Protective Effect Of Neuritin On The Damaged Spiral Ganglion Neurons In A Mongolian Gerbil Model

Objectives:The spiral ganglion neurons(SGNs)transmit the sound signals received by the hair cells to the auditory center and produce hearing.The quantity and function are important for maintaining normal hearing function.Limited by the limited regenerative capacity,SGNs are difficult to spontaneously regenerate and restore their original functions after injury.Various neurotrophic factors play an important role in this process.Neutritin,a neurite outgrowth factor,is a neurotrophic factor closely related to neural plasticity.Our previous research found that it plays an important role in promoting hair cell transdifferentiation and regeneration,and can protect nerve fibers from hair cell loss.Secondary injury indicates a potentially important role in maintaining the survival and function of SGNs.Based on the previous study of the function and mechanism of recombinant human Neuritin in the inner ear,this study aimed to screen and establish a deafness model of SGNs specific lesions using mongolian gerbils.To explore its protective effect on damaged SGNs in the cochlea,in order to provide a new scientific basis and effective strategy for the treatment of sensorineural deafness caused by the loss of SGNs.Methods:1.Detection of expression of Neuritin at the molecular level by qPCR and WB methods with cochlear tissue at different developmental stages.After frozen sectioning,the expression of Neuritin was detected by immunofluorescence,and determined the expression of Neuritin in the developmental stages of mongolian gerbils.2.Three ototoxic drugs(combined kanamycin and furosemide,neomycin,ouabain)were selected,and ABR detection and histomorphometric analysis were used to screen and identify specific lesions of mongolian gerbil cochlear SGNs.The deafness model was used to investigate the expression changes of Neuritin in the cochlea after deafness and to determine its correlation with the deafness model.3.In vitro,the cochlear Corti tissue was cultured,and different concentrations of exogenous Neuritin were added simultaneously with the ouabain injury to explore its protective effect on damaged cochlear SGNs.In vivo,using the damage animal model,different doses of exogenous Neuritin were instilled through a round window,and the effect of Neuritin on impaired hearing function was comprehensively evaluated by ABR detection and tissue level analysis.Results:1.It was found that Neuritin was expressed in different developmental stages of gerbil cochlea.The expression of E20 was lower and the expression of P1 to P56was continuously increased.It was mainly distributed in SGNs and Corti region.2.The results of different drug modeling showed that only low concentration of ouabain(1 mM)could cause the specific loss of SGNs in the cochlea,and the hearing threshold increased significantly,and the model was stable.Finally,this condition was selected to establish the injury model;further studies showed that after injury Down-regulation of Neuritin expression in the cochlea indicates a negative correlation with the injury model.3.In vitro studies showed that the number of SGNs per 100μm~2 in the Neuritin(16μg/mL)group and the Neuritin(32μg/mL)group was 13.2±1.2,16±2.6,significantly higher than the injury group 7.3±2.2,The number of nerve fibers per 100μm was 8.2±1.1,11.6±1.5,which was also significantly higher than that of the injury group by 4.6±1.1,and the arrangement was more neat;and the hair cells of each group were almost unaffected.In vivo,using the above damage model,the round window was instilled with Neuritin.The results showed that the 16kHz and 32 kHz hearing thresholds of the high frequency region of the Neuritin(16μg)group were 32.5±8.9 dB,61.3±3.5 dB,which was significantly lower than the damage group of 48.3±16.0.dB,85±5.5 dB.The hearing thresholds of the 8 kHz,16 kHz and 32 kHz in the high frequency region of the Neuritin(32μg)group were25±10.7 dB,31.3±9.9 dB,and 55±5.3 dB,all significantly lower than the injury group.The results of histomorphometry showed that the number of spiral ganglion cells and nerve fibers in the middle of the cochlear in the Neuritin treatment group was significantly higher than that in the injury group,which was consistent with the functional results.Conclusion:1.Neuritin is expressed during the development of the gerbil cochlea,especially in the juvenile to adult stage,and is mainly expressed in the SGNs and Corti regions of the cochlea.2.Low concentration of ouabain(1 mM)can cause specific loss of SGNs in the mongolian gerbil cochlea,with a significant increase in hearing threshold,model stability,and it was the best animal model selected for this study.The expression of Neuritin is negatively correlated with the injury model.3.In vitro and in vivo studies have shown that Neuritin significantly maintains the number and arrangement of spiral ganglion cells and nerve fibers in the gerbils of damaged gerbils,and effectively protects the high-frequency listening function of gerbils.