Name: a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening
Uploaded: 2014-10-06T15:00:00.000+00:00
Duration: 5 min 29 s
Description: Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

We thank Ellen Ytebrouck for her valuable information regarding method application in rat. This work was supported by the Research Council of the KU Leuven (OT09/22) and the Fund for Scientific Research-Flanders (FWO Vlaanderen). Jeroen Aerts is supported by a Ph.D. fellowship of the Agency for Innovation through Science and Technology Flanders (IWT Vlaanderen) and Julie Nys by a Ph.D. grant from FWO Flanders.

全ての実験は、KUルーベンの倫理調査委員会により承認され、2010年9月22日の欧州共同体理事会指令（63分の2010 / EU）に厳密に従ってとベルギーの法律（2013年5月29日のKB）であった。あらゆる努力は、動物の苦痛を最小限にし、動物の数を減らすために行われた。 1動物の治療と麻酔<ol><li>塩酸ケタミン（75 mg / ml）を生理食塩水でメデトミジン塩酸塩（1mg / kg）の混合物の腹腔内注射でマウスを麻酔。 </li><li>マウスが完全に鎮静されている保証するために鉗子でつま先をつまんで反射神経をチェックしてください。 </li><li>まぶたや綿棒を使って、目の周囲の領域を消毒するために70％エタノールを適用します。さらに鎮静の程度を評価するためにまぶた反射を確認してください。 </li></ol> 2アイの削除<ol><li>乾いた、動物が平らに常駐していることを確認して、滑らかな表面。 </li><li>湾曲した、鋸歯状の先端部（：0.5×0.4ミリメートル優先チップサイズ）鉗子を滅菌する。 </li><li>静かに眼球をソケットから変位して視神経が到達可能であるされるまで、鉗子で眼角（目の角）に押してください。 </li><li>目の後ろの鉗子ガイド。押しては、好ましくは、曲線の始点と鉗子のではない、非常に先端で、しっかりと視神経を開催しています。これはソケットから地球を持ち上げるようにし、完全な視神経をクランプするのに役立ちます。 </li><li>マウスは、平らな面にしたまま手が最も抵抗のある方に鉗子を保持した円の動きを作る。マウスは、手の動きの方向に応じて表面に沿ってスイングします。 </li><li>視神経が二つに絞られるまで漸増速度でこのアクションを実行します（通常は7〜15円の動きとの間に、約半分毎秒1フルターンまで）。したがって、デタッチ目玉です削除しました。 </li></ol> 3。術後ケア<ol><li> （まれ）出血の場合には、粘性の凝固および止血剤で軌道を埋める。 </li><li>腹腔内に生理食塩水にatipamezol塩酸塩を1mg / kgで注入して麻酔を元に戻します。 </li><li>痛みを和らげるために腹腔内に24時間毎メロキシカムの1mg / kgのを管理します。 </li><li>角膜の脱水を防ぐために、残りの眼に眼軟膏を適用します。 </li><li>動物は、加熱プレート上回復または体温を制御するために個別のケージに絶縁材料で動物をラップしてみよう。 </li><li>最低2日間毎日マウスの体重を測定します。重量の損失は、苦痛を示し、動物が完全に回復するまで、この場合には、メロキシカムの治療を継続することができる。 </li></ol>

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The authors have nothing to disclose.

