Name: vascular balloon injury and intraluminal administration in rat carotid artery
Uploaded: 2014-12-23T13:00:00.000+00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery at JoVE.com

We are grateful to Dr. Clowes for first developing and describing this method. We are also thankful to Dr. Tulis for his detailed protocol which has been fundamentally helpful to our previous, current and future work. This work was supported by grants R01HL097111 and R01HL123364 from the NIH to M.T., and by American Heart Association grant 14GRNT18880008 to M.T.
We would like to thank Rachel Newton for her expert technical support and for her valuable help during the filming process.

注：以下の実験のための動物の使用を見直し、施設内動物管理使用委員会（IACUC）によって承認されています。 1.術前手続き<ol style="margin-left: 40px;"><li>使用前に手術器具を滅菌する。 <ol><li>すべての手術器具を手術前24時間以下オートクレーブ。複数の手術が同日に行われている場合は、手術の間の乾燥ビーズ滅菌器によって楽器を殺菌。 </li></ol></li><li>フィルター滅菌し、使用前に生理食塩水を。 </li><li>ラットを秤量し、麻酔薬（ケタミン80 mg / kgをとキシラジン7 mg / kgを）の投与量を計算する。 </li><li>腹腔内（IP）麻酔薬を管理します。 <ol><li> 5〜10分で足指のピンチによる鎮静の妥当性を確認してください。鎮静が完了していない場合は、薬（ケタミン7ミリグラム/ kgおよびキシラジンは0.6mg / kg）を追加の小さな用量を投与。 </li><li>針が配信薬、イントラあたりに十分な深さであることを確認してくださいitoneally腹腔内に全体の薬液を提供する失敗は不十分な鎮静の原因になりますので。 注：注射部位の皮膚の潰瘍及び脱毛は、数日手術後不注意皮下（sc）薬液注入による表示されます。 </li></ol></li><li> 3ミリリットルの無菌食塩水を皮下（s。c。）注射する。滅菌綿棒を用いて、乾燥から角膜を防ぐために、両眼に眼軟膏を少量適用する。 </li><li>加熱装置を準備します。マイクロ波または水浴によって事前温熱パッド。 </li><li>外科プラットフォーム上で仰向け動物を置く。 <ol><li>腹側頸部領域で毛を削除します。 30秒待ってからガーゼで完全に拭き取って、綿棒で毛リムーバーを適用します。 </li><li>ポビドンヨードスクラブと70％エタノールで首を綿棒。 </li></ol></li><li>ガウン、ヘアカバー、外科マスク、眼鏡を含め、個人保護具を使用すること。 STERに置く無菌手術器具や消耗品を取り扱う前に最後に手術用手袋をILE。 </li><li>暴露だけ首領域との無菌手術用シートでラットをドレープ。 </li></ol> 2.外科的処置<ol style="margin-left: 40px;"><li>外科手術中に、15分毎でつま先のピンチによって、動物の鎮静深度を確認してください。動物はつま先のピンチに応答する場合、ケタミンとキシラジンの追加の小さな用量（初期投与量の10％）を追加します。 </li><li>左総頚動脈（CCA）を解剖<ol><li>首の真ん中にまっすぐ縦切開を作るためにメスを使用してください。皮膚切開のおおよその長さは、動脈1.5-2センチ部分を単離する目的のために1.5〜2センチ。長さは変更される場合があります研究の目的に依存します。 </li><li>露骨に皮膚から結合組織を解剖。鉗子のヒントを維持し、皮膚やその下の組織を穿刺しないように注意してください。 </li><li>長手方向にalon筋肉層を分析グラム気管の左側。 </li><li>筋層を開放すると、密着迷走神経と左CCAを視覚化する。ぶっきらぼうにストレッチ最小限に迷走神経を分離するために細心の注意を払って左頸動脈と一緒に解剖する。 </li><li>分岐点まで遠位にCCAを解剖。内頸動脈（ICA）と外頚動脈（ECA） - 慎重に分岐し、2つのブランチ解剖。 </li><li>動脈1.5-2センチ約部分が周囲の組織から分離されるまで、CCAの周りに解剖してください。 </li></ol></li><li>バルーン障害<ol><li>恒久的には約5ミリメートル離れて分岐からECA上の合字を作る。永久にECAとICAの分岐に近いECAの後頭部枝を連結。どんな場合にも恒久的に分岐し、ECAの合字の間場所、他の枝を結紮 - 例えば、上甲状腺ブランチを。すべての合字のために使用される縫合糸は4-0黒ですシルク。 CCAの近位端とICAの先端にクリップ。 注：ここで、血流が（CCAとICAに）クリッピングのいずれかを介して恒久的に（ECA上の）ライゲーションを介して、または一時的に停止されました。分岐部領域内の管腔内容物は、全身循環から単離されている。 </li><li>小マイクロはさみによってECA上の動脈切開切開を行います。切開は、遠位縫合糸の結び目の近くにあることを確認してください。生理食塩水と綿棒で清掃して血。 </li><li> ECA内腔に膨張していない2Fバルーンカテーテルを挿入します。 CCA内腔の近位にバルーンカテーテルを進める。クリップは留まるところ、その先端が到達するまで、近位にカテーテルを前進してください。 </li><li> 3方活栓のメスルアーロックにバルーン膨張デバイスを接続し、バルーンカテーテルに3方活栓の雄ルアーロックを接続します。 </li><li>徐々に直径の1.5倍に頸動脈を拡張させるために、約1.5気圧の圧力でバルーンを膨張させる。 </lI&gt; <li>ゆっくりと回転戻って分岐部にバルーンを引っ張る。 </li><li>バルーンを収縮し、近位端に戻ってそれを進める。再びそれを膨らませると引き戻し手順をさらに2回繰り返します。 </li><li>動脈内腔からバルーンカテーテルを引き出す。 </li></ol></li><li>試薬の管腔内投与（ 例えば、siRNAは、薬） 注記：ここでは、使用される試薬のいずれかをコードするのshRNAは、間質相互作用分子1（STIM1）または非標的shRNAを制御ターゲッティングレンチウイルス粒子を含有する溶液であった。 STIM1は、原形質膜のCa 2+チャネルの活性化を制御するERのCa 2+センサであり、増殖、遊走表現型12,30-33に血管平滑筋分化の間にアップレギュレートされる単一の膜貫通小胞体（ER）タンパク質である。 <ol><li>注射器（24 G 1.6センチ）に血管内オーバーザニードルカテーテルを取り付けます。テストREAGの吸引除去30μlのソリューションエント。 ECAの同じ切開にカテーテルを挿入します。 </li><li> CCAにカテーテル先端を進め、カテーテルを固定し、一時的に切開を閉じるためにECA上の単一結び目と縫合糸の部分を結ぶ。 </li><li> CCAの内腔に検査試薬溶液を注入する。 30分間の血管内腔内の溶液にしてください。生理食塩水で湿った露出した組織を維持し、湿ったガーゼでそれをカバーしています。 </li><li>インキュベーション後、残りの溶液を吸引する。その単一の結び目を緩め、カテーテルを撤回。 </li><li>動脈切開切開に対して近位縫合糸の切れ端でECAを結ぶ。分岐部にできるだけ近い結び目を作る。 </li></ol></li><li>傷をクローズアップ。 <ol><li>バルーン傷害とカテーテル導入は、血管の漏れやパンクの原因となりますので、ICA上のクリップを削除し、すべての漏れがあるかどうかを確認します。出血が観察される場合、ガーゼを適用し、出血を止めるために穏やかな圧力を加える。 </li><li>削除するCCA上の他のクリップ。 </li><li>出血の兆候がないことを確認し、すべてのクランプや他の手術器具を取り外します。余分な縫合糸を切り取ります。 </li><li>閉じる傷用いた皮膚縫合糸（4-0黒絹）。 Povidon-ヨウ素または他の抗敗血症性/殺菌/殺ウイルス剤を用いて、閉じた傷口のすべての側面に綿棒。滅菌生理食塩水の皮下を3mlを注入する</li></ol></li></ol> 3.術後の手順<ol style="margin-left: 40px;"><li>手術後の熱パッドの上にラットを保管してください。ラットに筋肉内注射（IM）を介して0.05​​ mg / kgをブプレノルフィンの1用量を投与。復旧手順の間、湿った動物の目と口を保つ。それは目を覚ましや通院になるまで動物を監視します。 </li><li>それが完全に回復するまで、すべての寝具なしできれいなケージに動物を置きます。回復後、動物を動物室に戻し、個別に収容される。 </li></ol>

