Wir sind dankbar, dass DRS. Diane Palmieri und Patricia Steeg von NCI für die Bereitstellung MDA-MB231Br Zellen. Wir danken Dr. Ralph Mason, Mr. Jason Reneau und Frau Ramona Lopes für technische und kollegiale Unterstützung. Diese Arbeit wurde zum Teil durch das DOD IDEA Awards W81XWH-08-1 bis 0583 und W81XWH-12-1 bis 0317 unterstützt. MRI-Experimente wurden in der Advanced Imaging Research Center, ein NIH BTRP # P41-RR02584 Anlage durchgeführt und Ultraschall-geführte intrakardiale Injektion wurde mit Visualsonics Vevo 770 unter 1S10RR02564801 durchgeführt.

Alle Tier Verfahren wurden von der Institutional Animal Care und Verwenden Ausschuss der University of Texas Southwestern Medical Center genehmigt. 1. Vorbereitung der Zellen MDA-MB231/Br-GFP <ol><li> Abrufen und Kultur MDA-MB231/Br-GFP die Zellen in DMEM-Medium mit 10% FBS, 1% Glutamin und 1% Penicillin / Streptomycin (freundlicherweise von Dr.. Palmieri und Steeg, NCI). </li><li> Beachten Sie den Zustand der Zellen und Farbe des Mediums, und ersetzen Medium alle 2-3 Tage. </li><li> Trypsinieren und sammeln Sie die Zellen, wenn sie 80% Konfluenz erreicht ist, wie in den Schritten skizziert 1.3.1-1.3.5: <ol><li> Entfernen Sie das alte Medium vollständig und mit 5 ml PBS (1x), um die Zellen vorsichtig waschen. Entfernen Sie die PBS von der Schale. </li><li> 1,5 ml Trypsin in die Schüssel, kippen die Schale vorsichtig, um sicherzustellen, dass alle die Zellen durch Trypsin bedeckt. Setzen Sie die Schale zurück in die Zelle und halten Sie sie Inkubator bei 37 ° C für 1 min. </li><li> Nehmen Sie die Schale von Inkubator und beobachtendie Zellen unter einem optischen Mikroskop, um die Zellen zu gewährleisten lösen sich von der Schale. </li><li> 3 ml Medium, um die Wirkung des Trypsins zu stoppen. Sammeln der Zellmischung in einem Zentrifugenröhrchen. Zentrifugieren der Mischung bei 2.000 UpM für 5 min. </li><li> Entfernen Sie das Medium vorsichtig resuspendieren und in 5 ml Serum-freiem Medium, bis sie homogen. </li></ol></li><li> Zählen geeignete Anzahl von Zellen und Resuspendieren sie in Serum-freiem DMEM-Medium mit einer Endkonzentration von 1,75 × 10 5 Zellen in 100 &amp;mgr; l Volumen. <ol><li> Nehmen Sie 50 ul Zellgemisch und 50 ul Trypanblau. Nach dem Mischen nehmen 10 ul Mischung und vorsichtig auf die Zellzählung Rutsche. Verwenden Sie mehrere Proben, um eine genaue Abschätzung der Zellzahl zu gewährleisten. </li><li> Halten Sie in der Zellzählung Rutsche, ein Ende zu einer Zeit und dem Zellzähler startet automatisch gezählt. Nehmen Durchschnitt aller Zählergebnisse und die Berechnung der Gesamtzellzahl. </li><li> Zentrifugieren Sie die Zellen wieder und wieder zu suspendieren sie indas entsprechende Volumen der serumfreien Medium in einer Endkonzentration von 1,75 × 10 5 Zellen/100 &amp;mgr; l Volumen führen. </li></ol></li><li> Platz Zellen auf Eis vor der intra Injektion. </li></ol> 2. Ultraschall-Imaging-guided Intra Injection <ol><li> Verwenden weiblichen Nacktmäusen (BALB / c nu / nu) zwischen 6-8 Wochen alt. </li><li> Log in dem Abbildungssystem. Initialisieren der Wandler 704 (40 MHz). </li><li> Starten Sie eine neue Studie und füllen Sie die Informationen. </li><li> Anästhesiert (3% isoflurane/100% O 2 in einer Induktionskammer) und während des gesamten Verfahrens über einen Nasenkonus, die Tiere mit Isofluran (2%) in 100% O 2 (1 dm 3 / min) zu halten. Anästhesie wird bestätigt, wenn kein Rückzugsreflex ist mit Zehen Prise beobachtet. </li><li> Stellen Sie die Temperatur der Abbildungstabelle auf 37 ° C. Kleben Sie die narkotisierten Maus zum beheizten Bildgebungstisch in Rückenlage. </li><li> Halten Sie die