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.

NOTE: The protocols used have been approved by our institutional Animal Research Ethics Board at McMaster University according to Canadian Council on Animal Care guidelines. Experiments were performed at the McMaster University Central Animal Facility.

1. Culturing LCRT Cells
<ol>
	<li>Culture LCRT cells in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 &#181;g/ml streptomycin. Maintain cells in T-150 tissue culture flasks at 37 &#176;C and 5% CO2. Passage cells when they form a 90% confluent monolayer (every 2-3 days, Figure 1).</li>
	<li>Pre-warm 1x phosphate buffered saline (PBS), 1x trypsin and medium in a 37 &#176;C water bath for 10 min prior to splitting the cells.</li>
	<li>Aspirate medium from the flask and rinse cells with 5 ml of 1x PBS.</li>
	<li>After rinsing, aspirate PBS and incubate cells with 2 ml of 1x trypsin until cells dissociate from the flask (~2 min).</li>
	<li>Resuspend cells in 8 ml medium (for a total of 10 ml cell suspension) and gently pipette up and down to break up clumps of cells.</li>
	<li>Maintain cells in a T-150 flask by seeding 1 ml cell suspension into 24 ml medium (for a total of 25 ml per T-150) and rock flask gently from side to side. Maintain cells at 37 &#176;C and 5% CO2 until next split.</li>
</ol>
2. Assessing Virus Replication and Cytotoxicity in LCRT Cells
<ol>
	<li>Virus Replication 
	NOTE: Virus constructs expressing a fluorescent tag, such as green fluorescent protein (GFP), under control of endogenous viral promoters facilitate visualization of virus infection and spread using fluorescence plate readers.
	<ol>
		<li>Seed LCRT cells into culture plates, leaving a well for counting. Seed cells such that they will be 80-90% confluent one day later. Use a concentration of 105 cells/ml (100 &#181;l per well) to produce the desired confluency one day later in 96-well flat bottom plates.</li>
		<li>The next day, pre-warm 1x PBS, 1x trypsin, complete and serum-free medium in a 37 &#176;C water bath for 10 min prior to starting the experiment.</li>
		<li>Aspirate medium from the counting well and rinse cells with 5 ml of 1x PBS by rocking it over the surface of the well.</li>
		<li>After rinsing, aspirate PBS and incubate cells with 2 ml of 1x trypsin until cells dissociate from the flask (~2 min).</li>
		<li>Resuspend the cells in the appropriate volume of complete medium to yield a cell density within the countable range using a hemocytometer. To ensure an accurate cell count, thoroughly mix the cell suspension prior to inoculating the hemocytometer by pipetting up and down.</li>
		<li>Determine the volume of virus stock required for infection at the desired multiplicity of infection (MOI). 
		Required Paque Forming Units (pfu) = number of plated cells * MOI (pfu/cell) 
		Volume of virus stock required = required pfu / virus stock titer (pfu/ml)</li>
		<li>Prepare virus inoculum in serum-free medium in tubes. Mix thoroughly by vortexing or pipetting before adding inoculum to the cells.</li>
		<li>Infect cells for 1 hr at 37 &#176;C, after which apply a maintenance overlay of DMEM + 1% FBS.</li>
		<li>Scan plates one and two days post infection (pi) to visualize GFP fluorescence.</li>
	</ol>
	</li>
	<li>Virus Cytotoxicity 
	NOTE: Perform the resazurin cytotoxicity assay under low light conditions as the compound is photosensitive. A well containing medium only should be included to correct for background fluorescence.
	<ol>
		<li>Prepare a 5% (v/v) solution of resazurin in 1x PBS. Mix the solution by pipetting.</li>
		<li>Aspirate medium from cells and apply the 5% resazurin solution. Include a well containing medium only to correct for background fluorescence.</li>
		<li>Incubate cells for 30 min at 37 &#176;C, after which read fluorescence using a fluorescence plate reader (excitation 530 nm, emission 595 nm).</li>
		<li>Analyze data relative to uninfected controls correcting for background fluorescence.</li>
	</ol>
	</li>
</ol>
3. Housing and Handling
<ol>
	<li>Housing and Diet
	<ol>
		<li>House cotton rats individually to decrease in-fighting in polycarbonate rat cages containing rodent bedding (1/8&#8221; corncob bedding), a section of PVC tubing no longer than 8 inches and nestlets as enrichment (Figure 2).</li>
		<li>Use a securable steel basket to sit overtop of the cage and contain rodent food and a water bottle. 
