Three-dimensional quantification of fibrosis and ossification after cochlear implantation via virtual re-sectioning: Potential implications for residual hearing.

Hearing preservation may be achieved initially in the majority of patients after cochlear implantation, however, a significant proportion of these patients experience delayed hearing loss months or years later. A prior histological report in a case of delayed hearing loss suggested a potential cochlear mechanical origin of this hearing loss due to tissue fibrosis, and older case series highlight the frequent findings of post-implantation fibrosis and neoosteogenesis though without a focus on the impact on residual hearing. Here we present the largest series (N = 20) of 3-dimensionally reconstructed cochleae based on digitally scanned histologic sections from patients who were implanted during their lifetime. All patients were implanted with multichannel electrodes via a cochleostomy or an extended round window insertion. A quantified analysis of intracochlear tissue formation was carried out via virtual re-sectioning orthogonal to the cochlear spiral. Intracochlear tissue formation was present in every case. On average 33% (SD 14%) of the total cochlear volume was occupied by new tissue formation, consisting of 26% (SD 12%) fibrous and 7% (SD 6%) bony tissue. The round window was completely covered by fibro-osseous tissue in 85% of cases and was associated with an obstruction of the cochlear aqueduct in 100%. The basal part of the basilar membrane was at least partially abutted by the electrode or new tissue formation in every case, while the apical region, corresponding with a characteristic frequency of < 500 Hz, appeared normal in 89%. This quantitative analysis shows that after cochlear implantation via extended round window or cochleostomy, intracochlear fibrosis and neoossification are present in all cases at anatomical locations that could impact normal inner ear mechanics.

Single-neuron labeling in the cat auditory nerve.

Single auditory nerve fibers in the cat were labeled intracellularly with horseradish peroxidase. The sample of fibers was selected to represent different response types over a wide range of characteristic frequencies. All 56 labeled neurons were found to be radial fibers innervating inner hair cells, suggesting that none of the single-unit data reported to date has been from the outer hair cell innervation. Differences in rates of spontaneous discharge and thresholds to tones among these labeled neurons were closely correlated with morphological differences in the caliber and location of their unmyelinated terminals on the body of the inner hair cell.

Hair-cell innervation by spiral ganglion cells in adult cats.

A horseradish peroxidase technique was used to trace the peripheral terminations of two types of ganglion cells in adult cats. It was found that large, usually bipolar ganglion cells end on inner hair cells and small, usually pseudomonopolar ganglion cells end on outer hair cells. Thus, a virtually complete segregation of afferent neural inputs from the two types of hair cells was directly confirmed.

Primary Neural Degeneration in the Human Cochlea: Evidence for Hidden Hearing Loss in the Aging Ear.

The noise-induced and age-related loss of synaptic connections between auditory-nerve fibers and cochlear hair cells is well-established from histopathology in several mammalian species; however, its prevalence in humans, as inferred from electrophysiological measures, remains controversial. Here we look for cochlear neuropathy in a temporal-bone study of "normal-aging" humans, using autopsy material from 20 subjects aged 0-89 yrs, with no history of otologic disease. Cochleas were immunostained to allow accurate quantification of surviving hair cells in the organ Corti and peripheral axons of auditory-nerve fibers. Mean loss of outer hair cells was 30-40% throughout the audiometric frequency range (0.25-8.0 kHz) in subjects over 60 yrs, with even greater losses at both apical (low-frequency) and basal (high-frequency) ends. In contrast, mean inner hair cell loss across audiometric frequencies was rarely >15%, at any age. Neural loss greatly exceeded inner hair cell loss, with 7/11 subjects over 60 yrs showing >60% loss of peripheral axons re the youngest subjects, and with the age-related slope of axonal loss outstripping the age-related loss of inner hair cells by almost 3:1. The results suggest that a large number of auditory neurons in the aging ear are disconnected from their hair cell targets. This primary neural degeneration would not affect the audiogram, but likely contributes to age-related hearing impairment, especially in noisy environments. Thus, therapies designed to regrow peripheral axons could provide clinically meaningful improvement in the aged ear.