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M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+rats+via+embryo+microinjection+of+zinc-finger+nucleases.">Knockout rats via embryo microinjection of zinc-finger nucleases.</a> Science. 325, 433 (2009).</li><li>Tesson, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Knockout+rats+generated+by+embryo+microinjection+of+TALENs.">Knockout rats generated by embryo microinjection of TALENs.</a> Nature Biotechnology. 29, 695-696 (2011).</li><li>Li, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heritable+gene+targeting+in+the+mouse+and+rat+using+a+CRISPR-Cas+system.">Heritable gene targeting in the mouse and rat using a CRISPR-Cas system.</a> Nature Biotechnology. 31, 681-683 (2013).</li><li>Potier, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+STIM1-+and+Orai1-dependent+store-operated+calcium+influx+through+ICRAC+in+vascular+smooth+muscle+cells:+role+in+proliferation+and+migration.">Evidence for STIM1- and Orai1-dependent store-operated calcium influx through ICRAC in vascular smooth muscle cells: role in proliferation and migration.</a> FASEB Journal : Official Publication Of The Federation Of American Societies For Experimental Biology. 23, 2425-2437 (2009).</li><li>Aubart, F. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+targeting+STIM1+suppresses+vascular+smooth+muscle+cell+proliferation+and+neointima+formation+in+the+rat.+Molecular+Therapy.">RNA interference targeting STIM1 suppresses vascular smooth muscle cell proliferation and neointima formation in the rat. Molecular Therapy.</a> The Journal Of The American Society Of Gene Therapy. 17, 455-462 (2009).</li><li>Berra-Romani, R., Mazzocco-Spezzia, A., Pulina, M. V., Golovina, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++handling+is+altered+when+arterial+myocytes+progress+from+a+contractile+to+a+proliferative+phenotype+in+culture.+American+journal+of+physiology.">Ca2+ handling is altered when arterial myocytes progress from a contractile to a proliferative phenotype in culture. American journal of physiology.</a> Cell Physiology. 295, C779-C790 (2008).</li><li>Bisaillon, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+STIM1/Orai1-mediated+calcium+influx+in+PDGF-induced+smooth+muscle+migration.+American+journal+of+physiology.">Essential role for STIM1/Orai1-mediated calcium influx in PDGF-induced smooth muscle migration. American journal of physiology.</a> Cell Physiology. 298, C993-C1005 (2010).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+carotid+artery+balloon+injury+model.">Rat carotid artery balloon injury model.</a> Methods In Molecular Medicine. 139, 1-30 (2007).</li><li>Zhang, W., Trebak, M., Szallasi, A., B&#237;r&#243;, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balloon+Injury+in+Rats+as+a+Model+for+Studying+TRP+Channel+Contribution+to+Vascular+Smooth+Muscle+Remodeling.">Balloon Injury in Rats as a Model for Studying TRP Channel Contribution to Vascular Smooth Muscle Remodeling.</a> T TRP Channels in Drug DiscoveryMethods in Pharmacology and Toxicology. , 101-111 (2012).</li><li>Tulis, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+morphometric+analyses+for+rat+carotid+balloon+injury+model).">Histological and morphometric analyses for rat carotid balloon injury model).</a> Methods In Molecular Medicine. 139, 31-66 (2007).</li></ol>

The authors declare that they have no competing financial interests.

ラット頸動脈バルーン損傷はよく2007 34 Tulisによって記載されている。それは、包括的に博士Tulisこの手順のすべての詳細を説明してきた。この手順を実行するに興味がある読者は非常にTulis &#39;プロトコルを読むことをお勧めします。しかし、我々は博士Tulisに同意していない一つのことがあります：代わりに生理食塩水または液体のいずれかの種類でバルーンを膨張させるのが、私たちは空気でそ?...

