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.

Effects of antioxidants on auditory nerve function and survival in deafened guinea pigs.

Based on in vitro studies, it is hypothesized that neurotrophic factor deprivation following deafferentation elicits an oxidative state change in the deafferented neuron and the formation of free radicals that then signal cell death pathways. This pathway to cell death was tested in vivo by assessing the efficacy of antioxidants (AOs) to prevent degeneration of deafferented CNVIII spiral ganglion cells (SGCs) in deafened guinea pigs. Following destruction of sensory cells, guinea pigs were treated immediately with Trolox (a water soluble vitamin E analogue)+ascorbic acid (vitamin C) administered either locally, directly in the inner ear, or systemically. Electrical auditory brainstem response (EABR) thresholds were recorded to assess nerve function and showed a large increase following deafness. In treated animals EABR thresholds decreased and surviving SGCs were increased significantly compared to untreated animals. These results indicate that a change in oxidative state following deafferentation plays a role in nerve cell death and antioxidant therapy may rescue SGCs from deafferentation-induced degeneration.

[The influence of glucocorticoids on the view of chicken's inner ear damage after exposure to wide-band-noise]. / Wplyw glikokortykoidów na obraz zniszczen komórek rzesatych ucha wewnetrznego kurczat poddanych ekspozycji na halas szerokopasmowy.

The aim of the study was to assess the influence of glucocorticoids on the view of hair cell regeneration process being in the chicken's inner ear (basilar papilla - BP) after exposure to wide-band noise at the level 120 dB (A) for 48 hours. We found that glucocorticoids given during and/or after exposure to the noise have a cytoprotective activity to the hair cells, they limitate the extensiveness and decrease the dynamics of hair cells injury. We observed that new "young" hair cells reappeared at the sensory epithelium on the 7th day after the end of exposure. Regenerated hair cells have immature, short and thick cilia and small apical surface area.

Supporting cell proliferation after hair cell injury in mature guinea pig cochlea in vivo.

In cold-blooded animals, lost sensory hair cells can be replaced via a process of regenerative cell proliferation of epithelial supporting cells. In contrast, in mammalian cochlea, receptor (hair) cells are believed to be produced only during embryogenesis; after maturity, sensory or supporting cell proliferation or regeneration are thought to occur neither under normal conditions nor after trauma. Using bromodeoxyuridine (BrdU) as a proliferation marker, we have assessed cell proliferation activity in the mature organ of Corti in the cochlea of young guinea pigs following severe damage to the outer hair cells induced by kanamycin sulfate and ethacrynic acid. Although limited, we have found BrdU-labeled nuclei in the regions of Deiters cells when BrdU is given for 3 days or longer. When BrdU is given for 10 days, at least one labeled nucleus can be observed in the organ of Corti in approximately half of the ears; proliferating cells typically appear as paired daughters, with one nucleus being displaced away from the basement membrane to the position expected of the hair cells. Double-staining with antibodies to cytokeratin, vimentin, and p27 have shown that the BrdU-labeled nuclei are located in cells phenotypically similar to Deiters cells. Most of the uptake of BrdU occurs 3-5 days following ototoxic insult, and the number of BrdU-labeled cells does not decrease until 30 days following insult. These findings indicate that Deiters cells in the mature mammalian cochlea maintain a limited competence to re-enter the cell cycle and proliferate after hair cell injury, and that they can survive at least for 1 month.

Different effects of intracochlear sensory and neuronal injury stimulation on expression of synaptic N-methyl-D-aspartate receptors in the auditory cortex of rats in vivo.