Role of alpha9 nicotinic ACh receptor subunits in the development and function of cochlear efferent innervation.

Cochlear outer hair cells (OHCs) express alpha9 nACh receptors and are contacted by descending, predominately cholinergic, efferent fibers originating in the CNS. Mice carrying a null mutation for the nACh alpha9 gene were produced to investigate its role(s) in auditory processing and development of hair cell innervation. In alpha9 knockout mice, most OHCs were innervated by one large terminal instead of multiple smaller terminals as in wild types, suggesting a role for the nACh alpha9 subunit in development of mature synaptic connections. Alpha9 knockout mice also failed to show suppression of cochlear responses (compound action potentials, distortion product otoacoustic emissions) during efferent fiber activation, demonstrating the key role alpha9 receptors play in mediating the only known effects of the olivocochlear system.

Blast-induced cochlear synaptopathy in chinchillas.

When exposed to continuous high-level noise, cochlear neurons are more susceptible to damage than hair cells (HCs): exposures causing temporary threshold shifts (TTS) without permanent HC damage can destroy ribbon synapses, permanently silencing the cochlear neurons they formerly activated. While this "hidden hearing loss" has little effect on thresholds in quiet, the neural degeneration degrades hearing in noise and may be an important elicitor of tinnitus. Similar sensory pathologies are seen after blast injury, even if permanent threshold shift (PTS) is minimal. We hypothesized that, as for continuous-noise, blasts causing only TTS can also produce cochlear synaptopathy with minimal HC loss. To test this, we customized a shock tube design to generate explosive-like impulses, exposed anesthetized chinchillas to blasts with peak pressures from 160-175 dB SPL, and examined the resultant cochlear dysfunction and histopathology. We found exposures that cause large >40 dB TTS with minimal PTS or HC loss often cause synapse loss of 20-45%. While synaptopathic continuous-noise exposures can affect large areas of the cochlea, blast-induced synaptopathy was more focal, with localized damage foci in midcochlear and basal regions. These results clarify the pathology underlying blast-induced sensory dysfunction, and suggest possible links between blast injury, hidden hearing loss, and tinnitus.

Divergent roles for thyroid hormone receptor beta isoforms in the endocrine axis and auditory system.

Thyroid hormone receptors (TRs) modulate various physiological functions in many organ systems. The TR alpha and TR beta isoforms are products of 2 distinct genes, and the beta 1 and beta 2 isoforms are splice variants of the same gene. Whereas TR alpha 1 and TR beta 1 are widely expressed, expression of the TR beta 2 isoform is mainly limited to the pituitary, triiodothyronine-responsive TRH neurons, the developing inner ear, and the retina. Mice with targeted disruption of the entire TR beta locus (TR beta-null) exhibit elevated thyroid hormone levels as a result of abnormal central regulation of thyrotropin, and also develop profound hearing loss. To clarify the contribution of the TR beta 2 isoform to the function of the endocrine and auditory systems in vivo, we have generated mice with targeted disruption of the TR beta 2 isoform. TR beta 2-null mice have preserved expression of the TR alpha and TR beta 1 isoforms. They develop a similar degree of central resistance to thyroid hormone as TR beta-null mice, indicating the important role of TR beta 2 in the regulation of the hypothalamic-pituitary-thyroid axis. Growth hormone gene expression is marginally reduced. In contrast, TR beta 2-null mice exhibit no evidence of hearing impairment, indicating that TR beta 1 and TR beta 2 subserve divergent roles in the regulation of auditory function.

Noise-induced cochlear synaptopathy in rhesus monkeys (Macaca mulatta).