バルーンカテーテルは、血管内のアテロームまたは血栓閉塞部位を広げる目的で、血管形成術の手順で使用する医療機器である。狭め血管内腔は、膨張バルーンによって開くように強制され、血液供給は、狭心症、心筋梗塞、および下肢痛のような下流の虚血症状を緩和するために連続的に復元される。それにもかかわらず、血管形成術の大成功は、そのような血管内腔（再狭窄）1の再狭小血管気圧外傷（バルーン損傷）、すなわち血管壁リモデリングを引き起こす力の結果として、多くの場合、術後の合併症によって減少されました。 多くの動物モデルは、研究者は、バルーン損傷に関連する血管壁リモデリング2のメカニズムを理解するための血管形成術を模倣開発されてきた。モデリングのために利用されるすべての動物種の中では、ラットは、最も頻繁にものを用いられる。 ℃ウサギ、イヌおよびブタにompared、ラットの利点は、その低コスト、使用が比較的容易とラットの生理学の現在の知識である。マウスは、遺伝子操作株の広い範囲で追加の利点を有するが、マウスの血管は、バルーンカテーテルを挿入するには小さすぎる。過去30年間にわたり、実験ラットは、研究者が3-6を改造新生内膜形成と血管を支える分子·細胞メカニズムのより良い理解を得ることができました。バルーン傷害を越え、血管リモデリングはまた、アテローム性動脈硬化症7,8、高血圧9、及び動脈瘤10などのほとんどの主要な血管疾患に関与している。このように、知識は、バルーン傷害モデルを通して得、一般に、全体的な血管壁の疾患の研究に有益である。 ラットバルーン傷害モデルの全体的な目標は、だけでなく、さらに血管疾患を理解するだけでなく、のための新規薬剤の効力を試験することである疾病コントロール11,12。再狭窄の現在の臨床薬物治療は、右血管形成術後の血管内腔を介して配置された薬剤溶出ステントにより適用される。動物モデルでは、新しいエージェント·テストのための効率がさらに経済的な方法は十分に開発されたローカル腔内灌流法である。候補このメソッドを使ってテストされている薬剤の小分子薬を含んで13,14、サイトカインまたは成長は15,16因子 、薬剤を操作する遺伝子（cDNAクローンは、siRNA など ） を 17-20、及び新規な医薬製剤21,22。 これまでに、ラットバルーン傷害モデルは、血管疾患/障害を研究するための最も有用なモデルの一つである。それは、最初のステップは、in vivoでのin vitroでの移動は、通常のように、ベンチからベッドサイドへの基本的なステップですが、それは最後の1であってはならない。ラットの実験の結果は、審議し、さらに人間への変換の前に特徴付けされる必要があるによる血管床および血管の解剖学的構造ならびにヒトおよびラットの23〜26との間の本質的な種差の差に臨床使用。それにもかかわらず、それはまだ翻訳医学研究に不可欠なツールである。このような研究は、遺伝的に改変されたラットの欠如によって制限されるために使用されるが、このようなジンクフィンガーとしての新規なゲノムアプローチは29ノックアウトラットを簡単にアクセスできるようにしている、27ヌクレアーゼTALENs 28およびCRISPR-Casのため、それはもはや問題ではなかった。

<table><tbody><tr><td>Fogarty balloon embolectomy catheters, 2 French&amp;nbsp;</td><td>Edwards Lifesciences, Germany</td><td>&amp;nbsp;120602F</td><td></td></tr><tr><td>Deltaphase Operating Board - Includes 2 Pads &amp;amp; 2 Insulators</td><td>Braintree Scientific, Inc.</td><td>39OP</td><td></td></tr><tr><td>LED light source</td><td>Fisher Scientific</td><td>12-563-501&amp;nbsp;</td><td></td></tr><tr><td>Hartmann Mosquito Forceps 4&amp;rdquo; curved</td><td>Apiary Medical, Inc. San Diego, CA</td><td>gS 22.1670</td><td></td></tr><tr><td>Crile Retractor 4&amp;rdquo; double ended</td><td>Apiary Medical, Inc.</td><td>gS 34.1934</td><td></td></tr><tr><td>Other surgical instruments</td><td>Roboz Surgical Instrument Company, Inc., Gaithersburg, MD</td><td></td><td></td></tr><tr><td>Peripheral Intravenous (I.V.) Cannula, 24 G</td><td>BD</td><td>381312</td><td></td></tr><tr><td>Ketamine HCl, 100 mg/ml, 10 ml</td><td>Ketaset- Patterson Vet</td><td>07-803-6637&amp;nbsp;</td><td></td></tr><tr><td>Xylazine (AnaSed), 20 mg/ml, 20 ml</td><td>Ketaset- Patterson Vet</td><td>07-808-1947</td><td></td></tr><tr><td>Buprenex, 0.3 mg/1 ml (5 Ampules/Box)</td><td>Ketaset- Patterson Vet</td><td>07-850-2280</td><td></td></tr><tr><td>Nair Baby Oil Hair Removal Lotion - 9 oz</td><td>Amazon/Walmart/CVS</td><td>N/A</td><td></td></tr><tr><td>Inflation Device</td><td>Demax Medical</td><td>DID30</td><td></td></tr><tr><td>D300 3-way Stopcock</td><td>B.Braun Medical Inc.</td><td>4599543</td><td></td></tr><tr><td>Artificial Tears Ointment&amp;nbsp;</td><td>Rugby Laboratories, Duluth, GA</td><td>N/A</td><td></td></tr></tbody></table>

