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.

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.

Fate of neural stem cells grafted into injured inner ears of mice.

Loss of sensory hair cells in the inner ear is a major cause of permanent hearing loss, since regeneration of hair cells rarely occurs in mammals. The aim of this study was to examine the potential of neural stem cell transplantation to restore inner ear hair cells in mice. Fetal neural stem cells were transplanted into the mouse inner ear after drug-induced injury. Histological analysis demonstrates that the majority of grafted cells differentiated into glial or neural cells in the inner ear. Strikingly, however, we show that grafted cells integrate in vestibular sensory epithelia and express specific markers for hair cells. This finding suggests that transplanted neural stem cells have the potential to differentiate and restore inner ear hair cells.

Extra inner hair cells in the developing rabbit cochlea.

Development of the rabbit inner ear was analysed with respect to the presence of extra inner hair cells (XIHCs) in phalloidin-impregnated cochleas of newborn rabbits 0, 3 and 5 days of age. The number of XIHCs ranged from four to 77. The distribution was asymmetrical with a peak in the apical 3 mm of the cochlea. There was no general disorganisation in the vicinity of the XIHCs, and the numbers were highly symmetrical between the two ears. The number was significantly larger (P<0.001) in newborns than in adults. There was no correlation between number of XIHCs and cochlear length, making it unlikely that the presence of XIHCs is due to lack of space in the ordinary row of IHCs. The possible relation of the XIHCs to growth factors, molecular genetics and regeneration is discussed.

Functional reorganization in chinchilla inferior colliculus associated with chronic and acute cochlear damage.

This paper describes some of the unexpected functional changes that occur in the inferior colliculus (IC) following noise- and drug-induced cochlear pathology. A striking example of this is the compensation that is seen in IC responsiveness after drug-induced selective inner hair cell (IHC) loss. Despite a massive reduction in the compound action potential (CAP) caused by partial IHC loss, the evoked potential amplitude from the IC shows little or no reduction. Acoustic trauma, which impairs cochlear sensitivity and tuning, also reduces the CAP amplitude. Despite this reduced neural input, IC amplitude sometimes increases at a faster than normal rate and the response amplitude is enhanced at frequencies below the hearing loss. Single unit recordings suggest the IC enhancement phenomenon may be due to the loss of lateral inhibition. After an acute traumatizing exposure to a tone located above the characteristic frequency (CF), approximately 50% of IC neurons show a significant increase in their spike rate, a significant expansion of the low frequency tail of the tuning curve and a significant improvement in sensitivity in the tail of the tuning curve. These changes suggest that IC neurons receive inhibition from a high frequency side band and that this inhibition is diminished by acoustic trauma above CF. To determine if side band inhibition was locally mediated, specific antagonist(s) to inhibitory neurotransmitters were applied and found to produce effects similar to acoustic trauma. The results suggest that lesioned-induced central auditory plasticity could contribute to several symptoms associated with sensorineural hearing loss such as loudness recruitment, tinnitus and poor speech discrimination in noise.

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.

Inner hair cell loss leads to enhanced response amplitudes in auditory cortex of unanesthetized chinchillas: evidence for increased system gain.

Carboplatin preferentially destroys inner hair cells (IHCs) in the chinchilla inner ear, while retaining a near-normal outer hair cell (OHC) population. The present study investigated the functional consequences of IHC loss on the compound action potential (CAP), inferior colliculus potential (ICP) and auditory cortex potential (ACP) recorded from chronically implanted electrodes. IHC loss led to a reduction in CAP amplitude that was roughly proportional to IHC loss. The ICP amplitude was typically reduced by IHC loss, but the magnitude of this reduction was generally less than that observed for the CAP. In contrast to the CAP and ICP, ACP amplitudes were generally not reduced following IHC loss. In some animals, the ACP amplitude remained at pre-carboplatin values despite substantial IHC loss. However, in other animals, IHC loss led to an increase ('enhancement') of ACP amplitude. ACP enhancement was greatest at 1-2 weeks post-carboplatin, returning towards baseline amplitudes at 5 weeks post-carboplatin. In other animals, the ACP remained enhanced up to 5 weeks post-carboplatin. We interpret the transient and sustained enhancement of ACP amplitude following partial IHC loss as evidence of functional reorganization occurring at or below the level of the auditory cortex. These results suggest that the gain of the central auditory pathway increases following IHC loss to compensate for the reduced input from the cochlea.

