Genetic and pharmacological intervention for treatment/prevention of hearing loss

Twenty years ago it was first demonstrated that birds could regenerate their cochlear hair cells following noise damage or aminoglycoside treatment. An understanding of how this structural and functional regeneration occurred might lead to the development of therapies for treatment of sensorineural hearing loss in humans. Recent experiments have demonstrated that noise exposure and aminoglycoside treatment lead to apoptosis of the hair cells. In birds, this programmed cell death induces the adjacent supporting cells to undergo regeneration to replace the lost hair cells. Although hair cells in the mammalian cochlea undergo apoptosis in response to noise damage and ototoxic drug treatment, the supporting cells do not possess the ability to undergo regeneration. However, current experiments on genetic manipulation, gene therapy, and stem cell transplantation suggest that regeneration in the mammalian cochlea may eventually be possible and may 1 day provide a therapeutic tool for hearing loss in humans. LEARNING OUTCOMES: The reader should be able to: (1) Describe the anatomy of the avian and mammalian cochlea, identify the individual cell types in the organ of Corti, and distinguish major features that participate in hearing function, (2) Demonstrate a knowledge of how sound damage and aminoglycoside poisoning induce apoptosis of hair cells in the cochlea, (3) Define how hair cell loss in the avian cochlea leads to regeneration of new hair cells and distinguish this from the mammalian cochlea where there is no regeneration following damage, and (4) Interpret the potential for new approaches, such as genetic manipulation, gene therapy and stem cell transplantation, could provide a therapeutic approach to hair cell loss in the mammalian cochlea.     

Cisplatin-induced increases in spontaneous neural activity in the dorsal cochlear nucleus and associated outer hair cell loss

Tinnitus is one of the consequences of cisplatin chemotherapy, but its underlying mechanisms are not well understood. Since it has been shown that cisplatin causes outer hair cell loss, it is possible that loss of these cells might induce tinnitus by increasing spontaneous activity in the central auditory system. To test this possibility, the present study examined the effects of cisplatin treatment on cochlear hair cells and on spontaneous neural activity in the dorsal cochlear nucleus of hamsters. Recordings, carried out approximately 1 month after cisplatin treatment, demonstrated significant increases in spontaneous activity across broad regions of the dorsal cochlear nucleus relative to levels in saline-treated controls. Histological results showed that cisplatin-treated animals also displayed dramatic loss of outer hair cells over most of the basal turn of the cochlea. Inner hair cells remained intact, although some evidence of damage to their stereocilia was evident. These findings indicate that cisplatin treatment causes abnormalities in spontaneous activity in the dorsal cochlear nucleus that are associated with widespread damage to outer hair cells. However, since some damage to inner hair cells was also observed, the role of inner hair cell injury in contributing to higher spontaneous activity cannot be ruled out.     

Changes in the audiograms of a nasopharyngeal cancer patient during the course of treatment: a temporal bone histopathological study

This report shows the changes that occurred in consecutive audiograms of a patient who underwent chemotherapy and radiotherapy for nasopharyngeal cancer and the histopathological examination of the temporal bones. Both conductive and sensorineural hearing loss developed, but followed different modes of progression. In the left ear, an air-bone gap appeared and deepened, while in the right ear, severe conductive hearing loss was present upon admission and improved after treatment. Sensorineural hearing loss was worse in the right ear, but deteriorated gradually in both ears. Histology revealed tumor invasion in the right temporal bone. Both middle ears showed effusion, but no radiation-induced changes that may be responsible for the conductive loss. The main changes in the cochlea were vascular stria degeneration, spiral ligament atrophy, and spiral ganglion cell depletion, while the hair cells were only occasionally missing. Apart from confirming what has been established previously by other authors, some interesting findings were observed: (1) in addition to the high frequency hearing loss typically caused by cis-platinum and by radiation, there was also low frequency hearing loss, and (2) the cochlear damage was most severe in the vascular stria and spiral ganglions, sparing the hair cells.     

