Name: lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice
Uploaded: 2016-12-29T13:00:00.000+00:00
Duration: 8 min 41 s
Description: Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

VP is a participant in the Charit&#233; Clinical Scientist Program, funded by the Charit&#233; - Universit&#228;tsmedizin Berlin and the Berlin Institute of Health. TB is an SNSF PostDoc Mobility fellow. The authors receive grant support from EinsteinStiftung/A-2012-153 to PV.

倫理の声明：動物を対象とした実験は、該当する場合、Landesamt fuerお大事にウントSoziales、ベルリン、ドイツ（G0298 / 13）とARRIVE基準によって定められたガイドラインおよび規則に従って行いました。 12週齢の雄のC57BL / 6Jマウスに本研究では、10-に使用しました。 1.横慢性頭蓋窓の準備<ol><li>皮下ケタミンの注射（90 mgの/ kg）およびキシラジン（10mg / kg）で麻酔を行います。痛みの刺激との適切な鎮静のためのテスト。 </li><li> 70％エタノールで手術器具および手術野を滅菌します。 </li><li>げっ歯類のシェーバーを使用して、目に首から頭皮の毛を削除します。 </li><li>定位フレームにヘッドを固定してください。 </li><li>脱水症状を避けるために、両眼にデクスパンテノール眼軟膏を使用してください。 </li><li>すべての毛を除去し、74.1パーセントのエタノールと10％2-プロプラノロールの3層で、それを滅菌する手術領域を清掃してください。 </li><li>正中線を実行しますメスを用いて目に首から切開。 </li><li> 4テンティング縫合糸で皮膚フラップにまたがります。 </li><li>頭筋が始まる点にメスでそ​​れを掻き落とし左半球に慎重に骨膜を削除します。 注：この製剤はまた、カバーガラスを固定する歯科接着剤、のための良好な接着領域を作成するのに役立ちます。 </li><li>マイクロドリルを用いて骨フラップの縁に頭蓋骨を薄くすることにより、直径4mmの前頭頭頂開頭術を実行します。熱損傷を回避するために掘削しながら、生理食塩水を適用します。 </li><li>カニューレと骨弁を高め、マイクロピンセットを使ってそれを削除します。 </li><li>食塩水で慎重かつ広範囲に灌漑。 </li><li>それは適切な一貫性を持っており、流体でなくなるまで、歯科用接着剤を混ぜる（ すなわち、接着剤はもはやスレッドを生成するべきではありません）。開頭術の周りの骨の上に置きます。 </li><li>準備された接着剤の6ミリメートル径のカバーガラスを置き、目でそれを修正歯科接着剤の電子レスト。それは防水であることを確認してください。接着剤は、ピンセットでそれをテストすることによって乾燥させ、ハードされるまで待ちます。 注：追加の灌漑は、硬化プロセスを加速します。 </li><li>テンティング縫合糸を外し、皮膚縫合糸で傷口を閉じます。 </li><li>マウスの脇腹の皮膚を引き上げます。針を皮下に挿入し、ゆっくりと体液バランスのために滅菌生理食塩水を皮下の0.5ミリリットルを代入します。 </li><li>手術後、90分間加熱した回復ケージに動物を飼います。動物は無人それらを離れる前に、完全に目を覚ましているまで待ってください。動物は完全に他の動物とのケージに戻す前に回収されるまで待ちます。 </li><li>ステップ1.16で説明したように、約12時間後に皮下生理食塩水の体積置換を繰り返します。 </li><li>常に手術（ 例えば、パラセタモール（10 mg / mlで）、または他の非ステロイド性抗炎症薬）の後に口腔内に強制飼養を介して、または直接に鎮痛を適用します。 </li><li>チェック<html>常に動物の状態、手術後、毎日、とは単純な食べ作ることと、手術後の重要な重量損失を回避するために、ペトリ皿の中で床の上につぶした動物性食品を提供します。 注：生体顕微鏡は、頭蓋窓の準備の後の最初の日に行うことができます。 </li><li>イソフルラン麻酔を適用し、ヘッドホルダに動物を固定します。皮膚の縫合糸を開き、綿棒と、滅菌生理食塩水でウィンドウをきれいにします。 