The authors would like to acknowledge support through the American Heart Association (10SDG2630139), the Welch Foundation and through the NIH Director&#8217;s New Innovator Grant (1DP2 OD008716-01). The authors would like to thank the services provided by the ICMB (Institute of Cellular and Molecular Biology) core facility and TherapeUTex at University of Texas at Austin.

注：このプロトコルに示されているすべてのメソッドは、施設内動物管理使用委員会によって承認されている。 手術台の調製<ol><li>解剖顕微鏡下で、手術台に温水を循環させるとともに加熱パッドを設定します。加熱パッド上にステンレス鋼製基板を置きます。ベースプレートと加熱パッド上の無菌吸収パッドを配置します。 </li><li>隣接無菌の吸収パッド上の無菌手術器具を配置します。傾斜した先の細いピンセット2対の角度付き鉗子二対の三止血鉗子つ開創ワイヤ、小さな外科用ハサミ、マイクロ剪刀つ血管形成ワイヤ（0.15インチ径）と6.0絹縫合糸の3~6インチのセグメントを集める。 </li><li>大腿動脈の曲に合わせて血管形成術線のラウンド終了を曲げる。これは、簡単に手術中にワイヤーを前進させるために行います。 </li></ol>の調製手術のためのマウス<ol><li> 2.5％のイソフルランの連続吸入により、マウスを麻酔。手順を通して、動物の状態を監視するようにしてください。それは完全に麻酔をかけていることを確認するために、マウスの足のピンチテストを実行します。ピンチ試験を投与した場合、動物が動かないことを確認してください。 </li><li>乾燥を防ぐために、動物の目に潤滑軟膏を適用します。サージカルテープを使用して吸収パッドに仰臥位で動物を固定します。ノーズコーンを経由して、2.5％イソフルランを投与し続けます。 </li><li>脱毛クリームを使用して、ミッドラインに足と腹部から毛を取り除く。水で十分に皮膚を洗い流す。脱毛は、手術の前に一日に行うことができる。クリームとの過度の治療は皮膚刺激につながる可能性があることに注意してください。 </li><li>すぐに手術の前に、皮膚を滅菌するために綿棒で脱毛した領域にポビドンヨードを適用します。 70％エタノールで脱毛し皮膚を洗い流すとドライ無菌の綿棒を持つ。 3回繰り返します。 </li><li>皮下注射を介してのカルプロフェン5mg / kgの術前投与量を投与する。 </li></ol>大腿動脈の3.絶縁<ol><li>小さな外​​科用ハサミを使用して、大腿動脈上の皮膚に湾曲した切開する。ぶっきらぼうに大腿動脈を見つけるために開創磁気リトラクター固定器を使用して周囲の組織を解剖し、固定します。無菌の綿棒を使用して生理食塩水を適用する灌漑用生理食塩水を使用して定期的に組織を湿らせます。 </li><li>ピンセットを用いて大腿動脈を分離します。静かに先の細いピンセットを用いて維管束から神経を分離する。静脈穿刺避け、神経を損傷しない。それを刺激避けるために離れてバンドルから神経を押してください。 </li><li>静かに大腿分岐の位置を、大腿動脈から大腿静脈を分離する。分岐部の領域を解剖するのが特に困難である。 </li><li> P分岐、ループ6.0絹の大腿動脈の下で縫合し、止血剤との安全にosterior。この近位の縫合糸は、動脈内の血流を制限するために使用される。 注：焼灼法に対するライゲーション法を行う際関係に若干のばらつきがある（ 図1及び図2を参照）。 </li><li>分岐部の遠位に、ループ6.0絹の大腿動脈の下で縫合し、止血剤で固定します。動脈の位置で、この遠位縫合糸を支援します。 </li><li>大腿動脈の筋肉の枝の下にループ2本の縫合糸は、それらを事前にタイや止血剤で固定します。生理食塩水で組織を湿らすることを忘れないでください。ワイヤ傷害の焼灼法を実施した場合、唯一のループ付き縫合糸は、筋枝に必要である。 </li></ol>大腿動脈ワイヤ傷害の4.性能<ol><li>近位縫合糸を引いて、大腿動脈への血流を制限します。少し遠位hemostを引っ張るATと止血動脈切開のためのサイトを公開するために枝を固定する。その周りに縫合糸を結びつけることによって筋肉の枝を結紮。 注：上向きの動脈をリフティングすると、より効果的に一人で水平に引っ張っよりも血流を制限します。 </li><li> 2本の縫合糸の間にメスで小枝を切除。マイクロはさみを使用して、分岐部の側枝に動脈切開を行う。マイクロハサミを安定させるための手術テープのロールにバランスのとれた鉗子を使用してください。 </li><li>先の細いピンセットを用いて動脈切開の存在を確認。静かに鉗子で動脈切開の開口部を持ち上げる。鉗子を用いて動脈切開にワイヤーの丸い端部を導入する。ワイヤの前進を容易にするために、シリンジを用いて領域にリドカインの1つまたは2つの滴を加える。 </li><li>ワイヤーは、近位縫合糸に到達すると、縫合糸を解放し、ワイヤの前進を妨げることができないように調整する。それをさらに進めることができないまでワイヤーを挿入します。目の先端電子線は鼠径靭帯の領域に停止する必要があります。 </li><li>ワイヤーは1分間大腿動脈に残るようにします。傷つけると大腿動脈の内皮をdenude 10回を1分後に、後退させソーイング運動ワイヤを前進させる。