滅菌野の1セットアップ<ol><li>キャップとマスクを含むドン適切な服装。時の処理室での目の保護具を着用してください。 </li><li>処理領域を準備します。処理室を使用する場合は、室内の正圧を作成するために、ファンの連続運転を維持する。 </li><li>すべての電源を収集します。有効期限は、パッケージの整合性、および滅菌インジケータを確認してください。 </li><li>手を洗うと非滅菌手袋を着用。 </li><li>滅菌野を確立するために、無菌キットを開きます。処理室を使用する場合は、テーブルの上にキットを配置し、無菌的にラップを開きます。このセグメントは、非無菌考えられているように無菌領域の一部としてラップの外側の1.5インチを利用しないでください。 </li><li>オープン無菌技術を使用し、あらかじめ作られたパックの既に一部がない限り無菌領域に無菌のアイテムをドロップします。 </li><li>それは無菌フィールドの上になるように顕微鏡を移動し、顕微鏡の光源をオンにし、必要に応じて接眼レンズを調整します。 </li><li> OBTAIN角膜組織は、準備無菌処理領域の横の場所（保存メディアに）取り除かれる。閲覧室のキャップをゆるめ、それが従事していないスレッドをチャンバーベースを見ての上に休ませ。非滅菌手袋を破棄します。 </li><li>タオルで滅菌手袋及び滅菌ガウンのいずれかのペアを開きます。 </li><li>外科スクラブを実行します。無菌ガウンを着用し、滅菌手袋。 </li><li>オープン計器箱すべての電源がアクセスできるようにします。作業領域を作成するためのオープン水分不透過ドレープ。 </li><li> 5ccのシリンジに針を取り付けます。止血鉗子を使用して、ベベルから曲げ1mmの配置することによって30 G針を準備し、ベースから別の曲げ1mmの（「S」形状を形成する）。 </li><li>シリンジにプランジャーを置き、指がプランジャーを締めて、トリパンブルー染色用ガラス注射器を組み立てます。カニューレを取り付けます。 </li><li>平衡塩類溶液（BSS）瓶にカニューレを取り付けます。 </li><li>真空ブロックからトレフィンパンチを切り離し、脇に置きます。顕微鏡の視野内に真空ブロックをセンタリング。 </li><li>顕微鏡の設定を調整することで焦点に真空ブロックを持参してください。 </li></ol>ドナー組織の準備のために2。手順<ol><li>バリアとして箔の正方形を使用して、チャンバの視聴リフト蓋。反対側の手で鉗子を使用して、静かに角膜を除去するために強膜リムによって角膜を持ってください。チャンバーを見るの蓋を交換してください。 </li><li>完全に、真空ブロック上に角膜上皮側を下に置き、吸引シリンジを押し下げ、それが中央に配置されていることを確認します。 （ 図1） </li><li>ガイドポストを用いた真空ブロックに軽く下側座リング。ゆっくりと吸引注射器のピストンを解放することによって真空に係合する。座席リングを取り外し、脇に置きます。 （ 図2）。 </li><li>デスメ膜を通ってゴールを部分的穿孔を実行します。 </li><li>組織が十分に可視化されるように、顕微鏡ズームとフォーカスを調整します。 </li><li&gt;真空ブロックを傾け、余分な培地を除去するために角膜輪部に配置された滅菌綿棒の槍を使用しています。 （ 図3） </li><li>得点デスメ膜の端部を染色するために角膜輪部で十分な汚れを配置します。汚れが60-90秒の内皮上に残るようにします。 （ 図4） </li><li>ステインを除去します。 <ol><li>角膜のオフとガーゼパッド上に汚れを注ぐために真空ブロックを傾けます。 </li><li>内皮に​​触れないように注意しながら、角膜輪部の領域から余分な汚れを除去するための手術用槍を使用してください。 （ 図5） </li></ol></li><li>過剰BSSを除去するために染色液、および滅菌綿棒の槍の角膜をリンスし、BSSの数滴を使用してください。 （ 図6） </li><li>乾燥から組織を防止するために、強膜のリムにBSSの1〜2滴を配置します。作業中は、必要に応じて繰り返します。 </li><li>デスメ膜を部分的に解剖を実行します。 <ol><li>チューブが接続するところから始めてオープン鉗子を用いた真空ブロックへのsは、スリキズの端に1チップを置き、静かに間質からデスメ膜と内皮を分離する。 （ 図7）。 </li><li>得点エッジから1ミリメートルを超えていないデスメ膜と内皮セパレートや周りの解剖360°を続ける。 （ 図8）。 </li><li>皮膚マーカーと真空ブロック上にドットを配置することによって、任意のマイクロ涙の場所をメモします。 注：トレフィンは、必要に応じて染色の第2の適用に続いて、デスメ膜を完全に貫通して浸透し、必要に応じてデスメ膜を通してのスコアを完了するために準備された30 G針の傾斜エッジを使用しなかった場合。デスメ膜の密着性が部分的な解剖を妨害されている場合は、必要に応じて、trephinatedエリア外の内皮を除去します。 </li></ol></li><li>スリキズと重なるあらゆる内皮タグを削除するには鉗子を使用してください。 </li> &lt;李&gt;は、以前にマークされたマイクロ涙の位置を考慮し、最大は6時位置になるように真空ブロックを回転させる。これは、フラップのヒンジになります。 </li><li>鉗子を使用して、12時位置にデスメ膜を把握する。静かにヒンジに向かって剥がすことによって間質からデスメ膜を分離する。スリキズからの分離2ミリメートルを停止することによって、ヒンジを作成します。 注：フラップの周縁部には、分離中にスクロールを開始します。 （ 図9）。 </li><li>静かに間質上の所定の位置にフラップをバック横たわっていた。内皮をunscrollと元の位置に戻してBSSと綿棒槍を使用してください。 （ 図10A-C） </li><li>フラップが所定の位置に留まるように滅菌綿棒の槍とデスメ膜と間質の間から残留流体を除去します。 （ 図11） </li><li>ヒンジ近くの強膜から余分な水分を吸収するために滅菌綿棒の槍を使用してください。描画するために、皮膚のマーカーを使用して、nは、ヒンジの中心を指す強膜リムに矢じり滅菌綿棒の槍を使用して余分なインクを除去する。 （ 図12） </li><li>真空ブロックに接続された注射器のピストンを押すことにより、真空を解除してください。真空が解除されている間、真空ブロックから角膜を除去するために鉗子を使用しています。 </li><li>バリアとして箔の正方形を使用して、チャンバの視聴リフト蓋。閲覧室に角膜を置き、内皮側を上にし、チャンバーを見るの蓋を交換してください。 </li><li>該当する場合は、マテ角膜とプロシージャのステップの上に繰り返します。第二の組織を処理する前に真空ブロックをきれいにガーゼを使用してください。 </li><li>閲覧室蓋（複数可）を締めて、処理領域から組織を除去する。 </li><li>バイオハザード廃棄物管理計画に従った項目の処分により無菌領域を打破する。汚染された手袋、ガウンを削除します。手を洗うと非滅菌手袋を着用。 </li><li>ポストカット細隙灯評価と角膜のスペキュラーマイクロスコープ（複数可）を実行するとの調査結果を記録する。 </li></ol>

