Gli autori desiderano ringraziare il Dr. Lubor Borsig (Istituto di Fisiologia, Università di Zurigo) per i suoi consigli sulla tecnica della perfusione polmonare. Questo lavoro è stato sostenuto dalle concessioni dal Krebsliga del Cantone di Zurigo, la L. Walter e Johanna Wolf Foundation, Zurigo, il Lydia Hochstrasser Foundation, Zurigo, il Fondo nazionale svizzero, SNF, Svizzera, Schweizerischer Verein Balgrist, e l&#39;Università di Zurigo.

1. In-situ perfusione polmonare e fissaggio <ol><li> Anestetizzare topi mediante iniezione ip di ~ 150 microlitri soluzione salina tamponata con fosfato (PBS) contenente 112,5 mg / kg (di peso corporeo) ketamina, 16,5 mg / kg xilazina, e 15 mg / kg acepromazina (o da un altro anestesia contenente antidolorifici adeguati). </li><li> Quando riflessi del mouse non sono più rispettate, fissarlo sul tavolo operatorio indietro verso il basso e bagnare il pelo con il 70% di etanolo lisci i capelli giù. </li><li> Aprire la pelle dall&#39;addome fino al collo e tirare ai lati o rimuoverlo. </li><li> Aprire il peritoneo e il torace ad una dimensione ampia. Evitare rotture di vasi grandi (ad esempio il jugularis vena) per evitare una perdita di efficienza perfusione successivo. </li><li> Tagliare il caudalis vena cava sotto il fegato. </li><li> Utilizzare una siringa da 20 ml dotato di un 21G - ago 24G per iniettare lentamente 10-15 ml PBS nel ventricolo destro del cuore battere fino al polmone è completamente bianco eil cuore smette di battere (asistolia). </li><li> Afferrare i vasi sanguigni di cui sopra il fegato e il diaframma con una pinza vascolare. </li><li> Utilizzare una siringa da 10 ml dotato di un 21G - ago 24G per iniettare ~ 2 ml del 3% paraformaldeide (PFA) in PBS nel ventricolo destro. </li><li> Scoprire la trachea all&#39;esterno del torace. Utilizzare la stessa siringa per iniettare ~ 3 ml del 3% in PBS PFA craniale (fuori del torace) nella trachea fino ai polmoni è gonfiato. Immediatamente dopo la rimozione dell&#39;ago, pizzichi via caudale trachea della puntura con una pinza vascolare e attendere per 10 minuti per consentire fissaggio. </li><li> Transect i vasi sanguigni di sopra della pinza vascolare, rimuovere la fascetta e iniettare ~ 2 ml PBS nel ventricolo destro per rimuovere l&#39;eccesso PFA. </li><li> Forare le parti inferiori di tutti i lobi polmonari con un ago, rimuovere la pinza vascolare alla trachea e iniettare 3-5 ml di una soluzione di PolyFreeze incorporamento medio / PBS 01:01 nella trachea caudale della puntura prima fino alla PFA nella polmonelobi è sostituita dalla soluzione. </li><li> Rimuovere il polmone tirandola il cuore con una pinza e da sezionare legamenti del tessuto connettivo, vasi sanguigni e la trachea. Mantenere il cuore collegato ai polmoni. </li><li> A seconda di come procedere con l&#39;analisi del tessuto polmonare continuare con i protocolli descritti nelle sezioni 2 o 3. Se si vuole fare entrambe le analisi, rimuovere un lobo polmonare e procedere come descritto al punto 3. Trattare i lobi rimanenti collegati al cuore, come descritto al punto 2. </li></ol> 2. Visualizzazione di lacZ-etichetta cellule