Questo studio è stato supportato da CCSRI di Grant # 700537 e CIHR Concessione # 84535 di JDL e NIH / NCI concedere # CA120711-01A1-01A1 e CA120711 ad AZ. Tutti gli esperimenti sono stati eseguiti in conformità con i regolamenti e linee guida della cura degli animali e utilizzo Comitato Istituzionale presso la University of California di San Diego, cura degli animali e Usa presso la University of Western Ontario.

1. Inoculazione di tumore in CAM embrione aviaria <ol><li> Preparare conchiglia meno embrioni di pollo fecondate, come descritto 8. </li><li> Il 9 ° giorno di sviluppo embrionale, assemblare un micro-iniettore con un 18-gauge collegato su siringa da 1 ml. Tagliare un pezzo di 5-6 centimetri di tubo Tygon (1 / 32 &quot;di diametro interno, 3 / 32&quot; di diametro esterno, 1 / 32 &quot;spessore della parete) e inserire con cautela bisello dell&#39;ago nel tubo. Circa 4-5 cm di tubo dovrebbe si estendono dalla punta dell&#39;ago (figura 1a). </li><li> Riempire la siringa e il tubo con la sospensione cellulare. Quindi, inserire un ago di vetro microiniezione alla fine del tubo e con attenzione rimuovere eventuali bolle d&#39;aria. </li><li> Iniettare Giorno 9 embrioni (figura 1b) in un ambito di dissezione con un illuminatore con 10,000-100,000 cellule del cancro come un bolo all&#39;interno del CAM (Figura 1c). Iniettare lentamente e con attenzione le cellule in modo che l&#39;ago è nel posto giusto per le cellule di formare un bolo visibile all&#39;interno della CAM. Le cellule che a goccia sulla superficie CAM può essere pulito con applicatore Kimwipe o altro. </li><li> Ritorna embrioni incubatore umidificato a 38 ° C al 60% di umidità e permettere tumore di crescere e vascolarizzano (fino a 7 giorni). </li></ol> 2. Preparazione di nanoparticelle <ol><li> Per preparare fluorescente nanoparticelle CPMV per l&#39;iniezione in embrione di pollo; diluire le nanoparticelle virale (come sintetizzato in 8 in PBS, pH 7,4, ad una concentrazione di 100 mg / ml miscela Vortex bene prima dell&#39;uso e centrifugare per un minuto di togliere qualunque. aggregati. sonicazione può essere utile anche a seconda coniugati e il grado di aggregazione. Stock di CPMVs fluorescente sono stabili per almeno 1 anno se conservato a 4 ° C al buio. Controllare periodicamente le scorte da mettere un paio di gocce su un vetrino e controllo per fluorescenza. microscopia elettronica a trasmissione (TEM) può essere utilizzato anche per determinare se le particelle rimaste intatte dopo coniugazione. </li><li> In breve, un microgrammo di nanoparticelle virale (in un volume finale di 2 mL) possono essere aggiunti alle reti in rame rivestito. Successivamente, aggiungere un uguale volume di acetato di uranile 2% per 1 minuto al microscopio elettronico (Philips EM 410). </li></ol> 3. Iniezione endovenosa di fluorescenza marcata con nanoparticelle virale <ol><li> Il Giorno 16, assemblare micro-iniettore come descritto sopra. Redigere il volume desiderato di nanoparticelle virale attraverso il tubo nella siringa (&gt; 200 mL). Rimuovere con attenzione eventuali bolle d&#39;aria. Inserire l&#39;ago di vetro microiniezione alla fine del tubo. Assicurarsi che l&#39;ago è lungo e affusolato possibile (Figura 2a). Se l&#39;ago è troppo schietto, non sarà in grado di penetrare l&#39;ectoderma e non riuscirà a penetrare il vaso. Se l&#39;ago è troppo forte, la punta crollerà quando penetrare nei tessuti. </li><li> Per via endovenosa iniettare 100 ml di 1 mg / ml di fluoresceina destrano (70.000 MW Da, Invitrogen) in embrione cuscinetto tumore distale dal sito desiderato da visualizzare (Figura 3d). Cannulazione successo della vena CAM è evidente dalla compensazione di sangue nel percorso del flusso di iniezione (Figura 2b). Valutare vascolare perdita 1 ora dopo l&#39;iniezione. </li><li> Iniettare per via endovenosa 50 ml di 1 mg / ml di soluzione di CPMV-Alexa Fluor 647 (CPMV AF-647) o pegilato CPMV-Alexa Fluor 647 (CPMV-PEG-AF 647) in embrioni contenenti i tumori con perdite vascolari distali dal sito desiderato essere visualizzati. </li><li> Tumori immagine subito e ogni ora dopo l&#39;iniezione utilizzando un microscopio Zeiss Examiner Z1 verticale dotata di un disco di Yokogawa filatura, Hamamatsu Imagem 9100-12 EM-CCD. </li></ol> 4. In tempo reale intravitale di imaging <ol><li> Per assemblare l&#39;unità di riproduzione embrione, applicare un sottile strato di grasso per vuoto attorno alla circonferenza del porto di imaging sul lato inferiore del coperchio della fotounità embrione, e si adattano a 18 mm di vetro coprioggetto sul porto. </li><li> Posizionare l&#39;embrione in modo che il coprioggetto coprirà l&#39;area desiderata per l&#39;imaging. Abbassare lentamente il coperchio fino a quando il coprioggetto fa solo contatto con l&#39;embrione, e quindi avvitare il coperchio sull&#39;unità per tenerlo