This work was supported by the KU Leuven PF &#8216;IMIR&#8217;, the FWO Research community on biology and ecology of bacterial and fungal biofilms (FWO: WO.026.11N) and by the FWO project G.0804.11. SK gratefully acknowledges KU Leuven for the PDMK 11/089 fellowship and FWO for the postdoctoral fellowship. We are grateful to Nico Vangoethem for his assistance with preparation of the figures. We would like to acknowledge Celia Lobo Romero for technical assistance during in vivo experimental procedures.

NOTE: All animal experiments were approved by the ethical committee of KU Leuven (project number 090/2013). Maintain animals in accordance with the KU Leuven animal care guidelines.
1. C. albicans Growth
<ol>
	<li>Twenty-four hours before the initiation of the animal experiment, prepare YPD plates by adding 10 g of yeast extract granulated, 20 g of bacteriological peptone, and 15 g of granulated agar. Make up volume to 900 ml with Milli-Q water and autoclave.</li>
	<li>Add 50 ml of sterile 40% glucose. Mix thoroughly and pour into Petri dishes. Leave agar plates to cool and solidify. 
	NOTE: In this study, use two strains, namely wild type C. albicans SC5314 &#8211;gLuc negative strain (referred to as WT)and C. albicans SKCA23 strain, which is a wild type C. albicans SC5314 transformed with Clp10::Act1p-gLUC59 plasmid 22.In this strain (named SKCA23-ACTgLuc) gLuc was fused to the endogenous PGA59 gene under the control of ACT1 (actin) promoter (this promoter is active in the yeast, as well as hyphal stage of fungal growth). This strain can be requested from the laboratory of Prof. Patrick Van Dijck, KU Leuven, Leuven, Belgium. Plasmid Clp10::Act1p-gLUC59 plasmid 22 was kindly donated by Prof. C. d&#8217;Enfert, Institute Pasteur, Paris, France.</li>
	<li>Maintain both strains in glycerol stock and store at -80 &#176;C.</li>
	<li>Prior to any experiment streak strains onto an YPD plate. Incubate plate at 37 &#176;C overnight.</li>
</ol>
2. Catheter Pieces Preparation
<ol>
	<li>Before the experiment, determine how many catheters are needed. It is possible to implant up to 6 catheter pieces per mouse (3 catheters on the left and 3 catheter on the right side of the animal (Figure 2A).</li>
	<li>Twenty-four hr prior to the animal surgery, open the package containing triple-lumen catheter under the biological safety cabinet. Remove all unnecessary parts with sterile tweezers and cut the part attached to the catheter with a sterile scalpel. Place ruler under the plastic package and cut polyurethane catheter pieces of exactly 1 cm according to the scale on the ruler (Figure 1). 
	NOTE: It is important to mention that this type of catheter piece is not phosphorescent and therefore suitable for BLI 18,19.</li>
	<li>Place a maximum of 15 cut catheter pieces (step. 2.2) into 2 ml microcentrifuge tubes. Always prepare 3 pieces extra, which are used to enumerate the amount of attached Candida cells on the device after the period of adhesion.</li>
	<li>Supplement catheters with approximately 1.8 ml of 100% fetal bovine serum (FBS).</li>
	<li>Vigorously vortex and add additional 100-200 &#956;l of 100% FBS. Completely cover all catheter pieces with serum.</li>
	<li>Incubate at 37 &#176;C overnight.</li>
</ol>
3. Animals and Suppression of the Immune System
<ol>
	<li>Keep female BALB/c mice (approximately 8 weeks of age) in individually ventilated filter top cages with free access to standard food and water ad libitum.</li>
	<li>Initiate suppression of the immune system 24 hr before animal surgery by adding dexamethasone (0.4 mg/L) to the drinking water of animals. In order to avoid any bacterial contamination of the host, supplement the drinking water with antibiotic, e.g., ampicillin sodium powder (0.5 g/L).</li>
	<li>Keep immunosuppression of animals during the entire experiment (up to 6 days).</li>
</ol>
4. Ex vivo C. albicans Adhesion on FBS-coated Polyurethane Substrates
<ol>
	<li>Transfer each serum-coated catheter piece into a fresh 1.5 ml microcentrifuge tube.</li>
	<li>Scrape off some C. albicans cells grown on YPD plates (Step 1) and suspend them in 1 ml of PBS.</li>
	<li>Dilute Candida cells (1:100) in a separate microcentrifuge tube. Take 10 &#956;l of the diluted sample and apply it on a cell counting chamber. Count at least 16 small squares.</li>
	<li>Prepare Candida cells (each strain separately) in RPMI 1640 medium to a final concentration of 5 x 104 cells/ml.</li>
	<li>Add 1 ml of cell suspension to each serum-coated catheter piece.</li>
