Name: preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo
Uploaded: 2018-05-08T12:30:00.000+00:00
Duration: 7 min 35 s
Description: Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

この作品は、エルンスト ・ ウント ベルタ Grimmke 財団とドイツの Ophthalmologische 協会 (犬) からの助成金によって支えられました。

本研究の実験プロシージャによって、動物ケア委員会のラインラント = プファルツ州、ドイツが承認されました。動物の世話は、眼科における動物の使用のためのビジョンと眼科 (ARVO) ステートメントで研究・ ビジョン研究制度のガイドラインと協会に適合。動物は、動物実験のための EU 指令 2010年/63/EU によると扱われました。生後 3-4 ヶ月男性の c57bl/6 j マウス (のジャクソンの実験室、バー ハーバー、私、米国) は、実験に使われました。動物は、標準条件 (温度 23 ± 2 ° C、湿度範囲 55 ± 10%、12 時間の明暗サイクル) の下で収容された、標準的なマウスの食事と水広告自由にアクセスしていた。
1. マウス網膜の分離
<ol><li>準備テーブルに解剖器具を置き、すべての楽器がクイック準備を有効にするかどうかはチェックします。</li>
	<li>CO2吸入によるマウスを犠牲に。</li>
	<li>スチールはさみとマウスの首をはねる、頭蓋骨 sagitally をはさみの同じ組み合わせを使用して 2 つの半分に切る、皮膚と脳目はさみ (図 1) を使用して取り外します。</li>
	<li>目はさみを使って軌道の骨だけを残して頭蓋骨骨を断ちます。</li>
	<li>冷たい促進バッファーを含んでいる解剖皿に軌道を転送します。</li>
	<li>氷上解剖皿を維持または 10 分ごとのバッファーを変更します。</li>
	<li>目はさみを使用して軌道の骨を切り取って、タイプ 4 の精密ピンセットと学生 Vannas 春はさみ (図 2) を使用して眼窩組織とともに眼球を慎重に取り外します。</li>
	<li>タイプ 5 の精密ピンセットと眼動脈の主な枝を損傷しないように注意を取って、Vannas capsulotomy はさみを使用して外眼筋、結合組織、ハーダー腺を慎重に取り外します。</li>
	<li>眼動脈眼窩枝は、最終的に 10-0 ナイロン縫合糸 (図 3) を使用して眼動脈の主な枝の近位端を縛る。</li>
	<li>4 精密ピンセット 10 s を使用して入力の 70% のエタノールを含むペトリ皿に目グローブを転送します。目グローブ解剖皿に戻し、新鮮なクレブス ソリューションでバッファーを置き換えます。角膜が白っぽいエタノール曝露後表示されます。</li>
	<li>30 ゲージ針周辺の角膜を穿刺そして Vannas capsulotomy はさみを使用して角膜を解剖します。優しく虹彩組織を切断、Vannas capsulotomy はさみ脈絡膜と網膜の間の 1 つの鋭い点を配置し、脈絡膜と強膜を切断します。(図 4) 視神経の周りいくつかの強膜の組織を残します。</li>
</ol>2. 灌流チェンバで網膜の取付
注: 網膜細動脈実験用灌流チェンバは自家製です。透明な貯水池で、流出管 (図 5) で構成されています。シリコン チューブの一方の端は histoacryl 接着剤と三方活栓に接続されているもう一方の端の部屋の下に釘付けに。三方活栓に新鮮なクレブス バッファーを含む注射器を接続し、バッファーで管を埋めます。
<ol><li>ニードル ホルダーとガラス ピペットを取るし、灌流チャンバーの下部に管の端にそれをプッシュします。100 μ m 径の先端を取得する 4 型ハサミ、ピペットの先端を破る。</li>
