The authors thank&#160;the financial support from FAPESP (S&#227;o Paulo Research Foundation, process number FAPESP #2013/07276-1 (CePID CePOF) and 2008/00601-6. We also thank Dr. Ana Paula Silva for providing the information about the CUR-based water-soluble salt.&#160;

The research protocol for the use of mice was approved by the Ethics Committee for Animal Use (case numbers 05/2008 and 09/2020) at the School of Dentistry, Araraquara, UNESP. C. albicans (ATCC 90028) was used as the reference strain. Six-week-old female Swiss mice (n = 45), with a body mass range of 20-30 g, were used for the present study. The animals were provided by S&#227;o Paulo State University, UNESP, Botucatu.
1. Preparation of PS and selection of the light source for aPDT
<ol>
	<li>Prepare the PS according to the specifications for the substance to be tested. 
	NOTE: In this study, a water-soluble mixture of curcuminoids (CUR-based water-soluble salt mixture12) was used as the PS (see Table of Materials and Figure 1). Dilutions at 20 &#181;M, 40 &#181;M, and 80 &#181;M (15.6, 29.2, and 58.4 mg/L, respectively) were prepared in ultra-pure water immediately before use and kept at 5 &#176;C.</li>
	<li>Select a light equipment with an appropriate wavelength (the same as that of the PS absorption) capable of illuminating the entire photosensitized target uniformly and simultaneously without heating the tissue. 
	&#8203;NOTE: In this study, a handpiece containing a light-emitting diode (LED, see Table of Materials), that was designed at the Instituto de F&#237;sica de S&#227;o Carlos (University of S&#227;o Paulo, S&#227;o Carlos, SP, Brazil), was used. The output power delivered by the light was 89.2 mW/cm2 at a blue wavelength of approximately 455 nm.</li>
</ol>
2. Preparation of C. albicans inoculum
<ol>
	<li>Keep the reference strain C. albicans (ATCC 90028) in yeast-peptone-glucose (YEPD, see Table of Materials) with 50% glycerol at -80 &#176;C until use.</li>
	<li>Thaw the frozen strain at room temperature, spread a 100 &#181;L aliquot on a Petri dish with Sabouraud dextrose agar (SDA) with chloramphenicol (see Table of Materials), and incubate at 37 &#176;C for 48 h.</li>
	<li>Transfer five colonies into 10 mL of yeast nitrogen broth with 2% glucose (YNBg) medium and grow aerobically at 37 &#176;C for 16 h.</li>
	<li>Dilute the yeast culture in fresh YNBg (1:10) and incubate aerobically at 37 &#176;C until the optical density reaches the mid-log phase of growth. 
	NOTE: Standardize the microbial growth curve for each laboratory previously. In the current research, cultures were allowed to incubate for around 8 h, until they reached the mid-log phase of growth as indicated by the optical density at 540 nm (OD540), with a mean value of 0.536 &#177; 0.062 arbitrary units (mean &#177; standard deviation [SD]).</li>
	<li>Centrifuge the culture at 5,000 x g at room temperature for 5 min, discard the supernatant and wash the pellet twice with the same volume of sterile phosphate-buffered saline (PBS; 0.136 M NaCl, 2 mM KCl, 1 mM KH2PO4, 10 mM Na2HPO4, pH 7.4).</li>
	<li>Use 100 &#181;L aliquots for performing four serial 10-fold dilutions in PBS, spread dilutions on SDA plates, and incubate at 37 &#176;C for 48 h for colony count. As a standard, fungal suspensions are used at concentrations of approximately 4.5 &#215; 107 colony forming units per milliliter (CFU/mL)].</li>
</ol>

