Characterization of mouse melanocytes reveals ultrastructural and immunological insights into the inner ear function.

Human Bartter syndrome IV is an autosomal recessive disorder characterized by congenital deafness and severe renal salt and fluid loss. It is caused by mutations in BSND, which encodes barttin, a beta-subunit of ClC-Ka and ClC-Kb chloride channels. Inner-ear-specific disruption of Bsnd in mice now reveals that the positive potential, but not the high potassium concentration, of the scala media depends on the presence of these channels in the epithelium of the stria vascularis. The reduced driving force for K(+)-entry through mechanosensitive channels into sensory hair cells entails a profound congenital hearing loss and subtle vestibular symptoms. Although retaining all cell types and intact tight junctions, the thickness of the stria is reduced early on. Cochlear outer hair cells degenerate over several months. A collapse of endolymphatic space was seen when mice had additionally renal salt and fluid loss due to partial barttin deletion in the kidney. Bsnd(-/-) mice thus demonstrate a novel function of Cl(-) channels in generating the endocochlear potential and reveal the mechanism leading to deafness in human Bartter syndrome IV.

Labyrinthitis ossificans (LO) is the pathological deposition of new bone within the lumen of the cochlea and labyrinth. This process occurs most commonly as a result of infection or inflammation affecting the otic capsule. Trauma and vascular compromise can also lead to neo-ossification within the otic capsule. The mechanism that regulates this process remains unestablished. This study details the end-stage histopathology in high-resolution plastic thin sections. Twenty Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3 followed by subcutaneous penicillin G procaine (8 days) and were painlessly sacrificed 3 months later. The cochleas were serially divided and sectioned for light and electron microscopy. Sixteen of 20 animals (27 of 40 cochleas) demonstrated LO. Cochlear damage was most extensive in the vestibule and basal turn and decreased toward the apex, which often appeared normal. The histopathologic findings consisted of 1) new bone, calcospherites, osteoid, and fibrosis without dense connective tissue or osteoblasts extending from the endosteal wall into the lumen of the vestibule and scala tympani; 2) areas of dense connective tissue and osteoid enclosed by epithelial cells conjoined with the organ of Corti, stria vascularis, spiral ligament, and vestibular (Reissner's) membrane; and 3) partial to complete loss of the organ of Corti, spiral ligament cell bodies, stria vascularis, and spiral ganglion cells. Osteoblastic activity was not demonstrated in end-stage ossification in LO in the gerbil model. Neo-ossification appears to occur by calcospherite deposition along collagen-like fibrils within osteoid. The destruction of the organ of Corti, spiral ganglion cells, stria vascularis, and cells of Reissner's membrane and the spiral ligament occurs even in the absence of ossification of the cochlear duct.

The inner ear of mammals consists of the cochlea, which is involved with the sense of hearing, and the vestibule and three semicircular canals, which are involved with the sense of balance. Although different regions of the inner ear contribute to different functions, the bony chambers and membranous ducts are morphologically continuous. The gross anatomy of the cochlea that has been related to auditory physiologies includes overall size of the structure, including volume and total spiral length, development of internal cochlear structures, including the primary and secondary bony laminae, morphology of the spiral nerve ganglion, and the nature of cochlear coiling, including total number of turns completed by the cochlear canal and the relative diameters of the basal and apical turns. The overall sizes, shapes, and orientations of the semicircular canals are related to sensitivity to head rotations and possibly locomotor behaviors. Intraspecific variation, primarily in the shape and orientation of the semicircular canals, may provide additional clues to help us better understand form and function of the inner ear.

Methods to study the development, anatomy, and function of the zebrafish inner ear across the life course.

Zebrafish has become an excellent model for studying the development and function of inner ear. We report here a zebrafish line in which claudin 7b (cldn7b) locus is interrupted by a Tol2 transposon at its first intron. The homozygous mutants have enlarged otocysts, smaller or no otoliths, slowly formed semicircular canals, and insensitiveness to sound stimulation. These abnormal phenotypes and hearing loss of inner ear could be mostly rescued by injection of cldn7b-mRNA into one-cell stage homozygous mutant embryos. Mechanistically, cldn7b-deficiency interrupted the formation of apical junction complexes (AJCs) in otic epithelial cells of inner ear and the ion-homeostasis of endolymph, which then led to the loss of proper contact between otoliths and normally developed hair cells in utricle and saccule or aberrant mechanosensory transduction. Thus, Cldn7b is essential for the formation and proper function of inner ear through its unique role in keeping an initial integrity of otic epithelia during zebrafish embryogenesis.

