その優秀な技術支援の Uli Heuter レオン デッカーとの優秀な編集協力、ピーター Kurdybacha ローマ ヴィツォレクに感謝したいと思います。

動物を対象とする手順がアーヘン工科大学、アリゾナ州 84 02.04.2012.A234 で承認された機関動物ケアおよび使用委員会 (IACUC).
 注: CD1 の背景を持つ成人男性野生型マウスを使用して、手順を説明します。食品、動物用の特殊なコントロールや治療への適切なアクセスを確保し、手術前後の専門研究所単位でマウスを飼うため。動物を購入した場合の手術を行う前に、1 週間を順化外。
 1 ドナーの準備 <ol><li>感染症を避けるために手術中に滅菌状態を維持するために滅菌材料・機器を使用します。</li>。
	<li>は、ボリューム イソフルランとフェイス マスクを通して酸素を 100% 1.5% の混合物に各マウスを麻酔します。仰臥位とテープでプラットフォーム オペレーティング テーブルにすべての足でマウスを置きます。その反射を確認するには、マウスを十分に麻酔、確かに後部のフィートをつまんでします。プロシージャの間に乾燥を防ぐために目の上眼軟膏を置きます</li>。
	<li>脱毛ジェルを使用して腹部からすべての髪を削除または髭剃りを使用します。無菌条件下で操作を実行します。クロルヘキシジンと滅菌水のスクラブを交互に腹部を消毒します</li>。
	<li>ハサミやメスで正中線腹部切開を介してドナー大動脈を削除します。右に手動で腸を撤回します。手動で軽くパウダー フリー手袋を使用する側に上腸を反映します。
	<ol><li>それを湿った保つため生理食塩水で湿らしたガーゼの部分に腸を配置します</li>。
		<li>離れて非常に慎重にピンセットで鈍的切離を使用して周囲の組織から腹部大動脈を解剖します</li>。
		<li>ピンセットで腎動脈と上大静脈からの分岐の間に大動脈のセグメントを区切ります</li>。
		<li>このセグメントを非常に慎重に 11-0 モノフィラメント縫合を使用のすべての小さな枝を確保します</li>。
		<li>大動脈を削除する前に下大静脈にヘパリンの 50 U を含む生理食塩水の溶液 0.5 ミリリットル (mL) を挿入します</li>。
		<li>大動脈のセグメントが削除された後の exsanguinate ドナー動物ができます</li>。
		<li>生理食塩水を完全にグラフトをすすいで氷冷生理食塩水の容器にすぐに転送します</li>。
	</ol></li></ol> 2。受信者の準備 <ol><li>ボリューム イソフルランとフェイス マスクを通して酸素を 100% 1.5% の混合物を持つ受信者の動物の麻酔を髪を削除し、(セクション 1) を駆除します。メスと骨盤に、剣から中間線切開を行い、腹部の壁を撤回します。プロシージャの間に乾燥を防ぐために目の上眼軟膏を置きます</li>。
	<li>ラップ食塩水内腸ガーゼを湿らせたし、動物に非常にゆっくり転置 &#39; s 右</li>。
	<li>Dissect 高齢者大動脈腎動脈の間無料の近位と遠位のピンセット分岐します</li>。
	<li>このセグメントを非常に慎重に 11-0 モノフィラメント縫合を使用のすべての小さな枝を確保します</li>。
	<li>6-0 シングル絹縫合糸で大動脈の近位および遠位部分をクランプします</li>。
	<li>クランプ間の中間に大動脈を分割し、ルーメン オープンをフラッシュするヘパリン生理食塩水で切口を灌漑します</li>。
	<li>は世話を正しく合わせてドナーと受信者 (大動脈の任意のねじれを避けるために 11-0 のモノフィラメントで縫合に続いて受信容器に挿入餌容器端と同所性同種の位置にグラフトを配置します。 図 1) 10.</li> <li>吻合部の検査を実施した後、合字を慎重に取り外します。まず遠位クランプをリリースします。この方が低気圧が近位部の高圧側を解放する前に、一緒に壁を保持します</li>。
	<li>はすぐに移植を灌流し、パルスが見えるを確認してください。絹の残党を慎重に取り外します。ドナーと受信者の大動脈の間最適なラップの長さは 1-2 mm.</li>
	<li>腹部の内容を腹腔内に戻り、全体を閉じる実行中 3-0 ポリグ リコール酸縫合糸で巻き</li>。
	<li>マウス ブプレノルフィンを与える (0.05 mg/kg 体重の皮下 (SC)) 麻酔を終了する前にします</li>。
	<li>放置しないでください動物それが完全に意識するまで。疼痛管理 buprenophine 0.05 mg/kg 体重で療法が SC 術後後 3 日間一日に三回管理機関監督機関による承認</li>。
	<li>組織採取、麻酔として受信者のマウスの上記の説明し、4% ホルムアルデヒド/PBS、続いてリン酸緩衝生理食塩水 (PBS) で血管のフラッシュ (pH 7.4 を =) 心臓穿刺によって。グラフトをそっと取り外します。4% ホルムアルデヒド/PBS で一晩固定後プロセス標本はさらに、パラフィンに埋め込む</li>。
</ol>