3. Induction of OC in mice
NOTE: The following methodology was previously described by Takakura et al.13 and reproduced by our group10,14, with some modifications.
<ol>
	<li>Randomly distribute mice in nine groups (n = 5), corresponding to the same number of treatments (Supplementary File 1). 
	NOTE: The oral cavity of mice should be negative for Candida growth. This needs to be checked by swabbing the oral cavity and plating the sample on SDA.</li>
	<li>Keep the animals in propylene boxes10(5 mice per box max.),&#160;with wood shavings for floor covering,&#160;in a temperature-controlled vivarium at 23 &#177; 2 &#176;C under a 12/12 h light/dark cycle. No specific enrichment needs to be provided.</li>
	<li>Maintain mice on extruded mouse diet and filtered water ad libitum.</li>
	<li>Subcutaneously inject the immunosuppressive drug, prednisolone&#160;(100 mg/kg body weight, see Table of Materials), for the first time on day one to all the members of groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20 &#181;M, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1). 
	NOTE: Keep mice from the same group in the same box and monitor them daily for any sign of stress and weight loss. Seek veterinary advice for analgesic or altered food/water if stress or weight loss are noted..</li>
	<li>Starting on day one and until the end of the experiment, provide tetracycline hydrochloride in drinking water at 0.83 mg/mL13.</li>
	<li>Inoculate mice tongues on day two, including groups C+L+ 20, C+L+ 40, C+L+ 80, C+L- 20, C+L- 40, C+L- 80, C-L+, and C-L- (Supplementary File 1), with the C. albicans inoculum. Do not inoculate those mice from the negative control group (NCtrl).
	<ol>
		<li>Sedate animals by intramuscular injection of 10 mg/kg chlorpromazine chloride (see Table of Materials) on each thigh muscle&#160;before carrying out inoculation.</li>
		<li>Perform intraoral inoculation of the mice&#160;by soaking a sterile swab (one per animal) into a freshly prepared (i.e., prepared immediately before inoculation) standardized suspension of C. albicans.</li>
		<li>Place sedated mice in a supine position. Gently pull their tongues out of their mouths using a sterile forceps. Rub each mouse&#39;s tongue with a soaked swab for 30 s. Monitor mice until they recover from sedation.</li>
	</ol>
	</li>
	<li>On day five, administer a complementary subcutaneous injection of prednisolone (100 mg/kg body weight) to maintain the immunosuppression. 
	&#8203;NOTE: This protocol is adequate to keep the infection for 7 days after intraoral inoculation. The protocol timeline is shown in Figure 2.</li>
</ol>
4. Antimicrobial Photodynamic Therapy and recovery of C. albicans from oral lesions
<ol>
	<li>On day seven, prior to treatment, anesthetize the animals using an intraperitoneal injection of ketamine hydrochloride (100 mg/kg body weight) combined with xylazine (10 mg/kg body weight) (see Table of Materials).</li>
	<li>Place each animal in a supine position on a pad device14,15equipped with stainless steel wires that can be looped around the incisors to keep the mouth open. 
	NOTE: Isothermal pads are recommended for warming the mice while they are sedated, preventing hypothermia at room temperature and avoiding mortality related to anesthesia15. Each anesthetized animal should be monitored by the lack of a withdrawal reflex when toes are pinched to confirm the anesthesia depth. One maintenance dose of ketamine at 1/3 the original dose (without xylazine) may be administered if needed.</li>
	<li>With the mandible and cheeks retracted, gently place the tongue outside the mouth.</li>
	<li>For mice from C+L+ groups, pipette 70 &#181;L of the PS on the dorsum of the tongue (at one of the tested concentrations). Maintain the mice in a dark environment for a duration of 20 min as a pre-irradiation period. Ensure that during this time, the tongue of each animal remains positioned inside the oral cavity to prevent the photosensitizer (PS) from being ingested.</li>
	<li>After the photosensitization period, pull the tongue out of the mouth for illumination. Place the LED device onto the dorsum of the tongue and illuminate at 37.5 J/cm2 (7 min with the present device) (Figure 3).</li>
	<li>For animals from the C+L- groups, pipette 70 &#181;L of the PS at one of the tested concentrations on the dorsum of the tongue, and keep these mice in the dark for 27 min.</li>
	<li>For animals from the C-L+ group (treated with light without any previous photosensitization), pipette 70 &#181;L of sterile saline solution on the dorsum of the tongue. Keep the mice in the dark for 20 min and then illuminate their tongues at 37.5 J/cm2 (7 min with the present device).</li>
	<li>For mice from the C-L- group (neither treated with PS nor with light), pipette 70 &#181;L of sterile saline solution on the dorsum of the tongue and keep them in the dark for 27 min.</li>
	<li>Recover C. albicans from the tongues of all mice immediately after treatment. Swab the dorsum of the tongue for 1 min with a sterile cotton swab.</li>
	<li>Place each sample swab in a tube containing 1 mL of sterile saline and vortex for 1 min to resuspend the microbial cells.</li>
	<li>Immediately perform 10-fold serial dilutions in PBS and plate 25 &#181;L aliquots over SDA plates in duplicate. Incubate the plates aerobically at 37 &#176;C for 48 h.</li>
	<li>After 48 h, use a digital colony counter (see Table of Materials) to determine yeast colony counts. Calculate C. albicans load (CFU/mL) for each animal.</li>
	<li>Transform CFU/mL values into log10 and analyze properly according to the design of the experiment and data assumptions. 
	NOTE: In the present investigation, Welch&#39;s one-way ANOVA and Games-Howell post-hoc test (&#945; = 0.05)16 was used.</li>
</ol>
5. Histopathological analyses
<ol>
	<li>On day eight, anesthetize mice with ketamine at 100 mg/kg combined with xylazine at 10 mg/kg&#160;via intraperitonial injection and euthanize them with an overdose of ketamine (200 mg/kg administered via intraperiotonial). Confirm death by checking for the absence of respiratory movement (apnea), and absence of heartbeat (asystole).</li>
	<li>Carefully pinch the tongue and pull it out of the animal&#39;s mouth. Using a manual scalpel, make an incision before the circumvallate papillae to surgically remove the entire tongue without causing any injuries to the anterior and central regions.</li>
	<li>Fix the tongue in 10% buffered formalin (pH 7.2-7.4) for 24 h and wash it in running water for 2 h.</li>
	<li>Proceed with the inclusion in the paraffin process: dehydration, clarification, and impregnation10,14.</li>
	<li>Cut serial sections (5 &#181;m thick) using a microtome, then affix the sections onto glass slides. Proceed to stain these sections using periodic acid-Schiff and hematoxylin (PAS-H) for histopathological examination and identifying fungi through observation under a light microscope.</li>
	<li>Ask a pathologist, preferably blind to the groups studied, to examine the tissue reaction due to C. albicans infection.</li>
	<li>Describe the histological characteristics of the tissue (epithelium and connective tissue), especially local inflammatory responses of varying intensities.</li>
</ol>

