Prevetive And Therapeutic Effects Of D-methionine On Noise-induced Hearing Loss

Objective: To study the preventive and therapeutic effects of D-methionine(D-Met)on noise-induced hearing loss(NIHL)in mice.Through the animal model,this study aims to investigate whether antioxidant D-Met has certain preventive and therapeutic effects on the cochlear tissue of the experimental animals on the levels of function,morphology,reactive oxygen species(ROS),apoptosis and connexin 26(Cx26),connexin 30(Cx30),and to provide further evidence for the pathogenesis of noise-induced hearing loss.Methods:1 To establish the animal model of noise-induced hearing loss: 40 healthy female Kunming mice were selected and randomly divided into four groups:(1)Control group(CG)(n=10)(2)Noise exposed group(NG)(n=10)(3)D-methionine before noise exposed group(D-MetBN)(n=10)(4)D-methionine after noise exposed group(D-MetAN)(n=10).The external ear,middle ear and inner ear of Kunming mice were primarily examined to exclude the factors that may affect the hearing function of inner ear.The mice of CG were non-noise exposed and intraperitioneal injected saline 400mg/Kg,twice a day,for 3 days.The mice of NG were exposed to white noise,the sound pressure level of 100 d B SPL in a ventilated sound exposure chamber for 8 hours over 3 days.The mice of D-MetBN were intraperitioneal injected400mg/Kg of D-Met before noise exposed,twice a day,for 3 days,then give100 dB SPL white noise.D-MetAN were exposed to 100 dB SPL white noise for 8 hours over 3 days,D-Met at 400mg/Kg per dose was administered 1hour after noise exposure,twice a day,for 3 days.2 Monitor changes of hearing functions in mice before and after noise exposure: Auditory brainstem response(ABR)was measured for each animalbefore exposure to noise,1 hour and 4 days after noise exposure,respectively.3 Changes of cochlear structure under microscope: hair cells were observed under optical microscope after staining with HE.Basilar membrane damage was also observed under laser confocal microscope after staining with Myosin-?.4 Immunohistochemical method: to test 4-hydroxynonenal(4-HNE)by Immunohistochemical method,with the purpose of evaluating the oxidative stress in each level.5 TUNEL apoptosis method: observed the apoptosis by the application of TUNEL method.6 Western blot method: to test the expression of Cx26 and Cx30 in each group by Western blot.Results:1 General observations: The mice in the CG grew well,kept moving and were sensitive to sound.The mice in the NG didn't like moving and weren't sensitive to sound.D-MetBN and D-MetAN grew well,kept moving and were sensitive to sound.2 The results of ABR test: There were no significant differences among the four groups in ABR test before experiment(P<0.05),the ABR threshold was(16.25±3.58)dB SPL in CG,(16.00±3.37)dB SPL in NG,(16.00±2.69)dB SPL in D-MetBN,(15.75±2.90)dB SPL in D-Met AN.After 1 hour noise exposure,the CG was(16.75±3.74)dB SPL,the NG was(58.75±6.80)dB SPL,D-MetBN was(52.75±9.01)dB SPL,D-MetAN was(60.00±5.14)dB SPL,there were statistical difference between the NG and the D-MetBN and D-MetAN(P<0.05).After noise exposure for 4 days,the ABR threshold was(16.50±3.16)dB SPL in CG,(36.25±7.48)d B SPL in NG,(23.00±3.29)dB SPL in D-Met BN,(23.25±3.13)dB SPL in D-MetAN,and there was a significant difference between the NG and the D-Met BN or D-MetAN(P<0.05),though no significant difference was seen in D-MetBN and D-MetAN(P>0.05).3 The results of HE staining and Myosin-? staining: The hair cells in theCG arranged orderly,and their structures were seen completely,no pathological changes were found.Numerous hair cells were lost in the NG,especially in the basal turn outer hair cells(OHC).Less hair cell lost were seen in the D-MetBN and D-MetAN,pathological changes like degeneration were found mainly in outer hair cell(OHC)and the inner hair cell(IHC)structure remained completed.4 The results of 4-HNE immunohistochemistry: The CG of cochlear basilar membrane,stria vascularis,spiral ganglion didn't express positive results.The positive results in the cochlear basilar membrane,stria vascularis,spiral ganglion of NG were obvious.there were less positive results found in the D-MetBN and D-MetAN than in NG.5 The results of TUNEL staining: The mice of CG didn't found TUNEL positive cells in the cochlear basilar membrane,stria vascularis,spiral ganglion.Many positive cells appeared in the cochlear tissue of NG,especially in the lateral wall.TUNEL positive cells were much less in the D-MetBN and D-MetAN than NG.6 The results of Cx26 and Cx30 western blot test: The expression of Cx26 and Cx30 in the NG was much lower than the CG,D-Met BN and D-MetAN,and there was no difference between the D-MetBN and D-MetAN.Conclusions:1 Noise-induced hearing loss mainly damage the organ of Corti of the inner and outer hair cells,especially the damage of the OHC,D-Met can protect against NIHL in both functional study and morphological study.2 Noise can lead to the NIHL by ROS and apoptosis,while D-Met has a protective effect on ROS and apoptosis.3 Cx26 and Cx30 may participate in NIHL,and D-Met can prevent their decline.4 D-methionine before noise exposed group and D-methionine after noise exposed group showed that the effects of D-methionine on the prevention and therapeutic of noise-induced hearing loss.