Gli autori ringraziano Christiane Pahrmann per il suo contributo. Un ringraziamento speciale a Ethicon, Norderstedt, Amburgo (Germania) per fornire il materiale di sutura. Finanziamento Sonja Schrepfer ha ricevuto un assegno di ricerca della Deutsche Forschungsgemeinschaft (DFG) (SCHR992 / 3 1 e SCHR992/4-1).] Il lavoro è stato supportato dal ISHLT Shumway Career Development Grant 2010 e il Research Funding Falk (Stanford University).

1. Arteria mammaria interna (IMA) Preparazione <ol><li> L&#39;endotelio arteriosa è denudato dal passaggio di un 2-francese catetere di Fogarty embolectomia arteriosa (Baxter Healthcare, Deerfield, IL, USA). Il catetere viene tirato attraverso la lunghezza intera imbarcazione due volte per assicurare danno endoteliale. </li><li> Utilizzare qualsiasi stent umano 8 millimetri di lunghezza e 2,5 mm-3 mm di diametro (ad esempio Translumina, Yukon stent). ATTENZIONE: Il diametro dello stent non deve superare il diametro del vaso di oltre il 10% per evitare stenosi pre-e post-stent. ATTENZIONE: Non accendere la lunghezza dello stent all&#39;interno dello stesso studio. </li><li> Distribuire la stent utilizzando la pressione appropriata palloncino (che è notato sul pacchetto stent) per ottenere il diametro desiderato. </li><li> Conservare stent IMA in 4 ° C RPMI + eparina (500 IE/10 ml) in ghiaccio fino al trapianto. </li></ol> 2. Preparazione degli animali RNU Nude (Crl: NIH-Foxn1rnu) ratti (300-350 g) sono HOUsed in condizioni convenzionali in scantainer armadi ventilati, alimentato standard di ratto chow e autoclavato libidum ad acqua. <ol><li> Anestetizzare ratto con isoflurano (2,5-3%) con una camera di induzione. </li><li> Radersi i capelli addominale e mettere il topo sul dorso e piazzare una maschera sul suo naso e bocca per mantenere l&#39;anestesia. </li><li> Disinfettare la zona addominale ampiamente la diffusione tramite Provo-iodio, prossimo utilizzo 80% etanolo - ripetere questa operazione due volte. Controllare riflessi pizzicare i piedi posteriori per essere sicuri che il ratto è sufficiente anestetizzato. </li><li> In vista microscopico, eseguire una laparotomia mediana superiore per esporre l&#39;aorta addominale sottorenale. </li><li> Mettere l&#39;intestino in un guanto salina idratata. Piegare il guanto intorno gli intestini per evitare la perdita di umidità. </li><li> Staccare l&#39;aorta dalla regione infrarenale alla biforcazione, con attenzione per non causare danni ai rami dei vasi. </li><li> Usare i morsetti micro per fermare il flusso aortico sangue. Posizionare ilmorsetto prossimale primo, seguito dal morsetto distale. </li><li> Rimozione di un app. 0,5-0,7 millimetri segmento di aorta e lavare l&#39;aorta rimanente con eparina (200 unità). </li><li> Prendere la IMA stent e accorciare alla lunghezza adeguata e posizionarlo nello spazio. </li><li> Collegare l&#39;IMA all&#39;aorta destinatario, eseguendo con suture 8-0 prolene sutura (Ethicon, Norderstedt, Germania). </li><li> Aprire con cautela il primo morsetto cranica e poi il morsetto caudale. </li><li> Dovrebbe esserci un impulso visibile nella trapiantato IMA e all&#39;estremità distale della aorta. </li><li> Posizionare gli intestini nuovamente dentro l&#39;addome. </li><li> Lavare l&#39;addome con pre-riscaldata soluzione salina sterile. </li><li> Chiudere lo strato muscolare della parete addominale con 6-0 punti di sutura