私たちの方法に従って成功した除核を行うためには、考慮すべき最も重要なステップは、1）適切なサイズの湾曲や鋸歯状の先端を有するピンセットを用いて; 2）滑らかで乾燥した表面上の摘出を実施する。 3）徐々少なくとも摩擦方向に円の動きをスピードアップ。 効果的な結果のためには、湾曲した鋸歯状の先端部（;：2.15のx 1.3ミリメートル：マウス、ラットのx 0.4ミリメートル0.5優先先端サイズ）を特徴とする、適切な鉗子を使用することが不可欠である。曲率は眼球変位後視神経への容易なアクセスを可能にし、円運動を行う際に、正しい手の配置が必要である。視神経を保持しているときに、必要なグリップを欠いているので、スムーズなヒントが推奨されていません。円の動きの間、適切に視神経を保持しないと、眼動脈、眼の貧しい離脱ひいては貧困再現性の破壊をもたらす。そのためには、 生体内での方法を適用した後、最大の動物福祉を保証するために、取り扱い鉗子の最適化のために安楽死させた動物に最初の練習にこの手法をお勧めします。成功した眼の摘出は、最近、手動で動物体を旋回し、鉗子を静止状態に保つことを除いて同じ手法を用いて研究室でラットにおいて行われた。 技術の一つの制限は、おそらく網膜を損傷する可能性がある。したがって、この方法は、組織学5を実行するために網膜を収集するにはあまり適していない。眼球がまぶたを削除するか、切断することなく、ソケットから変位される必要があるので、私たちの方法は、年齢を開いて目を投稿する制限されています。 げっ歯類を含む異なる種における眼の摘出は、日常的に、多くの場合、まぶたの除去を必要と視神経18,23-25 ​​を切断する別の方法を用いて行われる。これらのメトdsはより侵襲的である傾向があり、ここに記載の技術よりも高い学習曲線を持っている。取り外しやまぶたを縫合する必要性がなければ、手術後の回復時間は、より高い動物福祉と、より再現性のある結果をもたらし、最小化される。

目を削除し、それにより不可逆的感覚受容体表面（網膜）を破壊、視覚路に沿って感覚入力のかなりの損失を課す。若年及び成体視覚系における摘出モデルは、異なる視覚中心1-4の発達、可塑性および機能を理解する上で貴重であることが証明された。この感覚遮断の、分子細胞および生理学的結果は、皮質回路が対処し、経験ではそのような大規模な変更に対応して、その構造と機能をどのように変化するか正常な発達調節されているとどのように確立された洞察を提供することができます。 視覚的剥奪の異なる方法が存在し、それらはすべて、ビジョン関連の研究では、それらの具体的な利点があります。例えば、暗い飼育は、具体的にはまだそれが自発的な網膜活性に影響を与えない、視覚的に駆動アクティビティを排除します。同様に、蓋縫合糸またはアイパッチは、パターン化された映像設備を削除L入力自発活動を妨害することなく、彼らは、閉じた目を通して分散光の透過を可能にする。これらの方法は、可逆的であり、開発6-8中に皮質回路を彫刻における眼の入力のパターン化視覚および低レベルの相関の役割を理解する上で貴重であることが示されている。それは視神経9,10を構成網膜神経節細胞の入力の進行性の喪失を確立するため、緑内障の研究では、成体動物における視神経挫滅モデルが広く用いられている。目的は不可逆的に一度に自発的でパターン化された両方のビジョンを削除するときに、目とは、このように、網膜が完全に瞬時に取り除かれる一方、脱核、上、剥奪の適切な選択である。また、活動マッピング研究11,12における信号対雑音比を向上させることができる強固な眼内活動の不均衡を誘発する。目に摘出に応答して、機能的および構造的変化を比較する大証、例えば蓋縫合糸としてはあまり思い切った方法によって、剥奪した後、また、可塑性の恒常性とシナプス型の両方の自発網膜活動の役割に新たな洞察を公開する可能性があります。 除核は、直接網膜標的における栄養の影響の損失をトリガします。例えば、BDNFレベルが有意に外側膝状核（LGN）と成人の除核したラット13の上丘で下方制御されている。構造的リモデリングを媒介するメッセンジャー分子として機能する反応性酸素種もまた、成体ラットの視覚システム14の皮質下の構造で検出された。さらに、マウスの異なる皮質下の視覚ターゲット構造全体のミクログリアおよびアストログリアの活性化は、一週間15の具体的なポスト摘出時間枠で起こる。一緒に、グリア、構造的および分子レベルで異なる皮質下の応答における視神経求心路遮断をもたらす。これらの皮質下にもかかわらず効果は、必ずしも、皮質レベル16での効果を巻き込むません。奪わ視覚野への非視覚的な入力の強化の隣に他の感覚分野の修正を含む注目すべきは、クロスモーダル皮質可塑性は、単眼（ME）3,4,17,18および双眼（BE）1,17の両方の後に発生摘出。 別に視覚神経科学に貢献するから、求心路遮断のタイプとして除核は、中枢神経系の19の神経保護及び神経変性20-22特性のバランスを研究するために使用することができる。 摘出を実行するための異なる手順は、既に文献に記載されている。ラットとマウスにおけるin vivo私の ​​ためにいくつかの方法論が原因軌道筋肉や組織23〜25の不必要な切片にあまり簡単である。そのようなマハジャンらのような他の出版。（2011）5を用いて詳細なプロトコルを提供<html>遺伝子型 - 表現型の相関関係を研究するための、目の高スループット収集、おそらく検死のための鈍的切開。その目的のために、この方法は、便利で速い。一つは（生きた動物での）除核ではなく、目自体、次求心視覚路を勉強することを選択するときしかし、この方法は、in vivoでの摘出にはあまり適しています。このような設定では、後の摘出生存率は非常に重要である。また、 生体内の損傷や視神経の保存および眼窩組織における最小限のは有利で ​​ある。 ここで、私たちはファゲらにより記載されたものと類似の代替摘出方法を提示する（2008）26、すなわち、特定の有利な特性を提供する：それは、迅速な術後回復に関連しており、非常に低い学習しきい値によって特徴付けられる研究者のための。眼の形態学的または視覚的経路の研究：一般的に、異なる方法が、その後の研究の焦点に応じて相補的である。 ve_contentは&quot;&gt;要するに、摘出は恒常性とクロスモーダル脳の可塑性、グリア応答特性、および軸索の安定性の調査に向けてのビジョンの研究から適用することができます。この可視化の記事では、私たちがin vivoでの眼の摘出のために実行可能で信頼性の高い方法を実証マウス。