		NOTE: This cage setup will allow for safe and easy capture of the animals, with the placement of the enrichment tube against the end of the cage being of the utmost importance.</li>
	</ol>
	</li>
	<li>Handling
	<ol>
		<li>Handle cotton rats in the morning, prior to rounds by animal facility technicians to avoid exciting them before a procedure.</li>
		<li>During all procedures, wear thick leather gloves for protection.</li>
		<li>As the animals primarily remain in the enrichment tubes, use them to transfer the rats into a new cage during routine cleaning. Alternatively, open the cage slightly to allow the handler to reach their hand in, the animal can then be restrained by scruffing the skin just above the shoulders and pushing down. Practice care not to use excessive force as the animal may bite their tongue.</li>
		<li>Be patient and use a steady hand as the animals have a strong flight-or-fight response and will try to avoid capture by running and jumping out of the cage. Importantly, do not handle animals by the tail as degloving will occur.</li>
		<li>Trap the animals in their enrichments tubes over direct handling. This drastically decreases injuries and escaping.</li>
	</ol>
	</li>
</ol>
4. Capture and Anesthesia
<ol>
	<li>Capture
	<ol>
		<li>Wear thick leather gloves for protection during all procedures.</li>
		<li>Use a large clear plastic container with holes for air and a lid, an anesthetic induction chamber large enough to fit the container and a nose cone fitted to the gas output hose (Figure 3).</li>
		<li>Work in pairs to make the procedure more efficient and to decrease the exposure time of the animals to isoflurane, an inhalation anesthetic. Make sure one researcher is responsible for opening and replacing the steel cover on the cage and the lid of the induction chamber (handler #1) and their associate is responsible for capture of the animal in the tube and transport to the induction chamber (handler #2).</li>
		<li>Place the cage on a flat surface and remove the outer lid. Lift the steel feed tray slightly and slowly maneuver the enrichment tube so it is parallel with the sides of the cage and against the back. If necessary, use an object to maneuver the enrichment tube without opening the cage to avoid agitating the animal (handler #1).</li>
		<li>If the animal becomes agitated and leaves the tube, allow adequate time for the animal to relax and once again settle in the tube (handler #1).</li>
		<li>Slowly and deliberately lift the edge of the steel cover furthest from the enrichment tube, keeping the other end in contact with the cage. Make a space large enough for the plastic container (handler #2).</li>
		<li>In one smooth, quick motion, push the plastic container overtop of the enrichment tube. Maintain contact of the container with the side of the cage, trapping the animal in the tube. Perform steps 4.1.6 and 4.1.7 as quickly as possible (handler #2).</li>
		<li>Remove the steel feed tray and give the plastic container lid to handler #2 (handler #1). Slide the plastic lid between the side of the cage and the container, being mindful of the trapped animal&#8217;s appendages in the process. Do not seal the container as this will make the next step more difficult (handler #2).</li>
	</ol>
	</li>
	<li>Anesthesia
	<ol>
		<li>Make sure the container remains closed and transport the animal to the induction chamber. Quickly place the animal in the chamber and remove the container lid in one fluid motion (handler #2). Open and immediately replace the induction chamber lid (handler #1).</li>
		<li>Turn on the flow of isoflurane to the induction chamber (5 L/min) and monitor the animal for signs of lethargy, at which point quickly slide the animal from the tube and container, removing both from the induction chamber to facilitate gas circulation.</li>
		<li>When the rat is fully anesthetized, move it to the working surface and place the nose and mouth into the nose cone (Figure 3). The rat is fully anesthetized when it is unresponsive to a forceful toe pinch.</li>
		<li>Place vet petrolatum ophthalmic ointment on the animal&#39;s eyes to prevent dryness and abrasions. This is an essential step as cotton rats have large eyes that can be prone to infection if injury occurs.</li>
		<li>Carefully monitor and maintain a constant respiration rate and ensure that the animal&#8217;s nose remains in the nose cone fitting throughout the procedure. Adjust the flow rate of isoflurane appropriately. The amount of isoflurane required to anesthetize each animal will vary.</li>
		<li>Post-procedure, return the animal to its cage and ensure it regains full mobility and sternal recumbency.</li>
	</ol>
	</li>
</ol>
5. Preparation of LCRT Cells for Subcutaneous Tumor Formation
NOTE: One T-150 flask of LCRT (90% confluency) yields approximately 2 x 107 cells. Base the number of T-150 flasks required on the total number of cells needed. Seed extra flasks to ensure the total number of cells required is obtained and to accommodate cells lost during preparation and those needed for extra injections. Keep cells on ice whenever possible to prolong cell viability.
