The authors would like to acknowledge Jonathan Chou, MD for his assistance on preparation and editing of the final manuscript and Wenzhong Liu for the OCT data. We would also like to acknowledge support from the Macula Society Research Grant (AAF), support from an unrestricted grant to Northwestern University from Research to Prevent Blindness, Inc., New York, NY, USA, and support from NIH-EY019951.

全ての動物は、実験動物の管理と使用2013年版のガイドに従って処理され、ビジョンと眼科（ARVO）の眼科と視覚研究における動物の使用のための声明の研究のための協会、および動物実験によって承認されましたお手入れとノースウェスタン大学のための使用に関する委員会。 注意：次の手順は、1つの演算子で完全に行うことができます。しかし、より効率的に応じて分割するタスクを2つの演算子を用いて行われます。 1.レーザーとプリレーザー駅を準備<ol><li>それは簡単にアクセスすることができ、レーザスリットランプを配置します。レーザーをオンにして、事前に決定されたパラメータ（例えば、75μmのスポットサイズ、100 mWのパワー、100ミリ秒の持続時間）に設定してください。 注意：オペレータは、すべての動物、レーザー安全保護具及び適切なレーザー安全標識は、処置室の外に表示されている身に着けていることを確認。 <ol><li>いずれの実験動物を使用する前に、キャリブレーション、非実験マウスを使用して理想的なパラメータを見つけます。レーザパラメータは、使用するレーザに依存するであろう。理想のパラメータが適切に焦点を当てたときに一貫して「バブル」形成を引き起こす最も低いレーザパワーの設定によって定義されています。適切な「バブル」形成を伴う病変の例は、ビデオを参照してください。 </li></ol></li><li>麻酔、暖かい動物、組織ワイプ、およびすべての目薬（トロピカミド、テトラカイン、および人工涙液）がオペレータに手の届くところに簡単になるように事前にレーザーステーションを準備します。 <ol><li>暖かいその動物を確保することは、麻酔誘発性低体温を回避するために、第1のマウスに麻酔薬を注入する前に、温度（37°C）を修正するために予備加熱されます。 </li></ol></li><li>それは熱を保持し、レーザー手順が始まると暖かく保つことができるので、暖かい上でマウスの段階を置きます。 </li></ol> 2.マウスの麻酔とプリレーザー準備<ol><li>注入前に麻酔をる、それは奇形や角膜の明瞭さ（例えば白内障）を減少させる異常を持っていないことを確認するために、巨視的に目を検査します。 </li><li>マウスを計量。 </li><li>記録体重、性別、および動物ID番号。 </li><li>体重を使用して、機関によって与えられたガイドラインに基づいて、使用する麻酔薬の適切な量を計算する（例えば100ミリグラム/ kgの塩酸ケタミン、10mg / kgのキシラジンまたはトリブロモエタノールの250mg / kgで、20グラムのマウスのための0.20ミリリットルキシラジン/ケタミンカクテルを注入トリブロモエタノールのOR 0.25ミリリットル）。数学的なエラーを低減するために、1グラム単位で重量当たりの予め計算された投与量のテーブルがあります。 </li><li>首筋マウスとステップ2.4​​での計算に基づいて腹腔内に麻酔薬を注入します。 </li><li>暖かい動物の上でマウスを置き、マウスが完全につま先のピンチ（約3-5分）を介してペダル反射を確認することによって麻酔されるまで待ちます。 </li><li>ワイプ組織をロールアップし、aspiratを防止するために、マウスの鼻の領域を保護液体ロールオフのイオン。ロールの側でマウスおよび局所麻酔のためにそれぞれの眼に塩酸テトラカインの低下（約30μl）を配置します。解決策を有効にするために2分を待ちます。 </li><li>瞳孔散大のための局所トロピカミドの一滴と手順を繰り返し2.7。また、拡張のためのフェニレフリン塩酸塩（2.5％）を使用します。 </li><li>ソリューションを有効にするために2分待ち。この時間の間に暖かい上の動物を維持します。 </li><li>適切な時間が経過した後、すぐにマウスステージ上にマウスを置いて、スリットランプの顎当て上の段階を置きます。 </li><li>最低光の明るさにスリットランプをオンにして、瞳孔散大の程度を確認します。瞳が十分に（約2.5〜3ミリメートル）を拡張していない場合は、暖かい動物にマウスを返し、待ちます。また、トロピカミドの別のドロップを管理します。目が十分に拡張すると、レーザーの手順に進みます。 </li></ol> 3.レーザー手順注：他のPを確認してくださいersons部屋にレーザーで保護されたスリットランプアイピースから離れるとき保護用眼鏡を着用<ol><li>それは理想的な視神経の可視化（3.1.2を参照）のために配置されるように、マウスステージ上のマウスの位置を調整します。 <ol><li>それが水平になるよう、他の1つの側に頭と尾と、ランプの光をスリットに垂直なホルダー上でマウスを向けます。理想的なマウスの配置は、カバースリップが適用されると非常に簡単視神経の可視化を行います。 </li><li>それはレーザーオペレータに近いヘッドで〜170°の角度になるようにマウスをわずかに回します。 </li><li>そのマウスは、ランプをスリットにできるだけ近いが、それでもそれは安定しており、どこにオペレータの手が細かい操作のための十分なスペースを持っています位置に確認してください。 </li></ol></li><li>マウスが理想的に配置された後、25ミリメートル×25ミリメートルのガラスカバースリップ上の人工涙液の一滴を置きます。 <ol><li>マウスのOPPに人工涙液の一滴を置きosite目 - この目が水和されていることを確認し、白内障形成を遅らせるのに役立ちます。 </li></ol></li><li>親指とポインタ指の間カバーグラスの角を持ち、位置ガラスは両方の指の先端との間で圧搾されるようになっています。 </li><li>優しくサポートと手の安定化のために、動物の体の周り残りの3本の指を包みます。位置手ようにカバーガラスを容易に、マウスの眼に配置することができます。 <ol><li>手首は​​手の震えを減少させるために、しっかりした面に安定していることを確認してください。 </li></ol></li><li>安定した位置が得られると、慎重にマウスの眼に（まだ付着した人工涙の滴で）カバーガラスを押してください。 <ol><li>カバーガラスは、レーザ光散乱または反射を防止するために、レーザービームにできるだけ垂直に位置づけされていることを確認します。カバースリップは、角膜を平坦化するコンタクトレンズとして機能します。 </li></ol></li><li>トグルUNT集中スリットランプを通って、フリーハンドで見てIL網膜を可視化することができます。網膜は可視化位置に応じて淡黄色/赤色を持っていますが、明確な、赤い血管が見えるようになります。 </li><li>ゆっくりと慎重に視神経を視覚化するまで、マウスの頭部および/またはカバースリップを操作します。視神経は、それから放射複数の容器と色が黄色になります。 </li><li>オペレータは視神経の可視化を確認した後、ビームを集束レーザーをオンにします。 </li><li>レーザービームがオンにされた後、所望の位置に（視神経から約1ディスク径）ビームを集束レーザーを操縦。 </li><li>眼底のRPEにレーザー光の焦点を合わせます。適切な焦点は、最も鮮明かつ明確なレーザービームを有することにより達成されます。ビームを目指している場合は、楕円形やフォーカス、トグルスリットランプフォーカスまたは再位置カバーガラスの外に見えます。 </li><li>照準ビームがRPEに集束されると、レーザーの足のトリガーを用いたレーザ管理を開始します。 <ol><li>眼内彼を防止するために、網膜血管を避けてくださいmorrhage。 </li></ol></li><li>すぐにレーザー投与後の泡の出現を監視します。レーザーショットの輪郭がはっきりとどのような方法でかすんでいないでなければなりません。 <ol><li>レーザーショットは、泡の形成をもたらさない場合、または衝撃の面積は（対成功した影響を明確に、はっきりと定義された境界線不明確な円形境界線で曇った外観）かすんで見える、または出血がレーザー投与後に見られている場合、しないでください将来の分析のためにこれらの病変が含まれます。 </li></ol></li><li>繰り返しは、すべての希望CNV位置の3.10から3.12（通常は3で、6、9、および視神経の周りの12時の位置）を繰り返します。レーザー誘導は、視神経からほぼ同じ距離で適用された場合、リフォーカスは必要ありません。しかし、マウスの眼の網膜に存在する可能性が小さい変動の強い曲率に、ビームを再集束することは連続したレーザー投与の間必要になることがあります。 </li><li>ノートブックのレコードの電子の位置と結果ACHのレーザーショットの管理と結果（成功、かすんで、出血など ）、眼用の各投与ショットの。使用しないときは、スタンバイモードでレーザーを配置してください。 </li><li>必要に応じて安定化して、新しいカバースリップのための反対側の手を使用して、マウスのもう片方の目のための3.1から3.14を繰り返します。 </li><li>すべての所望のレーザショットが投与された後、レーザーをオフにして、スリットランプを。 </li><li>カバースリップを破棄し、麻酔からの回復のための暖かい上にマウスを置きます。肉眼傷害のために目を検査し、水和目を維持し、潜在的に将来の白内障の発症を予防するために人工涙液の液滴を配置します。マウスは麻酔から回復した後、ケージに戻します。 </li></ol><table fo:keep-together.within-page="1"><tbody><tr><td></td><td> AVERTIN </td><td> AVERTIN </td><td> AVERTIN </td><td> XYL / KET </td><td> XYL / KET </td></tr><tr><td> マウスの重量（g） </td><td> 用量（MG /キログラム） </td><tD&gt; 溶液濃度（mg / mlで） </td><td> 麻酔投与量（ミリリットル） </td><td> 用量（MG /キログラム） </td><td> 麻酔投与量（ミリリットル） </td></tr><tr><td> 15 </td><td> 250 </td><td> 20 </td><td> 0.1875 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.15 </td></tr><tr><td> 16 </td><td> 250 </td><td> 20 </td><td> 0.2 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.16 </td></tr><tr><td> 17 </td><td> 250 </td><td> 20 </td><td> 0.2125 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.17 </td></tr><tr><td> 18 </td><td> 250 </td><td> 20 </td><td> 0.225 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.18 </td></tr><tr><td> 19 </td><td> 250 </td><td> 20 </td><td> 0.2375 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.19 </td></tr><tr><td> 20 </td><td> 250 </td><td> 20 </td><td> 0.25</td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.2 </td></tr><tr><td> 21 </td><td> 250 </td><td> 20 </td><td> 0.2625 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.21 </td></tr><tr><td> 22 </td><td> 250 </td><td> 20 </td><td> 0.275 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.22 </td></tr><tr><td> 23 </td><td> 250 </td><td> 20 </td><td> 0.2875 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.23 </td></tr><tr><td> 24 </td><td> 250 </td><td> 20 </td><td> 0.3 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.24 </td></tr><tr><td> 25 </td><td> 250 </td><td> 20 </td><td> 0.3125 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.25 </td></tr><tr><td> 26 </td><td> 250 </td><td> 20 </td><td> 0.325 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.26 </td></tr><tr><td> 27 </td><td> 250 </td><tD&gt; 20 </td><td> 0.3375 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.27 </td></tr><tr><td> 28 </td><td> 250 </td><td> 20 </td><td> 0.35 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.28 </td></tr><tr><td> 29 </td><td> 250 </td><td> 20 </td><td> 0.3625 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.29 </td></tr><tr><td> 30 </td><td> 250 </td><td> 20 </td><td> 0.375 </td><td> 100mgの/ kgのケタミンを、 10mg / kgのキシラジン</td><td> 0.3 </td></tr></tbody></table> 表1：XyIKet投与チャート。

