Breanne Cuddington holds a fellowship from the Canadian Breast Cancer Foundation. This work was sponsored by operating grants from the Cancer Research Society and the Canadian Cancer Society Research Institute (formerly the Canadian Breast Cancer Research Alliance). We thank Ann Tollefson (Saint Louis University School of Medicine) for LCRT cells and Dr. Kathleen Delaney and Marion Corrick for technical assistance with cotton rat housing and sedation.

NOTA: I protocolli utilizzati sono stati approvati dal nostro istituzionale Animal Research Ethics Consiglio della McMaster University in base al Canadian sugli orientamenti per la cura degli animali. Gli esperimenti sono stati condotti presso l&#39;impianto di McMaster University Central Animal. 1. Le celle Culturing LCRT <ol><li> Cellule Culture LCRT in terreno Eagle modificato di Dulbecco (DMEM) supplementato con 10% siero bovino fetale (FBS), 2 mM L-glutammina, 100 U / ml di penicillina e 100 ug / ml di streptomicina. Mantenere le cellule in T-150 fiasche di coltura tissutale a 37 ° C e 5% di CO 2. Passaggio cellule quando formano un monostrato 90% confluenti (ogni 2-3 giorni, Figura 1). </li><li> Pre-caldo 1x tampone fosfato salino (PBS), 1x tripsina e medie in un bagno d&#39;acqua a 37 ° per 10 minuti prima di dividere le celle. </li><li> Aspirare il terreno dal pallone e lavare le cellule con 5 ml di 1x PBS. </li><li> Dopo il risciacquo, aspirare PBS e incubarecellule con 2 ml di tripsina 1x fino cellule dissociano dal pallone (~ 2 min). </li><li> Risospendere le cellule in 8 ml di mezzo (per un totale di sospensione cellulare 10 ml) e delicatamente pipettare su e giù per rompere grumi di cellule. </li><li> Mantenere cellule in un pallone T-150 mediante inseminazione di 1 ml di sospensione cellulare in 24 ml di mezzo (per un totale di 25 ml per T-150) e pallone roccia delicatamente da lato a lato. Mantenere le cellule a 37 ° C e 5% di CO 2 fino al prossimo divisione. </li></ol> 2. Valutare la replicazione del virus e citotossicità in cellule LCRT <ol><li> Replica Virus NOTA: costrutti virus che esprimono un tag fluorescenti, come la proteina verde fluorescente (GFP), sotto il controllo di promotori virali endogeni facilitano la visualizzazione di infezione da virus e diffondere con lettori di piastre a fluorescenza. <ol><li> Seed cellule LCRT in piastre di coltura, lasciando un bene per il conteggio. Cellule semi tale che essi saranno 80-90% confluenti un giorno in più. Utilizzare una concentrazione di 10 5 cells / ml (100 ml per pozzetto) per produrre la confluenza desiderato un giorno in più in 96 pozzetti piastre a fondo piatto. </li><li> Il giorno dopo, pre-caldo 1x PBS, 1x tripsina, terreno completo e privo di siero in un bagno d&#39;acqua a 37 ° per 10 minuti prima di iniziare l&#39;esperimento. </li><li> Aspirare il terreno dalle conteggio ben e risciacquo cellule con 5 ml di PBS 1x di oscillazione sopra la superficie del pozzetto. </li><li> Dopo il risciacquo, le cellule aspirare PBS e incubare con 2 ml di tripsina 1x fino cellule si dissociano dal pallone (~ 2 min). </li><li> Risospendere le cellule in volume appropriato di terreno completo per produrre una densità cellulare nell&#39;intervallo numerabile utilizzando un emocitometro. Per assicurare una conta cellulare accurata, mescolare la sospensione cellulare prima inoculando l&#39;emocitometro pipettando su e giù. </li><li> Determinare il volume del virus azione necessaria per l&#39;infezione alla molteplicità desiderata di infezione (MOI). Richiesto Paque Forming Unit (pfu) = numero di cellule placcato * MOI(Pfu / cell) Volume del virus magazzino required = richiesto pfu / virus magazzino titolo (pfu / ml) </li><li> Preparare inoculo virus in terreno privo di siero in tubi. Mescolare accuratamente con il vortex o pipettare prima di aggiungere inoculo alle cellule. </li><li> Infettare le cellule per 1 ora a 37 ° C, dopo di che applicare una sovrapposizione di DMEM + 1% FBS manutenzione. </li><li> Piastre di scansione uno e due giorni dopo l&#39;infezione (pi) di visualizzare GFP fluorescenza. </li></ol></li><li> Virus citotossicità NOTA: Eseguire il test di citotossicità resazurina in condizioni di scarsa illuminazione, come il composto è fotosensibile. Solo Un mezzo ben contenente dovrebbe essere inclusa per correggere fluorescenza di fondo. <ol><li> Preparare una soluzione al 5% (v / v) di resazurina in PBS 1x. Mescolare la soluzione pipettando. </li><li> Medio Aspirare dalle cellule e applicare la soluzione resazurina 5%. Includere un mezzo ben contenente solo per correggere per fluorescenza di fondo. </li><li> Incubare le cellule per 30 min a 37 ° C, dopoche leggere la fluorescenza utilizzando un lettore di piastre a fluorescenza (eccitazione 530 nm, emissione 595 nm). </li><li> Analizzare i dati relativi ai controlli non infetti correzione per fluorescenza di fondo. </li></ol></li></ol> 3. Housing and Handling <ol><li> Housing e dieta <ol><li> Ratti Cotton House individualmente a diminuire in-combattimenti in policarbonato gabbie ratto contenenti biancheria da letto di roditori (1/8 &quot;assestamento pannocchia), un tratto di non più di 8 pollici e nestlets come arricchimento tubi in PVC (Figura 2). </li><li> Usare un cestello in acciaio a protezione diretta a sedersi overtop della gabbia e contenere cibo roditori e una bottiglia d&#39;acqua. NOTA: Questa configurazione gabbia consentirà cattura sicuro e facile degli animali, con il posizionamento del tubo arricchimento contro l&#39;estremità dell&#39;essere gabbia della massima importanza. </li></ol></li><li> Trattamento <ol><li> Maniglia ratti cotone al mattino, prima di giro da parte dei tecnici degli impianti animale per evitare emozionante primauna procedura. </li><li> Durante tutte le procedure, indossare guanti di pelle di spessore per la protezione. </li><li> Come gli animali rimangono essenzialmente nei tubi di arricchimento, utilizzarli per trasferire i ratti in una nuova gabbia durante la pulizia di routine. In alternativa, aprire un po &#39;la gabbia per consentire al