この作品は、CKBのNIH（RO1 HL098228）からの助成金によって部分的にサポートされていました。

注：すべての動物の手続きは、全国の小児病院施設内動物管理使用委員会によって承認された。 1。グラフトづくり<ol><li>ヒュームフードの下に2ミリリットルのジオキサンに100mgのPは（LA / CL）を追加することにより、ε-カプロラクトンとL-ラクチド共重合体P（LA / CL）の溶液を作製。渦上に溶液を置き、完全に溶解する1〜1.5時間、連続して混ぜる。 </li><li>一方、ポリグリコール酸（PGA）のシートを削除冷凍庫から感じた、いくつかの5×8ミリメートルの部分を切り取る。また、単にフィルター上0.1〜10μlのピペットの先端を切り落とした。 </li><li>ピペットチップの先端部に19gの針（1.5長さ）を挿入し、PGAはマイクロピンセットを用いて、針の周りに感じ包む。 </li><li> 19 Gの針をその中に挿入されている間、慎重に、内腔が鈍い18 Gの針を使用して、近位部よりも直線的であるピペットチップの先端にフェルトを押してください。 </li><li>ピペット上からピペットチップに40μlのP（CL / LA）ソリューションを提供します。 PGAは、溶液と感じ飽和する。次いで、ピペットディスペンサーを用いて気泡を押し出す。 必要な場合には、このプロセスを繰り返す。 </li><li>場所の50ミリリットルチューブ内移植片および20分間-80℃の冷凍庫に入れてください。針の頭は下向きにしてください。 </li><li> 24時間凍結乾燥し、真空中にチューブを移す。空気の流れを持っているチューブの蓋を開けていることを確認してください。 </li><li>移植片を取り出し、針から削除します。 〜5ミリメ​​ートルのセクションを残して、移植片の両端をカットし、その形状を維持するために、針の上に戻ってそれらを置く。デシケーター中での移植片を保管してください。 </li><li>バイオセーフティーフードのO / Nの前に細胞播種のUV光の下で足場を置きます。 </li></ol> 2。骨髄単核細胞の回収および単離<ol><li>ケタミン/キシラジンの過剰摂取（ケタミン、200 mg / kgのキシラジンと、20 mg / kg）を持つマウスを安楽死させる。 </li><li>骨（大腿骨をAN削除する10 CCのRPMIでペトリ皿中10移植注入や場所のための3匹のマウスからDの脛骨）。 3のCC RPMIで新しいペトリ皿に25 G針で注射器を用いて骨とフラッシュ骨髄の両端をカットします。 15 CC管内に骨髄およびRPMI液を収集し、BMの残りの部分を収集するために、追加の2のCC RPMIでペトリ皿を洗う。 </li><li>サンプル（5〜10μl）を取り、 自動細胞カウンターまたは血球計数器を用いて細胞を数える。その結果を記録します。 </li><li> 15 CC遠心管に5のCCフィコールを入れて、骨髄およびRPMIソリューションを追加します。フィコールと混合するのを防ぐために非常に穏やかに溶液を入れる。 </li><li> 24℃で「ブレーキなし」と30分間528×gで遠心分離</li><li>上側のピンクの層を除去する。 MNC層であるミドルクリア層の図1を収集し、PBS 1時01分でそれを希釈する。 </li><li>遠心分離機24℃で10分間528×gで希薄MNCソリューション</li><li>上清およびジを削除5ミリリットルのPBSでペレットをリュート。 </li><li> 24℃で10分間、528×gでペレット溶液を遠心分離</li><li>上清を取り除きます。 RPMI中（〜200μL）の適切な量で、ペレットを希釈します。 </li><li> 5〜10μlのサンプルを取り、 自動細胞カウンターまたは血球計算板を用いて細胞を数える。その結果を記録します。 1より多くの時間をカウントし、細胞を繰り返して、平均セル数を計算する。 </li><li>のRPMIを使用してμlの1,000,000 cells/10に細胞濃度を希釈する。 </li></ol> 3。細胞播種<ol><li>のRPMIを削除して、5分間の管腔内5μLのRPMIを追加することで、予備湿潤足場。 </li><li>足場内腔へのステップ2.12からRPMI中で10μlの骨髄由来の単核細胞を加え、10分、細胞が足場に取り付けることができるようにするのを待ちます。 </li><li> 1cmの長さに19 G針をカットし、足場の形状を維持するために、足場の内腔に針を入れた。 24ウェルプレートに載せてください。&lt;/李&gt; <li>各ウェルに千μLのRPMIを加え、インキュベーター中で、O / Nインキュベートする。 </li></ol> 4。グラフト移植<ol><li>手術前に、すべての手術器具をオートクレーブ：細かいハサミ、3Xマイクロ鉗子、2倍のマイクロ血管クランプ、鉗子を適用する1Xクランプ、1Xマイクロニードルホルダー、1X春のはさみ、1Xリトラクターを1X。 </li><li> 6〜8週齢の雌C57BL / 6は、組織工学的血管移植片のレシピエントとして使用されている。そのケージからマウスを削除し、ケタミン/キシラジンカクテルで腹部の右下の象限（ケタミン、100 mg / kgのキシラジンと、10 mg / kg）を腹腔内注射により麻酔して、それを比較検討する。ケトプロフェン（5 mg / kgを、IPは）事前に麻酔鎮痛剤として使用されます。 </li><li>物語つまんで鎮静のレベルを確認し、腹部の毛をクリップ。無菌眼軟膏で目を潤滑し、パッド上の背側横臥位にマウスを置きます。ベタジンとアルコールパッドで腹部を消毒。 CO無菌ドレープでマウスをVERだけ切開領域を公開します。 </li><li>恥骨領域への剣状突起下から正中開腹切開を行い、自己保持リトラクターを挿入します。生理食塩水湿らせたガーゼで腸を包む。ぶっきらぼう大動脈大動脈と大静脈を定義します。 </li><li>近位大動脈と大静脈の遠位両側の2のマイクロ血管のクランプを配置し、ぶっきらぼうに大静脈トランセクト大静脈から大動脈を分離する。必要に応じて、テーパー針に10-0モノフィラメント縫合糸で腹部大動脈の枝を結紮。 </li><li>無菌10-0縫合糸を使用して吻合を終了するために、近位端および遠位端と下大静脈の介在移植片を移植する。マウスの解剖学に依存し、移植片、出荷までmmで、トリム。近位端および遠位端の両方で1ステッチで移植性を確保し、移植片の反対側から4-5 stichesで連続縫合を開始します。前面を終えた後、クランプおよび移植片を反転反対側に、グラフトの裏側を縫合。注入中に、急性血栓症を予防するために頻繁にヘパリン溶液で移植片をフラッシュします。 </li><li>近位クランプを外し、局所吸収性無菌止血剤を塗布して出血を制御します。出血が完全に停止すると、遠位クランプを取り外して、出血を同じように制御します。血液が移植片を流れて​​いることを確認してください。 </li><li>含まねじ切り針で6-0黒色ポリアミドモノフィラメント縫合糸を使用した2層に腹部の筋肉と皮膚を閉じます。 </li><li> 0.5ミリリットルの生理食塩水を皮下注射すると、マウスが完全にモバイルになるまで、温暖化パッド上で回復ケージにマウスを置きます。復旧時には、紙の寝具で新しいケージにマウスを返す。 48時間、鎮痛薬（イブプロフェン、30 mg / kgで、飲料水）を得た。 </li></ol>

