sのOCH Drottning Viktorias Frimurarstiftelse：この作品は、カロリンスカ研究所KID-資金調達、スウェーデン放射線防護機関、カロリンスカ研究所アイ研究財団、ガンOCH BERTIL Stohnes Stiftelse、聖エリックの眼科病院研究財団、Ögonfonden、KonungグスタフVによってサポートされていました。

1。紫外線照射<ol><li>暴露前の15分には、腹腔内注射で90 mg / kgのKetalar（ケタミン）及び10 mg / kg Rompun（キシラジン）の混合物で雌Sprague-Dawleyラットを麻酔。 </li><li>ラットの拘束に動物を置き、トランクスクイズ13を発生させることなく、ラットの固定までベルトを締めます。 </li><li>散瞳を誘導するために、ラットの両眼にMydriacyl（トロピカミド）、10 mg / mlのを植え付ける。 </li><li>片目が半値全幅は10nmと300nmの14でUVRの狭いビームに対して位置決めされるように動物を置き、黒いテープで反対側の眼を遮蔽する。 </li><li> 15分15のための300 nmにおけるサブスレッショルド·用量1 KJ / m 2の UVR-Bに一方的にラットを公開します。 </li></ol> 2。解剖<ol><li>予め決められた露光後インターバル（1、5、24および120時間）の後、炭で6週齢のラット（体重&lt;200グラム）を犠牲にするn個の二酸化窒息と頸椎脱臼。 </li><li>摘出するの目。その後、後方側を、目の角膜を上にして置き。次に、強膜に非常に近いレンズの損傷を防ぐために、強膜に対して接線方向にパンチ27ゲージのカニューレを適用することにより、最小限の穴とプッシュ。その後、強膜の後部をオフに持ち上げることができるまで、ちょうど縁の後ろに周方向に切断するために眼科手術用はさみのペアを使用しています。その後、鈍湾曲したピンセットでレンズを持ち上げます。 </li><li> 5〜10分を超えない平衡塩溶液中でレンズを保持し、顕微鏡下でレンズ赤道から毛様体の残骸を削除。 </li></ol> 3。定量的RT-PCR <ol><li> 350μlのRA1の溶解緩衝液（NucleoSpin RNA IIの全RNA単離キット）および2mlのエッペンドルフチュー​​ブに3.5μLのβ-メルカプトエタノールのミックスにレンズを置きます。 </li><li>室温で30分間の間、このミックスでレンズを保管してください。 </li><li>レンズをホモジナイズレンズの唯一のハード核まで針がレンズ（皮質、レンズのカプセルが溶解バッファー中で溶解されている）から残っている。ミックスから核を取り除きます。 </li><li>ストアサンプル直ちに-70℃ </li><li>サンプルを解凍し、プロトコル &quot;培養細胞や組織からの全RNA精製&quot;（NucleoSpin RNA IIの全RNA単離キット）に従う。 </li><li>ストアRNAサンプル-70℃ </li><li> 2分間、95℃、40サイクルのステップ2：各試料からのDNAの十分な除去を確認するには、以下の条件（ステップ1でPCRを実行し95℃で20秒、55℃20秒、72℃ 20秒のC;ステップ3：7分72℃）、p53のDNA特異的プライマーを用いて、フォワード5&#39;-ACCCTCTGACCTTTTTCCCA-3 &#39;と逆方向5&#39;-TGCTGGGATCTTAGGCACTC-3&#39;およびTaq DNAポリメラーゼ（dNTPack）、製造によるプロトコル。予想されたPCR産物は、243塩基対である。 </li><li> 243塩基対のDNA特異的なPCR産物を検出するために、1.5％アガロースゲル電気泳動を実行します。準備するにはTBE緩衝液中で1.5％アガロースゲルはアガロースの3.75グラムを取るとTBEバッファー250mlにそれを解決する。臭化エチジウム（0.5 mg / ml）を1.5％アガロースゲル溶液500mlで500μlを添加する。マイクロ波オーウェンに、混合物を加熱してアガロースを解決するためにかき混ぜる。 </li></ol>試料はいずれも1.5％アガロースゲル電気泳動上の任意のDNA特異的なPCR産物を明らかにするために持っていません。 <ol start="9"><li> RNAサンプルの濃度（1μL）および260 nm（RNA）と光度計ND-1000分光光度計で280nmの（タンパク質）におけるサンプルの吸光度比を測定します。 RNAサンプルの吸光度のタンパク質に対する比のRNAが2.0以上である場合、RNAサンプルは純粋です。 RNAサンプルの吸光度比が2.0より低い場合は、RNA精製ステップ3.5をやり直してください。 </li><li>プロトコル第1ストランド cDNA合成キット（RT-PCRのための第1ストランド cDNA合成キット）に続く1μgに相当するRNAサンプルの音量を取ると、cDNAを合成。 </li><li> -20℃でcDNAサンプルを保存（期間1年以上とするため、-70℃で保存）。 </li><li> iCycler MyiQ単色リアルタイムPCR検出システムで、定量的リアルタイムPCRを実行します。