著者はクリスティアーネ ・ Pahrmann の彼女の技術的な援助をありがちましょう。
社殿は、最大 Kade 財団によって支えられました。両手を使った受信助成他 Kröner 財団 (2012_EKES.04) とドイツ研究振興協会 (DE2133/2-1 _。S. s. は、ドイツ研究振興協会 (DFG; から研究助成を受けてください。SCHR992/3-1、SCHR992/4-1)。

動物実験動物の原則のためのガイドに準拠した人道的なケアを受けて、実験動物資源の研究所でを準備、健康の国民の協会によって公開されました。動物のすべてのプロトコルは、(「Amt こだわり健康を祝して und Verbraucherschutz、ハンザ (健康および消費者保護のためのオフィス)」) 責任がある地方自治体によって承認されました。
1. カテーテルの準備
注: はカテーテルについては材料の表を参照してください。
<ol><li>カテーテルをカテーテル ホルダーから取る。</li>
	<li>うち遠位ポートを通して内腔からガイドワイヤーを引き上げます。</li>
	<li>カテーテルの遠位端にシアノアクリ レート系接着剤のドロップを置きます。 注意: は、ラテックスやニトリル手袋を着用します。</li>
	<li>カテーテルの RX ポートにガイドワイヤを置き、遠位のポートに内腔進みます。その後、ガイドワイヤ少し引き終わりに 〜 5 mm のスペースを残して。</li>
	<li>乾燥して接着剤を許可する 5 分を待ちます。</li>
	<li>ガイドワイヤを移動、それは修正される必要があります。まだ携帯電話の場合は、手順 1.3-1.6 を繰り返します。</li>
	<li>1.5 mL 0.9% 生理食塩水 3 mL シリンジを入力し、バルーン膨張ポートに接続します。</li>
	<li>バルーンの膨張をテストするシリンジのプランジャーを押してください。0.6 mL のシリンジに生理食塩水を残します。</li>
</ol>2. マウス作製
<ol><li>重さ約 30 g 14 週齢で雄マウスを取得します。 注意: 動物実験動物研究所からの取得を使用しています。我々 はここで c57bl/6 j を使用しました。</li>
	<li>Anaesthetize 2.0 2.5 %isofurane (500 mL/分の酸素流量) とマウスに誘導室を使用します。</li>
	<li>加熱パッドの上にマウスを置くし、マウスの鼻と口を覆うマスクで麻酔を維持します。後部のフィートと反射の不在を確認する尻尾をつまんで麻酔の十分な深さを確認します。</li>
	<li>髪トリマーを使用して腹部の毛を削除します。</li>
	<li>後肢の足を広げ、テープを使用して自分の位置を修正します。</li>
	<li>続いて 80% エタノール、ポビドン ヨードを使用して腹部領域を消毒します。この手順をさらに 2 回繰り返します。</li>
	<li>手術部位は汚染を取得されないように手術用ドレープを使用します。に沿って皮膚や筋肉層を開く、リネア アルバシザー (またはメス) 腹部の臓器を公開します。</li>
	<li>0.9% 生理食塩水うるおい手袋で腸を置くし、湿潤を保つためにラップします。</li>
	<li>腹部大動脈上脂肪組織を除去するのに 2 つの微細鉗子を使用します。</li>
	<li>インスリン注射器 (30 G) を使用して、下大静脈 (IVC) にヘパリン溶液 (50 U/mL) の 250 μ L を注入し、全身の分布の 3 分を待ちます。ヘパリンが止血を抑制し、手術中に不要な凝固を防ぐ。</li>
	<li>2 つの鉗子を使用すると、その分岐とその発信の分岐血管まで高齢者大動脈を解剖します。</li>
	<li>高温師と固定の領域に配置すると予想される側血管を縛る。</li>
	<li>腎動脈のすぐ下に上行大動脈のクランプによって血流を停止します。</li>
	<li>遠位大動脈分岐のすぐ上に 2 番目のクランプを配置します。</li>
	<li>小さな水平切開血管に沿ってクランプの間の中間点にはさみを使用してを実行します。 注: 切開の大きさは船のまわりの 3 分の 1 に相当する必要があります。</li>
	<li>切開 (30 G) 針と注射器を挿入し、250 μ L ヘパリン溶液 (50 U/mL) と大動脈をフラッシュします。</li>
	<li>10-0 縫合糸を使用して、切開の各側に 1 つの単一の結び目を配置します。</li>
	<li>大動脈を切開に容器ガイドワイヤーを挿入して広げるし、容器を少し広げます。2 ~ 3 回拡張を繰り返します。</li>
	<li>0.9% 生理食塩水をカテーテルに潤いを与えます。</li>
	<li>大動脈に平面的なバルーンカテーテルを挿入し、大動脈に近位のクランプの方それを進めます。</li>
	<li>近位部のクランプに達し、慎重に近位のクランプを開き、~0.6 mL の生理食塩水を注入することで血液の漏出を防ぐためにバルーンを膨らませます。 注: 血管にバルーンを膨らませての比は 1.5: 1 です。</li>
	<li>約 2 cm の逆行性のカテーテルを進めます。</li>
	<li>注射器を外して少しバルーンをしぼませるながら戻って、拡張カテーテルを引き出します。</li>
	<li>カテーテル大動脈切開に達するとは、近位部のクランプを接続し直します。完全にバルーンを収縮し、それを削除します。</li>
	<li>30 G の注射器を使用して 250 μ L ヘパリン溶液 (50 U/mL) と大動脈をすすいでください。</li>
