埋め込みとスライド制作に関する貴重な技術支援をしてくれたアンジェラ・フロイント夫人に感謝します。また、動物実験外科研究所のタデウシュ・ストインスキー氏の獣医の仕事に対する洞察力に満ちた支援に感謝します。

実験は、科学的目的のために使用される動物の保護に関するドイツの動物福祉法(TSchG)および指令2010/63/EUに従って行われました。この研究の公式承認は、政府動物のケアと使用委員会によって付与されました (プロトコル No.: AZ 87-51.04.2010.A065;ランデサムト・フュル・ナートゥール、ウムヴェルト・ウント・ヴェルブラウヒャーシュッツ・ノルドハイン=ヴェストファーレン、レックリングハウゼン、ドイツ)。研究プロトコルは、実験動物のケアと使用のためのガイドに準拠しています。術後疼痛治療は、ドイツ実験動物科学会(GV-SOLAS)とイニシアチブ獣医痛み治療の勧告に基づいています。
1. 基本的なテクニックと一般的な手順
<ol><li>ホモ接合アポエを使用してください -/- スプレイグ・ドーリーラット。標準的な方法17を使用して、各動物の遺伝子型を特定する。</li>
	<li>同じ条件(21°C±2°C、湿度5%±60%、明暗サイクル12時間)で動物を保管し、水と食べ物への自由なアクセスを確保してください。</li>
	<li>清潔で無菌状態ですべての手順を実行します。</li>
	<li>ラットを麻酔したら、16倍の倍率で手術顕微鏡ですべての処置を行う。</li>
	<li>圧縮止止めには綿棒を使用してください。授乳中のリンゲル溶液を浸したガーゼ綿棒(5cm x 5cm)は、鼠径部を湿潤に保つのに役立ちます。</li>
	<li>廃棄物処理規則に従って、使用済み資材を処分する。</li>
</ol>2. 手術前の準備
<ol><li>手術を開始する前に、動物用医薬品を準備してください。特に明記されていない限り、すべての溶液を室温に保ちます。</li>
	<li>
処置の30分前に 、0.03-0.05 mg/kgブプレノルフィンを皮下に投与する。
</li>
	<li>ラットを腹腔内注射で麻酔薬を100mg/kg体重(BW)(S)ケタミンおよび8mg/kg BWキシラジンで麻酔します。</li>
	<li>計量スケールを使用してラットの体重を評価します。</li>
	<li>ラットを加熱パッドに置き、医療テープを使用して上肢と下肢を固定します。大腿動脈と大動脈の間に直線を作るように、左後肢を完全に伸ばし、背骨に沿ってラットを配置する。これは大動脈分岐を通して気球取付けられたステントを進めるのを容易にする。</li>
	<li>2 L/minの流量で97.5%酸素中1.5体積%イオブルランの吸入で麻酔を維持する。 注:ラットは挿管せずに自発的に呼吸することができます。</li>
	<li>意識不明の間に目の損傷を防ぐために眼軟膏を適用します。</li>
	<li>ラットの鼠径部と下腹部の部分から毛皮を剃り、ポビドネ-ヨウ素溶液で対応する皮膚を殺菌する。</li>
	<li>手術を開始する前に、尾端とデジタル間組織をつまんで麻酔の適切な深さを確認してください。</li>
</ol>3. 手術
<ol><li>皮膚と下層筋膜を開くために、左鼠径部に〜0.5\u20121 cmの内側切開を行います。</li>
	<li>脈動した左大腿動脈が同定されるまで、深部で鈍く解剖し、プローブする。</li>
	<li>非常に細かい鉗子を使用して、周囲の結合組織を穏やかに除去することによって大腿動脈を準備する。大腿神経にも大腿静脈も、動脈の内側に害を与えないように注意してください。</li>
	<li>大腿動脈の約1cmを準備します。慎重に容器の下に鉗子の先端を置き、そっと持ち上げます。</li>
	<li>動脈の遠位部および近位部分の下の4-0シルク縫合糸の糸片および形態スリング。外科用クランプの枝の間の2つの糸のスリングの両端をクランプします。動脈を制御するために外科用クランプを使用してください。血流を一時的に中断するために、スリングを軽く伸ばして持ち上げます。 注:組織の損傷につながる可能性があり、長時間の止血帯を避けるために速く働きます。</li>
	<li>鋭利なマイクロハサミを使用して、大腿動脈の真ん中で動脈切開術を行う。</li>
	<li>動脈切開術を通してガイドワイヤーを導入します。近位糸スリングに達したら、外科用クランプを動かして糸の張力を解放し、ガイドワイヤーを腹部大動脈に向かってさらに進める。 注: 取り扱いが容易な場合は、ワイヤカッターを使用してガイドワイヤをカットします。</li>
	<li>ダイヤフラムと腎動脈の間にガイドワイヤーの近位端を置きます。 注:ガイドワイヤーを進みすぎると、大動脈や心臓の損傷のリスクがあります。少なくとも最初の数匹の動物のためにガイドワイヤーとステントの適切な位置を確保するために腹部を開くことをお勧めします。</li>