	<li>マイクロ ピペットの先端に焦点を当てるし、経由で注射器をチューブに接続されているフラッシュします。毛細血管が残骸によって妨げられていません注意してください。閉塞の場合に、削除し、フラッシュ チューブ別マイクロ ピペットを挿入します。</li>
	<li>冷たいクレブス バッファーと灌流チャンバーし、ステレオ顕微鏡室を置きます。</li>
	<li>灌流チェンバに透明なプラスチックのプラットフォームを配置します。このプラットフォームは、視神経と眼動脈の幅 1.8 mm のインデントを持って、直径 1.6 mm の 2 つの鋼線が配置されます。次に、プラットフォームに 4.0 mm 外径内径 2.8 mm のステンレス製リングを配置します。</li>
	<li>スプーンを使用して細心の注意で、視神経と眼動脈の接続されている網膜を解剖室から灌流チェンバに転送します。この手順は、潜在的なを防ぐために重要な組織のストレッチとして、実験に支障をきたすことがあります。</li>
	<li>眼動脈の近位端の縫合部分を切断、マイクロ ピペットに 10-0 ナイロン縫合糸の前もって形成された 2 つのループを配置、ピペットと眼動脈を cannulate します。マイクロ ピペット (図 6) に動脈を結ぶ、罰金ポイント ピンセットを使用して透明なプラットフォーム上に網膜を配置します。</li>
	<li>優しく 2 つの細かい点ピンセットで前方レンズ被膜を開いて涙、レンズを抜くし、刀を使用して全体のレンズの被膜を断ちます。</li>
	<li>その後、網膜の視神経にパルス プラナから半分の距離に 4 つの放射状切開を作るし、下部にそれを修正する網膜上にステンレス製のリングを配置します。網膜は、(図 7) 実験の準備が整いました。</li>
</ol>3. 網膜細動脈の実験のための準備
<ol><li>光学顕微鏡の下で灌流チェンバを配置します。目的は 100 X 水浸対物レンズです。</li>
	<li>2 つビデのために- とアウト-flow をペリ ポンプに接続し、商工会議所でクレブス バッファー 95% O2と酸素し、37 ° C で 5% CO2炭酸循環100 と 120 mL/min の流量を使用します。</li>
	<li>貯留層シリコン ホースを介して三方活栓にピペットへ接続すると、シリコン チューブを接続します。それから、リザーバーは 50 mm Hg に対応する高さにクレブス バッファーがまだ三方活栓を開いていません。</li>
	<li>顕微鏡、網膜面に焦点を当てるの目標に目を通します。血管や赤血球を検索します。血管が完全に折りたたまれていること、時々 だけいくつかの赤血球は表示可能です。</li>
	<li>血管が見つかったら、それに焦点を当てるし、三方活栓を開きます。容器の直径がすぐに増やす必要があります、場合は、細動脈や小静脈の場合 centripetally、赤の血液細胞のいずれかを遠心に移動します。細動脈は直径同じ順序の静脈に比べて小さいです。</li>
	<li>よく見える壁と血管が見つかったら、次の 30 分 (図 8) の平衡期間の実験のためにも流用できます。平衡期間中に細動脈の弱い本質的な緊張の結果内腔の直径の減少の 10% 未満で開発します。</li>
</ol>4. 実験を実行します。
<ol><li>お風呂のソリューションに追加することによって、トロンボキサン擬態 9,11-dideoxy-9α 11α methanoepoxy プロスタグランジン F2α (U46619) と船の生存率をテストします。このエージェントは、≥ 10 の濃度で直径を休んでから約 50% によってマウス網膜細動脈を収縮-6 M。</li>
	<li>一度船を実行可能な確認は、ポンプを循環させることにより風呂からアゴニストを洗浄し、お風呂に新鮮なクレブス バッファーを供給します。</li>
</ol>

