Experimental Observation Of Influence Of Radiation On Distortion Product Otoacoustic Emission And The Prestin Protein Expression In The Mice Outer Hair Cells

Objective:1. To study the effect of single-dose of radiation on distortion product otoacoustic emission(DPOAE) and the Prestin protein expression in cochlear outer hair cells of mice.2. To study the effect of different doses of radiation on distortion product otoacoustic emission(DPOAE) and the Prestin protein expression in cochlear outer hair cells of mice.3. To explore the possible mechanism of radiotherapy causing sensorineural hearing loss(SNHL).Materials&Methods:1.MaterialsFour weeks old male Balb/c mice,which were normal, excluded the infection in external auditory canals and middle ears, weighing17-22g, the average was about20g, purchased from the Animal Center of Southern Medical University, were randomly divided into experimental groups and blank control group.2.Methods:2.1Establishment of mice model with SNHL after radiotherapyAll the mice were anaesthetized with2%sodium pentobarbital (0.3ml/100g) after weighted. They were kept in the supine position, and we defined the inner ear radiotherapy range by X-ray orientation.The experimental mice were irradiated on the right ears with stated doses of6MeV electrons from a linear accelerator (Varian2100, America). Mice were treated with the source skin distance of100cm, dose rate of400cGy/min and subcutaneous reference depth of1.5cm.All the mice undergoing radiotherapy were provided with sheet leads to protect the digestive canal and respiratory tract. Then we tested various index sign of DPOAE and the prestin protein expression in cochlear outer hair cells of mice.2.2In vivo acoustic emission assessments(DPOAE)All the mice were anaesthetized with2%sodium pentobarbital (0.3ml/100g) and the response amplitude and signal to noise ratio of distortion product otoacoustic emission(DPOAE) were recorded. A cubic distortion product of2fl-f2was recorded from frequency0.5-12kHz with f2/fl=1.22at intensity of L1/L2=80/80dB SPL. DPOAE was measured before and after exposured to the radiation.2.3Prestin Western blottingThe whole cochlea was crushed in a modified RIPA cell lysis buffer (10mM Tris,100mM NaCl,2mM EDTA,0.1%SDS,1%Triton X-100, protease inhibitor cocktail and1mM PMSF), frozen and sonicated. Lysates were centrifuged at12000g for10min. The supernatant was collected and denatured with Laemmli loading buffer for5min. Then, some of the cochlear lysates(200μl) were loaded at two cochleas per well onto the12%sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the proteins in the gel were electrotransferred onto an immunoblot PVDF membrane. The membrane was blocked with5%non-fat dry milk, and then incubated with anti-prestin antibodies (1:2000). After being washed with Tris-buffered saline with0.1%Tween20three times10min every time, themembrane was incubated with the secondary antibody(rabbit anti-goat IgG,1:3000) in PBS with5%non-fat dry milk. Finally, after being thoroughly washed with Tris-buffered saline with0.1%Tween20, membranes for measuring prestin expression were incubated with SuperSignal West Dura Extended Duration Substrate for2min and exposed to X-ray film to detect the ECL signal. P-actin was used as an internal control. The blots were scanned and measured by Biosens SC805blotting analysis software, and then normalized to β-actin.2.4The whole cochlear RNA extraction and real-time RT-PCR measurementThe cochlea was freshly isolated and the whole cochlea was homogenized with two cochleas per tube. Its total RNA was extracted using an Absolutely RNA Miniprep Kit (Biozol), following the manufacturers instructions, which included a DNase treatment to digest genomic DNA, and the RNA was purified by Recombinant DNase I (RNase-free)(TaKaRa). The obtained mRNA was converted to cDNA by First-Strand cDNA Synthesis Kit ReverTra Ace-α (Toyobo). Quantitative realtime PCR was performed by MyiQ real-time PCR detection system of ABI7500. The cycling conditions were95℃2min;95℃15s, and60℃40s, for45cycles for prestin amplification,reviewed the signal at60℃. The melting curve was measured at the end of recording. Each cochlear sample was repeatedly measured in triplicate and averaged. The cochlear cDNA from one of the normal cochlea was diluted and used to generate the standard curve. Universal18S served as an internal control. The relative quantities of gene expression tested were calculated from standard curves and normalized to the amount of18S mRNA.2.5Analysis of statistics All dates were statistically treated with statistical software package of SPSS13.0. The difference among different groups was analyzed by One-Way ANOVA test. The LSD and Dunnett’s T3test were used when the difference among groups was found.Results:1. The Balb/c mice model with SNHL after radiotherapy was successfully established.2. When it was exposured to a single dose of16Gy radiation:The response rates of DPOAE were low in all the mice before or after radiation exposure at0.5,1, and2kHz, and it was100%at3,4,6,8,10and12kHz. The DPOAE amplitudes of the mice on the3rd and7th day after RT were lower than those before radiation exposure at3,4,6,8,10and12kHz, which was also showed from the I/O curve of10and12kHz,and the amplitudes on the7the day were lower than those on the3rd day at most frequencies. The expressions of Prestin and Prestin mRNA after RT were parallel down-regulated as compared with those before exposure and the expressions of Prestin and Prestin mRNA on the3rd day after RT were lower than those on the7th day.3. The amplitudes of DPOAE of the mice after RT(8,10,12Gy) were lower than the normal mice at3,4,6,8,10and12kHz.The amplitudes decreased with the rising dose,which were significant difference in statistics at3,4,6,8and12kHz. Compared with the normal mice,the expression of Prestin protein and Prestin mRNA were parallelly down-regulated in the other three groups mice,which decreased with the rising dose.Conclusion:1. The structure and function in inner ear of the mice after exposured to a single large dose of radiation were injuried,which caused the expression of Prestin protein and Prestin mRNA down-regulated in outer hair cells after RT, and the auditory function was also injuried.2. The structure and function in inner ear and the auditory function of the mice after exposured to different doses of radiation were injuried, and the extant of damage was positively correlated with the dose.3. The mechanism of SNHL after RT maybe correlated with the function expression abnormalities of the Prestin protein in the outer hair cells after RT.