Les auteurs tiennent à remercier Christiane Pahrmann pour sa contribution. Un merci spécial à Ethicon, Norderstedt, Hambourg (Allemagne) pour fournir du matériel de suture. Financement Sonja Schrepfer a reçu une subvention de recherche de la Deutsche Forschungsgemeinschaft (DFG) (SCHR992 / 3 1 et SCHR992/4-1).] Le travail a été soutenu par le développement de carrière ISHLT Shumway Grant 2010 et la recherche de financement Falk (Université de Stanford).

1. L&#39;artère mammaire interne (IMA) Préparation <ol><li> L&#39;endothélium artériel est dénudée par le passage d&#39;un cathéter 2-françaises Fogarty embolectomie artérielle (Baxter Healthcare, Deerfield, IL, USA). Le cathéter est tiré à travers toute la longueur du navire à deux reprises pour assurer la lésion de l&#39;endothélium. </li><li> Utilisez n&#39;importe quel stent humaine de la longueur de 8mm et 2,5 mm à 3 mm de diamètre (par exemple Translumina, Yukon stent). ATTENTION: Le diamètre du stent ne doit pas dépasser le diamètre du vaisseau de plus de 10% pour éviter une sténose pré-et post-stent. ATTENTION: Ne mettez pas la longueur du stent dans la même étude. </li><li> Déployer le stent en utilisant la pression appropriée ballonnet (qui est indiquée sur le paquet de stent) pour atteindre le diamètre désiré. </li><li> Magasin stentées IMA dans le 4 ° C RPMI + héparine (500 IE/10 ml) sur la glace jusqu&#39;à la transplantation. </li></ol> 2. Préparation des animaux RNU Nu (Crl: NIH-Foxn1rnu) chez les rats (300-350 g) sont housed dans des conditions classiques dans scantainer ventilé armoires, une nourriture standard chez le rat et chow autoclavé libitum ad &#39;eau. <ol><li> Anesthésier rat avec de l&#39;isoflurane (2,5-3%) en utilisant une chambre d&#39;induction. </li><li> Raser les poils abdominaux et placez le rat sur son dos et placer un masque sur son nez et la bouche pour maintenir l&#39;anesthésie. </li><li> Désinfecter la région abdominale en utilisant largement Provo-iode, la prochaine utilisation d&#39;éthanol à 80% - répétez cette étape deux fois. Vérifiez réflexes pinçant les pattes de derrière pour être sûr que le rat anesthésié est suffisante. </li><li> En vertu de vue microscopique, effectuer une laparotomie médiane supérieure pour exposer l&#39;aorte abdominale. </li><li> Placez les intestins dans un gant de solution saline hydratée. Pliez le gant autour des intestins pour prévenir la perte d&#39;humidité. </li><li> Disséquer l&#39;aorte de la région de infrarénale à la bifurcation, attention à ne pas causer des dommages sur les branches des vaisseaux. </li><li> Utilisez des pinces micro pour arrêter le flux sanguin aortique. Placez lePince proximale en premier, suivi par la pince distale. </li><li> Supprimer une application. 0,5 au 0,7 mm segment aortique et rincer l&#39;aorte restante avec de l&#39;héparine (200 unités). </li><li> Prenez l&#39;IMA stent et le raccourcir à la longueur adéquate et le positionner dans l&#39;espace. </li><li> Connectez l&#39;IMA à l&#39;aorte destinataire, en exécutant des sutures en utilisant 8-0 prolène suture (Ethicon, Norderstedt, Allemagne). </li><li> Ouvrez avec précaution la première pince crânienne puis la pince caudale. </li><li> Il doit être une impulsion visible dans la transplanté IMA et à l&#39;extrémité distale de l&#39;aorte. </li><li> Placez les intestins dans l&#39;abdomen. </li><li> Rincer l&#39;abdomen avec préchauffé une solution saline stérile. </li><li> Fermez la couche musculaire de la paroi abdominale à l&#39;aide 6-0 prolène sutures en cours d&#39;exécution (Ethicon, Norderstedt, Allemagne). </li><li> Alors que le rat est