Temporary hearing threshold shift (TTS) resulting from a "benign" noise exposure can cause irreversible auditory nerve afferent terminal damage and retraction. While hearing thresholds and acute tissue injury recover within 1-2 weeks after a noise overexposure, it is not clear if multiple TTS noise exposures would result in cumulative damage even though sufficient TTS recovery time is provided. Here, we tested whether repeated TTS noise exposures affected permanent hearing thresholds and examined how that related to inner ear histopathology. Despite a peak 35-40 dB TTS 24 hours after each noise exposure, a double dose (2 weeks apart) of 100 dB noise (8-16 kHz) exposures to young (4-week-old) CBA mice resulted in no permanent threshold shifts (PTS) and abnormal distortion product otoacoustic emissions (DPOAE). However, although auditory brainstem response (ABR) thresholds recovered fully in once- and twice-exposed animals, the growth function of ABR wave 1( p-p ) amplitude (synchronized spiral ganglion cell activity) was significantly reduced to a similar extent, suggesting that damage resulting from a second dose of the exposure was not proportional to that observed after the initial exposure. Estimate of surviving inner hair cell afferent terminals using immunostaining of presynaptic ribbons revealed ribbon loss of ∼ 40 % at the ∼ 23 kHz region after the first round of noise exposure, but no additional loss of ribbons after the second exposure. In contrast, a third dose of the same noise exposure resulted in not only TTS, but also PTS even in regions where DPOAEs were not affected. The pattern of PTS seen was not entirely tonotopically related to the noise band used. Instead, it resembled more to that of age-related hearing loss, i.e., high frequency hearing impairment towards the base of the cochlea. Interestingly, after a 3rd dose of the noise exposure, additional loss of ribbons (another ≈ 25 %) was observed, suggesting a cumulative detrimental effect from individual "benign" noise exposures, which should result in a significant deficit in central temporal processing.