	<li>Vigorously vortex and ensure that the catheters are submerged in the medium and not floating on top.</li>
	<li>Incubate catheters at 37 &#176;C for 90 min (period of adhesion).</li>
	<li>Remove catheters with sterile tweezers and wash them twice with 1 ml of PBS. During this step make sure that the washing fluid goes through the lumen by keeping the catheter in vertical position while very gently flushing the catheters. Importantly, do not use a strong flow which may lead to the removal of attached cells.</li>
	<li>Transfer each washed catheter to a clean microcentrifuge tube (one piece per tube).</li>
	<li>Place on ice and keep there until surgery.</li>
</ol>
5. Anesthesia
<ol>
	<li>Prepare anesthesia by mixing 75 &#181;l of ketamine (100 mg/ml) with 100 &#181;l of medetomidine (1 mg/ml) and 825 &#181;l of sterile saline. Administer intraperitoneally (i.p.) 60-80 &#181;l of anesthetic cocktail per 10 g body weight, resulting in a dose of 45-60 mg/kg ketamine and 0.6-0.8 mg/kg medetomidine.</li>
	<li>For reversal of anesthesia, dilute 50 &#181;l atipamezole (5 mg/ml) in 4.95 ml saline, administer i.p. 100 &#181;l per 10 g body weight as antidote, resulting in a dose of 0.5 mg/kg.</li>
	<li>After injection of anesthesia, place the animal into a separate cage and wait until it is fully asleep.</li>
	<li>Confirm proper anesthetization of the animal by light skin pinch and toe pinch, which do not cause any damage to the skin. Any observed movement indicates that the animal is not sufficiently anesthetized to perform surgery. If this happens, wait a couple of minutes longer until the animal does not show any signs of movement upon skin or toe pinch.</li>
</ol>
6. Animal Surgery
<ol>
	<li>Transfer anesthetized animal from the cage on a clean tissue placed on the heating pad, pre-warmed to 37 &#176;C (Figure 2A, (1)). 
	NOTE: A cheaper alternative is to use electrically heated blankets. It is also possible to use isothermic pads, which must be warmed up in the microwave prior to the animal surgery.</li>
	<li>Apply ophthalmic ointment on the eyes.</li>
	<li>Shave the lower back of the animal with an electric razor. Remove all animal hairs and transfer animal on a clean tissue. Disinfect the skin (e.g., with 1% iodine isopropanol or 0.5% chlorhexidine in 70% alcohol) and leave the disinfected area to dry for approximately 1 min (Figure 2A, (2)).</li>
	<li>Make a small incision in the skin (one on the left and one on the right side of the animal) (approximately 0.5&#8211;1 cm) (Figure 2A, (3)).</li>
	<li>Dissect the subcutis with a scissor to create two subcutaneous tunnels. Each tunnel should be approximately 1.5 cm long and 1 cm wide.</li>
	<li>Insert three catheter pieces, previously infected with Candida, in each tunnel. Ensure that the catheters lie next to each other in a horizontal arrangement and that they do not cover each other to enable the implantation of six catheter fragments in total (Figure 2A, (4)).</li>
	<li>Close the incisions with sutures. Alternatively, use Dermabond to close the wound.</li>
	<li>Disinfect the wound very gently with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).</li>
	<li>Apply local anesthetic (xylocaine gel, 2%) directly on the wound.</li>
	<li>Administer reversal of anesthesia (protocol 5, step 2): intraperitoneally 100 &#181;l per 10 g body weight.</li>
	<li>Transfer animal to a clean cage previously placed on a heating plate. Keep the animal separate and warm until the animal is completely awake. Meanwhile, continue with the operation and implant of the next animal. Once all operated animals are fully awake. Transfer to one cage. Monitor animals regularly.</li>
</ol>
7. Bioluminescence Imaging: Preparation of Coelenterazine (CTZ), the Substrate for G. princeps luciferase
<ol>
	<li>Prepare fresh coelenterazine (CTZ) stock solution by dissolving 5 mg/ml CTZ in acidified ethanol or according to manufacturer&#8217;s instructions.</li>
	<li>Prepare 1.2 mM working solution by diluting the stock solution 1:10 in sterile PBS. 
	NOTE: Inject 100 &#181;l CTZ working solutions subcutaneously in the area surrounding the catheters. Use insulin syringes for subcutaneous injection of CTZ.</li>
	<li>Always keep CTZ in the dark (e.g., cover microcentrifuge tubes containing CTZ with aluminum foil). Store the stock solution at -80 &#176;C for the duration of the experiments.</li>
</ol>
8. Bioluminescence Imaging
<ol>
	<li>Initialize the BLI camera.</li>
	<li>Anesthetize the animals using an induction box. Use a gas mixture of isoflurane in oxygen, N2O/ O2, or air at 2-3%.</li>
