この作品のサポートは、健康補助金の国民の協会によって提供されていました＃R01 HL 108229、米国心臓協会サイエンティスト開発グラント＃0830352N、ハワードヒューズ医学研究所のキャリア開発賞、ブロードビューベンチャーズ、欧州共同体の第七次フレームワーク計画（助成金の下でFP7/2007-2013契約＃235689）、およびMGHウィリアムシュライヤーフェローシップ。

実験的大動脈腸骨動脈のアテローム性動脈硬化症の発生：in vivo動物モデルで 1）ベースライン血管造影およびバルーンの削剥<ol><li>ベースライン血管造影およびバルーンの削剥を取得する前に、ニュージーランドホワイトウサギは1週間のための高コレステロール血症（1％）食を供給されます。この動物は、ウサギの大動脈iliacs血管がヒト冠状動脈（2.5〜3.5ミリメートル）と同じ口径であり、2）高脂血症、バルーン傷害モデルが同じような炎症細胞（マクロファージが付いた炎症を起こしてアテローム性動脈硬化症を生成する1）などの翻訳関連性のために利用されている人間のアテローム性動脈硬化症のように）と分子（カテプシン）。 </li><li>コレステロール摂食に続いて、動物は、プロポフォールとケタミンで麻酔です。 1インチの腹側正中の首の切開は、サイズ15メス刃を用いて使用して行われます。鈍的切開の技術を使用して、気管の右側にある筋膜の下にある筋肉が露出している。左sternocephalicusの筋肉は、その結合組織の接合に沿って分離され、右総頸動脈を露出している。動脈は、迷走神経から分離されている。近位および遠位縫合糸ループは、撤回し、閉塞を可能にするために動脈に配置されます。 2mm厚1は動脈切開をベベル5フランス語（1.67ミリメートル外径が）血管シースを挿入し、ヘパリン（1000μ/mL、〜150units/kg）シースを介して動脈内に投与されているを介して行われます。 </li><li>造影剤（Ultravistが）その後、遠位大動脈と両方腸骨動脈の制御の血管造影を得るために2秒の期間にわたって（1〜2mLの）注入されます。 </li><li>腸骨大腿動脈と大動脈は、内皮削剥によって負傷している。標準的なX線透視法を用いて、3FRフォガティの塞栓摘除カテーテルは、遠位腸骨大腿動脈内に配置し、0.3から0.5コントラストのCC（50％contrast/50％生理食塩水）または空気で膨らませている。カテーテルは、その膨張した状態で近位に左腎動脈の離陸までの右腸骨と遠位大動脈に沿った距離を引き出される。バルーンの削剥に続いて、血管造影は、血管の開存性を文書化するために繰り返されます。血管造影に続いて、すべてのカテーテル及びシースが削除され、近右総頸動脈を結紮され、筋肉や筋膜が4 / 0吸収性縫合糸で縫合し、皮膚切開は4 / 0、非吸収性縫合糸で閉じられます。 </li><li>動物は、1用量の抗生物質（Cephazolin、0.5グラムIM）の投与で回復するために許可されています。は0.01 mg / kgのブプレノルフィンIM（必要に応じて日に二回）を含む鎮痛薬。動物は、その後4週間後のバルーンの削剥のための1％のコレステロールを続けています。週5で、動物は、0.3％コレステロール食に移行されています。 </li></ol>ウサギAtheromataの統合されたマルチモーダルイメージング 2）注射ナノセンサーを使用してタンパク質分解アクティブ炎症プラークの標識、血管造影、血管内超音波（IVUS）、およびウサギアテロームのin vivoで血管内NIRFイメージングで <ol><li>バルーン損傷およびイメージングの前に24時間以下の8週間は、ウサギは耳静脈を経由して静脈内500ナノモル/ kgのProsense/VM110（パーキンエルマー）を注入されています。 </li><li>注入24時間後、動物を麻酔し、動脈のアクセスは左総頸動脈（ステップ1.2参照）を介して取得されています。動脈内ヘパリン（150単位/ kg）に投与される。ベースライン血管造影は、上記のように得られる。 </li><li> IVUSカテーテルは臨床的冠動脈できる0.014インチワイヤーにロードされ、シースに挿入されます。透視を使って、線の放射線不透過性の先端は右腸骨動脈内に遠位方向に配置されている。 IVUSカテーテルは、標準的な臨床モノレールの技術を使用して近位腸骨動脈内に進めています。 </li><li> 100mmのプルバックが開始され、画像が記録されます。容器の長手方向の再建が得られ、内腔のプラークが識別されます。 </li><li>右腸骨動脈内に遠位方向に配置されているNIRFカテーテル11,12は、0.014インチワイヤー（モノレールシステム）にロードされ、カテーテルを慎重にシースとイメージングヘッドに挿入されます。 </li><li>複数の自動pullbacks（1 mm /秒縦引き戻し、毎分30発）が行われ、アテローム性動脈硬化症のゾーン内の蛍光シグナルが記載されています。画像が記録され、信号の範囲に基づいて適切なスケーリングと窓とのさらなる処理が実現されます。 </li></ol> 3）安楽死および ex vivo大動脈腸骨組織の分離<ol><li>安楽死は安楽死剤の1cc（390mgペントバルビタールナトリウムと50mgのフェニトインナトリウムの溶液）を、静脈内、単回注入で実現されます。 </li><li>下大静脈は、血液の透明になるまで動脈樹を、0.9％生理食塩水で灌流される。アテローム性動脈硬化大動脈および腸骨動脈を同定し、周囲の組織からフリー解剖されています。 liのほかに、小型の2 × 2 cmの断片バージョン、腎臓、脾臓、心臓も得られる。 </li><li>血管内NIRFイメージングカテーテルとex vivoで NIRFイメージングは、この段階で行うことができます。イメージングヘッドは右腸骨動脈または分岐部に配置されるまで、近位大動脈に血管が引き伸ばされているとNIRFカテーテルを再挿入されます。複数の自動pullbacksは（2.6参照）上記のように実行されます。 </li></ol>解離した大動脈および腸骨動脈の4） エクスビボ蛍光反射イメージング（金） <ol><li>解剖組織は、生理食塩水の10-20 CCに置かれ、金の分析（コダックイメージステーション4000MMプロ、ケアストリームヘルス社）のために輸送される。 </li><li>大動脈、腸骨血管が近似リアルタイムの長さやイメージに引き延ばされている複数の波長で得られる[白色光、緑色蛍光チャンネル（EX 495nmの、全角515 nm）を、Cy5の（元565 nmの、全角670 nm）およびCy7（EX波長650nm、全角760 nm）に]チャンネル。露光時間のシリーズは、各波長のために利用されている（0.1 - 30秒）と取得された画像は、DICOMや、さらなる分析のための16ビットスケーリングされていないTIFFファイルとしてエクスポートされます。陽性および陰性対照として、臓器（肝臓、脾臓、腎臓および心臓が）同じようなチャンネルと露光時間で撮像する。 </li><li>近赤外チャンネルの増加信号（780nm +）の領域は、アテローム性動脈硬化動脈に記載されています。 </li></ol>セクショニングと免疫組織化学的分析のため5）組織の埋め込み<ol><li>正常の領域（非損傷した組織、つまり、左腸骨動脈）とプラークの領域が識別され、組織の小5〜10ミリメートルのリングは、OCT（最適切断温度）メディアに埋め込まれています。ブロックは切片まで-80℃で保存されています。 </li><li>切片と免疫組織化学的分析のための標準的な技術が実行されます。