<ol>
	<li>Fernandez, M., Afshari, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+From+DLEK+to+DMEK.">Endothelial Keratoplasty: From DLEK to DMEK.</a> Middle East African Journal of Ophthalmology. 17 (1), 5-8 (2010).</li><li>Kruse, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Stepwise+Approach+to+Donor+Preparation+and+Insertion+Increases+Safety+and+Outcome+of+Descemet+Membrane+Endothelial+Keratoplasty.">A Stepwise Approach to Donor Preparation and Insertion Increases Safety and Outcome of Descemet Membrane Endothelial Keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Dapena, I., Moutsouris, K., Droutsas, K., Ham, L., van Dijk, K., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+'No-Touch'+Technique+for+Descemet+Membrane+Endothelial+Keratoplasty.">Standardized 'No-Touch' Technique for Descemet Membrane Endothelial Keratoplasty.</a> Archives of Ophthalmology. 129, 88-94 (2011).</li><li>Tourtas, T., Laaser, K., Bachmann, B., Cursiefen, C., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+Endothelial+Keratoplasty+versus+Descemet+Stripping+Automated+Endothelial+Keratoplasty.">Descemet Membrane Endothelial Keratoplasty versus Descemet Stripping Automated Endothelial Keratoplasty.</a> American Journal of Ophthalmology. 153 (6), 1082-1090 (2012).</li><li>Guerra, F., Anshu, A., Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Keratoplasty:+fellow+eyes+comparison+of+Descemet+stripping+automated+endothelial+keratoplasty+and+Descemet+membrane+endothelial+keratoplasty.">Endothelial Keratoplasty: fellow eyes comparison of Descemet stripping automated endothelial keratoplasty and Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (12), 1382-1386 (2011).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 2012 Eye Banking Statistical Report: Surgical Use and Indications for Corneal Transplant Statistical Report Analysis. , (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+stripping+endothelial+keratoplasty.">Descemet's stripping endothelial keratoplasty.</a> Current Opinion Ophthalmology. 18 (4), 290-294 (2007).</li><li>Woodward, M., Titus, M., Mavin, K., Shtein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+Donor+Tissue+Preparation+for+Endothelial+Keratoplasty.">Corneal Donor Tissue Preparation for Endothelial Keratoplasty.</a> Journal of Visualized Experiments. (64), e3847 (2012).</li><li>Price, M., Price, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet's+membrane+endothelial+keratoplasty+surgery:+update+on+the+evidence+and+hurdles+to+acceptance.">Descemet's membrane endothelial keratoplasty surgery: update on the evidence and hurdles to acceptance.</a> Current Opinion Ophthalmology. 24 (4), 329-335 (2013).</li><li>Venzano, D., Pagani, P., Randazzo, N., Cabiddu, F., Traverrso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Descemet+Membrane+air-bubble+separation+in+donor+cornea.">Descemet Membrane air-bubble separation in donor cornea.</a> Journal of Cataract and Refractive Surgery. 36 (12), 2022-2027 (2010).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DSEK: What you need to know about endothelial keratoplasty. , (2009).</li><li>Studeny, P., Sivekova, D., Lieheova, K., Vokrojova, M., Kuchynka, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+technique+of+lamellar+keratoplasty+(DMEK-S).">Hybrid technique of lamellar keratoplasty (DMEK-S).</a> Journal of Ophthalmology. 2013, 2013.254383 (2013).</li><li>Muraine, M., Gueudry, J., He, Z., Piselli, S., Lefevre, S., Toubeau, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+technique+for+the+preparation+of+corneal+grafts+for+Descemet+membrane+endothelial+keratoplasty.">Novel technique for the preparation of corneal grafts for Descemet membrane endothelial keratoplasty.</a> American Journal of Ophthalmology. 156 (5), 851-859 (2013).</li><li>Lie, J. T., Birbal, R., Ham, L., van der Wees, J., Melles, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor+tissue+preparation+for+Descemet+membrane+endothelial+keratoplasty.">Donor tissue preparation for Descemet membrane endothelial keratoplasty.</a> Journal of Cataract Refractive Surgery. 34 (9), 1578-1583 (2008).</li><li>Kruse, F. E., Laaser, K., Cursiefen, C., Heindl, L. M., Schlotzer-Schrehardt, U., Riss, S., Bachmann, B. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stepwise+approach+to+donor+preparation+and+insertion+increases+safety+and+outcome+of+Descemet+membrane+endothelial+keratoplasty.">A stepwise approach to donor preparation and insertion increases safety and outcome of Descemet membrane endothelial keratoplasty.</a> Cornea. 30 (5), 580-587 (2011).</li><li>Rudolph, M., Kaaser, L., Bachmann, B., Cursiefen, C., Epstein, D., Kruse, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+higher-order+aberrations+after+Descemet&#8217;s+membrane+endothelial+keratoplasty.">Corneal higher-order aberrations after Descemet&#8217;s membrane endothelial keratoplasty.</a> Ophthalmology. 119 (3), 528-535 (2012).</li><li>Price, F., Price, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+endothelial+keratoplasty.">Evolution of endothelial keratoplasty.</a> Cornea. 32, 28-32 (2013).</li><li>Dapena, I., Dapena, L., Dirisamer, M., Ham, L., Melles, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+acuity+and+endothelial+cell+density+following+Descemet+membrane+endothelial+keratoplasty+(DMEK).">Visual acuity and endothelial cell density following Descemet membrane endothelial keratoplasty (DMEK).</a> Arch Soc Esp Oftalmol. 86 (12), 395-401 (2011).</li></ol>

