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PubMed1983

The sensory epithelia of the human labyrinth. A freeze-fracturing and transmission electron microscopic study.

The end-organs of the human vestibular system were studied with transmission electron microscopic and freeze-fracturing techniques. The general morphology of the sensory epithelia was comparable to that of animals. The hair cells consisted of flask-shaped type I hair cells with the surrounding nerve calyx and rod-shaped type II hair cells contacted by several small bouton-shaped nerve endings. The hair cells were coupled to neighboring supporting cells by tight junctions at the lateroapical aspect. These were regular in appearance and of the moderately tight type. This type of tight junction has been found elsewhere in the inner ear and is believed to be competent to maintain its unique ion composition. The lateral surfaces of the supporting cells were coupled by gap junctions. The afferent nerve terminal of the type I hair cell, the so-called calyx, totally encompassed the hair cell. It could not be concluded on the basis of the present material whether the type I hair cell-calyx complex mediates electrical or chemical synaptic contact. The synapses of afferent nerves on type II hair cells, however, displayed typical signs of being chemically mediated. Synaptic bodies, similar to those seen in animals, were associated with the presynaptic membrane inside the hair cell. Membrane specializations comparable to those of animals were also seen in these synapses. Efferent nerve endings and synapses were not readily identified and were thus not discussed in this study.

Cell Membrane↗
PubMed2005

Influence of supporting cells on neuronal degeneration after hair cell loss.

In sensorineural hearing loss, hair cell loss is often followed by loss of cochlear nerve fibers, which can continue for years after the insult. The degree and time course of neuronal loss varies, but the reasons for this variation are unclear. The present study addresses this issue with a quantitative analysis of hair cell, supporting cell, and neuronal survival in animals with long-term survival of up to 5.5 years from two types of drug-induced hair cell loss: aminoglycoside antibiotics and platinum-containing chemotherapeutics. To complement the analysis of the effects of organ of Corti damage on neuronal survival, cases of primary neuronal degeneration, via auditory nerve section, are also assessed. Analysis shows that (1) long-term neuronal survival is enhanced when supporting cells in the inner hair cell (IHC) area remain intact; (2) after hair cell loss, the time course of neuronal loss is slower in the apex than in the base; (3) primary loss of cochlear nerve fibers does not lead to secondary degeneration of sensory cells or supporting cells in the organ of Corti; and (4) after auditory nerve section, there can be a massive reinnervation of the IHC region, especially in the apex. Results are consistent with the idea that supporting cells participate in the regulation of neuronal survival and neuronal sprouting in the organ of Corti.

Aminoglycosides↗
PubMed1987

Cupulogenesis and glycoconjugates in the labyrinthine ampulla as revealed by WGA-gold labeling.

We studied the distribution of wheat germ agglutinin (WGA)-bindable glycoconjugates in the vestibular ampulla of mongolian gerbils. WGA was conjugated with gold particles and applied to Lowicryl K4M sections of the ampulla. WGA-binding sites were found on the cupula and some of the secretory granules and Golgi apparatuses in the supporting cells of the sensory epithelia. The granules were seen to secrete into the endolymphatic space through reticular membrane. It is likely, therefore, that glycoconjugates are glycosylated at the Golgi apparatus in the supporting cells, stored in the granules, and secreted through the reticular membrane into the endolymphatic space to be used as a component of the cupula. The cell membranes of various cells, connective tissue filaments in the perilymphatic space and the cytoplasm of melanocytes were also labeled with WGA-gold.

Animals↗
PubMed1996

[Continuous proliferation of supporting cells and indications for hair cell differentiation in the inner ear of adult song birds with genetic cochlear hearing loss].

Our previous investigations demonstrated that the Belgian Waterslagers (BWS) canary (Serinus canarius) was affected by an inherited sensorineural hearing loss. Compared to normal canaries of others strains, hair cell numbers in these birds were reduced on average by 30%. Since other birds are able to replace similar hair cell numbers after cochlear trauma, we investigated if BWS have the potential for supporting cell proliferation with subsequent hair cell differentiation or if they lack the repair mechanisms known to operate in other birds. In the present study the S-phase marker bromodeoxyuridine (BrdU) was used to demonstrate DNA synthesis and thus cell proliferation. We found on average six labelled nuclei per basilar papilla in BWS. This number of proliferating cells was in accordance with previous estimates of newly generated hair cells as based on the frequency of immature-appearing hair cells observed by scanning electron microscopy. We conclude that the division of supporting cells in BWS precedes the differentiation of hair cells. In contrast to BWS we found on average only one supporting cell division per day in normal canaries of other strains. However, this supporting cell proliferation in normal birds is probably not related to a loss of hair cells and does not lead to the differentiation of new hair cells. Our data indicate that differentiation of hair cells after supporting cell division occurs only if the rate of supporting cell proliferation is increased above the normal low level (probably by the loss of hair cells). Since BWS do not repair their basilar papilla despite a 30% hair cell loss (as compared to normal canaries) although they continuously produce new hair cells, we suggest that the regulation of the regeneration process is abnormal.

