Bringhurst Meats (Berlin, NJ) is acknowledged for their genuine help in providing the swine eyeballs.

すべてのプロトコルは、施設内動物管理使用委員会該当する場合のポリシーに準拠して実施しました。 1.準備<ol><li> 75％のダルベッコ改変イーグル培地（DMEM）で安定化媒体を準備し、25％ハンクス平衡塩溶液（HBSS）。使用するまで-20℃でアリコートとストア、よく混ぜます。 </li><li>リン酸緩衝生理食塩水（PBS）を準備し、オートクレーブで滅菌します。溶液を室温で保管してください。実験（ウェル当たり5ミリリットル）の前に37℃にPBSを温め。 </li><li> PBS中の10％、20％および30％スクロースを準備します。殺菌のための独立した0.22μmのフィルターの上、個別にすべてのソリューションをフィルタリングします。 4℃でのソリューションを保存します。 </li><li> 90 mMの濃度にPBS中のヒスタミン原液を準備します。 0.22μmのフィルターを介して濾過することにより溶液を滅菌します。小分けし、-80℃で溶液を保存します。複数の凍結と融解の繰り返しは避けてください。 </li><li>実験の前に、PBS中の新鮮な4％パラホルムアルデヒド（PFA）を確認します。氷の上に置きます。 注意：ドラフト内でPFAを維持し、適切な保護具を着用します。 </li><li>安定化媒体（網膜あたり20 ml）を解凍します。 100 U / mlの最終濃度になるようにペニシリン - ストレプトマイシンを追加します。実験前インキュベーターで37℃まで媒体の暖かい部分（網膜当たり15ミリリットル）。氷の上の媒体の残りの部分を残します。 </li><li>氷上でHBSS（網膜当たり10ミリリットル）を配置します。 </li></ol> 2.網膜器官培養<ol><li>組織培養フードにサンプルを転送します。レンズ周りを切断することにより、アイカップを開きます。ブラシを使用して、静かに網膜を引っ張ることなく、硝子体液を除去します。ゆっくりと視神経乳頭が露出するようにブラシで切断縁から網膜を切り離します。カミソリを伴う重度の視神経と網膜をリリース。 </li><li>ペトリ皿に網膜を転送し、慎重に冷HBSSを使用して一度網膜をすすぎます。私にHBSS中にサンプルを置き、CE。ステップ2.1と、この工程を繰り返すことにより、必要な数の網膜を解剖。 </li><li>かみそりで対称的に半分に網膜をカット。穏やかにウェル当たり安定培地3mlで6ウェルプレートにサンプルを移します。 5％CO 2を含有する雰囲気のインキュベーター中で37℃で30分間平衡化を可能にします。 </li><li>ステップ2.3でのインキュベーションの間に以下の試薬を準備し、暖かい培地中の所望の濃度（450μM）にヒスタミン原液を希釈します。 250 ngの/ mlの最終濃度まで培地にLXA4（アルゴン下で保存されている）を溶解します。 注：LXA4の効果を測定する場合、他の半分はLXA4でヒスタミンを用いているが、単一の網膜から半分は、単独でヒスタミンに使用されます。 </li><li>慎重に安定化媒体を吸引除去し、各ウェル（ウェル当たり3ミリリットル）に調製した培地を追加します。 5％CO 2を含有する雰囲気のインキュベーター中で1時間、37℃でサンプルをインキュベートします。 </li><li>リンス一度暖かい、滅菌PBS中のサンプル。 </li></ol> 3.免疫染色のためのサンプルを準備します<ol><li>プレートから吸引し、PBS。 4％PFA（ウェルあたり3 ml）を加え、室温で15分間インキュベートします。 </li><li>迅速PFAを吸引。一度PBSを用いてサンプルをすすぎます。 10％スクロースにサンプル（ウェルあたり5ミリリットル）に移し、4℃で2時間〜4インキュベートします。 </li><li> 30％スクロース（ウェル当たり5ml）中にサンプルを移し、4℃で一晩インキュベートします。作動凍結培地を作製するために、20％ショ糖の2つの部分を有する市販の凍結培地の一部を混合します。気泡が消えるまで、一晩以上4°Cでそれを残します。 </li><li>作動凍結培地にサンプルを移し、室温で5分間平衡化を可能にします。長方形（5ミリメートルによっておよそ3ミリメートル）にそれぞれの網膜をトリミングします。 </li><li>慎重に円筒状容器（約1cm半径×2センチ高さ）devの中に含まれる作業の凍結培地にサンプルを移しますアルミ箔からised。網膜のスライスは、液体窒素でそれらを凍結する（セクショニング時に網膜の断面を提供するように配向）垂直であることを確認してください。使用するまで-80℃でブロックを保存します。 </li><li>項各14μmの厚さのスライスにクライオスタット上のブロックとは、ガラススライド上のセクションをマウントします。使用するまで-80℃でスライドを保存してください。 </li></ol> 4.免疫染色<ol><li>一度、5％ショ糖リン酸緩衝液（SPB、PBS中の5％スクロース、pH7.4）でセクションを洗ってください。室温で1時間（10％正常ヤギ血清および0.5％トリトンX-100で5％SPB）をブロッキング緩衝液中で切片をインキュベートすることによって非特異的結合部位をブロックします。 </li><li> 1時間：（ブロッキング緩衝液中で500 1に希釈し、GFAPまたはMAP2、）適切な一次抗体を用いて（スライド上）のセクションをインキュベートします。ソリューションを吸引。 5％SPBで3回洗浄します。内因性のIgGを染色するために、このステップをスキップします。 </li><li>対応するseconを持つセクションをインキュベート1時間：DARY抗体（ブロッキング緩衝液中で800 1に希釈シアニン/ FITC結合ロバ抗マウス/抗ウサギIgG）。 </li><li> 5％SPB 3回で十分セクションを洗ってください。メディア取り付けDAPIを含有し、カバースリップで覆うことでそれらを実装することにより、顕微鏡用の試料を準備します。 </li><li>グループ間など 、滞留時間、値を得るため、このようなレーザ強度と同一のパラメータの下で20Xレンズを通して画像を取り込む、レーザー共焦点顕微鏡を使用して。 525分の488 nmのように（FITCを検出するために）785分の561 nmのように緑のチャンネルのために（シアニン3を検出するために）赤チャンネルのために、447分の408 nmのように（DAPIを検出するために）青チャネルの励起/発光波長を設定します。 </li></ol> 5.画像解析<ol><li>次の基準12に従って漏れやすい血管の割合を定量化：数のセクションの平面内の内皮細胞の核を有する血管のみを含めます。それはAGを示している場合は、血管漏出性を検討します容器の周囲の免疫染色のradient。 </li><li>発現レベルを決定するために、関心領域を選択し、画像解析ソフトウェアを用いて、平均グレー値を測定します。 </li><li>プロセスの幅を測定するために、プロセスが異なっている部分（例えば外核層、ONL）のエリアを選択します。そして、このようなプロセスが見えると連続している光学フィールドを選択します。 、微視的な設定に応じてスケールバーを設定し、プロセス全体に線を描画して、測定を行います。 </li><li>プロセスの継続性を分析するために、関心の選択領域は、その近傍の分散で、各ピクセルを置き換えることによって、画像のエッジを強化する分散と呼ばれるフィルタを適用し、自動的に、コントラストと明るさを調整する行をカウントし、カウントを正規化エリア。 </li><li> 両側スチューデントt検定を用いて統計的有意性を計算します。 *、P &lt;0.05。 **、P &lt;0.01。 ***、P &lt;0.001。 P平均±SEMとして多くの結果を。 </li></ol>

