Study On The Protective Effect And Mechanism Of Mito-TEMPO Against Noise-Induced Hearing Loss

Objective: To establish a stable NIHL animal model by using noise exposure.To investigate whether mito-TEMPO(MT)has a protective effect against noise-induced hearing loss and cochlear injury in the rat model.To elucidate the mechanism by which MT exerts protective effective on hearing.Methods: The female adult SD rats with normal hearing were used in the study.The8-16 k Hz octave band noise was selected for exposure.Rats were exposed to noise of 110,112,115 or 120 d B SPL for 2 h,or 115 d B SPL for 1 h,respectively.ABR tests were performed to evaluate the threshold shift after the noise exposure.Noise-induced cochlear injury was investigated by HE staining of cochlear paraffin sections and the immunofluorescence histochemistry of cochlear surface preparations.After intraperitoneal injection of 1 mg/kg MT,the cochlear perilymph of the rats was sampled.The concentration of MT in the perilymph was detected using liquid chromatography tandem mass spectrometry(LC-MS/MS).Rats were randomly assigned to three groups: the normal control group,the vehicle-treated group,and the MT-treated group.The animals in the MT-treated group were injected intraperitoneally with 1 mg/kg MT,while the animals in the other two groups received injections of normal saline at the same time points.The animals in the vehicle-treated group and the MT-treated group were exposed to the 8-16 k Hz octave band noise at 112 d B SPL for 2 h.ABR was used to evaluate the compound threshold shift and the permanent threshold shift.The immunofluorescence histochemistry of the cochlear surface preparations and frozen sections were used for analyses of the protective effective of MT on noise-induced injury to hair cells,ribbon synapses and auditory nerve fibers.The immunofluorescence histochemistry were used for the analysis of mt DNA oxidative damage of the cochlea and TFAM-mt DNA interaction of the hair cells.The total DNA and total RNA of the cochlea were extracted and real time-PCR was used for the analysis of cochlear mt DNA copy number and Nd6 expression,respectively.ATP levels in the cochlea were measured.The total protein of the cochlea was extracted and immunoblotting was used for analyzing of the expression of PGC-1α/NRF-1/TFAM pathway and other molecules.Results: Exposure to the 8-16 k Hz octave band noise caused the most severe hearing loss at 16 k Hz.Using the 112 d B SPL noise exposure for 2 h,the average compound threshold shift and the permanent threshold shift were 44.7 d B and 34.7 d B,3 and 14 d after the noise exposure respectively.Fourteen days after noise exposure,immunofluorescence histochemistry of cochlear surface preparations showed that the acoustic trauma caused OHC loss and reduction of IHC ribbon synapses.HE staining of the cochlear paraffin sections showed that acoustic trauma resulted in decreased number of SGNs and atrophy of the stria vascularis.Fifteen minutes after the intraperitoneal injection of MT,the mass concentration of MT in the perilymph of the cochlea was determined by LC-MS/MS to be 18.54 μg/kg.Three and fourteen days after the noise exposure,the compound threshold shifts and the permanent threshold shifts at all tested frequencies of the animals in the MT-treated group were significantly smaller than those in the vehicle-treated group.Noise exposure resulted in the most OHC loss at the base turn of the cochlea in the vehicle-treated group,showing 81.5% of the OHC survival.MT-treated significantly reduced noise-induced OHC loss at the base and middle turns of the cochlea,and the OHC survival reached 95.2% at the base turn of the cochlea.The number of paired synapses of IHCs was significantly decreased in the vehicle-treated group.Treatment with MT significantly reduced the noise-induced loss of the paired synapses.The ANFs in the vehicle-treated group were fragmented and degenerated,and the density of ANFs decreased by more than 50%.Treatment with MT significantly recovered the density of ANFs after acoustic trauma.The integrity of nerve fibers was partially restored in the MT-treated group.After the noise exposure,the level of DHE and 4-HNE in the cochleae was rapidly increased.A dramatic increase of 8-OHd G production was observed in the cytoplasm of SGNs.The mt DNA copy number,Nd6 expression and ATP level in the cochlea were significantly reduced.Noise exposure resulted in decreased expression of TFAM and SOD2,and increased expression of Bax in the cochlea.Noise exposure also induced a reduction of TFAM-mt DNA interaction,leading to a significant increase of naked mt DNA in the cytoplasm of OHCs.Compared with the vehicle-treated group,the increase of DHE and 4-HNE was significantly inhibited in the MT-treated group.Treatment of MT significantly reduced noise-induced 8-OHd G production in SGNs,and improved the mt DNA copy number and Nd6 expression in the cochlea.MT treatment also significantly improved the ATP level,improved the expression of TFAM and SOD2,and inhibited the expression of Bax in the cochlea after the noise exposure.MT treatment partially recovered the TFAM-mt DNA interaction and significantly alleviated the increase of naked mt DNA in OHCs.Conclusion: A NIHL rat model of permanent threshold shift can be constructed by exposing to the 8-16 k Hz octave band noise at 112 d B SPL for 2 h.MT can enter the inner ear via systemic administration.MT can attenuate hearing impairment,OHC loss,ribbon synapse loss,and ANF degeneration induced by noise exposure.MT exerts protective effective against NIHL by improving the expression and mt DNA-binding ability of TFAM,alleviating mt DNA oxidative damage,maintaining mitochondrial biogenesis and ATP production in the cochlea.