	<li>ガイドワイヤーを介して2.25mm x 8mm(最大2.5mm x 8 mm)の圧着とバルーンマウントされた冠状動脈ステントを大腿動脈に導入し、腹部大動脈に進めます。</li>
	<li>ステントを大動脈分岐のすぐ上に置き、腎動脈の下に置きます。インフレシリンジシステムを使用して、バルーンカテーテルを15sの12気圧に膨らませてステントを展開します。</li>
	<li>バルーンカテーテルを収縮させ、使用中のステントに対するメーカーの推奨に従って負圧を維持します。</li>
	<li>ステントを所定の位置に残したまま、膨張したカテーテルをゆっくりと引き出します。</li>
	<li>カテーテルを取り出す直前に、切開部の上の糸ループに緊張を作り出し、外科的クランプで血流を中断します。次にバルーンカテーテルを取り外し、容器を直接近位にリゲートします。</li>
	<li>近位と遠位の糸を結んで大腿動脈を装い、動脈管内の十分な止血を確認する。副次動脈は、四肢へのさらなる灌流を保証する。</li>
	<li>10-0非蘇性縫合糸を使用して、動脈の上の筋肉を閉じるだけでなく、皮膚切開。</li>
</ol>4. ステント移植後の動物のケア
<ol><li>操作の直後に、ラットが温め空気(30\u201235 °C)と酸素供給を備えた特別な集中治療室のケージで60分間回復することを許可する。</li>
	<li>完全に回復するまで、動物を注意深く見てください。その後、ラットを通常のケージに移動します。水と食べ物へのアドリビタムアクセスを提供します。</li>
	<li>術後鎮痛を6-12時間ごとに0.03-0.05mg/kgブプレノルフィン.c(500μl NaCl)で6時間ごとに、臨床評価の下で合計72時間投与する。</li>
	<li>植え付けられたステントの血栓症を避けるためにクロピドグレル(15mg/kg)と混合食品を持っています。</li>
	<li>高コレステロール血症状態とプラーク形成を高めるために、生後6\u20128週に西洋の食事餌を開始し、安楽死まで続ける。必要であれば、正常なラットチャウを与えられた動物のコホートがコントロールとして機能することができる。</li>
</ol>5. 組織の収集と処理
<ol><li>指定された時点で組織の排泄を開始する前に、IACUCガイドラインに従って動物を安楽死させる。観察期間の終わりに組織学的分析のためにステント大オルタを収穫する。</li>
	<li>正中線の切開で腹部を開き、大間のステントセグメントと大間の隣接する非ステント部分を取り除き、それぞれ0.5cmを測定します。</li>
	<li>組織を4%緩衝ホルマリンの溶液に入れ、24時間固定します。</li>
	<li>ステント動脈組織をプラスチックに埋め込み、標準プロトコル18、19に従って組織学的および免疫組織化学的染色を行う。</li>
</ol>6. ヒストモルフォメトリック解析
<ol><li>適切な画像解析ソフトウェアを用いてコンピュータにリンクされた顕微鏡を用いて、ステント付大関係の近位、中間部、および遠位部分のヒストモルフォメトリクス解析を行う。</li>
	<li>外部弾性薄層(EEL、アドベンチシアとメディアの間)、内部弾性薄層(IEL、メディアとネオインティマの間)、およびグラフィック描画タブレットを備えたルーメンの輪郭をトレースします。これらの値から、ソフトウェアで EEL 面積、IEL 領域、およびルーメン領域を計算します。</li>
	<li>ステント・レステノーシス (ISR) の断面積率を計算します。 <img alt="Equation 1" src="/files/ftp_upload/61442/61442equ01.jpg">
</li>
	<li>合計のネオインティマル領域を計算します (Ai): <img alt="Equation 2" src="/files/ftp_upload/61442/61442equ02.jpg">
</li>
	<li>支柱と内腔の間の距離として、各ステントストラット上のネオインティマル厚さ(NIT)を測定します。ステント支柱間の NIT を IEL とルーメンの間の距離として測定します。 注: または、NIT を次のように計算します。 <img alt="Equation 3" src="/files/ftp_upload/61442/61442equ03.jpg"> ここで、PL および PIEL はルーメンと内部弾性層周長、それぞれ20です。</li>
	<li>スタディの要件に従って、追加の解析を実行します。</li>
</ol>

