Name: an acute retinal model for evaluating blood retinal barrier breach and potential drugs for treatment
Uploaded: 2016-09-13T15:00:00
Description: Watch this Scientific Journal Video about An Acute Retinal Model for Evaluating Blood Retinal Barrier Breach and Potential Drugs for Treatment at JoVE.com

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?...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="984">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">DMEM</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">11965-092</td>
			<td style="width:305px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">HBSS</td>
			<td style="width:269px;">Life Technologies&#160;</td>
			<td style="width:119px;">14170-112</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Sucrose</td>
			<td style="width:269px;">J.T.Baker</td>
			<td style="width:119px;">4072-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Histamine&#160;</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">H7125-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:269px;">Invitrogen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">PFA</td>
			<td style="width:269px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Freezing Media&#160;</td>
			<td style="width:269px;">Triangle Biomedical Sciences</td>
			<td style="width:119px;">TFM-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Normal Goat Serum&#160;</td>
			<td style="width:269px;">Rockland</td>
			<td style="width:119px;">D104-00-0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">Triton X-100</td>
			<td style="width:269px;">Sigma</td>
			<td style="width:119px;">T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">GFAP Antibody</td>
			<td style="width:269px;">Millipore</td>
			<td style="width:119px;">AB5804</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">MAP2 Antibody</td>
			<td style="width:269px;">EMD Millipore</td>
			<td style="width:119px;">MAB3418</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">FITC conjugated Donkey anti-rabbit IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">711-095-152</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Cy3 conjugated Donkey anti-mouse IgG</td>
			<td style="width:269px;">Jackson ImmunoResearch Laboratories, Inc.</td>
			<td style="width:119px;">715-165-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">mounting medium containing DAPI</td>
			<td style="width:269px;">Vector Laboratories, Inc.</td>
			<td style="width:119px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:291px;">Laser Confocal Microscope</td>
			<td style="width:269px;">Nikon</td>
			<td style="width:119px;">Eclipse Ti microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:291px;">ImageJ</td>
			<td style="width:269px;">National Institutes of Health</td>
			<td style="width:119px;">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 ...

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

Techniques for Processing Eyes Implanted with a Retinal Prosthesis for Localized Histopathological Analysis: Part 2 Epiretinal Implants with Retinal Tacks

網膜タックと2部網膜上インプラント：ローカライズされた病理組織学的分析のための人工網膜を移植した処理の目のための技術

Retinal prostheses for the treatment of certain forms of blindness are gaining traction in clinical trials around the world with commercial devices ...

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

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

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

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

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

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

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

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

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

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

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 ...