負荷は、1μlのcDNA試料は、TaqMan遺伝子発現マスターミックス、カスパーゼ3のためのTaqMan遺伝子発現アッセイを96ウェルプレートに三連の指示を製造する方法。負荷は、1μlのcDNA試料は、TaqMan遺伝子発現マスターミックス、18sのためのTaqMan遺伝子発現アッセイを持つ別の96ウェルプレートにトリプリケート命令（のTaqMan遺伝子発現アッセイプロトコル）を製造する方法。直列3ランダムに選ばれた非露光レンズからのcDNAの一部を希釈します。 96ウェルプレート中のサンプルからのcDNAと共に希釈を実行します。 </li><li>結果の定量化のための標準曲線法を使用します。しきい蛍光におけるサイクル数はMyiQソフトウェアの測定として使用されます。キャリブレータの相対濃度の関数としてしきい値でサイクル数を表現する標準曲線は、esです各プレートで連続希釈用tablished。 3非処理レンズ試料からのcDNAは、キャリブレーションのために使用された。測定されたcDNAサンプルの相対濃度を得るために標準曲線と各サンプルのサイクル数のしきい値を関連付けます。最後に、cDNAの内部統制18Sに対するcDNAターゲットの相対濃度を関連付けることにより、標的遺伝子の発現レベルを取得します。 </li></ol> 4。免疫組織化学的染色<ol><li>固定<ol><li> PBS中目（; pH7.4のリン酸緩衝生理食塩水）を解剖する。 </li><li> 20分間氷冷4％パラホルムアルデヒド（PFA）で満たされたチューブに目を入れた。前日に4％PFAを準備します。 4℃で保存し、使用時まで。 PFAは、約1週間新鮮です。 </li><li>マイクロピペットでPFAを削除します。その後、20分間氷冷PBSで洗浄する。 </li><li> ℃で一晩4でスクロース30％で目を入れた。 </li><li> （10月）最適切削温度で満たさカップに目を入れて、媒体（ティッシュ- Tek社）。セクショニングの適当な位置に目を置いた。 </li><li>ドライアイスの上に、OCT-培地中で凍結する。 </li><li> -70℃で保存し、使用時まで。 </li></ol></li><li>セクショニング<ol><li>適切な平面にクライオセクショニングの目の位置を決めます。 </li><li>各レンズの中央部から35μm厚半ば矢状断面をカットします。異なるセクションの同じ核を染色避けるために順次セクションの間には、少なくとも6つのセクションを破棄します。 </li><li>セクションがカットされたら、そっとその上にスライドを配置します。セクションでは、スライドに固執する必要があります。 </li><li>使用前に空気乾燥にスライドしたままにします。必要になるまでスライドを-20℃で保存することができます。 </li></ol></li><li>蛍光免疫組織化学<ol><li>サンプルは室温（5〜10分）を達成しましょう​​。 </li><li> PAP-ペン（Invitrogen）を用いて試験片の周りにエッジを描画する。 </li><li>しばらく（10分）の境界線に乾かします。 </li><li>顕微鏡スライドラベルを付けます。 </li><li> 1に水分補給×Pを15分間のBS。 </li><li> 30分間の標準ブロック·ソリューションで透過処理。 </li><li>標準ブロック·溶液中で1:3,000に希釈し、一次抗体（セルシグナリングテクノロジー株式会社ウサギポリクローナル、切断カスパーゼ-3抗体Asp175 966​​1）を追加します。 </li><li>一晩4℃で加湿チャンバーに保管してください。 </li><li> （3×5分）、ピペッティングによりまたは大浴場（&gt; 15分、1月2日変更）中に浸漬することにより、PBS中で洗浄します。 </li><li>標準ブロック·溶液中で1:300に希釈した二次抗体（特定の吸収/発光スペクトルを持つ抗ウサギ二次抗体）を追加します。 </li><li> （3×5分）、ピペッティングによりまたは大浴場（&gt; 15分、1月2日変更）中に浸漬することにより、PBS中で洗浄します。 </li><li> PAPペンスメアを拭き取ってください。 </li><li> 3時間室温で加湿チャンバー内で保管してください。光への曝露を避ける。 </li><li> Vectashieldのドロップを追加して、スライドにカバースリップを置く。泡を作ることは避けてください。 </li><li> Vectashield硬化（〜20分）をしましょう​​。 </li><li>セクションI保つ分析するまでダークをn。 </li><li>すべての制御セクションは、一次抗体の非存在下で処理されます。 </li><li>蛍光顕微鏡下で数時間以内に結果を探してください。 </li><li>片側核船首から各レンズの反対側の核弓に上皮細胞の核を数える。青のすべての水晶体上皮核と緑におけるカスパーゼ3陽性核を見て二次抗体の放出に適合する標準フィルタを表示するには、標準の青のフィルタを適用します。 </li><li>すべての水晶体上皮核および陽性核の数を記録します。セクションごとに細胞を三回を数える。 </li></ol></li></ol>

