著者は表現、心からケイティ Fink とリン Dondlinger のおかげで、この原稿の準備のために貴重な援助。
助成: NIH #R21-OD018309;#R56-HL065289;#R01 HL128242。

著者は競合する金融興味を持ってないです。

述べる導入で説明した、ようテレビ顕微鏡を使用し、分離抵抗動脈だけでなく、標準ラットモデル (とビデオで採用)、血管機能評価へのアプローチがまたで専門性の高い遺伝子設計されたラット系統は、小説を示し、これらのアプローチを活用した得ることができる強力な洞察力。アクティブ トーンを評価するこれらの強力な技術の使用と受動的な機械的性質抵抗小動脈内皮依存性規制を含...

多くの利用動脈導管動脈における血管機能の初期の研究の場合、大動脈です。太い動脈の力発生一般的に動脈のリング セグメントを組織内の力覚センにアタッチすることにより検討しました。大動脈の場合ヘリカル切削によってストリップ容器の平滑筋線維間の接続点との収縮によって発生する力の最良推定値を提供するために、力覚セン縦方向に指向されたので、その縦軸に沿って平滑筋。大動脈のヘリカル ストリップを切断するための標準的な方法は、血管の内腔にガラス棒を置いて、希望の角度で血管壁に切れ込み、カットは全体を生成する拡張された血管壁の露出の端の端につかまって容器のヘリカル ストリップ。その時点では、血管の内皮側だった容器ストリップを合力トランスデューサーに接続して、酸素の準備を水没前に破片を削除する一般的に消された、温度制御組織バス。最終的には、アプローチが Furchgott、Zawadski1、すなわち内皮の役割によって生理学の歴史の中で最も有名で重要な発見の一つにつながった由来リラックス因子 (EDRF)、その後に窒素酸化物で識別されます。血管機能を調節します。その発見につながる重要なイベントは調査官が外部表面と動脈の内皮側との接触を避けることでそのまま血管内皮細胞を維持し、大動脈のストリップは予想される展示していないことに気づいた状況アセチルコリン (ACh) が、ACh の応答の代わりにリラックスに収縮します。その観察に基づいて、調査官大動脈の標準はすばのストリップを (しかし、収縮力を生成できません) そのまま内皮と大動脈セグメントを接続いる「サンドイッチ」準備を開発し、ACh による変換緩和に収縮します。
この分野で現在広く使用されている 2 つの主要な進歩は、小さな抵抗動脈2,3でアクティブな収縮力を測定する製剤の開発(小腸間膜3のものなど) と抵抗動脈準備4,5,6を cannulated。最も早いレポートのいずれか、Mulvany とハルパーン3は自然発症高血圧ラット (SHR) の小腸間膜から分離抵抗血管の収縮力を勉強するワイヤー第 1 報準備の使い方を説明し、正常血圧 WKY コントロール。ワイヤー第 1 報システムの開発後、● キャニュレイテッド抵抗動脈準備は生体内での条件4,5,6に近い血管の研究を許可するように開発されました。 両方のアプローチは、貴重な結果を提供する、効果的な動脈の本質的なアクティブな調子を維持以上の追加のメリットがあります ● キャニュレイテッド動脈準備経壁圧と容器のレスポンス流量と血管内皮のせん断応力の変化の変化に筋活動を研究する研究者を許可して (を参照してくださいハルパーンとケリー6レビュー)。
本稿の主要な目標は重要なこれらのアクティブなトーンを調節するメカニズムに関する正確な情報を得るために分離、● キャニュレイテッド抵抗血管を用いたビデオ顕微鏡の昔ながらの手法を採用する方法を説明します血管、神経、体液、または実質のセルの独立に影響します。この基本情報は、採用の標準ラット モデルとの私たちの研究から例を新しい遺伝子ラット系統を設計、テレビで得られる血管の機能に関する洞察力の種類の考えを読者に提供します顕微鏡のアプローチ、およびすべてのコントロールおよび治験責任医師の選択、選択的近親交配によって生成される強力な新しいラット実験的モデルを含むと新開発の遺伝の実験グループを含む研究を用いることができます。エンジニア リング技術。
