著者は、技術的な喜びヤオ博士カレンアルトからのサポートだけでなく、NHMRCとNHFからの資金を承認したいと思います。

動物に関わるすべての実験はアルフレッド医学研究および教育分署動物倫理委員会（E / 2015分の1534 / B）によって承認されました。すべての外科的操作は、全身麻酔下で行われ、動物がどの段階でも痛みを経験しませんでした。記載されている全ての実験は、非回復しています。 1.準備<ol><li>フィルターペーパー（1ミリメートル×2ミリメートル）の薄​​いバンドをカットします。 </li><li>新たに4％の塩化第二鉄の2つの溶液を調製し（w / v）の6％（W / V）の脱イオン水に希釈しました。 0.22μmのを介して濾過し、PBS中のローダミン6G液0.3％を、準備します。 </li><li>シリンジラッパーの白いプラスチックの小片（5ミリメートル×1センチ）カット。 </li></ol>生体顕微鏡で観測された2腸間膜細動脈の血栓形成<ol><li> 10-12週齢の雄のC57BL / 6野生型マウスを秤量し、腹腔内注射しかしケタミンの混合物（100mg / kg）およびキシラジン（10mg / kg）で麻酔に応じ。つま先、尾および/または皮膚のピンチに反応し、角膜および眼瞼ピンチからの反応性、およびウィスカの動きが存在しないことによって麻酔の深さを監視します。必要に応じて、動物の麻酔を維持するために、ケタミンの第二の用量（50mg / kg）を注入します。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を適用します。 37℃に調整し、加熱パッド上に配置された小さなペトリ皿にマウスを置きます。 注：IP注射は、動物のための痛みをもたらす可能性があるが、この不快感の持続時間は（3秒未満）最小限になります。注射に伴う痛みや不快感は、経験豊富で有能な人材と適切な針のサイズ（25 G）を使用することによって最小化されるであろう。すべての手順の後、頸椎脱臼が続くケタミンとキシラジンの過剰投与を使用して、すべての動物を安楽死させます。 </li><li>皮膚に約3cmの腹部正中切開を行い、慎重に腹膜を切りました。 </li><li>ペットの側にマウスを置きますRI料理は、静かにその腸を体外に出す、慎重にペトリ皿の表面に適切な容器を持って来るために2綿棒で腸間膜を広げます。繊細なワイパーと正しく乾燥させます。 NOTE：腸間膜血管の動きを制限するために、パパベリンは、腸の蠕動運動を阻害するために使用することができます。 </li><li>血管を拡張し、白血球と血小板を標識するために29 Gシリンジでマウスの尾静脈にローダミン6G（0.3％）の30μLを注入するために温水にマウスの尾を浸します。 </li><li>倒立顕微鏡下でペトリ皿を置き、明視野チャネルを使用して、選択された細動脈に焦点を当てています。 </li><li> 6％（w / v）の塩化鉄（III）でろ紙のバンドを浸し二鉗子で動脈に濾紙を適用します。最初のものは、ろ紙、穏やかに関心のある領域にそれを押して第二のものを保持します。ろ紙の堆積後の最初の10秒で血栓形成を観察します。 注：それは非常にCOMMです囲まれた微小血管系とろ紙の堆積と優しく圧力の精度を傷つけるためにでこの問題を制限することが重要です。 </li><li>ろ紙、：（ピーク励起557 nmの発光ピーク576 nmのTRITCチャネル）蛍光顕微鏡により血栓形成を観察します。血栓にローダミン6Gおよびそれらの凝集を取り上げてきた循環白血球と血小板を観察することは、識別することは容易です。 </li><li>塩化鉄（III）への曝露の1分後にろ紙を脱いで、血栓の形成を監視し続けます。 PBSで容器を洗ってください。 </li><li>ローダミン6Gと血小板および白血球の標識で強調血栓の動的生成を観察し、記録します。画像をキャプチャし、血栓の大きさを測定します。本明細書に提示される画像は、TRITC蛍光チャネルにおける対物4X、ものの、倒立生体顕微鏡を用いて得ました。 </li><li> Fすべての手順をollowing、頸椎脱臼が続くケタミンとキシラジンの過剰摂取を用いて動物を安楽死させます。 </li></ol> 3.頸動脈血栓形成は、血流速測定により評価し<ol><li> 10〜12週齢の雄をC57BL / 6マウスを秤量し、腹腔内注射しかしケタミン（100mg / kg）およびキシラジン（10mg / kg）の混合物に応じて麻酔。麻酔下ながら乾燥を防ぐために、目に獣医軟膏を適用します。 </li><li> 37℃に調整し、加熱パッド上に、足に粘着テープを使用して、手術用顕微鏡下でマウスを修正しました。 </li><li> 1つのワイパーの外に小さな枕を作り、少し頭を上昇させるために、マウスの頭の下にそれをテープで固定します。 （上の歯を使用します）鼻をプルダウンするトングと糸ループを使用してください。これは、簡単にアクセスするための頸動脈の領域を露出します。 </li><li>ダウン胸骨へ、直接顎の下の皮膚の小さな深さ5mmの切開を行います。 </li><li> FAを分析SCIAと分岐上記左または右総頸動脈のいずれかの断片を分離します。 </li><li>慎重イン間の動脈とそれらを分離するための神経ピンセットをご紹介します。近い動脈に実行して神経を妨害し、血管への損傷を引き起こすことができるように頸動脈のあまり触れないように注意してくださいしないでください。動脈の5mm以上のセクションを分離します。 </li><li>任意の液体がFeCl 3をを妨害することを防ぐため、適切にワイパーと動脈の領域を乾燥させます。 </li><li>のFeCl 3は、周囲の組織に浸していないので、総頸動脈の単離された部分の下に小さな白いプラスチック片を入れてください。この目的のために、ゆっくりと動脈の下にプラスチック製の用紙をスライドさせ、次に第1のピースをもたらすために第二の鉗子を使用しています。 </li><li> 4％（w / v）の6％（w / v）の塩化第二鉄で濾紙を​​浸し、動脈の周りにそれをすべて置きます。 </li><li> 3分間の露光後、ろ紙を脱いで、WエリアをPBSで洗浄し、乾燥i番目のワイパー。 </li><li>損傷領域で容器の周りにドップラーフロープローブを配置し、流れの変化の記録を開始。成体マウスの健康な総頸動脈では、流れは、通常、約1ml /分です。注意！塩化第二鉄とプローブの接触は、任意の接触は避けるべきであるので、プローブを損傷します。本明細書に提示されるデータは、ナノドップラーフロープローブ0.5 PBSを備えた遷音速システム株式会社流量計、TS420血管周囲モジュールを得ました。 注：塩化第二鉄の濃度が異なるオクルージョン時間が生じる血栓形成の動態を変更することができます。このように、塩化第二鉄（w / v）の6％への曝露は、塩化第二鉄（w / v）の4％に曝露よりも​​速く閉塞をもたらします。 </li><li>すべての手順の後、頸椎脱臼が続くケタミンとキシラジンの過剰投与を使用して、すべての動物を安楽死させる、慎重にドップラープローブを清掃してください。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transendothelial+migration+of+ferric+ion+in+FeCl3+injured+murine+common+carotid+artery.">Transendothelial migration of ferric ion in FeCl3 injured murine common carotid artery.</a> Thromb Res. 118 (2), 275-280 (2006).</li><li>Bonnard, 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=Leukocyte+mimetic+polysaccharide+microparticles+tracked+in+vivo+on+activated+endothelium+and+in+abdominal+aortic+aneurysm.">Leukocyte mimetic polysaccharide microparticles tracked in vivo on activated endothelium and in abdominal aortic aneurysm.</a> Acta Biomater. 10 (8), 3535-3545 (2014).</li><li>Boulaftali, Y., Lamrani, L., 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=The+mouse+dorsal+skinfold+chamber+as+a+model+for+the+study+of+thrombolysis+by+intravital+microscopy.">The mouse dorsal skinfold chamber as a model for the study of thrombolysis by intravital microscopy.</a> Thromb Haemost. 107 (5), 962-971 (2012).</li><li>Konstantinides, S., Sch&#228;fer, K., Thinnes, T., Loskutoff, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasminogen+activator+inhibitor-1+and+its+cofactor+vitronectin+stabilize+arterial+thrombi+after+vascular+injury+in+mice.">Plasminogen activator inhibitor-1 and its cofactor vitronectin stabilize arterial thrombi after vascular injury in mice.</a> Circulation. 103 (4), 576-583 (2001).</li><li>Li, W., McIntyre, T. M., Silverstein, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferric+chloride-induced+murine+carotid+arterial+injury:+A+model+of+redox+pathology.">Ferric chloride-induced murine carotid arterial injury: A model of redox pathology.</a> Redox Biol. 1 (1), 50-55 (2013).</li></ol>

The authors have nothing to disclose.