<ol>
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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choroidal+Neovascularization+in+the+Rat+Induced+by+Adenovirus+Mediated+Expression+of+Vascular+Endothelial+Growth+Factor.">Choroidal Neovascularization in the Rat Induced by Adenovirus Mediated Expression of Vascular Endothelial Growth Factor.</a> IOVS. 41, 3582-3589 (2000).</li><li>Claybon, A., Bishop, A. J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+a+Mouse+Eye+for+a+Whole+Mount+of+the+Retinal+Pigment+Epithelium.">Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium.</a> JoVE. , 2563 (2011).</li><li>Espinosa-Heidmann, D. G., 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=Age+as+an+Independent+Risk+Factor+for+Severity+of+Experimental+Choroidal+Neovascularization.">Age as an Independent Risk Factor for Severity of Experimental Choroidal Neovascularization.</a> IOVS. 43, 1567-1573 (2002).</li><li>Zhu, Y., 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=Improvement+and+Optimization+of+Standards+for+a+Preclinical+Animal+Test+Model+of+Laser+Induced+Choroidal+Neovascularization.">Improvement and Optimization of Standards for a Preclinical Animal Test Model of Laser Induced Choroidal Neovascularization.</a> PLoS ONE. 9, e94743 (2014).</li><li>Weinstock, M., Stewart, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Occurrence+in+rodents+of+reversible+drug+induced+opacities+of+the+lens.">Occurrence in rodents of reversible drug induced opacities of the lens.</a> British J Ophthalmol. 45, 408-414 (1961).</li><li>Ridder III, W. H., Nusinowitz, S., Heckenlively, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+of+Cataract+Development+in+Anesthetized+Mice.">Causes of Cataract Development in Anesthetized Mice.</a> Exp Eye Res. 75, 365-370 (2002).</li></ol>

