Учебники / Hair Cell Regeneration, Repair, and Protection Salvi 2008

xii Volume Preface

supporting cells are destroyed. In Chapter 4, Dooling, Dent, Lauer, and Ryals go into considerable detail about actual recovery of hearing function following loss of hair cells. Behavioral measures of hearing, the gold standard, show almost complete recovery of function on simple measures such as threshold as well as highly sophisticated measures that involve discrimination of complex vocalizations.

The mechanisms involved in proliferation, differentiation, and regeneration are discussed in detail by Oesterle and Stone in Chapter 5. The roles that growth factor, intercellular signaling, intracellular signaling and differentiation factors play in proliferation, conversion, and repair are carefully considered. In Chapter 6, Forge and Van De Water consider ways to protect sensory hair cells from damage so that regeneration is not needed. The modes of cell death are reviewed and various strategies for blocking cell death such as antioxidants, inhibition of apoptosis, and small molecules that block genes or enzymes in the cell death pathway are considered. Finally, in Chapter 7, Rivolta and Holley discuss new experimental approaches that may aid in understanding cell death, cell repair, proliferation, and differentiation. The use of gene array technologies and inner ear cell lines may provide more efficient and comprehensive methods for understanding apoptosis, repair, and regeneration.

As is often the case, new volumes in the Springer Handbook of Auditory Research amplify and extend materials discussed in earlier volumes in the series. While the current volume concerns regeneration and repair, engineering methods have been quite successful in dealing with deafness. In particular, cochlear implants have been a widely used approach and this was covered in depth in Vol. 20 of the series, Cochlear Implants (edited by Zeng, Popper, and Fay). The genetics of the ear and of hearing loss was discussed in detail in Vol. 14, Genetics and Auditory Disorders (edited by Keats, Popper, and Fay). While the current volume focuses on hair cells, Vol. 23, Plasticity of the Auditory System (edited by Parks, Rubel, Fay, and Popper), includes chapters that consider overall plasticity at many levels of the auditory system. Mechanisms of damage to the auditory system is considered at length in in Vol. 31, Auditory Trauma, Protection, and Repair (edited by Schacht, Popper, and Fay). Finally, the physiology and function of sensory hair cells is discussed in many chapters of Vol. 27, Vertebrate Hair Cells (edited by Eatock, Fay, and Popper).

Richard J. Salvi, Buffalo, NY

Arthur N. Popper, College Park, MD

Richard R. Fay, Chicago, IL

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The next giant step toward restoring hearing to the profoundly deaf will involve regenerating the damaged biological structures in the inner ear, in particular the hair cells and spiral ganglion neurons. The major clinical advances in hearing and balance that will occur in the 21st century will involve biologically based medical innovations that were set into motion during the past few decades by the discovery of hair cell regeneration and the recognition that stem cells exist in many regions of the nervous system, including the inner ear.

1.2 Zeitgeist, Regeneration, and Repair

The history of science is often impeded by roadblocks, intellectual and technical, that hinder the advancement of our thinking and imagination. Our views of the world are often constrained by existing knowledge that is accepted as fact by the majority of scientists: the so-called Zeitgeist or spirit of the times. The collective knowledge and prevailing views of the majority can have a profound impact on how new scientific findings are viewed and interpreted for years or even centuries. New data and theories are often immediately rejected by the majority if the findings go against the prevailing view. Indeed, individual scientists may reject their own findings and consider them artifacts or experimental errors if the data contradict prevailing beliefs, opinions, or knowledge.

The ancient theory of sensory processing known as the “principle of likeness” postulated that sensory stimuli in the environment evoked activity within the sensory organ of the same kind (Gitter 1990; Sente 2004). On the basis of this principle, Empedocles proposed the theory of implanted air in the 4th century b.c. whereby sound in the environment evoked a similar activity within the ear to induce the sensation of hearing. The doctrine provided an intellectual framework, though a distorted one, for interpreting the gross anatomical and microscopic observations that were made over the next 2000 years. In the 1500s, Coiter rejected the theory of “implanted air” based on careful anatomical observations of the middle ear and Eustachian tube. Nevertheless, the principle of likeness persisted for another 100 years until it was finally put to rest by detailed examination of the inner ear with the compound microscope and advances in neurophysiology.