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The authors have nothing to disclose.

In this report, we present a powerful ex vivo acute retinal model of BRB dysfunction using the swine retina. This model system does not require special instruments and can be easily adapted under most laboratory settings. However, to obtain a successful result, several steps require close attention. After obtaining the eyeballs from the source, they must be kept at 4 &#176;C or on ice and processed as soon as possible. When the effect of a treatment is being analyzed, two halves of the same retina must be used -...

証拠の成長体は、血液網膜関門（BRB）1-5と血液脳関門（BBB）6,7との類似性が存在することをサポートしています。 BBBの妥協はしっかりと、このようなせん妄10として因果関係や、アルツハイマー病（AD）などの慢性神経変性疾患の診断マーカーとして8,9および急性の条件をリンクされています。潜在的な薬剤標的のためのこれらの病理と発見への機構的洞察は、一般的に脳​​の限られたアクセシビリティとネットワーク複雑さによって妨げられています。このようなin vivoイメージング11、脳の器官培養の12、初代細胞培養13,14と共培養システム15などの選択肢が生成されています。しかしながら、これらのモデルの多くは、細胞を同定するために特殊な器具、長い実験期間または複数のマーカーを必要とします。機能的および構造的BBBとBRBの間の類似性だけでなく、dyの間の相関関係2のsfunctionsは16-19を主張してきました。また、簡単にアクセス、明確に定義された細胞型および層状​​構造は、脳へのウィンドウとしてよく特徴付けられた網膜を許可しています。 BBB及びBRBの構造的および機能的な同一性を詳細に比較されていません。しかし、網膜の病状、特にBRBの違反は、またしっかりと糖尿病18-19とAD 21,22を含む、様々な疾患の進行と関連しています。したがって、機構を描写するだけでなく、潜在的な薬物をスクリーニングするだけでなく、BRBの機能不全システムを確立するために重要です。この報告書では、単純な急性網膜文化を使用して、BRBの機能不全を可能にするプロトコルが開発され、提示されています。 増加BBBの透過性およびAD様病理学的変化は、ヒスタミン、前炎症性メディエータ12とインキュベートし、脳の器官培養で確立されています。したがって、提示システムでは、ヒスタミンはAPPLましたBRBの機能不全を誘導するためにex vivoでの網膜培養をIED。このようなハツカネズミとボス牡牛座のようないくつかの種からの網膜は、テストされています。それらの商業的入手し、ヒト組織に似ていることに、新鮮な豚眼球はここで報告されたデータを提供するために利用されました。ヒスタミンおよび/ ​​または他の薬物とインキュベートした後、網膜は、免疫グロブリンG（IgG）、血液の主要なコンポーネントの1つとして、いくつかのタンパク質12の免疫染色による評価のために処理しました。グリア細胞繊維性酸性タンパク質（GFAP）、グリア活性化のための周知のマーカー。そして、微小管結合タンパク質2（MAP2）、微小管アセンブリのための不可欠なニューロン特異的細胞骨格タンパク質。さらに、網膜の層構造は、その幅と継続性の変化として、ミュラー細胞と神経節細胞のプロセスの詳細な分析を可能にします。このようにいくつかの追加のパラメータがの影響を評価するために利用可能ですBRB違反早い段階で、同様に潜在的な治療法の逆転の効果を評価します。 血管の漏れ（のBV）、グリア細胞の活性化および神経細胞の損傷応答：このプロトコルでは、スクリーニング薬の潜在的な反転効果は3つの観点から評価されています。いくつかの定量法は、例えば、免疫染色の強度、強調フィルタにより示される神経突起のプロセス及び連続の幅測定によって示される発現レベルを利用しています。より良い方法を説明するために、結果の解釈を助けるために、リポキシンA4（LXA4）、内因的炎症性損傷および減衰内皮機能不全23に対応して合成した化合物は、デモの目的のために選択されています。

