Name: intracranial implantation with subsequent 3d in vivo bioluminescent imaging of murine gliomas
Uploaded: 2011-11-06T14:00:00.000+00:00
Duration: 9 min 46 s
Description: Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

Wir möchten Dr. Joshua B. Rubin für das großzügige Geschenk der Plasmide für die Lentivirus-System sowie Mahil Rao für hilfreiche Anregung für die Vorbereitung der GL261-luc-Zellen erkennen. Wir bedanken uns bei Studenten Unterstützung Brain Tumor Research (SSBTR), The Barrow Neurological Foundation und The Wallace Foundation für ihre großzügige Unterstützung. Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her durch die Institutional Animal Care und Verwenden Ausschuss der St. Joseph-Hospital und Medical Center eingerichtet durchgeführt.

<ol>
	<li>Candolfi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+glioblastoma+models+in+preclinical+neuro-oncology:+neuropathological+characterization+and+tumor+progression.">Intracranial glioblastoma models in preclinical neuro-oncology: neuropathological characterization and tumor progression.</a> J Neurooncol. 85, 133-148 (2007).</li><li>Stafford, P., Abdelwahab, M. G., do, K. i. m., Preul, Y., Rho, M. C., M, J., Scheck, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ketogenic+diet+reverses+gene+expression+patterns+and+redudes+reactive+oxygen+species+levels+when+used+as+an+adjuvant+therapy+for+glioma.">The ketogenic diet reverses gene expression patterns and redudes reactive oxygen species levels when used as an adjuvant therapy for glioma.</a> Nutr Metab. 7, (2010).</li><li>Caliper Life Sciences, Inc. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Living Image Software Version 4.0. VivoVision Systems. , (2010).</li><li>Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (2001).</li><li>Jouanneau, E., Poujol, D., Gulia, S., Le, M. I., Blay, J. Y., Belin, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+are+essential+for+priming+but+inefficient+for+boosting+antitumour+immune+response+in+an+orthotopic+murine+glioma+model.">Dendritic cells are essential for priming but inefficient for boosting antitumour immune response in an orthotopic murine glioma model.</a> Cancer Immunol Immunother. 55, 254-267 (2006).</li></ol>

Freier Zugang zu diesem Artikel wird von Caliper Life Sciences gefördert.

Die Zelle Inokulum wird in einer Tiefe von 2,6 mm von der Oberfläche des Gehirns nach der Erstellung eines 0,4 mm Tasche infundiert. Um eine korrekte Platzierung und Tiefe der Nadel sorgen für eine X-ray kann aufgenommen mit einem C-Arm oder ähnliches Röntgenbild Intensivierung Gerät, jedoch ist diese optional. Komplikationen bei der Operation auftreten können, wenn das Tier nicht ordnungsgemäß betäubt, an welchem ​​Punkt kann das Tier während der Zellteilung Infusion zu bewegen. Dies kann zu Undichtigkeiten der Zelle Mix oder Blutungen aus Nadel zu verfolgen. Austreten von Zellen verursacht ektopische Wachstum von Tumorzellen. Es ist auch wichtig, nicht die Punktion der Ventrikel, was getan werden kann, wenn die BOHRLOCHRESERVOIR wird medial von der 2,3 mm in das Protokoll 4 beschrieben. Die richtige Platzierung der Nadel und Zelle verteilt wurde durch Infusion einer Maus mit 2 ul Methylenblau und seziert das Hirngewebe um die Position der infundierten Farbstoff überprüfen getestet. In diesem Protokoll haben wir die IVIS Spectrum in-vivo-Bildgebung und t verwendeter lebt Image Software (v 4.0) für die Verwendung mit diesem Instrument. (Caliper Life Sciences). Alle vergleichbaren in-vivo-Imaging-System und Bild-Analyse-Tools können verwendet werden, um ähnliche Ergebnisse zu erzielen. Diese Systeme bieten einige Vorteile gegenüber herkömmlichen Magnet-Resonanz-Tomographie (MRT), um das Wachstum eines experimentellen intrakraniellen Tumor zu folgen. Der offensichtlichste ist die relativen Kosten der 2-Instrumente - Tier MRT-Geräte sind wesentlich teurer und erfordern in der Regel die Dienste eines qualifizierten MRI-Techniker. In-vivo-Bildgebung, wie, was hier beschrieben, kann durch den Endverbraucher durchgeführt werden. Die Biolumineszenz Daten quantitative, während die Quantifizierung von MRT-Daten ist zeitaufwändig und etwas ungenau. Darüber hinaus zeigen MRT-Aufnahmen Ödemen und Entzündungen zusätzlich zu Tumorzellen und es kann schwierig sein, Tumor-Behandlung Wirkung zu trennen. Aus diesen Gründen erhalten genaue volumetrische Messungen des wachsenden Tumors kann eine Herausforderung sein. Biolumineszenz erfordert ATPDaher nur lebende Tumorzellen beitragen, die Tumorgröße Daten. Trotzdem gibt es einige Vorteile der MRT, ob eine Maschine zur Verfügung steht. Cells müssen nicht mit einem Biolumineszenz-Marker werden durch MRI sichtbar gekennzeichnet sein. Die Fähigkeit, peri-Tumor-Ödem visualisieren kann ein Vorteil für einige experimentelle Protokolle werden. Eine Stärke der beiden Technologien ist, dass mit einem nicht ausschließt, die Verwendung der anderen, so können Daten über das Wachstum des Tumors sowie das Vorhandensein von peri-Tumor-Ödemen und Entzündungen aus dem gleichen Tier gewonnen werden, wenn beide Technologien verfügbar sind an den Forscher.

