この作品は、米国心臓協会（EIA 0740129N）とNIH T32 HL90610によってサポートされていました。

<ol>
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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+arachidonic+acid-induced+thromboxane+generation+impairs+skeletal+muscle+arteriolar+dilation+with+genetic+dyslipidemia.">Increased arachidonic acid-induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia.</a> Microcirculation. 15, 621-631 (2008).</li><li>Baumbach, G. L., Hadju, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+and+composition+of+cerebral+arterioles+in+renal+and+spontaneously+hypertensive+rats.">Mechanics and composition of cerebral arterioles in renal and spontaneously hypertensive rats.</a> Hypertension. 21, 816-826 (1993).</li><li>Uchida, E., Bohr, D. F., Hoobler, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+rat+middle+cerebral+arterieal+to+reduced+PO2+and+prostacyclin.">Electrical and mechanical responses of rat middle cerebral arterieal to reduced PO2 and prostacyclin.</a> Am. J. Physiol. 276, H509-H516 (1994).</li></ol>

利害の衝突が宣言されません。

この一般的な手法は容易にほとんどの組織に適用することができますが、提示さプロトコルは、骨格筋の微小血管の分離、除去し、ダブルカニュレーションについて説明します。現在の原稿のために、用語 &quot;細動​​脈&quot;はアクティブなトーンを休んで、直径70から120ミクロンの範囲の抵抗血管を記述するために著者によって使用されており、臓器にも血流抵抗の調節に主要な貢献者であるか組織。 いくつかの修正と共に、このシステムは、特定の研究者のために複数のアプリケーションに装着することができ、容易に（例えば、膜電位17の決定のための貫通電極の使用）の深さの実験の多くを提供するために追加の技術の取り込みが可能になります。基本的な準備の重要なステップは、常に明確なままで、できるように、容器をカニューレを挿入するために使用される流入量と流出量ピペットがよく一致していることを保証されてい安易な方法で維持される流量と圧力。ピペットは、破片の分の部分で目詰まりすると、その流れを復元するためのヒントを非常に小さな部分を絶つ必要があるかもしれません。捜査官が慎重であれば、彼らはどちらuncloggedできないか、または血管のカニュレーションは非常に困難になる直径が十分に大きなポイントに到達するまで、ピペットは複数回再利用することができます。しかし、この手術と実験準備、細部への注意、および具体的な条件や要件に合わせて適切な変更の習得と、調査官は、容易に生理学的および薬理学の課題無数に応答して血管反応性を決定する経路を評価することができます。この製剤はまた、捜査官は、アクティブなトーン14の条件下での血管壁の力学の変化の基本的な分析を実行することができます。これは確かに非常に強力で適応性の高い技術であり、その一つである容易に、より基本的な応答のハイスループットスクリーニングに詳細な機序の研究の範囲にまたがるアプリケーション用に使用することができます。