gestore di raggiungere la loro mano, l&#39;animale può essere trattenuta scruffing pelle appena sopra le spalle e spingendo verso il basso. Prestare attenzione a non usare forza eccessiva, come l&#39;animale può mordere la lingua. </li><li> Siate pazienti e utilizzare una mano ferma, come gli animali hanno una forte risposta di volo o di lotta e cercheranno di evitare la cattura da correre e saltare fuori dalla gabbia. È importante sottolineare che, non maneggiare gli animali per la coda, come si verificherà degloving. </li><li> Trappola gli animali nel loro arricchimenti tubi sopra manipolazione diretta. Questo diminuisce drasticamente lesioni e fuga. </li></ol></li></ol> 4. Acquisizione e Anestesia <ol><li> Cattura <ol><li> Indossare guanti di pelle di spessore per protezionelitio durante tutte le procedure. </li><li> Utilizzare un grande contenitore in plastica trasparente con fori per l&#39;aria ed un coperchio, una camera di induzione anestetica abbastanza grande da contenere il contenitore ed un cono montato sul tubo di uscita del gas (Figura 3). </li><li> Lavorano in coppia per rendere la procedura più efficiente e per diminuire il tempo di esposizione degli animali isoflurano, un anestetico per inalazione. Assicurarsi un ricercatore è responsabile per l&#39;apertura e la sostituzione della copertura in acciaio sulla gabbia e il coperchio della camera di induzione (gestore 1 #) e loro socio è responsabile per la cattura dell&#39;animale nel tubo e trasporto alla camera di induzione (gestore # 2). </li><li> Posizionare la gabbia su una superficie piana e rimuovere il coperchio esterno. Sollevare il vassoio di alimentazione inox leggermente e lentamente manovrare il tubo arricchimento quindi è parallelo ai lati della gabbia e contro la parte posteriore. Se necessario, utilizzare un oggetto per manovrare il tubo arricchimento senza aprire la gabbia per evitare agitare l&#39;animale (gestirer # 1). </li><li> Se l&#39;animale diventa agitato e lascia il tubo, lasciare il tempo sufficiente per l&#39;animale per rilassarsi e ancora una volta stabilirsi nel tubo (handler 1 #). </li><li> Lentamente e deliberatamente sollevare il bordo della copertura in acciaio lontano dal tubo arricchimento, mantenendo l&#39;altra estremità a contatto con la gabbia. Fare uno spazio sufficiente per il contenitore di plastica (handler 2 #). </li><li> In un movimento rapido e uniforme, spingere il contenitore di plastica overtop del tubo arricchimento. Mantenere il contatto del contenitore con il lato della gabbia, intrappolando l&#39;animale nel tubo. Eseguire i passi 4.1.6 e 4.1.7 il più rapidamente possibile (handler 2 #). </li><li> Rimuovere il vassoio di alimentazione in acciaio e dare il coperchio del contenitore di plastica per Handler 2 # (handler 1 #). Scorrere il coperchio di plastica tra il lato della gabbia e il contenitore, memori di appendici dell&#39;animale intrappolato nel processo. Non sigillare il contenitore come questo renderà il prossimo passo più difficile (handler # 2). </li></ol></li><li> Anesthesia <ol><li> Assicurarsi che il contenitore rimane chiuso e trasportare l&#39;animale alla camera di induzione. Introdurre rapidamente l&#39;animale in camera e rimuovere il coperchio del contenitore in un unico movimento fluido (handler 2 #). Aprire e sostituire immediatamente il coperchio della camera di induzione (handler # 1). </li><li> Accendere il flusso di isoflurano alla camera di induzione (5 L / min) e monitorare l&#39;animale segni di letargia, a questo punto rapidamente scorrono l&#39;animale dal tubo e contenitore, rimozione sia dalla camera di induzione per facilitare la circolazione del gas. </li><li> Quando il ratto è completamente anestetizzato, spostarlo nella superficie di lavoro e posizionare il naso e la bocca nel cono (Figura 3). Il ratto è completamente anestetizzato quando non risponde ad un pizzico punta forte. </li><li> Posizionare vet vaselina pomata oftalmica sugli occhi dell&#39;animale per prevenire la secchezza e abrasioni. Questo è un passo essenziale come i ratti di cotone hanno grandi occhi che possono essere a rischio di infezione in caso di ferite. </li> &lt;li&gt; monitorare attentamente e mantenere un ritmo di respirazione costante e garantire che il naso dell&#39;animale rimane nel naso cono del raccordo durante tutta la procedura. Regolare il flusso di isoflurano appropriato. La quantità di isoflurano richiesta per anestetizzare ogni animale varierà. </li><li> Post-procedura, tornare l&#39;animale a sua gabbia e assicurarsi che riacquista piena mobilità e decubito sternale. </li></ol></li></ol> 5. Preparazione di cellule LCRT per sottocutaneo Tumor Formazione NOTA: Un pallone T-150 LCRT (90% di confluenza) produce circa 2 x 10 7 cellule. Basate il numero di T-150 flaconi necessari sul numero totale di cellule necessarie. Seme flaconi supplementari per garantire il numero totale di cellule viene ottenuta e per ospitare cellule perse durante la preparazione e quelli necessari per iniezioni supplementari. Mantenere le cellule in ghiaccio ogniqualvolta possibile prolungare la vitalità cellulare. <ol><li> Per raccogliere le cellule, a medio aspirato dal un fiascod cellule Sciacquare con 5 ml di 1x PBS. </li><li> Aspirare PBS e incubare cellule con 2 ml di 1x tripsina fino cellule si dissociano dal pallone (~ 2 min). </li><li> Risospendere le cellule in 8 ml di mezzo (per un totale di sospensione cellulare 10 ml) e delicatamente pipettare su e giù per rompere grumi di cellule. Continuare a raccogliere le cellule da palloni supplementari. </li><li> Piscina tutto sospensioni cellulari in un tubo conico, circa 4 T-150s per tubo da 50 ml. </li><li> Centrifugare la provetta a 160 x g per 10 min a 4 ° C. </li><li> Aspirare medio e risospendere il pellet cellulare in volume appropriato di PBS (10 ml PBS per T-150) per produrre una densità cellulare all&#39;interno della gamma numerabile utilizzando un emocitometro. Per assicurare una conta cellulare accurata, mescolare la sospensione cellulare prima di caricare l&#39;emocitometro