著者は、技術援助と支援の実験のためにさんりさ大塚に感謝します。この研究は部分的に教育、科学、スポーツ文化省によってサポートされていました、若手研究（B）、2010、22700476、及びKTのためにスズケン記念財団2010年、この研究費補助金は、研究開発によってサポートされていましたリビング物質の次世代統合シミュレーション、文部科学省次世代スーパーコンピュータ·プロジェクトの開発と利用の一部であり、JST ERATO末松ガス生物学プロジェクトによって一部インチ

1。フルオレセインイソチオシアネート異性体I（FITC）で標識赤血球我々が使用するすべての実験的なプロトコルは、慶應義塾大学医学部の動物ケア委員会によって承認された。 <ol><li>ドナーマウスを麻酔し、行った後、腹腔切開術は、ヘパリンの少量を含む1mlのシリンジで下大静脈から全血を取り消すことができます。マウスはすぐにペントバルビタールナトリウム注射（100 mg / kgのIP）によって安楽死させた。 </li><li>スイングバケットローターで400 グラムで5分間遠心分離によってPBS（pH7.4）で二回全血を洗う。 </li><li>の100mM Na 2 HPO 4、5 mlのFITCの10mgを溶解し、0.22μmの細孔を有する膜でそれをフィルタリングします。 </li><li>試験管に180 mMの0.25ミリリットルのNaH 2 PO 4、及び100mMのNa 2 HPO 4 1.5 mlの、3 mMのグルコースの0.15ミリリットルとFITC溶液1mlを混ぜます。 Rの0.2ミリリットルを追加します。BCペレットと混合するためのチューブをタップします。 </li><li> 4°Cで2時間チューブを保持し、PBSで2回ステンド赤血球を洗浄する。 </li><li>スライドの芝生の上でRBC懸濁液の少量を入れて、赤血球は健康に見えるかどうか（すなわち、その形状や大きさが正常であること）と蛍光強度が十分であること。 </li><li> pH7.4のPBS 0.9 mlの赤血球の0.1 mlを中断し、4℃で懸濁液を保つ°Cを注入するまで。 </li></ol> 2。酸素感受性色素の調製<ol><li> pH7.4のPBSの10mlのBSA 500 mgを溶解する。 </li><li> BSA溶液にパラジウム（II） - メソ - テトラ（4 - カルボキシフェニル）ポルフィン（PD-TCPP）の30 mgを追加し、一晩攪拌する。 </li><li> （省略可能）フリーのPd-TCPPからそれを分離する重力流クロマトグラフィーまたはスピンカラムを用いてBSAに結合したパラジウムTCPPを抽出します。 </li><li>パラジウムTCPP溶解除去し、0.22μmの細孔を有するメンブランフィルターで上清をフィルタリングするためのソリューションを遠心分離します。 &lt;/ LI&gt; <li> -20℃でチューブ内の店舗1mlのアリコート凍結融解の繰り返しを避けることができます。 </li></ol> 3。動物の準備<ol><li>微小循環の顕微鏡観察用の穴に比べて30 mm角の正方形のカバーガラス孔20の直径mmとし、テープでプラスチック板を準備します。 </li><li>マウスを麻酔し、肌に毛皮を削除し、準備。ヘパリン化PBSで満たされた10cmのポリエチレン（PE 10）カテーテルに接続されている30 G針を使用して、薬剤注入のための無駄な尾部にカテーテルを挿入します。 </li><li>正中切開した後、プラスチックプレート上に肝臓の主な小葉を拡張し、伏臥でマウスを置きます。 </li><li>呼吸と肝臓の移動と乾燥からそれを維持抑制するために肝葉の縁の周りにキッチンラップ（3ミリメートル×8 mm）とタイル一緒に小さなスリップを準備します。 </li><li>顕微鏡下での肝微小循環を（透過光で）観察すると、bていることを確認lood·フローは、少なくとも15分間の視野内にうっ滞がありません。 </li><li>徐々に血流の観察またはpO 2は測定用のPd-TCPP溶液0.2mlのために蛍光標識した赤血球の0.2ミリリットルを注入します。 FITC標識赤血球のこの量は、可視化領域内の循環内のすべての赤血球の1/50を占めることになる。 </li></ol> 4。血液の流れの可視化<ol><li>バンドパスフィルタ（450から490 nm）の1を通過した水銀灯の光でそれを照射することにより、FITCを励起する。 </li><li> CCDカメラによる蛍光像を記録します。 </li></ol> 5。はpO 2測定 PD-TCPP燐光は比較的弱く、高感度の検出器で検出する必要があります。すべての実験は暗い部屋で実行する必要があります。 <ol><li>パラジウムTCPPの吸収ピークは410 nmと532 nmであるので、第二高調波の波長532 nmの励起をお勧めします。 