Experimental Study On Photo-activated Disinfection In The Treatment Of Acute Pseudomembranous Stomatitis

Part One Establishment and evaluation of a mice model of acute pseud-omembranous stomatitis.Objective:To establish a mice model of acute pseudomembranous stomatitis and evaluate the model.Methods:Forty 6-week-old male ICR mice were randomly divided into two groups:blank control group（n=20）and model group（n=20）.Mice in b oth groups received immunosuppression.Model group was inoculated wi th SC5314 Candida albicans solution with a concentration of 1×10⁷CFU/mL on the dorsal tongue,while blank control group was smeared with normal saline on the dorsal tongue.On the third day after inoculation,t he clinical manifestations were observed,the score of leukoplakia area o f dorsal tongue and the fungal load of dorsal tongue were measured.Th e animals were killed for tissue load determination and histopathological examination to evaluate the establishment of acute pseudomembranous stomatitis model.Results:1.Before Candida albicans inoculation,there was no significant difference in body weight between the blank control group and the model group,but on the 3rd day after inoculation,the body weight of the model group was significantly lower than that of the blank control group（P<0.05）.2.Before Candida albicans inoculation,there was no obvious abnormality in the tongue of mice in the blank control group and model group.On the 3rd day after inoculation,the tongue of the blank control group was no erythema or white pseudomembranous.In the model group,15 mice had white pseudomembranous on the dorsal tongue（the lesion score was mainly 3 to 4points）,and 5 mice had normal tongue without erythema or white pseudomembranous.3.On the 3rd day after Candida albicans inoculation,the fungal load in the tongue of the blank control group was 0（not shown）.The dorsal lingual fungal load of 15 mice in the model group was 4.77±0.35Log₁₀CFU/mL,and that of 5 mice was 0（not shown）.4.On the 3rd day after Candida albicans inoculation,the Candida albicans load in liver,kidney and feces of mice in the blank control group was0（not shown）.In the model group,the tissue load of liver and kidney was 0（not shown）,and the fecal load of colon was 7.17×10⁴CFU/g.5.Histopathological examination:HE staining showed that the filamentous papilla of the tongue back of the blank control group was arranged neatly and the epithelial structure was regular.PAS staining showed that there were no Candida hyphae and spores in the blank control group.In the model group,a large number of hyphae and spores could be seen in the outer 1/3 of the keratinized layer or epithelium.The hyphae were long and slender rod-shaped,beaded or segmented,and most of them were vertical or at a certain angle to the surface of the epithelium.Conclusions:The model of acute pseudomembranous stomatitis was successfully established by oral inoculation of Candida albicans into immunosuppressed ICR mice.Part two Observation on the efficacy of PAD in the treatment of acute pseudomembranous stomatitis in mice.Objective:To explore the therapeutic effect of Photo-activated disinfection（PAD）on acute pseudomembranous stomatitis in mice,and to provide experimental basis for PAD in treating acute pseudomembranous stomatitis.Methods:According to the random number table method,45 mice were selected from the model and randomly divided into control group,PAD treatment group 1（treatment group 1）and PAD treatment group 2（treatment group 2）,with 15 mice in each group.The leukoplakia area of dorsal tongue was measured in all mice before treatment.Photo-activation disinfection technique based on PAD^TM Plus was used to treat mice in treatment group 1 and treatment group 2.Treatment group 1:1mg/mL toluidine blue solution was smeared on the back of tongue of mice,incubated for 1 minute and 750mW LED red light irradiation for 1 minutes.Treatment group 2:1mg/mL toluidine blue solution was smeared on the back of tongue of mice,incubated for 2 minutes and 750mW LED red light irradiation for 1 minutes.The mice in the control group did not receive any treatment.Fungal load of dorsal tongue was measured directly in the control group,and immediately after PAD treatment in the treatment group 1 and 2.48 hours after treatment,the clinical manifestations of the three groups of mice were observed,the leukoplakia area of the dorsal tongue and the fungal load of the dorsal tongue were determined,and the mice were killed for histopathological examination.The efficacy of PAD in the treatment of acute pseudomembranous stomatitis in mice was evaluated according to the lesions of the dorsal tongue,fungal load and histopathological changes.Results:1.Before PAD treatment,there was no significant difference in the area score of dorsal tongue leukoplakia among treatment group 1,treatment group2 and control group（P>0.05）.At 48 hours after PAD treatment,the score of dorsal tongue leukoplakia area in treatment group 1 and treatment group 2 was significantly lower than that in control group（P<0.05）,but there was no significant difference between treatment group 1 and treatment group 2（P>0.05）.2.Immediately after PAD treatment,the load of Candida albicans in treatment group 1 and treatment group 2 was significantly lower than that in control group（P<0.05）.There was no significant difference in Candida albicans load between treatment group 1 and treatment group 2（P>0.05）.At 48 hours after PAD treatment,the load of Candida albicans in treatment group 1 and treatment group 2 was significantly lower than that in control group（P<0.05）.There was no significant difference in Candida albicans load between treatment group 1 and treatment group 2（P>0.05）.3.Histopathology of posterior dorsal tongue treated by PAD:48 hours after treatment,HE staining showed that in the control group,the epithelial tissue thickened and the epithelial nailing process was irregular,the superficial neutrophils infiltrated,and a small abscess was formed in some locations.The epithelial structure of treatment group 1 and treatment group 2 was more regular,and there was no significant difference between the two treatment groups.PAS staining showed that a large number of mycelia were covered in the control group,and long rod-shaped or segmented hyphae invaded on the superficial surface of the epithelium.The number of hyphae in treatment group 1 and treatment group 2 was significantly less than that in the control group,and occasionally hyphae invaded the keratinized layer,but did not penetrate into the epidermis.There was no significant difference between the two treatment groups.Conclusions:1.Photo-activation disinfection technology can significantly reduce the fungal load on the dorsal tongue of mice with acute pseudomembranous stomatitis and relieve the symptoms of leukoplakia on the dorsal tongue of mice.2.The treatment of acute pseudomembranous stomatitis by 1mg/mL toluidine blue solution incubation for 1min and 750mW LED red light irradiation for 1min can achieve obvious therapeutic effect.When other parameters are constant,prolonging the incubation time of 1mg/mL toluidine blue solution will not improve the efficacy of acute pseudomembranous stomatitis.