in posizione (Figura 2c). </li><li> Aggiungere acqua riscaldata a 37 ° C all&#39;interno dell&#39;unità embrione di imaging fuori del piatto che contiene l&#39;embrione, e poi si porta il complesso sulla scena di un microscopio confocale verticale con la camera interna ambientali equilibrati a 37 ° C. </li><li> Posizionare l&#39;unità di riproduzione che contiene l&#39;embrione sotto il microscopio a fluorescenza Spinning disco confocale (Figura 2 sexies). L&#39;unità imaging terrà l&#39;embrione al suo posto e tenere il campo visivo durante l&#39;acquisizione di immagini fisse, consentendo sia tridimensionale stack Z e time-lapse immagini da catturare. Noi acquisire e analizzare tridimensionale time-lapse immagini utilizzando Perkin Elmer (ex Improvvisazione) pacchetto software Volocity (Figura 3a). Acquisire ad alta risoluzione tridimensionale pila di vascolarizzazione del tumore e circostanti per visualizzare detailed analisi strutturale a specifici tempi punti. Acquisire tridimensionale pile a intervalli regolari di tempo i punti di mappa dettagliata dei cambiamenti strutturali nel sistema vascolare del tumore. Tumori immagine ogni ora dopo l&#39;iniezione. </li><li> Quantificare l&#39;assorbimento delle nanoparticelle virale calcolando la media Alexa Fluor (AF) 647 del segnale all&#39;interno di regioni selezionate nel tumore o nello stroma (non tumorali zona) software per immagini utilizzando la quantificazione come Volocity (Perkin Elmer). Il tumore al rapporto stroma è stato calcolato dividendo la media AF 647 segnale nel tumore oltre la media AF 647 segnale nello stroma. Un tumore / stroma rapporto superiore a 1 indica che la nanoparticella è stato preso dal tumore. </li></ol> 5. Rappresentante dei risultati: Nell&#39;esempio qui descritto, abbiamo iniettato le cellule HT-29 cancro al colon a formare un bolo di circa 1 mm di grandezza entro il giorno 9 CAM di embrioni di pollo (Figura 1b). Dopo l&#39;inoculazione, gli embrioni sono stati coltivati ​​per 7 giorni in un incubatore umidificato per consentire una crescita sufficiente e la vascolarizzazione del tumore (Figura 1c). Gli embrioni sono stati iniettati per via endovenosa con un peso di destrano a basso molecolare per confermare vascolarizzazione del tumore, ei tumori sono stati visualizzati al microscopio Zeiss AxioExaminer Z1 verticale (Figura 2d). Dopo somministrazione endovenosa di CPMV-AF 647 o CPMV-PEG-AF 647 nanoparticelle (Figura 3a e b), ad alta risoluzione in tempo reale di imaging confocale (Figura 2 sexies) ha rivelato che sia CPMV e CPMV-PEG nanoparticelle rapidamente etichettato l&#39;intero sistema vascolare, ma l&#39;assorbimento di CPMV-PEG da parte del tumore è stata di circa 3 volte superiore CPMV dopo 12 ore (Figura 3a). L&#39;assorbimento relativo di tumore nanoparticelle è stata determinata utilizzando software per l&#39;analisi dell&#39;immagine (Volocity da Perkin Elmer). Regioni di interesse sono stati selezionati all&#39;interno e all&#39;esterno del tumore (nel vano stromali) e l&#39;intensità media di fluorescenza di ogni stato determinato. I dati vengono espressi come tumore / stroma rapporto. <img src="/files/ftp_upload/2808/2808fig1.jpg" alt="Figura 1"/> Figura 1. Microiniezione dei tumori nel CAM di un embrione aviaria. (A) L&#39;apparecchio microiniezione viene assemblata dai componenti, come indicato. (B) gli embrioni aviaria al giorno 9 sono pronti per essere inoculati con tumore quando la CAM si è diffusa per coprire l&#39;intera superficie. (C) Al giorno 16, il tumore è cresciuto fino a 1 cm di diametro (linea tratteggiata) ed è pronto per l&#39;iniezione di nanoparticelle. <img src="/files/ftp_upload/2808/2808fig2.jpg" alt="Figura 2"/> Figura 2. Iniezione di nanoparticelle e di imaging intravitale. Iniezione sotto un microscopio che mostra la dissezione (a) la punta dell&#39;ago microinjector pronto per essere iniettato nella vena CAM e (b) l&#39;ago microinjector inserito nella vena (indicato dalla freccia) e nanoparticelle iniettate nel flusso sanguigno (visto da compensazione di sangue). (C) Imaging unit contenente embrione aviaria con il coprioggetto interfacciato direttamente con il CAM. (D) Prima dell&#39;iniezione nanoparticelle, vascolarizzazione tumorale viene valutata utilizzando l&#39;imaging intravitale dopo l&#39;iniezione di fluorescina destrano. (E) Imaging unit contenente l&#39;embrione posizionato sul palcoscenico di un microscopio confocale in posizione verticale all&#39;interno di una temperatura regolata impostata recinto a 37 ° C. <img src="/files/ftp_upload/2808/2808fig3.jpg" alt="Figura 3"/> Figura 3. Visualizzazione intravitale di assorbimento delle nanoparticelle nei tumori umani. I tumori sono visualizzate 7 ore dopo l&#39;iniezione di (a) CPMV-AF647 e (b) CPMV-PEG-AF647. d) Escissione del tumore dall&#39;embrione aviaria per successive analisi.

