Slc26a4 expression prevents fluctuation of hearing in a mouse model of large vestibular aqueduct syndrome.

SLC26A4 mutations cause fluctuating and progressive hearing loss associated with enlargement of the vestibular aqueduct (EVA). SLC26A4 encodes a transmembrane anion exchanger called pendrin expressed in nonsensory epithelial cells of the lateral wall of cochlea, vestibular organs and endolymphatic sac. We previously described a transgenic mouse model of EVA with doxycycline (dox)-inducible expression of Slc26a4 in which administration of dox from conception to embryonic day 17.5 (DE17.5) resulted in hearing fluctuation between 1 and 3months of age. In the present study, we hypothesized that Slc26a4 is required to stabilize hearing in DE17.5 ears between 1 and 3months of age. We tested our hypothesis by evaluating the effect of postnatal re-induction of Slc26a4 expression on hearing. Readministration of dox to DE17.5 mice at postnatal day 6 (P6), but not at 1month of age, resulted in reduced click-evoked auditory brainstem response (ABR) thresholds, less fluctuation of hearing and a higher surface density of pendrin expression in spindle-shaped cells of the stria vascularis. Pendrin expression in spindle-shaped cells was inversely correlated with ABR thresholds. These findings suggest that stabilization of hearing by readministration of dox at P6 is mediated by pendrin expression in spindle-shaped cells. We conclude that early re-induction of Slc26a4 expression can prevent fluctuation of hearing in our Slc26a4-insufficient mouse model. Restoration of SLC26A4 expression and function could reduce or prevent fluctuation of hearing in EVA patients.

Failure of fluid absorption in the endolymphatic sac initiates cochlear enlargement that leads to deafness in mice lacking pendrin expression.

Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4(-/-), results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4(+/-) and Slc26a4(-/-) mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4(-/-) compared to Slc26a4(+/-) mice. In Slc26a4(+/-) and Slc26a4(-/-) mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4(+/-) mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4(-/-) mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.

Otolith crystals (in Carapidae): growth and habit.

The biomineralization of otoliths results mainly from the release of soluble Ca(2+), which is in turn precipitated as CaCO(3) crystals. In some Carapidae, sagittae sections have been shown to reveal a three-dimensional asymmetry with a nucleus close to the sulcal side, an unusual position. This study seeks to understand otolith formation in Carapus boraborensis. The unusual shape of the otolith is partly explained by the distribution of the epithelium cells, and particularly the sensory epithelium. Experimental evidence shows for the first time that aragonite growth takes place along the c-axis. These aragonite needles present two different habits. On the sulcal side is found the acicular form resulting from rapid growth during a short period of time. On the anti-sulcal side, the prismatic form seen there is due to a slower growth speed over longer periods. The otolith surface was observed each hour during a period of 24h in fishes reared in similar conditions. This allowed for the first time the direct observation on the otolith surface of the deposition of the two layers (L-zone and D-zone). In C. boraborensis, the organic-rich layer (D-zone) develops during the day, whereas the CaCO(3) layer (L-zone) seems to be deposited during the night.

Expression patterns of aquaporins in the inner ear: evidence for concerted actions of multiple types of aquaporins to facilitate water transport in the cochlea.

Water transport between the perilymph and endolymph is important in regulations of volume and osmotic pressure of the inner ear labyrinth. It is now known that expression of water channels (aquaporins or AQPs) in the cell membrane dramatically increases the ability of water to cross epithelial cells. The aims of the current study were to investigate the cellular localization of AQPs by immunolabeling, and to study the developmental expression and relative abundance of various subtypes of AQPs. We report here that AQP3, AQP7 and AQP9 were expressed in the inner ear. Specific subtypes of AQPs were found in discrete regions expressed by both epithelial cells and fibrocytes in cochlear and vestibular organs. Semi-quantitative measurements showed that AQP4 and AQP1 were the two most abundantly expressed AQP subtypes in the inner ear, and their expressions were dramatically upregulated during development. These data showed a highly localized and largely non-overlapping distribution pattern for different subtypes of AQPs in the inner ear, suggesting the existence of regional subtype-specific water transport pathways, and global regulation of water transport in the inner ear may require concerted actions of multiple types of AQPs.

