Researches Of Audiological Function, Inner Ear Morphology And Outer Hair Cells Prestin Protein Expression Characteristics After Early Periodof Radiation In Balb/C Mice

Background and purposeNasopharyngeal carcinoma (nasopharyngeal carcinoma, NPC) is the most common head and neck cancer in the southern of China, radiation therapy is the preferred method for the treatment of nasopharyngeal carcinoma, the5-year survival rate of patient with NPC after radiation therapy is31%-53%。 The radiation area includes outer ear, middle ear,and inner ear,so hearing loss after radiation is a common complication, which cause a great impact on the quality of life of patient after rehabilitation. Cochlear accepts radiation doses of up to96%when NPC patients accept radiation therapy,normal tissue will be damaged as well as cancer cells. Sensorineural hearing loss is one of common complications after radiotherapy, the major manifestation is progressive hearing loss, hearing loss is given to with high frequency, even there will be a total deafness. It is universally acknowledged that the cochlea will be injured after radiation, however, due to the difference of radiation dose, radiation time, radiation source and observation object, there is no unified standard about the critical radiation dose which cause sensorineural hearing loss.There is relatively few studies about the radiation effect on hearing pathway above cochlear at home and broad. At present, distortion product otoacoustic emission(DPOAE) is used for the monitoring of the cochlea function clinically, auditory brainstem response(ABR) is used for the monitoring of the auditory pathway above cochlea. The combination of both is more useful in determining the function of the auditory nerve pathways and integrity. We will explore the characteristics of the cochlea and auditory nerve pathway injury after different-dose and-time radiation through the monitoring of DPOAE and ABR in mice, then formulate reasonable radiation dose and provide more experimental basis for the mechanism of the inner ear injury caused by radiotherapy.As early as1970years scholars have observed the structure of the inner ear after radiation will appear different degree of change. Pathologic specimens is difficult to collect from the human body,thus experimental animals are used as research object to study the cochlear morphology change after radiotherapy. We will observe the influence of the difference-dose radiation and time on the microscopic structure and ultrastructure of cochlear in Balb/c Mice, then explore the morphological basis of mechanism of sensorineural hearing loss caused by radiation.The mechanism of SNHL caused by RT is still unclear, based on a lot of experimental researches, there are two kinds of theory about the mechanism. First, free radical reaction is induced by cell lipid peroxidation after RT; secondly, the vascular stria and micro-vessels are damaged. Prestin is the motor protein of the cochlear outer hair cell, recently, Chinese scholars found that the Prestin protein expression in cochlear outer hair cells may be associated with the mechanism of SNHL caused by RT. In present study we will observe the microstructural distribution of Prestin in outer hair cells and study the characteristic of expression in the early period after radiation in Balb/c mouse, then to explore the molecular biological characteristic of Prestin and the possible mechanism of sensorineural hearing loss caused by radiation, finally provide a primary suggestion for the prevention and treatment of SNHL after radiation.Methods1. the standard setBalb/c mice, male, age for4weeks; no outer ear deformity; no external auditory canal infection purulence,tympanic membrane integrity; ABR check which give sound stimulus intensity for click lOdBnHL can elicit Ⅰ-Ⅴ wave, the amplitude and peak latency are approximately normal. DPOAE examination given L1/L2=80/80stimulus intensity can get through.2. the preparation of radiation modelMice were weighed and given2%pentobarbital sodium solution for anesthesia by intraperitoneal injection of0.3ml/100g dose, prone position fixed. In the X line positioning, we determine the inner ear exposure area, then fix mice on the linear accelerator treatment bed. The United States Varian21006mev electron linear accelerator, the dose rate is400cGy/min, source skin distance is100cm.use the lead block to avoid respiratory tract, enteron radiation. All indicators were respectively detectedon the3rd and7th day after exposure to different doses of8,12,16Gy radiation.3. Audiology studySeventy four-week-old mice were randomly divided into four groups, one control group and three experimental groups, then each experimental group were randomly divided into two groups which were respectively measured DPOAE and ABR on the third and seventh day after exposure to different doses of8,12and16Gy radiation.4. Morphological studySixteen four-week-old mice were randomly divided into four groups, control group and three experimental groups, then every experimental group were randomly divided into two groups which were respectively sacrificed on the third and seventh day after exposure to different doses of8,12,16Gy radiation, then paraffin embedding, tissue section, HE staining and scanning electron microscope observation were performed after a cochlear specimen collected.5. Molecular biology studySeventy four-week-old mice were randomly divided into four groups, including one control group and three experimental groups, then every experimental group was randomly divided into