<table><tbody><tr><td>Ketamine hydrochloride (Anesketin)</td><td>Dechra Veterinary Products (Eurovet)</td><td>BE-V136516</td><td></td></tr><tr><td>Medetomidine hydrochloride (Domitor)</td><td>Orion Corporation (Janssen Animal Health)</td><td>BE-V151742</td><td></td></tr><tr><td>Atipamezol hydrochloride (Antisedan)</td><td>Orion Corporation (Elanco Animal Health)</td><td>BE-V153352</td><td></td></tr><tr><td>Antibiotics (cefazolin, Kefzol)</td><td>Eurocept Pharmaceuticals</td><td>BE 106267</td><td></td></tr><tr><td>Eye ointment (Fucithalmic)</td><td>Leo Pharma nv-sa</td><td>BE 144654</td><td></td></tr><tr><td>Moria MC31 Forceps - Serrated Curved</td><td>Fine Science Tools</td><td>11370-31</td><td>For application in the mouse. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td></tr><tr><td>Narrow Pattern Forceps - curved</td><td>Fine Science Tools</td><td>11003-13</td><td>For application in the rat. Any forceps with similar dimensions can be used as long as the tip is curved and serrated.</td></tr><tr><td>Hemostatic cotton wool</td><td>Qualiphar</td><td>N/A</td><td>Other hemostatic agents are equally suitable (&lt;em&gt;e.g.&lt;/em&gt;, Viscostat, #649, Ultradent Products)</td></tr></tbody></table>

a highly reproducible and straightforward method to perform in vivo ocular enucleation in the mouse after eye opening

