Name: establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment
Uploaded: 2015-06-05T15:00:00.000+00:00
Duration: 7 min 53 s
Description: Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

This work was supported by National Institutes of Health Grant to A.S.M. (EY021784).

以下のプロトコルは、トーマス·ジェファーソン大学制度動物実験委員会のガイドラインに及び視覚研究における動物の使用に関するARVO声明に準拠しています。 ex vivoで創傷文化用レンズの1セットアップと準備<ol><li>無菌層流フード内で3 100ミリメートルペトリ皿を置きます。 RTで、途中トリス/デキストロース緩衝液を用いてペトリ皿を2つ（140ミリモルのNaCl、5mMの塩化カリウム、0.7ミリモルのNa 2 PO 4、5mMのD-グルコース、8.25 mMトリス塩基、HClで7.4にpHをTDバッファー）フィル、第三の空を残します。プレ暖かい培養培地（メディア199 1％L-グルタミンおよび1％ペニシリン/ストレプトマイシンを補充した）37 O Cに 注： インビボで起こるように培養培地を、標準的な治癒創傷は、無血清です。しかし、創傷修復の培養物は、血清または他の因子を含む定義された培地条件で正常に成長させることができます。 </li><li>肥沃な胚Dを削除しますAYインキュベーターから15白色レグホンニワトリの卵（穏やかに揺らしながら37.7℃に保持） </li><li>洗浄瓶からの層流フード、70％エタノールでシェルのきれいな外で選択された卵を置きます。滅菌溶液および器具を使用して、層流フード内で無菌条件下で、以下のすべての手順を実施しています。 </li><li>空の100mmペトリ皿に卵と場所の内容を解読。標準鉗子と細かいハサミを使用して胚を刎ねます。 TDバッファーを含むペトリ皿にニワトリ胚の頭部を置き、適切に胚の残りを処分。必要に応じてもう15分より、短時間のTDバッファにニワトリ胚の頭を維持しません。 </li><li>シャーレの蓋にニワトリ胚ヘッドを配置します。高精度の鉗子を使用して、以下の順序で目からその添付硝子体液と一緒にレンズを取り外してください。目の奥に小さな開口部を作成するために、ピンセットで目の奥ピンチ。 </li><li>そして、W硝子体液を把握i番目の鉗子と穏やかローリング運動と硝子体に引っ張るが、取り付けられたレンズと硝子体が眼から追い出されます。 TDバッファを含む残りのペトリ皿に入れレンズ/硝子体。レンズは、もはやよりも30分間TDバッファに残ることを可能にします。 </li><li>解剖顕微鏡下で新たなペトリ皿の蓋にレンズを移動します。この点以降は、解剖顕微鏡下にあるすべての手順を実行します。高精度なピンセットで慎重にレンズティッシュを傷つけないよう注意しながら、鉗子のエッジを使用してレンズを追い出された任意の毛様体（色素性細胞）を離れて磨きます。 注：毛様体の取り外しは、レンズに内因性ではない細胞タイプは、創傷修復培養に含まれていないことを保証します。 </li><li>後方レンズカプセルとの関連から硝子体をピンチオフすることにより、高精度なピンセットで硝子体液からレンズを分離します。 </li><li>高精度の鉗子を使用して、小滴の中にレンズを転送します35ミリメートルの組織培養皿でTDバッファ（約200μL）の。 </li></ol> 2.実行モック白内障手術<ol><li>オリエント上に向け、レンズの前方側面に35mm皿でTDバッファのドロップでレンズ。 注：レンズの前面を簡単にレンズ上皮の前部と赤道域の境界を指摘し、組織内の密度の高いリングの存在によって同定されます。対照的に、水晶体線維細胞が結合している水晶体嚢の後部上のマークが存在しないことがあります。 </li><li>二つの高精度なピンセットを使用すると、それぞれの手で1鉗子で組織を把持することにより、前方レンズカプセルの中心、レンズ組織を囲む厚い基底膜、及びそれに関連する前方レンズ上皮で小切開（約850μm）を作成し、静かに反対方向に引っ張ります。 </li><li>レンズ組織の大部分を構成する繊維細胞塊を、削除、dislodレンズ上皮にその添付ファイルからそれを銀杏、ハイドロ溶出（ 図1Aにモデル化された古典的な白内障手術に使用されるアプローチ）により、レンズカプセルを囲みます。 </li><li> TDバッファー300μlの27.5 G針の先端で1mlシリンジを埋めます。前部レンズカプセルに作られた切開部に針の先端を挿入し、ほぼ中間レンズへ。 </li><li>ゆっくりレンズ繊維細胞塊にTDバッファーを注入する注射器を押し下げます。 50〜200μlのTD、決して300以上μLを注入します。上皮およびレンズカプセルから自分自身を緩め繊維細胞塊を観察します。 </li><li>高精度の鉗子を使用して、前方切開部位を介してレンズから緩め繊維細胞塊を除去。 注：この手順は、このサイトにちょうど隣接する繊維細胞が付着細胞を裸出されていたに後方レンズ基底膜カプセル、損傷した水晶体上皮を残します。 </li></ol> 3. Preparinex vivoでの文化のためのG傷ついたレンズ<ol><li>カプセルバッグの前面に5つのカットをすることによって、培養皿上で、上記の白内障手術、細胞側を上にした結果、レンズカプセルバッグを平らに。 </li><li>レンズの赤道まで元の切開部位に垂直にカットします。ダウン培養皿カプセル側に取り付けられた上皮細胞面を上にして、レンズカプセルの合成5「フラップ」をフラット化。星や花のような形状（ 図1B参照 ）を取るべきである今ex vivoで負傷したレンズに注意してください。 </li><li>星の各ポイントでピンセットでそっと下、皿にプレスをカプセルを保護することができます。これは、外植片の5つの最も外側の先端に小さなくぼみを作り、皿に持続的な添付ファイルになります。 注：この手順の間にカプセルを損傷することが可能であるので、それは、FLAの先端に近い皿にカプセルを確保することが重要ですできるだけPS、並びに可能なように固定ポイントの最小量を作るために（一般的に2つ、フラップごとに3つの最大）。 </li><li> 35ミリメートル皿からTDバッファを削除し、予め温めておいたメディアの1.5ミリリットルと交換してください。インキュベーター内でその蓋と場所（37℃、5％CO 2）で35ミリメートル皿をカバーしています。 </li></ol>嚢の創傷領域の再上皮化は、定量分析のために、水晶体上皮細胞の元の添付ゾーン（OAZ）から、発生セントラル移行ゾーン（CMZ）、の4分離。 注：細胞が傷害に反応してすぐにCMZ領域に移動し始めます。文化の中で初日によって十分な細胞は、マイクロ解剖23によるCMZとOAZの分離後、分子および生化学的解析のためCMZ全体に移行しています。このプロトコルは、カプセルの創傷領域から一度に1つのフラップ（OAZ）の除去を含みます。 <ol><li> demarを観察カチオン、OAZとCMZの間、解剖顕微鏡下ではっきりと見える（ 図2A参照 ）。二つの高精度なピンセットを使用して、行のいずれかの側に、OAZ / CMZ線、他にちょうど隣接する一方の端部の両方鉗子で把持（ 図2A、B、矢印参照）。 </li><li>片手を使用して、/ CMZの側に1つの鉗子は、一方/ピンセットで、優しくOAZ / CMZ線に沿ってOAZを引き出しながら、負傷した文化に保持し続けます。 CMZは、簡単にこの線に沿ってOAZから分離します。二つの領域が完全に分離されるまで、培養物全体の周りに、この線に沿って進みます。 </li><li>例えば、ウェスタンブロットまたは免疫共沈降23,25のようなRNA-、配列24または生化学的解析などの分子解析のための分離OAZとCMZの分画を研究。 </li></ol>