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Deviation of man from the usual pattern.</a> Circulation Research. 25 (6), 677-686 (1969).</li><li>Lowe, H. C., James, B., Khachigian, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+model+of+in-stent+restenosis:+rat+aortic+stenting.">A novel model of in-stent restenosis: rat aortic stenting.</a> Heart. 91 (3), 393-395 (2005).</li><li>Unthank, J. L., Nixon, J. C., Lash, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+adaptations+in+collateral+and+microvascular+resistances+after+ligation+of+the+rat+femoral+artery.">Early adaptations in collateral and microvascular resistances after ligation of the rat femoral artery.</a> Journal of Applied Physiology. 79 (1), 73-82 (1985).</li><li>Nevzati, E., 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=Biodegradable+Magnesium+Stent+Treatment+of+Saccular+Aneurysms+in+a+Rat+Model+-+Introduction+of+the+Surgical+Technique.">Biodegradable Magnesium Stent Treatment of Saccular Aneurysms in a Rat Model - Introduction of the Surgical Technique.</a> Journal of Visualized Experiments. (128), e56359 (2017).</li><li>Aquarius, R., Smits, D., Gounis, M. J., Leenders, W. P. J., de Vries, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+diverter+implantation+in+a+rat+model+of+sidewall+aneurysm:+a+feasibility+study.">Flow diverter implantation in a rat model of sidewall aneurysm: a feasibility study.</a> Journal of NeuroInterventional Surgery. 10 (1), 88-92 (2018).</li></ol>

著者らは開示するものは何もない。

このプロトコルは、ヒトサイズの冠状動脈ステントをapoE-/- ラットの腹部大動脈に移植する方法を説明する。いくつかの技術的な点は強調する価値があります。まず、ステントサイズと大オータのサイズの不一致を避けるべきです。ステントを小さすぎると、ステントのマラプ位置につながる可能性がありますが、大間に大きすぎるステントの埋め込みは、血管の過度の伸縮、引裂、...

経皮的冠動脈介入(PCI)は、冠状動脈ステントの展開と相まって、冠動脈疾患1の介入治療におけるゴールドスタンダードを表す。しかし、ステントの長期的な成功は、ステント内の新内組織の過剰増殖によって決定されるステント内レステノーシス(ISR)の発生によって制限され得る2,3。ISRは、冠状動脈バイパスまたは再PCIのいずれかで再介入を必要とするかもしれない。ISRの研究には様々な動物モデルが提案されており、それぞれが長所と短絡を特徴とする。最も一般的に使用されるブタ冠動脈およびウサギの腸動脈モデルの主な欠点は、ステント移植後のヒトと著しく類似した病変を発症する4、5であり、特に長期的な研究における物流上の困難、ならびに取り扱いや機器の制限を引き起こす大きな動物および住宅コストである。さらに、豚やウサギの細胞タンパク質に対する抗体の入手は限られています。一方、マウスモデルは、高スループットと再現性の大きな利点だけでなく、取り扱い、ハウジング、したがってコスト効率の容易さを提供します。さらに、より多くの抗体が利用可能である。しかし、アポリポタンパク質E欠損(apoE-/--)マウスはアテローム性動脈硬化症6、7、8の研究に広く使用されてきたが、ステントをマウスに移植するために小型化しなければならないため、ISRの研究には不向きであり、ステントの機械的特性を変化させる可能性がある。さらに、マウスの大動脈壁は、若いマウスでは50μm、古いマウスでは85μmの間で測定され、ステントは2気圧の低い圧力レベルを使用して展開する必要があり、ステント10のマラッピングにつながる可能性がある。しかしながら、ラットは、市販のヒト冠状ステントの移植を可能にし、大動脈ステント移植後に大きな動物と同様の血管治癒過程を示す、最初にLangeveldららによって報告された。この技術はもともと、血流の一時的な中断を達成するために大動脈の物理的な収縮を必要とする経腹的なアクセスを必要としました。潜在的に関連する血管損傷および炎症反応を避けるために、この技術は後にトランス腸骨アクセスの導入によって洗練され、さらに動物12の生存率が高くなる。
野生型ラットはアテローム硬化性病変13を発症しないため、アポエ-/-ラットは、転写活性化因子様エフェクターヌクレアーゼ(TALEN)14などのヌクレアーゼ技術を用いて生成されており、クラスター化された定期的に間隔をあけた短いパリンドローム反復(CRISPR/Cas9)15、およびジンクフィンガー(ZF)16。ApoE-/-ラットは2011年から市販されている。アテローム原性の背景を提供するapoE-/-ラットは、特にISRに関して、ヒトサイズの冠状動脈ステントのより現実的な評価を可能にする。
ここで、我々は、経大的なアクセス経路を介して、市販の薄いストラットコバルトクロム薬物溶出ステント(DES)を用いた方法を記述するが、ベアメタルステント(BMS)または生分解性ステントのような他のステントタイプの研究にも適用することができる。