摘出または眼の外科的除去は、一般的に、神経求心路遮断のためのモデルとして考えることができる。これは、哺乳動物の視覚系1-4に沿って視覚的に、クロスモーダル·発生の可塑性の異なる側面を研究するための貴重なツールを提供します。 ここでは、大人2名まで20日齢のマウスでは検証されたマウスの1の除去または両眼のためのエレガントで簡単な方法を実証する。簡単に言うと、消毒湾曲した鉗子は、目の後ろに視神経をクランプするために使用されます。その後、円の動きは、視神経を収縮し、眼球を除去するために行われる。この技術の利点は、高い再現性、無出血への最小限の、迅速な術後の回復や実験者のための非常に低い学習しきい値です。したがって、動物の大量の最小限の労力で操作し、処理することができる。技術の性質は、わずかに損傷を誘導する可能性が手順中の網膜。この副作用は、マハジャンらと比較して、この方法はあまり適しています。（2011）5ゴールは、網膜組織を収集し、分析することであれば。眼球がまぶたを削除せずにソケットから変位させる必要があるので - ：また、私たちの方法は、ポストアイ開口の年齢（13年以降P10マウス）に制限されています。この原稿に記載のin vivoでの除核技術は、最近成功したラットにおいて軽微な変更を加えて適用され、一般的にはげっ歯類の求心視経路を研究するために有用であると思われています。

図1に記載したプロトコルを用いて眼の正常な除去を示し、出血の非存在下または眼窩組織または眼窩（ 図1A、1B）に明らかな物理的損傷によって特徴付けられる。さらに、削除された眼は完全に無傷の地球儀（ 図1C）の指標と、滑らかな角膜、脈絡膜および光ディスクを持っています。私たちのプロトコルは、目と機械旋盤の後ろに視神経のクランプを包含しているため、削除された眼の視神経は、網膜（ 図1D）の基部にくびれている。周囲の脳領域（ 図1E）を損傷することなく、きれいなカットの視神経に記載されたプロシージャの結果を実行する。 単眼摘出は、活動マッピング（ 図2）との組み合わせで、一方が急激に反対映像設備で機能的または眼特定の入力領域を描写することができサル28のような高次の哺乳動物におけるマウス12,27、さらには眼優位コラムlの皮質。 マウスを用いた実験では、一方の除去（ME）または両眼（BE）は、ターゲットを絞った視覚刺激と組み合わせ、 をZif268のmRNAまたはc-Fos蛋白質発現レベルの検出は、視覚野12,27の地域ニューロンの活性化を明らかにするために適用した。視覚的に刺激された対照（ 図2A）とは対照的に、BEマウスは、視覚入力（ 図2B）の欠如を完了することにより、視覚野において基底活性を示した。このように、非視覚野（ すなわち 、より前方部分より後方のセクションの聴覚野における体性感覚皮質）との視覚との間に境界線が発見された。 1週間の生存時間で私のマウスからの結果は、反対側の視覚皮質に目特定の入力領域を可視化した。 2単眼駆動型の領域は、低活動だったとメディ位置アルと中央両眼ゾーン（ 図2C）の横。 <img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51936/51936fig1highres.jpg" width="500" /> 眼窩、取り出し、目と視神経の後の除核状態の図1。定性的評価。曲がったピンセット（A）は出血や損傷が眼窩（B）に観察されないと、眼を除去した後。角膜および脈絡膜（C、D）の通常の出現によって反映されるように除去された眼は、完全に無傷である。視神経は、目（D）を残す視神経乳頭に絞られている。脳の腹側の部分の検査は、きれいにカット視神経（アスタリスク）およびその他の構造（E）の明白な損傷を明らかにしている。 C、D中のスケールバー：1ミリメートル。 E中のスケールバー：5ミリメートル。 ：前方; L：左; P：後部; R：右。 図摘出によって明らかにされたマウス視覚野における2。機能が目を入力固有の下位区分 。目や皮質を接続黒と灰色の線は、網膜求心と目特定の入力領域の上の交差を表す。神経活動は、コントロールの冠状断面上で可視化（A）、（B）BE、そしてブレグマレベルの周りのZif268のためのin situハイブリダイゼーション （ISH） の放射性によるME（C）マウス（グレースケール）-3.40 mmである。コントロール動物（A）は、両半球での視覚野は、視覚的な刺激後に高活性を発現する。一方または両方の眼（群）を摘出した場合、活動信号の明らかな減少が奪わ皮質領域の対応に表示されます。単眼は（C）マウスは、高ACTIのゾーンを示して除核削除された眼の反対単眼ゾーンで減少した信号に囲まれた視覚野の両眼ゾーンでVITY。スケールバー：2ミリメートル。ヴァンブリュッセルら 12からの許可を得て転載。

