Effect of acute endolymphatic hydrops overload on the endolymphatic sac.

CONCLUSIONS: Homeostasis of endolymph volume is a complex mechanism, in which the endolymphatic sac (ES) may play an important role. OBJECTIVES: To elucidate the effect of acute endolymphatic hydrops (EH) on the ES and to gain further information about the volume and pressure regulative function of the ES. MATERIALS AND METHODS: Distilled water was injected into the middle ear cavity of adult CBA/J mice. The ESs were studied morphologically by light and transmission electron microscopy. RESULTS: Mild EH was found, particularly in the upper turn of the cochlea. Acute EH led to an increase in the size of the ES lumen, accompanied by collapse of the lateral intercellular spaces and dense perisaccular tissue, changes which had reversed 2 h after the injection.

Distribution of PLGA nanoparticles in chinchilla cochleae.

OBJECTIVES: To study the distribution of polylactic/glycolic acid-encapsulated iron oxide nanoparticles (PLGA-NPs) in chinchilla cochleae after application on the round window membrane (RWM). STUDY DESIGN AND SETTING: Six chinchillas (12 ears) were equally divided into controls (no treatments) and experimentals (PLGA-NP with or without magnetic exposure). After 40 minutes of PLGA-NP placement on the RWM, perilymph was withdrawn from the scala tympani. The RWM and cochleae were fixed with 2.5% glutaraldehyde and processed for transmission electron microscopy. RESULTS: Nanoparticles were found in cochleae with or without exposure to magnet forces appearing in the RWM, perilymph, endolymph, and multiple locations in the organ of Corti. Electron energy loss spectroscopy confirmed iron elements in nanoparticles. CONCLUSION: The nanoparticles were distributed throughout the inner ear after application on the chinchilla RWM, with and without magnetic forces. SIGNIFICANCE: PLGA-NP applied to the RWM may have potential for sustained therapy to the inner ear.

Spiral Form of the Human Cochlea Results from Spatial Constraints.

The human inner ear has an intricate spiral shape often compared to shells of mollusks, particularly to the nautilus shell. It has inspired many functional hearing theories. The reasons for this complex geometry remain unresolved. We digitized 138 human cochleae at microscopic resolution and observed an astonishing interindividual variability in the shape. A 3D analytical cochlear model was developed that fits the analyzed data with high precision. The cochlear geometry neither matched a proposed function, namely sound focusing similar to a whispering gallery, nor did it have the form of a nautilus. Instead, the innate cochlear blueprint and its actual ontogenetic variants were determined by spatial constraints and resulted from an efficient packing of the cochlear duct within the petrous bone. The analytical model predicts well the individual 3D cochlear geometry from few clinical measures and represents a clinical tool for an individualized approach to neurosensory restoration with cochlear implants.

Pathological alterations of strial capillaries in dominant white spotting W/Wv mice.

Dominant white spotting W/W(v) and W(v)/W(v) mice are well-known mutants that lack strial intermediate cells in their cochlea and manifest hereditary sensorineural hearing loss. We recently reported marked thickening of and IgG deposition on the basement membrane of strial capillaries in W/W(v) mutant mice, similar to observations made in aged animals and in animals with autoimmune sensorineural hearing loss. The present study aimed to clarify the age-dependent changes in these pathological findings of strial capillaries in the W/W(v) mice. Male WBB6F1 +/+ and dominant white spotting W/W(v) mutant mice were sacrificed by transcardiac perfusion with paraformaldehyde solution. The cochlear ducts were isolated and subjected to light- and electron-microscopy, immunohistochemistry, immunoelectron microscopy. Alternatively, lanthanum chloride tracer examination in the isolated cochlear ducts was performed in order to compare the permeability of the strial capillaries between +/+ and W/W(v) mice. In the W/W(v) mice, thickening of and IgG deposition on the basement membrane of strial capillaries were observed as early as 1 week after birth and became more noticeable with age. Deposited IgG was preferentially localized to the thickened basement membrane and was also observed in partially the intercellular space between adjacent of endothelial cells. In addition, pinocytotic vesicles both in the apical and basal lesions of such cells also showed IgG deposition. Lanthanum chloride was retained along apical plasma membrane of the endothelial cells in the +/+ mice but penetrated through the endothelial layer in the W/W(v) mice. These results indicate that active transport via pinocytotic vesicles as well as increased permeability of strial capillaries in the W/W(v) mice occur in the early stage after birth, resulting in the morphological alterations in the strial capillaries of these mice.

