Time course of organ of Corti degeneration after noise exposure.

From our permanent collection of plastic-embedded flat preparations of chinchilla cochleae, 22 controls and 199 ears from noise-exposed animals were used to determine when, postexposure, hair cell (HC) and supporting cell (SC) degeneration were completed. The exposed ears were divided into four groups based on exposure parameters: 0.5- or 4-kHz octave band of noise at moderate (M) or high (H) intensities. Postexposure survival ranged from <1 h to 2.5 y. Ears fixed ≤ 0-12 h postexposure were called 'acute'. For 'chronic' ears, postexposure survival was ≥7 d for groups 0.5M and 4M, ≥ 1 mo for the 4H group and ≥7 mo for the 0.5H group. The time course of inner-ear degeneration after noise exposure was determined from data in the 0.5H and 4H groups because these groups contained ears with intermediate survival times. Outer hair cells (OHCs) began dying during the exposure. OHC loss slowed down beyond 1 mo but was still present. Conversely, much inner hair cell loss was delayed until 1-3 wk postexposure. Outer pillar and inner pillar losses were present at a low level in acute ears but increased exponentially thereafter. These results are the first to demonstrate quantitatively that hair cells (HCs) and supporting cells (SCs) may continue to degenerate for months postexposure. With short postexposure survivals, the remaining SCs often had pathological changes, including: buckled pillar bodies, shifted Deiters' cell (DC) nuclei, detachment of DCs from the basilar membrane and/or splitting of the reticular lamina. These pathological changes appeared to allow endolymph and perilymph to intermix in the fluid spaces of the organ of Corti, damaging additional HCs, SCs and nerve fibers. This mechanism may account for some postexposure degeneration. In ears exposed to moderate noise, some of these SC changes appeared to be reversible. In ears exposed to high-level noise, these changes appeared to indicate impending degeneration.

Relation of focal hair-cell lesions to noise-exposure parameters from a 4- or a 0.5-kHz octave band of noise.

In a previous study, we examined the relation between total energy in a noise exposure and the percentage losses of outer (OHC) and inner (IHC) hair cells in the basal and apical halves of 607 chinchilla cochleae [Harding, G.W., Bohne, B.A., 2004a. Noise-induced hair-cell loss and total exposure energy: analysis of a large data set. J. Acoust. Soc. Am. 115, 2207-2220]. The animals had been exposed continuously to either a 4-kHz octave band of noise (OBN) at 47-108 dB SPL for 0.5h-36 d, or a 0.5-kHz OBN at 65-128 dB SPL for 3.5h-433 d. Interrupted exposures were also employed with both OBNs. Post-exposure recovery times ranged from 0 to 913 days. Cluster analysis was used to separate the data into three magnitudes of damage. The data were also separated into recovery times of 0 days (acute) and >0 days (chronic) and the apical and basal halves of the organ of Corti (OC). A substantial part of these hair-cell losses occurred in focal lesions (i.e., >or=50% loss of IHCs, OHCs or both over a distance of >or=0.03 mm). This aspect of the damage from noise was not included in the previous analysis. The present analysis describes, within the same three clusters, the apex-to-base distribution of 1820 focal lesions found in 468 of 660 (71%) noise-exposed cochleae. In these cochleae, OC length in mm was converted to percent distance from the apex. The lesion data were analyzed for location in percent distance from the apex and size (mm) of the lesions. In 55 of 140 (39%) non-noise-exposed, control OCs, there were 186 focal hair-cell lesions, the characteristics of which were also determined. Focal lesions with hair-cell loss >or=50% involved predominantly OHCs, IHCs only, or both OHCs and IHCs (i.e., combined OHC-IHC lesions). The predominantly OHC and combined lesions were pooled together for the analysis. The distributions of lesion location (in percent distance from the apex), weighted by lesion size (in percent of OC length) were tallied in 2%-distance bins. In controls, focal lesions were uniformly distributed from apex to base and 70% of them were pure IHC lesions. In cochleae exposed to the 4-kHz OBN, lesions were distributed throughout the basal half of the OC. In cochleae exposed to the 0.5-kHz OBN, lesions occurred in both halves of the OC. With continuous exposures, 74% of the lesions were predominantly OHC or combined lesions. With interrupted exposures, 52% of the lesions were OHC or combined lesions. Lesion size was generally larger in the chronic compared to acute cochleae with similar exposures. There was a minimum total energy at which focal lesions began to appear and slightly higher energies resulted in nearly all exposed cochleae having focal lesions.