<ol>
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<html>本稿では、UVR-B誘発白内障の間に発生する分子事象を研究する有効な方法について説明します。 生体 UVR誘発性白内障で使用可能なほとんどの情報がアルビノSprague-Dawleyラット7、16、17、18、19日の実験から得られたことを、考慮して、現在の研究ではアルビノSprague-Dawleyラットを使用することを決めた。ラットの年齢は6週齢であった。性別は男性...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント（オプション） </td></tr><tr><td> Ketalar </td><td>ファイザー</td><td> 150086 </td><td> 50 mg / mlの</td></tr><tr><td> Rompun </td><td>バイエル</td><td> 022545 </td><td> 20 mg / mlの</td></tr><tr><td> Oculentumシンプレックス</td><td> Apoteket、スウェーデン</td><td> 336164 </td><td> 5グラム</td></tr><tr><td> Mydriacyl </td><td>アルコン</td><td> 00352 </td><td> 10 mg / mlの</td></tr><tr><td>平衡塩類溶液</td><td>アルコン</td><td> 0007950055 </td><td></td></tr><tr><td> 3.5 ulのβ-メルカプトエタノール</td><td></td><td></td><td></td></tr><tr><td> NucleoSpin RNA IIの全RNA単離キット</td><td>マシュレ·ナーゲル社＆株式会社、デューレン、ドイツ</td><td> 740955.50 </td><td></td></tr><tr><td> p53のDNA特異的プライマー</td><td> biomers.net社</td><td>カスタムメード</td><td></td></tr><tr><td> Taq DNAポリメラーゼ、dNTPack </td><td>ロッシュ</td><td> 04 728 866 001 </td><td></td></tr><tr><td>アガロース</td><td>シグマ</td><td> A5093 </td><td></td></tr><tr><td>エチジウムブロマイド溶液0.5 mg / mlの</td><td>シグマ</td><td> E1385 </td><td></td></tr><tr><td>ナノドロップND-1000分光</td><td>光度計の製品</td><td></td><td></td></tr><tr><td> RT-PCRのための第1ストランド cDNA合成キット（AMV） </td><td>ロシュ·ダイアグノスティックス社</td><td> 11 483 188 001 </td><td></td></tr><tr><td> iCycler MyiQ単色リアルタイムPCR検出システム</td><td>バイオ·ラッドラボラトリーズ</td><td></td><td></td></tr> &lt;TR&gt; <td> 96ウェルプレート</td><td>ザルスタット</td><td> 72.1979.202 </td><td></td></tr><tr><td>のTaqMan遺伝子発現のマスターミックス</td><td>アプライドバイオシステムズ</td><td> 4369016 </td><td></td></tr><tr><td>カスパーゼ3のためのTaqMan遺伝子発現アッセイ</td><td>アプライドバイオシステムズ</td><td> Rn00563902_m1 </td><td></td></tr><tr><td> 18sのためのTaqMan遺伝子発現アッセイ</td><td>アプライドバイオシステムズ</td><td> Hs99999901_s1 </td><td></td></tr><tr><td> MyiQソフトウェア</td><td>バイオ·ラッドラボラトリーズ</td><td></td><td></td></tr><tr><td>切断されたカスパーゼ-3（Asp175） </td><td>セルシグナリング技術</td><td> 9661 </td><td></td></tr><tr><td>抗ウサギIgG抗体</td><td>アブカム</td><td> Ab6798 </td><td></td></tr><tr><td>スクロース</td><td>シグマアルドリッチ</td><td> 84097 </td><td></td></tR&gt; <tr><td> Vectashield </td><td>ベクターラボラトリーズ</td><td></td><td></td></tr><tr><td>ユニバーサル顕微鏡Axioplan 2イメージング</td><td>カールツァイス</td><td></td><td></td></tr><tr><td>パラホルムアルデヒド</td><td>シグマアルドリッチ</td><td> 441244 </td><td></td></tr><tr><td> 10×TBEバッファー</td><td>プロメガ</td><td> V4251 </td><td></td></tr></tbody></table>