24時間後、頭蓋窓は、イメージングを可能にする、その時点によって脳脊髄液で満たされるべきです。確立された顕微鏡プロトコル18を使用して撮影を行います。 </li></ol> 2.遠位にMCAo 注：のMCAO手順はCW調製後約5 Dを実行する必要があります。これは、脳卒中により誘導される免疫反応のCW製剤によって引き起こされる免疫反応からの干渉を最小限に抑えることができます。 &quot;SRC =&quot; /ファイル/ ftp_upload / 54701 / 54701fig1.jpg &quot;/&gt; 遠位にMCAoの1.概要図。 A.これは、オペアンプの前に船のいいの概要です。第1のバイポーラ接触後B.船。第2のバイポーラ接触後のC.は船。 D.完全に締め切られている船舶、についての概要。低倍率でのE.ファイナル概要。 <a href="http://ecsource.jove.com/files/ftp_upload/54701/54701fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <ol><li> 2/3 N 2 Oと1/3 O 2を 介して 1.5％イソフルラン- 1.0と2％イソフルランおよびメンテナンス-獣医のスタッフ（ 例えば、1.5での誘導と協議の上、麻酔マスクと適切な麻酔薬体制を使用してマウスをAnaesthetize気化器）。 </li><li> （剃ると毛を除去し、適切な薬剤と皮膚や周囲の毛を消毒例えば、70％のエチルアルコール）、その後、それを乾燥させます。 </li><li> 36.5℃でのマウスの体温を維持するためには、手術中に0.5℃、±しました。マウスの直腸温度に応じて加熱されたフィードバック制御加熱パッドは、強くお勧めします。 </li><li>横位置で動物を置きます。麻酔マスクで鼻を修正し、1.0にイソフルラン濃度を調整 - 1.5％。 </li><li>両眼に濡れた軟膏（デクスパンテノール）を適用します。 </li><li> CWの準備のために作られた皮膚切開を使用してください。 </li><li>優しく肌を分離し、その下側頭筋を識別します。 </li><li>高周波発生器のエネルギー調整（5から7 W）をバイポーラモードを使用します。 </li><li>電気凝固鉗子を使用して、慎重に完全に筋肉を除去することなく、筋肉のフラップを作成し、頭蓋骨から頭筋を取り除きます。 </li><li>レトロ眼窩に背、時間領域の吻側部分に透明頭蓋骨の下にMCAを特定リットル洞。 MCA分岐部が識別できない場合、容器最も吻側を特定しようとします。 </li><li>継続的に熱損傷を避けるために灌漑しながら、マイクロドリルでMCA枝上記薄い頭蓋骨。 </li><li>カニューレと骨を持ち上げて、マイクロピンセットでそれを削除します。 </li><li> 5 W. - 3に高周波発電機のエネルギーを減少させます</li><li>上から動脈に近づき、そっと容器を持ち上げることなく、両側のバイポーラ鉗子でそれに触れます。 </li><li>血管分岐部に近位および遠位動脈を凝固。 </li><li> 30秒間待ってから、血流が恒久的に中断されることを保証するために、静かに動脈をタッチします。再疎通が観察されている場合は電気凝固を繰り返します。 </li><li>可能な場合、骨欠損をカバーするために、筋肉の挿入で1または2のステッチと頭筋を修正しました。 </li><li>傷口を縫合し、加熱された回収ボックスに動物を配置します。すぐに揮発性ANEST後に一般的には、動物の回復hesia。 </li><li>ステップ1.16で説明したように、ボリュームの置換については、滅菌生理食塩水の皮下に0.5mlのを適用します。 </li><li>手術後、動物は90分間加熱回復ケージに滞在することができます。動物は無人それらを離れる前に意識しているまで待ってください。それらが完全に回復している場合にのみ、他の動物とのケージに戻します。 </li><li> 12時間後に、ステップ1.16で説明したように、ボリュームの置換を繰り返します。 </li><li>飲料水（ 例えば、パラセタモール（10 mg / mlで）、または他の非ステロイド性抗炎症薬） を介して、術後鎮痛を適用します。 </li><li>手術後、毎日の動物の病状を確認してください。食を簡素化し、術後の体重減少を最小限に抑えるために床の上にペトリ皿にマッシュポテト動物性食品を提供します。 </li></ol> 3.シャム治療<ol><li>上記の-含むCW準備-除く露出した中大脳ARを凝固しない説明したステップ1及び2に同一のすべての手順を実行しますテリ。 </li></ol>

