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

Vorremmo ringraziare il dottor Joshua B. Rubin per il generoso dono dei plasmidi per il sistema di lentivirus e Mahil Rao per il suggerimento utile per la preparazione della GL261-luc cellule. Ringraziamo gli studenti Sostenere Tumore Brain Research (SSBTR), The Barrow Neurological Fondazione e la Fondazione Wallace per il loro generoso sostegno. Esperimenti su animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla cura degli animali e del Comitato Istituzionale uso dell&#39;Ospedale San Giuseppe e Medical Center.

<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>

Accesso gratuito a questo articolo è sponsorizzato da Caliper Life Sciences.

L&#39;inoculo delle cellule è infuso ad una profondità di 2,6 mm dalla superficie del cervello dopo la creazione di una tasca 0,4 mm. Per garantire il corretto posizionamento e la profondità dell&#39;ago una radiografia può essere assunto con un C-Arm o simili a raggi X dispositivo di intensificazione dell&#39;immagine, ma questo è opzionale. Le complicanze della chirurgia possono insorgere se l&#39;animale non è adeguatamente sedato, a quel punto l&#39;animale può muoversi durante l&#39;infusione delle cellule. Questo può causare perdite di miscela di cellule o sanguinamento da pista ago. Perdita di cellule causa la crescita ectopica di cellule tumorali. E &#39;anche importante non forare il ventricolo, che può essere fatto se l&#39;è fatta burrhole mediale a mm 2,3 descritte nel protocollo 4. Il corretto posizionamento della diffusione ago e cellulari è stato testato infondendo un mouse con 2 colorante blu di metilene microlitri e sezionare il tessuto cerebrale per verificare la posizione del colorante infuso. In questo protocollo abbiamo utilizzato lo spettro IVIS imaging in vivo del sistema e tha Living software Image (v 4.0) progettato per essere utilizzato con questo strumento. (Caliper Life Sciences). Qualsiasi paragonabile sistema di imaging in vivo e gli strumenti di analisi delle immagini può essere usata per ottenere risultati simili. Questi sistemi offrono alcuni vantaggi rispetto ai tradizionali risonanza magnetica (MRI) per seguire la crescita di un tumore sperimentale intracranica. Il più evidente è il costo relativo dei 2 strumenti - Macchine per animali RM sono molto più costosi e di solito richiedono i servizi di un tecnico specializzato risonanza magnetica. Imaging in vivo, come ciò che è stato qui descritto può essere effettuata dall&#39;utente finale. I dati bioluminescenza è quantitativa, mentre la quantificazione dei dati MRI è in termini di tempo e un po &#39;inesatto. Inoltre, le immagini MRI mostrano edema e infiammazione, oltre alle cellule tumorali e può essere difficile separare l&#39;effetto del trattamento di tumore. Per queste ragioni ottenere accurate misurazioni volumetriche del tumore in crescita può essere una sfida. Bioluminescenza richiede ATP, Quindi solo le cellule tumorali vive contribuire ai dati dimensioni del tumore. Nonostante questo, ci sono alcuni vantaggi a risonanza magnetica se una macchina è disponibile. Le cellule non devono essere etichettati con un pennarello bioluminescenti per essere visualizzate alla risonanza magnetica. La possibilità di visualizzare peri-tumorali edema può essere un vantaggio per alcuni protocolli sperimentali. Un punto di forza di entrambe le tecnologie è che l&#39;utilizzo di uno non esclude l&#39;uso degli altri, quindi i dati possono essere ottenuti sulla crescita del tumore così come la presenza di peri-tumorali edema e infiammazione dello stesso animale, quando le due tecnologie sono disponibili al ricercatore.

