This work was funded by the National Institute of Health, the National Heart Lung and Blood Institute (NIH and NHLBI; HL125572-01A1) and the LSUHSC-S start up fund to F.N.E. Gavins.

このプロトコルとビデオで報告した実験はLSUHSC-S施設内動物管理使用委員会によって承認され、NIHのガイドラインに準拠しているました。 注：25体重の雄C57BL / 6マウス - 47 gをこの研究で使用しました。マウスは個別換気ケージで12時間の明/暗サイクルの下で、水に自由にアクセスさせて、標準的な固形飼料ペレット食で維持しました。手順は、滅菌技術（ 例えば 、滅菌手袋、滅菌器具）を用いて無菌条件下で行われます。 1.手術前の準備<ol><li>ケタミン（150ミリグラム/ kg）およびキシラジン（10mg / kg）の組み合わせを使用して麻酔を誘導腹腔内（IP）を注射しました。麻酔が達成された後、5分及び10分ごとに - 最初は足のピンチごとに3によって麻酔深度を監視します。動物が麻酔下にある間、乾燥を防ぐために無菌眼軟膏を管理します。ケタミン/キシラジンの初期投与量は、通常、AB持続します30-40分アウト。足のピンチに対する動物の応答により検証され、必要に応じて追加の用量を投与することができます。 </li><li>温度調節熱マットの上に仰臥位でマウスを置き、直腸プローブを使用して検証されて36.5±0.1℃、で体温を維持。 </li><li>首を剃ると、70％のエチルアルコールで皮膚を消毒します。 </li></ol> MCAの2閉塞（図1） <ol><li>アイリスストレートはさみを使用して、正中頸部切開を行い、血管を露出させるために軟組織を（リトラクターを使って）後退させます。 </li><li>迷走神経を損傷することなくデュモンの鉗子を使用して周囲の組織からCCAとECAを解剖。 </li><li> 6-0シルクを使用して、CCAの周りの緩い結び目を結ぶことにより、一時的な縫合糸を作ります。 </li><li> ECAの周りに永久縫合糸、しっかりと（CCAの分岐に対して遠位）血管を結紮することによって、それから延びる小型船を作ります。 </li><li>縫合糸AROを作りますCCAの分岐にECAの近位をUND。 </li><li>内頸動脈（ICA）と翼口蓋動脈（PPA）の周りの微小血管クリップを配置します。 </li><li>春のはさみを解剖マイクロを使用して、ECAの小さな切開を行い、180μmのシリコンチップ付きモノフィラメントを挿入します。フィラメントのより容易な操作のために可能な限り永久縫合糸へのカットはの近くにしてください。 </li><li>挿入されたフィラメントとECAの周りに一時的な縫合糸を締めて、微小血管クリップを削除します。 </li><li>永久、遠位縫合糸と春のはさみを解剖マイクロを使用して、フィラメントのエントリポイント間のECAをカットします。 </li><li> MCAに - （CCAの分岐を超えて10μmの約9）抵抗が感じられるまでICAを通してフィラメントを案内します。 注：フィラメントの大部分はまだ表示されている間あまり抵抗が感じられる場合には、フィラメントは、PPAを入力した可能性があります。この問題が発生した場合は、ゆっくり膿、分岐点に戻って、フィラメントを引っ張りますICAへの前進時間デュモンの鉗子を使用して、それがICAで可視化することができるまで、フィラメントを進めます。 </li></ol> 3.再灌流<ol><li> 30分の閉塞期間の後、静かにデュモンの鉗子を使用して、それを引き戻すことにより、フィラメントを除去し、ECAの開放端の周りに縫合糸を固定します。 </li><li>慎重デュモンの鉗子を使用して、合字を緩めて血流がCCAを通して再開されることにより、CCAの周りに一時的な縫合糸を取り外します。 </li><li>連続外科手術用縫合糸で切開を閉じます。皮膚の閉鎖は、連続又は中断どちら縫合することによって達成することができます。スキンステープルも許容方法です。 </li><li>ボリューム補充などの鎮痛カルプロフェンと生理食塩水を皮下の1ミリリットルをマウスに注入する（5ミリグラム/キログラム、SC）の外科的処置、追加の疼痛緩和がバック猫背含ま必要であることを示す兆候、ungroomedコートからの痛みや不快感の軽減のため、異常な、活性の低下姿勢、そして食欲を減少させました。 </li><li>温度コントローラーを使用して、規制ヒートランプを使用して加熱された30℃のケージ内の麻酔からの回復を通じてマウスを観察し、食べることを奨励するために、ケージの床にペトリ皿にマッシュ飼料を配置します。マウスは、再灌流期間中のケージ当たり1匹収容されます。 </li></ol> 4.シャム手術<ol><li>モノフィラメント挿入せずに、同じ手順に従うことをマウス。 </li></ol> 5.術後の神経学的スコア（表1;図2） <ol><li>神経学的全般を評価する18ポイントスコアリングシステムを用いて適切な再灌流期間の後、マウスを評価します。モーター;感覚;固有感覚。高い神経学的スコアは、神経学的機能の低下に相当します。 注：応答しないと歩くことができないと判断されたマウスは安楽死させます。人道的な安楽死のための他の基準は20％以上の体重減少を含む、呼吸ジ手術領域の周りのストレス、および感染症。 CO 2チャンバーは、安楽死のために使用され、死を確認するための物理的方法は、頸椎脱臼または開胸になります。 </li></ol>測定脳梗塞体積の6（図3） <ol><li>麻酔が達成された後5分および10分毎 - ケタミン（150ミリグラム/ kg）およびキシラジン（10mg / kg）を最初に足ピンチ毎に3によって麻酔のIP監視深注入の組み合わせを使用して麻酔を誘導します。 </li><li>仰臥位でマウスを置き、アイリスをまっすぐはさみを使用して腹膜に続く首に腹部から皮膚をカット。 </li><li>デュモンの鉗子を使用して胸骨を持ち上げ、心臓を露出するリブをカット。 </li><li>心臓を露出させ、止血の2ペアを使用して左と右胸を開きます。 </li><li>左心室に5ミリリットルの注射器に取り付けた26 G針を挿入し、右心房を切りました。室温通常の生理食塩水またはホスファで潅流TE緩衝食塩水（PBS）液が透明になるまで（通常3 - 5分）。 </li><li>アイリスストレートはさみとデュモンの鉗子を使用して慎重に離れて脳から頭蓋骨を削除します。 </li><li>嗅球と脳がマトリクス状に収まるようにカミソリの刃を使用して、小脳をカット。でもセグメントに脳をスライスし、この行列を使用します。クールな組織を維持するために、使用前に行列を冷やします。マトリックスに脳を置き、氷の上に置きます。 </li><li> 2かみそりの刃を使用して、2ミリメートルの冠状セグメントに脳をスライス。上から2ミリメートルを開始するカミソリの刃を使用して最初のカットを作成し、代わりにこのかみそりの刃を残します。最初の後ろにカットし、別の2ミリメートルを行います。付着した組織との最初のカミソリの刃を取り外します。すべての組織がスライスされるまで、このプロセスを繰り返します。 注：4があるはずです - 終了時に5つのセグメント。 </li><li>その後、浅い水浴aで配置され、2％2,3,5- triphenyltetrazaliumクロリド（TTC）を含む24ウェルプレートにセグメントを配置します20分間T 37℃です。個々のウェルに各セグメントを配置すると、それらがカットされた順序でそれらを維持するのに役立ちます。 TTC溶液が完全に組織セグメントをカバーしていることを確認してください。 10分には、すべてのスライスを裏返した後。 </li><li>新しい24ウェルプレートのウェルに10％ホルマリンの少量を置き、それが切断されたために、このプレートにセグメントを転送します。 </li><li>コンピュータにセグメントをスキャンし、ImageJの解析ソフト（NIH 1.57 Imageソフトウェア）6を用いて全脳切片6の割合として梗塞サイズを分析します。 <ol><li>面積測定を生成するために、梗塞領域の輪郭を描きます。次の領域を決定するために、全体の半球を測定します。対側半球の面積によって梗塞領域を分割し、梗塞体積を決定するために100を掛け。 </li></ol></li></ol>