Cochlear synaptopathy can result from various insults, including acoustic trauma, aging, ototoxicity, or chronic conductive hearing loss. For example, moderate noise exposure in mice can destroy up to ∼50% of synapses between auditory nerve fibers (ANFs) and inner hair cells (IHCs) without affecting outer hair cells (OHCs) or thresholds, because the synaptopathy occurs first in high-threshold ANFs. However, the fiber loss likely impairs temporal processing and hearing-in-noise, a classic complaint of those with sensorineural hearing loss. Non-human primates appear to be less vulnerable to noise-induced hair-cell loss than rodents, but their susceptibility to synaptopathy has not been studied. Because establishing a non-human primate model may be important in the development of diagnostics and therapeutics, we examined cochlear innervation and the damaging effects of acoustic overexposure in young adult rhesus macaques. Anesthetized animals were exposed bilaterally to narrow-band noise centered at 2 kHz at various sound-pressure levels for 4 h. Cochlear function was assayed for up to 8 weeks following exposure via auditory brainstem responses (ABRs) and otoacoustic emissions (OAEs). A moderate loss of synaptic connections (mean of 12-27% in the basal half of the cochlea) followed temporary threshold shifts (TTS), despite minimal hair-cell loss. A dramatic loss of synapses (mean of 50-75% in the basal half of the cochlea) was seen on IHCs surviving noise exposures that produced permanent threshold shifts (PTS) and widespread hair-cell loss. Higher noise levels were required to produce PTS in macaques compared to rodents, suggesting that primates are less vulnerable to hair-cell loss. However, the phenomenon of noise-induced cochlear synaptopathy in primates is similar to that seen in rodents.

Deletion of SLC19A2, the high affinity thiamine transporter, causes selective inner hair cell loss and an auditory neuropathy phenotype.

Mutations in the gene coding for the high-affinity thiamine transporter Slc19a2 underlie the clinical syndrome known as thiamine-responsive megaloblastic anemia (TRMA) characterized by anemia, diabetes, and sensorineural hearing loss. To create a mouse model of this disease, a mutant line was created with targeted disruption of the gene. Cochlear function is normal in these mutants when maintained on a high-thiamine diet. When challenged with a low-thiamine diet, Slc19a2-null mice showed 40-60 dB threshold elevations by auditory brainstem response (ABR), but only 10-20 dB elevation by otoacoustic emission (OAE) measures. Wild-type mice retain normal hearing on either diet. Cochlear histological analysis showed a pattern uncommon for sensorineural hearing loss: selective loss of inner hair cells after 1-2 weeks on low thiamine and significantly greater inner than outer hair cell loss after longer low-thiamine challenges. Such a pattern is consistent with the observed discrepancy between ABR and OAE threshold shifts. The possible role of thiamine transport in other reported cases of selective inner hair cell loss is considered.

Heat stress and protection from permanent acoustic injury in mice.

The inner ear can be permanently damaged by overexposure to high-level noise; however, damage can be decreased by previous exposure to moderate level, nontraumatic noise (). The mechanism of this "protective" effect is unclear, but a role for heat shock proteins has been suggested. The aim of the present study was to directly test protective effects of heat stress in the ear. For physiological experiments, CBA/CaJ mice were exposed to an intense octave band of noise (8-16 kHz) at 100 dB SPL for 2 hr, either with or without previous whole-body heat stress (rectal temperature to 41. 5 degrees C for 15 min). The interval between heat stress and sound exposure varied in different groups from 6 to 96 hr. One week later, inner ear function was assessed in each animal via comparison of compound action potential thresholds to mean values from unexposed controls. Permanent threshold shifts (PTSs) were approximately 40 dB in the group sound-exposed without previous heat stress. Heat-stressed animals were protected from acoustic injury: mean PTS in the group with 6 hr heat-stress-trauma interval was reduced to approximately 10 dB. This heat stress protection disappeared when the treatment-trauma interval surpassed 24 hr. A parallel set of quantitative PCR experiments measured heat-shock protein mRNA in the cochlea and showed 100- to 200-fold increase over control 30 min after heat treatment, with levels returning to baseline at 6 hr after treatment. Results are consistent with the idea that upregulation of heat shock proteins protects the ear from acoustic injury.

The spiral ganglion and Rosenthal's canal in beluga whales.