metastatiche del tumore in lobi polmonari Intere <ol><li> Posizionare il polmone in una tazza di plastica con coperchio di chiusura a tenuta e fissarla con 2% di formaldeide in PBS a temperatura ambiente per 30-60 min. </li><li> Rimuovere la soluzione di fissaggio e lavare accuratamente 3 volte con PBS. </li><li> Aggiungere almeno 10 ml preparata 5-Bromo-4-cloro-3-indolil-β-D-galattoside (X-Gal) soluzione colorante (X-Gal soluzione stock [40 mg / ml inDimetilformammide] 01:40 diluito in soluzione di base di colorazione, pH 7.1 (Tabella 1), al riparo dalla luce). </li><li> Mettere un pezzo di garza in cima al polmone nuoto di mantenere completamente nella soluzione e posizionare il coperchio solo vagamente sulla pentola per consentire lo scambio di aria. </li><li> Incubare a 37 ° C per 3-5 ore al riparo dalla luce. </li><li> Rimuovere X-Gal soluzione, lavare una volta con PBS per rimuovere i residui di X-Gal soluzione (opzionale) e aggiungere 4% PFA. </li></ol> 3. Visualizzazione di lacZ-etichetta cellule metastatiche del tumore nel polmone criosezioni <ol><li> Prefill uno stampo incorporamento etichettati per 1/3 con media non diluito inclusione PolyFreeze. Mettere il lobo polmonare in alto e riempire con l&#39;incorporamento medio fino a che il lobo è completamente coperto. Cercate di evitare le bolle. </li><li> Incubare i polmoni incorporati a 4 ° C per 20 min. </li><li> Poi congelare i polmoni incorporati lentamente in una miscela di ghiaccio secco e isopentano e conservare a -80 ° C. </li><li> Tagliare 7-10 micron criosezioni su un criostato e montarle su vetrini da microscopio SuperFrostPlus. </li><li> Incubare le sezioni immediatamente con X-Gal soluzione di colorazione a 37 ° C per 24 ore in una camera umidificata. </li><li> Sciacquare i vetrini in PBS 2 volte per 5 min e poi brevemente in acqua distillata. </li><li> Counter-macchia con rosso nucleare veloce per 10-30 sec, sciacquare in acqua distillata e montare i vetrini con Immu-Mount. </li></ol> 4. Risultati rappresentativi Asai et al. Riportato nell&#39;articolo originale sul Dunn-derivato topo modello LM8 OS che i tumori sc primarie derivate dalle cellule parentali Dunn, diversi da quelli derivati ​​dal altamente metastatico LM8 linea cellulare sub, non formano spontaneamente metastasi polmonari rilevabili in singenici topi C3H 14. Con le tecniche qui riportate, abbiamo inchiesta la formazione di metastasi in modelli murini Dunn/LM8 OS. Abbiamo approfittato di stabile lacZ-trasdotte Dunn e LM8 cellulares e di un protocollo di in-situ perfusione polmonare e fissaggio in topi. Immagini rappresentative di polmoni perfusi e non perfuso di topi iniettati sottocute con lacZ-trasdotte e non trasdotte controllo Dunn e LM8 cellule sono mostrati in Figura 1. Nei topi iniettati con cellule di controllo Dunn, metastasi macroscopiche e microscopiche rimasta non rilevabile nei polmoni non perfusi e perfuso (Figura 1A, I-IV). Ma, interessante, in topi iniettati con cellule Dunn-lacZ, X-gal colorazione blu rivelato micrometastatica foci di cellule singole o piccoli ammassi cellulari (&lt;0,1 mm) sulla superficie del non-perfusi polmoni (Figura 