この作品は1R03DK096151によって部分的にサポートされていました。

全てのマウスは、ジャクソン研究所（バーハーバー、メイン州）から購入し、NIHガイドラインとコロラド州デンバーIACUCの大学の承認を得て応じコロラド州デンバー、バーバラ・デイビス・センター動物施設の大学で病原体を含まない条件下で飼育しました。 1.ドナー腎臓収穫<ol><li> 、すべての楽器を滅菌手順を通して滅菌手袋を着用し、無菌領域を維持します。手術用顕微鏡を用いて、すべての手術を行ってください。 </li><li>外科ペントバルビタール麻酔（60mgの/ kgのIP）のドナーからのマウスドナーの腎臓を削除します。つま先ピンチによって麻酔の深さを確認し、呼吸数を観察します。 </li><li> 4ウェイの制約により、マウスを固定化後、ファークリップ。ポビドンヨードで皮膚を準備し、無菌の方法で、マウスを羽織ります。 </li><li> 2cmの正中線垂直腹部切開を行い、腹腔を入力してください。上方腸を撤回し、それを外部化胸の上。手順を通して無菌湿ったガーゼに包まれた腸を保管してください。 </li><li>大きな腹部の血管を識別し、それらを動員、任意の腰椎枝を特定し、焼灼または10/0ナイロン縫合糸でそれらを連結のいずれか。 </li><li>左腎血管へのすぐ遠位約2ミリメートル以上の鈍的切開によって離れて下大静脈（IVC）と腹部大動脈（AA）をいじめます。 10/0ナイロンで結紮し、腎血管から小動脈および静脈枝を分けます。 </li><li>今これらの構造の鈍的切開によって慎重に腎動脈から腎静脈を分離。これは、受信者IVCに端側吻合を形成するために使用される腎静脈の近位端にカレルパッチの正確な作成を可能にします。 </li><li>特定し、ライゲーションし、左副腎血管を分割します。これは、副腎大動脈へのアクセスを許可し、6-0絹縫合糸を結紮の準備のために大動脈の周りに配置されますが、この時点で結ばれていません。 </LI&gt; <li>周囲フェーシャから腎臓、血管および尿管を動員。右と連結するために腎臓を回す/任意の後方分岐を分けます。その後、左に腎臓を返します。 </li><li>今尿管に注意を向けます。腎門部を乱すことなく、尿管、血管を維持するために世話をして、周囲のfaciaから尿管を解放します。精管のレベルで尿管を分割します。腎臓は現在回復するための準備ができました。 </li><li>ゆっくりと、それによってドナーをheparinizing遠位IVCへのヘパリン300単位を注射します。副腎AAの周りに配置され、6-0絹縫合糸を下に結ぶとヘパリン生理食塩水（100 U / ml）を0.8ミリリットル遠位腹大動脈を介して、左腎臓を灌流。 </li><li>灌流は静脈カレルパッチを作成して停止した後はすぐに腎臓に逆流を排除します。 AAと下の腎動脈を明らかにし、腎臓に向かって腎静脈を撤回。 </li><li>腎動脈に隣接する大動脈かかととするの作成を分割図1に示すように、電子カフ。ドナーから腎臓、血管および尿管を外します。 </li><li>移植時まで事前に準備された受信者（0分冷 ​​虚血時間）に直接腎臓を削除するか、事前に決められた時間のための最適なソリューションで4 O℃で保存しました。 exsanguanationと頸椎脱臼によってドナーを安楽死させました。ドナー腎臓を回復するための合計時間は約です。 15〜20分。 </li></ol> 2.腎臓インプラント技術<ol><li>ペントバルビタールでレシピエントマウスを麻酔（60mgの/ kgのIP初期投与量、25mgの/ kgのIP補足用量必要な場合）。つま先ピンチによって麻酔の深さを確認し、呼吸数を観察します。 4ウェイの制約により、マウスを固定化し、眼に眼軟膏を適用し、その後の毛皮をクリップ。ポビドンヨードで皮膚を準備し、無菌の方法で、マウスを羽織ります。 </li><li> 2cmの正中線垂直腹部切開を行い、腹腔を入力してください。腸を撤回上方と胸へ外部化。手順を通して無菌湿ったガーゼに包まれた腸を保管してください。 </li><li>このとき、右腎摘出を行います。 6-0絹縫合糸で腎動脈と静脈を結紮。縫合糸に腎遠位を切り出します。 6-0絹糸で尿管を結紮した後、腎臓の近位に分割します。 </li><li>腎血管下の腹部大動脈と下大静脈（IVC）を分離します。吻合部位に比べて劣っその後大動脈の周りに4-0綿の絆を置き、IVCが優れました。 