Effects Of Long-term Exposure To Low-intensity Environmental Noise On Age-related Hearing Loss

Objective:With the rapid development of urbanization,urban residents have been exposed to environmental noise pollution for a long time.Previous studies have shown that environmental noise affects the function of the auditory cortex and cause behavioural disorders,but the effect of long-term exposure to environmental noise on the inner ear and hearing remains to be clarified,and the role of environmental noise in auditory aging remains unclear.This study intends to study the effect of noise and aging on the occurrence and development of deafness by giving long-term low-intensity broadband white noise to presbycusis model mice to simulate urban environmental noise,and to provide an important basis for the prevention and control of deafness.Methods:Eight-week-old adult C57BL/6J mice were exposed to continuous broadband white noise with a sound pressure level of 70 dB SPL for 8 hours a day for 3 months to simulate the impact of environmental noise on hearing.The control group was placed in a quiet isolation room at the same time.The noise group and the control group used the auditory brainstem response(ABR)to evaluate the hearing degree of mice in the frequency range of 2-32 k Hz at 1 month and 3 months of the experiment,and the amplitude of the ABR wave ? was measured to evaluate the neuro transmission function of the cochlear ribbon synapses.The mice were sacrificed after the ABR test,and the cochlea of each group of mice was taken,and the cochlear section and basilar membrane were prepared.Immunofluorescence staining confocal microscopy was used to observe the basilar membrane morphology and the number of inner hair cell ribbon synapses.The cochlear diagram was used to observe the relationship between the number of synapses and auditory function,and the density changes of spiral neuron cells were observed by HE staining.Scanning electron microscope was used to observe the morphology of inner and outer hair cell cilia.In the in vitro experiment,the cochlea of 3 days C57BL/6J young mice was taken out,the basilar membrane was separated for tissue culture in vitro,40 mg/mL D galactose was added for 48 hours to simulate the aging effect,and 0.5 mM NMDA and kainite mixture continued 12 hours to simulate the excitotoxic effect of glutamate.The immunofluorescence staining confocal microscope was used to observe the changes in the number of inner hair cell ribbon synapses and the number of spiral nerve fibers.After that,the basilar membrane tissues of each group were tested by Western blot and ELISA to study the molecular mechanism.The data obtained were analyzed statistically using Graph Pad Prism 5 software.After satisfying the normal distribution and the homogeneity of variance test,the independent sample t test was used for the comparison between the two groups,and the analysis of variance was used for the comparison of multiple groups.Results:1.ABR threshold: At the first month of the experiment,there was no significant difference between the noise group and the control group;at the third month of the experiment,in the high frequency range of 16 k Hz,24 k Hz,and 32 k Hz,the threshold of the unexposed 3 months was higher than the unexposed 1 month;At the same time,compared with the unexposed 3 months,the exposed 3 months has different degrees of rise in all frequency range thresholds,and the most concentrated range is the middle frequency band of 8-24 k Hz.2.The number of inner hair cell ribbon synapses: There was no significant difference between the unexposed 1 month and the exposed 1 month.Compared with the unexposed1 month,the unexposed 3 months showed a decrease from 50% to the end,and the difference was statistically significant.Compared with the unexposed 3 months,the loss of synapses in the exposed 3 months almost affected the entire length of the cochlea,the most serious of which was the middle part(30-70% from the top of the cochlea).This is in line with the changes in ABR.3.ABR wave ? amplitude at 90 dB SPL sound intensity: Almost all frequencies did not change significantly during the experiment for 1 month.Compared with the unexposed 1month,the amplitude of wave ? in the unexposed 3 months decreased at 16 k Hz,24 k Hz and 32 k Hz,and the difference was statistically significant.Compared with the unexposed3 months,the amplitude of the exposed 3 months was reduced in all frequency ranges,and the 8-24 k Hz frequency difference was the most significant.4.Basilar membrane morphology: Compared with normal 8-week-old mice,there was no significant change in hair cell arrangement,cilia morphology and spiral neuron density in the exposed 1 month and the exposed 3 months.5.The number of inner hair cell ribbon synapses and spiral nerve fibers in the in vitro experiment: After the combined intervention of D-galactose and glutamate receptor agonists,the number of ribbon synapses was significantly lower than that of the control group.There are also significant differences between the intervention group and the control group or the combined intervention group.The reduction of ribbon synapses was different among the groups,and the number of synapses in the combined intervention group changed the most.At the same time,the changes in the number of spiral nerve fibers were consistent with the ribbon synapses,and there were significant differences between the groups.The number of spiral nerve fibers in the combined intervention group was the lowest.6.The expression of NLRP3 inflammasome and related pathway cytokines: The expression of NLRP3 in each intervention group was significantly higher than that in the control group,and the difference between the groups was significant.In the combined intervention of D-galactose and glutamate receptor agonists group,the expression of NLRP3 rose the most.The expressions of other inflammatory factors that interact with NLRP3,caspase-1,IL-1?,IL-18 and TNF-?,increased most significantly in the combined intervention group.Conclusions:The results of this study showed that the hearing threshold increased at a higher frequency with age,and the corresponding inner hair cell ribbon synapses were also damaged near the bottom of the cochlea.However,the combined effect of long-term,low-intensity environmental noise and aging has led to different forms of hearing loss,mainly in the mid-range of basilar membrane,which has changed the pattern of age-related hearing loss and caused more extensive and serious damage.The number and function of ribbon synapses are also severely reduced in the middle range of the basilar membrane.At the same time,within a certain time frame,the loss of inner hair cell ribbon synapses is not accompanied by damage to other major structures of the basilar membrane,indicating that ribbon synapses are the early cochlear damage caused by this type of noise.It can be seen that the cumulative effect of environmental noise has aggravated the progress of age-related hearing loss in both degree and scope.In vitro models show that the superimposed effects of glutamate excitotoxicity and aging are manifested in the reduction of inner hair cell ribbon synapses and spiral nerve fibers,and this change is closely related to the inflammatory response of the basilar membrane.Therefore,long-term exposure to low-intensity environmental noise and the combined effects of aging can cause permanent hearing loss via the destruction of ribbon synapses.This study explains the pathogenesis of inner ear damage caused by environmental noise exposure,and provides a new direction for the prevention and treatment of hearing loss.