Destruction Of Hair Cells Research Articles

Effects of restricted basilar papillar lesions and hair cell regeneration on auditory forebrain frequency organization in adult European starlings.

The frequency organization of neurons in the forebrain Field L complex (FLC) of adult starlings was investigated to determine the effects of hair cell (HC) destruction in the basal portion of the basilar papilla (BP) and of subsequent HC regeneration. Conventional microelectrode mapping techniques were used in normal starlings and in lesioned starlings either 2 d or 6-10 weeks after aminoglycoside treatment. Histological examination of the BP and recordings of auditory brainstem evoked responses confirmed massive loss of HCs in the basal portion of the BP and hearing losses at frequencies >2 kHz in starlings tested 2 d after aminoglycoside treatment. In these birds, all neurons in the region of the FLC in which characteristic frequencies (CFs) normally increase from 2 to 6 kHz had CF in the range of 2-4 kHz. The significantly elevated thresholds of responses in this region of altered tonotopic organization indicated that they were the residue of prelesion responses and did not reflect CNS plasticity. In the long-term recovery birds, there was histological evidence of substantial HC regeneration. The tonotopic organization of the high-frequency region of the FLC did not differ from that in normal starlings, but the mean threshold at CF in this frequency range was intermediate between the values in the normal and lesioned short-recovery groups. The recovery of normal tonotopicity indicates considerable stability of the topography of neuronal connections in the avian auditory system, but the residual loss of sensitivity suggests deficiencies in high-frequency HC function.

Immunocytochemical Traits of Type IV Fibrocytes and Their Possible Relations to Cochlear Function and Pathology

One of the more consistent and least understood changes in the aging human cochlea is the progressive loss of fibrocytes within the spiral ligament. This report presents an animal model for type IV fibrocyte loss, along with immunocytochemical evidence that noise-induced loss of these cells may account for previously unexplained hearing losses. The remarkably low threshold for noise-induced loss of type IV fibrocytes, approximately 24 dB less than the threshold for adjacent hair cell destruction, may account for the prevalence of missing fibrocytes in humans. In mice, changes in the spectrum of traumatizing noise had little effect upon the site of loss of the fibrocytes, suggesting that the primary site of damage that induced the loss was the basal-most cochlear turn, a site expected to be damaged by all three noise bands. Type IV fibrocytes were found to immunostain for connective tissue growth factor (CTGF) and for transforming growth factor beta receptor 3, a receptor that is known to activate CTGF expression. Type IV fibrocytes lack immunostaining for adenosine triphosphatase and connexins that are key players in potassium ion uptake and transmission, which suggests that they play little, if any, role in potassium recycling from perilymphatic space to the endolymphatic space. Consequently, their loss probably does not directly reduce this process. Immunostaining for a receptor for CTGF, low-density-lipoprotein-related protein 1, indicated that CTGF acts as an autocrine and a paracrine agent within the cochlea. The lack of CTGF paracrine effects following noise-induced loss of type IV fibrocytes may account for previously unexplained hearing losses.

