Name: murine corneal transplantation: a model to study the most common form of solid organ transplantation
Uploaded: 2014-11-17T13:00:00.000+00:00
Duration: 9 min 3 s
Description: Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

The authors would like to thank the many individuals who have worked on and perfected this technique and have been responsible for the generation of many manuscripts both in this lab and others. This work was supported by National Institutes of Health Grant EY12707 (PMS) and an unrestricted grant from Research to Prevent Blindness to Department of Ophthalmology.

注：この手順で使用するすべての動物は、ビジョンの研究のための協会と眼科文の眼科と視覚研究における動物の使用のためだけでなく、セントルイス大学で動物の監督委員会によって定めるガイドラインに従って扱われます。 注：すべての手術器具やソリューションは、目の微生物感染を制限するために手術前に滅菌される。これは、動物は、この手順のいくつかの痛みを経験しない間、我々は、鎮痛薬を使用しないことに留意すべきである。すべての鎮痛薬は、抗炎症性であり、角膜移植応答は、炎症を伴うため、系抗炎症薬の使用は、角膜移植不全に関与しているかの要因を決定する我々の能力を危うくするので、この理由による。 1.麻酔<ol style="margin-left: 40px;"><li>ケタミン（86mgのIP注射によって、全身麻酔下で、ドナーとレシピエントマウスを置きます。98ミリグラム/ kg）およびキシラジン（13.04 mg / kgを）。 </li><li>通常、時間に30分を要する作業中は麻酔下でレシピエントマウスにしてください。その結果、常に意識を取り戻すの兆候のためのマウスを監視します。 </li><li>動物は乾燥を防ぐために麻酔をかけた後に手術を受けることはありません目にpuralube軟膏を適用します。 </li></ol> 2。角膜移植<ol style="margin-left: 40px;"><li>ドナー角膜ボタンを取得する。 <ol><li>動物が完全に麻酔したら、1％トロピカミドおよび2.5％塩酸フェニレフリンの点眼剤のカップルの投与により十分な散瞳を達成。 </li><li>水平に頑丈な可動支持体の上に置かれ、ボード上のドナー動物の頭を置きます。目は操作全体を通して水平な位置にあることを保証するために、首全体でテープのストリップで頭を固定します。 </li><li>アウト、先端がメチルブルーで染色した直径2mmのトレフィンを使用して、中央角膜移植部位を裏打ちする。 </li><li>鋭利な刃で、角膜に浸透し、ドナー内皮および基盤となるレンズへの損傷の可能性を減らすためにそれを深めるために前房内にのHealonを注入する。 </li><li> vannasはさみを持つドナー移植片を切除し、使用するまでハンクス平衡塩溶液を含む皿に置く。 </li><li>ドナー移植片が除去された後、CO 2吸入によりドナーマウスを安楽死させる。 </li></ol></li><li>移植床の準備。 <ol><li>受信者に対して2.1.2を通じて、2.1.1で説明したのと同じ手順を繰り返します。 </li><li>直径1.5mmのトレフィンを使用して、受信者の移植部位を概説。 </li><li>鋭利な刃で、角膜に浸透し、下層のレンズへの損傷の可能性を低減することを深めて前房へのHealonを注入する。 </li><li> vannasはさみを使って受信者から概説し、中央の角膜のボタンを外して捨てる。 </li></ol></li><li>縫合移植<ol><li>受取人の角膜のベッドの上で移植ドナーの角膜を置きます。十分なのHealonは、レンズとの直接接触による損傷からドナー内皮細胞を保護するために、ドナー角膜の下にあることを確認してください。 </li><li>超微細な先端マイクロピンセットを使用して、その後オフネクタイ、受信者の側への完全な厚さの90％の深さを持つドナーを通じて、ドナー側に11-0ナイロン縫合糸の最初の一口を置く。 </li><li>角膜が所定の位置に固定されると、角膜は8~10総縫合糸を有し、ドナー角膜がしっかりと整列し、受信者の角膜の移植床に取り付けられるようにmidcardinal結節縫合を行う。 </li></ol></li><li>前房の深化<ol><li>前房内にHBSSまたは気泡を注入することによって前房を深め、静かにセルローススポンジで漏れのため、角膜移植片の完全性をチェック。 注：前房を改質することができない場合、cは確率が高い移植された角膜の将来の評価が非常に困難になり、また、潜在的ドナー角膜内皮機能不全、したがって、移植の失敗につながるataract。 </li></ol></li><li>最終的評価<ol><li>瞳孔が丸いと前房の深さが正常であることを決定するために、目を守ってください。 注：瞳孔が丸いされていない場合、これは、虹彩を縫合中に破損したため、移植片が技術的失敗と見なされていることを示します。 </li><li>目に抗生物質軟膏を適用します。オプション：7-0絹縫合糸で眼瞼を閉じます。 </li><li>彼らは完全に目を覚ましていると、その後個別に手術後2日以上のためにそれらを収容するまで、マウスを観察します。 </li></ol></li></ol> 3.抜糸<ol style="margin-left: 40px;"><li>上記のように、蓋縫合糸が使用されているような場合には、マウスを麻酔し、48時間後に蓋縫合糸を除去する。 </li><li>術後7日目のマウスを麻酔。 suturを削除ES角膜移植を確保。縫合糸が除去され、動物が完全に目覚めた後は、そのケージに戻す。 </li></ol> 4.臨床評価<ol style="margin-left: 40px;"><li> 、白内障（レンズの曇り）、前房出血（前房中の血液）、適切な深さではない前房、あるいは角膜のかなりの不透明度を含む処置合併症の兆候に目を調べます。 「妥協」として、これらの合併症を証明するものを考えてみましょうとCO 2吸入によってそれらを安楽死させる。 <ol><li>無麻酔のマウスのすべての検査を実行します。一方、目のより良いビューを有効にするために目をproptoseできるように、このようにしてマウスを拘束片手でマウスを保持する。観測が完了すると、そのケージに動物を返す。 </li></ol></li><li>治療群に不慣れでは角膜移植の兆候を2〜3回、週に移植した角膜を評価するオブザーバを持っている拒絶反応または角膜移植の失敗。手術用顕微鏡またはこれらの観​​察のための水平細隙灯生体顕微鏡のいずれかを使用します。 <ol><li>以下のようにスケールが定義されている0〜5のスケールを使用して不透明度毎に角膜を評価する。 <ol><li>不透明度の兆候を持っていないもの角膜0~10のスコアを割り当てます。 </li><li>最小限の表面的な不透明性を示すもの角膜にスコア1を割り当てます。 </li><li>マイルドで深い不透明度を表示しますが根底にある瞳と虹彩はまだ識別可能である角膜へ2のスコアを割り当てます。 </li><li>虹彩は、瞳孔の余白を除いて詳細に見ることができない、請求間質不透明度を表示角膜3のスコアを割り当てる。 </li><li>密な間質不透明度を表示しない根本的な構造は閲覧できない場合角膜に4のスコアを割り当てます。 </li><li>完全に覆い隠さ瞳孔と虹彩との完全な不透明度と集中的な間質浮腫を表示角膜に5のスコアを割り当てます。 </li></ol></li><li><html>また、これを達成するために1〜8の評価尺度を使用して、血管浸潤（新血管形成）の程度ごとに角膜を評価する4つの等しい四分円からなるとして角膜を表示​​し、これらの象限のそれぞれの中の血管の量を決定するその象限の広範な血管新生のために0（無血管）から2の範囲で得点。最終的な血管新生スコアを計算するために各象限から個々のスコアを追加します。 </li><li>彼らは5週間までの時点で連続する2つの観察のための3のスコアを持っているかのように急性拒絶された角膜を分類します。 </li><li>その角膜5週目で透明であったマウスを分類しますが&gt; 45日の時点で混濁を開発後期長期角膜同種移植片拒絶を受けたように、2つの連続した​​時点で3のスコアで、移植を投稿してください。移植片生存を分析するために、カプラン - マイヤー生存曲線を使用する。 </li></ol></li></ol>モデル5.操作中左：40px; &quot;&gt; <li>脾臓からの単一細胞の調製。 <ol><li>単一細胞を調製するため、第一のドナーマウスを安楽死させる。その後、脾臓を除去します。 </li><li>セルストレーナーに脾臓を置き、それを3ミリリットルのシリンジからシリンジプランジャを混乱させる。 </li><li>ハンクス平衡塩類溶液10mlに再懸濁細胞を洗ってください。 </li><li>細胞懸濁液10μlを削除し、0.4％トリパンの10μlに青追加し、混ぜる。血球計数器にそれを追加し、中央のグリッド内のセルを数える。チューブ内の細胞数は、細胞数×10 4×2（トリパンブルーで希釈係数）10（チューブ内の容積）×である。 </li></ol></li><li>前房内への注入。 <ol><li>前述のようにマウスを麻酔。 </li><li>解剖顕微鏡を使用して注射を行う。各房内注入のために、10 6 0.005ミリリットルボリューム内の脾臓細胞と33 Gの針を取り付け0.25ミリリットルのマイクロシリンジを使用しています。 注：その他の操作モデルの特定の因子が、同所角膜同種移植手術後の果たし得る役割を決定するためのいずれかのアンタゴニストまたはアゴニストとして作用する試薬で動物を処理することによって行うことができる。 </li></ol></li></ol>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Topical+interleukin-1+receptor+antagonist+promotes+corneal+transplant+survival.">Topical interleukin-1 receptor antagonist promotes corneal transplant survival.</a> Transplantation. 63, 1501-1507 (1997).</li><li>Cunnusamy, K., Chen, P. W., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-17A-dependent+CD4+CD25++regulatory+T+cells+promote+immune+privilege+of+corneal+allografts.">IL-17A-dependent CD4+CD25+ regulatory T cells promote immune privilege of corneal allografts.