The Role Of Cochlear-Macrophages In Noise-induced Deafness

【Objective】To explore the changes of macrophages in the cochlea at different time points after noise exposure,and try to figure out the effect of macrophage phenotype changes on cochlear hair cell survival in vitro,preliminary exploring the possible mechanism of macrophage changes after noise exposure.【Methods and Materials】Cx3cr1GFP /-mice of 6-8 weeks were divided into two groups.The mice in the first group were directly exposed to octave-band noise at 110 d B(8-16Hz)for 2 hours.and the mice in the second group received the same noise exposure,but two days before the exposure and three days after the noise exposure,the mice was injected with Clodronate liposomes(0.2 m L / 20 g)via rat tail vein.At different time points(1 day,7 days,and 14 days)after noise exposure,the cochlea was obtained for immunofluorescence staining to observe the changes of macrophage.At the same time,the mouse auditory brainstem response(ABR)was detected,and mouse cochlear RNA was extracted at different time points.Macrophage markers were detected by RT-PCR.Cx3cr1 GFP /-mice of 1 month were selected to collecte femur and tibia bone marrow of both hind limbs,and then removed the red blood cells of bone marrow.Bone marrow were cultured on the BMDM growth medium(BMDM + 10% FBS + 10 ng /ml M-CSF)for seven days to induce bone marrow cells into macrophages.On the seventh day,LPS and IL-4 were administered to induce macrophages to differentiate towards the M1 phenotype and M2 phenotype.After 24 hours of culture,immunofluorescence staining and RT-PCR were used to further detect and verify M1 and M2 macrophage markers,and protein chip technology and antibody chip technology were used to detect the cytokines in the culture supernatants of M0,M1,and M2.In addition,the supernatant was taken and co-cultured with OC-1 cells for 24 hours.Cell death was detected by flow cytometry.The above experiments preliminary explored the damage of different phenotypic macrophages to the survival of hair cells.【Results】After exposure to noise,macrophages in the cochlea basement membrane drum stage began to increase,reaching a peak in 3-7 days,and gradually returned to normal after14 days,and its morphology gradually changed from slender and protruding to circular or quasi-circular.At the same time,the hearing of mice decreased and hair cell damage increased after noise.Injecting macrophage scavengers to inhibit the circulation of macrophages into the cochlea can significantly reduce the aggregation and activation of cochlear macrophages,reduceing cochlea hair cell loss and hearing loss.Bone marrow-derived macrophages were cultured in vitro and stimulated differentiation.M1 and M2 macrophage surface marker m RNA expression and the characteristics of inflammatory cytokines in the culture supernatant were confirmed to successfully induce M1 and M2 macrophages.The secreted supernatants of different phenotype macrophages were co-cultured with the cochlear hair cell line HEI-OC1 cells.It was found that the culture supernatants of M1 macrophages had a significant cytotoxic effect on HEI-OC1 cells,while the culture supernatants of M2 macrophages had no obvious killing effect on HEI-OC1 cells.【Conculsions】The microenvironment of the cochlea changed after the noise exposure,activating a large number of macrophages in the cochlear basement membrane.The activated macrophages may differentiate into different polarized phenotypes.In the early stage of noise exposure,macrophages were polarized into M1-like macrophage,which killed cochlea hair cells by secreting cytotoxic factors,thereby promoting cochlear hair cell damage and aggravating noise hearing loss.While macrophages might be polarized to M2-like macrophage in the late stage of noise,participating tissue repair after cochlear injury.