This study was supported by the NIH grant NS074559 (to C. K.). We thank all collaborators who contributed to our research articles that the present methodology report is based upon.

このプロトコルは、エモリー大学の施設内動物管理使用委員会（IACUC）によって承認され、ケアと実験動物の使用のための健康ガイドラインの国立研究所に追従されます。 1.セットアップ<ol><li>手術の前に少なくとも15分間、37℃で熱ポンプに接続された温暖化のパッドの上に手術台を準備します。手術台の上に3ミリリットルの注射器のバレルを使用して、首のロールを配置します。医療空気中2％イソフルランで麻酔ガスを準備します。 </li><li>オートクレーブした鉗子、はさみ、マイクロニードルホルダー、止血剤、綿棒や縫合糸を準備します。組織接着剤や眼軟膏を準備します。 </li><li>加熱ランプおよび直腸プローブと低酸素システムと温度コントローラを設定します。 92.5％N 2でバランス7.5％のO 2中の2％イソフルランで低酸素ガスを準備します。 </li><li>一時間手術前に、マウスを徐放メロキシカム（4.0ミリグラム/ kg）を皮下注射することによりanalgesizedされています。 </li></ol> &lt;Pクラス= &quot;jove_title&quot;&gt; 2。一過性脳低酸素虚血（図1B） <ol><li>動物が足のスクイズに応答しなくなるまで、3％イソフルランで麻酔導入室で22〜30グラムの重さの10〜13週齢の雄のC57BL / 6マウスを麻酔し、その後電子シェーバーを使用して、右の首の毛を削除します。 </li><li> 2 L / minの流量で医療空気中の2％イソフルランで接続された手術台の上にマウスを置きます。セキュア前肢は、医療テープを使用して、両側にネックロールに沿って伸ばし。 </li><li>アルコール、その後綿棒続いベタジンで切開するための外科手術部位を清掃してください。 </li><li>解剖顕微鏡下で、まっすぐ鉗子と約0.2センチ正中皮膚からの横方向のマイクロはさみを使用して0.5センチメートル右頸部切開を行います。 </li><li>右総頸動脈（RCCA）を露出させるために筋膜と組織を引き離すために微細な鋸歯状の鉗子のペアを使用してください。慎重に細かいなめらかなピンセットを使用して、迷走神経からRCCAを分離します。 </li><li>ライブRCCA上の2つのプレカット5-0絹縫合糸（解除可能）を結び目、その後、4-0ナイロンモノフィラメント縫合糸（ 図1C）を用いて皮膚を縫い合わせます。 </li><li>乾燥を防ぐために、両眼に眼軟膏を適用します。 </li><li>素早く低酸素システムにマウスを転送し、30分間、0.5 L /分の流速で7.5％のO 2中の2％イソフルランでフェイスマスクで鼻と口を置きます。 <ol><li>低酸素時には、37.5±0.5℃の直腸温度を制御する加熱ランプと温度コントローラを使用しています。 80〜120呼吸/分で呼吸速度を監視します。低酸素時に37℃以上の体温を維持することは、一貫性の脳梗塞を作成することが重要です。低呼吸数は、通常20分の低酸素症の後に発生します。フェイスマスクを外し、呼吸数は40に下回る場合、これは1〜2分を取り、30分間の低酸素症の期間にカウントされません通常の空気の供給を可能にします。 </li></ol></li><li>低酸素症の後、転送手術台のマウスとはRCCAから2本の縫合糸を解放します。組織接着剤を使用して傷を閉じて、ケージにマウスを返します。 2つのライブノットの両方が予期せず、低酸素後にリリースされた場合、動物を除外します。 </li><li>低酸素および麻酔から回復するために5〜10分間マウスを監視します。ケージに濡れた食品を置き、動物飼育施設に戻します。 注：THI後24時間で重度の旋回動作に軽度を示す動物は脳梗塞と相関しています。発作症状を持つほとんどの動物は、Th1 24時間後の時点前に死亡します。 </li></ol> 3.レーザースペックルコントラストイメージング注意：これはTHIモデルの本質的な手順ではないが、二次元のレーザースペックルコントラスト画像化システム16は、中または一時低酸素性虚血後の脳の血流（CBF）の変化を特徴づけるために使用することができます。 STの直後に、THIで、レコードをCB​​Fの変化を文書化することEP 2.6。あるいは、THI傷害後のCBFの回復を比較するために、これらの手順は、ステップ2.10以下を行うことができます。 <ol><li>腹臥位で麻酔したマウスを置き、頭蓋骨露出したが、未開封で、頭皮に約1cm長の正中切開を行います。 </li><li>製造業者のプロトコルに従って、血流画像化装置の下で両方の大脳半球にCBFを監視し、CCAO手術（ステップ2.6）の直後に脳血流の記録を開始。 50分間続けます。 </li><li> 16色のパレットで任意の単位でCBF画像を表示して、製造業者の指示（ 図2）は、次のMoorFLPIソフトウェアを使用してリアルタイムで選択した領域を分析します。 </li><li> CBF画像を記録した後、組織接着剤で頭皮を閉じて、ケージに動物を返します。 </li></ol> 4. tPAの管理<ol><li> 220〜300（溶媒または10mg / kgの組換えtPAを尾静脈で動物を注入＆＃956; 1ミリグラム/ M TPA）のL 0.5、1、またはtCCAoプラス低酸素症後4時間で（ 図4）。 </li></ol>いくつかの異なるオプションで5脳の損傷検出注：THI後、脳試料を採取1でマウスを安楽死させる、4または24時間にします。 <ol><li> in vivoでの塩化2,3,5-トリフェニルテトラゾリウム（TTC）が記載以前のようなTHI傷害後24時間目の方法で梗塞体積の定量化を行う。17 <ol><li>腹腔内transcardial灌流前に1.4 Mマンニトール溶液（〜0.1ミリリットル/ g体重）を30分で動物を注入します。 PBSでTranscardial散らすマウスを2％TTCを10mlました。 </li><li> 10分後に手術器具を用いた動物の脳を削除し、ビブラトームで、1mmの厚さに一晩固定し、セクションの4％パラホルムアルデヒド中に配置します。 </li><li>デジタル顕微鏡および定量によって4区分脳のスライドの一連のスナップImageJソフトウェアを使用して、損傷していない、反対側の半球の面積に対する梗塞領域（右側の白い部分）の割合としての梗塞体積をIFY。 </li></ol></li><li>あるいは、THI傷害後の1時間後に免疫蛍光法により血栓形成を行います。 <ol><li> OCT化合物および部分クライオスタットを用いて、12μmの厚さで脳内の固定脳をフリーズします。 </li><li>蛍光顕微鏡で蛍光を観察するために：（200 1）ヤギ抗ウサギアレクサFluro 488色素により以下：（100 1）ウサギ抗フィブリノゲン抗体を用いた脳のスライドをインキュベートします。 </li></ol></li><li>あるいは、THI傷害の後4時間で100μlの2％エバンスブルー色素の尾静脈注射により血管閉塞を行います。 <ol><li>マウスを安楽死させるとすぐにエバンスブルー注射後、4％パラホルムアルデヒド中に脳を除去するために頭を切りました。注：これは、両方の前足の青色とエバンスブルー循環のための5-10分を取り、後肢。 </li><li> Secti100ミクロンの厚さで固定された脳にスライディングミクロトームを用いて、蛍光顕微鏡で680 nmの発光フィルターを用いて蛍光を観察します。 </li></ol></li></ol>

