| Objective:Radiotherapy(RT) is used widly for tumours in the head and neck, RT causes side effects such as sensorineurial hearing loss(SNHL). In China, nasopharyngeal carcinoma(NPC) is common and the prevalence of SNHL after radiotherapy for NPC has been reported to be as high as 36%. Hearing loss may be severe enough to impair the quallity of life in patients. Radiation-induced SNHL is therefore an important clinical problem to doctors.To be able to manage radiation-induced ototoxicity appropriately, a good understanding of the cellular and molecular basis of SNHL is necessary. In this study,we established up radiation-induced cochlear damage model and observe the cochlear damage by the phathological, immunohistochemistry, RT-PCR methods. To explore the protective effects of N-Acetylcysteine (NAC) on cochlear damage occuring in guinea pigs exposed to irradiation.Methods: The study including three parts:Part one,the irradiation-induced damage model of cochlear was established. Part two, study irradiation-induced cochlear apoptosis in guinea pigs. Part three, protective effects of N-acetylcysteine on cochlear exposed to irradiation. The methods of part one, 48 guinea pigs were randomly divided into controll group and irradiation group. The controll group(8 guinea pigs) didn't received irradiation. The irradiation group(40 guinea pigs) received irradiation of 70 Gy one time,The right ear was radiation ear.The ABR were examined and the guinea pigs were sacrificed in 1,4,7,14 and 30 days after irradiation. All specimens were dehydrated, embeded in paraffin and serially cutted into 5μm slices. Sections were stained with haematoxylin and eosin and 2 of cochlear Corti'organ for scanning electron microscope (SEM). The methods of part two,130 guinea pigs were randomly divided into controll group and irradiation group. The controll group(26 guinea pigs) didn't received irradiation. The irradiation group (104 guinea pigs)received irradiation of 70Gy ,The right ear was radiation ear.The guniea pigs were sacrificed in 1,4,7 and 14 days after irradiation.Some examinations were done.①Inspect the level of SOD and MDA.②TUNEL assay was performed on cochlear organ.③the protein of Caspase3,Caspase8,Caspase9 were inspected by the immunohistochemistry method.④the protein of Caspase3,Caspase8,Caspase9 were inspected by the Western Blot method.⑤RT-PCR method examined the expression of Bax,Bcl-2 and Fas mRNA. Part three, 144 guinea pigs were randomly divided into 4 groups,36 guinea pigs were in every group.Controll group rececved neither NAC nor irradiation, irradiation group was exposed to total cranium irradiation of 70Gy. Irradiation and saline group received cranium irradiation of 70 Gy and saline solution through the round window, NAC group received cranium irradiation of 70 Gy and NAC through the round window.The right ear received radiation.The guinea pigs were sacrificed in 4 and 14 days after ABR examination, the cochlears were dehydrated, embeded in paraffin and serially cutted into 5μm slices. Sections were stained with immunohistochemistry and TUNEL assay. Two cochlear basal membrane were made into scanning electron microscope. SOD and MDA were examined.Results: Part one,①Gross specimen observation, the auditory sac had no purulent discharge in 1,4,7 days after irradiation of 70 Gy. Minority auditory sac had purulent discharge in 14days. All the auditory sac had Purulent discharge in 30 days.②ABR threshold value: ABR threshold(20.00±4.47) is slightly higher than that of the control group(11.66±2.58) in 1 day after irradiation, But the difference was not statistically significant(p>0.05). The threshold value compared with the control group increased in 4 days(25.00±10.48) after irradiation(p<0.05). The threshold value compared with the control group significantly increased in 7 days(46.66±9.31),14 days (55.00±5.47)after irradiation(p<0.01).No ABR wave were drawm of in 30 days.③The cochlear organ is normal in the controll group, changes in the spiral ganglion (cytoplasmmic and nuclear condensation,nucleus and neuron loss) in every time in irradiation group by haematoxylin and eosin stain; in the organ of Corti(hydropic and loss of outer hair cell and inner hair cell)in 14, 30 days; in stria vascularis (edima, vacuolization and loss of cells) in 14, 30 days.④The ciliums of hair cells had no loss in controll group by SEM, the ciliums of OHC had been put in'v'order, the ciliums of IHC put in an arch shape. In irradiation group, no obvious lodging of ciliums in 1 day. But the SEM showed the lodging, fusion,loss in the third OHC in 4 days. The level became more severious in 7,14 days. Most of ciliums loss in the OHC in 30 days after irradiation. The results of part two,①The SOD activity of cochlear decreased in 1 days(14.08±2.90 U/mgprot),4 days(11.83±2.74 U/mgprot),7 days(11.30±2.98 U/mgprot),14 days(8.70±1.29 U/mgprot ) after irradiation compared with the controll group(41.61±26.49 U/mgprot)(p<0.01). The MDA level was elevated in 4 days (0.66±0.26 nmol/mgprot),7 days (0.88±0.30 nmol/mgprot),14 days (0.84±0.13 nmol/mgprot)after irradiation conpared with the controll group(0.24±0.09 nmol/mgprot)(p<0.01). There was no singnificant difference on radiation-induced cochlear MDA in 1 day(0.49±0.10 nmol/mgprot) after irradiation (p>0.05).