My first encounter with the Zeitgeist blinded my thinking on hair cell regeneration. In the late 1970s, I had been studying the effects of acoustic trauma on the firing patterns of auditory nerve fibers in mammals. The dogma at the time was that hair cell loss was permanent and irreparable. At the 1978 meeting of the Acoustical Society of America, I attended a special session on comparative studies of hearing in vertebrates in which Robert Capranica reviewed his work on anurans. Capranica ended his presentation with a provocative finding showing that when ototoxic aminoglycosides were applied to the frog’s inner ear, they completely abolished neurophysiological activity from the ear (Capranica 1978). Surprisingly, the frog’s “hearing” recovered after a few weeks. This was an unexpected finding that defied any conventional explanation. Several thoughts raced through my mind. Were frog hair cells incapable of being destroyed

by aminoglycosides? Were aminoglycoside antibiotics capable of causing only transient damage to the hair cells or neurons? If frog hair cells could not be destroyed with aminoglycoside antibiotics, maybe they could be destroyed with high-level acoustic stimulation. To test the later hypothesis, we started collaborating with the Capranica lab. Capranica’s group drove the frogs from Ithaca, NY to our labs in Syracuse and we exposed the frogs to high-intensity impulse noise in the range of 155–165 dB peak sound pressure level (SPL). Exposures at these levels had caused massive hair cell loss in mammals, and I expected they would do the same in frogs. Afterwards, the frogs were driven back to Cornell University and 1–2 months later their hearing was tested via electrophysiological methods. Surprisingly, the auditory function of the noise-exposed frogs was completely normal. Did the frogs have a potent, long-lasting acoustic reflex that they were using to thwart our acoustic trauma? We tried more vigorous noise exposures several times, but nothing seemed to work. Because normal hearing always returned in the frogs, we eventually dropped the project, considering it a complete failure. Had we examined the frog’s inner ear immediately after treatment with aminoglycoside antibiotics or acoustic overstimulation, we most likely would have seen missing sensory hair cells after the traumatic event and if we had waited a few weeks we would have observed a normal sensory epithelium filled with newborn hair cells (Baird et al. 1993). I never imagined that hair cells could regenerate after aminoglycoside treatment or acoustic trauma and we missed the opportunity to discover hair cell regeneration in nonmammals. The frog trauma data did not conform to our view of the world and therefore was ignored. Less than a decade later, Corwin, Cotanche, Rubel, Ryals, and other showed that hair cells regenerated in the avian ear after acoustic overstimulation and ototoxicity (Corwin and Cotanche 1988; Ryals and Rubel 1988). A new chapter in auditory neuroscience had started. The Zeitgeist has shifted 180 degrees, and the possibility that hair cell regeneration could be stimulated to occur in mammals via gene therapy became a realistic and exciting possibility (Zheng and Gao 2000; Izumikawa et al. 2005). Looking backwards through the rear view mirror of time, it is interesting to note that hair cell regeneration in amphibians had already been discovered by Stone in the 1930s, but it was largely overlooked only to be rediscovered and embraced 50 years later (Stone 1933, 1937).

1.3 Regeneration in Fish and Amphibians

The first reports of hair cell regeneration date back to the 1930s when it was discovered that after tail amputation or reamputation, the amphibian lateral line organs on the body surface would grow back by forming a regenerative placode that migrated into the regenerating tail where it formed a neuromasts with new hair cells that received afferent innervation (Stone 1933, 1937; Speidel 1947; Wright 1947; Jones and Corwin 1996). These findings indicate that amphibians possess stem cells that self-renew and differentiate into hair cells and support cells.

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Historically, much of the research in auditory neuroscience has focused on the anatomy, physiology, and development of the mammalian auditory system. Unfortunately, many of the early studies in nonmammals were ignored or overlooked by mainstream auditory neuroscientists. Beginning in the early 1970s, renewed interest in the morphological development of the inner ear of amphibians and fish revealed the proliferation of new hair cells and the expansion of the sensory epithelium between birth and adulthood (Lewis and Li 1973; Li and Lewis 1979; Popper and Hoxter 1984). Studies with tritiated thymidine, an amino acid that is incorporated into the DNA of dividing cells, showed that new hair cells and supporting cells were added at the periphery of the sensory epithelium of elasmobranchs and amphibians (Corwin 1981, 1985). Authors of later studies found evidence of new hair cells and supporting cells within the hair cell epithelium of teleost fish, turtles, and chicken vestibular system (Jargensen and Mathiesen 1988; Lombarte and Popper 1994; Severinsen et al. 2003).