<ol>
	<li>Mukherjee, D., Patil, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+the+global+burden+of+stroke.">Epidemiology and the global burden of stroke.</a> World Neurosurg. 76 (6), 85-90 (2011).</li><li>Ebinger, M., Pr&#252;ss, H., 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=Effect+of+the+use+of+ambulance-based+thrombolysis+on+time+to+thrombolysis+in+acute+ischemic+stroke:+a+randomized+clinical+trial.">Effect of the use of ambulance-based thrombolysis on time to thrombolysis in acute ischemic stroke: a randomized clinical trial.</a> JAMA. 311 (16), 1622-1631 (2014).</li><li>Ebinger, M., Lindenlaub, S., 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=Prehospital+thrombolysis:+a+manual+from+Berlin.">Prehospital thrombolysis: a manual from Berlin.</a> J vis Exp. (81), e50534 (2013).</li><li>Bogousslavsky, J., Van Melle, G., Regli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Lausanne+Stroke+Registry:+analysis+of+1,000+consecutive+patients+with+first+stroke.">The Lausanne Stroke Registry: analysis of 1,000 consecutive patients with first stroke.</a> Stroke. 19 (9), 1083-1092 (1988).</li><li>Engel, O., Kolodziej, S., Dirnagl, U., Prinz, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+stroke+in+mice+-+middle+cerebral+artery+occlusion+with+the+filament+model.">Modeling stroke in mice - middle cerebral artery occlusion with the filament model.</a> J Vis Exp. (47), (2011).</li><li>Donnan, G. A., Fisher, M., Macleod, M., Davis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stroke.">Stroke.</a> Lancet. 371 (9624), 1612-1623 (2008).</li><li>Meairs, S., Wahlgren, N., 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=Stroke+research+priorities+for+the+next+decade--A+representative+view+of+the+European+scientific+community.">Stroke research priorities for the next decade--A representative view of the European scientific community.</a> Cerebrovasc Dis. 22 (2-3), 75-82 (2006).</li><li>Rosamond, W., Flegal, K., 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=Heart+disease+and+stroke+statistics--2007+update:+a+report+from+the+American+Heart+Association+Statistics+Committee+and+Stroke+Statistics+Subcommittee.">Heart disease and stroke statistics--2007 update: a report from the American Heart Association Statistics Committee and Stroke Statistics Subcommittee.</a> Circulation. 115 (5), 69-171 (2007).</li><li>Moskowitz, M. A., Lo, E. H., Iadecola, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+stroke:+mechanisms+in+search+of+treatments.">The science of stroke: mechanisms in search of treatments.</a> Neuron. 67 (2), 181-198 (2010).</li><li>Dirnagl, U., Iadecola, C., Moskowitz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathobiology+of+ischaemic+stroke:+an+integrated+view.">Pathobiology of ischaemic stroke: an integrated view.</a> Trends Neurosci. 22 (9), 391-397 (1999).</li><li>Dirnagl, U., 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=Found+in+Translation:+Preclinical+Stroke+Research+Predicts+Human+Pathophysiology,+Clinical+Phenotypes,+and+Therapeutic+Outcomes.">Found in Translation: Preclinical Stroke Research Predicts Human Pathophysiology, Clinical Phenotypes, and Therapeutic Outcomes.</a> Stroke. , (2014).</li><li>Prinz, V., Hetzer, A. -. M., 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=MRI+heralds+secondary+nigral+lesion+after+brain+ischemia+in+mice:+a+secondary+time+window+for+neuroprotection.">MRI heralds secondary nigral lesion after brain ischemia in mice: a secondary time window for neuroprotection.</a> J Cereb Blood Flow Metab. , (2015).</li><li>Shih, A. Y., Mateo, C., Drew, P. J., Tsai, P. S., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polished+and+reinforced+thinned-skull+window+for+long-term+imaging+of+the+mouse+brain.">A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.</a> J Vis Exp. (61), (2012).</li><li>Holtmaat, A., Bonhoeffer, 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=Long-term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long-term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Iadecola, C., 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=The+microcircualtion--fantastic+voyage:+introduction.">The microcircualtion--fantastic voyage: introduction.</a> Stroke. 44 (6), 83 (2013).</li><li>Blinder, P., Tsai, P. S., Kaufhold, J. P., Knutsen, P. M., Suhl, H., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cortical+angiome:+an+interconnected+vascular+network+with+noncolumnar+patterns+of+blood+flow.">The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow.</a> Nat Neurosc. 16 (7), 889-897 (2013).</li><li>Shih, A. Y., Driscoll, J. D., Drew, P. J., Nishimura, N., Schaffer, C. B., Kleinfeld, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+microscopy+as+a+tool+to+study+blood+flow+and+neurovascular+coupling+in+the+rodent+brain.">Two-photon microscopy as a tool to study blood flow and neurovascular coupling in the rodent brain.</a> J Cereb Blood Flow Metab. 32 (7), 1277-1309 (2012).</li><li>Cabrales, P., Carvalho, L. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+of+the+mouse+brain+microcirculation+using+a+closed+cranial+window.">Intravital microscopy of the mouse brain microcirculation using a closed cranial window.</a> J Vis Exp. (45), (2010).</li><li>Rosell, A., Agin, V., 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=Distal+occlusion+of+the+middle+cerebral+artery+in+mice:+are+we+ready+to+assess+long-term+functional+outcome.">Distal occlusion of the middle cerebral artery in mice: are we ready to assess long-term functional outcome.</a> Transl Stroke Res. 4 (3), 297-307 (2013).</li><li>Dorand, R. D., Barkauskas, D. S., Evans, T. A., Petrosiute, A., Huang, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+intravital+thinned+skull+and+cranial+window+approaches+to+study+CNS+immunobiology+in+the+mouse+cortex.">Comparison of intravital thinned skull and cranial window approaches to study CNS immunobiology in the mouse cortex.</a> Intravital. 3 (2), (2014).</li><li>Balkaya, M., 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=Assessing+post-stroke+behavior+in+mouse+models+of+focal+ischemia.">Assessing post-stroke behavior in mouse models of focal ischemia.</a> J Cereb Blood Flow Metab. 33 (3), 330-338 (2013).</li><li>Balkaya, M., Kr&#246;ber, J., Gertz, K., Peruzzaro, S., 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=Characterization+of+long-term+functional+outcome+in+a+murine+model+of+mild+brain+ischemia.">Characterization of long-term functional outcome in a murine model of mild brain ischemia.</a> J Neurosci Methods. 213 (2), 179-187 (2013).</li><li>Freret, T., Bouet, V., 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=Behavioral+deficits+after+distal+focal+cerebral+ischemia+in+mice:+Usefulness+of+adhesive+removal+test.">Behavioral deficits after distal focal cerebral ischemia in mice: Usefulness of adhesive removal test.</a> Beh Neurosci. 123 (1), 224-230 (2009).</li><li>Liu, S., Zhen, G., Meloni, B. P., Campbell, K., Winn, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RODENT+STROKE+MODEL+GUIDELINES+FOR+PRECLINICAL+STROKE+TRIALS+(1ST+EDITION).">RODENT STROKE MODEL GUIDELINES FOR PRECLINICAL STROKE TRIALS (1ST EDITION).</a> J Exp Stroke Trans Med. 2 (2), 2-27 (2009).</li><li>Florian, B., Vintilescu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+hypothermia+reduces+infarct+volume+in+aged+rats+after+focal+ischemia.">Long-term hypothermia reduces infarct volume in aged rats after focal ischemia.</a> Neurosci Lett. 438 (2), 180-185 (2008).</li><li>Noor, R., Wang, C. X., Shuaib, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+hyperthermia+on+infarct+volume+in+focal+embolic+model+of+cerebral+ischemia+in+rats.">Effects of hyperthermia on infarct volume in focal embolic model of cerebral ischemia in rats.</a> Neurosci Lett. 349 (2), 130-132 (2003).</li><li>Barber, P. A., Hoyte, L., Colbourne, F., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-regulated+model+of+focal+ischemia+in+the+mouse:+a+study+with+histopathological+and+behavioral+outcomes.">Temperature-regulated model of focal ischemia in the mouse: a study with histopathological and behavioral outcomes.</a> Stroke. 35 (7), 1720-1725 (2004).</li><li>Shin, H. K., Nishimura, M., 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=Mild+induced+hypertension+improves+blood+flow+and+oxygen+metabolism+in+transient+focal+cerebral+ischemia.">Mild induced hypertension improves blood flow and oxygen metabolism in transient focal cerebral ischemia.</a> Stroke. 39 (5), 1548-1555 (2008).</li><li>Kapinya, K. 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 (11), 1431-1435 (2002).</li><li>Gertz, K., Priller, J., 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=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 (10), 1132-1140 (2006).</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 (12), 1465-1478 (2006).</li></ol>