encore en anesthésie, injecter 4-5 mg / kg carprofène sous-cutanée. </li><li> Administrer une analgésie post-opératoire, le cas échéant (par exemple carprofène ou méloxicam) pendant 3 jours après SurgERY. </li></ol> 3. Les résultats représentatifs Pour l&#39;histologie, les échantillons ont été fixés dans du formol à 4%, déshydraté dans une série graduée de l&#39;alcool, et se sont infiltrés dans un mélange (MMA I) de méthacrylate de méthyle de 80% et 20% de dibutylphtalate pour un jour, je MMA avec 1% de peroxyde de benzoyle sec pour un jour, et de MMA I avec du peroxyde de benzoyle 3% de matière sèche (MMA III) pendant 1-2 jours à 4 ° C. Par la suite, les échantillons ont été polymérisés à l&#39;état frais MMA III en flacons de verre dans un bain d&#39;eau sur une base pré-polymérisé. Polymérisation lente a été réalisée en conservant les flacons à 26 ° C pendant la nuit, ce qui augmente la température à 28 ° C le lendemain matin, puis en augmentant graduellement la température de 0,5 ° C de plus de 12 h jusqu&#39;à ce que la polymérisation s&#39;est produite. Les blocs polymérisés ont été sectionnés à 5 micron d&#39;épaisseur en utilisant un MICROM HM 360 microtome équipé d&#39;un couteau en métal dur. Pour identifier l&#39;origine des cellules proliférantes (figure 1), diapositives étaient Stained avec des anticorps identifiant soit le vert rat protéine fluorescente (GFP) ou MHC-I et cellules musculaires lisses humaines. Pour ces études, l&#39;homme IMA a été incubée avec le gène rapporteur GFP nuit en utilisant des particules lentiviraux pour la transduction stable de cellules IMA. Cellules filles de séparation d&#39;origine humaine ont pu être identifiés en exprimant la GFP. Après déparaffinage, la chaleur épitope est effectuée par chauffage des diapositives dans une solution de récupération d&#39;antigène en utilisant un bateau à vapeur. L&#39;image-Ti FX signal de activateur peut être utilisé pour l&#39;étape de blocage. Les cellules d&#39;origine humaine sont identifiés en utilisant des anticorps monoclonaux de souris contre la GFP (1:100 dilué dans du diluant l&#39;anticorps primaire (Dako)), et encore marqué par de chèvre anti-IgG de souris, Alexa Fluor 488 (1:1000 dilué dans du diluant anticorps secondaire ). Les cellules musculaires lisses ont été marqués avec le lapin polycolonal anti-muscle lisse α-actine (1:100 dilué dans du diluant anticorps primaire), suivie par de chèvre anti-IgG de lapin, Alexa Fluor 555 (1:1000 dilbués sur place en diluants anticorps secondaires). Chaque étape d&#39;incubation anticorps est effectuée à 37 ° C pendant 1 heure avec trois fois PBS lavage entre les deux. Les noyaux sont colorés au DAPI pendant 10 minutes. Après le montage des diapositives à l&#39;aide Prolongez réactif antifading Or, les échantillons ont été analysés en utilisant la microscopie confocale. <img alt="Figure 1" src="/files/ftp_upload/3663/3663fig1.jpg" /> Figure 1. les cellules néo-intimale comme cellules musculaires lisses humaines A:. vert = anti GFP, le marquage de cellules d&#39;origine humaine; rouge = anti-humain de l&#39;actine des cellules musculaires lisses; bleu = DAPI, l&#39;identification des noyaux de cellules B: Vert = rat anti CMH-I, l&#39;étiquetage des cellules d&#39;origine chez le rat; rouge = anti-humain de l&#39;actine des cellules musculaires lisses; bleu = DAPI, l&#39;identification des noyaux de cellules. Les cellules en prolifération sont identifiés comme les cellules musculaires lisses et positives pour la GFP, mais négatif pour le rat molécule MHC-I. Par conséquent, les cellules proliférantes sont d&#39;origine humaine.

