Epigenetic Influences on Sensory Regeneration: Histone Deacetylases Regulate Supporting Cell Proliferation in the Avian Utricle

The sensory hair cells of the cochlea and vestibular organs are essential for normal hearing and balance function. The mammalian ear possesses a very limited ability to regenerate hair cells and their loss can lead to permanent sensory impairment. In contrast, hair cells in the avian ear are quickly regenerated after acoustic trauma or ototoxic injury. The very different regenerative abilities of the avian vs. mammalian ear can be attributed to differences in injury-evoked expression of genes that either promote or inhibit the production of new hair cells. Gene expression is regulated both by the binding of cis-regulatory molecules to promoter regions as well as through structural modifications of chromatin (e.g., methylation and acetylation). This study examined effects of histone deacetylases (HDACs), whose main function is to modify histone acetylation, on the regulation of regenerative proliferation in the chick utricle. Cultures of regenerating utricles and dissociated cells from the utricular sensory epithelia were treated with the HDAC inhibitors valproic acid, trichostatin A, sodium butyrate, and MS-275. All of these molecules prevent the enzymatic removal of acetyl groups from histones, thus maintaining nuclear chromatin in a “relaxed” (open) configuration. Treatment with all inhibitors resulted in comparable decreases in supporting cell proliferation. We also observed that treatment with the HDAC1-, 2-, and 3-specific inhibitor MS-275 was sufficient to reduce proliferation and that two class I HDACs—HDAC1 and HDAC2—were expressed in the sensory epithelium of the utricle. These results suggest that inhibition of specific type I HDACs is sufficient to prevent cell cycle entry in supporting cells. Notably, treatment with HDAC inhibitors did not affect the differentiation of replacement hair cells. We conclude that histone deacetylation is a positive regulator of regenerative proliferation but is not critical for avian hair cell differentiation.     

Vestibular hair cell pathology in the Shaker-2 mouse

The circling-waltzing behaviour of the Shaker-2 mouse is suggested, at least in part, to be of peripheral origin. In this hereditary inner ear disease, degeneration of hair cells type I has been observed showing specific pathologic features: rod-shaped inclusion bodies and sensory hair fusion. Later, the hair cells type I are expelled into the endolymphatic space. A large number of sensory cells type II are morphologically normal. The failure of earlier investigators to demonstrate pathological changes in the sensory epithelia of this animal is likely to be due to the use of light microscopical methods only.     

Neurogenin 1 Null Mutant Ears Develop Fewer, Morphologically Normal Hair Cells in Smaller Sensory Epithelia Devoid of Innervation

The proneuronal gene neurogenin 1 (ngn1) is essential for development of the inner-ear sensory neurons that are completely absent in ngn1 null mutants. Neither afferent, efferent, nor autonomic nerve fibers were detected in the ears of ngn1 null mutants. We suggest that efferent and autonomic fibers are lost secondarily to the absence of afferents. In this article we show that ngn1 null mutants develop smaller sensory epithelia with morphologically normal hair cells. In particular, the saccule is reduced dramatically and forms only a small recess with few hair cells along a duct connecting the utricle with the cochlea. Hair cells of newborn ngn1 null mutants show no structural abnormalities, suggesting that embryonic development of hair cells is independent of innervation. However, the less regular pattern of dispersal within sensory epithelia may be caused by some effects of afferents or to the stunted growth of the sensory epithelia. Tracing of facial and stato-acoustic nerves in control and ngn1 null mutants showed that only the distal, epibranchial, placode-derived sensory neurons of the geniculate ganglion exist in mutants. Tracing further showed that these geniculate ganglion neurons project exclusively to the solitary tract. In addition to the normal complement of facial branchial and visceral motoneurons, ngn1 null mutants have some trigeminal motoneurons and contralateral inner-ear efferents projecting, at least temporarily, through the facial nerve. These data suggest that some neurons in the brainstem (e.g., inner-ear efferents, trigeminal motoneurons) require afferents to grow along and redirect to ectopic cranial nerve roots in the absence of their corresponding sensory roots.     