CONCLUSIONS: The expression of synaptic N-methyl-D-aspartate (NMDA) receptors in the auditory cortex is dynamic and is bidirectionally regulated by auditory activity. Furthermore, the time course of changes in the level of NR2A protein differs after sensory and neuronal injury stimulation, which modulate different changes in synaptic plasticity. OBJECTIVE: To examine the effects of different types of auditory activity on the expression of synaptic NMDA receptors (NMDARs) in the auditory cortex of rats. MATERIAL AND METHODS: We prepared synaptosomes from the auditory cortices of postnatal Day 28 ototoxic-deafened Sprague-Dawley rats and postnatal Day 28 Sprague-Dawley rats subjected to noise trauma that were given various treatments and compared them to the synaptosomes of 1-6-week-old normal Sprague-Dawley rats. The expression of different NMDAR subunits in the synaptosomes was investigated by means of Western blotting. RESULTS: Changes in NR1 and NR2B proteins were not significant during different types of auditory activity. The level of NR2A protein increased remarkably during postnatal development and as a result of electrical intracochlear stimulation, auditory deprivation and noise trauma. Seventy-two h after a 2-h period of sensory electrical intracochlear stimulation, the expression of NR2A protein returned to the level caused by auditory deprivation. Seventy-two h after a 3-h period of noise trauma, elevation of the level of NR2A protein was unchanged. We also confirmed that elevation of the level of synaptic NR2A protein was sensitive to protein synthesis inhibitor and NMDAR antagonist. However, transcription inhibitor had no effect on NR2A protein expression.

Is there a close relationship between changes in amplitudes of distortion product otoacoustic emissions and hair cell damage after exposure to realistic industrial noise in guinea pigs?

In long-term experiments in awake guinea pigs (n = 12), distortion product otoacoustic emissions (DPOAEs) at various frequencies were measured repeatedly over 6-8 months. About 9 weeks after the first measurement, the animals were exposed to industrial noise (car industry, maximal intensity about 110 dB SPL) for 2 h. The amplitudes of DPOAE were measured prior to noise exposure and 10 min, 70 min, 1 day and 2 days after the noise exposure and then once every week. Three to four months after noise exposure, the animals were killed, and the cochleae were prepared for scanning electron microscopy. The row of inner hair cells (IHCs) was complete in all animals, while the rows of outer hair cells (OHCs) showed a considerable hair cell loss in some of the animals without a correlation to the change in amplitudes of DPOAE. However, a closer relationship between the decline of amplitudes of DPOAE and the number of missing and changed OHCs (fused stereocilia bundles, missing tip links) could be established. The number of lost OHC does not reflect the decline in DPOAE in all cases. This discrepancy must be considered when the degree of hearing loss needs to be established from changed DPOAE.

Round window gentamicin application: an inner ear hair cell damage protocol for the mouse.

It is important to develop an inner ear damage protocol for mice that avoids systemic toxicity and produces damage in a relatively rapid fashion, allowing for study of early cellular and molecular mechanisms responsible for hair cell death and those that underlie the lack of hair cell regeneration in mammals. Ideally, this damage protocol would reliably produce both partial and complete lesions of the sensory epithelium. We present a method for in vivo induction of hair cell damage in the mouse via placement of gentamicin-soaked Gelfoam in the round window niche of the inner ear, an adaptation of a method developed to study hair cell regeneration in chicks. A total of 82 subjects underwent the procedure. Variable doses of gentamicin were used (25, 50, 100 and 200 microg). Saline-soaked Gelfoam, sham-operations and the contralateral, non-operated cochlea were used as controls. Survival periods were 1, 3 and 14 days. Damage was assessed on scanning electron microscopy. We found that this method produces relatively rapid hair cell damage that varies with dose and can extend the entire length of the sensory epithelium. In addition, this protocol produces no systemic toxicity and preserves the contralateral ear as a control.

A mechanism for sensing noise damage in the inner ear.

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia. This may eventually lead to hair cell death and irreversible deafness. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca(2+)](i)) and elicit intercellular Ca(2+) waves. Thus an attractive hypothesis is that changes in [Ca(2+)](i), propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP(3)-sensitive intracellular stores in support cells.

Inner ear: Ca(2+)n you feel the noise?