pipettando su e giù. </li><li> Calcolare il numero totale di cellule: Numero totale delle cellule raccolte = conta delle cellule (cellule / ml) x risospensione volume (ml) </li><li> Determinareil volume di sospensione cellulare richiesto per tutte le iniezioni. Effettuare 2-3 dosi extra per esperimento. Un totale di 5 x 10 5 cellule LCRT iniettate sottocute formerà tumori palpabili entro 3-4 giorni. Numero totale di celle richieste = 5 x 10 5 cellule x numero totale di dosi Sospensione cellulare Volume richiesto (ml) = (cellule totali richiesti * somma dei volumi di iniezione) / (numero totale di cellule raccolte) </li><li> Dispensare il volume richiesto di sospensione cellulare in un tubo conico contenente PBS e mescolare accuratamente. Aliquota iniezioni singole (100 microlitri) in provette Eppendorf. Mantenere i tubi in ghiaccio durante la procedura di iniezione. </li></ol> 6. Iniezioni NOTA: Eseguire le procedure con due ricercatori, uno per eseguire le iniezioni, mentre gli altri monitor frequenza respiratoria dell&#39;animale e la condizione generale, mentre sotto anestesia. Utilizzare siringhe da insulina (29 G x 1/2 &#39;, 0,3 ml) per tutte le iniezioni e un nuovo ago perogni animale. <ol><li> Iniezioni sottocutanee <ol><li> Cattura e anestetizzare l&#39;animale (sezione 4). </li><li> Shave il sito di iniezione con tagliaunghie. Cotton rat pelliccia è spessa e richiede un trimmer acuto per ottenere una superficie liscia per preparazioni iniettabili. Pulire il sito di iniezione con il 70% di etanolo con un batuffolo di cotone e lasciarlo evaporare completamente prima di procedere. </li><li> Siringhe di carico (29 G x 1/2 &#39;, 0,3 ml) con le cellule redazione lentamente e costantemente. Se le bolle sono flick evidenti la siringa con una certa forza. Una volta che le bolle sono premendo lo stantuffo alto finché il liquido è nella parte superiore dell&#39;ago. </li><li> Sollevare la pelle al sito di iniezione (denominato tenting pelle) e inserire l&#39;ago smusso verso l&#39;alto. Assicurarsi che l&#39;ago si muove liberamente sotto la pelle per evitare l&#39;iniezione intramuscolare. </li><li> Espellere il contenuto della siringa in modo uniforme e lentamente. Estrarre il lato smusso ago verso il basso. </li></ol></li><li> Iniezioni intratumorale <ol><li> Scattare unad anestetizzare l&#39;animale (sezione 4). </li><li> Pulire il sito di iniezione con il 70% di etanolo con un batuffolo di cotone e lasciarlo evaporare completamente prima di procedere. </li><li> Siringhe di carico (29 G 1/2 x &#39;, 0,3 ml) con l&#39;inoculo del virus elaborando lentamente e costantemente tenendo l&#39;ago in posizione verticale. Se le bolle sono flick evidenti la siringa con una certa forza. Una volta che le bolle sono premendo poi pistone sino all&#39;inizio del liquido è in cima dell&#39;ago. </li><li> Inserire il lato dell&#39;ago fino smusso nel tumore ed espellere il contenuto della siringa in modo uniforme e lentamente mentre si muove l&#39;ago in un modello a ventaglio, parzialmente estratto l&#39;ago prima di ogni movimento per evitare la lacerazione del tumore. Estrarre il lato smusso ago verso il basso. NOTA: i tumori LCRT sottocutanee sono in rapida crescita, raggiungendo circa 100 mm 3 in 5-7 giorni. Inoltre, necrotiche e emorragiche centri spesso formano sulla superficie del tumore entro diversi giorni e richiedono camonitoraggio Reful (Figura 4). </li></ol></li><li> Iniezioni intraperitoneali <ol><li> Cattura e anestetizzare l&#39;animale (sezione 4). </li><li> Pulire il sito di iniezione con il 70% di etanolo con tamponi di cotone e lasciarlo evaporare completamente prima di procedere. </li><li> Siringhe di carico (29 G x 1/2 &#39;, 0,3 ml) con la droga, elaborando lentamente e costantemente. Se le bolle sono flick evidenti la siringa con una certa forza. Una volta che le bolle sono premendo poi pistone sino all&#39;inizio del liquido è in cima dell&#39;ago. </li><li> Inserire l&#39;ago nel quadrante in basso a destra dell&#39;addome. Tirare indietro lo stantuffo per garantire che il sangue o feci non sono aspirati, ciò indica errato posizionamento dell&#39;ago. In questo caso, ritirare l&#39;ago e preparare una nuova siringa. Quando l&#39;ago è posizionato correttamente, di espellere il contenuto della siringa in modo uniforme e lentamente. </li></ol></li></ol> 7. Tumore escissione e Necropsy <ol><li> Raccogliere e pulire alstrumenti l con etanolo al 70% prima dell&#39;eutanasia dell&#39;animale. </li><li> Euthanize l&#39;animale con il metodo desiderato, si raccomanda CO 2 inalazione (2 L / min per 5-10 min). Esaminare l&#39;animale per eventuali anomalie nella condizione fisica. </li><li> Posto l&#39;animale in decubito dorsale su una tavola di dissezione e pulire l&#39;animale con il 70% di etanolo. </li><li> Utilizzare le pinzette per sollevare la pelle al basso addome. Tagliare la pelle ei muscoli con le forbici e fare un&#39;incisione mediale corre lungo dell&#39;animale (ano al mento). </li><li> Tagliare la gabbia toracica facendo due tagli, uno lateralmente sul lato della gabbia toracica e uno attraverso lo sterno per esporre cuore e polmoni. Esaminare i lobi dei polmoni per eventuali metastasi 27,29. </li><li> Esaminare tutti gli organi per le anomalie e registrare eventuali cambiamenti di colore, la dimensione e la consistenza. Se necessario, gli organi incidere con un bisturi per esaminare tessuti interni. In particolare, esamina il fegato, i reni, la milza e il tratto gastrointestinale. </li><li> Controllare i linfonodi per metastasi e l&#39;allargamento 27,29. </li><li> Per raccogliere il tumore, fare incisioni fianchi sopra e sotto il tumore in modo che la pelle può essere tirato via dal corpo con le pinzette. Tenendo saldamente la pelle con una pinzetta, utilizzare un bisturi per rimuovere accuratamente il tumore tagliando tra tumore e derma (Figura 5). </li><li> Posizionare immediatamente il tumore in un contenitore etichettato del 10% formalina tamponata neutra. </li><li> A seconda delle dimensioni del tumore, consentire 1-2 giorni (≤ 2 mm piccole) o 5-6 giorni (&gt; 2 mm grandi) di fissaggio prima di preparare sezioni per l&#39;analisi istologica (figura 6). </li></ol>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I+trial+of+intravenous+CG7870,+a+replication-selective,+prostate-specific+antigen-targeted+oncolytic+adenovirus,+for+the+treatment+of+hormone-refractory,+metastatic+prostate+cancer.">A phase I trial of intravenous CG7870, a replication-selective, prostate-specific antigen-targeted oncolytic adenovirus, for the treatment of hormone-refractory, metastatic prostate cancer.