Name: an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks
Uploaded: 2017-02-09T14:00:00.000+00:00
Duration: 6 min 54 s
Description: Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

This work was supported by National Institutes of Health Grant 5-P20GM103629 to WLM and the Tulane Center for Aging. We would like to thank Matthew Nice for his help with editing the protocol text.

全ての動物実験および手順は、チューレーン大学の施設内動物管理使用委員会（IACUC）によって承認されました。 1.外科プロシージャを設定<ol><li>手術前にオートクレーブ機器、手術用消耗品、および文化用品。各ラットに対する外科用品が含まれます：1ドレープ、プレカット穴に1ドレープを（0.5インチ×1.5で）中央に、ガーゼパッド、および1吸収性アンダーパッド。手術器具が含まれます：数10ブレードで1メス、ピンセットの2ペア、および微細なハサミを。文化用品が含まれます：1ドレープ、ピンセットの1組、およびポリカーボネートフィルターで6ウェルプレートのインサートを用意しています。 </li><li>プレキシガラスのプラットフォーム、外科的なステージと70％エタノールで手術用卓上スペースを滅菌します。使用するまで無菌ボウルに外科的ステージを保管してください。 <ol><li> 100ミリメートル培養皿の中央に穴中1により約2で穿孔することによって、外科的ステージを作成します。次に、滑らかにするためにサンドペーパーを使用任意のシャープなエッジとは、組織のための隆起表面を作成するために、穴のエッジにシリコーン接着剤の層を追加します。 </li><li>代替的に、CADソフトウェアを使用して外科的ステージを設計し、3-D印刷（ 図1）によって行います。 </li></ol></li><li>ダウン滅菌吸収性アンダーパッドを配置し、その上にプレキシガラスのプラットフォームを築きます。吸収性アンダーパッドの隣に加熱パッド上で、プレカット穴なしで、ドレープを配置します。 </li><li>前加温滅菌リン酸緩衝生理食塩水（PBS）、37°C​​にメディアおよび生理食塩水。独立した培養皿に入れメディアとPBS加熱パッドの上、手術のセットアップの隣に50 mLのコニカルチューブに生理食塩水を配置します。 </li><li>すべてのパッケージは、すべての材料の滅菌処理を確実にするために、手術の開始に先立って開かれていることを確認します。この手順で使用する一般的なツールの完全なリストは、に記載されています 具体的な外科材料とツールの表 。 </li></ol> 2. MesenterY組織の収穫<ol><li>使用大人の雄Wistarラット（350±25グラム; 6 - 8週齢）。他の株およびラットの年齢を置換することができます。 </li><li>ケタミン（80 mg / kg体重）およびキシラジン（8 mg / kg体重）の筋肉内注射によりラットを麻酔。ラットが反射反応を確認するために足の指の間につまんで麻酔下であることを確認。どれがあってはなりません。このターミナル手続きのための先制鎮痛は必要ありません。 </li><li>腹部を剃ると脱毛クリームを使用して残りの毛を取り除きます。ポビドンヨードに続く70％イソプロピルアルコールで二回腹部の皮膚を拭いてください。ワイプのために、外科医は、手術部位の中心に開始すべきであると以前に滅菌ガーゼまたは滅菌綿棒の同じ部分でスクラビングされた領域を重ならないように円形に準備されたエリアの外に移動します。その後、無菌手術用セットアップに動物を転送し、プレキシガラスプラットの上に置きます形。 </li><li>胸骨下の1を開始し、腸の切開で1.25 - メスの刃を使用して、0.75を作ります。腸や腸間膜（皮膚の1層、結合組織の1層、および筋肉の1層）を穿刺しないように注意してください。 </li><li>切開オーバープレカット穴とドレープを配置し、ドレープの上に無菌手術用のステージを配置します。開口部は切開部と整列することを確認してください。外科的ステージ開口部を通って回腸を見つけて引き出しを滅菌綿棒アプリケータを使用してください。 </li><li>綿棒アプリケーターを使用してステージを8腸間膜Windows、およびWindows（ 図1）には触れないように気をつけて- 6を引き出します。組織は、通常、盲腸の近く始まる小腸の回腸の領域から採取されます。ソリューションを滴下し、無菌注射器を使用して、必要に応じて加温した滅菌生理食塩水で湿った露出された組織を保管してください。 </li><li>ペントバルビタールナトリウム（ラット当たり0.2 mL）を心臓内注射を介してラットを安楽死させます。私を削除する前にsenteric窓は、ラットが心を触診によって安楽死させていることを確認します。何のパルスがあってはなりません。 </li><li>ウィンドウをカットする脂肪パッドと細かいハサミをつかむためにピンセットを使用することによって、所望の腸間膜組織を削除します。窓周りの脂肪（2ミリメートル）の境界線を残します。メディアで一度温めた滅菌PBSで一度組織を洗浄し。 </li><li>腹腔に体外回腸を戻し、施設のガイドラインに従って動物を処分。 </li></ol>タイムラプス研究3.腸間膜組織培養<ol><li>無菌層流フードへの転送オートクレーブ処理培養用品（1.1節を参照）や組織。 </li><li>ポリカーボネートフィルター膜の上に、各組織を転送するためにピンセットを使用してください。血管系の損傷を防ぐために、脂肪パッドによって組織をつかみます。 </li><li>迅速ウィンドウに触れないように注意しながら、脂肪パッドを使用して、組織を広げます。 6ウェルプレートの底部に組織とインサートを反転し、メディアの3ミリリットル（ 図1でカバー</st栄&gt;）。この手順のために使用される典型的な培地は、1％のペニシリンストレプトマイシン（PenStrep）及び10％ウシ胎児血清（FBS）を有する最小必須培地（MEM）を含みます。メディアは、血管新生を刺激する他の血清および/または成長因子を補充することができます。 </li><li>までの5日間、標準的なインキュベーター条件の各組織と文化のための3.3（5％CO 2、37℃） -繰り返して、3.2を繰り返します。 </li></ol>腸間膜組織の4タイムラプスイメージング<ol><li>イメージングの日に、共役BSIレクチンで各ウェルに培地を補充し、30分間の標準的な培養条件下でインキュベートします。レクチンを含まない培地で二回組織を洗ってください。 BSI-レクチン染色は、培養中の最大3日間腸間膜組織に表示されたままになります。 </li><li>顕微鏡ステージにプレートを転送します。その形態やネットワーク構造に基づいて、血液やリンパ管を識別します。 </li><li>各組織上の所望のネットワーク領域を見つけて、画像を撮影します。イメージングのメモを取ります同じ領域を確保するための場所が、後続のイメージのためにキャプチャされます。電動ステージを使用している場合、座標を文書化します。 </li><li>インキュベーターに組織を戻し、所望の最終地点まで培養を続けます。希望の実験の時点に応じて、必要に応じて4.3 - 繰り返し、4.1を繰り返します。 </li></ol> 5.組織免疫標識<ol><li> BSI-レクチンラベリング <ol><li>メディアを有する2つのすすぎに続いて培地中1：40 FITC結合レクチン（6ウェルプレート中のウェルあたり2.5 mLの抗体溶液）を用いて37℃で30分間組織をインキュベートします。リンスの場合は、メディアを追加した後、すぐに交換してください。 </li></ol></li><li> ライブ/デッドラベリング <ol><li> 1 37℃で10分間組織をインキュベート500 2mMのエチジウムホモダイマー1及び1：メディアを有する2つのすすぎに続いて、メディア500、1mMのカルセインAM（6ウェルプレート中のウェルあたり2.5 mLの抗体溶液）。 </li></ol></li><li> BSI-レクチン/ NG2ラベリング <ol><li>スライドし、顕微鏡での組織を広げますE（1から2の組織/スライド）と乾燥させます。脂肪を切除するためにしっかりと下に押して、メスで余分な脂肪を取り除きます。 </li><li> -20℃で30分間、冷メタノールで組織を修正しました。 PBS（3×10分）で組織を洗います。 </li><li> 100ウサギポリクローナルNG2抗体および5％正常ヤギ血清（NGS）：一次抗体の標識のために1を用いて室温で1時間、組織をインキュベートします。 PBS（3×10分）で組織を洗います。 </li><li> 100ヤギ抗ウサギCY2結合抗体（GAR-CY2）および5％NGS：二次抗体の標識のために1を用いて室温で1時間、組織をインキュベートします。 PBS（3×10分）で組織を洗います。 </li><li> PBSで2回リンスに続いて、PBS中の午前1時40分FITC結合レクチンを用いて室温で30分間、組織をインキュベートします。リンスのために、PBSを追加し、すぐに交換してください。 </li><li>スライドをマウントするには、上部の50:50 PBSで組織およびグリセロール溶液と場所のカバースリップをカバーしています。マニキュアを使用して、スライドの端をシールします。 </li></ol></li><li> LYVE-1 / PECAMラベリング<ol><li>顕微鏡スライド上の組織を広げて（1から2の組織/スライド）と乾燥させます。脂肪を切除するためにしっかりと下に押して、メスで余分な脂肪を取り除きます。 </li><li> -20℃で30分間、冷メタノールで組織を修正しました。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li> 200マウスモノクローナルビオチン化CD31抗体と1：一次抗体の標識のために1ながら室温で1時間、組織をインキュベート100ウサギポリクローナルLYVE-1抗体をPBS + 0.1％サポニン+ 2％ウシ血清アルブミン（BSA）+ 5％NGS 。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li>二次抗体の標識のために、1を用いて室温で1時間、組織をインキュベート：PBS + 0.1％サポニン+ 2％BSA + 5％NGSで100 GAR-CY2：500 Cy3結合ストレプトアビジン抗体および1。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li> 50:50 PBSとグリセロール溶液でスライド、カバー組織をマウントして、上にカバースリップを配置します。マニキュアを使用して、スライドの端をシールします。 </li></ol></li><li> BrdU / BSI-レクチンラベリング <ol><li>メディアへの1mg / mLのBrdUのを追加し、BrdUの溶液で組織メディアを交換。 37℃で2時間インキュベートします。 </li><li>顕微鏡スライド上の組織を広げて（1から2の組織/スライド）と乾燥させます。