<ol>
	<li>Rowinska, Z., 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=Non-+invasive+in+vivo+analysis+of+a+murine+aortic+graft+using+high+resolution+ultrasound+microimaging.">Non- invasive in vivo analysis of a murine aortic graft using high resolution ultrasound microimaging.</a> Eur J Radiol. 81 (2), 244-249 (2012).</li><li>Daugherty, A. <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+atherosclerosis.">Mouse models of atherosclerosis.</a> Am J Med Sci. 323 (1), 3-10 (2002).</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>Daugherty, A., Cassis, L. A. <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+abdominal+aortic+aneurysms.">Mouse models of abdominal aortic aneurysms.</a> Arterioscler Thromb Vasc .Biol. 24 (3), 429-434 (2004).</li><li>Zernecke, A., Shagdarsuren, E., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemokines+in+atherosclerosis:+an+update.">Chemokines in atherosclerosis: an update.</a> Arterioscler Thromb Vasc Biol. 28 (11), 1897-1908 (2008).</li><li>Chereshnev, I., 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=Mouse+model+of+heterotopic+aortic+arch+transplantation.">Mouse model of heterotopic aortic arch transplantation.</a> J Surg Res. 111 (2), 171-176 (2003).</li><li>Koulack, J., McAlister, V. C., Giacomantonio, C. A., Bitter-Suermann, H., MacDonald, A. S., Lee, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+aortic+transplant+model+of+chronic+rejection.">Development of a mouse aortic transplant model of chronic rejection.</a> Microsurgery. 16 (2), 110-113 (1995).</li><li>Engelbrecht, G., Kahn, D., Duminy, F., Hickman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+rapid+technique+for+renal+transplantation+in+the+rat.">New rapid technique for renal transplantation in the rat.</a> Microsurgery. 13 (6), 340-344 (1992).</li><li>Baxter, K., Hao, P. M., Howden, B. O., Saunder, A., Jablonski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+technique+of+abdominal+heart+transplantation+in+the+rat.">Modified technique of abdominal heart transplantation in the rat.</a> J Heart Lung Transplant. 17 (11), 1057-1064 (1998).</li><li>Dambrin, C., Calise, D., Pieraggi, M. T., Thiers, J. C., Thomsen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice:+a+new+model+of+allograft+arteriosclerosis.">Orthotopic aortic transplantation in mice: a new model of allograft arteriosclerosis.</a> J Heart Lung Transplant. 18 (10), 946-951 (1999).</li><li>Siemionow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+microarterial+anastomoses:+end-to-end+versus+end-in-end+(sleeve)+technique.">Histopathology of microarterial anastomoses: end-to-end versus end-in-end (sleeve) technique.</a> J Hand Surg Am. 15, 619-625 (1990).</li><li>Charo, I. F., Ransohoff, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+many+roles+of+chemokines+and+chemokine+receptors+in+inflammation.">The many roles of chemokines and chemokine receptors in inflammation.</a> N Engl J Med. 354 (6), 610-621 (2006).</li><li>Sun, H., 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=Improved+surgical+technique+for+the+establishment+of+a+murine+model+of+aortic+transplantation.">Improved surgical technique for the establishment of a murine model of aortic transplantation.</a> Microsurgery. 18 (6), 368-371 (1998).</li><li>Guo, L., Agarwal, A., George, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+aortic+transplantation+in+mice+for+the+study+of+vascular+disease.">Orthotopic aortic transplantation in mice for the study of vascular disease.</a> J Vis Exp. (69), e4338 (2012).</li></ol>

