Role of BDNF and neurotrophic receptors in human inner ear development.

The expression patterns of the neurotrophin, brain-derived neurotrophic factor, BDNF, and the neurotrophic receptors-p75NTR and Trk receptors-in the developing human fetal inner ear between the gestational weeks (GW) 9 to 12 are examined via in situ hybridization and immunohistochemistry. BDNF mRNA expression was highest in the cochlea at GW 9 but declined in the course of development. In contrast to embryonic murine specimens, a decline in BDNF expression from the apical to the basal turn of the cochlea could not be observed. p75NTR immunostaining was most prominent in the nerve fibers that penetrate into the sensory epithelia of the cochlea, the urticule and the saccule as gestational age progresses. TrkB and TrkC expression intensified towards GW 12, at which point the BDNF mRNA localization was at its lowest. TrkA expression was limited to fiber subpopulations of the facial nerve at GW 10. In the adult human inner ear, we observed BDNF mRNA expression in the apical poles of the cochlear hair cells and supporting cells, while in the adult human utricle, the expression was localized in the vestibular hair cells. We demonstrate the highly specific staining patterns of BDNF mRNA and its putative receptors over a developmental period in which multiple hearing disorders are manifested. Our findings suggest that BDNF and neurotrophin receptors are important players during early human inner ear development. In particular, they seem to be important for the survival of the afferent sensory neurons.

Regulation of the perilymphatic-endolymphatic water shunt in the cochlea by membrane translocation of aquaporin-5.

Volume homeostasis of the cochlear endolymph depends on radial and longitudinal endolymph movements (LEMs). LEMs measured in vivo have been exclusively recognized under physiologically challenging conditions, such as experimentally induced alterations of perilymph osmolarity or endolymph volume. The regulatory mechanisms that adjust LEMs to the physiological requirements of endolymph volume homeostasis remain unknown. Here, we describe the formation of an aquaporin (AQP)-based "water shunt" during the postnatal development of the mouse cochlea and its regulation by different triggers. The final complementary expression pattern of AQP5 (apical membrane) and AQP4 (basolateral membrane) in outer sulcus cells (OSCs) of the cochlear apex is acquired at the onset of hearing function (postnatal day (p)8-p12). In vitro, hyperosmolar perfusion of the perilymphatic fluid spaces or the administration of the muscarinic agonist pilocarpine in cochlear explants (p14) induced the translocation of AQP5 channel proteins into the apical membranes of OSCs. AQP5 membrane translocation was blocked by the muscarinic antagonist atropine. The muscarinic M3 acetylcholine (ACh) receptor (M3R) was identified in murine OSCs via mRNA expression, immunolabeling, and in vitro binding studies using an M3R-specific fluorescent ligand. Finally, the water shunt elements AQP4, AQP5, and M3R were also demonstrated in OSCs of the human cochlea. The regulation of the AQP4/AQP5 water shunt in OSCs of the cochlear apex provides a molecular basis for regulated endolymphatic volume homeostasis. Moreover, its dysregulation or disruption may have pathophysiologic implications for clinical conditions related to endolymphatic hydrops, such as Ménière's disease.

Water permeability of the mammalian cochlea: functional features of an aquaporin-facilitated water shunt at the perilymph-endolymph barrier.

The cochlear duct epithelium (CDE) constitutes a tight barrier that effectively separates the inner ear fluids, endolymph and perilymph, thereby maintaining distinct ionic and osmotic gradients that are essential for auditory function. However, in vivo experiments have demonstrated that the CDE allows for rapid water exchange between fluid compartments. The molecular mechanism governing water permeation across the CDE remains elusive. We computationally determined the diffusional (PD) and osmotic (Pf) water permeability coefficients for the mammalian CDE based on in silico simulations of cochlear water dynamics integrating previously derived in vivo experimental data on fluid flow with expression sites of molecular water channels (aquaporins, AQPs). The PD of the entire CDE (PD = 8.18 × 10(-5) cm s(-1)) and its individual partitions including Reissner's membrane (PD = 12.06 × 10(-5) cm s(-1)) and the organ of Corti (PD = 10.2 × 10(-5) cm s(-1)) were similar to other epithelia with AQP-facilitated water permeation. The Pf of the CDE (Pf = 6.15 × 10(-4) cm s(-1)) was also in the range of other epithelia while an exceptionally high Pf was determined for an epithelial subdomain of outer sulcus cells in the cochlear apex co-expressing AQP4 and AQP5 (OSCs; Pf = 156.90 × 10(-3) cm s(-1)). The Pf/PD ratios of the CDE (Pf/PD = 7.52) and OSCs (Pf/PD = 242.02) indicate an aqueous pore-facilitated water exchange and reveal a high-transfer region or "water shunt" in the cochlear apex. This "water shunt" explains experimentally determined phenomena of endolymphatic longitudinal flow towards the cochlear apex. The water permeability coefficients of the CDE emphasise the physiological and pathophysiological relevance of water dynamics in the cochlea in particular for endolymphatic hydrops and Ménière's disease.