Evaluating the ototoxicity of topical piperacillin–tazobactam

With the increased use of ototopical ciprofloxacin solution, newly evolved bacterial fluoroquinolone resistance has also become more of a problem. The emergence of ciprofloxacin-resistant Pseudomonas aeruginosa (CRPA) has created a new therapeutic challenge in otology. We evaluated the ototoxicity of topical fortified piperacillin-tazobactam solution by performing experiments in young male albino guinea pigs (weight, 250-300 g each). Antimicrobial assay by direct contact test using scanning electron microscope was performed. Twenty guinea pigs (250-300 g) were treated with fortified piperacillin-tazobactam solution. In experimental group 1 (n=10), a gelfoam ball impregnated with 120 microl of fortified piperacillin-tazobactam was implanted on the round window membrane by a posterior approach. In experimental group 2 (n=10), 20 microl of fortified piperacillin-tazobactam was injected into the middle ear cavity via silicone tube with a small hole placed in the superior aspect of the bulla by drilling. The drug application through the tube was performed for 7 consecutive days. In control group (n=5), a gelfoam ball impregnated with 120 microl of gentamicin solution (80 mg/2 ml) was implanted on the round window membrane. The fortified piperacillin-tazobactam 20 microl treated CRPAs showed destruction of their cell membrane in antimicrobial assay by direct contact test. No significant difference in the mean auditory brainstem response (ABR) thresholds before and after drug administration was found for the experimental group. However, significant elevation of the mean ABR thresholds was found for the control group. In experimental group, scanning electron microscopy showed almost normal sterociliary arrangements and surface structure on the inner and outer hair cells. However, significant destruction of outer hair cells was identified in control group. This present study's data suggests that fortified piperacillin-tazobactam solution can be effectively used for topically treating CRPA otorrhea in those patients who suffer with chronic suppurative otitis media.

Pica Behavior Induced by Body Rotation in Mice

Objectives: To study whether rotational stimulus induced pica and whether the vestibular apparatus was necessary for obtaining rotation-induced pica in mice. Methods: Pica behavior in mice was investigated following 60 min of rotation once daily at 70 rpm (15 s on with 5 s off) for 3 consecutive days. After evaluating vestibular function and histology of vestibular epithelia, we examined rotation-induced kaolin intake, so-called pica, in sham-lesioned and chemically labyrinthectomized mice. Results: The labyrinthectomized mice exhibited loss of the contact righting and swimming capability while the destruction of hair cells of vestibular epithelia was observed. Moreover, mice subjected to rotation, but not labyrinthectomized mice, showed a significant increase in kaolin intake at the last 2 rotation sessions and the first postrotation session. Conclusions: The findings indicated that a functioning vestibular system is necessary for rotation-evocating pica in mice and thus pica can be a behavioral index of motion sickness in mice.

Aminoglycoside-Induced Degeneration of Adult Spiral Ganglion Neurons Involves Differential Modulation of Tyrosine Kinase B and p75 Neurotrophin Receptor Signaling

Aminoglycoside antibiotics induce sensorineural hearing loss by destroying hair cells of the organ of Corti, causing progressive secondary degeneration of primary auditory or spiral ganglion neurons (SGNs). Recent studies show that the p75 neurotrophin receptor (NTR) is aberrantly up-regulated under pathological conditions when the neurotrophin receptor tyrosine kinases (Trks) are presumptively down-regulated. We provide in vivo evidence demonstrating that degenerating SGNs induced an augmented p75NTR expression and a coincident reduction of TrkB expression in their peripheral processes. Nuclear transcription factors c-Jun and cyclic AMP response element-binding protein phosphorylated by p75NTR- and TrkB-activated signal pathways, respectively, also showed a corresponding differential modulation, suggesting an activation of apoptotic pathways, coupled to a loss of pro-survival neurotrophic support. Our findings identified brain-derived neurotrophic factor (BDNF) expression in hair and supporting cells of the adult cochlea, and its loss, specifically the mature form, would impair TrkB-induced signaling. The precursor of BDNF (pro-BDNF) is differentially cleaved in aminoglycoside-deafened cochleae, resulting in a predominant up-regulation of a truncated form of pro-BDNF, which colocalized with p75NTR-expressing SGN fibers. Together, these data suggest that an antagonistic interplay of p75NTR and TrkB receptor signaling, possibly modulated by selective BDNF processing, mediates SGN death in vivo.

