この作品は、科学、技術と韓国の教育省からのNIH助成HL87012（HJ）、HL75209（HJ）、UO1HL80711（DH、DG、HJ）とワールドクラスの大学プロジェクト（HJ）からの資金によって支えられている。

1。左頸動脈の部分結紮<ol><li>マウスは、生​​後〜8週で使用されています。マウスの体重は少なくとも18グラムであることが推奨されます。 </li><li>ビーズ機器滅菌器で30秒のための器具を滅菌し、冷却することができます</li><li>ケタミン（80mg/kg）とキシラジン（10mg/kgより少ない）IACUCによって承認された腹腔内（ip）注射または別の麻酔と麻酔を誘発する。腹腔内注射の場合は、腹部の左側の低い象限にマウスを注入する。 </li><li>完全に麻酔までの温暖化チャンバ内の場所のマウス。つま先のピンチに反応して麻酔の深さを確認してください。 </li><li>仰臥位（腹側の上向き）の位置に外科的ステージの上に置いてマウス、。上下のステージへの唯一の後肢足テープフォア足の手のひらを。 </li><li>下顎と胸骨の間に首に脱毛剤のリベラルな量（例えばNairさんを）適用する。優しくマッサージすべての毛が除去されるまで。 </li><li>脱毛剤を拭き取ってdepilatedエリアにBetadineの適当な量を塗布します。 </li><li>ライゲーションのための6から0絹縫合糸の1インチの長さの切片を作製します。各マウスは、縫合糸の2つが必要になります。 </li><li>鋭い小さなはさみを使って、首の中央に4〜6ミリメートル縦切開を行います。皮膚および基礎となる筋膜の両方をカットする必要があります。 </li><li>正中線から左へ、左耳下腺を移動し、露骨にちょうど気管の解剖を開始。あなたは簡単に脈動左総頸動脈を識別することができるはずです。 </li><li>もし分岐が見つかるまで尾側に総頸動脈に従ってください。 </li><li>外頸動脈、内頸動脈、後頭動脈と上甲状腺動脈：単刀直入に左総頸動脈から次の4つの遠位枝を露出させる分岐を細かく分析。これらの血管、特に優れた甲状腺は、壊れやすいことができます。あなたの郭清を伴う非常に注意してください。 </li><li> 6から0絹縫合糸を使用して優れた甲状腺上記外頸動脈を結紮する。これを行うには、動脈の下one鉗子を渡すと、他の鉗子の助けを借りて、プレカット縫合糸の一部をつかむ。ゆっくりと動脈の下渡す縫合糸を引く。結び目に巻き込まれて周囲の組織を避けてしっかりと結びます。あなたが効果的に、実質的に異なるモデルである完全なライゲーションを行うことになりますプロシージャの最後で、上甲状腺動脈を脱いで下にはネクタイ場合。 </li><li>同じテクニックを使用して1つの結び目で内頚動脈と後頭動脈を結紮する。 </li><li>縫合糸と上甲状腺動脈の開存の位置を確認してください。 </li><li>ティッシュメンドの少量を使用しておおよその皮膚と密接な肌。 </li><li>回復するまで、温暖化のチャンバー内にマウスを置きます。回復は通常30〜60分かかります。 </li><li>動物は、最初の24時間以内に苦痛になっている場合に使用される麻酔は、長引く痛みの軽減（すなわち、吸入イソフルラン）を提供しない場合やブプレノ​​ルフィン（0.1mg/kg）の単回投与は、ポスト手術直後に投与する必要があります。我々の経験では、麻酔のために投与ケタミン投与量は手術が経験豊富な外科医によって実行され、それ以上の鎮痛が必要とされていない十分な鎮痛を提供します。 </li></ol> 2。頸動脈超音波検査<ol><li>部分的なライゲーションが乱流を誘発したかどうかを検証するには、頸動脈の流れは、1日後にライゲーションを検討すべきである</li><li>イソフルランで麻酔を誘導する - 必要に応じて麻酔インダクションボックスを使用することができる</li><li>上の画像の段階、腹側で麻酔したマウスを置きます</li><li>イメージング段階で心電図センサーにテープの腕や脚</li><li> Nairさんのリベラルなアプリケーションで首を落葉させる（すでにライゲーションの手続きのために行われていない場合は、この手順にのみ必要です） </li><li>温度を記録するために直腸の温度計を挿入する</li><li>首にゲルエコー適用されます。 </li><li> B -モードで起動します。 </li><li>結像面は90度（プローブ上のノブは、マウスの鼻に向かって指していなければなりません）でなければなりません</li><li>画像が得られるまで、マウスの首へのプローブを下げます。 </li><li>ステージは左と右気管まで（ミッドライン構造）識別された操作</li><li>左総頸動脈を識別する</li><li>チルトイメージング段階ので角度が撮像面と頸動脈の間に作成されていること。この角度より大きい、強いドップラー信号になります。 </li><li>左総頸動脈の中央にあるパルス波ドップラーを置きます。 </li><li>必要に応じて角度補正を使用してください。 </li><li>右総頸動脈の測定を繰り返します。 </li><li>成功した部分的な頸動脈結紮では拡張期大動脈入口に向かって流れの逆転と、右側に比べて左総頸動脈の流れの全体的な減少（〜80〜90％）になります。 </li><li>いったん良好な画像が得られている、テープの解像度からマウスやフリーマウスオフジェルをエコーワイプ、麻酔をオフにするtraints。温めた回復室でマウスを置きます。 </li></ol> 3。頸動脈から内膜のRNA分離<ol><li>制度的IACUCプロトコルに従ってCO 2吸入によりマウスを犠牲にする</li><li>ペーパータオルにテープ足を。 </li><li>腹部から胸部の上部にマウスの皮膚を切り取ります。 </li><li>ハサミで胸郭下腹壁を開きます。 </li><li>鉗子で胸骨を持ち上げ、横隔膜をカットして、心臓を露出させる胸郭を切り取る。 </li><li>はさみで大静脈をカット。 </li><li>肺と肝臓が蒼白になるまで、左心室を通して10単位/ mLのヘパリンを含む生理食塩水で2〜3分間圧力を浸み込ませる（120 mmHgの）。 </li><li>首の皮膚をカットし、頸動脈が露出されるまで、すべての脂肪、筋肉、および結合組織を取り除く。 </li><li>解剖顕微鏡下でマウスを置きます。 </li><li>穿刺右第二灌流のための左頚動脈の結紮部位以下の穴。 </li><li>圧力は、左頸動脈がよくperusedていることを確認し、左心室を通して10単位/ mLのヘパリンを含む生理食塩水で〜1分間再灌流。 </li><li>微細な先端の鉗子と小さな春のはさみを使用して、慎重にcarotidsを取り巻く周囲の外膜の組織を削除します。この洗浄ステップ中にcarotidsを圧迫やストレッチをしないように注意してください。 </li><li>大動脈弓と頸動脈分岐部上記のライゲーションの点の間の左頸動脈を切断。 </li><li>右鎖骨下動脈の分岐点と頸動脈分岐部の間に右頸動脈を切断。 </li><li>氷冷HBSSを含む、35mmの培養皿にcarotidsを転送する。必要に応じて、慎重に残りの周囲の外膜組織を除去する。 </li><li>各頸動脈あたり150μlのQIAsolの溶解バッファー（Qiagen社）を充填して29グラム針とインスリンの注射器（3 / 10 mlの注射器）を準備する。 </li><li>慎重に頸動脈の一端に針の先端を挿入する。 </li><li>すぐに、頸動脈やピンセットで針の先端を保持している間（〜1秒）1.5mlチューブ（内膜の溶出液）にQIAsol溶解バッファー（150μl）をフラッシュする。 </li><li>頸動脈残り（メディア+外膜）一度HBSSで、1.5 mlチューブに入れ、そして液体窒素中でスナップ凍結をすすぐ。 </li><li>内膜の溶出液と凍結残ったが、製造元の指示に従ってmiRNeasyミニキット（QIAGEN）を用いて内膜またはメディア+外膜RNAの分離に使用されます。 </li></ol> 4。代表的な結果： 我々は部分的に結紮マウス左頸動脈超音波検査は、手順に従って一日行った。図1に示すように、成功した部分的なライゲーションは、血流速度を低減し、心拡張期に左総頸動脈の流れ（乱流）を逆にします。高脂肪食を伴うアポEノックアウト（KO）マウスの部分的なライゲーションは、2週間（図2）で、左総頸動脈の動脈（LCA）ではなく、反対側の右の頸動脈で堅牢なアテローム性動脈硬化症を誘発する。 - SMA（図3）などの中膜平滑筋細胞マーカー遺伝子のフリーながら頸動脈内膜から採取したRNAは、そのようなPECAM - 1などの内皮マーカー遺伝子が含まれています。 <img src="/files/ftp_upload/1861/1861fig1.jpg" alt="図1" /> 図1：頸動脈解剖学とライゲーションの回路図： 上甲状腺動脈（STA）が開いたままで左総頸動脈の三枝（LCA）[外頸動脈（ECA）、内頸動脈（ICA）、および後頭動脈（OA）]は、連結した。 <img src="/files/ftp_upload/1861/1861fig2.jpg" alt="図2" /> 図2：超音波画像は、流れの速度プロファイルを示し、拡張期の間にLCAに（矢印で示される）部分的なライゲーションが流れの反転を引き起こすことが明らかになった。 RCAの流れは、ライゲーションした後も変更されずに残る。 <img src="/files/ftp_upload/1861/1861fig3.jpg" alt="図3" /> 図3：部分的ライゲーションと高脂肪食は急速にアポKOマウスのLCAでアテローム性動脈硬化症を誘発アポKOマウスでは、部分的に連結し、1〜3週間の高脂肪食を与えられた。 LCAから凍結切片をオイルレッドOで染色した。 <img src="/files/ftp_upload/1861/1861fig4.jpg" alt="図4" /> 図4内膜RNA調製の方法。内膜RNAと内側+外膜（mは+ A）RNAをC57BL / 6マウスに偽手術RCAとLCAから得られた。 RNAはPECAM - 1とα- SMAは、内部コントロールとして18秒を使用するための半定量的RT - PCR（A）と定量PCR（B、C）により分析した。バーグラフは± SEM、n = 3の平均値です。