10-0ナイロン縫合糸でフィールド内の任意の腰椎血管を識別し、連結。 </li><li>コットンネクタイ、優れ続く最初の劣る結び目。このように、いくつかの血液が大動脈切開を作るより簡単に大動脈内に保持されています。 </li><li>大動脈の内腔を入力するように30G針を用いて大動脈切開を形成します。約2ミリメートルの長さを微マイクロハサミで切開を拡張します。 </li><li>ドナー大動脈ヒールアンドトウカフの端部から側部に到る吻合を作ります以下の方法で受信者の大動脈。ドナー大動脈および受信者の大動脈とネクタイの切開部の下角に10-0ナイロン縫合糸滞在ステッチを配置します。 </li><li>ドナー大動脈と腹部大動脈とネクタイの切開部の優れたコーナーの最初の反対の第2の10-0ナイロンを配置します。大動脈の側壁に劣るよりも優れてから実行している縫合線を作成し、以前に配置された滞在ステッチに対して結びます。そして、実行中のファッションやタイで内側壁を縫合。 </li><li>以下の方法で受信者IVCへのドナー腎静脈の端部から側部に到る吻合を行います。 30G針でIVCを穿刺し、細かいマイクロハサミで切開を拡張します。 10-0ナイロンでIVCの切開部の下位隅にドナー腎静脈を接続します。腎静脈と下大静脈とタイの間で実行されている縫合線を作成します。 注：これは、完全な厚さは、血管adventit含む縫合針の通過することも極めて重要ですIAと内膜が達成されます。エッジの裏返しにもシールと吻合の治癒を助ける内膜ツー内膜の接触があることを保証します。止血凝固剤が漏れを減少させるために有用であることができるが、我々は、外科医が代わりに良い技術に依存していることをお勧めします。 </li><li>吻合は「クリーン」であることを確認してください。それはステッチを配置する際に、対向壁がキャッチされていないこと、です。これは失敗した移植片および後肢麻痺に極端な例になりますことを流す重要な収縮を引き起こします。 注：別の極めて重要な要因は、吻合縫合線のテンションも最適であることを保証することです。緩すぎると不可逆的な漏れ、きつすぎるとなります流れるように狭窄があるでしょう。動脈側の場合は混雑した腎臓はなり静脈側の場合、これが、移植片の不十分な灌流になります。 </li><li>静脈の流れを再確立遠位4-0綿のネクタイをリリース。静脈の止血たら私たちの吻合は、近位4-0綿のネクタイを緩め、止血のために動脈吻合を観察徐々に観察されています。 </li><li>両方の吻合が安全と見なされているいったんマウスから綿の関係を削除します。ピアース二つの穴を作成し、20G針で膀胱。 </li><li>貫​​通孔を湾曲した鉗子の先端を通過し、2 10-0ナイロン縫合糸で膀胱壁に固定されて尿管の近位端とピンセットで膀胱を介してドナー尿管を引っ張ります。尿管が膀胱内に撤回し、2 10-0ナイロン縫合糸で穴を閉鎖することを可能にする第二の孔から突出した尿管の余分な長さをトリミング。 </li><li>腹部に腸を返します。実行中のファッションで5-0シルク縫合糸を使用して2つの層で腹壁を閉じます。 </li><li>決算時に輸液蘇生などの腹部に滅菌、温かい生理食塩水1.0mlのボーラスを管理し、通常の生理食塩水を皮下術後の0.8ミリリットルを注入。他のsupportiありませんVEの対策は手術中に必要とされます。 </li><li>温暖化の毛布の上に動物を回復します。総インプラント時間は約です。 35〜45分。このような手順の開始時と72時間後のオペアンプごとに6〜12時間でブプレノルフィン、0.05ミリグラム/キログラム、SC、0.1〜0.2ミリリットルとして鎮痛剤を投与します。 </li></ol> 3.対側腎摘出術<ol><li>プロトコル要件に応じて、数日間は、インプラント手順の後に、イソフルラン麻酔下で対側腎摘出（誘導のための5％吸入イソフルラン、メンテナンスのための1.5から2.0パーセント）を行います。 </li><li>腹腔を入力して、優しく、動物の右に腸を撤回。周囲の筋膜から左腎臓と鈍い解剖を公開します。 </li><li> 6-0絹縫合糸で腎動脈、静脈および尿管を結紮した後、縫合糸の上に腎臓を切除し、腎臓を削除します。総手術時間は約です。 10分。 </li></ol> 4.グラフト評価<ol><li>メジャー血清creatiアルカリ性ピクリン酸法（ヤッフェ反応）を使用して9。 </li></ol>

