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Conclusion: Mouse-induced pluripotent stem cells (iPSCs) could differentiate into hair cell-like cells and spiral ganglion-like cells after transplantation into mouse cochleae, but it cannot improve the auditory brain response (ABR) thresholds in short term., Objective: To evaluate the potential of iPSCs for use as a source of transplants for the treatment of sensorineural hearing loss (SNHL)., Methods: Establishing SNHL mice model, then injecting the iPSCs or equal volume DMEM basic medium into the cochleae, respectively. Immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR) were used to assess the survival, migration, differentiation of the transplanted iPSCs in cochleae and then recorded the ABR threshold in different time. Hematoxylin-eosin (HE) staining was used to observe the teratoma formation., Results: Four weeks after transplantation, CM-Di1-labeled iPSCs could be found in the modiolus and Rosenthal's canal (RC), and some of them could expressed auditory hair cell markers or spiral ganglion neuron makers in group A, but not found in group B and C. As to the ABR threshold, no significance differences were found between pre- with postoperative in group A or B. In our study, no teratoma was observed in the cochleae.

Conclusion: Triamcinolone acetonide crystalline suspension (e.g. Volon A) was not ototoxic to the auditory hair cells present within organ of Corti explants and protected them from an ototoxic molecule, i.e. 4-hydroxy-2,3-nonenal (HNE), that is produced within the organ of Corti as a result of oxidative stress-induced damage., Objectives: To test the corticosteroid, triamcinolone acetonide, for ototoxicity and otoprotective capacity in organ of Corti explants., Materials and Methods: Organ of Corti explants excised from 4-day-old rats were the test system, HNE was the ototoxin challenge. Hair cell integrity counts were performed with fluorescent microscopy on fixed explants stained with FITC-labeled phalloidin. Statistical significance was set at p<0.05., Results: Triamcinolone acetonide did not affect hair cell integrity in the organ of Corti explants and it provided a high level of protection of hair cells against the ototoxic effects of a damaging level of HNE as determined by hair cell density counts.

Conclusion: The Id3 gene is expressed in the developing cochlear tissue and participates in the development of cochlear progenitor hair cells during the embryonic stage., Objectives: Inhibitor of differentiation (Id3) is an important transcription factor expressed in the cochlear tissue and progenitor hair cells. It is likely to be involved in cell-cycle progression and the proliferation of cochlear progenitor hair cells during the embryonic stage. The dynamic expression of Id3 in the developing cochlear tissue is, however, poorly understood. In this study we sought to characterize the expression of Id3 in the developing cochlear tissue and to explore its role in the growth and proliferation of progenitor hair cells., Material and Methods: Expression of the Id3 gene in the developing cochlear tissue was examined by means of Northern blotting, in situ hybridization and immunohistochemistry. The effects of Id3 on primary cultures of otocyst epithelial cells and a progenitor hair cell line (OT12) were studied using specific antisense oligonucleotides., Results: The Id3 gene was expressed in the rapidly growing otocyst on embryonic Day 12 and specifically in the fundamental structures of the cochlea, e.g. the organ of Corti, spiral ganglions and stria vascularis, on postnatal Day 1. Inhibition of Id3 gene expression with antisense oligonucleotides in cultured otocyst epithelial cells and OT12 reduced DNA synthesis and cell-cycle progression, suggesting that Id3 participates in the proliferation of cochlear progenitor hair cells.