The very distal part of the basilar papilla in the chicken: a morphological approach.

The very distal part of the chicken basilar papilla was investigated by light and electron (scanning and transmission) microscopy. The rostral tip of the basilar papilla has a lenticular area with atypical sensory hair cells which are more similar to vestibular than to auditory cells. The structure of the lenticular area appears to be suitable for vestibular function or, more likely, for auditory perception at very low frequencies. Several hypotheses can be proposed to explain this very peculiar portion of the avian cochlea. It is difficult to consider it a continuously growing area since it remains stable in adulthood. A better explanation would be that there is an incomplete ontogenetic or phylogenetic process.

Quantitative immunogold localization of Na+,K(+)-ATPase alpha-subunit in the tympanic wall of rat cochlear duct.

Ultrastructural localization of Na+,K(+)-ATPase was quantitatively investigated in the tympanic wall of rat cochlear duct by use of the protein A-gold method, using an affinity-purified antibody against the alpha-subunit of rat kidney Na+,K(+)-ATPase. A moderate number of gold particles were found on the basolateral membrane of the interdental cells of the spiral limbus. A small number of gold particles were found on the basolateral surfaces of the border cells and Hensen's cells. On the inner and outer sensory hair cells, however, the plasma membranes were rarely labeled by gold particles. The general pattern of labeling densities in cochlear structures determined here and in a previous communication from our laboratory shows good correlation with the distribution of Na+,K(+)-ATPase activity as previously estimated biochemically, cytochemically, and autoradiographically.

Anionic sites on Reissner's membrane, stria vascularis, and spiral prominence.

We demonstrated anionic sites on the lateral wall of cochlear duct and Reissner's membrane (RM) of ICR mice by Lowicryl K4M resin post-embedding and poly-L-lysine-colloidal gold conjugate (PL-CG) as a polycationic probe. The basement membrane and endolymphatic cell surface of RM were labeled with PL-CG pH 2.5 and pH 1.0. However, the perilymphatic cell surface was not labeled. PL-CG pH 2.5 and pH 1.0 strongly labeled the endolymphatic surface of the spiral prominence epithelium (SP), whereas the endolymphatic surface of the marginal cell (MC) in the stria vascularis was not labeled. Pre-digestion with several glycosidases eliminated PL-CG labeling. Our result suggests that an anionic charge located on the basement membrane of RM is largely due to the presence of heparan sulfate, chondroitin sulfate, and hyaluronic acid. An anionic charge on the endolymphatic cell surface of RM was mainly dependent on the presence of heparan sulfate. An anionic charge on the SP epithelium was caused to a substantial degree by chondroitin sulfate. We obtained histochemical evidence that the glycoconjugate content of the MC surface was quite different from that of the endolymphatic cell surface of RM and SP. We also identified RM-MC and SP-MC junctions at the ends of the stria vascularis between the marginal cells and the other endolymphatic epithelial cells of the cochlear duct.

The development of stereociliary bundles in the cochlear duct of chick embryos.