テレビ顕微鏡アプローチの精度のおかげで準備 ● キャニュレイテッド動脈径変化の測定は、血管の内皮依存性および内皮非依存性のメカニズムに関する非常に貴重な情報を提供できます。リラクゼーションだけでなく、高血圧、塩分の高い食事やその他の実験的介入で発生している血管の制御機構に重要である (および時々 予期しない) 変更。さらに、圧径関係の測定が分離し、Ca2 +との処置で最大限にリラックスして抵抗血管を cannulated-無料のソリューションまたは薬理学的血管拡張薬、評価を使用できます動脈血管リモデリングにより、動脈機能に影響を与えることができます動脈の受動的な機械的性質の変化の重要な洞察を提供することができる受動的な応力-ひずみ関係7を計算するための構造変化アクティブな制御機構の独立の (またはに加えて) を変更します。また、分離抵抗血管の研究から得た情報をことができます LDF、個体レベルの8,9 組織灌流の評価のための実用的な方法を利用した得られた情報によって補われることに注意してくださいすることが重要です。 ,10GS1 の蛍光標識レクチンを用いた微小血管密度の評価から得た情報によっては、具体的には、小細動脈と毛細血管の11の基底膜の糖鎖に結合して,12します後者のメソッドは、船生体内で、たとえば非灌流を数えることで微小血管密度の推定に古典的な困難を受けにくい微小血管密度の非常に正確な見積もりを提供します。血管の細動脈のアクティブな閉鎖のための血流の停止場所。これらのアプローチが分離抵抗動脈微小循環レベルでの組織の血流の変化に与える機能的変化を関連付けるための重要な洞察力を提供できる一緒に使用する場合● キャニュレイテッド動脈技術と組み合わせてこれらの貴重なアプローチの使用のいくつかの例は、本稿も提供されます。
本稿はザイモグラムの Sprague-dawley ラットの動脈の血管の変化を評価するビデオ顕微鏡検査の技術の使用に焦点を当てください。ただし、これらのテクニックが非常に貴重な品種改良や遺伝子技術を使用して、編集によって作成された専門性の高い遺伝子組み換えラット系統の表現型の変化を解明する証明されていることに注意してくださいすることが重要です。本稿では、どのようにビデオ顕微鏡検査の技術の例は最も広くダール食塩感受性 (SS) ラットの近交系ラットの歪みを含むモデル数貴重なラットの血管機能に関する重要な情報を提供しているを提供します。塩の機密 hypertenson18,19,20,21,22,23; のメカニズムを研究するのに実験モデルを使用塩を区別しないブラウン ノルウェー (BN) ラット系統 SS ラットの選択的な育成によって作成されるコンソミック ラット。コンソミック ラット パネルでブラウンのドブネズミからすべての染色体は、ダール SS24,25,26遺伝的背景に個別に交雑をされています。コンソミック ラット パネルの使用は、血圧と血管反応性24,25,26 を含む他の表現型の塩の感受性に貢献する特定の染色体に関する貴重な手がかりを提供しています。 ,,2728。
SS ラットおよび個々 の BN の染色体を運ぶコンソミック ラットを用いた選択的な繁殖戦略もダール SS 遺伝的に個々 のブラウン ノルウェー染色体交雑の小さなセグメント狭窄コンジェニック系統の世代を有効にしています。背景22,29。これらは、非常に貴重な入力の特定の遺伝子を提供したり、血圧、腎障害、血管反応性22,29などの重要な生理学的変数に影響を与えることができる染色体の地域を絞り込みます。ラットの遺伝ツールボックスに別の強力な追加、編集テクニック ZFNs、転写活性化ようなエフェクター核酸分解酵素 (TALENS) を含む高度な遺伝子を利用したラット遺伝子ノックアウト モデルの開発と最近 CRISPR Cas913 ,14,15,16,17。ラットをノックアウトする遺伝子を有効にこれらの強力な技術の出現は非常に重要な開発、遺伝子ノックアウト研究 (および使用し続ける) マウスを使用しているためほとんど専ら。本論文では実験的なコンポーネント別 ● キャニュレイテッド動脈技術とビデオ顕微鏡マスター抗酸化と細胞保護転写を欠けてノックアウト ラットの生理学的制御メカニズムを評価するための値を示します係数、核因子 (赤血球由来 2) - のような - 2 (NRF2)30,31, Sprague-dawley 遺伝的背景17TALEN 技術を使用して開発されました。NRF2 遺伝子の損失の機能検証を提供し、NRF2 を介した抗酸化物質の直接のアップレギュレーションに基づく可能性がある貴重な治療アプローチをテストに使用されたビデオ顕微鏡技術の in vitro実験、防御。NRF 2 は、ビタミン C や E32など抗酸化物質の直接投与を含む臨床試験の期待はずれの結果に照らして、人間の血管の酸化ストレスを闘う相当な治療の重要性の。