The death of hair cells in the inner ear as a result of exposure to loud noise can lead to irreversible deafness. New work shows that the mammalian cochlea can sense noxious sounds and use Ca(2+) waves to rapidly propagate hair cell damage signals.

The clinical free radical scavenger, edaravone, protects cochlear hair cells from acoustic trauma.

It is known that reactive oxygen species have toxicity to the cochlea. We investigated the effect of edaravone, a free radical scavenger for clinical use, on the cochleae of guinea pigs subjected to acoustic trauma. We assessed auditory brainstem response (ABR) thresholds to evaluate cochlear function and observed the sensory epithelium. After noise exposure (130 dB SPL, 3 h), we observed that the auditory brainstem response threshold shift in edaravone-treated ears was significantly less than that in untreated ears. This result suggests that edaravone protected the cochleae from acoustic trauma.

[The protection of Tiron to the injury of organ of Corti cultured in vitro caused by exogenous H2O2 in mouse].

OBJECTIVE: In order to observe the action of H2O2 on inner and outer hair cells of organ of Corti in mouse, and discuss whether Tiron can effectively resistant the injury of free radicals in cultured cochlea in vitro or not. METHOD: Using the culture technology of organ of Corti in newborn mouse in vitro, established the model of the injury of inner and outer hair cells caused by exogenous H2O2. And observed the protection to the damage by H2O2 at different concentration of Tiron. RESULT: When the concentration of H2O2 is higher than 1.0 mmol/L, the loss of hair cells in the bottom, middle, and parietal turn of basilar membrane vary, the injury of bottom turn is more severe. In lower than 0.5 mmol/L group, the injury of hair cells is no relationship with the location. Adding Tiron (10 mmol/L) in culture can apparently decrease the injury of hair cells caused by H2O2. When the concentration of H2O2 is 0.01-1.0 mmol/L, Tiron can almost inhibit the loss of hair cells completely. CONCLUSION: There is apparently preventive effection of Tiron on organ of Corti cultured in vitro, and the mechanism of inhibiting injury caused by H2O2 is probably by clearing O2- and combining irony ion.

Tip link loss and recovery on chick short hair cells following intense exposure to sound.

Stereocilia tip links on chick short hair cells (SHCs) were counted in the 'patch' lesion produced by acoustic overstimulation. Tip links were also counted on tall hair cells (THCs) immediately superior to the lesion. Eight groups were studied with three exposed to intense sound for differing durations. Three other groups were allowed to recover from the longest exposure for different time periods. Tip link counts from non-exposed control hair cells came from two other groups. Chicks exposed for 4, 24 or 48 h to a 120-dB SPL 0.9-kHz pure tone showed SHC tip link loss of 30.3, 40.6, and 35.5%, respectively. Chicks exposed for 48 h were allowed to recover for 24, 96 or 288 h, and showed systematic tip link recovery to control levels. Tip link loss and recovery in THCs adjacent to the patch lesion were identical to that seen in SHCs. After 288 h of recovery, surviving SHCs were distinguished from newly regenerated SHCs in the patch lesion. A comparison of tip link presence in the surviving (74%) and regenerated (84%) SHCs revealed a significant difference. These results suggest that the process of tip link destruction and recovery following acoustic overstimulation is the same for THCs and SHCs. This observation is surprising based on differences in the degree of acoustic injury to THC and SHC regions of the papillae, and the difference between THC and SHC sensory hair bundle stimulation.

Hair cell regeneration: winging our way towards a sound future.

The discovery of hair cell regeneration in the inner ear of birds provides new optimism that there may be a treatment for hearing and balance disorders. In this review we describe the process of hair cell regeneration in birds; including restoration of function, recovery of perception and what is currently known about molecular events, such as growth factors and signalling systems. We examine some of the key recent findings in both birds and mammals.

Acoustic trauma induces reemergence of the growth- and plasticity-associated protein GAP-43 in the rat auditory brainstem.