<ol>
	<li>Deuse, T., Ikeno, F., Robbins, R. C., Schrepfer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+In-Stent+Restenosis:+An+Inexpensive,+Reliable,+and+Rapid+Preclinical+Model.">Imaging In-Stent Restenosis: An Inexpensive, Reliable, and Rapid Preclinical Model.</a> J. Vis. Exp. (31), e1346 (2009).</li><li>Oyamada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-iliac+rat+aorta+stenting:+a+novel+high+throughput+preclinical+stent+model+for+restenosis+and+thrombosis.">Trans-iliac rat aorta stenting: a novel high throughput preclinical stent model for restenosis and thrombosis.</a> J. Surg. Res. 166, e91-e95 (2011).</li><li>Chamberlain, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mouse+model+of+in+situ+stenting.">A novel mouse model of in situ stenting.</a> Cardiovascular research. 85, 38-44 (2010).</li><li>Deuse, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introducing+the+first+polymer-free+leflunomide+eluting+stent.">Introducing the first polymer-free leflunomide eluting stent.</a> Atherosclerosis. 200, 126-134 (2008).</li><li>Finn, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+healing+after+sirolimus,+paclitaxel,+and+bare+metal+stent+placement+in+combination+with+peroxisome+proliferator-activator+receptor+gamma+agonists:+requirement+for+mTOR/Akt2+in+PPARgamma+activation.">Differential healing after sirolimus, paclitaxel, and bare metal stent placement in combination with peroxisome proliferator-activator receptor gamma agonists: requirement for mTOR/Akt2 in PPARgamma activation.</a> Circulation research. 105, 1003-1012 (2009).</li><li>Tellez, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+bare+metal+stent+implantation+in+homozygous+LDL+receptor+deficient+swine+induces+a+neointimal+formation+pattern+similar+to+humans.">Coronary bare metal stent implantation in homozygous LDL receptor deficient swine induces a neointimal formation pattern similar to humans.</a> Atherosclerosis. 213, 518-524 (2010).</li><li>Suzuki, Y., Yeung, A. C., Ikeno, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-clinical+animal+model+in+the+translational+research+of+interventional+cardiology.">The pre-clinical animal model in the translational research of interventional cardiology.</a> JACC Cardiovasc. Interv. 2, 373-383 (2009).</li><li>Holt, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intimal+proliferation+in+an+organ+culture+of+human+internal+mammary+artery.">Intimal proliferation in an organ culture of human internal mammary artery.</a> Cardiovascular research. 26, 1189-1194 (1992).</li><li>Swanson, N., Javed, Q., Hogrefe, K., Gershlick, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+internal+mammary+artery+organ+culture+model+of+coronary+stenting:+a+novel+investigation+of+smooth+muscle+cell+response+to+drug-eluting+stents.">Human internal mammary artery organ culture model of coronary stenting: a novel investigation of smooth muscle cell response to drug-eluting stents.</a> Clin. Sci. (Lond). 103, 347-353 (2002).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