Effect of infrasound on cochlear damage from exposure to a 4 kHz octave band of noise.

Infrasound (i.e., <20 Hz for humans; <100 Hz for chinchillas) is not audible, but exposure to high-levels of infrasound will produce large movements of cochlear fluids. We speculated that high-level infrasound might bias the basilar membrane and perhaps be able to minimize noise-induced hearing loss. Chinchillas were simultaneously exposed to a 30 Hz tone at 100 dB SPL and a 4 kHz OBN at either 108 dB SPL for 1.75 h or 86 dB SPL for 24h. For each animal, the tympanic membrane (TM) in one ear was perforated ( approximately 1 mm(2)) prior to exposure to attenuate infrasound transmission to that cochlea by about 50 dB SPL. Controls included animals that were exposed to the infrasound only or the 4 kHz OBN only. ABR threshold shifts (TSs) and DPOAE level shifts (LSs) were determined pre- and post-TM-perforation and immediately post-exposure, just before cochlear fixation. The cochleae were dehydrated, embedded in plastic, and dissected into flat preparations of the organ of Corti (OC). Each dissected segment was evaluated for losses of inner hair cells (IHCs) and outer hair cells (OHCs). For each chinchilla, the magnitude and pattern of functional and hair cell losses were compared between their right and left cochleae. The TM perforation produced no ABR TS across frequency but did produce a 10-21 dB DPOAE LS from 0.6 to 2 kHz. The infrasound exposure alone resulted in a 10-20 dB ABR TS at and below 2 kHz, no DPOAE LS and no IHC or OHC losses. Exposure to the 4 kHz OBN alone at 108 dB produced a 10-50 dB ABR TS for 0.5-12 kHz, a 10-60 dB DPOAE LS for 0.6-16 kHz and severe OHC loss in the middle of the first turn. When infrasound was present during exposure to the 4 kHz OBN at 108 dB, the functional losses and OHC losses extended much further toward the apical and basal tips of the OC than in cochleae exposed to the 4 kHz OBN alone. Exposure to only the 4 kHz OBN at 86 dB produces a 10-40 dB ABR TS for 3-12 kHz and 10-30 dB DPOAE LS for 3-8 kHz but little or no OHC loss in the middle of the first turn. No differences were found in the functional and hair-cell losses from exposure to the 4 kHz OBN at 86 dB in the presence or absence of infrasound. We hypothesize that exposure to infrasound and an intense 4 kHz OBN increases cochlear damage because the large fluid movements from infrasound cause more intermixing of cochlear fluids through the damaged reticular lamina. Simultaneous infrasound and a moderate 4 kHz OBN did not increase cochlear damage because the reticular lamina rarely breaks down during this moderate level exposure.

Distribution of focal lesions in the chinchilla organ of Corti following exposure to a 4-kHz or a 0.5-kHz octave band of noise.

An octave band of noise (OBN) delivers fairly uniform acoustic energy over a specific range of frequencies. Above and below this range, energy is at least 30 dB SPL less than that within the OBN. When the ear is exposed to an OBN, hair-cell loss often occurs outside the octave band. The frequency location of hair-cell loss is evident when the percent distance from the apex of focal lesions is analyzed. Focal lesions involve substantial loss of outer hair cells (OHCs) only, inner hair cells (IHCs) only, or both OHCs and IHCs (i.e., combined lesions) in a specific region of the organ of Corti (OC). Data sets were assembled from our permanent collection of noise-exposed chinchillas as follows: (1) the sum of exposure duration and recovery time was less than or equal to 11 d; (2) the exposure level was less than or equal to 108 dB SPL; and (3) focal lesions were less than 1.5mm in length. The data sets included a variety of exposures ranging from high-level, short duration to moderate-level, moderate duration. The center of each focal lesion was expressed as percent distance from the OC apex. Means, standard deviations and medians were calculated for focal-lesion size resulting from exposure to a 4-kHz or a 0.5-kHz OBN. Histograms were then constructed from the percent-location data using 2.0% bins. For the 4-kHz OBN, 5% of the lesions were in the apical half of the OC and 95% were in the basal half. The mean lesion size was 1.68% of total OC length for OHC and combined focal lesions and 0.42% for IHC focal lesions. Most OHC and combined lesions occurred in the 5-7-kHz region, at and just above the upper edge of the OBN. Clusters of lesions were also found around 8 and 12 kHz. A cluster was present at and just below the lower edge of the OBN, as well as another in the 1.5-kHz region. For the 0.5-kHz OBN, 34% of the lesions were in the apical half of the OC and 66% were in the basal half. The mean lesion size was 0.93% for OHC and combined focal lesions and 0.32% for IHC focal lesions. OHC and combined focal-lesion distribution showed clusters at 0.25, 0.75 and 1.5 kHz in the apical half of the OC. In the basal half, the distribution of focal lesions was similar to that seen with the 4-kHz OBN (r=0.54). With both OBNs, most IHC focal lesions occurred in the basal half of the OC. High resolution power spectrum analysis of each OBN and non-invasive tests for harmonics and distortion products in a chinchilla were performed to look for exposure energy above and below the OBN. No energy was found that could explain the OC damage.

