Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

The authors have nothing to disclose.

全身低酸素症マウスでの（ すなわち、2時間11％O 2の）への単回暴露は「一過性」 -短い持続である低酸素プレコンディショニングの課題へのエピジェネティックな応答を意味tMCAo、29から脳を保護し、ベースラインの表現型は、内に復元され、日。低酸素プレコンディショニング刺激の繰り返しプレゼンテーションは劇的に神経保護表現型の持続時間を延ばす。6</sup...

成人障害と死因の第4位の主要な原因としては、脳卒中は、米国の成人人口が直面する最も衰弱性疾患状態の一つである。脳卒中の1動物モデルは、虚血性傷害を軽減する新しい方法の実験的調査を可能にし、脳卒中後の回復を改善します。そのようなトランスレーショナルリサーチのための新規な一道がプリコンディショニングされます。前処理は、その後、より重度の傷害からの損傷を低減するために、非損傷刺激の意図的な使用である。2低酸素プレコンディショニングは、 インビボおよびインビトロ試験の両方において脳卒中に対する保護を提供する脳内の多面的な変化をもたらすことが示されています。低比重低酸素しかし、低酸素に一回の露光のみ成体マウスにおける虚血に対する耐性未満の72時間の誘導、短期的な神経保護を提供しています3。でも14時間毎日のエクスポージャーの4週間後の4、Linら。 FOウントその神経保護は、一週間持続した。5繰り返し低酸素プレコンディショニング（RHP）頻度、期間、および低酸素曝露の強度の確率的変動によって特徴付けられます。単一プレコンディショニング課題とは対照的に、RHPはマウスで8週間まで持続脳保護表現型を誘導する。6 RHP、最終低酸素暴露後週間梗塞体積、血液脳関門（BBB）破壊、血管炎症、および白血球漏出を減少。 RHPは、具体的には、虚血性半球におけるB細胞集団を維持しながら、T細胞、単球、およびマクロファージ集団を減少させることにより、虚血性脳の炎症を減少させた。 図7は、実際には、RHPは、脳卒中を含む、任意のCNS ​​傷害の前に、マウスにおける免疫表現型を誘導しました。 RHP処理された健康なマウスから単離したRHP処理されたB細胞は、抗原提示および抗体産生の両方のダウンレギュレーションと、ユニークな抗炎症性の表現型を示しました。ザ炎症誘発性適応免疫機構の全体的な減少だけでなく、中枢神経系に特異的な炎症性疾患のための内因性の免疫抑制を誘導するためにRHPに優れた方法論になりますが、また前炎症性病変を含む全身損傷または疾患モデル。 RHPは一過性の中大脳動脈閉塞（tMCAo）以下の梗塞体積およびBBB破壊の両方を低減します。このような一般的に使用されるtMCAoとして脳卒中の動物モデルは、劇的に脳卒中の病態生理の理解だけでなく、より効果的な神経治療薬の設計を改善します。まず、小泉らによって開発された1986年に、8 tMCAo手順は、広く使用されているげっ歯類において脳卒中を誘導する方法と、再灌流後の炎症を調査するための好ましい方法の一つです。 tMCAoための方法が進化するにつれて、シリコーンコートフィラメントのより最近の使用は、他のモデル9,10 &lt;と比較して、くも膜下出血の危険性を減らします/ SUP&gt;残念ながらtMCAoは、多くの場合、梗塞量に大きなばらつきを生成してもして、信頼性を向上させる。11-13これらの研究のほとんどは、2,3,5-トリフェニルクロリド（TTC）で染色することによって冠状脳切片における梗塞領域の輪郭を描く、考え梗塞定量化のためのゴールドスタンダードは、鮮やかな、複製可能な結果を​​生成するための簡単​​で安価な方法であるためです。 TTCは、ミトコンドリア内に存在する脱水素酵素の基質として機能します。脳切片をTTC溶液にさらされると、ホルマザン、非水溶性の還元生成物は、生存可能なミトコンドリアの深い赤色の沈殿ところ、TTCを選択的に生細胞内に取り込まれます。そのため、虚血組織におけるミトコンドリア機能障害のため、この組織が ​​損傷を受け、健康な組織の分化を可能にする、白のまま。14 RHPはまた、虚血性半球におけるBBB破壊を減少させる。6したがって、同じB内のBBBの整合性の二重の定量化をTTCベース梗塞ボリュームと雨は、内因性の保護の完全な有効性に関する有用な情報、および未処理と処理された動物におけるBBB破壊および梗塞の間の潜在的な因果関係を提供する15の測定します。脳卒中の二次破砕BBBを介して末梢血の流入は、最終的には虚血性脳卒中の患者における感染と死亡率を増加させ、虚血性半球で白血球集団、炎症性サイトカイン、酸化ストレス、血管原性浮腫、出血性転換を増加させます。16,17動物モデルにおいてBBB破壊を測定する一般的な方法は、脳へのエバンスブルー（EB）色素漏出の定量化によるものである。15,18-21 EBを選択的にアルブミン、球状タンパク質を血清に結合する（MW = 65kDaの）それは無傷の動物でBBBを通過していません。22虚血性脳卒中後、EBは、脳に浸透し、光学濃度withiの測定を可能にする、620 nmで蛍光を発しますnは灌流が実質を負傷。22光学密度はEBは経心臓灌流によって死後皮質血管系から洗い流されたBBBの透過性に正比例します。 EBを投与した動物におけるTTC染色脳の即時処理により、梗塞容積及びBBB破壊の両方を効果的に定量することができます。これは、神経損傷およびBBBの破壊は、脳卒中後の脳の同時処理ではないことに留意すべきである、23,24ので屠殺時の選択は重要な考慮事項です。 詳細RHP方法、そのモデルヒト患者において、中大脳動脈閉塞、神経および血管脳卒中損傷エンドポイントを決定するための二重の組織学的方法を、一時的な動脈閉塞を誘導するためのtMCAo方法を以下のプロトコル。 TTCは、全体の梗塞体積の定量化を可能にする、細胞死および累積的組織損傷を測定します梅、EBはBBB損傷の半球定量化を提供します。