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脳卒中は、人口の高齢化との任意の社会の高まる重要性の主要な健康問題です。世界的には、ストロークが全死亡18の11.1％に相当する2010年の推定590万致命的なイベント、との死因の第2位です。脳卒中は障害調整生存年数（DALYs）の第三の主要な原因は、1990年19で5位から上昇し、2010年に世界的に失われたもである。これらの疫学データは、急性のより有効な治療法（虚血性）...

血栓溶解および再疎通は、臨床診療1急性虚血性脳卒中の最も効果的な治療法です。しかし、前臨床神経保護研究の大部分は、血管閉塞を除去すると脳血流量の急速な回復を生成したtPAの血栓によるない利点のほとんどを示す過渡メカニック閉塞モデル（腔内縫合中大脳動脈閉塞）で行いました。これは、脳卒中モデルの選択は、疑わしい患者2,3に神経保護療法を変換することが困難に、少なくとも部分的に寄与することが示唆されています。したがって、前臨床研究におけるtPAの応答性血栓塞栓性脳卒中モデルを採用したため増加コールがありますが、このようなモデルはまた、技術的な問題（説明を参照）4-7を有しています。ここでは、一方的な一過性低酸素性虚血性（THI）侮辱および静脈内のtPA療法8への応答に基づいて新たな血栓性脳卒中モデルについて説明します。 THIストロークモデルは、1960年9成体ラットを用いた実験のために発明されたLevineの手順（チャンバ内の過渡的低酸素への曝露が続く一方的な総頸動脈の永久結紮）に基づいて開発されました。オリジナルのレバイン手順はあいまいに色あせたため、それが唯一の変数脳の損傷を生じたが、それは1981年10新生児低酸素性虚血性脳症（HIE）のモデルとしてロバート·バンヌッチと彼の同僚によって再導入されたときと同じ侮辱は、げっ歯類の仔で一貫性のある神経病理の原因となった。近年では、いくつかの低酸素室11内の温度を調整することにより、成体マウスの研究者の再適応レヴァイン-バンヌッチモデル。これは、元レヴァインの手順で一貫性のない脳病変は、低酸素室に大人のげっ歯類の体温の変動から生じ得ることがもっともらしいです。この仮説を試験するために、我々は、低酸素ガスを投与することにより、レヴァイン手順を改変しましたフェイスマスクを介して、手術台12の上に、37℃でげっ歯類コア温度を維持します。予想されるように、ストリンジェントな体温調節が大きくHI誘発性脳病変の再現性を増加させました。 HI傷害はまた、凝固、オートファジー、とグレーと白質損傷13をトリガします。他の研究者らはまた、脳卒中後の炎症反応14を調査するためにHIモデルを使用しています。 HIストロークモデルのユニークな特徴、それは密接に血流のうっ滞を含む、血栓形成のウィルヒョウのトライアドに従うことで、内皮損傷（ 例えばによるHI-誘導される酸化ストレスに対する）、および凝固亢進（HI誘発性血小板活性化）（ 図1A）15。このように、HIモデルは、実世界の虚血性脳卒中に関連したいくつかの病態生理学的メカニズムを取り込むことができます。念頭に置いてこの考えでは、我々はさらに、国連の可逆的ライゲーションとHIモデルを洗練しましたilateral総頸動脈（したがって過渡HI傷害を作成する）、およびまたはエダラボンなしのtPA血栓溶解にその応答をテストしました。エダラボンは、すでに発症9の24時間以内の虚血性脳卒中を治療するために、日本で承認され、フリーラジカルスカベンジャーです。我々の実験は、短い30分の過渡HIは、血栓性梗塞をトリガし、その組み合わせのtPA-エダラボン処理が相乗便益8を与えることを示しました。ここでは、詳細な外科的手順および急性虚血性脳卒中の再灌流治療を最適化するために使用することができますTHIモデルの方法論的な考慮事項について説明します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>adult male mice</td>
			<td>Charles River</td>
			<td>C57BL/6&#160;</td>
			<td>10~13 weeks old (22~30 g)</td>
		</tr>
		<tr>
			<td>Mobile Laboratory Animal Anesthesia System</td>
			<td>VetEquip</td>
			<td>901807</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Medical air (Compressed) air tank</td>
			<td>Airgas</td>
			<td>UN1002</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Piramal Healthcare</td>
			<td>NDC 66794-013-25</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Multi-Station Lab Animal AnesthesiaSystem</td>
			<td>Surgivet</td>
			<td>V703501</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>7.5% O2 balanced by 92.5% N2 tank</td>
			<td>Airgas</td>
			<td>UN1956</td>
			<td>hypoxia system</td>
		</tr>
		<tr>
			<td>Temperature Controller with heating lamp&#160;</td>
			<td>Cole Parmer&#160;</td>
			<td>EW-89000-10</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Rectal probe</td>
			<td>Cole Parmer&#160;</td>
			<td>NCI-00141PG</td>