Differences in the cochlear degeneration pattern in the guinea pig as a result of gentamicin and cis-platinum intoxication

This study gives the results of a series of studies on inner ears carried out with the help of microdissections and surface preparations. The total degeneration patterns of the cochlea of the guinea pig due to two different ototoxic drugs were investigated with this technique. Clear differences between the results of the ototoxic drugs (gentamicin and cis-platinum) in the inner ear were observed. Gentamicin has a 'typical starting point' of degeneration on the basilar membrane. From this 'degeneration point' the loss of hair cells progressed towards the round window (fast) and the apex (slowly). The stria vascularis showed no signs of degeneration due to gentamicin. Reissner's membrane, on the other hand, showed cellular vacuolization of the endolymphatic cells. Cis-platinum (DDP) showed no 'degeneration point'; the loss of hair cells was found over the complete length of the basilar membrane, with a preference for the basilar turn. The stria vascularis, on the other hand, showed severe degenerative changes due to DDP, whereas Reissner's membrane showed no change. In using the microdissection technique we were in a position to discover these differences between the two ototoxic drugs.     

The relationship between noise-induced hearing loss and hair cell loss in rats

Noise-induced hearing loss (NIHL) and hair cell loss are known to show only a moderate correlation. One reason for this is that NIHL may reflect not only the sum of dead hair cells, but also the sum of impaired but still living hair cells. This report compares hair cell loss in different cochlear regions in rats with noise-induced compound action potential (CAP) threshold elevation at corresponding frequencies. CAP threshold elevation and hair cell loss were determined 4 weeks after noise exposure. In the apical turn (<35% from the apex) there was no hair cell loss even when a 60 dB CAP threshold elevation was induced. In the region of 40-60% from the apex in the middle turn, significant hair cell loss was not observed until CAP threshold elevation exceeded about 40-50 dB. This critical level decreased towards the basal turn. In the basal turn, outer hair cell (OHC) loss was observed in almost all of the noise-exposed rats, even in some cases without detectable NIHL, but inner hair cell (IHC) loss was still not observed until 50 dB threshold elevation. In the region of 75-90% from the apex related to the highest frequencies tested in this study (30-40 kHz), a linear NIHL/OHC loss relationship was observed. The results of this paper suggest that the high frequency hair cells in rat cochlea may die relatively rapidly after injury, leading to a linear relation between NIHL and hair cell loss, but that the low frequency hair cells may survive without auditory function.     

灰鼠畸变产物耳声发射改变和外毛细胞缺失程度的相关分析

目的 探讨灰鼠畸变产物耳声发射(DPOAE)幅值的降低与外毛细胞缺失率之间的关系.方法 联合应用顺铂和利尿酸钠,建立灰鼠耳蜗毛细胞损伤模型.12只灰鼠在静脉注射利尿酸钠(40 mg/kg)同时,腹腔注射顺铂(0.2 mg/kg),分别于用药前和用药后的1周、2周及3周在清醒状态下检测动物DPOAE,并在施行最后1次DPOAE检测后处死动物.常规制备灰鼠耳蜗基底膜铺片并进行全耳蜗毛细胞计数.计算用药前和处死前DPOAE幅值的改变程度,通过Pearson相关分析对DPOAE幅值的降低与外毛细胞缺失程度之间的相关性进行分析.结果 顺铂和利尿酸钠引起灰鼠耳蜗外毛细胞缺失的特点是病变始发于基底膜的底回并逐渐向顶回发展,外毛细胞的缺损程度沿着基底膜从底回向顶回逐渐减轻,而不同频率DPOAE幅值降低的程度亦遵循从高频向低频发展的规律,各频率DPOAE在注射顺铂和利尿酸钠后的幅值改变与基底膜上相应部位的外毛细胞缺损程度存在一定的对应关系(r=0.796,P<0.05).外毛细胞每损失1%,可引起约0.24 dB的DPOAE幅值降低.结论 在灰鼠耳蜗损伤模型中,从DPOAE幅值的变化可以大致评估耳蜗外毛细胞的缺失程度.     

Vestibular destruction by slow infusion of gentamicin into semicircular canals

Intratympanic or round window application of gentamicin is often used to alleviate disabling vertigo arising from unilateral Meniere's disease; however, treatment is often accompanied by hearing loss because the drug initially enters the cochlea before diffusing to the vestibular system. In order to enhance vestibular damage and reduce the risk of hearing loss, gentamicin was infused directly into the vestibular system. An osmotic pump containing 50, 100, 200 or 400 microg/ml of gentamicin was infused into the superior semicircular canal of the chinchilla for 7 days. Afterwards, vestibular damage was evaluated by measuring the decline in hair cell density in the utricle, saccule and superior semicircular canals. Auditory damage was assessed with distortion product otoacoustic emissions (DPOAE) and outer hair cell (OHC) and inner hair cell (IHC) loss. Infusion with the two lowest gentamicin concentrations resulted in significant hair cell loss and reduced duration of the nystagmus response, but had little or no effect on OHC or DPOAE. Higher doses of gentamicin damaged cochlear hair cells and reduced the DPOAE. In conclusion, slow infusion of a low dose of gentamicin into the semicircular canals mainly damages the vestibular hair cells and inactivates the nystagmus response without damaging cochlear hair cells or DPOAE.     