<table><tbody><tr><td>DMEM</td><td>Life Technologies&amp;nbsp;</td><td>11965-092</td><td></td></tr><tr><td>HBSS</td><td>Life Technologies&amp;nbsp;</td><td>14170-112</td><td></td></tr><tr><td>Sucrose</td><td>J.T.Baker</td><td>4072-05</td><td></td></tr><tr><td>Histamine&amp;nbsp;</td><td>Sigma</td><td>H7125-1G</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp;</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>PFA</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>Freezing Media&amp;nbsp;</td><td>Triangle Biomedical Sciences</td><td>TFM-5</td><td></td></tr><tr><td>Normal Goat Serum&amp;nbsp;</td><td>Rockland</td><td>D104-00-0050</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>GFAP Antibody</td><td>Millipore</td><td>AB5804</td><td></td></tr><tr><td>MAP2 Antibody</td><td>EMD Millipore</td><td>MAB3418</td><td></td></tr><tr><td>FITC conjugated Donkey anti-rabbit IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>711-095-152</td><td></td></tr><tr><td>Cy3 conjugated Donkey anti-mouse IgG</td><td>Jackson ImmunoResearch Laboratories, Inc.</td><td>715-165-150</td><td></td></tr><tr><td>mounting medium containing DAPI</td><td>Vector Laboratories, Inc.</td><td>H-1200</td><td></td></tr><tr><td>Laser Confocal Microscope</td><td>Nikon</td><td>Eclipse Ti microscope</td><td></td></tr><tr><td>ImageJ</td><td>National Institutes of Health</td><td>1.45s</td><td></td></tr></tbody></table>

an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An inflammatory factor, histamine, is demonstrated to compromise vessel integrity in the cultured retina through positive staining of IgG outside of the blood vessels. The effects of histamine itself and those of candidate drugs for potential treatments, such as lipoxin A4, are assessed using three parameters: blood vessel leakage via IgG immunostaining, activation of M&#252;ller cells via GFAP staining and change in neuronal dendrites through staining for MAP2. Furthermore, the layered organization of the retina allows a detailed analysis of the processes of M&#252;ller and ganglion cells, such as changes in width and continuity. While the data presented is with swine retinal culture, the system is applicable to multiple species. Thus, the model provides a reliable tool to investigate the early effects of compromised retinal vessel integrity on different cell types and also to evaluate potential drug candidates for treatment.

私たちは、ヒスタミンにより誘発されるBRBの侵害から保護する可能性のある潜在的な治療法を評価するため、低コスト、時間、効率的で使いやすいシステムを提案します。 IgGは対照網膜（ 図1A）で血管内に拘束されているが、モデルが正常に確立されたことを確認ヒスタミン曝露（ 図1B）の際に血管の外に漏れます。 <p class="jove_content" fo:...

Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

低コストで、簡単に使用できる強力なシステムは、ヒスタミンにより誘発される血液網膜関門の違反を改善する可能性のある潜在的な治療法を評価するために確立されています。血管漏出、ミュラー細胞の活性化および神経突起の連続性は、損傷応答および潜在的薬物、リポキシンA4との逆転を評価するために利用されます。

治療のために血液網膜関門の違反と潜在的な薬を評価するための急性網膜モデル

A low-cost, easy-to-use and powerful model system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach. An ...

an-acute-retinal-model-for-evaluating-blood-retinal-barrier-breach

Research

JoVE Journal

Medicine

7.1K ビュー.  Rowan University. 低コストで、簡単に使用できる強力なシステムは、ヒスタミンにより誘発される血液網膜関門の違反を改善する可能性のある潜在的な治療法を評価するために確立されています。血管漏出、ミュラー細胞の活性化および神経突起の連続性は、損傷応答および潜在的薬物、リポキシンA4との逆転を評価するために利用されます。 