The authors declare they have no competing financial interests.

成功したレーザー誘導後にレーザ送出し、得られたCNV病変の発達に影響を与えることができ、複数の要因があります。これらの要因には、のために制御し、最も信頼性の高い結果を得るために標準化されるべきです。これらの要因の中で最も適切なマウスの選択（遺伝子型、年齢、性別）、麻酔薬の選択、及びレーザーの設定です。 使用される特定のマウスモデルは、CN...

加齢黄斑変性症（AMD）は50 1-3歳以上の個人における失明の主要な原因の1つです。萎縮（「ドライ」）AMDおよび血管新生（「ウェット」）AMD：AMDは二つの形態に分類することができます。前者は、後者は、漏れ、出血および線維症を引き起こす外網膜層への脈絡膜の異常な血管の侵入によって特徴付けられる一方で、網膜色素上皮（RPE）、脈絡毛細管板、及び光受容体の地図状萎縮によって特徴付けられ、最終的にされています失明1,2につながります。二つの形式のうち、血管新生AMDは失明1の大半を占めています。その萎縮相手には現在医療3を証明したのに対し、幸いなことに、この形式は、非常に多くの効果的な薬理学的管理の選択肢を持っていません。血管新生型が容易に動物モデルにおいて再降伏されているため、また、それは基本Aのより広くアクセス可能となっています新しい治療法4を開発するために 、基礎となる病理学的メカニズムを探求するのMD研究。 実験的脈絡膜新生血管（CNV）の最初の動物モデルは、ライアンらによって開発されました。ヒト以外の霊長類5インチこのモデルは、血管新生AMDに見られるものと同様の血管新生が生じる局所炎症反応を引き起こしたレーザー光凝固を介してブルッフ膜の破裂を誘発しました。血管形成後のレーザー誘導の組織病 ​​理学的進行は、モデルの妥当性を確認した6血管新生AMDを、模倣することが判明しました。非ヒト霊長類は、ヒトに最も類似の解剖学的構造を提供していますが、残念ながら、簡単に遺伝子操作することができない、維持するために高価であり、疾患の進行7の遅い時間経過しています。対照的に、げっ歯類モデルは、はるかに費用対効果を維持するためであり、遺伝的に比較的容易に操作することができ、はるかに高速COUを持ちます疾患進行のRSE（実験はヶ月対週間の時間スケールで実施することができます）。それはアルビノ動物において視覚化することは非常に困難であるように、これらの実験は、着色されたげっ歯類において実施されるべきです。 後半に最初90の10でカンポキアーログループによって開発されたマウスのレーザー誘発CNVモデルは、最近の研究11-16の大部分において支配的な動物モデルに成長しました。 CNVのさらに複雑で不明瞭な病因に、レーザーモデルは、将来のヒトへの使用のための新しい治療法を評価するには、血管新生を駆動する分子メカニズムを研究に至るまで、滲出型AMDの研究のすべての側面に適用されています。例えば、サクライら 。そして、エスピノサ・Heidmann ら 。トランスジェニックマウスおよび薬理学的枯渇治療15,16を使用して CNVの発症に対するマクロファージの影響を調査するために、レーザーモデルを使用した。ジアーニら 。 Hoerster らと。画像を用いた光コヒーレンストモグラフィー（OCT）レーザー誘発CNVの進行を特徴づけるとOCTイメージング12,17に見られる所見に組織病 ​​理学的所見を比較するための努力にCNV。最後に、抗血管新生剤の硝子体内注射を含む研究は、ヒトでの試験のための前提条件として使用され、血管新生AMD今日10,18,19の管理に使用される抗VEGF薬の第一世代の開発に不可欠であったされています。 実験的CNVのための代替モデルは、CNVを誘導するための外科的方法を利用します。この手順では、血管新生8,20の原因となることを目標に、血管新生AMDに見られる増加したVEGF発現を模倣するために血管新生促進物質（VEGF、RPE細胞および/ ​​またはポリスチレンビーズの網膜下注射を過剰発現例えば組換えウイルスベクター）を注入することを含みます。しかしながら、この方法は、血管新生のより低い発生率を大幅にもたらします。これらの研究は、CNVであることを示しましたC57 / BL6マウスは、マウス8,14の同系統のレーザ光 ​​凝固法で見られる〜70％の成功率対注射の31％で発生します。これらの理由から、および非ヒト霊長類対げっ歯類を使用することの利点を与え、レーザー誘発CNVのマウスモデルは、ほとんどの血管新生AMDの研究実験8 CNVのための標準的な動物モデルとなっています。 マウスの目はで動作するように非常に小さい、繊細な組織です。網膜を視覚化する目の操縦は困難であり、習得が達成されるまで、多くの練習が必要です。このタスクは、それが優勢および非利き手で学習しなければならないという事実によって複雑になります。網膜を可視化するために必要な細かい動きが学習された後さらに、両手レーザーを作動フットペダルとの間の連携が重要です。本稿では、レーザー誘発CNV P​​ROCに関わる物理的な操作のすべてを学習する課題を蒸留しようとしましたオペレータは、このモデルで迅速な成功を達成するの​​を助けることになるガイドにedure。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>532 nm (green) argon ophthalmic laser</td>