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

<ol>
	<li>Vila, T., Sultan, A. S., Montelongo-Jauregui, D., Jabra-Rizk, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+candidiasis:+a+disease+of+opportunity.">Oral candidiasis: a disease of opportunity.</a> Journal of fungi (Basel, Switzerland). 6 (1), 15 (2020).</li><li>Sudbery, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Candida+albicans+hyphae.">Growth of Candida albicans hyphae.</a> Nature Reviews Microbiology. 9 (10), 737-748 (2011).</li><li>Moyes, D. L., Richardson, J. P., Naglik, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans-epithelial+interactions+and+pathogenicity+mechanisms:+scratching+the+surface.">Candida albicans-epithelial interactions and pathogenicity mechanisms: scratching the surface.</a> Virulence. 6 (4), 338-346 (2015).</li><li>Lopes, J. P., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+virulence+of+Candida+albicans.">Pathogenesis and virulence of Candida albicans.</a> Virulence. 13 (1), 89-121 (2022).</li><li>Quind&#243;s, G., 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=Therapeutic+tools+for+oral+candidiasis:+Current+and+new+antifungal+drugs.">Therapeutic tools for oral candidiasis: Current and new antifungal drugs.</a> Medicina oral, patologia oral y cirugia buccal. 24 (2), e172-e180 (2019).</li><li>Nishimoto, A. T., Sharma, C., Rogers, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+genetic+basis+of+azole+antifungal+resistance+in+the+opportunistic+pathogenic+fungus+Candida+albicans.">Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans.</a> Journal of Antimicrobial Chemotherapy. 75 (2), 257-270 (2020).</li><li>Gholami, L., Shahabi, S., Jazaeri, M., Hadilou, M., Fekrazad, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+antimicrobial+photodynamic+therapy+in+dentistry.">Clinical applications of antimicrobial photodynamic therapy in dentistry.</a> Frontiers in Microbiology. 13, 1020995 (2013).</li><li>Trigo-Gutierrez, J. K., Vega-Chac&#243;n, Y., Soares, A. B., Mima, E. G. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antimicrobial+activity+of+curcumin+in+nanoformulations:+a+comprehensive+review.">Antimicrobial activity of curcumin in nanoformulations: a comprehensive review.</a> International Journal of Molecular Sciences. 22 (13), 7130 (2021).</li><li>Santezi, C., Reina, B. D., Dovigo, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Curcumin-mediated+Photodynamic+Therapy+for+the+treatment+of+oral+infections-A+review.">Curcumin-mediated Photodynamic Therapy for the treatment of oral infections-A review.</a> Photodiagnosis and Photodynamic Therapy. 21, 409-415 (2018).</li><li>Dovigo, L. N., 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=Curcumin-mediated+photodynamic+inactivation+of+Candida+albicans+in+a+murine+model+of+oral+candidiasis.">Curcumin-mediated photodynamic inactivation of Candida albicans in a murine model of oral candidiasis.</a> Medical Mycology. 51 (3), 243-251 (2013).</li><li>Zangirolami, A. C., Carbinatto, F., Filho, J. D. V., Bagnato, V. S., Blanco, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+light-activated+curcumin+and+curcuminoids+films+for+catheters+decontamination.">Impact of light-activated curcumin and curcuminoids films for catheters decontamination.</a> Colloids and SurfacesB: Biointerfaces. 213, 112386 (2022).</li><li>Santezi, C., Tanomaru, J. M., Bagnato, V. S., J&#250;nior, O. B., Dovigo, L. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+curcumin-mediated+photodynamic+inactivation+to+reduce+oral+colonization.">Potential of curcumin-mediated photodynamic inactivation to reduce oral colonization.</a> Photodiagnosis Photodynamic Therapy. 15, 46-52 (2016).</li><li>Takakura, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+murine+model+of+oral+candidiasis+with+local+symptoms+characteristic+of+oral+thrush.">A novel murine model of oral candidiasis with local symptoms characteristic of oral thrush.</a> Microbiology and immunology. 47 (5), 321-326 (2003).</li><li>Mima, E. G., 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=Susceptibility+of+Candida+albicans+to+photodynamic+therapy+in+a+murine+model+of+oral+candidosis.">Susceptibility of Candida albicans to photodynamic therapy in a murine model of oral candidosis.