ビデオ: An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

網膜色素上皮生理学と病態の研究のための実験的モデル

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

神経科学

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

医学

Using Adeno-associated Virus as a Tool to Study Retinal Barriers in Disease

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes (ILM), and in conjunction with astrocytes, pericytes and endothelial cells they establish the blood-retinal barrier (BRB). BRB limits material transport between the bloodstream and the retina while the ILM acts as a basement membrane that defines histologically the border between the retina and the vitreous cavity. Labeling M&#252;ller cells is particularly relevant to study the physical state of the retinal barriers, as these cells are an integral part of the BRB and ILM. Both BRB and ILM are frequently altered in retinal disease and are responsible for disease symptoms.
There are several well-established methods to study the integrity of the BRB, such as the Evans blue assay or fluorescein angiography. However these methods do not provide information on the extent of BRB permeability to larger molecules, in nanometer range. Furthermore, they do not provide information on the state of other retinal barriers such as the ILM. To study BRB permeability alongside retinal ILM, we used an AAV based method that provides information on permeability of BRB to larger molecules while indicating the state of the ILM and extracellular matrix proteins in disease states. Two AAV variants are useful for such study: AAV5 and ShH10. AAV5&#160;has a natural tropism for photoreceptors but it cannot get across to the outer retina when administered into the vitreous when the ILM is intact (i.e., in wild-type retinas). ShH10 has a strong tropism towards glial cells and will selectively label M&#252;ller glia in both healthy and diseased retinas. ShH10 provides more efficient gene delivery in retinas where ILM is compromised. These viral tools coupled with immunohistochemistry and blood-DNA analysis shed light onto the state of retinal barriers in disease.

To investigate the blood-retinal barrier permeability and the inner limiting membrane integrity in animal models of retinal disease, we used several adeno-associated virus (AAV) variants as tools to label retinal neurons and glia. Virus mediated reporter gene expression is then used as an indicator of retinal barrier permeability.

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

M&#252;ller cells are the principal glial cells of the retina. Their end-feet form the limits of the retina at the outer and inner limiting membranes ...

Xenopusの細胞修復戦略を調査するための網膜変性の生成

Generating Retinal Injury Models in Xenopus Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the M&#252;ller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian M&#252;ller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

著者スポットライト:Xenopus Frogの網膜再生メカニズムの探索 

We have developed several protocols to induce retinal damage or retinal degeneration in Xenopus laevis tadpoles. These models offer the possibility of studying retinal regeneration mechanisms.

Xenopusオタマジャクシにおける網膜損傷モデルの生成

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

発生生物学

A Mouse Model of Retinal Ischemia-Reperfusion Injury Through Elevation of Intraocular Pressure

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, diabetic retinopathy, and retinal vascular occlusions. Rodent models of I/R injury are providing significant insights into mechanisms and treatment strategies for human I/R injury, especially with regard to neurodegenerative damage in the retinal neurovascular unit. Presented here is a protocol for inducing retinal I/R injury in mice through elevation of intraocular pressure (IOP). In this protocol, the ocular anterior chamber is cannulated with a needle, through which flows the drip of an elevated saline reservoir. Using this drip to raise IOP above systolic arterial blood pressure, a practitioner temporarily halts inner retinal blood flow (ischemia). When circulation is reinstated (reperfusion) by removal of the cannula, severe cellular damage ensues, resulting ultimately in retinal neurodegeneration. Recent studies demonstrate inflammation, vascular permeability, and capillary degeneration as additional elements of this model. Compared to alternative retinal I/R methodologies, such as retinal arterial ligation, retinal I/R injury by elevated IOP offers advantages in its&#160;anatomical specificity, experimental tractability, and technical accessibility, presenting itself as a valuable tool for examining neuronal pathogenesis and therapy in the retinal neurovascular unit.

This article describes a procedure for inducing retinal ischemia-reperfusion injury by elevated intraocular pressure in mice. Retinal ischemia-reperfusion injury by elevated intraocular pressure serves to model human pathologies characterized by compromised oxygen and nutrient delivery in the retina, enabling researchers to examine potential cellular mechanisms and treatments for human diseases of the retinal neurovascular unit.

眼圧の上昇を通じて、網膜虚血再灌流障害のマウスモデル

Retinal ischemia-reperfusion (I/R) is a pathophysiological process contributing to cellular damage in multiple ocular conditions, including glaucoma, ...

