Nous tenons à remercier le Dr Joshua B. Rubin pour le don généreux de plasmides pour le système de lentivirus ainsi que Mahil Rao pour suggestion utile à la préparation des cellules GL261-Luc. Nous remercions les étudiants Soutien recherche sur les tumeurs cérébrales (SSBTR), Barrow Neurological La Fondation et la Fondation Wallace pour leur généreux soutien. Expériences sur les animaux ont été effectuées en conformité avec les directives et les règlements énoncés par le soin des animaux et du Comité institutionnel utilisation de l&#39;Hôpital St. Joseph et Medical Center.

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

Accès gratuit à cet article est sponsorisé par Caliper Life Sciences.

L&#39;inoculum cellulaire est perfusée à une profondeur de 2,6 mm de la surface du cerveau après la création d&#39;une poche de 0,4 mm. Pour assurer un placement correct et la profondeur de l&#39;aiguille d&#39;une radiographie peut être prise à l&#39;aide d&#39;un C-Arm ou autre dispositif de l&#39;image aux rayons X s&#39;intensifie, mais ceci est optionnel. Les complications de la chirurgie peuvent survenir si l&#39;animal n&#39;est pas correctement sous sédation, à quel point l&#39;animal peut se déplacer pendant la perfusion de cellules. Cela peut provoquer une fuite de mélange de cellules ou des saignements de trajet de l&#39;aiguille. Fuite des cellules provoque une croissance ectopique de cellules tumorales. Il est également important de ne pas percer le ventricule qui peut être fait si le burrhole est fait en dedans de la 2,3 mm décrite dans le protocole 4. Placement correct de la propagation de seringues et de cellules a été testé en infusant une souris avec deux colorants méthylène ul bleu et la dissection du tissu cérébral pour vérifier l&#39;emplacement du colorant infusé. Dans ce protocole, nous avons utilisé le spectre IVIS l&#39;imagerie in vivo du système et tIl Living logiciel Image (v 4.0) conçu pour une utilisation avec cet instrument. (Caliper Life Sciences). Toute comparables dans le système d&#39;imagerie in vivo et des outils d&#39;analyse d&#39;image peut être utilisée pour obtenir des résultats similaires. Ces systèmes fournissent certains avantages sur l&#39;imagerie par résonance magnétique classique (IRM) pour suivre la croissance d&#39;une tumeur intracrânienne expérimentale. Le plus évident est le coût relatif des deux instruments - machines animales IRM sont beaucoup plus chers et nécessitent généralement des services d&#39;un technicien qualifié IRM. L&#39;imagerie in vivo, comme ce qui était décrit ici peut être fait par l&#39;utilisateur final. Les données quantitatives bioluminescence est, tandis que la quantification des données de l&#39;IRM prend beaucoup de temps et quelque peu inexacte. Par ailleurs, les images IRM montrent un œdème et l&#39;inflammation, en plus de cellules tumorales et il peut être difficile de séparer la tumeur de l&#39;effet du traitement. Pour ces raisons, l&#39;obtention de mesures précises de la tumeur en pleine croissance volumétrique peut être un défi. La bioluminescence nécessite de l&#39;ATP, Donc que les cellules tumorales de vie contribuent à la taille de la tumeur de données. Malgré cela, il ya certains avantages à l&#39;IRM si une machine est disponible. Les cellules n&#39;ont pas à être marqué avec un marqueur bioluminescent être visualisés par IRM. La capacité à visualiser péri-tumoral oedème peut être un avantage pour certains protocoles expérimentaux. A force de ces deux technologies est que l&#39;aide l&#39;un n&#39;empêche pas l&#39;utilisation de l&#39;autre, afin que les données peuvent être obtenus sur la croissance de la tumeur ainsi que la présence de zones péri-tumorale œdème et l&#39;inflammation du même animal, lorsque les deux technologies sont disponibles pour le chercheur.

