Name: a murine model of arterial restenosis: technical aspects of femoral wire injury
Uploaded: 2015-03-10T15:00:00.000+00:00
Duration: 6 min 42 s
Description: Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

This work was supported by a Wisconsin Partnership Program New Investigator Award (ID 2832), a National Heart, Lung, Blood Institute R01 Grant (HL-068673) and a T32 training Grant (HL-110853). We thank Dr. Melina Kibbe&#8217;s group at Northwestern University for providing helpful information.

注：倫理に関する声明：すべての手順は、実験動物の管理と使用に関する指針（全米アカデミープレス、第8版、2011）、およびウィスコンシン大学の施設内動物管理使用委員会によって承認されたプロトコルに準拠しています。すべての手術は（速度2ml /分流量、吸入を介して）、イソフルラン麻酔下で実施し、全ての努力が苦痛を最小限にするために行われた。動物を、徐々にCO 2で満たされたチャンバー内で安楽死させた。 全身麻酔と皮膚切開の1の誘導<ol><li> 23〜28グラムの12〜16週の年齢と体重と雄のC57BL / 6マウスを使用してください。閉回路の麻酔​​装置を通過イソフルランにより動物を麻酔。そのつま先をつまんで、適切な麻酔を確認してください。乾燥を防ぐために、目にいくつかの人工涙液軟膏を適用します。 </li><li>少し拉致左後肢膝関節のSLと仰臥位でマウスを置きますightly撓む。左大腿上部領域の周囲の毛を剃るとクロルヘキシジンで皮膚を消毒、滅菌した綿先端アプリケーターを用いて3回を適用した。手術中に滅菌楽器を保つためにホットビーズ滅菌器を使用してください。 </li><li>左足の上に滅菌ガーゼドレープを配置します。鼠径部（ 図1A）に膝関節からの左大腿内側に1.5センチメートルストレート、縦皮膚切開を行います。 </li></ol>大腿動脈と枝の2暴露メモ： 図1（b）は、大腿上部における総血管系の解剖学を示している。皮膚切開が適切に大腿内側の正中線に沿って行われたとき浅大腿動脈（SFA）は、解剖顕微鏡で観察することができる。 <ol><li>細かいピンセットでSFAの上にある薄い筋膜を開き、9-0ナイロン縫合糸とループSFA。 注：このプロセスでは、大腿神経が容易ように置かれたその壁は、それが簡単に穿孔と出血していることを非常に薄いので、IDEが、隣接する大腿静脈は細心の注意が必要です。キー技術は動脈自体に集中することです。限り切開が近い動脈に行われるように、任意の偶発的静脈の損傷が回避される。 </li><li> SFAがループされると、モスキート鉗子で縫合糸を保持し、やや下向きに引き出します。この操縦は簡単にさらに解剖になりますが、あまりにも強制的に引っ張らないでください動脈を損傷しないように。 </li><li> 9-0ナイロン縫合糸で総大腿動脈（CFA）を露出し、ループするように近位に動脈を解剖続行。 注：通常、上腹部動脈（ 図1B）に遠位の点でCFAのループは、電線を挿入するための十分です。同じようにモスキート鉗子でCFAの縫合糸ループを保持します。 </li><li>上向きのCFAの縫合糸ループを引いて、SFAの縫合糸ループを下方にSFAと深い大腿動脈（DFA）の分岐を明らかに。 DFA、CFAの主要な枝、大腿筋（ 図1B）に向かう。 注：ワイヤーは後でDFAから挿入されます。 </li><li>余分な慎重にループDFA隣接静脈を動脈の下に絡まっているため。静脈からの出血の場合には、ガーゼで30〜60秒程度の単純な圧縮は、止血をもたらすのに通常は十分である。 </li><li> DFAは ​​、9-0ナイロン縫合糸でループされると、すべての3つの縫合糸ループは、（ 図2A）が正しい位置にあることを確認してください。 </li><li>遠位に可能な限り分岐からポイントで動脈を結紮するDFAの解剖を続行します。余分な縫合糸をカットしますが、両端がDFAは、ワイヤの挿入を作るより簡単に安定させるモスキート鉗子で保持できるように一定の長さにしておきます。 </li></ol> 3.ワイヤー傷害を作成する<ol><li> DFAを経由して電線を挿入<ol><li>一時的な止血を確立するために、CFA、SFA、およびDFAの縫合糸をひずみ。 </li><li> DFに小さな穴を作るmicroscissorsとA。 0.015インチ径のワイヤーのために十分な大きさの穴を拡張するために湾曲したmicrotweezersの先端を挿入します（ 図2Bは、動脈とワイヤとの間のサイズの違いを示しています）。 <ol><li> DFAに穴を行う前に、CFAの縫合糸ループを緩め、静かにクランプ/鈍い先端がmicrotweezersでDFA数回絞る。この操縦ワイヤの挿入を容易にするために著しく動脈の直径を拡大する。 PBSで徹底的にワイヤーを加湿することもスムーズに挿入するために重要である。 </li><li> DFAに穴を作るときは、動脈壁をトリミングが、小さなV字型のニップを作成し、操作するための鋭い先端がmicrotweezersとフラップを挟まないでください。 </li></ol></li><li> DFAに適切に-拡張穴を通してワイヤーを挿入します。 CFAの周りに縫合糸ループを緩めながらゆっくりと腸骨動脈に向かって線を進める。 5〜8ミリメートルはほとんどの場合、すべてのCFAの長さをカバーするために十分である（ 図2C </strong&gt;）。動脈穿孔は通常、マウスの損失をもたらすように強制的にワイヤーを挿入しないでください。長さ約15ミリにワイヤを短くする処理に役立ちます。 </li></ol></li><li> 1分間の中にワイヤーを残し<ol><li>最適な長さにワイヤーを挿入した後、動脈壁を伸び過ぎ1分間放置する。乾燥を防ぐためにPBSワイヤおよび動脈を湿らせる。別の9-0ナイロン縫合糸は、この待ち時間の間にワイヤ挿入ポイントにDFA近位の周りに配置することができる。この縫合糸は、ワイヤの除去後DFAに穴を閉じるために後で使用される。 </li></ol></li><li>ワイヤーを取得し、血流を再開<ol><li>血管カニュレーション鉗子で線を持ち、ゆっくりと引き戻す。ワイヤーがスムーズに取得されるべきである。通常動脈壁へのワイヤの付着によって生じるいかなる抵抗を克服するために、穏やかに回転してワイヤーを引き戻すと便利です。ここでも、力強い操縦は、動物の損失につながる可能性があります。 </li><li> Aワイヤーを取り出すFTERは、9-0ナイロン縫合糸との分岐点でDFAを連結する。 SFAへの血液流出を危うく分岐、に近づきすぎ連結しないでください。 </li><li>正常な血流を再開するためにSFAとCFAの縫合糸ループを緩めます。 CFAは、ワイヤの挿入（ 図2D）で十分に展開されていることに注意してください。 </li></ol></li></ol> 4.皮膚閉鎖と術後ケア<ol><li>コントロールが出血。マイナーにじみ出るは、簡単な圧縮によって停止することができる。いくつかの主要な動脈採血を11-0ナイロン縫合糸でステッチが必要になります。これらの縫合動脈は、最終的には動脈閉塞になります。 </li><li>スキンステープラー（通常3-4ステープル）で皮膚創傷を閉じます。クロルヘキシジン外科ソリューションを適用することにより、滅菌創傷領域にしてください。 </li><li>術後の治療法を適用します。ブプレノルフィンの皮下注射でマウスの痛みを制御します。それはSUFを取り戻したまでは温暖化パッド上でマウスを保つ胸骨の横臥位を維持するために意識をficient。他の動物の会社のない清潔なケージ内のハウスには、完全に回復するまで。 </li></ol>