The authors have nothing to disclose.

ストロークが1世界中の長期障害と死の主な原因の一つです。急性期治療を超えて、脳卒中後の回復を促進し、強化するための新しいアプローチやメカニズムを調査すると、プライム医療目標7のまま。実験ストローク研究者が頻繁に局所脳虚血の齧歯類モデルを採用しています。実際には、一時的または恒久的にMCAoを誘導するモデルは、患者4における局所脳虚血の最...

Stroke is among the principal causes of long-term disability and death worldwide1, coming second after coronary heart disease. In addition, stroke is the primary cause of long-term disability, underscoring its tremendous socioeconomic impact6-8. Beyond acute treatment, investigating new approaches and mechanisms to accelerate and enhance recovery after stroke remains a prime medical goal7. 
In the last few decades, data from experimental stroke research has contributed substantially to understanding the complex pathophysiological cascades triggered by ischemia9,10. Excitotoxicity, apoptosis, peri-infarct depolarization, and inflammation have been identified as the most relevant mediators of cell death following focal cerebral ischemia. Moreover, using animal models of cerebral ischemia, important concepts, diagnostic modalities, and therapeutic approaches have been developed and validated (e.g., "penumbra" and thrombolysis)11.
The availability of experimental stroke models, combined with non-invasive imaging modalities (e.g., magnetic resonance imaging (MRI), computed tomography, or laser speckle contrast analysis), enables the researcher to investigate hyperacute and chronic pathophysiological changes induced by the ischemic insult in a longitudinal manner12. Along with studying the spatiotemporal profile of the evolving lesion, changes resembling neuronal plasticity can be investigated and correlated to functional outcomes and histological findings. Within the last few years, further methodological advances have been made using the combination of cerebral ischemia models and in vivo microscopy via cranial windows13. These new techniques allow investigators to analyze the neurovascular unit at the cellular and molecular level, with great analytic power in the acute, subacute, and chronic phases following focal cerebral ischemia14. Moreover, in vivo microscopy imaging of microcirculatory dynamics has revealed novel aspects of cerebral microvasculature function and angioarchitecture, with significant pathophysiological relevance15-17. 
In this protocol, we present how to perform a chronic CW preparation lateral to the sagittal sinus and how to surgically induce a distal stroke underneath the window. This mouse model can be applied to sequential imaging of acute, subacute, and chronic changes following focal cerebral ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

<table><tbody><tr><td>Binocular surgical microscope</td><td>Zeiss</td><td>Stemi 2000 C</td><td></td></tr><tr><td>Light source for microscope</td><td>Zeiss</td><td>CL 6000 LED</td><td></td></tr><tr><td>Heating pad with rectal probe</td><td>FST</td><td>21061-10</td><td></td></tr><tr><td>Stereotactic frame</td><td>Kopf</td><td>Model 930</td><td></td></tr><tr><td>Anaethesia system for isoflurane</td><td>Draeger</td><td></td><td></td></tr><tr><td>Isoflurane</td><td>Abott</td><td></td><td></td></tr><tr><td>Dumont forceps #5</td><td>FST</td><td>11251-10</td><td></td></tr><tr><td>Dumont forceps #7</td><td>FST</td><td>11271-30</td><td></td></tr><tr><td>Bipolar Forceps</td><td>Erbe</td><td>20195-501</td><td></td></tr><tr><td>Bipolar Forceps&amp;nbsp;</td><td>Erbe&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 20195-022</td><td></td><td></td></tr><tr><td>Microdrill</td><td>FST&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 18000-17&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td><td></td></tr><tr><td>Needle holder</td><td>FST</td><td>12010-14</td><td></td></tr><tr><td>5-0 silk suture</td><td>Feuerstein, Suprama</td><td></td><td></td></tr><tr><td>7-0 silk suture</td><td>Feuerstein,Suprama</td><td></td><td></td></tr><tr><td>8-0 silk suture</td><td>Feuerstein, Suprama</td><td></td><td></td></tr><tr><td>Veterinary Recovery Chamber</td><td>Peco Services</td><td>V1200</td><td></td></tr></tbody></table>

lateral chronic cranial window preparation enables in vivo observation following distal middle cerebral artery occlusion in mice

