Douglas A. Cotanche

Hair cell regeneration in the bird cochlea following noise damage or ototoxic drug damage

Hair cells are sensory cells that transduce motion into neural signals. In the cochlea, they are used to detect sound waves in the environment and turn them into auditory signals that can be processed in the brain. Hair cells in the cochlea of birds and mammals were thought to be produced only during embryogenesis and, once made, they were expected to last throughout the lifetime of the animal. Thus, any loss of hair cells due to trauma or disease was thought to lead to permanent impairment of auditory function. Recently, however, studies from a number of laboratories have shown that hair cells in the avian cochlea can be regenerated after acoustic trauma or ototoxic drug damage. This regeneration is accompanied by a repair of the sensory organ and associated tissues and results in a recovery of auditory function. In this review, we examine and compare the structural events that lead to hair cell loss after noise damage and ototoxic drug damage as well as the processes involved in the recovery of the epithelium and the regeneration of the hair cells. Moreover, we examine functional recovery and how it relates to the structural recovery. Finally, we investigate the evidence for the hypothesis that supporting cells in the basilar papilla act as the progenitor cells for the regenerated hair cells and examine the cellular events required to stimulate the progenitor cells to leave the quiescent state, re-enter the cell cycle, and divide.

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.

Hair cell and supporting cell response to acoustic trauma in the chick cochlea

Damage to the chick cochlea in response to progressively increased periods of noise exposure was studied with scanning electron microscopy. Ten day-old chick hatchlings were exposed to a 1500 Hz pure tone at 120 dB SPL for 4, 8, 12, 24, and 48 h. Measurements of hair cell and supporting cell surface areas within a defined region of the cochlea showed that the average hair cell surface area decreased over the first 12 h of exposure. Between 12 h and 48 h there was no significant change in hair cell surface area. Supporting cells showed a corresponding increase in surface area over the same period. Noise damage first appeared after 4 h of exposure as a localized expansion of supporting cell surfaces near the inferior edge of the basilar papilla (BP). Between 8 and 12 h of exposure the supporting cell surface area increased dramatically and was visible throughout the noise damaged region. Hair cell expulsion was first seen after 12 h. Exposure to noise for 24-48 h resulted in further expansion of supporting cells, extensive expulsion of hair cells from the BP, and the appearance of a strip of noise damage along the superior, edge of the BP.

Hair cell fate decisions in cochlear development and regeneration

The discovery of avian cochlear hair cell regeneration in the late 1980s and the concurrent development of new techniques in molecular and developmental biology generated a renewed interest in understanding the genetic mechanisms that regulate hair cell development in the embryonic avian and mammalian cochlea and regeneration in the mature avian cochlea. Research from many labs has demonstrated that the development of the inner ear utilizes a complex series of genetic signals and pathways to generate the endorgans, specify cell identities, and establish innervation patterns found in the inner ear. Recent studies have shown that the Notch signaling pathway, the Atoh1/Hes signaling cascade, the stem cell marker Sox2, and some of the unconventional myosin motor proteins are utilized to regulate distinct steps in inner ear development. While many of the individual genes involved in these pathways have been identified from studies of mutant and knockout mouse cochleae, the interplay of all these signals into a single systemic program that directs this process needs to be explored. We need to know not only what genes are involved, but understand how their gene products interact with one another in a structural and temporal framework to guide hair cell and supporting cell differentiation and maturation.

Potential role of BFGF and retinoic acid in the regeneration of chicken cochlear hair cells

Messenger RNAs (mRNA) of several growth factor receptors and relate genes were examined with reverse transcriptase polymerase chain reaction (RT-PCR) in normal and noise-damaged chicken basilar papillae (BP). Analysis of the amplification products indicated the presence of mRNAs for epidermal growth factor receptor (EGFR), fibroblast factor receptor (FGFR), insulin-like growth factor receptor (IGFR), insulin receptor (IR), retinoic acid receptor beta (RAR beta), retinoic acid receptor gamma (RXR gamma), and basic fibroblast growth factor (BFGF) in both normal and noise-damaged BP. The RT-PCR products generated were characterized by size and sequencing analysis to confirm the identities of the target molecules. The subcellular localization of the mature protein analogs for EGFR, FGFR, IGFR, RAR beta, and BFGF were identified using fluorescence immunocytochemistry and confocal laser scanning microscopy. These experiments indicated that EGFR is present in the stereociliary bundles in the hair cells, IGFR is not present in the cells of the BP, BFGF localizes in the nuclei of supporting cells in the BP, but not hair cells or hyaline cells, and that RAR beta localizes in the perinuclear regions of hair cells. The subcellular distributions of these proteins were consistent in both noise-damaged and control BP. FGFR, in contrast, changed its distribution in the tissue after noise damage. In normal BP, FGFR is concentrated in the stereocilia of hair cells. However, in damaged regions of noise-exposed chick cochleae, FGFR is heavily expressed in the expanded apical regions of the supporting cells. These findings suggest that BFGF and retinoic acid may potentially play a role in the mechanisms which regulate the regeneration of chicken cochlear hair cells.