<table>
	<tbody>
		<tr>
			<td>Diet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SNIFF High Fat diet + Clopidogrel (15 mg/kg)</td>
			<td>SNIFF Spezialdi&#228;ten GmbH, Soest</td>
			<td>custom prepared</td>
			<td>Western Diet</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drugs and Anesthetics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Essex Pharma</td>
			<td>997.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>ISOFLO (Isoflurane Vapor) vaporiser</td>
			<td>Eickemeyer</td>
			<td>4802885</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Forene Abbott</td>
			<td>B 506</td>
			<td></td>
		</tr>
		<tr>
			<td>Isotonic (0.9%) NaCl solution</td>
			<td>DeltaSelect GmbH</td>
			<td>PZN 00765145</td>
			<td></td>
		</tr>
		<tr>
			<td>Ringer&#39;s lactate solution</td>
			<td>Baxter Deutschland GmbH</td>
			<td>3775380</td>
			<td></td>
		</tr>
		<tr>
			<td>(S)-ketamine</td>
			<td>CEVA Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Medistar Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Consumable supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL syringes</td>
			<td>BD Plastipak</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL syringes</td>
			<td>BD Plastipak</td>
			<td>4606027V</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 prolene suture</td>
			<td>ETHICON</td>
			<td>N-2719K</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 silk suture</td>
			<td>Seraflex</td>
			<td>IC 158000</td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthen Eye and Nose Ointment</td>
			<td>Bayer Vital GmbH</td>
			<td>6029009.00.00</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton Gauze swabs</td>
			<td>Fuhrmann GmbH</td>
			<td>32014</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore silk tape</td>
			<td>3M</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Alcohol Skin Desinfection Solution</td>
			<td>Antiseptica GmbH</td>
			<td>72PAH200</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterican needle 18 G</td>
			<td>B. Braun</td>
			<td>304622</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterican needle 27 3/4 G</td>
			<td>B.Braun</td>
			<td>4657705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Paper</td>
			<td>commercially available</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps curved x1</td>
			<td>Fine Science Tools Inc.</td>
			<td>11151-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Graefe forceps straight</td>
			<td>Fine Science Tools Inc.</td>
			<td>11050-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder Mathieu</td>
			<td>Fine Science Tools Inc.</td>
			<td>12010-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Fine Science Tools Inc.</td>
			<td>14074-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Semken forceps</td>
			<td>Fine Science Tools Inc.</td>
			<td>11008-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors curved</td>
			<td>Fine Science Tools Inc.</td>
			<td>14029-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Small surgical scissors straight</td>
			<td>Fine Science Tools Inc.</td>
			<td>14028-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard pattern forceps</td>
			<td>Fine Science Tools Inc.</td>
			<td>11000-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas spring scissors</td>
			<td>Fine Science Tools Inc.</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica MZ9</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controlled heating pad</td>
			<td>Sygonix</td>
			<td>26857617</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment for stent implantation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Drug-eluting stent Xience 2,25mm x 8mm</td>
			<td>Abbott Vascular USA</td>
			<td>1009544-18</td>
			<td></td>
		</tr>
		<tr>
			<td>Guide wire Fielder XT PTCA guide wire: 0.014&#34; x 300cm</td>
			<td>ASAHI INTECC CO., LTD Japan</td>
			<td>AGP140302</td>
			<td></td>
		</tr>
		<tr>
			<td>Inflation syringe system</td>
			<td>Abbott 20/30 Priority Pack</td>
			<td>1000186</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue processing and analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% H2O2</td>
			<td>Roth</td>
			<td>9681</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Roth</td>
			<td>K928.1</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Giemsas Azur-Eosin-Methylenblau</td>
			<td>Merck</td>
			<td>109204</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Graphic Drawing Tablet</td>
			<td>WACOM Europe GmbH</td>
			<td>CTL-6100WLK-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Roti Histofix, Formaldehyd 4% buffered</td>
			<td>Roth</td>
			<td>P087</td>
			<td>Histology</td>
		</tr>
		<tr>
			<td>Technovit 9100</td>
			<td>Morphisto</td>
			<td>12225.K1000</td>
			<td>Histology</td>
		</tr>
	</tbody>
</table>

implantation of human-sized coronary stents into rat abdominal aorta using a trans-femoral access