Death pathways in noise-damaged outer hair cells.

Using morphological criteria, death pathways in outer hair cells (OHCs) were determined in chinchilla organs of Corti that had been exposed to a high- or moderate-level octave band of noise (OBN) centered at either 0.5 or 4-kHz. The specimens were part of our large collection of plastic-embedded flat preparations of chinchilla cochleae. Three death pathways were identified: (1) oncotic - swollen, pale-staining cell with a swollen nucleus, (2) apoptotic - shrunken, dark-staining cell with a pyknotic nucleus and (3) a newly defined third pathway - no basolateral plasma membrane but cellular debris arranged in the shape of an intact OHC with a nucleus deficient in nucleoplasm. To minimize the secondary loss of OHCs from the entrance of endolymph into the organ of Corti, the specimens used for quantitative analysis of death pathways had the following characteristics: (1) the level to which they were exposed was less than or equal to 95dB SPL, (2) the exposure duration was 6-216h, (3) fixation for microscopic examination took place in vivo 1-2h post-exposure and (4) there were no focal OHC lesions in the organs of Corti. Fifty-eight noise-exposed cochleae met these criteria. In these specimens, degenerating and missing OHCs were classified as to which death pathway the cells had followed or were following. Nine non-noise-exposed cochleae were also evaluated for OHC death pathways. The number of OHCs following the third death pathway was significantly greater in the noise-exposed cochleae than the non-noise-exposed cochleae for total exposure energies greater than those produced by 75dB SPL for 216h to a 0.5-kHz OBN and 57dB SPL for 48h to a 4-kHz OBN. In cochleae exposed to either octave band, OHCs dying by oncosis or apoptosis were uncommon.

The effect of an age-related hearing loss gene (Ahl) on noise-induced hearing loss and cochlear damage from low-frequency noise.

Inbred C57BL/6J mice carry two copies of an age-related hearing loss gene (Ahl). It has been shown that these mice begin losing high-frequency hearing at two months. Several functional studies have reported that the Ahl gene renders mice more susceptible to noise-induced hearing loss (NIHL) than strains which do not carry this gene [e.g., Hear. Res. 93 (1996) 181; Hear. Res. 155 (2001) 82; J. Assoc. Res. Otolaryngol. 2 (2001) 233]. Johnson et al. [Hear. Res. 114 (1997) 83] developed a congenic B6.CAST-+Ahl mouse which carries the wild-type allele from Mus musculus castaneus at the Ahl locus. Five each of young C57BL/6J males and females, and B6.CAST-+Ahl males were exposed to a 4-kHz octave band of noise at 108 dB SPL for 4 h. Non-noise-exposed mice of the same strains and age served as controls. The noise-exposed mice were functionally tested for ABR thresholds and DPOAE levels pre-exposure and three times post-exposure: 0 days to determine the magnitude of temporary threshold shift (TTS); 6 days to determine rate of recovery; and 20 days to determine the magnitude of permanent threshold shift (PTS). At 20 days post-exposure, the animals underwent cardiac perfusion to fix their cochleae. The isolated cochleae were embedded in plastic and dissected into flat preparations. By phase-contrast microscopy, each cochlea was evaluated from apex to base to quantify the losses of hair cells, nerve fibers and stria vascularis and to localize stereocilia damage. Functional data from each mouse were aligned with the cytocochleogram using the frequency-place map of Ou et al. [Hear. Res. 145 (2000) 111; Hear. Res. 145 (2000) 123]. Sizable variation in the magnitude of TTS, PTS and hair-cell loss was found among mice of the same genetic strain. The congenic B6.CAST-+Ahl male mice had significantly less TTS immediately post-exposure than C57BL/6J males or females but not less PTS or hair-cell losses at 20 days post-exposure. These results indicate that, at one month of age, mice carrying two copies of the Ahl gene have an increased susceptibility to TTS from a low-frequency noise before they have any indication of age-related hearing or hair-cell loss. However, this appeared not to be the case for PTS. The Ahl gene appears to play a role in susceptibility to NIHL but, other genes as well as systemic and local factors must also be involved.

