Regulatory Roles Of The Cochlear Redox Status In Hair Cell Decision To Die By Apoptosis Or Necrosis Following Exposure To Intense Noise

Two modes of hair cell (HC) death, apoptosis and necrosis, has been found to be involved in the pathogenesis of HC lesions following exposure to intense noise. These two modes of cell death, although coexist in cochlear lesions, have different patterns of the spatial distribution. Apoptotic OHCs appear primarily in the severely-damaged sections of the cochlea, whereas the necrotic OHCs spread out from the severely-damaged focal lesions to the adjacent less severely-damaged regions. Recent studies indicate that the oxidative status of cells is a regulatory factor in determination of the prevalence of either apoptosis or necrosis. In this study, we manipulated the antioxidant capacity of the cochlea using local application of buthionine sulfoximine (BSO, a selective inhibitor of glutathione synthetase) or GSH, and observed the effect of the glutathione change on generation of apoptosis and necrosis in the cochlea.1. Development of a novel method for cochlea perfusion: From the tympanic scala of the basal turn to the semicircularis posterior.We developed a cochlea perfusion method which facilitated a rapid and uniformed drug distribution in the cochlea. Specifically, two small holes were drilled in the chinchilla' cochlea, one in the basal turn near the round window and the other in the semicircularis posterior. Artificial perilymph (10μl) was perfused gently from the hole in basal turn at the rate of 1μl/min with a micro-injection pump. Upon completion of the perfusion, the two holes were closed with a tissue adhesive (3M). Auditory brain-stem responses (ABR) were measured before, immediately after, 4 and 24hrs after the cochlear perfusion. The ABR measurements revealed no significant threshold shifts after the cochlear perfusion. Moreover, there were no apoptotic, necrotic or missing hair cells observed 24 hrs after the perfusion. These results clearly indicate that this novel approach of cochlear perfusion is a reliable technique for cochlear drug application.2. Effects of the local administration of BSO or GSH on the hearing sensitivity and hair cell viability.Adult chinchillas were divided into two groups: the BSO group and the GSH group. The BSO-group animals received 10μl of BSO solution (60 mM) in the right cochlea and the same volume of artificial perilymph in left cochlea serving as controls. In the GSH group, 10μl of GSH solution (10 mM) was perfused into the right cochlea and the same volume of artificial perilymph was perfused into the left cochlea serving as controls. ABR thresholds were measured before and after the perfusion. The cochlear GSH levels were measured in both the treated and control ears (4h after perfusion in the BSO group, 30min after perfusion in the GSH group). We also observed the hair cell morphology after the BSO or GSH treatment using surface preparation. The results showed that the GSH level and the GSH/GSSG ratio in the BSO-treated ears were significantly decreased than those in the control ears. In contrast, the GSH level and the GSH/GSSG ratio in the GSH-treated ear were significantly increased than those of control ears (p<0.05). There was no detectable difference in the ABR thresholds between the BSO-treated and the control ears, and between the GSH-treated and the control ears. No signs of hair cell damage were observed in both groups after the cochlear perfusion.3. Effects of the cochlear redox status on hair cell death mode following exposure to intense noiseTwenty adult chinchillas were divided into two groups: the BSO group and the GSH group. In the BSO group, animals were exposed to a 4 kHz narrow band noise at 117 dB SPL for 2 hours after the BSO treatment. In the GSH group, animals were exposed the same noise beginning at 30 min after the GSH treatment. Four hours after the noise exposure, all animals were sacrificed and the cochleae were collected for pathological examination to determine the cell death pathways incurred. The results showed that the ratio of the numbers of necrotic to apoptotic HCs was significantly increased in the BSO treated ears, but decreased in the GSH treated ears, as compared with those observed in the control ears. Surprisingly, the overall level of HC damage, as indicated by the sum of apoptotic, necrotic and missing HCs, remained unchanged in the treated ears. These results suggest that reduction of the GSH level in HCs creates a microenvironment that is favored for the generation of necrotic cell death, but does not alter the overall level of the cell viability.