Mononuclear phagocytes migrate into the murine cochlea after acoustic trauma

Acoustic injury results in destruction of hair cells and numerous nonsensory cells of the cochlea. How these injured structures undergo repair is not well understood. This study was designed to examine the cochlea for the presence of mononuclear phagocytes after tissue injury caused by noise damage. We used octave band noise (8--16 kHz) at three levels (106, 112, and 120 dB) for 2 hours and studied the mice at 1, 3, 7, and 14 days after noise exposure to determine how noise affected hearing thresholds, hair cell number, and tissue injury in the cochlea. Furthermore, we assessed the cochlea for presence of inflammation by performing immunohistochemistry for CD45, common leukocyte antigen. We counted the number of CD45(+) cells that were present in the cochlea at the above-mentioned time points after noise. CD45 is present on all bone marrow-derived white blood cells and is not otherwise expressed in the inner ear. We found that, after noise exposure, there is a large increase in CD45(+) cells. These marrow-derived cells are concentrated in the spiral ligament and spiral limbus, areas that are known to be susceptible to acoustic injury. It is possible that this inflammatory response plays a role in propagating cellular damage in these areas. Immunohistochemistry demonstrates that these cochlear cells are derived from the monocyte/macrophage lineage and serve a phagocytic function in the inner ear.

Vestibular cytotoxicity in gentamicin-treated frogs: a preliminary report

Objective The aim of this study is to determine the immediate effects of intraperitoneal doses of gentamicin (GM) which would result in variable degrees of destruction of crista ampullary hair cells of frogs. This information will serve as a baseline guide to cell regeneration experiments on the damaged vestibular sense organ. Study design The American bullfrog was administered daily intraperitoneal doses of 50, 100, 150, and 200 mg/kg of GM for 7 days. Animals were sacrificed 1 day after the final injection for cytomorphic evaluation. Histologically processed posterior semicircular duct cristae were resin-embedded, and tissue samples were subjected to serial cross sectioning of the crista from the periphery to the central zone using glass knives in an ultramicrotome. Stained sections were analyzed in light microscope using ocular grid micrometry. The areal density of nuclear profiles of the vestibular sensory and supporting cells (sensory cells [SNCs] and supporting cells [SPCs], respectively; number per square millimeter) and the nuclear diameter of SNCs were manually determined. Results A 7-day administration of GM produced noticeable quantitative alteration of the posterior crista hair cells and SPCs. Histological analysis revealed a significant decrease in the density of SNCs and a concomitant increase in the density of SPCs (1-way analysis of variance). Conclusion and significance The cytomorphic data derived from this study show that 4 doses of intraperitoneal gentamicin administered to the bullfrog caused a decline in the areal density of sensory hair cells of the posterior canal crista ampullaris. Also noted was an increase in the density of adjacent SPCs. Although speculative, the increase in SPC population could be a harbinger of regeneration of the vestibular hair cells as suggested by other investigators in different species. The significance of present observations will be helpful to initiate future studies related to recovery of SNCs in a similarly damaged frog ampullary organ. Through a standardized quantitative approach to the study of SNCs and SPCs of the crista organ, the vestibulo-toxicity of newly developed drugs can be assessed.

MDL 28170 Attenuates Gentamicin Ototoxicity

Aminoglycosides are highly effective antibiotics; however, their clinical utility is severely limited by their nephrotoxic and ototoxic side-effects. The selective destruction of hair cells in the inner ear by aminoglycoside antibiotics is thought to arise from a carefully orchestrated programme of cell death involving caspases and calcium activated proteases or calpains. To more fully evaluate the role of calpains in aminoglycoside ototoxicity, we applied the cell permeant, selective calpain inhibitor MDL 28170, to gentamicin (GM) treated cochlear organotypic cultures and evaluated the degree of hair cell damage at various drug concentrations. Mean hair cell losses in cochlear cultures treated for 24hours with 250, 500 and 1000 μM of GM were 17, 64 and 81%, respectively. Cochlear cultures treated with 200 μM of MDL 28170 alone for 24hours had no adverse effects on hair cell survival. However, the two highest doses of MDL 28170 (500 and 1000 μM) resulted in disorganization of hair cell rows, stereocilia d...