</a> OralSurgery, Oral Medicine, Oral Pathology, Oral Radiology and Endodontology. 109, 392-401 (2010).</li><li>Solis, N. V., Filler, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+oropharyngeal+candidiasis.">Mouse model of oropharyngeal candidiasis.</a> Nature Protocol. 7 (4), 637-642 (2012).</li><li>Mar&#244;co, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> An&#225;lise Estat&#237;stica com o SPSS Statistics 25. , (2018).</li><li>Naglik, J. R., Fidel, P. L., Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+mucosal+Candida+infection.">Animal models of mucosal Candida infection.</a> FEMS Microbiology Letters. 283 (2), 129-139 (2008).</li><li>Samaranayake, Y. H., Samaranayake, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+oral+candidiasis+in+animal+models.">Experimental oral candidiasis in animal models.</a> Clinical Microbiology Reviews. 14, 398-429 (2001).</li><li>Chamilos, G., Lionakis, M. S., Lewis, R. E., Kontoyiannis, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mini-host+models+in+the+study+of+medically+important+fungi.">Role of mini-host models in the study of medically important fungi.</a> The Lancet Infectious Diseases. 7, 42-55 (2007).</li><li>Carmello, 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=Treatment+of+oral+candidiasis+using+Photodithazine-+mediated+photodynamic+therapy+in+vivo.">Treatment of oral candidiasis using Photodithazine- mediated photodynamic therapy in vivo.</a> PLoS One. 11 (6), e0156947 (2016).</li><li>Sakima, V. 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=Antimicrobial+photodynamic+therapy+mediated+by+curcumin-loaded+polymeric+nanoparticles+in+a+murine+model+of+oral+candidiasis.">Antimicrobial photodynamic therapy mediated by curcumin-loaded polymeric nanoparticles in a murine model of oral candidiasis.</a> Molecules. 23 (8), 2075 (2018).</li><li>Abe, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+glucocorticoid+antagonist,+mifepristone+affects+anti-Candida+activity+of+murine+neutrophils+in+the+presence+of+prednisolone+in+vitro+and+experimental+candidiasis+of+prednisolone-treated+mice+in+vivo.">A glucocorticoid antagonist, mifepristone affects anti-Candida activity of murine neutrophils in the presence of prednisolone in vitro and experimental candidiasis of prednisolone-treated mice in vivo.</a> FEMS Immunology and Medical Microbiology. 13 (4), 311-316 (1996).</li><li>Jones, J. H., Russell, C., Young, C., Owen, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tetracycline+and+the+colonization+and+infection+of+the+mouths+of+germ-free+and+conventionalized+rats+with+Candida+albicans.">Tetracycline and the colonization and infection of the mouths of germ-free and conventionalized rats with Candida albicans.</a> Journal Antimicrobial Chemotherapy. 2 (3), 247-253 (1976).</li><li>Russell, C., Jones, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+oral+inoculation+of+Candida+albicans+in+tetracycline-treated+rats.">Effects of oral inoculation of Candida albicans in tetracycline-treated rats.</a> Journal of Medical Microbiology. 6 (3), 275-279 (1973).</li><li>Teichert, M. C., Jones, J. W., Usacheva, M. N., Biel, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+of+oral+candidiasis+with+methylene+blue-+mediated+photodynamic+therapy+in+an+immunodeficient+murine+model.">Treatment of oral candidiasis with methylene blue- mediated photodynamic therapy in an immunodeficient murine model.</a> OralSurgery, Medicine, Pathology, Radiology and Endodontology. 93, 155-160 (2002).</li><li>Totti, M. G. A., Santos, E. B., Almeida, O. P., Koga-Ito, C. Y., Jorge, A. O. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+candidosis+by+Candida+albicans+in+normal+and+xerostomic+mice.">Oral candidosis by Candida albicans in normal and xerostomic mice.</a> Brazilian Oral Research. 18, 202-207 (2004).</li><li>Hidalgo, K. J. R., 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=Antimicrobial+photodynamic+therapy+in+combination+with+nystatin+in+the+treatment+of+experimental+oral+candidiasis+induced+by+Candida+albicans+resistant+to+fluconazole.">Antimicrobial photodynamic therapy in combination with nystatin in the treatment of experimental oral candidiasis induced by Candida albicans resistant to fluconazole.</a> Pharmaceuticals (Basel). 12 (3), E140 (2019).</li></ol>

