著者らは、部分的に研究活動をサポートし、FP7 BiophotonicPlusプロジェクト「LITE」が付与されたトスカーナ地方（POR CREO FESR 2007-2013、坂東ユニコR＆S 2012）から資金提供を受けFORTEプロジェクト、EU FP7 ECHORD ++実験LA-ROSESに感謝したいですトスカーナ地域による。

研究は、病院の倫理委員会の承認を得て、将来に向かって行きました。インフォームドコンセントを得ました。研究は、ヘルシンキ宣言の教義を遵守していました。 1.ドナー内皮準備<ol><li>室温でローカルアイバンクにより調製したドナー角膜を使用してください。 </li><li>手術室では、その送達コンテナからドナー角膜を引き出し、角膜の輸送に使用されている組織保存と栄養のためのソリューションを離れて設定します。 </li><li>液体が流れ、可変圧力を作成することができますその中に人工前房、上のドナー角膜 - 強膜リムを配置。組織保持ヘッドで角膜をカバーしています。 </li><li>角膜の保存と栄養の液体で満たされたシリンジに3ウェイコネクタを介して人工前房（AAC）を接続します。 </li><li>所定の位置にしっかり保持器を保持するために圧縮リングを使用してください。 </li><li> SYRを使用してください（1.7参照）を最適圧力に達するまで組織保存溶液で前房を満たす：房内の圧力を維持するために、AACに接続インゲ。コネクタを閉じます。 </li><li>指で前方のチャンバー内の圧力をテストします。 （範囲60〜70 mmHgの中で）右内の圧力に到達するまでの内部の圧力を変更します。 3ウェイコネクタを閉じます。 </li><li>光コヒーレンスThomography（OCT）とドナー角膜の厚さを測定します。機器の光学系の前に固定された位置に、その角膜と前房を維持した後、全厚-OCT取得を取ります。 </li></ol>ドナー内皮の2フェムト秒レーザーの準備<ol><li>フェムト秒レーザ装置の下でドナー角膜の圧平を実行します。後部サイドカット、完全な層状と前サイドカット：3その後のカット​​で、ドナー組織を切断するfemtosecレーザーを使用してください。 3カットのために、以下のパラメータを設定します。<ol><li>フルラメラカット（ラスターパターン、アウトスタート）の場合は、必ず95ミクロンを減算、ドナー角膜（1.8で説明したように10月で測定）の最も薄い点に対応する切り込み深さを設定します。範囲（作業深さに応じて）0.8〜0.9μJにパルスエネルギーを設定します。 8.7ミリメートルに直径を設定します。 2ミクロンの接線スポット分離を設定します。 2μmのラジアルスポット分離を設定します。 </li><li>前方サイドカットについては、前回の完全ラメラカット30μmより深い後部の深さを設定します。 2.10μJにパルスエネルギーを設定します。 8.6ミリメートルの直径。層分離しながら3以上3μm以下にスポット分離、。 </li><li>後部サイドカットのために、後側の前方深さを見つける前方フルラメラカット30μmよりをカット。 900ミクロンの後方の深さを設定します。 2.10μJにパルスエネルギーを設定します。 8.3ミリメートルの直径。 2ミクロンのスポット分離; 2ミクロンの層分離。 </li></ol></lI&gt; </ol> 3.受信者の目の準備<ol><li>手術のために患者を準備します。刃を予め較正4.0ミリメートルと12時1 4.00ミリメートル角膜切開を行います。 2時位置に配置された1.2ミリメートル予め較正刃と1輪部穿刺を作ります。 30°刺すナイフで6時位置に別の輪部穿刺を行います。 </li><li> 2時穿刺を介して、患者の前室に前室のメンテナを挿入します。 </li><li>専用フックで1 8.2ミリメートルの直径の円形descemetorexisを実行します。 </li><li>後部間質からデスメ膜および内皮を除去し、デスメフックで組織を削除します。 </li></ol> 4.発色団の準備<ol><li> 1.5ミリリットルマイクロチューブにインドシアニングリーン粉末1mgを置きます。 </li><li>滅菌水の9 mgの追加（ICG水溶液はw / wの10％です）。 </li><li>手動金属攪拌機を備えたICG粉末と水を混ぜます。 </lI&gt; </ol>ドナー内皮の5染色<ol><li>インジェクタ表面に接触する内側と、ビジネスサービスインジェクタの広い部分にドナー内皮を置きます。 </li><li>スパチュラを使用して、その周辺部に、発色団溶液でドナー内皮の内側を染色します。正しく染色された組織は、均一な緑がかった色をしています。ステップ5.3を起動する前に3分を待ちます。 </li><li>ビジネスサービスインジェクタの前方部分にドナー内皮を引き出し、ロールバックされます。 </li></ol> 6.ドナー内皮の挿入<ol><li>内皮レンチキュラのための非外傷性の同軸鉗子を使用して、折り畳まれたドナー内皮をつかみ、挿入します。 </li><li>前房のメンテナを削除します。 </li><li>ナイロン10.0単一シュティッヒと角膜切開と（ステップ3.1で実行）穿刺を縫合。 </li><li>ドナー組織を展開し、受信者の角膜に対してそれを押すように気泡を注入します。気泡必見カンプletely前房の空間を埋めます。 </li></ol> 7.レーザー溶接<ol><li>フックやヘラで角膜の外側から気泡を移動させると内側から、受信者の角膜の中心にドナーレンチキュラを配置します。 </li><li>開口数（NA）として0.22で、300μmのコア径の滅菌光ファイバを装備した810 nmの発光ダイオードレーザーを使用します。 </li><li>眼球外ファイバ先端を維持し、透明な角膜組織を通して、染色された内皮に向かってレーザ光を実現します。ファイバチップは、非接触構造です。 </li><li>単一スポット発光モード、70〜80ミリ秒のパルス持続時間、パルス当たり35〜40ミリジュール：レーザーは、次の設定を使用します。彼は-Neが上のビームを目指しています。 </li><li>ドナー内皮の染色周囲に単一のレーザスポットを提供します。順次スポットを配信：最終的な側面は、ドナーレンチキュラの周辺のスポットの環です。 2 ADJA間の距離セントスポットセンタースポット径のダブルです。 </li><li> 0.3％トブラマイシンおよび0.1％デキサメタゾン眼科用懸濁液と一緒に、患者の目にコンタクトレンズを適用します。 </li></ol>

