Degeneration of cochlear SGNs caused by salicylate
Aspirin, whose active ingredient is sodium salicylate, has been extensively used as an anti-inflammation medicine. When administered at high doses (≥ 1 mM CSF), salicylate reliably induces temporary hearing loss and tinnitus. While salicylate-induced hearing loss is commonly considered to be completely reversible some data suggest that prolonged treatment might damage the inner ear. To investigate its potential ototoxicity, we treated cochlear organotypic cultures from postnatal day 3 rats with salicylate doses between 1 and 10 mM for 48 h. Afterwards the samples were fixed and stained with neurofilament to label spiral ganglion neurons (SGN), phalloidin to label hair cells plus other fluorescent markers of apoptosis. Hair cells showed no signs of damage even with salicylate doses as high as 10 mM. In contrast, pathological changes were clearly present in SGN. SGN showed obvious salicylate-induced histopathologies characterized by a dose-dependent shrinkage of the SGN soma plus degeneration and fragmentation of the fibers. The average (n=807) SGN cross sectional area decreased from 205 μm 2 in control cultures to 143, 116 and 91 μm 2 in cultures treated with, 1 mM, 3 mM and 5 mM salicylate-treated groups respectively. A statistically significant reduction in SGN size was seen with salicylate doses as low as 1 mM. TUNEL staining, an indication of DNA fragmentation, and caspase staining of salicylate-treated samples indicated the SGN death was occurring by apoptosis. To test the hypothesis that salicylate induces SGN degeneration by enhancing N-methyl-D-aspartate (NMDA) currents, we blocked NMDA receptors with a high concentrations (10 mM) of magnesium. Magnesium partially blocked the salicylate-induced decrease in SGN size; however, the 10 mM dose alone induced some shrinkage of the SGN soma. To identify potential cell death pathways involved in SGN degeneration, we used a quantitative RT-PCR apoptosis gene array to detect changes in apoptosis gene expression in SGN treated with 5 mM salicylate. Among the 84 apoptosis genes on the array, 8 genes (Tp53, Birc3, Tnfrsf5, Casp7, Nfkb1, Fas, Lta, Tnfsf10) were significantly up-regulated after a 3-h treatment, while one gene (Pycard) was down-regulated. After a 6-h treatment, only one gene (Nol3) was significantly up-regulated and two genes (Cideb, Lhx4) were down-regulated. After a 12-h treatment, two genes (Il10, Gadd45a) were significantly up-regulated and four (Prok2, Card10, Ltbr, Dapk1) were down-regulated. Our results suggested that high doses of salicylate can induce caspase-mediated SGN degeneration in postnatal cochlear cultures. Magnesium block experiments suggest that SGN degeneration appears to be mediated in part by the influx of calcium through NMDA receptor; however, other cell death signaling pathways are likely to be involved. Apoptotic genes, particularly members of TNF family, appear to play an important role in SGN degeneration during the early period of SGN degeneration. This is the first report to identify cell death pathways involved in SGN degeneration induced by a physiologically relevant dosage of salicylate.