Presbycusis is a complex of high frequency hearing loss and disproportionate loss of speech discrimination that is seen concomitantly with physical signs of aging. Among the most extensively characterized strains of mice that show an early hearing loss is the C57B16/J strain, a strain that shows early onset of high frequency hearing loss at age 6 months and complete hearing loss by 1 year of age. The histopathology of this strain consists of loss of hair cells and spiral ganglion neurons in the basal turn, with a progression of loss of hair cells and ganglion neurons towards the apical portion of the cochlea as the animal ages. The process of aging has been extensively studied and although details differ in various organisms the consensus today is that oxidative stress, i.e. free radical-mediated tissue damage, is one of the core mechanisms of aging. Aerobic metabolism results in the creation of hydrogen peroxide and reactive oxygen species. These are normally detoxified by a variety of enzymes and free radical scavengers, including superoxide dismutase (SOD), catalase and glutathione. To determine whether oxidative stress plays a role in the pathophysiology of hearing loss in this mouse model of presbycusis we determined the relative change in mRNA production for selected free radical detoxifying enzymes in the C57B16/J mouse cochlea. Using semi-quantitative RT-PCR with tubulin mRNA as a control, relative levels of antioxidant enzyme mRNAs were determined. There was an overall increase in SOD1 mRNA levels when comparing 1 and 9 month time points, and a transient increase in the expression level of catalase mRNA. B6.CAST+ Ahl mice, which carry the C57B16/J genome but receive their Ahl gene from CAST mice, do not show these alteractions in antioxidant enzyme production. Our results suggest that at an age of 9 months, at which point significant hearing loss has developed, the C57B16/J mouse cochlea is exposed to increased levels of free radicals and that the Ahl gene of the C57B16/J mouse mediates this decrease in protective enzymes and therefore increase in levels of oxidative stress.

The organ of Corti is highly ordered, with a single row of inner hair cells and three rows of outer hair cells. The number of hair cells produced was thought to be limited by the time of their terminal mitosis (i.e. E14 in the mouse). However, exogenous application of retinoic acid has been shown to stimulate the formation of supernumerary hair cells in organ of Corti explants from E13 to E16 mouse embryos. Using late embryonic and neonatal rat organ of Corti explants, we investigated the potential for production of supernumerary hair cells in more mature auditory sensory epithelia. When newborn rat organ of Corti explants were cultured under control conditions, an area of supernumerary hair cells was observed in a segment of organ of Corti that was at the junction between the basal and middle turns. In these areas of supernumerary hair cells the number of hair cells increased per unit of length, but remained constant per surface unit, further demonstrating the supernumerary character of this phenomenon. Organ of Corti explants treated with epidermal growth factor (EGF) showed a 50% increase in the length of the organ of Corti segment containing supernumerary hair cells. Upregulation of supernumerary hair cell formation by EGF was found to start and be maximal at birth (P0) and to disappear by 2 days after birth (P2). Treatment of EGF stimulated P0 explants with an antimitotic drug, cytosine arabinoside (ARAc), demonstrated that the production of supernumerary hair cells occurred independently of cell division.

Cochlear outer hair cells (OHC) are commonly assumed to function as mechanical effectors as well as sensory receptors in the organ of Corti (OC) of the inner ear. OHC in vitro and in organ explants exhibit mechanical responses to electrical, chemical or mechanical stimulation which may represent an aspect of their effector process that is expected in vivo. A detailed description, however, of an OHC effector operation in situ is still missing. Specifically, little is known as to how OHC movements influence the geometry of the OC in situ. Previous work has demonstrated that the motility of isolated OHCs in response to electrical stimulation and to K(+)-gluconate is probably under voltage control and causes depolarisation (shortening) and hyperpolarization (elongation). This work was undertaken to investigate if the movements that were observed in isolated OHC, and which are induced by ionic stimulation, could change the geometry of the OC. A synchronized depolarization of OHC was induced in guinea pig cochleae by exposing the entire OC to artificial endolymph (K+). Subsequent morphometry of mid-modiolar sections from these cochleae revealed that the distance between the basilar membrane (BM) and the reticular lamina (RL) had decreased considerably. Furthermore, in the three upper turns OHC had significantly shortened in all rows. The results suggest that OHC can change their length in the organ of Corti (OC) thus deforming the geometry of the OC. The experiments reveal a tonic force generation within the OC that may change the position of RL and/or BM, contribute to damping, modulate the BM-RL-distance and control the operating points of RL and sensory hair bundles. Thus, the results suggest active self-adjustments of cochlear mechanics by slow OHC length changes. Such mechanical adjustments have recently been postulated to correspond to timing elements of animal communication, speech or music.

