We appreciate Feifei Zhang for the helpful discussions. We are grateful to Jing Gao and Yixuan Sun for the technical assistance and to Zhengshuai Liu and Fengguang Ma for the animal studies.

全ての動物実験プロトコルは、生物科学のための栄養科学研究所、上海研究所、中国科学院（上海、中国）での制度的動物のケアと使用委員会によって承認されました。 1. DIOマウスモデル<ol><li> HFHS送り <ol><li> 40キロカロリー％の脂肪と40キロカロリー％のスクロースを含んでHFHSで8週齢の雄性C57BL / 6マウスを養います。 12時間明暗サイクル条件でそれらを収容します。 </li><li> 4 16週間HFHS食事送り状態でマウスを保管してください。体重を毎週チェックしてください。 </li></ol></li><li> 身体組成分析と、擬似カラー画像解析 <ol><li>身体組成分析器分析をマウスに施します。 <ol><li>プラスチックチューブ内に意識し、ない麻酔したマウスを固定します。先に2に記載のように、体脂肪量、除脂肪体重、およびNMRシステムを用いて流体を測定します。 </li></ol></li><li> MRIシステムを用いて、各マウスの全身をスキャンします。 <ol><li>腹腔内注射（25μL/ g）を介して、1％のアベルチンでマウスを麻酔し、特殊なガラス管に画像解析を行います。 注：イメージングのプロセスは、各マウスでほぼ0.5時間持続します。測定が完了すると、それらのケージにマウスを返します。 </li></ol></li><li>背側、中間、腹側の層にマウスの全身をスキャン。垂直方向に肝臓をスキャンします。 =セクションの厚さは= 3ミリメートル、TE = 14.8ミリ、TR = 400ミリ秒、FOVRead = 100ミリメートル、FOVPhase = 100ミリメートル、およびスキャン数、ミクロン192×256 = K・スペース：0.55 Tのマグネットを使用して、次の機器パラメータマウスのすべてのグループに同じ磁気条件を使用して8.スキャン。 </li><li>信号強度9に記載の擬似カラー画像にグレースケール画像の地図。 </li></ol></li><li> マウスの安楽死 <ol><li> dissecti滅菌このような鉗子やハサミなどngのツール。 </li><li>ペーパータオル上で十分なイソフルランを注ぎ、イソフルランは、ガラスジャーに揮発しましょう。マウスをイソフルランにより安楽死させていることを確認します。 </li><li>オペレーティングテーブルの上にマウスを修正しました。 75％エタノールで各腹部をスプレーハサミを使って剣状突起の真皮中の切開を行い、腹膜を露出させるために手で正中線を通って長手方向真皮を引き裂きます。 </li><li>ピンセットで剣状突起をクランプし、内臓を露出するために、リブの底部を通って腹膜縦横を切りました。 </li><li>心臓穿刺によってEDTAでコーティングされたチューブ内の血液を採取します。 </li><li>肝臓に沿って横隔膜をカットし、慎重に細かいハサミを使用してenterocoeliaから肝臓を削除します。