Damage to inner ear structure during cochlear implantation: Correlation between insertion force and radio-histological findings in temporal bone specimens.

Cochlear implant insertion should be as least traumatic as possible in order to reduce trauma to the cochlear sensory structures. The force applied to the cochlea during array insertion should be controlled to limit insertion-related damage. The relationship between insertion force and histological traumatism remains to be demonstrated. Twelve freshly frozen cadaveric temporal bones were implanted with a long straight electrodes array through an anterior extended round window insertion using a motorized insertion tool with real-time measurement of the insertion force. Anatomical parameters, measured on a pre-implantation cone beam CT scan, position of the array and force metrics were correlated with post-implantation scanning electron microscopy images and histological damage assessment. An atraumatic insertion occurred in six cochleae, a translocation in five cochleae and a basilar membrane rupture in one cochlea. The translocation always occurred in the 150- to 180-degree region. In the case of traumatic insertion, different force profiles were observed with a more irregular curve arising from the presence of an early peak force (30 ± 18.2 mN). This corresponded approximately to the first point of contact of the array with the lateral wall of the cochlea. Atraumatic and traumatic insertions had significantly different force values at the same depth of insertion (p < 0.001, two-way ANOVA), and significantly different regression lines (y = 1.34x + 0.7 for atraumatic and y = 3.37x + 0.84 for traumatic insertion, p < 0.001, ANCOVA). In the present study, the insertion force was correlated with the intracochlear trauma. The 150- to 180-degree region represented the area at risk for scalar translocation for this straight electrodes array. Insertion force curves with different sets of values were identified for traumatic and atraumatic insertions; these values should be considered during motorized insertion of an implant so as to be able to modify the insertion parameters (e.g axis of insertion) and facilitate preservation of endocochlear structures.

Macromolecular organization and fine structure of the human basilar membrane - RELEVANCE for cochlear implantation.

INTRODUCTION: Cochlear micromechanics and frequency tuning depend on the macromolecular organization of the basilar membrane (BM), which is still unclear in man. Novel techniques in cochlear implantation (CI) motivate further analyses of the BM. MATERIALS AND METHODS: Normal cochleae from patients undergoing removal of life-threatening petro-clival meningioma and an autopsy specimen from a normal human were used. Laser-confocal microscopy, high resolution scanning (SEM) and transmission electron microscopy (TEM) were carried out in combination. In addition, one human temporal bone was decellularized and investigated by SEM. RESULTS: The human BM consisted in four separate layers: (1) epithelial basement membrane positive for laminin-ß2 and collagen IV, (2) BM "proper" composed of radial fibers expressing collagen II and XI, (3) layer of collagen IV and (4) tympanic covering layer (TCL) expressing collagen IV, fibronectin and integrin. BM thickness varied both radially and longitudinally (mean 0.55-1.16 µm). BM was thinnest near the OHC region and laterally. CONCLUSIONS: There are several important similarities and differences between the morphology of the BM in humans and animals. Unlike in animals, it does not contain a distinct pars tecta (arcuate) and pectinata. Its width increases and thickness decreases as it travels apically in the cochlea. Findings show that the human BM is thinnest and probably most vibration-sensitive at the outer pillar feet/Deiter cells at the OHCs. The inner pillar and IHCs seem situated on a fairly rigid part of the BM. The gradient design of the BM suggests that its vulnerability increases apical wards when performing hearing preservation CI surgery.

Deiters cells tread a narrow path--the Deiters cells-basilar membrane junction.

Deiters cells extend from the basilar membrane to the reticular lamina and, together with pillar cells and outer hair cells, structurally define the micro-architecture of the organ of Corti. Studying vibrotome sections of the mouse organ of Corti with confocal and scanning electron microscopy we found that the basal pole of every Deiters cell, independently of their position in the organ of Corti and along the cochlear spiral, attached to the basilar membrane within a 15.1 ± 0.3 µm-wide stripe running the length of the cochlear spiral adjacent to the row of outer pillar cells. All Deiters cells' basal poles had similar diameter and general morphology, and distributed on the stripe in a precise arrangement with a center-to-center distance of 7.1 ± 0.3 µm between neighbor cells of the same row and 5.9 ± 0.4 µm for neighbor cells in adjacent rows. Complete detachment of Deiters cells revealed an elliptical imprint on the top surface of the basilar membrane consisting of a smaller central structure with a very smooth surface surrounded by a rougher area, suggesting the presence of two different anchoring junctions. These previously unidentified morphological features of Deiters cells could be critical for the mechanical response of the organ of Corti.