あまり重症の怪我のために、ワイヤは動脈の内と外に引っ張られたり、わずか1分間の動脈にワイヤーを残している回数を減少させる。 </li><li>ゆっくりとワイヤーを撤回。ワイヤーの丸い端が近位縫合糸を経過すると、近位縫合糸を引くことによって動脈への流れを制限する。完全にワイヤーを撤回。 </li></ol> 5.筋肉支店を連結<ol><li>筋肉の枝に残っている縫合糸を締めます。これは動脈切開からの出血防止します。筋肉の枝に流れを戻し、血液がそこから漏れていないことを確認。筋肉の枝に縫合糸から端をトリミング。 </li></ol> 6.代替方法：ローカライズされたカウ動脈切開のクタリゼーション注：別のアプローチは、筋肉枝のライゲーションを回避し、より大きなメイン大腿動脈を通して血管アクセスを可能にするために取ることができる。 <ol><li> 4.1から出発して、近位縫合糸を引くことにより大腿動脈への流れを制限する。遠位縫合糸と動脈切開（ 図1と図2）のためのサイトを公開するために少し筋肉の枝を固定して縫合糸を引き出します。 </li><li>マイクロはさみを使用して、大腿動脈と筋肉の枝の分岐点での動脈切開を行う。切開は、動脈の側で行われる場合には、焼灼するのがより容易であり得る。マイクロハサミを安定させるための手術テープのロールにバランスのとれた鉗子を使用してください。 </li><li>焼灼の成功をテストするには、近位の縫合糸を緩めて大腿動脈への流れを復元する。出血が動脈切開から発生する場合は、焼灼を繰り返します。焼灼が成功した場合、血流が再され動脈切開の遠位に格納されている。 </li><li>ワイヤーを紹介し、4章で説明したように、ワイヤ傷害を行う。 </li><li>大腿動脈への血流を制限し続ける。少なくとも6インチ離れてマウスから先の細い焼灼を熱し。焼灼先端が冷えると、切開を閉じるために動脈切開の側面を軽くそれを適用する。 </li><li>焼灼の成功をテストするには、近位の縫合糸を緩めて大腿動脈への流れを復元する。出血が動脈切開から発生する場合は、焼灼を繰り返します。焼灼が成功した場合、血流が動脈切開の遠位に復元される。一時的な近位および遠位の関係を削除します。 </li></ol>血管周囲ドラッグデリバリーパッチの7。移植<ol><li>血管周囲の薬剤パッチの作成​​11以前の研究で、または類似の方法を用いて説明したように作成することができる。無菌のハサミを使用して薬物送達パッチのバリア領域の角を鈍ら</li><li>薬物送達パットを配置動脈に直面して薬物放出側に負傷した大腿動脈の上にCH。必要に応じて、パッチの位置を改善するために鉗子を使用しています。 </li></ol> 8.創傷閉鎖と回復<ol><li>正方形の結び目からなるシンプルな中断された縫合糸で傷口を閉じます。不連続縫合糸は、動物が縫合糸を削除しようとする場合には、創傷閉鎖を延長。 </li><li>麻酔をオフにして、セットアップから動物を削除する。動物が温暖化パッド上に回復することを許可する。 </li><li>マウスの回復を監視し続ける。それは閉じたままであることを確実にするために、毎日切開部位を確認してください。手術後の痛みの治療のために、2日間カルプロフェンの5〜10mg / kgの皮下注射、手術後12時間ごとに、その後12時間ごとに管理する。痛みは最初の2日間を超えて継続した場合、さらに鎮痛薬の指示のために獣医師に相談してください。 </li></ol>組織学9.収穫大腿動脈<ol><li>手術後28日目に、マウスに、二酸化炭素の安楽死を行う。 NOTE：二酸化炭素の適切な流量は毎分室10〜30％を置換する必要があり、使用されるチャンバーのサイズに応じて変化するであろう。横隔膜と心臓穿刺の切断が安楽死の二法として行われるべきである。 </li><li>サージカルテープを使用して仰臥位でマウスを固定します。最初の外科的切開が行われた大腿動脈、上の切開を行います。 </li><li>負傷した動脈と対側脚から無傷の動脈の両方のために、ぶっきらぼうに解剖し、大腿動脈を見つけるためにリトラクターと磁気リトラクター固定器を使用して周囲の組織に固定します。灌漑用の生理食塩水を使用して定期的に組織を湿らせます。無菌の綿棒を使用して生理食塩水を適用します。動脈を損傷しないように注意してください。 </li><li>大腿動脈は腹部大動脈元動脈切開部位から単離された後に、ワンセグを結ぶ動脈切開の元のサイトの近くに絹縫合のメント。この縫合糸は、大腿動脈の最遠位端を特定し、試料の取り扱いを容易にするのに役立ちます。 </li><li>大腿動脈を切除するためのマイクロ解剖ハサミを使用する。縫合糸の1切開遠位てください。腹部大動脈の横に、大腿動脈の反対側の端に他の切開を行います。 </li><li>生理食塩水を含有するガラスシャーレに切除された動脈を転送します。過剰結合組織や脂肪を除去するためにさらに動脈を解剖。静かに生理食塩水ですすぐことによって内腔から血液を除去する。 </li><li> 10％緩衝ホルマリンのバイアルに動脈を転送します。穏やかに48時間揺らしながら4℃でバイアルに保管してください。 </li><li>それは組織学のために処理されるまで保存するために、70％エタノールに固定動脈を転送します。 </li><li>パラフィンブロックとセクション染色のためのブロックの中で動脈を埋め込みます。 </li><li>傷害および内膜HYPの程度を評価するために組織化学的および免疫化学染色を行いますerplasia。 注：代表的な結果のために、我々は、弾性層板や他の動脈のコンポーネントを視覚化するために、核および全体的な形態またはモバットのペンタクロム染色を可視化するためにヘマトキシリン​​およびエオシンを使用していました。 </li></ol>