			<td>temperature controllers</td>
		</tr>
		<tr>
			<td>Dissecting microscope&#160;</td>
			<td>Olympus&#160;</td>
			<td>SZ40</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Heat pump with warming pad</td>
			<td>Gaymar&#160;</td>
			<td>TP700</td>
			<td>surgical setup</td>
		</tr>
		<tr>
			<td>Fine curved forceps (serrated)</td>
			<td>FST</td>
			<td>11370-31</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Fine curved forceps (smooth)</td>
			<td>FST</td>
			<td>11373-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro scissors</td>
			<td>FST</td>
			<td>15000-03</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>micro needle holders</td>
			<td>FST</td>
			<td>12060-01</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>Halsted-Mosquito hemostats</td>
			<td>FST</td>
			<td>13008-12</td>
			<td>surgical instrument</td>
		</tr>
		<tr>
			<td>5-0 silk suture&#160;</td>
			<td>Harvard Apparatus</td>
			<td>624143</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>4-0 Nylon monofilament suture</td>
			<td>LOOK</td>
			<td>766B</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Tissue glue</td>
			<td>Abbott Laboratories</td>
			<td>NC9855218</td>
			<td>surgical supplies</td>
		</tr>
		<tr>
			<td>Puralube Vet ointment</td>
			<td>Fisher</td>
			<td>NC0138063&#160;</td>
			<td>eye dryness prevention&#160;</td>
		</tr>
		<tr>
			<td>MoorFLPI-2 blood flow imager</td>
			<td>Moor</td>
			<td>780-nm laser source</td>
			<td>Laser Speckle Contrast Imaging</td>
		</tr>
		<tr>
			<td>Mannitol</td>
			<td>Sigma</td>
			<td>M4125</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)&#160;</td>
			<td>Sigma</td>
			<td>T8877</td>
			<td>in-vivo TTC</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Stoelting</td>
			<td>51425</td>
			<td>brain section for in-vivo TTC&#160;</td>
		</tr>
		<tr>
			<td>Digital microscope</td>
			<td>Dino-Lite</td>
			<td>AM2111</td>
			<td>whole-braina imaging</td>
		</tr>
		<tr>
			<td>O.C.T compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-rabbit Alexa Fluro 488</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>Immunohistochemistry</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Vibratome</td>
			<td>ultrapro 5000</td>
			<td>brain section for IHC</td>
		</tr>
		<tr>
			<td>Evans blue</td>
			<td>Sigma</td>
			<td>E2129</td>
			<td>Detecting vascular perfusion</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Electron Microscopy Sciences</td>
			<td>5000</td>
			<td>brain section for histology</td>
		</tr>
		<tr>
			<td>Avertin (2, 2, 2-Tribromoethanol)</td>
			<td>Sigma</td>
			<td>T48402</td>
			<td>euthanasia</td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
	</tbody>
</table>

