Name: Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis
Uploaded: 2023-10-27T14:40:00
Description: Watch this Scientific Journal Video about Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis at JoVE.com

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 a...

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. 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T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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 ...

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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.