<table border="0" cellpadding="0" cellspacing="0" width="830">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:249px;">Material/ Equipment</td>
			<td style="width:171px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:283px;">Comments/Description</td>
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			<td height="220" style="height:220px;width:249px;">Flowmeters, regulators</td>
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			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5&#34; x 1&#34; x 1&#34; metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.</td>
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			<td height="26" style="height:26px;width:249px;">21% O2 tank</td>
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			<td style="width:127px;">OX USP200</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:249px;">11% O2 tank</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
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			<td height="26" style="height:26px;width:249px;">8% O2 tank&#160;</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">15L induction chambers</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">941454</td>
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			<td height="40" style="height:40px;width:249px;">Moor Laber Dopper Flow&#160;</td>
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			<td style="width:127px;">moorVMS-LDF1-HP</td>
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			<td height="20" style="height:20px;width:249px;">High Intensity Illuminator&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Zoom Stereo Microscope&#160;</td>
			<td style="width:171px;">NIkon</td>
			<td style="width:127px;">SMZ800</td>
			<td style="width:283px;">Other surgical microscopes may be used.&#160;</td>
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		<tr height="100">
			<td height="100" style="height:100px;width:249px;">Kent Scientific Right Temperature CODA</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">Discontinued</td>
			<td style="width:283px;">Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Hovabator Incubator</td>
			<td style="width:171px;">Stromberg&#39;s</td>
			<td style="width:127px;">2362-E</td>
			<td style="width:283px;">Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&#160;</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:249px;">V010 Anesthesia system&#160;</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">901807</td>
			<td style="width:283px;">Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">250 mL isoflurane&#160;</td>
			<td style="width:171px;">Butler Schein</td>
			<td style="width:127px;">NDC-11695</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">D-6 Vet Trim Animal Cordless Trimmer&#160;</td>
			<td style="width:171px;">Andis&#160;</td>
			<td style="width:127px;">#23905</td>
			<td style="width:283px;">Replacement blades are available from Andis (#23995)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Betadine&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">19-898-867&#160;</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Q-tips</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Gauze Pads</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">67622</td>
			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Surgical drape</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">GM300&#160;</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">Silk Sutures&#160;</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
			<td style="width:127px;">SP115</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">Nylon Sutures</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
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			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Durmont #5 forceps (2)&#160;</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">11251-35</td>
			<td style="width:283px;">Angled 45&#176;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Surgical Scissors</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">14028-10</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">3mm Vannas</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">INS600177</td>
			<td style="width:283px;">Straight blade</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Hartman Hemostats&#160;</td>
			<td style="width:171px;">Fine Scientific Tools</td>
			<td style="width:127px;">13002-10</td>
			<td style="width:283px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Occluding filaments</td>
			<td style="width:171px;">Washington University</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Evans Blue</td>
			<td style="width:171px;">Sigma Aldritch</td>
			<td style="width:127px;">E2129-10G</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Filter Paper&#160;</td>
			<td style="width:171px;">Sigma Aldritch</td>
			<td style="width:127px;">WHA1001150</td>
			<td style="width:283px;">150 mm, circles, Grade 1&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Weigh Boats</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">02-202-101</td>
			<td style="width:283px;">2.5&#34; diameter</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.9% Sodium Chloride Injection USP&#160;</td>
			<td style="width:171px;">Baxter Pharmaceutics&#160;</td>
			<td style="width:127px;">2B1321</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.3cc insulin syringe with 29 g needle</td>
			<td style="width:171px;">Becton Dickinson Labware</td>
			<td style="width:127px;">309301</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Flat bottom restrainer&#160;</td>
			<td style="width:171px;">Braintree Scientific&#160;</td>
			<td style="width:127px;">FB&#160;M</td>
			<td style="width:283px;">2.0&#34; diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">TTC</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:127px;">T8877</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">10X PBS, pH 7.4</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">BP399-20</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:249px;">Water Bath</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Styrofoam board</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Large Syringe Kit</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">P-SYRKIT-LG</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Perfusion Pump</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">NE-300&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">60 cc syringe</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">NC9203256</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">27g winged infusion set</td>
			<td style="width:171px;">Kawasumi Laboratories, Inc</td>
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			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">20 ml scintillation vial</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">50-367-126</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Stainless steel spatula</td>
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			<td style="width:127px;">14-373-25A</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Alto acrylic 1.0 mm mouse brain, coronal</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.21 mm stainless steel blades, 25 pk</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">4% paraformaldehyde</td>
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			<td style="width:127px;">SC-281692</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Superfrost microscope slides&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">HP Scanjet G4050</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">ImageJ&#160;</td>
			<td style="width:171px;">National Institute of Health</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Analytical Balance</td>
			<td style="width:171px;">Mettler Toledo&#160;</td>
			<td style="width:127px;">XSE 205U</td>
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			<td height="20" style="height:20px;width:249px;">Precision Compact Oven&#160;&#160;</td>
			<td style="width:171px;">Thermo Scientific&#160;</td>
			<td style="width:127px;">PR305225M</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">1.7 mL microcentrifuge tubes (Eppendorfs)</td>
			<td style="width:171px;">Denville Scientific&#160;</td>
			<td style="width:127px;">C2170</td>
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			<td height="20" style="height:20px;width:249px;">Formamide</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">BP228-100</td>
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			<td height="20" style="height:20px;width:249px;">96-well plates</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">07-200-9</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Epoch Microplate Spectrophotometer&#160;</td>
			<td style="width:171px;">BioTek&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
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quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice

脳卒中の実験動物モデルは、ストロークの病態を理解し、より効果的な治療戦略を開発するための貴重なツールです。反復的な低酸素プレコンディショニング（RHP）2週間のプロトコルは、焦点虚血性脳卒中のマウスモデルにおいて、中枢神経系（CNS）損傷に対する長期保護を誘導します。 RHPは期間（2または4時間）と強度（8％と11％のO 2）の両方に変化する低酸素症に9確率的エクスポージャーで構成されています。 RHPは、内因性CNS-保護表現型の長期的な誘導を示唆し、梗塞体積、血液脳関門（BBB）の破壊、および低酸素への最後の暴露後の数週間のための脳卒中後の炎症反応を減少させます。梗塞体積及びBBB破壊の二重定量化のための方法は、RHPまたは他の推定上の神経保護剤を有するマウスにおける神経血管保護を評価するのに有効です。成人男性のスイスウェブスターマウスを21％OにRHPまたは期間と同等の曝露によって予め調整しました。 <suB&gt; 2（ すなわち、室内空気）。 60分一過中大脳動脈閉塞（tMCAo）は、最後の低酸素曝露後の2週間誘導しました。閉塞および再灌流の両方が経頭蓋レーザドップラー流量測定によって確認しました。再灌流後の二十二時間、エバンスブルー（EB）を尾静脈注射を介して投与しました。 2時間後、動物をイソフルランの過剰投与、脳切片を2,3,5-塩化トリフェニルテトラゾリウム（TTC）で染色したことにより屠殺しました。梗塞容積を定量しました。次に、EBをtMCAo後にBBB破壊を決定するために、48時間かけて組織から抽出しました。要約すると、RHPは他のCNSベースおよびプロ炎症性全身疾患状態のための翻訳可能性のある、マウスにおける脳卒中損傷から長期内因性神経血管保護を誘導するために、最小限のコストで、複製することができる単純なプロトコルです。

本研究では、10週齢がRHPに無作為化の開始時であった（N = 10）25の雄Swiss Websterマウスまたは21％のO 2（N = 15）のグループが含まれていました。二週間最終RHP曝露後、外科的処置は盲検基により行い、日の間相殺します。 tMCAoに続いて、1マウスは、術後の回復中に死亡し、再灌流CBF基準を満たしていなかったので、1マウスは研究から除外しました。両方除外マウスは、21％O 2群?...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

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

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 ...

quantification-neurovascular-protection-following-repetitive-hypoxic

Research

JoVE Journal

Medicine

13.6K ビュー.  University of Texas Southwestern Medical Center. 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.

ビデオ: Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion 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 ...

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 ...

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 ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

P10ラットにおける新生児脳卒中の一過性中大脳動脈閉塞モデル

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...