C. albicans has been associated with oral and esophageal infections in individuals with an immunocompromised state, diabetes mellitus, prolonged use of antibiotics, and poor oral hygiene1,3. The study of human infectious diseases requires both in vitro and in vivo investigations before clinical trials can be safely and accurately designed. The present study describes a method for establishing a murine model of OC, which can be used to e...

Oral candidiasis (OC) is the main fungal infection of the oral cavity; it is caused by the overgrowth of Candida spp. Predisposing factors for OC include endocrine dysfunction, the use of broad-spectrum antibiotics, radio- and chemotherapy, nutritional deficiencies, xerostomia (low salivary flow), denture use, poor hygiene, and, especially, immunosuppression1. Among Candida species, Candida albicans is the most prevalent and virulent one; it is found as a commensal species in the human body and as an opportunistic pathogen. C. albicans has the ability to change its morphology from commensal yeasts (blastopores) to pathogenic filaments (hyphae and pseudohyphae)2. The filamentous forms, especially the hyphae, can invade the host epithelium by endocytosis or active penetration, causing infection3. Other virulence factors of C. albicans include adhesion, biofilm formation, and secretion of lipolytic and hydrolytic enzymes and toxins, such as lipases, phospholipases, proteinases, and candidalysin4.
OC treatments involve the use of antifungal agents, especially topical polyenes and azoles (nystatin and miconazole)5. However, they only show short-term efficacy, and recurrence is frequent. In addition, the overuse of antifungals has given rise to the problem of antifungal resistance development and spread6. Therefore, alternative therapies are needed, such as antimicrobial Photodynamic Therapy (aPDT), which combines a photosensitizer (PS) and light at an appropriate wavelength (the same as that of the PS absorption) in the presence of oxygen. PSs are bound to or taken up by cells and, when activated by light, produce reactive oxygen species (ROS) that are toxic to sensitized cells7.
In aPDT, one of the photosensitizers (PSs) employed is curcumin (CUR), a naturally occurring compound extracted from the rhizomes of the turmeric plant (Curcuma longa L.). Curcumin possesses numerous therapeutic attributes, including anti-inflammatory, antioxidant, anticancer, and antimicrobial capabilities8,9. A prior investigation found that aPDT utilizing CUR effectively diminished C. albicans in a murine model of oral candidiasis without causing any harm to the host&#39;s tissues10. CUR is the main curcuminoid extracted from turmeric, but other polyphenols, such as demethoxycurcumin and bis-demethoxycurcumin, are also found in this plant. Curcuminoid-mediated aPDT demonstrated antibacterial activity against biofilms of Staphylococcus aureus grown in catheters11. However, to the best of our knowledge, its antifungal activity against C. albicans remains unclear. Therefore, in this study, we evaluated aPDT mediated by a curcuminoid salt against C. albicans in a murine model of OC.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>C. albicans</td>
			<td>ATCC (Rockville, Md, USA)</td>
			<td>90028</td>
			<td>Used to prepare the Candida inoculum</td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td>Eppendorf&#160;Centrifuge 5804/5804R,B. Braun, Melsungen, Hesse, Germany</td>
			<td>022628146 (NA)</td>
			<td>Used to prepare the Candida inoculum</td>
		</tr>
		<tr>
			<td>Chlorpromazine chloride 2 mg/mL</td>
