この作品は、補助金協定nー201024とN ° 202213（欧州脳卒中ネットワーク）の下で欧州共同体の第七次フレームワーク計画（FP7/2007-2013）によって賄われていた。今回調達した資金は、BundesministeriumこだわりBildungウントForschung（脳卒中研究ベルリンセンター）、およびドイツ学術振興（Exzellenzcluster NEUROCURE）​​によって与えられた。 著者は、アニメーションを生成するための操作とエルケルートヴィヒ（シャリテビデオサービス）の間にサポートのためにMareike Thielkeを（実験的神経用Dep.、シャリテベルリン）に感謝いたします。

高い品質と再現性を保証するために、我々は、標準作業手順（SOP）の使用をお勧めします。このビデオでは、当研究室で開発し、使用されるなど、公開された標準操作手順書（SOP）が適用されます1。 1。大脳動脈閉塞<ol><li>マウスは、獣医スタッフに相談に適切な麻酔薬体制で麻酔です。 （1.5例：誘導 - 1.0 2％イソフルランおよびメンテナンス - 2 / 3 N2Oと気化器を使用して3分の1 O2におけるイソフルラン1.5％）。 <ol><li>マウスの体温は36.5℃で± 0.5℃の加熱プレートと手術中に維持されます。マウスの直腸温に応じて温めフィードバック制御された加熱パッドは、強くお勧めします。 </li><li>適切なエージェント（例えば70％エチルアルコール）を皮膚と周囲の毛を消毒し、その後、それを乾燥させてください。 </li></ol></li><li>正中首の切開が行われ、軟組織を離れて引っ張られている。 </li><li>左総頸動脈（LCCAは）慎重に周囲の神経（迷走神経に悪影響を及ぼすことなく）と結紮が6.0/7.0文字列を使用して作られているからフリー解剖される。 5.0文字列を使用することもできます。 </li><li>左外頸動脈（LECAが）その後、分離され、第結び目が行われます。 </li><li>次に、左内頸動脈（LICA）が隔離され、結び目は6.0フィラメントを用いて製造されています。 </li><li>左内頸動脈（LICA）と左の翼口蓋動脈（LPA）の良いビューを取得した後、両方の動脈は血管クリップを使用して、クリップされます。 </li><li>それはLECAとLICAに分岐する前に小さな穴がLCCAにカットされます。それは、クリップで止まるまで、シリコンの硬化剤の混合物（下記参照）でコーティングされた8.0ナイロン製モノフィラメントは、その後、LICAに導入される。注目は、後頭動脈を入力しないように支払われる必要があります。 （図1） </li><li>フィラメントは、ウィリス動脈輪におけるLMCAの起源を閉塞するLICAに挿入されている間クリッピング動脈が開かれます。 </li><li> LICAのサード結び目は位置にフ​​ィラメントを修正するために閉じている。 </li><li>マウスは、皮下のボリューム補充として生理食塩水0.5mlを受け取る。鎮痛のために、リドカインゲルを局所的に創傷に適用されます。 </li><li>再灌流が意図されている場合は、マウスが30のために滞在 - 温水ケージで90分の閉塞、傷が小さい縫合クリップで閉じることができる。その後、第二麻酔が行われ、ICAのサード結び目は瞬間的に開かれ、フィラメントが引き出される。 </li><li>残りの縫合糸が短縮され、皮膚は外科用縫合糸で構成されている。 </li><li>すべての動物は、上記のように2番目のボリューム補充を受ける。 </li><li>動物は、体温をコントロールするために2時間加熱したケージに入れています。 <ol><li>動物は不快の徴候のための手術後に毎日チェックする必要があります。マウスはいくつかの術後の体重減少を示すことができる。彼らは食べることを奨励するために床に置かれたシャーレでつぶした食べ物を受け取ります。食品は7日間毎日置き換えられます。 </li></ol></li></ol> 2。偽手術<ol><li>偽の操作のためにフィラメントがLMCAを閉塞するために挿入し、インスタント再灌流（1.8）を可能にするために直ちに撤回されている。第二麻酔を含めて - その後の操作は、脳虚血（1.14 1.9）受けている動物で実行されるのと同じです。 </li></ol> 3。フィラメントの調製<ol><li>フィラメントの無菌性を考慮する必要があります。滅菌装置だけでなく、フィラメントの適切​​な取扱いの使用は、その後無菌手術のための前提条件です。フィラメントの消毒は、一般的な滅菌法の多くは、フィラメントの品質を悪化させる可能性があるので、難しいです。しかし、放射線などのメソッドは、UV光またはγ線、または化学滅菌で、例えば、エチレンオキサイドのような高度に反応性ガスと、例えば、適用可能です。 </li><li> 8.0ナイロンフィラメントは、顕微鏡下で11mmの長さに切断され</li><li>フィラメントの先端はXantopren M粘膜とアクティベーターNF Optosilの硬化剤の混合物と8mmの長さにわたって完全に均等にコーティングする必要があります。 </li></ol> 4。代表的な結果異なる運動や行動障害の結果は、血流制限の持続時間に応じて。脳虚血の30〜60分後にはどちらも、ほとんどの場合動物は横方向プッシュ対する抵抗の減少を示し、運動の障害に起因する旋回。前里の屈筋の位置などの軽度病変のマニフェストMBS。これらを簡単に観察可能な標識は、操作の成功のための基本的なスコアとして使用することができます。2 形態学的病変は、組織や磁気共鳴イメージング（MRI）のいずれかを用いて評価することができる。虚血の30分は、線条体に限定、主に神経細胞死を引き起こすのに対し、中大脳動脈の閉塞60分は、線条体と新皮質の両方を含む領域に組織pannecrosisを生成する。3（図2）梗塞体積の面では、我々は期待する一連のオペレーションの30％未満の標準偏差低い。 60分後に20％ - 死亡率は、虚血および10の30分後に約5％の閉塞時間によって異なります。 別の最小限の侵襲の可能性は、その成功の直接制御を可能にする動作時のレーザードップラーフローメトリー（LDF）の使用です。個々の動物、10〜削減で- preocclusion値の20％は明らかに局所的脳虚血の正常な誘導を示している4 LDFだけ血液の量的変化を（パーセンテージで）測定できるので、しかし、LDFは、個体間比較の方法として使用することはできません。小さいと限定された組織サンプルボリューム内の流れ。それは血流の減少の空間的な範囲についての情報を与えるものではありません5。 歩行分析6,7、ロータロッド8、ポールテスト9,10、接着剤除去試験11,12、階段のテスト13,14、ラダーのラングのテスト15,16とモリス水迷路を含む脳卒中後の行動的側面 ​​を評価するには、いくつかのテストが、あります。 17。すべてのこれらのテストでは、MCAOにさらさマウスは少なく、正常対照動物より行います。 <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="図1" /> マウスの脳を供給する血管のアーキテクチャの図は、1。スキーム（バックグラウンドに描かれた）。異なる株は、例えば、後頭動脈はしばしば内頸動脈から離れ、バリエーションが表示されることがあります。 <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="図2" /> 図2。近MCAOモデルにおける再灌流の異なる時間点後の典型的な病変の大きさの模式図。途中で、機能的活動とMCAO後の脳血流の典型的なコースが描かれている。 （MCAO：中大脳動脈閉塞、LDF：レーザードップラー血流量測定）