Ultraschall-Gel bei 37 ° C vor dem IMAlterung. Wenden Sie das Gel auf die Brust der Maus. </li><li> Montieren Sie den Schwinger in die Halterung. Senken Sie den Wandler, bis die gewünschte Bildtiefe erreicht ist. Bewegen der Plattform, bis die linke Herzkammer mit der aufsteigenden Aorta als orientierungs (1A) identifiziert. Sperren Sie die Bühne, wenn eine klare Sicht des linken Ventrikels wird visualisiert. </li><li> Zeichnen 100 ul Zellgemisch in eine 1 ml Spritze mit einer 22 G-Nadel. Platzieren und befestigen Sie die Spritze an der Spritze montieren. </li><li> Bewegen Sie die Spritze nach vorn in Richtung der Maus Brust und vorsichtig bewegen sie von Seite zu Seite, bis die Nadelspitze in der Bildfeld (vor dem Betreten der Maus). </li><li> Stellen Sie die Nadel Höhe und Winkel, um zu zielen Sie die linke Herzkammer. </li><li> Dringen Sie die Spritzennadel in inter Raum sofort durch die Haut-und Muskelschichten in den linken Ventrikel unter der Leitung von Ultraschall-Bildgebung. Eine Angabe der erfolgreichen Einführung der Nadel in den linken Ventrikelcle ist der Rückfluss von frischen arteriellen Blut (rosa Farbe im Gegensatz zu den dunkelroten venösen Blut) in die Spritze </li><li> Spritzen Sie die Zellgemisch langsam (Abbildung 1B). </li><li> Nach Abschluss ziehen Sie die Nadel, heben Sie den Wandler, reinigen Sie die Ultraschall-Gel mit angefeuchteten Gaze und Klebeband entfernen. </li><li> Bereiten Sie einen sauberen Käfig mit einem vorgewärmten Pad. </li><li> Legen Sie das Tier auf dem Pad und beobachten Sie das Tier bis zur vollständigen Genesung. </li><li> Überwachung des Verhaltens des Tieres alle 24 h für zwei Tage. </li><li> Allgemeine Bedingungen und neurologischen Anzeichen von Komplikationen bei Versuchsmäusen werden routinemäßig überwacht. </li></ol> 3. MRT-Überwachung von intrakraniellen Tumorentwicklung <ol><li> Verwenden Sie ein 9,4 Tesla Magneten an intrakraniellen Entwicklung von Hirnmetastasen zu überwachen. </li><li> Initiieren MRI zwei Wochen nach der Tumorimplantation und wiederholen Sie einmal pro Woche für bis zu drei Wochen. </li><li> Sedieren die Tiere mit 3% Isofluran und pflegen sie unter Genral Anästhesie (Isofluran 1,5%). </li><li> Überwachung und die Tierkörpertemperatur und Atmung während des Experiments konstant zu halten. </li><li> Hochauflösende Multislice (14 Scheiben mit 1 mm dick, kein Spalt) T 1 - und T 2-gewichtete koronale Bilder, für die Region aus der Frontallappen an der hinteren Schädelgrube, werden mit den folgenden Parametern erworben: T 1-gewichtete Bilder: mehrere Spin-Echo-Scheibe (REM), TR / TE = 400 ms msec/20, Matrix: 256 x 256, FOV 20 x 20 mm, in der Ebene Auflösung: 78 x 78 um 2. T 2-gewichtete Bilder: Fast-Spin-Echo mehrere Scheibe (FSEMS) Sequenzen, TR / TE = 2500 msec/48 ms, 8 Echozüge, Matrix: 256 x 256, FOV 20 x 20 mm, in der Ebene Auflösung: 78 x 78 um 28,9. Tumor-Läsionen erscheinen heller als normale Hirngewebe auf T 2-gewichteten Bildern. </li><li> Die Tumorgröße ist auf T 2-gewichtete Bilder manuell umreißt die Zubeh bestimmtCING Teil der Masse auf jedes Bild mit Hilfe von MATLAB-Programme von uns 8 geschrieben. Gegeben meisten der Tumordurchmesser kleiner sind als die Schichtdicke (1 mm), wird die Größe des Tumors nach ebene Fläche anstatt das Volumen dargestellt. </li></ol> 4. H &amp; E-Färbung Bestätigung der Metastasen <ol><li> Opfere die Mäuse sofort nach der letzten MR-Scan, sezieren die tumortragenden Gehirne und binde sie in OCT-Medium und in -80 ° C eingefroren </li><li> Abschnitt eine Serie von 10 um dicke koronale Hirnproben mit Kryostaten. </li><li> Führen H &amp; E-Färbung auf den Gehirnschnitten. </li></ol>