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Nessun conflitto di interessi dichiarati.

La membrana corioallantoidea (CAM) dell&#39;embrione aviaria è un modello utile per valutare la dinamica vascolare e la farmacocinetica di tumori umani. La struttura e la posizione del CAM consente l&#39;acquisizione di immagini di alta qualità e si adatta di molti generi di xenotrapianti tumorali senza procedure chirurgiche invasive. Inoltre, xenotrapianti tumorali del cancro impiantati nella membrana corioallantoidea diventare vascolarizzato entro 7 giorni, offrendo un rapido, poco costoso e mezzo semi-high-throughput per valutare l&#39;accumulo di nanoparticelle nei tessuti tumorali. Dal xenotrapianti cancro impiantati nel CAM di shell-meno embrioni di pollo sono accessibili al ottica ad alta risoluzione di un microscopio confocale o verticale epifluorescenza, informazioni contestuali e temporali per quanto riguarda l&#39;assorbimento delle nanoparticelle nel sistema vascolare del tumore può essere facilmente ottenuto. Xenotrapianti cancro in questo modello tendono a crescere lateralmente attraverso la CAM, provocando tumori che sono di grandi dimensioni pur rimanendo meno di 200 m di profondità. Questo li rende particolarmente adatto per l&#39;imaging intravitale perché standard di microscopi epifluorescenza può effettivamente penetrare l&#39;intera massa tumorale. Al contrario, i tumori impiantati in entrambi i siti superficiali o ortotopico all&#39;interno del mouse proliferano in tre dimensioni, rendendo difficile localizzare con precisione le nanoparticelle in profondità all&#39;interno di questi tumori con tecniche non invasive. Abbiamo utilizzato questo modello per valutare la diffusione di punti quantici, liposomi, nanoparticelle di ossido di ferro e in un certo numero di xenotrapianti tumorali umani, mettendo in evidenza il potenziale di questo modello per essere adatto per l&#39;analisi in vivo di una vasta gamma di formulazioni di nanoparticelle.

