Study On The Relationship Between Neomycin-induced Differential Up-regulation Of NOX2 And The Vulnerability Of Cochlea Outer Hair Cells

Sensorineural hearing loss(SNHL)is a familiar clinical disorder that leads to verbal communication disorders,which seriously affects the quality of life of patients.Although there are many clinical strategies to treat sensorineural hearing loss,the effects of those treatments are unsatisfactory.Therefore,the prevention of sensorineural deafness has a significance in solving the problem.With the development of molecular biology to identify the molecular regulatory network of the cochlear hair cell damage and to find the genes that effectively prevent the deafness,will be a new direction for the future study of sensorineural deafness.Most patients with sensorineural deafness present the high frequency hearing loss.The high-frequency hearing loss implies the vulnerability of outer hair cells(OHCs)in the basal turn.Ototoxic drugs or noise treatment of experimental animals,also induce high frequency hearing loss,and the damage degree of OHCs increased gradually from the apical turn to the basal turn.The above phenomena indicate that OHCs in the basal turn are more vulnerable than OHCs in the apical turn,but the underlying mechanism has not been fully elucidated.Understanding the mechanism of the differential vulnerability of OHCs in different parts of the cochlea can help to better understand the mechanism of OHCs damage and provide fundamental basis for the prevention and treatment.Previous studies have shown that the occurrence and development of sensorineural deafness are closely related to oxidative damage.Ototoxic drugs,noise exposure and aging can increase the level of reactive oxygen species(ROS)in the cochlea which can cause damage and death of OHCs.In view of the above,we infer that the commonly seen high frequency hearing loss may be partly related to the differential sensitivity to oxidative stress of OHCs in different turns.Namely,the basal OHCs which response to high frequency acoustic signal might be more sensitive to ototoxic drug-induced oxidative injury than apical OHCs which response to low frequency acoustic signal.Objectives:1)To isolate and collect sufficient number of morphologically mature OHCs for further single cell transcriptome sequencing.2)To analyze the gene expression differences of the basal and apical OHCs using single cell transcriptome sequencing technology and identify the genes that might be related to their differential vulnerabilities.3)To further study the correlation between candidate genes and the vulnerability of OHCs,providing the basis for the prevention and treatment of the injury of OHCs.Methods:1.The basal and apical OHCs of the cochlea were isolated from postnatal 9 days SD rats and were collected by micromanipulation.2.Single cell transcriptome sequencing technology was used to sequence the basal and apical OHCs.The sequencing results were analyzed and screened for oxidative stress-related genes.By viewing literature,NOX2 was selected as candidate genes and its relationship with high frequency hearing loss was further explored.3.The expression of NOX2 in control cochlea OHCs as well as neomycin-treated OHCs were observed via immunofluorescence.4.To test if inhibition of NOX2 could reduce neomycin induced ototoxicity,DPI was used in in vivo studies to inhibit NOX2 function,ABR test and basilar membrane stretch was used to evaluate the hearing loss and OHCs damage.In in vitro studies of basilar membrane culture,gp91 ds-tat was used to inhibit NOX2 function and DHE was used to detect ROS level and cleaved caspase-3 and TUNEL to detect apoptosis.Basilar membrane stretch preparation was used to observe the OHCs losses to test the protective effect of NOX2 inhibition.Results:1 Isolation and capture of OHCs in the apical turn and basal turn.Through isolation of OHCs of SD rats of different ages,we found that postnatal 9 days SD rats were suitable for researches of apical and basal OHCs.As the degree of ossification increases,the number of OHCs that can be isolated from the basilar membrane,especially OHCs in the basal turn,decreased sharply.OHCs of P9 SD rats were in standard cylindrical shape,similar to OHCs of adult rats in morphology and a certain number of OHCs can be isolated enough for transcriptome sequencing.OHCs in the basal turn and apical turn from P9 rats were isolated and captured via micro manipulation with glass electrode under microscope.Three groups of samples of OHCs in basal and apical turn were collected with each sample containing 30 OHCs.2 Single cell transcriptome sequencing and bioinformatics analysis.Transcriptome sequencing were done using collected samples of OHCs in the basal turn and apical turn.According to the results,13392 transcripts were detected in apical OHCs and 13140 transcripts were detected in basal OHCs,among which 11245 transcripts were expressed in both basal and apical OHCs while 2147 transcripts were found to exclusively express in apical OHCs and 1895 in basal OHCs.Statistical analyses showed that a total of 107 genes expressed differentially between OHCs in the basal turn and apical turn,including 52 genes expressed at a higher level in basal turn and 55 genes expressed at a higher level in apical turn.Among the differentially expressed genes,7 genes were closely related with oxidative stress which was thought to play important roles in ototoxicity.After viewing literature,we focused on NOX2 and further explored its role in OHCs vulnerability.3 NOX2 expression in cochlea OHCs and its regional up-expression in OHCs after neomycin treatment.Using immunofluorescence and Western blot,we observed the expression of NOX2 in control cochlea OHCs.NOX2 was evenly expressed in OHCs among different turns,at a relatively low level.However,after neomycin treatment,NOX2 was dominantly induced in OHCs in the basal turn.In vivo and in vitro studies demonstrated that inhibition of NOX2 significantly alleviated neomycin-induced hearing loss and OHCs damages,as seen from both the cleaved caspase-3 and TUNEL staining.Moreover,gp91 ds-tat delivery and DHE staining results showed that NOX2-derived ROS was responsible for neomycin ototoxicity.Conclusion:In this study,we for the first time acquired the transcriptome dataset of rats basal and apical OHCs and performed subsequent bioinformatics anal yses.After screening the differentially expressed genes between basal and apical turn and reviewing literature concerning oxidative stress,we picked up several candidates that might be involved in the differences in OHCs vulnerabilities and finally focused on the relationship between NOX2 and OHCs vulnerability.Our results showed that regional up-expression of NOX2 and subsequent increase of ROS in OHCs of the basal turn is an important factor contributing to the vulnerability of OHCs there,which should shed light on the prevention of hearing loss induced by aminoglycoside antibiotics.