局所脳虚血（ すなわち、虚血性脳卒中）は、神経機能の深刻な損失に、その結果、運動および認知障害のホストにつながる、主要な脳損傷を引き起こす可能性があります。ストロークが1世界中の長期障害と死の主な原因の一つであるとして、その高い有病率は、深刻な健康負担をもたらします。神経機能の回復は、ほとんどの場合、唯一の部分です。これまでに、治療オプションが原因で血栓溶解2,3ための狭い時間ウィンドウに、特に、非常に制限されます。脳卒中からの回復を加速するための方法を決定することは、プライム医療目標です。しかしながら、これは、回復プロセス中に不十分な機構的洞察により妨げられてきました。実験ストローク研究者が頻繁に局所脳虚血の齧歯類モデルを採用しています。急性期を越えて、脳卒中の研究は、脳虚血次亜急性および慢性期にますます集中しています。ほとんどのストロークの研究者は、永続的またはトラン適用しますマウスまたはラットにおけるMCAのトランジェント閉塞。患者では、MCAの閉塞は、虚血性脳卒中4の最も頻繁な原因の一つです。フィラメントモデルを用いて、MCAの近位閉塞のほか、遠位MCAの外科的閉塞は実験的ストローク研究5の中で最も頻繁に使用されるモデルは、おそらくです。 MCAブランチ（lenticulo-線条動脈の分岐まで）遠位の閉塞は、通常、線条体を惜しみ、主に新皮質に影響を与えます。血管閉塞は、永久的または一時的であることができます。長期転帰に対する病変体積と非常に低い死亡率の高い再現性は、このモデルの主な利点です。ここでは、矢状静脈洞に慢性頭蓋窓（CW）準備横を実行するために、その後どのように開頭術アプローチを使用して、ウィンドウの下に遠位脳卒中を引き起こす外科的方法を示しています。このアプローチは、ビアの虚血後の急性および慢性の変化の連続画像形成のために適用することができます落射照明、共焦点レーザー走査、及び二光子生体顕微鏡。

タイムライン及び代表的な結果を図2および図3に示されています。上矢状静脈洞（ 図2 B、C、D）経験豊富な外科医によって行われる非常に低い死亡率および罹患率率の結果に小さな頭蓋窓の横で頭蓋窓の準備、。 10匹の動物の全てが生存し、そしてすべての慢性CWも28日目、手術後、高品質の画像化に使用することができます。創?...

Watch this Scientific Journal Video about Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice at JoVE.com

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

遠位中大脳動脈の分岐（MCAO）の外科的閉塞は実験的ストローク研究で頻繁に使用されるモデルです。この原稿は、マウスの縦生体顕微鏡検査のための機会を提供しています横頭蓋窓の挿入と組み合わせて永久にMCAoの基本的な手法について説明します。

横慢性頭蓋窓の準備を有効<em&gt;インビボ</em&gt;マウスの遠位中大脳動脈閉塞後に観測

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

lateral-chronic-cranial-window-preparation-enables-vivo-observation

Research

JoVE Journal

Neuroscience

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

11.6K ビュー.  Charité-Universitätsmedizin. 遠位中大脳動脈の分岐（MCAO）の外科的閉塞は実験的ストローク研究で頻繁に使用されるモデルです。この原稿は、マウスの縦生体顕微鏡検査のための機会を提供しています横頭蓋窓の挿入と組み合わせて永久にMCAoの基本的な手法について説明します。 

ビデオ: Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

神経科学

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 ...