<table border="1"><tbody><tr><th> Reagente Nome / attrezzature </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti </th></tr><tr><td> Uova fecondate livorno </td><td> Frey `s Hatchery, St. Jacobs </td><td> N / A </td><td></td></tr><tr><td> Dremel utensile rotante </td><td> Dremel </td><td></td><td> Può utilizzare qualsiasi modello </td></tr><tr><td> Ruote di taglio Dremel no. 36 </td><td> Dremel </td><td> 409 </td><td></td></tr><tr><td> Sportsman hatcher </td><td> Berry Hill </td><td> 1550HA </td><td></td></tr><tr><td> Sportsman incubatore </td><td> Berry Hill </td><td> 1502EA </td><td></td></tr><tr><td> Etanolo </td><td></td><td></td><td> 70% (vol / vol) </td></tr><tr><td> Polistirolo pesa barche </td><td> VWR </td><td> 12577-01 </td><td></td></tr><tr><td> Piazza Petri </td><td> Simport, VWR </td><td> 25378-115 </td><td></td></tr><tr><td> Rubbermaid contenitore in gomma con coperchio </td><td> Guillevin </td><td> RH3-228-00-BLU </td><td> fori in lati </td></tr><tr><td> Verticale pipetta estrattore </td><td> David Kopf Instruments </td><td> modello 720 </td><td> Impostazioni: 16,3 (riscaldamento) e 2.3 (solenoide) </td></tr><tr><td> Sodio tubi in vetro borosilicato capillari </td><td> Sutter strumento </td><td> BF100-58-10 </td><td> (OD, 1,0 mm, ID 0,58 millimetri, 10 cm lunghezza) </td></tr><tr><td> 1X Dulbecco modificato (DMEM) </td><td> Invitrogen </td><td> 11995073 </td><td></td></tr><tr><td> Dulbecco </td><td> Invitrogen </td><td> 14190250 </td><td> pH 7,4 </td></tr><tr><td> PBS (D-PBS) (1X), liquido </td><td></td><td></td><td></td></tr><tr><td> Tripsina, 0,05% (1X) 4NA con EDTA, liquido </td><td> Invitrogen </td><td> 25300054 </td><td></td></tr><tr><td> Siero bovino fetale </td><td> Invitrogen </td><td> 12483-020 </td><td> Calore inattivare </td></tr><tr><td> Emocitometro </td><td> Hausser scientifico, VWR </td><td> 15170-090 </td><td></td></tr><tr><td> Centrifuga </td><td> Eppendorf modello 5810R </td><td> 5811 000.010 </td><td></td></tr><tr><td> Messa a punto di forcipe </td><td> VWR </td><td> 25607-856 </td><td></td></tr><tr><td> Tygon R-3603 tubi </td><td> VWR </td><td> 63009-983 </td><td> 50 ft (1/32-inch diametro interno, 3/32-inch diametro esterno, spessore parete 1/32-inch </td></tr><tr><td> Aghi ipodermici per preparazioni iniettabili 18-gauge aghi </td><td> BD </td><td> 305195 </td><td> Scatola da 100 </td></tr><tr><td> Siringhe da 1 ml per preparazioni iniettabili </td><td> BD </td><td> 309602 </td><td> Scatola da 100 </td></tr><tr><td> Fibra ottica microscopio illuminatore </td><td> Amscope </td><td> HL 250-AY </td><td> 150W </td></tr><tr><td> Kimwipes </td><td> VWR </td><td> 10805-905 </td><td></td></tr><tr><td> Semi V. unguiculata (California occhio nero no. 5) </td><td> Burpee </td><td> 51771A </td><td></td></tr><tr><td> Luci di crescita interna </td><td> Sunlite, Alimentazione giardiniere </td><td></td><td></td></tr><tr><td> Metil-PEO4 NHS-estere </td><td> Perforare </td><td> PI22341 </td><td></td></tr><tr><td> MPEG-NHS, PEG succinimidile estere, MW 2000 </td><td> NANOCS </td><td> PEG1-0002 </td><td></td></tr><tr><td> Alexa Fluor 647 acido carbossilico (succinimidile estere) </td><td> Invitrogen </td><td> A20006 </td><td></td></tr><tr><td> Oregon verde 488 Estere di succinimidile * 6-isomero * </td><td> Invitrogen </td><td> O-6149 </td><td></td></tr><tr><td> Dimetilsolfossido (DMSO) </td><td> Sigma </td><td> D8418 </td><td></td></tr><tr><td> Monoidrogeno fosfato dibasico </td><td> Sigma </td><td> 379980 </td><td> K2HPO4 (per il buffer phopshate) </td></tr><tr><td> Diidrogeno fosfato monobasico </td><td></td><td> P5655 </td><td> KH2PO4 (per il buffer phopshate) </td></tr><tr><td> Superose 6 Dimensioni esclusione colonna </td><td> GE Healthcare delle scienze della vita </td><td> 17-0673-01 </td><td></td></tr><tr><td> Akta Explorer 100 Cromatografo </td><td> GE Amersham Pharmacia </td><td> WS-AKTA100 </td><td></td></tr><tr><td> Akta kit di alta portata </td><td> GE Healthcare delle scienze della vita </td><td> 18-1154-85 </td><td></td></tr><tr><td> Saccarosio </td><td> Sigma </td><td> S0389 </td><td></td></tr><tr><td> Ultracentrifuga </td&gt; <td> Beckman </td><td></td><td></td></tr><tr><td> Ti rotore SW 28 </td><td> Beckman </td><td> 342204 </td><td> Altalena secchio </td></tr><tr><td> 50,2 Ti rotore </td><td> Beckman </td><td> 337901 </td><td> Angolo fisso </td></tr><tr><td> Amicon Ultra-15 Unità di centrifuga Filtro </td><td> Millipore </td><td> UFC910008 </td><td> 100 kDa tagliare </td></tr><tr><td> In disuso </td><td> Biorad </td><td></td><td></td></tr><tr><td> destrano, fluoresceina, 70.000 MW, anionici </td><td> Invitrogen </td><td> D1823 </td><td></td></tr><tr><td> Spinning microscopio a fluorescenza confocale disco </td><td> Quorum; Yokogawa CSU 10, Yokogawa </td><td> N / A </td><td></td></tr><tr><td> Epifluorescenza a grande campo microscopio </td><td> Quorum; Zeiss Axio Examiner, Zeiss </td><td> N / A </td><td></td></tr><tr><td> Hamamatsu Imagem 9100-12 EM-CCD </td><td> Quorum; Hamamatsu </td><td> N / A </td><td></td></tr><tr><td> Unità di temperatura custodia per microscopio </td><td> Materie plastiche di precisione </td><td> N / A </td><td></td></tr><tr><td> Vuoto grasso </td><td> VWR </td><td> 59344-055 </td><td></td></tr><tr><td> Coprioggetto di vetro circolare nr. 1 (18 mm) </td><td> VWR </td><td> 16004-300 </td><td></td></tr><tr><td> Volocity software </td><td> Perkin Elmer </td><td></td><td></td></tr><tr><td> Chick embrione recinto </td><td></td><td> abitudine fabbricata </td><td></td></tr><tr><td> Forbici delicato </td><td> VWR </td><td> 25608-203 </td><td></td></tr><tr><td> Formalina </td><td> Bioshop </td><td> FOR201.500 </td><td> Uso in fumehood </td></tr><tr><td> Taglio ottimale </td><td> Fisher; Tissue Tek </td><td> 1437365 </td><td></td></tr><tr><td> Temperatura (OCT) </td><td></td><td></td><td></td></tr><tr><td> Stampi di plastica </td><td> Pescatore </td><td> 22-038217 </td><td></td></tr><tr><td> VWR VistaVision HistoBond adesivo diapositive </td><td> VWR </td><td> 16004-406 </td><td></td></tr><tr><td> Prolungare oro con DAPI </td><td> Invitrogen </td><td> P36931 </td><td></td></tr><tr><td> Lame monouso per criostato </td><td> Pescatore </td><td> 12-634-2 </td><td></td></tr><tr><td> Criostato </td><td> Leica CM 3050 S </td><td> 14047033518 </td><td></td></tr></table>