			<td>IRIDEX</td>
			<td>GLx</td>
			<td>any ophthalmic 532 nm (green) argon laser can be used</td>
		</tr>
		<tr>
			<td>slit lamp</td>
			<td>Carl Zeiss</td>
			<td>30SL-M</td>
			<td>any slit lamp can be used as long as it is compatible with the laser</td>
		</tr>
		<tr>
			<td>tribromoethanol</td>
			<td>Sigma</td>
			<td>T48402-25G</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>tert-amyl alcohol</td>
			<td>Sigma</td>
			<td>152463-1L</td>
			<td>used to make anesthetic</td>
		</tr>
		<tr>
			<td>amber glass vials + septa</td>
			<td>Wheaton</td>
			<td>WH-223696</td>
			<td>tribromoethanol storage</td>
		</tr>
		<tr>
			<td>tissue wipes</td>
			<td>VWR</td>
			<td>82003-820</td>
			<td>miscellaneous&#160;</td>
		</tr>
		<tr>
			<td>1% tropicamide</td>
			<td>Falcon Pharmaceuticals</td>
			<td>RXD2974251</td>
			<td>pupillary dilation</td>
		</tr>
		<tr>
			<td>0.5% tetracaine hydrochloride</td>
			<td>Alcon</td>
			<td>&#160;0065-0741-12</td>
			<td>topical anesthesia</td>
		</tr>
		<tr>
			<td>artificial tears</td>
			<td>Alcon</td>
			<td>&#160;58768-788-25</td>
			<td>hydration</td>
		</tr>
		<tr>
			<td>heat therapy pump (for animal warming)</td>
			<td>Kent Scientific</td>
			<td>HTP-1500</td>
			<td>used to maintain animal body temp</td>
		</tr>
		<tr>
			<td>warming pad</td>
			<td>Kent Scientific</td>
			<td>TPZ-0510EA</td>
			<td>maintains animal body temperature</td>
		</tr>
		<tr>
			<td>30 gauge insulin needles</td>
			<td>BD</td>
			<td>328418</td>
			<td>IP anesthesia injection</td>
		</tr>
		<tr>
			<td>scale</td>
			<td>American Weigh Scale</td>
			<td>AWS-1KG-BLK</td>
			<td>mouse weighing</td>
		</tr>
		<tr>
			<td>cover slip (25 mm x 25 mm)</td>
			<td>VWR</td>
			<td>48366089</td>
			<td>flatten cornea to visualize mouse retina</td>
		</tr>
		<tr>
			<td>xylazine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>ketamine</td>
			<td>obtained from institution</td>
			<td>obtained from institution</td>
			<td>anesthesia</td>
		</tr>
		<tr>
			<td>Volocity</td>
			<td>PerkinElmer</td>
			<td></td>
			<td>used for volumetric re-construction</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td>used for image analysis</td>
		</tr>
	</tbody>
</table>

