この研究は、文部科学省(MEXT)から科学研究助成(KAKENHI)(18K09424から栗原敏秀、20K18393から三輪幸宏)の支援を受けた。

ここに記載されている方法はすべて、慶應義塾大学医学部のIACUC(IACUC)の教育機関で承認されています。
1. 手術器具・動物の準備
<ol><li>オートクレーブ手術器具を70%エチルアルコールに保ちます。新しい外科手術の前に、70%のエチルアルコールを使用して慎重に外科用器具を洗浄する。</li>
	<li>雄BALB/cAJc1マウス(生後6週、26~28kg)を、手術前、手術中、手術後の無菌状態を維持するために、特異的病原体フリー(SPF)の部屋に準備します。</li>
</ol>2. 一過性両側性大動脈閉塞(tBCCAO)
<ol><li>ミダゾラム(40μg/100 μL)、メデトミジン(7.5 μg/100 μL)およびブトルファンノール酒石酸塩(50μg/100 μL)の組み合わせで腹腔内注射を介して麻酔下にマウスを入れて、前述の8,9と呼ばれる「MMB」と呼ばれる。マウスの背中のスキンを保持して、マウスが完全に麻酔状態になるまでマウスが目をぶつけないようにします。<ol>
<li>麻酔の深さを判断するには、マウスのつま先を応答がなくなるまでつまんで、その方法は完全な麻酔10をチェックするために一般的に使用される。 注:一般的に、マウスが眠りに落ちるためには5分未満が必要です。全身麻酔のための適切なレシピは、機関によって異なる場合があります。</li>
	</ol>
</li>
	<li>麻酔下で目の乾燥を防ぐために、0.1%精製したヒアルロン酸ナトリウムアイドロップ溶液を1滴ずつ目に塗布します。</li>
	<li>背中にマウスを置き、粘着テープを使用してマウスの足を固定します。</li>
	<li>手術前に70%エチルアルコールを使用してマウスの首の領域を消毒する。 注:毛皮の追加のクリッピングは、これが原因で、その後の皮膚の炎症11、12を引き起こす可能性があるため、実行されませんでした。</li>
	<li>首の矢状切開をブレードで行う(図1)。 注意:切開は首、胸骨、気管の間の正中線で行う必要があります。</li>
	<li>唾液腺を慎重に2つの鉗子を使って分離し、それらを動員して基礎となるCCAを視覚化する。</li>
	<li>右のCCAをそれぞれの迷走神経とそれに付随する静脈から慎重に分離し、その構造を損なうことなく、CCAの下に2つの6-0シルク縫合糸を置きます。2つのネクタイをしっかりと結び、血流を遮断します(図1)。 注:手順中に、小さな静脈が損傷する可能性があります。出血が見られる場合は、CCAを明確に可視化するために拭き取る必要があります。</li>
	<li>左のCCAを構造に害を与えることなく、それぞれの迷走神経とそれに付随する静脈から注意深く見つけ、クランプで2秒間左CCAを閉塞させる(図1)。 注:クランプ用の部位をマークするには、左CCAの下に6-0シルク縫合針を配置する必要があります。</li>
	<li>左CCAを再開した後、6-0シルク縫合糸による首の縫合傷を、細菌感染を阻害するために、頸部に抗生物質(50μL)のダブを適用する。 注:左CCAを再開するときに動脈壁を損傷しないようにクランプを柔らかく取り外します。</li>
	<li>マウスをマウスに0.75mg/kg注入し、深部麻酔から回復したマウスを助けます。マウスをプリヒーテッドパッドでマウスケージに戻します。 注:マウスが胸骨の不備を維持するのに十分な意識を取り戻すまで、マウスを放置しないでください。</li>
	<li>マウスが目を覚ますときの痛みの管理のために、マウスに0.4mg/kgのブトルファンノール酒を注入します。 注: プロトコルはここで一時停止することができます。tBCCAOを成功させるための最初のヒントとして、マウスの眼瞼垂動が観察される(図2)。</li>
	<li>安楽死のために、マウスにMMB混合物の3倍を注入し、実験のためにそれらを犠牲にする。</li>
</ol>3. 一般的な観測(生存率と眼瞼下垂)
<ol><li>手術後、0日目(手術後)、1、3、7のすべての死因の生存率を確認してください。</li>
	<li>4点の評価スケールで眼瞼垂れを評価する:1 = 垂れ下がらない、2 = 軽度の垂れ下がり(〜50%)、3 = 重度の垂れ下がり(50%以上)、4 = 眼の排出による重度の垂れ下がり。</li>
</ol>4. レチン血性輸血