A time-sequence study was made of the early ultrastructural changes of the stria vascularis and Reissner's membrane in the guinea pig after obliteration of the endolymphatic sac and duct. Pathological alterations of both the stria vascularis and Reissner's membrane were found to start in the apex of the cochlea. The morphological changes of the stria vascularis were characterized by an increase of vesicles in the marginal cells and by intercellular edema, followed by vacuolization and atrophy of marginal and intermediate cells. In Reissner's membrane extensive gaps in the mesothelial cell layer were observed together with intracellular pathology of the epithelial cells. The significance of these ultrastructural changes in the stria vascularis and Reissner's membrane with regard to the pathophysiology of the endolymphatic hydrops is discussed.

Connexin proteins form transmembranous gap junction channels that connect adjacent cells. Connexin26 and connexin30 have been previously shown to be strongly expressed in the inner ear of adult rats and to be mainly colocalized. Because intercellular connections by gap junction proteins are crucial for maturation of different tissues, we investigated the developmental expression of connexin26 and connexin30 in pre- and postnatal rats using immunocytochemistry. In the rat otocyst, staining for connexin26 as well as for connexin30 appeared at the 17th day of gestation. However, at this stage, expression of connexin30 was low and restricted to the neurosensory epithelium. Beginning from the 3rd postnatal day connexin26 and -30 were expressed with highest immunoreaction in the spiral limbus, the neurosensory epithelium, and between the stria vascularis and the spiral ligament. Beginning from postnatal day 12 the staining pattern resembled that of adult animals, with additional strong staining between all fibrocytes of the spiral ligament. Double labeling experiments demonstrated strongest colocalization of both connexins between the stria vascularis and the spiral ligament. These results demonstrate that development of the cochlear gap junction system precedes the functional maturation of the rat inner ear, which takes place between the 2nd and 3rd postnatal week. In the cochlea of a 22-week-old human embryo, connexin26 and connexin30 could be detected in the lateral wall, suggesting that both connexins also play a crucial role in function of the human inner ear.

Cations were precipitated with potassium antimonate in the cochlea of the guinea pig and electron microscopy was used to analyze the distribution of the formed reaction products. Differences in precipitate density between neighboring cells in Reissner's membrane, in the stria vascularis and in the limbus are described. Electron spectroscopic imaging was performed to obtain information about the spatial distribution of the precipitates and their elemental composition.

To investigate the distribution and role of alpha, beta and gamma subunits of epithelial sodium channel (ENaC) in the cochlea and endolymphatic sac of guinea pig. The expression of alpha-, beta- and gamma-ENaC subunits proteins was studied by immunohistochemistry with the specific polyclonal rabbit antibodies against the alpha, beta and gamma subunits of rat ENaC. Alpha-ENaC mRNA was detected by in situ hybridization with digoxin labeled cDNA probe. All three subunits of ENaC, alpha-, beta- and gamma-, were widely distributed in the labyrinth. In the cochlea, strong labeling of alpha-ENaC protein was found in the spiral limbus, and to a less extent, in the spiral ligament, organ of Corti and Reissner's membrane. The immunoreactivity of beta-ENaC was observed in the spiral ligament, spiral limbus, spiral ganglion, organ of Corti and Reissner's membrane with a less intensity than that of alpha-ENaC. Gamma-ENaC was presented primarily in the superior part of the spiral ligament, spiral limbus, spiral ganglion, and weakly in the organ of Corti and Reissner's membrane. In the endolymphatic sac, intensive immunoreactivities of all three subunits were seen in the epithelial cells and the subepithelial cells at similar intensity. Alpha-ENaC mRNA was localized in the spiral limbus, the inferior part of spiral ligament, stria vascularis, and epithelial cells and subepithelial cells of endolymphatic sac. Different subunits of the ENaC expressed in various cell regions of the cochlea and endolymphatic sac in distinct patterns may form the functional sodium channel to regulate the endolymph, thus serve to maintain homeostasis in inner ear.

The recessive mouse mutant headbobber (hb) displays the characteristic behavioural traits associated with vestibular defects including headbobbing, circling and deafness. This mutation was caused by the insertion of a transgene into distal chromosome 7 affecting expression of native genes. We show that the inner ear of hb/hb mutants lacks semicircular canals and cristae, and the saccule and utricle are fused together in a single utriculosaccular sac. Moreover, we detect severe abnormalities of the cochlear sensory hair cells, the stria vascularis looks severely disorganised, Reissner's membrane is collapsed and no endocochlear potential is detected. Myo7a and Kcnj10 expression analysis show a lack of the melanocyte-like intermediate cells in hb/hb stria vascularis, which can explain the absence of endocochlear potential. We use Trp2 as a marker of melanoblasts migrating from the neural crest at E12.5 and show that they do not interdigitate into the developing strial epithelium, associated with abnormal persistence of the basal lamina in the hb/hb cochlea. We perform array CGH, deep sequencing as well as an extensive expression analysis of candidate genes in the headbobber region of hb/hb and littermate controls, and conclude that the headbobber phenotype is caused by: 1) effect of a 648 kb deletion on distal Chr7, resulting in the loss of three protein coding genes (Gpr26, Cpmx2 and Chst15) with expression in the inner ear but unknown function; and 2) indirect, long range effect of the deletion on the expression of neighboring genes on Chr7, associated with downregulation of Hmx3, Hmx2 and Nkx1.2 homeobox transcription factors. Interestingly, deletions of the orthologous region in humans, affecting the same genes, have been reported in nineteen patients with common features including sensorineural hearing loss and vestibular problems. Therefore, we propose that headbobber is a useful model to gain insight into the mechanisms underlying deafness in human 10qter deletion syndrome.