YAGパルスレーザー2：この波長はNdによって生成することができます。 </li><li>倒立顕微鏡上の適切なポートにYAGレーザーと、焦点面の中央の位置にビームスポットを調整する：NDからのビームを供給します。空間分解能はピンホールを介してビームを渡すことによって変更されるレーザーのスポットサイズに依存します。 </li><li>燐光を検出するために、顕微鏡の他のポートへのロングパスフィルター（&gt; 620 nm）と光電子増倍管（PMT）を添付してください。 PMTは、特に700 nm付近に、赤の波長に敏感でなければなりません。 </li><li>燐光信号（200 kHzでサンプリングされた500点）をサンプリングし、崩壊の指数関数をフィッティングすることにより、減衰の時定数を計算します。 </li><li>キャリブレーション用のPO 2 150 mmHgで、0 mmHgでのPd-TCPPソリューションのサンプルの2セットを準備します。酸素の有無を生成するためにサンプリングするために亜ジチオン酸ナトリウムの体積の1％を追加します。 7.4でサンプルのpHと温度を保つ校正時37℃トゥーレ。 </li><li>スターン-ボルマー定数k qと無酸素の発光減衰時間式のτ0（1）3を修正します 。 37°Cと7.4のpHでのPd-TCPPソリューションと我々のケースでは、K Qは 374 mmHgで-1 s -1であり、τ0は 0.74ミリ秒です。これらの測定パラメータは、機器（すなわち、レーザー、検出器、その他の光学機器）、および信号処理方法によって異なりますので、キャリブレーションは、各測定システム4で必要とされる。 τ0 /τ= 1 + K Q•τ0•pO 2は （1） </li><li>顕微鏡ステージ上にプレートの上にマウスを設定し、肝微小循環を観察し、レーザスポットの点にターゲット微小血管の位置を調整します。 </li></ol> 6。肝炎モデル<ol><li>一晩水に自由にアクセスの高速マウス。 </li><li>準備するDMSO中のアセトアミノフェンmlの溶液（APAP）あたり20 mgである。 </li><li> 1 ml/100グラム体重（すなわち、200 mg / kg体重）で腹腔内に注入します。注入後、マウスが食料と水5への無料アクセスを与えることができます。 </li><li>注射後24時間は、マウスを麻酔し、正中切開を行うことによって肝臓を公開します。肝炎が達成されている場合は、中心周辺の地域の壊死が肉眼で容易に見えるようになります。 </li></ol> 7。代表的な結果肝微小循環のサンプル画像は、（a）透過光像であり、（b）の蛍光画像である図1に示されています。個々のFITC標識赤血球をビデオムービーで観察され、ポータル静脈、正弦波、中央静脈の血流の方向で認識されます。 図2に示すように、X100の対物レンズを介して中央静脈を照射するレーザスポットはapproximでした直径ately10μmである。肝微小循環における血管内のpO 2は、雄C57BL / 6マウス、および図3（a）で測定したpO 2は 、ポータルと中央静脈の血管径との関係を示しています。正弦波を通過しながら、ポータルからの中央静脈にpO 2は勾配が効率的にその赤血球リリース酸素を示しています。 図3（b）に示すように、平均pO 2は、ポータル船舶、正弦波では48.2 mmHgで、中央静脈で38.9 mmHgで59.8 mmHgであった。 我々はAPAP腹腔内に注入することにより急性肝炎を生成する場合は、血管内のpO 2は有意に肝微小循環の各部分で上昇していた：P正弦波と中心静脈（図3（c））における門脈およびp &lt;0.01 &lt;0.05。 <img src="/files/ftp_upload/3996/3996fig1.jpg" alt="図1" /> 図1低倍率I魔道士は、（a）透過光および（b）蛍光と肝微小循環を示している。 <img src="/files/ftp_upload/3996/3996fig2.jpg" alt="図2" /> 図2はpO 2測定の対象となる中心的な静脈を照射するレーザスポットのイメージ。 <img src="/files/ftp_upload/3996/3996fig3.jpg" alt="図3" /> 健康な対照マウスのアセトアミノフェン誘発肝傷害マウスの肝微小循環の図3。pO 2は勾配。 （1）PO 2対ポータル静脈（PV）と中央静脈（CV）の血管径を示し、（b）は正弦波（S）を介してPVからCVへの酸素勾配を示しています。 PVとCVの間にはpO 2の違いは、肝細胞または他の実質細胞の酸素消費量を反映しています。 （C）APAP投与により誘導される肝炎の状態で、PO 2が大幅にPV、S、およびCVとはpO 2で上昇したことを示しています勾配は、肝酸素消費量が危険にさらされたことを示す、減少した。