ハッサンTausif博士Navasujaチャンドラセカランを開示することは何もない。博士。ミアン、Shtein、ウッドワードはミシガンアイバンクのための医療ディレクターである。ミスター物知りさん、タイタス​​、さんとジョンソンは中西部アイバンクの従業員です。

角膜内皮細胞層を脱水を通して、角膜透明度を維持する上で重要な役割を果たしている。内皮細胞の疾患には、重大な視力障害をもたらす可能性がある。 DMEKはデスメ膜と内皮細胞層からなるドナー移植片で、罹患内皮細胞を置き換えることを目指しています。無傷のDMおよび適切な内皮細胞の数がドナー組織標本移植組織の生存に重要である。ドナー組織の準備中にDMを引き裂くことは、組?...

内皮角膜移植（EK）が急速に2012年6年に米国で実行されたすべての移植の50％以上を占めて内皮疾患の治療のための選択の手順として全層角膜移植（PKP）に置き換えられています。PKPは、高い乱視との長時間の視覚リハビリテーションによって制限されている屈折不安定、地球の整合性と縫合関連の合併症の損失。ディープラメラ内皮角膜移植（DLEK）を含む、初期の世代のEK手順は、部分的な厚さのドナー組織の移植による角膜実質の手動解剖を含んでいた。これらの手順は、無傷の角膜表面を維持しながら、ドナー内皮組織を移植することに成功したが、技術的に困難であったと不規則な角膜ラメラインタフェース1に限定最良矯正視力が得られた。選択した現在のEK手順は、角膜クラーを提供していますデスメストリップ内皮角膜移植（DSEK）ですビジョンの迅速な回復とITYは、術後の屈折異常や非点収差を最小限に抑えて、角膜表面7を維持しながら、外傷性創傷裂開のリスクを減少させる。この手順は、新しい内皮細胞層を含む部分的な厚ドナー角膜組織と罹患したデスメ膜および内皮および交換の除去を含む。ドナー角膜は8移植さ後部組織を調製するためにマイクロケラトームで解剖されている。人目銀行によるドナー組織の調製急速な成長に尽力して、間違いなく、この方法の成功を収めている。 2005年から2012年の間に米国のDSEK手続における近16倍の増加が、現在、アイバンク6により調製される組織のほぼ半分に対応する。 DSEKは20/40以上に視力を向上させることができますが、ほとんどの患者は、間質インターフェースヘイズ、増加した光に20分の20のビジョンを達成しないドナー間質組織7の添加から飛散。 EK手術法は、ドナーデスメ膜（DM）と内皮の移植を伴うデスメ膜内皮角膜移植（DMEK）に進んでいる。この手順では、20分の20のビジョンを達成するための患者の割合が高く、より高速視力回復、および内皮除去9の低いリスクを持っています。しかし、ドナー組織の準備が原因シンナー移植片の寸法により困難である。

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:268px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:216px;">VisionBlue&#160;</td>
			<td style="width:268px;">D.O.R.C., Zuiland, The Netherlands</td>
			<td style="width:119px;">NA</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Barron vacuum punch</td>
			<td style="width:268px;">Katena, Denville, New Jersey</td>
			<td style="width:119px;">K20-2112</td>
			<td></td>
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</table>

corneal donor tissue preparation for descemet's membrane endothelial keratoplasty

デスメ膜内皮角膜移植（DMEK）は基底膜（デスメ膜）と一緒に、単一の細胞層、角膜内皮が、受信者の後部間質3上に導入された角膜移植の一形態である。追加のドナー間質が導入されたデスメストリップ自動内皮角膜移植（DSAEK）とは異なり、不自然な間質対間質インターフェイスが作成されません。その結果、角膜の自然な解剖学的構造は、改善された回復時間および視力4を可能にする可能な限り保存される。内皮角膜移植（EK）は、内皮機能不全の治療のための選択の手順である。 EKの利点は小さいため、周辺切開1の使用急速な視力の回復、眼の完全性と最小限の屈折率変化の保存が含まれる。 DSAEKは、部分的な厚さ間質および内皮を用いて調製ドナー組織を利用しています。急速SUCCESおよびこの手順の利用は、アイバンクの準備プレカット組織の利用可能性に起因することができます。ドナー組織のアイバンクの準備の利点は、製造の間、組織の穿孔の場合には、バックアップドナー組織の手術室と可用性における特殊な装置の必要性の排除が含まれる。また、アイバンク技術者によるドナー組織の大量調製は、ドナー組織の改善された品質を提供することができる。 DSAEK起因ドナーとレシピエントの角膜の間質界面の構築に最善の矯正視力が限られている場合があります。 DMEKで唯一のドナーデスメ膜と内皮の移植にこのインタフェースの除去は、視覚的な転帰を改善し、EK 5の後の合併症を減少させることができる。 DSAEK、長期的な成功とDMEKの受け入れと同様にプレカットの入手のしやすさに依存している、アイバンクは、ドナー組織を作成した。ここでは、いくつかのゴシックを減少させることができるドナー組織の準備に段階的アプローチを提示ERSアイバンクがDMEK移植片を提供することに直面しています。