<ol>
	<li>Schouten, L. 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=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. 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=The+biology+of+brain+metastases-translation+to+new+therapies.">The biology of brain metastases-translation to new therapies.</a> Nat. Rev. Clin. Oncol. , (2011).</li><li>Lockman, P. R., 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=Heterogeneous+blood-tumor+barrier+permeability+determines+drug+efficacy+in+experimental+brain+metastases+of+breast+cancer.">Heterogeneous blood-tumor barrier permeability determines drug efficacy in experimental brain metastases of breast cancer.</a> Clin. Cancer Res. 16, 5664-5678 (2010).</li><li>Yoneda, T., 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=A+bone-seeking+clone+exhibits+different+biological+properties+from+the+MDA-MB-231+parental+human+breast+cancer+cells+and+a+brain-seeking+clone+in+vivo+and+in.">A bone-seeking clone exhibits different biological properties from the MDA-MB-231 parental human breast cancer cells and a brain-seeking clone in vivo and in.</a> 16, 1486-1495 (2001).</li><li>Palmieri, D., 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=Her-2+overexpression+increases+the+metastatic+outgrowth+of+breast+cancer+cells+in+the+brain.">Her-2 overexpression increases the metastatic outgrowth of breast cancer cells in the brain.</a> Cancer Res. 67, 4190-4198 (2007).</li><li>Percy, D. B., 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+characterization+of+changing+blood-tumor+barrier+permeability+in+a+mouse+model+of+breast+cancer+metastasis:+a+complementary+magnetic+resonance+imaging+approach.">In vivo characterization of changing blood-tumor barrier permeability in a mouse model of breast cancer metastasis: a complementary magnetic resonance imaging approach.</a> Invest. Radiol. 46, 718-725 (2011).</li><li>Zhou, 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=Longitudinal+MRI+evaluation+of+intracranial+development+and+vascular+characteristics+of+breast+cancer+brain+metastases+in+a+mouse+model.">Longitudinal MRI evaluation of intracranial development and vascular characteristics of breast cancer brain metastases in a mouse model.</a> PLoS One. 8, (2013).</li><li>Zhou, 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=Dynamic+near-infrared+optical+imaging+of+2-deoxyglucose+uptake+by+intracranial+glioma+of+athymic+mice.">Dynamic near-infrared optical imaging of 2-deoxyglucose uptake by intracranial glioma of athymic mice.</a> PLoS One. 4, (2009).</li><li>Zhang, R. 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Biol. 530, 19-27 (2003).</li><li>Gril, B., 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=Pazopanib+reveals+a+role+for+tumor+cell+B-Raf+in+the+prevention+of+HER2++breast+cancer+brain+metastasis.">Pazopanib reveals a role for tumor cell B-Raf in the prevention of HER2+ breast cancer brain metastasis.</a> Clin. Cancer Res. 17, 142-153 (2010).</li></ol>

Keine Interessenskonflikte erklärt.