著者は、彼らは競合する金銭的な利益があることを宣言します。

この研究の前にマウスでの他の各種の移植モデルは徹底的に分析3,6,7,10,13,14.私たちの条件にマッチした従来のエンド-エンド縫合方法と比較してスリーブ吻合部の高い信頼性を示したと Dambrinらによって変更とスリーブを用いた大動脈移植モデルが選択されました。1,10。
この手法は、クロス クランプ時間かなり短縮術中大動脈へのダメージを最小限に抑えることと多くの点で有利です。血栓症の発生率が低いは、容器口径ドナーと受信者の間の潜在的な不一致を避けることに加えられなかった。上記の観察はこの手法をマウスの大動脈移植における血管疾患を調査するため非常に作る。
超音波が移植後 8 週間が実行されたフォロー アップ研究で有意な変化は認められなかった.これは手術中に大動脈への損傷が最小限1だろうと仮定を確認しました。
この記事で紹介した移植術は障害血管整合性とその機能の両方を保証します。したがって、この実験的移植モデルが退行性血管疾患遺伝子改変マウスでの将来の分子および薬理学的調査のための貴重なツールとして可能性があります判断できます。
ビデオのガイドすることができますこの単純な動静脈動脈モデルの使用を示す教材として機能することと、血管病態の多くの重要な問題についてさらに実り多い議論に貢献することと考えています。この非常に急速な吻合法は、遺伝子組み換えマウスで血管疾患を勉強するに使えます。移植併用動脈瘤モデルでの変更としても使えます。
プロシージャの間に重要なポイントがあります。縫合自体を配置することは、最も重要なステップです。外科医は、ドナーとレシピエントの適切な配置による大動脈の任意のねじれを避けるために世話をするが。クランプは、吻合部の検査後慎重に削除されます。遠位クランプは、まず近位部の高圧側のリリース前に壁を保持している低圧の結果常に解放する必要があります。正しく次のリリースのシーケンスの結果を出血でしょう。

移植自体によって引き起こされる特定の血管反応と arteriogenic 環境に接続されている特定の全身的要因の識別をできるマウス大動脈移植モデルに細心の注意を支払われている先行研究で指摘したよう1,2,3。 ここで重要な役割を果たしている主要な要因は、ノックアウト、トランスジェニック マウスの可用性です。このようなモデルに関与識別および退行性血管疾患、動脈硬化や動脈瘤形成4,などの開発に関連付けられている新しい病態生理学的経路を決定する可能性を提供しています5。
それは、本質的な虚血/再灌流を移植中に移植用血管への損傷が表示されますは注目に値するです。したがって、移植の整合性や術後の期間中に予期しない炎症性反応に関する特定の問題の発生がおそらく血管の変性疾患3 の病態生理学的変化を除外除外できません。、4,5,6,7。スリーブ吻合は直径 1 ミリ以下の血管の動脈吻合のため代替エンドで方法で、大動脈に続いて合わせられたラットの腎・心臓移植で正常に適用されています。Dambrinらによるマウスの移植8,9,10,11。
スリーブの移植技術を用いた大動脈損傷は非常に技術的な失敗率が低く、平均でわずか 20 分を持続的な原因を最小限に抑えます。以前の研究結果は、スリーブ法1を用いた移植後体内の isograft の構造と機能性を実証しています。Dambrinら説明短い学習曲線の成功率は 78% を終わった後10。血栓症などの合併症はまれですが、たとえば Engelbrechtらがラット8腎移植でスリーブを用いた血栓症を観察しなかったです。
スリーブ吻合とマウス大動脈移植モデルは、iso/同種移植血管反応を研究する迅速かつ簡単なツールです。このビデオは、当研究室で実施する大動脈移植手順を示しています。この移植モデル血管変性疾患の根本的な病理メカニズムを定義する際に役に立つかもしれませんし、分子および薬理学的介入の12のそれ以上の評価に貢献するかもしれない。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Halsey Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12001-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5-45 Forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-35&#8232;</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps -</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Lexer-Baby Scissors</td>
			<td>Fine Science Tools</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Micro Needle Holder</td>
			<td>Fine Science Tools</td>
			<td>12060-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Aesculap, Germany</td>
			<td>Typ OC498R</td>
			<td></td>
		</tr>
		<tr>
			<td>Castroviejo Suture Forceps</td>
			<td>Geuder, Germany</td>
			<td>19015</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 silk black (Silk)</td>
			<td>Deknatel, Research Triangle Park NC, USA</td>
			<td>18020-60- FST</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 monofilament (Ethilon)</td>
			<td>Ethicon, Norderstedt, Germany</td>
			<td>EH7438G</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 polyglycolic acid suture (Serafit)</td>
			<td>Serag-Wiessner, Naila, Germany</td>
			<td>60203214</td>
			<td></td>
		</tr>
		<tr>
			<td>Isofluorane</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.9% saline</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Any genericon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bepanthene eye and nose cream</td>
			<td>Bayer, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscop</td>
			<td>Zeiss</td>
			<td>Opmi MDO/S5</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaporiser</td>
			<td>Eickemeyer</td>
			<td>TEC3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasound</td>
			<td>Vevo, Canada</td>
			<td>770,2100</td>
			<td></td>
		</tr>
	</tbody>
</table>

