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In Vivo Determination of Reduced Thiols in Rat Cerebellum Paraflocculus Following Salicylate-Induced Tinnitus by Fluorescence

Lingzhi Zhao,Zhao Li,Liu Zhao,Chenxiao Zhang

The increasing number of patients suffering from tinnitus may be explained by an imbalance in the antioxidant defense system and reactive oxygen species formation, suggesting the importance of the redox homeostasis in this condition. Endogenous biothiols play important roles in maintaining redox homeostasis. Hence, to understand the role of biothiols in the pathological process of tinnitus, this study demonstrates an in vivo method for monitoring the concentrations of biothiols in the paraflocculus of the rat cerebellum during tinnitus induced by the injection of salicylate. Resorufin-based fluorescent probe 1 was used as the selective probe with in vivo microdialysis. Probe 1 was premixed with microdialysates from the paraflocculus of the rat cerebellum and transferred into a cuvette for continuous-flow fluorescence. A linear relationship between the fluorescence and the concentrations of cysteine, homocysteine, and glutathione was from 1 to 15 µM, with detection limits of 128, 148, and 183 nM, respectively. The basal level of total reduced biothiols in the paraflocculus of the rat cerebellum microdialysate was determined to be 10.62 ± 1.34 µM based on the calibration curve for homocysteine. The injection of sodium salicylate into the animals significantly decreased the reduced biothiol concentrations in the paraflocculus of the rat cerebellum from 60 min, reaching 39.5 ± 6.20% of the basal level. In contrast, at 120, 180, and 300 min, the concentrations of reduced biothiols were 55.73 ± 9.35, 58.26 ± 9.56, and 91.69 ± 6.91% of the basal level, respectively. These results imply that the decrease in reduced biothiols in the paraflocculus of the rat cerebellum may be associated with salicylate-induced tinnitus. This study offers a new platform for in vivo monitoring of reduced biothiols in the paraflocculus of the rat cerebellum following salicylate-induced tinnitus and may be useful for the investigation of this condition.