Anatomical basis of drug delivery to the inner ear.

The isolated anatomical position and blood-labyrinth barrier hampers systemic drug delivery to the mammalian inner ear. Intratympanic placement of drugs and permeation via the round- and oval window are established methods for local pharmaceutical treatment. Mechanisms of drug uptake and pathways for distribution within the inner ear are hard to predict. The complex microanatomy with fluid-filled spaces separated by tight- and leaky barriers compose various compartments that connect via active and passive transport mechanisms. Here we provide a review on the inner ear architecture at light- and electron microscopy level, relevant for drug delivery. Focus is laid on the human inner ear architecture. Some new data add information on the human inner ear fluid spaces generated with high resolution microcomputed tomography at 15 µm resolution. Perilymphatic spaces are connected with the central modiolus by active transport mechanisms of mesothelial cells that provide access to spiral ganglion neurons. Reports on leaky barriers between scala tympani and the so-called cortilymph compartment likely open the best path for hair cell targeting. The complex barrier system of tight junction proteins such as occludins, claudins and tricellulin isolates the endolymphatic space for most drugs. Comparison of relevant differences of barriers, target cells and cell types involved in drug spread between main animal models and humans shall provide some translational aspects for inner ear drug applications.

Efferent neurotransmitters in the human cochlea and vestibule.

CONCLUSION: Current neurotransmission models based on animal studies on the mammalian inner ear not always reflect the situation in human. Rodents and primates show significant differences in characteristics of efferent innervation as well as the distribution of neuroactive substances. OBJECTIVE: Immunohistochemistry demonstrates the mammalian efferent system as neurochemically complex and diverse: several neuroactive substances may co-exist within the same efferent terminal. Using light and electron microscopic immunohistochemistry, this study presents a comparative overview of the distribution patterns of choline acetyltransferase (ChAT), the acetylcholine synthesizing enzyme, GABA, CGRP, and enkephalins within the peripheral nerve fiber systems of the human inner ear. MATERIALS AND METHODS: Human temporal bones were obtained post mortem and prepared according to a pre-embedding immunohistochemical technique to detect immunoreactivities to ChAT, GABA, CGRP, leu- and met-enkephalins at the electron microscopic level. RESULTS: Immunoreactivities of all the antigens were present within both the lateral and medial efferent systems of the cochlea, whereas only ChAT, GABA, and CGRP were detected in efferent pathways of the vestibular end organs.

Smooth muscle in the annulus fibrosus of the tympanic membrane in bats, rodents, insectivores, and humans.

The annulus fibrosus and its attachment to the bony tympanic ring were studied in a series of mammals. In the pallid bat, Antrozous pallidus, there is an extensive plexus of large interconnected blood sinuses in the part of the annulus that borders the tympanic bone. The spaces between the sinuses are packed with smooth muscle cells. Most of the cells have a predominately radial orientation; they extend from the bony tympanic sulcus to a dense collagenous matrix (apical zone) where radially oriented fibers of the pars tensa are confluent with the annulus. The muscles and vessels constitute a myovascular zone. A structurally similar myovascular zone is also present in the European hedgehog. In rodents, the annulus lacks the large interconnected blood sinuses but many small vessels are present. Smooth muscle is concentrated in the broad area of attachment of the annulus to the tympanic bone. In the gerbil, smooth muscle seems to be concentrated in the central part of the width of the annulus where it is attached to bone and radiates toward the tympanic membrane. In humans collections of radially oriented smooth muscle cells were found in several locations. The smooth muscle in all species studied appears to form a rim of contractile elements for the pars tensa. This arrangement suggests a role in controlling blood flow and/or creating and maintaining tension on the tympanic membrane.

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-ß2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-ß2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-ß2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Temporal bone characteristics in Meniere's disease.

The petrous portion of the temporal bone in patients with Meniere's disease differs from that of healthy individuals mainly in its lack of periaqueductal pneumatization and its consequently short and narrow vestibular aqueduct. Diminished pneumatization may have an impact upon the tomographic reproducibility of the aqueduct. A total lack of periaqueductal pneumatization is prevalent in long-standing Meniere's disease. Tomography may serve as a tool by providing a basis for the choice of surgical procedure. Roentgenologic and histologic studies have indicated that the pars rugosa of the endolymphatic sac in normals mainly is housed inside the distal part of the vestibular aqueduct. In patients with Meniere's disease, the sac might be located outside the aqueduct and therefore deprived of the functions of the loose and highly vascular tissue normally surrounding it within the aqueduct. This might influence the total vascular supply of the sac, thereby interfering with its resorptive and immunodefensive functions.