Prolene esecuzione (Ethicon, Norderstedt, Germania). </li><li> Mentre il ratto è ancora in anestesia, iniettare 4-5 mg / kg per via sottocutanea Carprofen. </li><li> Amministrare analgesia post-chirurgica a seconda dei casi (ad esempio Carprofen o Meloxicam) per 3 giorni dopo Surgery. </li></ol> 3. Risultati rappresentativi Per l&#39;istologia, i campioni sono stati fissati in formalina 4%, disidratati in una serie graduata di alcol, e infiltrati in una miscela (MMA I) del 80% e 20% metilmetacrilato dibutilftalato per 1 giorno, MMA I con 1% di perossido di benzoile per secco 1 giorno, e MMA io con 3% di perossido di benzoile secco (MMA III) per 1-2 giorni a 4 ° C. Successivamente, i campioni sono stati polimerizzati in fresco MMA III in fiale di vetro in un bagno di acqua su un pre-polimerizzato base. Polimerizzazione lenta è stato raggiunto mantenendo le fiale a 26 ° C per una notte, aumentando la temperatura a 28 ° C la mattina successiva, e poi aumentare gradualmente la temperatura di 0,5 ° C per 12 h fino a polimerizzazione avvenuta. I blocchi polimerizzate sono stati sezionati a 5 um di spessore con un microtomo microM HM 360 dotato di un coltello carburo di tungsteno. Per identificare l&#39;origine delle cellule in proliferazione (figura 1), slides sono state colorate con anticorpi che identificano sia la proteina fluorescente verde ratto (GFP) o MHC-I e cellule muscolari lisce. Per questi studi, IMA umana è stato incubato con il gene reporter GFP ore, usando particelle lentivirali per la trasduzione di cellule IMA stabile. Cellule figlie Divisione di origine umana potrebbe essere identificato esprimendo GFP. Dopo deparaffinizzazione, indotta dal calore epitopo recupero viene eseguito riscaldando i vetrini in una soluzione di antigene recupero utilizzando un piroscafo. La Image-iT FX segnale enhancer può essere usato per la fase di blocco. Le cellule di origine umana sono identificati con gli anticorpi monoclonali murini contro la GFP (diluito 1:100 nel diluente dell&#39;anticorpo primario (Dako)), e ulteriormente marcato con capra anti-IgG di topo, Alexa Fluor 488 (1:1000 diluito in diluente anticorpo secondario ). Le cellule muscolari lisce sono state contrassegnate con il polycolonal coniglio anti-muscolo liscio α-actina (1:100 diluito nel diluente anticorpo primario), seguita da capra-anti-IgG di coniglio, Alexa Fluor 555 (1:1000 diluito in diluenti anticorpo secondario). Ogni passaggio di incubazione anticorpo viene effettuata a 37 ° C per 1 ora con tre volte in PBS tra lavaggio. I nuclei sono colorati con DAPI per 10 minuti. Dopo il montaggio delle diapositive utilizzando Prolungare reagente oro antifade, i campioni sono stati analizzati usando la microscopia confocale. <img alt="Figura 1" src="/files/ftp_upload/3663/3663fig1.jpg" /> Figura 1. Le cellule neointimale le cellule muscolari lisce A:. Verde = anti GFP, l&#39;etichettatura, le cellule di origine umana, rosso = anti umane actina delle cellule muscolari lisce, blu = DAPI, individuando nuclei delle cellule B: Verde = anti ratto MHC-I, l&#39;etichettatura cellule di origine ratto, rosso = anti umane actina delle cellule muscolari lisce, blu = DAPI, individuando nuclei delle cellule. Cellule in proliferazione sono identificate come cellule muscolari lisce e positive per GFP, ma negativo per la molecola MHC-I di ratto. Pertanto, le cellule proliferanti sono origine umana.