	<li>10-0 縫合糸を用いた大動脈切開を閉じる。場所は、腹側に 1 つか 2 つのステッチに続いて、各側面にステッチを中断しました。</li>
	<li>遠位クランプを開きます。出血の場合、クランプを閉じて、追加ステッチを配置します。</li>
	<li>慎重に近位のクランプを開きます。</li>
	<li>それをサポートし、任意の出血を止める縫合に 2 つ綿棒を配置します。</li>
	<li>それを維持するために縫合を吸収性止血剤を配置します。 注: 大動脈脈が切開部から遠位表示されます。</li>
	<li>腹部に腸を配置します。</li>
	<li>あらかじめ 37 ° C に加温されている滅菌生理食塩水 0.9%、腹腔内を洗浄します。</li>
	<li>6-0 実行縫合糸を使用して腹部の筋肉層を閉じます。</li>
	<li>5-0 実行縫合糸で皮膚を閉じます。</li>
	<li>目を覚ますには、マウスを許可する前にカルプロフェンの 4-5 mg/kg を皮下注入します。それは意識を得ていますまで、動物を監視、胸骨の横臥を維持します。完全な復旧までケージの中だけで動物をしてください。</li>
	<li>3 日間の鎮痛薬として飲用水 (50 mg/100 mL) に Metamizole を追加し、毎日動物を監視します。通常甘いようなマウスは metamizole の味し、手術後すぐに飲み始めます。優先、徐放性注射剤は metamizole の代わりに使用できます。 注: このモデルの観察期間は 28 日です。</li>
</ol>3. 病理組織学的
<ol><li>2.2 に 2.9 の手順で説明するように、マウスを準備することによって、28 日後バルーン損傷大動脈を収穫します。</li>
	<li>はさみを使用してバルーン損傷大動脈 (間分岐と腎血管上 0.3 mm) を削除し、その心を切り出してマウスを安楽死させます。</li>
	<li>0.9% と血管の内腔を洗い流す塩化ナトリウム。</li>
	<li>一晩 4% パラホルムアルデヒド (PFA) で収穫された容器を修正し、5 時間の 2 時間、2 時間、1 時間の 80% エタノール、95% エタノール 70% エタノールで始まるエタノールそして 100% のエタノール濃度の増加で脱水します。その後、孵化させなさいキシレンのサンプル 2 時間 3 回、サンプルはパラフィンを浸透させる前に。 注: 0.9% の収穫の容器をフラッシング代わりに、塩化ナトリウム 4% とフラッシュすることが PFA。 注意: PFA とキシレン有毒である、特別な注意して処理する必要があります。</li>
	<li>パラフィン サンプルを埋め込み、ミクロトームを用いた 5 μ m 厚のスライスに切る。</li>
	<li>5 分間 3 回キシレンとスライドを deparaffinize します。</li>
	<li>エタノールの減少シリーズを使用して組織スライドを水分補給します。100% のエタノール 2 回の 95%、80%、70% のエタノールの 3 分後、5 分で開始します。</li>
	<li>説明18としてマッソンのヒアリン染色でスライドを染色します。</li>
	<li>2 回 10 分ずつの 100% エタノールで汚されたスライドを脱水します。2 回 10 分ずつのキシレンでクリアし、メディアをマウントでマウントします。</li>
	<li>明視野顕微鏡でスライドを表示します。概要図や詳細な観察のための 0.35 の開口と 20 x 倍率とレンズの 5 倍の倍率と 0.12 の開口レンズを使用します。</li>
</ol>4. 蛍光顕微鏡
<ol><li>エタノールの減少シリーズを使用して組織スライドを水分補給します。100% のエタノール 2 回の 95%、80%、70% のエタノールの 3 分後、5 分で開始します。</li>
	<li>20 分間蒸し器で抗原検索ソリューション内のスライドを加熱することによって抗原検索を実行します。</li>
	<li>室温までスライドを冷ます。</li>
	<li>スライドをリン酸緩衝生理食塩水 (PBS) で 3 回洗浄した後に、30 分のセクションにブロッキング液の抗原を適用します。</li>
	<li>3 回 PBS で 5 分用のスライドを洗います。</li>
	<li>一次抗体の希釈剤で希釈した一次抗体とのセクションを孵化させなさい。 注: 右の濃度と潜伏期間は、各抗体で別々 に選択する必要があります。</li>
	<li>3 回自由な抗体を削除する PBS で 5 分用のスライドを洗います。</li>
	<li>二次抗体の希釈剤で希釈済み共役二次抗体とセクションを孵化させなさい。 注: 右の濃度と潜伏期間は、各抗体で別々 に選択する必要があります。</li>
	<li>5 分用のスライドを 3 回洗浄することにより非連結抗体を削除します。</li>
	<li>4', 6-diamidino-2-phenylindole (DAPI) 15 分; を使用して細胞核を counterstain します。最終的な DAPI 濃度が 350 をする必要があります nM。</li>
	<li>蛍光抗体法対応ソリューションを装着でのスライドをマウントします。 注: は間違って実装ソリューションを使用して蛍光信号が見えなくなります。</li>
	<li>蛍光顕微鏡でスライドを表示します。1.3 の開口で 40 倍の倍率を使用します。</li>
</ol>