Watch this Scientific Journal Video about A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening at JoVE.com

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

また、脱核と呼ばれる目の除去は、それが不可逆的な部分（単眼）または完全な（両眼）視力低下を誘導するため、視覚的なクロスモーダル、および哺乳動物の視覚系に沿って発達可塑性の側面を研究するための有用な戦略を提供しています。ここでは、 生体内で脱核に実行するには、非常に再現性と簡単な方法について説明します。

実行するには非常に再現性と簡単な方法<em&gt;インビボ</emアイ開口後のマウスにおける&gt;眼摘出

Enucleation or the surgical removal of an eye can generally be considered as a model for nerve deafferentation. It provides a valuable tool to study the ...

a-highly-reproducible-straightforward-method-to-perform-vivo-ocular

Universidad San Sebastian

Research

JoVE Journal

Biology

A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

23.2K ビュー.  KU Leuven - University of Leuven. また、脱核と呼ばれる目の除去は、それが不可逆的な部分（単眼）または完全な（両眼）視力低下を誘導するため、視覚的なクロスモーダル、および哺乳動物の視覚系に沿って発達可塑性の側面を研究するための有用な戦略を提供しています。ここでは、 生体内で脱核に実行するには、非常に再現性と簡単な方法について説明します。 

ビデオ: A Highly Reproducible and Straightforward Method to Perform In Vivo Ocular Enucleation in the Mouse after Eye Opening

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

リモートハイスループット表現型解析のためのマウスの目の摘出

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

医学

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

マウスのビデオ眼球運動記録法

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

神経科学

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

生体のような器官型マウス網膜Wholemountの文化で

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

生物学

Monocular Visual Deprivation and Ocular Dominance Plasticity Measurement in the Mouse Primary Visual Cortex

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the cortex to the contralateral eye to a stimulus is much stronger than the response of the ipsilateral eye in the binocular segment of the mouse primary visual cortex (V1). During the mammalian critical period, suturing the contralateral eye will result in a rapid loss of responsiveness of V1 cells to contralateral eye stimulation. With the continuing development of transgenic technologies, more and more studies are using transgenic mice as experimental models to examine the effects of specific genes on ocular dominance (OD) plasticity. In this study, we introduce detailed protocols for monocular visual deprivation and calculate the change in OD plasticity in mouse V1. After monocular deprivation (MD) for 4 days during the critical period, the orientation tuning curves of each neuron are measured, and the tuning curves of layer four neurons in V1 are compared between stimulation of the ipsilateral and contralateral eyes. The contralateral bias index (CBI) can be calculated using each cell&#39;s ocular OD score to indicate the degree of OD plasticity. This experimental technique is important for studying the neural mechanisms of OD plasticity during the critical period and for surveying the roles of specific genes in neural development. The major limitation is that the acute study cannot investigate the change in neural plasticity of the same mouse at a different time.

Here, we present detailed protocols for monocular visual deprivation and ocular dominance plasticity analysis, which are important methods for studying the neural mechanisms of visual plasticity during the critical period and the effects of specific genes on visual development.

マウス初等視覚皮質における単眼視外剥離と眼優位可塑性測定

Monocular visual deprivation is an excellent experimental paradigm to induce primary visual cortical response plasticity. In general, the response of the ...