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W., Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Rat+and+Mouse+Carotid+Artery+Surgery:+Injury+&amp;+Remodeling+Studies.">Experimental Rat and Mouse Carotid Artery Surgery: Injury &amp; Remodeling Studies.</a> ISRN Minim Invasive Surg. 2013, (2013).</li><li>Iafrati, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+inhibits+the+vascular+injury+response+in+estrogen+receptor+alpha-deficient+mice.">Estrogen inhibits the vascular injury response in estrogen receptor alpha-deficient mice.</a> Nat Med. 3 (5), 545-548 (1997).</li><li>Sullivan, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estrogen+inhibits+the+response-to-injury+in+a+mouse+carotid+artery+model.">Estrogen inhibits the response-to-injury in a mouse carotid artery model.</a> J Clin Invest. 96 (5), 2482-2488 (1995).</li><li>Yin, Y., Zhao, X., Fang, Y., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carotid+artery+wire+injury+mouse+model+with+a+nonmicrosurgical+procedure.">Carotid artery wire injury mouse model with a nonmicrosurgical procedure.</a> Vascular. 18 (4), 221-226 (2010).</li><li>Nam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li><li>Nam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+disturbed+flow-induced+atherosclerosis+in+mouse+carotid+artery+by+partial+ligation+and+a+simple+method+of+RNA+isolation+from+carotid+endothelium.">A model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of RNA isolation from carotid endothelium.</a> J Vis Exp. (40), (2010).</li><li>Kuhlmann, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Implantation+of+a+carotid+cuff+for+triggering+shear-stress+induced+atherosclerosis+in+mice.">Implantation of a carotid cuff for triggering shear-stress induced atherosclerosis in mice.</a> J Vis Exp. (59), (2012).</li><li>Carmeliet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+wound+healing+and+neointima+formation+induced+by+perivascular+electric+injury+in+mice.">Vascular wound healing and neointima formation induced by perivascular electric injury in mice.</a> Am J Pathol. 150 (2), 761-776 (1997).</li></ol>