The dimensions and composition of stereociliary rootlets in mammalian cochlear hair cells: comparison between high- and low-frequency cells and evidence for a connection to the lateral membrane.

The sensory bundle of vertebrate cochlear hair cells consists of actin-containing stereocilia that are thought to bend at their ankle during mechanical stimulation. Stereocilia have dense rootlets that extend through the ankle region to anchor them into the cuticular plate. Because this region may be important in bundle stiffness and durability during prolonged stimulation at high frequencies, we investigated the structure and dimensions of rootlets relative to the stereocilia in apical (low-frequency) and basal (high-frequency) regions of rodent cochleae using light and electron microscopy. Their composition was investigated using postembedding immunogold labeling of tropomyosin, spectrin, beta-actin, gamma-actin, espin, and prestin. The rootlets have a thick central core that widens at the ankle, and are embedded in a filamentous meshwork in the cuticular plate. Within a particular frequency region, rootlet length correlates with stereociliary height but between regions it changes disproportionately; apical stereocilia are, thus, approximately twice the height of basal stereocilia in equivalent rows, but rootlet lengths increase much less. Some rootlets contact the tight junctions that underlie the ends of the bundle. Rootlets contain spectrin, tropomyosin, and beta- and gamma-actin, but espin was not detected; spectrin is also evident near the apical and junctional membranes, whereas prestin is confined to the basolateral membrane below the junctions. These data suggest that rootlets strengthen the ankle region to provide durability and may contact with the lateral wall either to give additional anchoring of the stereocilia or to provide a route for interactions between the bundle and the lateral wall.

Functional and morphological effects of fotemustine on the auditory system of the rat.

OBJECTIVE: This study aimed to elucidate the potential inner-ear effects of fotemustine, a chemotherapeutic agent which crosses the blood-brain barrier and is used in the treatment of primary and metastatic brain tumours and metastatic melanoma. METHODS: This study utilised distortion product otoacoustic emissions and transmission electron microscopy in order to conduct electrophysiological and morphological assessments, using a rat experimental model. Twelve ears of six male rats were examined two months following intraperitoneal slow infusion of fotemustine (100 mg/m2 or 7.4 mg/kg). Pre- and post-treatment measurements were compared. Finally, electron microscopy was performed on three rat temporal bones. RESULTS: After infusion of fotemustine, distortion product otoacoustic emissions revealed a significant reduction in signal-to-noise ratios only at 3600 Hz (from 11.95 +/- 7.52 to -0.26 +/- 9.45 dB) and at 3961 Hz (from 18.09 +/- 7.49 to 6.74 +/- 12.11 dB) (referenced to 2f1 - f2). Transmission electron microscopy of the temporal bone revealed ultrastructural changes in the outer hair cells, stria vascularis and cochlear ganglion at the cochlear basal turn. The ganglion cell perikarya were unaffected. CONCLUSIONS: Fotemustine was administered via intraperitoneal slow infusion in a rat experimental model. Twelve ears of six survivors, from 10 rats, were evaluated at the second month. Fotemustine was determined to have a potential for ototoxicity at 3600 and 3961 Hz. Three randomly chosen rats underwent electron microscopy for morphological analysis. Morphological effects in the cochlear basal turn were observed. Oedematous intracytoplasmic spaces and perivascular areas of the stria vascularis, as well as distorted chromatin content, were detected, thereby suggesting potential ototoxic effects for this agent. Further experimental and clinical studies are required in order to determine whether the effect seen in this pilot study is reversible, and to analyse effects in humans.

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.

Sharpened cochlear tuning in a mouse with a genetically modified tectorial membrane.

