倫理声明：すべての実験は、施設内動物管理使用委員会によって承認された。動物のケアは施設のガイドラインに従ってた。 ICR雌マウス（7から8週齢、25〜30グラム）はテルアビブ大学医学部のサックラー学部、エアコン、光制御動物施設に収容した。用語で、動物を麻酔の過剰投与で安楽死させた。 1. Fibred共焦点蛍光顕微鏡（FCFM）校正<ol><li>デバイスの電源を入れます。 </li><li>マイクロプローブ（mini0 / 30）を接続します。 </li><li>製造元の指示に従ってデバイスのキャリブレーション。 </li></ol> 2.マウスイメージングのための準備<ol><li>両方のKetaset（100mg / kg）およびXYL-M2（6ミリグラム/ kg）を皮下注射することにより麻酔。つま先ピンチに無反応によって適切な麻酔を確認します。 </li><li>するために、脚の付け根下の皮膚を切開<html>大腿動脈血管を明らかにする。切開以下の生理食塩水で湿った切開部位にしてください。 </li><li>バッグ（または手袋）を使用して尾を熱し、約30秒間（触れないほど熱くない）暖かい水で満たされた。尾静脈に注射針（30 G、1/2インチ）を挿入し、これに1mlシリンジを取り付けることにより、FITCデキストラン（造影剤）の生理食塩水または化学療法剤のいずれかの投与のための静脈内（IV）シャントを準備。静脈が生理食塩水を注入して開いていることを確認。 注：FITCデキストランのIV投与（高分子量; 100μL; 10 mg / mlの、2000 kDa）がFCFMによる大腿骨微小血管系を可視化が容易になります。ドキソルビシン（100μlを、8 mg / kgを、アドリアマイシン）又は生理食塩水は、後述予熱した尾静脈にIVで投与される。 </li><li>ポリスチレンステージ上に仰向けにマウスを置きます。パッドにマウスを固定し、外科ダクトテープを使用して位置を維持する。 </li></ol>中およびドキソルビシンまたは生理食塩水を投与した後のFCFMによる大腿血管のタイトル &quot;&gt; 3。イメージング注：（1）マイクロプローブ（mini0 / 30）本研究で使用した繊維質の共焦点顕微鏡は、二つのユニットから構成されている。 （2）レーザスキャニングユニット（LSU-488;波長488nm）。 <ol><li> LSU 488 nmの波長のレーザーを使用して分析し、すべての時間のラップを実行します。 注記：メインユニット検出器をろ過検出する（500から650 nm）を発する蛍光を。取得した画像は、その後、再構成及び12フレーム/秒の速度で表示されます。 </li><li>針から慎重に注射器を外し、FITCデキストランを含む新しいシリンジを取り付けます。 FITCデキストランの（IV）​​100μLを管理します。 </li><li>対応する画像を得るために、z軸調整後、ビューの適切なフィールドへのマイクロプローブ（mini0 / 30）にシフトして固定する。明確な集束信号がVISIになるまでフェードアウトするための最初のシグナルを待つBLE。 </li><li>短い安定化期間（〜30秒）のベースラインの血流を記録します。その後、ドキソルビシンまたは生理食塩水のいずれかを含む、針に別のシリンジを接続します。 IV100μlのドキソルビシンまたは生理食塩水を投与し。 </li><li> 20分間連続的に注入FITC-デキストランの流れを監視します。 FCFM関連ソフトウェアでは、血管を測定するために、上部のルーラー上の直径ボタンを使用して分類し、それらのような小さな（&lt;15ミクロン）または大（&gt; 15ミクロン）。 </li><li>麻酔過剰摂取で動物を安楽死させる。 </li></ol>