vascular balloon injury and intraluminal administration in rat carotid artery

ラットでの頸動脈バルーン傷害モデルは、よく20年以上のために確立されている。それは、血管平滑筋分化、新生内膜形成および血管リモデリングに関与する分子や細胞メカニズムを研究するための重要な方法のまま。雄Sprague-Dawleyラットをこのモデルのために最も頻繁に用いられる動物である。女性ホルモンは血管疾患に対して保護的であり、したがって、このプロシージャにバリエーションを導入として雌ラットは好ましくない。左頸動脈は、一般的に、陰性対照として右の頸動脈に怪我をされている。左頸動損傷は、内皮をdenudesとs血管壁を膨張させる膨張したバルーンによって引き起こされる。損傷後、そのような薬理学的化合物および遺伝子またはshRNA転送のいずれかの使用のような潜在的な治療戦略を評価することができる。典型的には遺伝子またはshRNA転送のため、血管内腔の負傷した部分が局所的にvで30分間形質導入され損傷した血管壁の送達および発現のためのタンパク質またはshRNAのいずれかをコードするIRAL粒子。増殖性血管平滑筋細胞を表す新生内膜肥厚は、通常、損傷後2週間でピーク。容器は、主に細胞および分子細胞シグナル伝達経路の解析、並びに遺伝子及びタンパク質発現のために、この時点で収穫される。容器はまた、意図された実験の目的に応じて、特定のタンパク質または経路の発現及び/又は活性化の開始を決定するために、以前の時点で収穫することができる。容器は、組織学的染色、免疫組織化学、タンパク質/ mRNAのアッセイ、および活性アッセイを用いて特徴付けし、評価することができる。同じ動物からの無傷の右頸動脈は理想内部対照である。分子および細胞パラメータにおける傷害誘導変化は、内部右側対照動脈に損傷動脈を比較することによって評価することができる。同様に、治療法は傷を負う比較することによって評価することができますコントロールにDと治療動脈は唯一の動脈を負傷した。

二週間損傷後、頸動脈を採取し、切片化し、形態学的分析に付す。動脈は、クロス切断し、H＆Eで染色している（ 図1、図2B、Cおよび3）。ラット頸動脈の壁はピンク色の線として表示され弾性板の4層が含まれています。最外部薄層、外弾性板（EEL）と最も内側の薄層、内弾性板（IEL）の間の領域は、メディア平滑筋層（ 図1）である。 IELの内側の面積は内膜、無傷の...

Watch this Scientific Journal Video about Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery at JoVE.com

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

このプロトコルは、ラット頚動脈上の管腔内損傷を引き起こすと今後新生内膜過形成を誘発するためにバルーンカテーテルを使用しています。これは、傷害に応答する脈管リモデリングのメカニズムを研究するための十分に確立されたモデルである。また、広く、潜在的な治療的アプローチの有効性を決定するために使用される。

ラット頸動脈における血管バルーン傷害と管腔内投与

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

vascular-balloon-injury-intraluminal-administration-rat-carotid

Research

JoVE Journal

Medicine

16.3K ビュー.  State University of New York College of Nanoscale Science and Engineering (SUNY CNSE). このプロトコルは、ラット頚動脈上の管腔内損傷を引き起こすと今後新生内膜過形成を誘発するためにバルーンカテーテルを使用しています。これは、傷害に応答する脈管リモデリングのメカニズムを研究するための十分に確立されたモデルである。また、広く、潜在的な治療的アプローチの有効性を決定するために使用される。 

ビデオ: Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

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 ...