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この記事は、myointimal 過形成の開発を研究するマウスモデルを示し、根本的な病理学的プロセスの探査と新しい薬や治療法のテストをことができます。
このプロトコルでは最も重要なステップは、大動脈の剥離です。特別な注意は、過剰な侵食が動脈瘤の形成とモデルが失敗につながる、このステップの間にお支払いください。その一方で、侵食が十分に実行された場合...

冠動脈および末梢動脈における動脈狭窄患者1の死亡率と罹患率に大きな影響があります。1 つの根本的な病理メカニズムは myointima 過形成 (MH)、増加増殖、移行、および血管平滑筋細胞 (SMC)2からの細胞外マトリックス蛋白質の合成によって特徴付けられる。SMC は容器のメディア層にあり、内腔の表面への刺激に移行します。刺激信号は成長因子、サイトカイン、細胞間連絡、脂質、細胞外マトリックス成分、機械的せん断をストレッチ力3,4,5,6あります。病理学的または医原性、血管壁の損傷血管内皮細胞と平滑筋細胞の損傷を引き起こすと炎症反応を刺激し、MH7につながります。
異なる動物モデルが現在利用できる myointima 増殖症動脈損傷を研究です。豚のような大型の動物や犬共有同様の動脈と冠動脈解剖学人間の利点を持っているし、特に血管形成技術、手順、およびデバイス8を調査し研究に適しています。ただし、豚モデルは、犬のみが軽度の血管傷害11レスポンス高い血栓9,10の欠点を持っています。さらに、特別な住宅、機器、およびスタッフは、高コストで接続されているし、常に、教育機関で利用されていないすべての大型動物モデルが必要です。実験動物は、ラットやマウスに含まれます。ラットと比較して、マウスは、低コストの利点とさまざまなモデルをノックアウトの存在があります。このビデオで説明されているモデルは、アポ-/-マウス西部飼料動脈硬化血管12血管形成術の臨床設定を忠実に再現すると併用できます。以前のモデルは、ワイヤー傷害13、流体乾燥14、春15、またはカフ傷害16を介して血管損傷を誘発しました。開発と MH の憲法にけがの性質に大きく影響するので臨床場面を模倣する最良の方法は、バルーン カテーテルを使用して血管損傷を誘発します。
この記事ではマウスのバルーンカテーテルと MH を誘導する手法について述べる.RX ポート (図 1 a) バルーンカテーテル (1.2 mm × 6 mm) の使用する内膜層のと同時に、船の overdistension の誘導にこする。これらの要因の両方が MH の開発のための重要なトリガーです。このモデルの観測時間は 28 日17歳です。