Frequency tuning in the cochlea is determined by the passive mechanical properties of the basilar membrane and active feedback from the outer hair cells, sensory-effector cells that detect and amplify sound-induced basilar membrane motions. The sensory hair bundles of the outer hair cells are imbedded in the tectorial membrane, a sheet of extracellular matrix that overlies the cochlea's sensory epithelium. The tectorial membrane contains radially organized collagen fibrils that are imbedded in an unusual striated-sheet matrix formed by two glycoproteins, alpha-tectorin (Tecta) and beta-tectorin (Tectb). In Tectb(-/-) mice the structure of the striated-sheet matrix is disrupted. Although these mice have a low-frequency hearing loss, basilar-membrane and neural tuning are both significantly enhanced in the high-frequency regions of the cochlea, with little loss in sensitivity. These findings can be attributed to a reduction in the acting mass of the tectorial membrane and reveal a new function for this structure in controlling interactions along the cochlea.

[The influence of glucocorticoids on the view of chicken's inner ear damage after exposure to wide-band-noise]. / Wplyw glikokortykoidów na obraz zniszczen komórek rzesatych ucha wewnetrznego kurczat poddanych ekspozycji na halas szerokopasmowy.

The aim of the study was to assess the influence of glucocorticoids on the view of hair cell regeneration process being in the chicken's inner ear (basilar papilla - BP) after exposure to wide-band noise at the level 120 dB (A) for 48 hours. We found that glucocorticoids given during and/or after exposure to the noise have a cytoprotective activity to the hair cells, they limitate the extensiveness and decrease the dynamics of hair cells injury. We observed that new "young" hair cells reappeared at the sensory epithelium on the 7th day after the end of exposure. Regenerated hair cells have immature, short and thick cilia and small apical surface area.

Morphologic changes in round window membrane after topical hydrocortisone and dexamethasone treatment.

HYPOTHESIS: Are all glucocorticoids supposed to have the same effect on the round window membrane? BACKGROUND: Interest in glucocorticoids for topical treatment of inner ear diseases is increasing. The safety of such treatment should therefore be an important consideration before clinical use. METHODS: In this study the authors investigated the morphology of the round window membrane after topical instillation of dexamethasone or hydrocortisone into the middle ear. Twenty Sprague-Dawley rats were used. Five rats received 5 doses, and five rats 10 doses, of 1 microg (20 microl) dexamethasone in the right ear, and five others were given 5 doses, and five rats 10 doses, of 2% (20 microl) hydrocortisone solution, also in the right ear. Membrane morphology was studied in both light microscopy and transmission electron microscopy. The thickness of exposed membranes was measured and compared with that of control membranes. RESULTS: Thickening and microscopically signs of inflammation were observed in hydrocortisone-exposed membranes but not in dexamethasone-exposed membranes, which did not differ morphologically from those in control ears. CONCLUSION: Although hydrocortisone has anti-inflammatory properties, it seems to provoke inflammation in the round window membrane after topical instillation. Dexamethasone had no such effects, however.

The presence and arrangement of type II collagen in the basilar membrane.

Previous studies demonstrating the presence of collagen II in the basilar membrane have used a biochemical approach or have used immunohistochemistry at the light microscopic level. In this investigation both the presence and arrangement of collagen II were demonstrated at the ultrastructural level using pre- and post-embedding immunoelectron microscopy. Labeling was dependent on the development of protocols to expose epitopes while maintaining identifiable ultrastructure. Both positive and negative controls indicate that the labeling was specific for collagen II. Collagen II was detected in the fibrous sheet of the pars tecta and in the two fibrous layers of the pars pectinata. It was detected in situ and on isolated individual 10-12 nm fibrils. The presence of collagen II in all the fibrous layers of the basilar membrane places constraints on the biomechanical properties of this important structure.

Immunolabeling type II collagen in the basilar membrane, a pre-embedding approach.

This paper describes the development of a protocol that can be used to detect collagen II in the healthy adult basilar membrane (BM) at the electron microscopic level. This protocol required aggressive epitope exposure techniques to break the crosslinks that bind the collagen molecules tightly into fibrils and to remove a dense mat of ground substance that surrounds the fibrils. On the other hand, the steps had to be carefully controlled to preserve BM ultrastructure and the collagen II epitopes that are typically labile. These requirements were satisfied by introducing a targeted crosslink breakage method and by regulating the duration of epitope exposure based on changes in tissue appearance observed with differential interference contrast microscopy. High levels of immunolabeling were achieved by substituting tissue preservation techniques for most or all of fixation; this was important because fixation reduces antigenicity directly and impedes epitope exposure. When these techniques were combined with more traditional trypsin and pepsin treatments, the result was dense immunolabeling and preservation of ultrastructure that allowed accurate localization of the immunolabeling. This pre-embedding immunoelectron microscopic method is the first to be carried out on the BM and may be adaptable to future studies of the BM as well as other tissues with similar molecular composition.