Bien que différents dans des modèles in vivo de recherche sont en vigueur pour enquêter sur le développement de l&#39;hyperplasie intimale après pose de stent, ces modèles sont encore face à des obstacles à surmonter traductionnelles. En outre, les modèles animaux de grande taille sont les conditions de logement coûteux et spéciaux ainsi que du matériel chirurgical n&#39;est pas disponible pour tous les laboratoires. L&#39;utilisation d&#39;un IMA humain pour é...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Le numéro de catalogue </td><td> Commentaires </td></tr><tr><td> 2 sonde de Fogarty française </td><td> Baxter Healthcare, Deerfield, IL, Etats-Unis </td><td> 120602F </td><td></td></tr><tr><td> Yukon stent </td><td> Translumina GmbH, Hechingen, Allemagne </td><td></td><td> Utilisez le stent de votre choix en fonction de votre protocole de l&#39;étude </td></tr><tr><td> Milieu RPMI </td><td> Biochrom </td><td> Nr.F1275 </td><td></td></tr><tr><td> héparine </td><td> Baxter </td><td> 2B0953 </td><td></td></tr><tr><td> isoflurane </td><td> Abbé </td><td> B506 </td><td></td></tr><tr><td> Provo-iode </td><td> Betadine Puredue Pharma </td><td> EAN: 5995327165830 </td><td></td></tr><tr><td> 80% éthanol </td><td> Geyer </td><td> ETV 80/0500 </td><td></td></tr><tr><td> Micro pince </td><td> Harvard Apparatus </td><td> PY2-61-0186 </td><td></td></tr><tr><td> Les sutures 8-0 </td><td> Johnson &amp; Johnson, </td><td> 2808G </td><td></td></tr><tr><td> Les sutures 6-0 </td><td> Johnson &amp; Johnson, </td><td> 1698 H </td><td></td></tr><tr><td> Carprofène </td><td> Vet Feizer </td><td> PZN: 0110208 </td><td></td></tr><tr><td> Metamizol </td><td> Ratiopharm </td><td></td><td></td></tr><tr><td> Target Retrieval Solution, pH 9 </td><td> Dako </td><td> S2368 </td><td></td></tr><tr><td> Image-iT FX signal de enhancer </td><td> Invitrogen </td><td> I36933 </td><td></td></tr><tr><td> monoclonal de souris anti-GFP anticorps </td><td> Couleurs de la vie BD </td><td> 632381 </td><td></td></tr><tr><td> diluant l&#39;anticorps primaire </td><td> Dako </td><td> S3022 </td><td></td></tr><tr><td> de chèvre anti-IgG de souris, Alexa Fluor 488 </td><td> Invitrogen </td><td> A11017 </td><td></td></tr><tr><td> diluant l&#39;anticorps secondaire </td><td> Dako </td><td> S0809 </td><td></td></tr><tr><td> lapin polycolonal anti-muscle lisse α-actine </td><td> Abcam, </td><td> ab5694 </td><td></td></tr><tr><td> de chèvre anti-IgG de lapin, Alexa Fluor 555 </td><td> Invitrogen </td><td> A21430 </td><td></td></tr><tr><td> Prolongez réactif antifading Or </td><td> Invitrogen </td><td> P36930 </td><td></td></tr></tbody></table>

human internal mammary artery (ima) transplantation and stenting: a human model to study the development of in-stent restenosis

Préclinique in vivo dans des modèles de recherche pour étudier les processus pathobiologiques et physiopathologiques dans le développement de l&#39;hyperplasie intimale après stenting navire sont cruciales pour 1,2 approches de translation. Les modèles animaux utilisés couramment comprennent les souris, les rats, les lapins et les cochons 3-5. Cependant, la traduction de ces modèles dans des milieux cliniques reste difficile, étant donné que ces processus biologiques sont déjà étudié dans les vaisseaux des animaux, mais jamais réalisée auparavant dans des modèles de recherche de l&#39;homme 6,7. Dans cette vidéo, nous démontrons un nouveau modèle humanisé de combler cette lacune en translation. La procédure indiquée est reproductible, facile et rapide à réaliser et est adapté pour étudier le développement de l&#39;hyperplasie intimale et l&#39;applicabilité des stents diverses. Cette vidéo montre comment effectuer la technique stent dans les vaisseaux de l&#39;homme suivie d&#39;une greffe chez des rats immunodéprimés, etidentifie l&#39;origine des cellules proliférantes comme des humains.

