Nós gostaríamos de agradecer o Dr. Joshua B. Rubin pelo dom generoso de os plasmídeos para o sistema de lentivírus, assim como Rao Mahil para sugestão útil na preparação das células GL261-luc. Agradecemos a Estudantes Apoio à Pesquisa do tumor cerebral (SSBTR), The Barrow Neurological Foundation e da Fundação Wallace, por seu apoio generoso. Experimentos em animais foram realizados de acordo com as diretrizes e regulamentos estabelecidos pelo Institutional Animal Care e do Comitê Use do Hospital São José e Centro Médico.

<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>

Acesso livre para este artigo é patrocinado pela Caliper Life Sciences.

O inóculo de células é infundida a uma profundidade de 2,6 mm da superfície do cérebro depois de criar uma bolsa de 0,4 mm. Para assegurar a colocação adequada e profundidade da agulha um raio-X pode ser tomada através de um C-Arm ou outro dispositivo de imagem semelhante de raios-X intensificando, no entanto, este é opcional. Complicações da cirurgia podem surgir se o animal não está devidamente sedado, altura em que o animal pode se mover durante a infusão de células. Isto pode causar vazamento de mistura de células ou sangramento da trilha da agulha. Vazamento de células provoca o crescimento de células tumorais ectópica. Também é importante para não perfurar o ventrículo o que pode ser feito se o burrhole medial é feita para o 2,3 mm descritos no protocolo 4. Colocação adequada da propagação agulha e celular foi testada através da infusão de um mouse com 2 mL azul de metileno e dissecando o tecido cerebral para verificar a localização do corante infundida. Neste protocolo temos utilizado a Spectrum IVIS in vivo imagens de sistema e tele Vivendo software de imagem (v 4.0) projetado para uso com este instrumento. (Caliper Life Sciences). Qualquer comparável in vivo do sistema de imagem e ferramentas de análise de imagem pode ser usada para obter resultados semelhantes. Estes sistemas oferecem algumas vantagens sobre a ressonância tradicional magnética (MRI) para acompanhar o crescimento de um tumor experimental intracraniana. O mais óbvio é o custo relativo dos dois instrumentos - máquinas de ressonância magnética animais são muito mais caros e, normalmente, necessitam dos serviços de um técnico especializado MRI. In vivo de imagens, como o que foi descrito aqui pode ser feito pelo usuário final. Os dados bioluminescência é quantitativa, enquanto quantificação de dados de MRI é demorado e um pouco inexata. Além disso, imagens de ressonância magnética mostram edema e inflamação, além de células tumorais e pode ser difícil separar tumor do efeito do tratamento. Por estas razões a obtenção de medições precisas volumétrica do tumor em crescimento pode ser um desafio. Bioluminescência requer ATP, Portanto somente as células tumorais que vivem contribuir para os dados de tamanho do tumor. Apesar disso, existem algumas vantagens de MRI se uma máquina está disponível. As células não precisam ser rotulados com um marcador bioluminescente para ser visualizado por MRI. A capacidade de visualizar peri-tumoral edema pode ser uma vantagem para alguns protocolos experimentais. A força das duas tecnologias é que o uso de uma não exclui a utilização do outro, portanto os dados podem ser obtidos no crescimento do tumor, bem como a presença de peri-tumoral edema e inflamação do mesmo animal, quando as duas tecnologias estão disponíveis para o pesquisador.

<table border="1"><tbody><tr><td> Nome do reagente ou fornecimento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> GL261-luc2 Bioware Ultra </td><td> Caliper Life Sciences </td><td> GL261-luc2 </td><td></td></tr><tr><td> Modificado Dulbecco 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> Soro fetal bovino (FCS) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Fosfato Buffered Saline (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> Ratos C57BL/6-cBrd/cBrd/Cr </td><td> NCI-Frederick </td><td></td><td></td></tr><tr><td> Lágrimas AKWA Pomada Oftálmica Lubrificante </td><td> Akorn Inc </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (cloridrato de cetamina) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (cloridrato de xilazina) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> Instrumento estereotáxica Pequenos Animais </td><td> Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump com SYS-MICRO4 Controlador </td><td> Precision Instruments mundo </td><td> UMP3-1 </td><td> Se não estiver disponível, é possível para infundir manualmente </td></tr><tr><td> 10μl seringa com 26 gauge agulha chanfrada </td><td> Precision Instruments mundo </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison Pinça </td><td> Precision Instruments mundo </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> Cabo da lâmina cirúrgica </td><td> BD </td><td> 371030 </td><td></td></tr><tr><td> Lâmina tamanho 15 </td><td> BD </td><td> 371315 </td><td></td></tr><tr><td> Sutura Vicryl 4-0 </td><td> Ethicon </td><td> VCP496G </td><td></td></tr><tr><td> Cera de osso </td><td> Medline </td><td> DYNJBW25 </td><td></td></tr><tr><td> Iodo-povidine Sticks Swab </td><td> Medline </td><td> MD93901 </td><td></td></tr><tr><td> D-Luciferina sal de potássio </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr><tr><td> Forâneo (isoflurano) </td><td> Baxter </td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Microscópio Pentero </td><td> Carl Zeiss, Inc. </td><td></td><td> Qualquer microscópio cirúrgico será suficiente </td></tr><tr><td> Xenogen Spectrum IVIS com sistema de anestesia opcional </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