1A, vi). In- perfusione situ e la fissazione dei polmoni ulteriormente migliorato la rilevabilità di Dunn-lacZ micrometastasi (Figura 1A, viii). Tuttavia, per conseguenza macroscopica foci non è stato osservato (Figura 1A, V, VII). &lt;/ P&gt; Nei topi iniettati con cellule di controllo LM8, traslucidi, appena rilevabile macrometastatic focolai più grandi di 0,1 mm di diametro sono stati rilevati a non perfusi polmoni (Figura 1B, i). Perfusione del polmone (Figura 1B, iii) non ha migliorato la rilevazione dei fuochi. Tuttavia, in topi iniettati con LM8-lacZ celle multiple X-Gal macchiato macro-blu (Figura 1B, v) e micrometastasi (Figura 1B, vi) sono stati rilevati sulla superficie del non-perfusi organi. Inoltre, la perfusione dei polmoni ulteriormente migliorato l&#39;individuazione delle macro e micrometastasi (Figura 1B, vii - viii). Di conseguenza, micro e macrometastasi divenne visibile una densità maggiore e un numero maggiore, principalmente a causa della traslucenza del tessuto perfuso in cui foci sotto la superficie dell&#39;organo divenne anche visibile. Un ulteriore hanalisi utilizzando criosezioni istologico di tessuto polmonare confermato la rilevabilità miglioramento delle metastasi polmonari nei topi iniettati con lacZ-trasdotte Dunn e LM8 cellule. Nei topi con tumori primari derivati ​​da Dunn-lacZ o LM8-lacZ cellule, a differenza di topi con tumori primari delle cellule di controllo, rispettivi micrometastasi o addirittura singola cella foci erano riconosciuti in sezioni polmonari (Figura 2). Inoltre, macrometastasi erano anche più chiaramente visibile in topi iniettati con LM8-lacZ cellule che negli animali iniettati con il controllo LM8 cellule (Figura 2C, D). <img alt="Figura 1" src="/files/ftp_upload/4162/4162fig1.jpg" /> Figura 1. Rilevamento di metastasi spontanee di non trasdotte (I-IV) e lacZ-trasdotte (v-viii) Dunn (A) e LM8 (B) in cellule non-perfuso (I, II, V, VI) e perfuso (III, IV, VII, VIII) polmoni nei topi C3H. Metastasi a rappresentante X-Gal-staINED polmoni interi (V, VII in A e B) e rispettivi primi piani (VI, VIII in A e B) appaiono blu. Macrometastatic foci (&gt; 0,1 mm) negli organi dei topi iniettati con i tag LM8 cellule (i-iv in B) sono indicati da un asterisco. Le aree indicate nei primi piani sono rappresentativi per i polmoni interi. Barre di scala indicano 1 mm di immagini di polmoni interi e 0,1 mm di primi piani. <a href="http://www.jove.com/files/ftp_upload/4162/4162fig1large.jpg" target="_blank">Clicca qui per ingrandire la figura</a> . <img alt="Figura 2" src="/files/ftp_upload/4162/4162fig2.jpg" /> Figura 2. Rilevamento di Dunn e LM8 micrometastasi in criosezioni polmonari. Sezioni di tessuto polmonare ottobre incorporato state incubate in X-Gal soluzione di colorazione a 37 ° C per 24 ore in una camera umidificata e poi colorate con rosso nucleare fast. Frecce indicano micrometastatica foci riconosciuto in sezioni polmonari di topi iniettati con Dunn-lacZ (B) o LM8-lacZ cellule (D). Micrometastases rimasta non rilevabile in sezioni polmonari di topi iniettati con non trasdotte Dunn (A) o LM8 cellule (C). Barre di scala indicano 0,1 mm.