<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>

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Sebbene in diversi modelli di ricerca in vivo esistenti per indagare lo sviluppo di iperplasia intimale dopo il posizionamento dello stent, questi modelli ancora affrontando ostacoli traduzionali da superare. Inoltre, i modelli animali di grandi dimensioni sono condizioni abitative costosi e speciali, nonché attrezzature chirurgiche non è disponibile per tutti i laboratori. Utilizzo di un IMA umano per studiare lo sviluppo delle risorse umane proliferazione intimale e la ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> 2 catetere di Fogarty francese </td><td> Baxter Healthcare, Deerfield, IL, USA </td><td> 120602F </td><td></td></tr><tr><td> Yukon stent </td><td> Translumina GmbH, Hechingen, Germania </td><td></td><td> L&#39;uso dello stent di vostra scelta in base al protocollo di studio </td></tr><tr><td> RPMI supporti </td><td> Biochrom </td><td> Nr.F1275 </td><td></td></tr><tr><td> eparina </td><td> Baxter </td><td> 2B0953 </td><td></td></tr><tr><td> isoflurane </td><td> Abate </td><td> B506 </td><td></td></tr><tr><td> Provo-iodio </td><td> Betadine Puredue Pharma </td><td> EAN: 5995327165830 </td><td></td></tr><tr><td> 80% ethanol </td><td> Geyer </td><td> ETV 80/0500 </td><td></td></tr><tr><td> Micro pinza </td><td> Harvard Apparatus </td><td> PY2-61-0186 </td><td></td></tr><tr><td> Suture 8-0 </td><td> Johnson &amp; Johnson, </td><td> 2808G </td><td></td></tr><tr><td> Suture 6-0 </td><td> Johnson &amp; Johnson, </td><td> 1698 H </td><td></td></tr><tr><td> Carprofen </td><td> Feizer Vet </td><td> PZN: 0110208 </td><td></td></tr><tr><td> Metamizol </td><td> Ratiopharm </td><td></td><td></td></tr><tr><td> Obiettivo soluzione di recupero, pH9 </td><td> Dako </td><td> S2368 </td><td></td></tr><tr><td> Image-iT FX potenziatore di segnale </td><td> Invitrogen </td><td> I36933 </td><td></td></tr><tr><td> monoclonale di topo anti-GFP anticorpo </td><td> BD viventi colori </td><td> 632381 </td><td></td></tr><tr><td> diluente anticorpo primario </td><td> Dako </td><td> S3022 </td><td></td></tr><tr><td> capra anti-IgG di topo, Alexa Fluor 488 </td><td> Invitrogen </td><td> A11017 </td><td></td></tr><tr><td> diluente anticorpo secondario </td><td> Dako </td><td> S0809 </td><td></td></tr><tr><td> polycolonal di coniglio anti-muscolo liscio α-actina </td><td> Abcam, </td><td> ab5694 </td><td></td></tr><tr><td> capra anti-IgG di coniglio, Alexa Fluor 555 </td><td> Invitrogen </td><td> A21430 </td><td></td></tr><tr><td> Prolungare reagente oro antifade </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

Preclinica in modelli di ricerca in vivo per studiare processi patofisiologici nei patobiologico e lo sviluppo di iperplasia intimale dopo stenting nave sono cruciali per traslatorio 1,2 approcci. I modelli animali comunemente usati includono topi, ratti, conigli e maiali 3-5. Tuttavia, la traduzione di tali modelli in contesti clinici rimane difficile, dal momento che tali processi biologici sono già studiati in vasi di animali, ma mai eseguite prima in modelli di ricerca umani 6,7. In questo video si dimostra un nuovo modello umanizzato per superare questo divario traduzionale. Il procedimento illustrato è riproducibile, facile, veloce e per eseguire ed è adatto per studiare lo sviluppo di iperplasia intimale e l&#39;applicabilità di stent diverse. Questo video mostra come eseguire la tecnica dello stent nei vasi umani, seguita da trapianto in topi immunodeficienti, eidentifica l&#39;origine delle cellule proliferanti come umano.

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

Questo video mostra un modello per studiare lo sviluppo di iperplasia intimale dopo la distribuzione stent utilizzando una nave umano (IMA) in un modello di ratto immunodeficienti.

Umano arteria mammaria interna (IMA) Trapianti e Stenting: un modello umano per studiare lo sviluppo della ristenosi in-stent

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

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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 Views.  TSI-Lab, Germany. Questo video mostra un modello per studiare lo sviluppo di iperplasia intimale dopo la distribuzione stent utilizzando una nave umano (IMA) in un modello di ratto immunodeficienti.

Video: 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.

Trapianto ortotopico aortica: un modello di ratto per studiare lo sviluppo di vasculopatia cronica

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

Ricerca

Medicina

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.

Utilizzando la Stimolazione Magnetica Transcranica per studiare il sistema neuromuscolare umano

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.

Trapianto di cellule staminali in un In vitro Simulato ischemia / riperfusione Modello

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.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

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 modello murino di impianto di stent a livello dell&#39;arteria carotidea per lo Studio della restenosi

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 umano<em&gt; Ex Vivo</em&gt; Placca aterosclerotica modello per studiare Lesione Biologia

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.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

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.

Murini trapianto di cornea: Un modello per studiare la forma più comune di trapianto di organi solidi

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.

Istituzione di un mieloma multiplo umano Xenograft modello nel pollo per studiare la crescita del tumore, invasione e l&#39;angiogenesi

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.

Induzione di aterosclerosi accelerata nei topi: il modello "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. ...