The differentiation of hair cell stereociliary bundles was investigated during early stages of embryonic development in the chick cochlear duct. The ultrastructural characteristics of the differentiating stereocilia and the position of the hair cells at the time of their differentiation were determined with scanning (SEM) and transmission (TEM) electron microscopy. Stereocilia were first identifiable with SEM as early as embryonic day 6 (stage 29) in only the distal region of the basilar papilla. By embryonic day 7.5 (stage 32) stereocilia were detected with both SEM and TEM on hair cells located in the distal two-thirds of the basilar papilla. Stereociliary bundles were recognizable throughout the entire basilar papilla by embryonic day 9 (stage 35). At this stage the hair cells exhibited a distal-to-proximal gradient in the cell surface area and the number of stereocilia on each hair cell. These results suggest that there is a distal-to-proximal wave of hair cell differentiation which occurs at a very early time period in the development of the chick cochlea. Both the timing and the direction of the stereociliary differentiation contrast with previous ultrastructural reports of avian hair cell development, yet compare favorably with the patterns of functional development in the auditory system.

A previously unknown hair cell epithelium in the pigeon cochlea: the papilla chaotica.

A previously unknown sensory epithelium can be found on the medial wall of the apical part of the pigeon cochlear duct. It comprises about 200 hair cells. These are not arranged in any regular pattern, shape and orientation of the ciliary bundles even differ in neighbouring hair cells. We therefore propose the term papilla chaotica.

Na+,K+-ATPase activity and ultrastructural localization in the tegmentum vasculosum in the cochlea of the duckling.

The tegmentum vasculosum of the avian cochlear duct mimics the stria vascularis of the mammalian cochlear duct in both location and structure, and previous studies indicate that it may be its functional counterpart with regard to endolymph synthesis. In the present study, we report on the enzymatic activity and ultrastructural localization of the Na+,K+-ATPase in the tegmentum vasculosum of the duckling. Na+,K+-ATPase activity was determined by measuring K+-dependent, ouabain-sensitive p-nitrophenyl phosphatase (p-NPPase) activity in homogenates of dissected regions of the cochlear duct. The ultrastructural localization of the Na+,K+-ATPase was identified using K+-dependent, ouabain-sensitive, p-NPPase cytochemistry. Specific enzyme activity was localized primarily in homogenates of the tegmentum vasculosum (2.27 micromol p-nitrophenyl phosphate/mg protein/min) when compared to homogenates of the entire cochlear duct (0.69 micromol p-nitrophenyl phosphate/mg protein/min). Reaction product for p-NPPase was localized primarily along the basolateral plasma membrane folds of the dark cells. The cytochemical deposits appeared to be located exclusively on the cytoplasmic side of the plasma membrane. The light cells were devoid of reaction product. Biochemical and cytochemical localization of p-NPPase activity on the basolateral plasma membrane folds of the dark cells of the tegmentum vasculosum in conjunction with the ultrastructural morphology of these cells is compatible with a Na+,K+-ATPase-dependent ion transport function related to endolymph synthesis.

Ultrastructure and blood supply of the tegmentum vasculosum in the cochlea of the duckling.

The tegmentum vasculosum of the duckling consists of a highly folded epithelium which extends over the dorsal and lateral walls of the cochlear duct, separating the scala media from the scala vestibuli. This epithelium consists of two distinct cell types, dark cells and light cells, and is well vascularized. The surface of the epithelium is formed by a mosaic of alternating dark and light cells. The goblet-shaped dark cells have an electron-dense, organelle-rich cytoplasm, and are expanded basally by extensive basolateral plasma membrane infoldings, within which are numerous mitochondria. Dark cells are isolated from each other and from the capillaries within the epithelium by intervening light cells. In contrast, columnar light cells exhibit an electron-lucent, organelle-poor cytoplasm and may extend from the underlying capillaries to the endolymphatic surface. Light cells contain abundant, coated endocytic vesicles on their apical surfaces and are bound, apically, to other light cells or to dark cells by tight junctions and desmosomes. Laterally, light cells are linked to each other either by complex, fluid-filled membrane interdigitations or by extensive gap junctions. Plasma membrane interdigitations and obvious, fluid-filled intercellular spaces characterize the lateral borders between light and dark cells. Vascular corrosion casting reveals the three-dimensional anatomy of the cochlear vasculature. A continuous arteriolar loop fed by anterior and posterior cochlear arterioles encircles the cochlear duct. The rich capillary beds of the tegmentum vasculosum are supplied by arching arterioles arising from this loop. These capillaries are the continuous type and are situated primarily within the core of the epithelium or along its border with the scala vestibuli. The structure and blood supply of the tegmentum vasculosum are characteristic of an epithelium involved in active transport.