以下の脂質測定のために肝臓組織の3枚をカット：H＆E染色のためのa）は、6×3×3ミリメートル、b）はオイルレッドO染色のための6×3×3ミリメートル、及びクロロホルムのための肝臓のc）に20-40ミリグラム/メタノール抽出。 </li></ol></li><li> H＆E染色 <ol><li> 4℃で一晩4％リン酸緩衝ホルマリン、酢酸で肝臓を修正した後、パラフィンワックスに埋め込みます。 </li><li>以前に3、10を説明したように、パラフィン切片（5μm）をカットし、H＆E染色用ガラススライド上にマウントします。 </li></ol></li><li> オイルレッドO染色 <ol><li>リージェント準備<ol><li> 10％ホルマリン、60％イソプロパノール、ヘマトキシリン、およびグリセロールゼリーとオイルレッドO作業溶液を準備します。イソプロパノール100mLでオイルレッドO粉末0.5gを溶解し、0.5％のストック溶液を作るために60℃の浴中で加熱。 </li><li> 2の比率（のddH 2 Oの3ストック溶液のmL及び2 mLの）：3で二回蒸留水（ddH 2 O）で希釈します。ワーキング溶液を濾過し、室温で少なくとも10分間放置しました。 </li></ol></li><li> APにおける最適切断温度（OCT）化合物中の肝臓を埋め込みますラスチック埋め込みボックス。 30分間-20℃でそれをフリーズします。スライド上の組織をマウントし、凍結ミクロトームで8μmのセクションにそれをカットし、室温で30分間置きます。 </li><li>染色槽の底部にペーパータオルを置き、紙タオルを濡らし、10％ホルマリンを少量注ぎます。染色用浴槽のご利用にスライドを置き、4℃で5分間、ホルムアルデヒド蒸気中で凍結切片を固定します。 </li><li> 3秒間の染色用浴槽の60％イソプロパノールでスライドを浸します。一回繰り返します。 </li><li>すべての組織をカバーし、15分間インキュベートするスライドにオイルレッドO作業溶液を数滴を追加します。室温で光から組織を保護します。 </li><li> 3秒間の染色用浴槽内の新しい60％イソプロパノールでスライドをすすぎます。二回繰り返します。 </li><li>静かに二回蒸留水を介してスライドをすすぎ、組織の周りに水を除去します。組織を乾燥しないように注意してください。 </li><li> 1分間ヘマトキシリンでスライドを置き、5月10日のために水道水で軽くすすぎます分。静かに二回蒸留水でスライドを洗浄し、カバースリップを置くために準備ができるまで、蒸留水に保管してください。 </li><li>電子レンジでグリセロールゼリーを加熱し、組織の上に数滴を置きます。ゆっくり上にカバースリップを配置し、気泡を形成しないように注意してください。 </li><li>顕微鏡下で染色を観察し、20Xと40Xの目的を使用して特性の画像をキャプチャします。 </li></ol></li><li> 肝臓脂質の測定 <ol><li>新しい2 mLのプラスチック製遠心管に解剖し、肝臓組織を置きます。ホモジナイザーでPBS 1mLに均質化し、新しい15-mLのガラス管に、溶解物を移します。 </li><li> 5クロロホルム/メタノールmLの（2：1、v / v）の追加ソリューション、ボルテックス1分間激しく混合し、そして氷上で2時間放置。 </li><li> 4℃で10分間、1650×gで遠心分離します。上部メタノール層、中間のタンパク質ディスク、およびボトムクロロホルムおよび脂質：混合物を3層に分離する必要があります。 </li><li>トラン<html>ガラスパスツールピペットを使用して、新しいガラス管に下相sfer。塔底留分の一滴を取得する必要はありません。氷の上に置き、チューブを設定します。 </li><li>積極的に前のチューブ、ボルテックスに残った上清画分にクロロホルムの4のMgCl 2および1.5 mLの600μLのを追加します。 4℃で10分間、1,650×gで30分間遠心分離氷上でインキュベートします。 </li><li>パスツールピペットを用いて、第1下相管を下相を兼ね備えています。上部と中間層を捨てます。 </li><li> 37℃の浴中で窒素ガス下でクロロホルムを蒸発させます。 1％トリトンX-100 /クロロホルム200μLで十分ガラス管の側面を洗浄（v / v）です。窒素ガス下で蒸発します。最終エマルジョンを作るためにも200のddH 2 OのμLと渦を有する脂質を溶解させます。 </li><li>製造業者のプロトコルに従って、トリグリセリドまたはコレステロールキットを使用して、総トリグリセリド（TG）及びコレステロール（TC）レベルを測定します外部参照&quot;&gt; 3。 注記：タンパク質定量3によって決定される脂質値は、最初のホモジネート中のタンパク質濃度のものに正規化されます。 </li></ol></li></ol> 2.初代マウス肝細胞モデル<ol><li> マウス初代肝細胞の単離 <ol><li> （腹腔内に10μL/ g）を6％抱水クロラールで各マウスを麻酔。 </li><li>以前に10を説明したように、初代肝細胞を分離します。オイルレッドO染色のために6ウェルプレートにコラーゲンコーティングされたガラスカバースリップ上で細胞を増殖させます。脂質抽出のために、10cmの細胞培養ディッシュ10上の細胞を播種します。 </li></ol></li><li> 細胞処理およびオイルレッドO染色 <ol><li>飢餓培地2mLで培地を交換し、37℃で24時間インキュベートします。 30 mMグルコースおよび100nMのインスリンでそれを扱います。 24時間37℃で培養インキュベーター中でそれをインキュベートします。 </li><li>培地を吸引除去します。 PBSの2 mLを加え、培地を洗浄するために穏やかに旋回。一回繰り返します。 </li><li>ゴムバンド付きスライドにカバースリップを修正しました。外表面に付着した細胞を公開します。 （ステップ1.5）上記のようにオイルレッドO染色を実行します。 注：脂質蓄積の基礎レベルは、血清飢餓の後、高グルコース+インスリン治療で刺激します。脂質滴は、オイルレッドO染色により可視化されます。 </li></ol></li><li> 脂質の測定 <ol><li>同じ条件（ステップ2.2.1）で、高グルコース+インスリンで肝細胞を扱います。各10cmの細胞培養皿に1mLのPBSを追加します。細胞を掻き落とし、100μLのアリコートをタンパク質定量のために、新鮮な1.5 mLチューブに転送され、そこから新たな15-mLのガラス管の中にそれらを収穫。 </li><li>細胞の残りの900μLに：クロロホルム/メタノール4mLの（1、V / V 2）を追加します。おおよそ20ビープ音のための超音波処理した後、激しく渦解放します細胞中の脂質。氷上で2時間混合物をインキュベートします。脂質抽出（ステップ1.6）を実行します。 </li></ol></li></ol>

