| Noise-induced hearing loss(NIHL)is one of the most common forms of non-hereditary sensorineural hearing loss(SNHL)except presbycus.With the development,the incidence rate is increasing year by year,which brings a heavy burden to the daily communication and physical and mental health of individuals in modern life.According to WHO data in 2020,more than 360 million people worldwide are affected by noise-induced deafness.The Centers for Disease Control(CDC)recently counted hearing impairment among American adults under 70 years old,and there are approximately 26 million people.The prevalence of Noise Induced Hearing Loss(NIHL)is 15%,and more than 16%of American adolescents with hearing impairment over 12 years old are caused by excessive noise exposure.The hearing impairment of young children and the elderly is also prone to autism and depression.Noise-induced hearing loss seriously threatens people’s health and is an important medical research field related to the long-term social and economic development of our country.Unlike conductive hearing loss,for sensorineural hearing loss,such as hearing loss caused by noise and ototoxic drugs,or presbycusis,due to the unclear mechanism,there is a lack of effective drugs for clinical treatment.Previous studies have found that long-term noise exposure can damage cochlear spiral ganglion cells,but the expression of its purinergic receptor protein is not clear.It is urgent to carry out research on the identification and expression regulation of purinergic receptors in spiral ganglion cells.The role of purinergic signaling molecules in the cochlear mechanism has been confirmed,including the establishment of synaptic transmission during the ontogeny of the cochlea,the signaling of noise damage in the supporting cells of the Corti organ,and the regulation of electrochemical stability based on acoustic conduction.state.ATP is an extracellular signal molecule,which is involved in almost all aspects of development,pathophysiology,neurotransmission,and neuromodulation.The receptor that uses the final cleavage product of ATP as a ligand is called P1 purinergic receptor(abbreviated as P1);the receptor that uses ATP and its analogues as ligands is called P2 purinergic receptor(abbreviated as P2).It is widely distributed in the body and is the main purinergic signal.P2 receptors include two families:ionotropic P2X and G protein coupled P2Y receptors.P2X receptors(representing ATP-gated ion channels)are subdivided into seven subtypes(P2X1 to P2X7);P2Y receptors include At least eight subtypes(P2Y1,P2Y2,P2Y4,P2Y6,P2Y11,P2Y12,P2Y13 and P2Y14).Before the development of the auditory system begins,Spiral Ganglion Neurons(SGNs)exhibit spontaneous activity,which establishes synaptic transmission with the central auditory pathway in the brain,and the purinergic signaling mechanism is the basis of this process.After exposure to noise,the up-regulation and down-regulation of ATP receptor protein expression can be detected,which indicates that P2 receptors may participate in noise damage and be regulated by noise.Due to the new coronavirus pneumonia(Corona Virus Disease 2019,COVID-19),some foreign antibodies cannot be ordered or are out of stock.Therefore,the types of P2 proteins in this study are limited to P2X2,P2X3,P2X4,P2X7,P2Y2,P2Y4.The novelty of this study lies in the use of isolated SGNs staining for morphological research,the use of medium and high intensity white noise to stimulate the cochlea for a long time to establish a noise-induced deafness animal model,observe the location,expression and changes of P2 receptors in cochlear SGNs,so as to explore SGNs The relationship between purine receptor expression regulation and noise-induced hearing loss provides a theoretical basis for clarifying the therapeutic target of SNHL.PART ⅠStudy on the expression morphology of P2 receptor in SGNs of normal guinea pig cochleaObjective:Innovative experimental methods for morphological research of isolated SGNs,and clearly observe the expression of P2 receptor protein in normal guinea pig cochlea SGNs,providing more intuitive and reliable experimental techniques for morphological research,and lays a foundation for further research on the role of P2 receptors in the process of hearing damage.Methods:Ten adult guinea pigs weighing 200-250g were selected,and the auditory brainstem response(ABR)was measured before the samples were collected,and then frozen sections and immunofluorescence staining of isolated cells were performed,and SGNs were labeled with NF-200.Confocal microscope imaging was used to observe P2 protein expression.Results:Combined with the