<table border="1"><tbody><tr><td> Nome del reagente o fornitura </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> GL261-luc2 Bioware Ultra </td><td> Caliper Life Sciences </td><td> GL261-luc2 </td><td></td></tr><tr><td> Dulbecco modificato (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> Di siero fetale bovino (FCS) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Soluzione salina tamponata con fosfato (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Cr Mice </td><td> NCI-Frederick </td><td></td><td></td></tr><tr><td> AKWA Tears lubrificante unguento oftalmico </td><td> Akorn Inc </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (cloridrato di ketamina) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (cloridrato xilazina) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> Piccoli Animali Strumento Stereotassica </td><td> Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump con SYS-Micro4 controller </td><td> Mondo Strumenti di precisione </td><td> UMP3-1 </td><td> Se non disponibile, è possibile infondere manualmente </td></tr><tr><td> Siringa 10μl con 26 gauge smussato aghi </td><td> Mondo Strumenti di precisione </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison Pinza </td><td> Mondo Strumenti di precisione </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 di precisione Glide Needle </td><td> Beckton, Dickinson and Company (BD) </td><td> 305198 </td><td></td></tr><tr><td> Lama maniglia chirurgico </td><td> BD </td><td> 371030 </td><td></td></tr><tr><td> Taglia 15 Lama </td><td> BD </td><td> 371315 </td><td></td></tr><tr><td> 4-0 sutura Vicryl </td><td> Ethicon </td><td> VCP496G </td><td></td></tr><tr><td> Bone Wax </td><td> Medline </td><td> DYNJBW25 </td><td></td></tr><tr><td> Povidine Iodio-tampone Sticks </td><td> Medline </td><td> MD93901 </td><td></td></tr><tr><td> D-Luciferin Potassio Salt </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr><tr><td> Foraneo (isoflurano) </td><td> Baxter </td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero Microscopio </td><td> Carl Zeiss, Inc. </td><td></td><td> Ogni microscopio operatorio sarà sufficiente </td></tr><tr><td> Xenogen Spectrum IVIS con sistema di anestesia opzionale </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

Il mouse glioma 261 (GL261) è riconosciuto come un sistema di modello in vivo che riassume molte delle caratteristiche del glioblastoma multiforme umano (GBM). La linea cellulare è stata originariamente indotta da iniezione intracranica di 3-metil-cholantrene in un C57BL / 6 ceppo di topi singenici 1; quindi, immunologicamente competenti C57BL / 6 topi possono essere utilizzati. Mentre usiamo GL261, il seguente protocollo può essere utilizzato per l&#39;impianto e il monitoraggio di qualsiasi modello intracranica del tumore del mouse. GL261 cellule sono state progettate per esprimere stabilmente lucciola luciferasi (GL261-luc). Abbiamo anche creato la più brillante GL261-luc2 linea cellulare di trasfezione stabile del gene luc2 espressa dal promotore CMV. Topi C57BL/6-cBrd/cBrd/Cr (variante albino di C57BL / 6) dal National Cancer Institute, Frederick, MD sono stati utilizzati per eliminare l&#39;attenuazione della luce causata da pelle nera e pelliccia. Con l&#39;uso di C57BL albino / 6 topi, imaging in vivo utilizzando la Spectrum IVIS in vivo iml&#39;invecchiamento del sistema è possibile dal giorno di impianto (Caliper Life Sciences, Hopkinton, MA). Il GL261 e GL261-luc-luc2 linee cellulari hanno mostrato lo stesso comportamento in vivo come genitori GL261 cellule. Alcune delle caratteristiche istologiche condivise presenti nel GBM umano e questo modello di topo sono: necrosi tumorale, pseudopalisades, neovascolarizzazione, invasione, ipercellularità, e l&#39;infiammazione 1. Prima di animali l&#39;impianto sono stati anestetizzati con una iniezione intraperitoneale di ketamina (50 mg / kg), xilazina (5 mg / kg) e buprenorfina (0,05 mg / kg), inserito in un apparato stereotassico e una incisione è stata fatta con un bisturi il linea mediana del cranio. Un burrhole è stata fatta posteriore 0,1 millimetri al bregma e 2,3 millimetri a destra della linea mediana. Un ago è stato inserito ad una profondità di 0.4mm 3mm ritirato ad una profondità di 2,6 mm. Due ml di GL261-luc o GL261-luc2 cellule (10 7 cellule / ml) sono state infuse nel corso di 3 minuti. Il burrhole è stato chiusobonewax e con l&#39;incisione è stata suturata. In seguito l&#39;impianto stereotassico le cellule bioluminescenti sono rilevabili dal giorno di impianto e il tumore può essere analizzato utilizzando la funzione di ricostruzione delle immagini 3D dello strumento Spectrum IVIS. Gli animali ricevono una iniezione sottocutanea di 150 mg luciferina / kg di peso corporeo 20 minuti prima di imaging. Massa tumorale è quantificato mediante bioluminescenza medio del tumore nel tempo. Portatori di tumore nei topi sono stati osservati quotidianamente per valutare la morbilità e sono stati sacrificati quando uno o più dei seguenti sintomi sono presenti: letargia, incapacità di deambulare, la postura ingobbita, il mancato sposo, anoressia con conseguente perdita&gt; 10% del peso. I tumori sono stati evidenti in tutti gli animali della necroscopia.