経皮的冠動脈介入(PCI)は、冠状動脈ステントの展開と相まって、冠動脈疾患の介入治療におけるゴールドスタンダードを表す。インステント・レステノーシス(ISR)は、ステント内の新内皮組織の過剰増殖によって決定され、ステントの長期的な成功を制限する。様々な動物モデルが、ブタ冠動脈およびウサギの腸骨動脈モデルが最も頻繁に使用されている、ステント内修復(ISR)の基礎となる病態生理学的プロセスを解明するために使用されてきた。Ming モデルは、高スループット、取り扱いとハウジングの容易さ、再現性、および分子マーカーの幅広い可用性の利点を提供します。アポリポプロテインE欠損(apoE-/-) マウスモデルは、心血管疾患の研究に広く使用されています。しかし、ステントは、機械的および(潜在的に)生物学的特性の重要な変化を伴うマウスに移植されるように小型化する必要があります。apoE-/- ラットの使用は、アポエ-/- ラットとしてこれらの欠点を克服できると同時に、アテロゲン性表現型を提供しながら、ヒトサイズの冠状動脈ステントの評価を可能にする。これにより、ステント注入後のISRを調査するための優れた信頼性の高いモデルになります。ここでは、例えば、市販のヒト冠状動脈ステントをapoE-/- バックグラウンドを有するラットの腹部大動脈へのトランスポンテーションを、トランス・フェモラル・アクセスを用いて詳細に説明する。

このプロトコルは、トランス大腿部アクセス経路を用いたラットの腹部大動脈におけるステント移植について説明する(図1)。この動物モデルの最初の中心点は、人間サイズの冠状動脈ステントの展開を可能にすることです。市販の圧着およびバルーン付着冠状動脈ステントは、ラットの腹部大動脈に配置することができる。したがって、加えて、ステント展開の原理は?...

Watch this Scientific Journal Video about Implantation of Human-Sized Coronary Stents into Rat Abdominal Aorta Using a Trans-Femoral Access at JoVE.com

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

このプロトコルは、トランス・フィスモラル・アクセスを用いて、apoE-/- バックグラウンドを有するラットの腹部大動脈へのヒト冠動脈ステントの移植を記述する。他の動物モデルと比較して、マウスモデルは高い効率、再現性、取扱いおよびハウジングの容易さ、および分子マーカーの広い利用可能性の利点を運ぶ。

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 ...

implantation-human-sized-coronary-stents-into-rat-abdominal-aorta

Research

JoVE Journal

Medicine

4.1K ビュー.  University Hospital RWTH Aachen. このプロトコルは、トランス・フィスモラル・アクセスを用いて、apoE-/- バックグラウンドを有するラットの腹部大動脈へのヒト冠動脈ステントの移植を記述する。他の動物モデルと比較して、マウスモデルは高い効率、再現性、取扱いおよびハウジングの容易さ、および分子マーカーの広い利用可能性の利点を運ぶ。

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

The C-seal: A Biofragmentable Drain Protecting the Stapled Colorectal Anastomosis from Leakage

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL varies between 2.5 and 20% 2-5. Over the years, many strategies aimed at lowering the incidence of anastomotic leakage have been examined6, 7. 
The cause of AL is probably multifactorial. Etiological factors include insufficient arterial blood supply, tension on the anastomosis, hematoma and/or infection at the anastomotic site, and co-morbid factors of the patient as diabetes and atherosclerosis8. Furthermore, some anastomoses may be insufficient from the start due to technical failure.
Currently a new device is developed in our institute aimed at protecting the colorectal anastomosis and lowering the incidence of AL. This so called C-seal is a biofragmentable drain, which is stapled to the anastomosis with the circular stapler. It covers the luminal side of the colorectal anastomosis thereby preventing leakage.
The C-seal is a thin-walled tube-like drain, with an approximate diameter of 4 cm and an approximate length of 25 cm (figure 1). It is a tubular device composed of biodegradable polyurethane. Two flaps with adhesive tape are found at one end of the tube. These flaps are used to attach the C-seal to the anvil of the circular stapler, so that after the anastomosis is made the C-seal can be pulled through the anus. The C-seal remains in situ for at least 10 days. Thereafter it will lose strength and will degrade to be secreted from the body together with the gastrointestinal natural contents.
The C-seal does not prevent the formation of dehiscences. However, it prevents extravasation of faeces into the peritoneal cavity. This means that a gap at the anastomotic site does not lead to leakage.
Currently, a phase II study testing the C-seal in 35 patients undergoing (colo-)rectal resection with stapled anastomosis is recruiting. The C-seal can be used in both open procedures as well as laparoscopic procedures. The C-seal is only applied in stapled anastomoses within 15cm from the anal verge. In the video, application of the C-seal is shown in an open extended sigmoid resection in a patient suffering from diverticular disease with a stenotic colon.