医学

2-Vessel Occlusion/Hypotension: A Rat Model of Global Brain Ischemia

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ischemia produces damage to specific brain regions shown to be highly sensitive to ischemia 1. Hippocampal neurons have higher sensitivity to ischemic insults compared to other cell populations, and specifically, the CA1 region of the hippocampus is particularly vulnerable to ischemia/reperfusion 2.
The design of therapeutic interventions, or study of mechanisms involved in cerebral damage, requires a model that produces damage similar to the clinical condition and in a reproducible manner. Bilateral carotid vessel occlusion with hypotension (2VOH) is a model that produces reversible forebrain ischemia, emulating the cerebral events that can occur during cardiac arrest and resuscitation. We describe a model modified from Smith et al. (1984) 2, as first presented in its current form in Sanderson, et al. (2008) 3, which produces reproducible injury to selectively vulnerable brain regions 3-6. The reliability of this model is dictated by precise control of systemic blood pressure during applied hypotension, the duration of ischemia, close temperature control, a specific anesthesia regimen, and diligent post-operative care. An 8-minute ischemic insult produces cell death of CA1 hippocampal neurons that progresses over the course of 6 to 24 hr of reperfusion, while less vulnerable brain regions are spared. This progressive cell death is easily quantified after 7-14 days of reperfusion, as a near complete loss of CA1 neurons is evident at this time.
In addition to this brain injury model, we present a method for CA1 damage quantification using a simple, yet thorough, methodology. Importantly, quantification can be accomplished using a simple camera-mounted microscope, and a free ImageJ (NIH) software plugin, obviating the need for cost-prohibitive stereology software programs and a motorized microscopic stage for damage assessment.

Bilateral carotid occlusion coupled with systemic hypotension produces global brain ischemia in the rat, resulting in damage to the hippocampus with reproducible severity. Animal subjects are impaired with predictable patterns of brain damage, they recover expediently, and mortality rates are comparatively low.

2血管閉塞/低血圧：グローバル脳虚血のラットモデル

Cardiac arrest followed by resuscitation often results in dramatic brain damage caused by ischemia and subsequent reperfusion of the brain. Global brain ...

Vascular Gene Transfer from Metallic Stent Surfaces Using Adenoviral Vectors Tethered through Hydrolysable Cross-linkers

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hr&#160;and consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

These studies report on reversible attachment of adenoviral gene vectors to coatless metal surfaces of stents and model mesh disks. Sustained release of transduction-competent viral particles contingent upon hydrolysis of cross-linkers used for vector immobilization results in a durable site-specific transgene expression in vascular cells and in stented arteries.

加水分解性架橋剤を介してアデノウイルスベクターテザーを使用した金属ステント表面からの血管の遺伝子導入

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

ラット頸動脈バルーン損傷モデルは、抗血管リモデリングの治療薬をテストします

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Reproducible Arterial Denudation Injury by Infrarenal Abdominal Aortic Clamping in a Murine Model

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These complications can be attributed to impairments in re-endothelialization within the wound margins. Yet, the cellular and molecular mechanisms of re-endothelialization remain to be defined. While several animal models to study re-endothelialization after arterial denudation are available, few are performed in the mouse because of surgical limitations. This undermines the opportunity to exploit transgenic mouse lines and investigate the contribution of specific genes to the process of re-endothelialization. Here, we present a step-by-step protocol for creating a highly reproducible murine model of arterial denudation injury in the infrarenal abdominal aorta using external vascular clamping. Immunocytochemical staining of injured aortas for fibrinogen and &#946;-catenin demonstrate the exposure of a pro-thrombotic surface and the border of intact endothelium, respectively. The method presented here has the advantages of speed, excellent overall survival rate, and relative technical ease, creating a uniquely practical tool for imposing arterial denudation injury in transgenic mouse models. Using this method, investigators may elucidate the mechanisms of re-endothelialization under normal or pathological conditions.

Understanding the cellular and molecular mechanisms of re-endothelialization following arterial denudation injury is of paramount importance in preventing thrombosis and restenosis of arteries. Here we describe a protocol for reproducible arterial denudation injury of the infrarenal abdominal aorta. The procedure was developed to investigate the underlying mechanisms that regulate endothelial regeneration using mouse models.

マウスモデルにおける下腹部大動脈クランプすることにより、再現性動脈侵食に起因けが

Percutaneous vascular interventions uniformly result in arterial denudation injuries that subsequently lead to thrombosis and restenosis. These ...