In vitro growth of human endolymphatic sac cells: a transmission electron microscopic and immunohistochemical study in patients with vestibular schwannoma and Ménière's disease.

HYPOTHESIS: Human endolymphatic sac cells have been notoriously difficult to maintain in culture. It was hypothesized that an in vitro environment intended for growth of keratinocytes would also be suitable for human endolymph sac cells. BACKGROUND: Studies on cell physiology of human endolymphatic sac cells have been hampered by difficulties in maintaining them in culture. METHODS: Human endolymphatic sac cells were taken from 10 patients during translabyrinthine skull base surgery for vestibular schwannoma, one of whom also had Ménière's disease. Cell lines of proliferating epithelial cells were obtained after trypsinization and growth in a 3:1 mixture of Dulbecco's modified Eagle medium and Ham's F12 medium supplemented with 10% fetal calf serum. Fibroblast overgrowth was counteracted by the use of so-called cloning rings. During various stages, cells were investigated with transmission electron microscopy and/or immunohistochemistry. RESULTS: Proliferation took place after 2 to 3 days of primary cell culture. The cells were cytokeratin-positive and pleomorphic, and they had abundant polarized microvillus-like projections, numerous coated cytoplasmic pits and vesicles, and a well-developed rough endoplasmic reticulum. CONCLUSION: Cell lines of proliferating human endolymphatic sac cells can be produced with the technique described here and may be a valid tool in studies of human endolymph sac physiology.

Developmental and cellular expression pattern of epithelial sodium channel alpha, beta and gamma subunits in the inner ear of the rat.

Endolymphatic ion composition in the adult inner ear is characterized by high K(+) and low Na(+) concentration. This unique ion composition is essential for proper functioning of sensory processing. Although a lot has been learned in recent years about molecules involved in K(+) transport in inner ear, the molecules involved in Na(+) transport are only beginning to emerge. The epithelial Na(+) channel (ENaC) is a highly selective Na(+) channel that is expressed in many Na(+)-reabsorbing tissues. The aim of our study was to investigate whether ENaC is expressed in inner ear of rats and could account for Na(+) reabsorption from endolymph. We detected mRNA for the three channel-forming subunits (alpha, beta and gamma ENaC) in cochlea, vestibular system and endolymphatic sac. mRNA abundance increased during the first 12 days of life in cochlea and vestibular system, coinciding with decreasing Na(+) concentration in endolymph. Expression was strongest in epithelial cells lining scala media, most notably Claudius' cells. As these cells are characterized by a very negative resting potential they would be ideally suited for reabsorption of Na(+). mRNA abundance in endolymphatic sac decreased during the first 6 days of life, suggesting that ENaC might be implicated in reabsorption of endolymph in the endolymphatic sac of neonatal animals. Together, our results suggest that the epithelial Na+ channel is a good candidate for a molecule involved in Na(+) homeostasis in inner ear.

Postnasal maturation of the human endolymphatic sac.

OBJECTIVE/HYPOTHESIS: The objective of this study was to trace the postnatal development of the human endolymphatic sac. The endolymphatic sac starts as a pouch-like structure during the fetal period in the human but later develops into a complex network of tubules in its mature form. STUDY DESIGN: Observational study. METHODS: Light microscopic examination of the morphology of the endolymphatic sac in a population of temporal bones. RESULTS: Examination of 28 temporal bones from 19 infants and children reveals that endolymphatic sac morphological maturation is usually completed in the first year of life, although it may occur as late as 4 years of age. There was good correlation of maturation levels between sides when pairs of temporal bones were evaluated. Immature pouch-like endolymphatic sacs may appear as late as 4 years of age. CONCLUSIONS: Endolymphatic sac maturation to its adult tubular form is usually completed by the first year. This finding contrasts with other studies examining the growth of the endolymphatic sac to adult size after birth. Histological evidence suggests that the endolymphatic sac, although not morphologically mature by appearance, may still be participating at some level of functioning.

Morphology of the developing human endolymphatic sac.