<ol>
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I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessments+in+the+rodent+stroke+model.">Functional assessments in the rodent stroke model.</a> Exp Transl Stroke Med. 2 (13), (2010).</li></ol>

The authors have nothing to disclose.

20年前の概念ので、フィラメントの挿入を含むヒトの脳卒中のMCAOモデルは、研究の膨大な数に使用されています。これは、それが脳卒中（ すなわち、虚血性脳卒中）の最も一般的な形で臨床的に何が起こるかを模倣したという事実のために主です。線条体は大脳皮質よりも虚血に対してより敏感であり、そのようなものとして、虚血時間の長さは、線条体と背皮質の両方が影響を受?...

脳卒中は、病気から4分ごとに死んで一人で、世界的な死因の第5位です。 80万人のアメリカ人は、患者のためだけでなく、その家族のためだけでなく、壊滅的であるストローク毎年、苦しみます。脳卒中は成人の身体障害の主な原因であり、歳出は非常にいくつかの治療オプションが利用可能であるにもかかわらず$ 36.5 10億のオーダーであると推定されます。 組織プラスミノーゲンアクチベーター（tPA）は唯一の食品医薬品局（FDA）は、虚血性脳卒中のための薬のライセンスを取得しています。脳卒中の発症から3~6時間以内に患者に投与し、そしてこれらのケースでは、患者2のわずか4％に利益をもたらす場合は、それだけに有効です。したがって、脳卒中の再現可能な、臨床的に関連する動物モデルは、この疾患の潜在的な治療戦略および治療法の開発を助けるために使用されることが必須です。 in vitroでのことに注意することが重要です</em&gt;のモデルは、脳機能障害の特定の側面をモデル化するのに有用な一方で、脳卒中後の脳と周辺で発生する複雑な生理学的相互作用を反復することができません。その結果、in vivoでのモデルが不可欠です。 脳卒中の最も一般的なタイプは、全ストロークの87％を占め、原点における虚血性です。他のストロークは脳内出血（9％）とくも膜下出血（4％）であり、中大脳動脈（MCA）の塞栓によって最も頻繁に引き起こされます。これは、破砕になるために脳に入る層の血流を引き起こすMCAのルートにある著名な曲線、に起因するものです。 MCAは、内頸動脈（ICA）から生じ、大脳基底核と一次運動や感覚皮質を含む前頭葉、頭頂と側頭葉の側面、に分岐し、プロジェクトの横方向溝に沿ってルート。ウィリス動脈輪はある大脳動脈によって作成されます脳動脈と後部の通信動脈に接続されています。 MCAoの腔内フィラメントまたは縫合糸モデルは、最も広く脳卒中の研究で使用されるの一つです。しかし、このモデルの異なるバリエーションがいくつかあり、これらはマイクロフィラメントが外頸動脈に挿入されているかどうかに基づいている（ECA、ロンガ法と呼ばれる）3、またはそれはICAに挿入されたかどうか（小泉と称される方法）4。ロンガの方法で、それが永久に5を接続する必要があり、そのECAありながらフィラメントは、CCAの切開部からの出血を防ぐために削除された場合は小泉の方法では、手術の側の総頸動脈（CCA）は、恒久的に接続する必要があります。私たちは、これははるかに優れおよび虚血性脳卒中のより臨床的に関連する外科的なモデルであると感じるようにここでロンガの方法が使用されます。また、特にロンガ法によるシリコン先端モノフィラメントの使用は、非常に生産します多くの場合、不完全閉塞および/またはくも膜下出血6を生成難鈍化しモノフィラメントとは対照的に、再現可能にMCAo。 腔内フィラメント法は、永久的または一時的閉塞4,6のモデルとして使用することができます。過渡モデルを実行するために、フィラメントは、虚血の期間（ 例えば、30分、60分、または2時間）後に除去され、そして再灌流が起こることが許されます。このモデルは、ある程度、ヒトにおける血栓塞栓性血餅を溶解するために、自発的または治療的介入（ 例えば 、tPAの投与）後の血流の回復をシミュレートします。永久的なモデルでは、フィラメントは、単に時間の期間（ 例えば 、24時間）のための場所に放置されているので、何の再灌流は発生しません。腔内フィラメント法の別の利点は、開頭術が頭蓋骨をそのまま残すことを可能にすると、頭蓋内圧や温度の変化を回避し、実行する必要がないという事実です。

