A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

Incubazione 1. Egg <ol><li> Ottenere 8 giorni vecchi specifici uova embrionate esenti da organismi patogeni. </li><li> Mettere le uova in rotazione vassoio dell&#39;uovo, estremità stampati rivolti verso l&#39;alto, e posizionare il vassoio rotante all&#39;interno di un incubatore uovo. Incubare le uova per 48 ore a 36 ° C e 50% di umidità. </li></ol> 2. Far cadere il CAM e apertura le uova <ol><li> Raccogliere le uova da incubatrice. Luogo uova in portauova, timbrato rivolta verso l&#39;alto, e portare alla cappa a flusso laminare. </li><li> Spegnere le luci in camera e il cappuccio. </li><li> Tenere l&#39;estremità stampata di uovo leggermente al candler uovo per esporre la vascolarizzazione del CAM nonché il sacco aereo. NOTA: Il Candler uovo è una fonte di luce usato per visualizzare l&#39;embrione in via di sviluppo e sacco aereo associati e vascolare. </li><li> Mettere un segno di matita tra due grandi vasi sanguigni. </li><li> Accendere le luci e utilizzare una puntina sterile per ottenere un foro sulla punta dell&#39;uovo sopra il sacco aereo (fine stampati) e un foro al pemark ncil. Non spingere la puntina tutto il percorso attraverso; un foro di circa 3 mm di profondità è generalmente sufficiente. </li><li> Spegnere le luci. Stringere la lampadina di sicurezza, premendo con forza l&#39;estremità aperta contro il buco sopra la sacca d&#39;aria. Applicare il vuoto per tirare l&#39;aria nel foro in corrispondenza del segno di matita, causando il CAM a cadere lontano dal serbatoio in segno di matita. </li><li> Controllare che il sacco d&#39;aria si è spostato dalla fine timbrata del uovo al foro matita marcata con il candler. Se non lo ha, utilizzare il bottone per fare entrambi i fori leggermente più profondo e poi ri-applicare il vuoto con la lampadina di sicurezza. </li><li> Accendere le luci. Tenendo l&#39;uovo in una mano, accendere lo strumento rotativo Dremel con una ruota di taglio 15/16 pollici attaccato e fare due tagli trasversali solo abbastanza in profondità per tagliare attraverso il guscio, ma non abbastanza in profondità per tagliare tutta la strada fino al depresso CAM. Fare i tagli intorno a 2 cm di lunghezza e 1 cm di distanza l&#39;uno dall&#39;altro. Fare i tagli sufficientemente ampia in modo che un anello di silicone 1 cm di diametro può passare attraversoma più sottile rispetto alla larghezza del nastro adesivo standard. </li><li> Successivamente, fare 1 taglio longitudinale e tra le estremità dei due tagli trasversali toccando leggermente il disco di taglio per il guscio. </li><li> Far scorrere pinza sterile sotto il pezzo di guscio e parallelamente alla shell. Prendete il pezzo shell con le pinze e rimuovere l&#39;intero pezzo in modo pulito. </li><li> Posizionare lo scotch sopra la finestra appena aperta, facendo attenzione a piegare una estremità su se stesso per facilità di rimozione. </li><li> Luogo uova indietro in incubatrice nel vassoio uovo (non nei rack uovo rotanti) fino al momento dell&#39;inoculazione. </li></ol> 3. Inoculazione <ol><li> Mantenere seminterrato miscela proteina di membrana, quali matrigel, in ghiaccio (per prevenire la polimerizzazione) e posto in cappuccio. </li><li> Utilizzare 2 mM EDTA per staccare le cellule (4 - 5 ml per pallone T75) e posto fiasche nel tessuto cultura incubatore per 5 min. </li><li> Risospendere le cellule staccate nei media e contare con un emocitometro e trypan blu. Utilizzare tra 500.000 t2.000.000 o celle per l&#39;innesto sulla CAM, seconda