脂肪を切除するためにしっかりと下に押して、メスで余分な脂肪を取り除きます。 </li><li> -20℃で30分間、冷メタノールで組織を修正しました。 PBS（3×10分）で組織を洗います。 </li><li> 37℃で1時間、2 M HCl中の組織DNAを変性させます。 PBS + 0.1％サポニン（3×10分）で組織を洗います。 </li><li> PBS + 0.1％サポニン+ 2％BSA + 5％NGSで100モノクローナルマウス抗BrdU：一次抗体の標識のために、1を用いて室温で1時間、組織をインキュベートします。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li>二次抗体標識のために、1を室温で1時間、組織をインキュベート100ヤギ抗マウスCy3結合抗体をPBS + 0.1％サポニン+ 2％BSA + 5％NGS中（GAM-のCy3）を。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li&gt;をPBSで2回リンスに続いてPBSで1：40 FITC結合レクチンを用いて室温で30分間、組織をインキュベートします。 </li><li>スライドをマウントするには、上部の50:50 PBSとグリセロールソリューションと場所カバースリップを有する組織をカバーしています。マニキュアを使用して、スライドの端をシールします。 </li></ol></li><li> BSI-レクチン/ CD11bのラベリング <ol><li>顕微鏡スライド上の組織を広げて（1から2の組織/スライド）と乾燥させます。脂肪を切除するためにしっかりと下に押して、メスで余分な脂肪を取り除きます。 </li><li> -20℃で30分間、冷メタノールで組織を修正しました。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li> PBSで100マウス抗ラットのCD11b + 0.1％サポニン+ 2％BSA + 5％NGS：一次抗体の標識のために1を用いて室温で1時間、組織をインキュベートします。洗浄は、PBS + 0.1％サポニン（3×10分）を有する組織します。 </li><li>二次抗体の標識のために1を用いて室温で1時間、組織をインキュベート：PBS + 0.1％サポニン+ 2％BSA + 5％NGSで100 GAM-のCy3。 PBSで組織を洗います+ 0.1％サポニン（3×10分）。 </li><li> PBSで2回リンスに続いて、PBS中の午前1時40分FITC結合レクチンを用いて室温で30分間、組織をインキュベートします。 </li><li>スライドをマウントするには、上部の50:50 PBSとグリセロールソリューションと場所カバースリップを有する組織をカバーしています。マニキュアを使用して、スライドの端をシールします。 </li></ol></li></ol>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+mechanical+stresses+in+microvascular+remodeling.">The role of mechanical stresses in microvascular remodeling.</a> Microcirculation. 3 (2), 143-165 (1996).</li><li>Kadohama, T., Nishimura, K., Hoshino, Y., Sasajima, T., Sumpio, B. E. <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+different+types+of+fluid+shear+stress+on+endothelial+cell+proliferation+and+survival.">Effects of different types of fluid shear stress on endothelial cell proliferation and survival.</a> J Cell Physiol. 212 (1), 244-251 (2007).</li><li>Milkiewicz, M., Brown, M. D., Egginton, S., Hudlicka, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+shear+stress,+angiogenesis,+and+VEGF+in+skeletal+muscles+in+vivo.">Association between shear stress, angiogenesis, and VEGF in skeletal muscles in vivo.</a> Microcirculation. 8 (4), 229-241 (2001).</li><li>Corliss, B. A., Azimi, M. S., Munson, J. M., Peirce, S. M., Murfee, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages:+An+Inflammatory+Link+Between+Angiogenesis+and+Lymphangiogenesis.">Macrophages: An Inflammatory Link Between Angiogenesis and Lymphangiogenesis.</a> Microcirculation. 23 (2), 95-121 (2016).</li></ol>