<ol>
	<li>Tan, D. T., Dart, J. K., Holland, E. J., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+transplantation.">Corneal transplantation.</a> Lancet. 379 (9827), 1749-1761 (2012).</li><li>El Husseiny, M. A., Manero, F., Gris, O., Elies, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+Review+and+Update+of+Surgical+Treatment+for+Corneal+Endothelial+Diseases.">Historical Review and Update of Surgical Treatment for Corneal Endothelial Diseases.</a> Ophthalmol Ther. , (2014).</li><li>Rossi, 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=Laser+tissue+welding+in+ophthalmic+surgery.">Laser tissue welding in ophthalmic surgery.</a> J Biophotonics. 1 (4), 331-342 (2008).</li><li>Pini, R., 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=Combining+femtosecond+laser+ablation+and+diode+laser+welding+in+lamellar+and+endothelial+corneal+transplants.">Combining femtosecond laser ablation and diode laser welding in lamellar and endothelial corneal transplants.</a> Proc. SPIE Ophthalmic Technologies XVIII. 6 (844), 684411-1-684411-7 (2008).</li><li>Rossi, 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=All-laser'+endothelial+corneal+transplant+in+human+patients.">All-laser' endothelial corneal transplant in human patients.</a> Proc. SPIE Ophthalmic Technologies XXII. 8209, 82091O-1-82091O-3 (2012).</li><li>Rossi, F., Pini, R., Menabuoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+and+model+analysis+on+the+temperature+dynamics+during+diode+laser+welding+of+the+cornea.">Experimental and model analysis on the temperature dynamics during diode laser welding of the cornea.</a> J Biomed Opt. 12 (1), 014031-1-014031-7 (2007).</li><li>Menabuoni, L., 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=Laser-assisted+corneal+welding+in+cataract+surgery:+retrospective+study.">Laser-assisted corneal welding in cataract surgery: retrospective study.</a> J Cataract Refract Surg. 33 (9), 1608-1612 (2007).</li><li>Buzzonetti, L., 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=Laser+Welding+in+Penetrating+Keratoplasty+and+Cataract+Surgery+in+Pediatric+Patients.+Early+Results.">Laser Welding in Penetrating Keratoplasty and Cataract Surgery in Pediatric Patients. Early Results.</a> J Cataract Refract Surg. 39 (12), 1829-1834 (2013).</li><li>Menabuoni, L., 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=The+'anvil'+profile+in+femtosecond+laser-assistedpenetrating+keratoplasty.">The 'anvil' profile in femtosecond laser-assistedpenetrating keratoplasty.</a> Acta Ophthalmol. 91 (6), e494-e495 (2013).</li><li>Canovetti, 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=Laser-assisted+penetrating+keratoplasty:+one+year&#8217;s+results+in+patients,+using+a+laser-welded+&#8220;anvil&#8221;-profiled+graft.">Laser-assisted penetrating keratoplasty: one year&#8217;s results in patients, using a laser-welded &#8220;anvil&#8221;-profiled graft.</a> Am J Ophthalmol. 158 (4), 664-670 (2014).</li></ol>