a mouse model for laser-induced choroidal neovascularization

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch&#8217;s membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. 
First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. 
The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD.
Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

CNV病変の定量化は、CNVの血管を標識するために免疫蛍光染色を使用してフラットマウント脈絡膜の分析を介して実行することができます。組織標本の二つの最も頻繁に使用される方法は、すぐに動物の犠牲、または内皮細胞のマーカーで死後の免疫染色の前に灌流を介して行わFITC-デキストランラベルです。これらの方法の両方を詳細13,14,21に以前に記載されている、それぞれ、それ...

Watch this Scientific Journal Video about A Mouse Model for Laser-induced Choroidal Neovascularization at JoVE.com

A Mouse Model for Laser-induced Choroidal Neovascularization

Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

レーザ誘起脈絡膜血管新生のためのマウスモデル

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) ...

a-mouse-model-for-laser-induced-choroidal-neovascularization

Research

JoVE Journal

Medicine

17.4K ビュー.  Northwestern University Feinberg School of Medicine. Here, we present the mouse laser-induced choroidal neovascularization (CNV) protocol, an experimental model that re-creates the vascular hallmarks of neovascular age-related macular degeneration (AMD). Once mastered, it can reliably and effectively induce CNV as a model system to test various experimental measures.

ビデオ: A Mouse Model for Laser-induced Choroidal Neovascularization

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

ビジュアル欠陥を特徴づけるために慢性高眼圧症のレーザー誘起マウスモデル

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

医学

Detecting Abnormalities in Choroidal Vasculature in a Mouse Model of Age-related Macular Degeneration by Time-course Indocyanine Green Angiography

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal vasculature of various eye diseases such as age-related macular degeneration (AMD). ICGA is especially useful to image the posterior choroidal vasculature of the eye due to its capability of penetrating through the pigmented layer with its infrared spectrum. ICGA time course can be divided into early, middle, and late phases. The three phases provide valuable information on the pathology of eye problems. Although time-course ICGA by intravenous (IV) injection is widely used in the clinic for the diagnosis and management of choroid problems, ICGA by intraperitoneal injection (IP) is commonly used in animal research. Here we demonstrated the technique to obtain high-resolution ICGA time-course images in mice by tail-vein injection and confocal scanning laser ophthalmoscopy. We used this technique to image the choroidal lesions in a mouse model of age-related macular degeneration. Although it is much easier to introduce ICG to the mouse vasculature by IP, our data indicate that it is difficult to obtain reproducible ICGA time course images by IP-ICGA. In contrast, ICGA via tail vein injection provides high quality ICGA time-course images comparable to human studies. In addition, we showed that ICGA performed on albino mice gives clearer pictures of choroidal vessels than that performed on pigmented mice. We suggest that time-course IV-ICGA should become a standard practice in AMD research based on animal models.

Indocyanine Green Angiography (or ICGA) performed by tail vein injection provides high quality ICGA time course images to characterize abnormalities in mouse choroid.