Growth of a fish ear: 1. Quantitative analysis of hair cell and ganglion cell proliferation

Proliferation (or addition) of inner ear sensory hair cells continues for a long time postembryonically in cartilaginous and bony fishes, and in amphibians. In contrast, proliferation only occurs during embryonic development in birds and mammals. However, detailed quantitative data on hair cell addition are not available for bony fishes. In order to quantify the extent of proliferation, we determined the number of sensory hair cells on the saccular sensory epithelium in specimens of the cichlid fish Astronotus ocellatus (the oscar) ranging from 2.0 to 19.0 cm in standard length (0.9-343 g). Ganglion cells were counted using serial sections of the saccular branch of the eighth nerve in animals of the same size range. The saccular macula of a 2.0 cm long (0.9 g) Astronotus contains approximately 5500 sensory hair cells; fish from 16 to 19 cm long have over 170 000 hair cells. The increase in number of sensory cells and the increase in both length and weight of the animals studied were statistically correlated (r2 = 0.8). The relative densities of saccular sensory cells in different epithelial regions remained constant in animals from 2.0 to 17 cm; in larger animals the cell density decreased somewhat. Based upon very conservative estimates of the rate of growth of Astronotus, we calculate that an average of 167 hair cells/day are added during the time when the cell population of the saccule increases. Ganglion cell number also increased approximately 4.8 times in the range of fish studied. The smallest animals in our study had about 150 ganglion cells per saccular epithelium, while the largest fish had over 600 ganglion cells. We estimate that the average ratio of hair cells to afferent fibers increases from about 30:1 in the smallest fish to over 300:1 in the largest animals.     

Distribution of gentamicin by immunofluorescence in the guinea pig inner ear

Summary We studied the distribution of gentamicin in the inner ear, brain and kidney of the guinea pig following intraperitoneal administration or perfusion of gentamicin through the perilymphatic space. The resulting histopathologcial changes were examined by immunofluorescence using antigentamicin antiserum. After perfusion of gentamicin through the perilymphatic space, specific fluorescence was found in the cochlea, and was especially prominent in the outer hair cells, basilar membrane and basilar crest. Although no fluorescence was observed in the cochlea following intraperitoneal administration of high doses of gentamicin, type I hair cells in the vestibule were seen to be selectively stained with the antibody. Furthermore, some of the vestibular ganglion cells, Purkinje cells and unidentified nuclei in the brain stem were also stained. In particular, fine granules showing relatively intense fluorescence were recognized in the cytoplasm of the stained cells. In the cortex of kidney, only proximal tubular cells were stained with intense fluorescence. Our results suggest that the aminoglycoside antibiotics have two sites of action: one is the cell membrane of the sensory hair cells and the other is the cytoplasm.This study was supported in part by a grant from the Ministry of Education, Science and Culture of Japan, and by a Research Grant for Specific Diseases from the Ministry of Health and Welfare to the Acute Profound Deafness Research Committee of Japan     

Unbiased stereologic type I and type II hair cell counts in human utricular macula

OBJECTIVE: The objective of the study was to obtain unbiased estimates of the total number of type I and type II hair cells in human utricular macula from individuals with documented normal vestibular function. STUDY DESIGN: Application of unbiased stereology using microdissected human temporal bone specimens was conducted in an observational study. METHODS: Postmortem temporal bones were obtained from 10 normal patients (age range, 42-96 y; mean age, 82 y). The utricular maculae were microdissected, embedded in plastic, and cut into serial 2-microm sections. Unbiased estimates of the total number of type I and type II hair cells were obtained using the physical fractionator technique of stereology. RESULTS: The average total number of hair cells was 27,508 (CV = 11%) consisting of 17,326 (coefficient of variation [CV] = 11%) type I hair cells and 10,182 (CV = 13%) type II hair cells. The ratio of type I to type II hair cells was 1.70:1. In the age range of the study, there was no statistically significant correlation between hair cell counts and age. CONCLUSIONS: Morphometric studies of the human utricular sensory epithelium can be accomplished using unbiased stereology on microdissected specimens. There was no effect of age on total hair cell counts or on the ratio of type I to type II hair cells in the age range of the study. Further studies on younger subjects are needed to establish the effect of age. The results from the present study are closely aligned with prior studies that estimated total hair cell counts using surface mount preparations. The current data represent the first total type I and type II hair cell counts in human utricular neuroepithelium.     

Stem/Progenitor Cells Derived from the Cochlear Sensory Epithelium Give Rise to Spheres with Distinct Morphologies and Features