The authors declare that they have no competing financial interests.

「すべてのレーザー「内皮移植は低侵襲性角膜移植に独創的なアプローチです。 プロトコル内で説明するすべての手順は、滅菌手袋、ガウン、マスクとキャップの使用などの手術中に一般的な慣行である衛生と消毒の手順を観察し、手術室で行いました。 ICG溶液は、すぐにドナー内皮の染色におけるその適用前に、手術室で調製しました。 ICG粉末、水および染色溶液?...

本研究では、ドナー組織と受信者のベッドの上に溶接する近赤外ダイオードレーザーを製造するためのフェムト秒レーザーの使用に基づいて内皮角膜移植への独創的なアプローチを提示します。ドナー角膜の術中測定が正しくドナー組織の大きさを設計する必要があります。内皮角膜内皮疾患1,2の治療に貫通角膜移植を置き換えるために提案されています。この技術の主な利点は、手術、移植片拒絶のリスクが減少し、目の整合性の保存時に角膜移植、減少麻酔を貫通に対して、より高速視力回復です。主なリスク要因は、手術後のドナーレンチキュラー転位です。標準的な技術は、それが気泡の注入によって維持され、その最終的な位置にドナー内皮を挿入することによって実行されます。何の縫合糸があるため、機械的な生物物理学的および次元キャラの使用されていません内皮のcteristics。また、視力の回復が原因で、移植厚い組織に主な理由は、ドナーとレシピエントの組織との間のミスマッチのために制限することができます。 ここでは、これらの主要な問題を克服することができる内皮角膜移植を行う際の手順を紹介します。ドナー内皮はレーザー溶接技術を用いて、その最終位置に確保することができます。これは、制御され、ローカライズされた光熱過程である：それは、ドナー/レシピエントの界面に誘導することができます。これは、過去10年間に研究し、角膜を貫通および内皮3-5の移植で提案されています。近赤外光（波長810 nm）の低電力ダイオードレーザーによって放射され、創傷部位での生体組織に向かって送出されます。角膜は、この波長に対して透明であり、当然ながら、レーザ光を吸収するために、この組織を作るためには、発色団でそれを染色する必要があります。提案された染料は、無菌飽和でありますインドシアニングリーン（ICG）のd水溶液。我々は、角膜組織が ​​適切にこのICG製剤で染色されている場合、それは810ナノメートル6に吸収ピークを示すことを実証しました。また、ICGは、広く臨床診断で使用され、その安全性は既にヒト被験体において実証されています。染色された角膜は、ダイオードレーザー光のエネルギーを吸収し、得られた主な効果は、溶接部位での制御された温度上昇です。熱的効果は無染色の組織において誘導されていません。温度拡張機能は、冷却時の傷の壁のすぐclosuringで、間質コラーゲンで可逆的熱変性を誘導します。このレーザ溶接効果がまず白内障手術7,8及び角膜移植9,10を貫通するに実証されました。私たちはこの論文で提示されている最適化されたアプローチは、内皮角膜移植に応用するために研究されています。 提案された手術では、単一のレーザスポット（ミリ秒の持続十）AReは、スポット寸法（直径ミクロンの数百）内に局在光熱効果で、その結果、組織に送達：誘導された効果は、ドナー/ホストインターフェイスに閉じ込められたコラーゲンの光凝固からなる、ハードレーザー溶接です。溶接部位でのコラーゲンの変性の結果は、このように標準的な技術（ステッチ）で取得することは不可能である縫合効果を提供する、ドナーとホスト組織との間に強い接着力です。組織は、短いフォローアップ（1ヶ月）に彼の自然な外観を取り戻し、ドナー/宿主組織との間の接着は、治癒期の非常に早い段階で提供溶接することによって改善されます。 厚いドナー組織の移植、すなわち内皮角膜移植の他の主要な危険を回避するために、intrasurgical光コヒーレンストモグラフィー（OCT）を使用する：正しいカットプロファイルで設計することができるように、商用デバイスは、ドナー角膜の厚さを測定しますザfemtosecレーザー。提案された「すべてのレーザー「内皮移植は、このように、この低侵襲手術の臨床結果を改善するように思われます。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Indocyanine Green</td>
			<td>Pulsion Medical Systems, Germany</td>
			<td>ICG-PULSION (http://www.pulsion.com/international-english/perfusion/icg-pulsion/)</td>
			<td>Alternative product: IC-GREEN, Akorn&#160; Inc., Lake Forest, Illinois- US (http://www.icginjection.com/)</td>
		</tr>
		<tr>