<ol>
	<li>To harvest cells, aspirate medium from the flask and rinse cells with 5 ml of 1x PBS.</li>
	<li>Aspirate PBS and incubate cells with 2 ml of 1x trypsin until cells dissociate from the flask (~2 min).</li>
	<li>Resuspend cells in 8 ml medium (for a total of 10 ml cell suspension) and gently pipette up and down to break up clumps of cells. Continue to harvest cells from additional flasks.</li>
	<li>Pool all cell suspensions into one conical tube, approximately 4 T-150s per 50 ml conical tube.</li>
	<li>Centrifuge the tube at 160 x g for 10 min at 4 &#176;C.</li>
	<li>Aspirate medium and resuspend the cell pellet in the appropriate volume of PBS (10 ml PBS per T-150) to yield a cell density within the countable range using a hemocytometer. To ensure an accurate cell count, thoroughly mix the cell suspension prior to loading the hemocytometer by pipetting up and down.</li>
	<li>Calculate the total number of cells: 
	Total number of cells harvested = cell count (cells/ml) x resuspension volume (ml)</li>
	<li>Determine the volume of cell suspension required for all injections. Make 2-3 extra doses per experiment. A total of 5 x 105 LCRT cells injected subcutaneously will form palpable tumors within 3-4 days. 
	Total number of cells required = 5 x 105 cells x total number of doses 
	Volume cell suspension required (ml) = (total cells required * sum of injection volumes) / (total number of cells harvested)</li>
	<li>Pipet the required volume of cell suspension into a conical tube containing PBS and mix thoroughly. Aliquot individual injections (100 &#181;l) into Eppendorf tubes. Maintain tubes on ice during injection procedure.</li>
</ol>
6. Injections
NOTE: Perform procedures with two researchers, one to perform the injections while the other monitors the animal&#8217;s respiration rate and general condition while under anesthesia. Use insulin syringes (29 G x 1/2&#8217;, 0.3 ml) for all injections and a new needle for each animal.
<ol>
	<li>Subcutaneous injections
	<ol>
		<li>Capture and anesthetize the animal (section 4).</li>
		<li>Shave the injection site using clippers. Cotton rat fur is thick and requires a sharp trimmer to get a smooth surface for injections. Clean the injection site with 70% ethanol using a cotton swab and allow it to evaporate completely before proceeding.</li>
		<li>Load syringes (29 G x 1/2&#8217;, 0.3 ml) with the cells by drawing up slowly and steadily. If bubbles are evident flick the syringe with some force. Once the bubbles are at the top push the plunger until the liquid is at the top of the needle.</li>
		<li>Lift the skin at the injection site (referred to as skin tenting) and insert the needle bevel side up. Make sure the needle moves freely under the skin to avoid injecting intramuscularly.</li>
		<li>Expel the contents of the syringe evenly and slowly. Withdraw the needle bevel side down.</li>
	</ol>
	</li>
	<li>Intratumoral injections
	<ol>
		<li>Capture and anesthetize the animal (section 4).</li>
		<li>Clean the injection site with 70% ethanol using a cotton swab and allow it to evaporate completely before proceeding.</li>
		<li>Load syringes (29 G x 1/2&#8217;, 0.3 ml) with the virus inoculum by drawing up slowly and steadily while holding the needle in an upright position. If bubbles are evident flick the syringe with some force. Once the bubbles are at the top push then plunger until the liquid is at the top of the needle.</li>
		<li>Insert the needle bevel side up into the tumor and expel the contents of the syringe evenly and slowly while moving the needle in a fan-like pattern, partially withdrawing the needle before each movement to prevent laceration of the tumor. Withdraw the needle bevel side down. 
