| Background:Sensorineural Hearing Loss(SNHL)is the most common type of hearing loss,referring to hearing loss due to pathology of the cochlea,auditory nerve or central nervous system,for which there is a lack of targeted treatment.Neurotrophin-3(NT-3)and brain-derived neurotrophic factor(BDNF)have been found to play an important role in the maturation and regeneration of hair cells.Neuritin(Nrn1)was discovered in 1993 by the Israeli scientist Nedivi and is a common downstream effector protein of NT3 and BDNF,which is expressed in cochlear hair cells,supporting cells and spiral ganglia,thus Neuritin may have a role in hair cell regeneration.In this study,we propose to investigate the protective effect of exogenous Neuritin on sensorineural deafness using a model of sensorineural deafness and its mechanism.Aims:1.To establish an in vivo model of sensorineural deafness;2.To investigate the protective effects of Neuritin on auditory function and cochlear tissue in a mouse model of SNHL by administering recombinant human Neuritin protein through a round window;3.To investigate the molecular mechanism of the protective effects of recombinant human Neuritin protein on the prevention and treatment of SNHL.Methods:1.A model of sensorineural deafness was established:C57BL/6 mice with hearing threshold below 40 d B were screened and treated with subcutaneous injection of Kanamycin(KM)(1000 mg/kg)and half an hour later with intraperitoneal injection of Furosemide(Fur)(300 mg/kg),and then test by auditory brainstem response(ABR).Immunofluorescence(IF)staining of mouse cochlear tissue was performed to observe the changes in the number and morphology of hair cells(HCs);2.C57BL/6 mice were pretreated with 2μL of 16 mg/m L Neuritin in the cochlear round window,and then were modeled with a combination of kanamycin and furosemide.Changes in hearing function were detected at 1d,3d and 7d after moulding by ABR to investigate the protective effects of Neuritin on hearing function and cochlear tissue in a mouse model of sensorineural deafness;3.The mice C57BL/6 postnatal day 3(P3)cochlear culture were prepared under a microscopeand 3 m M gentamicin was administered to establish a model of hair cell injury.Five groups:normal control group,injury group,exogenous administration of Neuritin+injury group,exogenous administration of Neuritin+injury+FGF signalling pathway inhibitor(PD173074),and exogenous administration of Neuritin+injury+FGF signalling pathway inhibitor(PD173074)were used to investigate the molecular mechanisms underlying the effects of Neuritin.Results:1.The results of SNHL modeling showed that:1d,3d and 7d after modeling with kanamycin sulfate(1000 mg/kg)and furosemide(300 mg/kg),ABR testing of hearing function revealed that the overall hearing function thresholds in the neuritin-treated group were significantly higher than those in the control group,with hearing thresholds of around 80d B in the low-frequency region(8k HZ),mid-frequency region(16k HZ)and high-frequency region(32k HZ)(P<0.05).2.The IF results showed that the hair cells in the normal group were neatly arranged in one row of inner hair cells and three rows of outer hair cells.At 1d,3d and 7d after moulding,there was significant loss of hair cells,almost loss of outer hair cells and disordered morphology of inner hair cells.3.The results of in vivo experiments showed that the hearing threshold of Neuritin pretreated mice was 10-15 d B lower than that of control mice at 1d after the drug loss moulding(P<0.05).In the Neuritin pretreatment group,the number of cochlear SGNs was higher than that of the control group,and the thickness of SVs in the parietal,middle and basal gyri was thicker than that of the control group(P<0.05).The cochlea of Neuritin pretreated mice was thicker than that of the control group(P<0.05).In vitro experiments showed that the number of hair cells was higher in the Neuritin-treated group than in the drug-injured group(P<0.05),and specific markers of hair cells,proliferating cells and supporting cells(Myo7a~+Ed U~+Sox2~+)appeared in the parietal gyrus of the Corti organ,in addition,in the parietal gyrus of the Corti organ,new hair cells were found to be encapsulated with nerve fibres in the Neuritin-treated group,but not in the damaged group;a small number of specific markers of proliferating and hair cells(Myo7a~+Ed U~+)were found after treatment with the FGF signalling pathway inhibitor PD173074,whereas no specific markers of proliferating and hair cells(Myo7a~+Ed U~+)were observed after treatment with PD173074 and Jaggedl,no triple-or double-positive cells were observed.Conclusions:1.Co-administration of kanamycin with furosemide caused significant hearing loss in adult mice with substantial hair cell loss,meeting the criteria for sensorineural deafness modeling;2.An in vivo model showed that Neuritin antagonised the damage to auditory hair cells caused by ototoxic drugs,reduced the number of spiral ganglia and atrophied the thickness of the vascular striae,acting as a protection against hearing function;3.The in vitro model showed that Neuritin could promote the proliferation of damaged cochlear support cells and the regeneration of hair cells,and the new hair cells were wrapped around the nerve fibres,suggesting that Neuritin may promote the proliferation of support cells to differentiate into hair cells and provide an environment for hair cells to function.pathway. |
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