ヘマトキシリン​​およびエオシン染色、ラム- 11およびカテプシンBの染色が行われている。 </li></ol>マルチモーダル画像の分析と統合（血管造影、IVUS、NIRFとFRI） NIRFと金のイメージ6）の処理<ol><li> NIRFと金（赤外線780nmのチャネル付近で撮影）pullbacksから画像データを含むDICOMファイルはそれぞれ、MATLABとOsiriXはソフトウェアを使用して処理されます。信号強度の全範囲を表示するために適切なウィンドウが達成されます。最後の画像は、TIFFファイルとしてエクスポートされます。 </li><li>ファイルは、標準的な画像処理ソフトウェア（基調講演を使用することができる）にインポートされます。画像は、基準点（血管造影、腸骨の分岐、および腎動脈上すなわち椎骨）に基づいて整列されます。正常な血管とプラークの領域が識別されます。 </li><li>興味（ROI）の領域は手動で追跡（プラークの正常組織や地域のため）と信号強度が金とNIRF画像の両方に対して、それぞれ、OsiriXは、MATLABを用いて取得されていることを意味している。適切なトレースを導くために、容器の長手方向のIVUS画像が使用され、正常な血管とプラークの同定が容易に識別されます。 </li><li>ターゲットへのバックグラウンド（TBR）の比率は、プラークのゾーンに対して計算されます。 </li></ol>代表的な結果： 上記のプロトコルの完了時に、我々は、識別することができますし、大動脈および腸骨血管内で炎症性プラークの拡張カテプシンプロテアーゼ活性の領域を特徴づける。起動可能なナノセンサー（Prosense/VM110）の注入は、私たちはタンパク質分解アクティブなプラークを識別することができます。これらは、近赤外チャンネル（750 nm）で金を用いて画像のように明るいまたは信号激しいゾーンで表示されます。 NIRFのpullbacksはNIRF信号の解剖学的に登録を許可IVUSと金とのアライメントによって増加信号強度と相関する。計算されたプラークのTBRは、金とNIRFから入手している（図3を参照してください：NIRF TBR 4.2意味、金TBR 2.9意味）と同様であった。明るいプラークの免疫組織化学的分析は、RAM - 11とプラークの領域（データは示さず）のカテプシンB活性の強い存在を確認。 <img alt="図1" src="/files/ftp_upload/2257/2257fig1.jpg" /> 図1。 1D NIRFセンシングアプローチ6の臨床的可能性を拡張するには、2D NIRFカテーテルの概略 、我々は新たな血管内イメージングのための2次元NIRF -カテーテルを構築した。11,12特注のカテーテルは、光ファイバ（125ミクロンの直径に収納で構成されていますポリエチレンチューブで：2.9F）それは、750 nmのレーザー励起源を使用して点灯します。レーザー光が繊維軸に対して90度の角度で放出される。システムは真の2次元画像を得るために付随する360度のイメージングと長手方向の引き戻しを有効にするには2つの自動化されたモータ（回転と並進）を利用しています。画像はあくまで参考11から許可を得て使用。 <img alt="図2" src="/files/ftp_upload/2257/2257fig2.jpg" /> 図2。ナノセンサーの回路図を実証プロテアーゼを介した活性化、Prosense/VM110。参考文献10より許可を得て使用されるイメージ。 <img alt="表1" srC =&quot;/ files/ftp_upload/2257/2257table1.jpg&quot;/&gt; 図3。 生体とex vivoでのプラークTBRs（バックグラウンド比ターゲットへ） の