</a> Mol Ther. 14 (1), 107-117 (2006).</li><li>Freytag, S. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+I+study+of+replication-competent+adenovirus-mediated+double+suicide+gene+therapy+for+the+treatment+of+locally+recurrent+prostate+cancer.">Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer.</a> Cancer Res. 62 (17), 4968-4976 (2002).</li><li>Benjamin, R., Helman, L., Meyers, P., Reaman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phase+I/II+dose+escalation+and+activity+study+of+intravenous+injections+of+OCaP1+for+subjects+with+refractory+osteosarcoma+metastatic+to+lung.">A phase I/II dose escalation and activity study of intravenous injections of OCaP1 for subjects with refractory osteosarcoma metastatic to lung.</a> Hum Gene Ther. 12 (12), 1591-1593 (2001).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), 3-5 (1994).</li><li>Tsai, J. C., Garlinghouse, G., McDonnell, P. J., Trousdale, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+animal+model+of+adenovirus-induced+ocular+disease.+The+cotton+rat.">An experimental animal model of adenovirus-induced ocular disease. The cotton rat.</a> Arch Ophthalmol. 110 (8), 1167-1170 (1992).</li><li>Ginsberg, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+model+for+investigating+the+molecular+pathogenesis+of+adenovirus+pneumonia.">A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia.</a> Proc Natl Acad Sci U S A. 88 (5), 1651-1655 (1991).</li><li>Russell, S. J., Peng, K. W., Bell, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncolytic+virotherapy.">Oncolytic virotherapy.</a> Nat Biotechnol. 30 (7), 658-670 (2012).</li><li>Mossman, K. L., Saffran, H. A., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+mutants+are+hypersensitive+to+interferon.">Herpes simplex virus ICP0 mutants are hypersensitive to interferon.</a> J Virol. 74 (4), 2052-2056 (2000).</li><li>Mossman, K. L., Smiley, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+and+ICP34.5+counteract+distinct+interferon-induced+barriers+to+virus+replication.">Herpes simplex virus ICP0 and ICP34.5 counteract distinct interferon-induced barriers to virus replication.</a> J Virol. 76 (4), 1995-1998 (2002).</li><li>Hummel, J. L., Safroneeva, E., Mossman, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+ICP0-Null+HSV-1+and+interferon+signaling+defects+in+the+effective+treatment+of+breast+adenocarcinoma.">The role of ICP0-Null HSV-1 and interferon signaling defects in the effective treatment of breast adenocarcinoma.</a> Mol Ther. 12 (6), 1101-1110 (2005).</li><li>Papp, Z., Middleton, D. M., Mittal, S. K., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mucosal+immunization+with+recombinant+adenoviruses:+induction+of+immunity+and+protection+of+cotton+rats+against+respiratory+bovine+herpesvirus+type+1+infection.">Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.</a> J Gen Virol. 78 (11), 2933-2943 (1997).</li><li>Papp, Z., Babiuk, L. A., Baca-Estrada, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+pre-existing+adenovirus-specific+immunity+on+immune+responses+induced+by+recombinant+adenovirus+expressing+glycoprotein+D+of+bovine+herpesvirus+type+1.">The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.</a> Vaccine. 17 (7-8), 933-943 (1999).</li><li>Mittal, S. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+systemic+and+mucosal+immune+responses+in+cotton+rats+immunized+with+human+adenovirus+type+5+recombinants+expressing+the+full+and+truncated+forms+of+bovine+herpesvirus+type+1+glycoprotein+gD.">Induction of systemic and mucosal immune responses in cotton rats immunized with human adenovirus type 5 recombinants expressing the full and truncated forms of bovine herpesvirus type 1 glycoprotein gD.</a> Virology. 222 (2), 299-309 (1996).</li><li>Steel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syngeneic+Cotton+Rat+Cancer+Model+for+Replicating+Adenoviral+Vectors.">Syngeneic Cotton Rat Cancer Model for Replicating Adenoviral Vectors.</a> Molecular Therapy. 13 (1), 123 (2006).</li><li>Toth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Hum Gene Ther. 16 (1), 139-146 (2005).</li><li>Toth, K., Spencer, J. F., Wold, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent,+semi-permissive+cotton+rat+tumor+model+for+the+evaluation+of+oncolytic+adenoviruses.">Immunocompetent, semi-permissive cotton rat tumor model for the evaluation of oncolytic adenoviruses.</a> Methods Mol Med. 130, 157-168 (2007).</li><li>Steel, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocompetent+syngeneic+cotton+rat+tumor+models+for+the+assessment+of+replication-competent+oncolytic+adenovirus.">Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus.</a> Virology. 369 (1), 131-142 (2007).</li><li>Workenhe, S. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunogenic+HSV-mediated+oncolysis+shapes+the+antitumor+immune+response+and+contributes+to+therapeutic+efficacy.">Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.</a> Mol Ther. 22 (1), 123-131 (2014).</li><li>Sobol, P. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+antiviral+immunity+is+a+determinant+of+the+therapeutic+success+of+oncolytic+virotherapy.">Adaptive antiviral immunity is a determinant of the therapeutic success of oncolytic virotherapy.</a> Mol Ther. 19 (2), 335-344 (2011).</li><li>Prince, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cotton+Rat+in+Biomedical+Research.">The Cotton Rat in Biomedical Research.</a> Animal Welfare Information Center Newsletter. 5 (2), (1994).</li></ol>