Establishment and characterization of cisplatin-resistant sublines of the human squamous carcinoma cell line HLac 79

Though various chemotherapy protocols lead to considerable response rates in squamous cell head and neck cancer (SCHNC), the overgrowth of a tumor cell phenotype which no longer responds to clinically achievable drug concentrations regularly impairs definite tumor control. In order to investigate mechanisms of drug resistance towards one of the most active agents in SCHNC we established four Cisplatin (CDDP)-resistant sublines (DDP1-DDP4) of the recloned human SCHNC cell line HLac 79. The 50% inhibitory drug concentration (IC50) of CDDP as determined by the colorimetric MTT-assay was increased by the factors 2.7 (DDP1), 3.3 (DDP2), 5.1 (DDP3), and 6.4 (DDP4) in the respective sublines. Three subpopulations contained significantly elevated glutathione (GSH) levels by the factors 1.4 (DDP3), 1.7 (DDP2), and 2.4 (DDP4) compared to the maternal line (50.2 nM/mg protein). DDP4 showed increased activity of gamma-glutamyl-transpeptidase (1.83 vs. 1.21 mU/mg protein), and DDP2 and DDP4 showed increased activity of GSH-S-transferase (35.6 and 51.9 vs. 25.1 mU/mg protein). Concerning both GSH-peroxidase and GSH-reductase no significant differences between the HLac 79 subpopulations were observed. Intracellular CDDP accumulation determined by neutron activation analysis revealed reduced drug uptake in DDP3 and DDP4 (60% and 76% of control value).     

Regenerative Medizin in der Therapie der Innenohrschwerhörigkeit

Regenerative medicine offers the prospect of causal treatment of sensorineural hearing loss. In humans, the loss of sensory hair cells is irreversible and results in chronic hearing loss. Other vertebrates, particularly birds, have the capability to spontaneously regenerate lost sensory hair cells and restore hearing. In the bird model, regeneration of hair cells is based on the proliferation of supporting cells. In mammals, supporting cells have lost their proliferative capacity and are terminally differentiated. To gain an understanding about regeneration of hair cells in mammals, cell division of supporting cells has to be controlled. Gene disruption of the cell cycle inhibitor p27(Kip1) allows supporting cell proliferation in the organ of Corti in vivo. Furthermore, in vitro studies indicate that newly generated cells may differentiate into hair cells after p27(Kip1) disruption. Other current methods to induce hair cell regeneration include the gene transfer of Math1 and transplantation of stem cells to the inner ear.     

Cochlear hair cell loss in single-dose versus continuous round window administration of gentamicin

CONCLUSIONS: Gentamicin-induced cochlear hair cell loss depends on local middle ear administration kinetics and the total drug dose. Single-dose gentamicin instillation in the middle ear is associated with a high variation in hair cell loss. OBJECTIVE: To compare the effects of single-dose and continuous round window administration of gentamicin on cochlear hair cell loss in a guinea pig model. MATERIAL AND METHODS: Two methods for drug administration to the inner ear were used. In groups of five animals, a total dose of 0.8 or 3.2 mg of gentamicin was either instilled as a single dose directly into the round window niche or administered continuously over a 1-week period using a pump-catheter system. Continuous administration was achieved by means of a posterior tympanotomy and subcutaneous placement of an osmotic pump fitted with a catheter. The tip of the catheter was fixed in the round window niche. One group of five animals served as controls and received a saline infusion. The animals were sacrificed after 1 week and hair cell loss was determined microscopically after dissection and phalloidin labelling of the basilar membrane and organ of Corti. RESULTS: Quantitation of cochlear hair cell loss revealed a dose-dependent effect of gentamicin. With both treatment modalities the higher dose induced a higher percentage of hair cell loss. There was inner and outer hair cell loss in all four groups that received gentamicin. With the single-dose instillation, hair cell loss was distributed irregularly from the round window membrane towards the cochlear apex, whereas continuous administration induced hair cell loss close to the round window membrane. Single-dose instillation induced greater hair cell loss than continuous administration at the same dose. The inter-individual variation in hair cell loss was highest following single-dose instillation.     

