本研究では、アーヘン工科大学、ドイツ（：START 690753 &quot;ArterialisationvenöserGefäßeダイ&quot;タイトル）医学部の研究推進プログラム &quot;START&quot;によって賄われていた。私たちは、彼女の優秀な編集支援のため夫人メアリー·ジョアンBlümichに感謝したいと思います。

1。実験動物<ol><li>動物保護FELASAのガイドラインに従って、実行されるすべての実験のために地域の倫理委員会の承認を得る。 </li><li>いかなる商業ブリーダー（ 例えばチャールズリバーWiga GmbHはSulzfeld、Germany）から購入することが可能な雌Sprague-Dawleyラットを使用しています。私たちは7匹術後3,6および12週目に屠殺し、その各々の3つのグループに分かれて21頭を使用していました。購入時には、動物は12週齢であると250〜300グラムの重量を量るべきである。 </li><li> 21の周囲温度を有する気候制御室で動物を飼う℃、食品や水への無料アクセスを相対湿度60％と12時間明暗サイクル。 </li></ol> 2。麻酔や手術の準備<ol><li> 1L /分の酸素流量で設定され、麻酔器に接続されているプレキシグラスボックスを使用して、全身麻酔を誘導し、イソフルラン気化器ワットi番目の5パーセント麻酔ガス。 </li><li>動物の深い感覚の反射が抑制されているとすぐに1.5％イソフルラン気化器で麻酔を維持します。 </li><li>体の暖まる皿の上に仰臥位でラットを固定します。 </li><li> 胸骨柄下2cmに顎角から、脱毛クリームで、その毛皮を押し削除して、それぞれの動物の動作領域を準備します。 </li><li>手術領域、動物および外科医の前面に用意面積に着目して、無菌手術野を準備します。 </li><ul><li>消毒剤でベンチを清掃してください。 </li><li>手術時手洗い溶液（ 例えばクロルヘキシジンと70％イソプロピルアルコール）の複数のアプリケーションを使用して切開部位を準備します。クロルヘキシジン、第一、第二アルコールと、最終的には、スクラブのソリューションを適用する。 </li><li>以前にwを洗浄されている使い捨ての手術着と手術用手袋、またはその代わりに、試験用手袋を着用してくださいi番目の外科消毒。 </li></ul><li>操作手順で滅菌マイクロサージェリーの楽器を使用しています。 </li><li> 10から20倍の倍率で手術用顕微鏡（ワイルドHeerbrugg M650、ガイス、スイス）を使用します。 </li></ol> 3。解剖学 図1は、ラットの首の解剖を表しています。 1a）の右胸鎖乳突筋の切断前に内臓 。 1b）の右胸鎖乳突筋の切断後の内臓 。 4。カフの準備プラスチックチューブ（Portex、内径0.75ミリメートル、外径0.94ミリメートル、スミスメディカル、英国）から手で3mmの長さで袖口をカット。これらの袖口は、プラスチックチューブ、カフ量の半分から成るカフハンドルの完全なボリュームを含む管状カフ本体で構成されています。カフ本体とカフハンドルの両方が均等に経度であるべきグラム。カフハンドルは外科手術中にカフを操作するために不可欠です。カフを容器の上に滑った後は、ミニブルドッグクランプは、出血を防ぐ位置にカフを維持し、安定性を確保するために、カフハンドルと容器に同時に配置されます。 5。カフテクニック<ol><li>容器の上にカフを滑る。 </li><li>その直後、容器を裏返す、カフ本体の外側の上にそれを引くと7から0絹糸結紮で固定します。このように、カフの内腔は内皮細胞で覆われている。その後、カフ本体の上に静脈グラフトをスリップし、新しい7から0絹糸結紮で固定します。血栓形成を防止するためには、所定の位置に動脈を保持しているタイに連絡から血を避けるために、カフ余裕に非常に近い静脈グラフトタイを固定するために不可欠です。これは、血流方向による遠位吻合するために重要です。しかし、著者によれば、それは考慮されません同じ理由から、近位吻合するのに不可欠です。 </li></ol> 6。外科的処置右外頸静脈の6.1準備<ol><li>直接胸骨柄上約2.5センチ長い正中頸部切開を加えます。 </li><li>周囲の組織から右外頸静脈から1cm長セグメントを完全に分離</li><li> 7から0絹縫合糸と静脈支流をライゲーションする。 </li></ol>右総頸動脈の6.2準備<ol><li>胸鎖乳突筋の準備をして、途中でそれを横断する。 </li><li>動脈または迷走神経を圧迫しないように確認しながら、慎重に右総頸動脈を準備します。ダウン腕頭動脈に頸動脈分岐部からそれを動員する。 </li><li>その後、切断前に7から0絹縫合糸で真ん中に2回を連結。 </li><li>ナイロンカフconsistiスリップカフハンドルが（ 図2b）容器の内側にあることを確認することが重要であることによりボディと総頸動脈の遠位端にハンドル（ 図2a）のng、。 </li><li>まさに安定性（ 図2c）を確保するためのカフ·ハンドル上のミニブルドッグクランプ付き遠総頚動脈のカフの端をクランプします。 </li><li>続いて、得られた動脈の切り株がカフ本体と同じくらいの長さにする必要がありそれによってリガチャー、真後ろに動脈を切った。 </li><li>ヘパリン生理食塩液（20 IUヘパリン/ mlの0.9％NaCl）を使って動脈内腔を洗浄します。後で使用するために適切な位置に置いておき結紮ループを設定します。 </li><li>動脈切り株をプルアップし、カフ本体オーバースリーブのようにそれをスリップ。 </li><li>その後、ライゲーションループと結び目しっかり引き出します。 </li><li>その袖の抜け動脈を防ぐには、トリックを実装するかもしれません：右手sと動脈壁と縫合糸の両方を把握するimultaneouslyと左手のピンセットで（ 図2d）ノット（ 図2e）を作る。 </li><li>近位の動脈断端（ 図2f）についても同じ手順を繰り返します。 </li></ol> 6.3静脈グラフトポジショニング<ol><li>クランプ外頸静脈の消費税1センチメートル長セグメント</li><li> 2カフ、動脈が静脈介在のために互いから離れすぎているので、おおよそこの手順の正しい距離は3-0 VICRYL-縫合糸を使用しているお客様べき各ミニブルドッグクランプの周りにループ。 </li><li>十字両方縫合。 </li><li>続いて、カフ、動脈切り株（ 図2G）の適切な位置決めを可能にし、反対方向に縫合糸を引く</li><li>ヘパリン生理食塩水で静脈部分を洗浄します。 </li><li>カフ遠位頚動脈の上に以前に逆転静脈部分の一方の端をスリップし、正しい位置に固定シルク7から0リガチャーとる。 </li><li>静脈の周囲が完全に均等に把握する必要があることをここで注意することが重要です。 </li><li>ヘパリン生理食塩溶液で静脈セグメントをリンスした後、近位頸動切り株の上に静脈セグメントのもう一方の端をスリップ。 </li><li>ライゲーション（ 図2H）を完了; </li><li>それによって妨げられていない血流と静脈グラフトの動脈化（ 図2I）が可能遠頚動脈切り株にしてから、近位1の最初のミニブルドッグクランプ他の後に1を削除します。血流や脈動がグラフトと総頸動脈（視覚的に評価し）内で観察された場合手術は成功したとみなされます。 </li></ol> 6.4。創縫合皮下縫合実行については、4から0 VICRYLを使用しています。皮膚縫合のためにランニングを3-0 VICRYLを使用しています。 7。リカバリおよび術後管理&lt;/ P&gt; <ol><li>動物は赤外線加温ランプ下で平らな紙の寝具に麻酔から回復しましょう​​。すべての動物が正常に見えるまでの間、家畜の保持領域に手術動物を返しません。 </li><li>術後2日間毎に8月12日HRは0.03から0.05 mg / kg体重scの濃度でブプレノルフィン（Temgesic）を管理する。ブプレノルフィンはちょうどイソフルラン誘導前に投与することができます。 </li><li>彼らの一般的な条件や、病気や感染症の徴候について術後の動物を監視します。 </li></ol> 8。血流測定血管周囲flowprobe（3ミリメートル、MA3PSB）で：移植の術後の開存性を研究遷音速血流計測システム（TS420、トランソニックシステムズヨーロッパBV、マーストリヒト、オランダモデル）を用いて、血流測定で証明された。システムは、ドップラー原理に基づいています。 9。代表的な結果 Fによってaithfully命令は前述のまま、手術の平均持続時間は約65分であった。動物は、優れた物理的条件を提示し、実験全体を通して予想通り体重が増えた：操作の開始時点で動物は312の重量を量った±20.39グラム。 3週間の犠牲時点で、手術後の動物は6週目で、325.2±28.7グラムの重量を量った333±39.9グラム、12週の時点で362.22±29グラム。 いいえ感染は手術部位と先進ない傷では観察されなかった。操作傷は予想された時間で完全に治癒した。神経制御は、脳梗塞や運動の欠乏の徴候を示さなかった。動物は頸動脈で運転され、2つの外頚静脈のいずれかが削除され、自動移植として使用されていたので、後で重要なポイントだった。術後疼痛緩和の治療に全くアレルギー反応は認められなかった。 &gt;全ての動物は、実験全体を通して警戒し、社会的なされた。食べ物や水を得ることに関する彼らの習慣は、全体の時間を通して変わらなかった。 すべての実験の生存率は犠牲の時点で100％であった。グラフト開存率は、最初の10個の手術動物のための60％、残りの11の動物のためのそれに応じて82％であった。合計グラフト開存率は71％であった。 arterialized静脈グラフトにおける血流が犠牲の時点で測定し、8歳の±3犠牲時点の間の統計的有意差なしの3ミリリットル/分であった。 <img src="/files/ftp_upload/4124/4124fig1.jpg" alt="図1" /> 図1ラットの首の解剖学。 1a）の右胸鎖乳突筋の切断前に内臓 。 1b）の右胸鎖乳突筋の切断後の内臓 。 e_content &quot;のfo：キープtogether.withinページ=&quot; always &quot;を&gt; <img src="/files/ftp_upload/4124/4124fig2.jpg" alt="図2" /> カフ遠総頸動脈の断端のクランプc）の工程と、b）カフは総頸動脈の遠位端上に滑って、カフ本体とカフハンドルから成る）カフd）の外転：2術中顕微鏡写真を図カフ上の総頸動脈の切り株と、e）リガチャーと外転総頸動脈の断端を固定して、f）の両方のカフ遠位お ​​よび近位の総頸動脈の切り株の外転後の内臓 、カフ2の近似g）は十字テクニック総頸動脈の切り株、h）を静脈介入後の内臓は、i）静脈介在グラフトの再灌流後の内臓 。 <img src="/files/ftp_upload/4124/4124fig3.jpg" alt="図3" /> 図3： 内臓の概略図静脈介在グラフトの再灌流後。 <a href="http://www.jove.com/files/ftp_upload/4124/4124fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。