Temporary DPOAE level shifts, ABR threshold shifts and histopathological damage following below-critical-level noise exposures.

DPOAE temporary level shift (TLS) at 2f(1)-f(2) and f(2)-f(1), ABR temporary threshold shift (TTS), and detailed histopathological findings were compared in three groups of chinchillas that were exposed for 24 h to an octave band of noise (OBN) centered at 4 kHz with a sound pressure level (SPL) of 80, 86 or 92 dB (n=3,4,6). DPOAE levels at 39 frequencies from f(1)=0.3 to 16 kHz (f(2)/f(1)=1.23; L(2) and L(1)=55, 65 and 75 dB, equal and differing by 10 dB) and ABR thresholds at 13 frequencies from 0.5 to 20 kHz were collected pre- and immediately post-exposure. The functional data were converted to pre- minus post-exposure shift and overlaid upon the cytocochleogram of cochlear damage using the frequency-place map for the chinchilla. The magnitude and frequency place of components in the 2f(1)-f(2) TLS patterns were determined and group averages for each OBN SPL and L(1), L(2) combination were calculated. The f(2)-f(1) TLS was also examined in ears with focal lesions equal to or greater than 0.4 mm. The 2f(1)-f(2) TLS (plotted at f(1)) and TTS aligned with the extent and location of damaged supporting cells. The TLS patterns over frequency had two features which were unexpected: (1) a peak at about a half octave above the center of the OBN with a valley just above and below it and (2) a peak (often showing enhancement) at the apical boundary of the supporting-cell damage. The magnitudes of the TLS and TTS generally increased with increasing SPL of the exposure. The peaks of the TLS and TTS, as well as the peaks and valleys of the TLS pattern moved apically as the SPL of the OBN was increased. However, there was little consistency in the pattern relations with differing L(1), L(2) combinations. In addition, neither the 2f(1)-f(2) nor f(2)-f(1) TLS for any L(1), L(2) combination reliably detected focal lesions (100% OHC loss) from 0.4 to 1.2 mm in size. Often, the TLS went in the opposite direction from what would be expected at focal lesions. Recovery from TLS and TTS was also examined in seven animals. Both TLS and TTS recovered partially or completely, the magnitude depending upon exposure SPL.

Noise-induced hair-cell loss and total exposure energy: analysis of a large data set.

The relation between total noise-exposure energy, recovery time, or rest during the exposure and amount of hair-cell loss was examined in 416 chinchillas. The exposures were octave bands of noise (OBN) with a center frequency of either 4 kHz at 47-108 dB sound pressure level (SPL) for 0.5 h to 36 d, or 0.5 kHz at 65-128 dB SPL for 3.5 h to 432 d. Recovery times varied from 0 to 365 d. With both OBNs, some animals were exposed on interrupted schedules. Hair-cell loss as a function of age in nonexposed animals (N = 117) was used to correct for sensory-cell loss due to aging. For both OBNs, the ears (N = 607) were separated into three subsets to characterize the primary hair-cell loss from noise and the secondary post-exposure loss and to determine if rest during the exposure decreased loss. Cluster and regression analyses were performed on data from the basal and apical halves of the cochlea to determine the specific rates for these three factors. It was found that: (1) when the OBN was above a critical level, there was no relation between total energy and hair-cell loss; (2) below a critical level, there were highly significant log-linear relations between total energy and hair-cell loss, but not at rates predicted by the equal-energy hypothesis; (3) rest periods during either OBN exposure reduced hair-cell loss; more so for the 4 kHz OBN than the 0.5 kHz OBN; (4) except for the highest exposure levels, the majority of outer hair cell loss from the 4 kHz OBN occurred after the exposure had terminated, while that from the 0.5 kHz OBN occurred during the exposure; and (5) a majority of the inner hair cell loss from both OBNs occurred post-exposure.