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著者らは、開示することは何もありません。

TEVGのマウスモデルは、新組織形成と狭窄の開発の細胞および分子メカニズムを研究するための貴重なツールです。シードされたBM-MNCが移植11に播種した細胞の組織学的およびSEM画像の両方で示された。細胞播種の効率はまた、DNAアッセイ7を用いて示された。このモデル系を用いて、我々は、細胞播種が私たち人間の臨床試験3の故障の主なモードだっTEVG狭窄の開発の...

先天性心疾患は、米国の出生の約8％に影響を与える深刻な状況である。先天性心疾患または2.4千あたりの出生とのそれらの乳児の約25％が、自分の人生1の最初の年に侵襲的な治療を必要とする。先天性心疾患のための最も有効な治療は、再建手術である。残念ながら、現在利用可能な血管導管の使用に起因する合併症は術後の罹患率と死亡率の最も重要な原因である。 この問題に対処するために、我々は、臨床使用のために第2組織工学血管移植片（TEVGs）を開発した。 TEVGsは、自家骨髄由来の、単核細胞（BM-MNCを）を播種し、先天性心臓手術のための静脈の導管として移植生分解性ポリエステルチューブから構築した。結果は、フォローアップの1〜3年で優れた開存率を示したが、狭窄の重要な発生率<s3,4&gt;まで。これは、血管新組織形成とTEVG狭窄の開発の基礎となるメカニズムのよりよい理解が必要であることは明らかであった。より良いTEVGsの開発や狭窄開発のメカニズムを理解するためには、ヒツジモデルは5,6を作成しました。このモデルでは、TEVGs正常生体血管に変換され、天然の静脈の形態と機能の両方において同様であった。大型動物モデルの使用はTEVGsの臨床使用量を助け、重要な前臨床情報を提供する上で良い最初のステップでした。しかし、大規模な動物モデルを用いTEVGsの血管新組織形成の細胞および分子メカニズムの完全な理解は原因種特異的分子ツールが不足しているため血管細胞の表現型の分子特性の限界に制限されています。これらの欠点を克服するために、TEVGsのマウスモデルは、マウス遺伝学の急速な進歩とその広範なmoleculaの理由によって開発されました短縮された時間スケールの付加的な利点を持つRの特性評価。 マウスのIVC介在モデルを忠実に大型動物やヒトで発生する新生血管形成の過程をで要約するが、はるかに短い時間経過6-9以上。ここで、生分解性の足場を用いた小規模な移植片の製造のための詳細なプロトコルは、BM-MNCの回収と単離、足場上のBM-MNCの播種、およびマウスモデルにおける移植片植え込みについて説明した。

<table><tbody><tr><td>Polyglycolic acid (PGA) felt</td><td>Biomedical Structures</td><td></td><td>Custome ordered</td></tr><tr><td>Pipet tip, 0.1-10 &amp;mu;l&amp;nbsp;</td><td>Fisher Sientific</td><td>02-707-456</td><td></td></tr><tr><td>Lyophilizer&amp;nbsp;</td><td>Labconco</td><td>7070020</td><td></td></tr><tr><td>RPMI medium 1604</td><td>Gibco</td><td>11875-093</td><td></td></tr><tr><td>Petri dish</td><td>BD</td><td>353003</td><td></td></tr><tr><td>24-well plate</td><td>Corning</td><td>3526</td><td></td></tr><tr><td>15 cc tube&amp;nbsp;</td><td>BD</td><td>352096</td><td></td></tr><tr><td>Ficoll</td><td>Sigma</td><td>10831-100ml</td><td>Also called &#039;Histopaque&#039;</td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Littauer bone cutter 4.5&quot; Straight</td><td>Roboz</td><td>RS-8480</td><td>For BM harvesting</td></tr><tr><td>Forceps 4.5&quot;</td><td>Roboz</td><td>RS-8120</td><td>For BM harvesting</td></tr><tr><td>Scissors 4.5&quot;</td><td>Roboz</td><td>RS-5912</td><td>For BM harvesting</td></tr><tr><td>Microscope</td><td>Leica</td><td>M80</td><td></td></tr><tr><td>C57BL/6J (H-2b), Female</td><td>Jackson Laboratories</td><td>664</td><td>8-12 weeks</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Hospira Inc.</td><td>NDC 0409-2053</td><td></td></tr><tr><td>Xylazine sterile solution</td><td>Akorn Inc.