Reproducible Motor Deficit Following Aortic Occlusion in a Rat Model Of Spinal Cord Ischemia

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing and treating this complication using experimental models of spinal cord ischemia. The model described here demonstrates varying degrees of paraplegia that relate to the length of occlusion following thoracic aortic occlusion in a rat spinal cord ischemia model.
A 2-Fr. balloon-tipped catheter was advanced through the femoral artery into the descending thoracic aorta until the catheter tip was placed at the left subclavian artery in anesthetized male Sprague-Dawley rats. Spinal cord ischemia was induced by inflating the catheter balloon. After a set period of occlusion (9, 10, or 11 min), the balloon was deflated. Neurologic assessment was performed using the motor deficit index at 24 h after surgery, and the spinal cord was harvested for histopathological examination.
Rats that underwent 9 min of aortic occlusion showed mild and reversible motor impairment in the hind limb. Rats subjected to 10 min of aortic occlusion presented with moderate but reversible motor impairment. Rats subjected to 11 min of aortic occlusion displayed complete and persistent paralysis. The motor neurons in the spinal cord sections were more preserved in rats subjected to shorter duration of aortic occlusion.
Researchers can achieve a reproducible hind limb motor deficit following thoracic aortic occlusion using this spinal cord ischemia model.

This study demonstrates the technique to make a minimally invasive and easily reproducible model of spinal cord ischemia in rats. Various degrees of hind limb motor deficit can be produced by controlling the aortic occlusion time.

脊髄虚血ラットモデルにおける大動脈閉塞後の再現性のある運動障害

Spinal cord ischemia is a fatal complication following thoracoabdominal aortic aneurysm surgery. Researchers can investigate the strategies for preventing ...

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 ...

Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of coronary artery disease. In-stent restenosis (ISR) is determined by an excessive proliferation of neointimal tissue within the stent and limits the long-term success of stents. A variety of animal models have been used to elucidate pathophysiological processes underlying in-stent restenosis (ISR), with the porcine coronary and the rabbit iliac artery models being the most frequently used. Murine models provide the advantages of high throughput, ease of handling and housing, reproducibility, and a broad availability of molecular markers. The apolipoprotein E deficient (apoE-/- ) mouse model has been widely used to study cardiovascular diseases. However, stents must be miniaturized to be implanted into mice, involving important changes of their mechanical and (potentially) biological properties. The use of apoE-/- rats can overcome these shortcomings as apoE-/- rats allow for the evaluation of human-sized coronary stents while at the same time providing an atherogenic phenotype. This makes them an excellent and reliable model to investigate ISR after stent implantation. Here, we describe, in detail, the implantation of commercially available human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access.

This protocol describes the implantation of human coronary stents into the abdominal aorta of rats with an apoE-/- background using a trans-femoral access. Compared with other animal models, murine models carry the advantages of high throughput, reproducibility, ease of handling and housing, and a broad availability of molecular markers.

トランス・大腿部アクセスを用いたヒトサイズの冠動脈ステントのラット腹部大動脈への移植

Percutaneous coronary intervention (PCI), combined with the deployment of a coronary stent, represents the gold standard in interventional treatment of ...

深部静脈治療研究のためのげっ歯類静脈形成術バルーンモデル

Rodent Inferior Vena Cava Venoplasty Balloon Model

Balloon venoplasty is a commonly used clinical technique to treat deep vein stenosis and occlusion as a consequence of trauma, congenital anatomic abnormalities, acute deep vein thrombosis (DVT), or stenting. Chronic deep venous obstruction is histopathologically characterized by thrombosis, fibrosis, or both. Currently, no direct treatment is available to target these pathological processes. Therefore, a reliable in vivo animal model to test novel interventions is necessary. The rodent survival inferior vena cava (IVC) venoplasty balloon model (VBM) allows the study of balloon venoplasty in non-thrombotic and post-thrombotic conditions across multiple time points. The local and systemic effect of coated and uncoated venoplasty balloons can be quantified via tissue, thrombus, and blood assays such as real-time polymerase chain reaction (RT-PCR), western blot, enzyme-linked immunosorbent assay (ELISA), zymography, vein wall and thrombus cellular analysis, whole blood and plasma assays, and histological analysis. The VBM is reproducible, replicates surgical human interventions, can identify local vein wall-thrombi protein changes, and allows multiple analyses from the same sample, decreasing the number of animals required per group.

著者スポットライト:げっ歯類の静脈形成術バルーンモデルを利用して、深部静脈の外科的介入をシミュレートする

The protocol presents a survival rodent model to test venoplasty balloon (VB) interventions in non-thrombotic, thrombotic, and post-thrombotic deep veins.