Name: handling of the cotton rat in studies for the pre-clinical evaluation of oncolytic viruses
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Description: Watch this Scientific Journal Video about Handling of the Cotton Rat in Studies for the Pre-clinical Evaluation of Oncolytic Viruses at JoVE.com

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 pen...

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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. 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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

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

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.

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

Research

JoVE Journal

Medicine

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

Ex Vivo Infection of Live Tissue with Oncolytic Viruses

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

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

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

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

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

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

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

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

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 ...

Pre-clinical Model 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 ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

Valutazione emodinamica invasiva per il sistema ventricolare destro e l'ipertensione arteriosa polmonare indotta dall'ipossia nei topi

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...