間質からのDMおよび内皮の適切な分離はDMEK用プレカット組織を調製するのに不可欠です。 65±6歳（範囲55〜75）、2616±321細胞/ mm 2の5.9±2.3日（範囲1-10）および内皮細胞密度の処理時間に死（ECD）（の平均年齢五十ドナー角膜範囲2,049-3,247）は、二つのアイバンクの技術者によって外科調製技術の実現可能性および再現性を評価した。成功率は、最初の25の場合には72％と76％の全体的な?...

Watch this Scientific Journal Video about Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty at JoVE.com

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

内皮角膜移植は、進歩がそれぞれ連続して1シンナー移植片の移植につながるように継続的に進化する手術法です。ここでは、内皮および移植のためのドナー2強角膜ボタンの中から、デスメ膜を安全かつ反復可能な分離を可能にする制御された手動の組織切開する手法を提示する。

デスメ膜内皮角膜移植のための角膜ドナー組織の準備

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

corneal-donor-tissue-preparation-for-descemet-s-membrane-endothelial

Research

JoVE Journal

Medicine

15.2K ビュー.  University of Michigan. 内皮角膜移植は、進歩がそれぞれ連続して1シンナー移植片の移植につながるように継続的に進化する手術法です。ここでは、内皮および移植のためのドナー2強角膜ボタンの中から、デスメ膜を安全かつ反復可能な分離を可能にする制御された手動の組織切開する手法を提示する。 

ビデオ: Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Single Port Donor Nephrectomy

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology. Since that time, the use of single port surgery for nephrectomy has expanded to include donor nephrectomy. Over the next two years the technique was adopted for many others types of nephrectomies to include donor nephrectomy. We present our technique for single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in over 100 patients and add this experience to our experience of over 1000 laparoscopic nephrectomies. With the proper equipment and technique, single port donor nephrectomy can be performed safely and effectively in the majority of live donors. We have found that our operative times and most importantly our transplant outcomes have not changed significantly with the adoption of the single port donor nephrectomy. We believe that single port donor nephrectomy represents a step forward in the care of living donors.

Single port laparoscopic surgery is changing the standard of care in surgical care like nothing since the laparoscopic technique was introduced 20 years ago. We present out technique of single port donor nephrectomy using the Gelpoint device. We have successfully performed this surgery in 100 patients.

シングルポートのドナー腎摘出

In 2007, Rane presented the first single port nephrectomy for a small non-functioning kidney at the World Congress of Endourology.  Since that time, the ...

医学

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

内皮角膜移植のために角膜ドナー組織の準備

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

Ex Situ Normothermic Machine Perfusion of Donor Livers

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous perfusion of organs provides the opportunity to improve organ quality and allows ex situ viability assessment of donor livers prior to transplantation. This video article provides a step by step protocol for ex situ normothermic machine perfusion (37 &#176;C) of human donor livers using a device that provides a pressure and temperature controlled pulsatile perfusion of the hepatic artery and continuous perfusion of the portal vein. The perfusion fluid is oxygenated by two hollow fiber membrane oxygenators and the temperature can be regulated between 10 &#176;C and 37 &#176;C. During perfusion, the metabolic activity of the liver as well as the degree of injury can be assessed by biochemical analysis of samples taken from the perfusion fluid. Machine perfusion is a very promising tool to increase the number of livers that are suitable for transplantation.

Ex Situ Normothermic Machine Perfusion of Donor Livers

Here we present a protocol describing oxygenated ex situ machine perfusion of donor liver grafts. This article contains a step by step protocol to procure and prepare the liver graft for machine perfusion, prepare the perfusion fluid, prime the perfusion machine and perform oxygenated normothermic machine perfusion of the liver graft.