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 ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

Stroke is the third cause of mortality and the leading cause of disability in the World. Ischemic stroke accounts for approximately 80% of all strokes. However, the thrombolytic tissue plasminogen activator (tPA) is the only treatment of acute ischemic stroke that exists. This led researchers to develop several ischemic stroke models in a variety of species. Two major types of rodent models have been developed: models of global cerebral ischemia or focal cerebral ischemia. To mimic ischemic stroke in patients, in whom approximately 80% thrombotic or embolic strokes occur in the territory of the middle cerebral artery (MCA), the intraluminal middle cerebral artery occlusion (MCAO) model is quite relevant for stroke studies. This model was first developed in rats by Koizumi et al. in 1986 1. Because of the ease of genetic manipulation in mice, these models have also been developed in this species 2-3.
	Herein, we present the transient MCA occlusion procedure in C57/Bl6 mice. Previous studies have reported that physical properties of the occluder such as tip diameter, length, shape, and flexibility are critical for the reproducibility of the infarct volume 4. Herein, a commercial silicon coated monofilaments (Doccol Corporation) have been used. Another great advantage is that this monofilament reduces the risk to induce subarachnoid hemorrhages. Using the Zeiss stereo-microscope Stemi 2000, the silicon coated monofilament was introduced into the internal carotid artery (ICA) via a cut in the external carotid artery (ECA) until the monofilament occludes the base of the MCA. Blood flow was restored 1 hour later by removal of the monofilament to mimic the restoration of blood flow after lysis of a thromboembolic clot in humans. The extent of cerebral infarct may be evaluated first by a neurologic score and by the measurement of the infarct volume. Ischemic mice were thus analyzed for their neurologic score at different post-reperfusion times. To evaluate the infarct volume, staining with 2,3,5-triphenyltetrazolium chloride (TTC) was usually performed. Herein, we used cresyl violet staining since it offers the opportunity to test many critical markers by immunohistochemistry. In this video, we report the MCAO procedure; neurological scores and the evaluation of the infarct volume by cresyl violet staining.

The intraluminal middle cerebral occlusion model in mice is herein presented. The extent of cerebral infarct is evaluated by a neurologic score and cresyl violet staining, an alternative staining to TTC, offering the great advantage to test in parallel many interest markers.

腔内MCAOのマウスモデル：クレシルバイオレット染色による脳梗塞の評価

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

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 ...

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.

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 ...

Two-vessel Occlusion Mouse Model of Cerebral Ischemia-reperfusion

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse model is useful for investigating the pathophysiology of cerebral ischemia-reperfusion and determining potential therapeutic strategies for patients with stroke. Variations in the anatomy of the circle of Willis of C57BL/6 mice affects their infarct volume after cerebral-ischemia-induced injury. Studies have indicated that distal MCA occlusion (MCAO) can overcome this problem and result in a stable infarct size. In this study, we establish a two-vessel occlusion mouse model of cerebral ischemia-reperfusion through the interruption of the blood flow to the right MCA. We distally ligate the right MCA and right common carotid artery (CCA) and restore blood flow after a certain period of ischemia. This ischemia-reperfusion injury induces an infarct of stable size and a behavioral deficit. Peripheral immune cells infiltrate the ischemic brain within the 24 h infiltration period. Additionally, the neuronal loss in the cortical area is less for a longer reperfusion duration. Therefore, this two-vessel occlusion model is suitable for investigating the immune response and neuronal recovery during the reperfusion period after cerebral ischemia.

A mouse model of cerebral ischemia-reperfusion is established to investigate the pathophysiology of stroke. We distally ligate the right middle cerebral artery and right common carotid artery and restore blood flow after 10 or 40 min of ischemia.

2 血管閉塞マウス脳虚血・再灌流モデル

In this study, a middle cerebral artery (MCA) occlusion mouse model is employed to study cerebral ischemia-reperfusion. A reproducible and reliable mouse ...

経頭蓋中大脳動脈閉塞マウスモデルの確立

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

著者スポットライト:脳卒中研究における革新的な技術と将来の方向性 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

高齢マウスの脳卒中転帰を研究するための修正経頭蓋中大脳動脈閉塞モデル

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

横慢性頭蓋窓の準備を有効

要約

さらに動画を探す

この動画の章

クローズ頭蓋窓を使用してマウス脳の微小循環の生体顕微鏡

中大脳動脈閉塞のマウスモデル

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

腔内MCAOのマウスモデル：クレシルバイオレット染色による脳梗塞の評価

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

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

2 血管閉塞マウス脳虚血・再灌流モデル

高齢マウスの脳卒中転帰を研究するための修正経頭蓋中大脳動脈閉塞モデル

高齢マウスの脳卒中転帰を研究するための修正経頭蓋中大脳動脈閉塞モデル

高齢マウスの脳卒中転帰を研究するための修正経頭蓋中大脳動脈閉塞モデル