Effects Of Sound Conditioning On Acute Acoustic Trauma And The Mechanism Of Its Protection

Part one: Protection of Sound Conditioning against Acute Acoustic Trauma in Acoustic Sensibility and Its Effects on Cochlear ConstructionObjective: To investigate the protection of sound conditioning against acute acoustic trauma in acoustic sensibility and its effects on cochlear construction.Methods: 20 healthy, adult, Sprague-Dawley rats which are only male were randomly divided into four groups such as the control group, the sound conditioning group, the noise exposure group and the sound conditioning plus noise exposure group(5 rats in each group). The noise exposure group was exposed to white noise at 115 d B SPL for 6 hours a day in 2 consecutive days and in sound conditioning group, all the animals were exposed to pure tone 500 Hz at 85 d B SPL for 24 hours; while the control group was given the sham-exposure and the animals in sound conditioning plus noise exposure group weregiven to take a rest for 3 hours between sound conditioning and noise exposure. Before and after sound conditioning and(or) noise exposure, auditory brainstem response(ABR) was available to investigate the protection of sound conditioning against acute acoustic trauma in acoustic sensibility. Furthermore, we respectively adopted stretched preparation of basilar membrane stained by FICT-phalloidin and scanning electron microscopy to confirm the effects of sound conditioning and noise exposure on outer hair cell. Meanwhile, we applied transmission electron microscope to investigate the effects of sound conditioning and noise exposure on mitochondria in spiral ganglion neuron cells.Results: After click sound and pure tone stimulus, the outcomes of auditory brainstem response(ABR) showed that the hearing thresholds of sound conditioning plus noise exposure group were lower than ones of noise exposure group and there were statistically different between(P<0.05), while there was no statistically significant difference of hearing threshold between control group and sound conditioning group(P>0.05). Furthermore, the results of basilar membrane-stretched preparation stained by FITC-phalloidin and scanning electron microscopy showed that sound conditioning could functionally result in reconstruction of outer hair cell such as shortness and less thinness of cilia, malalignment and looseness of outer hairs and so on; compared with noise exposure group, there were obviously less hair cell loss in both base turn and middle turn of basilar membrane in sound conditioning plus noise exposure group along with shortness and less thinness of cilia, malalignment and looseness of outer hairs(P<0.05), while there was no obviously hair cell loss in apex turn of basilar membrane in sound conditioning plus noise exposure group along with the same reconstruction of outer hair cell(P>0.05). In addition, the results of transmission electron microscope showed that sound conditioning could also increase the quantity of mitochondrion of spiral ganglion neuron cells(P<0.05), enhance the electron density in mitochondrion of spiral ganglion neuron cells and narrow the interspace of mitochondrion, while noise exposure could result in the shrinkage of spiral ganglion neuron cells, less mitochondria of spiral ganglion neuron cells, the lower electron density, larger interspace and the formation of vacuole in mitochondrion.Conclusion: Sound conditioning could protect against acute acoustic trauma in acoustic sensibility and couldn’t result in rat’s hearing loss. Furthermore, it could lessen hair cell loss caused by noise exposure in both base turn and middle turn of basilar membrane, but it’s not the case in apex turn of basilar membrane. In addition, it could also increase the quantity of mitochondrion, enhance its function of spiral ganglion neuron cells, and reverse the injury and disfunction of mitochondrion piral ganglion neuron cells caused by noise exposure.Part two: Mechanism of Sound Conditioning on Protection against Acute Acoustic Trauma in VivoObjective: To rationally clarify molecular mechanisms on regulation of mitochondria and its function in spiral ganglion neuron cells caused by sound conditioning, and hopefully develop sound conditioning as new therapy and draw new theory for prevention of acute acoustic trauma in clinic.Methods: 80 healthy, male, adult Sprague-Dawley rat were randomly divided into control group, sound conditioning group, noise exposure group and sound conditioning plus noise exposure group(20 rats in each group). The noise exposure group was exposed to white noise at 115 d B SPL for 6 hours a day in 2 consecutive days and in sound conditioning group, all the animals were exposed to pure tone 500 Hz at 85 d B SPL for 24 hours; while the control group was given the sham-exposure and the animals in sound conditioning plus noise exposure group were given to take a rest for 3 hours between sound conditioning and noise exposure. After sound conditioning and(or) noise exposure, we used RT-PCR, Western blot and Immunofluorescence to detect Hsp70, Bmi1, SOD1, SOD2 and AKT1 expression in spiral ganglion neuron cell of different groups.Results: Western blot analysis and Immunofluorescence revealed that noise exposure down-regulated the expression of Hsp70, Bmi1, SOD1, SOD2 in spiral ganglion neuron cell, while sound conditioning increased Hsp70, Bmi1, SOD1 and SOD2 expression in spiral ganglion neuron cell and resulted in transposition of Bmi1 into mitochondria. Theseresults indicated that sound conditioning could reverse the down-regulations of Hsp70, Bmi1, SOD1 and SOD2 expression caused by noise exposure in spiral ganglion neuron cell. Meanwhile RT-PCR showed that compared with control group, Hsp70, Bmi1, SOD1 and SOD2 m RNA expression obviously increased in spiral ganglion of sound conditioning group.Conclusion: Sound conditioning could induce the up-regulation of Hsp70 m RNA and protein expression in spiral ganglion neuron cell, and enhance protective stress reaction of spiral ganglion neuron. Meanwhile up-regulation of Bmi1, SOD1, and SOD2 m RNA and protein expression in spiral ganglion neuron cell as well as transposition of Bmi1 into mitochondria might take part in the protection of sound conditioning against acute acoustic trauma. Especially there is coincident on the up-regulations of Hsp70, Bmi1, SOD1 and SOD2 expression in spiral ganglion neuron cell after sound conditioning.Part three: Mechanism of Sound Conditioning on Protection against Acute Acoustic Trauma in VitroObjective: To further confirm molecular signal pathway on regulation of mitochondria and its function in spiral ganglion neuron cells caused by sound conditioning, and clearly verify that the antioxidant response genes of spiral ganglion neuron such as SOD1 and SOD2 might be regulated by Hsp70 and Bmi1.Methods: Cochlear spiral ganglion tissues were isolated from postnatal 0-3 day rats to culture in serum-free medium for 1day. Then spiral ganglion neuron cells were randomly divided into control and Bmi1 inhibitor groups. In Bmi1 inhibitor group, spiral ganglion neuron cells were cultured with 1μ M PTC-209 solution dissolved by DMSO, while in control group, they were cultured with the same dose of DMSO. Two days later, they were stained by immunofluorescence and Hsp70, Bmi1, SOD1 and SOD2 expression in spiral ganglion neuron cell were observed under fluorescence microscope.Results: Immunofluorescence revealed that there was obviously less red fluorescence of SOD1 and SOD2 expression in PTC-209 treated spiral ganglion neuron cells comparedwith control group, while that there was no difference of Hsp70 expression between the two groups. It indicated that Bmi1 positively regulates SOD1 and SOD2 expression but not Hsp70 expression in spiral ganglion neuron cell.Conclusion: The antioxidant response genes of spiral ganglion neuron such as SOD1 and SOD2 might be regulated by Hsp70 and Bmi1.