			<td>Compounding pharmacy, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;&#160;</td>
			<td>Used to sedate animals during candida inoculation</td>
		</tr>
		<tr>
			<td>Curcumin-based water-soluble salt</td>
			<td>PDTPharma, Cravinhos, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Consisting of 53.4% of natural curcumin, and&#160; 46.6% of other curcuminoids (demethoxycurcumin and bis-demethoxycurcumin). Prepared in water and N-MethylD-Glucamine (final average molecular weight of 730.32 g.mol&#8722;1)</td>
		</tr>
		<tr>
			<td>Digital colony counter</td>
			<td>CP 600 Plus, Phoenix Ind Com Equipamentos Cient&#237;ficos Ltda, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to count colonies on agar plates</td>
		</tr>
		<tr>
			<td>Extruded mouse chow</td>
			<td>Benelab food, Industry Qualy Animal Nutrition and Commerce Ltda., Lind&#243;ia, S&#227;o Paulo State, Brazil.</td>
			<td>&#160;-</td>
			<td>Used for the feeding of the mice</td>
		</tr>
		<tr>
			<td>Ketamine Hydrochloride 10%</td>
			<td>Ketamina Agener, Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A, Embu-Gua&#231;u, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to anesthetize animals before&#160; treatments and for euthanasia</td>
		</tr>
		<tr>
			<td>Light-emitting diode handpiece (prototype)</td>
			<td>Instituto de F&#237;sica de S&#227;o Carlos, University of S&#227;o Paulo, S&#227;o Carlos, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Fabricated with LXHL-PR09, Luxeon III Emitter, Lumileds Lighting, San Jose, California, USA</td>
		</tr>
		<tr>
			<td>Methylprednisolone acetate 40 mg</td>
			<td>DEPO-MEDROL, Pfizer, New York</td>
			<td>&#160;-&#160;</td>
			<td>Used as an immunosuppressant</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica&#160;Microsystems, Bannockburn, IL, USA</td>
			<td>SM2500</td>
			<td>Used to cut the serial sections of the tongues</td>
		</tr>
		<tr>
			<td>Propylene boxes (cages housing) H13 x L20 x D30 cm</td>
			<td>Bonther Equipaments, Ribeir&#227;o Preto, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used to keep the animals throughout&#160; the experimental period</td>
		</tr>
		<tr>
			<td>Sabouraud Dextrose Agar with Chloramphenicol</td>
			<td>HiMedia, Mumbai, India</td>
			<td>MM1067-500G</td>
			<td>Culture medium for yeast growth (agar)</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Spectrophotometer Kasvi K37-VIS , S&#227;o Jos&#233; dos Pinhais, PR, Brazil</td>
			<td>K37-VIS&#160;</td>
			<td>Used to standardize the inoculum concentration</td>
		</tr>
		<tr>
			<td>Tetracycline hydrochloride&#160;</td>
			<td>Compounding pharmacy, Araraquara, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Antibiotic given to induce oral dysbiosis</td>
		</tr>
		<tr>
			<td>Wood shavings</td>
			<td>J.R. Wood Shavings, Comerce of Sawdust Ltda., Conchal, S&#227;o Paulo State, Brazil</td>
			<td>&#160;-</td>
			<td>Used for floor covering inside the housing boxes</td>
		</tr>
		<tr>
			<td>Xylazine 2%</td>
			<td>Calmiun, Uni&#227;o Qu&#237;mica Farmac&#234;utica Nacional S/A, Embu-Gua&#231;u, SP, Brazil</td>
			<td>&#160;-&#160;</td>
			<td>Used in combination with ketamine for anesthesia</td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Broth&#160;</td>
			<td>Difco, InterLab, Detroit, MI, USA</td>
			<td>DF0919-07-3&#160;</td>
			<td>Culture medium for yeast growth (broth)</td>
		</tr>
		<tr>
			<td>Yeast Peptone Dextrose Broth</td>
			<td>NutriSelect Basic, Sigma Aldrich</td>
			<td>Y1375</td>
			<td>Culture medium for maintaining the strains at -80&#176;C and grow</td>
		</tr>
	</tbody>
</table>