</td><td>NADA# 139-236</td><td></td></tr><tr><td>Ketoprofen</td><td>Fort Dodge Animal Health</td><td>NDC 0856-4396-01</td><td></td></tr><tr><td>Ibuprofen</td><td>PrecisionDose</td><td>NDC 68094-494-59</td><td></td></tr><tr><td>Heparin sodium</td><td>Sagent Pharmaceticals</td><td>NDC 25021-400</td><td></td></tr><tr><td>Saline solution (sterile 0.9% sodium chloride)</td><td>Hospira Inc.</td><td>NDC 0409-0138-22</td><td></td></tr><tr><td>0.9% Sodium chloride injection</td><td>Hospira Inc.</td><td>NDC 0409-4888-10</td><td></td></tr><tr><td>Petrolatum ophthalmic ointment</td><td>Dechra Veterinary Products</td><td>NDC 17033-211-38</td><td></td></tr><tr><td>Iodine prep pads</td><td>Triad Disposables, Inc.</td><td>NDC 50730-3201-1</td><td></td></tr><tr><td>Alcohol prep pads</td><td>McKesson Corp.</td><td>NDC 68599-5805-1</td><td></td></tr><tr><td>Cotton tipped applicators</td><td>Fisher Scientific</td><td>23-400-118</td><td></td></tr><tr><td>Fine scissor</td><td>FST</td><td>14028-10</td><td></td></tr><tr><td>Micro-adson forcep</td><td>FST</td><td>11018-12</td><td></td></tr><tr><td>Clamp applying forcep</td><td>FST</td><td>00072-14</td><td></td></tr><tr><td>S&amp;amp;T Vascular clamp</td><td>FST</td><td>00396-01</td><td></td></tr><tr><td>Spring scissors</td><td>FST</td><td>15008-08</td><td></td></tr><tr><td>Colibri retractors</td><td>FST</td><td>17000-04</td><td></td></tr><tr><td>Dumont #5 forcep</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>Dumont #7 - fine forceps</td><td>FST</td><td>11274-20</td><td></td></tr><tr><td>Dumont #5/45 forceps</td><td>FST</td><td>11251-35</td><td></td></tr><tr><td>Tish needle holder/forceps</td><td>Micrins</td><td>MI1540</td><td></td></tr><tr><td>Black polyamide monofilament suture, 10-0</td><td>AROSurgical Instruments Corporation</td><td>TI638402</td><td>For suturing the graft</td></tr><tr><td>Black polyamide monofilament suture, 6-0</td><td>AROSurgical Instruments</td><td>SN-1956</td><td>For musculature and skin closure</td></tr><tr><td>Non-woven sponges</td><td>McKesson Corp.</td><td>94442000</td><td></td></tr><tr><td>Absorbable hemostat</td><td>Ethicon</td><td>1961</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309604</td><td></td></tr><tr><td>18 G 1.5 in, Needle</td><td>BD</td><td>305190</td><td></td></tr><tr><td>25 G 1 in, Needle</td><td>BD</td><td>305125</td><td></td></tr><tr><td>30 G 1 in, Needle</td><td>BD</td><td>305106</td><td></td></tr><tr><td>Warm water recirculator</td><td>Gaymar</td><td>TP-700</td><td></td></tr><tr><td>Warming pad</td><td>Gaymar</td><td>TP-22G</td><td></td></tr><tr><td>Trimmer</td><td>Wahl</td><td>9854-500</td><td></td></tr></tbody></table>