1.4 Regeneration in Birds

All the hair cells in the avian and mammalian cochlea ear arise during embryogenesis (Ruben 1967; Tilney et al. 1986; Katayama and Corwin 1989). Therefore the discovery that damaged hair cells in the chicken cochlea were replaced by newborn hair cells after acoustic trauma or aminoglycoside ototoxicity (Cruz et al. 1987; Girod et al. 1991; Hashino et al. 1992) generated considerable excitement and greatly accelerated research on this topic, as reflected by hundreds of articles on this topic during the last two decades (Corwin and Cotanche 1988; Ryals and Rubel 1988). Unlike those in the cochlea, hair cells in the normal avian vestibular system show a low level of hair cell regeneration that is balanced by ongoing hair cell death (Jargensen and Mathiesen 1988; Kil et al. 1997). In both the avian cochlea and vestibular organs, hair cell loss serves as the trigger that initiates a carefully orchestrated process of supporting cell proliferation and differentiation; however, both processes are confined to the damaged region (Girod et al. 1989; Hashino et al. 1995; Stone and Rubel 2000). These results suggest that newborn hair cells suppress the proliferation of additional hair cells, possibly through cell–cell contacts. The appearance of the pairs of dividing cells suggested that one member of the offspring differentiates into a hair cell while the other becomes a supporting cell, thereby replenishing the pool of progenitor cells. While proliferation and differentiation of supporting cells play a role in regeneration (Hashino and Salvi 1993), new hair cells appear in the newt in the presence of mitotic blockers (Taylor and Forge 2005). During avian hair cell regeneration, fewer than half of support cells and hair cells in the regenerated region are labeled with mitotic markers (Roberson et al. 1996). These results suggest that many new hair cells arise from phenotypic conversion (Taylor and Forge 2005). Conversion may also play an important role in generating new hair cells in birds and amphibians (Adler and Raphael 1996; Steyger et al. 1997).

1.5 Neurons and Other Structures Involved with Avian Hair Cell Regeneration

After acoustic trauma or aminoglycoside damage, newborn hair cells develop and migrate toward the luminal surface of the sensory epithelium and primitive stereocilia bundles emerge from their apical surface. The stereocilia bundles begin to develop, but even after 1 month, the bundle organization and orientation are still immature. It takes approximately 90 days before the stereocilia bundles on the regenerated hair cells regain their normal appearance and orientation in young chicks (Duckert and Rubel 1990, 1993). However, when hair cell regeneration was studied in adult birds, the bundles were still disoriented after a 142-day recovery period (Marean et al. 1993).

Approximately 3 days after the regenerated hair cells appear, afferent and efferent synapses can be seen contacting the basal pole of the cell (Ryals and Westbrook 1994) thereby providing the neural circuitry for delivering information to and from the brain. However, careful anatomical analyses raise some cautionary notes. The number of cochlear ganglion cells shows a mild-to- moderate decline between 30 and 90 days postexposure and possibly longer (Ryals et al. 1989). The factors mediating the loss of cochlear ganglion cells after acoustic trauma are currently unknown. The loss could be due to excitotoxic damage (Sun et al. 2001) or temporary lack of neurotrophic support between the time when the original hair cells were lost and the newborn hair cells had fully matured (Ernfors et al. 1995; Fritzsch et al. 1997). In cases in which severe excitotoxicity has occurred, cochlear ganglion cells do not appear to regenerate (Sun et al. 2001). Acoustic trauma also damages the upper fibrous layer and lower honey comb layer of the tectorial membrane. The lower honeycomb layer regenerates within a week or two; however, the upper fibrous layer does not regenerate (Cotanche 1987; Trautwein et al. 1996). Importantly, in cases of severe acoustic trauma in which there is extensive damage to both hair cells and supporting cells, hair cell regeneration fails to occur due to the destruction of progenitor cells (Muller et al. 1996).

The tegmentum vasculosum in the avian ear plays an important role in generating the endolymphatic potential (EP) and establishing the high potassium concentration in the endolymph. Acoustic trauma damages the dark cells in the tegmentum vasculosum of adult quail, but the tegmentum completely recovers after 4 days (Poje et al. 1995). Some results suggest that the repair of the tegmentum vasculosum plays an important role in the recovery of hearing after acoustic trauma.

1.6 Regeneration in the Mammalian Inner Ear

In the mammalian cochlea, where the supporting cells are highly specialized, there is no convincing evidence of proliferation or hair cell regeneration (Meyers and Corwin, Chapter 2) except under special conditions designed to stimulate proliferation and regeneration (Oesterle and Stone, Chapter 5). In the vestibular

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system of adult animals, there is evidence for limited hair cell regeneration after aminoglycoside treatment. New hair cells with immature stereocilia bundles reminiscent of regenerating avian hair cells have been observed in guinea pigs and human vestibular epithelia (Forge et al. 1993, 1998; Warchol et al. 1993). Many of the new hair cells presumably differentiated from supporting cells because the numbers of support cells were reduced in regions of regeneration. Cell proliferation most likely plays a minor role in producing new hair cells because there were relatively few [3H]thymidine-labeled cells in the damaged region. Finally, one cannot rule out the possibility that some of the new hair cells with immature stereocilia are actually hair cell bodies that have survived the ototoxic insult and are in the process of rebuilding their stereocilia (Sobkowicz et al. 1995; Zhao et al. 1996; Schneider et al. 2002; Rzadzinska et al. 2004; Forge and Van de Water, Chapter 6).