curcuminoid-mediated antimicrobial photodynamic therapy on a murine model of oral candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Oral candidiasis is highly prevalent, especially in removable dental processes over years in immunosuppressed individuals. The treatment with antifungals has drawbacks, like recurrence and resistance. So our group has been researching therapeutic alternatives for over 20 years.As per clinical animal models are as important as the steps prior to clinical trials, we seek to establish a murine model of oral candidiasis. Antimicrobial photodynamic therapy, or APDT, is a prompt alternative that may be widely used as an alternative to the current antimicrobial treatments. It requires a combination of photosensitizer, light and oxygen to produce antimicrobial effects against a broad spectrum of oral microorganisms, including those resistant to conventional medications.Curcumin could be a promising photosensitizer against candida biofilms. However, it is a hydrophobic polyphenol with low solubility in water and bioavailability. Thus, studies have been conducted to enhance the efficacy of curcumin mediated APDT by using drug delivery systems or associating the photosensitizer with antimicrobial peptides.The extensive use of antifungals has resulted in the global emergence of resistance in candida species, and subsequent treatment failures. Thus controlling candida biofilms and combat antifungal resistance by photodynamic action has been our main focus of research. The murine model of oral candidiasis we developed can be used to evaluate the pathogenesis of oral infections by candida albicans, and the efficacy of alternative antifungal approaches.We used the model to evaluate the efficacy of APDT mediated by a water soluble curcuminoid compound.

