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.

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.

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.

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.

The subcellular distribution of aquaporin 5 in the cochlea reveals a water shunt at the perilymph-endolymph barrier.

Aquaporins are membrane water channel proteins that have also been identified in the cochlea. Auditory function critically depends on the homeostasis of the cochlear fluids perilymph and endolymph. In particular, the ion and water regulation of the endolymph is essential for sensory transduction. Within the cochlear duct the lateral wall epithelium has been proposed to secrete endolymph by an aquaporin-mediated flow of water across its epithelial tight junction barrier. This study identifies interspecies differences in the cellular distribution of aquaporin 5 (AQP5) in the cochlear lateral wall of mice, rats, gerbils and guinea pigs. In addition the cellular expression pattern of AQP5 is described in the human cochlea. Developmental changes in rats demonstrate longitudinal and radial gradients along the cochlear duct. During early postnatal development a pancochlear expression is detected. However a regression to the apical quadrant and limitation to outer sulcus cells (OSCs) is observed in the adult. This developmental loss of AQP5 expression in the basal cochlear segments coincides with a morphological loss of contact between OSCs and the endolymph. At the subcellular level, AQP5 exhibits polarized expression in the apical plasma membrane of the OSCs. Complementary, the basolateral membrane in the root processes of the OSCs exhibits AQP4 expression. This differential localization of AQP5 and AQP4 in the apical and basolateral membranes of the same epithelial cell type suggests a direct aquaporin-mediated transcellular water shunt between the perilymph and endolymph in the OSCs of the cochlear lateral wall. In the human cochlea these findings may have pathophysiological implications attributed to a dysfunctional water regulation by AQP5 such as endolymphatic hydrops (i.e. in Meniere's disease) or sensorineural hearing loss (i.e. in Sjögren's syndrome).

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.

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.

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.

High resolution deletion analysis of constitutional DNA from neurofibromatosis type 2 (NF2) patients using microarray-CGH.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder whose hallmark is bilateral vestibular schwannoma. It displays a pronounced clinical heterogeneity with mild to severe forms. The NF2 tumor suppressor (merlin/schwannomin) has been cloned and extensively analyzed for mutations in patients with different clinical variants of the disease. Correlation between the type of the NF2 gene mutation and the patient phenotype has been suggested to exist. However, several independent studies have shown that a fraction of NF2 patients with various phenotypes have constitutional deletions that partly or entirely remove one copy of the NF2 gene. The purpose of this study was to examine a 7 Mb interval in the vicinity of the NF2 gene in a large series of NF2 patients in order to determine the frequency and extent of deletions. A total of 116 NF2 patients were analyzed using high-resolution array-comparative genomic hybridization (CGH) on an array covering at least 90% of this region of 22q around the NF2 locus. Deletions, which remove one copy of the entire gene or are predicted to truncate the schwannomin protein, were detected in 8 severe, 10 moderate and 6 mild patients. This result does not support the correlation between the type of mutation affecting the NF2 gene and the disease phenotype. This work also demonstrates the general usefulness of the array-CGH methodology for rapid and comprehensive detection of small (down to 40 kb) heterozygous and/or homozygous deletions occurring in constitutional or tumor-derived DNA.

A 3-D model of membrane specializations between human auditory spiral ganglion cells.

A three-dimensional model of the cell membrane contact area was made between two large spiral ganglion cells (type 1 cells) from a cell cluster in a normal human cochlea. The freshly fixed cochlea had been removed during skull base surgery, processed, and sectioned for ultrastructural analysis. 400 consecutive serial thin sections were prepared from the apical portion where the nerve cell density is high and cell clusters are numerous. A cell cluster is defined as a conglomerate of two or more nerve cell bodies, surrounded by common Schwann cells. Direct physical contact between ganglion cell membranes (ephapse) was possible, in places where adjacent cells lacked a separating Schwann cell layer (gaps). One such gap was selected, and observed in 57 of 90 consecutive sections. Membrane specializations, observed in 36 sections, were found to be of principally three different types namely: (1) symmetrical, (2) asymmetrical, and (3) asymmetrical subplasmalemmal. The functional properties of these membrane specializations are still unknown. Asymmetrical densities were seen on one or other of the two cell membranes. A graphic model based on serial thin sections was made to illustrate the gap area. Superficially membrane specializations were seen to form small disk-like areas varying in size, the largest measuring 3 x 2 microm. It is speculated whether these unique formations between human spiral ganglion cells, which have not been observed in other species, may constitute interactive electrotonic or ephaptic transmission pathways. These may be in the low-frequency region and may increase plasticity and signal acuity related to the coding of speech.

Ultrastructural analysis of 20 intraosseous endolymphatic sacs from patients with cerebello-pontine angle tumours. A surgically obtained control material for histopathological studies.

OBJECTIVE: The purpose of this work was to collect a surgically obtained, freshly fixed material of the human intraosseous endolymphatic sac. This biopsy material was used to describe the normal ultrastructure as well as to serve as a control material for histopathological studies on Ménière's disease in particular. METHOD: The specimens, obtained during surgery for cerebello-pontine angle tumours, were fixed by immersion and then prepared by routine methods for transmission electron microscopy. The ultrastructural analysis was focused on intraluminal content, epithelial cell layer, subepithelial space, and morphological signs of immunological activity. The ultrastructure was analysed in relation to inner ear sensory function, tumour diagnosis, and patient's age and sex. RESULTS: As it was possible to obtain numerous specimens with an intact bony shell, the intraluminal substance could be analysed. Two separate epithelial cell types are described: one less abundant, often lighter and mitochondria-rich cell type; the other, often darker, epithelial cell with fever mitochondrias. Some of the latter cell types showed signs of active secretion. The subepithelial space was characterized by loose connective tissue adjacent to the epithelial lining, being more dense toward the bone. Elastic fibres were seen surrounding the entire endolymphatic sac. Macrophages in the intraluminal space and lymphocytes in the epithelial and subepithelial layers are described. No distinct morphology correlating to inner ear sensory function, tumour diagnosis, or patient's age and sex was revealed. CONCLUSIONS: This study confirms previously described, extensive variations in form and structure of the human endolymphatic sac. Various factors, such as surgical trauma, previous treatment, and processing method, can affect the ultrastructure and must be taken into consideration. The specimens described in this work appear to constitute a good control material for histopathological study of the human endolymphatic sac. It is still necessary to obtain large control materials such as this, as surgical specimens from patients with Ménière's disease are uncommon.