implantation of inferior vena cava interposition graft in mouse model

骨髄単核細胞（のBMC）を播種した生分解性の足場は、しばしば、先天性心臓異常を治療するための再建手術のために使用される。長期臨床結果は、狭窄の有意な発生率が、優れた開存率を示した。血管新組織形成の細胞および分子機構を調査し、組織工学血管移植片（TEVGs）に狭窄発症を予防するために、我々は、約1mmの内径を有する移植片のマウスモデルを開発した。まず、ポリグリコール酸不織布から製造生分解性の管状の足場から組み立てたTEVGs、ε-カプロラクトンとL-ラクチド共重合体でコーティングされたメッシュと感じました。足場は、次いで、凍結乾燥機に入れ、24時間真空にし、細胞播種するまでデシケーター中に保存した。次に、骨髄を、ドナーマウスから採取し、単核細胞を、密度勾配遠心分離によって単離した。第三に、約100万個の細胞であった足場に播種し、O / Nインキュベート最後に、播種された足場は、その後、C57BL / 6マウスにおける大動脈大静脈介在移植片として移植した。注入された移植片は、血栓塞栓合併症や動脈瘤の形成の証拠がなく、優れた開存性（&gt; 90％）を示した。このマウスモデルは、理解で私たちを助け、TEVGに新組織形成の細胞および分子メカニズムを定量化します。

TEVG注入の概略図を図1に示されている。骨髄は、ドナーマウスから採取し、単核細胞を密度遠心分離を用いて単離し、その後、生分解性足場上に播種する。シードされた足場は、O / Nインキュベートし、下大静脈の介在移植片としてレシピエントマウスに移植した。 図2は、PGA-P（CL / LA）骨格の走査型電子顕微鏡を示す。内径は約1mmであり、壁の...