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総頸動脈で外頚静脈グラフトを介在させるには、高度なマイクロサージェリー技術です。このビデオでは、初心者外科医は実験動物の低い廃棄物と一緒に最小の時間で、このテクニックを習得するのに役立ちます。 それは、過度に深い麻酔ステージを回避うまく制御することができ、外科医に十分な時間が動作することができますので、吸入麻酔は、この操作のための?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td>編んだシルク黒7から0 </td><td>エチコン製品</td><td> 892100 </td><td></td></tr><tr><td>ビクリル3から0 </td><td>エチコン製品</td><td> J285G </td><td></td></tr><tr><td> 4から0ビクリル</td><td>エチコン製品</td><td> J284G </td><td></td></tr><tr><td> Portex非滅菌ナイロンチューブ0.75ミリメートル、0.94ミリメートルファインボアポリエチレン（ポリエチレン）チューブ</td><td> Portex英国</td><td> 800/200/175/100 </td><td></td></tr><tr><td>イソフルオラン5パーセント</td><td></td><td></td><td>すべてgenericon </td></tr><tr><td>ヘパリン</td><td></td><td></td><td>すべてgenericon </td></tr><tr><td> 0.9％生理食塩水</td><td></td><td&gt; </td><td>すべてgenericon </td></tr><tr><td>ブプレノルフィン</td><td></td><td></td><td>すべてgenericon </td></tr></tbody></table>

training a sophisticated microsurgical technique: interposition of external jugular vein graft in the common carotid artery in rats