<ol><li>マウスの左心室にFITC-dextran(25mg/mL)の200μLを注入し、マウスの眼科の血管13,14における輸血の観察に一般的に使用される。</li>
	<li>循環後2分、目を欠核化し、4%パラホルムアルデヒドに1時間固定する。このレティナを注意深く得て平らに取り付け、先に説明した15を、蛍光顕微鏡を介して調べた。</li>
	<li>4倍の倍率でレティナルマウント全体の写真を撮り、マージアナライザを使用して単一にマージします。</li>
	<li>NIH フィジー/ImageJ ソフトウェアの容器分析ツールを使用して、浸透した領域を測定します。</li>
</ol>5. ウェスタンブロット
<ol><li>tBCCAOの3時間及び6時間後、マウスの目を得て、すぐに冷たいPBSを含むペトリ皿に移して、レチナを単離する。</li>
	<li>レティナを分離した後、前述の9のようにウェスタンブロッティングを行う。</li>
	<li>低酸素誘導因子-1α(HIF-1α;一般的な低酸素マーカー)およびβアクチン(内部ローディングコントロール)に対する抗体を一晩インキュベートし、続いてHRP共役二次抗体をインキュベートする。化学発光を介して信号を可視化.</li>
</ol>6. 定量PCR (qPCR)
<ol><li>6、12および24時間後に、qPCRについて得られたレチナを処理し、前述の17.</li>
	<li>リアルタイム PCR システムを介して qPCR を実行します。使用するプライマーを 表 1に示します。ΔΔCT 法により、異なるトランスクリプトのレベル間の折り畳み変化を計算します。</li>
</ol>7. 免疫検査(IHC)
<ol><li>tBCCAOの3日後、マウスの目を得てパラフィンに埋め込む。</li>
	<li>パラフィン埋め込んだ目をミクロトームで切り、目の切片を得る。</li>
	<li>前に説明した5μmの厚さの目の切片を脱パラフィン化し、染色する13.</li>
	<li>グリア線維性酸性タンパク質(GFAP;レチナにおけるアストロサイトおよびミュラー細胞の信頼できるマーカー)に対する抗体と一晩のインキュベート、続いてアレクサフルオール555結合二次抗体のインキュベーション。</li>
	<li>DAPI(4′,6-ジミジノ-2-フェニリンドール)を使用して、レチナの核を染色します。蛍光顕微鏡で信号を可視化。</li>
	<li>前述の13、18:0=信号なし、1 = ガングリオン細胞層(GCL)の正のグリア端フィートの少ない、GCLから外核層(ONL)に到達する標識されたプロセスの数が少なく、3 = GCLからONLに到達する最もラベル付きされたプロセスで、4点の評価スケールでの形態スコア付けを評価する。</li>
</ol>8. 電解法(ERG)
<ol><li>tBCCAOの3日と7日後に、ガンツフェルトドーム、取得システムおよびLED刺激装置を用いてERGを行う、前述の9.</li>
	<li>一晩の暗い適応の後、薄暗い赤色光の下でMMBの組み合わせでマウスを麻酔する。</li>
	<li>0.5%のトロピックアミドと0.5%フェニレフリンの混合溶液を使用して瞳孔を拡張する。</li>
	<li>アクティブ電極をコンタクトレンズに置き、参照電極を口に入れます。</li>
	<li>各動物の両眼からERG応答を得る。</li>
	<li>様々な刺激で暗い適応の下でスコトピック応答を記録する。</li>
	<li>ベースラインから波の最低点までの波の振幅を測定します。</li>
	<li>波の最も低い点からb波のピークまでのb波の振幅を測定します。</li>
	<li>ヒートパッドを使用して手順中にすべてのマウスを暖かく保ちます。</li>
</ol>9. 光コハレンス断層撮影(OCT)
<ol><li>tBCCAO の 2 週間後に、前に報告された8,9のように、SD-OCT システムを使用して OCTを実行します。</li>
	<li>測定のために、対象マウスは0.5%のトロピックアミドと0.5%フェニレフリンの混合溶液によりマイドリア症を、MMBの混合物による全身麻酔にする。</li>
	<li>赤道スライスのen-faceスキャンからBスキャン画像を取得します。</li>
	<li>視神経の頭部から0.2、0.4および0.6mmのレティナを調べてください。</li>
	<li>レチナル神経繊維層(NFL)から外部制限膜(ELM)までのレチナル厚さを測定し、個々のマウスのレチナル厚として測定値の平均を考える。</li>
	<li>結果をスパイダー ダイアグラムとしてプロットします。</li>
</ol>