<ol>
	<li>Chow, A. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthracyclines+cause+endothelial+injury+in+pediatric+cancer+patients:+a+pilot+study.">Anthracyclines cause endothelial injury in pediatric cancer patients: a pilot study.</a> J Clin Oncol. 24 (6), 925-928 (2006).</li><li>Nuver, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+chemotherapy-induced+cardiovascular+changes+in+patients+with+testicular+cancer.">Acute chemotherapy-induced cardiovascular changes in patients with testicular cancer.</a> J Clin Oncol. 23 (36), 9130-9137 (2005).</li><li>Vos, F. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+effects+of+cytotoxics:+balance+between+desired+and+unwanted+effects.">Endothelial cell effects of cytotoxics: balance between desired and unwanted effects.</a> Cancer Treat Rev. 30 (6), 495-513 (2004).</li><li>Kerbel, R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term='Accidental'+anti-angiogenic+drugs.+anti-oncogene+directed+signal+transduction+inhibitors+and+conventional+chemotherapeutic+agents+as+examples.Eur.">'Accidental' anti-angiogenic drugs. anti-oncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples.Eur.</a> J Cancer. 36 (10), 1248-1257 (2000).</li><li>Soultati, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+vascular+toxicity+from+chemotherapeutic+agents:+preclinical+evidence+and+clinical+implications.">Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications.</a> Cancer Treat Rev. 38 (5), 473-483 (2012).</li><li>Tempelhoff, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+coagulation+during+adjuvant+epirubicin/cyclophosphamide+chemotherapy+in+patients+with+primary+operable+breast+cancer.">Blood coagulation during adjuvant epirubicin/cyclophosphamide chemotherapy in patients with primary operable breast cancer.</a> J Clin Oncol. 14 (9), 2560-2568 (1996).</li><li>Ben Aharon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+vascular+toxicity--targeting+potential+pathways+may+reduce+procoagulant+activity.">Doxorubicin-induced vascular toxicity--targeting potential pathways may reduce procoagulant activity.</a> PLoS One. 8 (9), e7515 (2013).</li><li>Laemmel, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibered+confocal+fluorescence+microscopy+(Cell-viZio)+facilitates+extended+imaging+in+the+field+of+microcirculation.+A+comparison+with+intravital+microscopy.">Fibered confocal fluorescence microscopy (Cell-viZio) facilitates extended imaging in the field of microcirculation. A comparison with intravital microscopy.</a> J Vasc Res. 41 (5), 400-411 (2004).</li><li>Al-Gubory, K. H., Houdebine, L. M. <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+green+fluorescent+protein-expressing+cells+in+transgenic+animals+using+fibred+confocal+fluorescence+microscopy.">In vivo imaging of green fluorescent protein-expressing cells in transgenic animals using fibred confocal fluorescence microscopy.</a> Eur J Cell Biol. 85 (8), 837-845 (2006).</li><li>Bar-Joseph, H., et al. <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+bioimaging+as+a+novel+strategy+to+detect+doxorubicin-induced+damage+to+gonadal+blood+vessels.">In vivo bioimaging as a novel strategy to detect doxorubicin-induced damage to gonadal blood vessels.</a> PLoS One. 6 (9), e23492 (2011).</li><li>Kaushal, V., Kaushal, G. P., Mehta, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+toxicity+of+anthracyclines+on+cultured+endothelial+cells.">Differential toxicity of anthracyclines on cultured endothelial cells.</a> Endothelium. 11 (5-6), 253-258 (2004).</li><li>Kim, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+platelet+cytotoxicity:+a+new+contributory+factor+for+doxorubicin-mediated+thrombocytopenia.">Doxorubicin-induced platelet cytotoxicity: a new contributory factor for doxorubicin-mediated thrombocytopenia.</a> J Thromb Haemost. 7 (7), 1172-1183 (2009).</li><li>Walsh, J., Wheeler, H. R., Geczy, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tissue+factor+on+human+monocytes+by+cisplatin+and+adriamycin.">Modulation of tissue factor on human monocytes by cisplatin and adriamycin.</a> Br J Haematol. 81 (4), 480-488 (1992).</li><li>Kotamraju, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doxorubicin-induced+apoptosis+in+endothelial+cells+and+cardiomyocytes+is+ameliorated+by+nitrone+spin+traps+and+ebselen.+Role+of+reactive+oxygen+and+nitrogen+species.">Doxorubicin-induced apoptosis in endothelial cells and cardiomyocytes is ameliorated by nitrone spin traps and ebselen. Role of reactive oxygen and nitrogen species.</a> J Biol Chem. 275 (43), 33585-33592 (2000).</li><li>Vasquez-Vivar, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+nitric+oxide+synthase-dependent+superoxide+generation+from+adriamycin.">Endothelial nitric oxide synthase-dependent superoxide generation from adriamycin.</a> Biochemistry. 36 (38), 11293-11297 (1997).</li></ol>

化学療法誘発性血管毒性を評価することにより、リアルタイムでの刺激に応答して血管系の動態を可視化が困難であるため困難である。多くの臨床研究が、いくつかの化学療法は、直接血管損傷を引き起こすことを示唆されている、まだこの毒性のメカニズムと特性はまだ解明されていない。私たちはここに8-10説明したように繊維質共焦点蛍光顕微鏡の構成マウスでの化学療法の潜?...