</a> J. Immunol. 186, 6737-6745 (2011).</li><li>Fu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+depletion+as+a+mechanism+for+the+immune+privilege+of+corneal+allografts.">Arginine depletion as a mechanism for the immune privilege of corneal allografts.</a> Eur. J. Immunol. 41, 2997-3005 (2011).</li><li>Medina, C. A., Rowe, A. M., Yun, H., Knickelbein, J. E., Lathrop, K. L., Hendricks, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Azithromycin+treatment+increases+survival+of+high-risk+corneal+allotransplants.Cornea.">Azithromycin treatment increases survival of high-risk corneal allotransplants.Cornea.</a> , 32-658 (2013).</li><li>Cho, Y. K., Zhang, X., Uehara, H., Young, J. R., Archer, B., Ambati, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+Endothelial+Growth+Factor+Receptor+1+morpholino+increases+graft+survival+in+a+murine+penetrating+keratoplasty.">Vascular Endothelial Growth Factor Receptor 1 morpholino increases graft survival in a murine penetrating keratoplasty.</a> Invest. Ophthalmol. Vis. Sci. 53, 8458-8471 (2012).</li><li>Kim, H. K., Choi, J. A., Uehara, H., Zhang, X., Ambati, B. K., Cho, Y. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presurgical+corticosteroid+treatment+improves+corneal+transplant+survival+in+mice.">Presurgical corticosteroid treatment improves corneal transplant survival in mice.</a> Cornea. 32, 1591-1598 (2013).</li><li>Yamazoe, K., Yamazoe, K., Shimazaki-Den, S., Shimazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+factors+for+corneal+graft+recovery+after+severe+corneal+graft+rejection+following+penetrating+keratoplasty.">Prognostic factors for corneal graft recovery after severe corneal graft rejection following penetrating keratoplasty.</a> BMC Ophthalmol. 13, 5 (2013).</li><li>Panda, A., Vanathi, M., Kumar, A., Dash, Y., Priya, 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+graft+rejection.">Corneal graft rejection.</a> Surv. Ophthalmol. 52, 375-396 (2007).</li><li>Patel, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graft+survival+and+endothelial+outcomes+in+the+new+era+of+endothelial+keratoplasty.">Graft survival and endothelial outcomes in the new era of endothelial keratoplasty.</a> Exp. Eye Res. 95, 40-47 (2012).</li><li>Anshu, A., Price, M. O., Tan, D. T., Price, F. W. <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:+a+revolution+in+evolution.">Endothelial keratoplasty: a revolution in evolution.</a> Surv. Ophthalmol. 57, 236-252 (2013).</li></ol>