Effects of high intensity pure tone stimulation on the endolymphatic sac. Correlations between cochlear morphology and endolymphatic sac response.

A systematic study of the effects of acoustic overstimulation on the endolymphatic sac (ES) in the guinea pig was performed. The ES was studied with light and transmission electron microscopy after exposure of the animals to a 3.85 kHz pure tone of 108 dB SPL or 120 dB SPL for 22.5 min (sound energy 9.4 and 150 Pa2 X h, respectively). The damage pattern in the organ of Corti was studied after various post-exposure times with SEM and correlated with the morphological characteristics of the ES in the same ear. This was made possible by using a modified technique for histological processing. In ears with induced structural abnormalities to the organ of Corti, the ES displayed few morphological changes without obvious signs of accumulation of cell debris within the lumen. Initially an increase in the amount of freely floating cells was found which persisted for at least 24 h. The role of the ES for disposal and digestion of locally produced degeneration products within the cochlea after acoustically generated structural damage is discussed.

The endolymphatic sac and inner ear homeostasis. II: Effect of glycerol on the sensory end organs with or without colchicine pretreatment.

The effects of glycerol and colchicine on the sensory end organs of the inner ear were investigated in mice. Glycerol alone induced a widening of the intercellular spaces lining vestibular dark and transitional cells as well as the marginal cells of the stria vascularis. This was noted within 30 min after the injection of glycerol and was normalized again within 4 h after the injection. Colchicine induced some morphological changes in the inner ear sensory cells, such as dissociation of Golgi complexes etc. These isolated glycerol or colchicine injections did not cause any signs of inner ear functional impairment. Treatment with glycerol following pretreatment with colchicine, however, induced marked inner ear dysfunction with impaired sense of balance and audition. The inner ear morphology revealed a combination of changes as compared with what was observed after isolated treatment with glycerol or colchicine i.e. edema of the stria vascularis, and vestibular dark and transitional cells as well as dissociation of Golgi complexes in the sensory cells. The cochlea showed moderate endolymphatic hydrops. These findings indicate that colchicine affects the inner ear fluid regulating mechanisms which may lead to severe functional derangement after additional glycerol treatment. It is conceivable that the present experiment may serve as a useful model for further studies on inner ear changes related to endolymphatic hydrops and Ménière's disease.

The endolymphatic sac and inner ear homeostasis. I: Effect of glycerol on the endolymphatic sac with or without colchicine pretreatment.

The combined effects of glycerol and colchicine on the endolymphatic sac were investigated in mice. Glycerol induced signs of secretion from the epithelium with formation of secretory granules in the light epithelial cells. Other characteristics of the epithelial lining were also changed resulting in an increased widening of the lateral intercellular spaces, a partial collapse of the lumen and with a deposition of a stainable substance within the lumen. This reaction lasted from 30 min to 24 h following the injection. Pretreatment with colchicine was found to decrease or inhibit the glycerol-induced secretion of macromolecules into the sac. The lumen collapsed but frequently there was no presence of stainable substance. Animals treated with both glycerol and colchicine showed marked signs of inner ear malfunction which could indicate that the secretory activity in the sac might be closely related to the regulation of inner ear fluid homeostasis and that functional disturbances in this system may lead to disorders of inner ear function.

The endolymphatic sac in a mouse mutant with cochleo-saccular degeneration. Electrophysiological and ultrastructural correlations.

The response of the endolymphatic sac to a disturbance in endolymph homeostasis was investigated by examining the sac in a mouse mutant, viable dominant spotting, which is known to exhibit primary strial dysfunction and cochleo-saccular degeneration. The function of the vascular stria was assessed by measuring the endocochlear potential and the sacs were then studied by light and transmission electron microscopy. The endolymphatic sac was found to be morphologically abnormal in these mutants, the main abnormality being the presence of granular epithelial cells, which showed clear histological signs of secretory activity. A stainable precipitate, believed to be secreted by the granular cells, was observed in the lumen of the endolymphatic sac in the mutants. The findings strengthen the view that the sac is involved in the regulation of endolymph volume and pressure.

Subcellular changes in the endolymphatic sac after administration of hyperosmolar substances.