Direct inner ear infusion of dexamethasone attenuates noise-induced trauma in guinea pig

The protective effect of dexamethasone (DEX) against noise-induced trauma, as reflected in hair cell destruction and elevation in auditory brainstem response (ABR) sensitivity, was assessed in guinea pigs. The animals were administered DEX (1, 10, 100, and 1000 ng/ml) or artificial perilymph (AP) via a mini-osmotic pump directly into scala tympani and, on the fourth day after pump implantation, exposed to 120 dB SPL octave band noise, centered at 4 kHz, for 24 h. Animals receiving DEX demonstrated a dose-dependent reduction in noise-induced outer hair cell loss (significant at 1, 10 and 100 ng/ml DEX animals compared to AP control animals) and a similar attenuation of the noise-induced ABR threshold shifts, observed 7 days following exposure (significant at 100 ng/ml DEX animals compared to AP control animals). These physiological and morphological results indicate that direct infusion of DEX into the perilymphatic space has protective effects against noise-induced trauma in the guinea pig cochlea.

Caspase Activation in Hair Cells of the Mouse Utricle Exposed to Neomycin

Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating neomycin-induced hair cell death in this preparation.

Vocal Memory and Learning in Adult Bengalese Finches with Regenerated Hair Cells

Critical learning periods are common in vertebrate development. In many birds, song learning is limited by a critical period; juveniles copy songs from adult birds by forming memories of those songs during a restricted developmental period and then using auditory feedback to practice their own vocalizations. Adult songs are stable over time regardless of exposure to other birds, but auditory feedback is required for the maintenance of stable adult song. A technique was developed to reversibly deafen Bengalese Finches by destruction and regeneration of inner ear auditory hair cells. With this approach, we asked two questions about the plasticity of song information stored in the adult brain. First, do adult birds store memories or "templates" of their songs that exist independent of auditory reinforcement? Such memories could be used to control vocal output by acting as fixed models of song to which ongoing vocalizations are matched. Second, can adult song learning, which does not normally occur in this species, be induced by removing and then restoring hearing? Studying changes in adult song behavior during hair cell loss and regeneration revealed two findings: (1) adult birds store memories or templates of their songs that exist independent of auditory input and can be used to restore normal vocal behavior when hearing is restored; (2) under experimental circumstances, adult birds can be induced to acquire song material from other birds. Results suggest that, in Bengalese Finches, the degree of behavioral and neural plasticity in juvenile and adult birds may be less distinct that previously thought.

HSV amplicon-mediated neurotrophin-3 expression protects murine spiral ganglion neurons from cisplatin-induced damage.

Ototoxicity is a major dose-limiting side effect of cisplatin (DDP) administration due to its propensity to induce destruction of hair cells and neurons in the auditory system. Previous studies demonstrated that TrkC-expressing spiral ganglion neurons (SGN) are protected from the cytotoxic effects of DDP by localized delivery of the trophic factor neurotrophin-3 (NT-3). Successful in vivo implementation of such a therapy requires the development of an efficient gene delivery vehicle for expression of NT-3 within the cochlea. To this end, we constructed a herpes simplex virus (HSV) amplicon vector that expressed a c-Myc-tagged NT-3 chimera (HSVnt-3myc). Helper virus-free vector stocks were initially evaluated in vitro for their capacity to direct expression of NT-3 mRNA and protein. Transduction of cultured murine cochlear explants with HSVnt-3myc resulted in production of NT-3 mRNA and protein up to 3 ng/ml as measured over a 48-h period in culture supernatants. To determine whether NT-3 overexpression could abrogate DDP toxicity, cochlear explants were transduced with HSVnt-3myc or a murine intestinal alkaline phosphatase-expressing control vector, HSVmiap, and then exposed to cisplatin. HSVnt-3myc-transduced cochlear explants harbored significantly greater numbers of surviving SGNs than those infected with control virus. These data demonstrate that amplicon-mediated NT-3 transduction can attenuate the ototoxic action of DDP on organotypic culture. The potency of NT-3 in protecting spiral ganglion neurons from degeneration suggests that in vivo neurotrophin-based gene therapy may be useful for the prevention and/or treatment of hearing disorders.