<ol>
	<li>Dzau, V. J., Braun-Dullaeus, R. C., Sedding, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+proliferation+and+atherosclerosis:+new+perspectives+and+therapeutic+strategies.">Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.</a> Nat Med. 8 (11), 1249-1256 (2002).</li><li>Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+hyperplasia+in+murine+models.">Intimal hyperplasia in murine models.</a> Curr Drug Targets. 9 (3), 251-260 (2008).</li><li>Ferns, G. A., Avades, T. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanisms+of+coronary+restenosis:+insights+from+experimental+models.">The mechanisms of coronary restenosis: insights from experimental models.</a> Int J Exp Pathol. 81 (2), 63-88 (2000).</li><li>Sata, 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=A+mouse+model+of+vascular+injury+that+induces+rapid+onset+of+medial+cell+apoptosis+followed+by+reproducible+neointimal+hyperplasia.">A mouse model of vascular injury that induces rapid onset of medial cell apoptosis followed by reproducible neointimal hyperplasia.</a> J Mol Cell Cardiol. 32 (11), 2097-2104 (2000).</li><li>Xu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+arteriosclerosis:+from+arterial+injuries+to+vascular+grafts.">Mouse models of arteriosclerosis: from arterial injuries to vascular grafts.</a> Am J Pathol. 165 (1), 1-10 (2004).</li><li>Lv, L., Meng, Q., Ye, M., Wang, P., Xue, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT4+deficiency+protects+against+neointima+formation+following+arterial+injury+in+mice.">STAT4 deficiency protects against neointima formation following arterial injury in mice.</a> J Mol Cell Cardiol. 74C, 284-294 (2014).</li><li>Zhu, B., Kuhel, D. G., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apolipoprotein+E+inhibits+neointimal+hyperplasia+after+arterial+injury+in+mice.">Apolipoprotein E inhibits neointimal hyperplasia after arterial injury in mice.</a> Am J Pathol. 157 (6), 1839-1848 (2000).</li><li>Fu, Y., Duru, E. A., Davies, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+metabolic+syndrome+on+the+response+to+arterial+injury.">Effect of metabolic syndrome on the response to arterial injury.</a> J Surg Res. , (2014).</li><li>Kuhel, D. G., Zhu, B., Witte, D. P., Hui, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinction+in+genetic+determinants+for+injury-induced+neointimal+hyperplasia+and+diet-induced+atherosclerosis+in+inbred+mice.">Distinction in genetic determinants for injury-induced neointimal hyperplasia and diet-induced atherosclerosis in inbred mice.</a> Arterioscler Thromb Vasc Biol. 22 (6), 955-960 (2002).</li><li>Cooley, 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=Mouse+strain+differential+neointimal+response+in+vein+grafts+and+wire-injured+arteries.">Mouse strain differential neointimal response in vein grafts and wire-injured arteries.</a> Circ J. 71 (10), 1649-1652 (2007).</li><li>Ishigami, N., 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=Deficiency+of+CuZn+superoxide+dismutase+promotes+inflammation+and+alters+medial+structure+following+vascular+injury.">Deficiency of CuZn superoxide dismutase promotes inflammation and alters medial structure following vascular injury.</a> J Atheroscler Thromb. 18 (11), 1009-1017 (2011).</li></ol>