<ol>
	<li>Skoskiewicz, M., Chase, C., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+transplants+between+mice+of+graded+immunogenetic+diversity.">Kidney transplants between mice of graded immunogenetic diversity.</a> Transplant Proc. 5 (1), 721-725 (1973).</li><li>Kalina, S. L., Mottram, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microsurgical+technique+for+renal+transplantation+in+mice.">A microsurgical technique for renal transplantation in mice.</a> Aust N Z J Surg. 63 (3), 213-216 (1993).</li><li>Han, W. R., Murray-Segal, L. J., Mottram, P. L. <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+for+kidney+transplantation+in+mice.">Modified technique for kidney transplantation in mice.</a> Microsurgery. 19 (6), 272-274 (1999).</li><li>Ge, F., Gong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+successfully+establishing+a+kidney+transplant+in+a+mouse+model.">Strategies for successfully establishing a kidney transplant in a mouse model.</a> Exp Clin Transplant. 9 (5), 287-294 (2011).</li><li>Martins, P. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Learning+curve,+surgical+results+and+operative+complications+for+kidney+transplantation+in+mice.">Learning curve, surgical results and operative complications for kidney transplantation in mice.</a> Microsurgery. 26 (8), 590-593 (2006).</li><li>Corry, R. J., Winn, H. J., Russell, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+hearts+in+mice.+The+role+of+H-2D,+H-2K,+and+non-H-2+antigens+in+rejection.">Primarily vascularized allografts of hearts in mice. The role of H-2D, H-2K, and non-H-2 antigens in rejection.</a> Transplantation. 16 (4), 343-350 (1973).</li><li>Zhang, Z., Chen, H., Qian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+Transplantation+in+Mice.">Kidney Transplantation in Mice.</a> Experimental Organ Transplantation. , 45-64 (2013).</li><li>Zhang, L., Moskovitz, M., Piscatelli, S., Longaker, M. T., Siebert, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+study+of+different+angled+end-to-side+anastomoses.">Hemodynamic study of different angled end-to-side anastomoses.</a> Microsurgery. 16 (2), 114-117 (1995).</li><li>Liu, Q., Mirc, D., Fu, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+mechanisms+of+thrombosis+in+intact+bent+microvessels+of+rat+mesentery.">Mechanical mechanisms of thrombosis in intact bent microvessels of rat mesentery.</a> J Biomech. 41 (12), 2726-2734 (2008).</li><li>Chesnutt, J. K., Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tortuosity+triggers+platelet+activation+and+thrombus+formation+in+microvessels.">Tortuosity triggers platelet activation and thrombus formation in microvessels.</a> J Biomech Eng. 133 (12), 121004 (2011).</li><li>Han, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+buckling+within+soft+surrounding+tissue+generates+tortuosity.">Blood vessel buckling within soft surrounding tissue generates tortuosity.</a> J Biomech. 42 (16), 2797-2801 (2009).</li><li>Gutierrez-Diaz, J. A., 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=Intraluminal+thrombus+and+neointimal+hyperplasia+after+microvascular+surgery.">Intraluminal thrombus and neointimal hyperplasia after microvascular surgery.</a> Surg Neurol. 24 (2), 153-159 (1985).</li><li>Khouri, R. K., Cooley, B. C., Kenna, D. M., Edstrom, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombosis+of+microvascular+anastomoses+in+traumatized+vessels:+fibrin+versus+platelets.">Thrombosis of microvascular anastomoses in traumatized vessels: fibrin versus platelets.</a> Plast Reconstr Surg. 86 (1), 110-117 (1990).</li><li>Johnson, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+platelet+deposition+at+the+human+microvascular+anastomosis:+effect+on+downstream+platelet+deposition+to+intact+and+injured+vessels.">Initial platelet deposition at the human microvascular anastomosis: effect on downstream platelet deposition to intact and injured vessels.</a> Plast Reconstr Surg. 90 (4), 650-658 (1992).</li></ol>