<table>
	<tbody>
		<tr>
			<td>SS Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS/JHsd/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.5BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 5BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>SS.13BN Consomic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS-Chr 13BN/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-BN Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13hmgc41-D13)hmgc23/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSA Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat77-D13rat105/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Ren1-SSB Congenic Rat</td>
			<td>Medical College of Wisconsin</td>
			<td>SS.BN-(D13rat124-D13rat101/Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Nrf2(-/-) Knockout Rat and Wild Type Littermates</td>
			<td>Medical College of Wisconsin</td>
			<td>SD-Nfe212em1Mcwi strain</td>
			<td>Contact Dr. Aron Geurts (ageurts@mcw.edu)</td>
		</tr>
		<tr>
			<td>Low Salt Rat Chow (0.4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113755</td>
			<td></td>
		</tr>
		<tr>
			<td>High Salt Rat Chow (4% NaCl)-AIN-76A</td>
			<td>Dyets, Inc.</td>
			<td>113756</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorado Video Caliper</td>
			<td>Colorado Video, Inc.</td>
			<td>Model 308</td>
			<td></td>
		</tr>
		<tr>
			<td>Video Camera</td>
			<td>Hitachi</td>
			<td>KPM1AN</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Life Science</td>
			<td>CKX41</td>
			<td></td>
		</tr>
		<tr>
			<td>Television Monitor</td>
			<td>Panasonic</td>
			<td>WVBM1410</td>
			<td></td>
		</tr>
		<tr>
			<td>Pressure Transducers</td>
			<td>Stoelting</td>
			<td>56360</td>
			<td></td>
		</tr>
		<tr>
			<td>Blood Pressure Display Unit</td>
			<td>Stoelting</td>
			<td>50115</td>
			<td></td>
		</tr>
		<tr>
			<td>Cannulated Artery Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>CH-1</td>
			<td>Single vessel chamber for general use</td>
		</tr>
		<tr>
			<td>Temperature Controller for Single Chamber</td>
			<td>Living Systems Instrumentation</td>
			<td>TC-09S</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas Dispersion Tube, Miniature,Straight</td>
			<td>Living Systems Instrumentation</td>
			<td>GD-MS</td>
			<td>Provides aeration in the vessel bath</td>
		</tr>
		<tr>
			<td>Gas Exchange Oxygenator, Miniature</td>
			<td>Living Systems Instrumentation</td>
			<td>OX</td>
			<td>Allows gas exchange with perfusate</td>
		</tr>
		<tr>
			<td>Laser-Doppler Flowmeter</td>
			<td>Perimed</td>
			<td>PeriFlux 5000 LDPM</td>
			<td></td>
		</tr>
		<tr>
			<td>GS1 Lectin</td>
			<td>Vector Labs</td>
			<td>RL-1102</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillary Tubes for Micropipettes</td>
			<td>Fredrich Haer Co.</td>
			<td>27-33-1</td>
			<td>2 mm ODX1 mm ID</td>
		</tr>
		<tr>
			<td>Verticle Pipette Puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon suture material (10/0)-3 PLY</td>
			<td>Ashaway Line and Twine Manufacturing Co.</td>
			<td>114-ANM-10</td>
			<td>Single strands of 3 ply nylon suture teased out for use on vessels</td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps-Inox</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas Scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Protandim</td>
			<td>Protandim</td>
			<td></td>
			<td>NRF2 Inducer: Contact Dr. Joe McCord (JOE.MCCORD@UCDENVER.EDU)</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Bioreagents</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Fisher Chemical</td>
			<td>S233-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose (d-glucose) anhydrous</td>
			<td>Fisher Chemical</td>
			<td>D16-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4-7H2O)</td>
			<td>Sigma Aldrich</td>
			<td>M1880-500 G</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2-2 H2O)</td>
			<td>Sigma</td>
			<td>C5080-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate-Monobasic (NaH2PO4)</td>
			<td>Sigma</td>
			<td>S0751-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Fisher Chemical</td>
			<td>P217-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA)</td>
			<td>Sigma</td>
			<td>ED255-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of vascular control mechanisms utilizing video microscopy of isolated resistance arteries of rats