We explored the consequences of unilateral acoustic trauma to intracochlear and central nervous system structures in rats. An acoustic trauma, induced by applying click stimuli of 130 dB (sound pressure level; SPL) for 30 minutes, resulted in an instant and permanent threshold shift of 95.92 +/- 1.08 dB (SEM) in the affected ear. We observed, as a consequence, a structural deterioration of the organ of Corti. Deprivation-dependent changes of neurons of the auditory brainstem were determined using antibodies against neurofilament and the growth-associated protein GAP-43 and compared with those following cochleotomy, studied earlier. By 231 days posttrauma, spiral ganglion cell bodies and their processes were almost entirely lost from all cochlear regions with destroyed organ of Corti. In the lateral superior olive (LSO) ipsilateral to the trauma, cell bodies of lateral olivocochlear neurons turned transiently GAP-43 positive within the first 1.5 years posttrauma. The time course of emergence and disappearance of this population of neurons was similar to that found after cochleotomy. Additionally, after noise trauma, principal cells in contralateral LSO and in medial superior olive (MSO) on both sides of the brainstem developed an expression of GAP-43 that began 3 and 16 days posttrauma, respectively, and lasted for at least 1 year. Such cells were rarely observed after cochleotomy. An unequivocal rise in GAP-43 immunoreactivity was also found in the neuropil of the inferior colliculus and the ventral cochlear nucleus, both preferentially on the acoustically damaged side. We conclude that the degree and specific cause of sudden unilateral deafness entail specific patterns of plasticity responses in the auditory brainstem, possibly to prevent the neural network dedicated to locate sounds in the environment from delivering erroneous signals centralward.

Therapeutic effect of parenteral magnesium on noise-induced hearing loss in the guinea pig.

We have recently demonstrated in the guinea pig that preventive dietary magnesium supplement can significantly reduce impulse noise induced hearing loss by on average 18 dB. The purpose of the present study was to examine whether magnesium might also have a therapeutic effect on noise trauma. Anesthetized guinea pigs were exposed to an impulse noise series (1/s) of L(peak) 167 dB (L(eq,ls) 127 dB) for 38 min. The permanent hearing threshold shift (PTS) was determined one week post-exposure, using auditory brainstem response audiometry at a frequency range of 0.5-32 kHz. The total magnesium concentrations of perilymph (PL), cerebrospinal fluid and blood were analysed by atomic absorption spectrometry at different times of treatment. In a first set of experiments, animals on a low initial magnesium status were injected either of 4 different dose levels of magnesium (1.14-3.42 mmol MgSO4 s.c./kg per day) for 3 days or saline as a placebo. The treatment was started immediately after the exposure. The magnesium groups received drinking water with an additive of 39 mmol MgCl2/l for one week and the placebo group tap water (0.43 mmol Mg/l) alone. A dose level of 2.85 mmol Mg has proved to be most effective and reduced the PTS by 13-20 dB compared to the placebo group. The magnesium concentrations increased to above 4 mmol/l in serum and to 1.2 mmol/l in PL during the first 3 days of this treatment. In a second set of experiments, we tested the dependence of the therapeutic efficacy on the post-exposure time of onset of the optimal treatment (1 min, 2 h and 4 h), using guinea pigs on normal initial magnesium status. In the 1 min-group, the reduction of hearing loss was similar to that found in the first series. The therapeutic effect decreases with the length of time elapsed between the end of exposure and the beginning of treatment. In a few animals, hair cell stereocilia were examined using scanning electron microscopy. The results also revealed a magnesium related reduced susceptibility of hair cell stereocilia to impulse noise exposure.

Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity.