Watch this Scientific Journal Video about Implantation of Inferior Vena Cava Interposition Graft in Mouse Model at JoVE.com

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

細胞および分子新組織形成の我々の知識を向上させるために、TEVGのマウスモデルは、最近開発されました。グラフトは、C57BL / 6マウスにおける大動脈大静脈介在移植片として移植した。このモデルは、私たちの臨床研究で達成されるものと同様の結果を達成するが、はるかに短縮され、時間経過とともに。

マウスモデルにおける下大静脈介在移植の移植

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

implantation-of-inferior-vena-cava-interposition-graft-in-mouse-model

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

11.8K ビュー.  Nationwide Children's Hospital. 細胞および分子新組織形成の我々の知識を向上させるために、TEVGのマウスモデルは、最近開発されました。グラフトは、C57BL / 6マウスにおける大動脈大静脈介在移植片として移植した。このモデルは、私たちの臨床研究で達成されるものと同様の結果を達成するが、はるかに短縮され、時間経過とともに。 

ビデオ: Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

マウスにおける異所心臓移植

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

生物学

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

変更されたカフ法を用いたマウス子宮頸心臓移植モデル

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

医学

Transplantation of Pulmonary Valve Using a Mouse Model of Heterotopic Heart Transplantation

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed results. However, the cellular and molecular mechanisms of the neotissue development, valve thickening, and stenosis development are not researched extensively. To answer the above questions, we developed a murine heterotopic heart valve transplantation model. A heart valve was harvested from a valve donor mouse and transplanted to a heart donor mouse. The heart with a new valve was transplanted heterotopically to a recipient mouse. The transplanted heart showed its own heartbeat, independent of the recipient&#8217;s heartbeat. The blood flow was quantified using a high frequency ultrasound system with a pulsed wave Doppler. The flow through the implanted pulmonary valve showed forward flow with minimal regurgitation and the peak flow was close to 100 mm/sec. This murine model of heart valve transplantation is highly versatile, so it can be modified and adapted to provide different hemodynamic environments and/or can be used with various transgenic mice to study neotissue development in a tissue engineered heart valve.

In order to understand the cellular and molecular mechanisms underlying neotissue formation and stenosis development in tissue engineered heart valves, a murine model of heterotopic heart valve transplantation was developed. A pulmonary heart valve was transplanted to recipient using the heterotopic heart transplantation technique.

異心臓移植のマウスモデルを用いた肺動脈弁の移植

Tissue engineered heart valves, especially decellularized valves, are starting to gain momentum in clinical use of reconstructive surgery with mixed ...

Heterotopic Cervical Heart Transplantation in Mice

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies this model by avoiding challenging suture anastomoses of small vessels thereby reducing warm ischemia time. In comparison to abdominal graft implantation the cervical model is less invasive and the implanted graft is easily accessible for further follow-up examinations. Anastomoses are performed by pulling the ascending aorta of the graft over the cuff with the recipient&#8217;s common carotid artery and by pulling the main pulmonary artery over the cuff with the external jugular vein. Selection of appropriate cuff size and complete mobilization of the vessels are important for successful revascularization. Ischemia-reperfusion (I/R) injury can be minimized by perfusing the graft with a cardioplegic solution and by hypothermia. In this article, we provide technical details for a simplified and improved cuff technique, which should allow surgeons with basic microsurgical skills to perform the procedure with a high success rate.

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

マウスにおける異子宮頸心臓移植

The heterotopic cervical heart transplantation in mice is a valuable tool in transplant and cardiovascular research. The cuff technique greatly simplifies ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

下大静脈狭窄: 深部静脈血栓症のマウスモデル

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

免疫・感染学

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

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

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

静脈介在モデル：バイパスグラフト開存を研究するための適切なモデル

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

Mouse Heterotopic Cervical Cardiac Transplantation Utilizing Vascular Cuffs

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after transplantation, and the impact of immunomodulatory therapies on graft rejection. Heterotopic cervical heart transplantation in mice was first described in 1991 using sutured anastomoses and subsequently modified to include cuff techniques. This modification allowed for improved success rates, and since then, there have been multiple reports that have proposed further technical improvements. However, translation into more widespread utilization remains limited due to the technical difficulty associated with graft anastomoses, which requires precision to achieve adequate length and caliber of the cuffs to avoid vascular anastomotic twisting or excessive tension, which can result in damage to the graft. The present protocol describes a modified technique for performing heterotopic cervical cardiac transplantation in mice which involves cuff placement on the recipient's common carotid artery and the donor's pulmonary artery in alignment with the direction of the blood flow.