The adult human endolymphatic sac (ES) has been described as a complex network of interconnected tubules. Embryologic examination describes the human ES as a single-lumen, pouch-like structure. Transition from saccular shape to tubules during the entire fetal period has not been previously reported. Tubular ES structure is thought to be unique to humans. Animal investigations describe similar saccular appearance, but without tubules in mature sacs. The authors examined 45 human fetal temporal bones to trace ES development and reviewed six types of animal sacs. Results in humans reveal tubular structure as early as 26 weeks' gestation. Maturation variably occurred in the fetal period and postnatally. For the first time, the tubular system is noted in the animal, the rhesus monkey. These findings suggest that the tubular system may represent more advanced specialized function.

Pre- and postnatal expression of glycoconjugates (3-fucosyl-N-acetyllactosamine and HNK-1 epitopes) in the mouse inner ear.

The distribution of two glycoconjugates 3-fucosyl-N-acetyllactosamine (CD15) and HNK-1 epitope (CD57) in the inner ear of the NMRI mouse was analysed from the eighth day of gestation to the 16th day after birth. CD15 epitope distribution is developmentally regulated. The up- and down-regulation of expression, the change in the number of cells which are positive, the ingrowth of CD15-positive cells and their distribution, intracellular and/or cell-surface-associated expression, all assume a characteristic appearance at each developmental stage. Distribution of CD57 documented the nerve outgrowth and formation of the innervation of the vestibular apparatus and cochlear duct. Correlation between CD15 and CD57 expression patterns revealed differences in the interaction of the ingrowing fibres and epithelial tissue between the vestibular organ and the cochlea and differences in the development of the cristae and maculae.

Expression of the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine (CD 15) in the adult guinea pig inner ear.

The expression of the CD 15 (3-fucosyl-N-acetyl-lactosamine) epitope was immunohistochemically studied on paraffin sections of adult guinea pig inner ears. Two regions of the inner ear expressed the epitope for CD 15: the tectorial membrane of the cochlea and the endolymphatic sac. The upper part of the main body of the tectorial membrane was deeply stained. In the rugosal and distal part of the endolymphatic sac several unevenly distributed cells showed strong intra- and extracellular localization of the CD15 epitope. The CD15 epitope is associated with a transduction structure (tectorial membrane) and with a "volume regulating" compartment (endolymphatic sac) and may be involved in the maintenance of the structural integrity of both.

Murine endolymphatic sac development in tissue culture: an in vitro model for sac function.

Numerous studies have attempted to elucidate the function of the mammalian endolymphatic sac (ELS). All of these studies have been performed on in vivo specimens and are thus influenced by humoral and tissue factors extraneous to the sac. In contrast, an in vitro model would provide an opportunity to study ELS cells in a carefully controlled environment. This report presents our experience with tissue culturing the murine endolymphatic sac removed from 16 and 18 gestational day fetuses. Light (LM) and transmission electron microscopical (TEM) evaluations of the developing endolymphatic sac were performed over periods of one, four, and seven days in tissue culture. In order to confirm growth and maturation, three-dimensional reconstructions from serial sections of the cultured ELS were made and compared with published accounts of in vivo murine ELS development for equivalent periods of time. Both whole and dissected otocysts were grown in tissue culture and compared with one another. Two different tissue culture medias were investigated, each with and without the addition of collagenase, used to soften the dense fibrous capsule of the otocyst and thus facilitate dissection and histological preparation. The impact of collagenase and the tissue culture medias on endolymphatic sac growth were studied. Results demonstrated that murine ELS cells were able to differentiate and mature in tissue culture, as confirmed by LM, TEM, and three-dimensional reconstructions. After an initial delay, in vitro maturation of cells in tissue culture paralleled normal in vivo growth and in some specimens appeared to show accelerated maturation. This in vitro model should prove useful in efforts to define ELS function and in providing a technique for tissue culturing human ELS from normal and diseased ears.

Light microscopic analysis of the gravireceptor in Xenopus larvae developed in hypogravity.

The paper describes an investigation of the influence of gravity on the early differentiation of gravity receptors in Xenopus embryos and larvae. There is evidence that the expression of crystals in the saccus endolymphaticus was statistically greater when the embryos developed in near weightlessness (hypogravity) than on earth. The function of these crystals is unknown but they may contribute to the functioning of the vestibular apparatus.

The pre- and postnatal maturation of the epithelium in the endolymphatic sac. An electron microscopic survey.