<ol>
	<li>Andersson, J., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+immunity+and+atherosclerosis.">Adaptive immunity and atherosclerosis.</a> Clin Immunol. 134, 33-46 (2010).</li><li>Calfon, M. A., Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+near-infrared+fluorescence+molecular+imaging+of+atherosclerosis:+toward+coronary+arterial+visualization+of+biologically+high-risk+plaques.">Intravascular near-infrared fluorescence molecular imaging of atherosclerosis: toward coronary arterial visualization of biologically high-risk plaques.</a> Journal of Biomedical Optics. 15, 011107-011107 (2010).</li><li>Chen, J., Tung, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vivo+Imaging+of+Proteolytic+Activity+in+Atherosclerosis.">In Vivo Imaging of Proteolytic Activity in Atherosclerosis.</a> Circulation. 105, 2766-2771 (2002).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Imaging+of+Cardiovascular+Disease.">Molecular Imaging of Cardiovascular Disease.</a> Circulation. 116, 1052-1061 (2007).</li><li>Jaffer, F. A., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+and+Multimodality+Molecular+Imaging:+Insights+Into+Atherosclerosis.">Optical and Multimodality Molecular Imaging: Insights Into Atherosclerosis.</a> Arterioscler Thromb Vasc Biol. 29, 1017-1024 (2009).</li><li>Jaffer, F. A., Vinegoni, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-Time+Catheter+Molecular+Sensing+of+Inflammation+in+Proteolytically+Active+Atherosclerosis.">Real-Time Catheter Molecular Sensing of Inflammation in Proteolytically Active Atherosclerosis.</a> Circulation. 118, 1802-1809 (2008).</li><li>Kim, D. -. E., Kim, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease+Imaging+of+Human+Atheromata+Captures+Molecular+Information+of+Atherosclerosis,+Complementing+Anatomic+Imaging.">Protease Imaging of Human Atheromata Captures Molecular Information of Atherosclerosis, Complementing Anatomic Imaging.</a> Arterioscler Thromb Vasc Biol. 30, 449-456 (2010).</li><li>Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammation+in+atherosclerosis.">Inflammation in atherosclerosis.</a> Nature. 420, 868-874 (2002).</li><li>Naghavi, M., Libby, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Vulnerable+Plaque+to+Vulnerable+Patient:+A+Call+for+New+Definitions+and+Risk+Assessment+Strategies:+Part+I.">From Vulnerable Plaque to Vulnerable Patient: A Call for New Definitions and Risk Assessment Strategies: Part I.</a> Circulation. 108, 1664-1672 (2003).</li><li>Weissleder, R., Tung, C. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+tumors+with+protease-activated+near-infrared+fluorescent+probes.">In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.</a> Nat Biotech. 17, 375-375 (1999).</li><li>Razansky, R. N., Rosenthal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Near-infrared+fluorescence+catheter+system+for+two-dimensional+intravascular+imaging+in+vivo.">Near-infrared fluorescence catheter system for two-dimensional intravascular imaging in vivo.</a> Optics Express. 18, 11372-11381 (2010).</li><li>Jaffer, F. A., Calfon, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-Dimensional+Intravascular+Near-Infrared+Fluorescence+Molecular+Imaging+of+Inflammation+in+Atherosclerosis+and+Stent-Induced+Vascular+Injury.">Two-Dimensional Intravascular Near-Infrared Fluorescence Molecular Imaging of Inflammation in Atherosclerosis and Stent-Induced Vascular Injury.</a> Journal of the American College of Cardiology. 57, 2516-2526 (2011).</li></ol>