using the sleeve technique in a mouse model of aortic transplantation - an instructional video

スリーブを用いた同所性同種大動脈弁移植は、唯一の 10-20% の故障率で大動脈への損傷を低減します。スリーブ メソッドを使用してマウスの大動脈を吻合するために時間は短くて簡単な平均 20 分、iso/同種移植の研究を許可します。次の資料では、私たちの研究室で使用される大動脈移植手順について説明します。マウスは、1.5% ボリューム イソフルランとフェイス マスクを通して 100% の酸素の混合物で麻酔をかけられました。この時点で、大動脈腎動脈とその分岐間のセグメントは、上大静脈、自由に準備と clampedat から分離された単一の絹糸と近位および遠位のセグメント。大動脈の削除する前に、ヘパリンを含む食塩は下大静脈に注入されました。大動脈クランプの間カットされたし、生理食塩水ヘパリン溶液を用いて内腔をフラッシュします。モノフィラメント縫合糸で袖のテクニックは、同所性同種位置で腹部大動脈を移植するために使用されました。

血栓症の高いリスクがあったが、マウスがない観測の身体障害を持つ 15-30 分以内麻酔から回復しました。超音波解析が行われた術後フォロー。野生型マウスの研究で使用されるは、内腔の寸法変化を起こしません。したがって、狭窄も動脈瘤の形成が観察されました。移植動物は血管壁 (図 2) のプラーク開発を展示できませんでした。
プラーク, 平滑筋細胞とマクロファージの集積を決定する immunohistology など従来の汚損プロシージャを使用できます。本研究では組織学的免疫組織化学染色をグラフトの整合性をテストする接ぎ木後 6 週間行った。組織染色 (ヘマトキシリンとエオシン (HE) および免疫組織化学染色 (平滑筋アクチン (SMA) とマクロファージ (MAC2)) (図 3) を見せてくれた平滑筋細胞、内皮細胞のライニング、および no の分布パターンをそのまま内膜の細胞の蓄積。グラフト血管 (図 3) で検出された重要な病変や細胞の活性化がないことが示唆されました。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/54915/54915fig1.jpg"> 
図 1: 袖の手法。腹部大動脈は、袖のテクニックを使用して移植されました。この手順ではドナーの大動脈は浅刺さ餌容器内に存在と同所性同種の位置に置かれました。<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/54915/54915fig2.jpg"> 
図 2: 術中超音波画像と。移植移植 (A)、(B) 三次元超音波および B モード表示 (C) 後 6 週間の術中 (剖検) ビューの例です。術後のフォロー アップは、超音波を用いて行った。写真は、ルーメンの次元で変化なしでグラフトの開存を表示.また、狭窄や動脈瘤の形成は認められなかった。<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/54915/54915fig3.jpg"> 
図 3: 組織学的および免疫組織化学画像。移植後 6 週間で移植した動物の組織学的および免疫組織化学の代表的なイメージ。組織によって移植された大動脈に大きな病変認められなかった (ヘマトキシリンとエオシン、彼は、100 倍の倍率、スケールバー = 50 μ m)、免疫組織染色 (平滑筋アクチン SMA (赤)、またはマクロファージ MAC2 (緑) 200 X 倍率、スケール バー =25 μ m)。核カウンターで染色 DAPI (青).<a href="//ecsource.jove.com/files/ftp_upload/54915/54915fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video at JoVE.com