The C-seal is a biofragmentable drain protecting the stapled colorectal anastomosis. In this video, details of the manufacturing, deployment and use are shown.

C -シール：漏れから結腸直腸器械吻合を保護Biofragmentableドレイン

Colorectal anastomotic leakage (AL) is a serious complication in colorectal surgery leading to high morbidity and mortality rates1. The incidence of AL ...

医学

Rat Heterotopic Abdominal Heart/Single-lung Transplantation in a Volume-loaded Configuration

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft involves anastomosis of donor inferior vena cava (IVC) to recipient IVC, and donor ascending aorta (Ao) to recipient abdominal Ao. The right upper and middle lung lobes are preserved and function as conduits for blood flow from right heart to left heart.
There are several advantages to using this technique, and it lends itself to a broad range of applications. Because the graft is transplanted in a configuration that allows for dyamic volume-loading, cardiac function may be directly assessed in vivo. The use of pressure-volume conductance catheters permits characterization of load-dependent and load-independent hemodynamic parameters. The graft may be converted to a loaded configuration by applying a clamp to the recipient&#8217;s infra-hepatic IVC. We describe modified surgical techniques for both donor and recipient operations, and an ideal myocardial protection strategy. Depending on the experimental aim, this model may be adapted for use in both acute and chronic studies of graft function, immunologic status, and variable ventricular loading conditions. The conducting airways to the transplanted lung are preserved, and allow for acute lung re-ventilation. This facilitates analysis of the effects of the mixed venous and arterial blood providing coronary perfusion to the graft.
A limitation of this model is its technical complexity. There is a significant learning curve for new operators, who should ideally be mentored in the technique. A surgical training background is advantageous for those wishing to apply this model. Despite its complexity, we aim to present the model in a clear and easily applicable format. Because of the physiologic similarity of this model to orthotopic transplantation, and its broad range of study applications, the effort invested in learning the technique is likely to be worthwhile.

We describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The transplant configuration results in a partially loaded graft circulation, allowing direct functional assessment. This model may be employed for acute or chronic studies of function and immunologic status of the transplanted graft.

ラット異腹部ボリュームにロードされた構成でのハート/シングル肺移植

Herein, we describe a novel technique for heterotopic abdominal heart-lung transplantation (HAHLT) in rats. The configuration of the transplant graft ...

Ultrasound Based Assessment of Coronary Artery Flow and Coronary Flow Reserve Using the Pressure Overload Model in Mice

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow reserve (CFR) in humans. Reduced CFR is accompanied by marked intramyocardial and pericoronary fibrosis and is used as an indication of the severity of dysfunction. This study explores, step-by-step, the real-time changes measured in the coronary flow velocity, CFR and systolic to diastolic peak velocity (S/D) ratio in the setting of an aortic banding model in mice. By using a Doppler transthoracic imaging technique that yields reproducible and reliable data, the method assesses changes in flow in the septal coronary artery (SCA), for a period of over two weeks in mice, that previously either underwent aortic banding or thoracotomy. 
During imaging, hyperemia in all mice was induced by isoflurane, an anesthetic that increased coronary flow velocity when compared with resting flow. All images were acquired by a single imager. Two ratios, (1) CFR, the ratio between hyperemic and baseline flow velocities, and (2) systolic (S) to diastolic (D) flow were determined, using a proprietary software and by two independent observers. Importantly, the observed changes in coronary flow preceded LV dysfunction as evidenced by normal LV mass and fractional shortening (FS). 
The method was benchmarked against the current gold standard of coronary assessment, histopathology. The latter technique showed clear pathologic changes in the coronary artery in the form of peri-coronary fibrosis that correlated to the flow changes as assessed by echocardiography. 
The study underscores the value of using a non-invasive technique to monitor coronary circulation in mouse hearts. The method minimizes redundant use of research animals and demonstrates that advanced ultrasound-based indices, such as CFR and S/D ratios, can serve as viable diagnostic tools in a variety of investigational protocols including drug studies and the study of genetically modified strains.