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Series B, Biological. 359, 777-784 (2004).</li><li>Nodder, S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+in+embryos:+a+review.">Wound healing in embryos: a review.</a> Anatomy and embryology. 195, 215-228 (1997).</li><li>Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+repair+and+regeneration.">Wound repair and regeneration.</a> Nature. 453, 314-321 (2008).</li></ol>

The authors declare that they have no competing financial interests.

Here is described a technique for preparing a culture model of wound repair that involves performing an ex vivo cataract surgery on chick embryo lenses after their removal from the eye. The lens epithelium responds to this clinically relevant wounding with a repair process that closely mimics that which occurs in vivo, and shares features with wound repair in other epithelial tissues2,4. While the protocol is straightforward and simple to follow, performing mock cataract surgery with embryoni...

臨床的に関連する、モック白内障手術は、ここで説明するex vivoでの上皮の創傷治癒モデルは、損傷に応答して、上皮組織の修復を制御する機構を調査するためのツールを提供するために開発されました。このモデルを作成する際にを目的とした主な機能は、1を含む）と密接画像へのカルチャ設定で負傷、2）修理の調節要素を調節のしやすさ、および3）能力修復プロセスへのインビボ応答をレプリケート条件を提供します、その全体を、リアルタイムで。課題は、したがって、細胞のネイティブの微小環境では、上皮創傷修復を研究することが可能とされた培養モデルを作成し、操作することでした。この創傷修復モデルの利用可能性は、修復プロセスを調節するマトリックスタンパク質、サイトカインおよびケモカインからの内因性シグナル伝達の手がかりを識別するための新たな可能性を開きます。また、モデルがどのように検査するための理想的ですnは上皮が創傷領域2,3を 、再上皮化する集団シートとして負傷した上皮4の集団移動を導くで機能創傷端に間葉リーダー細胞の系統を決定するために移動することができます。また、このモデルでは、効果的な創傷治癒を促進し、異常な創傷修復5を防ぐことができる治療薬を同定する際のプラットフォームを提供します。 今日創傷修復プロセスについて知られているものの大部分を提供してきた文化およびin vivoの両方で利用可能な創傷修復モデル、の数が既にあります。このような角膜6-12や皮膚13-17のような動物損傷モデルでは、からの寄与を含むプロセスに関与することができたすべての修復メディエーターの文脈で負傷に組織の反応を研究する機会があります血管系と神経系。しかし、experiを操作するには限界がありますインビボでの精神状態、それが継続的に時間をかけて、in vivoでの修復応答のイメージング研究を行うことがまだ可能ではありません。対照的に、そのようなスクラッチ傷のようなほとんどのインビトロ創傷修復培養モデルは、容易に操作することができ、経時的に追跡が、in vivoでの組織の創傷治癒を研究する環境的文脈を欠いています。 ex vivoでのモデルは、プロセスの任意の時点で修理の分子制御因子を調節する能力と結合し、細胞の微小環境のコンテキスト内で時間かけて連続的に損傷修復過程を研究するという利点を提供するが、これらに適合するいくつかのモデルが存在しますパラメータ。 ここでは、生理的創傷の上皮組織の応答を再現する文化を癒し、再現性の高いex vivoでの上皮の傷を生成する手順を説明します。組織源、 エクスビボ MOCとしてニワトリ胚のレンズを使用しましたk個の白内障手術が行われます。レンズは無血管、神経支配、および関連する間質18,19の自由ではない、それは自己完結型の厚い基底膜カプセル内にあるので、これらの研究のために使用するのに理想的な組織です。ヒト疾患において、白内障手術は、レンズの混濁による視力喪失に対処し、かつレンズの大部分を含むレンズ繊維細胞塊の除去を含みます。次の白内障手術のビジョンは、人工の眼内レンズを挿入することによって復元されます。白内障手術の手順は、繊維細胞を除去して、繊維細胞によって占有されていたレンズカプセルの後部領域の再上皮化することによって応答する各レンズ上皮における損傷応答を誘導します。白内障手術では、ほとんどの傷の修復反応のように、時にはレンズに事後Capsuとして知られている筋線維芽細胞の出現に関連する創傷治癒応答に異常な線維化の成果は、そこに発生しますル混濁20-22。白内障手術創傷治癒モデルを生成するには、白内障手術の手順は、生理学的損傷を生成するためにニワトリ胚の眼から取り出したレンズで模倣されます。レンズ上皮細胞に囲まれた非常に一貫性のある円形の創傷領域のレンズ繊維細胞の結果の顕微除去。この細胞集団は、しっかりとレンズの基底膜カプセルに取り付けられたままであり、外科的処置により負傷されます。上皮細胞は、リーダーセル1として修復プロセスで知られているビメンチン豊富な間葉細胞の集団が率いる傷を癒すために、内因性の基底膜の剥皮領域に移行します。このモデルでは損傷に対する上皮の応答が容易に可視化することができ、細胞の微小環境のコンテキスト内で経時的に追跡しました。細胞は、創傷修復に関与すると予想される分子の発現または活性化の変更に容易にアクセスできます。目の強力な機能モデルは分離し、創傷治癒の枠組みの中でマイグレーション固有の変化を研究する能力です。研究のために熟成一致エクスビボ創傷治癒培養多数を調製する能力は、このモデルの別の利点です。したがって、このモデル系は、創傷治癒プロセスに対するそれらの効果について、創傷修復のメカニズムと試験治療薬を離れていじめるするユニークな機会を提供します。 エキソビボモック白内障手術モデルは損傷修復のメカニズムを研究するための重要なリソースを提供し、幅広い適用性を有することが期待されます。