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我々は、開示することは何もありません。

組織に酸素を提供することは微小循環の重要な役割であり、論文の多くは、微小血管の解剖学とレオロジー6に関連する疾患について説明します。肝血流量は動脈と静脈血の組み合わせです。肝臓への血流の約30％が門脈によって提供された肝動脈と他の70％で提供されるだけでなく酸素化血液は、脾臓、消化管7からの不十分な酸素血静脈流出である。そのような脂肪肝、衝撃...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td>パラジウム（II）メソ - テトラ（4 - カルボキシフェニル）ポルフィン</td><td>フロンティアサイエンティフィック株式会社</td><td> PdT790 </td></tr><tr><td>フルオレセインイソチオシアネート異性体I </td><td>シグマアルドリッチ（株） </td><td> F7250 </td></tr><tr><td>アセトアミノフェン</td><td>シグマアルドリッチ（株） </td><td> A7085 </td></tr></tbody></table> <table border="1"><tbody><tr><td> 機器の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>光電子増倍管</td><td>浜松ホトニクス</td><td> H10722-20 </td></tr></tbody></table>

visualization and analysis of blood flow and oxygen consumption in hepatic microcirculation: application to an acute hepatitis model

肝酸素消費量が比較的高いですが、肝臓の血液供給の約70％が胃腸管および脾臓由来の門脈血を酸素化されているため、肝臓の酸素需要と供給の間にかなりの食い違いがあります。酸素は、正弦波を介して中央静脈に門脈終末枝から流れる血液によって肝臓に配信され、これは肝小葉内の酸素勾配になります。酸素勾配は、肝微小循環におけるアップストリームおよびダウンストリームの酵素の発現を伴う重要な物理パラメータであるが、肝微小循環の酸素消費量を測定するための技術の欠如は、肝臓での酸素代謝に関わるメカニズムの解明を遅らせました。そこで我々は肝微小循環を可視化するためにFITC標識赤血球を使用し、そこに血管中の酸素分圧を測定​​するためにレーザーアシストりんを使用していました。 NoncontaCTと連続的な光学的測定は、血流速度、血管径、肝臓での酸素消費量に関連した酸素勾配を定量化することができます。急性肝炎モデルでは、我々は中心周辺のゾーン内の肝細胞壊死が酸素分圧をシフトアップと酵素の発現に影響を​​与える可能性があることを示し、我々はポータルと中央静脈の両方で増加した酸素分圧が正弦波で減少した酸素勾配を観察したマウスにアセトアミノフェンを投与することにより行われ門脈ゾーンインチ結論として、測定の肝血行動態および酸素消費のために私たちの光学的方法では、肝疾患に関連するメカニズムを明らかにすることができます。

Watch this Scientific Journal Video about Visualization and Analysis of Blood Flow and Oxygen Consumption in Hepatic Microcirculation: Application to an Acute Hepatitis Model at JoVE.com

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

光学系は、FITC標識赤血球と肝微小循環を可視化するために、レーザーアシストりんと血管中の酸素分圧を測定​​するために開発されました。このメソッドは、微小血管の構造、直径、血流速度、および酸素分圧を分析することにより、生理学的および病理学的メカニズムを調べるために使用することができます。

肝微小循環における血流と酸素消費量の可視化と分析：急性肝炎モデルへの応用

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

visualization-analysis-blood-flow-oxygen-consumption-hepatic

Research

JoVE Journal

Medicine

12.5K ビュー.  Keio University. 光学系は、FITC標識赤血球と肝微小循環を可視化するために、レーザーアシストりんと血管中の酸素分圧を測定​​するために開発されました。このメソッドは、微小血管の構造、直径、血流速度、および酸素分圧を分析することにより、生理学的および病理学的メカニズムを調べるために使用することができます。

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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

マウス生物発光肝腫瘤モデル

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

医学

Heterotopic Auxiliary Rat Liver Transplantation With Flow-regulated Portal Vein Arterialization in Acute Hepatic Failure

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the failing native liver has regenerated.1-3 The APOLT-method, the orthotopic implantation of auxiliary segments- averts most of the technical problems. However this method necessitates extensive resections of both the native liver and the graft.4 In 1998, Erhard developed the heterotopic auxiliary liver transplantation (HALT) utilizing portal vein arterialization (PVA) (Figure 1). This technique showed promising initial clinical results.5-6 We developed a HALT-technique with flow-regulated PVA in the rat to examine the influence of flow-regulated PVA on graft morphology and function (Figure 2).
A liver graft reduced to 30 % of its original size, was heterotopically implanted in the right renal region of the recipient after explantation of the right kidney.&#160; The infra-hepatic caval vein of the graft was anastomosed with the infrahepatic caval vein of the recipient. The arterialization of the donor&#8217;s portal vein was carried out via the recipient&#8217;s right renal artery with the stent technique. The blood-flow regulation of the arterialized portal vein was achieved with the use of a stent with an internal diameter of 0.3 mm. The celiac trunk of the graft was end-to-side anastomosed with the recipient&#8217;s aorta and the bile duct was implanted into the duodenum. A subtotal resection of the native liver was performed to induce acute hepatic failure. 7
In this manner 112 transplantations were performed. The perioperative survival rate was 90% and the 6-week survival rate was 80%. Six weeks after operation, the native liver regenerated, showing an increase in weight from 2.3&#177;0.8 g to 9.8&#177;1 g. At this time, the graft&#8217;s weight decreased from 3.3&#177;0.8 g to 2.3&#177;0.8 g.
We were able to obtain promising long-term results in terms of graft morphology and function. HALT with flow-regulated PVA reliably bridges acute hepatic failure until the native liver regenerates.

Auxiliary liver transplantation provides a temporary support in acute hepatic failure, until regeneration of the failing liver. The heterotopic auxiliary liver transplantation (HALT) with portal vein arterialization (PVA) renders sufficient liver function. We developed an analogous technique in the rat, to examine the influence of the portal vein arterialization on the morphology and function of the graft.

急性肝障害における流れ調節性門脈動脈血と異補助ラット肝移植

In acute hepatic failure auxiliary liver transplantation is an interesting alternative approach. The aim is to provide a temporary support until the ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

くも膜下出血の急性および後期後遺症の研究のためのウサギの血液シャントモデル：技術的側面

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

Contrast Enhanced Ultrasound Imaging for Assessment of Spinal Cord Blood Flow in Experimental Spinal Cord Injury

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an important target for neuroprotective therapies. Although several techniques have been described to assess SCBF, they all have significant limitations. To overcome the latter, we propose the use of real-time contrast enhanced ultrasound imaging (CEU). Here we describe the application of this technique in a rat contusion model of SCI. A jugular catheter is first implanted for the repeated injection of contrast agent, a sodium chloride solution of sulphur hexafluoride encapsulated microbubbles. The spine is then stabilized with a custom-made 3D-frame and the spinal cord dura mater is exposed by a laminectomy at ThIX-ThXII. The ultrasound probe is then positioned at the posterior aspect of the dura mater (coated with ultrasound gel). To assess baseline SCBF, a single intravenous injection (400 &#181;l) of contrast agent is applied to record its passage through the intact spinal cord microvasculature. A weight-drop device is subsequently used to generate a reproducible experimental contusion model of SCI. Contrast agent is re-injected 15 min following the injury to assess post-SCI SCBF changes. CEU allows for real time and in-vivo assessment of SCBF changes following SCI. In the uninjured animal, ultrasound imaging showed uneven blood flow along the intact spinal cord. Furthermore, 15 min post-SCI, there was critical ischemia at the level of the epicenter while SCBF remained preserved in the more remote intact areas. In the regions adjacent to the epicenter (both rostral and caudal), SCBF was significantly reduced. This corresponds to the previously described &#8220;ischemic penumbra zone&#8221;. This tool is of major interest for assessing the effects of therapies aimed at limiting ischemia and the resulting tissue necrosis subsequent to SCI.