Mouse cardiac transplantation models represent valuable research tools for studying transplantation immunology. The present protocol details mouse heterotopic cervical cardiac transplantation that involves the placement of cuffs on the recipient's common carotid artery and the donor's pulmonary artery trunk to allow for laminar blood flow.

血管カフを用いたマウスヘテロトピック頸部心臓移植

Murine models of cardiac transplantation are frequently utilized to study ischemia-reperfusion injury, innate and adaptive immune responses after ...

A Rabbit Venous Interposition Model Mimicking Revascularization Surgery using Vein Grafts to Assess Intimal Hyperplasia under Arterial Blood Pressure

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor because of the acceleration of intimal hyperplasia due to the exposure to arterial blood pressure. The present protocol is designed for the establishment of experimental venous intimal hyperplasia by interposing rabbit jugular veins to the ipsilateral carotid arteries. The protocol does not require surgical procedures deep in the body trunk and the extent of the incision is limited, which is less invasive for the animals, allowing long-term observation after implantation. This simple procedure enables researchers to investigate strategies to attenuate the progression of intimal hyperplasia of the implanted vein grafts. Using this protocol, we reported the effects transduction of microRNA-145 (miR-145), which is known to control the phenotype of vascular smooth muscle cells (VSMCs) from the proliferative to the contractile state, into harvested vein grafts. We confirmed the attenuation of intimal hyperplasia of vein grafts by transducing miR-145 before implantation surgery through the phenotype change of the VSMCs. Here we report a less invasive experimental platform to investigate the strategies that can be used to attenuate intimal hyperplasia of vein grafts in revascularization surgeries.

The present protocol aims to experimentally create venous intimal hyperplasia by subjecting veins to arterial blood pressure for developing strategies to attenuate venous intimal hyperplasia following revascularization surgery using vein grafts.

静脈移植を用いた血管形成術を模したウサギ静脈間位置モデルによる動脈血圧下の内膜過形成の評価

Although vein grafts have been commonly used as autologous grafts in revascularization surgeries for ischemic diseases, the long-term patency remains poor ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the established technique for a modified blood circuit reconstruction in a heterotopic abdominal heart transplantation model is presented. This method uses the intrathoracic inferior vena cava (IIVC) instead of the pulmonary artery of the donor heart for the anastomosis to the inferior vena cava of the recipient. It is facilitating and improving success rates for abdominal heart transplantation in mice.

A novel technique for blood circuit reconstruction in a heterotopic abdominal mouse heart transplantation model is demonstrated.

腹部マウス心臓移植モデルにおける血液回路再建

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

A Modified Cuff Technique for Mouse Cervical Heterotopic Heart Transplantation Model

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for investigating the mechanism of cardiac allograft rejection in preclinical studies because of their high homology with human genes. This understanding would help develop unique approaches to improving patients&#39; long-term survival treated with cardiac allografts. In a mouse model, abdominal donor heart implantation is commonly performed with an end-to-end&#160;anastomosis to the recipient&#39;s aorta and inferior vena cava using stitches. In this model, the donor&#39;s heart is implanted by end-to-end anastomosis to the recipient&#39;s carotid artery and jugular vein by the modified-Cuff technique. The transplantation surgery is performed without stitching and thus may increase the survival of the recipient since there is no interference with the blood supply and venous reflux of the lower body. This mouse model would help investigate the mechanisms underlying the immunological and pathological (acute/chronic) rejection of cardiac allografts.

In the present protocol, a mouse heart transplantation model is used for investigating the mechanism of cardiac allograft rejection. In this heterotopic heart transplantation model, operation efficiency is improved, and the survival of cardiac grafts is ensured by a cervical end-to-end anastomosis of heart implantation using a modified Cuff technique.

マウス頸部異所性心臓移植モデルのための修正カフ法

Cardiac allograft rejection limits the long-term survival of patients after heart transplantation. A mouse heart transplantation model is ideal for ...