two groups that were respectively killed to collect specimens of cochlear on the3rd and7th day after exposure to different doses of8,12,16Gy radiation. These cochleas were embedded in paraffin, and then cut into sections. The sections were immunostained with anti-Prestin antibodies. The distribution of Prestin in outer hair cells was observed under optical microscopy and the density of Prestin positive expression was calculated quantitatively by Imageproplus.6. Statistical analysisMeans±tandard errors of the mean (SEM) were calculated of all parameters measured. One-way ANOVA was used to detect statistically significant differences between the changes in one control group and three experimental groups. All statistical tests and graphics were made using SPSS13.0, and p<0.05was the criterion for statistical significance.Results1. There were not significantly difference the DPOAE amplitudes at all frequencies after exposure to different doses of8,12and16Gy on the3rd day after radiation between each experimental group and control group (P>0.05). However the DPOAE amplitudes of each experimental group were significantly lower than the ones of control group on7th day at8,10and12kHz (P<0.05), but no significant difference at4and6kHz between each experimental group and control group. The DPOAE amplitudes reduced gradually with radiation dose increasing (P<005). The response thresholds of ABR increased significantly on3rd day after exposure to different doses of8,12and16Gy radiation in experimental groups (P<0<05), however a recovery to normal occurred on7th day after exposure. The wave Ⅰ and Ⅱ latenciesof ABR in each experimental group were significantly longer than the one in control group on3rd day after exposure (P<0.05), however a recovery to normal occurred on7th day after exposure.2. Inner hair cells and outer hair cells arranged in order, no lodging, disorder and missing in control group under scanning electron microscope. Slight lodgy and disorder occurred in inner cells and outer hair cells were missing by chance in exposed animals, irrespective of irradiation dose. 3. Prestin expressed highly in the lateral membrane and expressed lowly in the cytoplasm of outer hair cells above the nucleus. The density of Prestin protein expression in outer hair cells of the basal turn was not significant difference compared with control group after exposure to different doses of8,12,16Gy radiation, but up-regulation of Prestin protein expression occurred in the apex turn.Conclusions1. The impairment of radiation with a lump-sum of different doses on hearing function in micemay occur on7th day after exposure, especiallyin high frequencies. The hearing impairment may primarily occur in the retro-cochlea auditory pathway, which maygenerally be reversible dysfunction. The time when auditory nerve pathways above cochlear appear to change and tolerance dose of radiation need to be further experimental studies.By understanding that,we will find a reliable objective predictors,establish reasonable radiotherapy plan,which can reduce the occurrence of sensorineural hearing loss after radiotherapy and improve the quality of the survival of patients.2. Slight changes of ultrastructure of Balb/c Mice occurred in the early period after equal to and less than16Gy radiation. The research results show that cochlear morphology structure change was not obvious,but cochlea morphology damage increase gradually with the increase of irradiation dose and time,the basal turn was damaged more severely than the apical turn.These results provide morphology evidence for the clinical features that radioactive auditory damage often is given priority to with high frequency hearing,also prompt cochlear hearing function is sensitive to the effect of ionizing radiation,and possible earlier than morphological changes,and show obvious quantity-effect and time-effect of the positive phase relations.The results of this study provide the morphological basis of animal experiment for the mechanism of sensorineural hearing loss caused by radiotherapy for patients with the head-neck cancer.3. Prestin protein expresses mainly in the lateral membrane above the nucleus; Prestin protein may be responsible for the mechanism of injury to inner ear caused by radiation.Prestin is the motor protein of the cochlear outer hair cell. In response to changes in membrane potential, the cylindrical outer hair cell rapidly alters its length and stiffness. These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by inner hair cells. So the mammalian hearing have a characteristic of high sensitivity, broad auditory domain and sensitive frequency selective hearing. Prestin protein maybe play a part of role in the mechanism of inner ear damage caused by radiation, extensive study and long-term observation research should be made to explore other molecular basis related with the mechanism.The inner ear damage is one of complications after patient suffered from nasopharyngeal carcinoma treated with radiation, which seriously influence the quality of life after radiotherapy. At present, according to the possible mechanism of inner ear damage caused by radiotherapy, the antioxidants and inhibit apoptosis of drugs are applied through the middle ear locally, which has certain curative effect. Further studies should be carried out to investigate the molecular biological mechanisms of radioactive inner ear damage and then to identify better methods to prevent and cure the deafness complication, improved the quality of life of patients with nasopharyngeal carcinoma.