<ol>
	<li>Korshunov, V. A., Berk, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-induced+vascular+remodeling+in+the+mouse:+a+model+for+carotid+intima-media+thickening.">Flow-induced vascular remodeling in the mouse: a model for carotid intima-media thickening.</a> Arterioscler Thromb Vasc Biol. 23 (12), 2185-2191 (2003).</li><li>Sullivan, C. J., Hoying, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow-dependent+remodeling+in+the+carotid+artery+of+fibroblast+growth+factor-2+knockout+mice.">Flow-dependent remodeling in the carotid artery of fibroblast growth factor-2 knockout mice.</a> Arterioscler Thromb Vasc Biol. 22 (7), 1100-1105 (2002).</li><li>Nam, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+carotid+ligation+is+a+model+of+acutely+induced+disturbed+flow,+leading+to+rapid+endothelial+dysfunction+and+atherosclerosis.">Partial carotid ligation is a model of acutely induced disturbed flow, leading to rapid endothelial dysfunction and atherosclerosis.</a> Am J Physiol Heart Circ Physiol. 297 (4), H1535-H1543 (2009).</li></ol>

この手順では、外頸動脈、内頸動脈、後頭動脈と上甲状腺動脈を含む、総頸動脈のすべての支店の適切な識別が不可欠です。示すように超音波を使用してフローのパターンを検証することも重要です。当社の経験豊富な手で、500以上のマウスの超音波の研究に基づいて部分的な結紮手術の私達の成功率は90％よりも優れています。頸動脈内膜RNA調製には、まず血流のステップの後に頸動脈の?...

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<tr>
<td>Normal Saline (0.9% sodium chloride)</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>2B1323</td>
<td>&nbsp;</td>
<tr>
<td>Insulin syringe (3/10 ml 29g syringe)</td>
<td>&nbsp;</td>
<td>Tyco Health care</td>
<td>8881600145</td>
<td>&nbsp;</td>
<tr>
<td>HBSS</td>
<td>&nbsp;</td>
<td>Cellgro, Mediatech, Inc.</td>
<td>21-023</td>
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<td>miRNeasy mini kit (including, QIAzol)</td>
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<td>QIAGEN</td>
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<td>Vevo 770 High-Resolution Micro-Imaging System</td>
<td>&nbsp;</td>
<td>Visualsonics</td>
<td>a.Visualsonics 770 system b.Imaging stage c.Vevo integrated rail system d.RMV scan head for mice</td>
<td>or similar system</td>
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<td>6-0 Silk Suture, sterile</td>
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<td>s-1172 c2</td>
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<td>Small scissors</td>
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<td>RS-5675</td>
<td>or similar</td>
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<td>Dissecting Forceps</td>
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<td>Roboz</td>
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<td>Tissue Mend II</td>
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<td>Webster Vet Supply</td>
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a model of disturbed flow-induced atherosclerosis in mouse carotid artery by partial ligation and a simple method of rna isolation from carotid endothelium

