1. Herstellung des Kontrastmittels <ol><li> Für FITC-Dextran 70 kDa, die injizierten Dosis ist 500 mg / kg (10 mg FITC-Dextran in 0,1 ml Kochsalzlösung für eine Maus mit einem Gewicht von 20 g verdünnt). </li><li> Das Mittel sollte nicht zu lange ausgesetzt werden, um Licht. Bleich zu vermeiden, empfiehlt es sich, das Röhrchen mit Aluminiumfolie abzudecken. </li></ol> 2. Anästhesie <ol><li> Mäuse wurden durch eine intraperitoneale Injektion einer Mischung von 1:4 von Xylazin (Rompun 2%, Bayer, Puteaux, Frankreich) und Ketamin (Ketamin 500, Virbac, Carros, Frankreich), die jeweils 66 mg / kg und 264 mg / kg für eine 20 g-Maus. </li></ol> 3. Vorbereitung der Orgel in der Nähe <ol><li> Wir rasierte Mäuse am Ort von Interesse (z. B. über einen subkutanen Tumor). Tier Haar ist oft, wenn Autofluoreszenz weiß. Wenn schwarz, absorbiert es Licht. </li><li> Die Haut vor der Orgel, die abgebildet werden wurde eingeschnitten. Es ist wichtig, zu warten, bis die bleeding hat vor der Injektion des Kontrastmittels gestoppt, sonst wird es im Blut austreten und verunreinigen das Bild. </li></ol> 4. Erwerb <ol><li> Das Kontrastmittel wurde entweder durch die Halsschlagader oder die Schwanzvene injiziert. Es gibt keine oder nur eine geringe Hintergrundsignal in dem in Abwesenheit eines fluoreszierenden Kontrastmittels beobachtet Organ. </li><li> Die Sonde wurde vor dem abzubildenden Organ platziert. In unserer Studie war der Tumor. </li><li> Der Laser eingeschaltet, um den Tumor zu beleuchten und sehen die Fluoreszenz in den Kapillaren. </li><li> Der Tumor wurde manuell durch Bewegen der Sonde in einem sehr langsamen Bewegung während der Aufnahme, um die Kapillare Netzwerk visualisieren erforscht. Es ist wichtig, eine ruhige Hand zu halten, und diese Technik erfordert ein wenig Erfahrung. In unserer Studie erlaubt dieser erste Schritt Quantifizierung der funktionellen Kapillardichte. </li><li> Der zweite Schritt war die dynamische Erfassung über die Zeit. Für diese Studie verwendeten wir ein 70 kDa-Dextran-FITC. Es gibt keine interstitielle Leckage in den meisten normalen Organen, aber es ist in Tumoren. Um Bilder von der gleichen Stelle über die Zeit (wie in unserem Fall) zu erwerben, ist es wichtig, ein System einzurichten, um die Sonde auf dem Gebiet von Interesse aufrecht zu erhalten. Dies wurde durch eine handgefertigte Unterstützung, um die Sonde zu halten gemacht, und indem man ein bisschen Ultraschall-Gel auf der Spitze der Sonde. Vor der Aufnahme wurde Zeit für die Stabilisierung der Sonde in Kontakt mit dem Tumor platziert. Sobald die Position gesichert war, gab es nur minimale Bewegung aufgrund der Maus die Atmung. Der Laser eingeschaltet, um Bilder 3 alle 30 s für 20 min aufgezeichnet, um die Anwesenheit kapillares Leck zu erfassen. Es wurde zwischen jeder Aufnahme drehte sich um Kontrastmittel Bleich reduzieren. </li><li> In unserem Experiment wurden die Mäuse am Ende des Verfahrens für die histologische Analyse von Tumoren geopfert. </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>

Wir haben nichts zu offenbaren.

Die Untersuchung der Tumormikrozirkulation unverzichtbar geworden für das Verständnis der Pathophysiologie von Tumorwachstum, Verbreitung und Ansprechen auf die Therapie ein. Optisches Abbildungs ​​ist eine der Techniken, die verwendet werden können, um die Kapillaren zu beobachten unter Verwendung eines fluoreszierenden Kontrastmittels und morphologischen (funktionelle Kapillardichte) und funktionellen (Index Leckage) Parameter zu quantifizieren. Die Fluoreszenz Mikroskopi...

