Establishment Of Mouse Model Of Otomycosis And The Expressions Of TLR2 And TLR4 In External Auditory Canal Of Otomycosis Model Rats

Part Ⅰ Establishment and Pathological Analysis of a Mouse Model of OtomycosisObjective: establish an animal model of otomycosis to study the natural progress and clinical features of the disease,and do histopathological examinations at different stages of fungal infection of the external auditory canal.In order to establish a basis for other study of otomycosis.Methods: Totally,36 wistar rats were randomly and equally divided into 3 groups(12 rats in each group):immunosuppressive group,general experimental group and control group.Before the start of the experiment,rats in the immunosuppressive group were intraperitoneally injected with dexamethasone(30mg/kg)for 3 consecutive days.Scratch the bilateral external auditory canal of the rats slightly until reddened with a scrub stick,rats in immunosuppressive group and general experimental group are inoculated with Aspergillus fumigatus suspension of 1*10^9 CFU/ml(50ul)in bilateral external auditory canal,at the same time,rats in the control group are inoculated with normal saline with same dosage.The rats were observed daily by electro-otoscopy and were graded and recorded every other day according to their conditions and statistical analysis.On the 7th,14 th and 21 st day of the experiment,randomly selected rats in each group cut their external auditory canal tissues to observed and analysis by histopathology.All experimental data were analyzed and processed by SPSS 24.0 statistical software.Results: the clinical scores of the three groups were statistically analyzed:The average clinical scores of the immunosuppressive group and the general experimental group were both higher than those of the control group,and the difference was statistically significant(p < 0.01).The average clinical scores of the immunosuppressive group were higher than that of the general experimental group,and the difference was statistically significant(p < 0.05).Analyzed the clinical scores in different stages and found the clinical scores of the immunosuppressive group and the general experimental group were significantly increased on about the 14 th day of the experiment,and then decreased.Histology and image analysis showed that the skin of the external auditory canal of the model rats with otomycosis mycosis presented stratified squamous epithelium hyperplasia,accompanied by hyperkeratosis on the surface,extended epithelial horns in some areas,proliferation and expansion of subepidermal capillaries,and infiltration of inflammatory cells in some parts.Conclusion:1.The rat model of otomycosis successfully established in immunosuppressed rats and normal rats inoculated with Aspergillus fumigatus suspension.2.The natural course of otomycosis presented a change trend of aggravation first and then alleviation.The infection was most severe on the 7th day of the experiment but improved significantly on 21 st day,showing a certain degree of self-healing tendency.Part Ⅱ Immunohistochemical Method was Used to Detect the Expressions of TLR2 and TLR4 in External Auditory Canal of Otomycosis Model Rats.Objective: Observe the distribution and expression of TLR2 and TLR4 in different groups and disease courses of rats models of otomycosis,and discuss their possible roles in the pathogenesis of otomycosis,so as to provide experimental basis for clinical studies.Methods: Analysis rats external auditory canal tissues through IHC method to test the expression of TLR2 and TLR4 in immunosuppressive group,general experimental group and control group at the experimental process of 7 days,14 days,21 days,grade and assessed the results by morphology and image analysis system,analysis and processing rank data and AOD value of TLR2 and TLR4 expression by SPSS 24.0 statistical software.Results: Observing the immunohistochemical sections,it was found that TLR2 and TLR4 were varying degrees expression in the cuticle,granular layer,pricle cell layer and the cytoplasm and membrane of the basal layer of the external auditory canal skin,and the stratum spinosum was the most obvious.The positive expression rates of TLR2 and TLR4 in the modeling group(immunosuppressive group,general experimental group)were 100%(p < 0.01),in the control group were about 50% and were mainly concentrated in the early stage of the experiment.The expression intensity of TLR2 and TLR4 in the modeling group was significantly higher than that in the control group(p <0.01).Analyzed AOD values of the three groups and found the mean values of TLR2 and TLR4 in the immunosuppressive group and the ordinary experimental group were higher than those in the control group,and the difference was statistically significant(p < 0.01).There was no statistically significant difference between the mean values of TLR2 and TLR4 in the immunosuppressive group and the ordinary experimental group(p > 0.05).In the modeling group,the expression intensity of TLR4 was significantly correlated with that of TLR2(p < 0.01).Conclusion:1.TLR2 and TLR4 were mainly and different degrees expressed in the cuticle,granular layer,pricle cell layer and the cytoplasm and membrane of the basal layer of the external auditory canal skin of mouse model of otomycosis,and the stratum spinosum was the most obvious.2.The expressions of TLR2 and TLR4 were significantly increased in the animal models of otomycosis,and the change trend was basically consistent with the inflammatory progression of otomycosis.This indicates that TLR2 and TLR4 are involved in the pathogenesis of external auditory meatus mycosis.3.The expressions of TLR4 are stronger than that of TLR2 in the rat model of otomycosis.