<ol>
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著者は競合する金融興味を持ってないです。

マウス網膜における血管反応の測定は、網膜血管のサイズが小さいため困難です。紹介したテクニックを持つ網膜細動脈は透過光顕微鏡による可視化します。これは、孤立した網膜は半透明なので可能です。テクニックの利点は、高光学解像度です。計算された空間分解能は 11 px/μ m です。ただし、白色光を使用する光学系の実際の解像度は 200 と 300 nm、アッベの回折限界で説明されている?...

網膜の血流障害は、糖尿病網膜症、高血圧性網膜症、緑内障1,2,3などの様々 な眼疾患の病態に関与しています。したがって、網膜の血管反応性の測定を目的とした研究がこれらの疾患の病態を理解することが重要、新しい治療法を開発するために近づきます。
マウスのゲノムに遺伝子操作の可能性があるためマウス心血管系4研究の広く使用されている動物モデルとなっています。しかし、網膜血管 (≤ 30 μ m) のサイズが小さいためマウス網膜における血管反応性の測定は難しい。たとえば、生体内計測表面技術の光学解像度で限られている、まさに搭載時 ≤ 30 μ m 径未満の少量の血液で直径または血の流れの変化を検出することがのみできます。蛍光染料または適応光学スキャン ・光検眼鏡5,6を使用して共焦点顕微鏡など、追加の高度なデバイスまた、ローカルの識別を目的とした体内測定の解釈は網膜血管のメカニズムは、麻酔薬、によって混同することができますシグナリング全身血圧と球後血管の影響で変更します。
したがって前のヴィヴォ高光学解像度とマウスの網膜の血管の反応を測定する方法を開発しました。ここに記載した技術光顕微鏡を介して送信される網膜細動脈の描出が可能します。ラットにも使用することができます、この方法は、眼血管研究の技術をターゲット遺伝子の利点へのアクセスを提供します。

<table><tbody><tr><td>Steel Scissors</td><td>Carl Roth GmbH</td><td>3576.1</td><td>1x 140 mm</td></tr><tr><td>Eye Scissors</td><td>Geuder</td><td>G-19390</td><td>1x straight, 10.5 cm</td></tr><tr><td>Precision tweezers, straight with fine tips</td><td>Carl Roth GmbH</td><td>LH68.1</td><td>2x type 4</td></tr><tr><td>Precision tweezers, straight with extra fine tips</td><td>Carl Roth GmbH</td><td>LH53.1</td><td>2x type 5</td></tr><tr><td>Vannas capsulotomy scissors</td><td>Geuder</td><td>19760</td><td>1x straight, 77 mm</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91501-09</td><td>1x curved,</td></tr><tr><td>&lt;em&gt;Barraquer&lt;/em&gt; Needle Holder</td><td>Geuder</td><td>G-17500</td><td>1x curved, 120 mm</td></tr><tr><td>Needle</td><td>Becton, Dickinson and Company</td><td>305128</td><td>1x 30 G</td></tr><tr><td>Glass Capillaries (for producing micropipettes)</td><td>Drummond Scientific Company</td><td>9-000-1211</td><td>1x (1.2 x 0.8 mm; outer/inner diameter)</td></tr><tr><td>Nylon Suture</td><td>Alcon</td><td>198001</td><td>1x 10-0</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>153066</td><td>1x 35 mm diameter</td></tr><tr><td>Nunclon cell culture dish</td><td>Thermo Fisher Scientific</td><td>172931</td><td>1x 100 mm diameter</td></tr><tr><td>Discofix C</td><td>Braun</td><td>16500C</td><td>10 cm</td></tr><tr><td>Histoacryl adhesive</td><td>B. Braun Surgical, S.A.</td><td>1050052</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pericyclic pump&amp;nbsp; (CYCLO II)</td><td>Carl Roth GmbH</td><td>EP76.1</td><td>1x</td></tr><tr><td>Vertical Pipette Puller Model 700C</td><td>David Kopf Instruments</td><td></td><td>1x</td></tr><tr><td>Microscope (Vanox-T AH-2)</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Water immersion objective LUMPlanFL, 1.0 NA</td><td>Olympus</td><td></td><td>1x</td></tr><tr><td>Digital camera (TK-C1381)</td><td>JVC</td><td></td><td>1x</td></tr><tr><td>Perfusion chamber</td><td>self-made</td><td></td><td>1x</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Drugs and Solutions&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ethanol</td><td>Carl Roth GmbH</td><td>K928.4</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>5239.1</td><td></td></tr><tr><td>Kalium chloride (KCl)</td><td>Carl Roth GmbH</td><td>6781.1</td><td></td></tr><tr><td>Kalium dihydrogen phosphate (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>3904.2</td><td></td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>261.2</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Carl Roth GmbH</td><td>9265.2</td><td></td></tr><tr><td>Sodium hydrogen carbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Carl Roth GmbH</td><td>0965.3</td><td></td></tr><tr><td>&amp;alpha;-(D)-(+)- Glucose monohydrate</td><td>Carl Roth GmbH</td><td>6780.1</td><td></td></tr><tr><td>9,11-dideoxy-9&amp;alpha;,11&amp;alpha;-methanoepoxy prostaglandin F2&amp;alpha; (U-46619)</td><td>Cayman Chemical</td><td>16450</td><td></td></tr><tr><td>Acetylcholine chloride</td><td>Sigma-Aldrich</td><td>A6625-25G</td><td></td></tr></tbody></table>

preparation steps for measurement of reactivity in mouse retinal arterioles ex vivo