a thrombotic stroke model based on transient cerebral hypoxia-ischemia

脳卒中の研究は、臨床実践に神経保護療法を変換するときに、多くの挫折に耐えてきました。これとは対照的に、現実世界の治療因子（tPAの血栓溶解）はめったに前臨床ストローク研究を支配する機械的閉塞ベースの実験モデルで利益を、生成しません。ベンチとベッドサイドの間のこの分割は、前臨床ストローク研究でのtPA応答モデルを採用する必要性を示唆しています。この目的のために、簡単かつ反応性のtPA血栓性脳卒中モデルを考案し、ここで説明されています。 37.5±0.5℃で、動物の直腸温度を維持しながら、このモデルは、30分間、成体マウスにおいて、フェイスマスクを介して片側の総頸動脈および7.5％酸素の送達の一過性閉塞から成ります。片側頚動脈または低酸素の可逆的連結各のみ一過性脳血流を抑制しているが、両方の損傷の組合せが持続する再灌流障害、フィブリンおよび血小板沈着、および大infarを引き起こし中大脳動脈が提供する地域におけるCT。重要なことには0.5で、組換えtPAの、尾静脈注射、1、または4時間後THI（10mg / kg）は死亡率および梗塞サイズの時間依存的な減少をもたらしました。この新たな脳卒中モデルが単純であり、実験結果を比較するために実験室を横切って標準化することができます。また、頭蓋骨切除術または予め形成された塞栓を導入することなく、血栓症を誘発します。これらのユニークなメリットを考えると、THIモデルは、前臨床ストローク研究のレパートリーに有用な付加です。

二次元レーザスペックルコントラストイメージング（LSCI）16は、30分の過渡片側頚動脈閉塞（tCCAO）、低酸素状態に30分間露光（7.5％酸素）、および30分片側によって脳血流（CBF）の変化を比較しました低酸素症（THI）の下で連結を頸動脈。この実験は、tCCAOが通常酸素下で迅速に頸動脈閉塞（ 図2AのR）のリリース後85％以上に回復し、ベースライン値の〜50％に頸動脈結紮...

Watch this Scientific Journal Video about A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia at JoVE.com

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

一過性脳低酸素性虚血に基づく血栓性脳卒中モデル

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

a-thrombotic-stroke-model-based-on-transient-cerebral-hypoxia-ischemia

Research

JoVE Journal

Medicine

14.7K ビュー.  The Emory University School of Medicine. Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients. 

ビデオ: A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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 ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

くも膜下出血のマウスモデル

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

脳Microslicesの興奮毒性刺激<em&gt;インビトロ</em&gt;脳卒中のモデル

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

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 ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

マウス脳低酸素性虚血の間に同時PET / MRIイメージング

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...