Expressions Of MeCP2, BDNF And Atoh1 In The Cochlea And Cochlear Nuclei Of BALB/c Mice With Age-related Hearing Loss

Objective:On the basis of auditory function measurements and cochlear morphological evaluation in BALB/c mice with different ages, in the present study,we attempt to investigate the characteristics of methyl-CpG-binding protein 2(MeCP2),brain-derived neurotrophic factor(BDNF) and atonal homolog 1(Atoh1) expressions in the cochlea and cochlear nuclei of BALB/c mice with different ages,so as to explore the possible roles of these genes on age-related hearing loss,and to lay a foundation for further analyzing the aging-related genes of auditory system and finding the molecular mechanisms of presbycusis.Method:①Through auditory brain stem response(ABR) measurements,cochlear surface preparation/hair cell counting and scanning electronic microscopy,the 8 kHz auditory thresholds,hair cell losses and morphological changes of organ of Corti were comparatively investigated in 3-,6-,12- and 18-month-old BALB/c mice.②The expression and distribution of MeCP2 protein in the cochlea and cochlear nuclei of BALB/c mice with different ages were detected and analyzed by means of immunohistochemical staining.③The levels of MeCP2 protein in the cochlea and cochlear nuclei of mice with different ages were detected and analyzed via Western blot assay.④Detection and analysis of the mRNA expression of MeCP2,BDNF and Atoh1 in the cochlea and cochlear nuclei were carryied out by RT-PCR on 3-,6-,12- and 18-month- old mice,respectively.Results:①ABR measurements demonstrated that auditory thresholds at 8 kHz in 3-,6- and 12-month-old mice were(25±5.1), (52±6.7) and(93±7.5) dB SPL,respectively.In eighteen-month-old mice, the 8 kHz auditory responses at 120 dB SPL could almost not be dectected.The outer hair cell(OHC) loss was mainly observed in the basal turn of the cochlea in 6-month-old mice and got deteriorated with increase of age.In 12-month-old mice,the whole OHCs were almost absent at basal and middle turn,and inner hair cells(IHC) were markedly missing.Scanning electronic microscopy showed that ultrastructural characteristics of the stereocilia On the outer and inner hair cells were short and disarrayed bundles,hair fusion and stereocilia bundles loss. These changes got aggravated with aging.②Immunohistochemical staining showed that MeCP2 expressed in both cochlea and cochlear nuclei of BALB/c mice,predominantly expressing in the cell nuclei.The positive staining became weaker following the age increase.③Western blot assay showed that the levels of MeCP2 protein in the cochlea and cochlear nuclei tissues of mice decreased with aging.④Results of RT-PCR demonstrated that the mRNA expression of MeCP2,BDNF and Atohl decreased as the age increasing.The optical density(OD) ratio of MeCP2,BDNF and Atoh1 was significantly lower in 6-,12- and 18-month-old mice than in 3-month-old mice.Statistics analysis showed that the mRNA expression between each two groups had a significant difference(p＜0.01).Furthermore,it was verified that the gene sequence of mRNA was identical to that of RT-PCR by PCR and BLAST.Conclusion:①Hearing loss,hair cell missing and stereocilia bundle damage in BALB/c mice gradually become heavier with aging.②MeCP2 was expressed both in mouse cochlea and cochlear nuclei,predominantly expressing in cell nuclei,and the expression levels age-relatedly decreased.③The mRNA expression of MeCP2,BDNF and Atoh1 gradually decreased as the age increasing.The expression of those three genes was highly consistent.④Expression of MeCP2 and its related genes,BDNF and Atoh1,decrease following the age increasing, indicating that they may play an immportant role in the development of age-related hearing loss.