The cellular development of the endolymphatic sac was studied in the CBA/CBA mouse, starting from day 10 of gestation following the different stages of maturation up to an adult age of one month. The first immature cylindrical cells lining the future sac in several cell layers are seen at day 12 of gestation. At day 18 of gestation, a true sac appears and a floccular precipitate is frequently found in its lumen together with signs of increased activity in the still immature epithelial cells. Approximately one day before birth the first signs of the future light and dark cells can be distinguished. At day 4 post partum the cells are more differentiated with some showing signs of secretory activity indicating that these cells start to function at this stage. Eight days after birth differentiation into distinguishable almost mature light and dark cells is seen. Two days later these epithelial cells have obtained a fully mature appearance. At 14 days after birth widened lateral intercellular spaces separating the epithelial cells can be visualized and a few free floating cells are found in the sac lumen. The sac epithelium is thus considered to have completed its maturation process at this stage.

The development of the endolymphatic duct and sac. A light microscopical study.

The development and maturation of the endolymphatic sac were studied in the CBA/CBA mouse. The otocyst is developed at gestational day 10 and the primitive endolymphatic sac is present as a large slit-like appendage at day 12 of gestation. At day 18 the endolymphatic sac is clearly detached from the rest of the otocyst, forming a true sac. The epithelial lining consists of only one layer of immature cells containing large vesicles. The endolymphatic sac is surrounded by a rich network of vessels. One day before birth, the epithelial lining is uneven and the first signs of differentiation into light and dark cells is visible. This situation is more pronounced 2 days post partum when the sac also seems to be filled with a stainable material. At day 6 post partum the otic capsule fuses around the sac, forming the vestibular aqueduct. At 14 days post partum the sac is mature, with clearly developed light and dark cells and widened lateral intercellular spaces, constituting the rugose epithelium. The lumen is filled with a stainable precipitate and a few free-floating cells.

Dimensional anatomy of the vestibular aqueduct and the endolymphatic sac (rugose portion) in human temporal bones. Statistical analysis of 79 bones.

The purpose of this study was to investigate the anatomy of the vestibular aqueduct (VA) and rugose portion (RP) of the endolymphatic sac. Serial horizontal sections of 79 normal temporal bones of individuals aged 20 to 102 years were used. Medial view graphic reconstruction of the VA was performed for each specimen to determine the area, length, angle and position of the VA. The relationship between the width of the VA and the area of the VA, and the relationship between the degree of development of the VA and the degree of development of the temporal bone (periaqueductal pneumatization and the otic capsule in the periaqueductal region) were also investigated. Measurements of the RP were also made and further histological study of the RP was performed on 30 selected specimens. We found that 1) the VA in adult temporal bone varies in size and can be classified as hypoplastic, normoplastic, or hyperplastic; 2) many of the VAs in each group have similar sizes, although they vary in length, angle, or position; 3) all or most of the RP was located within the VA in all but one specimen, which had a hypoplastic VA (in this specimen more than half of the entire RP extended into the posterior cranial fossa); 4) the histology of the RP was characteristic in each type of VA; and 5) the degree of development of the VA seems to correlate with the degree of development of the otic capsule in the periaqueductal region.

Postnatal development of the vestibular aqueduct and endolymphatic sac.

The purpose of this study was to gain basic information about the postnatal development of the vestibular aqueduct (VA) and the endolymphatic sac (ES). For this study, serial horizontal sections of 31 normal temporal bones of individuals whose ages ranged from 0 to 13 years were used. Medial view graphic reconstruction of the VA and rugose portion (RP) of the ES was performed in every case for analysis of the VA and RP. The findings of this study revealed the following new information about the postnatal development of the VA and ES. 1) The VA and RP undergo significant growth postnatally up to age 3 years. 2) In the newborn, individual variations in the VA and RP already exist and at age 3 years significantly wide individual variations which can be classified into three groups (hypoplastic, normoplastic, hyperplastic) may be recognized. 3) Hypoplastic VAs are of two types: one is fairly elongated and tubelike while the other is short and funnel-shaped. The tubelike VA seems to be the prenatal form. 4) The changes that occur with development postnatally in the area of the VA are more closely related to the changes that occur in the length of the external aperture of the VA than they are to the changes that occur in the length of the VA. 5) Development of the area of the VA is closely correlated with development of the area of the RP.