Nonmammalian vertebrates regenerate lost sensory hair cells by means of asymmetric division of supporting cells. Inner ear or lateral line supporting cells in birds, amphibians, and fish consequently serve as bona fide stem cells resulting in high regenerative capacity of hair cell-bearing organs. Hair cell regeneration does not happen in the mammalian cochlea, but cells with proliferative capacity can be isolated from the neonatal cochlea. These cells have the ability to form clonal floating colonies, so-called spheres, when cultured in nonadherent conditions. We noticed that the sphere population derived from mouse cochlear sensory epithelium cells was heterogeneous, consisting of morphologically distinct sphere types, hereby classified as solid, transitional, and hollow. Cochlear sensory epithelium-derived stem/progenitor cells initially give rise to small solid spheres, which subsequently transition into hollow spheres, a change that is accompanied by epithelial differentiation of the majority of sphere cells. Only solid spheres, and to a lesser extent, transitional spheres, appeared to harbor self-renewing stem cells, whereas hollow spheres could not be consistently propagated. Solid spheres contained significantly more rapidly cycling Pax-2-expressing presumptive otic progenitor cells than hollow spheres. Islet-1, which becomes upregulated in nascent sensory patches, was also more abundant in solid than in hollow spheres. Likewise, hair cell-like cells, characterized by the expression of multiple hair cell markers, differentiated in significantly higher numbers in cell populations derived from solid spheres. We conclude that cochlear sensory epithelium cell populations initially give rise to small solid spheres that have self-renewing capacity before they subsequently convert into hollow spheres, a process that is accompanied by loss of stemness and reduced ability to spontaneously give rise to hair cell-like cells. Solid spheres might, therefore, represent the most suitable sphere type for cell-based assays or animal model transplantation studies aimed at development of cell replacement therapies.     

Volume fraction and ultrastructure of age pigment in the saccular epithelium of old mice

Age pigment in the sensory and supporting cells was a prominent characteristic distinguishing old saccules from young. However, the pigment was not distributed uniformly throughout the sensory epithelium but displayed cell-specific patterns of accumulation. The highest cytoplasmic volume density was in the old supporting cells followed by type I hair cells and then type II hair cells. The most common form of pigmented inclusion seen in old type I hair cells was a cluster of granules resembling melanin. This form was never seen in type II hair cells or supporting cells where a form containing a lipid-like droplet was prevalent. The differences in the amount of age pigment and the forms accumulated probably reflects metabolic differences between the three cell types.     

Distribution of Na,K-ATPase α Subunits in Rat Vestibular Sensory Epithelia

The afferent encoding of vestibular stimuli depends on molecular mechanisms that regulate membrane potential, concentration gradients, and ion and neurotransmitter clearance at both afferent and efferent relays. In many cell types, the Na,K-ATPase (NKA) is essential for establishing hyperpolarized membrane potentials and mediating both primary and secondary active transport required for ion and neurotransmitter clearance. In vestibular sensory epithelia, a calyx nerve ending envelopes each type I hair cell, isolating it over most of its surface from support cells and posing special challenges for ion and neurotransmitter clearance. We used immunofluorescence and high-resolution confocal microscopy to examine the cellular and subcellular patterns of NKAα subunit expression within the sensory epithelia of semicircular canals as well as an otolith organ (the utricle). Results were similar for both kinds of vestibular organ. The neuronal NKAα3 subunit was detected in all afferent endings—both the calyx afferent endings on type I hair cells and bouton afferent endings on type II hair cells—but was not detected in efferent terminals. In contrast to previous results in the cochlea, the NKAα1 subunit was detected in hair cells (both type I and type II) but not in supporting cells. The expression of distinct NKAα subunits by vestibular hair cells and their afferent endings may be needed to support and shape the high rates of glutamatergic neurotransmission and spike initiation at the unusual type I-calyx synapse.     

Hair Cell Generation by Notch Inhibition in the Adult Mammalian Cristae

Balance disorders caused by hair cell loss in the sensory organs of the vestibular system pose a significant health problem worldwide, particularly in the elderly. Currently, this hair cell loss is permanent as there is no effective treatment. This is in stark contrast to nonmammalian vertebrates who robustly regenerate hair cells after damage. This disparity in regenerative potential highlights the need for further manipulation in order to stimulate more robust hair cell regeneration in mammals. In the utricle, Notch signaling is required for maintaining the striolar support cell phenotype into the second postnatal week. Notch signaling has further been implicated in hair cell regeneration after damage in the mature utricle. Here, we investigate the role of Notch signaling in the mature mammalian cristae in order to characterize the Notch-mediated regenerative potential of these sensory organs. For these studies, we used the γ-secretase inhibitor, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), in conjunction with a method we developed to culture cristae in vitro. In postnatal and adult cristae, we found that 5 days of DAPT treatment resulted in a downregulation of the Notch effectors Hes1 and Hes5 and also an increase in the total number of Gfi1⁺ hair cells. Hes5, as reported by Hes5-GFP, was downregulated specifically in peripheral support cells. Using lineage tracing with proteolipid protein (PLP)/CreER;mTmG mice, we found that these hair cells arose through transdifferentiation of support cells in cristae explanted from mice up to 10 weeks of age. These transdifferentiated cells arose without proliferation and were capable of taking on a hair cell morphology, migrating to the correct cell layer, and assembling what appears to be a stereocilia bundle with a long kinocilium. Overall, these data show that Notch signaling is active in the mature cristae and suggest that it may be important in maintaining the support cell fate in a subset of peripheral support cells.     