<table border="1"><tbody><tr><td> 試薬および器具 </td><td> 会社 </td><td> コメント/カタログ＃ </td></tr><tr><td>血管商工会議所</td><td>カスタム</td><td>デイブEick（MCW） </td></tr><tr><td>温水循環水浴</td><td> PolyScienceとハーケ</td><td>ハーケDC 10 </td></tr><tr><td>ピペット</td><td>フレデリックHaer·アンド·カンパニー</td><td>キャピラリーチューブ2.0ミリメートル外径×内径1.0 mm（27-33-1） </td></tr><tr><td>圧力監視装置</td><td>世界の精密機器</td><td></td></tr><tr><td>水ジャケットリザーバー</td><td>カスタム</td><td></td></tr><tr><td>外部光源</td><td>世界の精密機器</td><td> Novaflex </td></tr><tr><td>ピペットプラー</td><td>ミクロ·インスツルメンツ</td><td> PMP102マイクロピペットプラー</td></tr><tr><td>完全相補手術器具のメンター</td><td>ファイン科学ツール</td><td>デュモン</td></tr><tr><td>ウルトラファイン鉗子</td><td>ファイン科学ツール</td><td>イノックス＃5 </td></tr><tr><td>シルク縫合糸</td><td> Ethilon </td><td> ＃10から0または9から0 </td></tr><tr><td>ステレオ顕微鏡</td><td>オリンポス</td><td>オリンパスSZ-11 </td></tr><tr><td>アナログビデオキャリパー</td><td> Boeckeler </td><td>を介してコントローラ（VIA-100） </td></tr><tr><td>高解像度アナログカメラ</td><td>パナソニック</td><td> GP-MF 602 </td></tr><tr><td>酸素タンク</td><td>地域の</td><td> 21％のバランスの窒素および5％CO 2のバランス窒素</td></tr><tr><td>チューブ</td><td>タイゴン</td><td></td></tr><tr><td>排出ポンプ</td><td>コー​​ルパーマーインストゥルメント（株） </td><td></td></tr><tr><td>変更されたラットのPSS </td><td>以下のレシピを参照してください。 </td><td></TD&gt; </tr><tr><td>バン·ブレーメンの弛緩PSS </td><td>以下のレシピを参照してください。 </td><td></td></tr></tbody></table> 表1。図に示され孤立した微小血管ステーションのセットアップの主要なコンポーネントのリストが表示されます。 <table border="1"><tbody><tr><td> 変更されたラットPSSレシピ </td><td> PSSの2リットルを作成するには </td><td> 20X塩ストック（2L） </td><td> 20Xバッファーストック（2L） </td></tr><tr><td> NaClを</td><td></td><td> 278.0グラム</td><td></td></tr><tr><td>塩化カリウム</td><td></td><td> 14.0グラム</td><td></td></tr><tr><td> MgSO 4を -7H 2 O </td><td></td><td> 11.5グラム</td><td></td></tr><tr><td>のCaCl 2-H 2 O </td><td></td><td> 9.4グラム</td><td></td></tr><tr> &lt;TD&gt; NaHCO 3を </td><td></td><td></td><td> 80.8グラム</td></tr><tr><td> EDTA </td><td></td><td></td><td> 0.4グラム</td></tr><tr><td>のNaH 2 PO 4 </td><td> 0.28グラム</td><td></td><td></td></tr><tr><td>グルコース</td><td> 1.98グラム</td><td></td><td></td></tr><tr><td> 20倍の塩ストック</td><td> 100 mLの</td><td></td><td></td></tr><tr><td> 20Xバッファーストック</td><td> 100 mLの</td><td></td><td></td></tr><tr><td>蒸留水</td><td> 1800 mLの</td><td></td><td></td></tr></tbody></table> 表2。孤立した微小血管のプロトコルで使用される標準の生理食塩水（PSS）のレシピ。 レシピへのコメント：塩ストックとバッファストックの2 L 2 Lを作成します。これらのC使用されていないときに冷蔵なりますが、PSSを準備する前によく、多くの場合、それらを横に振る。追加成分は、最終的なPSSの準備の時に追加されます。 <table border="1"><tbody><tr><td> バン·ブレーメンの弛緩PSS </td><td> PSSの2リットルを作成するには </td><td> 20X塩ストック（1L） </td><td> 20X緩衝在庫（1L） </td></tr><tr><td> NaClを</td><td></td><td> 107.4グラム</td><td></td></tr><tr><td>塩化カリウム</td><td></td><td> 7.0グラム</td><td></td></tr><tr><td> MgSO 4を -7H 2 O </td><td></td><td> 5.76グラム</td><td></td></tr><tr><td>のMgCl 2·6H 2 O </td><td></td><td> 81.32グラム</td><td></td></tr><tr><td> NaHCO 3を </td><td></td><td></td><td> 40.4グラム</td></tr><tr><td> EDTA </td><td></td><td></td><td> 0.2グラム</td></tr><tr><td> EGTA </td><td></td><td></td><td> 15.22 </td></tr><tr><td>のNaH 2 PO 4 </td><td> 0.28グラム</td><td></td><td></td></tr><tr><td>グルコース</td><td> 1.98グラム</td><td></td><td></td></tr><tr><td> 20倍の塩ストック</td><td> 100 mLの</td><td></td><td></td></tr><tr><td> 20Xバッファーストック</td><td> 100 mLの</td><td></td><td></td></tr><tr><td>蒸留水</td><td> 1800 mLの</td><td></td><td></td></tr></tbody></table> 表3。ゼロアクティブなトーンの条件下で分離された微小血管のプロトコルで使用されるバン·ブレーメンの弛緩生理食塩水（PSS）のレシピ。 レシピへのコメント：塩株式の1 Lを作成する緩衝在庫のND 1 L。これらは使用されていないときに冷蔵なりますが、よく、多くの場合、PSSを準備する前にそれらを振ることができます。追加成分は、最終的な弛緩PSSの準備の時に追加されます。

the ex vivo isolated skeletal microvessel preparation for investigation of vascular reactivity