Contrast Enhanced Ultrasound imaging is a reliable in-vivo tool for quantifying spinal cord blood flow in an experimental rat spinal cord injury model. This paper contains a comprehensive protocol for application of this technique in association with a contusion model of thoracic spinal cord injury.

実験的脊髄損傷における脊髄血流の評価のための強化された超音波画像コントラスト

Reduced spinal cord blood flow (SCBF) (i.e., ischemia) plays a key role in traumatic spinal cord injury (SCI) pathophysiology and is accordingly an ...

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

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

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

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

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

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

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 ...

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic study in the liver. Here, numerous assays are described to comprehensively measure the phenotype and parameters of hepatic steatosis in mouse and hepatocyte models. 
Combining the physiological, histological, and biochemical methods, this system can be used to assess the progress of hepatic steatosis. In vivo, the measurements of body weight and nuclear magnetic resonance (NMR) provide a general understanding of mice in a non-invasive manner. Hematoxylin and Eosin (H&amp;E) and Oil Red O staining determine the histological morphology and lipid deposition of liver tissue under nutrient overload conditions, such as high-fat diet feeding. Next, the total lipid contents are isolated by chloroform/methanol extraction, which are followed by a biochemical analysis for triglyceride and cholesterol. Moreover, mouse primary hepatocytes are treated with high glucose plus insulin to stimulate lipid accumulation, an efficient in vitro model to mimic diet-induced hyperglycemia and hyperinsulinemia in vivo. Then, the lipid deposition is measured by Oil Red O staining and chloroform/methanol extraction. Oil Red O staining determines intuitive hepatic steatotic phenotypes, while the lipid extraction analysis determines the parameters that can be analyzed statistically. The present protocols are of interest to scientists in the fields of fatty liver diseases, insulin resistance, and type 2 diabetes.

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

Here, optimized methods to generate in vivo and in vitro models of hepatic steatosis and to analyze the steatotic phenotypes and related physiological parameters are described.

の最適化分析<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;脂肪肝

Establishing a system of procedures to qualitatively and quantitatively characterize in vivo and in vitro hepatic steatosis is important for metabolic ...

A Chronic Cardiac Ischemia Model in Swine Using an Ameroid Constrictor

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but regardless of the approach, an in vivo model is needed to test each treatment. The pig is one of the most used large animal models for cardiovascular disease. Its heart is very similar in anatomy and function to that of a human. The ameroid placement technique creates an ischemic area of the heart, which has many useful applications in studying myocardial infarction. This model has been used for surgical research, pharmaceutical studies, imaging techniques, and cell therapies.
There are several ways of inducing an ischemic area in the heart. Each has its advantages and disadvantages, but the placement of an ameroid constrictor remains the most widely used technique. The main advantages to using the ameroid are its prevalence in existing research, its availability in various sizes to accommodate the anatomy and size of the vessel to be constricted, the surgery is a relatively simple procedure, and the post-operative monitoring is minimal, since there are no external devices to maintain. This paper provides a detailed overview of the proper technique for the placement of the ameroid constrictor.

The purpose of this protocol is to demonstrate the placement of a delayed constricting device (an ameroid constrictor) around a coronary artery in a swine model. This device creates an ischemic area of the heart that is useful for studying new diagnostic imaging techniques and new methods of treatment.

アメロイド収縮を使用して豚の慢性心筋虚血モデル

Cardiovascular disease remains the number one cause of mortality in the United States. There are numerous approaches to treating these diseases, but ...

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

細胞外フラックス アナライザーを使用してプライマリの培養マウス新生児心筋酸素消費量の分析

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.