The authors declare no competing financial interests.

ワイヤ傷害手順は、我々は12-16週齢の雄C57BL / 6マウスを使用する限り、それらの解剖学的構造が類似しているように4本研究では、マウスのすべての株に適用可能である。私たちは私たちの予備的研究から学んだように、10週より若いマウスの大腿動脈は、多くの場合、線挿入は非常に困難であることが非常に小さい。一方、16週間以上経過したマウスにおける電線挿入は、技術的に容?...

In the era of expanded application of endovascular treatments for various cardiovascular diseases, restenosis after angioplasty is one of the major problems for patients undergoing such treatments. Damage to the vascular endothelium at the time of angioplasty, in concert with the atherosclerotic background, induces excessive smooth muscle cell proliferation in the medial layer, resulting in neointimal hyperplasia.1,2 A viable animal model that recapitulates post-angioplasty neointimal hyperplasia is, thus, important not only for the investigation of disease mechanisms but also for the development of effective therapeutics to treat this pathology.
Mice represent an excellent model animal to recapitulate neointimal hyperplasia for the following reasons: the genetic backgrounds of experimental mice are well established; a wide variety of genetically-modified strains are available;2 obtaining littermates of the same background is easy; and the cost of the animals is relatively low. Arterial ligation model and wire injury model are the two most common mouse models of mechanically-induced neointimal hyperplasia. The arterial ligation model is easy to create, but physiologically dissimilar to the actual angioplasty procedure. The wire injury model closely mimics actual angioplasty procedures but is technically difficult due to the small size of mouse arteries.2,3 Sata et al. first described a wire injury method for mouse femoral arteries based on the anatomical structure of the vasculature and the use of proper-sized flexible wire. Utilizing this technique, they succeeded in reproducibly inducing neointimal hyperplasia in various strains of mice.4
Although femoral wire injury is a well-established model, some of the technical aspects of the technique are highly challenging compared to other models such as ligation.5 The purpose of this paper is to describe our mouse wire injury model procedures in detail, which is a modified version of Sata&#8217;s original method. We have made two main modifications: 1) Looping only the arteries, and 2) No lidocaine use.