血管不全と通常の網膜血流の変化、様々 な視力を脅かす眼疾患、糖尿病網膜症、高血圧性網膜症と緑内障などの発症の主な要因です。したがって、網膜微小血管の準備、基本的な病態生理学的メカニズムを記述する生理学的および薬理学的研究とデザイン疾患の治療に極めて重要なツールです。眼科研究マウス モデルの普及にもかかわらず網膜血管反応性に関する研究がこの種の不足しています。この矛盾の主な理由はであるこれらの網膜血管のサイズが小さいため挑戦的な分離のプロシージャ 〜 ≤ 内腔の直径 30 μ m。前のヴィヴォ近く生理条件下でマウス網膜血管反応の研究をできる分離および準備の技術を設けてこれらの網膜微小血管の機能研究のための直接分離の問題を回避するために.現在実験準備は特にマウス網膜細動脈を参照、ただしこの方法容易にラットから微小血管を利用します。

U-46619 は、c57bl/6 j 背景の野生型マウスから網膜細動脈の濃度依存性血管収縮反応を生産しました。10-6 M の濃度で内腔の直径の減少安静時直径から ≈50% であった。図 9 aは、1 つの網膜細動脈の代表的な濃度応答曲線を示します。細動脈 U46619 と preconstricted、累積アセチルコリン投与誘発濃度依存性内腔の直径上昇 ≈ 25 %10-5 M、そ?...

Watch this Scientific Journal Video about Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo at JoVE.com

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

多くのビジョンを脅かす眼疾患は、網膜血管の機能不全に関連付けられます。したがって、網膜血管応答の測定は、根本的な病態生理学的メカニズムを調査することが重要です。この記事では、マウスの網膜細動脈の分離と血管作動性物質の血管径に及ぼす影響を評価するために準備のための詳しいプロトコルについて説明します。

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...

preparation-steps-for-measurement-reactivity-mouse-retinal-arterioles

Research

JoVE Journal

Medicine

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

7.1K ビュー.  University Medical Center of the Johannes Gutenberg University Mainz. 多くのビジョンを脅かす眼疾患は、網膜血管の機能不全に関連付けられます。したがって、網膜血管応答の測定は、根本的な病態生理学的メカニズムを調査することが重要です。この記事では、マウスの網膜細動脈の分離と血管作動性物質の血管径に及ぼす影響を評価するために準備のための詳しいプロトコルについて説明します。

ビデオ: Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

マウス網膜を使用してCNSにおける血管再生の評価

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

神経科学

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 ...

医学

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.

A low-cost, easy-to-use and powerful system is established to evaluate potential treatments that could ameliorate blood retinal barrier breach induced by histamine. Blood vessel leakage, M&#252;ller cell activation and the continuity of neuronal processes are utilized to assess the damage response and its reversal with a potential drug, lipoxin A4.

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 ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

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 ...

Quantification of Vascular Parameters in Whole Mount Retinas of Mice with Non-Proliferative and Proliferative Retinopathies

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, gliosis&#160;and a progressive change in vascular function and structure. Although the onset of the retinopathies is characterized by subtle disturbances in visual perception, the modifications in the vascular plexus are the first signs detected by clinicians. The absence or presence of neovascularization determines whether the retinopathy is classified as either non-proliferative (NPDR) or proliferative (PDR). In this sense, several animal models tried to mimic specific vascular features of each stage to determine the underlying mechanisms involved in endothelium alterations, neuronal death and other events taking place in the retina. In this article, we will provide a complete description of the procedures required for the measurement of retinal vascular parameters in adults and early birth mice at postnatal day (P)17. We will detail the protocols to carry out retinal vascular staining with Isolectin GSA-IB4 in whole mounts for later microscopic visualization. Key steps for image processing with Image J Fiji software are also provided, therefore, the readers will be able to measure vessel density, diameter and tortuosity, vascular branching, as well as avascular and neovascular areas. These tools are highly helpful to evaluate and quantify vascular alterations in both non-proliferative and proliferative retinopathies.