経時インドシアニングリーン血管造影による加齢黄斑変性症のマウスモデルにおける脈絡膜血管系の異常を検出する

Indocyanine Green&#160;Angiography (or ICGA) is a technique performed by ophthalmologists to diagnose abnormalities of the choroidal and retinal ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

マウス角膜血管新生のアルカリ熱傷損傷モデル

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

A Mouse Fetal Skin Model of Scarless Wound Repair

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs across species but is age dependent. The transition from a scarless to scarring phenotype occurs in the third trimester of pregnancy in humans and around embryonic day 18 (E18) in mice. However, this varies with the size of the wound with larger defects generating a scar at an earlier gestational age. The emergence of lineage tracing and other genetic tools in the mouse has opened promising new avenues for investigation of fetal scarless wound healing. However, given the inherently high rates of morbidity and premature uterine contraction associated with fetal surgery, investigations of fetal scarless wound healing in vivo require a precise and reproducible surgical model. Here we detail a reliable model of fetal scarless wound healing in the dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

During mammalian development, early gestational skin wounds heal without a scar. Here we detail a reliable and reproducible model of fetal scarless wound healing in the cutaneous dorsum of E16.5 (scarless) and E18.5 (scarring) mouse embryos.

瘢痕創傷修復のマウス胎児皮膚モデル

Early in utero, but not in postnatal life, cutaneous wounds undergo regeneration and heal without formation of a scar. Scarless fetal wound healing occurs ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

新生児マウス脊髄圧縮損傷モデル

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

虹彩ルベオーシスのマウス モデルとして虹彩新生血管を穿刺誘発

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

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

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

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

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

A Novel Approach to Monitoring Graft Neovascularization in the Human Gingiva

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids direct contact with the measured area, it is suitable for monitoring blood flow changes during wound healing in human patients. Vestibuloplasty is periodontal surgery to the oral vestibule, aiming to restore vestibular depth with simultaneous enlargement of the keratinized gingiva. In this special clinical case, a split thickness flap was elevated at the first upper premolar and a xenogenic collagen matrix was adapted to the resulting recipient bed. LSCI was used to monitor the re- and neovascularization of the graft and the surrounding mucosa for one year. A protocol is introduced for the correct adjustment of microcirculation measurement in the oral mucosa, highlighting difficulties and possible failures.
The clinical case study presented demonstrated that &#8212; following the appropriate protocol &#8212; LSCI is a suitable and reliable method for following up microcirculation in a healing wound in the human oral mucosa and gives useful information on graft integration.

This study introduces a protocol for measuring microcirculation in human oral mucosa by laser speckle contrast imaging. The monitoring of wound healing after vestibuloplasty combined with a xenogenic collagen graft is presented on a clinical case.

ひと歯肉移植血管新生を監視への新しいアプローチ

Laser speckle contrast imaging (LSCI) is a novel method for measuring superficial blood perfusion over large areas. Since it is non-invasive and avoids ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

胆管TNBS輸液による慢性膵炎のマウスモデル

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...

Controlled Semi-Automated Laser-Induced Injuries for Studying Spinal Cord Regeneration in Zebrafish Larvae

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes this animal model very useful for studying spinal cord injury and regeneration. The standard protocol for inducing such lesions is to transect the dorsal part of the trunk manually. However, this technique requires extensive training and damages additional tissues. A protocol was developed for laser-induced lesions to circumvent these limitations, allowing for high reproducibility and completeness of spinal cord transection over many animals and between different sessions, even for an untrained operator. Furthermore, tissue damage is mainly limited to the spinal cord itself, reducing confounding effects from injuring different tissues, e.g., skin, muscle, and CNS. Moreover, hemi-lesions of the spinal cord are possible. Improved preservation of tissue integrity after laser injury facilitates further dissections needed for additional analyses, such as electrophysiology. Hence, this method offers precise control of the injury extent that is unachievable manually. This allows for new experimental paradigms in this powerful model in the future.

The present protocol describes a method to induce tissue-specific and highly reproducible injuries in zebrafish larvae using a laser lesion system combined with an automated microfluidic platform for larvae handling.

ゼブラフィッシュ幼虫の脊髄再生を研究するための制御された半自動レーザー誘発傷害

Zebrafish larvae possess a fully functional central nervous system (CNS) with a high regenerative capacity only a few days after fertilization. This makes ...