The authors have no competing financial interests.

ここで説明する角膜移植のマウスモデルは、因子が最良の拒絶15,17,18,20、26-30と角膜の受け入れ21-25両方に関連付けられているかを予測するモデルでのヒト角膜同種移植片拒絶を研究する研究者を可能に同種移植片。患者が治療又は予防のいずれか拒絶31のいずれかに局所または全身ステロイド治療を与えられたヒト角膜移植とは異なり、このモデルは、典型的には、?...

角膜移植は、ヒトで行わ移植の最も成功したと一般的なタイプの一つです。この手術が行われる理由は、損傷、感染症1、または非感染性角膜疾患2の他の形態の結果である。アメリカのアイバンク協会からフィギュアは、46000の上には、（：restoresight.org/eye_banks/eye_banks.htmlでのWebサイトを参照してください）​​2011年に行われたことを示している。その成功の指標は、同種移植片、角膜1年間故障率10〜15％の範囲で5年での成功率が70％3-8を超えているということである。多くの研究が示したように、角膜の同種移植の成功は、直接目に免疫学的特権部位であるという事実に関連している。免疫特権部位として角膜の状態を担当する要因は、角膜内の両方血管およびリンパ管の欠如は、抗原提示細胞の相対的欠如、suppres角膜によって産生される因子を含むの免疫エフェクターfuntions 9-15、MHCの低発現は16抗原、およびFasLの17-20の発現。 しかし、成功のためのこれらの移植片を素因これらの要因にもかかわらず、それらは拒絶3-7を受けない。したがって、この拒絶反応を媒介するそれらのメカニズムを理解するだけでなく、拒絶反応を防止するために、様々な治療法をテストする非常に重要である。そのために、我々はここで、制御の実験環境での角膜移植を研究するために20年以上にわたって使用されている角膜移植のマウスモデルを記述する。移植応答が最終的な移植組織が ​​失敗したか成功したかどうかを決定する協調して働いて、さまざまな要因が関与するので、任意のインビトロモデルにおけるこれらの要因の重要性を理解することは不可能である。結果的に、無傷の動物を用いた研究が成功またはfailuどちらにとって重要などのような要因を決定するために必要とされている移植された組織の再。 動物の他の種は角膜移植を研究するために使用されてきたが、他の種を用いて比較した場合、マウスモデルは、いくつかの利点を有する。最初は、特定の遺伝子を発現するかまたは機能移植におけるより良い研究することができる特異的な免疫学的因子の発現を欠くように遺伝子標的化されたマウスの多くの株が存在することである。また、動物の多くの他の種は存在しないマウスに特異的な多くの試薬（因子を中和する組換え因子および抗体の両方）がある。これらの要因が存在するため、このモデルは、急性角膜の同種移植片の応答15、17,18,20 -29に関与し、関連する因子を同定するために広く使用されている。さらに、角膜移植に関与する因子の多くは、他の組織の移植において機能的であることが知られている。

<table><tbody><tr><td>Zeiss Surgical Microscope</td><td>Zeiss</td><td>Rebuilt</td><td></td></tr><tr><td>1 ml Syringe</td><td>BD</td><td>305122</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>309657</td><td></td></tr><tr><td>10 ml Syringe</td><td>BD</td><td>309602</td><td></td></tr><tr><td>Vannus Scissors</td><td>Stortz</td><td>E-3387</td><td></td></tr><tr><td>11-0 Sutures</td><td>Alcon</td><td>717939M</td><td></td></tr><tr><td>Trephine 2.0 mm</td><td>Katena</td><td>K 2-7520</td><td></td></tr><tr><td>Trephine 1.5 mm</td><td>Katena</td><td>K 2-7510</td><td></td></tr><tr><td>Tricaine Hydrochloride 0.5%</td><td>Alcon</td><td>NDC 0065-0741-12</td><td></td></tr><tr><td>Healon</td><td>Abbott</td><td>Healon OVD</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-20</td><td></td></tr><tr><td>7-0 Sutures</td><td>Alcon</td><td>8065</td><td></td></tr><tr><td>2.5% Phenylephrine HCl</td><td>Alcon</td><td>NDC 61314-342-02</td><td></td></tr><tr><td>1% Tropicamide</td><td>Bausch &amp;amp; Lomb</td><td>NDC-24208-585-59</td><td></td></tr><tr><td>Hamilton Syringe</td><td>Hamilton</td><td>7654-01</td><td></td></tr><tr><td>33 gauge needle</td><td>Hamilton</td><td>90033</td><td></td></tr><tr><td>Cell Strainer (100 &amp;mu;m nylon)</td><td>BD Falcon</td><td>352360</td><td></td></tr><tr><td>Hemocytometer</td><td>Cardinal Health</td><td>B3175</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma</td><td>T8154</td><td></td></tr></tbody></table>

murine corneal transplantation: a model to study the most common form of solid organ transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

角膜移植のマウスモデルが正常角膜同種移植片拒絶19-23と角膜同種移植の受け入れ13、15,16,18、24-27両方のメカニズムを特徴づけるために20年以上にわたって使用されてきた。このモデルは、FasLのを欠く動物は、角膜の同種移植片15を受け入れることができなかったという点で、角膜の同種移植片の受け入れにFasL発現の重要性を確立するために使用した。また、血管内?...