Angiogenese ist ein komplexer Prozess 1, die die Bildung neuer Blutgefäße aus bereits vorhandenen Fahrzeugen beinhaltet. Pathologische Veränderungen im Gewebe Mikrozirkulation, von Arteriolen, Kapillaren und Venen besteht, sind in einer großen Palette von Krankheiten, wie Krebs, Entzündungen oder Diabetes in Verbindung gebracht. Es ist daher wichtig, Methoden zu quantitativ beurteilen Mikrogefäßstruktur und-funktion zu entwickeln. Bildgebung ermöglicht die Untersuchung von Mikrogefäßen in einem nicht-oder mikroinvasive Weise in Echtzeit und in vivo und wiederholten Messungen im Laufe der Zeit in dem gleichen Tier 2. Derzeit wird die dynamische kontrastverstärkte (DCE) Bild 3 häufig verwendet, um Gewebe-Mikrozirkulation zu beurteilen. Dynamischen kontrastverstärkten Bildgebung ist eine Technik, die im Laufe der Zeit die Bioverteilung eines Tracers intravenös injiziert folgt. Von dieser Übernahme kann quantitative Parameter extrahiert reflektierende Gewebe Gefäß werden. DCE-Bildgebungwurde am häufigsten mit CT, MRT oder Ultraschall verwendet werden. Allerdings sind diese bildgebenden Verfahren nicht zulassen, dass die direkte Betrachtung der Mikrogefäße, da ihre Auflösung, anders als bei der Verwendung von bestimmten experimentelle Geräte bleibt meist makroskopische. In diesem Papier, schlagen wir vor, das Gefäßsystem des Tumors auf der mikroskopischen Skala und in vivo-Studie mit dynamischen kontrastverstärkten optischen Bildgebung, mit fibered konfokalen Videomikroskopie. Wir verwendeten eine makromolekulare Kontrastmittel (FITC-Dextran), die ausschließlich in Behältern oder Lecks durch die endotheliale Schranke in das Interstitium bleibt, nach seinem Molekulargewicht und die Eigenschaften des Endothels der untersuchten Gewebe 4. Dies ermöglichte die Untersuchung der beiden Mikrogefäßstruktur, durch die korrekte Abgrenzung Gefäße und Durchlässigkeit der Kapillaren, durch auslaufende und der sich in das Interstitium.

<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

Fibered konfokalen Fluoreszenz in-vivo-Bildgebung mit einem Faseroptikbündel verwendet das gleiche Prinzip wie Fluoreszenz konfokalen Mikroskopie. Es kann Fluoreszenz-in situ-Elemente durch die optischen Fasern zu erregen, und dann erfassen einige der emittierten Photonen, über den gleichen optischen Fasern. Die Lichtquelle ist ein Laser, der das Anregungslicht durch ein Element innerhalb des Faserbündels sendet und wie es über die Probe scannt, erstellt ein Bild Pixel für Pixel. Da diese Überprüfung ist sehr schnell, indem sie mit speziellen Bildverarbeitungssoftware können die Bilder in Echtzeit mit einer Frequenz von 12 Bildern / sec erhalten werden. Wir entwickelten eine Technik, um quantitativ zu charakterisieren Kapillare Morphologie und Funktion mit einem konfokalen Fluoreszenz-Videomikroskopie Gerät. Der erste Schritt in unserem Experiment wurde auf 5 sec Filme aufnehmen in den vier Quadranten des Tumors, die kapillare Netzwerk visualisieren. Alle Filme wurden mit einer Software verarbeitet (ImageCell, Mauna Kea Technologie, Paris), die eine automatische Segmentierung von Schiffen zu einem gewählten Durchmesser (10 um in unserem Fall) führt. So konnten wir die funktionalen Kapillardichte &quot;, die das Verhältnis zwischen der gesamten Gefäßfläche und der Gesamtfläche des Bildes zu quantifizieren. Dieser Parameter war ein Surrogatmarker für die mikrovaskuläre Dichte, mit der Pathologie Tools in der Regel gemessen. Der zweite Schritt war, um Filme des Tumors aufnehmen über 20 Minuten, um ein Auslaufen der makromolekularen Kontrastmittel durch die Kapillarwand in das Interstitium zu quantifizieren. Durch Messung des Verhältnisses der Signalintensität im Interstitium gegenüber der in den Behältern wurde eine Index Leckage &quot;erhalten wird, als Surrogatmarker für die Durchlässigkeit der Kapillaren wirkt.