Coronary flow reserve (CFR) is useful for assessment of myocardial oxygen demand and evaluation of cardiovascular risk. This study establishes a step-by-step transthoracic Doppler echocardiographic (TTDE) method for longitudinal monitoring of the changes in CFR, as measured from coronary artery in mice, under the experimental pressure overload of aortic banding.

マウスの圧負荷モデルを用いた冠動脈の流れと冠血流準備の超音波ベースのアセスメント

Transthoracic Doppler echocardiography (TTDE) is a clinically useful, noninvasive tool for studying coronary artery flow velocity and coronary flow ...

Creation of Abdominal Adhesions in Mice

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or intraabdominal infection. The precise mechanisms underlying adhesion formation are poorly understood. Many compounds and physical barriers have been tested for their ability to prevent adhesions after surgery with varying levels of success. The mouse and rat are important models for the study of abdominal adhesions. Several different techniques for the creation of adhesions in the mouse and rat exist in the literature. Here we describe a protocol utilizing abrasion of the cecum with sandpaper and sutures placed in the right abdominal sidewall. The mouse is anesthetized and the abdomen is prepped. A midline laparotomy is created and the cecum is identified. Sandpaper is used to gently abrade the surface of the cecum. Next, several figure-of-eight sutures are placed into the peritoneum of the right abdominal sidewall. The abdominal cavity is irrigated, a small amount of starch is applied, and the incision is closed. We have found that this technique produces the most consistent adhesions with the lowest mortality rate.

Abdominal adhesions that form after surgery are a major cause of pain, infertility, and hospitalization and reoperation for small bowel obstruction. Our surgical procedure for creating abdominal adhesions in mice is a reliable tool to study the mechanisms underlying the formation of adhesions.

マウスの腹部癒着の作成

Abdominal adhesions consist of fibrotic tissue that forms in the peritoneal space in response to an inflammatory insult, typically surgery or ...

A Model of Cardiac Remodeling Through Constriction of the Abdominal Aorta in Rats

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and fluid retention. Changes in myocardial structure, electrical conduction, and energy metabolism develop with heart failure, leading to contractile dysfunction, increased risk of arrhythmias, and sudden death. Hypertensive heart disease is one of the key contributing factors of cardiac remodeling associated with heart failure. The most commonly-used animal model mimicking hypertensive heart disease is created via surgical interventions, such as by narrowing the aorta. Abdominal aortic constriction is a useful experimental technique to induce a pressure overload, which leads to heart failure. The surgery can be easily performed, without the need for chest opening or mechanical ventilation. Abdominal aortic constriction-induced cardiac pathology progresses gradually, making this model relevant to clinical hypertensive heart failure. Cardiac injury and remodeling can be observed 10 weeks after the surgery. The method described here provides a simple and effective approach to produce a hypertensive heart disease animal model that is suitable for studying disease mechanisms and for testing novel therapeutics.

A rat model of abdominal aortic constriction that induces cardiac hypertrophy and remodeling is described. An efficient, highly-reproducible, and minimally-invasive method is used to provide a simple yet useful platform for research in myocardial hypertrophy and dysfunction.

ラットの腹部大動脈の収縮を通じて心臓リモデリングのモデル

Heart failure is one of the leading causes of death worldwide. It is a complex clinical syndromethat includes fatigue, dyspnea, exercise intolerance, and ...

Murine Echocardiography of Left Atrium, Aorta, and Pulmonary Artery

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic pressure through echocardiography, as well as to obtain measurements of the Aorta and PA diameter in mice.
Mice older than 10 d of age can be analyzed using the present technique. The technique is composed of 3 main steps: set-up, image acquisition, and image analysis. The set-up step consists of getting the mouse anesthetized with 1% isoflurane, shaving it, and taping it in a supine position to a heated EKG board where the image acquisition will take place. The image acquisition step consists of learning to identify the cardiac structures and obtaining all the required images with its correspondent probe and axis in order to be able to calculate volumes and diameters. The image analysis step consists of measuring the previously acquired images with the aid of computer software.
Advantages of the proposed technique include a fast (15 min) procedure that would allow the researcher to evaluate interventions in a non-invasive, non-terminal approach and therefore follow the same mouse over time; each mouse can be used as its own control. This fact plus having the same operator perform all the acquisition and analysis for the entire experiment minimizes the limitation of operator-dependency. The present methodology is useful for mouse researchers in cardiovascular and pulmonary medicine.