Spiral Ligament Pathology: A Major Aspect of Age-Related Cochlear Degeneration in C57BL/6 Mice

Data from systematic, light microscopic examination of cochlear histopathology in an age-graded series of C57BL/6 mice (1.5-15 months) were compared with threshold elevations (measured by auditory brain stem response) to elucidate the functionally important structural changes underlying age-related hearing loss in this inbred strain. In addition to quantifying the degree and extent of hair cell and neuronal loss, all structures of the cochlear duct were qualitatively evaluated and any degenerative changes were quantified. Hair cell and neuronal loss patterns suggested two degenerative processes. In the basal half of the cochlea, inner and outer hair cell loss proceeded from base to apex with increasing age, and loss of cochlear neurons was consistent with degeneration occurring secondary to inner hair cell loss. In the apical half of the cochlea with advancing age, there was selective loss of outer hair cells which increased from the middle to the extreme apex. A similar gradient of ganglion cell loss was noted, characterized by widespread somatic aggregation and demyelination. In addition to these changes in hair cells and their innervation, there was widespread degeneration of fibrocytes in the spiral ligament, especially among the type IV cell class. The cell loss in the ligament preceded the loss of hair cells and/or neurons in both space and time suggesting that fibrocyte pathology may be a primary cause of the hearing loss and ultimate sensory cell degeneration in this mouse strain.     

豚鼠前庭感觉上皮损伤中细胞凋亡的观察

细胞凋亡对细胞增殖、器官发生和功能维持起着重要作用。一定剂量的庆大霉素连续注射，造成豚鼠前庭器官损伤。采用半薄切片，透射电镜（TEM）和TUNEL（TdT－modidedbiotin-dUTPNick-endlabeling，末端脱氧核苷酸转移酶介导的生物素标记）原位杂交技术，特异标记DNA片段3′－OH末端，原位显示凋亡细胞。在半薄切片和TEM观察中发现两种类型的细胞损伤方式：①毛细胞肿胀，胞浆空泡化，细胞体从顶端表面挤出；②毛细胞在上皮内变性，显示出细胞凋亡的形态特征，包括细胞核凝缩，核膜消失，成碎块状，并由支持细胞吞噬。原位杂交显示：细胞凋亡标记阳性细胞主要分布在上皮表层，较高水平标记主要发生于给药后第3到7天。提示细胞凋亡是内耳前庭感觉细胞损伤的一种重要方式，凋亡的主动发生可能是一种潜在的介入方式来减少氨基甙类抗生素对毛细胞造成的急性损伤，并与感觉上皮损伤后的修复过程有关。     

Distribution and time course of hair cell regeneration in the pigeon utricle.

Vestibular and cochlear regeneration following ototoxic insult from aminoglycoside antibiotics has been well documented, particularly in birds. In the present study, intraotic application of a 2 mg streptomycin paste was used to achieve complete vestibular hair cell destruction in pigeons (Columba livia) while preserving regenerative ability. Scanning electron microscopy was used to quantify hair cell density longitudinally during regeneration in three different utricular macula locations, including the striola, central and peripheral regions. The utricular epithelium was void of stereocilia (indicating hair cell loss) at 4 days after intraotic treatment with streptomycin. At 2 weeks the stereocilia began to appear randomly and mostly in an immature form. However, when present most kinocilia were polarized toward the developing striola. Initially, regeneration occurred more rapidly in the central and peripheral regions of the utricle as compared to the striola. As regeneration proceeded from 2 to 12 weeks, hair cell density in the striola region equaled the density noted in the central and peripheral regions. At 24 weeks, hair cell density of the central and peripheral regions was equal to normal values, however the striola region had a slightly greater hair cell density than that observed for normal animals.     