	<li>After induction, maintain anesthesia in the induction box and in the imaging chamber at 1.5-2%.</li>
	<li>Before starting the imaging session, place the imaging plate in position A, which corresponds to a FOV of 10 cm. Ensure that the right position of the anesthesia outlets and animal by placing a sleeping animal in the box.</li>
	<li>Take few photographs until animal is in the desired imaging position, in the FOV right under the camera.</li>
	<li>Prepare two insulin syringes each containing 100 &#181;l of the CTZ working solution.</li>
	<li>Place one animal on the bench and keep it asleep by means of a nose cone providing gas anesthesia.</li>
	<li>Bring the needles of the syringes in a place surrounding catheters subcutaneously and inject the CTZ simultaneously on top of the catheters.</li>
	<li>Immediately after injection, place the animal on the warm plate in the camera box and start the bioluminescence image acquisition.</li>
	<li>Acquire consecutive scans with acquisition times ranging from 20 to 60 sec (depending on the signal intensity) until the maximum signal intensity is reached. During the acquisition of the next frame, measure the BLI signal intensity of the previously acquired frames by placing a ROI over each catheters trio and measuring the photon flux through this ROI.</li>
	<li>Repeat from step 7 for the next animal(s).</li>
	<li>After imaging, return animals to their cage. Repeat BLI during the course of an experiment for longitudinal, non-invasive follow-up of biofilm formation.</li>
	<li>Analyze the BLI data using Living Image software. Place a rectangular ROI of fixed size over each catheter trio and measure the photon flux (radiance) through each ROI. Repeat this for every animal.</li>
	<li>Report the BLI signal intensity of each catheter trio as photon flux per second. Represent the BLI data on a logarithmic scale by plotting the mean and SD of the photon flux per second for each group. Perform statistical analysis on the log10 transformed data.</li>
</ol>
9. Catheter Explant
<ol>
	<li>Prepare microcentrifuge tubes containing 1 ml of PBS, one for each catheter device and keep them on ice.</li>
	<li>Euthanize the animals by cervical dislocation (Figure 2B, (1)).</li>
	<li>Disinfect the skin of the back with 0.5% chlorhexidine in 70% alcohol or with iodine isopropanol (1%).</li>
	<li>Make an incision (approximately 3 cm) above the catheters.</li>
	<li>Cut subcutaneous tissue and remove the catheter fragments one by one from under the subcutaneous tissue using sterile tweezers (Figure 2B, (2)).</li>
	<li>Handle catheter gently in vertical position and wash it twice with 1 ml of sterile PBS. Place each catheter piece into a separate microcentrifuge tube (prepared in Step 1).</li>
</ol>
10. Quantification of Biofilm-associated Cells by Colony Forming Units Count (CFUs) and Statistical Analyses of Results
<ol>
	<li>Sonicate catheters previously placed into 1 ml of PBS for 10 min at 40,000 Hz in a water bath sonicator and place them on ice.</li>
	<li>After sonication, vigorously vortex for 30 sec and place again on ice.</li>
	<li>Prepare two additional microcentrifuge tubes containing 900 &#956;l of PBS.</li>
	<li>Make 1:10 and 1:100 dilutions from your original sample (the one which contains the catheter) and keep all microcentrifuge tubes on ice.</li>
	<li>Plate 100 &#181;l of the original samples, 1:10 and 1:100 dilutions on YPD agar plates in duplicate.</li>
	<li>Incubate plates for 2 days at 37 &#176;C and count CFUs.</li>
	<li>Count the colonies grown on YPD agar plates (countable amount of colonies, maximum 300 colonies/plate) and multiply them by the proper dilution factor (1x-original, 10x or 100x dilution). Further multiply each catheter piece by 10x, which corresponds to the final amount of colonies in 1 ml tube containing the catheter. Bring the final amount of colonies to log10 cells/catheter piece with standard deviation (SD). Express data as a mean &#177; SD.</li>
	<li>Place all values for each catheter and specific group into a spreadsheet and/or statistical program, to perform the above-mentioned calculations and statistical analyses. Indicate specific groups to compare, i.e. WT vs. mutant or 2 days vs. 6 days biofilm formation and perform unpaired t-test and ANOVA with Tukey post-test on the log10-transformed data.
	<ol>
		<li>Express the level of significance by a p value. In this study indicate significance if p value &#60;0.05, represented as follows: * p &#60;0.05, **p &#60;0.005, ***p &#60;0.0005.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