We have presented a method for performing vascular injury in mice and delivery therapeutic compounds to the injured region through a perivascular cuff. The ligation method for femoral and carotid arteries has been described in conventional methods papers and characterized extensively11-14,19-23 and we present an alternative method for achieving the same vascular injury that is less technically demanding procedure that can often be used in younger mice. One of the chief advantages of the using a mouse wire inju...

Arterial injury and inflammation caused by angioplasty and stent implantation can induce neointimal hyperplasia that contributes to the thickening of the arterial wall, a process known as restenosis.1,2 The formation of restenosis is major mode of failure for interventions such as angioplasty and stenting with bare metal stents.3 Due to recent concerns with the inhibition of vascular healing in arteries treated with drug eluting stents, there is also a need to find compounds that can inhibit restenosis while maintaining vascular healing and re-endothelialization.4-7 In addition, while stents have had success in the coronary vasculature, percutaneous interventions of all types in the peripheral arteries continue to fail at a higher rate due to restenosis.8-10 Mouse models of surgical interventions allow the use of powerful genetic manipulations that can provide mechanistic insight into the mechanisms underlying the failure of clinical therapies and can provide an initial test bed for compounds to inhibit intimal hyperplasia. 
Here, we describe a mouse model of vascular injury that allows the testing of therapeutic compounds to inhibit neointimal hyperplasia and assess whether their effects on re-endothelialization following endothelial denudation. A key challenge in executing vascular injury in mice is the technical skill needed to obtain vascular access and to restore flow to the injured artery following the wire injury. For this reason, simple arterial ligation models have been used to study neointimal hyperplasia in mice that do not require endovascular manipulations but are easier to implement.11 However, this type of surgical model differs substantially from the mechanical and biological aspects of a percutaneous intervention, lacking key aspects including arterial wall stretch, endothelial denudation and luminal blood flow following injury. 
We present two methods for obtaining and closing vascular access for wire injury of the femoral artery in mice. The first technique is the conventional method described by several groups previously and uses vascular access through a side branch of the femoral artery.12-14 This method requires older, larger mice and more surgical skill to implement the endoluminal access through the smaller artery. It also requires the ligation of the muscular branch of the femoral artery following the procedure. The second method we describe uses an arteriotomy in the branch point of the main and side branch and thereby allows for a larger access to the artery for performing wire injury. In this method, the arteriotomy is closed using controlled local cauterization that leaves both branches with blood flow following the procedure. The conventional method is applicable to mice of at least 20 weeks of age while the alternative method can be used in mice of at least 15 weeks of age. In both methods, the wire creates arterial stretch and abrasion leading to injury and endothelial denudation. Following either procedure a perivascular drug delivery patch can be implanted that allows the delivery of compounds to alter the response to injury. The use of the drug delivery patch allows mice to be used as a test bed for new compounds to inhibit restenosis through perivascular therapies.15,16