<table><tbody><tr><td>Fixed Core Wire Guide</td><td>Cook</td><td>G02426</td><td>Diameter 0.015 inch, Straight</td></tr><tr><td>Dilation Forceps</td><td>Roboz</td><td>RS-4927</td><td>Curved, blunt tips</td></tr><tr><td>Dumont Tweesers #5</td><td>World Precision Instruments</td><td>14095</td><td>Straight, sharp tips</td></tr><tr><td>Dumont Vessel Cannulation Forceps</td><td>World Precision Instruments</td><td>503373</td><td></td></tr><tr><td>McPherson-Vannas Scissors</td><td>World Precision Instruments</td><td>501234</td><td></td></tr><tr><td>Mosquito Forceps</td><td>World Precision Instruments</td><td>501291</td><td></td></tr><tr><td>Ethilon Nylon Suture 9-0</td><td>Ethicon</td><td>7717G</td><td>9-0, Black Nylon Monofilament</td></tr><tr><td>Micro AROSuture, Sterile 11-0, 70 micrometers, MET Point</td><td>AROSurgical</td><td>VT4A00N07</td><td>11-0, Black Nylon Monofilament</td></tr><tr><td>3M Precise Multi-Shot DS Disposable Skin Stapler</td><td>3M</td><td>DS-25</td><td></td></tr></tbody></table>

a murine model of arterial restenosis: technical aspects of femoral wire injury

アテローム性動脈硬化症により引き起こされる心血管疾患は、先進国における死亡の主な原因である。動脈硬化性プラークの開発や確立プラークの破裂による血管内腔の狭窄は、このような心筋梗塞や脳卒中などの様々な病的状態につながる正常な血流を遮断する。診療所では、そのような血管形成術などの血管内の手順は、一般的に内腔を再度開くために行われている。しかし、これらの治療は、必然的に、過剰な治癒反応と血管狭窄（​​再狭窄）を引き起こすルーメン内に延びて、新生内膜プラークの発達を誘発する、血管壁、ならびに血管内皮を損傷する。再狭窄は、アテローム性動脈硬化症のための血管内治療の失敗の主要な原因である。したがって、再狭窄の前臨床動物モデルは、病態生理学的機序ならびに脈管介入翻訳アプローチを調査するために極めて重要である。いくつかのネズミexperimeうち、それは密接に内皮および血管壁の両方を傷つける血管形成術に似ているので、NTALモデルは、大腿動脈ワイヤ傷害は広く血管形成術後の再狭窄の研究のために最も適していると認められている。しかし、多くの研究者が、技術的難易度の高い程度に、このモデルを利用して困難である。金属ワイヤが顕著新生を誘導するのに十分な損傷を生成するために、ワイヤの約3倍薄い大腿動脈に挿入する必要があるため、これは主にある。ここでは、効果的に、このモデルの主要な技術的困難を克服するために不可欠の手術の詳細を説明します。 、提示された手順に従って実行することによって、マウスの大腿動脈ワイヤ傷害が容易になる。慣れた後、全体の手順は、20分以内に完了することができる。

手術後4週間、ワイヤー損傷大腿動脈を収穫し、新生内膜形成を分析するためにパラフィンブロック標本を作る。上記のように全身麻酔を誘導する。すぐに胸を開き、左心室に20ゲージの翼状針を挿入します。点滴ボトルにラインを接続します。 4％パラホルムアルデヒド（通常は10〜20ミリリットル、各）に続いて、PBSの点滴を経由して灌流固定を行います。動物は、ワイヤ損傷CFAを取?...

Watch this Scientific Journal Video about A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury at JoVE.com

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

再狭窄のマウス大腿動脈ワイヤ傷害モデルは、技術的に困難である。このプロトコルでは、我々は成功し、再狭窄の研究のための一貫性の新生内膜を誘導するために、ワイヤ傷害を実施するための不可欠なキー技術的な詳細を示している。

動脈再狭窄のマウスモデル：大腿ワイヤー傷害の技術的な側面

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to ...

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Research

JoVE Journal

Medicine

20.1K ビュー.  University of Wisconsin School of Medicine and Public Health. 再狭窄のマウス大腿動脈ワイヤ傷害モデルは、技術的に困難である。このプロトコルでは、我々は成功し、再狭窄の研究のための一貫性の新生内膜を誘導するために、ワイヤ傷害を実施するための不可欠なキー技術的な詳細を示している。 

ビデオ: A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

後脚虚血症のマウスモデル

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

生物学

Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. Its unique anatomy makes the spinotrapezius useful for investigation of ischemic injury and subsequent microvascular remodeling. Here, we demonstrate an arteriolar ligation model in the murine spinotrapezius muscle that was developed by our research team and previously published1-3. For certain vulnerable mouse strains, such as the Balb/c mouse, this ligation surgery reliably creates skeletal muscle ischemia and serves as a platform for investigating therapies that stimulate revascularization. Methods of assessment are also demonstrated, including the use of intravital and confocal microscopy. The spinotrapezius is well suited to such imaging studies due to its accessibility (superficial dorsal anatomy) and relative thinness (60-200 &mu;m). The spinotrapezius muscle can be mounted en face, facilitating imaging of whole-muscle microvascular networks without histological sectioning. We describe the use of intravital microscopy to acquire metrics following a functional vasodilation procedure; specifically, the increase in arterilar diameter as a result of muscle contraction. We also demonstrate the procedures for harvesting and fixing the tissues, a necessary precursor to immunostaining studies and the use of confocal microscopy.