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Nessun conflitto di interessi dichiarati.

I risultati qui presentati nel modello di topo Dunn/LM8 OS dimostrare la potenza del metodo appena stabilito che combina l&#39;X-Gal colorazione lacZ-etichetta cellule tumorali con l&#39;in-situ perfusione / fissaggio del tessuto polmonare. Questa combinazione delle due tecniche permette la rilevazione elevata sensibilità di lesioni micrometastatica fino a livello di singola cellula e migliora anche la visualizzazione macrometastasi sulla superficie polmonare (Figura 1) e in sezioni polmonari (Figura 2). Mentre l&#39;X-Gal colorazione permette inoltre la rilevazione di (micro) metastasi in altri organi, in-situ perfusione / fissazione migliora la rilevabilità di foci metastatici in tessuti diversi dal polmone solo leggermente a causa del colore naturale sangue e tessuti correlati di questi organi 13. Anche se la perfusione è diretta ad un altro organo, ad esempio il fegato, la rimozione del sangue potrebbe migliorare solo parzialmente dal contrastoween X-Gal colorazione e il colore naturale dell&#39;organo. Tuttavia, il metodo è applicabile a qualsiasi tipo di etichetta lacZ-cellule tumorali, migliorare notevolmente la rilevabilità di metastasi polmonari fino al livello di dormienti monocellulari micrometastasi e consente una quantificazione semplice e affidabile di macro e micrometastasi. Un limite di questo metodo e tutte le altre tecniche che si basano su geni reporter luciferasi, comprese le proteine ​​fluorescenti, e la stabilità del transgene. Come mostrato in figura 2d, non tutte le cellule tumorali all&#39;interno foci macrometastatic sono di colore blu, che indica una mancanza di beta-galattosidasi. Questo potrebbe essere correlato a necrosi, ma è più probabilmente dovuta ad una perdita di espressione del transgene. Abbiamo osservato che il Dunn e LM8 cellule sono molto efficaci nel down-regulation di lacZ e transgeni altri anche in continua selezione per l&#39;espressione. Abbiamo quindi commutata in studi recenti per l&#39;murine K12e K7M2 e gli umani HOS, 143B e SaOS-2 linee cellulari di osteosarcoma, tutte mantenute stabili lacZ espressione in vitro e in vivo fino al 100% nel tempo. Una volta stabile lacZ-espressione è garantita, questa tecnica può essere applicata in studi con gene-manipolati cellule tumorali, per esempio per investigare meccanicamente il processo di colonizzazione tessuto 13 e per lo sviluppo e la sperimentazione di nuove terapie obiettivo di eradicazione delle lesioni metastatiche 15 , 16. Inoltre, può servire come punto di riferimento per il miglioramento delle attuali tecniche di imaging radiologici, come la PET, (micro) TC e RM, utilizzati per la diagnosi precoce delle lesioni metastatiche. In uno studio PET recente (inedito) con traccianti diversi abbiamo verificato in vivo polmone rilevato metastasi ex vivo successivamente con il protocollo descritto. In uno studio in corso con un nuovo animale piccolo micro-CT (SkyScan) siamo così lontani abper rilevare le metastasi polmonari in vivo fino ad una dimensione di 0,5 mm ed ex vivo fino a 0,3 mm, ma relative ad una risoluzione di 0,1 mm. Curiosamente, questo è il limite che abbiamo impostato per distinguere macro-di micrometastasi con il metodo combinato di perfusione in situ e X-Gal colorazione. Questo sottolinea ancora una volta la sensibilità e l&#39;utilità di questa tecnica semplice e conveniente.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Narketan10 (ketamina) </td><td> Vétoquinol AG </td><td></td><td> 100 mg / ml soluzione </td></tr><tr><td> Xylazin (xilazina) </td><td> Streuli Pharma AG </td><td></td><td> 20 mg / ml soluzione </td></tr><tr><td> Prequilan (acepromazina) </td><td> Fatro SpA </td><td></td><td> 10 mg / ml soluzione </td></tr><tr><td> Bulldog Tipo Serrefine pinza vascolare </td><td> Strumenti Scienza Belle </td><td> 18051-35 </td><td> curvo, mm 35 </td></tr><tr><td> Tazza di plastica con coperchio </td><td> Semadeni </td><td> 2988 </td><td> 25 tazze ml con coperchio </td></tr><tr><td> 5-Bromo-4-cloro-3-indolil-β-D-galattoside (X-Gal); polvere </td><td> Axxora </td><td> ALX-582-002-G005 </td><td> sciogliere in N, N-di-metilformammide a 40 mg / ml, negozio protetto dalla luce a -20 ° C </td></tr><tr><td> Colorazione soluzione basica, equilibrata a pH 7,1, memorizzato protetto dalla luce a 4 ° C per max. 1 mese </td><td> fatto da sé </td><td></td><td> 100 ml / L 10xPBS, 1,64 g / L K3Fe (CN) 6, 2,1 g / L K4Fe (CN) 6, 2 ml / L 1M MgCl2, 2 ml / L 10% NP40, 1 ml / L 10% di sodio desossicolato- </td></tr><tr><td> PolyFreeze Embedding Media </td><td> Polysciences; Distributore svizzero: Brunschwig </td><td> 19636-1 </td><td></td></tr><tr><td> Incorporamento stampo </td><td> Polysciences </td><td> 18646A-1 </td><td></td></tr><tr><td> Leica CM1850 criostato </td><td> Leica Microsystems </td><td></td><td></td></tr><tr><td> SuperFrost diapositive </td><td> Menzel </td><td> J1800AMNZ </td><td></td></tr><tr><td> Nucleare-Fast Red - soluzione di solfato di alluminio </td><td> Divisione Chroma Waldeck GmbH &amp; Co KG distributor: Medite </td><td> 84-0241-00 </td><td></td></tr><tr><td> Immu-Mount </td><td> Thermo Electron, distributore svizzero: Histocom </td><td> 9990412 </td><td></td></tr></tbody></table> Tabella 1. Reagenti e materiale.

