The Mechanism Of Immune Response Involved In Cochlear Sensory Epithelial Cells Injury Induced By Targeted Knockout Of Gjb2 Gene And Its Intervention

PART Ⅰ Establish a mouse model of targeted knockout of Gjb2 gene in inner ear supporting cells and observation of the damage pattern of inner ear sensory epithelial cellsObject:To establish a variety of specific inner ear support cell GJB2 gene knockout mouse models and preliminarily explore the changes of hearing function and hair cell injury patterns in the above mouse models.Methods:CX26^flox/floxmice were hybridized with Lgr5-Cre ER and Fgfr3-Cre ER mice to obtain CX26^flox/flox;Lgr5-Cre ER and Cx26^flox/flox;Fgfr3-Cre ER mice.Mouse model of targeted knockout of Cx26 in inner ear supporting cells were obtained by subcutaneous injection of tamoxifen（TMX）for two consecutive days after birth（P0,P1）.CX26^flox/flox;Lgr5-Cre ER and Cx26^flox/flox;Fgfr3-Cre ER mice were used as the experimental group,and litters of CX26^flox/floxmice served as control group.Immunofluorescence staining was used to detect CX26 knockout in cochlea.Immunofluorescence staining was used to detect the spatial distribution characteristics of CX26 in different inner ear supporting cells at different time points in the early development of the mouse cochlea.The hearing of P18mice was measured by detecting the auditory brainstem response（ABR）.Immunofluorescence staining was used to observed the survival of hair cells and support cells.Resin sections were stained with toluidine blue to observe the morphology of Corti apparatus.HE staining of frozen sections for counting spiral ganglion cells（SGN）.The spatial structure of Corti apparatus was observed by scanning electron microscopy,and the ultrastructure of supporting cells was observed by transmission electron microscopy.Results:In postnatal day（P3）of normal mice,Cx26 was expressed preferentially between adjacent DCs in the same row in Deiter’s cells（DCs）,and there was almost no Cx26expression between different rows of DCs.At P5,this longitudinal Cx26 expression between DCs in the same row is still dominant.At P7,CX26 becomes evenly distributed on the DC.CX26^flox/flox;Lgr5-Cre ER mice with basal membrane Lgr5-positive cell CX26knocked out was successfully constructed.At P18,no obvious hearing impairment was observed in CX26^flox/flox;Lgr5-Cre ER mice.Degeneration of the third row of outer hair cell（OHC3）was observed in the basal turn of CX26^flox/flox;Lgr5-Cre ER mice,but the corresponding DC cells could still survive.Cx26^flox/flox;Fgfr3-Cre ER mice showed high frequency hearing loss,Significant hair loss was found in the basal turn,without significant loss of corresponding DC.Further observation was made by transmission electron microscopy,no obvious developmental disorder and ultrastructural abnormality were observed in organ of Corti in Cx26^flox/flox;Lgr5-Cre ER and Cx26^flox/flox;Fgfr3-Cre ER mice.Conclusion:At P3-P5,CX26-related gap junction channels were preferentially established between longitudinal DCs.Targeted knockout of inner ear supporting cells Cx26 in the early postnatal period will leads to the degeneration of outer hair cells and hearing loss.The specific knockout of DC cell Cx26 will not affect the development of column cells and the structure of the organ of Corti.PART Ⅱ Macrophage-related immune responses are involved in cochlear epithelial damage in the targeted knockout of Gjb2 gene mouse modelObject: To explore the mechanism of macrophage-related immune response involved cochlear epithelial damage induced by targeted knockout of Gjb2 gene in supporting cellsMethods: CX26 flox /flox mice were hybridized with Fgfr3-Cre ER mice to obtain Cx26flox/flox;Fgfr3-Cre ER mice.Mouse model of targeted knockout of Cx26 in inner ear supporting cells were obtained by subcutaneous injection of tamoxifen（TMX）for two consecutive days after birth（P0、P1）.The morphology and distribution patterns of macrophages in the inner ear of mice in the knockout group were observed by using dual mononuclear macrophage molecular marker（CD45+CX3CR1）.Transcription levels of various inflammatory cytokines in cochlea were detected by PCR.HE staining of frozen sections for counting spiral ganglion cells（SGN）.The expression of inflammatory chemokine CX3CL1 was detected by frozen