The effects of hyperosmolar substances on the ultrastructure of the endolymphatic sac were studied in mice. Fifteen minutes after intravenous injection of urea or glycerol, subcellular changes in the endolymphatic sac were observed. These consisted of the occurrence of abundant cytoplasmic granules with a floccular or lamellar material, or both, in the light epithelial cells. Similar material was also present in the lumen of the endolymphatic sac, suggesting a common source and increased secretory activity. Mannitol caused similar changes, though less pronounced. The possibility that the alterations in the fine structure of the endolymphatic sac may be associated with a reduction in the hydrostatic fluid pressure in the rest of the labyrinth is discussed.

Synapses on human spiral ganglion cells: a transmission electron microscopy and immunohistochemical study.

A transmission electron microscopy (TEM) study and synaptophysin immunoreactivity analysis of neurons in the human spiral ganglion was performed with particular emphasis on the demonstration of synapses. The study was based on surgical biopsy material obtained during transcochlear meningioma surgery. Vesiculated nerve endings of unmyelinated nerve fibers occurred frequently on the small ganglion cells at all levels. The nerve terminals exhibited abundant clear synaptic vesicles but also dense-core vesicles. Multisynaptic contact sites were also seen with fibers of the intraganglionic spiral bundle (IGSB). Complex associations of synapses could be demonstrated, including several synaptic terminals in conjunction with contact sites or an adherent type of junctions on large ganglion cells. These contact sites exhibited membrane densities which were symmetric or asymmetric, changed their polarity recurrently over their extension from one cell to the other and back and lacked clear synaptic vesicles. This suggests the existence of connections between efferents, belonging to the olivocochlear bundle, and both small and large ganglion cells. Thus, both the inner and outer hair cell system may be under the influence of efferent innervation in the human spiral ganglion. The morphology and course of synaptophysin-positive nerve fibers indicated that synaptic contacts within the spiral ganglion, as observed under the electron microscope, may be abundant. These results indicate that complex neural processing may occur at the level of the spiral ganglion in man.

Structural/audiometric correlations in a human inner ear with noise-induced hearing loss.

A morphological analysis was performed on a human cochlea removed during skull base surgery. The patient experienced a noise-induced hearing loss following 30 years of mechanical exposure. The tissue was processed according to the block surface technique and the organ of Corti, osseous spiral lamina and spiral ganglion were analyzed at different levels. There was a circumscribed lesion approx. 10 mm from the round window extending to about 13 mm. At this site, the dominant pathological feature was the loss of outer hair cells that was comprehensive in the centermost area and partial in the peripheral region of the damage. The degradation of inner hair cells was less severe with signs of cell atrophy yet with limited loss. Outer pillar cells were often collapsed leading to deformation of the acoustic ridge. The Deiters cells were often present and physically interactive with remaining nerve fibers. In the reticular lamina, surgical manipulation and dissection resulted in tears which may be attributed to a reduction of intercellular strength between cells. In the damaged area, there was a 45% loss of myelinated nerve fibers measured at the osseous spiral lamina. Pathological changes could not be observed in the spiral ganglion with certainty although the type II cells innervating the outer hair cells were often difficult to discern.

Morphological changes of the endolymphatic sac induced by microinjection of artificial endolymph into the cochlea.

Morphological changes of the endolymphatic sac were analyzed in guinea pigs following microinjection of artificial endolymph into the cochlea or withdrawal of a quantity of native endolymph. Injections were performed into the second turn of scala media with a micro-pump at a rate of 60-100 nl/min, lasting for a period of 4, 7. 5, 15 or 18 min. In withdrawal experiments, endolymph was aspirated from the second cochlear turn over a period of 8 min. For each procedure the contralateral (non-treated) ear served as a histological control. Following artificial endolymph injections of 7. 5 min or more there was an almost total absence of the normal intraluminal homogeneous substance (HS) on the injected side. Our observations suggest that the disappearance of the HS occurs by both enzymatic and macrophagic activity. After endolymphatic withdrawals the ES was found to contain increased amounts of HS. The results could suggest that the volume of fluid in the ES, and hence the volume of the entire membranous labyrinth, may be regulated by a dynamic relationship between active secretion and enzymatic degradation of a lumen-expanding substance that is intimately related to the intraluminal macrophages. The exact mechanism governing these regulatory systems, and their relationship to ion and water movements across the epithelium of the sac, remain to be elucidated.

Quantitative evaluation of cochlear neurons and computer-aided three-dimensional reconstruction of spiral ganglion cells in humans with a peripheral loss of nerve fibres.