2. Physiological Function After Avian Hair

Cell Regeneration

Although avian hair cells regenerate, it was not clear if there would be complete recovery of physiological function (Saunders and Salvi, Chapter 3) because of the loss of cochlear ganglion neurons and lack of regeneration in the upper fibrous layer of the tectorial membrane after acoustic trauma. Gross potentials, evoked potentials, single-unit recordings, and otoacoustic emissions have all been used to assess different aspects of auditory function.

When hair cells are destroyed with aminoglycoside antibiotics, physiological thresholds generally recover to normal levels at low frequencies whereas at high frequencies thresholds only partially recover, resulting in residual thresholds shifts (Tucci and Rubel 1990; Chen et al. 1993). Recovery of physiological thresholds lags behind the emergence of regenerated hair cells by 3–4 months.

2.1 Physiological Thresholds

In cases of severe acoustic trauma where there is complete destruction of supporting cells and sensory cells on the basilar membrane, there is little or no recovery of the compound action potential (CAP) or the spontaneous or sound-evoked discharge patterns of cochlear ganglion neurons (Muller et al. 1996, 1997). However, in cases of moderate acoustic trauma where there is almost complete hair cell regeneration, physiological thresholds measured with different techniques such as the CAP, auditory evoked response, and singlefiber recordings from cochlear ganglion neurons all show significant threshold elevation (40–60 dB) immediately after the exposure followed by complete or nearly complete recovery 1–4 months postexposure (Henry et al. 1988; Chen et al. 1996; Saunders et al. 1996; Muller et al. 1997). Single fiber spontaneous discharge rates, phase-locking, and frequency tuning are nearly normal after a few weeks or months postexposure. However, discharge rate-intensity functions

and two-tone rate suppression continue to exhibit residual deficits after 1 month of recovery (Saunders et al. 1996; Chen et al. 2001; Lifshitz et al. 2004; Furman et al. 2006). These deficits may be related to the lack of regeneration of the upper fibrous layer of the tectorial membrane.

The endolymphatic potential (EP), which is approximately +18 mV in adult birds, provides a positive driving force for moving potassium down its concentration gradient into the hair cells. The effects of acoustic trauma and aminoglycosides on the EP appear to be different in young chicks compared to adults. The EP in young chicks shows a large decline after acoustic trauma and then recovers over time, consistent with other physiological measures (Poje et al. 1995). However, acoustic trauma and aminoglycosides failed to produce a decrease in the positive EP in adult chickens, suggesting the EP was more resistant in adults (Chen et al. 1995; Trautwein et al. 1997).

2.2 Otoacoustic Emissions and Avian Hair

Cell Regeneration

Distortion product otoacoustic emissions (DPOAEs), a nonlinear response presumably generated by an active process in the hair cells, show a significant decline immediately postexposure in both young chicks and adult animals. DPOAE amplitudes completely recover in young chicks after acoustic trauma and aminoglycoside damage (Ipakchi et al. 2005). However, DPOAE amplitude only partially recovers in adult chickens; residual deficits are evident in adults chicken even after a 16-week recovery period (Trautwein et al. 1996; Chen et al. 2001). It was originally suggested that the persistent reduction in DPOAE amplitude in adults might be related to residual damage to the upper fibrous layer of the tectorial membrane; however, this explanation may not be valid because the fibrous layer is also missing in young chicks that show complete recovery. Collectively, the DPOAE and EP data suggest that the ears of young birds are more resistant or better able to recover from acoustic trauma than are adult birds.

DPOAEs are thought to arise from an active feedback mechanism that enhances the sensitivity and frequency selectivity of the inner ear. In mammals, the active feedback mechanism, or electromotile response, arises from the motor protein, prestin, located in the lateral wall of outer hair cells (OHCs) (Hofstetter et al. 1997; Zheng et al. 2000a; Liberman et al. 2002). Because birds have robust DPOAEs, but lack OHCs, can the short and tall hair cells in avian ears also generate an electromotile response? Surprisingly, when AC current is applied to the avian ear, a strong, electrically evoked otoacoustic emission emerges from the cochlear into the ear canal (Chen et al. 2001). Selective destruction of avian hair cells with kanamycin greatly reduces otoacoustic emission amplitude. Emission amplitude partially recovers as the hair cells regenerate, suggesting that avian hair cells are the source of the electrically evoked emission. Unlike that of mammals, however, the avian hair cell soma does not elongate or contract in response to electrical stimulation (He et al. 2003). Thus, avian electrically