発生生物学

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the present protocol, mice expressing green fluorescent protein (gfp) under the promoter of Cx3cr1 (BALB/c-Cx3cr1gfp/gfp) were used to image microglia cells in vivo in the retina. Microglia are resident macrophages of the retina and have been implicated in several retinal diseases1,2,3,4,5,6. This protocol provides a detailed approach for generation of retinal B-scans, with SD-OCT, and imaging of microglia cell distribution in Cx3cr1gfp/gfp mice with SLO in vivo, using an ophthalmic imaging platform system. The protocol can be used in several reporter mouse lines. However, there are some limitations to the protocol presented here. First, both SLO and SD-OCT, when used in the high-resolution mode, collect data with high axial resolution but the lateral resolution is lower (3.5 &#181;m and 6 &#181;m, respectively). Moreover, the focus and saturation level in SLO is highly dependent on parameter selection and correct alignment of the eye. Additionally, using devices designed for human patients in mice is challenging due to the higher total optical power of the mouse eye compared to the human eye; this can lead to lateral magnification inaccuracies7, which are also dependent on the magnification by the mouse lens among others. However, despite that the axial scan position is dependent upon lateral magnification, the axial SD-OCT measurements are accurate8.

In Vivo Imaging of Cx3cr1gfp/gfp Reporter Mice with Spectral-domain Optical Coherence Tomography and Scanning Laser Ophthalmoscopy

This protocol describes how high-resolution imaging techniques such as spectral domain optical coherence tomography and scanning laser ophthalmoscopy can be utilized in small rodents, using an ophthalmic imaging platform system, to obtain information on retinal thickness and microglial cell distribution, respectively.

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1gfp/gfpレポーター マウスの画像

Spectral domain optical coherence tomography (SD-OCT) and scanning laser ophthalmoscopy (SLO) are extensively used in experimental ophthalmology. In the ...

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 ...

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural changes. These diseases affecting the fundus always exhibit typical abnormalities in certain cell types in the retina, including photoreceptor cells, retinal ganglion cells, cells in the retinal blood vessels, and cells in the choroidal vascular cells. Noninvasive, highly efficient, and adaptable imaging techniques are required for both clinical practice and basic research. Image-guided optical coherence tomography (OCT) satisfies these requirements because it combines fundus photography and high-resolution OCT, providing an accurate diagnosis of tiny lesions as well as important changes in the retinal architecture. This study details the procedures of data collection and data analysis for image-guided OCT and demonstrates its application in rodent models of choroidal neovascularization (CNV), optic nerve crush (ONC), light-induced retinal degeneration, and experimental autoimmune uveitis (EAU). This technique helps researchers in the eye field to identify rodent retinal structural changes conveniently, reliably, and tractably.

The protocol presented here details the procedures of data collection and data analysis for image-guided optical coherence tomography (OCT) and demonstrates its application in multiple rodent models of ocular diseases.

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

げっ歯類の目の微小解剖

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

マウスの精密アルカリ熱傷のための修正生検パンチ法

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

著者スポットライト:角膜混濁の治療と再生の研究の進歩 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

実行するには非常に再現性と簡単な方法

要約

さらに動画を探す

この動画の章

リモートハイスループット表現型解析のためのマウスの目の摘出

マウスのビデオ眼球運動記録法

生体のような器官型マウス網膜Wholemountの文化で

マウス初等視覚皮質における単眼視外剥離と眼優位可塑性測定

生体内でマルチモーダルイメージングとマウスレーザー誘発脈絡膜新生血管モデルの解析

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1^gfp/gfpレポーターマウスの画像

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

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

げっ歯類の目の微小解剖

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

実行するには非常に再現性と簡単な方法

要約

さらに動画を探す

この動画の章

リモートハイスループット表現型解析のためのマウスの目の摘出

マウスのビデオ眼球運動記録法

生体のような器官型マウス網膜Wholemountの文化で

マウス初等視覚皮質における単眼視外剥離と眼優位可塑性測定

生体内でマルチ モーダル イメージングとマウス レーザー誘発脈絡膜新生血管モデルの解析

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1gfp/gfpレポーター マウスの画像

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

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

げっ歯類の目の微小解剖

マウスモデルにおけるパンチトレフィン法を用いた角膜および辺縁部のアルカリ損傷誘導

生体内でマルチモーダルイメージングとマウスレーザー誘発脈絡膜新生血管モデルの解析

生体内でスペクトル領域光干渉断層法と走査レーザー眼底Cx3cr1^gfp/gfpレポーターマウスの画像

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