The authors have nothing to disclose.

この移植技術をマスターすることは困難であるが、一度それは非常に強力な研究ツールで達成しました。患者外科医/研究者は小さな動物モデルでそれ以上に、任意の外科的手技を習得するための鍵である、細部および技術の整合性に注意することによって報われる。マウスの腎臓移植​​をマスタリングの技術的な問題は、多くのひだであり、それは他の小動物の移植モデルでの経験は、こ...

1973年以来、マウスにおける腎移植モデルは、貴重な研究ツールとなっているが、技術的な問題は、その広範な使用を妨げてきました。年間でいくつかの論文は、この手順の改善/改良を詳細に公開されています。主に血管新生した固形臓器移植のモデルとして、この手順は、また、1973年6でラッセルの研究所で考案された異心臓移植モデルにおそらく秒です。どちらのモデルも、同種異系拒絶反応、遅延移植片機能の開発に検索するには自分自身を貸しますそして、虚血再灌流障害。 腎臓移植に報告するための最も一般的な問題の一つは、我々はまた、我々の研究室で経験した動脈血栓症4,5,7の比較的高い発生率です。そこで我々は、血栓形成の文献レビューを行い、場合によっては、この技術的な問題の原因を見つけるために着手したとも考案します可能な解決策。血栓症の最も可能性の高い原因は、血液がドナー腎動脈の上、次にドナー腎大動脈に、受信者の大動脈から取り多少曲がりくねった道です。この経路は、血小板活性化および血栓形成を引き起こし得る、腎動脈の乱れを引き起こします。最近の観察と関連文献8-14の検索に基づいて、我々は0％に血栓症を減少させた新しい技術を思い付きました。 ここに記載の技術は、施設は、血流を改善し、動脈ヒールアンドトウカフの形成以前に報告の手法によって異なると著しく血栓形成を減少させます。カフ未満45 O大動脈（ 図1Aおよび1B）の長手方向軸に対してある角度で、腎動脈口の面を横切る赤外線の腎動脈を分割して形成されています。この長さは約2ミリメートルカフをもたらします。静脈カレルパッチをrを横断することによって形成されますIVCにエナール静脈ことにより、カフの直径を増加させます。赤外線腎ドナー腹部大動脈ヒールアンドトウカフは、レシピエントの腹部大動脈およびドナー腎静脈/ IVCパッチに吻合端側である受信者の腹部下大静脈（IVC）に吻合端側であります。尿管は、その後に導入され、Hanら3に記載されているように、膀胱に固定されます。 この研究のためにのみ温虚血時間（ すなわち 、ノー冷たい虚血）で処理されていない移植が比較されます。この場合、暖かい虚血は、レシピエント（ステップ以下2.11）、ドナーの腎臓を通る血流の停止（以下ステップ1.11）、および移植片の再潅流からの時間を指します。冷虚血は、腎臓を灌流されていないインプラント手順の開始まで冷蔵で維持される時間を指します。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Instrument</td>
			<td>Roboz #</td>
			<td>Fine Science Tools #</td>
			<td>Arosurgical #</td>
		</tr>
		<tr>
			<td>Straight micro-dissecting forcep #5</td>
			<td>RS-5015</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved micro-dissecting forcep #7</td>
			<td>RS-5047</td>
			<td>11297-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved serrated forcep</td>
			<td>RS-5137</td>
			<td>11052-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas micro-dissecting scissors, short</td>
			<td>RS-5610</td>
			<td></td>
			<td>09.140.08</td>
		</tr>
		<tr>
			<td colspan="2">Micro-dissecting scissors, straight, sharp, long</td>
			<td></td>
			<td>11.602.11</td>
		</tr>
		<tr>
			<td>Micro spring handle needle holder</td>
			<td></td>
			<td></td>
			<td>11.549.15</td>
		</tr>
		<tr>
			<td>Straight mosquito forcep</td>
			<td></td>
			<td>91308-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, straight, blunt</td>
			<td>RS-5962</td>
			<td>14078-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors, curved, blunt</td>
			<td>RS-5981</td>
			<td>14079-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro retractor</td>
			<td>RS-6540</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument tray, 10&#8221; x 6 &#189;&#8221; x &#190;&#8221;</td>
			<td>RT-1350S</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silk suture, 5/0, 22.5m spool</td>
			<td></td>
			<td>18020-50</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Suture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10/0 nylon</td>
			<td></td>
			<td></td>
			<td>T4A10Q07</td>
		</tr>
		<tr>
			<td>5/0 silk</td>
			<td></td>
			<td></td>
			<td>E19A05N</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gloves</td>
			<td></td>
			<td>Drapes</td>
			<td></td>
		</tr>
		<tr>
			<td>Biogel from Medex Supply</td>
			<td></td>
			<td>Precept, #64-9012-9</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes</td>
			<td></td>
			<td>Cotton applicators</td>
			<td></td>
		</tr>
		<tr>
			<td>B-D 1cc insulin, #329424</td>
			<td></td>
			<td colspan="2">Fisher-brand, #23-400-100</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PDI, #B40600</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4/0 Cotton ties</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Domestic cotton autoclaved with instruments</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