新生内膜過形成は、末梢動脈のバイパス手術で同様に動静脈瘻の動脈冠動脈バイパス手術において非常に重要であるarterialized静脈の狭窄の1主な原因です。1-5は、総頸動脈の静脈グラフト挿入の実験手順カフの手法を用いて新生内膜過形成とそれに対処するための治療法の選択肢の病因を調べるために、いくつかの研究プロジェクトに適用されています6-8カフは新生内膜過形成の発達、それによって血管吻合改造を防止し、移植片内で乱流を誘発します。 優れた静脈静脈グラフトを使用すると、静脈動脈化実験のための確立された小動物モデルである。9月11日これは、現在のプロトコルが確立された頚静脈グラフト介在第一Zou らによって記載された技術。、9と同様に他人を指します12月14日 </sup&gt;それにもかかわらず、これらの引用小動物プロトコルは複雑である。 手続きを簡素化するために、必要な実験動物の数を最小限にするため、ビデオトレーニングで詳細な動作プロトコルが表示されます。このビデオでは、カフテクニックと静脈グラフト介在両方を学ぶ初心者外科医を助ける必要があります。これにより、右外頸静脈は、それぞれ21日目、42および84に屠殺した3つの等しいグループに分類21雌性Sprague Dawleyラットの総頚動脈のカフの技術で移植した。自動移植が行われたため、特筆すべきは、NOドナー動物は、必要ありませんでした。生存率は犠牲の時点で100％であった。また、グラフト開存率は、最初の10個の手術動物のための60％、残りの11の動物のための82％であった。屠殺時に血液の流れが8±3ミリリットル/分であった。結論として、この外科的プロトコルはかなり簡素化され、このコンピュータを最適化し、標準化licated手順。それによって彼らは速く専門知識を獲得し、実験動物の無駄な犠牲を避けるために貢献し、可能な落とし穴を説明して、初心者の外科医に、簡単なステップバイステップの指示を与える。

Watch this Scientific Journal Video about Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats at JoVE.com

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

新生内膜過形成はarterialized静脈の狭窄症の主な原因です。右外頸静脈は、Sprague Dawleyラットの総頚動脈のカフ手法を用いてグラフト化されることにより、我々は新しいプロトコルを提案する。生存率は犠牲の時点で100％であった。

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 ...

training-sophisticated-microsurgical-technique-interposition-external

Research

JoVE Journal

Medicine

20.3K ビュー.  University Hospital RWTH Aachen. 新生内膜過形成はarterialized静脈の狭窄症の主な原因です。右外頸静脈は、Sprague Dawleyラットの総頚動脈のカフ手法を用いてグラフト化されることにより、我々は新しいプロトコルを提案する。生存率は犠牲の時点で100％であった。

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

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 ...

医学

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 ...

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, ...

Vein Interposition Model: A Suitable Model to Study Bypass Graft Patency

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. Research models for bypass graft failure are essential for a better understanding of pathobiological and pathophysiological processes during graft patency loss. Large animal models, such as pigs or sheep, resemble human anatomical structures but require special facilities and equipment. This video describes a rat vein interposition model to investigate vein graft patency loss. Rats are inexpensive and easy to handle. Compared to mouse models, the convenient size of rats permits better operability and enables a sufficient amount of material to be obtained for further diverse analysis. In brief, the inferior epigastric vein of a donor rat is harvested and used to replace a segment of the femoral artery. Anastomosis is conducted via single stitches and sealed with fibrin glue. Graft patency can be monitored non-invasively using duplex sonography. Myointimal hyperplasia, which is the main cause for graft patency loss, develops progressively over time and can be calculated from histological cross sections.

This video demonstrates a model to study the development of myointimal hyperplasia after venous interposition surgery in rats.

静脈介在モデル：バイパスグラフト開存を研究するための適切なモデル

Bypass grafting is an established treatment method for coronary artery disease. Graft patency continues to be the Achilles heel of saphenous vein grafts. ...

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, ...

Phase Contrast Magnetic Resonance Imaging in the Rat Common Carotid Artery

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous studies have shown that abnormal blood flow may be associated with systemic vascular risk. Thus, PC-MRI can facilitate the translation of data obtained from animal models of cardiovascular diseases to pertinent clinical investigations. In this report, we describe the procedure for measuring blood flow in the common carotid artery (CCA) of rats using cine-gated PC-MRI and discuss relevant analysis methods. This procedure can be performed in a live, anesthetized animal and does not require euthanasia after the procedure. The proposed scanning parameters yield repeatable measurements for blood flow, indicating excellent reproducibility of the results. The PC-MRI procedure described in this article can be used for pharmacological testing, pathophysiological assessment, and cerebral hemodynamics evaluation.

The overall goal of this procedure is to measure the blood flow in the rat common carotid artery by using noninvasive phase contrast magnetic resonance imaging.

ラットの総頸動脈で位相コントラスト磁気共鳴イメージング

Phase contrast magnetic resonance imaging (PC-MRI) is a noninvasive approach that can quantify flow-related parameters such as blood flow. Previous ...

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 ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

過期治療アプリケーションを用いてラット頸動脈圧制御区分バルーン損傷

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...