臨床的証拠は、化学療法のいくつかのクラスは、レイノー現象、高血圧、心筋梗塞、脳血管発作、および肝静脈occlusivedisease 1,2によって明らかに血管の病理の多様を引き出すことを示しています。 「「偶発的」抗血管形成薬」が本来そのために3-5のために開発されたが、わずか「担保を課すことにより、腫瘍細胞を排除するように設計されていないが、可能性の血管新生阻害剤として作用する従来の化学療法剤を記載比較的新しい用語である可能な3として正常細胞へのダメージ」。血清バイオマーカーを用いた臨床試験で観察されたように、いくつかの化学療法はvasculo-毒物として暗示されています。これらのうち剤（例えばシクロホスファミドなど）、白金（シスプラチンなど）の化合物およびアントラサイクリン1,2,5-7アルキル化されている。 急性心血管合併症がresulとして発生する可能性があります化学療法によって誘発される血管毒性のトン。彼らは遅く、血管罹患率のリスク増加のためのアテローム性動脈硬化症およびアカウントのような慢性疾患に進行することがある。しかし、取り付け臨床的証拠にもかかわらず、指定された研究の血管毒性のメカニズムを強調し、したがって、それらが与える正確な病因のさらなる解明が保証さの不足がある。 化学療法誘発血管毒性のメカニズムを明らかにすることで主要な課題は、 生体内で血管機能を調査する複雑さに由来する。我々は、ここで血流および船舶の特性を捕捉することができ、マウスにおけるインビボ分子イメージングにおける高分解能のプラットフォームを記述する。リアルタイムで、並びに同じ個体内の時間の期間にわたって、それらを以下：このプラットフォームは、直接処理により誘導される血管作用の検出を容易にする。

<table><tbody><tr><td>general anesthesia</td><td>Fort Dodge Animal Health, IA, USA and Biove Laboratories, France</td><td></td><td>100 mg/kg ketaset and 6 mg/kg XYL-M2</td></tr><tr><td>depilatory cream (Veet)</td><td>ReckittBenckiser, Bristol, UK</td><td></td><td></td></tr><tr><td>30 G, 1/2 inch needle attached to 1 ml syringe</td><td></td><td></td><td></td></tr><tr><td>FITC dextran (10 mg/ml; MW 2,000 kDa)</td><td>Sigma</td><td>FD2000S</td><td>100 &amp;mu;l volume</td></tr><tr><td>Doxorubicin</td><td>Teva, Israel</td><td></td><td>8 mg/kg, Adriamycin</td></tr><tr><td>paclitaxel</td><td>Taro, Israel</td><td></td><td>1.2 mg/kg, Medexel</td></tr><tr><td>saline</td><td></td><td></td><td></td></tr></tbody></table>

chemotherapy-induced vascular toxicity - real-time in vivo imaging of vessel impairment

化学療法の特定のクラスは、血管の罹患率のリスク増加に患者を素因となることが、長期的な条件に進行することが急性血管の変化を及ぼすことがあります。しかし、取り付け臨床的証拠とはいえ、そこに血管毒性の明確な研究の不足があり、したがって、血管/心臓血管障害の異質グループの病因はまだ解明されていない。また、血管毒性を根底にある機構は、完全に筋細胞傷害を誘導するように関連して、化学療法誘発性の心毒性の原理と異なっていてもよい。我々は、抗癌治療の潜在的な急性血管毒性を評価するために、生体内分子イメージングプラットフォーム 、リアルタイムを確立した。 私たちは、限られた臓器内の血管系を可視化するのに適したマウスでのin vivoで 、高解像度の分子イメージング、および参照BLOのプラットフォームを設定している各個人が自身の対照として役立つのに対し、同じ個人内のOD船。血管壁は、エンド臓器損傷の初期事象かもしれ血管毒性のユニークなメカニズムを表す、ドキソルビシン投与後に損なわれた。本明細書において、繊維質の共焦点蛍光顕微鏡（FCFM）ベースのイメージングの方法は、動物被験体における細胞および細胞下レベルでの生理学的現象を理解するために革新的なモードを提供する、記載されている。