<table border="1"><tbody><tr><td> Name des Reagenzes oder Lieferung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> GL261-luc2 Bioware Ultra- </td><td> Caliper Life Sciences </td><td> GL261-luc2 </td><td></td></tr><tr><td> Dulbecco Modified Eagle Medium (DMEM) </td><td> Invitrogen </td><td> 10313039 </td><td></td></tr><tr><td> Geneticin (G418) </td><td> Gibco (Invitrogen) </td><td> 11811-023 </td><td></td></tr><tr><td> Fetal Calf Serum (FCS) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Phospate Buffered Saline (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Cr Mäuse </td><td> NCI-Frederick </td><td></td><td></td></tr><tr><td> AKWA Tränen Lubricant Augensalbe </td><td> Akorn Inc </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (Ketamin Hydrochlorid) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (Xylazinhydrochlorid) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> Small Animal stereotaktische Instrument </td><td> Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump mit SYS-Micro4-Controller </td><td> World Precision Instruments </td><td> UMP3-1 </td><td> Falls nicht vorhanden, ist es möglich, manuell ziehen </td></tr><tr><td> 10 &amp;mgr; l-Spritze mit 26 Gauge abgeschrägte Nadel </td><td> World Precision Instruments </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison Zange </td><td> World Precision Instruments </td><td> 500092 </td><td></td></tr><tr><td> Penfield Dissector </td><td> Codman </td><td> 65-1015 </td><td></td></tr><tr><td> 16g 1 ½ Precision Glide Needle </td><td> Beckton, Dickinson and Company (BD) </td><td> 305198 </td><td></td></tr><tr><td> Chirurgische Klinge Griff </td><td> BD </td><td> 371030 </td><td></td></tr><tr><td> Größe 15 Blatt </td><td> BD </td><td> 371315 </td><td></td></tr><tr><td> 4-0 Vicryl Naht </td><td> Ethicon </td><td> VCP496G </td><td></td></tr><tr><td> Knochenwachs </td><td> Medline </td><td> DYNJBW25 </td><td></td></tr><tr><td> Povidin-Jod-Swab Sticks </td><td> Medline </td><td> MD93901 </td><td></td></tr><tr><td> D-Luciferin Kaliumsalz </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr><tr><td> Forane (Isofluran) </td><td> Baxter </td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero Mikroskop </td><td> Carl Zeiss, Inc. </td><td></td><td> Jeder OP-Mikroskop genügt </td></tr><tr><td> Xenogen IVIS Spectrum mit optionalem Anästhesie-System </td><td> Caliper Life Sciences </td><td></td><td></td></tr></tbody></table>

intracranial implantation with subsequent 3d in vivo bioluminescent imaging of murine gliomas