Blockade of c-Jun N-terminal kinase pathway attenuates gentamicin-induced cochlear and vestibular hair cell death

The ototoxic action of aminoglycoside antibiotics leading to the loss of hair cells of the inner ear is well documented. However, the molecular mechanisms are poorly defined. We have previously shown that in neomycin-exposed organotypic cultures of the cochlea, the c-Jun N-terminal kinase (JNK) pathway--associated with stress, injury and apoptosis--is activated in hair cells and leads to their death. We have also shown that hair cell death can be attenuated by CEP-1347, an inhibitor of JNK signalling [Pirvola et al., J. Neurosci. 20 (2000) 43-50]. In the present study, we demonstrate that gentamicin-induced ototoxicity leads to JNK activation and apoptosis in the inner ear hair cells in vivo. We also show that systemic administration of CEP-1347 attenuates gentamicin-induced decrease of auditory sensitivity and cochlear hair cell damage. In addition, CEP-1347 treatment reduces the extent of hair cell loss in the ampullary cristae after gentamicin intoxication. Particularly, the inner hair cells of the cochlea and type I hair cells of the vestibular organs are protected. We have previously shown that also acoustic overstimulation leads to apoptosis of cochlear hair cells and that CEP-1347 can attenuate noise-induced sensory cell loss. These results suggest that activation of the JNK cascade may be a common molecular outcome of cellular stress in the inner ear sensory epithelia, and that attenuation of the lesion can be provided by inhibiting JNK activation.     

Effects of exposing gonadectomized and intact C57BL/6J mice to a high-frequency augmented acoustic environment: Auditory brainstem response thresholds and cytocochleograms

Gonadectomized and surgically intact adult C57BL/6J (B6) mice of both sexes were exposed for 12h nightly to a high-frequency augmented acoustic environment (AAE): repetitive bursts of a half-octave noise band centered at 20 kHz, 70 dB SPL. The effects of sex, gonadectomy, and AAE treatment on genetic progressive hearing loss (exhibited by B6 mice) were evaluated by obtaining auditory brainstem response thresholds at ages 3-, 6-, and 9-months; hair cell counts (cytocochleograms) were obtained at 9 months. A sex difference in the rate of genetic progressive hearing loss in B6 mice (observed by earlier studies) was confirmed, with females exhibiting a faster rate of threshold elevations and more severe loss of hair cells at age 9 months. Gonadectomy had no consistent effects on the rate or severity of hearing loss in non-exposed mice of either sex. An unexpected finding was that the high-frequency AAE treatment caused additional ABR threshold elevations and hair cell loss. In an earlier study, the same high-frequency AAE treatment on DBA/2J mice ameliorated hearing loss. The most severe AAE-induced losses occurred in surgically intact females, suggesting a potentiating effect of ovarian hormone(s).     

Glutathione content and gamma-glutamyltranspeptidase activity in squamous cell head and neck cancer xenografts

Drug resistance is a major problem in chemotherapy of squamous cell head and neck cancers (SCHNC). Since glutathione (GSH) plays a crucial role in mediating tumor cell resistance against various toxic insults, GSH metabolism in SCHNC xenografts was investigated. Xenografts from lymph node metastases contained markedly higher GSH concentrations compared with those derived from the corresponding primary lesions. After subcurative chemotherapy with cisplatin (DDP), a significant increase of both GSH levels and gamma-glutamyltranspeptidase activity (gamma-GT) was gained in tumor HT1M. Tumor HT3M showed high concentrations of GSH and gamma-GT, although these latter concentrations did not increase following chemotherapy with DDP. These findings suggest a possible impact of GSH metabolism on both the formation of metastases and the phenomenon of drug resistance in SCHNC.     

Synaptophysin immunohistochemistry during vestibular hair cell recovery after gentamicin treatment