improved visualization of lung metastases at single cell resolution in mice by combined in-situ perfusion of lung tissue and x-gal staining of lacz-tagged tumor cells

Metastasi è la principale causa di morte nella maggior parte dei tipi di cancro e di conseguenza un obiettivo principale nella ricerca sul cancro. Tuttavia, il rilevamento di micrometastasi di immagini radiologica e il successo nella loro eradicazione terapeutico rimangono limitate. Mentre modelli animali hanno dimostrato di essere strumenti preziosi per la ricerca sul cancro 1, il controllo / visualizzazione di micrometastasi rimane una sfida e una valutazione imprecisa di diffusione metastatica in studi preclinici porta potenzialmente a risultati deludenti negli studi clinici 2. Di conseguenza, vi è un grande interesse per affinare i metodi per consentire finalmente rilevamento riproducibile e affidabile di metastasi al livello di singola cellula nel tessuto normale. L&#39;obiettivo principale è quindi sulle tecniche, che consentono l&#39;individuazione delle cellule tumorali in vivo, come la micro-tomografia computerizzata (micro-CT), la tomografia ad emissione di positroni (PET), bioluminescenza o imaging di fluorescenza &lt;sup&gt; 3,4. Attualmente stiamo ottimizzando queste tecniche per il monitoraggio in vivo della crescita tumorale primario e metastasi in modelli diversi osteosarcoma. Alcune di queste tecniche può essere utilizzato anche per l&#39;analisi ex vivo di metastasi accanto metodi classici come qPCR 5, 6 o FACS diversi tipi di colorazione istologica. Come valore di riferimento, abbiamo stabilito nel presente studio la trasfezione stabile o trasduzione di cellule tumorali con il gene lacZ codifica l&#39;enzima batterico β-galattosidasi che metabolizza il substrato cromogeno 5-bromo-4-cloro-3-indolil-beta-D -galattopiranoside (X-Gal) in un colorante blu insolubile indaco 7 e permette altamente sensibile e selettivo colorazione istochimica blu di cellule tumorali in vivo ex tessuti mouse per livello di singola cellula, come qui mostrato. Questo è uno strumento a basso costo e non attrezzature ad alta intensità, che consente la validazione precisa di metastasi 8 in studi di valutazione di un nuovoterapie ticancer 9-11. Un fattore limitante di X-gal colorazione è il basso contrasto, ad esempio, consanguinei colorazione rosso di ben tessuti vascolarizzati. In tessuto polmonare questo problema può essere risolto in situ perfusione polmonare, una tecnica che è stata recentemente istituita dal Borsig et al. Perfusi 12 anni che i polmoni di topi in anestesia per eliminarli dal sangue e per fissare e incorporarli in situ sotto inflazione attraverso la trachea. Questo metodo impedisce anche il collasso del polmone e mantiene quindi la morfologia funzionale alveoli polmonari, che migliora la qualità del tessuto per l&#39;analisi istologica. Nel presente studio, si descrive un nuovo protocollo, che sfrutta una combinazione di X-gal colorazione lacZ cellule tumorali che esprimono e in-situ perfusione e fissazione del tessuto polmonare. Questo protocollo permette raffinato alta sensibilità di rilevazione di singole cellule metastatiche nel polmone e permesso in un recente studio di detect &quot;dormienti&quot; micrometastasi polmonari in un modello murino 13, che è stato originariamente descritto di essere non-metastatico 14.

Watch this Scientific Journal Video about Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tum

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Il nuovo protocollo riportato nel presente studio permette la rilevazione selettiva delle metastasi polmonari con una risoluzione singola cellula nei topi combinata<em&gt; In-situ</em&gt; Perfusione polmonare e la fissazione e colorazione X-Gal di<em&gt; LacZ</em&gt;-Tagged cellule tumorali.