リアルタイムでの生体内連続撮影では、 ここで使用される撮像装置は、脈管構造および化学療法などの種々の刺激に対する応答の可視化を可能にするプローブを備えた共焦点顕微鏡を、繊維質、高精細である。この方法は、深い血管または器官の画像化を容易にすることができるが、それはプローブのための小さな切開を必要とするので、低侵襲性である。?...

Watch this Scientific Journal Video about Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment at JoVE.com

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 ...

chemotherapy-induced-vascular-toxicity-real-time-vivo-imaging-vessel

Research

JoVE Journal

Medicine

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

7.4K ビュー.  Tel Aviv University. 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.

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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

MicroCTを使用して造影血管イメージング

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

医学

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

免疫・感染学

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 Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

新規高分解能<em生体内で&gt;</em腫瘍増殖と治療に頭蓋内構造物の動的応答を研究する&gt;イメージング技術

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

マイクロ流体工学に基づく血管新生ヒト微小腫瘍モデルの準備

Establishing a Physiologic Human Vascularized Micro-Tumor Model for Cancer Research

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical cancer research and therapeutic development.&#160;To overcome this problem, we have developed the vascularized microtumor (VMT), or tumor chip, a microphysiological system that realistically models the complex human tumor microenvironment. The VMT forms de novo within a microfluidic platform by co-culture of multiple human cell types under dynamic, physiological flow conditions. This tissue-engineered micro-tumor construct incorporates a living perfused vascular network that supports the growing tumor mass just as newly formed vessels do in vivo. Importantly, drugs and immune cells must cross the endothelial layer to reach the tumor, modeling in vivo physiological barriers to therapeutic delivery and efficacy. Since the VMT platform is optically transparent, high-resolution imaging of dynamic processes such as immune cell extravasation and metastasis can be achieved with direct visualization of fluorescently labeled cells within the tissue. Further, the VMT retains in vivo tumor heterogeneity, gene expression signatures, and drug responses. Virtually any tumor type can be adapted to the platform, and primary cells from fresh surgical tissues grow and respond to drug treatment in the VMT, paving the way toward truly personalized medicine. Here, the methods for establishing the VMT and utilizing it for oncology research are outlined. This innovative approach opens new possibilities for studying tumors and drug responses, providing researchers with a powerful tool to advance cancer research.

著者スポットライト:トランスレーショナルがん研究のモデルとしてのヒト血管新生微小腫瘍の作成 

This protocol presents a physiologically relevant tumor-on-a-chip model to perform high-throughput basic and translational human cancer research, advancing drug screening, disease modeling, and personalized medicine approaches with a description of loading, maintenance, and evaluation procedures.

がん研究のための生理学的ヒト血管新生微小腫瘍モデルの確立

A lack of validated cancer models that recapitulate the tumor microenvironment of solid cancers in vitro remains a significant bottleneck for preclinical ...

がん研究

マウスにおける腫瘍のガイド下接種とマルチモーダルイメージング

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

著者スポットライト:高解像度生体内イメージングとMRIを統合して、定位放射線治療計画を強化する

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

化学療法誘発性血管毒性 - リアルタイム

要約

さらに動画を探す

この動画の章

MicroCTを使用して造影血管イメージング

乳癌のマウスモデルにおいて腫瘍微小環境における薬剤応答のイメージングを生きる

生きたマウスにおける血管炎症及び血栓形成の間に活性化内皮上に異型血小板、好中球の相互作用のリアルタイムイメージング

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

新規高分解能

がん研究のための生理学的ヒト血管新生微小腫瘍モデルの確立

がん研究のための生理学的ヒト血管新生微小腫瘍モデルの確立

がん研究のための生理学的ヒト血管新生微小腫瘍モデルの確立

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル

トランスレーショナルがん研究における生体内顕微鏡検査と磁気共鳴画像法の併用のための背側Skinfold Window Chamber腫瘍マウスモデル