孤立した微小血管の準備は、このように血流抵抗が1から5まで様々な要因の寄与を制御する血管径の検査を可能にするex vivoでの製剤である、と。これは、大規模な測定では、最初に内田らによって記述されていた15数十年前に古典的な実験の準備です。この最初の記述は主にバージニア6-8の大学で博士ブライアンDulingの研究室で、広範囲に変更され、強化された技術の基礎を提供し、我々は以下のページで現在のアプローチを提示します。この準備は、特に任意の微小血管としてラットの薄筋の動脈を参照してくださいますが、基本的な準備が容易に種を9月13日を越え、ほぼすべての他の組織または器官から分離された船舶に適用することができます。孤立した微小血管の機械的（すなわち、次元）の変更が容易に評価することができます生理の広範な配列（例えば、低酸素症、血管内の圧力、せん断）または薬理学的な課題への応答、および無傷たが、ex vivoで 、組織に統合された応答を構成する機構要素への洞察を提供することができます。また、血管内の圧力（筋）など、他のソースからの貢献の多くの制御を許可しながら、この方法の重要性は、血行動態、自律神経支配、それは微小血管径の統合された規制上の影響の容易な操作のためにできることです（例えば、せん断応力）、内皮依存性または独立した刺激、ホルモン、および実質の影響は、部分的なリストを提供します。適切な実験条件下で、適切な目標と、これは、 インビボまたは容易に広範な全身の変数の容易な制御のために許可されていない現場組織/臓器の準備、 の上の利点として機能することができます。 マこの準備のJOR制限は本質的に強みの結果である。定義により、これらの血管の動作は血管抵抗の調節に最も重要な貢献者の多くはこのように、神経体液性、代謝などを含む、削除されている、研究者が避けるために注意を促している条件の下で検討されている過この調製物を利用して収集されるデータの解釈と外挿。この準備に関して懸念の他の重要なエリアは、そのような内皮細胞ライニングや血管平滑筋などの細胞成分を損傷し、エラーの変数ソースを導入することができるような非常に簡単であるということです。それが強く、個々の治験責任医師はプロトコルのコースを通じて準備の品質を確保するための適切な測定、実験開始時と定期的に両方を利用することをお勧めします。

Watch this Scientific Journal Video about The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity at JoVE.com

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

不定冠詞<em&gt; ex vivoで</em&gt;準備は血管の血管作動性刺激に対する反応と受動的な壁の力学を介して基本的な構造特性の評価の両方を尋問のために最大の薄筋の抵抗動脈の分離に記載されています。

ザ ex vivoで</em血管反応性の調査のために>隔離された骨格微小血管の準備

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

the-ex-vivo-isolated-skeletal-microvessel-preparation-for

Nanyang Technological University

Research

JoVE Journal

Biology

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

10.6K ビュー.  West Virginia University. 不定冠詞 ex vivoで準備は血管の血管作動性刺激に対する反応と受動的な壁の力学を介して基本的な構造特性の評価の両方を尋問のために最大の薄筋の抵抗動脈の分離に記載されています。

ビデオ: The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

イントラ不可欠な顕微鏡による骨格筋のin vivo微小循環測定における

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

生物学

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

レンチウイルスを介したノックダウン中生体外で</emヒト造血幹細胞の>赤血球

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

隔離された骨格筋の収縮、倦怠感や交互の ex vivo での評価

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

小説<em&gt;エクスビボ</emマウス胎児ハート&gt;文化法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

単離された血液灌流ラットの肺の準備にシングル微小血管透過性を定量化する

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

マウスの心臓変力作用の調​​査のために分離された乳頭筋の収縮の測定

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

網膜の低温電子顕微鏡セクション全体-マウント、および微小血管の免疫組織化学的可視化の低張性分離血管準備

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

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 ...

Assessment of Vascular Tone Responsiveness using Isolated Mesenteric Arteries with a Focus on Modulation by Perivascular Adipose Tissues

Altered vascular tone responsiveness to pathophysiological stimuli contributes to the development of a wide range of cardiovascular and metabolic diseases. Endothelial dysfunction represents a major culprit for the reduced vasodilatation and enhanced vasoconstriction of arteries. Adipose (fat) tissues surrounding the arteries play important roles in the regulation of endothelium-dependent relaxation and/or contraction of the vascular smooth muscle cells. The cross-talks between the endothelium and perivascular adipose tissues can be assessed ex vivo using mounted blood vessels by a wire myography system. However, optimal settings should be established for arteries derived from animals of different species, ages, genetic backgrounds and/or pathophysiological conditions.

The protocol describes the use of wire myography to evaluate the transmural isometric tension of mesenteric arteries isolated from mice, with special consideration of the modulation by factors released from endothelial cells and perivascular adipose tissues.

ザ ex vivoで隔離された骨格微小血管の準備