linea cellulare. Ottimizzare il numero corretto di esperimenti preliminari. </li><li> Volume di misura necessaria per numero di cella appropriata e centrifugare a 500 xg per 5 min. </li><li> Gettare il surnatante e poi risospendere pellet cellulare in 40 μ l di PBS ++ e 20 μ l di miscela membrana basale. NOTA: A questo punto, le cellule possono essere trattati con farmaci o altri agenti di test come necessario per esperimenti risospendendo nella cellula / seminterrato sospensione miscela membrana. In alternativa, gli agenti possono essere pipettati sul cellulare / seminterrato sospensione miscela membrana dopo l&#39;innesto. </li><li> Recupera portauova con le uova dal termostato e posto nella cappa a flusso laminare. </li><li> Staccare il nastro della finestra, recuperare un anello di silicone 1 mm dal tappo di una fiala criogenico con pinze sterili, e rilasciare l&#39;anello attraverso la finestra. </li><li> Preparare la soluzione di cellule l 60 μ di PBS ++ e membrana basale Mixture direttamente al centro del cilindro di silicone appoggiato sul CAM. </li><li> Re-tape finestra e spostare portauova torna incubatrice. A questo punto, fare attenzione quando si spostano le uova, come l&#39;anello e la sospensione cellulare può facilmente diventare sfollati. </li><li> Lasciare i tumori a crescere per 5 a 7 giorni. </li></ol> 4. raccolta tumori <ol><li> Mettere sotto-pad in cappuccio. Preparare 35 x 10 mm piatti e contenitori paraformaldeide come necessario per l&#39;istologia. Mettere 1 ml di PBS ++ in ogni piatto mm 35 x 10. </li><li> Mettere sacchetto in cappa. Prendete le forbici dissezione sterili e inserire l&#39;estremità appuntita nell&#39;uovo al foro vicino alla fine timbrato. </li><li> Tagliare tutto l&#39;uovo intero longitudinalmente. NOTA: il CAM e il tumore con l&#39;anello dovrebbero attenersi per la metà superiore del guscio con la finestra. </li><li> Utilizzando forbici dissezione e pinze, sezionare il tumore dal CAM e posizionare tumore nel piatto mm 35 x 10. Misurare e pesare i tumori. Tumori fissi in paraformaldehyli de e poi paraffina incorporare per l&#39;uso in istologia ed immunoistochimica esperimenti. </li></ol> 5. (Facoltativo): Tumore delle cellule Dissociazione <ol><li> Usare le forbici dissezione per tritare il tumore nel piatto mm 35 x 10. Versare tumore tritati in una provetta da centrifuga da 15 ml e rimuovere quanto più PBS ++ possibile attraverso pipettaggio. </li><li> Aggiungere 2 ml di collagenasi (1 mg / ml in PBS ++), girare il tubo più volte, e incubare tubo a 37 ° C per almeno 30 min (fino a diverse ore). </li><li> Aggiungere 5 ml media per ciascuna provetta e sospendere nuovamente accuratamente pipettando su e giù 20 - 40x. Pipettaggio accurata è fondamentale per la dissociazione cellulare completo. </li><li> Lasciare sedimento di stabilirsi, e trasferire il surnatante in una nuova provetta. Centrifugare a 500 xg per 5 minuti. Rimuovere il surnatante, lasciando dietro di pellet dissociato. NOTA: A questo punto, pellet cellulari possono essere utilizzati per vari esperimenti (citofluorimetria, lisi per uso in Western blotting, isolamento dell&#39;RNA, etc). Totale conteggio delle cellule tumorali può essere ottenuta anche utilizzando un emocitometro e trypan colorazione blu. Si noti che, cellule di forma ovale più piccole sono cellule di pollo dal CAM e non devono essere considerati come cellule tumorali. Ci dovrebbe essere un numero minimo di contaminare cellule non tumorali (CAM) se il tumore è sezionato dal CAM accuratamente. </li></ol>