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

マウスでスリーブを用いた同所性同種大動脈移植モデルを提案します。それは非常に急速な吻合法のことで、血管疾患の研究に用いることができます。

大動脈弁移植 - 教則ビデオのマウスモデルでスリーブを用いた

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

using-sleeve-technique-mouse-model-aortic-transplantation-an

Research

JoVE Journal

Medicine

8.9K ビュー.  Ruhr- University Bochum. マウスでスリーブを用いた同所性同種大動脈移植モデルを提案します。それは非常に急速な吻合法のことで、血管疾患の研究に用いることができます。

ビデオ: Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

同所性大動脈移植：慢性血管障害の発生研究のラットモデル

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

医学

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

のマウスモデル子宮内で移植

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

血管疾患の研究のためのマウスにおける同所性大動脈移植

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

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 ...

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 ...

Creation and Transplantation of an Adipose-derived Stem Cell (ASC) Sheet in a Diabetic Wound-healing Model

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients with impeded blood flow or with large wounds might be prolonged. Cell-based therapies have appeared as a new technique for the treatment of diabetic ulcers, and cell-sheet engineering has improved the efficacy of cell transplantation. A number of reports have suggested that adipose-derived stem cells (ASCs), a type of mesenchymal stromal cell (MSC), exhibit therapeutic potential due to their relative abundance in adipose tissue and their accessibility for collection when compared to MSCs from other tissues. Therefore, ASCs appear to be a good source of stem cells for therapeutic use. In this study, ASC sheets from the epididymal adipose fat of normal Lewis rats were successfully created using temperature-responsive culture dishes and normal culture medium containing ascorbic acid. The ASC sheets were transplanted into Zucker diabetic fatty (ZDF) rats, a rat model of type 2 diabetes and obesity, that exhibit diminished wound healing. A wound was created on the posterior cranial surface, ASC sheets were transplanted into the wound, and a bilayer artificial skin was used to cover the sheets. ZDF rats that received ASC sheets had better wound healing than ZDF rats without the transplantation of ASC sheets. This approach was limited because ASC sheets are sensitive to dry conditions, requiring the maintenance of a moist wound environment. Therefore, artificial skin was used to cover the ASC sheet to prevent drying. The allogenic transplantation of ASC sheets in combination with artificial skin might also be applicable to other intractable ulcers or burns, such as those observed with peripheral arterial disease and collagen disease, and might be administered to patients who are undernourished or are using steroids. Thus, this treatment might be the first step towards improving the therapeutic options for diabetic wound healing.

Creation and Transplantation of an Adipose-derived Stem Cell ASC Sheet in a Diabetic Wound-healing Model

Adipose-derived stem cells (ASCs) are easily isolated and harvested from the fat of normal rats. ASC sheets can be created using cell-sheet engineering and can be transplanted into Zucker diabetic fatty rats exhibiting full-thickness skin defects with exposed bone and then covered with a bilayer of artificial skin.

作成および糖尿病創傷治癒モデルにおける脂肪由来幹細胞 (ASC) シートの移植

Artificial skin has achieved considerable therapeutic results in clinical practice. However, artificial skin treatments for wounds in diabetic patients ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

変更されたカフを用いた膵臓移植マウス モデル

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

改変非縫合カフ技術を用いたマウス子宮頸動脈大動脈移植モデル

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...