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著者は、彼らが競合する金融利害関係を持たないことを宣言します。 資金調達：この作品は、中国の国家自然科学基金（番号81270930、31471129、および31671224）と百タラント中国科学院のプログラム（2013OHTP04）YLへの助成金によってサポートされていました

NAFLDは、メタボリックシンドローム、肥満、インスリン耐性、または2型糖尿病（T2DM）11に関連付けられている進行性肝疾患のシリーズです。 NAFLDの顕著な特徴は、脂肪症、肝細胞における脂質の蓄積です。ここでは、方法のスペクトル表現型およびDIOマウスおよびマウス初代肝細胞を用いて、肝臓脂肪症のパラメータを特徴付けるために提示されています。この手順は、NAF...

肥満は、先進国と途上国で急成長している健康問題です。 NAFLD患者1で30と100パーセントの間の範囲の有病率で、頻繁に非アルコール性脂肪肝疾患（NAFLD）に関連付けられた共存条件の一つであることが報告されています。脂肪肝と肥満との間の強い相関関係を、食餌誘発性肥満（DIO）マウスモデルが広くNAFLD 2、3、4、5、6の開発に関連した複雑な分子機構を研究するために使用されます。肝臓脂肪症は、NAFLDの最も初期の段階であり、非アルコール性脂肪肝炎（NASH）、肝硬変、そして最終的に、肝臓癌7に進行することができます。したがって、この方法の全体的な目標は、肝脂肪変性条件の動物および細胞モデルを生成し、PRします効率的で正確な脂質測定のための詳細なプロトコルをovide。これらのモデルと測定値はまた、インスリン抵抗性および2型糖尿病のような他の代謝性疾患の研究に有用です。 肥満は、NAFLDのための主要な危険因子の一つであると同定されているように、洋風高脂肪食を模高脂肪、高スクロース食（HFHS）は、マウスにおいて肥満を誘導するために使用されます。その後、肝臓脂肪症の程度は、異なる方法を用いて評価することができます。まず、核磁気共鳴（NMR）を用いて、体重及び体組成の分析は、時間の供給中の脂質の蓄積を示します。脂肪量および除脂肪体重は、非侵襲性リアルタイム方法で定量することができます。 また、磁気共鳴イメージング（MRI）は、全身および脂肪の肝臓分布の両方を示すために使用されます。 MRI分析の階調信号が読みやすい擬似カラー画像に変換し、強度のできますグレースケールや色は半定量化可能です。この技術は、生きた動物における脂質蓄積を測定するためのユニークな利点を提供します。第二に、肝臓の組織学的分析は、肝臓脂肪症を決定するための最も一般的に使用される方法です。オイルレッドO染色は、肝細胞における脂肪滴のサイズと位置を示している、ヘマトキシリンおよびエオシン（H＆E）染色は、このような肝細胞形態学およびマクロファージ浸潤として、組織学的情報を提供します。第三に、クロロホルム/メタノール抽出を使用して、脂質含有量の分析は、肝臓脂質の正確かつ定量的な測定値です。総トリグリセリドおよびコレステロールレベルは、生化学的方法を用いて測定することができます。重要なのは、脂質抽出分析及びオイルレッドO染色はまた、遺伝子操作または薬学的に処理された肝細胞で使用することができます。 本発明の方法の利点は、肝臓の脂肪変性のモデルを生成するために、複数の最適化されたアプローチのその使用でありますそして総合インビボおよびインビトロの両方の表現型を特徴付けます。 DIOマウスモデルは、ヒトの脂肪肝疾患の病理学および代謝表現型を再現することができます。ヒトにおける他の代謝パラメータは、ウェル8のように、このモデルで複製することができます。高グルコース+インスリンに応答して脂肪変性肝細胞モデルの生成は、効率的で便利であり、費用と時間のかかるマウスの仕事の限界を克服します。まとめると、これらの方法は、栄養過負荷時の肝脂質障害およびインスリン抵抗性の研究のための十分かつ不可欠です。

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			<td height="20" style="height:20px;width:216px;">Infinity Cholesterol kit</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Penicillin / Streptomycin</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">FBS</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:163px;">10099141</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">PBS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">R21-040-CVR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">HBSS</td>
			<td style="width:183px;">cellgro</td>
			<td style="width:163px;">20-021-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Insulin</td>
			<td style="width:183px;">TOCRIS Bioscience</td>
			<td style="width:163px;">3435</td>
			<td>dissolve in PBS, 1mM for stock</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Glucose</td>
			<td style="width:183px;">Sigma</td>
			<td style="width:163px;">G8270-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Microscope</td>
			<td style="width:183px;">Olympus</td>
			<td style="width:163px;">BX53</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Peristaltic pump</td>
			<td style="width:183px;">Longerpump</td>
			<td style="width:163px;">BT100-2J</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">10cm cell culture dish</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">420167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">6-well-plate</td>
			<td style="width:183px;">Corning</td>
			<td style="width:163px;">3516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">BCA assay</td>
			<td style="width:183px;">Beyotime</td>
			<td style="width:163px;">P0010</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Nuclear Magnetic Resonance</td>
			<td style="width:183px;">Niumag technology</td>
			<td style="width:163px;">MiniQMR23-060H-I</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">High fat high surcose diet(HFHS)</td>
			<td style="width:183px;">Research Diets</td>
			<td style="width:163px;">D12327</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

図1Aに示すように、マウスの体重は約固形飼料給餌群よりも1.5倍であるHFHS送り、16週間後に45±1.2 gまで増加しました。 NMR体組成物は、脂肪量およびマウスの除脂肪量を示す分析する（ 図1B）に示されています。全身の、肝臓の脂肪分布は、MRIによって測定された、ライブ、意識のあるマウスにおける代表的な疑似カラー画像は、 図1C-D<...