<table border="1"><tbody><tr><td> Nom du réactif ou de la fourniture </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> GL261-luc2 Bioware Ultra </td><td> Caliper Life Sciences </td><td> GL261-luc2 </td><td></td></tr><tr><td> Eagle modifié par Dulbecco (DMEM) </td><td> Invitrogen </td><td> 10313039 </td><td></td></tr><tr><td> Généticine (G418) </td><td> Gibco (Invitrogen) </td><td> 11811-023 </td><td></td></tr><tr><td> Sérum de veau fœtal (SVF) </td><td> Invitrogen </td><td> 26140079 </td><td></td></tr><tr><td> Phosphate saline tamponnée (PBS) </td><td> Invitrogen </td><td> 70011044 </td><td></td></tr><tr><td> Souris C57BL/6-cBrd/cBrd/Cr </td><td> NCI-Frederick </td><td></td><td></td></tr><tr><td> AKWA larmes onguent ophtalmique Lubrifiant </td><td> Akorn Inc </td><td> 17478-062-35 </td><td></td></tr><tr><td> Ketaset (chlorhydrate de kétamine) </td><td> Wyeth </td><td> 11570775 </td><td></td></tr><tr><td> Sedazine (chlorhydrate de xylazine) </td><td> Wyeth </td><td> 10031894 </td><td></td></tr><tr><td> Petit instrument stéréotaxique animaux </td><td> Kopf Instruments </td><td> 900 </td><td></td></tr><tr><td> UltraMicroPump par SYS-Micro4 Controller </td><td> Instruments de précision du monde </td><td> UMP3-1 </td><td> S&#39;il n&#39;est pas disponible, il est possible d&#39;insuffler manuellement </td></tr><tr><td> Seringue avec 10 ul de calibre 26 biseautés aiguille </td><td> Instruments de précision du monde </td><td> SGE010RNS </td><td></td></tr><tr><td> Adison forceps </td><td> Instruments de précision du monde </td><td> 500092 </td><td></td></tr><tr><td> Penfield Dissecteur </td><td> Codman </td><td> 65-1015 </td><td></td></tr><tr><td> 16g 1 ½ Précision Glide aiguille </td><td> Beckton, Dickinson and Company (BD) </td><td> 305198 </td><td></td></tr><tr><td> Poignée lame chirurgicale </td><td> BD </td><td> 371030 </td><td></td></tr><tr><td> Taille 15 Lame </td><td> BD </td><td> 371315 </td><td></td></tr><tr><td> 4-0 suture Vicryl </td><td> Ethicon </td><td> VCP496G </td><td></td></tr><tr><td> La cire d&#39;os </td><td> Medline </td><td> DYNJBW25 </td><td></td></tr><tr><td> Bâtons Swab Povidine-iode </td><td> Medline </td><td> MD93901 </td><td></td></tr><tr><td> Sel de potassium D-luciférine </td><td> Caliper Life Sciences </td><td> 122796 </td><td></td></tr><tr><td> Forane (isoflurane) </td><td> Baxter </td><td> 1001936060 </td><td></td></tr><tr><td> OPMI Pentero Microscope </td><td> Carl Zeiss, Inc </td><td></td><td> Toute microscope chirurgical suffira </td></tr><tr><td> Xenogen IVIS spectre avec un système d&#39;anesthésie option </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

