| Objective: Recently,hearing Loss has become one of the most important reasons affecting global human health.According to the results of 2013 World Health Organization(WHO Word Health Organization)survey data,there are approximately 360 million hearing impaired patients worldwide,accounting for about 5.3% of the total population,of which there are about 32 million children with hearing impairment under the age of 15.According to the etiology,hearing loss can be divided into congenital and acquired.Among them,congenital hearing loss mainly includes hereditary deafness,whereas acquired hearing loss mainly includes noise-induced hearing loss(NIHL),ototoxic drug-induced hearing loss,and presbycusis and so on.Thus,if genes related with hearing loss could be comfirmed as soon as possible,the diagnosis and assessment of disease risk will be of great significance for ultimately reducing the incidence of hearing loss in the population and improving the quality of human beings.Ultimate cause of hearing loss is blocking the auditory conduction pathway.And the auditory conduction pathway could be mainly divided into the peripheral auditory pathway and the auditory central pathway.The peripheral auditory pathway: sound passes through the external ear canal and passes through the eardrum.Then the sound transmits to the middle ear,and send the sound signal to the oval window via ossicles,and stimulate the organ of Corti through the lymph fluid.The central nervous system: after the outer hair cells receiving the sound stimulation,the sound signal is amplified to stimulate the inner hair cells to encode sound signals through ribbon synapses,and then transmit the signals to bipolar cells,which could be transmited to the auditory central system.They first reach the cochlear nucleus of the cerebrum,and most of the emitted fibers cross the cerebrum to the contralateral side in the pontine.Folding up to the outer olive nucleus,it is called the lateral thalamus.The fibers of the lateral thalamus go up through the dorsal lateral part of the midbrain,and most of the fibers stop in the hypothalamus and pass from the hypothalamus to the left and right medial geniculate bodies Information.There are also a few lateral thalamus that stop directly on the medial geniculate body,and a few snail nerves do not cross the anterior and posterior nucleus fibers and enter the ipsilateral side.Finally,it transmits acoustic information to the auditory cortex of the temporal lobe via medial geniculate body.In the auditory pathway,cochlear ribbon synapses have become a hotspot in the field of otoneuroscience.Unlike the central nervous synapses,the cochlear ribbon synapses are the first synapses in the process of transmitting sound signals to the auditory central system.And the structure is called “ribbon synapses” because of its spatial distribution.The conduction of hearing from the periphery to the center depends on the rapid and precise release of neurotransmitters within the ribbon synapses,which also determines the quality and quantity of sound signal transmission.In the auditory pathway,the inner cochlear hair cells are responsible for converting the mechanical vibrations of sound waves into electrical signals,and finally projected to the central neurons through cochlear ribbon synapses.Therefore,any destructive factors such as noise,ototoxic drugs,aging,etc.,inducing damaging changes in ribbon synaptic structure and function could have a significant impact on the encoding of sound.At present,hidden hearing loss is a disease whose main pathological mechanism is cochlear ribbon synaptic damage.Clinically,patients have no significant change in hearing threshold,but they perform a significant reduction in speech recognition rate in a noisy environment.And basic research shows that after moderate noise exposure,animal models could induce temporary threshold shifts,irreversible damage of cochlear ribbon synapses,and irreversible reduction of ABR I amplitude,consistent with clinical manifestations.Therefore,it could be used as Noise-induced Hidden Hearing Loss(NIHHL)model.At present,according to the clinical manifestations of NIHHL,the majority of scholars believe that the mechanism might be that the medium-level noise only damaging the high-threshold,low-spontaneous auditory nerve fibers,while almost does not damage the low-threshold and high-spontaneous discharge rate auditory nerve fibers.The function of the high-threshold,low-spontaneous rate of the auditory nerve fibers is mainly to regulate the speech recognition rate in a noisy environment.Therefore,it was believed that the medium-intensity noise in hidden hearing loss directly damages the ribbon synapses between inner hair cells and the high-threshold,low-spontaneous rate auditory nerve fibers.And then induce disconnection between the high-threshold,lowspontaneous rate of the auditory nerve to the inner hair cells to lose their function,which leads to a reduction in the speech recognition rate of patients with hidden hearing loss in a noisy environment.And it could induce temporary threshold shifts,irreversible damage to cochlear ribbon synapses,and a significant decrease in ABR I wave height threshold amplitude in the animal model.However,the molecular mechanism still keeps largely unknown.The fibroblast growth factor 22(Fgf22)gene was first cloned in 2001,which is closely related to mammalian embryonic brain development,the formation,maintenance,and regulation of excitatory synapses in the central nervous system.And a recent study has found that Fgf22 gene could protect hearing loss from gentamicin ototoxicity by maintaining the number of cochlear ribbon synapses.Thus,it is speculated that Fgf22 might be also closely related to the cochlear ribbon synapses in the auditory system.Therefore,our study aims to further explore the function of Fgf22 in the auditory pathway and its possible mechanism,as well as whether Fgf22 gene is closely related to NIHHL and its molecular mechanism.At present,transcriptomics research like RNA-seq is very popular in the field of basic scientific research.The main reason is that it could reveal the cellular and tissue molecular