Watch this Scientific Journal Video about Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis at JoVE.com

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 ...

optimized-analysis-of-in-vivo-and-in-vitro-hepatic-steatosis

Research

JoVE Journal

Medicine

Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

15.8K ビュー.  Chinese Academy of Sciences. 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. 

ビデオ: Optimized Analysis of In Vivo and In Vitro Hepatic Steatosis

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

There is a considerable discrepancy between oxygen supply and demand in the liver because hepatic oxygen consumption is relatively high but about 70% of the hepatic blood supply is poorly oxygenated portal vein blood derived from the gastrointestinal tract and spleen. Oxygen is delivered to hepatocytes by blood flowing from a terminal branch of the portal vein to a central venule via sinusoids, and this makes an oxygen gradient in hepatic lobules. The oxygen gradient is an important physical parameter that involves the expression of enzymes upstream and downstream in hepatic microcirculation, but the lack of techniques for measuring oxygen consumption in the hepatic microcirculation has delayed the elucidation of mechanisms relating to oxygen metabolism in liver. We therefore used FITC-labeled erythrocytes to visualize the hepatic microcirculation and used laser-assisted phosphorimetry to measure the partial pressure of oxygen in the microvessels there. Noncontact and continuous optical measurement can quantify blood flow velocities, vessel diameters, and oxygen gradients related to oxygen consumption in the liver. In an acute hepatitis model we made by administering acetaminophen to mice we observed increased oxygen pressure in both portal and central venules but a decreased oxygen gradient in the sinusoids, indicating that hepatocyte necrosis in the pericentral zone could shift the oxygen pressure up and affect enzyme expression in the periportal zone. In conclusion, our optical methods for measuring hepatic hemodynamics and oxygen consumption can reveal mechanisms related to hepatic disease.

An optical system was developed to visualize hepatic microcirculation with FITC-labeled erythrocytes and to measure the partial pressure of oxygen in the microvessels with laser-assisted phosphorimetry. This method can be used to investigate physiological and pathological mechanisms by analyzing microvascular structure, diameter, blood flow velocity, and oxygen tension.

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 ...

医学

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

細胞追跡のためのin vivoでの19 F MRI における

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Monitoring of Systemic and Hepatic Hemodynamic Parameters in Mice

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. However, due to the small size of the mouse, technical details of the intraoperative monitoring procedure suitable for the mouse were rarely described. Previously we have reported a monitoring procedure to obtain hemodynamic parameters for rats. Now, we adapted the procedure to acquire systemic and hepatic hemodynamic parameters in mice, a species ten-fold smaller than rats. This film demonstrates the instrumentation of the animals as well as the data acquisition process needed to assess systemic and hepatic hemodynamics in mice. Vital parameters, including body temperature, respiratory&#160;rate&#160;and heart&#160;rate were recorded throughout the whole procedure. Systemic hemodynamic parameters consist of carotid artery pressure (CAP) and central venous pressure (CVP). Hepatic perfusion parameters include portal vein pressure (PVP), portal flow rate as well as the flow rate of the common hepatic artery (table 1). Instrumentation and data acquisition to record the normal values was completed within 1.5 h. Systemic and hepatic hemodynamic parameters remained within normal ranges during this procedure.
This procedure is challenging but feasible. We have already applied this procedure to assess hepatic hemodynamics in normal mice as well as during 70% partial hepatectomy and in liver lobe clamping experiments. Mean PVP after resection (n= 20), was 11.41&#177;2.94 cmH2O which was significantly higher (P&#60;0.05) than before resection (6.87&#177;2.39 cmH2O). The results of liver lobe clamping experiment indicated that this monitoring procedure is sensitive and suitable for detecting small changes in portal pressure and portal flow rate. In conclusion, this procedure is reliable in the hands of an experienced micro-surgeon but should be limited to experiments where mice are absolutely needed.