塩化第二鉄誘発される血栓症モデルは、優れた研究ツールです。この研究で示されるように、実装が非常に容易であり、生体顕微鏡またはドップラー流量計と組み合わせて使用​​する場合には、血栓形成の良好なリアルタイムモニタリングを提供します。長時間露光とのFeCl 3の濃度を調整する、それはまた、非閉塞または閉塞のいずれか血栓を生成する可能性を提供しています。 <...

血栓症および抗血栓薬の有効性の評価の開発に関与するメカニズムの研究は、十分に実験動物モデルを確立が必要です。大型動物モデルは、それらが齧歯類1よりヒトに類似大型船を提供するように使用される最初でした。しかし、高コストは、必要な大きな施設とそれらを操作する上での困難は、遺伝的にそれらの使用に大きな欠点であり、げっ歯類の予備試験は決定的な結果を2与えた後に大型動物は、現在後期前臨床研究に制限されています。トランスジェニックおよびノックアウト株の広い可用性とインビボ試験のために必要な抗血栓薬の量を最小限に抑え、その小さなサイズで、マウスは主に血栓症の研究のために使用されています。したがって、血栓障害のいくつかのモデルは、マウス3で開発されてきました。 多くの確立された血栓症モデルがintimを混乱させる血栓4の形成を誘導する血流のサブ内皮細胞外マトリックスの暴露に続いて、血管壁の層。血栓は/及び凝固カスケード5を活性化する組織因子の曝露または血小板の活性化を引き起こすコラーゲンの曝露から生じ得ます。いくつかの技術が、初期血管損傷を達成するために使用されます。 Pierangeli らは大腿静脈6のマイクロサージェリーツールを使用して機械的破壊モデルを開発しました。 Kikushi ら緑色光（540 nm）の7と関心の血管壁の特定の励起に続いて内皮細胞の脂質二重層内に蓄積する光反応性化合物（ローズベンガル）を投与することからなるモデルを説明しました。損傷はまた、短い高強度のパルスレーザー照射8によって誘導することができます。別の技術は、最初にラットの頸動脈に設立します塩化第二鉄（FeCl 3を ）9の局所適用に構成されています。この場合、容器の露出は、内皮細胞10の脂質過酸化及び破壊を引き起こすのFeCl 3で生成されたフリーラジカルに起因します。損傷は、血小板粘着及び凝集、並びに白血球の動員を誘発するいくつかの接着分子の発現を誘導します。これは、白血球、特に好中球は、血栓11につながる血液凝固カスケードの活性化に重要な役割を果たしていることが実証されています。この方法は、凝固カスケードを再現するのに適しています。研究者らは、ヒトにおける血栓症は、主に病気の例で発生しているのに対し、このマウスモデルにおいて、血栓症は、通常、健康な血管において誘導され、心に留めておく必要があります。アテローム性動脈硬化の血管。 このモデルは、実装が非常に簡単であり、また、マウスにおいて有効であるように、それは今、主に使用される血栓症のモードですin vivo試験での小動物用L。また、この技術は、血管の様々な血栓の形成を誘導する可能性を提供します。ターゲット容器は大口径（頸動脈、大腿、大静脈）または小径（腸間膜、精巣挙筋）12-14の動脈または静脈することができます。より最近では、それはまた、脳卒中15のモデルを開発するために、近位中大脳動脈で使用されました。血栓形成は、直接血小板および白血球の蛍光標識した後、生体顕微鏡検査によって観察または温度プローブまたはドップラープローブ12,16,17との血流の減少を測定することによってモニターすることができます。このような閉塞時間、血栓形成時間または血栓サイズなど、い​​くつかのパラメータは、検討してもよいです。容器間の生理的な違いは、得られた血栓の大幅な変動に結果を検討しました。そのため、研究者らは、通常、彼らがmeasuするパラメータに応じて標的血管を選択します再および/またはそれらが調査する疾患の設定。典型的には、頸動脈のモデルは、大静脈の研究は、深部静脈血栓症の研究のために、より関連性の高いのに対し、心筋梗塞や脳卒中に関連するアテローム血栓症の研究のためのより適切です。異なる容器のアクセスも血栓の成長を測定するために使用される方法を決定します。例えば、腸間膜血管が生体内顕微鏡観察および血栓形成のダイナミクスの研究のためのこのモデルは非常に適して作るのアクセスが容易です。頸動脈は、血流の測定を可能にし、閉塞性血栓症を研究するための優れたモデルを提供しアクセスしにくいが、大きいです。 塩化第二鉄誘発される血栓症モデルは、この病理学の理解に多大な進歩を提供してきました。これは、血栓形成18,19におけるフォン·ヴィレブランド因子の役割に焦点を当てた多くの研究で使用されています。遺伝MODIと組み合わせfication技術は、それは、血栓性疾患に関与する多くの特定の遺伝子の同定を可能にしました。ラムラニら 。例えばJAK2 V617F遺伝子のノックインが不安定血餅20の加速形成に関連していることが示されています。 Zhang らは、P2Y12血小板受容体の生理的意義を調査し、血小板に特異的にこの受容体を過剰発現するトランスジェニックマウスのみを、FeCl 3を 21で損傷した腸間膜動脈においてより迅速で安定した血栓の形成を示したことを実証しました。