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			<td height="22" style="height:22px;width:436px;">Male C57BL/6 mice</td>
			<td style="width:291px;">Jackson Laboratory, Bar Harbor, ME</td>
			<td style="width:397px;">#000664</td>
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			<td height="22" style="height:22px;width:436px;">Ketamine Hydrochloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Shreveport, LA</td>
			<td>67457-108-10</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Xylazine</td>
			<td style="width:291px;">Akorn, Inc, Lake Forest, IL</td>
			<td>NADA# 139-236</td>
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			<td height="22" style="height:22px;width:436px;">DC temperature control system</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-8D</td>
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			<td height="22" style="height:22px;width:436px;">Mini rectal thermistor probe</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-80-5D-02</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Heating pad</td>
			<td style="width:291px;">FHC, Bowdoin, ME</td>
			<td>40-90-2-06</td>
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			<td height="22" style="height:22px;width:436px;">Clippers</td>
			<td style="width:291px;">Amazon, Bellevue, WA</td>
			<td>#64800</td>
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			<td height="22" style="height:22px;width:436px;">70% ethanol</td>
			<td style="width:291px;">Worldwide Medical Products, Bristol, PA</td>
			<td>#51011023</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Dissecting microscope</td>
			<td style="width:291px;">Olympus, Center Valley, PA</td>
			<td>SZ40</td>
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			<td height="22" style="height:22px;width:436px;">Iris scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11251-20</td>
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			<td height="22" style="height:22px;width:436px;">Dumont forceps (45&#176; bent tip)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>11297-00</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Micro vessel clip</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18055-05</td>
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			<td height="22" style="height:22px;width:436px;">Micro dissecting spring scissors (straight)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>14088-10</td>
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			<td height="22" style="height:22px;width:436px;">Retractors (blunt)</td>
			<td style="width:291px;">Fine Science Tools, Foster City, CA</td>
			<td>18200-11 (Helen used 17022-13)</td>
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			<td height="22" style="height:22px;width:436px;">Cotton tipped applicators</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>23-400-100</td>
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			<td height="22" style="height:22px;width:436px;">Gauze sponges</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>#9023</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">6-0 silk braided surgical suture</td>
			<td style="width:291px;">Roboz, Gaithersburg, MD</td>
			<td>SUT-1073-11</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">0.9% sodium chloride</td>
			<td style="width:291px;">Morris &#38; Dickson, Lake Forest, IL</td>
			<td>0409-4888-20</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:436px;">6-0 medium MCAO suture (silicon rubber coated monofilament)</td>
			<td style="width:291px;">Doccol Corporation, Sharon, MA</td>
			<td>6023PKRe</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Sofsilk 6-0 silicone coated braided silk</td>
			<td style="width:291px;">Covidien, Mansfield, MA</td>
			<td>SUT-14-1</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Carprofen</td>
			<td style="width:291px;">Pfizer, New York, NY</td>
			<td>NADA# 141-199</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Puralube</td>
			<td style="width:291px;">Dechra, Norwich, UK</td>
			<td>NDC 17033-211-38</td>
			<td></td>
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		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Physitemp temperature controller</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>TCAT-2AC</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Heat lamp</td>
			<td style="width:291px;">Harvard Apparatus, Holliston, MA</td>
			<td>HL-1</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Laser doppler probe</td>
			<td style="width:291px;">AD Instruments, Colorado Springs, CO</td>
			<td>MSP100XP</td>
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			<td height="22" style="height:22px;width:436px;">24-well plates</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>#353226</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Phosphate buffered saline (PBS)</td>
			<td style="width:291px;">Life Technologies, Carlsbad, CA</td>
			<td>20012-050</td>
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			<td height="22" style="height:22px;width:436px;">Single edge razor blades</td>
			<td style="width:291px;">Fisher Scientific, Waltham, MA</td>
			<td>12-640</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">2,3,5-triphenyltetrazalium chloride (TTC)</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>T8877-50G</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Mouse brain matrix slicer</td>
			<td style="width:291px;">Braintree Scientific, Braintree, MA</td>
			<td>BS-A 5000C</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">Water bath</td>
			<td style="width:291px;">VWR, Radnor, PA</td>
			<td>#182</td>
			<td></td>
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			<td height="22" style="height:22px;width:436px;">10% formalin</td>
			<td style="width:291px;">Sigma Aldrich, St. Louis, MO</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Image J analysis software</td>
			<td style="width:291px;">NIH, Bethesda, MD</td>
			<td>free download</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:436px;">Retractor</td>
			<td style="width:291px;">Medical Device Purchase, Newcastle, CA</td>
			<td>MP-740</td>
			<td></td>
		</tr>
	</tbody>
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surgical approach for middle cerebral artery occlusion and reperfusion induced stroke in mice