Die Maus Gliom 261 (GL261) als in-vivo-Modell erkannt, dass rekapituliert viele der Funktionen des menschlichen Glioblastoma multiforme (GBM). Die Zelllinie wurde ursprünglich von intrakraniellen Injektion von 3-Methyl-cholantrene in eine C57BL / 6 syngenen Mausstamm 1 induzierte, deshalb immunologisch kompetenten C57BL / 6 Mäuse verwendet werden kann. Während wir GL261 verwenden, können das folgende Protokoll für die Implantation und Überwachung jeder intrakraniellen Maus-Tumor-Modell verwendet werden. GL261-Zellen wurden stabil exprimieren, Leuchtkäfer-Luciferase (GL261-luc) entwickelt. Wir schuf auch die helleren GL261-luc2 Zelllinie durch stabile Transfektion der luc2 Gen aus dem CMV-Promotors exprimiert. C57BL/6-cBrd/cBrd/Cr Mäusen (Albino-Variante der C57BL / 6) von der National Cancer Institute, Frederick, MD wurden verwendet, um das Licht Dämpfung durch schwarze Haut und Fell zu eliminieren. Mit dem Einsatz von Albino C57BL / 6 Mäusen, in-vivo-Bildgebung mit dem IVIS Spectrum in vivo imAltern ist ab dem Tag der Implantation (Caliper Life Sciences, Hopkinton, MA) möglich. Die GL261-luc und GL261-luc2 Zelllinien zeigten die gleichen In-vivo-Verhalten der Eltern GL261-Zellen. Einige der gemeinsamen histologischen Merkmale in der menschlichen GBM und das Maus-Modell beinhalten: Tumor-Nekrose, pseudopalisades, Neovaskularisation, Invasion, Hyperzellularität und Entzündungen 1. Vor der Implantation Tiere wurden durch eine intraperitoneale Injektion von Ketamin (50 mg / kg), Xylazin (5 mg / kg) und Buprenorphin (0,05 mg / kg), in einem stereotaktischen Apparat und einen Einschnitt gelegt betäubt wurde mit einem Skalpell über die aus kranialen Mittellinie. Ein BOHRLOCHRESERVOIR war 0.1mm posterior zum Bregma und 2,3 mm nach rechts von der Mittellinie aus. Eine Nadel wurde bis in eine Tiefe von 3mm und zurückgezogen 0.4mm bis zu einer Tiefe von 2,6 mm eingesetzt. Zwei ul der GL261-luc oder GL261-luc2 Zellen (10 7 Zellen / ml) wurden im Verlauf von 3 Minuten infundiert. Die BOHRLOCHRESERVOIR wurde geschlossenmit bonewax und der Schnitt genäht. Nach stereotaktische Implantation der Biolumineszenz-Zellen sind ab dem Tag der Implantation nachweisbar und der Tumor kann unter Verwendung des 3D-Bildrekonstruktion Merkmal der IVIS Spectrum Instrument sein. Tiere erhalten eine subkutane Injektion von 150μg Luciferin / kg Körpergewicht 20 min vor der Bildgebung. Tumorlast wird unter Verwendung bedeuten Tumor Biolumineszenz im Laufe der Zeit. Tumor-tragende Mäuse wurden täglich auf Morbidität beurteilen beobachtet und wurden eingeschläfert, wenn einer oder mehrere der folgenden Symptome vorhanden sind: Lethargie, das Versagen der ambulate, gekrümmte Haltung, das Versagen der Bräutigam, Anorexie was bei&gt; 10% Gewichtsverlust. Die Tumore wurden offensichtlich in all der Tiere bei der Obduktion.