Changes in distortion product otoacoustic emissions during prolonged noise exposure.

The aim of this study was to evaluate the reduction in 2f1-f2 distortion product otoacoustic emission (DPOAE) amplitude resulting from prolonged noise exposures. A group of five chinchillas was exposed continuously to an octave-band noise centered at 4.0 kHz for a total of 42 days, 6 days at each of seven exposure levels. Exposure level increased in 8-dB steps from 48 to 96 dB SPL. DPOAE input-output (I/O) functions were measured at octave intervals over a range of primary tone f2 frequencies between 1.2 and 9.6 kHz. Measurements were obtained (1) pre-exposure, (2) during days 3-6 of each 6-day exposure, and (3) 4 weeks after the final exposure. Continuous noise exposure caused a reduction in DPOAE amplitude that was greatest at f2 frequencies within and above (3.4-6.8 kHz) the octave-band noise exposure. For these f2 frequencies, DPOAE amplitudes decreased as exposure level increased up to approximately 72-80 dB SPL; higher exposure levels failed to cause any further reduction in DPOAE amplitude. The noise level at which DPOAE amplitude began to decrease was approximately 50 dB SPL. Above this critical level, DPOAE amplitude decreased 1.3 dB for every dB increase in noise level up to approximately 75 dB SPL.

Brain-derived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss.

Destruction of auditory hair cells results in the secondary degeneration of auditory neurons. This is because of the loss of neurotrophic factor support from the auditory hair cells, namely neurotrophin 3, which is normally produced by the inner hair cells. Both in vitro and in vivo studies have shown that delivery of either neurotrophin 3 or brain-derived neurotrophic factor to these neurons can replace the trophic support supplied by the hair cells and prevent their degeneration. To prevent the degeneration of auditory neurons that occurs after neomycin destruction of the auditory hair cells we used a replication defective herpes simplex-1 vector (HSVbdnflac) to transfect the gene for brain-derived neurotrophic factor into the damaged spiral ganglion. Four weeks after the HSVbdnflac therapy we were able to detect stable functional production of brain-derived neurotrophic factor that supported the survival of auditory neurons and prevented the loss of these neurons because of trophic factor deprivation-induced apoptosis.

Effects of selective inner hair cell loss on auditory nerve fiber threshold, tuning and spontaneous and driven discharge rate.

Current theories assume that the outer hair cells (OHC) are responsible for the sharp tuning and exquisite sensitivity of the ear whereas inner hair cells (IHC) are mainly responsible for transmitting acoustic information to the central nervous system. To further evaluate this model, we used a single (38 mg/kg) or double dose (38 mg/kg, 2 times) of carboplatin to produce a moderate (20-28%) or severe (60-95%) IHC loss while sparing a large proportion of the OHCs. The surviving OHCs were functionally intact as indicated by normal cochlear microphonic (CM) potentials and distortion product otoacoustic emissions (DPOAE). Single-unit responses were recorded from auditory nerve fibers to determine the effects of the moderate or severe IHC loss on the output of the surviving IHCs. Most neurons that responded to sound in the single-dose group had normal or near-normal thresholds and normal tuning. Relatively few neurons in the double-dose group responded to sound because of the severe IHC loss. The neurons that did respond to sound had narrow tuning curves. Some neurons in the double-dose group also had thresholds that were within the normal range, but most had thresholds that were elevated a mild-to-moderate degree. These results indicate that intact IHCs can retain relatively normal sensitivity and tuning despite massive IHC loss in surrounding regions of the cochlea. However, the spontaneous and driven discharge rates of neurons in the carboplatin-treated animals were significantly lower than normal. These changes could conceivably be due to sublethal damage to surviving IHCs or to postsynaptic dysfunction in the auditory nerve.