The distribution of Na+, K(+)-ATPase, Ca(++)-ATPase, carbonic anhydrase, and calcium-binding proteins were investigated immunohistochemically in paraffin sections of guinea pig inner ears. Marginal cells of the stria vascularis, type II fibrocytes of the spiral ligament, and cells in supralimbal and suprastrial regions, were positive for Na+, K(+)-ATPase. Type I fibrocytes of the spiral ligament were positive for Ca(++)-ATPase, carbonic anhydrase, calmodulin and osteopontin. In the vestibular system, dark cells were positive for Na+, K(+)-ATPase. However, these cells and subepithelial fibrocytes were negative for Ca(++)-ATPase, carbonic anhydrase, and the calcium-binding proteins. In the endolymphatic sac, epithelial cells in intermediate and distal portions were positive for Na+, K(+)-ATPase, but the reaction was less than that in the stria. The same endolymphatic sac cells that were positive for Na+, K(+)-ATPase were also positive for Ca(++)-ATPase and calcium-binding proteins, but negative for carbonic anhydrase. The presence of Ca(++)-ATPase and calcium-binding proteins in the type I fibrocytes of the spiral ligament suggests that these cells are involved in mediating Ca++ regulation. Lower levels of Na+, K(+)-ATPase and the co-existence of Ca(++)-ATPase and calcium-binding proteins in the epithelial cells of the endolymphatic sac indicate that these cells have a distinctive role in ion transport that is different from that of the cells of the stria vascularis and vestibular dark cells.

The present paper summarizes the main stages in the maturation of the cochlear neurosensory structures in humans. New trends in the field of the functional development of the cochlea, as well as some recent experimental data are also discussed.

Several studies have reported variations in shape and size of stereociliary bundles and in a limited number of observations have associated them to type I and type II hair cells. A systematic study has been undertaken for which a technique was developed in order to identify both cell types and their corresponding hair bundles. Numerous fissures were obtained in saccular epithelia and observed in scanning electron microscopy. Saccular type I and type II hair cells in the guinea pig were found to have distinctive hair bundles. The tallest stereocilia of almost all type I cells were longer than 6 microns, and were shorter in the striola compared to the periphery. In contrast, the tallest stereocilia of almost all type II cells were shorter than 6 microns and were not found to vary notably in size from the striola to the periphery. Hair bundles with stereocilia organized in straight or in staggered rows were found for both types of cells across the whole saccular epithelium, with no apparent particular distribution. Possible physiological significance of differences in hair bundles is discussed.

A histological study was made to determine the cochlear neuronal populations of 20 human ears having hearing loss caused by developmental defects. The neuronal populations ranged from 7677 in an ear with Mondini dysplasia to 30 753 in an ear with DiGeorge's syndrome, the norm for young human subjects being 35 000 neurons. The length of the cochlear (spiral) ganglion varied from 7.3 mm to 14.8 mm, the norm for human subjects being 12 mm. The sensorineural hearing losses in all cases were attributable to malformation or degeneration of the sensorineural structures. The hearing loss was moderate in one case of Alport's syndrome, severe in one case of Usher's syndrome and in one case of severe Mondini dysplasia; it was profound in one case of maternal rubella, one of congenital deafness of unknown cause, another case of severe Mondini dysplasia and one of Down's syndrome (Trisomy 21). One case of mild Mondini dysplasia and one of DiGeorge's syndrome were known to have normal hearing. In 4 other cases hearing status was absent.

The human organ of Corti was investigated with the freeze fracturing technique with the purpose of analysing membrane specializations. Tight junctions were found on hair cells as well as on supporting cells. Inner and outer hair cells were coupled to the supporting cells by rather extensive tight junctions. The tight junctions between the Deiter's cells were comparable to those of the hair cells, while the tight junctions between the Hensen's cells were considerably less extensive. Gap junctions were present coupling all supporting elements in the organ of Corti, small ones preferably in the apical regions of the cells and large ones in the basal region.