The murine model of OC showed typical white patches and pseudomembranes on the tongue of all infected mice (Figure 4A). C. albicans recovered from C-L- animals confirmed tissue colonization by this microorganism (values ranged from 1.62 x 104 to 4.80 x 105 CFU/mL). As expected, animals from the NCtr group did not show any tissue alterations or colony growth after sampling (Figure 4B).
aPDT decreased the ...

Watch this Scientific Journal Video about Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis at JoVE.com

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

curcuminoid-mediated-antimicrobial-photodynamic-therapy-on-murine

Research

JoVE Journal

Immunology and Infection

Induction of Oral Candidiasis and Antimicrobial Photodynamic Therapy in a Murine Model

To begin, randomly distribute the mice whose oral cavity is negative for candida growth into nine groups, each corresponding to a different concentration of curcuminoids with or without light. On the first day, subcutaneously inject the immunosuppressive drug prednisolone to all members of the groups. Provide tetracycline hydrochloride at the concentration of 0.83 milligrams per milliliter in drinking water from day one till the end of the experiment.On the second day, administer chlorpromazine chloride intramuscularly on each thigh muscle to sedate the mouse for intraoral inoculation. Next, immerse a sterile swab into a freshly-prepared, standardized suspension of candida albicans. After placing the mouse in a supine position, rub its tongue with a soaked swab for 30 seconds.On the fifth day, administer a complimentary subcutaneous injection of prednisolone to maintain immunosuppression. On the seventh day, prior to treatment, place the anesthetized mouse in a supine position on a PAD device equipped with threads. Loop the thread around the incisors to keep the mouth open.Dispense 70 microliters of the photosensitizer onto the dorsum of the tongue of each mouse belonging to the C+L+groups. Then, move the tongue back inside the oral cavity and keep the mouse in a dark environment for 20 minutes as a pre-irradiation period. After the photosensitization period, place the LED device on the dorsum of the tongue and illuminate it for seven minutes.Apply photosensitizer to the C+L-groups as demonstrated and keep the mice in the dark for 27 minutes. Immediately after treatment swab the dorsum of the tongue for one minute, using a sterile cotton swab to retrieve candida albicans from the tongue. Place each sample swab into a tube containing one milliliter of sterile saline and vortex for one minute to resuspend the microbial cells.Without delay, perform ten-fold serial dilutions in PBS and plate 25 microliters aliquot onto Sabouraud Dextrose Agar plates in duplicate. incubate the plates aerobically at 37 degrees Celsius for 48 hours. After 48 hours, use a digital colony counter to determine yeast colony counts.Calculate the load of candida albicans for each animal. On the eighth day, after properly euthanizing the animal, proceed to surgically remove the tongue, perform the tongue removal ahead of the circumvallate papillae to ensure the entire tongue is removed. Finally, proceed with histopathological analysis of the isolated tongue.The murine model of oral candidiasis showed typical white patches and pseudo membranes on the tongue of infected mice. Antimicrobial photodynamic therapy reduced candida albicans viability at 80 micromolar curcuminoids concentration. The tongues of uninfected animals showed normal healthy tissues, including:intact lamina propria, basal membrane, and filiform papillae.In the C-L-group, yeast and filaments were observed in the keratinized epithelial layer. A mild inflammatory response was present in the underlying connective tissue. In contrast, mice treated with antimicrobial photodynamic therapy mediated by 80 micromolar curcuminoids had fewer fungal cells in the keratinized layer of the tongue epithelium.

581 Views.  São Paulo State University (UNESP).  This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Summary