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J., Prass, K., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isoflurane+induced+prolonged+protection+against+cerebral+ischemia+in+mice:+a+redox+sensitive+mechanism.">Isoflurane induced prolonged protection against cerebral ischemia in mice: a redox sensitive mechanism.</a> Neuroreport. 13, 1431-1435 (2002).</li><li>Endres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+stroke+protection+by+physical+activity.">Mechanisms of stroke protection by physical activity.</a> Ann Neurol. 54, 582-590 (2003).</li><li>Gertz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+activity+improves+long-term+stroke+outcome+via+endothelial+nitric+oxide+synthase-dependent+augmentation+of+neovascularization+and+cerebral+blood+flow.">Physical activity improves long-term stroke outcome via endothelial nitric oxide synthase-dependent augmentation of neovascularization and cerebral blood flow.</a> Circ Res. 99, 1132-1140 (2006).</li><li>Richter, S. H., Garner, J. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments.">Environmental standardization: cure or cause of poor reproducibility in animal experiments.</a> Nat Methods. 6, 257-261 (2009).</li><li>Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+systemic+immune+alterations+after+murine+focal+ischemia:+immunodepression+versus+immunomodulation.">The spectrum of systemic immune alterations after murine focal ischemia: immunodepression versus immunomodulation.</a> Stroke. 40, 2849-2858 (2009).</li><li>Meisel, C., Schwab, J. M., Prass, K., Meisel, A., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+injury-induced+immune+deficiency+syndrome.">Central nervous system injury-induced immune deficiency syndrome.</a> Nat Rev Neurosci. 6, 775-786 (2005).</li><li>Engel, O., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Infection+Before+and+After+Stroke:+Investigating+New+Targets.">Models of Infection Before and After Stroke: Investigating New Targets.</a> Infect Disord Drug Targets. 9, (2010).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26, 1465-1478 (2006).</li><li>Macleod, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+laboratory+practice:+preventing+introduction+of+bias+at+the+bench.">Good laboratory practice: preventing introduction of bias at the bench.</a> Stroke. 40, 50-52 (2009).</li><li>Group, S. T. A. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standards+regarding+preclinical+neuroprotective+and+restorative+drug+development.">Recommendations for standards regarding preclinical neuroprotective and restorative drug development.</a> Stroke. 30, 2752-2758 (1999).</li></ol>