よく知られている密接な関係にもかかわらず、アテローム発生する乱流を結ぶ直接的な証拠が欠けていた。我々は最近、急性マウス頸動脈に、、乱流の2つの重要な特性を低くし、振動流の条件を誘導することを示すために頸動脈部分的ライゲーション法[1,2]の修正版を使用している。このモデルを使用して、我々は、乱流が実際にアポEノックアウトマウスで迅速かつ堅牢なアテローム性動脈硬化症の開発[3]を導くことを示す直接的な証拠を提供している。我々はまた、マウス頸動脈内膜からの高純度を持つ内皮RNA調製の方法[3]を開発した。このマウスモデルと方法を使用して、我々は、その部分的なライゲーションが4週間でこのようなintraplaque血管新生などの複雑病変形成の機能と共に高脂血症のマウスモデルでの二週間で、堅牢かつ迅速なアテローム形成に続いて、週に内皮機能障害を引き起こすが見つかりました。この急速な<em&gt; in vivoで</em&gt;モデルと内皮RNAの調製法は、血管生物学と疾患の流量依存性の調節の分子メカニズムを決定するために使用することができる。また、それが大幅に削減研究期間における内皮機能障害とアテローム性動脈硬化症をターゲットとする様々な治療的介入をテストするために使用することができる。

Watch this Scientific Journal Video about A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium at

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

これは、マウスの総頚動脈のintraplaque新血管形成（4週間）と（二週間で）乱流の条件とその後のアテローム性動脈硬化症の発展の原因となる部分的な頸動脈結紮術を、説明します。我々はまた、高純度内皮RNAを提供する、頸動脈内膜からのRNA分離の新しい方法を説明します。

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 ...

a-model-disturbed-flow-induced-atherosclerosis-mouse-carotid-artery

Research

JoVE Journal

Medicine

28.1K ビュー.  Emory University. これは、マウスの総頚動脈のintraplaque新血管形成（4週間）と（二週間で）乱流の条件とその後のアテローム性動脈硬化症の発展の原因となる部分的な頸動脈結紮術を、説明します。我々はまた、高純度内皮RNAを提供する、頸動脈内膜からのRNA分離の新しい方法を説明します。

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

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

マウスでのせん断応力誘発アテローム性動脈硬化症をトリガするための頸動脈カフの注入

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

ラットの総頚動脈で外頚静脈グラフトの介在：洗練された顕微鏡下手術の訓練

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

A Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

再狭窄の研究のための頸動脈内ステント留置のマウスモデル

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

マウス肺挿管の簡単な方法

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

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 ...

Catheterization of the Carotid Artery and Jugular Vein to Perform Hemodynamic Measures, Infusions and Blood Sampling in a Conscious Rat Model

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. Intra-tracheal inoculation of a known amount of bacteria has been successfully used to reproduce the pathogenesis of pneumonia which then develops into sepsis. Monitoring hemodynamic parameters and providing standard clinical treatment including infusion of antibiotics, fluids and drugs to maintain blood pressure is critical to simulate routine supportive care in this model but to do so requires both arterial and venous vascular access. The video details the surgical technique for implanting carotid artery and common jugular vein catheters in an anesthetized rat. Following a 72 hr recovery period, the animals will be re-anesthetized and connected to a tether and swivel setup attached to the rodent housing which connects the implanted catheters to the hemodynamic monitoring system. This setup allows free movement of the rat during the study while continuously monitoring pressures, infusing fluids and drugs (antibiotics, vasopressors) and performing blood sampling.

Vascular accesses to measure hemodynamics, provide fluids and perform blood sampling are important to any small animal model study. We present a technique for implanting catheters into the carotid artery and the common jugular vein in an anesthetized rat for connecting to a system to perform monitoring, infusions and sampling.

配慮ラットモデルにおける血行動態の措置、煎茶や血液サンプリングを実行するために頸動脈と頸静脈のカテーテル

The success of a small animal model to study critical illness is, in part, dependent on the ability of the model to simulate the human condition. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric chloride injury is now a well-established technique to rapidly and accurately induce the formation of thrombi in exposed veins or artery of small and large diameter. This model has played a key role in the study of the pathophysiology of thrombosis, in the discovery and validation of novel antithrombotic drugs and in the understanding of the mechanism of action of these new agents. Here, the implementation of this technique on a mesenteric vessel and carotid artery in mice is presented. The method describes how to label circulating leukocytes and platelets with a fluorescent dye and to observe, by intravital microscopy on the exposed mesentery, their accumulation at the injured vessel wall which leads to the formation of a thrombus. On the carotid artery, the occlusion caused by the clot formation is measured by monitoring the blood flow with a Doppler probe.

The FeCl3 induced thrombosis model in mice is described herein. A method to monitor thrombus growth by intravital microscopy observation on a mesenteric vessel and by blood flow measurement in the carotid artery is presented.

塩化第二鉄誘発性頚動脈および腸間膜血管に血栓症のマウスモデル

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

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, ...