著者らは開示するものは何もない。

この研究では、単純な縫合糸とクランプを使用したtBCCAOが、眼科虚血およびそれに伴う筋機能障害を引き起こす可能性があることを示している。さらに、我々は、レチナル虚血のマウスモデルの開発のための我々の現在のプロトコルは、2、3、7の陰部虚血損傷モデルの開発のための他の以前のプロトコルと比較してより簡...

このレティナは、視覚機能のための神経感覚組織である。視覚機能には相当量の酸素が必要であるため、このレティナは体内で最も酸素要求の多い組織の1つとして知られている。このレティナは、酸素が血管を介して送達される血管疾患の影響を受けやすい。糖尿病性網膜症や網膜血管(静脈または動脈)閉塞などの血管疾患の様々なタイプは、網膜虚血を誘発することができます。レチナル虚血の病理的メカニズムを調べるには、再現性と臨床的に関連する、レチナル虚血の実験モデルが必要と考えられる。中大脳動脈閉塞(MCAO)は、インビボ歯止め薬の実験用虚血2,3の開発に最も一般的に利用される方法である内無血フィラメントの挿入による。MCAに対する眼動脈(OpA)の近接性のために、MCAOモデルは、網膜虚血4、5、6の病態生理を理解するために同時に使用される。脳虚血を、レチナル虚血と共に誘導するために、長いフィラメントは通常、一般的な頸動脈(CCA)または外頸動脈(ECA)の切開を通して挿入される。これらの方法は、実行するのが困難であり、手術を完了するのに長い時間(1つのマウスで60分以上)を必要とし、手術後の結果に高い変動性をもたらす7。これらの懸念を改善するために、より良いモデルを開発することが依然として重要です。
本研究では、短い一過性の両側CCA閉塞(tBCCAO)を針とクランプで使用してマウスの裂精虚血を誘導し、陰部における虚血性傷害の典型的な結果を分析した。このビデオでは、tBCCAO の手順のデモンストレーションを行います。