Animals↗
PubMed2006

Distinct population of hair cell progenitors can be isolated from the postnatal mouse cochlea using side population analysis.

In mammals, the permanence of hearing loss is due mostly to the incapacity of the cochlea to replace lost mechano-receptor cells (i.e., hair cells [HCs]). The generation of new HCs from a renewable source of progenitors is a principal requirement for developing a cell therapy within this sensory organ. A subset of stem cells, termed side population (SP), has been identified in several tissues of mammals. The ATP-binding cassette transporter Abcg2/Bcrp1 contributes to the specification of the SP phenotype and is proposed as a universal marker for stem/progenitor cells. A defining character of these SP cells is a high efflux capacity for Hoechst dye. Here, we demonstrate that Abcg2 transporter is expressed with two other stem/progenitor cell markers (i.e., Nestin and Musashi1) in distinct and overlapping domains of the supporting cells within the postnatal cochlea. We have developed and describe a fluorescence-activated cell sorting (FACS) technique that enables the purification of a discrete subpopulation of SP-supporting cells from the early postnatal mouse cochlea based on their ability to exclude Hoechst dye. These FACS-isolated cells can divide and express markers of stem/progenitor cells such as Abcg2, a determinant of the SP phenotype, and Musashi1, a neural stem/progenitor cell marker. These markers can differentiate cells expressing markers of HCs and supporting cells in vitro. Our observation that these SP cells are capable of differentiating into HC-like cells implies a possible use for such cells (i.e., the replacement of lost auditory HCs within damaged cochlea).

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMedDate not supplied

Heregulin enhances regenerative proliferation in postnatal rat utricular sensory epithelium after ototoxic damage.

Hair cell loss due to acoustic and ototoxic damage often leads to hearing and balance impairments. Although a spontaneous event in chicks and lower vertebrates, hair cell replacement occurs at a much lower frequency in mammals presumably due to a very low rate of supporting cell proliferation following injury. We report here that heregulin, a member of the neuregulin family, dramatically enhances proliferation of supporting cells in postnatal rat utricular epithelial sheet cultures after gentamicin treatment, as revealed by bromo-deoxyuridine (BrdU) immunocytochemistry. A dose-dependent study shows that the maximal effects of heregulin are achieved at 3 nM. The mitogenic effects of heregulin are confirmed in utricular whole mount cultures. Autoradiography of the utricular whole mount cultures shows that heregulin also enhances the number of tritiated thymidine-labeled cells within the hair cell layer. TaqMan quantitative RT-PCR analysis and immunocytochemistry reveal that heregulin and its binding receptors (ErbB-2, ErbB-3 and ErbB-4) are expressed in the inner ear sensory epithelium. Of several ligands activating various ErbB receptors, including heregulin, neuregulin-3, beta-cellulin, heparin binding-epidermal growth factor (HB-EGF), transforming growth factor-alpha (TGF-alpha) and EGF, heregulin shows the most potent mitogenic effects on supporting cells. Because neuregulin-3 that signals only through ErbB-4 does not show an effect, these data suggest that activation of the ErbB-2-ErbB-3 heterodimeric complexes, rather than ErbB-4, is critical for the proliferative response in the utricular sensory epithelium. In addition, gentamicin treatment induces an upregulation of heregulin mRNA. Considered together, heregulin may play an important role in hair cell regeneration following ototoxic damage.

Animals↗
PubMed1998

Effect of membrane tension on gap junctional conductance of supporting cells in Corti's organ.

The effects of turgor pressure-induced membrane tension on junctional coupling of Hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques. Turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions. Both positive pipette pressure and extracellular applications of hypotonic solutions, which caused cell size to concomitantly increase, uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance. These changes were, in many cases, reversible and repeatable. Intracellular application of 50 microM H-7, a broad-based protein kinase inhibitor, and 10 mM BAPTA did not block the uncoupling effect of positive turgor pressure on inner ear gap junctions. The transjunctional conductance at a holding potential of -80 mV was 53.6 +/- 5.8 nS (mean +/- SEM, n = 9) and decreased approximately 40% at a turgor pressure of 1.41 +/- 0.05 kPa. Considering the coincident kinetics of cell deformation and uncoupling, we speculate that mechanical forces work directly on gap junctions of the inner ear. These results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis.

