1。造影剤の調製<ol><li> FITC-デキストラン70kDaのために、注射用量は500 mg / kgの（FITC-デキストラン10mgを体重20gのマウスについての生理食塩水を0.1mlに希釈した）である。 </li><li>エージェントは、光に長すぎるさらしてはならない。漂白を回避するために、アルミホイルでチューブを覆うことが推奨される。 </li></ol> 2。麻酔<ol><li>マウスをキシラジンの1:4の混合物の腹腔内注射により麻酔した（ロンプン2％、バイエル、ピュトー、フランス）とケタミン（ケタミン500、Virbacの、カロス、フランス）、それぞれ66 mg / kgで264 mg / kgの20グラムのマウス用。 </li></ol> 3。関心のある器官の調製<ol><li>私たちは、（例えば、皮下腫瘍の上）興味のある場所でマウスを剃毛。動物の毛は、多くの場合、白の自動蛍光性である。ときは黒、それが光を吸収する。 </li><li>画像化される器官に面する皮膚を切開した。それはbleedinまで待つことが重要ですGは、それ以外の場合は、血液中に漏出し、画像を汚染する、造影剤を注入する前に停止しました。 </li></ol> 4。買収<ol><li>造影剤は、頚静脈又は尾静脈のいずれかを介して注射した。蛍光造影剤の非存在下で観察される器官でないか、またはほとんどバックグラウンド信号が存在しない。 </li><li>プローブは、画像化される器官の前方に配置した。我々の研究では、これは腫瘍であった。 </li><li>レーザーは、腫瘍を照射し、毛細血管の蛍光を見るためにスイッチを入れた。 </li><li>腫瘍は毛細血管網を可視化するために記録しながら非常にゆっくり移動プローブを移動させることにより手動で調査した。これは、着実に手を維持することが重要であり、この技術はほとんど経験が必要です。我々の研究では、この最初のステップは、機能的な毛細管密度の定量化を可能にした。 </li><li>第二段階は、時間の経過とともにダイナミックな買収だった。この研究のために、我々は70kDaのFITC-デキストランを使用。ほとんどの正常臓器には間質漏れはありませんが、腫瘍であります。 （我々の場合のように）時間をかけて同じ場所の画像を取得するためには、関心領域にプローブを維持するためにシステムを設定することが重要です。これは、プローブを保持するために手作りのサポートを使用することにより、前記プローブの先端に超音波ゲルのビットを配置することによって行った。記録する前に、時間は、腫瘍と接触して配置されているプローブを安定化するために費やされた。位置を固定した後、マウスの呼吸のために最小限の動きがありました。レーザは毛細血管漏出の存在を検出するために20分間3画像30秒毎に記録するためにスイッチを入れた。これは、造影剤の退色を低減するために、各記録の間にオフにされた。 </li><li>我々の実験において、マウスを腫瘍の組織学的分析のための手順の終了時に屠殺した。 </li></ol>

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fundamental+concepts+of+the+angiogenic+process.">Fundamental concepts of the angiogenic process.</a> Curr Mol Med. 3 (7), 643-651 (2003).</li><li>Laemmel, E., Genet, M., Le Goualher, G., Perchant, A., Le Gargasson, J. F., Vicaut, E. <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>Charnley, N., Donaldson, S., Price, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+angiogenesis.">Imaging angiogenesis.</a> Methods Mol Biol. 467, 25-51 (2009).</li><li>Faye, N., Fournier, L., Balvay, D., Taillieu, F., Cuenod, C., Siauve, N., Clement, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+contrast+enhanced+optical+imaging+of+capillary+leakage.">Dynamic contrast enhanced optical imaging of capillary leakage.</a> Technol Cancer Res Treat. 10 (1), 49-57 (2011).</li><li>Kurose, I., Kubes, P., Wolf, R., Anderson, D. C., Paulson, J., Miyasaka, M., Granger, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+nitric+oxide+production.+Mechanisms+of+vascular+albumin+leakage.">Inhibition of nitric oxide production. Mechanisms of vascular albumin leakage.</a> Circ Res. 73 (1), 164-171 (1993).</li><li>Faye, N. F. L., Balvay, D., Thiam, R., Orliaguet, G., Clement, O., Dewachter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macromolecular+capillary+leakage+is+involved+in+the+onset+of+anaphylactic+hypotension.">Macromolecular capillary leakage is involved in the onset of anaphylactic hypotension.</a> Anesthesiology. , (2012).</li><li>Faye, N., Fournier, L., Balvay, D., Thiam, R., Orliaguet, G., Clement, O., Dewachter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Macromolecular Capillary Leakage Is Involved in the Onset of Anaphylactic Hypotension. Anesthesiology. 117 (5), 1072-1079 (2012).</li><li>Tozer, G. M., Kanthou, C., Baguley, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+tumour+blood+vessels.">Disrupting tumour blood vessels.</a> Nat Rev Cancer. 5 (6), 423-435 (2005).</li><li>Ntziachristos, V., Schellenberger, E. A., Ripoll, J., Yessayan, D., Graves, E., Bogdanov, A., Josephson, L., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+antitumor+treatment+by+means+of+fluorescence+molecular+tomography+with+an+annexin+V-Cy5.5+conjugate.">Visualization of antitumor treatment by means of fluorescence molecular tomography with an annexin V-Cy5.5 conjugate.</a> Proc Natl Acad Sci U S A. 101 (33), 12294-12299 (2004).</li><li>Cuccia, D. J., Bevilacqua, F., Durkin, A. J., Merritt, S., Tromberg, B. J., Gulsen, G., Yu, H., Wang, J., Nalcioglu, O. <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+quantification+of+optical+contrast+agent+dynamics+in+rat+tumors+by+use+of+diffuse+optical+spectroscopy+with+magnetic+resonance+imaging+coregistration.">In vivo quantification of optical contrast agent dynamics in rat tumors by use of diffuse optical spectroscopy with magnetic resonance imaging coregistration.</a> Appl Opt. 42 (16), 2940-2950 (2003).</li></ol>