murine kidney transplant technique

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique using the donor aorta to form the arterial anastomosis instead of the renal artery was developed and reported in 1993 by Kalina and Mottram 2 with a further advancement coming from the same laboratory in 1999 3. While one can become proficient in this model, a search of the literature reveals that many labs still experience a high proportion of graft loss due to arterial thrombosis. We describe here a technique that was devised in our laboratory that vastly reduces the arterial thrombus reported by others 4,5. This is achieved by forming a heel-and-toe cuff of the donor infra-renal aorta that facilitates a larger anastomosis and straighter blood flow into the kidney.

この手術法は、単純な移植片生存/拒絶の研究、または非常に複雑な実験プロトコルのいずれかを可能にします。以下の図では、この改良された動脈吻合の技術を使用することの利点を実証します。この技術を用いて、我々は著しく生産性を増加させる0％と35％の動脈血栓症の発生率を減少させました。我々は維持し、同じ0％血栓症の結果で1年以上のためにこの技術を使用している。 ?...

Watch this Scientific Journal Video about Murine Kidney Transplant Technique at JoVE.com

Murine Kidney Transplant Technique

The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis.

マウスの腎臓移植​​技術

The first mouse kidney transplant technique was published in 19731 by the Russell laboratory. Although it took some years for other labs to become ...

Research

JoVE Journal

Medicine

15.8K ビュー.  University of Colorado, Denver. The goal of this manuscript is to describe the steps required to perform a kidney transplant in a mouse, paying particular attention to the details of the arterial anastomosis. 