Low-frequency 'conditioning' provides long-term protection from noise-induced threshold shifts in chinchillas.

Studies have shown that loss of auditory sensitivity caused by exposure to high-level acoustic stimuli can be significantly reduced by pre-exposing the subject to moderate-level acoustic stimuli. Although the protective effects of such 'conditioning' exposures have been well documented, very little is known about the persistence of conditioning-induced protection, or about the biological mechanisms underlying it. In the present study, the persistence of conditioning-induced protection was examined in chinchillas by imposing either a 30- or 60-day recovery period between conditioning (10 days of exposure to 0.5 kHz noise at 90 or 95 dB, 6 h/day) and high-level (0.5 kHz noise at 106 dB for 48 h) exposures. Comparisons of threshold shifts between conditioned animals and control animals exposed only to high-level noise indicated that conditioning provided significant protection from noise-induced threshold shifts for at least 2 months. Conditioned animals sustained outer hair cell losses similar to controls, ranging from 15 to 30% in the apical half of the cochlea. The results suggest that low-frequency conditioning can trigger long-lasting changes in cochlear homeostasis rather than temporary changes in physiology or reductions in susceptibility to hair cell loss in chinchillas.

Interaural correlations in normal and traumatized cochleas: length and sensory cell loss.

Sizable intraspecies variations have been found in both the length of the organ of Corti (OC) and the amount of damage resulting from exposure to a particular ototraumatic agent. These variations have made it difficult to address certain research questions such as the susceptibility of the previously injured ear to further damage. If intra-animal correlation is high, the variability problem could be circumvented by using the two ears from a given animal for different aspects of the same study. Therefore, correlation coefficients were calculated for OC length and for percentage of missing inner (IHCs) and outer hair cells (OHCs) in a large sample of chinchillas which included controls and animals which had been exposed to noise or treated with ionizing radiation. The correlation coefficients were +0.96 for OC length, +0.93 for IHC loss, and +0.97 for OHC loss.

Cochlear hair cell damage from intermittent noise exposure in young and adult guinea pigs.

A comparative study of age-dependent damage to cochlea from intermittent noise exposure was carried out on five-week-old and one-year-old pigmented guinea pigs. Hair cell loss in the organ of Corti was studied after five weeks' exposure to a pure tone stimulus (95 dB SPL at 2 kHz, one hour per day for five weeks). The noise-induced damage was sharply limited to the 7- and 11-mm marks from the apex. Damage was more marked in younger guinea pigs and was distinct from natural age-induced cell loss. When the 7- to 11-mm zone was further analyzed, outer hair cell damage appeared highly significant in both age groups but more severe in younger animals. Inner hair cell damage in this area was severe in both groups but statistically insignificant.

Total energy and critical intensity concepts in noise damage.

Groups of chinchillas were given a series of noise exposures of approximately equal energy ranging from 22 minutes at 120 dB SPL to 150 days at 82 dB. For all exposures involving levels of 112 dB or less, the same average permanent hearing losses (15-20 dB) and degree of outer hair cell destruction (8-10%) resulted, thus confirming the validity of the total energy principle for assessing the hazard associated with single continuous exposures at moderate levels. The 22-minute, 120-dB exposure, however, produced a 60-dB hearing loss and massive hair cell destruction (70-80%), indicating that some critical level had been exceeded, thus producing acoustic trauma. Further histological study suggests that the massive destruction is a result of breaks in the organ of Corti, produced by severe mechanical stress, that permit the mixture of endolymph with perilymph, thus creating a hostile environment for the hair cells.