動物実験はLandesamt福二お大事にウントSoziales、ベルリン、ドイツで定められたガイドラインおよび規制に準拠して行った。

一過性のモデルは、ここで紹介する近位MCA閉塞18,19は、患者の虚血性脳卒中の最も一般的なタイプのいずれかを模倣する。20は変数の再灌流時間に基づいて、モデルは、一過性脳虚血発作（TIA）から大型まで被害の異なるグレードを提供しています虚血半球の主要な部分を含む梗塞。これは、脳卒中後の研究者がさまざまな病態生理学的メカニズムを研究することができます。20,21 手術は短時間で行われ、再現性の高い病変を生成することができます。それにもかかわらず、これは交絡因子の徹底したコントロールを必要とする。運用テクニック22小さな違いが梗塞に異なった効果を説明しうる。さらに23,24、脳血管解剖の分散のために、異なるマウス系統は、異なる結果を示す。25,26ボディを温度がより小さい病変27、より重度の障害に温熱療法につながる低体温で、神経学的損傷に影響を与えます。28したがって、温度制御やメンテナンスが、このモデルでは、関連性の高いです。加えて29血圧と血液ガスの結果の重要な交絡因子であるとする必要がある監視される。迅速かつ最小限の侵襲的な方法（非侵襲的血圧測定、適切かつ容易にアクセス可能な採血部位）の30,31の使用は推奨されます。いくつかの神経保護効果を持っている、および/ ​​または血管拡張薬となる可能性があるため、麻酔薬の選択はイソフルランなどのように、、また非常に重要である。そのため32、麻酔への曝露が可能と標準化されただけ短くしてください。我々は、より長い15分間の手術を受けた動物を除外する。 手術部位をシェービングすることmicroabrasionsと炎症を生成し、髪のフラグメントを解放します。これにより、さらに炎症を増加させ、脳卒中病態に影響を与える可能性があるローカル感染を、促進することがあります。住宅の条件、濃縮の特に使用は、脳卒中転帰に影響を及ぼす可能性がありますし、標準化と研究の報告書に記載されている必要があります。再現性で環境エンリッチメントとその効果の6,33,34使用は議論の問題である。35もう一つの重要な交絡因子がある追加の罹患率と死亡率の増加につながる特に虚血36の長い時間の後に感染の脳卒中誘発される危険性、。37,38感染症は5〜約3日目での症状のようになると、これはそのようなモデルでのフォローアップ長期的に重要な影響をもたらします。 脳卒中、標準化と品質管理のために新たな治療戦略の開発に関連する結果を生成するには、前臨床トランスレーショナルストロークの研究で非常に重要である39&quot;優良試験所&quot;40,41義務付けては。： <ol><li>使用する動物の適切かつ詳細な説明; </li><li>期待される効果の大きさのサンプルサイズの計算とレポート作成; </li><li>試験前に設定した選択基準と除外基準、; </li><li>グループへの割り当てのランダム化。 </li><li>研究者に関して割付けの隠蔽; </li><li>分析から除外動物を報告。 </li><li>結果の盲検評価; </li><li>関心と研究の資金の潜在的な競合を報告。 </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical scissors (skin cut)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>14028-10</td>
<td>&nbsp;</td>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors (according to Vannas)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Applying forceps for micro clamps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00072-14</td>
<td>&nbsp;</td>
<tr>
<td>Micro vascular clamp (e.g. S&amp;G B1-V)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00396-01</td>
<td>Also Serrefine possible</td>
</tr>
<tr>
<td>2 Hemostats according to Hartmann</td>
<td>&nbsp;</td>
<td>FST</td>
<td>13002-10</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder (according Olsen-Hegar or other)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>12002-14</td>
<td>&nbsp;</td>
<tr>
<td>Standard anatomical forceps (for wound closure)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>5/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for vascular ligatures</td>
</tr>
<tr>
<td>6/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for skin suture, 5/0 also possible</td>
</tr>
<tr>
<td>Lidocaine (e.g. Xylocain Gel 2%)</td>
<td>&nbsp;</td>
<td>Astra Zeneca</td>
<td>&nbsp;</td>
<td>or other local pain relief</td>
</tr>
<tr>
<td>Dissecting microscope (stereo microscope), magnification 6x to max. 40x</td>
<td>&nbsp;</td>
<td>Leica Zeiss</td>
<td>MZ6 Stemi2000C</td>
<td>&nbsp;</td>
<tr>
<td>Cold light source</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>KL1500</td>
<td>&nbsp;</td>
<tr>
<td>Temperature feedback controlled heating pad system</td>
<td>&nbsp;</td>
<td>FST</td>
<td>21052-00</td>
<td>With mouse pad and small probe</td>
</tr>
<tr>
<td>Anaethesia system for isoflurane</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Abott</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Recovery Chamber</td>
<td>&nbsp;</td>
<td>Peco Services</td>
<td>V1200</td>
<td>Heated cage</td>
</tr>
<tr>
<td>8.0 nylon filament</td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>TEL181005</td>
<td>for coating</td>
</tr>
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cutting filament in pieces</td>
</tr>
<tr>
<td>Ruler</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To cut correct length of filament</td>
</tr>
<tr>
<td>Xantopren M Mucosa</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Activator Universal Plus</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>Optosil - Xantopren</td>
</tr>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>A soft underlay for storing and grasping the uncoated filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. swabs, foam, &#x2026;</td>
</tr>
<tr>
<td>Receptacle for new build filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. petri dish, flexible foam,&#x2026;</td>
</tr>		</tbody>
		</table>
		</div>