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>S271-3</td><td>Use at 140 mM in TD Buffer</td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>P217-500</td><td>Use at 5 mM in TD Buffer</td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma</td><td>S0876</td><td>Use at 0.7 mM in TD Buffer</td></tr><tr><td>D-glucose (Dextrose)</td><td>Fisher Scientific</td><td>D16-500</td><td>Use at 0.5 mM in TD Buffer</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP152-1</td><td>Use at 8.25 mM in TD Buffer</td></tr><tr><td>Hydrochloric acid</td><td>Fisher Scientific</td><td>A144-500</td><td>Use to pH TD buffer to 7.4</td></tr><tr><td>Media 199</td><td>GIBCO</td><td>11150-059</td><td></td></tr><tr><td>L-glutamine</td><td>Corning/CellGro</td><td>25-005-CI</td><td>Use at 1% in Media199</td></tr><tr><td>Penicillin/streptomycin</td><td>Corning/CellGro</td><td>30-002-CI</td><td>Use at 1% in Media199</td></tr><tr><td>100 mm petri dishes</td><td>Fisher Scientific</td><td>FB0875711Z</td><td></td></tr><tr><td>Stericup Filter Unit</td><td>Millipore</td><td>SCGPU01RE</td><td>Use to filter sterilize Media</td></tr><tr><td>Dumont #5 forceps (need 2)</td><td>Fine Science Tools</td><td>11251-20</td><td></td></tr><tr><td>35 mm Cell Culture Dish</td><td>Corning</td><td>430165</td><td></td></tr><tr><td>27 G 1 ml SlipTip with precision glide needle</td><td>BD</td><td>309623</td><td></td></tr><tr><td>Fine Scissors</td><td>Fine Science Tools</td><td>14058-11</td><td></td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>91100-12</td><td></td></tr><tr><td>Other Items Needed: General dissection instruments,&amp;nbsp; fertile white leghorn chicken eggs, check egg incubator (humidified, 37.7&amp;deg;C), laminar flow hood, binocular stereovision dissecting microscope</td><td></td><td></td><td></td></tr></tbody></table>

establishment of a clinically relevant ex vivo mock cataract surgery model for investigating epithelial wound repair in a native microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

エクスビボモデルは、細胞の天然の微小環境において創傷治癒過程を研究するために作成しました細胞の天然の微小環境内の上皮の創傷治癒の調節に関与するメカニズムを調べるために、臨床的に関連するex vivoでの模擬白内障手術のモデルが作成されました。このモデルは、その固有の特性に起因する多くの利点を提供する水晶体組織から作成されます：1...

Watch this Scientific Journal Video about Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment at JoVE.com

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

臨床的に関連の確立<em&gt;エクスビボ</emネイティブ微小環境における上皮創傷修復を調査するための&gt;モック白内障手術モデル

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

establishment-clinically-relevant-ex-vivo-mock-cataract-surgery-model

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

6.5K ビュー.  Thomas Jefferson University. Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment. 

ビデオ: Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Human Ex vivo Wound Model and Whole-Mount Staining Approach to Accurately Evaluate Skin Repair

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic wound treatments are inadequate, while available pre-clinical models poorly predict the clinical efficacy of new therapies. Here we describe a high throughput, pre-clinical model to assess multiple aspects of the human skin repair response. Partial thickness wounds were created in human ex vivo skin and cultured across a healing time course. Skin wound biopsies were collected in fixative for the whole-mount staining procedure. Fixed samples were blocked and incubated in primary antibody, with detection achieved via fluorescently conjugated secondary antibody. Wounds were counterstained and imaged via confocal microscopy before calculating percentage wound closure (re-epithelialization) in each biopsy. Applying this protocol, we reveal that 2 mm excisional wounds created in healthy donor skin are fully re-epithelialized by day 4-5 post-wounding. On the contrary, closure rates of diabetic skin wounds are significantly reduced, accompanied by perturbed barrier reformation. Combining human skin wounding with a novel whole-mount staining approach allows a rapid and reproducible method to quantify ex vivo wound repair. Collectively, this protocol provides a valuable human platform to evaluate the effectiveness of potential wound therapies, transforming pre-clinical testing and validation.

Here we demonstrate an optimized technique for assessing wound repair using ex vivo human skin combined with a whole-mount staining approach. This methodology provides a pre-clinical platform for the evaluation of potential wound therapies.

皮膚修復を正確に評価するヒトEx vivo創傷モデルと全山染色法

Chronic non-healing wounds, which primarily affect the elderly and diabetic, are a significant area of clinical unmet need. Unfortunately, current chronic ...