The authors acknowledge there are no financial conflicts of interest related to this research.

Cotton rats are highly excitable and have a strong flight response. Therefore, special care should be taken to minimize any undue stress on the animal. The cage setup described will allow for safe and easy capture of the animals, with the placement of the enrichment tube being of the utmost importance. When setting up cages, ensure that the enrichment tubes meet the size and shape requirements, and are placed in proper orientation in the cage. It is also important to ensure that any technicians who might be aiding in ani...

Virus oncolitici (OV) replicano selettivamente nelle cellule tumorali sfruttando differenze biochimiche tra cellule normali e tumorali 1,2. Ci sono due tipi di VU: quelli che non necessitano di una mutazione per raggiungere oncolisi selettivo, denominato naturalmente allo wild-type virus e quelli che devono essere progettati per ottenere oncolisi selettiva. La collezione di mutazioni all&#39;interno di un determinato tipo di tumore determina la natura del vantaggio di crescita selettiva su cellule normali per OV 2. La sicurezza e vantaggio di utilizzare VU è stata dimostrata in studi clinici 3-7. Nonostante i progressi nel campo della viroterapia oncolytic esistono spazi vuoti tra i risultati pre-clinici e clinici, suggerendo che i modelli migliori sono necessari per valutare l&#39;efficacia antitumorale di VU. Bovine herpesvirus 1 (BHV-1) è un membro della famiglia Herpesviridae, e Alphaherpesviridae sottofamiglia. BHV-1 initiAtes bovina complesso malattie respiratorie nei bovini, manifestando in una grande varietà di sintomi che assomiglia ad un brutto raffreddore 8,9. BHV-1 si lega fissaggio e ingresso recettori utilizzati da HSV-1, come eparan solfato e nectin-1 10. Tuttavia, si lega CD155 nel luogo di nectin-2 10. BHV-1 ha una gamma di ospiti molto ristretta tale che sia in grado di entrare e avviare la replica in cellule murine normali e trasformate 3,4,10 efficiente. Questo rende l&#39;uso di modelli murini convenzionali problematico. La capacità di oncolytic BHV-1 è stata dimostrata in vitro 11,12. BHV-1 ha dimostrato di avviare la replica e uccidere le cellule tumorali umane da una varietà di origini istologici, comprese le cellule del cancro al seno e il cancro al seno avvio cellule 11,12. Tuttavia, la capacità antitumorale di BHV-1 deve essere valutata in vivo nel contesto di un ospite immunocompetente. Adenovirus umano (Ad), per il qualeci sono 57 sierotipi identificati, più comunemente provoca malattie respiratorie negli esseri umani. Ad vettori oncolitici sono stati valutati per la loro efficacia antitumorale con diversi avanzare in studi clinici 13-15. Nonostante i dati pre-clinici promettenti, i risultati clinici sono stati inferiori alle aspettative. Modelli xenotrapianto di tumori umani sono in genere utilizzati per studiare l&#39;efficacia antitumorale di vettori Ad, anche se esibiscono attenuate le risposte immunitarie al virus 16,17. Inoltre, i modelli murini singenici sono non-permissive all&#39;infezione annuncio, rendendo la valutazione della risposta immunitaria con questi modelli impraticabile 17,18. Il sistema immunitario dell&#39;ospite è stato identificato come il meccanismo più influente attraverso il quale VU suscitano tumorali morte cellulare 19. Risposte antitumorali tra tolleranti e l&#39;antigene associato al tumore non-tolleranti (TAA) modelli differiscono e possono influenzare notevolmente il successo della terapia OV. L&#39;HSV-1 OV KM100 (ICP0 N212VP16 nel 1814 20) 20,21 suscitato regressione del tumore nel 80% dei topi portatori di tumore in un murino antigene Polyoma Medio T modello di cancro mammario 22. Tuttavia, in HER-2 modelli / neu, l&#39;efficacia antitumorale di KM100 variava tra il 20% completa regressione nei topi singenici e tumore stasi transgenici, topi HER2-tolleranti. Insieme, questi dati evidenziano l&#39;importanza di valutare pienamente VU utilizzando modelli animali che meglio ricapitolano il paesaggio immunitario umano per comprendere appieno le caratteristiche che determinano il successo terapeutico. Il ratto di cotone (Sigmodon hispidus), indigeni del Nord e Sud America, è più comunemente usato come modello di infezione da virus respiratorio sinciziale (come rivisto in 5). Ratti di cotone sono utilizzati anche in-BHV-1 contro la ricerca di vaccinazione come ricapitolano la patologia associata a malattia respiratoria bovina complessa 6,23. Inoltre, BHV-1 infezione di ratti di cotoneè immunogenico, inducendo mucosa sostenuta e risposte immunitarie sistemiche 6,23-25. Le linee cellulari sono stati derivati ​​da fibrosarcoma spontanea e osteosarcomi