ビデオ: Murine Kidney Transplant Technique

A Method for Murine Islet Isolation and Subcapsular Kidney Transplantation

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize their blood glucose levels, islet transplantation has been proposed to be a potential treatment for type 1 diabetes 1,2. More recently, advances in human islet transplantation have further strengthened this view 1,3. However, two major limitations prevent islet transplantation from being a widespread clinical reality: (a) the requirement for large numbers of islets per patient, which severely reduces the number of potential recipients, and (b) the need for heavy immunosuppression, which significantly affects the pediatric population of patients due to their vulnerability to long-term immunosuppression. Strategies that can overcome these limitations have the potential to enhance the therapeutic utility of islet transplantation.
Islet transplantation under the mouse kidney capsule is a widely accepted model to investigate various strategies to improve islet transplantation. This experiment requires the isolation of high quality islets and implantation of islets to the diabetic recipients. Both procedures require surgical steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol. We also briefly discuss different transplantation models: syngeneic, allogeneic, syngeneic autoimmune, and allogeneic autoimmune.

Transplantation of isolated islets has been proposed to be a potential treatment for type 1 diabetes. Here we describe a method to isolate islets from mouse pancreata and transplant them to the subcapsular space of the kidney.

マウス膵島の単離と被膜下腎移植のための方法

Since the early pioneering work of Ballinger and Reckard demonstrating that transplantation of islets of Langerhans into diabetic rodents could normalize ...

医学

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 ...

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for liver transplantation die without receiving an organ transplant or are delisted for disease progression. One strategy to increase the donor pool is the utilization of marginal grafts, such as fatty livers, grafts from older donors, or donation after cardiac death (DCD). The current preservation technique of cold static storage is only poorly tolerated by marginal livers resulting in significant organ damage. In addition, cold static organ storage does not allow graft assessment or repair prior to transplantation.
These shortcomings of cold static preservation have triggered an interest in warm perfused organ preservation to reduce cold ischemic injury, assess liver grafts during preservation, and explore the opportunity to repair marginal livers prior to transplantation. The optimal pressure and flow conditions, perfusion temperature, composition of the perfusion solution and the need for an oxygen carrier has been controversial in the past.
In spite of promising results in several animal studies, the complexity and the costs have prevented a broader clinical application so far. Recently, with enhanced technology and a better understanding of liver physiology during ex vivo perfusion the outcome of warm liver perfusion has improved and consistently good results can be achieved.
This paper will provide information about liver retrieval, storage techniques, and isolated liver perfusion in pigs. We will illustrate a) the requirements to ensure sufficient oxygen supply to the organ, b) technical considerations about the perfusion machine and the perfusion solution, and c) biochemical aspects of isolated organs.

Technique of Subnormothermic Ex Vivo Liver Perfusion for the Storage, Assessment, and Repair of Marginal Liver Grafts

Marginal grafts, such as fatty livers, grafts from older donors, or livers retrieved after cardiac death (DCD) tolerate conventional, cold static storage only poorly. We developed a novel model of subnormothermic ex vivo liver perfusion for preservation, assessment, and repair of marginal liver grafts prior to transplantation.

Subnormothermicのテクニック<em&gt;生体外</em&gt;肝臓ストレージ、評価のための灌流、およびマージナル肝移植片の修復

The success of liver transplantation has resulted in a dramatic organ shortage. In most transplant regions 20-30% of patients on the waiting list for ...

Murine Heterotopic Heart Transplant Technique

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become proficient in and utilize this technique, it is now widely used by many laboratories around the world. A significant refinement to the original technique was developed and reported in 2001 by Niimi. Described here are the techniques that have evolved over more than a decade in the hands of three surgeons (Plenter, Grazia, Pietra) in our center. These techniques are now being passed on to a younger generation of surgeons and researchers.
Based largely on the Niimi experience, the procedures used have evolved in the fine details - details which we will endeavor to relate here in such a way that others may be able to use this very useful model. Like Niimi, we have found that a video aid to learning is a priceless resource for the beginner.

The manuscript describes the steps required to perform the heterotopic heart transplant in the mouse.

ネズミ異心臓移植技術

It is now over forty years since this technique was first reported by Corry, Wynn and Russell. Although it took some years for other labs to become ...