An in vivo tracer study of noise-induced damage to the reticular lamina.

An in vivo tracer was used to determine if the reticular lamina and/or the cell membranes abutting the endolymphatic space are temporarily disrupted after intense noise exposure (4-kHz OBN, 108-dB SPL, 1.75 h). Using a double-barreled micropipette, the endolymphatic potential (EP) was recorded and artificial endolymph containing 10% carbon particles was injected into the endolymphatic space either 0 days or 28 days post-exposure. The cochleae were fixed 30-45 min post-injection, then dehydrated, embedded in plastic and dissected as flat preparations. Damage in the organ of Corti (OC) was quantified, the location of carbon was determined, and some OC segments were then sectioned radially. EP averaged 72+/-5 mV in five controls. These cochleae had carbon tracer in the endolymphatic space only. Four of five noise-exposed chinchillas examined 3-4 h post-exposure had a low EP (30+/-6 mV). The cochleae from these 0-day animals had several focal lesions in which nearly all outer hair cells had just degenerated. At these lesions, carbon was attached to cell membranes and debris between the reticular lamina and basilar membrane. By transmission electron microscopy, discontinuities were found in the apical membranes of sensory and supporting cells. Carbon particles were found in the cytoplasm of these cells. Four of five animals examined at 28 days had an average EP of 70+/-11 mV. The cochleae from these animals had multiple lesions in the basal turn, all of which were healed by phalangeal scars or squamous epithelial cells. In these cochleae, no carbon was found within the OC. Acute disruption of the reticular lamina and the apical membranes of sensory and supporting cells from noise appears to be a major mechanism to account for degeneration in the cochlea that spreads or continues for days to weeks post-exposure.

DPOAE level shifts and ABR threshold shifts compared to detailed analysis of histopathological damage from noise.

A detailed comparison of 2f(1)-f(2) distortion product otoacoustic emission (DPOAE) level shifts (LS) and auditory brainstem response (ABR) threshold shifts with noise-induced histopathology was conducted in chinchillas. DPOAE levels (i.e., L(1) and L(2)) at f(1) and f(2), respectively, ranged from 55-75 dB sound pressure level (SPL), with f(2)/f(1)=1.23, 6 points/octave, f(2)=0.41-20 kHz, and ABR thresholds at 0.5-20 kHz, 2 points/octave, were determined pre-exposure. The exposure was a 108 dB SPL octave band of noise centered at 4 kHz (1-1.75 h, n=6) or 80-86 dB SPL (24 h, n=5). DPOAE LSs (magnitude pre- minus post-exposure) and ABR threshold shifts (TS) were determined at 0 days and up to 28 days post-exposure. The cochleae were fixed, embedded in plastic and dissected into flat preparations. The length of the organ of Corti (OC) was measured; missing inner (IHC) and outer (OHC) hair cells counted; stereocilia damage rated; and regions of OC and nerve-fiber loss determined. Cytocochleograms were made showing functional loss and structural damage with the LS and TS overlaid. Some unexpected results were obtained. First, the best correlation of LS with histopathology required plotting the DPOAE data at f(1) with respect to the chinchilla-place map. The best correlation of TS was with IHC and nerve-fiber loss. Second, wide regions of up to 10% scattered OHC loss in the apical half of the OC showed little or no LS. Third, with the 108 dB SPL noise, there was 20-40 dB of recovery for DPOAEs at mid-high frequencies (3-10 kHz) in eight of 12 cochleae where there was 70-100% OHC loss in the basal half of the OC. The largest recovery at mid-high frequencies occurred in regions where the OC was entirely missing. Fourth, with the 80-86 dB SPL noise, there was no LS at small focal lesions (100% loss of OHCs over 0.4 mm) when the frequency place of either f(1) or f(2) was within the lesion but not both. There was no correlation of LS with OHC stereocilia loss, fusion or disarray. These results suggest that, after noise exposure, DPOAEs at mid-high frequencies can originate from or be augmented by generators located at someplace other than the frequency place of f(2), possibly the basal 20% of the OC when this region is intact. Also, noise-induced DPOAE LSs seemed to reflect differing mechanisms for temporary and permanent hearing loss.