Ultrastructural findings of the macula utriculi in a case of a petrous apex cholesteatoma: a comparison with findings in a patient with an acoustic neuroma

The morphological characteristics of the vestibular sensory cells of the macula utriculi obtained during surgery in a patient with a petrous apex cholesteatoma were examined using scanning and transmission electron microscopy. Findings were compared to cells studied in a patient with acoustic neuroma. Scanning electron microscopy showed that compared to the apparently normal cells in the acoustic neuroma case, most sensory cells in the cholesteatoma case had large cuticular plates, irregular locations of cilia and no clear polarizations. Supporting cells showed profuse short microvilli on the whole surface. With transmission electron photomicrographs, type I hair cells were not seen and certain morphological changes were observed in type-1I-like cells and supporting cells. We presume that the degenerative changes in the vestibular epithelia were due to circulatory disturbances and/or direct pressure applied to the vestibular nerve at the internal auditory canal, with subsequent involvement of the macula utriculi.     

The aging vestibular hair cell

The ultrastructure of aging vestibular hair cells of the guinea pig can be abnormal even though they appear morphologically normal by light microscopy. In only a few of the hair cells studied did nonspecific changes occur. In general, the age-related changes involved specific structures in the hair cells: aggregations of lipofuscin pigments, multivesiculated bodies, disintegration of the cuticular plate, and rod-shaped inclusions from the cuticle area into the hair cell. Furthermore, sensory hairs are generally present when the cuticular plate shows an advanced disintegration. Nerve calyces can be ultrastructurally changed without morphologic changes in the adjacent hair cell. Type I hair cells show age-related morphologic changes more often than do type II hair cells.     

A scanning electron microscopic study of vestibular organ malformation following prenatal gamma irradiation

Summary Pregnant CBA/CBA mice were exposed to 1 and 2 Gy whole-body gamma irradiation on the 13th and 16th gestational days, respectively. The litters were born on the 21st day of gestation and were tested for vestibular function at the age of 1 month. The animals were then sacrificed and their inner ears were analyzed by scanning electron microscopy. No disturbances of vestibular function were noted in the animals studied. However, the cristae ampullares showed severe malformations as regards their gross shape, with irregularities of their outer contours. Type I hair cells seemed to be more severely changed than Type II hair cells, with fusion of sensory hairs, giant hair formation and bulging of the cuticular plate. In certain sites the hair cells were totally missing. These derangements were usually located in the central areas of the cristae ampullares and in the striolar portion of the maculae utriculi. The morphological damage found showed a dose-dependent, time-related pattern.Supported by grants from the Swedish Medical Research Council (12X-0720) and from the Karolinska Institute     

Solitary Hair Cells Are Distributed Throughout the Extramacular Epithelium in the Bullfrog's Saccule

The frog inner ear contains eight sensory organs that provide sensitivities to auditory, vestibular, and ground-borne vibrational stimuli. The saccule in bullfrogs is responsible for detecting ground- and air-borne vibrations and is used for studies of hair cell physiology, development, and regeneration. Based on hair bundle morphology, a number of hair cell types have been defined in this organ. Using immunocytochemistry, vital labeling, and electron microscopy, we have characterized a new hair cell type in the bullfrog saccule. A monoclonal antibody that is specific to hair cells revealed that a population of solitary hair cells exists outside the sensory macula in what was previously thought to be nonsensory epithelium. We call these extramacular hair cells. There are 80–100 extramacular hair cells in both tadpole and adult saccules, which extend up to 1 mm from the edge of the sensory macula. The extramacular hair cells have spherical cell bodies and small apical surfaces. Even in adults, the hair bundles of the extramacular cells appear immature, with a long kinocilium (6–9 μm) and short stereocilia (0.5–2 μm). At least 90% of extramacular hair cells are likely to be innervated as demonstrated by labeling of nerve fibers with an antineurofilament antibody. The extramacular hair cells may differentiate in regions just beyond the edge of the macula at an early stage in development and then be pushed out via the interstitial growth of the epithelium that surrounds the macula. It is also possible that they may be produced from cell divisions in the extramacular epithelium that has not been considered capable of giving rise to hair cells.     