The use of animal models, and especially rodent models, for studies dedicated to microbial biofilms is very important as the host immune system is an essential factor in biofilm formation that in vitro models cannot account for. In this study, we describe a relatively straightforward subcutaneous C. albicans biofilm mouse model, which can be easily adopted in a research laboratory and does not require strong technical skills. This model was originally developed to study Staphylococcus epidermidis</e...

Candida albicans is a commensal organism, which can be found at different sites of healthy individuals, for example on the skin or as a part of the gastrointestinal and vaginal flora. However, in hospitalized, and especially immunocompromised patients, it may cause a wide range of infections 1. In such individuals, the weakened immune system allows Candida cells to disseminate into the bloodstream and to invade deeper tissues causing life-threatening infections. In addition, the presence of abiotic substrates such as central venous and urinary catheters, artificial heart valves and joints may provide a niche for Candida attachment 2. Adhesion to such substrates is a prerequisite for further biofilm development, which represents a layer of yeast and hyphal cells embedded in extracellular polymeric material, mainly consisting of polysaccharides 2. C. albicans catheter &#8211;associated infections are associated with high mortality rate. A general characteristic of biofilms is their decreased susceptibility to known antifungals, such as azoles 3,4. Only newer classes of antifungal drugs, such as echinocandins and liposomal formulation of amphotericin B proved to be active against catheter-associated infections 5-7. Because of biofilm resilience to antifungals, therapeutic approaches are very limited, often leading to catheter removal and its subsequent replacement as a sole solution.
Most of our current understanding of C. albicans biofilm development originates from in vitro studies on abiotic substrates such as polystyrene, or plastics used for the manufacture of above-mentioned devices, i.e., silicone, polyurethane 2. These models are quite advanced and try to mimic the situation in vivo as closely as possible. However, these systems do not involve the continuous blood flow and the immune system of the host. This resulted in the development of in vivo model systems, such as the central venous catheter (CVC) model 8-10, the denture stomatitis model of oral candidiasis 11 and a murine model for catheter-associated candiduria 12. Additionally, C. albicans biofilm development was studied in vivo on the mucosal surfaces, such as those from the vagina 13 and oral cavity 14. Our laboratory contributed with the establishment of a subcutaneous C. albicans biofilm model, which is based on the implant of infected catheter pieces on the back of Sprague Dawley rats 15. This model was successfully used in our laboratory to test biofilm susceptibility to fluconazole and echinocandin drugs 5,16, to study the effect of combinatorial therapy of diclofenac and caspofungin 17. More recently, we adapted this system for use in BALB/c mice 18,19. In comparison with other in vivo models, the main advantage of this subcutaneous model is the possibility to study multiple biofilms per animal developed inside the lumen of implanted catheter pieces.
To reduce the number of laboratory animals, we have adapted this model to study the development of C. albicans biofilms non-invasively by using bioluminescence imaging (BLI) 18,19. This method proved to be a powerful technique, which can be used to quantify biofilms by measuring the specific BLI signal at the region of interest (in our case the area of implanted catheters), avoiding animal sacrifice. In comparison to bacteria, which can express both the gene and the substrate required for the bioluminescence reaction due to the introduction of a specific lux operon 20, most of the eukaryotic organisms, including C. albicans, are dependent on the heterologous expression of a luciferase gene coupled with the external administration of a specific substrate, such as D-luciferin or coelenterazine 21. Probably due to the presence of the fungal cell wall and C. albicans morphogenesis, the intracellular delivery of the substrate for the luciferase enzyme was a main challenge 21. In order to solve this problem, Enjalbert et al. 22 engineered a strain where a synthetic C. albicans codon-optimized version of the gene for the naturally secreted Gaussia princeps luciferase (gLuc) was fused to to the C. albicans PGA59 gene, a GPI- anchored cell wall protein. Because of the presence of luciferase at the cell wall, problems concerning the intracellular availability of the substrate could be avoided. This particular system was used to study superficial infections caused by C. albicans 22. Very recently, BLI was also used to follow the progression of oropharyngeal candidiasis and its possible treatment 23. Such findings support the use of BLI as a promising technique to study infections caused by free-living cells but also device-associated infections.
In this study, we describe the C. albicans biofilm development on polyurethane catheter pieces in BALB/c mice and its quantification using BLI. We provide a detailed protocol of in vitro colonization of polyurethane catheters during the period of adhesion followed by implantation in mice and subsequent biofilm development in live animals. Apart from measuring the BLI signal emitted by the C. albicans cells, we also determine the colony forming units for comparison with the standard technique for biofilm fungal load quantification.