<table border="0" cellpadding="0" cellspacing="0" width="956">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Name</td>
			<td style="width:239px;">Company</td>
			<td style="width:239px;">Catalog Number</td>
			<td style="width:239px;">Comments</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Straight spring wire, 0.15&#8221; diameter</td>
			<td style="width:239px;">Cook</td>
			<td style="width:239px;">G02426</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High Temperature cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">HIT1</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">High-temperature fine tip for cautery</td>
			<td style="width:239px;">Bovie Medical Corp.</td>
			<td style="width:239px;">H101</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Micro-scissors</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">15000-13</td>
			<td style="width:239px;">For performance of arteriotomy</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Angled fine-tipped forceps</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">11251-35</td>
			<td style="width:239px;">For blunt dissection of vascular bundle</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Angled forceps</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5069</td>
			<td style="width:239px;">For clearing tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Surgical scissors</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-5840</td>
			<td style="width:239px;">For cutting skin</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-10</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Retractor wire</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-05</td>
			<td style="width:239px;">Attached to retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Base plate</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-03</td>
			<td style="width:239px;">For use with retractor</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Magnetic retractor fixator</td>
			<td style="width:239px;">Fine Science Tools</td>
			<td style="width:239px;">18200-01</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Needle Holder</td>
			<td style="width:239px;">Roboz</td>
			<td style="width:239px;">RS-7822</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Hemostatic forceps</td>
			<td style="width:239px;">Biomedical Research Instruments, Inc.</td>
			<td style="width:239px;">34-1000</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Dissecting microscope</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">EMZ-5TR</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Microscope light source</td>
			<td style="width:239px;">Meiji Techno</td>
			<td style="width:239px;">FT191</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Warm water recirculator</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-500</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Reusable heating pad</td>
			<td style="width:239px;">Gaymar</td>
			<td style="width:239px;">TP-R 22G</td>
			<td style="width:239px;">For maintaining mouse body temperature</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lidocaine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">4.0 Vicryl suture with half circle needle</td>
			<td style="width:239px;">Ethicon</td>
			<td style="width:239px;">J494G</td>
			<td style="width:239px;">For post-surgical wound closure</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Sterile cotton-tipped applicators</td>
			<td style="width:239px;">Puritan</td>
			<td style="width:239px;">25-806-2WC</td>
			<td style="width:239px;">For application of depilatory cream and absorbing fluids</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Depilatory cream</td>
			<td style="width:239px;">Nair</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Isoflurane</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Betadine</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">6.0 braided silk suture</td>
			<td style="width:239px;">Teleflex Medical</td>
			<td style="width:239px;">4-S</td>
			<td style="width:239px;">For isolation of femoral artery during surgery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">0.9% sodium chloride</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;">For irrigating tissues</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Gel eye lubricant</td>
			<td style="width:239px;">Various</td>
			<td style="width:239px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Glass petri dish</td>
			<td style="width:239px;">Pyrex</td>
			<td>3160-60</td>
			<td>For femoral artery harvest</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">10% buffered formalin</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">70% ethanol</td>
			<td style="width:239px;">Various</td>
			<td></td>
			<td style="width:239px;">For fixation of femoral artery</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Bouin&#39;s fluid</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td></td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcian blue, 1%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-01</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alkaline alcohol</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-02</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Orcein, 0.2%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-03</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Hematoxylin alcoholic, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-04</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Ferric chloride, 10%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-05</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Lugol&#39;s Iodine</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-06</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:239px;">Woodstain scarlet-acid fuchsin working solution</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-07</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Acetic acid, 0.5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>Various</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Phosphotungstic acid, 5%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-09</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:239px;">Alcoholic saffron, 6%</td>
			<td style="width:239px;">Electron Microscopy Sciences</td>
			<td>26385-10</td>
			<td style="width:239px;">For Movat&#39;s Pentachrome staining</td>
		</tr>
	</tbody>
</table>

murine model of femoral artery wire injury with implantation of a perivascular drug delivery patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

ワイヤー損傷後、新生内膜過形成は、時間をかけて開発し、一般的に14〜28日後に検査される。 図3の組織学的結果を示すように、マウスにおける内膜過形成の堅固な生成に、この作業のリードに記載された技術は、無傷の大腿動脈は、無傷の弾性ラメラと通常の厚さ及び外周を実証する。負傷した大腿動脈は、内膜過形成を示し、弾性ラメラが劣化し、後の時点で再内皮のデモ?...