水産庁 - 元コンサルタント、VisEn医療、謝礼、ボストンサイエンティフィック

炎症を起こして高リスクまたは脆弱なプラークは、心筋梗塞の大多数の可能性が高い責任があります。症状の発症に先立ってこのようなプラークの同定は、両方の結果を予測し、薬物療法を導く臨床的に重要な意味を持っています。このようなX線血管造影などの従来の冠状動脈の画像診断法は、通常、内腔の狭窄の特性評価ではなく、ハイリスク、通常は非狭窄病変の基礎となる生物学的プ...

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Prosense 750</td>
<td>&nbsp;</td>
<td>Visen Medical</td>
<td>VM110</td>
<td>500 nmol/kg IV injection</td>
</tr>
<tr>
<td>Heparin Sodium</td>
<td>&nbsp;</td>
<td>APP Pharmaceuticals</td>
<td>401586D</td>
<td>&nbsp;</td>
<tr>
<td>Cephazolin</td>
<td>&nbsp;</td>
<td>NovaPlus</td>
<td>46015683</td>
<td>&nbsp;</td>
<tr>
<td>Lidocaine HCL 2%</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC 0409-4277-01</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine</td>
<td>&nbsp;</td>
<td>Bedford Laboratories</td>
<td>NDC 55390-100-10</td>
<td>&nbsp;</td>
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<td>Ketamine</td>
<td>&nbsp;</td>
<td>Hospira</td>
<td>NDC 0409-2051-05</td>
<td>&nbsp;</td>
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<td>High Cholesterol Diet 1%</td>
<td>&nbsp;</td>
<td>Research Diets</td>
<td>C30293</td>
<td>&nbsp;</td>
<tr>
<td>HIgh Cholesterol Diet 0.3%</td>
<td>&nbsp;</td>
<td>Research Diets</td>
<td>C30255</td>
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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.

Watch this Scientific Journal Video about In vivo Near Infrared Fluorescence NIRF Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis at JoVE.co

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

プラークの生物学の2次元血管内分子イメージングのための我々は詳細に新しい近赤外蛍光（NIRF）カテーテルを<em&gt; in vivoで</em&gt;。 NIRFのカテーテルは、プラーク熱心な起動可能とターゲット近赤外蛍光色素の存在に報告することによって、炎症など主要な生物学的プロセスを可視化することができます。カテーテルは臨床工学と電力要件を利用し、ヒトの冠状動脈のアプリケーションを対象としています。以下の調査研究では、小説を利用したマルチモーダルイメージング戦略を説明<em&gt; in vivoで</em&gt;血管内NIRFイメージにカテーテルとタンパク質分解アクティブ炎症ウサギatheromataにおける炎症性プラークを定量化する。

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 ...

in-vivo-near-infrared-fluorescence-nirf-intravascular-molecular

Research

JoVE Journal

Medicine

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

17.8K ビュー.  Harvard Medical School. プラークの生物学の2次元血管内分子イメージングのための我々は詳細に新しい近赤外蛍光（NIRF）カテーテルを in vivoで。 NIRFのカテーテルは、プラーク熱心な起動可能とターゲット近赤外蛍光色素の存在に報告することによって、炎症など主要な生物学的プロセスを可視化することができます。カテーテルは臨床工学と電力要件を利用し、ヒ?...

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

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

医学

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

マウスでのせん断応力誘発アテローム性動脈硬化症をトリガするための頸動脈カフの注入

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration1, 2. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient4. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities9, 10. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations11. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse)12-14. Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans10,11, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

A non-invasive means to evaluate the success of myoblast transplantation is described. The method takes advantage of a unified fusion reporter gene composed of genes whose expression can be imaged with different imaging modalities. Here, we make use of a fluc reporter gene sequence to target cells via bioluminescence imaging.

移植筋前駆細胞を標的とする分子イメージング

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle ...

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite fluorescent in situ elements through the optical fibers, and then record some of the emitted photons, via the same optical fibers. The light source is a laser that sends the exciting light through an element within the fiber bundle and as it scans over the sample, recreates an image pixel by pixel. As this scan is very fast, by combining it with dedicated image processing software, images in real time with a frequency of 12 frames/sec can be obtained.
We developed a technique to quantitatively characterize capillary morphology and function, using a confocal fluorescence videomicroscopy device. The first step in our experiment was to record 5 sec movies in the four quadrants of the tumor to visualize the capillary network. All movies were processed using software (ImageCell, Mauna Kea Technology, Paris France) that performs an automated segmentation of vessels around a chosen diameter (10 &mu;m in our case). Thus, we could quantify the 'functional capillary density', which is the ratio between the total vessel area and the total area of the image. This parameter was a surrogate marker for microvascular density, usually measured using pathology tools.
The second step was to record movies of the tumor over 20 min to quantify leakage of the macromolecular contrast agent through the capillary wall into the interstitium. By measuring the ratio of signal intensity in the interstitium over that in the vessels, an 'index leakage' was obtained, acting as a surrogate marker for capillary permeability.