Active control of sensory hair mechanics implied by susceptibility to media that induce contraction in muscle

The motion of individual sensory hair bundles in the crista ampullaris was studied quantitatively by subjecting them to a brief jet of fluid in response to which they would swing away and then return by elasticity inherent at their insertion point. This motion was studied in media that would induce relaxation or contraction in a muscle system. The motion became severely restricted under conditions promoting contraction. Similar results were obtained by application to the organ of an ionophore that has the capacity to enter the cell membrane and allow influx of calcium ions. There was no effect of the ionophore in the absence of calcium ions. These results indicate that the sensory cells in the ear may possess a contractile machinery situated at the input end of the cell in the region of the sensory hairs and cuticular plate. The functional implication is that the mechanical input properties of the hair cells, and thus their excitability, can be under physiological control. It further implies that hair cells can produce a mechanical output in response to sensory or synaptic stimuli.     

Rods of actin filaments in type I hair cells of the shaker-2 mouse

Summary The shaker-2 mouse with inherited inner ear disease suffers from deafness and a shaking-waltzing behavior. The hair cell type I in cristae ampullares and maculae utriculi show a specific pathology, featuring fusion of the stereocilia and presence of a rod-shaped inclusion body. The inclusion body is composed of filaments that could be identified as the protein actin by the method of decoration with subfragment S-1 of myosin. The functional polarity was determined, and S-1 fragments were found to point apically, that is, from the nucleus up toward the cuticular plate. These observations are identical to those earlier described in the waltzing guinea pig. It is concluded that the identical pathology at a cellular level in two different species may indicate a pathologic disorder in a process fundamental to the normal development of this type of hair cell.Supported by grants from the Swedish Medical Research Council (no 04X-02461 and 12X-00720), The Ragnar and Torsten Söderberg's Foundation and the Foundation Tysta Skolan     

Expression of glycoconjugates in the mouse inner ear after prenatal irradiation

Summary Irradiation of the murine fetal inner ear is known to produce damage both to the vestibular and cochlear parts in the adult mouse. Fluorescein-labelled lectins were used to reveal possible differences in the glycoconjugate content between normal and irradiated inner ears. In the vestibular part, the otoconia showed the highest uptake of labelled sugars. This uptake was weaker after irradiation when compared to non-irradiated specimens. The type I hair cells in the ampulla and in the utricle showed a weaker uptake, but no labelling was demonstrated in the type II hair cells compared to the non-irradiated controls. In the cochlear part of the inner ear almost no uptake of fluorescent-binding lectins could be demonstrated in the irradiated groups except for in the tectorial membrane. In the endolymphatic sac no uptake was shown after prenatal irradiation. These findings are discussed and correlated to the already known damage of the inner ear following prenatal irradiation.     

Transient receptor potential channels in the inner ear: presence of transient receptor potential channel subfamily 1 and 4 in the guinea pig inner ear

Conclusion. The results of this study indicate that transient receptor potential subfamily 1 (TRPV1) may play a functional role in sensory cell physiology and that TRPV4 may be important for fluid homeostasis in the inner ear. Objective. To analyze the expression of TRPV1 and -4 in the normal guinea pig inner ear. Material and methods. Albino guinea pigs were used. The location of TRPV1 and -4 in the inner ear, i.e. cochlea, vestibular end organs and endolymphatic sac, was investigated by means of immunohistochemistry. Results. Immunohistochemistry revealed the presence of TRPV1 in the hair cells and supporting cells of the organ of Corti, in spiral ganglion cells, sensory cells of the vestibular end organs and vestibular ganglion cells. TRPV4 was found in the hair cells and supporting cells of the organ of Corti, in marginal cells of the stria vascularis, spiral ganglion cells, sensory cells, transitional cells, dark cells in the vestibular end organs, vestibular ganglion cells and epithelial cells of the endolymphatic sac.     

The inner ear and the in vitro system

Summary The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.Supported by grants from Karolinska Institutet, The Swedish Medical Research Council (grant no 12X-720) and The Swedish Society of Medical Sciences     

Energy dispersive X-ray microanalysis of individual yestibular hair cells

Summary Microprobe analysis was performed at the cellular and subcellular levels of type I and type II vestibular hair cells. In principle the same types of elemental histograms were found in the two types of hair cells studied. High concentrations of Cl and K were detected in stereocilia, whereas calcium was found when analyzing stereocilia and the supranuclear cytoplasm.Supported by grants from the Swedish Medical Research Council (12X-720 and 12X-7305), the Swedish Society for Medical Sciences, the Foundation Tysta Skolan, and the Ragnar and Torsten Söderberg Foundation