With the increase of human activity and corresponding increase in anthropogenic sounds in marine waters of the Arctic, it is necessary to understand its effect on the hearing of marine wildlife. We have conducted a baseline study on the spiral ganglion and Rosenthal's canal of the cochlea in beluga whales (Delphinapterus leucas) as an initial assessment of auditory anatomy and health. We present morphometric data on the length of the cochlea, number of whorls, neuron densities along its length, Rosenthal's canal length, and cross-sectional area, and show some histological results. In belugas, Rosenthal's canal is not a cylinder of equal cross-sectional area, but its cross-section is greatest near the apex of the basal whorl. We found systematic variation in the numbers of neurons along the length of the spiral ganglion, indicating that neurons are not dispersed evenly in Rosenthal's canal. These results provide data on functionally important structural parameters of the beluga ear. We observed no signs of acoustic trauma in our sample of beluga whales.

Central projections of auditory-nerve fibers of differing spontaneous rate. I. Anteroventral cochlear nucleus.

Auditory nerve fibers have been subdivided into three functional groups (Liberman, M.C. [1978] J. Acoust. Soc. Am. 63:442-455) differing in acoustic sensitivity and spontaneous discharge rate (SR). Using intracellular injection of horseradish peroxidase, the present study analyzes the projections of these three neuronal subclasses to the various subdivisions of the anteroventral cochlear nucleus (AVCN) and to the different cell types found therein. The average number of swellings and number of cells contacted decreased from low- to medium- to high-SR groups. However, these differences in terminal elaboration were not evenly distributed throughout the AVCN. The small cell cap was almost exclusively innervated by low- and medium-SR fibers, i.e., those with the highest acoustic thresholds. Within anterior AVCN, spherical-cell innervation was seen from all SR groups, whereas almost all multipolar cell innervation was from low- and medium-SR fibers. In the posterior AVCN, multipolar-cell innervation was equally likely from all SR groups, whereas globular cells were preferentially contacted by high-SR fibers. These SR-based trends in cochlear nucleus innervation help explain some of the known physiological properties of cell-types in each subdivision. They also suggest that additional physiological study of the small cell cap may be key in elucidating the functional significance of the low-SR population.

Central projections of auditory nerve fibers of differing spontaneous rate, II: Posteroventral and dorsal cochlear nuclei.

Response properties of auditory nerve fibers (ANFs), including threshold sensitivity, vary systematically with spontaneous discharge rate (SR) (Liberman, M.C.: J. Acoust. Soc Amer. 63:442-455, 1978). Thus, an understanding of the mechanisms underlying signal transformation in the cochlear nucleus (CN) must include a description of any SR-based difference in ANF projections. This study is the second of a pair describing the CN projections of intracellularly labeled ANFs of known SR, the first of which summarized projection to the anteroventral CN (Liberman, M.C.: J. Comp. Neurol. 313:240-258, 1991). For each swelling from each labeled fiber, the position (within CN subdivisions), the size, and the type of cell contacted (if determinable) was noted: roughly one in four labeled swellings appeared in intimate contact with the soma or proximal dendrites of a CN cell. In all such cases, cell size and swelling size were measured. As reported for auteroventral cochlear nucleus, the ANF innervation of the small-cell regions of posteroventral CN (PVCN) was almost exclusively by low- and medium-SR fibers. Other significant SR-based trends in ANF projections included 1) a tendency for high-SR fibers to contact larger cells in PVCN, 2) a meager projection of low- and medium-SR fibers to octopus cells, and 3) a tendency in the dorsal CN (DCN) for low-SR terminals to end closer to the fusiform cell layer than high-SR terminals. There were no significant SR-based difference in ANF swelling sizes in any subdivision. A consideration of the average cell sizes, ANF swelling sizes and estimated numbers of ANFs of different CF and SR converging on each CN cell help explain some of the differences in response transformation associated with different cell types in the CN.

Afferent innervation of outer hair cells in adult cats: I. Light microscopic analysis of fibers labeled with horseradish peroxidase.

