Protective effect of Resveratrol and N-acetylcysteine combination on kanamycin-induced ototoxicity in rats

Abstract

Aminoglycoside antibiotics are known to have ototoxic effects and may induce sensorineural hearing loss. Oxidative stress and inflammation have been suggested as key mechanisms. This study investigated the protective effect of a combination of Resveratrol and N-acetylcysteine (NAC), which has antioxidant and cytoprotective properties, against kanamycin-furosemide ototoxicity. Two month-old male Wistar rats (n=44) were divided into Resveratrol+NAC and control saline groups. Resveratrol, 10mg/kg and NAC, 400mg/kg per day, was given for 5 days by intraperitoneal injection. A gelatine sponge soaked in 75μl of a concentrated solution of kanamycin (200mg/ml) and furosemide (50mg/ml) was applied on the right ear round window by bullostomy in the second day of treatment. Hearing was assessed with auditory brainstem response (ABR) tests, before and 5, 14 and 21 days after the beginning of the treatment. Cochlear samples were taken for molecular and morphological evaluation 5 and 21 days after the beginning of the treatment. PCR reactions were performed to evaluate expression of 84 genes related to oxidative stress and antioxidant defense, using RT2 Prolifer™ PCR array PARN-065Z, and also the expression of pro and anti-inflammatory cytokines with Taqman specific probes (Il1b, Il4, Il6, Il10, Tnfα, Tgfβ1 and Foxp3). Paraffin and frozen cochlear section were employed for Nissl staining, TUNEL, Phalloidin, Myosin VIIa and SOX2 immunohistochemistry. Functional results indicated that local administration of kanamycin and furosemide induced a moderate to severe threshold shift, especially for high frequencies. Resveratrol and NAC partially protected the inner ear from ototoxic damage, but this beneficial effect did not continue after the end of the treatment. Gross histology from right ears 21 days after surgery showed characteristic lesions after ototoxic insult, mainly disruption of Corti structure and loss of hair cells in the basal cochlear region. Gene expresion results suggested that treatment with Resveratrol and NAC might protect against ototoxicity via mediating expression of genes responsible for regulating ROS metabolism and oxygen transport, and also expression of proinflammatory cytokines. These results suggested that administration of antioxidant drugs like resveratrol and NAC could be useful to protect from ototoxicity during aminoglycoside therapy.