Ultrastructural localization of megalin in the rat cochlear duct.

Megalin is an endocytic receptor predominantly expressed in the kidney proximal tubule cells. In the present study, localization of megalin was examined using a post-embedding immunogold method in the rat cochlear duct. Marginal cells of the stria vascularis were labeled on the apical surface, but not on the basolateral surface. This localization pattern resembles kidney proximal tubule cells. Immunoreactivity was also detected on various other cells, including epithelial cells of the spiral prominence and epithelial cells of Reissner's membrane. In contrast, virtually no gold particles were seen on intermediate cells and basal cells of the stria vascularis, mesothelial cells of Reissner's membrane or fibrocytes in the lateral wall. Also unlabeled were cells in the tympanic wall of the cochlear duct, including sensory cells and supporting cells of the organ of Corti. The present findings show the involvement of megalin in endocytosis of marginal cells and are suggestive of different uptake mechanisms for aminoglycosides in the kidney proximal tubule cells and in the cochlear sensory cells.

Development of the human stria vascularis.

Fifteen human fetal cochleas were investigated by light microscopy and transmission electron microscopy in order to observe the development of the stria vascularis. The earliest signs of strial cell differentiation take place during the 11th week of gestation. Subsequently, the first stages of the stria vascularis development occur quickly. At week 14 the three types of cells, namely, marginal, intermediate and basal cells are discernable. Moreover at this stage, signs of specific activity are already present. The adult-like appearance of the stria vascularis is reached by week 21 but its maturation is completed only during the last trimester of pregnancy. This is in good agreement both with the development of the organ of Corti structures and with the maturation of the human auditory function.

Anatomical correlates of the passive properties underlying the developmental shift in the frequency map of the mammalian cochlea.

As the cochlea develops, the cells in the basal cochlea become sensitive to progressively higher frequencies. To identify features of cochlear morphology that may underlie the place code shift, measurements of infant and adult gerbil cochleas were made at both the light and electron microscopic levels. The measurements included areas of the cochlear duct, basilar membrane, and organ of Corti, height and width of the basilar membrane, thickness of the tympanic cover layer, thickness of the upper and lower basilar membrane fiber bands, and optical density of the basilar membrane. The results indicated that basilar membrane dimensions do not change as the place code shifts and that regions that code for the roughly the same frequency (e.g., approximately 11.2 kHz) at different ages can have basilar membranes of very different dimensions. In contrast, the size of the organ of Corti and the thickness of fiber bands inside the basilar membrane do change in ways consistent with the shift in the frequency map.

Atrophy of outer hair cell stereocilia and hearing loss in hydropic cochleae.

We have earlier described selective atrophy of short and middle stereocilia on outer hair cells of the three upper cochlear turns in hydropic cochleae of guinea pigs. The present study describes sequential early stages of stereocilia degeneration leading to this specific atrophy. Comparison of the morpho-pathology with the ultimate CAP audiograms taken before sacrifice indicated a close association between the low frequency hearing loss and this atrophy of stereocilia. The atrophy appeared to be associated first with the short and then the middle stereocilia of the 2nd and 3rd rows of outer hair cells between 0.5 kHz and 2 kHz and with time included the 1st row of all outer hair cells of the upper cochlear turns down to the 8 kHz region.

Ultrastructural and electrophysiological maturation of the chick tegmentum vasculosum.