我々は、開示することは何もありません。

腫瘍微小循環の研究は、腫瘍成長、播種および治療​​1への応答の病態生理学を理解する上で必要不可欠となっている。光学イメージングは​​、蛍光造影剤を用いて毛細血管を観察し、形態学的（機能的毛細血管密度）および機能（インデックス漏れ）のパラメータを定量化するために使用することができる技術の一つである。 我々は本研究で使用した蛍光?...

血管新生は、既存の血管からの新たな血管の形成を含む複雑なプロセス1である。細動脈、毛細血管、および細静脈からなる組織の微小循環における病理学的変化は、例えば、癌、炎症、または糖尿病などの疾患の広い範囲に関与している。これは、定量的に微小血管の構造および機能を評価するための方法を開発することが不可欠である。画像化は、リアルタイムでおよびインビボで 、又は非マイクロ侵襲的に微小血管の研究を可能にし、同じ動物において2時間にわたって反復測定。 現在、ダイナミック造影（DCE）イメージング3は、一般的に、組織の微小循環を評価するために使用される。ダイナミック造影画像化を経時的に静脈内注射したトレーサーの生体内分布を、以下の技術である。この買収により、定量的なパラメータは、組織の血管新生を反映して抽出することができる。 DCEイメージング最も頻繁にCT、MRIまたは超音波で使用されている。しかし、これらのイメージング技術は、特定の実験装置を用いた以外、その解像度は、ほとんどの場合、巨視的なままであるため、微小血管の直接観察することはできません。 本論文では、繊維質共焦点ビデオ顕微鏡で、ダイナミック造影光学イメージングを用いて微細なスケールでそして生体内で腫瘍血管系を研究することを提案する。我々は、その分子量および組織の内皮細胞の特性を研究し4によれば、間質への内皮バリアを介して血管又は漏れ内で排他的に残る高分子造影剤（FITC-デキストラン）を使用した。これが漏れて間質に蓄積することにより、正確に血管を描出することにより、両方の微小血管構造の研究、および毛細血管の透過性を可能にした。

<table border="1">
<tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Insulin serynge 
 Myjector 1ml 
 29G</td>
 <td>Terumo Europe </td>
 <td>BS-05M2913</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorescein isothiocyanate-dextran 70 kDa </td>
 <td>Sigma-Aldrich</td>
 <td>01619HH</td>
 <td>100 mg/mL 
 diluted in saline</td>
 </tr>
 <tr>
 <td>Fibered confocal videomicroscopy</td>
 <td>Cellvizio - MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Calibration and Cleaning Kit for LEICAFCM1000</td>
 <td>Leica Microsystems</td>
 <td>LSU-488</td>
 <td>Store at 4 &deg;C</td>
 </tr>
 <tr>
 <td>Probe ProFlexTM Z</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Mosaicing software</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Vessel detection software</td>
 <td>MaunaKea Technologies</td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

in vivo imaging of tumor angiogenesis using fluorescence confocal videomicroscopy

光ファイバ束と繊維質共焦点蛍光in vivoイメージング、蛍光共焦点顕微鏡と同じ原理を使用しています。それは、光ファイバを介してその場要素で蛍光を励起し、同じ光ファイバを介して 、放出された光子のいくつかを記録することができます。光源は、ファイバ束内の要素を介して励起光を送信し、それが試料上を走査するように、画素によって画像画素を再現レーザである。このスキャンは、専用の画像処理ソフトウェアと組み合わせることにより、非常に高速であるように、12フレーム/秒の周波数にリアルタイムで画像を得ることができる。 我々は、共焦点蛍光ビデオ顕微鏡装置を用いて、定量的にキャピラリーの形態及び機能を特徴付けるための技術を開発した。我々の実験の最初のステップは、毛細血管網を視覚化するために、腫瘍の4象限に5秒の動画を撮影することでした。すべての映画は、ソフトウェア（Iを用いて処理したmageCell、選ばれた直径（ここでは10ミクロン）の周囲に血管の自動化されたセグメント化を行い、マウナ·ケア·テクノロジー、パリフランス）。したがって、我々は、総血管面積画像の総面積との比である「機能毛細血管密度を &#39;、定量化できた。このパラメータは、通常、病理学ツールを使用して測定し、微小血管密度の代理マーカーであった。 第二段階は、間質への毛細血管壁を介して高分子造影剤の漏出を定量化するために20分間かけて腫瘍の映画を記録することであった。血管におけるその上間質におけるシグナル強度の比を測定することによって、「インデックスリーク &#39;が毛細血管の透過性のための代理マーカーとして働く得た。