			<td>Femtosecond Laser&#160;</td>
			<td>Abbott Medical Optics,&#160; Abbott Laboratories Inc. Abbott Park, Illinois, USA</td>
			<td>iFS150 (http://www.abbottmedicaloptics.com/products/refractive/ilasik/ifs-advanced-femtosecond-laser)</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical Coherence Tomography (OCT)</td>
			<td>Carl-Zeiss Meditec, Dublin, California- US (http://www.zeiss.com/meditec/en_de/home.html)</td>
			<td>Visante</td>
			<td></td>
		</tr>
		<tr>
			<td>Diode Laser</td>
			<td>E.l.En. Group s.pa., Calenzano-FI, Italy (http://www.elengroup.com/en/divisions/medical)</td>
			<td>Mod. WELD 800</td>
			<td></td>
		</tr>
		<tr>
			<td>Artificial Anterior Chamber</td>
			<td>CORONET, corneal graft products. Network Medical Products Ltd. Coronet House, Kearsley Road, Ripon, North Yorkshire, HG4 2SG, UK</td>
			<td>&#160;Artificial Anterior Chamber (A.A.C.) with large and small tissue-retaining heads. Code 51-935 (http://www.networkmedical.co.uk/ophthalmic_artificial_anterior_chamber.html)</td>
			<td></td>
		</tr>
		<tr>
			<td>Solution for tissue preservation and nutrition&#160;&#160;</td>
			<td>&#160;AL.CHI.MI.A. Srl, Viale Austria 14, 35020 - Ponte S. Nicol&#242; - PD 
			&#160;ITALY</td>
			<td>Carry-C media for corneal deturgescence and transport at room temperature - 12 x 50 ml (http://www.alchimiasrl.com/en/organ-culture-at-31&#176;-c-eb/carry-c-eb)</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

「全てのレーザ」外科手術を低侵襲角膜移植を行うことが提案されています。手順は、（ 図1参照）を実行するのは簡単である：標準内皮移植に関して、角膜の厚さを測定ドナー組織を染色し、レーザー光を送達する唯一のステップが追加されます。達成される利点は、主に、数分の増加の外科時間を補償します。ドナー角膜の厚さとドナーレンチキュラー寸法をカスタマイズ?...

Watch this Scientific Journal Video about An â€œAll-laserâ€ Endothelial Transplant at JoVE.com

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 ...

Research

JoVE Journal

Medicine

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

9.3K ビュー.  Italian National Research Council. 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.

ビデオ: An &#8220;All-laser&#8221; Endothelial Transplant

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 ...

医学

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 ...

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.