Quantitative data on human cochlear neuronal elements were collected from various regions in five patients with high-tone hearing loss due to presbycusis and in two patients with normal hearing. The number of nerve fibers was assessed in the spiral lamina and in the inner acoustic meatus together with counts of spiral ganglion cells. The results show that the number of neurons decreased peripherally, i.e., with increasing distance from the central nervous system in patients with high-tone hearing loss due to presbycusis. In two patients with normal hearing no significant difference in the number of neurons was found in the lamina spiralis as compared to the inner acoustic canal. Computer-aided 3-dimensional reconstruction of the human spiral ganglion displayed large bipolar neurons (type I cells), but also large ganglion cells with one missing axon. The results may indicate that a slow retrograde degeneration occurs from the periphery towards the spiral ganglion in presbycusis. Transmission electron microscopy analysis of freshly fixed human spiral ganglions displayed interneural connections. It is speculated whether a trophic supply from other neurons at the level of the spiral ganglion can prevent or delay further degeneration of the central axon.

Selective aspects of human pathology in high-tone hearing loss of the aging inner ear.

Accompanied with aging, the thresholds for high frequency sounds may elevate and result in a progressive hearing loss described as presbycusis. Based on correlations between audiometric measures of aged patients and histologic findings garnered from postmortem examinations, four types of presbycusis have been characterized: sensory-neural, neural, strial, and conductive [Schuknecht, H.F., Gacek, M.R., 1993. Ann. Otol. Rhinol. Laryngol. 102, 1--16]. Otopathologic changes to the inner ear as a direct function of age, however, remain controversial. The focus of this investigation involves the pathological impact on remaining sensory structures in patients having sensory--neural degeneration. The current study presents seven human temporal bones extracted from patients aged 53--67 years with high-tone hearing loss and with no known history of extraordinary environmental events involving head or noise trauma, acoustic overstimulation, or ototoxicity. In previously published findings of these specimens, all but one temporal bone failed to demonstrate a meaningful correlation between audiometric measurements and loss of functional hair cell populations with secondary retrograde degeneration of nerve fibers. Using the block surface method, electron microscopic micrographs demonstrate ultrastructural changes in the cuticular plate, stereocilia, pillar cells, stria vascularis, and the spiral ligament. In all pathological specimens, the greatest incidence of degeneration was seen at the cuticular plate. Conclusively, our findings present three implications in the aging human cochlea: firstly, audiometric measures that represent a high-tone hearing loss may take various forms with respect to ultrastructural patterns of degeneration and surviving structures; secondly, the incidence of lipofuscin and lysosome granules does not correlate with the degree of hearing loss and; thirdly, as shown only in guinea pigs [Anniko, M., 1988. Scanning Microsc. 2, 1035--1041], high-tone hearing loss can be associated with deformation of the cuticular plate.

Size variations in the lateral intercellular spaces of the endolymphatic sac induced by dietary factors.

Since much evidence suggests that the endolymphatic sac is responsible for endolymph resorption, and that the endolymphatic sac lateral intercellular spaces which are lined by the energy-dependent transport complex NA+,K(+)-ATPase are important in this process, we sought to evaluate the effects of dietary salt and a food extract that inhibits the activity of Na+,K(+)-ATPase on lateral intercellular space size. Animals fed this food factor and a high-sodium diet had significantly smaller endolymphatic sac lateral intercellular spaces than those animals fed only a high-sodium diet (analysis of variance with Scheffe's multiple comparison test, P less than 0.001). Animals fed a high-sodium diet had significantly larger endolymphatic sac lateral intercellular spaces than those animals fed a control diet only (analysis of variance with Scheffe's multiple comparison test, P less than 0.001). Results of this study suggest that dietary sodium affects endolymphatic sac fluid dynamics and that other food factors may regulate sodium metabolism, and therefore endolymphatic sac function.

A technique for reembedding celloidin sections for electron microscopy.

A new technique for reembedding celloidin sections of human temporal bones for transmission electron microscopy is described. It consists of four steps: 1. loosening of celloidin sections from glass slides with use of xylene and dissection of the area of interest, 2. removal of celloidin with use of clove oil, 3. staining with 1% osmium tetroxide and 1% tannic acid, and 4. embedding in epoxy resin. Autolytic changes were seen due to poor fixation. TEM of reembedded celloidin sections of optimally fixed tissue revealed that the celloidin-embedding procedure affected ultrastructural preservation to some degree. This included less well-preserved cell membranes and some increased electron density of the cytosol decreasing the EM resolution of intracytoplasmic organelles. The technique allows TEM analysis of the intact labyrinth at all regions in the same specimen without dissection of the fragile tissue components of the membranous labyrinth. This might make the technique useful for processing freshly fixed human inner ear tissue and temporal bones for ultrastructural histopathological analysis.