Nos gustaría agradecer al Dr. Joshua B. Rubin por el generoso regalo de los plásmidos para el sistema de lentivirus, así como Rao Mahil por sugerencia útil en la preparación de las células GL261-luc. Agradecemos a los estudiantes Apoyo a la investigación del tumor cerebral (SSBTR), la Fundación Neurológica Barrow y la Fundación Wallace por su generoso apoyo. Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Cuidado de Animales institucional y el empleo de Hospital St. Joseph y el 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>

Acceso libre a este artículo es patrocinado por Caliper Life Sciences.

El inóculo de células se infunde a una profundidad de 2,6 mm de la superficie del cerebro después de la creación de una bolsa de 0,4 mm. Para asegurar la colocación adecuada y la profundidad de la aguja de una radiografía se puede tomar con un brazo-C o similar de rayos X de dispositivos de imagen intensificando, sin embargo, esto es opcional. Las complicaciones de la cirugía pueden surgir si el animal no está adecuadamente sedado, momento en el que el animal puede moverse durante la infusión de células. Esto puede causar fugas de la mezcla de células o sangrado de trayecto de la aguja. La fuga de las células causa el crecimiento ectópica de las células tumorales. También es importante no perforar el ventrículo que se puede hacer si el burrhole se hace por dentro de la de 2,3 mm se describe en el protocolo 4. La colocación correcta de la aguja y propagación de células se puso a prueba mediante la infusión de un ratón con dos l colorante azul de metileno y la disección de los tejidos cerebrales para verificar la ubicación del colorante infundido. En este protocolo se ha utilizado el espectro IVIS de imágenes in vivo del sistema y tque estar de software de imagen (v 4.0), diseñado para su uso con este instrumento. (Caliper Life Sciences). Cualquier sistema de imagen comparable en vivo y herramientas de análisis de imagen se puede utilizar para obtener resultados similares. Estos sistemas ofrecen algunas ventajas sobre las tradicionales imágenes por resonancia magnética (MRI) para seguir el crecimiento de un tumor intracraneal experimental. El más obvio es el costo relativo de los dos instrumentos - máquinas de resonancia magnética de los animales son mucho más caros y suelen requerir los servicios de un técnico de MRI especializada. Imágenes in vivo, como lo que se describe aquí se puede hacer por el usuario final. Los datos de la bioluminiscencia es cuantitativo, mientras que la cuantificación de los datos de la RM es mucho tiempo y un poco inexacta. Además, imágenes de resonancia magnética muestran edema y la inflamación, además de las células tumorales y puede ser difícil separar el efecto del tratamiento de tumores. Por estas razones, la obtención de precisas mediciones volumétricas del crecimiento del tumor puede ser un desafío. Bioluminiscencia requiere ATP, Por lo tanto, sólo las células tumorales de vida contribuyen a los datos del tamaño del tumor. A pesar de esto, hay algunas ventajas de la RM, si una máquina está disponible. Las células no tienen que estar etiquetados con un marcador bioluminiscente a visualizar por resonancia magnética. La capacidad de visualizar el edema peri-tumoral puede ser una ventaja para algunos protocolos experimentales. Una de las ventajas de ambas tecnologías es que el uso de uno no excluye el uso de la otra, por lo que se pueden obtener datos sobre el crecimiento del tumor, así como la presencia de peri-tumoral edema y la inflamación del mismo animal, cuando ambas tecnologías están disponibles para el investigador.

<table border="1"><tbody><tr><td> Nombre del reactivo o el suministro </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> GL261-luc2 Bioware ultra </td><td> Caliper Life Sciences </td><td> GL261-luc2 </td><td></td></tr><tr><td> Dulbecco Eagle modificado (DMEM) </td><td> Invitrogen </td><td> 10313039 </td><td></td></tr><tr><td> Geneticina (G418) </td><td> Gibco (Invitrogen) </td><td> 11811-023 </td><td></td></tr><tr><td> Suero fetal bovino (FCS) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Fosfato (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> C57BL/6-cBrd/cBrd/Cr Ratones </td><td> NCI-Frederick </td><td></td><td></td></tr><tr><td> AKWA lágrimas lubricantes oftálmicos pomada </td><td> Akorn Inc </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (clorhidrato de ketamina) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (clorhidrato de xilazina) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> Pequeños Animales de instrumentos estereotáxica </td><td> Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump con SYS-Micro4 Controller </td><td> Mundial de Instrumentos de Precisión </td><td> UMP3-1 </td><td> Si no está disponible, es posible inyectar manualmente </td></tr><tr><td> 10μl jeringa con aguja de calibre 26 biselados </td><td> Mundial de Instrumentos de Precisión </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison Pinzas </td><td> Mundial de Instrumentos de Precisión </td><td> 500092 </td><td></td></tr><tr><td> Penfield disector </td><td> Codman </td><td> 65-1015 </td><td></td></tr><tr><td> 16 g 1 ½ precisión Glide aguja </td><td> Beckton Dickinson and Company (BD) </td><td> 305198 </td><td></td></tr><tr><td> Manejar la hoja quirúrgica </td><td> BD </td><td> 371030 </td><td></td></tr><tr><td> Tamaño de la hoja 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> La cera de hueso </td><td> Medline </td><td> DYNJBW25 </td><td></td></tr><tr><td> Yodo povidona palillos de hisopo </td><td> Medline </td><td> MD93901 </td><td></td></tr><tr><td> D-luciferina de sal de potasio </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr><tr><td> Foráneo (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> Cualquier microscopio quirúrgico es suficiente </td></tr><tr><td> Xenogen IVIS del espectro con el sistema opcional de la anestesia </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