<table>
	<tbody>
		<tr>
			<td>Atipamezole hydrochloride</td>
			<td>Zenoaq</td>
			<td>Antisedan</td>
			<td>For anti-anesthesia</td>
		</tr>
		<tr>
			<td>Applied Biosystems 7500 Fast</td>
			<td>Applied Biosystems</td>
			<td>-</td>
			<td>For qPCR</td>
		</tr>
		<tr>
			<td>Butorphanol tartrate</td>
			<td>Meiji Seika Pharma</td>
			<td>Vetorphale</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>BZ-II Analyzer</td>
			<td>KEYENCE</td>
			<td>-</td>
			<td>For an image merge</td>
		</tr>
		<tr>
			<td>BALB/cAJc1</td>
			<td>CLEA</td>
			<td>-</td>
			<td>Mouse strain</td>
		</tr>
		<tr>
			<td>&#946;-Actin (8H10D10) Mouse mAb</td>
			<td>CST</td>
			<td>3700</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>Clamp Forcep</td>
			<td>World Precision Instruments</td>
			<td>WPI 500451</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Dumont forceps #5</td>
			<td>Fine Science Tools</td>
			<td>11251-10</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>DAPI solution</td>
			<td>Dojindo</td>
			<td>340-07971</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Envisu SD-OCT system</td>
			<td>Leica</td>
			<td>R4310</td>
			<td>For OCT</td>
		</tr>
		<tr>
			<td>FITC-dextran</td>
			<td>Merk</td>
			<td>FD2000S</td>
			<td>For retinal blood perfusion</td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>KEYENCE</td>
			<td>BZ-9000</td>
			<td>For fluorescence detection</td>
		</tr>
		<tr>
			<td>Gatifloxacin hydrate</td>
			<td>Senju Pharmaceutical</td>
			<td>Gachifuro</td>
			<td>For anti-bacterial infection</td>
		</tr>
		<tr>
			<td>GFAP Monoclonal Antibody (2.2B10)</td>
			<td>Thermo</td>
			<td>13-0300</td>
			<td>For IHC</td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td>Marukan</td>
			<td>RH-200</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>HIF-1&#945; (D1S7W) XP Rabbit mAb</td>
			<td>CST</td>
			<td>36169</td>
			<td>For western blot</td>
		</tr>
		<tr>
			<td>ImageQuant LAS 4000 mini</td>
			<td>GE Healthcare</td>
			<td>-</td>
			<td>For chemiluminescence</td>
		</tr>
		<tr>
			<td>Midazolam</td>
			<td>Sandoz K.K</td>
			<td>SANDOZ</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Microtome Tissue-Tek TEC 6</td>
			<td>Sakura</td>
			<td>-</td>
			<td>For sectioning</td>
		</tr>
		<tr>
			<td>Medetomidine</td>
			<td>Orion Corporation</td>
			<td>Domitor</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Handaya</td>
			<td>HS-2307</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>PuREC</td>
			<td>MAYO Corporation</td>
			<td>-</td>
			<td>For ERG</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>Fine Science Tools</td>
			<td>91460-11</td>
			<td>For surgery</td>
		</tr>
		<tr>
			<td>Sodium hyaluronate</td>
			<td>Santen Pharmaceutical</td>
			<td>Hyalein</td>
			<td>For eye lubrication</td>
		</tr>
		<tr>
			<td>Tropicamide/Penylephrine hydrochloride</td>
			<td>Santen Pharmaceutical</td>
			<td>Mydrin-P</td>
			<td>For mydriasis</td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Natsume</td>
			<td>E12-60N2</td>
			<td>For surgery</td>
		</tr>
	</tbody>
</table>

a murine model of ischemic retinal injury induced by transient bilateral common carotid artery occlusion

糖尿病性網膜症、網膜静脈または動脈の閉塞、眼性虚血症候群などの多様な血管疾患は、網膜虚血を引き起こす可能性があります。レチナル虚血の病理的メカニズムを調べるには、関連する実験モデルを開発する必要がある。解剖学的に、主な眼の血液供給血管は眼動脈(OpA)であり、OpAは一般的な頸動脈(CCA)の内頚動脈に由来する。したがって、CCAの破壊は効果的に、虚血の原因となりうる。ここでは、一過性両側性一般的な頸動脈閉塞(tBCCAO)によるレチナル虚血のマウスモデルを確立し、右CCAを6-0シルク縫合糸で結び付け、クランプを介して左CCAを一時的に2秒間閉塞させ、tBCCAOが急性レチナル虚血を誘発し、性間機能障害を引き起こす可能性があることを示した。現在の方法は、手術針とクランプのみを使用することによって手術器具への依存を減らし、中大脳動脈閉塞のマウスモデルによく見られる予期せぬ動物の死を最小限に抑えるために閉塞時間を短縮し、一般的な再発性虚血所見の再現性を維持する。このモデルは、マウスにおける虚血性網膜症の病態生理を調べるのに利用でき、さらにインビボ薬物スクリーニングに使用することができる。

2分間のFITC-dextranの全身循環後、恥動マウスおよびtBCCAO操作マウスにおける左右のレチナの眼管切開を調べた(補足図1)。FITC-dextranは、tBCCAO操作マウスのシャム操作マウスと左レチナの両方で完全に見えるが、tBCCAO操作マウスの右左部内で部分的に検出可能であった。
tBCCAOの後、眼瞼垂れを調べた(図2)。右目は軽度(スコア2;75%)を示したと?...