The auditory pathway potentials have been recorded from a series of 35 cats, and the stria vascularis and Reissner's membrane examined with the electron microscope. The ages varied from the first postnatal day to at least 6 years. 30 of the animals had white coats, and 45 of the ears lacked potentials. The basal processes of the marginal cells fail to develop in pathological ears, and appear displaced towards the endolymphatic surface by a layer of grossly swollen, chromophobe cells. These features are already present at the 3 day stage, and persist until the third week. Thereafter, increasing electron density of all cell types is found, with progressive reduction in strial thickness. Swelling of the capillary endothelial cells and descent of Reissner's membrane are found in the first coil at 2 weeks. Obliteration of capillaries, and development of vacuolar cytoplasmic outgrowths from the vestibular membrane mesothelial cells occur at later stages. These findings are discussed and compared with the pathological changes produced by a variety of endogenous 2nd exogenous factors in both man and animals.

Recent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ has not been characterized. We used a reporter mouse in which cells expressing NT3 also express beta-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3 expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult (P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular and cochlear sense organs, Reissner's membrane, saccular membrane, and the dark cells adjacent to canal organs. With increasing age, staining disappeared in most cell types but remained relatively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea, by P0 there is a longitudinal gradient (apex > base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar > extrastriolar regions and supporting cells > hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular systems and that supporting cells are the main source of this factor in the adult.

The aim of the present study was to show the morphological degeneration at the ultrastructural level, after damaging the stria vascularis experimentally. The acute lesions, after acute atoxyl intoxication, occur after about 12 hours, and begin as a degeneration of both the marginal and the intermediate cells, whereas the basal cells remain mainly unaffected. The severely damaged marginal or intermediate cells may become loosened from the stria vascularis and rejected from it into the endolymphatic space. Under such conditions the basal cells may line the surface facing the endolymph, although this occurs very rarely. Intially, there may be a slight bulging of Reissner's membrane, but soon the membrane becomes depressed, and sometimes a total collapse occurs, with Reissner's membrane flattened over the tectorial membrane against the organ of Corti. It is only seldom that Reissner's membrane touches the strial surface. Mitochondrial degeneration with formation of intramitochondrial inclusion bodies is an interesting early finding in the damage pattern.

Complement is known to relate to many inflammatory reactions. C4a, C3a and C5a, known as anaphylatoxins, are known to cause strong inflammatory reactions. In this study, the role of anaphylatoxins on the pathogenesis in the cochlea was examined. On hundred forty six male Harley guinea pigs, weighing about 350 grs, all susceptible to preyer's reflex, were used in this study. Anaphylatoxins were made from guinea pig serum treated with zymosan, and inoculated into the carotid artery of the guinea pigs. Parts of these animals were sacrificed and examined at ten minutes, one day, two days, three days, seven days, ten days and fifteen days after injection of anaphylatoxins. Pathological changes in inner ears were observed by light microscopy. After 10 minutes, inner ears were found morphologically normal. After one day, inner ears were found to be almost morphologically normal but the stria vascularis was observed with cystic formation. After two days, cystic formations in the stria vascularis were enlarged and Reissner's membranes were collapsed in some other animals. After three days, the stria vascularis in the various cochlear turns except in the basal turn, were extremely atrophied, some cochlear nerves showed degeneration and some cochlea showed endolymphatic hydrops. After seven days, ten days and fifteen days, the morphological changes showed atrophy in the stria vascularis similar to the results observed on the third days. Atrophy in the stria vascularis was improved gradually with time, but the degeneration of the cochlear nerve was not improved. Opinions have been divided on the cause of inner ear disease including Meniere's disease. Many authors have reported that infectious diseases, for example mumps, measles and cytomegalovirus infection, have caused human sensorineural hearing loss. These diseases have been reported to result in atrophy in the stria vascularis, degeneration of the cochlear nerve and some other pathological changes. In this study, it was clearly observed that the atrophy of the stria vascularis, the endolymphatic hydrops and other morphological changes were caused by introduction of anaphylatoxins. These results were similar to the pathological changes observed in inner ear diseases in human beings. Therefore, inflammatory substances, including anaphylatoxins, were closely related to the cause of inner ear diseases. The animal model used in this report is considered to be important for elucidating the pathogenesis of inner ear diseases.

Endolymphatic pseudohydrops of the cochlear apex