脳卒中は、世界中の主要な死亡原因であり、長期的な成人の障害の主要な原因の一つであり続けています。ストロークの約87％が起源における虚血性であり、中大脳動脈（MCA）の領土で発生します。現在、食品医薬品局（FDA）は、この壊滅的な疾患の治療のための薬物を承認し、組織プラスミノーゲンアクチベーター（tPA）です。しかし、tPAが投与のための小さな治療窓があり（3から6時間）、および実際にそれを受けた患者の4％でのみ有効です。現在の研究は、潜在的な治療標的を見つけるために脳卒中の病態生理を理解することに焦点を当てています。したがって、信頼性のあるモデルが重要であり、MCA閉塞（MCAO）モデルは、虚血性脳卒中の最も臨床的に関連する外科的モデルとみなされる（さらに腔内フィラメントまたは縫合糸モデルと呼ばれる）、およびかなり非侵襲的かつ容易に再現可能です。典型的には、MCAOモデルは、特にによるマウスと、げっ歯類で使用されていますこの種のために利用可能なすべての遺伝的変異に。ここでは、成功し、信頼性と再現性のあるデータを生成するために、マウスでは（再灌流で）MCAOモデルを実行する方法について説明します（とビデオに存在します）。

マウスは、（24時間と1週間がここに提示されているが、再灌流の長さを変化させることができる）再灌流の期間に続いて30分にMCAo誘発性脳虚血（ 図1）を受けました。 MCAo時の死亡率は、（約2％）最小でした。ポスト虚血、（最初の24時間以内）の死亡率は約26％でした。 レーザードップラー流量測定は、にMCA...

Watch this Scientific Journal Video about Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice at JoVE.com

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

脳卒中の病態生理学を理解するためには、信頼性のあるモデルを使用することが重要です。この論文は、マウスにおいて最も頻繁に使用される脳卒中モデルのいずれかを説明する、中大脳動脈閉塞（MCAO）モデルと呼ばれる再潅流と（また腔内フィラメントまたは縫合モデルと呼ばれます）。

マウスの中大脳動脈閉塞再灌流誘発性の脳卒中のための外科的アプローチ

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

surgical-approach-for-middle-cerebral-artery-occlusion-reperfusion

Research

JoVE Journal

Medicine

21.4K ビュー.  Louisiana State University. 脳卒中の病態生理学を理解するためには、信頼性のあるモデルを使用することが重要です。この論文は、マウスにおいて最も頻繁に使用される脳卒中モデルのいずれかを説明する、中大脳動脈閉塞（MCAO）モデルと呼ばれる再潅流と（また腔内フィラメントまたは縫合モデルと呼ばれます）。 

ビデオ: Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

マウスにおけるモデリングのストローク - フィラメントモデルと中大脳動脈閉塞

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

医学

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 ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

マウスにおけるレーザードップラー流量計の指針と虚血性脳卒中に対する腔内中大脳動脈閉塞（MCAO）モデル

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Bilateral Common Carotid Artery Occlusion as an Adequate Preconditioning Stimulus to Induce Early Ischemic Tolerance to Focal Cerebral Ischemia

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.

There is accumulating evidence, that ischemic preconditioning (PC) &#x2013; a non-damaging ischemic challenge to the brain - confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.

局所脳虚血への早期虚血耐性を誘導するために適切な前処理刺激として二国間の総頚動脈閉塞

There is accumulating evidence, that ischemic preconditioning - a non-damaging ischemic challenge to the brain - confers a transient protection to a ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

ラットにおけるレーザードップラー血流計の指針と虚血性脳卒中のエンドセリン-1誘発性中大脳動脈閉塞モデル

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

エンドセリン-1誘発性中大脳動脈閉塞中大脳動脈を可視化するために頭蓋窓を利用

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

腔内中大脳動脈閉塞のラット脳卒中モデルにおける脳血流モニタリングのための最適化されたシステム

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

マウスにおけるモデリングのストローク：遠位中大脳動脈の永久的な凝固

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

ラットにおける同種血血栓による虚血性脳卒中のための塞栓中大脳動脈閉塞（MCAO）

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

マウスにおける反復低酸素プレコンディショニングと過渡中大脳動脈閉塞後に神経血管保護の定量

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...