composition through the acquisition of intact transcription factors and their changes in cells under specific developmental stages or physiological conditions.In addition,understanding the significance of development and disease or its pathogenesis could be revealed directly at the molecular level.Therefore,it is currently widely used.Among them,long non-coding RNA(long non-coding RNA,lnc RNA)is a type of nonprotein coding RNA larger than 200 nucleotides,which play an important role in various diseases by regulating transcription and so on.Therefore,this study will use RNA-seq technology to further analysis the molecular mechanisms involved in this study.Methods: 1.To study the spatial and temporal expression characteristics of Fgf22 gene in auditory pathways by immunofluorescence in normal adult mice;it was used to determine the spatial and temporal expression characteristics of Fgf22 gene in the auditory pathway through cochlear basement membrane staining on P0,P7,P14,P28;2.After constructing Fgf22 gene knockout mice model by CRISPR / Cas9 technology,we first identified the mouse genotype via PCR and gene sequencing,to confirm that the Fgf22 gene knockout was successfully built up.And then we observed and evaluated the growth and development characteristics such as reproductive ability and weight of mice with different genotypes;3.To define the acoustic phenotype of the Fgf22 gene knockout mouse model by Auditory Brain Response(ABR)at 4k Hz,8k Hz,16 k Hz,32 k Hz;4.After giving 100 d B of SPL white noise for 2 hours of moderate-intensity noise exposure,we aimed to observe the differences in audiological performance between Fgf22 knockout mice and wild-type mice with NIHHL,and to observe the changes of cochlear ribbon synapses using immunefluorescence;5.To construct a mouse model of NIHHL,and set the NE-1st group(NIHHL model),NE-2nd group(NIHL model),and Control group,respectively.And detect ABR threshold changes,the ABR I wave amplitude changes,as well as the changes in the number of cochlear ribbon synapses by immunofluorescence at 100 d B SPL white noise for 2 hours on Day1,Day7 and P14 after noise exposure;6.Cochlear tissue samples were collected to perform RNA-seq analysis and RNAseq technology was used to determine the main regulatory pathways and target genes for NIHHL,and further verified by q RT-PCR.Finally,it might be concluded that the possible mechanism of Fgf22 gene defect in the NIHHL;Results: 1.Expression characteristics of Fgf22 gene in mouse auditory pathway during the development of mouse cochlea;(1)Temporal expression characteristics: Fgf22 is firstly observed in inner hair cells,gradually appears in outer hair cells,and finally is also observed in spiral ganglion cells at P28,where synapses are actively expressed.Cochlear ribbon synapses mostly located between the inner cochlea hair cells and type I auditory nerve fibers;(2)Spatial expression characteristics: In adult mouse cochlea,we found that Fgf22 gene is expressed in inner hair cells,outer hair cells and spiral ganglion cells,which is consistent with developmental expression results.2.Constructed Fgf22 gene knockout mice by CRISPR / Cas9 technology;(1)Genotype verification results showed that Fgf22 gene knockout mice was built successfully; (2)There was no significant difference in hearing function between wild-type mice and Fgf22 kockout mice.However,Fgf22 knockout mice were found to be more sensitive compared with wilde type mice after white noise exposure at 100 d B SPL for 2 hours,which performing decreased cochlear ribbon synapses,the decreased amplitude of the ABR I wave compared with control group with significant differences;3.Successfully constructed NIHHL model induced by 100 d B SPL white noise exposure for 2 hours;(1)Normal C57 BL / 6J adult mice showed temporary threshold shift,decreased wave I amplitude of ABR,and significantly reduced number of cochlear ribbon synapses after two weeks of exposure to 100 d B white noise for 2 hours,which indicating it could be used as an animal model of NIHHL with significant difference compared with control group;(2)In normal C57 BL / 6J adult mice,it showed permanent threshold shifts,significantly decreased ABR wave I amplitudes,and significantly decreased the number of cochlear ribbon synapses 2 weeks later after 100 d B white noise exposure for 2 hours,which could be used as NIHHL model.4.Identify possible regulatory pathways and target genes for NIHHL via RNA-seq;(1)Analysis of RNA-seq and lnc RNA-m RNA co-expression analysis revealed that m RNA GNAS and its upstream lnc RNA Sept7 were significantly increased in the NE-1st group,and the difference was statistically significant;(2)The Adrenergic Signaling path is found to have the lowest p-value after analysis.Conclusion: 1.Our study has fistly found that during the development of mouse cochlea,Fgf22 firstly was expressed in inner hair cells,gradually appeared in outer hair cells,and finally was also expressed in spiral ganglion cells at P28.In adult mouse cochlea,we found that Fgf22 gene expressed in inner hair cells,outer hair cells and spiral ganglion cells,which was consistent with developmental expression results;2.CRISPR / Cas9 technology was used to construct Fgf22 gene knockout mice successfully.It was found that there was no significant difference in hearing function compared with wild type mice under normal circumstances.However,Fgf22 gene knockouts mice showed a decreased cochlear ribbon synapses and a significantly decreased I wave amplitude of ABR after exposing to white noise at 100 d B SPL for 2 hours compared with wild-type mice;3.Normal C57 BL / 6J adult mice showed temporary threshold shift,significantly decreased ABR wave I amplitude,and significant decreased in the number of cochlear ribbon synapses two weeks later after exposure to 100 d B white noise for 2 hours,which could be used as the NIHHL;4.Based on RNA-seq and lnc RNA-m RNA co-expression analysis,it was proposed that Fgf22 might regulate the occurrence and development of NIHHL via Adrenergic Signaling pathway with m RNA GNAS and its upstream lnc RNA Sept7. |
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