characterization of molecular mechanisms of in vivo uvr induced cataract

白内障は、世界1の失明の主要な原因である。世界保健機関（WHO）は、光の伝達を妨げ、眼のレンズの混濁として白内障が定義されています。白内障は、糖尿病、喫煙、紫外線照射（UVR）、アルコール、電離放射線、ステロイドや高血圧に関連付けられている多因子疾患である。強い実験2-4および疫学的証拠は、UVR 5,6は白内障引き起こすことがあります。我々は麻酔7及び非麻酔動物8の両方でB UVR誘発白内障のための動物モデルを開発しました。 白内障のための唯一の治療法は手術ですが、この治療法はすべてにアクセスすることはできません。それは10年間の白内障の発症の遅延が50％9で白内障手術の必要性を減らすことができると推定されている。白内障の発症を遅延させるには、それは白内障形成のメカニズムを理解し、効果的な予防戦略を見つけるために必要とされるegies。白内障開発のためのメカニズムの中で、アポトーシスは、ヒトおよび動物10の白内障の開始に重要な役割を果たしている。我々の焦点は、最近白内障開発8,11,12するためのメカニズムとしてレンズのアポトーシスとなっています。これはアポトーシス経路上のUVRの効果の理解は白内障防ぐための新たな医薬品の発見の可能性を提供することが予想されます。 本稿では、実験的にUVR-Bへのin vivoでの暴露にによって誘導することができる方法白内障について説明します。さらにRT-PCR法と免疫組織化学は、UVR-B誘発白内障の分子メカニズムを研究するためのツールと​​して提示されます。

測定値の変動のさまざまな源は分散分析を用いて推定された、それは動物ごとに3つの測定値を考慮すると測定のための分散は動物のそれの15％のオーダーであることがわかった。したがって、レンズ全体の分析を考慮すれば、精度を向上させることはできません。直交テストは120時間の待ち時間の間隔対短い待ち時間間隔のカスパーゼ-3メッセージの統計学的に有意なコントラストを明らか?...

Watch this Scientific Journal Video about Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract at JoVE.com

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

白内障は世界の失明の主要な原因である。太陽紫外線（UVR）は白内障開発のための主要な危険因子である。遠くUVR-B誘発白内障の動物モデルが開発されました。 UVR、定量的RT-PCR法と免疫組織化学への暴露：この記事では、白内障の調査のための方法を説明します。

の分子機構のキャラクタリゼーション<emインビボ&gt;</em&gt; UVR誘発性白内障

Cataract is the leading cause of blindness in the world 1. The World Health Organization defines cataract as a clouding of the lens of the eye which ...

characterization-molecular-mechanisms-vivo-uvr-induced

Research

JoVE Journal

Medicine

Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

9.8K ビュー.  Karolinska Institutet. 白内障は世界の失明の主要な原因である。太陽紫外線（UVR）は白内障開発のための主要な危険因子である。遠くUVR-B誘発白内障の動物モデルが開発されました。 UVR、定量的RT-PCR法と免疫組織化学への暴露：この記事では、白内障の調査のための方法を説明します。