della ghiandola mammaria (LCRT) e ossa (CCRT e VCRT), rispettivamente, 26. Cotone ratti sono stati utilizzati per valutare l&#39;efficacia in vivo di vettori Ad oncolytic come sono suscettibili all&#39;infezione annuncio ed esporre patologia simile all&#39;uomo 27-29. L&#39;uso di modelli immunocompromessi per la valutazione pre-clinica di VU non sono solo meno indicativo di risposte cliniche alla terapia ma non tengono conto del ruolo del sistema immunitario in viroterapia oncolitica 30,31. Pertanto, la singenici e modelli tumorali cotone ratto-tolleranti di carcinoma mammario e osteosarcoma sono modelli pertinenti a cui valutare l&#39;efficacia pre-clinica di VU, quali BHV-1 e Ad che non può essere studiata utilizzando modelli murini convenzionali.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium&#160;</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>May use any brand&#160;</td>
		</tr>
		<tr>
			<td>1X Phosphate Buffered Saline&#160;</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab, filter to sterilize</td>
		</tr>
		<tr>
			<td>200 mM L-glutamine</td>
			<td>Gibco</td>
			<td>25030164</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>100x Antibiotic-Antimycotic&#160;</td>
			<td>Gibco</td>
			<td>15240-062</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Quality Biological Inc.</td>
			<td>110-001-101HI</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>T-150cm2 tissue culture flask</td>
			<td>Fisher Scientific</td>
			<td>14-826-80</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>1X TypLE Express</td>
			<td>Life Technologies</td>
			<td>12604-013</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well cell culture plate, flat bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-29</td>
			<td>May use any brand, must be tissue culture treated</td>
		</tr>
		<tr>
			<td>alamarBlue</td>
			<td>Life Technologies</td>
			<td>DAL1025</td>
			<td>May use an alternative reagent for determination of cell viability</td>
		</tr>
		<tr>
			<td>8640 Teklad 22/5 Rodent diet</td>
			<td>Harlan&#160;</td>
			<td>8640</td>
			<td></td>
		</tr>
		<tr>
			<td>1/8&#8221; corncob rodent bedding</td>
			<td>Harlan</td>
			<td>7092</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestlets</td>
			<td>Ancare</td>
			<td>-</td>
			<td>Made of pulped virgin cotton fiber, dust-free and autoclavable</td>
		</tr>
		<tr>
			<td>50 mL Conical tubes</td>
			<td>Fisher Scientific</td>
			<td>14-432-22</td>
			<td>May use any brand, must be sterile</td>
		</tr>
		<tr>
			<td>Isoflurane USP, 99.9 %, inhalation anesthetic</td>
			<td>Pharmaceutical Partners of Canada Inc.</td>
			<td>M60302</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td></td>
			<td></td>
			<td>Can prepare in lab</td>
		</tr>
		<tr>
			<td>10 % Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Name of Material/Equipment</td>
			<td>Company</td>
			<td>Catalog #</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>NAPCO NapFlow 1200 Class II A/B3 Biosafety Microbiological Safety Cabinet (cell culture hood)</td>
			<td>NAPCO</td>
			<td>Model used not currently available</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Thermo Fisher Scientific Precision Heated Water Bath</td>
			<td>Fisher Scientific</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Reichert Bright-line Hemacytometer</td>
			<td>Sigma-Aldrich</td>
			<td>Z359629</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Typhoon Trio BioAnalyzer&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Tecan Safire2 Multi-detection Microplate Reader</td>
			<td>Tecan</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Allegra 6R benchtop centrifuge</td>
			<td>Beckman Coulter</td>
			<td>366816</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Table Top Anaesthesia machine</td>
			<td>VetEquip</td>
			<td>Model used not currently available&#160;</td>
			<td>May use any brand, must be portable</td>
		</tr>
		<tr>
			<td>Wahl Peanut Mini Clippers</td>
			<td>Wahl</td>
			<td></td>
			<td>May use any brand of small clippers</td>
		</tr>
		<tr>
			<td>Insulin syringes 29 G x 1/2&#39;, 0.3 mL</td>
			<td>BD</td>
			<td>329464</td>
			<td>May use any brand. Insulin syringes are recommended as they make injections easier through the rat&#8217;s tough skin.&#160;</td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>MedPro</td>
			<td>018-425</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Sharp-Pointed Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>8940</td>
			<td>May use any brand</td>
		</tr>
		<tr>
			<td>Dissecting Tissue Forceps</td>
			<td>Fisher Scientific</td>
			<td>13-812-41</td>
			<td>May use any brand</td>
		</tr>
	</tbody>
</table>

handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact. For this strategy to be successful, recent studies have shown that involvement of the host immune system is essential. Therefore, oncolytic virotherapy should be evaluated within the context of an immunocompetent model. Furthermore, the study of antitumor therapies in tolerized animal models may better recapitulate results seen in clinical trials. Cotton rats, commonly used to study respiratory viruses, are an attractive model to study oncolytic virotherapy as syngeneic models of mammary carcinoma and osteosarcoma are well established. However, there is a lack of published information on the proper handling procedure for these highly excitable rodents. The handling and capture approach outlined minimizes animal stress to facilitate experimentation. This technique hinges upon the ability of the researcher to keep calm during handling and perform procedures in a timely fashion. Finally, we describe how to prepare cotton rat mammary tumor cells for consistent subcutaneous tumor formation, and how to perform intratumoral and intraperitoneal injections. These methods can be applied to a wide range of studies furthering the development of the cotton rat as a relevant pre-clinical model to study antitumor therapy.

A causa della natura estremamente eccitabile di ratti cotone, essendo a conoscenza e utilizzando procedure ottimizzate per ridurre lo stress degli animali faciliterà nel loro uso come modello animale preclinico. L&#39;uso di tecniche di manipolazione anche minimizzare i rischi per il ricercatore. Quando si utilizzano topi cotone è indispensabile per mantenere la calma. I ratti sono altamente eccitabile e tenteranno di sfuggire alla loro gabbia. L&#39;uso di un tubo di arricchimento e nestl...

Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model.

Manipolazione del Cotton Rat in studi per la valutazione pre-clinica di oncolitici virus

Oncolytic viruses are a novel anticancer therapy with the ability to target tumor cells, while leaving healthy cells intact.  For this strategy to be ...

handling-cotton-rat-studies-for-pre-clinical-evaluation-oncolytic

Research

JoVE Journal

Medicine

9.2K Views.  McMaster University. Cotton rats are extremely excitable and have a strong flight-or-fight response. A handling method optimized to reduce the stress of the animals is described which will make cotton rats more accessible as a preclinical model. 

Video: Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness of a variety of oncolytic platforms including HSV, Reovirus, and Vaccinia OVs as treatment for cancer are currently underway2-5. One key characteristic of oncolytic viruses is that they can be genetically modified to express reporter transgenes which makes it possible to visualize the infection of tissues by microscopy or bio-luminescence imaging6,7. This offers a unique advantage since it is possible to infect tissues from patients ex vivo prior to therapy in order to ascertain the likelihood of successful oncolytic virotherapy8. To this end, it is critical to appropriately sample tissue to compensate for tissue heterogeneity and assess tissue viability, particularly prior to infection9. It is also important to follow viral replication using reporter transgenes if expressed by the oncolytic platform as well as by direct titration of tissues following homogenization in order to discriminate between abortive and productive infection. The object of this protocol is to address these issues and herein describes 1. The sampling and preparation of tumor tissue for cell culture 2. The assessment of tissue viability using the metabolic dye alamar blue 3. Ex vivo infection of cultured tissues with vaccinia virus expressing either GFP or firefly luciferase 4. Detection of transgene expression by fluorescence microscopy or using an In Vivo Imaging System (IVIS) 5. Quantification of virus by plaque assay. This comprehensive method presents several advantages including ease of tissue processing, compensation for tissue heterogeneity, control of tissue viability, and discrimination between abortive infection and bone fide viral replication.

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Oncolytic viruses are promising for cancer therapeutics. The ability to ascertain the infectability of live tissue specimens obtained from patients prior to treatment is a unique advantage of this therapeutic approach. This protocol describes how to process tissues for ex vivo infection with oncolytic virus and subsequent viral quantification.

Ex infezione Vivo del tessuto Live con virus oncolitici

Oncolytic Viruses (OVs) are novel therapeutics that selectively replicate in and kill tumor cells1. Several clinical trials evaluating the effectiveness ...