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To properly leverage this greater translatability, there is a need to devise reproducible methods of drawing quantitative data from these images. Optical coherence tomography (OCT) imaging can resolve retinal histology at micrometer resolution and reveal functional differences in vascular blood flow. Here, we delineate noninvasive vascular readouts that we use to characterize pathological damage post vascular insult in an optimized mouse model of retinal vein occlusion (RVO). These readouts include live imaging analysis of retinal morphology, disorganization of retinal inner layers (DRIL) measure of capillary ischemia, and fluorescein angiography measures of retinal edema and vascular density. These techniques correspond directly to those used to examine patients with retinal disease in the clinic. Standardizing these methods enables direct and reproducible comparison of animal models with clinical phenotypes of ophthalmic disease, increasing the translational power of vascular injury models.

In Vivo Vascular Injury Readouts in Mouse Retina to Promote Reproducibility

Here, we present three data analysis protocols for fluorescein angiography (FA) and optical coherence tomography (OCT) images in the study of Retinal Vein Occlusion (RVO).

生体内 再現性を促進するためのマウス網膜における血管損傷の読み出し

Advancements in ophthalmic imaging tools offer an unprecedented level of access to researchers working with animal models of neurovascular injury. To ...

Retinal Pathophysiological Evaluation in a Rat Model

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal detachment, breakdown of the blood-retinal barrier (BRB), and retinal angiogenesis. An in vivo rat model is a valuable experimental tool to examine the changes in the structure and function of the retina. We propose three different experimental techniques in the rat model to identify morphological changes of retinal cells, retinal vasculature, and compromised BRB. Retinal histology is used to study the morphology of various retinal cells. Also, quantitative measurement is performed by retinal cell count and thickness measurement of different retinal layers. A BRB breakdown assay is used to determine the leakage of extraocular proteins from the plasma to vitreous tissue due to the breakdown of BRB. Fluorescence angiography is used to study angiogenesis and leakage of blood vessels by visualizing retinal vasculature using FITC-dextran dye.

Diabetic retinopathy is one of the leading causes of blindness. Histology, blood-retinal barrier breakdown assay, and fluorescence angiography are valuable techniques to understand the pathophysiology of the retina, which could further enhance the efficient drug screening against diabetic retinopathy.

ラットモデルにおける網膜病態生理学的評価

A posterior segment eye disease like diabetic retinopathy alters the physiology of the retina. Diabetic retinopathy is characterized by a retinal ...

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

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

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

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

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

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

治療のために血液網膜関門の違反と潜在的な薬を評価するための急性網膜モデル

要約

さらに動画を探す

この動画の章

網膜色素上皮生理学と病態の研究のための実験的モデル

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

Xenopusオタマジャクシにおける網膜損傷モデルの生成

Xenopusオタマジャクシにおける網膜損傷モデルの生成

Xenopusオタマジャクシにおける網膜損傷モデルの生成

眼圧の上昇を通じて、網膜虚血再灌流障害のマウスモデル

生体内再現性を促進するためのマウス網膜における血管損傷の読み出し

ラットモデルにおける網膜病態生理学的評価

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

治療のために血液網膜関門の違反と潜在的な薬を評価するための急性網膜モデル

要約

さらに動画を探す

この動画の章

網膜色素上皮生理学と病態の研究のための実験的モデル

血液脳関門の破壊のラットモデルは、対象となる神経血管治療を許可する

病における網膜障壁を研究するツールとしてアデノ随伴ウイルスを使用して、

Xenopusオタマジャクシにおける網膜損傷モデルの生成

Xenopusオタマジャクシにおける網膜損傷モデルの生成

Xenopusオタマジャクシにおける網膜損傷モデルの生成

眼圧の上昇を通じて、網膜虚血再灌流障害のマウスモデル

生体内 再現性を促進するためのマウス網膜における血管損傷の読み出し

ラットモデルにおける網膜病態生理学的評価

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

生体内再現性を促進するためのマウス網膜における血管損傷の読み出し

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