<table>
	<tbody>
		<tr>
			<td>10-0 Ethilon suture</td>
			<td>Ethicon</td>
			<td>2814G</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL Syringe</td>
			<td>BD Medical</td>
			<td>309658</td>
			<td></td>
		</tr>
		<tr>
			<td>37% HCl</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 prolene suture</td>
			<td>Ethicon</td>
			<td>EH7229H</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 prolene suture</td>
			<td>Ethicon</td>
			<td>8706H</td>
			<td></td>
		</tr>
		<tr>
			<td>Acid Fuchsin</td>
			<td>Sigma-Aldrich</td>
			<td>F8129-25G</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Antigen retrieval solution</td>
			<td>Dako</td>
			<td>S1699</td>
			<td></td>
		</tr>
		<tr>
			<td>Azophloxin</td>
			<td>Waldeck</td>
			<td>1B-103</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Bepanthen Eye and Nose ointment</td>
			<td>Bayer</td>
			<td>1578675</td>
			<td>Eye ointment</td>
		</tr>
		<tr>
			<td>Betadine Solution</td>
			<td>Betadine Purdue Pharma</td>
			<td>NDC:67618-152</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J</td>
			<td>Charles River</td>
			<td>Stock number 000664</td>
			<td></td>
		</tr>
		<tr>
			<td>Clamp applicator</td>
			<td>Fine Science Tools</td>
			<td>18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen 3</td>
			<td>abcam</td>
			<td>ab7778</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Thermo Fischer</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-Goat IgG AF555</td>
			<td>Invitrogen</td>
			<td>A21432</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF488</td>
			<td>Invitrogen</td>
			<td>A11055</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Donkey anti-Rabbit IgG AF555</td>
			<td>Invitrogen</td>
			<td>A31572</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Ethanol 70%</td>
			<td>Th. Geyer</td>
			<td>2270</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Th. Geyer</td>
			<td>2295</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Th. Geyer</td>
			<td>2246</td>
			<td></td>
		</tr>
		<tr>
			<td>FAP</td>
			<td>abcam</td>
			<td>ab28246</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Forceps fine</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps standard</td>
			<td>Fine Science Tools</td>
			<td>11023-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial Acetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>537020</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>WAHL</td>
			<td>8786-451A ARCO SE</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Rotexmedica</td>
			<td>PZN 3862340</td>
			<td>25.000 I.E./mL</td>
		</tr>
		<tr>
			<td>High temperature cautery kit</td>
			<td>Bovie</td>
			<td>18010-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Image-iT FX Signal Enhancer</td>
			<td>Invitrogen</td>
			<td>I36933</td>
			<td>Blocking solution</td>
		</tr>
		<tr>
			<td>Light Green SF</td>
			<td>Waldeck</td>
			<td>1B-211</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Microsurgical clamp</td>
			<td>Fine Science Tools</td>
			<td>18055-04</td>
			<td>Micro-Serrefine - 4mm</td>
		</tr>
		<tr>
			<td>MINI TREK Coronary Dilatation Catheter 1.20 mm x 6 mm / Rapid-Exchange</td>
			<td>Abbott</td>
			<td>1012268-06U</td>
			<td></td>
		</tr>
		<tr>
			<td>Molybdatophosphoric acid hydrate</td>
			<td>Merck</td>
			<td>1.00532.0100</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>NaCl 0,9%</td>
			<td>B.Braun</td>
			<td>PZN 06063042 Art. Nr.: 3570160</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Fine Science Tools</td>
			<td>12075-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Novaminsulfon</td>
			<td>Ratiopharm</td>
			<td>PZN 03530402</td>
			<td>Metamizole</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Waldeck</td>
			<td>1B-221</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Leica biosystems</td>
			<td>REF 39602004</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS pH 7,4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA 4%</td>
			<td>Electron Microscopy Sciences</td>
			<td>#157135S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S solution</td>
			<td>Serva Electrophoresis</td>
			<td>33427</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Primary antibody diluent</td>
			<td>Dako</td>
			<td>S3022</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold Mounting solution</td>
			<td>Thermo Fischer</td>
			<td>P36930</td>
			<td>Mounting solution for immunofluorescence stained slides</td>
		</tr>
		<tr>
			<td>Replaceable Fine Tip</td>
			<td>Bovie</td>
			<td>H101</td>
			<td></td>
		</tr>
		<tr>
			<td>Resorcin-Fuchsin Weigert</td>
			<td>Waldeck</td>
			<td>2E-30</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Rimadyl</td>
			<td>Pfizer</td>
			<td>400684.00.00</td>
			<td>Carprofen</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors Vannas-style</td>
			<td>Fine Science Tools</td>
			<td>15000-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Secondary antibody diluent</td>
			<td>Dako</td>
			<td>S0809</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast acting Adhesive MINIS 3x1g</td>
			<td>UHU</td>
			<td>45370</td>
			<td>Cyanoacrylate</td>
		</tr>
		<tr>
			<td>Slide Rack</td>
			<td>Ted Pella</td>
			<td>21057</td>
			<td></td>
		</tr>
		<tr>
			<td>SM22</td>
			<td>abcam</td>
			<td>ab10135</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>SMA</td>
			<td>abcam</td>
			<td>ab21027</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Staining dish</td>
			<td>Ted Pella</td>
			<td>21075</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Leica</td>
			<td>M651</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabotamp fibrillar</td>
			<td>Ethicon</td>
			<td>431962</td>
			<td>Absorbable hemostat</td>
		</tr>
		<tr>
			<td>Transpore Surgical Tape</td>
			<td>3M</td>
			<td>1527-1</td>
			<td></td>
		</tr>
		<tr>
			<td>U-100 Insulin syringe</td>
			<td>BD Medical</td>
			<td>324825</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Dilator</td>
			<td>Fine Science Tools</td>
			<td>18603-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Vitro-Clud</td>
			<td>Langenbrinck</td>
			<td>04-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Weigerts iron hematoxylin Kit</td>
			<td>Merck</td>
			<td>1.15973.0002</td>
			<td>Trichrome staining</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Th. Geyer</td>
			<td>3410</td>
			<td></td>
		</tr>
	</tbody>
</table>