modeling stroke in mice - middle cerebral artery occlusion with the filament model

ストロークは、特に先進国では、死と成人の障害の最も一般的な原因のひとつです。しかし、現在までの治療の選択肢は非常に限られています。新たな治療アプローチの必要性を満たすために、実験的脳卒中の研究は、しばしば局所脳虚血の齧歯類モデルを採用しています。ほとんどの研究は、マウスやラットにおける中大脳動脈（MCA）の永続的または一時的な閉塞を使用してください。 腔内縫合のテクニック（そのフィラメントまたは糸のモデルと呼ばれる）を介して中大脳動脈（MCA）の近位閉塞は、おそらく実験的脳卒中研究において最も頻繁に使用されるモデルです。腔内MCAOモデルは、比較的非侵襲的な方法でMCA領域の再現性の一時的または永続的な虚血を誘導するという利点があります。腔内のアプローチは、この動脈の全体領土の血の流れが中断される。フィラメント閉塞は、このように逮捕者は、大脳基底核を供給lenticulo - 線条体動脈、近位に流れます。フィラメントは、皮質と線条体における再現性の病変でMCAの結果の閉塞と永久的または一時的のどちらでもかまいません。対照的に、遠位誘導モデル（lenticulo - 線条体動脈の分枝には）MCA閉塞は、通常、線条体を惜しまないし、主に新皮質を含む。さらに、これらのモデルはクレイニエクトミを必要としません。この記事で紹介モデルでは、シリコンコーティングされたフィラメントは、総頸動脈に導入され、それが中大脳動脈の起源をブロックウィリスのサークルに内頸動脈に沿って進んだ。患者では、中大脳動脈の閉塞は虚血性脳卒中の最も一般的な原因の一つです。虚血の間隔を変化することは再灌流の時点に応じて、このモデルでは自由に選択できるので、重要度の度合いも異なる虚血性病変を製造することができる。吸蔵フィラメントの除去は、少なくとも部分的にモデルヒトの血栓塞栓血栓の自発的または治療因子（tPA）溶解後の血流の回復によって再灌流。 このビデオでは、基本的なテクニックだけでなく、このモデルの予測値を制限することが主な落とし穴と交絡因子を提示する。

Watch this Scientific Journal Video about Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model at JoVE.com

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

中大脳動脈の糸状閉塞は、マウスでは虚血性脳卒中を研究するための一般的なモデルです。

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 ...

modeling-stroke-mice-middle-cerebral-artery-occlusion-with-filament

Research

JoVE Journal

Medicine

91.0K ビュー.  Center for Stroke Research Berlin, Charité Universitätsmedizin. 中大脳動脈の糸状閉塞は、マウスでは虚血性脳卒中を研究するための一般的なモデルです。

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

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 ...

医学

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 ...

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 ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

持続的な障害と皮質虚血を研究するために高齢ラットにおける一般的な頸動脈閉塞と永久遠位中大脳の実行

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

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

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 ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.