Lele was adequate to maintain typical vestibular function in mice.Cochlear MorphologyCochlear morphology was investigated in homozygous mice (i.e., Slc26a4tm2Dontuh/tm2Dontuh) and compound heterozygous mice (i.e., Slc26a4tm1Dontuh/tm2Dontuh). The cochlear morphologies of wildtype mice plus the profoundly deaf Slc26a4tm1Dontuh/tm1Dontuh mice have been also compared. Abnormal morphological findings in Slc26a4tm1Dontuh/tm1Dontuh mice, like serious endolymphatic hydrops with dilatation with the scala media (Fig. 3B), significant atrophy from the stria vascularis (Fig. 3B), and degeneration of your cochlear hair cells (Fig. 3E), were not observed in Slc26a4tm2Dontuh/ tm2Dontuh mice (Fig. 3C and 3F) and Slc26a4tm1Dontuh/tm2Dontuh mice (Fig. 3D and 3G).Vestibular MorphologyThe vestibular morphology was investigated in homozygous mice (i.e., Slc26a4tm2Dontuh/tm2Dontuh) (Fig. 4A, 4B, and 4C) and compound heterozygous mice (i.e., Slc26a4tm1Dontuh/tm2Dontuh) (Fig. 4D, 4E, and 4F). Each mice showed normal morphologicalPLOS 1 | plosone.orgMouse Model with SLC26A4 p.H723R Mutationfindings and amount of otoconia in the vestibule. Fluorescence confocal microscopy revealed that vestibular hair cells in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice have been not degenerated (Fig. 4G and 4I). SEM revealed typical otoconia in the utricle in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice (Fig. 4H and 4J).Immunolocalization and Expression of PendrinWe then investigated the expression of pendrin inside the cochlea of Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice (Fig. 5A) by immunolocalization. In each strains of mice, pendrin was generally distributed in the spiral prominence and root cells, indicating that the expression of pendrin was not affected by the p.H723R mutation in mice. Compared using the wild-type mice, no substantial distinction in the molecular weight (Fig. 5B) or the expression level (Fig. 5C) of pendrin have been observed in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice by western blotting analyses.Kcnj10 ExpressionWe then investigated the expression of Kcnj10 (GeneID: 16513) in Slc26a4+/+mice, Slc26a4tm2Dontuh/tm2Dontuh mice, and Slc26a4tm1Dontuh/tm2Dontuh mice by real-time PCR (for mRNA expression) and western blotting (for protein expression) analyses. It has been demonstrated that Slc26a4-depleted mice showed decreased Kcnj10 expression, which contributes for the failure of endocochlear potential generation [27]. KCNJ10 (GeneID: 3766) mutations have already been observed in EVA patients by way of digenic inheritance with SLC26A4 mutations [28].2-(Difluoromethyl)pyridin-4-amine Purity In this study, the stria vascularis of P15 mouse cochleae from Slc26a4+/+ mice, Slc26a4tm2Dontuh/tm2Dontuh mice, and Slc26a4tm1Dontuh/tm2Dontuh mice were isolated by microdissection, and total RNA and protein extracted from these tissue fractions have been applied for real-time PCR and quantitative immunoblot analyses.7,8-Difluoronaphthalen-1-ol Order Compared with the wild-type mice, no substantial differences inside the mRNA (Fig.PMID:33452148 5D) or protein levels of Kcnj10 (Fig. 5E) had been observed in Slc26a4tm2Dontuh/tm2Dontuh mice and Slc26a4tm1Dontuh/tm2Dontuh mice.Noise Exposure ExperimentsWe then attempted to induce audiologic phenotypes in transgenic mice with noise exposure [29]. Neither Slc26a4tm2Dontuh/tm2Dontuh mice nor Slc26a4tm1Dontuh/tm2Dontuh mice showed a significantly greater shift in hearing thresholds at all frequencies 30 min and at day 1, two, 3, 7, and 14 soon after noise exposure than Slc26a.