収集したデータを用いて、定量的に微小循環を反射異なるパラメータを分析することができる。 我々は、（高分子蛍光造影剤フルオレセインイソチオシアネート-デキストランの注射後、 インビボ繊維質共焦点蛍光ビデオ顕微鏡システム（Cellvizio、マウナケア技術、パリ、フランス2）を用いてのbalb-cマウスに移植された結腸腫瘍の末梢血管ネットワーク<em...

Watch this Scientific Journal Video about In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy at JoVE.com

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

本論文では、ダイナミック造影蛍光ビデオ顕微鏡を用いてインビボで腫瘍の微小血管を分析する手法を提案する。二つの定量的なパラメータを取得した：機能性腫瘍の血管分布を反映した毛細血管密度、および内皮の壁の漏出インデクス反映漏れ。

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 ...

in-vivo-imaging-tumor-angiogenesis-using-fluorescence-confocal

Research

JoVE Journal

Medicine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

10.6K ビュー.  Université Paris Descartes Sorbonne Paris Cité, INSERM UMR-S970. 本論文では、ダイナミック造影蛍光ビデオ顕微鏡を用いてインビボで腫瘍の微小血管を分析する手法を提案する。二つの定量的なパラメータを取得した：機能性腫瘍の血管分布を反映した毛細血管密度、および内皮の壁の漏出インデクス反映漏れ。 

ビデオ: In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

マウスモデルにおける乳がんの脳転移の腫瘍の低酸素動態の in vivo生物発光イメージングに

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

磁気共鳴イメージング、体積CT画像と超音波を用いた乳がんの骨転移のヌードラットモデルにおける血管新生のマルチモーダルイメージング

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

ヒアルロン酸ナトリウムの網膜下注入によるげっ歯類における網膜剥離モデル

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

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. ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

蛍光SAPC-DOPSナノベシクルを用いて脳腫瘍、関節炎のin vivo光イメージング中

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

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 ...

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with distinct functions and gene expression profiles. The roles of microglial activation in health, injury and disease remain incompletely understood due to their dynamic and complex regulation in response to changes in their microenvironment. Thus, it is critical to non-invasively monitor and analyze changes in microglial activation over time in the intact organism. In vivo studies of microglial activation have been delayed by technical limitations to tracking microglial behavior without altering the CNS environment. This has been particularly challenging during chronic neurodegeneration, where long-term changes must be tracked. The retina, a CNS organ amenable to non-invasive live imaging, offers a powerful system to visualize and characterize the dynamics of microglia activation during chronic disorders.
This protocol outlines methods for long-term, in vivo imaging of retinal microglia, using confocal ophthalmoscopy (cSLO) and CX3CR1GFP/+ reporter mice, to visualize microglia with cellular resolution. Also, we describe methods to quantify monthly changes in cell activation and density in large cell subsets (200-300 cells per retina). We confirm the use of somal area as a useful metric for live tracking of microglial activation in the retina by applying automated threshold-based morphometric analysis of in vivo images. We use these live image acquisition and analyses strategies to monitor the dynamic changes in microglial activation and microgliosis during early stages of retinal neurodegeneration in a mouse model of chronic glaucoma. This approach should be useful to investigate the contributions of microglia to neuronal and axonal decline in chronic CNS disorders that affect the retina and optic nerve.

In Vivo Dynamics of Retinal Microglial Activation During Neurodegeneration: Confocal Ophthalmoscopic Imaging and Cell Morphometry in Mouse Glaucoma

Microglia activation and microgliosis are key responses to chronic neurodegeneration. Here, we present methods for in vivo, long-term visualization of retinal CX3CR1-GFP+ microglial cells by confocal ophthalmoscopy, and for threshold and morphometric analyses to identify and quantify their activation. We monitor microglial changes during early stages of age-related glaucoma.

共焦点検眼鏡イメージングとマウス緑内障における細胞の形態計測：神経変性中に網膜ミクログリアの活性化のインビボダイナミクス

Microglia, which are CNS-resident neuroimmune cells, transform their morphology and size in response to CNS damage, switching to an activated state with ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

共焦点光学イメージングによるローズベンガル光血栓<em&gt;インビボ</em&gt;：単一の容器ストロークのモデル

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...