balloon-based injury to induce myointimal hyperplasia in the mouse abdominal aorta

動物モデルの使用は、MH、動脈狭窄症の主要な原因の 1 つのより良い理解のために不可欠です。この記事では、大動物で確立された容器損傷モデルに匹敵するマウス バルーン侵食モデルを示しています。バルーン カテーテルを大動脈侵食モデルは臨床設定を模倣し、匹敵する病態と生理学的な変化に 。簡潔に、実行後水平切開腹部大動脈で、バルーンカテーテル、血管に挿入、水増し、逆行性導入します。バルーンのインフレは内膜傷害と容器の overdistension に します。カテーテルを抜去後、大動脈切開は単一 stiches を閉鎖します。この資料に示すモデルは再生可能で、行うには、簡単、迅速かつ確実に確立することができます。特に経済的な方法で適用することができます高価な実験的治療剤の評価に適しています。別のノックアウト マウス系統を用いた MH 開発の別の遺伝子の影響を評価できます。

バルーン侵食は、マウスで MH の開発研究に適したモデルです。動物手術から回復を示し、優れた物理的な条件術後。このモデルは、50 マウス外科的処置のための 3% の死亡率よりも少ないに設立しました。図 1 b-Cは、主な手術手順を表示します。に沿って皮膚切開後、 linea alba、腹部大動脈を識別します。顕微鏡視下ク?...