[Alterations of the basal membrane in middle ear cholesteatoma]. / Alteraciones de la membrana basal en el colesteatoma de oído medio.

Cholesteatoma epithelium is characterized by a keratinocyte disregulation accompanied by destruction of the ossicles and other bony parts of the temporal bone. Immunohistochemical methods using antibodies to fibronectin, tenascin and metalloproteinases were used to assess the alterations of the instrinsic and extrinsic components of the basement membrane. Spatial orientation of the basement membrane was preserved in histological sections. Collagen type IV, tenascin, fibronectin, basic fibroblastic growth factor (bFGF), and matrix metalloproteinases (MMPs) are related to the matrix, perimatrix of normal or pathological tissues. They were studied immunohistologically in twenty cholesteatomas, eight samples of normal auditory canal skin, and six specimens of normal middle ear mucosa. Cholesteatomas displayed alterations of the basal membrane, with presence of MMPs and a linear immunoreactivity for collagen type IV and laminin, disrupted in areas with intense inflammation. The electronic microscope revealed protrusions, duplications, thickening and disruptions of the lamina densa of the basement membrane. Thus, we conclude, that MMPs and bFGF could play an important role maintaining the proliferative activity and the aggressive behaviour of cholesteatoma in the middle ear.

Clinical aspects of round window membrane permeability under normal and pathological conditions.

Current research and an overall review of 25 years of round window membrane studies are presented. The approach, rationale and concepts that have evolved from these studies are described. Ultrastructural studies of the round window membrane of humans, monkeys, felines and rodents have disclosed three basic layers: an outer epithelium, a middle core of connective tissue and an inner epithelium. Interspecies variations are mainly in terms of thickness, being thinnest in rodents and thickest in humans. Morphologic evidence suggests that the layers of the round window participate in resorption and secretion of substances to and from the inner ear, and that the membrane could play a role in the defense system of the ear. Different substances, including antibiotics and tracers, when placed in the middle ear side traverse the membrane. Tracers placed in perilymph become incorporated into the membrane by the inner epithelial cells. Permeability is selective and factors affecting permeability include size, concentration, electrical charge, thickness of the membrane and tacilitating agents. Passage of substances through the membrane is by different pathways, the nature of which is seemingly decided at the outer epithelium of the membrane. Round window membrane studies have provided increased knowledge of the anatomy and function of this structure, as well as new insights into pathology and pathogenesis. The concepts that have evolved from these studies are potentially useful for understanding middle and inner ear interactions, and for eventual drug delivery (based on permeability) to the inner ear.

Reversible and irreversible damage to cochlear afferent neurons by kainic acid excitotoxicity.

Kainic acid (KA) selectively damages afferent synapses that innervate, in chickens, mainly tall hair cells. To better understand the nature of KA-induced excitotoxic damage to the cochlear afferent neurons, KA, at two different concentrations (0.3 or 5 mM), was injected directly into the inner ear of adult chickens. Pathologic changes in the afferent nerve ending and cell body were evaluated with light and transmission electron microscopy at various time points after KA application. The compound action potential (CAP) and cochlear microphonic (CM) potential were recorded to monitor the physiologic status of the afferent neurons and hair cells, respectively. Hair cell morphology and function were essentially normal after KA treatment. However, afferent synapses beneath tall hair cells were swollen within 30 minutes after KA at both low (KA-L) and high (KA-H) doses. In the KA-L group, the swelling disappeared within 1 day and the morphology of the postsynaptic region returned to near normal condition. In the KA-H group, by contrast, the vacant region beneath tall hair cells remained evident even 20 weeks after KA. The number of cochlear ganglion neurons in the KA-H group decreased progressively from 1 to 8-20 weeks, whereas hair cells in the basilar papilla remained morphologically intact out to 20 weeks after KA. There was no significant change in neuron number in the KA-L group. Temporal changes in the CAP amplitude paralleled the anatomic changes, although the CAP only partially recovered. These results suggest that KA induces partially reversible damage to cochlear afferent neurons with low KA concentration; above this level, KA triggers irreversible, progressive neurodegeneration.