The authors declare that they have no competing financial interests.

このプロトコルは、微小血管ネットワークの成長のタイムラプスイメージングのためのex vivoでのツールとしてラットの腸間膜培養モデルを使用するための方法を提示します。我々の研究室での以前の研究は、1のための我々のモデル）、血管新生8、2）リンパ管8、3）周皮細胞-内皮細胞の相互作用8、および4）抗血管新生薬物検査<sup...

微小血管ネットワークの成長とリモデリングは、組織機能のための共通の分母、創傷治癒、および複数の病状であり、重要なプロセスは、既存の1、2から新たな血管の成長として定義され、血管形成です。組織は新しい血管を設計するか、血管新生に基づく治療を設計するため、血管新生に関与する細胞のダイナミクスの重要性を理解することが重要です。しかしながら、この方法は複雑です。これは、微小血管ネットワーク内の特定の位置で変化し、複数の細胞型（ 例えば 、内皮細胞、平滑筋細胞、周皮細胞、マクロファージ、幹細胞）と、複数のシステム（リンパ管ネットワーク及びニューラルネットワーク）を含むことができます。 in vitroモデルは、血管新生3に関与する異なるセル間の関係を調べることに大いに貢献してきたが、それらの生理学的関連性はtheiのために弱体化することができます<html>限られた複雑さと、彼らは密接にin vivoでのシナリオを反映していないという事実rを。これらの制限、三次元培養系3、 エクスビボ組織モデル4を克服するために、マイクロ流体システム5、6、および計算モデル7は、近年開発され導入されています。しかし、無傷の微小血管ネットワークエクスビボでの血管形成を調査するタイムラプス機能を持つモデルの必要性が依然として存在します。複雑さのレベルの血管新生研究のための新たなタイムラプスモデルの確立は、血管新生を調節する根底にあるメカニズムを理解し、治療法を改善するための貴重なツールを提供します。 無傷の微小血管ネットワークを介して血管新生のex vivoでの調査を可能にする潜在的なモデルは、ラット腸間膜培養モデルであります&gt; 8。最近の研究では、血液とリンパ微小血管ネットワークは培養後に生存し続けることが実証されています。さらに重要なことは、ラット腸間膜培養モデルは、機能的な周皮細胞 - 内皮細胞の相互作用、血液およびリンパ管内皮細胞コネクション、タイムラプス撮影を調査するために使用することができます。本稿の目的は、タイムラプスイメージング法のために私たちのプロトコルを提供することです。当社の代表的な結果は、血清と血管新生の刺激後に生き続けると組織特異的血管新生反応と同様に内皮細胞追跡研究を定量化するため、このメソッドの使用例を提供する複数の細胞型を文書化します。