②TUNEL assay: cell nuclear present brown in the apoptosis cell,and blue in non-apoptosis cell. No apoptosis cell can be seen in controll group(0.00±00). In irradiation group,there were no obvius apoptosis cell in Corti'organ and stria vascularis,but more apoptosis cells in SGC. The ratio of apoptosis cells in SGC was on the increase with the time prolonged. There was no difference on ratio of apoptosis between the controll group and irradiation group(1 day)(5.20±1.40). There was a singnificant difference on radiation-induced ratio of apoptosis in SGC in 4 days(16.00±1.10),7 days(19.00±4.20),14 days(32.00±8.70) after irradiation (p>0.05).③The protein expression of Caspase3,Caspase8,Caspase9 were obvious in SGC,and not obvious in Corti'organ and stria vascularis(SV). There was a singnificant difference on radiation-induced average optical density value of caspase8 in spiral ganglion cell (p<0.01) in 1 days(0.12±0.01),4 days(0.23±0.04),7 days(0.11±0.01),14 days(0.21±0.03) in irradiation group. Radiation-induced optical density value of caspase3 in SGC were also difference from controll group in the 1 days(0.10±0.02 )(p<0.05), there was significant difference between two groups in 4 days(0.11±0.02),7 days, (0.14±0.01)14 days(0.10±0.01 )(p<0.01). There was a singnificant difference on radiation-induced average optical density value of caspase9 in spiral ganglion cell (p<0.01) in 4 days(0.22±0.03),7 days(0.35±0.02),14 days(0.29±0.02) in irradiation group.④There was a singnificant difference on radiation-induced gray value of caspase3 of cochlear in 1 days(0.59±0.09),4 days(0.58±0.19),14 days(0.54±0.05) in irradiation group versus controll group(0.23±0.07) by Western Blot test (p<0.01). There was a singnificant difference on radiation-induced gray value of caspase9 of cochlear in 1 days(2.06±0.24),4 days(2.05±0.13),14 days(1.94±0.25) in irradiation group versus controll group(0.46±0.09) by Western Blot test (p<0.01).There was a singnificant difference on radiation-induced gray value of caspase8 of cochlear in 1 days(0.68±0.09),4 days(0.75±0.19),7 days(0.95±0.25) in irradiation group versus controll group(0.17±0.02) by Western Blot test(p<0.01).⑤There was a singnificant difference on radiation-induced gray scale value of Fas mRNA in cochlear in 1 day(2.13±1.23) in irradiation group(p<0.05), a singnificant difference in 4 days (2.92±2.06 )(p<0.01). The gray scale value of Bax mRNA in irradiation group in 1 days(1.90±1.20),4 days(1.81±1.54) increased versus controll group(0.65±0.42)(p<0.01).The results of part three,①ABR threshold value: ABR threshold value of controll group(11.66±2.58 dBnHL) was lower than simple irradiation group(p<0.01) in 4 days(25.00±10.48 dBnHL),14 days(55.00±5.47 dBnHL). There was no difference between the NAC group(4 days)( 26.25±8.53 dBnHL) and simple irradiation group(4 days). But the value of ABR of NAC group (14 days) (41.25±11.08 dBnHL) decreased versus simple irradiation group(p<0.01). There was no difference between the physiological saline group(4 ,14 days) and simple irradiation group(4,14 days).②The SEM showed the lodging, fusion,loss in the third day in OHC ciliums in 4 days in simple group and in saline group. But the ciliums of hair cells had no clear loss in NAC group(4 days). The level of loss in ciliums became more severious in 14 days after irradiation in simple and saline group. But the ciliums of hair cells had no clear loss in NAC group(14 days).③The level of MDA of NAC groupin 4 days(0.40±0.20 nmol/mgprot)decreased versus simple irradiation group(0.66±0.26 nmol/mgprot)(p<0.05). The level of MDA of NAC group in 14 days (0.33±0.05 nmol/mgprot)decreased versus simple irradiation group(0.84±0.13 U/mgprot nmol/mgprot)(p<0.01). The level of SOD in NAC group(17.27±3.80 U/mgprot) increased versus simple irradiation group in 4 days(11.84±2.74 U/mgprot) after irradiation.(p<0.01). The level of SOD in NAC group(10.65±2.95 U/mgprot ) increased versus simple irradiation group(8.7±1.29 U/mgprot)in 14 days after irradiation.(p<0.05).④The average optical density value of caspase3, 8, 9 in spiral ganglion in simple irradiation group significantly increased versus the normal group respectively(in 4 ,14 days). The average optical density value of caspase3, 8, 9 in spiral ganglion in NAC group significantly decreased versus the irradiation group respectively(4 ,14 days). There was no difference between the physiological saline group and simple irradiation group(4 ,14 days)(p>0.05).⑤TUNEL assay:No apoptosis cell can be seen in normal group(0.00±0.00). The ratio of apoptosis cell in SGC in NAC group in 4 days(6.40%±1.80),14 days(7.80%±1.79) decreased versus simple irradiation group in 4 days(6.40%±1.80),14 days(32.00%±8.70 )(p<0.01). The number of apoptosis cells was on the increase with the time prolonged.Conclusions:1. 70Gy 60Coγ-ray could lead to the damage of hearing function and cochlear organ. The damage became severious with the time prolonged.2. Irradiation could cause SOD activity decreased and MDA level increased post-irradiation.3. Irradiation might cause caspase3,8,9 expression increase in SGC.4. Apoptotic gene expression including that of Fas and Bax were observed in irradiation-induced cochlear organ.5. Mitochrondrial apotosis and extrinsic apoptosis pathway might has regarded to play role in irradiation-induced cochlear damage.6. NAC might reduced radiation-induced cochlear damage on irradiation in guinea pigs.
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