Watch this Scientific Journal Video about A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion at JoVE.com

A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

ここでは、単純な縫合糸とクランプを用いた一過性両側共通頸動脈閉塞によるレチナル虚血のマウスモデルについて述べている。このモデルは、心血管異常によって引き起こされる虚血の病的メカニズムを理解するのに有用である。

一過性両側性頸動脈閉塞による虚血性筋炎損傷のマウスモデル

Diverse vascular diseases such as diabetic retinopathy, occlusion of retinal veins or arteries and ocular ischemic syndrome can lead to retinal ischemia. ...

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7.4K ビュー.  Keio University School of Medicine. ここでは、単純な縫合糸とクランプを用いた一過性両側共通頸動脈閉塞によるレチナル虚血のマウスモデルについて述べている。このモデルは、心血管異常によって引き起こされる虚血の病的メカニズムを理解するのに有用である。

ビデオ: A Murine Model of Ischemic Retinal Injury Induced by Transient Bilateral Common Carotid Artery Occlusion

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.

This describes a partial carotid ligation surgery, which causes disturbed flow conditions and subsequent atherosclerosis development (in two weeks) with intraplaque neo-vascularization (in four weeks) in the mouse common carotid artery. We also describe a novel method of RNA isolation from the carotid intima, providing high purity endothelial RNA.

部分的ライゲーションによるマウス頸動脈における乱流誘発アテローム性動脈硬化症と頸動脈内皮からのRNA単離の方法における簡便な方法のモデル

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified ...

医学

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 ...

Adult Mouse Venous Hypertension Model: Common Carotid Artery to External Jugular Vein Anastomosis.

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model creating an artificial fistula between the common carotid artery (CCA) and the external jugular vein (EJV) has been widely described and proved technically feasible. This construct provokes a consistent cerebral venous hypertension (CVH), and therefore has helped studying the contribution of venous hypertension to formation, clinical symptoms, and prognosis of brain AVMs and dural AVFs. Equivalent mice models have been only scarcely described and have shown trouble with stenosis of the fistula. An established murine model would allow the study of not only pathophysiology but also potential genetic therapies for these cerebrovascular diseases.
We present a model of arteriovenous fistula that produces a durable intracranial venous hypertension in the mouse. Microsurgical anastomosis of the murine CCA and EJV can be difficult due to diminutive anatomy and frequently result in a non-patent fistula. In this step-by-step protocol we address all the important challenges encountered during this procedure. Avoiding excessive retraction of the vein during the exposure, using 11-0 sutures instead of 10-0, and making a carefully planned end-to-side anastomosis are some of the critical steps. Although this method requires advanced microsurgical skills and a longer learning curve that the equivalent in the rat, it can be consistently developed.
This novel model has been designed to integrate transgenic mouse techniques with a previously well-established experimental system that has proved useful to study brain AVMs and dural AVFs. By opening the possibility of using transgenic mice, a broader spectrum of valid models can be achieved and genetic treatments can also be tested. The experimental construct could also be further adapted to the study of other cerebrovascular diseases related with venous hypertension such as migraine, transient global amnesia, transient monocular blindness, etc.

We describe a method for creating a reliable model of cerebral venous hypertension in the adult mouse. This model has been widely described and tested in the rat. This new counterpart in the mice opens the possibility of using genetic modified animals and thereby broadens the applications of the model.

成体マウス静脈性高血圧モデル：外頸静脈吻合部の総頸動脈。

The understanding of the pathophysiology of brain arteriovenous malformations and arteriovenous fistulas has improved thanks to animal models. A rat model ...

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

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

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

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

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

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 ...