Electrical stimulation of the auditory nerve: single neuron strength-duration functions in deafened animals.

Destruction of cochlear hair cells initiates degenerative changes within auditory nerve fibres (ANFs), including loss of peripheral processes and demyelination of the cell body. These changes are likely to affect the biophysical processes involved in action potential generation to an electrical stimulus. We measured the strength-duration relationship in acutely deafened (100% ANF survival) versus long-term deafened cochleae (approximately 15% ANF survival) by recording from single neurons in the central nucleus of the inferior colliculus (ICC). Input/output functions were constructed for 22 ICC neurons in response to stimulation of the auditory nerve using biphasic current pulses of 20-1000 micros/phase. Strength-duration curves were derived and found to be of the same general form for both acute and long-term deafened cochleae. While there was an increase in rheobase for neurons from long-term versus acute deafened cochleae, this increase was not statistically significant (p=0.097). In contrast, chronaxie--which is related to the membrane time constant--was significantly shorter in the long-term deafened cochleae (p = 0.004). This presumably reflects a shift in the site of action potential initiation to the larger diameter, heavily myelinated central axon as a result of the pathology. These changes in the site of action potential generation have implications for the delivery of charge to ANFs via cochlear implants.

Response of spiral ganglion neurones to cochlear hair cell destruction in the guinea pig.

Loss of ganglion cells after hair cell destruction in the mammalian cochlea continues to occur over a long period of time, with the possibility of more than one factor contributing to this process. Despite the absence of hair cells, some ganglion cells are, however, able to survive for considerable periods of time. Because functional ganglion cells are crucial to the successful use of cochlear implants, a better understanding of the response of these cells to injury is required so that their loss can be prevented or ameliorated. Quantitative light microscopy, electron microscopy and immunocytochemical techniques were used to examine the response of type I spiral ganglion neurones to hair cell destruction, in guinea pigs at 1, 3, 6 and 30 weeks survival following intracochlear injection with gentamicin. The time course of ganglion cell loss was determined, while a closer examination of those cells able to survive was carried out. A significant early loss of large numbers of ganglion cells was followed by a further significant loss of these cells by 30-week survival. At the same time a decrease in the numbers of central fibres was also observed. Surviving ganglion cells have little or no perikaryal myelin, an appearance resembling that of type I ganglion cells at 55 days gestation. Ganglion cells surviving the initial loss were also found to have a significantly larger soma size than controls although this was not maintained and at 30 weeks survival the few remaining cells were similar in size to that of controls. The growth associated protein GAP 43 was upregulated in surviving ganglion cells at 3 weeks survival, but appeared diminished by 6 weeks survival. These features may indicate a survival response in ganglion cells and may provide a basis on which to develop appropriate means to prevent their loss.

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.

Dizocilpine attenuates streptomycin-induced vestibulotoxicity in rats

NMDA receptor mediated excitotoxicity contributes substantially to aminoglycoside antibiotic-induced cochlear damage. Since vestibular as well as cochlear hair cells have glutamatergic synapses, aminoglycoside-induced vestibulotoxicity may also have an excitotoxic component. This hypothesis was tested by examining the effects of the uncompetitive NMDA receptor antagonist dizocilpine on streptomycin-induced vestibulotoxicity. Streptomycin-treated rats exhibited almost complete destruction of sensory hair cells in the crista ampullaris, vestibular impairment in the drop test, and hyperkinesia. Concurrent treatment with dizocilpine not only rescued a substantial population of sensory hair cells in the cristae, but prevented the attendant hyperkinesis and vestibular impairments. These results indicate that excitotoxic mechanisms contribute to aminoglycoside-induced vestibulotoxicity and that NMDA antagonists may be useful in attenuating aminoglycoside ototoxicity.

The effect of ruby laser light on ex vivo hair follicles: clinical implications.