Outer spiral fibers (OSFs), the afferent innervation of the outer hair cells (OHCs), were retrogradely labeled following horseradish peroxidase injections into the cat's auditory nerve. The peripheral branching patterns of 85 OSFs from adult cochleas were reconstructed. Fibers contacted OHCs via terminal or en passant swellings; however, the latter were seen exclusively in the apical half of the cochlea. Many OSFs also gave off branches ending on structures other than OHCs. Fibers in the cochlear apex were much more highly branched than in the base. Most fibers contacted only one row of OHCs, and more fibers contacted row 1 than row 2 or row 3 OHCs. Third-row fibers were the most highly branched in all cochlear regions. These results are consistent with a growing body of morphological evidence that suggests that the peripheral branching patterns of OSFs may be fundamentally similar in all mammalian ears.

Afferent innervation of outer hair cells in adult cats: II. Electron microscopic analysis of fibers labeled with horseradish peroxidase.

Outer spiral fibers (OSFs) were retrogradely labeled with injections of horseradish peroxidase. After the peripheral arborization patterns were reconstructed at the light microscopic level (Simmons and Liberman, '88), restricted regions of selected fibers were analyzed via electron-microscopic reconstruction of serial sections. The ultrastructural data in the present study suggested that the contact between the outer hair cell (OHC) and the terminal swellings of OSFs corresponds to the afferent synapse described in numerous other ultrastructural studies. The en passant swellings that contacted OHCs also appeared to be points of synaptic contact. However, en passant synapses were not always associated with a swelling of the OSF at the point of contact: thus, the light-microscopic reconstructions probably underestimate the numbers of synapses. OSF branches terminating well below the OHCs were seen to end most commonly in intimate contact with the Deiters' cells. Membrane specialization was occasionally seen at this point of contact; however, the specialization was sufficiently undifferentiated to preclude identification.

Intracellular labeling of auditory nerve fibers in guinea pig: central and peripheral projections.

Auditory-nerve fibers (ANFs) in the cat have been subdivided according to spontaneous rate (SR), with high-SR fibers showing the lowest thresholds. Cochlear terminals of the three SR groups differ in caliber and synaptic position around the inner hair cell (Liberman [1982b] Science 216:1239-1241); central terminals differ in degree of branching and in which subregions of the cochlear nucleus (CN) are targeted (Liberman [1991] J. Comp. Neurol. 313:240-258). The present study investigates whether these SR-based differences in ANF connections are unique to the cat. Thirty ANFs from 15 guinea pigs were intracellularly labeled after measuring characteristic frequency, threshold, and SR. Labeled cochlear projections showed significant SR-based differences in axonal caliber, with low- and medium-SR fibers 20-40% thinner than those of high-SR fibers for both peripheral and central (modiolar) axons. Spatial segregation in the inner hair cell area could not be assessed; however, the peripheral axons in the osseous spiral lamina showed the same SR-based organization reported for the cat (Kawase and Liberman [1992] J. Comp. Neurol. 319:312-318). Labeled central projections also showed significant SR-based differences. Low- and medium-SR fibers: 1) were more highly branched, 2) sent significantly more terminals to the small-cell cap region of the CN, and 3) produced endbulb terminals (on spherical cells) that were significantly more complex than high-SR fibers. All of these SR-based trends for both central and peripheral projections are analogous to those reported in the cat, and, thus, may represent a fundamental organizational principle of the mammalian ear.

Spatial organization of the auditory nerve according to spontaneous discharge rate.

Auditory-nerve fibers in mammals have been classified into three functional subclasses according to spontaneous discharge rate (SR). In cat, the peripheral terminals of these SR groups are segregated around the sensory cell circumference (Liberman, '82, Science 216:1239-1241). The present study shows that this spatial segregation is at least partly maintained through the peripheral axonal course from sensory cell to spiral ganglion. Analysis of intracellularly labeled auditory-nerve fibers shows that peripheral axons and cell bodies of low- and medium-SR fibers tend to be found closer to scala vestibuli than high-SR fibers. Since low- and medium-SR fibers tend to be thinner, this SR-based segregation can also be demonstrated as a fiber-caliber gradient in the osseous spiral lamina. The issue of SR-based spatial segregation is relevant to reports that ganglion cells near scala vestibuli project to different regions of the cochlear nucleus than cells near scala tympani (Leake and Snyder, '89, J. Comp. Neurol. 281:612-629). Combining the results of the two studies suggests that there may be some SR-based spatial segregation of inputs to the cochlear nucleus.

Morphometry of intracellularly labeled neurons of the auditory nerve: correlations with functional properties.

Single auditory-nerve fibers were injected with horseradish peroxidase after their tuning properties, characteristic frequencies, and spontaneous discharge rates were measured. From these functional properties virtually all other aspects of auditory-nerve response can be predicted. Labeled fibers were reconstructed from the point of peripheral termination on cochlear hair cells to the point at which they enter the cochlear nucleus. Several morphological properties were measured at the light-microscopic level, including axonal diameter, axonal length, internodal distances, cell-body area, and cell-body shape. All of these parameters were correlated, though some weakly, with characteristic frequency. However, only axonal diameter was correlated with spontaneous discharge rate.

Ultrastructural differences among afferent synapses on cochlear hair cells: correlations with spontaneous discharge rate.

The major class of cochlear afferent fibers, the type-I or radial-fiber (RF) population, has been subdivided into three functional groups according to spontaneous discharge rate (SR): those with low SR have the highest acoustic thresholds, high SR fibers have the lowest thresholds and medium SR fibers are of intermediate sensitivity (Liberman [1978] J. Acoust. Soc. Amer. 63:442-455). Existing evidence from intracellular labeling studies at the light microscopic level (Liberman [1982a] Science 216:1239-1241) suggests that a single cochlear inner hair cell makes synaptic contact with representatives of all three functional groups; however, low and medium SR fibers are spatially segregated from high SR fibers around the hair cell circumference, and low and medium SR fibers are smaller in caliber than those with high SR. The present study extends to the ultrastructural level the structure-function correlations available via intracellular labeling. Analysis is based on serial section reconstruction of the synaptic contacts between 11 radial fibers of known SR and their target hair cells. Results suggest systematic differences in synaptic ultrastructure among fibers of the three SR groups: with decreasing SR, the size and complexity of the synaptic body (a presynaptic specialization characteristic of the peripheral afferent synapses in all hair cell systems and some other peripheral receptors) tend to increase, as does the associated number of synaptic vesicles. The possible functional significance of these trends is discussed in the context of other known structural and functional differences among the three SR groups.

Morphological subclasses of lateral olivocochlear terminals? Ultrastructural analysis of inner spiral bundle in cat and guinea pig.

The lateral olivocochlear efferent pathway terminates in vesicle-filled swellings in the inner spiral bundles under inner hair cells (IHCs) and has been suggested to include at least two chemically distinct subclasses (see, e.g., Vetter et al. [1991] Synapse 7:21-43). In the present study, the ultrastructure and peripheral targets of vesicle-filled swellings in the IHC area of the cat and guinea pig cochleas were quantitatively analyzed to determine 1) whether morphological subclasses could be defined based on swelling size or on the density, size or shape of clear and dense-cored vesicles and 2) whether swellings with different postsynaptic targets differed morphologically. In both cat and guinea pig, all swellings contained large, round, clear vesicles and a variable number of dense-core vesicles. Although evidence of clear-cut subclasses was not compelling, the smallest swellings tended to be rich in dense-core and poor in clear vesicles and rarely formed synaptic contacts. Most of the larger swellings, which tended to contain few dense-core vesicles and a rich complement of clear round vesicles, formed synapses with radial afferent fibers. However, there were no morphological differences between swellings contacting afferents originating on the modiolar vs. pillar sides of the IHC (the source of afferents with low and high spontaneous discharge rates, respectively). We conclude that 1) if distinct gamma-amino butyric acid (GABA)ergic and cholinergic subclasses of lateral olivocochlear (LOC) fibers exist, then the vesicle morphology of their terminals does not differ as it does in the central nervous system and that 2) if peptide neurotransmitters, such as calcitonin gene-related peptide and enkephalins, are packaged in dense-core vesicles, then the LOC terminals synapsing with IHC afferent fibers are not particularly rich in these peptides.