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves focal endothelial-selective transgenesis, thereby allowing an investigator to determine the biological roles of endothelial-expressed transgenes and to quantify the in vivo transcriptional activity of DNA sequences in large artery endothelial cells. The method uses surgical isolation of rabbit common carotid arteries and an arteriotomy to deliver a transgene-expressing viral vector into the arterial lumen. A short incubation period of the vector in the lumen, with subsequent aspiration of the lumen contents, is sufficient to achieve efficient and durable expression of the transgene in the endothelium, with no detectable transduction or expression outside of the isolated arterial segment. The method allows assessment of the biological activities of transgene products both in normal arteries and in models of human vascular disease, while avoiding systemic effects that could be caused either by targeting gene delivery to other sites (e.g. the liver) or by the alternative approach of delivering genetic constructs to the endothelium by germ line transgenesis. Application of the method is limited by the need for a skilled surgeon and anesthetist, a well-equipped operating room, the costs of purchasing and housing rabbits, and the need for expertise in gene-transfer vector construction and use. Results obtained with this method include: transgene-related alterations in arterial structure, cellularity, extracellular matrix, or vasomotor function; increases or reductions in arterial inflammation; alterations in vascular cell apoptosis; and progression, retardation, or regression of diseases such as intimal hyperplasia or atherosclerosis. The method also allows measurement of the ability of native and synthetic DNA regulatory sequences to alter transgene expression in endothelial cells, providing results that include: levels of transgene mRNA, levels of transgene protein, and levels of transgene enzymatic activity.

In Vivo Gene Transfer to the Rabbit Common Carotid Artery Endothelium

This method is to introduce a transgene into the endothelium of rabbit carotid arteries. Introduction of the transgene allows the assessment of the biological role of the transgene product either in normal arteries or disease models. The method is also useful for measuring activity of DNA regulatory sequences.

生体内でウサギの一般的な頚動脈内皮細胞への遺伝子導入

The goal of this method is to introduce a transgene into the endothelium of isolated segments of both rabbit common carotid arteries. The method achieves ...

A Rabbit Model of Durable Transgene Expression in Jugular Vein to Common Carotid Artery Interposition Grafts

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, intimal hyperplasia, and atherosclerosis. The goal of this article is to demonstrate a method for placing bilateral venous interposition grafts in a rabbit, then transducing the grafts with a gene transfer vector that achieves durable transgene expression. The method allows the investigation of the biological roles of genes and their protein products in normal vein graft homeostasis. It also allows the testing of transgenes for the activities that could prevent vein graft failure, e.g., whether the expression of a transgene prevents the neointimal growth, reduces the vascular inflammation, or reduces atherosclerosis in rabbits fed with a high-fat diet. During an initial survival surgery, the segments of right and left external jugular vein are excised and placed bilaterally as reversed end-to-side common carotid artery interposition grafts. During a second survival surgery, performed 28 days later, each of the grafts is isolated from the circulation with vascular clips and the lumens are filled (via an arteriotomy) with a solution containing a helper-dependent adenoviral (HDAd) vector. After a 20-min incubation, the vector solution is aspirated, the arteriotomy is repaired, and flow is restored. The veins are harvested at time points dictated by individual experimental protocols. The 28-day delay between the graft placement and the transduction is necessary to ensure the adaptation of the vein graft to the arterial circulation. This adaptation avoids rapid loss of transgene expression that occurs in vein grafts transduced before or immediately after grafting. The method is unique in its ability to achieve durable, stable transgene expression in grafted veins. Compared to other large animal vein graft models, rabbits have advantages of low cost and easy handling. Compared to rodent vein graft models, rabbits have larger and easier-to-manipulate blood vessels that provide abundant tissue for analysis.

This method describes the placement of interposition vein grafts in rabbits, the transduction of the grafts, and the achievement of durable transgene expression. This allows the investigation of physiological and pathological roles of transgenes and their protein products in grafted veins, and testing of gene therapies for vein graft disease.

耐久性のある遺伝子発現により一般的な頚動脈移植に頸静脈の家兎モデル

Vein graft bypass surgery is a common treatment for occlusive arterial disease; however, long-term success is limited by graft failure due to thrombosis, ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

塩化第二鉄によるイヌ総頚動脈血栓症: 血管損傷の大動物モデル

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

マウスにおける一般的な頸動脈の修理で再灌流を許可する中大脳動脈閉塞

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...