We demonstrate a novel arterial ligation model in murine spinotrapezius muscle, including a step-by-step procedure and description of required instrumentation. We describe the surgery and relevant outcome measurements relating to vascular network remodeling and functional vasodilation using intravital and confocal microscopy.

微小血管機能およびリモデリングに動脈結紮の影響を評価するためにネズミSpinotrapeziusモデル

The murine spinotrapezius is a thin, superficial skeletal support muscle that extends from T3 to L4, and is easily accessible via dorsal skin incision. ...

医学

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 ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

発生と光学投影トモグラフィを用いたマウスにおける動脈病変の3次元定量

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

血管周囲ドラッグデリバリーパッチの移植に大腿動脈ワイヤ傷害のマウスモデル

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

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. ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

マウス後肢モデルにおける動脈形成中の白血球動員モニタリングのための多光子生体内イメージング

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

High-Resolution Three-Dimensional Imaging of the Footpad Vasculature in a Murine Hindlimb Gangrene Model

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering from its most severe form, chronic limb-threatening ischemia (CLTI), face substantial impairments to daily living, including chronic pain, limited walking distance without pain, and nonhealing wounds. Preclinical models have been developed in various animals to study PAD, but mouse hindlimb ischemia remains the most widely used. There can be significant variation in response to ischemic insult in these models depending on the mouse strain used and the site, number, and means of arterial disruption. This protocol describes a unique method combining femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase (NOS) inhibitor to reliably induce footpad gangrene in Friend Virus B (FVB) mice that resembles the tissue loss of CLTI. While traditional means of assessing reperfusion such as laser Doppler perfusion imaging (LDPI) are still recommended, intracardiac perfusion of the lipophilic dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) is used to label the vasculature. Subsequent whole-mount confocal laser scanning microscopy allows for high-resolution, three-dimensional (3D) reconstruction of footpad vascular networks that complements traditional means of assessing reperfusion in hindlimb ischemia models.

The present protocol describes a unique, clinically relevant model of peripheral arterial disease that combines femoral artery and vein electrocoagulation with the administration of a nitric oxide synthase inhibitor to induce hindlimb gangrene in FVB mice. Intracardiac DiI perfusion is then used for high-resolution, three-dimensional imaging of the footpad vasculature.

マウス後肢壊疽モデルにおける足蹠血管系の高解像度3次元イメージング

Peripheral arterial disease (PAD) is a significant cause of morbidity resulting from chronic exposure to atherosclerotic risk factors. Patients suffering ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

ネズミ大動脈挫傷：効率的<em&gt;インビボ</em&gt;平滑筋細胞の増殖と内皮機​​能のモデル

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Establishing a Diaphyseal Femur Fracture Model in Mice

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age and overall health status of the patient, failures can occur, leading to delayed union or nonunion. Due to the increasing number of fractures resulting from high-energy trauma and aging, the development of innovative therapeutic strategies to improve bone repair based on the combination of skeletal/mesenchymal stem/stromal cells and biomimetic biomaterials is urgently needed. To this end, the use of reliable animal models is fundamental to better understanding the key cellular and molecular mechanisms that determine the healing outcomes. Of all the models, the mouse is the preferred research model because it offers a wide variety of transgenic strains and reagents for experimental analysis. However, the establishment of fractures in mice may be technically challenging due to their small size. Therefore, this article aims to demonstrate the procedures for the surgical establishment of a diaphyseal femur fracture in mice, which is stabilized with an intramedullary wire and resembles the most common bone repair process, through cartilaginous callus formation.

This protocol describes a surgical procedure for the establishment of a diaphyseal fracture in the femur of mice, which is stabilized with an intramedullary wire, for fracture healing studies.

マウスにおける骨幹大腿骨骨折モデルの確立

Bones have a significant regenerative capacity. However, fracture healing is a complex process, and depending on the severity of the lesions and the age ...