We have examined the ultrastructural and electrophysiological events associated with the embryonic development of the tegmentum vasculosum, the ion-transporting epithelium in the chick cochlear duct. The cytodifferentiation of the light and dark cells in the epithelium from embryonic day 6 through post-hatching day 7 was studied with transmission electron microscopy. The predominant ultrastructural change in the developing tegmentum vasculosum was the elaboration of the complex basolateral infoldings on the dark cells from embryonic day 11 through post-hatching day 7. The relationship between the development of the endocochlear transepithelial electrical potential difference (PD) and the cytodifferentiation of the tegmentum vasculosum was examined with in vitro studies. Microelectrode impalements of the scala media showed that the positive endocochlear PD was first detectable on embryonic day 20 but did not reach a mature value of +16 mV until post-hatching day 7. Maturation of the endocochlear PD paralleled the time during which the dark cells in the tegmentum vasculosum displayed the most extensive increase in basolateral infoldings. This correlation suggests that the development of the endocochlear PD may result from an increase in the number of Na+-K+ pump sites located on the basolateral infoldings of the dark cells.

Electron microscopic autoradiographic demonstration of inositol incorporation in the cochlea.

[3H]Inositol was perilymphatically perfused into the guinea pig cochlea and the sites of its incorporation were autoradiographically identified by electron microscope. In the organ of Corti, distinct areas of inositol incorporation were observed at sites enriched with cytoskeletal proteins. In addition, a wide range of cell organelles incorporated inositol. Cell membrane/subsurface cisterna complex of the outer hair cells and the cell membrane/microvilli complex incorporated inositol more avidly than did other cell membranes. In the stria vascularis, intermediate cells incorporated inositol more frequently than cells in other areas.

Stage dependent development of intraocular cochlear grafts.

The intraocular grafting technique was employed to test whether the peripheral hearing organ, the cochlea, is capable of survival and an organized development in total isolation from the temporal bone. Rat cochleae obtained from gestation day 16, postnatal day 1 and 7 were chosen for transplantation into the anterior chamber of the eye of adult Sprague-Dawley rats. The grafts were maintained in the anterior chamber for 6, 10, or 15 weeks survival time. The salient features of this study is that 1) cochlear structures survive and, 2) the cochlear structures develop beyond their pre-grafted stage as determined from light and electron micrographs. In the present study, the grafts obtained at gestation day 16 (GD 16) and postnatal day 1 gave a much higher rate of survival and development than the postnatal day 7 grafts. In addition, grafts maintained for either 6 or 10 weeks had a better survival rate than those grafts left for 15 weeks. It is estimated from light and electron micrographs that the gestation day 16 otocysts that were maintained for 10 weeks, developed to the equivalent of a postnatal day 10 cochlea. The grafts obtained from postnatal day one rats developed to the equivalent of approximately 14 days after birth. Interestingly, in the absence of synaptic contact, the inner and outer hair cells were capable of survival, differentiation and maturation. It remains to be determined if the spiral ganglion cells require additional neurotrophic factors for survival in the anterior chamber of the eye.

Differentiation of the rat stria vascularis.

The differentiation of the rat stria vascularis (SV) was investigated by light- and electron microscopy and by immunocytochemistry. Loss of the basal lamina at the epithelial-mesenchymal interface of SVs as indicated by immunoreactions of laminin and fibronectin induces the formation of vascular feet by basal infoldings of the marginal cells (MCs), and the development of the strial capillaries (SCs) by mesenchymal cells in a manner of vasculogenesis is progressing at the same time. The production of fibronectin in the rough endoplasmic reticulum of mesenchymal cells and the involvement of this glycoprotein in a mechanical linkage between the vasoformative mesenchymal cells and endothelial ones of the SCs are indicated by immunocytochemistry. The plasma membrane of the marginal cells (MCs) begins to show immunoreactions of Na+.K+ ATPase at postnatal day 5 and is conjugated to each other by tight junctions at postnatal day 14. The apical tubules of the differentiating MCs do not seem to be involved in the endocytotic activity but are involved in the plasma membrane supply for the rapid differentiation.