In contrast to conventional static cold preservation (0-4 &#176;C), ex situ machine perfusion may provide better preservation of donor livers. Continuous ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene and cell therapies are highly promising alternative approaches for CVD treatment. However, the broad clinical application of gene therapy is greatly limited by the lack of suitable gene delivery systems. The development of appropriate gene delivery vectors can provide a solution to current challenges in cell therapy. In particular, existing drawbacks, such as limited efficiency and low cell retention in the injured organ, could be overcome by appropriate cell engineering (i.e., genetic) prior to transplantation. The presented protocol describes the efficient and safe transient modification of endothelial cells using a polyethyleneimine superparamagnetic magnetic nanoparticle (PEI/MNP)-based delivery vector. Also, the algorithm and methods for cell characterization are defined. The successful intracellular delivery of microRNA (miR) into human umbilical vein endothelial cells (HUVECs) has been achieved without affecting cell viability, functionality, or intercellular communication. Moreover, this approach was proven to cause a strong functional effect in introduced exogenous miR. Importantly, the application of this MNP-based vector ensures cell magnetization, with accompanying possibilities of magnetic targeting and non-invasive MRI tracing. This may provide a basis for magnetically guided, genetically engineered cell therapeutics that can be monitored non-invasively with MRI.

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

This manuscript describes the efficient, non-viral delivery of miR to endothelial cells by a PEI/MNP vector and their magnetization. Thus, in addition to genetic modification, this approach allows for magnetic cell guidance and MRI detectability. The technique can be used to improve the characteristics of therapeutic cell products.

準備と<em&gt;インビトロ</em磁化のmiR修飾内皮細胞の&gt;キャラクタリゼーション

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

分割角膜ボタンを用いてのブタ角膜内皮器培養モデル

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Rodent Heart and Brain Tissue Preparation for Digital Macro Photography after Ischemia-reperfusion

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue preparation and subsequent image capture are steps performed immediately after the ischemia-reperfusion (IR) experiment and must be performed in a timely manner and with appropriate care. For the evaluation of IR-induced damage in the heart and brain, this paper describes 2,3,5-triphenyl-2H-tetrazolium chloride (TTC)-based staining followed by macro photography. Scientific macro photography requires controlled lighting and an appropriate imaging setup. The standardized methodology ensures high-quality, detailed digital images even if a combination of an inexpensive up-to-date digital camera and macro lens is used. Proper techniques and potential mistakes in sample preparation and image acquisition are discussed, and examples of the influence of correct and incorrect setups on image quality are provided. Specific tips are provided on how to avoid common mistakes, such as overstaining, improper sample storage, and suboptimal lighting conditions. This paper shows the appropriate methodology for rat heart and brain tissue slicing and staining and provides guidelines for establishing lighting and camera setups and photography techniques for high-resolution image acquisition.

Presented here is a protocol for the standardized methodology of rodent tissue preparation after the ischemia-reperfusion experiment and guidelines for establishing lighting and camera setups for high-resolution image acquisition. This method is applicable to all experimental small-animal organ photography.

虚血再灌流後のデジタルマクロ写真のためのげっ歯類の心臓および脳組織の準備

Macro photography is applicable for imaging various tissue samples at high magnification to perform qualitative and quantitative analyses. Tissue ...

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

角膜創傷治癒を研究するための上皮擦過傷モデル

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

A Porcine Heterotopic Heart Transplantation Protocol for Delivery of Therapeutics to a Cardiac Allograft

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts and complications such as primary graft dysfunction and graft rejection. The recent clinical use of an ex vivo perfusion device in cardiac transplantation introduces a unique opportunity for treating cardiac allografts with therapeutic interventions to improve function and avoid deleterious recipient responses. Establishing a translational, large-animal model for therapeutic delivery to the entire allograft is essential for testing novel therapeutic approaches in cardiac transplantation. The porcine, heterotopic heart transplantation model in the intraabdominal position serves as an excellent model for assessing the effects of novel interventions and the immunopathology of graft rejection. This model additionally offers long-term survival for the pig, given that the graft is not required to maintain the recipient's circulation. The aim of this protocol is to provide a reproducible and robust approach for achieving ex vivo delivery of a therapeutic to the entire cardiac allograft prior to transplantation and provide technical details to perform a survival heterotopic transplant of the ex vivo perfused heart.

We present a protocol for utilizing a normothermic ex vivo sanguinous perfusion system for the delivery of therapeutics to an entire cardiac allograft in a porcine heterotopic heart transplant model.

心臓同種移植への治療薬の送達のためのブタヘテロトピック心臓移植プロトコル

Cardiac transplantation is the gold standard treatment for end-stage heart failure. However, it remains limited by the number of available donor hearts ...