Regeneration of hair cells in the vestibulocochlear system of birds and mammals

Regeneration of hair cells leads to a structural and functional recovery in the mature avian vestibular and auditory sensory epithelia. This regeneration replaces hair cells that have been lost as a result of noise damage, ototoxic drug poisoning, or other trauma. Recent findings suggest that it may be possible to induce a similar mechanism for repair in the vestibular and auditory epithelia of mammals, including humans.

Migration of hyaline cells into the chick basilar papilla during severe noise damage

Severe acoustic damage in the chick cochlea causes a destruction of both hair cells and supporting cells in a localized area on the basilar papilla. In this region, the sensory cells are replaced by a layer of flattened epithelial cells. We have employed scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) to examine the structure and cytoskeletal changes involved in this process. Immunocytochemical staining for actin indicates that the flattened cells are derived from the hyaline cells normally located along the inferior edge of the basilar papilla. In control cochleae the hyaline cells contain dense bundles of actin filaments that anchor into the basal surface of the cells. The hyaline cells appear to redistribute into the severely damaged region by extending the actin bundles at their basal surfaces. Moreover, the efferent nerves that normally form a network among the hyaline cells move into the severely damaged area along with the hyaline cells. In moderately damaged cochleae, where only hair cells are lost, the hyaline cells do not spread into the damaged region. The functional role of this hyaline cell migration is unknown, but it may be involved in maintenance or repair of the severely damaged cochlea.

Distribution of nerve fibers in the basilar papilla of normal and sound-damaged chick cochleae

Epifluorescent light microscopy and confocal laser scanning microscopy were employed to visualize the distribution of nerve fibers in whole‐mount preparations of normal and sound‐damaged chick basilar papillae (BP). In normal cochleae, we identified a consistent pattern of nerve processes that ran transversely across the BP. The transverse processes increase in number from the proximal to the distal ends of the epithelium. However, when the processes are separated into populations of thin fibers and thick bundles, the thin fibers are more prevalent in distal regions whereas thick bundles are more extensive in proximal regions. Furthermore, the thick bundles form an elaborate longitudinal network in the border cell and hyaline cell region. Based on these data and on other previous studies, the thin fibers appear to be afferent nerves and the thick bundles represent efferent nerves. When birds are exposed to acoustic trauma, the normal pattern and number of nerve processes is not altered by levels of sound that produce moderate levels of damage, i.e., damage that leads to hair cell loss and regeneration. However, the nerve pattern is disrupted by severe levels of damage that destroy both hair cells and supporting cells. These findings indicate that the level of sound exposure that induces hair cell regeneration may damage the synaptic endings associated with the lost hair cells, but that the nerve processes that give rise to these endings remain intact within the sensory epithelium. In contrast, severe damage destroys both the hair cells and their associated nerve fibers.

Video-enhanced DIC images of the noise-damaged and regenerated chick tectorial membrane

Exposure of the chick cochlea to acoustic overstimulation results in a loss of hair cells and a disruption of the tectorial membrane. With time, new hair cells are produced to replace those that are lost and, concurrently, a new tectorial membrane is regenerated. Previous studies of tectorial membrane regeneration examined tissues that were fixed and processed for scanning and transmission electron microscopy. This processing results in a considerable shrinkage of the membrane, and, therefore, it was unclear how the noise damage and subsequent regeneration affected the unfixed, in situ structure of the tectorial membrane. We have recently developed techniques for studying the unfixed tectorial membrane with video-enhanced differential-interference-contrast (DIC) light microscopy. Exposure to a 1500-Hz pure tone at 120 dB SPL for 24 h causes localized damage to the hair cells and tectorial membrane in the mid-proximal region of the basilar papilla. Examination of the unfixed membrane immediately after noise exposure shows that the damage to the tectorial membrane is actually caused by the acoustic trauma and is not an artifact of fixation. After 14 days of recovery, a thick, honeycomb of new matrix has grown from the supporting cells in the basilar papilla and has formed new connections with the stereocilia of surviving and regenerating hair cells. Moreover, this new honeycomb has fused with the remainder of the surrounding, undamaged tectorial membrane, thus reestablishing a continuity in the structure of the membrane across both the damaged and undamaged regions of the basilar papilla.

SEM analysis of the developing tectorial membrane in the chick cochlea

The development of the tectorial membrane in the embryonic chick cochlea was studied using scanning electron microscopy. Chick embryos ranged in age from embryonic day 7 (E7) to post-hatching day 15. Our studies revealed that a fine filamentous matrix arose on the apical surface of the basilar papilla at approximately E7. This matrix was secreted by the supporting cells which encircled the hair cells. By E9, the early matrix had increased in volume but remained filamentous in structure, except at the inferior edge of the basilar papilla where it was condensed into a layer of laterally-oriented columns. At E9 the TM exhibited an additional layer of matrix, called the amorphous component. It appeared to originate from the homogene cell population, and attached to the early columnar matrix at the inferior edge of the basilar papilla. The two components of the TM were separated by a longitudinal ridge, called the track, which marked the inferior edge of the amorphous component. As the cochlea developed, the basilar papilla increased in width, the columnar component elongated and the track appeared to recede. These morphological findings point to separate developmental origins for the two components of the tectorial membrane.