		NOTE: Subcutaneous LCRT tumors are fast-growing, reaching approximately 100 mm3 in 5-7 days. Furthermore, necrotic and hemorrhagic centers often form on the surface of the tumor within several days and require careful monitoring (Figure 4).</li>
	</ol>
	</li>
	<li>Intraperitoneal injections
	<ol>
		<li>Capture and anesthetize the animal (section 4).</li>
		<li>Clean the injection site with 70% ethanol using cotton swabs and allow it to evaporate completely before proceeding.</li>
		<li>Load syringes (29 G x 1/2&#8217;, 0.3 ml) with the drug by drawing up slowly and steadily. If bubbles are evident flick the syringe with some force. Once the bubbles are at the top push then plunger until the liquid is at the top of the needle.</li>
		<li>Insert the needle into the right lower quadrant of the abdomen. Pull back on the plunger to ensure that blood or feces are not aspirated, this indicates incorrect placement of the needle. If this occurs, withdraw the needle and prepare a new syringe. When the needle is correctly placed, expel contents of the syringe evenly and slowly.</li>
	</ol>
	</li>
</ol>
7. Tumor Excision and Necropsy
<ol>
	<li>Gather and clean all tools with 70% ethanol prior to euthanasia of the animal.</li>
	<li>Euthanize the animal by the desired method, CO2 inhalation (2 L/min for 5-10 min) is recommended. Examine the animal for any abnormalities in body condition.</li>
	<li>Place the animal in dorsal recumbency on a dissection board and clean the animal with 70% ethanol.</li>
	<li>Use tweezers to lift the skin at the lower abdomen. Cut through the skin and muscle using scissors and make a medial incision running the length of the animal (anus to chin).</li>
	<li>Cut the rib cage by making two cuts, one laterally up the side of the ribcage and one across the sternum to expose the heart and lungs. Examine the lobes of the lung for any metastases 27,29.</li>
	<li>Examine all organs for abnormalities and record any changes in color, size, and consistency. If necessary, incise the organs with a scalpel to examine internal tissues. Specifically, examine the liver, kidneys, spleen and gastrointestinal tract.</li>
	<li>Inspect the lymph nodes for metastases and enlargement 27,29.</li>
	<li>To collect the tumor, make flank incisions above and below the tumor such that the skin can be pulled away from the body with tweezers. While firmly holding the skin with tweezers, use a scalpel to carefully remove the tumor by cutting between the tumor and dermis (Figure 5).</li>
	<li>Immediately place the tumor in a labeled container of 10% neutral buffered formalin.</li>
	<li>Depending on the size of the tumor, allow 1-2 days (&#8804; 2 mm, small) or 5-6 days (&#62; 2 mm, large) for fixing before preparing sections for histological analysis (Figure 6).</li>
</ol>

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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. 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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...

Oncolytic viruses (OV) selectively replicate in tumor cells by exploiting biochemical differences between normal and tumor cells 1,2. There are two types of OVs: those that do not require a mutation to achieve selective oncolysis, referred to as naturally occurring wild-type viruses and those which must be engineered to achieve selective oncolysis. The collection of mutations within a given tumor type determines the nature of the selective growth advantage over normal cells for an OV 2. The safety and benefit of using OVs has been demonstrated in clinical trials 3-7. Despite advancements in the field of oncolytic virotherapy there exist gaps between pre-clinical and clinical results, suggesting that better models are needed to evaluate the antitumor efficacy of OVs.