ビデオ: Characterization of Molecular Mechanisms of In vivo UVR Induced Cataract

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading to an accumulation of lipid-laden intra-luminal plaque, smooth muscle cell proliferation and progressive narrowing of the vessel lumen. The formation of such vulnerable plaques prone to rupture underlies the majority of cases of acute myocardial infarction. The complex molecular and cellular inflammatory cascade is orchestrated by the recruitment of T lymphocytes and macrophages and their paracrine effects on endothelial and smooth muscle cells.1
Molecular imaging in atherosclerosis has evolved into an important clinical and research tool that allows in vivo visualization of inflammation and other biological processes. Several recent examples demonstrate the ability to detect high-risk plaques in patients, and assess the effects of pharmacotherapeutics in atherosclerosis.4 While a number of molecular imaging approaches (in particular MRI and PET) can image biological aspects of large vessels such as the carotid arteries, scant options exist for imaging of coronary arteries.2 The advent of high-resolution optical imaging strategies, in particular near-infrared fluorescence (NIRF), coupled with activatable fluorescent probes, have enhanced sensitivity and led to the development of new intravascular strategies to improve biological imaging of human coronary atherosclerosis.
Near infrared fluorescence (NIRF) molecular imaging utilizes excitation light with a defined band width (650-900 nm) as a source of photons that, when delivered to an optical contrast agent or fluorescent probe, emits fluorescence in the NIR window that can be detected using an appropriate emission filter and a high sensitivity charge-coupled camera. As opposed to visible light, NIR light penetrates deeply into tissue, is markedly less attenuated by endogenous photon absorbers such as hemoglobin, lipid and water, and enables high target-to-background ratios due to reduced autofluorescence in the NIR window. Imaging within the NIR 'window' can substantially improve the potential for in vivo imaging.2,5
Inflammatory cysteine proteases have been well studied using activatable NIRF probes10, and play important roles in atherogenesis. Via degradation of the extracellular matrix, cysteine proteases contribute importantly to the progression and complications of atherosclerosis8. In particular, the cysteine protease, cathepsin B, is highly expressed and colocalizes with macrophages in experimental murine, rabbit, and human atheromata.3,6,7 In addition, cathepsin B activity in plaques can be sensed in vivo utilizing a previously described 1-D intravascular near-infrared fluorescence technology6, in conjunction with an injectable nanosensor agent that consists of a poly-lysine polymer backbone derivatized with multiple NIR fluorochromes (VM110/Prosense750, ex/em 750/780nm, VisEn Medical, Woburn, MA) that results in strong intramolecular quenching at baseline.10 Following targeted enzymatic cleavage by cysteine proteases such as cathepsin B (known to colocalize with plaque macrophages), the fluorochromes separate, resulting in substantial amplification of the NIRF signal. Intravascular detection of NIR fluorescence signal by the utilized novel 2D intravascular NIRF catheter now enables high-resolution, geometrically accurate in vivo detection of cathepsin B activity in inflamed plaque.
In vivo molecular imaging of atherosclerosis using catheter-based 2D NIRF imaging, as opposed to a prior 1-D spectroscopic approach,6 is a novel and promising tool that utilizes augmented protease activity in macrophage-rich plaque to detect vascular inflammation.11,12 The following research protocol describes the use of an intravascular 2-dimensional NIRF catheter to image and characterize plaque structure utilizing key aspects of plaque biology. It is a translatable platform that when integrated with existing clinical imaging technologies including angiography and intravascular ultrasound (IVUS), offers a unique and novel integrated multimodal molecular imaging technique that distinguishes inflammatory atheromata, and allows detection of intravascular NIRF signals in human-sized coronary arteries.

In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

炎症性プラークの赤外蛍光（NIRF）血管内分子イメージング、マルチモーダルアプローチの近くin vivoでの動脈硬化のイメージングに

The vascular response to injury is a well-orchestrated inflammatory response triggered by the accumulation of macrophages within the vessel wall leading ...

医学

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

Muscle strains are one of the most common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and physical exam alone, however the clinical presentation can vary greatly depending on the extent of injury, the patient's pain tolerance, etc. In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Biological markers, such as serum creatine kinase levels, are typically elevated with muscle injury, but their levels do not always correlate with the loss of force production. This is even true of histological findings from animals, which provide a "direct measure" of damage, but do not account for all the loss of function. Some have argued that the most comprehensive measure of the overall health of the muscle in contractile force. Because muscle injury is a random event that occurs under a variety of biomechanical conditions, it is difficult to study. Here, we describe an in vivo animal model to measure torque and to produce a reliable muscle injury. We also describe our model for measurement of force from an isolated muscle in situ. Furthermore, we describe our small animal MRI procedure.

An in vivo Rodent Model of Contraction-induced Injury and Non-invasive Monitoring of Recovery

An in vivo animal model of injury is described. The method takes advantage of the subcutaneous position of the fibular nerve. Velocity, timing of muscle activation, and arc of motion are all pre-determined and synchronized using commercial software. Post injury changes are monitored in vivo using MR imaging/spectroscopy.

