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。

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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

Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

肝微小循環における血流と酸素消費量の可視化と分析：急性肝炎モデルへの応用

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of ...

医学

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

などの酸素グルコース欠乏および再酸素化<em&gt;インビトロ</em&gt;虚血再灌流傷害モデル血液脳関門の機能障害を研究するための

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

Quantitative Fundus Autofluorescence for the Evaluation of Retinal Diseases

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks performed by the RPE are phagocytosis and processing of outer photoreceptor segments through lysosome-derived organelles. These degradation products, stored and referred to as lipofuscin granules, are composed partially of bisretinoids, which have broad fluorescence absorption and emission spectra that can be detected clinically as fundus autofluorescence with confocal scanning laser ophthalmoscopy (cSLO). Lipofuscin accumulation is associated with increasing age, but is also found in various patterns in both acquired and inherited degenerative diseases of the retina. Thus, studying its pattern of accumulation and correlating such patterns with changes in the overlying sensory retina are essential to understanding the pathophysiology and progression of retinal disease. Here, we describe a technique employed by our lab and others that uses cSLO in order to quantify the level of RPE lipofuscin in both healthy and diseased eyes.

The retinal pigment epithelium (RPE) supports the sensory retina through recycling visual cycle byproducts, which accumulate as lipofuscin. These products are autofluorescent and can be qualitatively imaged in vivo. Here, we describe a method to quantitatively image RPE lipofuscin using confocal scanning laser ophthalmoscopy.

網膜疾患の評価のための定量的眼底自己蛍光

The retinal pigment epithelium (RPE) is juxtaposed to the overlying sensory retina, and supports the function of the visual system. Among the tasks ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

ウサギにおける網膜下手術のためのステッププロトコルによってステップ

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

腸血液関門と微生物叢製品の肝代謝を評価するためのIn Vivo法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Oxygen-Induced Retinopathy Model for Ischemic Retinal Diseases in Rodents

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR model induction and its readouts in both mice and rats. Retinal neovascularization is induced in OIR by exposing rodent pups either to hyperoxia (mice) or alternating levels of hyperoxia and hypoxia (rats). The primary readouts of these models are the size of neovascular (NV) and avascular (AVA) areas in the retina. This preclinical in vivo model can be used to evaluate the efficacy of potential anti-angiogenic drugs or to address the role of specific genes in the retinal angiogenesis by using genetically manipulated animals. The model has some strain and vendor specific variation in the OIR induction which should be taken into consideration when designing the experiments.

Oxygen-induced retinopathy (OIR) can be used to model ischemic retinal diseases such as retinopathy of prematurity and proliferative diabetic retinopathy and to serve as a model for proof-of-concept studies in evaluating antiangiogenic drugs for neovascular diseases. OIR induces robust and reproducible neovascularization in the retina that can be quantified.

げっ歯類における虚血性網膜症の酸素誘導網膜症モデル

One of the commonly used models for ischemic retinopathies is the oxygen-induced retinopathy (OIR) model. Here we describe detailed protocols for the OIR ...

Retinal Organoid Induction System for Derivation of 3D Retinal Tissues from Human Pluripotent Stem Cells

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to differentiate into all types of retinal cells, even mini-retinal tissues, hold huge promises for patients with these diseases and many opportunities in disease modeling and drug screening. However, the induction process from hPSCs to retinal cells is complicated and time-consuming. Here, we describe an optimized retinal induction protocol to generate retinal tissues with high reproducibility and efficiency, suitable for various human pluripotent stem cells. This protocol is performed without the addition of retinoic acid, which benefits the enrichment of cone photoreceptors. The advantage of this protocol is the quantification of EB size and plating density to significantly enhance the efficiency and repeatability of retinal induction. With this method, all major retinal cells sequentially appear and recapitulate the main steps of retinal development. It will facilitate downstream applications, such as disease modeling and cell therapy.

Here we describe an optimized retinal organoid induction system, which is suitable for various human pluripotent stem cell lines to generate retinal tissues with high reproducibility and efficiency.

ヒト多能性幹細胞からの3Dレチナル組織の導出のためのレチナルオガノイド誘導システム

Retinal degenerative diseases are the main causes of irreversible blindness without effective treatment. Pluripotent stem cells that have the potential to ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

ヒト以外の霊長類モデルにおける変性網膜疾患の網膜色素上皮移植

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...