Animals↗
PubMed2002

REST mRNA expression in normal and regenerating avian auditory epithelium.

Hair cells (HCs) and supporting cells (SCs) in the auditory epithelium initially arise from a sheet of undifferentiated cells. Although much has been learned about the initial steps leading to the fate determination of HCs and SCs, respectively, little is known about what molecular events 'finalize' cell fate determination. We investigated the role of repressor element-1 (RE-1) silencing transcription factor (REST), whose inactivation is known to be a requirement for a cell to assume a neuronal identity. Here we show by in situ hybridization (ISH) that SCs express REST messenger RNA (mRNA) but sensory HCs lack detectable expression. Using a more sensitive reverse transcription-polymerase chain reaction assay, however, we detected the presence of a neuron-specific splice variant in the epithelium, suggesting that HCs express REST mRNA at levels too low to be detectable by ISH. In regenerating auditory epithelium, we found that REST mRNA was expressed and upregulated in all remaining cells in the damaged region of the epithelium, consistent with its expression pattern during development prior to neurogenesis. Surprisingly, REST mRNA was also upregulated in SCs in the apical, undamaged region of the epithelium, and readily detectable by ISH in the HCs in this region. This finding suggests that the grossly undamaged region of the epithelium is in fact biochemically altered towards a 'less developed' state. Our results indicate that REST inactivation is an important step in finalizing HC fate in the chick inner ear.

Animals↗
PubMed1994

Differences along the place-frequency map in the structure of supporting cells in the gerbil cochlea.

The function of supporting cells was investigated by comparing their morphologic adaptations at six different places in the gerbil cochlea. The volume of Deiters cells and tectal (cover) cells increased whereas that of Boettcher and Claudius cells decreased from base to apex. Deiters cells in the basal region tuned between 40 and 20 kHz lacked the unique rosette complex seen in regions encoding frequencies at or below 10 kHz. Deiters cells in high frequency regions differed from those at lower frequency places in several other ways: they possessed more apical microtubules, a larger basal microtubule stalk, mitochondria in the basal compartment, apical mitochondria that were unassociated with plasmalemma and more symmetric, and a less elaborately folded apicomedial plasmalemma enveloping fewer nerves. The tectal cells covering the outer tunnel appeared unlike Hensen cells in location and structure and differed further in exhibiting more variability with position in the cochlea. These covering cells in regions encoding high frequencies (20 and 40 kHz) extended a thin process medially that formed the roof of the outer tunnel and connected with the phalanx of the third Deiters cell. The tectal cells exclusively in places at 10 kHz or below projected numerous fimbriae into the outer tunnel. Hensen cells lateral to the cover cells also differed with frequency in showing abundant apical microvilli and mitochondria and basal juxtaposition to Boettcher cells only in the 40 to 20 kHz region. The observed structural differences provide evidence for functional variability along the place-frequency map. They attest to greater ion resorption from the outer tunnel by Deiters and tectal cells in low to mid frequency regions, and for greater ion exchange between endolymph and perilymph by Hensen, Boettcher and outer sulcus cells in regions of the cochlea encoding high frequencies. Amplification of the Deiters cells' microtubule system in the base of the cochlea possibly imparts increased stiffness to these cells and enhances transmission of mechanical energy at high frequency.

Acoustic Stimulation↗
PubMed2005

Regional estimates of hair cells and supporting cells in the human crista ampullaris.

Regional estimates of type I and type II vestibular hair cells (HC) and supporting cell (SC) numbers were obtained from the horizontal crista ampullaris by using design-based stereology in human. Cristae were microdissected from temporal bones obtained post-mortem (N=16, age range 26-98 years). Three groups were made according to age: group 1, n=5, ages between 26 and 67 years, average age 51 years; group 2, n=4, average age 84 years; and group 3, n=7, average age 94 years. For group 1, the average total HC number was 8,005+/-214, corresponding to 4,119+/-107 type I HC, 3,886+/-117 type II HC, and 10,274+/-224 SC. The type I:type II HC ratio was 1.06+/-0.01, and HC density was 0.80 cells/100 microm2. For group 2, the average total HC number was 7,074+/-489, corresponding to 3,733+/-212 type I HC, 3,341+/-314 type II HC, and 9,321+/-858 SC. The type I:II HC ratio was 1.12+/-0.06, and HC density was 0.75 cells/100 microm2. For group 3, the average HC number was 6,009+/-327, corresponding to 3,380+/-223 type I HC, 2,628+/-235 type II HC, and 10,185+/-182 SC. The type I:II HC ratio was 1.34+/-0.10, and HC density was 0.63 cells/100 microm2. A significant decline in type I, type II, and total HC number and density was found in groups 2 and 3, with individuals exceeding the average human life span.