The following protocol describes the methodology for the acquisition and analysis of echocardiographic images used to obtain the Left Atrial Volume (LAV), Aorta (Ao) diameter, and Pulmonary Artery (PA) diameter in mice. This technique is a non-invasive, non-terminal procedure that allows assessment of the cardiopulmonary function.

左心房、大動脈、肺動脈のマウス心エコー

The present methodology teaches the investigator how to measure and use the LAV as a surrogate of chronic elevations in Left Ventricular diastolic ...

Patch Angioplasty in the Rat Aorta or Inferior Vena Cava

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, we developed a rat model of patch angioplasty that can be used in either a vein or an artery, creating a patch venoplasty or a patch arterioplasty, respectively. Technical aspects of this model are discussed. The infra-renal IVC or aorta are dissected and then clamped proximally and distally. A 3 mm venotomy or arteriotomy is performed in the infrarenal inferior vena cava or aorta of 6 to 8 week-old Wistar rats. A bovine pericardial patch (3 mm x 1.5 mm x 0.6 mm) is then used to close the site using a 10-0 nylon suture. Compared to arterial patches, venous patches show increased neointimal thickness on postoperative day 7. This novel model of pericardial patch angioplasty can be used to examine neointimal hyperplasia on vascular biomaterials, as well as to compare the differences between the arterial and venous environments.

We have established a model of pericardial patch angioplasty that can be used in either small-diameter veins or arteries. This model can be used to compare venous and arterial neointimal hyperplasia formation.

ラット大動脈または下大静脈内のパッチ血管形成術

Pericardial patches are commonly used in vascular surgery to close vessels. To facilitate studies of the neointimal hyperplasia that forms on the patch, ...

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery (ALCAPA)

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ischemia and infarction in children. If left untreated, it results in a 90% mortality rate in the first year of life. In patients who survive to the adulthood, the coronary steal phenomenon and retrograde left-sided coronary flow provide a substrate for chronic subendocardial ischemia, which may lead to left ventricular dysfunction, ischemic mitral regurgitation, malignant ventricular arrhythmias, and sudden cardiac death. The average age of life-threatening presentation is 33 years and of sudden cardiac death 31 years. Therefore, surgical correction is highly recommended as soon as the diagnosis is made, regardless of age. In adult-type ALCAPA originating from the right-facing sinus of the pulmonary artery, direct re-implantation of the ALCAPA into the aorta is the more physiologically sound repair technique to re-establish the dual-coronary perfusion system and is recommended. This protocol describes the technique of direct re-implantation of adult-type ALCAPA into the aorta.

Direct Re-implantation of Left Coronary Artery into the Aorta in Adults with Anomalous Origin of Left Coronary Artery from the Pulmonary Artery ALCAPA

Surgical correction of ALCAPA is highly recommended, regardless of age or the degree of intercoronary collateralization. This protocol presents a technique for the direct re-implantation of adult-type ALCAPA into the aorta to re-establish the dual-coronary perfusion. Whenever feasible, direct re-implantation is preferred to other surgical correction techniques.

肺動脈から左冠状動脈の異常原点と成人における大動脈内に左冠状動脈の直接再注入（ALCAPA）

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) is a rare congenital anomaly which is one of leading causes of myocardial ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

マウスの腹部大動脈 Myointimal 過形成を誘導するために気球を利用した傷害

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

Identifying Coronary Artery Calcification on Non-gated Computed Tomography Scans

Coronary artery calcification (CAC) provides an objective measure of coronary artery disease and can readily be identified on non-gated computed tomography (CT) scans with a high correlation with gated cardiac CT scans. This standardized protocol takes a step-wise approach to not only optimizing an image for the identification of calcification but also to distinguishing CAC from other common causes of calcification in the cardiac silhouette. Recognition of CAC on non-gated CT scans helps to identify a very powerful prognostic factor that can influence therapeutic interventions or downstream diagnostic testing without requiring a gated cardiac scan. These non-gated CT scans are often acquired as part of the routine care of the patient, and this data is readily available without another dose of&#160;ionizing radiation. This protocol allows for the precise and accurate extraction of this data for the purposes of retrospective data analysis in clinical research studies, but also in the clinical evaluation and management of patients.

Here, we present a protocol to reliably and systematically identify coronary artery calcification (CAC) on non-gated computed tomography (CT) scans of the chest or abdomen. CAC provides an objective measure of coronary artery disease for both research and clinical purposes.