Ricerca

Medicina

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

L&#39;uso di Farmacologiche-sfida fMRI in ricerca pre-clinica: Applicazione del sistema 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Valutazione pre-clinica della tirosina chinasi inibitori per il trattamento di leucemia acuta

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis. In recent years, oncolytic virotherapies have proven themselves to be potentially powerful tools to fight malignancy. Due to the unique dual blood supply in the liver, it is possible to apply therapies locally to orthotopic liver tumors, which are predominantly fed by arterial blood flow. We have previously demonstrated that hepatic arterial delivery of oncolytic viruses results in safe and efficient transduction efficiency of multifocal HCC lesions, resulting in significant prolongation of survival in immune competent rats. This procedure closely mimics the application of transarterial embolization in patients, which is the standard palliative care provided to many HCC patients. The ability to administer tumor therapies through the hepatic artery in rats allows for a highly sophisticated preclinical model for evaluating novel viral vectors under development. Here we describe the detailed protocol for microdissection of the hepatic artery for infusion of oncolytic virus vectors to treat orthotopic HCC.

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

Transarteriosa somministrazione di oncolitici virus per locoregionale Terapia delle ortotopico HCC in Rats

Hepatocellular carcinoma (HCC) is a disease with limited treatment options and poor prognosis.  In recent years, oncolytic virotherapies have proven ...

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pre-clinica ortotopico Modello murino di Human cancro alla prostata

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

Use of a Low-flow Digital Anesthesia System for Mice and Rats

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers utilize a syringe pump to directly administer volatile anesthetics into a gas stream. Unlike a traditional vaporizer, it can be used at very low flow rates, making it ideal for use on mice and rats. 
The equipment's capability to use low flow rates could result in a substantial cost savings due to the reduced need for anesthetic agents, compressed gas, and charcoal scavenging filters1. A lower flow rate means less waste of anesthetic gas and likely reduces the risk of anesthetic exposure to laboratory personnel. Thus, the high levels of precision and safety associated with direct injection vaporizers, along with a reduced need for anesthetic agents, compressed gas, and charcoal filters are beneficial for research requiring small animal anesthesia. 
The goal of this protocol is to demonstrate the use of a syringe-driven direct injection vaporizer as part of a digital, low-flow anesthesia system. The direct injection vaporizer is capable of accurately delivering anesthesia at very low flow rates compared to a traditional vaporizer, making it a promising alternative for controlled gas anesthetic delivery to rodents.

Here, we present a protocol to more safely and efficiently administer anesthetic gas to mice using a digital, low flow anesthesia system utilizing a syringe-driven direct injection vaporizer.

L&#39;utilizzo di un basso flusso di sistema per anestesia digitale per topi e ratti

A traditional vaporizer depends on flowing gas and atmospheric pressure for passive anesthetic vaporization. Newly developed direct injection vaporizers ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Analizzando Effetti benefici dei supplementi nutrizionali sulle funzioni intestinali epiteliali barriera durante Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

We describe a surgical procedure in an anesthetized rat model for determining the muscle tone and viscoelastic properties of the tongue. The procedure involves specific stimulation of the hypoglossal nerves and application of passive Lissajous force/deformation curves to the muscle.

Valutazione in vivo delle proprietà meccaniche e viscoelastiche del linguaggio del ratto

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

A Pre-Clinical Porcine Model of Orthotopic Heart Transplantation

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart failure. Multiple small-animal models of heart transplantation have been used to study the acute and long-term effects of novel therapies. However, few are tested and demonstrated success in clinical trials. It is of critical importance to evaluate new therapies in a clinically relevant large-animal model for efficient and reliable translation of basic studies&#39;&#160;findings. Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and previously used to investigate novel cardioprotective strategies. This procedure focuses on acute ischemia-reperfusion injury and is a reliable method to investigate novel interventions which have been tested and validated in smaller experimental models, such as the murine model. We demonstrate its usefulness in assessing cardiac performance during the early post-transplantation period and other potential possibilities enabled by the model.

Here, we describe a pre-clinical large-animal (porcine) model of orthotopic heart transplantation that has been firmly established and utilized to investigate novel cardioprotective strategies.

Un modello pre-clinico di trapianto di cuore ortotopico

Fifty-years following the first successful report, cardiac transplantation remains the gold-standard treatment for eligible patients with advanced heart ...

Pre-clinical Model of Cardiac Donation after Circulatory Death

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after circulatory death (DCD) is a solution to address this limited availability, but due to a period of prolonged warm ischemia and the risk of tissue injury, its routine use in cardiac transplantation is seldom seen. In this manuscript we provide a detailed protocol closely mimicking current clinical practices in the context of DCD with continuous monitoring of heart function, allowing for the evaluation of novel cardioprotective strategies and interventions to decrease ischemia-reperfusion injury.
In this model, the DCD protocol is initiated in anesthetized Lewis rats by stopping ventilation to induce circulatory death. When systolic blood pressure drops below 30 mmHg, the warm ischemic time is initiated. After a pre-set warm ischemic period, hearts are flushed with a normothermic cardioplegic solution, procured, and mounted onto a Langendorff ex vivo heart perfusion system. Following 10 min of initial reperfusion and stabilization, cardiac reconditioning is continuously evaluated for 60 min using intraventricular pressure monitoring. A heart injury is assessed by measuring cardiac troponin T and the infarct size is quantified by histological staining. The warm ischemic time can be modulated and tailored to develop the desired amount of structural and functional damage. This simple protocol allows for the evaluation of different cardioprotective conditioning strategies introduced at the moment of cardioplegia, initial reperfusion and/or during ex vivo perfusion. Findings obtained from this protocol can be reproduced in large models, facilitating clinical translation.

This protocol shows a simple and flexible approach for the evaluation of new conditioning agents or strategies to increase the feasibility of cardiac donation after circulatory death.

Modello preclinico di donazione cardiaca dopo la morte circolatoria

Cardiac transplantation demand is on the rise; nevertheless, organ availability is limited due to a paucity of suitable donors. Organ donation after ...