evaluation of nanoparticle uptake in tumors in real time using intravital imaging

Le attuali tecnologie per l&#39;imaging del tumore, come l&#39;ecografia, risonanza magnetica, PET e CT, sono in grado di produrre immagini ad alta risoluzione per la valutazione di assorbimento delle nanoparticelle nei tumori a livello microscopico 1,2,3, mettendo in evidenza l&#39;utilità di un modello di xenotrapianto adeguata in cui per eseguire analisi dettagliate assorbimento. Qui, usiamo imaging ad alta risoluzione intravitale per valutare l&#39;assorbimento delle nanoparticelle in xenotrapianti di tumori umani in un modello di embrione modificato senza guscio di pollo. Il modello di embrione di pollo è particolarmente adatto per queste analisi in vivo in quanto supporta la crescita di tumori umani, è relativamente poco costoso e non richiede anestesia o la chirurgia 4,5. Le cellule tumorali forma pienamente xenotrapianti vascolarizzato entro 7 giorni una volta impiantati nella membrana corioallantoidea (CAM) 6. I tumori risultanti sono visualizzati da non invasive di imaging in tempo reale e ad alta risoluzione che può essere mantenuta per un massimo di 72 ore con un impatto minimo su entrambi l&#39;host o sistemi di tumore. Nanoparticelle con una vasta gamma di formati e formulazioni somministrato distale al tumore possono essere visualizzati e quantificabili, perché il flusso attraverso il flusso sanguigno, fuoriuscito da vasi sanguigni del tumore che perde, e si accumulano nella sede del tumore. Descriviamo qui l&#39;analisi delle nanoparticelle derivate da virus del mosaico del Cowpea (CPMV) decorati con coloranti fluorescenti nel vicino infrarosso e / o polimeri di polietilene glicole (PEG) 7, 8, 9,10,11. Dopo somministrazione per via endovenosa, queste nanoparticelle virali sono rapidamente internalizzati dalle cellule endoteliali, con conseguente etichettatura globale del sistema vascolare sia all&#39;esterno che all&#39;interno del tumore 7,12. PEGilazione delle nanoparticelle virale aumenta la loro emivita plasmatica, si estende il loro tempo in circolazione, e migliora in ultima analisi, il loro accumulo nei tumori attraverso la maggiore permeabilità e ritenzione (EPR) effetto 7, 10,11. La velocità e il grado di accumulo di nanoparticelle in un tumore è misurata nel tempo utilizzando un software di analisi delle immagini. Questa tecnica fornisce un metodo per visualizzare e quantificare sia le dinamiche di nanoparticelle nei tumori umani.