This film demonstrates how to acquire systemic and hepatic hemodynamics in mice. The whole monitoring includes acquisition of vital parameters, systemic blood pressure, central venous pressure, common hepatic artery flow rate, and portal vein pressure as well as the portal flow rate in mice.

マウスにおける全身および肝血行動態パラメータの監視

The use of mouse models in experimental research is of enormous importance for the study of hepatic physiology and pathophysiological disturbances. ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

がん薬感の有効性を評価<em&gt;インビトロ</em&gt;と<em&gt;インビボ</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. Despite the discovery of key regulators of uterine contractility, this process is still not fully understood. Transgenic mice provide an ideal model in which to study parturition. Previously, the only method to study uterine contractility in the mouse was ex vivo isometric tension recordings, which are suboptimal for several reasons. The uterus must be removed from its physiological environment, a limited time course of investigation is possible, and the mice must be sacrificed. The recent development of radiometric telemetry has allowed for longitudinal, real-time measurements of in vivo intrauterine pressure in mice. Here, the implantation of an intrauterine telemeter to measure pressure changes in the mouse uterus from mid-pregnancy until delivery is described. By comparing differences in pressures between wild type and transgenic mice, the physiological impact of a gene of interest can be elucidated. This technique should expedite the development of therapeutics used to treat myometrial disorders during pregnancy, including preterm labor.

Intrauterine Telemetry to Measure Mouse Contractile Pressure In Vivo

This manuscript provides a protocol for implanting telemeters in the mouse for the purpose of measuring intrauterine pressures during pregnancy.

マウス収縮圧力を測定するために子宮内テレメトリ<em&gt;インビボ</em

A complex integration of molecular and electrical signals is needed to transform a quiescent uterus into a contractile organ at the end of pregnancy. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

ヒト腫瘍異種移植片からマウス細胞の枯渇は大幅に標的細胞の下流分析を向上させます

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

In VitroとIn Vivo検出、ひと細胞、線虫、マウス Mitophagy の

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Preparation Of Gushukang (GSK) Granules for In Vivo and In Vitro Experiments

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang (GSK) granules, a marketed prescription in China, have bone-protective effects in treating POP. Before administration to the body, one standard preparation procedure is commonly required, which aims to promote the release of active constituents from raw herbs and enhance the pharmacological effects as well as therapeutic outcomes. This study proposes a detailed protocol for using GSK granules in in vivo and in vitro experimental assays. The authors first provide a detailed protocol to calculate the animal-appropriate dosages of granules for in vivo investigation: weighing, dissolving, storage, and administration. Second, this article describes protocols for micro-CT scanning and the measurement of bone parameters. Sample preparation, protocols for running the micro-CT machine and quantification of bone parameters were evaluated. Third, serum-containing GSK granules are prepared, and drug-containing serum is extracted for in vitro osteoclastogenesis and osteoblastogenesis. GSK granules were intragastrically administered twice per day to rats for three consecutive days. Blood was then collected, centrifuged, inactivated, and filtered. Finally, serum was diluted and used for performing osteoclastogenesis and osteoblastogenesis. The protocol described here can be considered a reference for pharmacological investigations of herbal prescription medicines, such as granules.

Preparation Of Gushukang GSK Granules for In Vivo and In Vitro Experiments

This article provides a detailed protocol for preparing a working solution of Gushukang granules for animal studies and GSK granule containing serum for in vitro experiments. This protocol can be applied to pharmacological investigations of herbal medicines as well as prescriptions for both in vivo and in vitro experiments.

インビボおよびインビトロ実験用グシュカン(GSK)顆粒の調製

Traditional Chinese herbal medicine plays a role as an alternative method in treating many diseases, such as postmenopausal osteoporosis (POP). Gushukang ...

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

インビトロおよび創傷治癒の生体内分析のためのスクラッチ移行アッセイとドーサルスキンフォールドチャンバー

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...