Watch this Scientific Journal Video about Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta at JoVE.com

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

この記事は、大動脈バルーン傷害の後 myointimal 過形成 (MH) の開発を研究するマウスモデルを示します。

マウスの腹部大動脈 Myointimal 過形成を誘導するために気球を利用した傷害

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

balloon-based-injury-to-induce-myointimal-hyperplasia-mouse-abdominal

Research

JoVE Journal

Medicine

9.5K ビュー.  University Heart Center. この記事は、大動脈バルーン傷害の後 myointimal 過形成 (MH) の開発を研究するマウスモデルを示します。

ビデオ: Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL varies between 2.5 and 20% 2-5. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7. 
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.

The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.

C -シール：漏れから結腸直腸器械吻合を保護Biofragmentableドレイン

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

医学

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

マウスにおける急性肺障害を誘発するために圧力制御された換気

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

CYP2D6の動物モデル：マウスにおける自己免疫性肝炎を誘導する方法

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

弱視における抑制の測定と治療

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

マウス角膜血管新生のアルカリ熱傷損傷モデル

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Inducing Myointimal Hyperplasia Versus Atherosclerosis in Mice: An Introduction of Two Valid Models

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque formation and stenosis, as observed for example in ischemic heart disease. Two highly reproducible mouse models&#160;&#8211;&#160;both resulting in artery stenosis but each underlying a different pathway of development&#160;&#8211; are introduced here. The models represent the two most common causes of artery stenosis; namely one mouse model for each myointimal hyperplasia, and atherosclerosis are shown. To induce myointimal hyperplasia, a balloon catheter injury of the abdominal aorta is performed. For the development of atherosclerotic plaque, the ApoE -/- mouse model in combination with western fatty diet is used. Different model-adapted options for the measurement and evaluation of the results are named and described in this manuscript. The introduction and comparison of these two models provides information for scientists to choose the appropriate artery stenosis model in accordance to the scientific question asked.

This video shows two models of intimal plaque development in murine arteries and emphasizes the differences in myointimal hyperplasia and atherosclerosis.

マウスにおけるアテローム性動脈硬化症に対する筋内膜過形成を誘発する。つの有効なモデルの導入

Various in vivo laboratory rodent models for the induction of artery stenosis have been established to mimic diseases that include arterial plaque ...

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are still occurring, particularly in reconstructive flap surgery. Multiple experimental flap models have been developed to analyze underlying causes and mechanisms and to investigate treatment strategies to prevent ischemic complications. The limiting factor of most models is the lacking possibility to directly and repetitively visualize microvascular architecture and hemodynamics. The goal of the protocol was to present a well-established mouse model affiliating these before mentioned lacking elements. Harder et al. have developed a model of a musculocutaneous flap with a random perfusion pattern that undergoes acute persistent ischemia and results in ~50% necrosis after 10 days if kept untreated. With the aid of intravital epi-fluorescence microscopy, this chamber model allows repetitive visualization of morphology and hemodynamics in different regions of interest over time. Associated processes such as apoptosis, inflammation, microvascular leakage and angiogenesis can be investigated and correlated to immunohistochemical and molecular protein assays. To date, the model has proven feasibility and reproducibility in several published experimental studies investigating the effect of pre-, peri- and postconditioning of ischemically challenged tissue.

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

マウスの背部皮脂商工における虚血性組織損傷：皮膚弁モデル急性永続虚血を調査する

Despite profound expertise and advanced surgical techniques, ischemia-induced complications ranging from wound breakdown to extensive tissue necrosis are ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

マウスの腹部癒着の作成

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

マウスモデルにおける下腹部大動脈クランプすることにより、再現性動脈侵食に起因けが

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

ラットの腹部大動脈の収縮を通じて心臓リモデリングのモデル

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...