このプロトコルの分離脳抵抗動脈 (と他の船舶) の血管機能を評価するための in vitroテレビ顕微鏡の使い方について説明し、レーザー ドップラー血流計 (LDF を使用して組織の血流を評価するための手法について説明します) と蛍光を用いた微小血管密度グリフォニア simplicifolia (GS1) レクチンをラベル付けします。現在の勉強方法貫圧力発生体内抵抗血管を分離、実質細胞の影響のない状態で生体内での研究と分子から得られる情報との間の重要なリンクを提供個体レベルでの統合的な応答に限られた洞察力を提供する還元主義的アプローチ。LDF とテクニックは、GS1 の蛍光標識レクチンと細動脈と毛細血管を選択的に識別するためには、分離抵抗血管の研究から得られた知識を拡張する捜査官を可能にする実用的なソリューションを提供します。本稿血管生理学一般実験モデルとしてラットの病理学の基礎的知識を得るためにこれらの技術の応用と専門の様々 な提供することができます「デザイナー」ラット系統遺伝子組み換え特定の遺伝子が重要な血管表現型に与える影響の重要な洞察力。ノックアウト マウス モデルで開発した科学的前提の厳しさを拡大する選択的な繁殖戦略と、ラットの遺伝子ノックアウト モデルを生産するための新しい技術によって開発されたラット系統でこれらの貴重な実験的アプローチを活用し、人の知識をよく理解の生理学的背景とサイズが大きいため生体への適合性、関連性の高い動物モデルを拡張します。

● キャニュレイテッド抵抗血管の培養顕微鏡は、小さな抵抗動脈 (および大きい細動脈) のアクティブなトーンに影響を及ぼす要因の検討経壁圧正常生体内で実質細胞の不在影響します。種々 の血管拡張薬と血管収縮薬の刺激に血管と通常 PSS、Ca2 +の経壁圧上昇に対する筋の反応性を評価するとともに-無料 PSS を出納し、superfusate の終わりに追?...

Watch this Scientific Journal Video about Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats at JoVE.com

Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

本稿では、ラット脳の抵抗血管での血管の機能を評価するためのビデオ顕微鏡検査のプロトコル体外について説明します。原稿もレーザー レーザードップラー血流計を用いた蛍光に分類されたレクチンと組織潅流と微小血管密度を評価するための手法を説明します。

分離抵抗性ラットの動脈のビデオ顕微鏡を用いた血管制御機構の評価

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other ...

evaluation-vascular-control-mechanisms-utilizing-video-microscopy

Research

JoVE Journal

Medicine

9.2K ビュー.  Marquette University. 本稿では、ラット脳の抵抗血管での血管の機能を評価するためのビデオ顕微鏡検査のプロトコル体外について説明します。原稿もレーザー レーザードップラー血流計を用いた蛍光に分類されたレクチンと組織潅流と微小血管密度を評価するための手法を説明します。

ビデオ: Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia polycythemia sequence (TAPS)1,2. Injection studies of twin placentas have shown that such anastomoses are almost invariably present in monochorionic twins and extremely rare in dichorionic twins1. Three types of anastomoses have been documented: from artery to artery, from vein to vein and from artery to vein. Arterio-venous (AV) anastomoses are unidirectional and are referred to as "deep" anastomoses since they proceed through a shared placental cotyledon, whereas arterio-arterial (AA) and veno-venous (VV) anastomoses are bi-directional and are referred to as "superficial" since they lie on the chorionic plate. Both TTTS and TAPS are caused by net imbalance of blood flow between the twins due to AV anastomoses. Blood from one twin (the donor) is pumped through an artery into the shared placental cotyledon and then drained through a vein into the circulation of the other twin (the recipient). Unless blood is pumped back from the recipient to the donor through oppositely directed deep AV anastomoses or through superficial anastomoses, an imbalance of blood volumes occurs, gradually leading to the development of TTTS or TAPS. The presence of an AA anastomosis has been shown to protect against the development of TTTS and TAPS by compensating for the circulatory imbalance caused by the uni-directional AV anastomoses1,2. 
Injection of monochorionic placentas soon after birth is a useful mean to understand the etiology of various (hematological) complications in monochorionic twins and is a required test to reach the diagnosis of TAPS2. In addition, injection of TTTS placentas treated with fetoscopic laser surgery allows identification of possible residual anastomoses3-5. This additional information is of paramount importance for all perinatologists involved in the management and care of monochorionic twins with TTTS or TAPS. Several placental injection techniques are currently being used. We provide a simple protocol to accurately evaluate the presence of (residual) vascular anastomoses using colored dye injection.