Watch this Scientific Journal Video about Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation at JoVE.com

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

murine-corneal-transplantation-model-to-study-most-common-form-solid

Research

JoVE Journal

Medicine

9.6K ビュー.  Saint Louis University. Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

ビデオ: Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

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

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

変更されたカフ法を用いたマウス子宮頸心臓移植モデル

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

医学

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

マウス腎臓移植：同種移植片拒絶のモデル

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

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 ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

マウス全層皮膚移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

免疫・感染学

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

マウスにおける異所のための新しい顕微モデル、エン圏胸壁、胸腺、および心臓移植

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

Orthotopic Hind Limb Transplantation in the Mouse

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and translational research in the field of transplantation medicine. A vast array of reagents is available exclusively in this setting, including mono- and polyclonal antibodies for both diagnostic and interventional applications. In addition, a vast number of genotyped, inbred, transgenic, and knock out strains allow detailed investigation of the individual contributions of humoral and cellular components to the complex interplay of an immune response and make the mouse the gold standard for immunological research. 
Vascularized Composite Allotransplantation (VCA) delineates a novel field of transplantation using allografts to replace "like with like" in patients suffering traumatic or congenital tissue loss. This surgical methodological protocol shows the use of a non-suture cuff technique for super-microvascular anastomosis in an orthotopic mouse hind limb transplantation model. The model specifically allows for comparison between established paradigms in solid organ transplantation with a novel form of transplants consisting of various different tissue components. Uniquely, this model allows for the transplantation of a viable vascularized bone marrow compartment and niche that have the potential to exert a beneficial effect on the balance of immune acceptance and rejection. This technique provides a tool to investigate alloantigen recognition and allograft rejection and acceptance, as well as enables the pursuit of functional nerve regeneration studies to further advance this novel field of transplantation.

This novel model for orthotopic hind limb transplantation in the mouse, applying a non-suture cuff technique for super-microvascular anastomosis, provides a powerful tool for in&#160;vivo mechanistic immunological research related to vascularized composite allotransplantation (VCA).

マウスで同所後肢移植

In vivo animal model systems, and in particular mouse models, have evolved into powerful and versatile scientific tools indispensable to basic and ...

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 ...

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. ...

A Modified Murine Heterotopic Heart Transplant Protocol Matching Contemporary Standards of Aseptic Technique, Anesthesia, and Analgesia

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ transplantation. While the heterotopic vascularized murine cardiac transplantation model was initially utilized in studies of graft rejection using combinations of mismatched inbred mouse strains, access to genetically modified strains and therapeutic modalities can provide powerful new preclinical insights. Fundamentally, the surgical methodology for this technique has not changed since its development, especially with respect to important factors such as aseptic technique, anesthesia, and analgesia, which make material impacts on postsurgical morbidity and mortality. Additionally, improvements in perioperative management are expected to provide improvements in both animal welfare and experimental outcomes. This paper reports upon a protocol developed in collaboration with a subject matter expert in veterinary anesthesia and describes the surgical technique with an emphasis on perioperative management. Additionally, we discuss the implications of these refinements and provide details on troubleshooting critical surgical steps for this procedure.

The present paper describes a modified technique for heterotopic vascularized cardiac transplantation with updated aseptic technique, analgesia, and anesthesia.

無菌技術、麻酔、および鎮痛の現代の基準に一致する修正マウス異所性心臓移植プロトコル

The development of experimental models of cardiac transplantation in animals has contributed to many advances in the fields of immunology and solid organ ...

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 ...

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

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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

マウス腎臓移植：同種移植片拒絶のモデル

マウスの腎臓移植技術

マウス全層皮膚移植

マウスにおける異所のための新しい顕微モデル、エン圏胸壁、胸腺、および心臓移植

マウスで同所後肢移植

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

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

無菌技術、麻酔、および鎮痛の現代の基準に一致する修正マウス異所性心臓移植プロトコル

分割角膜ボタンを用いてのブタ角膜内皮器培養モデル

ネズミ角膜移植：固体臓器移植の最も一般的な形態を研究するためのモデル

要約

さらに動画を探す

この動画の章

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

マウス腎臓移植：同種移植片拒絶のモデル

マウスの腎臓移植​​技術

マウス全層皮膚移植

マウスにおける異所のための新しい顕微モデル、エン圏胸壁、胸腺、および心臓移植

マウスで同所後肢移植

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

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

無菌技術、麻酔、および鎮痛の現代の基準に一致する修正マウス異所性心臓移植プロトコル

分割角膜ボタンを用いてのブタ角膜内皮器培養モデル

マウスの腎臓移植技術