Verwendung der gesammelten Daten, könnten wir verschiedene Parameter quantitativ zu analysieren reflektierenden Mikrozirkulation. Wir untersuchten in vivo die periphere Gefäßnetz eines Darmtumors in Balb-c-Mäuse mit einem konfokalen Fluoreszenzvideomikroskopie fibered System (Cellvizio, Maunakea Technologie, Paris, France 2), nach der Injektion eines makromolekularen fluoreszierenden Kontrastmittels Fluoresceinisothiocyanat-Dextran (implantiert FITC-Dextran) mit einem...

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

In diesem Beitrag stellen wir eine Methode, um Tumormikrogefäßen in vivo Analyse mit dynamischen Kontrastmittel-Fluoreszenz-Videomikroskopie. Zwei quantitative Parameter wurden erworben: funktionelle Kapillardichte was die Durchblutung des Tumors und Leckage Index spiegelt die Undichtigkeit der endothelialen Wand.

In-vivo-Imaging der Tumorangiogenese mit Fluoreszenz-Videomikroskopie Konfokale

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

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Research

JoVE Journal

Medicine

In vivo Imaging of Tumor Angiogenesis using Fluorescence Confocal Videomicroscopy

10.6K Aufrufe.  Université Paris Descartes Sorbonne Paris Cité, INSERM UMR-S970.  In diesem Beitrag stellen wir eine Methode, um Tumormikrogefäßen in vivo Analyse mit dynamischen Kontrastmittel-Fluoreszenz-Videomikroskopie. Zwei quantitative Parameter wurden erworben: funktionelle Kapillardichte was die Durchblutung des Tumors und Leckage Index spiegelt die Undichtigkeit der endothelialen Wand. 

Serie: 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.

In vivo Near Infrared Fluorescence (NIRF) intravaskuläre Molecular Imaging von entzündlichen Plaque, einen multimodalen Ansatz, um Imaging der Atherosklerose

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

Forschung

Medizin

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.

In-vivo-Biolumineszenz-Bildgebung von Tumorhypoxie Dynamics of Breast Cancer Hirnmetastasen in einem Mausmodell

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.

Multimodale Bildgebung von Angiogenese in einem Nude Rattenmodell der Brustkrebs Knochenmetastasen mittels Magnetresonanztomographie, volumetrische Computertomographie und Ultraschall

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.

A Novel Hochauflösende<em&gt; In vivo</em&gt; Bildtechnik, um die Dynamik der intrakranielle Strukturen zu Tumorwachstum und Therapeutics Studieren

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.

Modell Netzhautablösung in Nagetiere durch subretinale Injektion von Natriumhyaluronat

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.

In-vivo-19 F-MRT für die Zellverfolgung

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.

In Vivo Optical Imaging von Hirntumoren und Arthritis mit fluoreszierenden SapC-DOPS Nanovesicles

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.

Validierung der Nanobody und Antikörper-basierte<em&gt; In-vivo-</em&gt; Tumorxenotransplantat NIRF-Bildgebung Experimente bei Mäusen verwenden<em&gt; Ex vivo</em&gt; Durchflusszytometrie und Mikroskopie

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.

In Vivo Dynamics of Retinal Mikrogliaaktivierung Während Neurodegeneration: Confocal Ophthalmoskopische Imaging and Cell Morphometrie in Maus Glaucoma

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.

Rose Bengal Photothrombosis von konfokalen optischen Imaging<em&gt; In Vivo</em&gt;: Ein Modell des Einzelfahrzeug Stroke

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