Twin-to-twin transfusion syndrome and twin anemia polycythemia sequence are two potentially devastating problems in perinatal medicine. Both disorders occur only in monochorionic twins and result from unbalanced blood flow through placental vascular anastomoses. We provide a simple protocol to accurately evaluate the presence of vascular anastomoses using colored dye injection of placental vessels after birth.

カラー染料を使用して単一絨毛膜性の胎盤における血管吻合の正確かつ簡単に評価

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

医学

Training a Sophisticated Microsurgical Technique: Interposition of External Jugular Vein Graft in the Common Carotid Artery in Rats

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in peripheral arterial bypass surgery as well as in arteriovenous fistulas.1-5 The experimental procedure of vein graft interposition in the common carotid artery by using the cuff-technique has been applied in several research projects to examine the aetiology of neointimal hyperplasia and therapeutic options to address it. 6-8 The cuff prevents vessel anastomotic remodeling and induces turbulence within the graft and thereby the development of neointimal hyperplasia.
Using the superior caval vein graft is an established small-animal model for venous arterialization experiment.9-11 This current protocol refers to an established jugular vein graft interposition technique first described by Zou et al., 9 as well as others.12-14 Nevertheless, these cited small animal protocols are complicated.
To simplify the procedure and to minimize the number of experimental animals needed, a detailed operation protocol by video training is presented. This video should help the novice surgeon to learn both the cuff-technique and the vein graft interposition. Hereby, the right external jugular vein was grafted in cuff-technique in the common carotid artery of 21 female Sprague Dawley rats categorized in three equal groups that were sacrificed on day 21, 42 and 84, respectively. Notably, no donor animals were needed, because auto-transplantations were performed. The survival rate was 100 % at the time point of sacrifice. In addition, the graft patency rate was 60 % for the first 10 operated animals and 82 % for the remaining 11 animals. The blood flow at the time of sacrifice was 8&plusmn;3 ml/min. In conclusion, this surgical protocol considerably simplifies, optimizes and standardizes this complicated procedure. It gives novice surgeons easy, step-by-step instruction, explaining possible pitfalls, thereby helping them to gain expertise fast and avoid useless sacrifice of experimental animals.

Neointimal hyperplasia is the primary cause of stenosis in arterialized veins. We propose a new protocol whereby the right external jugular vein is grafted using the cuff technique in the common carotid artery of Sprague Dawley rats. The survival rate was 100 % at the time point of sacrifice.

ラットの総頚動脈で外頚静脈グラフトの介在：洗練された顕微鏡下手術の訓練

Neointimal hyperplasia is one the primary causes of stenosis in arterialized veins that are of great importance in arterial coronary bypass surgery, in ...

Assessing Myogenic Response and Vasoactivity In Resistance Mesenteric Arteries Using Pressure Myography

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic response is a key regulator of local blood flow. In isobaric conditions small resistance arteries develop sustained constriction known as myogenic tone (MT), which is a major determinant of systemic vascular resistance (SVR). Hence, ex vivo&#160;pressurized preparations of small resistance arteries are major tools to study microvascular function in near-physiological states. To achieve this, a freshly isolated intact segment of a small resistance artery (diameter ~260 &#956;m) is mounted onto two small glass cannulas and pressurized. These arterial preparations retain most in vivo characteristics and permit assessment of vascular tone in real-time. Here we provide a detailed protocol for assessing vasoactivity in pressurized small resistance mesenteric arteries from rats; these arteries develop sustained vasoconstriction - approximately 25% of maximal diameter - when pressurized at 70 mmHg. These arterial preparations may be used to study the effect of investigational compounds on relationship between intra-arterial pressure and vasoactivity and determine changes in microvascular function in animal models of various diseases.

