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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Medicine

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

20.3K 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

Murine Bioluminescent Hepatic Tumour Model

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are administered specifically to the liver to induce a localised liver tumour, via mobilisation of the spleen and splitting into two, leaving intact the vascular pedicle for each half of the spleen. Lewis lung carcinoma cells that constitutively express the firefly luciferase gene (luc1) are inoculated into one hemi-spleen which is then resected 10 minutes later. The other hemi-spleen is left intact and returned to the abdomen. Liver tumour growth can be monitored by bioluminescence imaging using the IVIS whole body imaging system. Quantitative imaging of tumour growth using IVIS provides precise quantitation of viable tumour cells. Tumour cell death and necrosis due to drug treatment is indicated early by a reduction in the bioluminescent signal. This mouse model allows for investigating the mechanisms underlying metastatic tumour-cell survival and growth and can be used for the evaluation of therapeutics of liver metastasis. 

This article describes a procedure for the induction of orthotopic bioluminescent liver tumours in mice, and subsequent analysis of tumour growth confined to the liver using live whole body luminescence imaging.

Modelo de tumor hepático murino Bioluminescent

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

Medicina

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

In vivo dupla Substrato Bioluminescent imagem

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Implantation of a Carotid Cuff for Triggering Shear-stress Induced Atherosclerosis in Mice

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce atherosclerosis (reviewed in 1 and 2). The role of shear stress has been extensively studied in vitro investigating the influence of flow dynamics on cultured endothelial cells 1,3,4 and in vivo in larger animals and humans 1,5,6,7,8. However, highly reproducible small animal models allowing systematic investigation of the influence of shear stress on plaque development are rare. Recently, Nam et al. 9 introduced a mouse model in which the ligation of branches of the carotid artery creates a region of low and oscillatory flow. Although this model causes endothelial dysfunction and rapid formation of atherosclerotic lesions in hyperlipidemic mice, it cannot be excluded that the observed inflammatory response is, at least in part, a consequence of endothelial and/or vessel damage due to ligation.
In order to avoid such limitations, a shear stress modifying cuff has been developed based upon calculated fluid dynamics, whose cone shaped inner lumen was selected to create defined regions of low, high and oscillatory shear stress within the common carotid artery 10. By applying this model in Apolipoprotein E (ApoE) knockout mice fed a high cholesterol western type diet, vascular lesions develop upstream and downstream from the cuff. Their phenotype is correlated with the regional flow dynamics 11 as confirmed by in vivo Magnetic Resonance Imaging (MRI) 12: Low and laminar shear stress upstream of the cuff causes the formation of extensive plaques of a more vulnerable phenotype, whereas oscillatory shear stress downstream of the cuff induces stable atherosclerotic lesions 11. In those regions of high shear stress and high laminar flow within the cuff, typically no atherosclerotic plaques are observed.
 
In conclusion, the shear stress-modifying cuff procedure is a reliable surgical approach to produce phenotypically different atherosclerotic lesions in ApoE-deficient mice.

The constricting cuff presented in this article is designed to induce atherosclerosis in the murine common carotid artery. Due to the conical shape of its inner lumen the implanted cuff generates well-defined regions of low, high and oscillatory shear stress triggering the development of atherosclerotic lesions of different inflammatory phenotypes.

Implantação de um Cuff Carotid para Triggering Shear-estresse aterosclerose induzida em camundongos

It is widely accepted that alterations in vascular shear stress trigger the expression of inflammatory genes in endothelial cells and thereby induce ...

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 Murine Model of Stent Implantation in the Carotid Artery for the Study of Restenosis

Despite the considerable progress made in the stent development in the last decades, cardiovascular diseases remain the main cause of death in western countries. Beside the benefits offered by the development of different drug-eluting stents, the coronary revascularization bears also the life-threatening risks of in-stent thrombosis and restenosis. Research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes. Here, we describe a rapid and accessible procedure of stent implantation in mouse carotid artery, which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings.