Enzyme-dissociated chinchilla cochlear sensory cells maintained excellent cellular integrity for two hours or more when kept in the tissue culture medium. The healthy appearing sensory cells maintained their refractile characteristics. Early signs of cell stress were shown by the aggregation of nuclear chromatin, clustering of cytoplasmic contents in the central portion of the supranuclear region of the outer hair cells, and Brownian motion of the mitochondria. The ultrastructural morphology of the enzyme-dissociated outer hair cells was generally well maintained, but with such subtle changes as increased vesicle formation, condensation of mitochondrial matrix, nuclear chromatin aggregation, and vacuole formation. Some of the vesicles present in the infranuclear region are interpreted to be neural synaptic vesicles that have accumulated in the cell, or they may be dilated microsomes or vesicles formed from the degenerating cytoplasmic membrane system (ER system) as a sign of cell stress or degeneration.

Methods for counting vestibular ganglion cells and determining the densities of hair cells and intraepithelial basophilic inclusions (IBI) in samples of cross-sectioned vestibular sensory epithelia are described. Data obtained by means of these methods in vestibular sensory epithelia and Scarpa's ganglia in individual temporal bones from subjects at different ages are presented. Both vestibular hair cells and nerve cells in Scarpa's ganglia are found numerically reduced in ears of aged individuals. Changes in the vestibular sensory epithelia appear to precede those seen in Scarpa's ganglion. The incidence of intraepithelial basophilic inclusions correlates with degeneration in the respective vestibular sensory epithelia. There are no striking differences in hair cells densities of the different vestibular sense organs of the same ear and from subjects at about the same age.

The cytodifferentiation and maturation of vestibular hair cells, and the pathological development of vestibular type I hair cells, were followed in a strain of the waltzing guinea pig. The hair cells are first identified on the 30th gestational day. During the next 7-10 days the number of hair cells increases considerably in parallel with cytodifferentiation: the development of stereocilia, the cuticular plate, the afferent nerve terminals and the formation of synaptic contacts. On the 48th gestational day efferent nerve terminals are identified as are also a large number of fully developed nerve calyces. Differentiation into hair cells type I and type II was clearly evident at this stage. The first pathological finding appeared on the 38th gestational day as fusion of stereocilia. Actin filament rods in type I hair cells are identified with certainty on the 60th gestational day. In parallel with the in vivo development, inner ear explants (30-50th gestational days) were cultured in vitro. In this material actin filament rods were found already in 30th gestational day explants cultured 8 days in vitro. At this stage also hair cell protrusion and sensory hair fusion occurred.

The outer hair cells and their nerve endings in the basal and third turns of the guinea pig cochlea were reconstructed three-dimensionally from serial thin sections by means of computer graphics, and morphometric data were obtained. The number of nerve endings in the third turn was two to three times greater than that in the basal turn. Many afferent and efferent terminals in the third turn did not demonstrate synaptic specialization. Presynaptic dense bodies were missing in the majority of outer hair cells in both basal and third turns. The morphologic arrangement of the subsurface cisternae and efferent fiber synapses on the side and base of the outer hair cells suggests a close functional relationship. The nerve fibers and cisternae may be involved in the contractile process of the cells. The volume of the outer hair cells in the first row of the basal turn was about 656 micron 3, and third turn, 1358 micron 3. The total count of mitochondria in the outer hair cells of the first row in the basal turn was 1425, and 1963 in the third turn. The density of mitochondria in the sensory cell in the basal turn was higher. The highest density was seen in the infranuclear region. The mitochondrial distribution patterns suggest that metabolic activity of the outer hair cells is higher in the basal turn than in the third turn and the energy requirement is greatest in the region close to nerve endings.

Round window cochlear microphonic recordings from 33 guinea pigs were obtained prior to and following exposure to a 4 kHz pure tone at levels ranging from 124 dB to 140 dB for 5 to 80 minutes. When electro-physiological evaluation was complete, the animals were killed and tje cochlear tissues prepared using a soft-surface preparation technique. Experimental and control animals were mixed and randomly assessed without prior knowledge of the groups to which the specimens belonged. Histopathological observations were quantified and computer analysed. Statistical analyses suggest that noise exposure results in an overall decrease in blood flow to the cochlea with slight intracochlear increases in flow, perhaps compensatory in nature, to locations corresponding to the 4 kHz exposed region. Results implied that the initial localized hair cell damage known to occur as a result of overexposure to a discrete pure tone is not caused by a decreased blood flow. Relationships between electro-physiological and vascular changes and noise 'dose' are discussed.