Watch this Scientific Journal Video about Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging at JoVE.com

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Vi presentiamo un nuovo approccio per quantificare la localizzazione delle nanoparticelle nei vasi di tumori umani xenotrapiantati utilizzando dinamica, imaging in tempo reale intravitale in un modello di embrione aviaria.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

evaluation-nanoparticle-uptake-tumors-real-time-using-intravital

Nanyang Technological University

Research

JoVE Journal

Medicine

18.2K Views.  University of Western Ontario. Vi presentiamo un nuovo approccio per quantificare la localizzazione delle nanoparticelle nei vasi di tumori umani xenotrapiantati utilizzando dinamica, imaging in tempo reale intravitale in un modello di embrione aviaria.

Video: Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multispettrali in tempo reale immagini a fluorescenza per il rilevamento intraoperatoria del linfonodo sentinella in Oncologia Ginecologica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

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Medicina

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Rilevamento MicroRNA in tumori della prostata dal Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury (IRI) of the Rat Cremaster Muscle

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, intestinal ischemia as well as following transplant and cardiovascular surgery.1 Reperfusion of previously ischemic tissue, while essential for the prevention of irreversible tissue injury, elicits excessive inflammation of the affected tissue. Adjacent to the production of reactive oxygen species, activation of the complement system and increased microvascular permeability, the activation of leukocytes is one of the principle actors in the pathological cascade of inflammatory tissue damage during reperfusion.2, 3 Leukocyte activation is a multistep process consisting of rolling, firm adhesion and transmigration and is mediated by a complex interaction between adhesion molecules in response to chemoattractants such as complement factors, chemokines, or platelet-activating factor.4
While leukocyte rolling in postcapillary venules is predominantly mediated by the interaction of selectins5 with their counter ligands, firm adhesion of leukocytes to the endothelium is selectin-controlled via binding to intercellular adhesion molecules (ICAM) and vascular cellular adhesion molecules (VCAM).6, 7 
Gold standard for the in vivo observation of leukocyte-endothelial interaction is the technique of intravital microscopy, first described in 1968.8
Though various models of IRI (ischemia-reperfusion injury) have been described for various organs, 9-12 only few are suitable for direct visualization of leukocyte recruitment in the microvascular bed on a high level of image quality.8 
We here promote the digital intravital epifluorescence microscopy of the postcapillary venule in the cremasteric microcirculation of the rat 13 as a convenient method to qualitatively and quantitatively analyze leukocyte recruitment for IRI-research in striated muscle tissue and provide a detailed manual for accomplishing the technique. We further illustrate common pitfalls and provide useful tips which should enable the reader to truly appreciate, and safely perform the method.
In a step by step protocol we depict how to get started with respiration controlled anesthesia under sufficient monitoring to keep the animal firmly anesthetized for longer periods of time. We then describe the cremasteric preparation as a thin flat sheet for outstanding optical resolution and provide a protocol for leukocyte imaging in IRI that has been well established in our laboratories.

Real-time Digital Imaging of Leukocyte-endothelial Interaction in Ischemia-reperfusion Injury IRI of the Rat Cremaster Muscle

Digital intravital epifluorescence microscopy of postcapillary venules in the cremasteric microcirculation is a convenient method to gain insights into leukocyte-endothelial interaction in vivo in ischemia-reperfusion injury (IRI) of striated muscle tissue. We here provide a detailed protocol to safely perform the technique and discuss its applications and limitations.

In tempo reale Imaging digitale di leucociti-endotelio in ischemia-riperfusione (IRI) del muscolo Cremaster Rat

Ischemia-reperfusion injury (IRI) has been implicated in a large array of pathological conditions such as cerebral stroke, myocardial infarction, ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Movimento Riqualificazione con feedback in tempo reale delle prestazioni

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

In Vivo Imaging ottico dei tumori cerebrali e artrite Uso fluorescente SAPC-DOPS nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemioterapia-indotta vascolare Tossicità - in tempo reale<em&gt; In vivo</em&gt; Imaging di Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

In tempo reale citotossicità saggi in Human Sangue intero

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

In tempo reale a raggi X Imaging del polmone volumi di fluido in neonatale topo polmone

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...

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 ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...