section immunostaining.Results: Targeted supporting cells Gjb2 gene knockout mice showed external hair cell damage in the basal gyrus accompanied by basilar membrane macrophage activation.The number of macrophages in the damaged area of hair cells increased significantly,the average size macrophages in the basal region of the KD group was larger than that of the control group.At P60,hair cell damage in the knockout group progressed to the middle turn,and the activation range of basilar membrane macrophages was expanded accordingly.Regression analysis showed that the degree of hair cell loss was significantly correlated with the number of recruited macrophages.The results of PCR showed that CX3CL1 m RNA was significantly up-regulated in the knockout group,while m RNA levels of other common inflammation-related cytokines TNF-α,IL-1β,CX3CR1,CCL2,CCR2,ICAM1 and TLR4 were not significantly changed.Frozen-section immunofluorescence results showed that the fluorescence signal of CX3CL1 protein in DC cells of mice in the knockout group was significantly increased.At P60,Degeneration of spiral ganglion neuron weas observed in knockout group.The number of macrophages in the damaged area of spiral ganglion increased significantly,and the expression of CX3CL1 also increased.Conclusion: The macrophage-related immune response is involved in the degeneration process of cochlear sensory epithelial cells induced by Cx26 deletion.Degeneration of sensory epithelial cells is accompanied by recruitment and activation of basilar membrane macrophages in a mouse model with specific knockout of the Gjb2 gene.The recruitment and activation of macrophages in the injured area may be related to the increased expression of CX3CL1.PART Ⅲ Protections of glucocorticoids on hearing loss and cochlear sensory cell damage in the inner ear induced by targeted knock out support cell Gjb2geneObject: To explore the protective effect and mechanism of glucocorticoid on hearing loss and sensory cell damage induced by specific knockout of GJB2 gene in inner ear.Methods: Two mouse models of targeted knockout of inner ear supporting cell CX26 were constructed.The experimental animals were divided into Control group,knockout group（KD）,Control + Dexamethasone group（C+DEX）,Knockout + Dexamethasone group（KD+DEX）.Mice in C+DEX and KD+DEX groups were intraperitoneally injected with5mg/kg dexamethasone on P10-P18 every other day,and the control group and the knockout group were given the same dose of normal saline.At P18,ABR test was performed to assess the hearing and hair cell counts were used to detect hair cell damage patterns.Cochlea basilar membrane of P3-P5 mice was isolated and cultured to establish a cochlear sensory cell injury model induced by glucose oxidase（GO）.The basilar membrane was divided into Control group（C）,Glucose oxidase group（GO）,Control +Dexamethasone group（C+DEX）,Glucose oxidase + Dexamethasone group（GO+DEX）.Basilar membrane was collected after 24 h culture,and hair cell counts were used to detect hair cell damage patterns.Results: At P18,Fgfr3-Cx26 KD mice showed high frequency hearing loss,significant hair cell degeneration was observed in the basal turn of basilar membrane.High frequency hearing loss was reduced in the dexamethasone intervention group（KD+DEX）compared to the knockout group.Degeneration of hair cell was also significantly reduced in the basal turn.The protection of dexamethasone on Cx26 knockout induced sensory cell injury in the inner ear was also observed in the Lgr5-Cx26 KD model.In the basilar membrane culture experiment,significantly hair cell loss was observed in the GO treatment group,and the degeneration of hair cells in the apical turn was significantly reduced in the GO + DEX group.Conclusion: In targeted supporting cells Cx26 knockout mouse models,we observed degeneration of outer hair cells in the basal turn and high-frequency hearing loss.Systemic administration of dexamethasone partially saved hearing loss and reduced hair cell death in the knockout group.It is further suggested that the immune response of the inner ear is involved in the CX26 knockout induced sensory epithelial cell injury of the inner ear,and intervention of the immune response of the inner ear can reduce the sensory epithelial cell injury and hearing loss.