アン in vivoで</em収縮で誘起された損傷と回復の非侵襲的モニタリングの>齧歯類モデル

Muscle strains are one of the most  common complaints treated by physicians. A muscle injury is typically diagnosed from the patient history and  physical ...

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. ...

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the extracellular matrix (ECM). Characterizing the biology of this phenotypically distinct group of cells could substantially improve our understanding of early events during the metastatic cascade.

Tumor invasion is a dynamic process facilitated by bidirectional interactions between malignant epithelium and the cancer associated stroma. In order to examine cell-specific responses at the tumor stroma-interface we have combined organotypic co-culture and laser micro-dissection techniques.
Organotypic models, in which key stromal constituents such as fibroblasts are 3-dimentioanally co-cultured with cancer epithelial cells, are highly manipulatable experimental tools which enable invasion and cancer-stroma interactions to be studied in near-physiological conditions.

Laser microdissection (LMD) is a technique which entails the surgical dissection and extraction of the various strata within tumor tissue, with micron level precision.
By combining these techniques with genomic, transcriptomic and epigenetic profiling we aim to develop a deeper understanding of the molecular characteristics of invading tumor cells and surrounding stromal tissue, and in doing so potentially reveal novel biomarkers and opportunities for drug development in CRC.&#160;&#160;&#160;

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molecular profiling of laser microdissected cells and stroma from synthetic, tissue-engineered colorectal cancer models represents a novel and manipulatable approach to characterize and dissect the distinctive biology at the interface between tumor cells at the invasive front and cancer associated stromal cells. &#160;

大腸癌細胞の3次元モデルにおける侵襲性腫瘍微小環境の分子プロファイリングと<em&gt;生体外</em&gt;線維芽細胞

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

化学療法誘発性血管毒性 - リアルタイム<em&gt;インビボ</em船舶減損の&gt;イメージング

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

マウスモデルにおけるUTIとCAUTIの樹立とその性状

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

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 ...

A Doxorubicin-Induced Murine Model of Dilated Cardiomyopathy In Vivo

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac performance in the absence of hypertension, valvular, congenital, or ischemic heart diseases, and which may be related to infection, autoimmune or metabolic abnormalities, or family inheritance. It can progress into congestive heart failure with a poor prognosis. Doxorubicin (Dox) is widely employed as a chemotherapeutic drug, but its use is limited because it causes DCM-like changes of the myocardium. Its myocardial toxicity is attributed to oxidative stress, chronic inflammation, and cardiomyocyte apoptosis. A model of DCM exploiting these Dox-induced DCM symptoms has not been established.

Described is a protocol to establish a Doxorubicin-induced dilated cardiomyopathy (DCM) model in mice via long-term intraperitoneal injection of Doxorubicin.

インビボにおける拡張型心筋症のドキソルビシン誘発性マウスモデル

Dilated cardiomyopathy (DCM) refers to a spectrum of heterogeneous myocardial disorders characterized by ventricular dilation and depressed cardiac ...

Generation and Characterization of Right Ventricular Myocardial Infarction Induced by Permanent Ligation of the Right Coronary Artery in Mice

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In contrast to the extensively used mouse myocardial infarction (MI) model generated by left coronary artery ligation, the RVI mouse model is rarely employed due to the difficulty associated with model generation. Research on the mechanisms and treatment of RVI-induced RV remodeling and dysfunction requires animal models to mimic the pathophysiology of RVI in patients. This study introduces a feasible procedure for RVI model generation in C57BL/6J mice. Further, this model was characterized based on the following: infarct size evaluation at 24 h after MI, assessment of cardiac remodeling and function with echocardiography, RV hemodynamics assessment, and histology of the infarct zone at 4 weeks after RVI. In addition, a coronary vasculature cast was performed to observe the coronary arterial arrangement in RV. This mouse model of RVI would facilitate the research on mechanisms of right heart failure and seek new therapeutic targets of RV remodeling.

There are several differences between the right and left ventricles. However, the pathophysiology of right ventricular infarction (RVI) has not been clarified. In the present protocol, a reproducible method for RVI mouse model generation is introduced, which may provide a means to explain the mechanism of RVI.

マウスにおける右冠動脈の永久結紮による右心室心筋梗塞の生成と特徴付け

Right ventricular infarction (RVI) is a common presentation in clinical practice. Severe RVI can lead to fatal hemodynamic dysfunction and arrhythmia. In ...

Methods Article

の分子機構のキャラクタリゼーション