<table><tbody><tr><td>Drape</td><td>Cardinal Health</td><td>4012</td><td>12&amp;rdquo; x 12&amp;rdquo; Bio-Shield Regular Sterilization Wraps</td></tr><tr><td>Scalpel Handle</td><td>Roboz Surgical Instrument</td><td>RS-9843</td><td>Scalpel Handle, #3; Solid; 4&quot; Length</td></tr><tr><td>Sterile Surgical Blade</td><td>Cincinnati Surgical</td><td>0110</td><td>Stainless Steel; Size 10</td></tr><tr><td>Culture Dish (60 mm)</td><td>Thermo Scientific</td><td>130181</td><td>10/Sleeve</td></tr><tr><td>Graefe Forcep (curved tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5135</td><td>Micro Dissecting Forceps; Serrated; Slight Curve; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Graefe Forcep (straight tweezers)</td><td>Roboz Surgical Instrument</td><td>RS-5130</td><td>Micro Dissecting Forceps; Serrated, Straight; 0.8 mm Tip Width; 4&quot; Length</td></tr><tr><td>Noyes Micro Scissor</td><td>Roboz Surgical Instrument</td><td>RS-5677</td><td>Noyes Micro Dissecting Spring Scissors;&lt;br/&gt; Straight, Sharp-Blunt Points; 13 mm Cutting Edge; 0.25 mm Tip Width, 4 1/2&quot; Overall Length</td></tr><tr><td>Gauze Pads</td><td>FisherBrand</td><td>13-761-52</td><td>Non-Sterile Cotton Gauze Sponges; 4&quot; x 4&quot; 12-Ply</td></tr><tr><td>Cotton-Tippled Applicators</td><td>FisherBrand</td><td>23-400-124</td><td>6&quot; Length; Wooden Shaft; Single Use Only</td></tr><tr><td>6-Well Plate</td><td>Fisher Scientific</td><td>08-772-49</td><td>Flat Bottom with Low Evaporation Lid; Polystyrene; Non-Pyrogenic</td></tr><tr><td>Sterile Syring 5 mL</td><td>Fisher Scientific</td><td>14-829-45</td><td>Luer-Lok Tip</td></tr><tr><td>Sterile Bowl</td><td>Medical Action Industries Inc.</td><td>01232</td><td>32 oz. Peel Pouch; Blue; Sterile Single Use</td></tr><tr><td>6-Well Plate Inserts (CellCrown Inserts)</td><td>SIGMA</td><td>Z681792-3EA</td><td>6-Well Plate Inserts; Non-Sterile</td></tr><tr><td>Polycarbonate Filter Membrane</td><td>SIGMA</td><td>TMTP04700</td><td>Isopore Membrane Filter; Polycarbonate; Hydrophilic; 5.0&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, White Plain</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments/Description&lt;/strong&gt;</td></tr><tr><td>Beuthanasia</td><td>Schering-Plough Animal Health Corp. Union (Ordered from MWI Veterinary Supply)</td><td>MWI #: 011168</td><td>Active Ingredient: Per 100 mL, 390 mg pentobarbital sodium, 50 mg phenytoin sodium&amp;nbsp;</td></tr><tr><td>Ketamine</td><td>Fort Dodge Animal Health (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Kateset 100 mg/mL</td></tr><tr><td>Xylazine</td><td>LLOYD. Inc. (Ordered from MWI Veterinary Supply)</td><td>MWI #: 000680</td><td>Anased 100 mg/mL</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7122</td><td></td></tr><tr><td>PBS</td><td>Invitrogen</td><td>14040-133</td><td></td></tr><tr><td>MEM</td><td>Invitrogen</td><td>11095080</td><td></td></tr><tr><td>PenStrep</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>FBS</td><td>Invitrogen</td><td>16000-044</td><td></td></tr><tr><td>BSA</td><td>Jackson ImmunoResearch</td><td>001-000-162</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>SIGMA</td><td>S7900-100G</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>Fisher Scientific</td><td>S25372</td><td></td></tr><tr><td>Povidone-Iodine</td><td>Operand</td><td>82-226</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>SIGMA</td><td>320331</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>67-56-1</td><td></td></tr><tr><td>Glycerol</td><td>Fisher Scientific</td><td>56-81-5</td><td></td></tr><tr><td>FITC-conjugated Lectin</td><td>SIGMA</td><td>L9381-2MG</td><td></td></tr><tr><td>Anti-NG2 Chondroitin Sulfate Proteoglycan Antibody</td><td>SIGMA</td><td>AB5320</td><td></td></tr><tr><td>PECAM (CD31) Antibody</td><td>BD Biosciences</td><td>555026</td><td></td></tr><tr><td>LYVE-1 Antibody</td><td>AngioBio Co.</td><td>11-034</td><td></td></tr><tr><td>Goat Anti-Rabbit Cy2-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>111-585-144</td><td></td></tr><tr><td>Goat Anti-Mouse Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>115-227-003</td><td></td></tr><tr><td>Streptavidin Cy3-conjugated Antibody</td><td>Jackson ImmunoResearch</td><td>016-160-084</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit</td><td>Invitrogen</td><td>L3224</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Jackson ImmunoResearch</td><td>005-000-121</td><td></td></tr><tr><td>5-Bromo-2&#039;-Deoxyuridine</td><td>SIGMA</td><td>B5002</td><td></td></tr><tr><td>Monoclonal Mouse Anti-Bromodeoxyuridine&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Clone Bu20a</td><td>Dako</td><td>M074401-8</td><td></td></tr><tr><td>Mouse Anti-Rat CD11b&amp;nbsp;</td><td>AbD Serotec</td><td>MCA275R</td><td></td></tr></tbody></table>