results of frozen section and isolated cell staining,P2X2,P2X3,P2X4,P2X7,P2Y2,P2Y4 are all expressed in SGNs,and all expressed in the cell,and isolated cell staining is more antigenic than frozen section.Strong,the shape observation is clearer.Among them,P2X2,P2Y2,P2Y4 are expressed strongly in SGNs,while P2X3,P2X4,and P2X7 are expressed weakly.Conclusions:This study found for the first time that ATP receptor subtypes P2X2,P2X3,P2X4,P2X7,P2Y2,P2Y4 are expressed in guinea pig SGNs,that is,the expression of P2 receptor is not only expressed in guinea pig cochlear sensory cells and supporting cells,but also expressed in nerve cells.The isolated SGNs observation and staining used in the study is a novel technique for observing morphology,which avoids tissue masking,and can directly observe the expression sites of P2 receptors in SGNs more clearly,combined with frozen sections to correct the localization of the observed tissue provides a theoretical and experimental basis for the next step in studying the changes in the expression of P2 receptors in SGNs after noise damage.PART ⅡThe effect of noise damage on the expression of P2 receptor in guinea pig cochlea SGNsObjective:To clarify the expression changes of P2 receptors in cochlea SGNs after noise exposure in guinea pigs,and to explore the relationship between the changes of P2 receptors in spiral ganglion cells and noise-induced hearing loss.Methods:ABR screened 10 normal-hearing healthy adult male Dunkin Hartley guinea pigs,exposed to white noise(120 dB SPL,3 hours a day,10 consecutive days),and performed ABR audiometry on the 7th and 10th day of the noise exposure.Take the material after the exposure.Then the frozen section and immunofluorescence staining of isolated cells were performed,SGNs were labeled with NF-200,and the P2 receptor expression was observed using confocal microscope imaging.Results:Fluorescence results showed that the expression of P2X2 and P2X3 in SGNs increased after noise exposure,while the expression of P2X4,P2X7,P2Y2,and P2Y4 were downregulated.The increased expression of P2X3 and the downregulation of P2X4 and P2Y2 were statistically significant(P<0.05).Conclusions:Before and after 120 dB SPL white noise exposure,the expression of P2 receptors in SGNs is up-regulated(P2X3)or down-regulated(P2X4,P2Y2).It is speculated that the changes in purinergic receptors in SGNs may affect the Ca2+balance to reduce the sensitivity of hearing after noise stimulation.To achieve the prevention and treatment effect of NIHL.PART Ⅲ Verification of P2 receptor expression difference in guinea pig cochlea SGNs before and after noise damageObjective:To determine the differences in P2 receptor expression in guinea pig cochlea SGNs before and after noise exposure at the protein and RNA levels,and to further verify the results of morphological studies.Methods:ABR screened 20 normal-hearing healthy adult male Dunkin Hartley guinea pigs,exposed to white noise(120 dB SPL,3 hours a day,10 consecutive days),and performed ABR audiometry on the 7th and 10th days of the noise exposure.Take the material after the exposure.Western blot and fluorescence real-time quantitative PCR were performed.The difference of P2 receptor expression in cochlear SGNs before and after noise exposure was analyzed by gray value and 2-△△CT value.Results:The expression of P2X2,P2X3,P2X4,P2X7,P2Y2,P2Y4 and other receptor proteins in the cochlea SGNs of guinea pigs before and after noise exposure.The expression of P2 receptors in SGNs was up-regulated or down-regulated after noise exposure compared with before noise exposure.The expression of P2X3 after noise exposure was significantly higher than that before noise exposure(P<0.05),and the expression of P2X4 and P2Y2 after noise exposure was significantly lower than before noise exposure(P<0.05).Fluorescence real-time quantitative PCR was used to detect the mRNA levels of guinea pig cochlea SGNs in the normal group and the noise group.The change trend of the mRNA level of P2X2,P2X3,P2X4,P2X7,P2Y2,P2Y4 was consistent with the protein expression trend,and the mRNA level of P2X7,P2Y2,P2Y4 the changes are significantly different(P<0.05).Conclusions:Through Western blot and fluorescence real-time quantitative PCR studies,P2X3 expression was up-regulated after noise exposure,and P2X4 and P2Y2 were down-regulated,which was consistent with the results of cellular immunofluorescence staining,further indicating that P2 receptors play an important role in noise-induced hearing loss.It can provide a theoretical basis for exploring the therapeutic target of SNHL. |
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