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

In this paper, we present a method to analyze tumor microvessels in vivo using dynamic contrast-enhanced fluorescence videomicroscopy. Two quantitative parameters were acquired: functional capillary density reflecting the vascularity of the tumor, and index leakage reflecting the leakiness of the endothelial wall.

蛍光共焦点ビデオ顕微鏡を使用して腫瘍血管新生のin vivoイメージング

Fibered confocal fluorescence in vivo imaging with a fiber optic bundle uses the same principle as fluorescent confocal microscopy. It can excite ...

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

ヒューマン<em&gt;生体外</em&gt;動脈硬化性プラークのモデルは、病変生物学を研究する

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery (BCS). Optical imaging, in particular near-infrared fluorescence (NIRF) imaging, might reduce the frequency of positive surgical margins following BCS by providing the surgeon with a tool for pre- and intraoperative tumor localization in real-time. In the current study, the potential of NIRF-guided BCS is evaluated using tissue-simulating breast phantoms for reasons of standardization and training purposes.
Breast phantoms with optical characteristics comparable to those of normal breast tissue were used to simulate breast conserving surgery. Tumor-simulating inclusions containing the fluorescent dye indocyanine green (ICG) were incorporated in the phantoms at predefined locations and imaged for pre- and intraoperative tumor localization, real-time NIRF-guided tumor resection, NIRF-guided evaluation on the extent of surgery, and postoperative assessment of surgical margins. A customized NIRF camera was used as a clinical prototype for imaging purposes.
Breast phantoms containing tumor-simulating inclusions offer a simple, inexpensive, and versatile tool to simulate and evaluate intraoperative tumor imaging. The gelatinous phantoms have elastic properties similar to human tissue and can be cut using conventional surgical instruments. Moreover, the phantoms contain hemoglobin and intralipid for mimicking absorption and scattering of photons, respectively, creating uniform optical properties similar to human breast tissue. The main drawback of NIRF imaging is the limited penetration depth of photons when propagating through tissue, which hinders (noninvasive) imaging of deep-seated tumors with epi-illumination strategies.

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

乳がん手術における潜在的近赤外蛍光イメージングアプリケーションを評価するための組織シミュレーファントム

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

マウス内視鏡検査のため<em&gt;インビボ</em&gt;発がんと腸の評価のマルチモーダルイメージング創傷治癒および炎症

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.
First we describe how to generate subcutaneous tumors in mice, using antigen-negative cell lines as negative controls and antigen-positive cells as positive controls in the same mice for intraindividual comparison. We outline how to administer intravenously near-infrared fluorophore labelled (AlexaFluor680) antigen-specific nanobodies and conventional antibodies. In vivo imaging was performed with a small-animal NIRF-Imaging system. After the in vivo imaging experiments the mice were sacrificed. We then describe how to prepare the tumors for parallel ex vivo analyses by flow cytometry and fluorescence microscopy to validate in vivo imaging results.
The use of the near-infrared fluorophore labelled nanobodies allows for non-invasive same day imaging in vivo. Our protocols describe the ex vivo quantification of the specific labeling efficiency of tumor cells by flow cytometry and analysis of the distribution of the antibody constructs within the tumors by fluorescence microscopy. Using near-infrared fluorophore labelled probes allows for non-invasive, economical in vivo imaging with the unique ability to exploit the same probe without further secondary labelling for ex vivo validation experiments using flow cytometry and fluorescence microscopy.

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional antibodies.

ナノボディ及び抗体ベースの検証<em&gt;インビボ</em使用してマウスにおける&gt;腫瘍異種移植NIRFイメージング実験<em&gt;エクスビボ</em&gt;フローサイトメトリーおよび顕微鏡

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

直接脳血栓塞栓を可視化するための複合近赤外蛍光イメージングおよびマイクロコンピュータ断層撮影

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

マウスにおける加速アテローム性動脈硬化症の誘導:ワイヤー傷害モデル

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...