Watch this Scientific Journal Video about Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch at JoVE.com

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

murine-model-femoral-artery-wire-injury-with-implantation

Research

JoVE Journal

Medicine

17.9K ビュー.  University of Texas at Austin. We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis. 

ビデオ: Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

自動ワイヤの筋運動記録法を用いた腸間膜動脈の収縮と弛緩の研究

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

医学

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

再狭窄の研究のための頸動脈内ステント留置のマウスモデル

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

左前下の動脈の結紮を通して心筋虚血再灌流傷害のマウスモデル

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

A Simple Critical-sized Femoral Defect Model in Mice

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all limb bone fractures fail to heal completely due to the extent of the trauma, disease, or age of the patient. Our ability to improve bone regenerative strategies is critically dependent on the ability to mimic serious bone trauma in test animals, but the generation and stabilization of large bone lesions is technically challenging. In most cases, serious long bone trauma is mimicked experimentally by establishing a defect that will not naturally heal. This is achieved by complete removal of a bone segment that is larger than 1.5 times the diameter of the bone cross-section. The bone is then stabilized with a metal implant to maintain proper orientation of the fracture edges and allow for mobility. 
Due to their small size and the fragility of their long bones, establishment of such lesions in mice are beyond the capabilities of most research groups. As such, long bone defect models are confined to rats and larger animals. Nevertheless, mice afford significant research advantages in that they can be genetically modified and bred as immune-compromised strains that do not reject human cells and tissue.
Herein, we demonstrate a technique that facilitates the generation of a segmental defect in mouse femora using standard laboratory and veterinary equipment. With practice, fabrication of the fixation device and surgical implantation is feasible for the majority of trained veterinarians and animal research personnel. Using example data, we also provide methodologies for the quantitative analysis of bone healing for the model.

Animal models are frequently employed to mimic serious bone injury in biomedical research. Due to their small size, establishment of stabilized bone lesions in mice are beyond the capabilities of most research groups. Herein, we describe a simple method for establishing and analyzing experimental femoral defects in mice.

マウスの簡単な重要なサイズの大腿欠陥モデル

While bone has a remarkable capacity for regeneration, serious bone trauma often results in damage that does not properly heal. In fact, one tenth of all ...

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.

The mouse femoral artery wire injury model of restenosis is technically challenging. In this protocol we show the key technical details essential for successfully performing wire injury to induce consistent&#160;neointima for studies of restenosis.

動脈再狭窄のマウスモデル：大腿ワイヤー傷害の技術的な側面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

Murine Model of Intestinal Ischemia-reperfusion Injury

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, hypotension, necrotizing enterocolitis, bowel transplantation, trauma and chronic inflammation. Intestinal ischemia-reperfusion (IR) injury is a consequence of acute mesenteric ischemia, caused by inadequate blood flow through the mesenteric vessels, resulting in intestinal damage. Reperfusion following ischemia can further exacerbate damage of the intestine. The mechanisms of IR injury are complex and poorly understood. Therefore, experimental small animal models are critical for understanding the pathophysiology of IR injury and the development of novel therapies.
Here we describe a mouse model of acute intestinal IR injury that provides reproducible injury of the small intestine without mortality. This is achieved by inducing ischemia in the region of the distal ileum by temporally occluding the peripheral and terminal collateral branches of the superior mesenteric artery for 60 min using microvascular clips. Reperfusion for 1 hr, or 2 hr after injury results in reproducible injury of the intestine examined by histological analysis. Proper position of the microvascular clips is critical for the procedure. Therefore the video clip provides a detailed visual step-by-step description of this technique. This model of intestinal IR injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

Here we describe the detailed procedure of intestinal ischemia-reperfusion in mice which results in reproducible injury without mortality to encourage the standardization of this technique across the field. This model of intestinal ischemia-reperfusion injury can be utilized to study the cellular and molecular mechanisms of injury and regeneration.

腸管虚血 - 再灌流傷害のマウスモデル

Intestinal ischemia is a life-threatening condition associated with a broad range of clinical conditions including atherosclerosis, thrombosis, ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

塩化第二鉄によるイヌ総頚動脈血栓症: 血管損傷の大動物モデル

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

過期治療アプリケーションを用いてラット頸動脈圧制御区分バルーン損傷

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...