Watch this Scientific Journal Video about Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis at JoVE.com

Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Cette vidéo montre un modèle pour étudier le développement de l&#39;hyperplasie intimale après le déploiement du stent aide d&#39;un vaisseau humain (IMA) dans un modèle de rat immunodéprimé.

Human artère mammaire interne (IMA) à la transplantation et stenting: un modèle humain pour étudier le développement de la resténose intra-stent

Preclinical in vivo research models to investigate pathobiological and pathophysiological processes in the development of intimal hyperplasia after vessel ...

human-internal-mammary-artery-ima-transplantation-stenting-human

Research

JoVE Journal

Medicine

Human Internal Mammary Artery (IMA) Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

11.2K vues.  TSI-Lab, Germany. Cette vidéo montre un modèle pour étudier le développement de l'hyperplasie intimale après le déploiement du stent aide d'un vaisseau humain (IMA) dans un modèle de rat immunodéprimé.

Vidéo: Human Internal Mammary Artery IMA Transplantation and Stenting: A Human Model to Study the Development of In-Stent Restenosis

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Orthotopique aortique transplantation: un modèle de rat pour étudier le développement de la vasculopathie chronique

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Recherche

Médecine

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Utilisant la stimulation magnétique transcrânienne à l&#39;étude du système neuromusculaire de l&#39;homme

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Transplantation de cellules souches dans un In vitro Simulation d&#39;ischémie / reperfusion modèle

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

L&#39;homme neuroendocriniennes lignées cellulaires tumorales comme un modèle tridimensionnel pour l&#39;étude de la thérapie des tumeurs neuro-endocrines humaines

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic lesions. Mouse models are an important tool to investigate inflammatory processes in atherogenesis, but these models suffer from the phenotypic and functional differences between the murine and human immune system. In vitro cell experiments are used to specifically evaluate cell type-dependent changes caused by a substance of interest, but culture-dependent variations and the inability to analyze the influence of specific molecules in the context of the inflammatory compound in atherosclerotic lesions limit the impact of the results. In addition, measuring levels of a molecule of interest in human blood helps to further investigate its clinical relevance, but this represents systemic and not local inflammation. Therefore, we here describe a plaque culture model to study human atherosclerotic lesion biology ex vivo. In short, fresh plaques are obtained from patients undergoing endarterectomy or coronary artery bypass grafting and stored in RPMI medium on ice until usage. The specimens are cut into small pieces followed by random distribution into a 48-well plate, containing RPMI medium in addition to a substance of interest such as cytokines or chemokines alone or in combination for defined periods of time. After incubation, the plaque pieces can be shock frozen for mRNA isolation, embedded in Paraffin or OCT for immunohistochemistry staining or smashed and lysed for western blotting. Furthermore, cells may be isolated from the plaque for flow cytometry analysis. In addition, supernatants can be collected for protein measurement by ELISA. In conclusion, the presented ex vivo model opens the possibility to further study inflammatory lesional biology, which may result in identification of novel disease mechanisms and therapeutic targets.

A Human Ex Vivo Atherosclerotic Plaque Model to Study Lesion Biology

Atherosclerosis is a chronic inflammatory process. This manuscript illustrates an easy to use ex vivo model to investigate fresh carotid or coronary artery plaques. The ex vivo model allows for the investigation of potential substances on the inflammatory milieu in human atherosclerotic lesions and results can be analyzed by various methods.

Un humain<em&gt; Ex Vivo</em&gt; Plaque athéroscléreuse modèle pour étudier la biologie de la lésion

Atherosclerosis is a chronic inflammatory disease of the vasculature. There are various methods to study the inflammatory compound in atherosclerotic ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Murin de transplantation de la cornée: Un modèle pour étudier la forme la plus courante de la transplantation d&#39;organes solides

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Mise en place d&#39;un modèle de xénogreffe humain de myélome multiple dans le poulet pour étudier la croissance tumorale, l&#39;invasion et l&#39;angiogenèse

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Induction de l’athérosclérose accélérée chez les souris : le modèle « Wire-Injury »

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...