In der vorliegenden Studie haben wir gezeigt, dass Ultraschall bildgesteuerten linksventrikuläre Injektion gewährleistet die Genauigkeit, so dass jedes Tier in dieser Studie entwickelte Gehirn-Metastasen und kein Tier Tod beobachtet. Die Schönheit des bildgeführten Injektion ist, dass der Weg der Nadelpenetration von Haut und schließlich in den linken Ventrikel überwacht und unter Bild, das sich von dem manuellen Verfahren strengen anatomischen Strukturen erfordern zu folgen ist, eingestellt werden. <p class="...

Hirnmetastasen ist die häufigste Krebserkrankung bei intrakraniellen Erwachsene. Die Prognose ist sehr schlecht, mit einer medianen Überlebens von 4-6 Monaten auch bei aggressiven Behandlung. Brustkrebs ist eine der drei großen Primärtumoren mit einer hohen Morbidität von Hirnmetastasen 3.1. Mehrere Gehirn-tropischen Brustkrebslinien sind in der Lage sich entwickelnde Gehirn Metastasen nach intra oder intracarotid Injektion 4. Direkte Injektion von Tumorzellen in den linken Ventrikel kann die Lunge Kapillarbett zu umgehen und somit die Häufigkeit der Bildung Hirnmetastasen bei Minimierung viszerale Metastasen. Der MDA-MB231Br Linie ist eine der am weitesten verbreiteten menschlichen Brustkrebslinien Hirnmetastasen in Nagetiermodellen 5,6 zu entwickeln. Wie viele andere Studien 4,7, haben wir eine manuelle Prozedur intrakardialer Injektion in unseren früheren Studien durchgeführt. Es wurde jedoch nur 50% Erfolgsquote bei der manuellen erhaltenEinspritzung und ein Bruchteil der Mäuse starben aus den wiederholten invasiven Verfahren, wenn vor Versuchen fehlgeschlagen. Hier stellen wir die Verwendung eines bildgesteuerten Verfahren, um die Injektion von Hirnsuchbrustkrebszellen in den linken Ventrikel von athymischen Mäusen zu sichern. Längshochauflösende MRT wird angewendet, um intrakranielle Entwicklung von Hirnmetastasen zu folgen.

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		<tr height="21">
			<td height="21" style="height:21px;width:413px;">DMEM</td>
			<td style="width:136px;">HyClone</td>
			<td style="width:87px;">SH30022.01</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Trypsin</td>
			<td style="width:136px;">Mediatech</td>
			<td style="width:87px;">25-051-C1</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
			<td style="width:136px;">Biorad</td>
			<td style="width:87px;">TC10</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
			<td style="width:136px;">Baxter International Inc.</td>
			<td style="width:87px;">1001936060</td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
			<td></td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
			<td style="width:87px;"> </td>
			<td style="width:147px;"> </td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">O.C.T Compound</td>
			<td>Sakura Finetek USA</td>
			<td style="width:87px;">4583</td>
			<td style="width:147px;"> </td>
		</tr>
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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

Brustkrebs Hirnmetastasen, bei 30% der Patientinnen mit Brustkrebs im Stadium IV auftreten, wird mit hoher Sterblichkeit. Die mediane Überlebens ist nur 6 Monate. Es ist kritisch, geeignete Tiermodelle haben die hämodynamischen Ausbreitung der metastatischen Zellen im klinischen Szenario zu simulieren. Hier stellen wir die Verwendung von Kleintierultraschallbildgebung, um eine genaue Einspritzung von Gehirn tropischen Brustkrebszellen in den linken Ventrikel von athymischen nackten Mäusen führen. Längs-MRT zur Beurteilung intrakranieller Initiierung und das Wachstum von Hirnmetastasen eingesetzt. Ultraschall-gesteuerte intrakardiale Injektion gewährleistet nicht nur eine genaue Einspritzung und hiermit eine höhere Erfolgsquote, sondern auch deutlich verringert Sterblichkeit im Vergleich zu unseren bisherigen manuellen Verfahren. In vivo hochauflösende MRT erlaubt die Visualisierung von hyperintens multifokale Läsionen, so klein wie 310 um Durchmesser auf T2-gewichteten Bildern 3 Wochen nach der Injektion. Follow-up-MRT zeigt intrakraniellen Tumorwachstum und die erhöhte Anzahl von Metastasen, die während des gesamten Gehirns zu verteilen.