A model of stent implantation in mouse carotid artery is described. Compared to other similar methods, this procedure is very rapid, simple and accessible, offering the possibility to study in a convenient way the vascular wall reaction to different drug-eluting stents and the molecular mechanisms of restenosis.

Um modelo murino de implante de stent na artéria carótida para o Estudo da reestenose

Despite  the considerable progress made in the stent development in the last decades,  cardiovascular diseases remain the main cause of death in western ...

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 ...

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions preclinically. However, valid assessment of pathological alterations often requires histological analysis, and when performed ex vivo, necessitates death of the animal. Therefore in conventional experimental settings, intra-individual follow-up examinations are rarely possible. Thus, development of murine endoscopy in live mice enables investigators for the first time to both directly visualize the gastrointestinal mucosa and also repeat the procedure to monitor for alterations. Numerous applications for in vivo murine endoscopy exist, including studying intestinal inflammation or wound healing, obtaining mucosal biopsies repeatedly, and to locally administer diagnostic or therapeutic agents using miniature injection catheters. Most recently, molecular imaging has extended diagnostic imaging modalities allowing specific detection of distinct target molecules using specific photoprobes. In conclusion, murine endoscopy has emerged as a novel cutting-edge technology for diagnostic experimental in vivo imaging and may significantly impact on preclinical research in various fields.

Murine Endoscopy for In Vivo Multimodal Imaging of Carcinogenesis and Assessment of Intestinal Wound Healing and Inflammation

Small animal imaging techniques allow serial diagnostic examinations and therapeutic interventions in vivo. Recently, the scope of applications has significantly widened and currently includes assessment of colonic tumor development, wound healing and monitoring of inflammation. This protocol illustrates these diverse potential applications of murine endoscopy.

Murino Endoscopia para<em&gt; In Vivo</em&gt; Cura Multimodal imagem de Carcinogênese e Avaliação de Intestinal ferida e inflamação

Mouse models are widely used to study pathogenesis of human diseases and to evaluate diagnostic procedures as well as therapeutic interventions ...

Murine Model of Femoral Artery Wire Injury with Implantation of a Perivascular Drug Delivery Patch

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. While these therapies lead to the rapid restoration of blood flow, these technologies remain limited by restenosis in the case of bare metal stents and angioplasty, or reduced healing and possibly enhanced risk of thrombosis in the case of drug eluting stents. A key pathophysiological mechanism in the formation of restenosis is intimal hyperplasia caused by the activation of vascular smooth muscle cells and inflammation due to arterial stretch and injury. Surgeries that induce arterial injury in genetically modified mice are useful for the mechanistic study of the vascular response to injury but are often technically challenging to perform in mouse models due to the their small size and lack of appropriate sized devices. We describe two approaches for a surgical technique that induces endothelial denudation and arterial stretch in the femoral artery of mice to produce robust neointimal hyperplasia. The first approach creates an arteriotomy in the muscular branch of the femoral artery to obtain vascular access. Following wire injury this arterial branch is ligated to close the arteriotomy. A second approach creates an arteriotomy in the main femoral artery that is later closed through localized cautery. This method allows for vascular access through a larger vessel and, consequently, provides a less technically demanding procedure that can be used in smaller mice. Following either method of arterial injury, a degradable drug delivery patch can be placed over or around the injured artery to deliver therapeutic agents.

We describe a surgical technique that produces wire injury in the femoral artery of mice to induce neointimal hyperplasia to serve as a model testing system for the perivascular delivery of therapeutic compounds for the inhibition of restenosis.

Modelo murino de artéria femoral Injury fio com a implantação de uma droga patch Entrega perivascular

Percutaneous interventions including balloon angioplasty and stenting have been used to restore blood flow in vessels with occlusive vascular disease. ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depleção de células do rato de xenoenxertos de tumor humano Melhora Significativamente Análise Downstream de células alvo

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Visualização Quantitativa de Infiltrado de Leucócitos em um Modelo Murino de Miocardite Fulminante por Microscopia de Folha de Luz

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...