フィブリン分解プロセスにおける組織型プラスミノーゲン活性化因子（tPA）およびウロキナーゼ型プラスミノーゲン活性化因子（uPA）の重要な役割は、この方法22において検討されています。さらにこのモデルはまた、 インビボで多くの新規薬剤のフィブリン溶解能力をテストする簡単かつ正確な方法を提供します。例えば、Wang らは、目のためにこのモデルを使用しています活性化血小板23を標的と新規組換えプラスミノーゲン活性化因子のE前臨床検証。この方法は、ダニ、吸血コウモリ、及び蚊の唾液から、またはターゲット24〜27の固有の識別とヘビの毒から単離された治療用タンパク質の検証を可能にしました。これらの例は、塩化第二鉄モデルの汎用性を実証します。本稿では、二つの方法に焦点を当て、二つの異なる容器の種類に塩化第二鉄誘発される血栓症を研究します。腸間膜血管および頸動脈。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Whatman chromatography paper</td>
			<td>GE Healthcare</td>
			<td>3030917</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) chloride 40 % (w/v)</td>
			<td>VWR</td>
			<td>24212.298</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine 6G</td>
			<td>Sigma</td>
			<td>R4127</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope&#160;</td>
			<td>Olympus</td>
			<td>IX81</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital black-and-white camera&#160;</td>
			<td>Olympus</td>
			<td>XM10</td>
			<td></td>
		</tr>
		<tr>
			<td>Doppler flowmeter</td>
			<td>Transonic</td>
			<td>TS420</td>
			<td></td>
		</tr>
		<tr>
			<td>Nano-doppler flow probe</td>
			<td>Transonic</td>
			<td>0.5 PBS</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Hospira&#160;</td>
			<td>0409-2051-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine (Rampun)</td>
			<td>Bayer</td>
			<td>75313&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Sarstedt</td>
			<td>82.1472</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe (29 G)</td>
			<td>BD Ultra-Fine</td>
			<td>326103</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton tipped applicators</td>
			<td>BSN medical</td>
			<td>211827A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynek dysilk sutures</td>
			<td>Dynek Pty Ltd</td>
			<td>CS30100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffer saline (PBS)</td>
			<td>Gibco life technologies</td>
			<td>21600-069</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Kirchner</td>
			<td>T60</td>
			<td></td>
		</tr>
	</tbody>
</table>

ferric chloride-induced thrombosis mouse model on carotid artery and mesentery vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

ローダミン6Gの蓄積を明らかにする腸間膜の蛍光生体顕微鏡観察は、FeCl 3をすることによって損傷した血管壁に沿って、血小板と白血球を標識しました。部分的な血栓形成の進行は200ミクロンの腸間膜血管（ 図1）でモニタされます。血栓がゆっくりと表示され、FeCl 3をへの暴露の最初の分（ 図1、tは60秒=）の後に明らかに識別可能です。 40秒のFeCl <s...