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

Impianto intracranico di GL261 cellule in C57BL / 6 topi produce gliomi maligni che ricapitolano molte delle caratteristiche del glioblastoma multiforme umano. Abbiamo usato GL261 cellule che esprimono stabilmente luciferasi per permetterci di utilizzare<em&gt; In vivo</em&gt; Imaging per seguire la progressione del tumore. L&#39;intervento e 3D<em&gt; In vivo</em&gt; Immagini sono dimostrati.

Impianto intracranica con 3D successivi In Vivo Bioluminescent Imaging dei gliomi Murine

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 ...

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Universidad de Cartagena UDEC

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

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

Video: 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.

Stabilire intracranica xenotrapianti Brain Tumor Con successiva analisi della crescita tumorale e risposta alla terapia con Imaging bioluminescenza

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

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Neuroscienze

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 Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

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

Medicina

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.

Fluorescenza tomografia molecolare per l'Imaging In Vivo di Glioblastoma xenotrapianti

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 ...

Ricerca sul cancro

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.

Stereotassica impianto intracranico e<em&gt; In vivo</em&gt; Imaging Bioluminescent di xenotrapianti di tumori in un sistema modello del mouse di 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.

Un romanzo ad alta risoluzione<em&gt; In vivo</em&gt; Tecnica di imaging per studiare la risposta dinamica di strutture intracraniche alla crescita del tumore e Terapeutica

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.

Un ortotopico Glioblastoma modello di mouse mantenimento di vincoli Cervello parenchimale fisiche e adatto per intravitale due fotoni Microscopia

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

Tracciamento in tempo reale di nanoparticelle nei tumori cerebrali utilizzando 2P-IVM nei topi

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.

Autore in primo piano: Strategie di imaging multimodale per ottimizzare la somministrazione di farmaci e la diagnosi precoce nel trattamento del glioblastoma

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.

Microscopia intravitale a due fotoni del glioblastoma in un modello murino

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>Nanoparticelle di ossido di ferro per la somministrazione di terapia guidata da immagini nel glioblastoma murino

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>Autore in primo piano: Terapie innovative contro il cancro con nanoparticelle di ossido di ferro per il trattamento del glioblastoma

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.

Consegna di nanoparticelle di un carico utile di oligonucleotidi in un modello animale multiforme di glioblastoma

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 ...

Impianto intracranica con 3D successivi In Vivo Bioluminescent Imaging dei gliomi Murine

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Stabilire intracranica xenotrapianti Brain Tumor Con successiva analisi della crescita tumorale e risposta alla terapia con Imaging bioluminescenza

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

Fluorescenza tomografia molecolare per l'Imaging In Vivo di Glioblastoma xenotrapianti

Stereotassica impianto intracranico e

Un romanzo ad alta risoluzione

Un ortotopico Glioblastoma modello di mouse mantenimento di vincoli Cervello parenchimale fisiche e adatto per intravitale due fotoni Microscopia

Microscopia intravitale a due fotoni del glioblastoma in un modello murino

Microscopia intravitale a due fotoni del glioblastoma in un modello murino

Microscopia intravitale a due fotoni del glioblastoma in un modello murino

Consegna di nanoparticelle di un carico utile di oligonucleotidi in un modello animale multiforme di glioblastoma