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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

Diversi passaggi chiave di questo protocollo molto probabilmente rappresentano il miglioramento della sopravvivenza embrionale così come una maggiore affidabilità della crescita tumorale. Lasciando il CAM lontano dal serbatoio applicando aspirazione al sacco aereo è meno invasivo rispetto ad altri metodi esistenti (utilizzando un ago per rimuovere l&#39;albumina dall&#39;uovo, smussa, ecc). Utilizzando una puntina sterile per creare due piccoli fori necessari per questo metodo è la parte più impegnativa del protoco...

Il carcinoma epatocellulare (HCC) è la 3 ° causa principale di mortalità per cancro nel mondo 1. Attualmente solo il 30% dei pazienti con carcinoma epatico sono ammissibili per i trattamenti chirurgici potenzialmente curative 2, e la chemioterapia sistemica non è efficace 3. Pertanto, vi è una pressante necessità clinica insoddisfatta di terapie HCC romanzo, e lo sviluppo di sistemi modello adatti efficiente sperimentazione di nuovi agenti. Il corioallantoidea test pulcino membrana (CAM) fornisce una riproducibilità, costo-efficacia, e veloce metodo di medio-throughput di testare potenziali farmaci antitumorali in vivo. Il saggio CAM è stato ampiamente utilizzato per studiare l&#39;angiogenesi 4. E &#39;stato anche sviluppato con successo in un modello di xenotrapianto di tumore di tumori, tra cui 5 glioblastoma, cancro al pancreas 6, il melanoma 7-9, e osteosarcoma 10-11. Sia in ovo 12 ed ex ovo13 tecniche sono state utilizzate in letteratura, con dettagli variano da protocollo protocollo. Una sfida importante per il modello CAM xenotrapianto è relativamente alta incidenza di morte embrionale dopo la manipolazione delle uova, con tassi di mortalità embrione di pulcino pubblicati vanno 25-50 per cento 11-14. In questo articolo, si descrive lo sviluppo di un modello di xenotrapianto in ovo di HCC che produce in modo affidabile la crescita di tridimensionali, tumori vascolarizzati che istologicamente assomigliano HCC indifferenziata. Abbiamo adattato un protocollo prima descritto da Ossowski et al. 14 e abbiamo raggiunto pulcino embrionale tassi di sopravvivenza fino al 93% con altissima attecchimento del tumore.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

La membrana corioallantoidea pulcino (CAM) comincia a sviluppare il giorno 7 dopo la fecondazione e matura di giorno 12. Il CAM è naturalmente immunodeficienti e altamente vascolarizzato, che lo rendono un sistema ideale per tumore impianto. Inoltre, il CAM contiene proteine ​​della matrice extracellulare, come fibronectina, laminina, collagene, integrina alfa (v) beta3, e MMP-2, che lo rendono un modello attraente per studiare invasione tumorale e metastasi. Gli scienziati hanno a lungo approfittato della fisiologia del CAM usandolo come modello di angiogenesi. Più di recente, il test CAM è stato modificato per lavorare come in vivo xenotrapianto sistema modello per vari tipi di cancro che colma il divario tra il lavoro di base in vitro e modelli di cancro animali più complessi. Il test permette CAM per lo studio della crescita tumorale, terapie anti-tumorali, e percorsi molecolari pro-tumorali in un sistema biologicamente rilevante che è sia in termini di costi e di tempo efficace. Qui, descriviamo lo sviluppo di Xen CAMModello ograft del carcinoma epatocellulare (HCC), con tassi di sopravvivenza embrionali fino al 93% e tumore affidabile take che porta alla crescita dei tumori vascolarizzati tridimensionali.

Immagini rappresentative di passaggi chiave nel protocollo sono mostrati qui. Figura 1A mostra l&#39;uso del candler di visualizzare l&#39;embrione in via di sviluppo, il sacco aereo, e la vascolarizzazione della CAM. Figura 1B-1C mostrano il processo di caduta del CAM facendo i due fori e quindi applicando pressione negativa con il bulbo della sicurezza, e la Figura 1D mostra una cancellati correttamente CAM con una bolla d&#39;aria sotto il foro matit...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Il<em&gt; In Ovo</em&gt; Chick corioallantoidea membrana (CAM) Saggio come un efficiente dello xenotrapianto Modello di carcinoma epatocellulare

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

34.9K Views.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Video: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Ricerca

Medicina

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modello di mouse ortotopico di anaplastico della tiroide Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Gli<em&gt; In ovo</em&gt; CAM-saggio come Xenograftmodel per Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generazione di sottocutaneo e intraepatica umane carcinoma epatocellulare xenotrapianti in topi immunodeficienti

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

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 ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Confocale Time lapse imaging come un metodo efficiente per la valutazione citocompatibilità di compositi dentali

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

A &quot;paziente-Like&quot; ortotopico Singenico modello murino della carcinoma epatocellulare Metastasi

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Progettazione CAD/CAM di guide chirurgiche per ricostruzione mascellari utilizzando un approccio in-House

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Predire la risposta di trattamento alle terapie immagine-guida utilizzando Machine Learning: un esempio per Trans-arteriosa trattamento dell'epatocarcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...