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			<td height="42" style="height:42px;width:329px;">Name of Reagent</td>
			<td style="width:199px;">Company</td>
			<td style="width:113px;">Catalog Number</td>
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			<td height="42" style="height:42px;width:329px;">Yeast extract granulated</td>
			<td style="width:199px;">Merck</td>
			<td style="width:113px;">MERC1.03753.0500</td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Bacteriological peptone</td>
			<td style="width:199px;">Oxoid</td>
			<td style="width:113px;">LP037B</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Agar granulated</td>
			<td style="width:199px;">Difco</td>
			<td style="width:113px;">214530</td>
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			<td height="21" style="height:21px;width:329px;">D-(+)-glucose</td>
			<td style="width:199px;">Fluka</td>
			<td style="width:113px;">49159-5KG</td>
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			<td height="25" style="height:25px;">Phosphate buffered saline&#160;</td>
			<td style="width:199px;">Prepared in the laboratory</td>
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			<td>for 1L of 10x PBS: 80 g NaCl, 2 g KCl, 14.4 g Na2HPO4, 2.4 g KH2PO4</td>
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			<td height="42" style="height:42px;width:329px;">RPMI1640 with L-glutamine and without sodium carbonate&#160;</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">R6504-1L</td>
			<td>Prepare according the protocol for Candida albicans drug susceptibility testing&#160;</td>
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			<td height="21" style="height:21px;width:329px;">3-(N-Morpholino)propanesulfonic acid (MOPS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">M1254</td>
			<td>MOPS is used to adjust the pH of RPMI medium (pH 7.0)</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">fetal bovine serum (FBS)</td>
			<td style="width:199px;">Sigma</td>
			<td style="width:113px;">F7524</td>
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			<td height="42" style="height:42px;width:329px;">Polyurethane tripe-lumen intravenous catheter piece (2.4 mm diameter, Certofix Trio S730)&#160;</td>
			<td style="width:199px;">BBraun</td>
			<td style="width:113px;">CV-15703</td>
			<td>Polyurethane part cut into 1 cm pieces</td>
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			<td height="21" style="height:21px;width:329px;">Dexamethasone</td>
			<td style="width:199px;">Fagron SAS, France</td>
			<td style="width:113px;">611139</td>
			<td>Immunosuppressant (stock solution 10 mg/ml)</td>
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			<td height="42" style="height:42px;width:329px;">Ampicillin&#160;</td>
			<td style="width:199px;">Duchefa Biochemie, The Netherlands</td>
			<td style="width:113px;">A0104</td>
			<td>Antibacterial prophylaxis</td>
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			<td height="21" style="height:21px;width:329px;">Ketamine 1000</td>
			<td>Pfizer</td>
			<td style="width:113px;">804 119</td>
			<td>Anesthetic</td>
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			<td height="21" style="height:21px;width:329px;">Domitor</td>
			<td>Pfizer</td>
			<td style="width:113px;">134737-1</td>
			<td>Anesthetic</td>
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			<td height="21" style="height:21px;width:329px;">Antisedan</td>
			<td>Pfizer</td>
			<td style="width:113px;">134783-2</td>
			<td>Reversal of anesthesia</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylocaine gel (2%) - this is Linisol</td>
			<td style="width:199px;">AstraZeneca</td>
			<td style="width:113px;">352 1206</td>
			<td>Local anesthetic for the skin</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Terramycin/ polymyxin-b ophthalmic ointment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>To prevent drying and infection of eyes</td>
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		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Coelenterazine&#160;</td>
			<td style="width:199px;">Prolume (Nanolight)</td>
			<td style="width:113px;">NF-CTZ-FB</td>
			<td>Light sensitive agent (must be kept in the dark)</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Iodine isopropanol (1%)</td>
			<td style="width:199px;">3M&#8482; DuraPrep&#8482;&#160;</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.5 % chlorhexidine in 70 % alcohol.&#160;&#160;</td>
			<td style="width:199px;">Cedium</td>
			<td style="width:113px;"></td>
			<td>Disinfectant for the skin</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Equipment</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Cell counting chamber</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:329px;">Insulin syringes (0.3 ml)</td>
			<td style="width:199px;">Terumo Myjector 29G</td>
			<td style="width:113px;">324826</td>
			<td>For injection of coelenterazine</td>
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			<td height="21" style="height:21px;width:329px;">Electric razor</td>
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			<td>For small animals</td>
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			<td height="21" style="height:21px;width:329px;">Sterile surgical tools</td>
			<td style="width:199px;"></td>
			<td style="width:113px;"></td>
			<td>Scissors, 2 pairs of tweezers, scalpel</td>
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			<td height="21" style="height:21px;width:329px;">Heating pad</td>
			<td style="width:199px;">Leica</td>
			<td style="width:113px;">14042321474</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin suture</td>
			<td style="width:199px;">Johnson&#38;Johnson</td>
			<td style="width:113px;">K890H</td>
			<td>Surgical thread, needle</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Water bath sonicator</td>
			<td>Branson 2210</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">BLI camera (IVIS Spectrum)&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;">IVISSPE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Living Image software&#160;</td>
			<td style="width:199px;">Perkin Elmer, Alameda</td>
			<td style="width:113px;"></td>
			<td>(version 4.2)&#160;</td>
		</tr>
	</tbody>
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candida albicans biofilm development on medically-relevant foreign bodies in a mouse subcutaneous model followed by bioluminescence imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