発生生物学

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

胚性創傷チック角膜における瘢痕のない組織再生の調査

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

角膜上皮摩耗眼はりのモデルとして角膜創傷治癒

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

医学

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 ...

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

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

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

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

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

An Epithelial Abrasion Model for Studying Corneal Wound Healing

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

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

角膜創傷治癒を研究するための上皮擦過傷モデル

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

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮の機械的および化学的損傷のためのEx Vivoおよびin vivo動物モデル

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched in epitheliotropic growth factors that are essential in corneal wound healing. Unlike AS, PRP is based on a differential centrifugation system, yielding more platelet-derived growth factors. Autologous conditioned serum (ACS) not only preserves the preparation of AS and PRP, but also focuses on immune-modulating properties, which are important in inflammatory diseases.
The lack of standardized protocols and high preparation costs are limitations for the clinical application of ACS. This video experiment demonstrates a standard operating procedure for preparing modified autologous conditioned serum (mACS) eye drops. First, glycerol was added into heparin syringes as the blood cell stabilizer during hypoxic incubation. To activate the blood cells, a 4 h incubation at 37 &#176;C was initiated. Then, the blood samples were centrifuged at 3,500 &#215; g for 10 min at room temperature. After filtration of the supernatant through a 0.22 &#181;m filter, the mACS eye drops were fully prepared.
A tentative try-out of the therapeutic effect of mACS showed that it may have competitive advantages over conventional AS in the corneal wound healing in ex vivo mouse eyes. The AS used in this study was prepared according to published studies and the clinical practice in our hospital. Therefore, the efficacy of mACS on ocular surface diseases could be evaluated in future research through in vivo animal studies and clinical trials.

Production of Modified Autologous Conditioned Serum and Ex Vivo Assessment of Its Healing Potential in Murine Corneal Epithelium

This article describes a protocol to simplify the process and render the preparation of autologous conditioned serum (ACS) less expensive. No special syringes or surface-coated glass beads are needed. Moreover, the modified ACS (mACS) has competitive advantages over conventional autologous serum in the corneal wound healing of murine eyes ex vivo.

改変自家馴化血清の作製とマウス角膜上皮における治癒能の ex vivo 評価

Human blood-derived topical therapies have been a boon to clinicians in recent decades. Autologous serum (AS) and platelet-rich plasma (PRP) are enriched ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

All animal organs, from the skin to eyes to intestines, are covered with sheets of epithelial cells that allow them to maintain homeostasis while protecting them from infection. Therefore, it is not surprising that the ability to repair epithelial wounds is critical to all metazoans. Epithelial wound healing in vertebrates involves overlapping processes, including inflammatory responses, vascularization, and re-epithelialization. Regulation of these processes involves complex interactions between epithelial cells, neighboring cells, and the extracellular matrix (ECM); the ECM contains structural proteins, regulatory proteins, and active small molecules. This complexity, together with the fact that most animals have opaque tissues and inaccessible ECMs, makes wound healing difficult to study in live animals. Much work on epithelial wound healing is therefore performed in tissue culture systems, with a single epithelial cell-type plated as a monolayer on an artificial matrix. Clytia hemisphaerica (Clytia) provides a unique and exciting complement to these studies, allowing epithelial wound healing to be studied in an intact animal with an authentic ECM. The ectodermal epithelium of Clytia is a single layer of large squamous epithelial cells, allowing high-resolution imaging using differential interfering contrast (DIC) microscopy in living animals. The absence of migratory fibroblasts, vasculature, or inflammatory responses makes it possible to dissect the critical events in re-epithelialization in vivo. The healing of various types of wounds can be analyzed, including single-cell microwounds, small and large epithelial wounds, and wounds that damage the basement membrane. Lamellipodia formation, purse string contraction, cell stretching, and collective cell migration can all be observed in this system. Furthermore, pharmacological agents can be introduced via the ECM to modify cell:ECM interactions and cellular processes in vivo. This work shows methods for creating wounds in live Clytia, capturing movies of healing, and probing healing mechanisms by microinjecting reagents into the ECM.

Characterizing Epithelial Wound Healing In Vivo Using the Cnidarian Model Organism Clytia hemisphaerica

This paper describes a method to create wounds in the epithelium of a live Clytia hemisphaerica medusa and image wound healing at a high resolution in vivo. Additionally, a technique to introduce dyes and drugs to perturb signaling processes in the epithelial cells and extracellular matrix during wound healing is presented.

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