This article describes a well-established and reproducible lectin stain assay for the whole mount retinal preparations and the protocols required for the quantitative measurement of vascular parameters frequently altered in proliferative and non-proliferative retinopathies.

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

Retinopathies are a heterogeneous group of diseases that affect the neurosensory tissue of the eye. They are characterized by neurodegeneration, ...

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 ...

Measuring Retinal Vessel Diameter from Mouse Fluorescent Angiography Images

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. Mutations in the microphthalmia-associated transcription factor (Mitf) gene show hypopigmentation, microphthalmia, retinal degeneration, and in some cases, blindness. In vivo imaging of the mouse retina by noninvasive means is vital for eye research. However, given its small size, mouse fundus imaging is difficult and might require specialized tools, maintenance, and training. In this study, we have developed a unique software enabling analysis of the retinal vessel diameter in mice with an automated program written in MATLAB. Fundus photographs were obtained with a commercial fundus camera system following an intraperitoneal injection of a fluorescein salt solution. Images were altered to enhance contrast, and the MATLAB program permitted extracting the mean vascular diameter automatically at a predefined distance from the optic disk. The vascular changes were examined in wild-type mice and mice with various mutations in the Mitf gene by analyzing the retinal vessel diameter. The custom-written MATLAB program developed here is practical, easy to use, and allows researchers to analyze the mean diameter and mean total diameter, as well as the number of vessels from the mouse retinal vasculature, conveniently and reliably.

Mouse retinal vasculature is particularly interesting in understanding the mechanisms of vascular pattern formation. This protocol automatically measures the diameter of mouse retinal vessels from fluorescent angiography fundus images at a fixed distance from the optic disk.

マウス蛍光血管造影画像からの網膜血管径の測定

It is important to study the development of retinal vasculature in retinopathies in which abnormal vessel growth can ultimately lead to vision loss. ...

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the choroidal vessels supply the photoreceptors. Alterations in retinal perfusion contribute to numerous sight-threatening disorders, including diabetic retinopathy, glaucoma and retinal branch vein occlusions. Understanding the molecular mechanisms involved in the control of blood flow through the retina and how these are altered during ocular disease could lead to the identification of new targets for the treatment of these conditions. Retinal arterioles are the main resistance vessels of the retina, and consequently, play a key role in regulating retinal hemodynamics through changes in luminal diameter. In recent years, we have developed methods for isolating arterioles from the rat retina which are suitable for a wide range of applications including cell physiology studies. This preparation has already begun to yield new insights into how blood flow is controlled in the retina and has allowed us to identify some of the key changes that occur during ocular disease. In this article, we describe methods for the isolation of rat retinal arterioles and include protocols for their use in patch-clamp electrophysiology, calcium imaging and pressure myography studies. These vessels are also amenable for use in PCR-, western blotting- and immunohistochemistry-based studies.

Isolation of Retinal Arterioles for Ex Vivo Cell Physiology Studies

This manuscript describes a straightforward protocol for the isolation of arterioles from the rat retina that can be used in electrophysiological, calcium imaging and pressure myography studies.

Ex Vivo細胞生理学研究のため網膜細動脈の隔離

The retina is a highly metabolically active tissue that requires a substantial blood supply. The retinal circulation supports the inner retina, while the ...

生物学

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

要約

さらに動画を探す

この動画の章

マウス網膜を使用してCNSにおける血管再生の評価

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、

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

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

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

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

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

マウス蛍光血管造影画像からの網膜血管径の測定

Ex Vivo細胞生理学研究のため網膜細動脈の隔離

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

マウス網膜細動脈Ex Vivoでの反応性の測定の準備手順

要約

さらに動画を探す

この動画の章

マウス網膜を使用してCNSにおける血管再生の評価

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、

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

Ex Vivoのレティナル組織サンプルにおけるミトコンドリア呼吸と解糖の測定

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

不拡散性網膜症を有するマウスの全型網膜における血管パラメータの定量化

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

マウス蛍光血管造影画像からの網膜血管径の測定

Ex Vivo細胞生理学研究のため網膜細動脈の隔離

細胞外のフラックスを用いた Explanted 網膜組織のエネルギー代謝の測定

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