In the present study, morphometric and immunohistochemical techniques were used to evaluate the degree of synaptic recovery in the chinchilla crista sensory epithelia during various post-gentamicin-treatment periods of hair cell loss and recovery. For this purpose, two groups of animals were treated with Gelfoam pellets impregnated with 50 micro g of gentamicin implanted in the perilymphatic space within the otic capsule of the superior semicircular canal. Animals were sacrificed 1, 2 and 4 weeks after treatment. The degree of synaptic reinnervation was evaluated in the horizontal crista of the first group of animals using immunohistochemical techniques and antibodies against synaptophysin, a marker for synaptic reinnervation and synaptogenesis. Quantification of immunoreactivity in this group was made in the mid-region of the crista using the NIH 'Image' program. The second group of animals was used for quantification of the number of hair cells and supporting cells in the horizontal crista. In the normal sensory epithelium, synaptophysin immunoreactivity was found in the areas corresponding to the known distribution of afferent and efferent nerve terminals. Immunoreactivity was predominantly located within the afferent calyces of type I hair cells. No immunoreactivity was found in the supporting cells. Seven days after treatment there was a significant loss of hair cells and synaptophysin-stained area (SSA). In the mid-region of the crista the loss of synaptophysin immunoreactivity was quantitatively the greatest within the central zone of this region (93%) while the loss of hair cells was the smallest. These results suggest that afferent and efferent nerve terminals were also severely affected by the ototoxic treatment. Four weeks after treatment corresponding to the end of the recovery phase of gentamicin ototoxicity, there was a proportional increase in the number of hair cells and of the degree of SSA in the mid-region of the crista. The number of hair cells recovered to 58% with a recovery of SSA to 54% of normal. These results suggest that a greater fraction of synaptophysin expression within the sensory epithelium depends on the presence of afferent calyceal endings, which are greatly affected by gentamicin. Also, these results demonstrate a significant level of reinnervation of the newly regenerated hair cells, forecasting the potential for functionality of the regenerated hair cells.     

Serum antibody response and germinal centre reaction in the upper respiratory tract after antigen challenge in mice

The difference in the hearing threshold before and after treatment with cis-diaminnedichloroplatinum (DDP) is analysed in 69 patients. Hearing loss due to DDP treatment is mainly limited to 8,000 Hz and the incidence is about 40%. The effect of DDP is dose-related, although even at the lowest dose 20% of the patients are affected. The age of the patients is not an important factor. Loss of hearing due to DDP treatment occurs independent of any pre-existent hearing loss although those patients with great pre-existent hearing loss do not show a further loss. Of the patients with hearing loss, 39% show a difference of 20 dB or more between the left and the right ear. Hearing loss due to DDP is of minor importance compared with many of the other side-effects of DDP.     

Selective Inner Hair Cell Dysfunction in Chinchillas Impairs Hearing-in-Noise in the Absence of Outer Hair Cell Loss

Poorer hearing in the presence of background noise is a significant problem for the hearing impaired. Ototoxic drugs, ageing, and noise exposure can damage the sensory hair cells of the inner ear that are essential for normal hearing sensitivity. The relationship between outer hair cell (OHC) loss and progressively poorer hearing sensitivity in quiet or in competing background noise is supported by a number of human and animal studies. In contrast, the effect of moderate inner hair cell (IHC) loss or dysfunction shows almost no impact on behavioral measures of hearing sensitivity in quiet, when OHCs remain intact, but the relationship between selective IHC loss and hearing in noise remains relatively unknown. Here, a moderately high dose of carboplatin (75 mg/kg) that produced IHC loss in chinchillas ranging from 40 to 80 % had little effect on thresholds in quiet. However, when tested in the presence of competing broadband (BBN) or narrowband noise (NBN), thresholds increased significantly. IHC loss >60 % increased signal-to-noise ratios (SNRs) for tones (500–11,300 Hz) in competing BBN by 5–10 dB and broadened the masking function under NBN. These data suggest that IHC loss or dysfunction may play a significant role in listening in noise independent of OHC integrity and that these deficits may be present even when thresholds in quiet are within normal limits.     

Hair cell loss in the aged guinea pig cochlea

The various effects of ageing on the auditory system, collectively termed presbycusis, are being studied across a wide range of animal species, including humans. One contributing factor to presbycusis is thought to be losses of the sensory hair cells in the cochlea. In this study, hair cell counts were obtained from cochleas of pigmented guinea pigs (Cavia porcellus) at ages ranging from 11 days to 4 years 7 months, using scanning electron microscopy to visualize the organ of Corti. Representative samples of the basal, middle and apical turn of the cochlea were photographed for analysis. Hair cell loss was observed, even in young animals. However, the loss was greater in the aged animals, but was not distributed evenly throughout the length of the cochlea. No significant loss of hair cells was seen in the basal (high frequency) or middle turn of the cochlea of the aged animals. In the apical (low frequency) turn, there was a significant loss of hair cells in all rows of outer hair cells (up to around 20%), and was most severe in the third row. There was no loss of apical inner hair cells in the aged animals.