Ref-1 And NF-κB Expression On The Spiral Ganglion Of Guinea Pig Cochlea With Gentamicin-induced Ototoxic Damage

Objective To investigate the expression and dynamic changes of Ref-1 and NF-κB on the spiral ganglion of guinea pig cochlea with gentamicin-induced ototoxic damage and prevention with antioxidant sodium salicylate, and to explore the roles of Ref-1 and NF-κB in the ototoxic mechanism of aminoglycoside antibiotics.Methods Twenty-eight healthy male guinea pigs with normal Preyer's reflex weighed from 250 to 350 g were involved in this study. All the animals were randomly divided into 7 groups and received intraperitoneal injections according to their arranged group. GroupⅠ(control), treated with normal saline. GroupⅡ(GM3d), treated with gentamicin for 3 days. GroupⅢ(GMSA3d), gentamicin in combination with sodium salicylate, 3 days. GroupⅣ(GM7d), gentamicin alone, 7 days. GroupⅤ(GMSA7d), gentamicin plus sodium salicylate, 7 days. GroupⅥ(GM14d), gentamicin alone, 14 days. GroupⅤ(GMSA14d), gentamicin plus sodium salicylate, 14 days. Changes of Ref-1 and NF-κB expression on the spiral ganglion were detected through paraffinembedded immunohistochemistry. The positive staining cells in the plasma and nucleus were accounted under the light microscope and 10 visual fields of 400 magnification for each cochlea were observed.Results A slightly positive reaction for Ref-1 and NF-κB staining was found in the cochlea of the control group, mainly shown in the cytoplasm. After treated with gentamicin alone or gentamicin in combination with sodium salicylate, the expression of Ref-1 and NF-κB immunoreactivity prominently increased, especially in the cytonucleus. The results of quantitative oberservation displayed that the cell counts of nuclear-positive-staining for Ref-1 expression in GM3d, GM7d, GM14d, GMSA3d, GMSA7d, GMSA14d were (5.20±1.21), (25.20±1.23), (14.80±0.79), (9.20±1.23), (29.40±1.49) and (16.60±1.07) per visual field, respectively. These data showed that the positive staining cells in each experimental group significantly increased in comparison with the control group [(1.40±1.07) per visual field] (P＜0.05). The cell counts of positive staining for NF-κB in GM3d, GM7d, GM14d, GMSA3d, GMSA7d, GMSA14d were (6.60±1.07), (29.00±1.49), (18.00±1.49), (10.00±1.49), (34.60±1.96), (21.00±1.49) per visual field, respectively, showing that each experimental group had a significant increment of NF-κB positive cells compared with the control group [(3.20±0.79) per visual field]. The differences were statistically significant (P＜0.01).Conclusions A slightly positive reaction for Ref-1 and NF-κB staining was found in the cochlea of normal guinea pigs, mainly observed in the cytoplasm of the spiral ganglion. At the early period of gentamicin administration, upregulation and translocation to the nucleus of Ref-1 and NF-κB were shown. Simutaneous administration of antioxidant sodium salicylate could further significantly increase the expression of Ref-1 and NF-κB. At the seventh day of administration, the expression of Ref-1 and NF-κB immunoreactivity on the spiral ganglion reached the highest, mainly in the cytonuclei. However, along with heavier damage of the cochlea, the positive staining and translocation to the nucleus decreased at the fourteenth day. These results indicated that Ref-1 and NF-κB may play roles to prevent the cochlea from gentamicin-induced oxidative damage.