Watch this Scientific Journal Video about Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel at JoVE.com

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

塩化第二鉄誘発性頚動脈および腸間膜血管に血栓症のマウスモデル

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

ferric-chloride-induced-thrombosis-mouse-model-on-carotid-artery

Research

JoVE Journal

Medicine

17.3K ビュー.  Baker IDI Heart and Diabetes Institute. The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

ビデオ: Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

部分的ライゲーションによるマウス頸動脈における乱流誘発アテローム性動脈硬化症と頸動脈内皮からのRNA単離の方法における簡便な方法のモデル

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

医学

Mouse Complete Stasis Model of Inferior Vena Cava Thrombosis

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE). In the United States (U.S.), the high morbidity and mortality rates make VTE a serious health concern 1-2. After heart disease and stroke, VTE is the third most common vascular disease 3. In the U.S. alone, there is an estimated 900,000 people affected each year, with 300,000 deaths occurring annually 3. A reliable in vivo animal model to study the mechanisms of this disease is necessary.
The advantages of using the mouse complete stasis model of inferior vena cava thrombosis are several. The mouse model allows for the administration of very small volumes of limited availability test agents, reducing costs dramatically. Most promising is the potential for mice with gene knockouts that allow specific inflammatory and coagulation factor functions to be delineated. Current molecular assays allow for the quantitation of vein wall, thrombus, whole blood, and plasma for assays. However, a major concern involving this model is the operative size constraints and the friability of the vessels. Also, due to the small IVC sample weight (mean 0.005 grams) it is necessary to increase animal numbers for accurate statistical analysis for tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent (ELISA), zymography, vein wall and thrombus cellular analysis, and whole blood and plasma assays 4-8.
The major disadvantage with the stasis model is that the lack of blood flow inhibits the maximal effect of administered systemic therapeutic agents on the thrombus and vein wall.

The mouse complete stasis model of inferior vena cava thrombosis yields quantifiable amounts of vein wall tissue and thrombus. It has proven useful for evaluating interactions between the vein wall and the occlusive thrombus and in assessing the progression from acute to chronic inflammation.

下大静脈血栓症のマウス完全な停滞のモデル

Venous thromboembolism (VTE) includes both deep vein thrombosis (DVT) and pulmonary embolism (PE).  In the United States (U.S.), the high morbidity and ...

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

中大脳動脈閉塞のマウスモデル

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成体マウス静脈性高血圧モデル：外頸静脈吻合部の総頸動脈。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

ラット頸動脈における血管バルーン傷害と管腔内投与

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

ラット頸動脈バルーン損傷モデルは、抗血管リモデリングの治療薬をテストします

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Ferric Chloride-induced Murine Thrombosis Models

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, diabetes, obesity, cancer and chronic autoimmune rheumatologic disorders. Thrombi are the cause of most heart attacks, strokes and extremity loss, making thrombosis an extremely important public health problem. Since these thrombi stem from inappropriate platelet activation and subsequent coagulation, targeting these systems therapeutically has important clinical significance for developing safer treatments. Due to the complexities of the hemostatic system, in vitro experiments cannot replicate the blood-to-vessel wall interactions; therefore, in vivo studies are critical to understand pathological mechanisms of thrombus formation. To this end, various thrombosis models have been developed in mice. Among them, ferric chloride (FeCl3) induced vascular injury is a widely used model of occlusive thrombosis that reports platelet activation and aggregation in the context of an aseptic closed vascular system. This model is based on redox-induced endothelial cell injury, which is simple and sensitive to both anticoagulant and anti-platelets drugs. The time required for the development of a thrombus that occludes blood flow gives a quantitative measure of vascular injury, platelet activation and aggregation that is relevant to thrombotic diseases. We have significantly refined this FeCl3-induced vascular thrombosis model, which makes the data highly reproducible with minimal variation. Here we describe the model and present representative data from several experimental set-ups that demonstrate the utility of this model in thrombosis research.

We report a refined procedure of the ferric chloride (FeCl3)-induced thrombosis models on carotid and mesenteric artery as well as vein, characterized efficiently using intravital microscopy to monitor time to occlusive thrombi formation.

塩化第二鉄誘発性マウス血栓症モデル

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

ラットの総頸動脈で位相コントラスト磁気共鳴イメージング

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

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, ...