Una migliore visualizzazione delle metastasi polmonari a risoluzione singola cella in Mouse di combinata<em&gt; In-situ</emPerfusione&gt; del tessuto polmonare e X-Gal colorazione del<em&gt; LacZ</em&gt;-Tagged cellule tumorali

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

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Research

JoVE Journal

Medicine

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

17.2K Views.  Balgrist University Hospital, Zurich. Il nuovo protocollo riportato nel presente studio permette la rilevazione selettiva delle metastasi polmonari con una risoluzione singola cellula nei topi combinata In-situ Perfusione polmonare e la fissazione e colorazione X-Gal di LacZ-Tagged cellule tumorali.

Video: Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. As this technology expands and improves, the ability to potentially evaluate and improve the quality of substandard lungs prior to transplant is a critical need. In order to more rigorously evaluate these approaches, a reproducible animal model needs to be established that would allow for testing of improved techniques and management of the donated lungs as well as to the lung-transplant recipient. In addition, an EVLP animal model of associated pathologies, e.g., ventilation induced lung injury (VILI), would provide a novel method to evaluate treatments for these pathologies. Here, we describe the development of a rat EVLP lung program and refinements to this method that allow for a reproducible model for future expansion. We also describe the application of this EVLP system to model VILI in rat lungs. The goal is to provide the research community with key information and &#8220;pearls of wisdom&#8221;/techniques that arose from trial and error and are critical to establishing an EVLP system that is robust and reproducible.

Method of Isolated Ex Vivo Lung Perfusion in a Rat Model: Lessons Learned from Developing a Rat EVLP Program

Ex-Vivo Lung Perfusion (EVLP) has allowed lung transplantation in humans to become more readily available by enabling the ability to assess organs and expand the donor pool. Here, we describe the development of a rat EVLP program and refinements that allow for a reproducible model for future expansion.

Metodo di isolato<em&gt; Ex Vivo</em&gt; Lung Perfusion in un modello di ratto: Lezioni apprese da Sviluppo di un programma EVLP Rat

The number of acceptable donor lungs available for lung transplantation is severely limited due to poor quality. Ex-Vivo Lung Perfusion (EVLP) has allowed ...

Ricerca

Medicina

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

Tumore primario e isolamento delle cellule MEF per studiare Lung Metastasi

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Valutazione del polmone metastasi in mouse mammarie tumorali modelle da Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Aprire tracheostomia gastrico acido aspirazione Modello murino di lesioni polmonari acute Risultati in massima acuta non letale Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

Generazione di colture del tessuto polmonare umano 3D (3D-LTC) per la modellazione di malattia

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone smooth muscle cell lineage tracing mice.

Analisi quantitativa della composizione cellulare nelle lesioni aterosclerotiche avanzate del muscolo liscio delle cellule Lineage-Tracing topi

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Indurre lesioni polmonari acute nei topi mediante instillazione diretta del lipopolioaccaride intracheale

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ quality exacerbated by excessive geographic transportation distance and stringent donor organ acceptance criteria pose limitations to current LTx efforts. Ex situ lung perfusion (ESLP) is an innovative technology that has shown promise in attenuating these limitations. The physiologic ventilation and perfusion of the lungs outside of the inflammatory milieu of the donor body affords ESLP several advantages over traditional cold static preservation (CSP). There is evidence that negative pressure ventilation (NPV) ESLP is superior to positive pressure ventilation (PPV) ESLP, with PPV inducing more significant ventilator-induced lung injury, pro-inflammatory cytokine production, pulmonary edema, and bullae formation. The NPV advantage is perhaps due to the homogenous distribution of intrathoracic pressure across the entire lung surface. The clinical safety and feasibility of a custom NPV-ESLP device have been demonstrated in a recent clinical trial involving extender criteria donor (ECD) human lungs. Herein, the use of this custom device is described in a juvenile porcine model of normothermic NPV-ESLP over a 12 h duration, paying particular attention to management techniques. Pre-surgical preparation, including ESLP software initialization, priming, and de-airing of the ESLP circuit, and the addition of anti-thrombotic, anti-microbial, and anti-inflammatory agents, is specified. The intraoperative techniques of central line insertion, lung biopsy, exsanguination, blood collection, cardiectomy, and pneumonectomy are described. Furthermore, particular focus is paid to anesthetic considerations, with anesthesia induction, maintenance, and dynamic modifications outlined. The protocol also specifies the custom device's initialization, maintenance, and termination of perfusion and ventilation. Dynamic organ management techniques, including alterations in ventilation and metabolic parameters to optimize organ function, are thoroughly described. Finally, the physiological and metabolic assessment of lung function is characterized and depicted in the representative results.