Technique of Porcine Liver Procurement and Orthotopic Transplantation using an Active Porto-Caval Shunt

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation waiting list die without receiving an organ transplant or are delisted due to disease progression. Even after a successful transplantation, rejection and side effects of immunosuppression remain major concerns for graft survival and patient morbidity. 
Experimental animal research has been essential to the success of liver transplantation and still plays a pivotal role in the development of clinical transplantation practice. In particular, the porcine orthotopic liver transplantation model (OLTx) is optimal for clinically oriented research for its close resemblance to human size, anatomy, and physiology. 
Decompression of intestinal congestion during the anhepatic phase of porcine OLTx is important to guarantee reliable animal survival. The use of an active porto-caval-jugular shunt achieves excellent intestinal decompression. The system can be used for short-term as well as long-term survival experiments. The following protocol contains all technical information for a stable and reproducible liver transplantation model in pigs including post-operative animal care.

Experimental animal research plays a pivotal role in the development of clinical transplantation practice. The porcine orthotopic liver transplantation model (OLTx) closely resembles human conditions and is frequently used in clinically oriented research. The following protocol contains all information for a reliable porcine OLTx model using an active porto-caval-jugular shunt.

アクティブポルト大静脈シャントを使用して、ブタ肝臓調達と同所移植の技術

The success of liver transplantation has resulted in a dramatic organ shortage. Each year, a considerable number of patients on the liver transplantation ...

Mouse Model of Alloimmune-induced Vascular Rejection and Transplant Arteriosclerosis

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune system of the recipient causes damage of the arterial wall and dysfunction of endothelial cells and smooth muscle cells. This triggers a pathological repair response that is characterized by intimal thickening and luminal occlusion. Understanding the mechanisms by which the immune system causes vasculature rejection and TA may inform the development of novel ways to manage graft failure. Here, we describe a mouse aortic interposition model that can be used to study the pathogenic mechanisms of vascular rejection and TA. The model involves grafting of an aortic segment from a donor animal into an allogeneic recipient. Rejection of the artery segment involves alloimmune reactions and results in arterial changes that resemble vascular rejection. The basic technical approach we describe can be used with different mouse strains and targeted interventions to answer specific questions related to vascular rejection and TA.

We describe a protocol for aortic interposition grafting in mice. The goal of the protocol is to provide a model with which to study pathological processes and therapeutic strategies relevant to alloimmune reactions in arteries and the resultant arterial changes that contribute to organ transplant failure.

同種免疫により誘導される血管拒絶反応および移植動脈硬化症のマウスモデル

Vascular rejection that leads to transplant arteriosclerosis (TA) is the leading representation of chronic heart transplant failure. In TA, the immune ...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a serious problem. Therefore, the acceptance criteria for organ donors have been extended leading to the usage of marginal kidney grafts. These marginal organs tolerate cold storage poorly resulting in increased preservation injury and higher rates of delayed graft function. To overcome the limitations of cold storage, extensive research is focused on alternative normothermic preservation methods.
Ex vivo normothermic organ perfusion is an innovative preservation technique. The first experimental and clinical trials for ex vivo lung, liver, and kidney perfusions demonstrated favorable outcomes.
In addition to the reduction of cold ischemic injury, the method of normothermic kidney storage offers the opportunity for organ assessment and repair. This manuscript provides information about kidney retrieval, organ preservation techniques, and isolated ex vivo normothermic kidney perfusion (NEVKP) in a porcine model. Surgical techniques, set up for the perfusion solution and the circuit, potential assessment options, and representative results are demonstrated.

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to &#160;transplantation.

正常温度の<em&gt;エクスビボ</em&gt;腎臓の保全のための腎臓の灌流は移植前の移植片

Kidney transplantation has become a well-established treatment option for patients with end-stage renal failure. The persisting organ shortage remains a ...

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An &amp;#8220;All-laser&amp;#8221; Endothelial Transplant

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

「オールレーザー」内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

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 ...

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. ...

マウスの腎臓移植​​技術

マウスの腎臓移植技術