Pressure myography is used to assess vasoactivity of small arteries that develop sustained constriction when pressurized. This manuscript provides a detailed protocol to assess in isolated segments of small mesenteric arteries from rats, vasoactivity and the effect of intraluminal pressure on vascular diameter.

圧力筋運動記録法を用いた抵抗腸間膜動脈における筋原性応答および血管作用を評価します

Small resistance arteries constrict and dilate respectively in response to increased or decreased intraluminal pressure; this phenomenon known as myogenic ...

Computerized Dynamic Posturography for Postural Control Assessment in Patients with Intermittent Claudication

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic conditions. As part of a diagnostic assessment, the early detection of postural deficits is important so that appropriate and targeted interventions can be prescribed. The Sensory Organization Test (SOT) on the EquiTest determines an individual&#39;s use of the sensory systems (somatosensory, visual, and vestibular) that are responsible for postural control. Somatosensory and visual input are altered by the calibrated sway-referenced support surface and visual surround, which move in the anterior-posterior direction in response to the individual&#39;s postural sway. This creates a conflicting sensory experience. The Motor Control Test (MCT) challenges postural control by creating unexpected postural disturbances in the form of backwards and forwards translations. The translations are graded in magnitude and the time to recover from the perturbation is computed.
Intermittent claudication, the most common symptom of peripheral arterial disease, is characterized by a cramping pain in the lower limbs and caused by muscle ischemia secondary to reduced blood flow to working muscles during physical exertion. Claudicants often display poor balance, making them susceptible to falls and activity avoidance. The Ankle Brachial Pressure Index (ABPI) is a noninvasive method for indicating the presence of peripheral arterial disease and intermittent claudication, a common symptom in the lower extremities. ABPI is measured as the highest systolic pressure from either the dorsalis pedis or posterior tibial artery divided by the highest brachial artery systolic pressure from either arm. This paper will focus on the use of computerized dynamic posturography in the assessment of balance in claudicants.

Individuals with intermittent claudication exhibit poor balance compared to healthy controls. Computerized dynamic posturography is an objective method for measuring an individual's postural responses to balance disturbances. This provides an objective reflection of the person&#x2019;s ability to respond to situations under which sensory stimuli are altered and unexpected perturbations occur.

間歇性跛行患者における姿勢制御評価のためのコンピュータ化されたダイナミックPosturography

Computerized dynamic posturography with the EquiTest is an objective technique for measuring postural strategies under challenging static and dynamic ...

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 ...

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical specimens, have been applied to uncover these resistance mechanisms to facilitate follow-up care. Although these approaches have led to clinically relevant discoveries, difficulties in attaining the relevant patient material or in deconvoluting the genomic data collected from these specimens have severely hampered the path towards a cure. To this end, we here describe a tool for expeditious discovery that may guide improvement in first-line therapies and alternative clinical management strategies. By coupling preclinical in vitro or in vivo drug selection with next-generation sequencing, it is possible to identify genomic structural variations and/or gene expression alterations that may serve as functional drivers of resistance. This approach facilitates the spontaneous emergence of alterations, enhancing the probability that these mechanisms may be observed in the patients. In this protocol we provide guidelines to maximize the potential for uncovering single nucleotide variants that drive resistance using adherent lines.

Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms

Drug resistance to targeted therapeutics is widespread and the need to identify mechanisms of resistance--prior to or following clinical onset--is critical for guiding alternative clinical management strategies. Here, we present a protocol to couple derivation of drug-resistant lines in vitro with sequencing to expedite discovery of these mechanisms.

の実装<em&gt;インビトロ</em&gt;薬剤耐性アッセイ：臨床的に関連性のメカニズムを暴くための可能性を最大化

Although targeted therapies are initially effective, resistance inevitably emerges. Several methods, such as genetic analysis of resistant clinical ...

