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.