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.

Embryonic and postnatal accumulation of homogeneous substance in the endolymphatic sac in the guinea pig.

OBJECTIVE: The endolymphatic sac (ES) of vertebrates contains varying amounts of a homogeneous substance (HS) that stains deeply with basic aniline dyes. Histochemically, HS is characterized as a carbohydrate-protein complex, being both neutral and acidic in nature. In the present study, deposition of HS in the ES was studied in the guinea pig from the 3rd week of gestation to 104 weeks postnatally in order to find out if HS accumulates with age, at which point during embryonic development this substance appears, if its presence is correlated to the sense of hearing and if the amount of substance in the left versus right ear of one and the same animal is correlated to any degree. METHODS: Sixty-nine endolymphatic sacs were evaluated in 38 guinea pigs. The ES specimens were sectioned for light and transmission electron microscopy and the amount of HS filling was categorized in four groups: none, low, medium and a high level of substance. RESULTS: The substance was not discerned until after 7 weeks of gestation, when it filled only a minor part of the distal ES lumen. At 9 weeks gestation the nature of the substance altered, becoming homogeneous, as visualized by osmium-toluidine blue staining and approximately filling the distal half of the luminal space. In the postnatal period, 65% of ES specimens were filled with HS to the intermediate or proximal ES, whereas only 6.5% of the ES specimens were devoid of the substance. The extent of filling of the ES in the prenatal temporal bones was significantly less than postnatally (P < 0.0001, chi2-test). The extent of postnatal filling was not correlated with age. Left and right ears were closely correlated in one and the same animal. Phagocytic cells were often found at the border between clear endolymph and stainable substance. CONCLUSION: The appearance of HS seemed to coincide temporally with the onset of hearing during the prenatal period indicating that it could play a part in normal inner ear functioning in the guinea pig. The close correlation regarding the level of the HS in the left and right ear, both pre- and postnatally could reflect a general symmetry in endolymph pressure-volume conditions within the inner ear fluid systems, as well as in the environmental hydrostatic pressure in the posterior cranial fossa.

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.

On a novel type of neuron with proposed mechanoreceptor function in the human round window membrane--an immunohistochemical study.

An immunohistochemical study was performed on surgically obtained human fresh cochlear tissue, using synaptophysin antibodies. After immediate aldehyde fixation and decalcification in Na-EDTA serial cryosections were made of the cochlea including the round window membrane (RWM). Apart from highly specific immunostaining of spiral ganglion cells and unmyelinated nerve fibers an immunoreactive neuroreceptor could be demonstrated at the postero-medial insertion of the RWM. The perikaryon showed intense synaptophysin immunoreativity with a distal process projecting into the fibrous stroma of the RWM displaying structural specializations suggestive of a mechanoreceptor function. It is speculated whether the neuroreceptor may be involved in the proprioception and/or mechanoreception of tensile forces generated within the lamina propria during displacement of the yielding RWM in the bony labyrinth. Such a function could be important for the regulation of perilymph pressure.

Middle ear mucosa changes following exposure to Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (1 microgram/20 microliters) was instilled into the middle ear cavity in the rat. Morphological changes of the mucosa were analyzed after various intervals using light (LM) and transmission electron microscopy (TEM). An inflammatory cell reaction was seen in which there was a predominance of polymorphonuclear leukocytes (PMNs) and mononuclear cells that were mostly lymphocytes and monocytes/macrophages. Epithelial cells often showed signs of metaplasia and hyperplasia, followed by degradation through necrosis and/or apoptosis, resulting in denudation of the submucosal layer. A characteristic feature was the physical interaction between intraepithelial lymphocytes and epithelial cells. Lymphocytes adhering to the cell coat of epithelial cells showed signs of directed secretion that seemed to end in necrosis or apoptosis of the target cell. These changes occurred simultaneously with phagocytosis of cells and cell debris by PMNs and macrophages. Ultrastructural analysis suggested that intraepithelial, lymphocyte-mediated cytolysis and apoptosis may be important events in degradation of the epithelium following exposure to Pseudomonas sp. exotoxin. These findings indicate that denuding of the lamina propria may facilitate the penetration of toxin into the labyrinth, thus explaining subsequent inner ear damage.

Stereocilia-like structures in the endolymphatic sac in Ménière's disease and acoustic neuroma.

The vestibular aqueduct was surgically removed in 3 patients undergoing labyrinthectomy due to severe Ménière's disease (MD). Stereocilia-like structures were found in the luminal contents of the endolymphatic sac (ES) in all of these patients. The ES from 18 patients with acoustic neuroma were used as controls. In 1 of these, numerous stereocilia-like structures were found in the ES and in 3 additional patients, a few isolated cilia-like structures were disclosed. The findings may suggest an ongoing hair cell degeneration in the inner ear that is more advanced in patients with MD. The data also suggest that the endolymphatic duct is patent and that a longitudinal flow of endolymph also occurs in patients with MD.