Normothermic Negative Pressure Ventilation Ex Situ Lung Perfusion: Evaluation of Lung Function and Metabolism

This paper describes a porcine model of negative pressure ventilation ex situ lung perfusion, including procurement, attachment, and management on the custom-made platform. Focus is made on anesthetic and surgical techniques, as well as troubleshooting.

Perfusione polmonare ex situ di ventilazione a pressione negativa normothermica: valutazione della funzione polmonare e del metabolismo

Lung transplantation (LTx) remains the standard of care for end-stage lung disease. A shortage of suitable donor organs and concerns over donor organ ...

Lung Rapid Recovery Procurement Combined with Abdominal Normothermic Regional Perfusion in Controlled Donation after Circulatory Death

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years confirm that the outcomes after lung transplantation from cDCD are similar to those from brain death donors; however, the utilization of lungs from asystole donors remains low. Several reasons may be involved: different legal frameworks among countries and centers with different premortem interventions, inadequate lung donor care before procurement, or even poor experience with cDCD procedures and protocols.
Initially, the rapid recovery technique was commonly employed for the procurement of thoracic and abdominal organs in cDCD, but, in the last decade, abdominal normothermic regional perfusion (ANRP) with extracorporeal membrane oxygenation devices has become a useful method to restore blood flow to abdominal organs, allowing their quality improvement and their functional assessment prior to transplantation. This makes the donation procedure more complex and generates doubts about injury to the grafts due to dual temperature.
The aim of this article is to describe a protocol based on a single center experience with Maastricht III donors combining lung cooling rapid recovery in the thorax and abdominal normothermic regional perfusion. Tips and tricks focused on premortem interventions and lung procurement procedure techniques are explained. This may help to minimize the reluctance among professionals to use this combined technique and encourage other donor centers to use it, despite the increased complexity of the procedure.

The protocol combines a lung cooling rapid recovery technique with abdominal normothermic regional perfusion for abdominal graft procurement in controlled asystole donors, which is a safe and useful method to expand the donor pool.

Approvvigionamento di recupero rapido polmonare combinato con perfusione regionale normomica addominale nella donazione controllata dopo morte circolatoria

Controlled donation after circulatory death (cDCD) has contributed to increasing donor numbers all over the world. Experiences published in the last years ...

Imaging polmonare innovativo con sezioni di vibratomo incorporate in agarosio

Automated Vibratome Sectioning of Agarose-Embedded Lung Tissue for Multiplex Fluorescence Imaging

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural support for sectioning, pieces of lung tissue are commonly embedded in paraffin or OCT compound and cut with a microtome or cryostat, respectively. A more recent technique, known as precision-cut lung slices, adds structural support to fresh lung tissue through agarose infiltration and provides a platform to maintain primary lung tissue in culture. However, due to epitope masking and tissue distortion, none of these techniques adequately lend themselves to the development of reproducible advanced light imaging readouts that would be compatible across multiple antibodies and species. 
To this end, we have developed a tissue-processing pipeline, which utilizes agarose embedding of fixed lung tissue, coupled to automated vibratome sectioning. This facilitated the generation of lung sections from 200 &#181;m to 70 &#181;m thick, in mouse, pig, and human lungs, which require no antigen retrieval, and represent the least "processed" version of the native isolated tissue. Using these slices, we reveal a multiplex imaging readout capable of generating high-resolution images whose spatial protein expression can be used to quantify and better understand the mechanisms underlying lung injury and regeneration.

Autore in primo piano: Studio dell'espressione proteica spaziale utilizzando sezioni di tessuto polmonare incorporate in agarosio 

We have developed a tissue-processing technique utilizing a vibratome and agarose-embedded lung tissue to generate lung sections, thereby permitting the acquisition of high-resolution images of lung architecture. We employed immunofluorescence staining to observe spatial protein expression using specific lung structural markers.

Sezionamento automatizzato con vibratomo del tessuto polmonare incorporato nell'agarosio per l'imaging a fluorescenza multiplex

Due to its inherent structural fragility, the lung is regarded as one of the more difficult tissues to process for microscopic readouts. To add structural ...