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 ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Optimized Griess Reaction for UV-Vis and Naked-eye Determination of Anti-malarial Primaquine

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening infections caused by P. vivax and ovale. However, PMQ has unwanted adverse effects that lead&#160;to acute hemolysis in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency. There is a need to develop simple and reliable methods for PMQ determination with the purpose of dosage monitoring. In early 2019, we have reported an UV-Vis and naked-eye based approach for PMQ colorimetric quantification. The detection was based on a Griess-like reaction between PMQ and anilines, which can generate colored azo products. The detection limit for direct measurement of PMQ in synthetic urine is in the nanomolar range. Moreover, this method has shown great potential for PMQ quantification from human serum samples at clinically relevant concentrations. In this protocol, we will describe the technical details regarding the syntheses and characterization of colored azo products, the reagent preparation, and the procedures for PMQ determination.

This protocol describes a novel colorimetric method for antimalarial primaquine (PMQ) detection in synthetic urines and human serums.

UV-Visに対する最適化されたグリース反応と抗マラリアプリマキンの肉眼判定

Primaquine (PMQ), an important anti-malarial drug, has been recommended by the World Health Organization (WHO) for the treatment of life-threatening ...

Organ Ischemia-Reperfusion Injury by Simulating Hemodynamic Changes in Rat Liver Transplant Model

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, including ischemia-reperfusion injury (IRI) of extrahepatic organs. This model requires numerous experiments and devices. The duration of anhepatic phase is closely related to the time to develop IRI after transplantation. In this experiment, we used hemodynamic changes to induce extrahepatic organ damage in rats and determined the maximum tolerance time. The time until the most severe organ injury varied for different organs. This method can easily be replicated and can also be used to study IRI of the extrahepatic organs after liver transplantation.

This paper provides a detailed description of how to build an animal model of the anhepatic phase (liver ischemia) in rats to facilitate basic research into ischemia-reperfusion injury after liver transplantation.

ラット肝移植モデルにおける血球力学的変化をシミュレートすることによる臓器虚血再灌流損傷

Orthotopic liver transplantation (OLT) in rats is a tried and proven animal model used for preoperative, intraoperative, and postoperative studies, ...

TBase - an Integrated Electronic Health Record and Research Database for Kidney Transplant Recipients

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory values, medical reports, radiology and pathology data) via standardized interfaces with manual data entry during routine treatment (e.g., clinical notes, medication list, and transplantation data). By this means, a comprehensive database for KTR is created with benefits for routine clinical care and research. It enables both easy everyday clinical use and quick access for research questions with highest data quality. This is achieved by the concept of data validation in clinical routine in which clinical users and patients have to rely on correct data for treatment and medication plans and thereby validate and correct the clinical data in their daily practice. This EHR is tailored for the needs of transplant outpatient care and proved its clinical utility for more than 20 years at Charit&#233; - Universit&#228;tsmedizin Berlin. It facilitates efficient routine work with well-structured, comprehensive long-term data and allows their easy use for clinical research. To this point, its functionality covers automated transmission of routine data via standardized interfaces from different hospital information systems, availability of transplant-specific data, a medication list with an integrated check for drug-drug interactions, and semi-automated generation of medical reports among others. Key elements of the latest reengineering are a robust privacy-by-design concept, modularity, and hence portability into other clinical contexts as well as usability and platform independence enabled by HTML5 (Hypertext Markup Language) based responsive web design. This allows fast and easy scalability into other disease areas and other university hospitals. The comprehensive long-term datasets are the basis for the investigation of Machine Learning algorithms, and the modular structure allows to rapidly implement these into clinical care. Patient reported data and telemedicine services are integrated into TBase in order to meet future needs of the patients. These novel features aim to improve clinical care as well as to create new research options and therapeutic interventions.

TBase combines an electronic health record with an innovative research database for kidney transplant recipients. TBase is built upon an in-memory database platform, connected to different hospital systems, and used for regular outpatient care. It automatically integrates all relevant clinical data including transplantation-specific data creating a unique research database.

TBase - 腎臓移植レシピエントのための統合された電子健康記録と研究データベース

TBase is an electronic health record (EHR) for kidney transplant recipients (KTR) combining automated data entry of key clinical data (e.g., laboratory ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

内血網膜バリア透過性を評価する ためのin vitro モデルとしての内皮細胞トランスサイトーシスアッセイ

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...