La souris gliome 261 (GL261) est reconnu comme un système modèle in vivo qui récapitule plusieurs des caractéristiques du glioblastome humain (GBM). La lignée cellulaire a été initialement provoquée par l&#39;injection intracrânienne de 3-méthyl-cholantrene dans un C57BL / 6 souche de souris syngéniques 1; donc immunologiquement compétentes souris C57BL / 6 peut être utilisé. Bien que nous utilisions GL261, le protocole suivant peut être utilisé pour l&#39;implantation et le suivi de tous les modèles tumoraux de souris intracrânienne. GL261 cellules ont été conçues pour exprimer de façon stable la luciférase de luciole (GL261-Luc). Nous avons également créé le plus lumineux de ligne GL261-luc2 cellule par transfection stable du gène exprimé luc2 du promoteur CMV. Souris C57BL/6-cBrd/cBrd/Cr (variante albinos de C57BL / 6) de l&#39;Institut national du cancer, Frederick, MD ont été utilisés pour éliminer le atténuation de la lumière causée par la peau noire et la fourrure. Avec l&#39;utilisation de C57BL albinos / 6 souris; l&#39;imagerie in vivo en utilisant le spectre IVIS in vivo imle vieillissement du système est possible à partir du jour de l&#39;implantation (Caliper Life Sciences, Hopkinton, MA). Le GL261-Luc et GL261-luc2 lignées cellulaires ont montré le même comportement in vivo que le GL261 parentale cellules. Certaines des caractéristiques histologiques partagé présent dans GBM humain et ce modèle de souris comprennent: nécrose tumorale, pseudopalisades, néovascularisation, une invasion, une hypercellularité et l&#39;inflammation 1. Avant l&#39;implantation d&#39;animaux ont été anesthésiés par une injection intrapéritonéale de kétamine (50 mg / kg), la xylazine (5 mg / kg) et la buprénorphine (0,05 mg / kg), placé dans un appareil de stéréotaxie et une incision a été faite avec un bistouri au cours de la ligne médiane du crâne. Un burrhole postérieure a été faite à l&#39;0.1mm 2.3mm bregma et à droite de la ligne médiane. Une aiguille est insérée à une profondeur de 0,4 mm 3mm et retiré à une profondeur de 2,6 mm. Deux ul du GL261-Luc ou GL261-luc2 cellules (10 7 cellules / ml) ont été perfusés au cours de 3 minutes. Le burrhole a été ferméavec bonewax et l&#39;incision a été suturée. Après l&#39;implantation stéréotaxique les cellules bioluminescentes sont détectables à partir du jour de l&#39;implantation et la tumeur peut être analysée en utilisant la fonction image 3D de reconstruction de l&#39;instrument Spectrum IVIS. Les animaux reçoivent une injection sous-cutanée de 150μg luciférine / kg de poids corporel 20 min avant l&#39;imagerie. Charge tumorale est quantifiée en utilisant la bioluminescence tumorale moyenne au cours du temps. Souris porteuses de tumeurs ont été observés quotidiennement pour évaluer la morbidité et ont été euthanasiés lorsque un ou plusieurs des symptômes suivants sont présents: la léthargie, incapacité à déambuler, une posture voûtée, non marié, l&#39;anorexie entraîne une perte&gt; 10% du poids. Les tumeurs ont été évident dans tous les animaux présents sur l&#39;autopsie.

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

L&#39;implantation intracrânienne d&#39;GL261 cellules en souris C57BL / 6 produit gliomes malins qui récapitulent plusieurs des caractéristiques de glioblastome humain. Nous avons utilisé des cellules exprimant de façon stable GL261 luciférase pour nous permettre d&#39;utiliser<em&gt; In vivo</em&gt; Imagerie pour suivre la progression tumorale. La chirurgie et la 3D<em&gt; In vivo</em&gt; Imagerie sont démontrés.

Implantation intracrânienne avec 3D ultérieures In Vivo Bioluminescent imagerie des gliomes murin

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 vues.  Barrow Neurological Institute of St. Joseph’s Hospital and Medical Center. 中文

Vidéo: 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.

Modèle murin Bioluminescent tumeurs hépatiques

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

Recherche

Médecine

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 double Substrat 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 ...

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.

Implantation d&#39;une manchette carotidienne pour le déclenchement de cisaillement-stress athérosclérose induite chez la souris

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.

Implantation stéréotaxique intracrânienne et<em&gt; In vivo</em&gt; Imagerie bioluminescente de xénogreffes tumorales dans un système de modèle de souris de glioblastome 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 modèle murin d&#39;implantation d&#39;une endoprothèse dans l&#39;artère carotide pour l&#39;étude de la resténose

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 haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

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.

Murin endoscopie pour<em&gt; In Vivo</em&gt; Guérison multimodale d&#39;imagerie de la cancérogenèse et de l&#39;évaluation de la plaie intestinale et l&#39;inflammation

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.

Modèle murin de l&#39;artère fémorale fil des blessures à l&#39;implantation d&#39;un périvasculaire 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.

Appauvrissement des cellules de souris de xénogreffes de tumeurs humaines améliore de manière significative l&#39;analyse en aval des cellules cibles

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.

Visualisation quantitative de l&#39;infiltration de leucocytes dans un modèle murin de myocardite fulminante par microscopie à feuilles lumineuses

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