Ultrasound Assessment of Flow-Mediated Dilation of the Brachial and Superficial Femoral Arteries in Rats

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of endothelial function can be achieved by employing the flow-mediated dilation technique, typically performed in the brachial or superficial femoral artery. Briefly, a blood pressure cuff placed distal to an ultrasound probe is inflated to a suprasystolic pressure, which results in limb ischemia. After 5 min of occlusion the cuff is deflated, resulting in reactive hyperemia and increases in wall shear rate that signal vasodilatory molecules to be released from the endothelium eliciting vasodilation. Despite the thousands of studies performing flow-mediated dilation in humans, surprisingly, no studies have performed this technique non-invasively in living rats. Considering the recent shift in focus to translational research, the establishment of guidelines for non-invasive measurement of flow-mediated dilation in rats and other rodents would be extremely valuable. In the following article, a protocol is presented for the non-invasive measurement of flow-mediated dilation in brachial and superficial femoral arteries of rats, as those sites are most commonly measured in humans.

Non-invasive assessment of endothelial function in humans can be determined by the flow-mediated dilation technique. Although thousands of studies have used this technique, no study has performed this technique non-invasively in rats. The following article describes non-invasive measurement of flow-mediated dilation in the brachial and superficial femoral arteries of rats.

ラットにおける上腕と浅大腿動脈のフロー依存性血管拡張の超音波評価

Arterial vasodilation to increases in wall shear rate is indicative of vascular endothelial function. In humans, the non-invasive measurement of ...

Using the Sleeve Technique in a Mouse Model of Aortic Transplantation - An Instructional Video

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the aorta in mice using the sleeve method was short and easy averaging 20 min, permitting studies of iso/allo grafts. The following article describes the aortic transplantation procedure used in our laboratory. The mice were anesthetized with a mixture of 1.5% volume isoflurane and 100% oxygen through a face mask. At this point, the segment of the aorta between the renal arteries and its bifurcation was separated from the vena cava, freely prepared and clampedat the proximal and distal segments with a single silk suture. Prior to the removal of the aorta, a saline solution containing heparin was injected into the inferior vena cava. Then the aorta was cut between the clamps and a saline heparin solution was used to flush the lumen. The sleeve technique with monofilament sutures was used in order to transplant the abdominal aorta in the orthotopic position.

We present an orthotopic aortic transplantation model using the sleeve technique in mice. It is a very rapid anastomosis method, which can be employed in studies of vascular disease.

大動脈弁移植 - 教則ビデオのマウスモデルでスリーブを用いた

Orthotopic aortic transplantation using the sleeve technique reduces injury to the aorta with failure rate of only 10-20%. The time to anastomose the ...

A Cell Culture Model of Resistance Arteries

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and signaling processes that can control vascular tone and blood pressure. As it is a projection from either the endothelial or smooth muscle cell, and due to its small size (on average, an area of ~1 &#181;m2), the MEJ is difficult to study in isolation. However, we have developed a cell culture model called the vascular cell co-culture (VCCC) that allows for in vitro MEJ formation, endothelial cell polarization, and dissection of signaling proteins and processes in the vascular wall of resistance arteries. The VCCC has a multitude of applications and can be adapted to suit different cell types. The model consists of two cell types grown on opposite sides of a filter with 0.4 &#181;m pores in which the in vitro MEJs can form. Here we describe how to create the VCCC via plating of cells and isolation of endothelial, MEJ, and smooth muscle fractions, which can then be used for protein isolation or activity assays. The filter with intact cell layers can be fixed, embedded, and sectioned for immunofluorescent analysis. Importantly, many of the discoveries from this model have been confirmed using intact resistance arteries, underscoring its physiological relevance.

A cell culture model of resistance arteries is described, allowing for the dissection of signaling pathways in endothelium, smooth muscle, or between endothelium and smooth muscle (the myoendothelial junction). The selective application of agonists or protein isolation, electron microscopy, or immunofluorescence can be utilized using this cell culture model.

抵抗血管の細胞培養モデル

The myoendothelial junction (MEJ), a unique signaling microdomain in small diameter resistance arteries, exhibits localization of specific proteins and ...

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Here, we present a powerful and physiological model to study the molecular mechanisms underlying gut hormone secretion and intestinal absorption &#8212; the isolated perfused rat small intestine.

免震を用いた消化管ホルモン分泌のメカニズム潅流ラット小腸

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, ...