El ratón glioma 261 (GL261) es reconocido como un sistema en el modelo in vivo que resume muchas de las características del glioblastoma multiforme humano (GBM). La línea celular fue inducida inicialmente por inyección intracraneal de 3-metil-cholantrene en un C57BL / 6 cepa de ratón singénico 1, por lo tanto, inmunocompetentes ratones C57BL / 6 se puede utilizar. Mientras que utilizamos el GL261, el siguiente protocolo se puede utilizar para la implantación y seguimiento de cualquier modelo de ratón de tumores intracraneales. GL261 células fueron diseñados para expresar de forma estable la luciferasa de luciérnaga (GL261-luc). También hemos creado el más brillante GL261-luc2 de líneas celulares mediante transfección estable del gen luc2 expresa del promotor de CMV. Ratones C57BL/6-cBrd/cBrd/Cr (variante albina de C57BL / 6) del Instituto Nacional del Cáncer, Frederick, MD se utiliza para eliminar la atenuación de la luz causada por la piel y el pelo negro. Con el uso de C57BL albino / 6 ratones; imágenes in vivo utilizando el espectro IVIS en vivo imel envejecimiento del sistema es posible a partir del día de la implantación (Caliper Life Sciences, Hopkinton, MA). El GL261-luc y GL261-luc2 líneas celulares mostraron el mismo comportamiento in vivo como la de sus padres GL261 células. Algunas de las características compartidas histológicos presentes en GBMs humanos y este modelo de ratón son: necrosis tumoral, pseudopalisades, la neovascularización, la invasión, hipercelularidad, y la inflamación 1. Antes de la implantación de los animales fueron anestesiados con una inyección intraperitoneal de ketamina (50 mg / kg), xilacina (5 mg / kg) y buprenorfina (0,05 mg / kg), colocado en un aparato estereotáctico y una incisión se realizó con un bisturí sobre la la línea media del cráneo. A burrhole se hizo posterior a la de 0,1 mm y 2,3 mm bregma a la derecha de la línea media. Una aguja se inserta a una profundidad de 0,4 mm y 3 mm de retirarse a una profundidad de 2,6 mm. Dos l de GL261-luc o GL261-luc2 células (10 7 células / ml) fueron infundidos en el transcurso de 3 minutos. El burrhole se cerrócon bonewax y la incisión fue suturada. Tras el implante estereotáctico de las células bioluminiscentes son detectables a partir del día de la implantación y el tumor puede ser analizada utilizando la función de reconstrucción de imágenes 3D de los instrumentos de espectro IVIS. Los animales reciben una inyección subcutánea de 150μg luciferina / kg de peso corporal de 20 minutos antes de la imagen. La carga tumoral se cuantifica utilizando bioluminiscencia tumor significa que el paso del tiempo. Los ratones portadores del tumor fueron observados diariamente para evaluar la morbilidad y fueron sacrificados cuando uno o más de los siguientes síntomas están presentes: letargo, falta de deambular, postura encorvada, falta de novio, anorexia resulta en la pérdida&gt; 10% del peso. Los tumores fueron evidentes en todos los animales en la 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

Implantación intracraneal de GL261 células en ratones C57BL / 6 produce los gliomas malignos que recapitular muchas de las características del glioblastoma multiforme humano. Hemos utilizado GL261 células que expresan establemente la luciferasa que nos permita utilizar<em&gt; En vivo</em&gt; Imágenes para seguir la progresión del tumor. La cirugía y 3D<em&gt; En vivo</em&gt; Imágenes se muestran.

La implantación intracraneal con posterioridad 3D En Vivo Bioluminiscentes imagen 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 ...

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

Research

JoVE Journal

Medicine

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

20.3K vistas.  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

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.

Murino hepática Bioluminiscente modelo tumoral

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

Investigación

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.

En vivo doble sustrato bioluminiscentes imagen

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.

Implantación de un manguito de la carótida para provocar esfuerzo cortante aterosclerosis inducida en ratones

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.

La implantación estereotáctica intracraneal y<em&gt; En vivo</em&gt; Radiología bioluminiscente de xenoinjertos de tumores en un sistema modelo de ratón de 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.

Un modelo murino de la implantación de un stent en la arteria carótida para el Estudio de la reestenosis

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.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

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; En Vivo</em&gt; Healing Multimodal Imaging de Carcinogénesis y Evaluación de la herida y la inflamación intestinal

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 la arteria femoral Lesiones Alambre con implantación de un perivascular Drug Delivery Patch

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.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

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.

Visualización Cuantitativa del Infiltrado de Leucocitos en un Modelo Murino de Miocarditis Fulminante por Microscopía de Hoja Ligera

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