Watch this Scientific Journal Video about Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas at JoVE.com

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intrakranielle Implantation von GL261-Zellen in C57BL / 6 Mäusen produziert maligne Gliome, dass viele Merkmale der menschlichen Glioblastoma multiforme zu rekapitulieren. Wir verwendeten GL261-Zellen stabil exprimiert Luziferase, damit wir verwenden<em&gt; In vivo</em&gt; Bildgebung zu folgen Tumorprogression. Die Operation und 3D<em&gt; In vivo</em&gt; Bildgebung demonstriert.

Intrakranielle Implantation mit anschließender 3D In Vivo Bioluminescent Imaging von Murine Gliome

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

intracranial-implantation-with-subsequent-3d-vivo-bioluminescent

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Research

JoVE Journal

Medicine

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

20.4K Aufrufe.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

Serie: Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Gründung Intrakranielle Brain Tumor Xenografts mit anschließender Analyse des Tumorwachstums und Ansprechen auf die Therapie mit Biolumineszenz-Bildgebung

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

Forschung

Neurowissenschaften

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 ...

Medizin

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting immunodeficient mice subcutaneously with cancer cells and measuring the tumor mass after it becomes visible and palpable. Orthotopic injections of cancer cells aim to introduce the xenograft in the microenvironment that most closely resembles the tissue of origin of the tumor being studied. Brain cancer research requires intracranial injection of cancer cells to allow the tumor formation and analysis in the unique microenvironment of the brain. The in vivo imaging of intracranial xenografts monitors instantaneously the tumor mass of orthotopically engrafted mice. Here we report the use of fluorescence molecular tomography (FMT) of brain tumor xenografts. The cancer cells are first transduced with near infrared fluorescent proteins and then injected in the brain of immunocompromised mice. The animals are then scanned to obtain quantitative information about the tumor mass over an extended period of time. Cell pre-labeling allows for cost effective, reproducible, and reliable quantification of the tumor burden within each mouse. We eliminated the need for injecting imaging substrates, and thus reduced the stress on the animals. A limitation of this approach is represented by the inability to detect very small masses; however, it has better resolution for larger masses than other techniques. It can be applied to evaluate the efficacy of a drug treatment or genetic alterations of glioma cell lines and patient-derived samples.

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Orthotopic intracranial injection of tumor cells has been used in cancer research to study brain tumor biology, progression, evolution, and therapeutic response. Here we present fluorescence molecular tomography of tumor xenografts, which provides real-time intravital imaging and quantification of a tumor mass in preclinical glioblastoma models.

Fluoreszenz Molekulare Computertomographie für die In-Vivo Bildgebung des Glioblastoms Xenotransplantate

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Krebsforschung

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Stereotaktische Intrakranielle Implantation und<em&gt; In vivo</em&gt; Bioluminescent Imaging der Tumorxenotransplantaten in einem Maus-Modell-System des Glioblastoma multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Ein Orthotopische Glioblastoma Maus-Modell Pflege Gehirn Parenchymale Körperliche Einschränkungen und geeignet für Zwei-Photonen-Intravitalmikroskopie

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Echtzeit-Tracking von Nanopartikeln in Hirntumoren mittels 2P-IVM bei Mäusen

Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM).
The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications.
This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Autor im Rampenlicht: Multimodale Bildgebungsstrategien zur Optimierung der Wirkstoffverabreichung und Früherkennung bei der Behandlung von Glioblastomen

We present a novel approach for two-photon microscopy of the tumor delivery of fluorescent-labeled iron oxide nanoparticles to glioblastoma in a mouse model.

Zwei-Photonen-Intravitalmikroskopie des Glioblastoms im Mausmodell

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the ...

<meta charset="utf8"></head><body>Eisenoxid-Nanopartikel für die bildgestützte Therapieverabreichung beim murinen Glioblastom

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

<meta charset="utf8"></head><body>Autor im Rampenlicht: Innovative Krebstherapien mit Eisenoxid-Nanopartikeln zur Behandlung von Glioblastomen

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Nanopartikel-Verabreichung einer Oligonukleotid-Nutzlast in einem Glioblastom-Multiforme-Tiermodell

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Intrakranielle Implantation mit anschließender 3D In Vivo Bioluminescent Imaging von Murine Gliome

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