an ex vivo method for time-lapse imaging of cultured rat mesenteric microvascular networks

血管新生は、既存の血管か​​らの新たな血管の成長として定義され、内皮細胞、周皮細胞、平滑筋細胞、免疫細胞、及びリンパ管および神経との調整を含みます。マルチセル、マルチシステムの相互作用は、生理学的に関連する環境における血管形成の調査を必要とします。 in vitro細胞培養モデルの使用は、機械的な洞察を提供してきつつ、一般的な批判は、それらが微小血管ネットワークに関連する複雑さを再現しないことです。このプロトコルの目的は、培養ラット腸間膜組織における血管新生の刺激の前と後に無傷の微小血管ネットワークのタイムラプス比較を行う能力を実証することです。培養組織は、その階層を維持する微小血管ネットワークが含まれています。免疫組織化学的標識化は、内皮細胞、平滑筋細胞、周皮細胞、血管およびリンパ管の存在を確認します。でdditionは、BSI-レクチンと組織を標識すると、増加した毛細血管の発芽と血管密度によって特徴づけられる血清または成長因子刺激の前と後に、ローカルネットワーク領域の経時比較を可能にします。一般的な細胞培養モデルと比較すると、この方法は、内皮細胞系統の研究および生理学的に関連する微小血管ネットワークにおける組織特異的血管新生薬物評価のためのツールを提供します。

培養3日後、組織を、ラット腸間膜培養モデル（ 図2A）で微小血管系の生存能力を実証するために生/死生存率/細胞毒性キットを用いて標識しました。腸間膜中に存在する細胞の大部分は、内皮細胞が微小血管セグメントにおけるそれらの位置に基づいて同定された培養物中で生存可能のままでした。内皮細胞増殖はまた、レクチン/ BrdU標識（ 図2D）...

Watch this Scientific Journal Video about An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks at JoVE.com

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

アン<em&gt; ex vivoで</em&gt;メソッド培養ラット腸間膜微小血管ネットワークのタイムラプスイメージングのための

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

an-ex-vivo-method-for-time-lapse-imaging-cultured-rat-mesenteric

Research

JoVE Journal

Developmental Biology

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

6.8K ビュー.  Tulane University. Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis. 