Several clinical studies on the efficacy of ruby laser-assisted hair removal have reported that regrowth of hair after treatment is common. One of the reasons for the regrowth of hair is the incomplete destruction of germinative hair cells due to the insufficient penetration of the ruby laser in the skin. It was the aim of this study to estimate the extent of damage to the hair follicles after one ruby laser treatment and to determine whether the ruby laser destroyed the bulbs and the bulge regions of hair follicles. The extent of laser damage in hair shafts was determined by serial examination of six specimens of ex vivo scalp skin lasered with the Chromos 694 Depilation Ruby Laser at 14 J per square centimeter and 20 J per square centimeter. Another nine specimens of ex vivo scalp skin were similarly lasered, and monoclonal antibody LP2K was used to identify the bulge regions of the hair follicles using the immunoperoxidase technique. Damage to the bulge region was assessed from consecutive specimens, which were stained with hematoxylin-eosin stain. The mean depth of laser damage sustained by hair follicles was 1.34 mm (14 J per square centimeter) and 1.49 mm (20 J per square centimeter) underneath the skin surface. Most of the laser damage involved the bulge regions but fell short of the hair bulbs. The laser damage did not seem to extend far enough down the hair shafts to result in permanent hair destruction. The clinical implications of this finding are discussed.

4-Methylcatechol, a Potent Inducer of Nerve Growth Factor Synthesis, Protects Spiral Ganglion Neurons from Aminoglycoside Ototoxicity: Preliminary Report

The degeneration of spiral ganglion neurons after hair cell destruction following aminoglycoside ototoxicity is associated not only with the direct effect on the neurons, but also with a loss of neurotrophic factors provided by auditory hair cells. The neurotrophic factors, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), have been reported to be effective in enhancing neuronal survival against aminoglycoside ototoxicity in vivo . To determine whether 4-methylcatechol (4-MC), a potent inducer of nerve growth factor synthesis, can protect spiral ganglion neurons after aminoglycoside treatment in vivo , we studied the spiral ganglion neurons of gentamicin (GM)-treated mice with or without 4-MC administration. We found that the number of surviving spiral ganglion neurons of 4-MC-treated animals was significantly greater than for those treated with GM alone. Our results suggest that 4-MC promotes synthesis of NGF (and/or other neurotrophins), which enhances spiral ganglion neuron survival after gentamicin treatment.

Brain-derived neurotrophic factor gene therapy prevents spiral ganglion degeneration after hair cell loss: Second Place—Resident Clinical Science Award 1997

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. (Otolaryngol Head Neck Surg 1998;119:7-13.)

Is the round window membrane a portal for bacterial toxins? A putative mechanism for sensorineural hearing loss following acute otitis media

Aim: To investigate the hypothesis that bacterial toxins produced in the middle ear during acute otitis media (AOM), travel via the round window membrane (RWM) to cause cochlear damage and hence sensorineural hearing loss (SNHL). Methods: We investigated toxins produced by the two most common pathogens isolated from middle ear effusions in children. These were Pneumolysin, a product of S. pneumoniae and a cytoplasmic extract of Haemophilus influenza. Both electrophysiological measurements and morphological investigations were made after these toxins were placed on the RWM of guinea pigs. Control experiments were done using artificial perilymph as a substitute for the toxins. In each experiment one of the cochleas served as an internal control. Results: Losses in both the Cochlear Microphonics (CM) and Compound Action Potential (CAP) were observed indicating impaired hair cell function resulting in SNHL. Morphological studies showed hair cell destruction. Examination of the RWM exposed to pneumolysin showed disruption of this three-layered structure. Conclusions: The RWM is a probable portal for access to the inner ear by substances produced during AOM. Sensorineural hearing loss could result from the type of damage observed. More experiments are needed to verify this hypothesis and to determine the extent of damage that can be caused by these substances, especially with regard to the position of the damaged hair cells. Most of the damage observed occurred in the basal turn. Careful audiological examination (especially at high frequencies) of children who suffer with AOM might reveal a population of children with sensorineural hearing loss which might otherwise go undetected.