In this study, we show the surgical procedure of catheter implant and explant during in vivo C. albicans biofilm development in a mouse. Moreover, we display the quantification of mature biofilms not only by classical CFUs enumeration but also by BLI.
As shown in Figure 1A, non-phosphorescent polyurethane catheter pieces were cut into 1 cm devices and subsequently coated with serum. This step is very important because it allows Candida cells to attach to the ...

Watch this Scientific Journal Video about Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging at JoVE.com

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

candida-albicans-biofilm-development-on-medically-relevant-foreign

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

12.9K Views.  Institute of Botany and Microbiology, VIB, KU Leuven. We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Video: Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 9.4 T MRI.

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently labelled structures in large biological specimens, and is down to cellular resolution. Meanwhile, the constant refinement of underlying protocols and the enhanced availability of specialized commercial systems enable us to investigate the microstructure of whole mouse organs and even allow for the characterization of cellular behavior in various live-cell imaging approaches. Here, we describe a protocol for the spatial whole-mount visualization and quantification of the CD45+ leukocyte population in inflamed mouse hearts. The method employs a transgenic mouse strain (CD11c.DTR)that has recently been shown to serve as a robust, inducible model for the study of the development of fulminant fatal myocarditis, characterized by lethal cardiac arrhythmias. This protocol includes myocarditis induction, intravital antibody-mediated cell staining, organ preparation, and LSFM with subsequent computer-assisted image post-processing. Although presented as a highly-adapted method for our particular scientific question, the protocol represents the blueprint of an easily adjustable system that can also target completely different fluorescent structures in other organs and even in other species.

Here, we describe a light-sheet microscopy approach to visualize the cardiac CD45+ leukocyte infiltrate in a murine model of aseptic fulminant myocarditis, which is induced by the intratracheal diphtheria toxin treatment of CD11c.DTR mice.

Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

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A Neonatal Mouse Spinal Cord Compression Injury Model

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography