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.     

Functional recovery in the avian ear after hair cell regeneration.

Trauma to the inner ear in birds, due to acoustic overstimulation or ototoxic aminoglycosides, can lead to hair cell loss which is followed by regeneration of new hair cells. These processes are paralleled by hearing loss followed by significant functional recovery. After acoustic trauma, functional recovery is rapid and nearly complete. The early and major part of functional recovery after sound trauma occurs before regenerated hair cells become functional. Even very intense sound trauma causes loss of only a proportion of the hair cell population, mainly so-called short hair cells residing on the abneural mobile part of the avian basilar membrane. Uncoupling of the tectorial membrane from the hair cells during sound overexposure may serve as a protection mechanism. The rapid functional recovery after sound trauma appears not to be associated with regeneration of the lost hair cells, but with repair processes involving the surviving hair cells. Small residual functional deficits after recovery are most likely associated with the missing upper fibrous layer of the tectorial membrane which fails to regenerate after sound trauma. After aminoglycoside trauma, functional recovery is slower and parallels the structural regeneration more closely. Aminoglycosides cause damage to both types of hair cells, starting at the basal (high frequency) part of the basilar papilla. However, functional hearing loss and recovery also occur at lower frequencies, associated with areas of the papilla where hair cells survive. Functional recovery in these low frequency areas is complete, whereas functional recovery in high frequency areas with complete hair cell loss is incomplete, despite regeneration of the hair cells. Permanent residual functional deficits remain. This indicates that in low frequency regions functional recovery after aminoglycosides involves repair of nonlethal injury to hair cells and/or hair cell-neural synapses. In the high frequency regions functional recovery involves regenerated hair cells. The permanent functional deficits after the regeneration process in these areas are most likely associated with functional deficits in the regenerated hair cells or shortcomings in the synaptic reconnections of nerve fibers with the regenerated hair cells. In conclusion, the avian inner ear appears to be much more resistant to trauma than the mammalian ear and possesses a considerable capacity for functional recovery based on repair processes along with its capacity to regenerate hair cells. The functional recovery in areas with regenerated hair cells is considerable but incomplete.     

Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma

Damage to the tectorial membrane caused by acoustic trauma was examined with scanning and transmission electron microscopy immediately after exposure and at selected time points over a 10 day recovery period. At 0 h of recovery the structure of the tectorial membrane overlying the region of hair cell damage was severely disrupted and connections between the membrane and the basilar papilla were lost. By 24 h of recovery, regeneration of the tectorial membrane was evident in the secretion of new matrix materials by the supporting cells of the basilar papilla. By 10 days of recovery a new honeycomb-like matrix had replaced the segment of damaged tectorial membrane, re-established connections with hair cell stereocilia and become fused with adjacent regions of undamaged tectorial membrane. However, the regenerated segment included only the honeycomb-like structure of the lower layer of the normal tectorial membrane. The laterally-oriented fibers which form the upper layer of the membrane were not regenerated over the damaged region. These findings indicate that the tectorial membrane is regenerated in parallel with the hair cells during recovery from acoustic trauma but the full extent of this recovery and its effect on cochlear function are not yet clear.     

Sensory hair cell death and regeneration: two halves of the same equation

PURPOSE OF REVIEW: Sensory hair cells are susceptible to ototoxic damage from a variety of sources, including antibiotic treatment. Unfortunately, this often results in permanent hearing and/or balance problems in humans. By understanding how sensory hair cells die in response to aminoglycoside treatment, preventive strategies may be developed. This review will discuss some of the key recent findings in sensory hair cell death and regeneration. RECENT FINDINGS: Aminoglycosides induce hair cell death through the initiation of apoptosis. Early and late stages of hair cell apoptosis have been defined, and several of the key molecules involved in the cascade have been identified. Moreover, specific inhibitors of apoptosis rescue hair cells from death and preserve function. Hair cell death has been shown to induce regeneration through supporting cell transdifferentiation, proliferation, and new hair cell differentiation in birds and lower vertebrates. Regeneration in the mammalian cochlea does not occur spontaneously, but genetic manipulation of cell cycle genes, induction of new hair cells through gene therapy, and introduction of stem cells into damaged cochleas suggest that repair and replacement of lost hair cells in the organ of Corti may be possible. Finally, continuing investigations of the mouse, zebrafish, and human genomes may one day enable manipulation of the cochlea so that functional regeneration is readily available as a therapeutic intervention. SUMMARY: The discovery that hair cells can regenerate in birds and other nonmammalian vertebrates has fueled a wide range of studies to find ways to restore hearing and balance in mammals. The demonstration that apoptosis and proliferation are coupled as controlling factors in regeneration and the advent of new approaches such as gene therapy, stem cell transplantation, and genomics may lead to methods for inducing hair cell regeneration and repair in the mammalian cochlear and vestibular systems.     

cAMP-induced Auditory Supporting Cell Proliferation is Mediated by ERK MAPK Signaling Pathway

Sensorineural hearing deficiencies result from the loss of auditory hair cells. This hearing loss is permanent in humans and mammals because hair cells are not spontaneously replaced. In other animals such as birds, this is not the case. Damage to the avian cochlea evokes proliferation of supporting cells and the generation of functionally competent replacement hair cells. Signal transduction pathways are clinically useful as potential therapeutic targets, so there is significant interest in identifying the key signal transduction pathways that regulate the formation of replacement hair cells. In a previous study from our lab, we showed that forskolin (FSK) treatment induces auditory supporting cell proliferation and formation of replacement hair cells in the absence of sound or aminoglycoside treatment. Here, we show that FSK-induced supporting cell proliferation is mediated by cell-specific accumulation of cyclic adenosine monophosphate (cAMP) in avian supporting cells and the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. By a combination of immunostaining and pharmacological analyses, we show that FSK treatment increases cAMP levels in avian auditory supporting cells and that several ERK MAP inhibitors effectively block FSK-induced supporting cell proliferation. Next, we demonstrate by Western blotting and immunostaining analyses the expression of several ERK MAPK signaling molecules in the avian auditory epithelium and the cell-specific expression of B-Raf in avian auditory supporting cells. Collectively, these data suggest that FSK-induced supporting cell proliferation in the avian auditory epithelium is mediated by increases of cAMP levels in supporting cells and the cell-specific expression of the ERK MAPK family member B-Raf in supporting cells.     

Anatomical correlates of functional recovery in the avian inner ear following aminoglycoside ototoxicity

Tucci and Rubel have demonstrated functional recovery of the chick cochlea following aminoglycoside ototoxicity. The cochleae of these same animals were examined by scanning electron microscopy (SEM) in order to further understand this recovery process. Hatchling chicks were given daily doses of gentamicin for 10 days. Auditory-evoked potential measurements and examination of the cochlea by scanning electron microscopy were performed after survival periods of 5 days to 20 weeks. After 5 days of gentamicin exposure, there was near complete basal hair cell loss associated with a high-frequency hearing loss. Apical progression of damage with a broad-band hearing loss occurred over 4 weeks. At 20-weeks, hair cell counts were normal with a small high-frequency hearing loss. Hair cell regeneration played a major role in the functional recovery of the cochlea.     

Hair cell regeneration in the avian cochlea: if it works in birds, why not in man?

Hearing loss caused by cochlear hair cell loss is the most common process afflicting the hearing impaired. Recent studies in the avian cochlea following ototoxic drug and noise damage have demonstrated a remarkable capacity for anatomical and functional recovery. Hair cell regeneration has been shown to play a major role in this recovery process. Future studies may one day make hair cell regeneration or transplantation possible in man.     

鸡内耳盖膜的超微结构观察

为了解鸟纲内耳盖膜的超微结构及其与毛细胞、支持细胞的关系，应用扫描电镜和透射电镜对鸡内耳盖膜的超微结构进行观察。发现：扫描电镜下盖膜横断面呈三角形，内有许多空洞，内淋巴面呈网状；透射电镜下见盖膜由微丝及肢状基质构成，听毛细胞的最高排静纤毛及动纤毛插入盖腹中，支持细胞的微绒毛通过絮状细丝与盖膜呈锚状连接。这种连接既有弹性又十分牢固，可在一定范围内限制盖膜运动。由此推测鸟纲内耳与哺乳纲内耳在感受声刺激的方式上有所不同。     

Hair cell regeneration and recovery of auditory thresholds following aminoglycoside ototoxicity in Bengalese finches

Birds regenerate auditory hair cells when original hair cells are lost. Regenerated hair cells become innervated and restore hearing function. Functional recovery during hair cell regeneration is particularly interesting in animals that depend on hearing for vocal communication. Bengalese finches are songbirds that depend on auditory feedback for normal song learning and maintenance. We examined the structural and functional recovery of the Bengalese finch basilar papilla after aminoglycoside ototoxicity. Birds were treated with the ototoxic aminoglycoside, amikacin, daily for 1 week. Treatment resulted in hair cell loss across the basal half of the basilar papilla and corresponding high frequency hearing loss. Hair cell regeneration and recovery of auditory brainstem responses were compared in the same animals. Survival times following treatment were between 1 day and 12 weeks. Analysis of structural recovery at weekly intervals indicated that hair cells in the Bengalese finch papilla require a maximum of 1 week to regenerate and appear with immature morphology at the epithelial surface. An additional 6 days are required for adult-like morphology to develop. Repopulation of the damaged region was complete by 8 weeks. Recovery of auditory thresholds began 1 week after treatment and reached asymptote by 4 weeks. Slight residual threshold shifts at 2.0 kHz and above were observed up to 12 weeks after treatment. Direct comparison of structural and functional recovery indicates that auditory thresholds recover maximally before a full complement of hair cells has regenerated.     

Structural relationships of the unfixed tectorial membrane

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.     

Histopathological differences between temporary and permanent threshold shift

The structural changes associated with noise-induced temporary threshold shift (TTS) were compared to the damage associated with permanent threshold shift (PTS). A within-animal paradigm involving survival-fixation was used to minimize problems with data interpretation from interanimal variability in response to noise. Auditory brainstem response thresholds for clicks and tone pips were determined pre- and 1-2 h post-exposure in 11 chinchillas. The animals were exposed for 24 h to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 86 dB. Three animals (0/0-day) had both cochleas terminal-fixed 2-3 h post-exposure. Two animals (27/27-day) had threshold shifts determined every other day for 1 week, every week thereafter, and underwent terminal-fixation of both cochleas 27 days after exposure. Six animals (0/n-day) had threshold shifts determined in both ears upon removal from the noise; their left cochlea was then survival-fixed 2-3 h post-exposure. Threshold shifts were determined in their right ear every 2-3 days until their hearing either returned to pre-exposure values or stabilized at a reduced level at which time their right cochlea was terminal-fixed (4-13 days post-exposure). All cochleas were prepared as plastic-embedded flat preparations. Missing hair cells were counted and supporting cells and nerve fibers were evaluated throughout the organ of Corti using phase-contrast microscopy. Post-exposure, all animals had moderate TTSs in their left and right ears which averaged 43 dB for 4-12 kHz. In the 0/0-day animals, the only abnormality which correlated with TTS was a buckling of the pillar bodies. In the 0/n-day animals, their left cochlea (survival-fixed 2-3 h post-exposure) had outer hair cell (OHC) stereocilia which were not embedded in the tectorial membrane in the region of the TTS whereas OHC stereocilia were embedded in the tectorial membrane throughout the cochleas of non-noise-exposed, survival-fixed controls. Three of six right cochleas (terminal-fixed 4-13 days post-exposure) from the 0/n-day animals developed a PTS and two of these cochleas had focal losses of inner and outer hair cells and afferent nerve fibers at the corresponding frequency location. The other cochlea with PTS had buckled pillars in the corresponding frequency region. These results suggest that with moderate levels of noise exposure, buckling of the supporting cells results in an uncoupling of the OHC stereocilia from the tectorial membrane which results in a TTS. The mechanisms resulting in TTS appear to be distinct from those that produce permanent hair cell damage and a PTS.     

支持细胞在哺乳动物耳蜗发育及功能中的作用

耳蜗的感觉上皮细胞有毛细胞和支持细胞两种类型.许多研究旨在探讨听力损失的基本机制,重点针对毛细胞和螺旋神经元,较少关注支持细胞.目前对支持细胞作用的了解已从单纯的结构支持扩展到耳蜗毛细胞分化发育、耳蜗离子稳态、毛细胞感音功能的调节以及突触发生发育、毛细胞损伤修复的多个方面.本文对支持细胞在哺乳动物耳蜗发育及功能中的关键作用做一综述.     

Hair cell regeneration in the chicken cochlea following aminoglycoside toxicity.

Hair cell loss in the avian cochlea partially recovers following both acoustic trauma and aminoglycoside intoxication. DNA labeling with tritiated thymidine has shown that the restoration of cell number following acoustic trauma results from the production of new hair cells by mitotic division. The purpose of the present study was to determine if mitosis also contributes to the recovery of hair cell number which occurs following aminoglycoside intoxication. Chickens received daily injections of either gentamicin sulfate or distilled water for 10 consecutive days. During the latter 7 days of this period, all birds were also injected with [3H]thymidine. Following postinjection survival periods of 3 or 6 days, one papilla from each bird was processed for autoradiography and the other for scanning electron microscopy (SEM). Incorporation of [3H]thymidine was seen over hair cells and support cells in experimental papillae in regions of hair cell loss. No labeling was seen outside of damaged regions or in the papillae of control birds. SEM showed that damaged regions in experimental birds contained cells similar in appearance to developing auditory hair cells in avian embryos. These results show that the restoration of hair cell number following aminoglycoside toxicity results from the production of new cells by mitosis.     

Possible precursors of regenerated hair cells in the avian cochlea following acoustic trauma

Hair cell regeneration following acoustic trauma to the avian cochlea has been documented using DNA labeling with tritiated thymidine. The goal of this study was to identify potential precursor cell populations for regenerating hair cells. Chicks were exposed in pairs to a 1500 Hz pure tone at 120 dBSPL for 18 h. The animals received repeated injections of 3H-thymidine over a survival period of 6, 15, or 24 h, 3 days or 30 days after the completion of noise exposure. One cochlea from each animal was processed for autoradiography and the other for scanning electron microscopy. Labeled, regenerated hair cells were present by 3 days after exposure and recovery from injury was nearly complete by 30 days. Examination of animals in short survival groups suggest that two precursor populations may exist. For inferior sensory epithelial damage, cuboidal or hyaline epithelial cells appear to serve as the precursor cell population for the regeneration of both hair cells and supporting cells. With isolated superior damage, however, supporting cells may be the precursor population.     

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.     

Acoustic overstimulation alters the morphology of the tectorial membrane

After a permanent threshold shift was induced by exposing guinea pigs to a 1 kHz pure tone at 105 dB(A) for 72 h, light microscopic observations of freshly dissected and stained tectorial membranes showed an increased waviness and clumping of the fibers of the middle zone. Hensen's stripe was not seen as a continuous dense structure running through the middle zone but was at times discontinuous and curved. As measured from cross-sections of the cochlea, the thickness of the tectorial membrane was decreased after acoustic overstimulation. The stereocilia of the inner and outer hair cells lie directly under the middle zone. Visual detection levels of threshold of tectorial membrane movement was determined by stimulating the marginal zone of the tectorial membrane of isolated cochlear coils by an oscillating water jet. After acoustic overstimulation the tectorial membrane became more compliant. The tectorial membrane abnormalities were restricted to the regions of the cochlea that demonstrated a 40–50 dB hearing loss.     

Guinea pig tectorial membrane profile in an in vitro cochlear preparation

The guinea pig cochlea was examined under high-magnification light microscopy in an in vitro preparation. After extraction of the otic capsule, the bulla was opened widely and a small hole made into the fourth turn of the scala vestibuli. The organ of Corti was visualized under artificial endolymph at 600 X magnification. Added 1-micron titanium dioxide particles settled on the upper surface of the transparent tectorial membrane. Particle positions showed that much of this upper surface lay in a flat sheet that extended centrifugally almost to the Hensen's cells, giving the impression it was attached there. The sheet extended at least to the level of the inner hair cells, where a tectorial membrane thickness of about 40 micron was reached. Titanium dioxide particles were seen regularly in immediate proximity to the hair cell cilia, indicating that scala media is continuous with the subtectorial space. Upon mechanical manipulation, Hensen's cells proved to be extremely cohesive and elastic. It is suggested that hair cell stereocilia provide major mechanical connections for the tectorial membrane.     

Hair cell regeneration after local application of gentamicin at the round window of the cochlea in the pigeon

Hair cells in the basilar papilla of birds have the capacity to regenerate after injury. Methods commonly used to induce cochlear damage are systemic application of ototoxic substances such as aminoglycoside antibiotics or loud sound. Both methods have disadvantages. The systemic application of antibiotics results in damage restricted to the basal 50% of the papilla and has severe side effects on the kidneys. Loud sound damages only small parts of the papilla and is restricted to the short hair cells. The present study was undertaken to determine the effect of local aminoglycoside application on the physiology and morphology of the avian basilar papilla. Collagen sponges loaded with gentamicin were placed at the round window of the cochlea in adult pigeons. The time course of hearing thresholds was determined from auditory brain stem responses elicited with pure tone bursts within a frequency range of 0.35–5.565 kHz. The condition of the basilar papilla was determined from scanning electron micrographs. Five days after application of the collagen sponges loaded with gentamicin severe hearing loss, except for the lowest frequency tested, was observed. Only at the apical 20% of the basilar papilla hair cells were left intact, all other hair cells were missing or damaged. At all frequencies there was little functional recovery until day 13 after implantation. At frequencies above 1 kHz functional recovery occurred at a rate of up to 4 dB/day until day 21, beyond that day recovery continued at a rate below 1 dB/day until day 48 at the 5.6 kHz. Below 1 kHz recovery occurred up to day 22, the recovery rate was below 2 dB/day. A residual hearing loss of about 15–25 dB remained at all frequencies, except for the lowest frequency tested. At day 20 new hair cells were seen on the basilar papilla. At day 48 the hair cells appeared to have recovered fully, except for the orientation of the hair cell bundles. The advantage of the local application of the aminoglycoside drug over systemic application is that it damages almost all hair cells in the basilar papilla and it has no toxic side effects. The damage is more extensive than with systemic application.     

Characterization of supporting cell phenotype in the avian inner ear: implications for sensory regeneration

The avian inner ear possesses a remarkable capacity for the regeneration of sensory receptors after acoustic trauma or ototoxicity. Most replacement hair cells are created by renewed cell division within the sensory epithelium, although some new hair cells may also arise through nonmitotic mechanisms. Current data indicate that epithelial supporting cells play an essential role in regeneration, by serving as progenitor cells. In order to become progenitors, however, supporting cells may need to undergo partial dedifferentiation. In this review, I describe molecules that are expressed by supporting cells in the avian ear. Although a number of these molecules are likely to be critical to the maintenance of the supporting cell phenotype, we presently know very little about phenotypic changes in supporting cells during the early phase of regeneration.     

Responses of auditory nerve fibers innervating regenerated hair cells after local application of gentamicin at the round window of the cochlea in the pigeon.

Hair cells in the basilar papilla of birds have the capacity to regenerate after injury. There is also functional recovery of hearing after regeneration of the hair cells. The present study was undertaken to determine the effect of local aminoglycoside application on the physiology of auditory nerve fibers innervating regenerated hair cells. Collagen sponges loaded with gentamicin were placed at the round window of the cochlea in adult pigeons. The local application of gentamicin-loaded collagen sponges resulted in total hair cell loss over at least the basal 62% of the basilar papilla. According to the pigeon cochlear place-frequency map (Smolders, Ding-Pfennigdorff and Klinke, Hear. Res. 92 (1995) 151-169), frequencies above 0.3 kHz are represented in this area. Physiological data on single auditory nerve fibers were obtained 14 weeks after gentamicin treatment. The response properties showed the following characteristics when compared to control data: CF thresholds (CF = characteristic frequency) were elevated in units with CF above 0.15 kHz, sharpness of tuning (Q10dB) was reduced in units with CF above 0.38 kHz, low-frequency slopes of the tuning curves were reduced in units with CF above 0.25 kHz, high frequency slopes of the tuning curves were reduced in units with CF above 0.4 kHz, spontaneous firing rate was reduced in units with CF above 0.38 kHz, dynamic range of rate-intensity functions at CF was reduced in units with CF above 0.4 kHz and the slopes of these rate-intensity functions were elevated in units with CF above 0.4 kHz. Maximum discharge rate was the only parameter that remained unchanged in regenerated ears. The results show that the response properties of auditory nerve fibers which innervate areas of the papilla that were previously devoid of hair cells are poorer than the controls, but that action potential generation in the afferent fibers is unaffected. This suggests that despite structural regeneration of the basilar papilla, functional recovery of the auditory periphery is incomplete at the level of the hair cell or the hair cell-afferent synapse.