Bovine herpesvirus type 1 (BHV-1) is a member of the Herpesviridae family, and Alphaherpesviridae subfamily. BHV-1 initiates bovine respiratory disease complex in cattle, manifesting in a wide variety of symptoms resembling a bad cold 8,9. BHV-1 binds attachment and entry receptors used by HSV-1, such as heparan sulfate and nectin-1 10. However, it binds CD155 in the place of nectin-2 10. BHV-1 has a very narrow host range such that it is unable to efficiently enter and initiate replication in normal and transformed murine cells 3,4,10. This makes the use of conventional murine models problematic. The oncolytic capacity of BHV-1 has been demonstrated in vitro11,12. BHV-1 has been shown to initiate replication in and kill human tumor cells from a variety of histological origins, including breast cancer cells and breast cancer initiating cells 11,12. However, the antitumor capacity of BHV-1 must be evaluated in vivo within the context of an immunocompetent host.
Human Adenovirus (Ad), for which there are 57 identified serotypes, most commonly causes respiratory illness in humans. Oncolytic Ad vectors have been evaluated for their antitumor efficacy with several advancing into clinical trials 13-15. Despite promising pre-clinical data, clinical results have fallen short of expectations. Human tumor xenograft models are typically used to study the antitumor efficacy of Ad vectors, although they exhibit attenuated immune responses to the virus 16,17. Furthermore, syngeneic murine models are non-permissive to Ad infection, making the evaluation of host immune responses using these models impractical 17,18.
The host immune system has been identified as the most influential mechanism by which OVs elicit tumor cell death 19. Antitumor responses between tolerized and non-tolerized tumor-associated antigen (TAA) models differ and can greatly impact the success of OV therapy. The HSV-1 OV KM100 (ICP0n212VP16in181420)20,21 elicited tumor regression in 80% of tumor-bearing mice in a murine Polyoma Middle T antigen mammary cancer model 22. However, in HER-2/neu models, the antitumor efficacy of KM100 varied between 20% complete regression in syngeneic mice and tumor stasis in transgenic, HER2-tolerized mice. Together these data highlight the importance of fully evaluating OVs using animal models that best recapitulate the human immune landscape to fully understand what features determine therapeutic success.
The cotton rat (Sigmodon hispidus), indigenous to North and South America, is most commonly used as a model of respiratory syncytial virus infection (as reviewed in 5). Cotton rats are also used in anti-BHV-1 vaccination research as they recapitulate the pathology associated with bovine respiratory disease complex 6,23. Furthermore, BHV-1 infection of cotton rats is immunogenic, inducing sustained mucosal and systemic immune responses 6,23-25. Cell lines have been derived from spontaneous fibrosarcoma and osteosarcomas of the mammary gland (LCRT) and bone (CCRT and VCRT), respectively 26. Cotton rats have been used to evaluate the in vivo efficacy of oncolytic Ad vectors as they are susceptible to Ad infection and exhibit similar pathology to humans 27-29. The use of immunocompromised models for the pre-clinical evaluation of OVs are not only less indicative of clinical responses to therapy but they fail to take into account the role of the immune system in oncolytic virotherapy 30,31. Therefore, the syngeneic and tumor-tolerized cotton rat models of mammary carcinoma and osteosarcoma are relevant models in which to evaluate the pre-clinical efficacy of OVs, such as BHV-1 and Ad which cannot be studied using conventional murine models.

<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.

Due to the extremely excitable nature of cotton rats, being familiar with and utilizing procedures optimized to reduce the stress of the animals will ease in their use as a pre-clinical animal model. Use of proper handling techniques will also minimize risk to the researcher.
When using cotton rats it is imperative to stay calm. The rats are highly excitable and will attempt to escape their cage. Use of an enrichment tube and nestlets will minimize escape attempts. Figure 2 sh...

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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