Calpains, a family of calcium-activated proteases that breakdown proteins, kinases, phosphatases and transcription factors, can promote cell death. Since leupeptin, a calpain inhibitor, protected against hair cell loss from acoustic overstimulation, we hypothesized that it might protect cochlear and vestibular hair cells against gentamicin (GM) ototoxicity. To test this hypothesis, mouse organotypic cultures from the cochlea, maculae of the utricle and the crista of the semicircular canal (P1-P3) were treated with different doses of GM (0.1-3 mM) alone or in the presence of leupeptin (0.1-3 mM). The percentage of outer hair cells (OHCs) and inner hair cells (IHCs) decreased with increasing doses of GM between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced GM-induced damage to IHCs and OHCs; this protective effect was dose-dependent. GM also significantly reduced hair cell density in the crista and utricle in a dose-dependent manner between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced hair cell loss in the crista and utricle for GM concentrations between 0.1 and 3 mM. These results suggest that one of the early steps in GM ototoxicity may involve calcium-activated proteases that lead to the demise of cochlear and vestibular hair cells.

[Regeneration of ciliated cells in the internal ear]. / La régénération des cellules ciliées de l'oreille interne.

Hair cells are the mechanotranducer transforming the sound into a bioelectrical signal. Hair cell and supporting cell productions are completed during early embryonic development of the mammalian cochlea. In mammalian, after an injury, no hair cell replacement is observed, as opposed to birds, where regenerative mechanisms produce new sensory cells and restore the auditory function. However, a production of hair cells occurs in the mammalian sensory epithelium. Progenitor cells, isolated from newborn rats, proliferate and differentiate in hair cells and supporting cells. Supernumerary hair cells also arise in the cultured organ of Corti. This model is used to investigate the role of cell cycle regulator molecules and cell-cell interaction. The persistence of sensory cell progenitors in adult mammalian organ of Corti and the understanding of the mechanisms leading to the production of hair cells, in the developing cochlea, open the prospect of hair cell regeneration in the mature inner ear.

Degeneration in the cochlea after noise damage: primary versus secondary events.

PURPOSE: To determine if noise damage in the organ of Corti is different in the low- and high-frequency regions of the cochlea. MATERIALS AND METHODS: Chinchillas were exposed for 2 to 432 days to a 0.5 (low-frequency) or 4 kHz (high-frequency) octave band of noise at 47 to 95 dB sound pressure level. Auditory thresholds were determined before, during, and after the noise exposure. The cochleas were examined microscopically as plastic-embedded flat preparations. Missing cells were counted, and the sequence of degeneration was determined as a function of recovery time (0-30 days). RESULTS: With high-frequency noise, primary damage began as small focal losses of outer hair cells in the 4-8 kHz region. With continued exposure, damage progressed to involve loss of an entire segment of the organ of Corti, along with adjacent myelinated nerve fibers. Much of the latter loss is secondary to the intermixing of cochlear fluids through the damaged reticular lamina. With low-frequency noise, primary damage appeared as outer hair cell loss scattered over a broad area in the apex. With continued exposure, additional apical outer hair cells degenerated, while supporting cells, inner hair cells, and nerve fibers remained intact. Continued exposure to low-frequency noise also resulted in focal lesions in the basal cochlea that were indistinguishable from those resulting from exposure to high-frequency noise. CONCLUSIONS: The patterns of cochlear damage and their relation to functional measures of hearing in noise-exposed chinchillas are similar to those seen in noise-exposed humans. Thus, the chinchilla is an excellent model for studying noise effects, with the long-term goal of identifying ways to limit noise-induced hearing loss in humans.

Lack of calbindin-D28k does not affect hearing level or survival of hair cells in acoustic trauma.

Calbindin is a cytosolic calcium-binding protein abundant in the hair cells of the inner ear and in distinct neurons of the auditory pathway. It is suggested to speed the return of potentially toxic calcium levels to normal. In this study, we show the basic hearing functions and the result of noise trauma from the calbindin null mutant mice generated by gene targeting. Auditory brainstem evoked response and distortion product otoacoustic emissions appear similar as in the control group. A moderate noise-induced trauma produced a similar loss of hair cells in calbindin null mutant mice than in wild-type controls. The result suggests that although calbindin is abundant in hair cells, it is not essential for the main hearing function and it does not provide physiological protection against a moderate noise-induced inner ear trauma in mice.