl- and HOBr-specific dye aminophenyl fluorescein (APF) and the primary peroxidase substrate hydrogen peroxide. Upon subsequent flow cytometry analysis all peroxidase-positive cells (neutrophils, monocytes, eosinophils) are distinguishable and their halogenating peroxidase activity can be quantified. Since APF staining may be combined with the application of cell surface markers, this protocol can be extended to specifically address leukocyte sub-fractions. The method is applicable to detect HOCl and HOBr production both in human and in rodent leukocytes.
Given the widely and diversely discussed immunological role of these enzymatic products in chronic inflammatory diseases, this protocol may contribute to a better understanding of the immunological relevance of leukocyte-derived heme peroxidases.

This protocol describes the quick enrichment of leukocytes from small blood samples for a subsequent specific determination of the halogenating peroxidase activity within the cells. The method can be applied to human and non-human material and may contribute to the evaluation of new inflammatory markers.

白血球に富んだ血液サンプル中のハロゲンペルオキシダーゼ活性の迅速かつ具体的な評価

In this paper a protocol for the quick and standardized enrichment of leukocytes from small whole blood samples is described. This procedure is based on ...