Adult↗
PubMed1996

[DNA replication and cellular proliferation in the noise damaged basilar papillar of chicks].

An acoustic trauma model for study of the repair of the auditory epithelia was established in postnatal 8-14 days chicks. The animals were continuously exposed to the wide band noise at 115 dB (A) for 72 hours. The tracer of DNA replication-[3H] thymidine (3H-dR) was given in vivo or in vitro during or after exposure. The basilar papilla were cultured and processed with autoradiographic technique. The results demonstrated that at 48 hours following initial labelling of tracer, the incorporation of 3H-TdR was found over the nucli of supporting cells (or precursor cells) in the injured region of basilar papilla. The labelled hair cells appeared at 96 hours near the labelled supporting cells. Until the fifth to sixth days of culturing, the regenerated hair cells were still immature morphologically. No sign of labelled hair cells were detected in the control animals. Our findings indicated the potential capability of cellular proliferation and DNA replication in damaged auditory epithelia in vitro. Some kind of supporting cells in damaged region could be the precursors for regenerating hair cells. The positive cells labelled with 3H-TdR migrated to the surface of basilar papilar and differentiated into hair cells eventually. Therefore, it could be speculated that a homogeneous relationship may exist between labelled supporting cells and labelled hair cells.

Animals↗
PubMed2006

Transdifferentiation and its applicability for inner ear therapy.

During normal development, cells divide, then differentiate to adopt their individual form and function in an organism. Under most circumstances, mature cells cannot transdifferentiate, changing their fate to adopt a different form and function. Because differentiated cells cannot usually divide, the repair of injuries as well as regeneration largely depends on the activation of stem cell reserves. The mature cochlea is an exception among epithelial cell layers in that it lacks stem cells. Consequently, the sensory hair cells that receive sound information cannot be replaced, and their loss results in permanent hearing impairment. The lack of a spontaneous cell replacement mechanism in the organ of Corti, the mammalian auditory sensory epithelium, has led researchers to investigate circumstances in which transdifferentiation does occur. The hope is that this information can be used to design therapies to replace lost hair cells and restore impaired hearing in humans.

Animals↗
PubMed1990

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.

Animals↗
PubMed2004

Mechanical properties of sensory and supporting cells in the organ of Corti of the guinea pig cochlea--study by atomic force microscopy.

Mammalian hearing is refined by amplification of the motion of the cochlear partition. To understand the cochlear amplification, mechanical models of the cochlea have been used. When the dynamic behavior of the cochlea is analyzed by a model, elastic properties of the cells in the organ of Corti must be determined in advance. Recently, elastic properties of outer hair cells (OHCs) and pillar cells have been elucidated. However, those of other cells have not yet been clarified. Therefore, in this study, using an atomic force microscope (AFM), elastic properties of Hensen's cells, Deiters' cells and inner hair cells (IHCs) in the apical turn and those in the basal and second turns were estimated. As a result, slopes indicative of cell elastic properties were (8.9 +/- 5.8) x 10(3) m(-1) for Hensen's cells (n = 30), (5.5 +/- 5.3) x 10(3) m(-1) for Deiters' cells (n = 20) and (3.8 +/- 2.6) x 10(3) m(-1) for IHCs (n = 20), and Young's modulus were 0.69 +/- 0.45 kPa for Hensen's cells and 0.29 +/- 0.20 kPa for IHCs. There was no significant difference between elastic properties of each type of cell in the apical turn and those in the basal and second turns. However, it was found that there is a significant difference between Young's moduli of cells estimated in this study and those of the OHCs and pillar cells reported previously.

Animals↗
PubMed1981

Immunofluorescence localizations of proteins in semithin 0.2--1 micron frozen sections of the ear. A report of improved techniques including gelatin encapsulation and cryoultramicrotomy.

The present work describes a high resolution technique for locating proteins in frozen sections of the inner ear by immunofluorescence. Dissected organs are encapsulated in gelatin, and sections 0.1--1 micron thick are cut at --100 degrees C in a cryoultramicrotome. These are labelled with antibodies against two cytoskeletal proteins, actin and tubulin. Actin, which had previously only been described in the sensory cells, is found in the supporting cells as well. Tubulin is identified in the supporting cells and in outer spiral nerve fibres.

Actins↗