Mit der hohen räumlichen Auflösung der MRT (78 in der Ebene um Auflösung) können hyperintense Läsionen so klein wie 310 um im Durchmesser (Abbildung 2) identifiziert werden. Da die Metastasen in dieser Studie sind sehr klein und die Entwicklung der Nekrose und Ödem ist minimal, die hyperintense Läsion auf T 2-gewichteten Bildern die Tumormasse wirklich vertreten. Längs-MRT-Studien ermöglichen in vivo nicht-invasive Beurteilung des Tumorwachstums....

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Ein Brustkrebs Hirnmetastasen Mausmodell mit Ultraschall bildgesteuerten Injektion von intra MDA-MB231/Br-GFP Zellen etabliert. Entwicklung von Hirnmetastasen multifokale längs mit hochauflösenden 9,4 T MRT überwacht.

Ultraschall-Imaging-guided Intra Injection zur Entwicklung eines Mausmodells von Brustkrebs Hirnmetastasen durch Längs MRI Gefolgt

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.7K Aufrufe.  University of Texas Southwestern Medical Center.  Ein Brustkrebs Hirnmetastasen Mausmodell mit Ultraschall bildgesteuerten Injektion von intra MDA-MB231/Br-GFP Zellen etabliert. Entwicklung von Hirnmetastasen multifokale längs mit hochauflösenden 9,4 T MRT überwacht. 

Serie: Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

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 ...

Forschung

Medizin

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-geführten Störung der Blut-Hirn-Schranke mit Hilfe der transkraniellen fokussiertem Ultraschall in einem Rattenmodell

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

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 ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Initiation Metastasierende Mammakarzinom durch Targeting des duktalen Epithel mit Adenovirus-Cre: Eine neue transgene Maus-Modell der Brustkrebs

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

Quantifizierung der Brustkrebs-Zelle Invasivität Mit einem Drei-dimensionale (3D) Modell

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Akute Brain Trauma in Mäusen Gefolgt durch Längs Zwei-Photonen-Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Einer präklinischen Mausmodell der Lebermetastasen

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Orthotopische Injektion von Brustkrebszellen in das Brustfettpolster von Mäusen, das Tumorwachstum zu studieren.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm-Entwicklung über die medizinisch relevanten Fremdkörper in einem Maus-Modell durch subkutane Biolumineszenz Imaging Gefolgt

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

High-frequency ultrasound (HFUS) is widely employed as a non-invasive method for imaging internal anatomic structures in experimental small animal systems. HFUS has the ability to detect structures as small as 30 &#181;m, a property that has been utilized for visualizing superficial lymph nodes in rodents in brightness (B)-mode. Combining power Doppler with B-mode imaging allows for measuring circulatory blood flow within lymph nodes and other organs. While HFUS has been utilized for lymph node imaging in a number of mouse&#160; model systems, a detailed protocol describing HFUS imaging and characterization of the cervical lymph nodes in mice has not been reported. Here, we show that HFUS can be adapted to detect and characterize cervical lymph nodes in mice. Combined B-mode and power Doppler imaging can be used to detect increases in blood flow in immunologically-enlarged cervical nodes. We also describe the use of B-mode imaging to conduct fine needle biopsies of cervical lymph nodes to retrieve lymph tissue for histological&#160; analysis. Finally, software-aided steps are described to calculate changes in lymph node volume and to visualize changes in lymph node morphology following image reconstruction. The ability to visually monitor changes in cervical lymph node biology over time provides a simple and powerful technique for the non-invasive monitoring of cervical lymph node alterations in preclinical mouse models of oral cavity disease.

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also demonstrated.