ビデオ: An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

in vitroおよびin vivoモデルでは血管壁の下でフローの状況に細菌付着を研究するために、

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

免疫・感染学

Imaging Neutrophils and Monocytes in Mesenteric Veins by Intravital Microscopy on Anaesthetized Mice in Real Time

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in vivo is crucial for understanding the molecular basis of leukocyte transendothelial migration and interaction with vascular endothelium. One powerful approach involves intravital microscopy on transgenic mice expressing fluorescent proteins in cells of interest.
Here we present a protocol for imaging monocytes and neutrophils in the CX3CR1gfp/wt mouse i.v. injected with orange dye-labeled neutrophils with an inverted confocal microscope. Time-lapse movies gathered from 30 min&#160;to several hours of imaging allow the analysis of leukocyte behavior in mesenteric veins under both steady state and inflammatory conditions. We also describe the steps to locally induce blood vessel inflammation with TLR2/TLR1 agonist Pam3SK4 and monitor the subsequent recruitment of neutrophils and monocytes.
The presented technique can also be used to monitor other populations of leukocytes and investigate molecules implicated in leukocyte recruitment or trafficking using other stimuli or transgenic mice.

We detail a protocol to monitor the behavior of neutrophils and monocytes in mesenteric veins under steady state and inflammatory conditions using intravital confocal microscopy on anaesthetized mice.

リアルタイムで麻酔したマウスに生体顕微鏡により腸間膜静脈に好中球および単球のイメージング

Efficient immune response is dependent on rapid mobilization of blood leukocytes to the site of infection or injury. Investigating leukocyte migration in ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

ラット間膜の血管新生アッセイ

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

生物学

Rat Mesentery Exteriorization: A Model for Investigating the Cellular Dynamics Involved in Angiogenesis

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease conditions1, 2. Network growth is commonly attributed to angiogenesis, defined as the growth of new vessels from pre-existing vessels. The angiogenic process is also directly linked to arteriogenesis, defined as the capillary acquisition of a perivascular cell coating and vessel enlargement. Needless to say, angiogenesis is complex and involves multiple players at the cellular and molecular level3. Understanding how a microvascular network grows requires identifying the spatial and temporal dynamics along the hierarchy of a network over the time course of angiogenesis. This information is critical for the development of therapies aimed at manipulating vessel growth.
The exteriorization model described in this article represents a simple, reproducible model for stimulating angiogenesis in the rat mesentery. It was adapted from wound-healing models in the rat mesentery4-7, and is an alternative to stimulate angiogenesis in the mesentery via i.p. injections of pro-angiogenic agents8, 9. The exteriorization model is attractive because it requires minimal surgical intervention and produces dramatic, reproducible increases in capillary sprouts, vascular area and vascular density over a relatively short time course in a tissue that allows for the two-dimensional visualization of entire microvascular networks down to single cell level. The stimulated growth reflects natural angiogenic responses in a physiological environment without interference of foreign angiogenic molecules. Using immunohistochemical labeling methods, this model has been proven extremely useful in identifying novel cellular events involved in angiogenesis. Investigators can readily correlate the angiogenic metrics during the time course of remodeling with time specific dynamics, such as cellular phenotypic changes or cellular interactions4, 5, 7, 10, 11.

This article describes a simple model for stimulating angiogenesis in the rat mesentery. The model produces dramatic increases in capillary sprouting, vascular area and vascular density over a relatively short time course in a tissue that allows en face visualization of entire microvascular networks down to the single cell level.

ラット腸間膜具象化：血管新生に関与する細胞のダイナミクスを調べるためのモデル

Microvacular network growth and remodeling are critical aspects of wound healing, inflammation, diabetic retinopathy, tumor growth and other disease ...

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 ...

医学

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

マウス胎児腸全体培養システム<em&gt;生体外</em&gt;シグナル伝達経路の操作と絨毛開発の3次元ライブイメージング

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

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

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

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

マウス心臓における毛細血管、細動脈、および周囲細胞の動的測定とイメージング

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

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

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

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 ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

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

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

ラット腸間膜に微小血管透過性の調節を調査するために、微小灌流法

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

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 ...

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