O mouse glioma 261 (GL261) é reconhecido como um sistema modelo in vivo que recapitula muitas das características de glioblastoma multiforme humano (GBM). A linhagem de células foi originalmente induzida pela injeção intracraniana de 3-metil-cholantrene em uma linhagem C57BL / 6 do mouse singeneicos 1 e, portanto, imunologicamente competentes camundongos C57BL / 6 pode ser usado. Enquanto usamos GL261, o protocolo a seguir podem ser usados ​​para a implantação e monitoramento de qualquer modelo de mouse intracraniana tumor. GL261 células foram projetadas para expressar estavelmente firefly luciferase (GL261-luc). Criamos também o mais brilhante linha GL261-luc2 celular por transfecção estável do gene luc2 expressa do promotor CMV. Camundongos C57BL/6-cBrd/cBrd/Cr (variante albina de C57BL / 6) do National Cancer Institute, Frederick, MD foram utilizados para eliminar a atenuação da luz causada por pele negra e pele. Com o uso de C57BL albino / 6 ratos; em imagem in vivo utilizando o espectro IVIS in vivo imo envelhecimento do sistema é possível a partir do dia da implantação (Caliper Life Sciences, Hopkinton, MA). O GL261 e GL261-luc-luc2 linhagens celulares mostraram o mesmo comportamento em vivo como o dos pais GL261 células. Algumas das características compartilhadas histológico presente em GBMS humano e este modelo de camundongo incluem: necrose tumoral, pseudopalisades, neovascularização, invasão, hipercelularidade e inflamação 1. Antes da implantação animais foram anestesiados com uma injeção intraperitoneal de cetamina (50 mg / kg), xilazina (5 mg / kg) e buprenorfina (0,05 mg / kg), colocado em um aparelho estereotáxico e foi feita uma incisão com um bisturi sobre o linha média do crânio. A burrhole foi feito posterior ao bregma 0,1 milímetros e 2,3 milímetros para a direita da linha média. A agulha foi inserida a uma profundidade de 0,4 milímetros de 3mm e retirado a uma profundidade de 2,6 milímetros. Dois mL de GL261-luc ou GL261-luc2 células (10 7 células / ml) foram infundidos ao longo de três minutos. O burrhole foi fechadacom bonewax ea incisão foi suturada. Após a implantação estereotáxica as células bioluminescentes são detectáveis ​​a partir do dia da implantação e do tumor podem ser analisadas usando o recurso de reconstrução de imagem 3D do instrumento IVIS Spectrum. Animais recebem uma injeção subcutânea de 150μg luciferina / kg de peso corporal 20 min antes da imagem. Carga tumoral é quantificado usando bioluminescência médio do tumor ao longo do tempo. Tumor-rolamento camundongos foram observados diariamente para avaliar a morbidade e foram sacrificados quando um ou mais dos seguintes sintomas estão presentes: insuficiência letargia, de deambular, postura arqueada, a falta de noivo, resultando em anorexia&gt; perda de 10% do peso. Os tumores eram evidentes em todos os animais em necropsia.

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

Implantação intracraniana de GL261 células em camundongos C57BL / 6 produz gliomas malignos que recapitulam muitas das características de glioblastoma multiforme humano. Usamos GL261 células expressando estavelmente luciferase para nos permitir usar<em&gt; In vivo</em&gt; Imagem para acompanhar a progressão do tumor. A cirurgia e 3D<em&gt; In vivo</em&gt; Imagens são demonstradas.

Implantação intracraniana com 3D subseqüentes In Vivo Bioluminescent imagem de Gliomas murino

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

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

Vídeo: 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.

Estabelecer xenoenxertos tumor intracraniano Cérebro com posterior análise do crescimento tumoral e resposta à terapia com imagem Bioluminescência

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

Neurociência

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.

Na imagem de bioluminescência vivo Tumor de mama Dynamics Hipóxia metástase do cancro cerebral em um modelo de rato

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.

Fluorescência Molecular Tomography para a imagem latente na Vivo de 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 ...

Pesquisa do câncer

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.

Implantação estereotáxica intracraniana e<em&gt; Em vivo</em&gt; Imaging Bioluminescent de xenoenxertos de tumores em um Sistema Modelo de Rato 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 de alta resolução<em&gt; In vivo</em&gt; Técnica de Imagem para estudar a resposta dinâmica de estruturas intracranianas ao crescimento do tumor e Terapêutica

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.

Um Glioblastoma Rato modelo ortotópico Manter restrições Cérebro parênquima físicas e Adequado para intravital dois fótons Microscopia

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

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Criando anatomicamente precisos e reprodutíveis intracraniana Xenoenxertos de tumores cerebrais humanos

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Rastreamento em tempo real de nanopartículas em tumores cerebrais usando 2P-IVM em camundongos

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 em destaque: Estratégias de imagem multimodal para otimizar a administração de medicamentos e a detecção precoce no tratamento do 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 Intravital de Dois Fótons de Glioblastoma em Modelo Murino

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

Implantação intracraniana com 3D subseqüentes In Vivo Bioluminescent imagem de Gliomas murino

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