Keratin filament deployment and cytoskeletal networking in a sensory epithelium that vibrates during hearing.

The intricate and spatially precise ways in which keratin intermediate filaments are deployed in certain cochlear epithelial cells, called supporting cells, suggests that these filaments make a micromechanically important contribution to the functional design of the guinea pig organ of Corti. Filament arrays that include keratins 8, 18, and 19 are confined mainly to regions close to the ends of large transcellular microtubule bundles in supporting cells. These cells and their microtubule bundles link sensory hair cells to a specialized basement membrane that vibrates during hearing. The keratin filament arrays apparently help anchor the ends of the microtubule bundles to cell surfaces. Filaments are concentrated at the apices and bases of most cells that contact hair cells. Substantial arrays of adherens junctions link the apices of these cells. Hence, keratin filaments may contribute to a cytoskeletal network that distributes mechanical forces from cell to cell and that coordinates the displacement of neighboring hair cells. However, high concentrations of keratin filaments have not been detected at the apices of one of the supporting cell types, which apparently has a mechanical role that is different from that of the others. Transmission electron microscopy has revealed previously undescribed filament networks at all the locations where the binding of antibodies to keratins is most marked. There is evidence that intercellular linkage of the keratin networks via their association with actin-containing meshworks and adherens junctions is more extensive than linkage provided by desmosomes.

Hair cell regeneration after local application of gentamicin at the round window of the cochlea in the pigeon.

Hair cells in the basilar papilla of birds have the capacity to regenerate after injury. Methods commonly used to induce cochlear damage are systemic application of ototoxic substances such as aminoglycoside antibiotics or loud sound. Both methods have disadvantages. The systemic application of antibiotics results in damage restricted to the basal 50% of the papilla and has severe side effects on the kidneys. Loud sound damages only small parts of the papilla and is restricted to the short hair cells. The present study was undertaken to determine the effect of local aminoglycoside application on the physiology and morphology of the avian basilar papilla. Collagen sponges loaded with gentamicin were placed at the round window of the cochlea in adult pigeons. The time course of hearing thresholds was determined from auditory brain stem responses elicited with pure tone bursts within a frequency range of 0.35-5.565 kHz. The condition of the basilar papilla was determined from scanning electron micrographs. Five days after application of the collagen sponges loaded with gentamicin severe hearing loss, except for the lowest frequency tested, was observed. Only at the apical 20% of the basilar papilla hair cells were left intact, all other hair cells were missing or damaged. At all frequencies there was little functional recovery until day 13 after implantation. At frequencies above 1 kHz functional recovery occurred at a rate of up to 4 dB/day until day 21, beyond that day recovery continued at a rate below 1 dB/day until day 48 at the 5.6 kHz. Below 1 kHz recovery occurred up to day 22, the recovery rate was below 2 dB/day. A residual hearing loss of about 15-25 dB remained at all frequencies, except for the lowest frequency tested. At day 20 new hair cells were seen on the basilar papilla. At day 48 the hair cells appeared to have recovered fully, except for the orientation of the hair cell bundles. The advantage of the local application of the aminoglycoside drug over systemic application is that it damages almost all hair cells in the basilar papilla and it has no toxic side effects. The damage is more extensive than with systemic application.

Noise-induced transient microlesions in the cell membranes of auditory hair cells.

Several types of nonauditory cells recover from transitory mechanically induced microlesions in their cell membranes. We report evidence that hair cells in the auditory papilla of the alligator lizard suffered similar membrane wounding when exposed to noise loud enough to induce a temporary threshold shift. Lucifer yellow, a molecular marker that does not normally penetrate through the cell membrane into the cytoplasm, was introduced into the extracellular fluid bathing the basolateral membrane of the hair cells. We assessed the effect of loud noise on the function of the ear by measuring compound action potentials of the auditory nerve before exposure to the noise, immediately after cessation of the noise, and after recovering overnight. Hair cells that were exposed to the noise took up much more Lucifer yellow than hair cells that were not exposed. We propose that the Lucifer yellow entered the hair cells via noise-induced lesions in their cell membranes, and that the cells were able to survive and recover functionally.

Fine structure of the basilar papilla of the emu: implications for the evolution of avian hair-cell types.

The morphology of the basilar papilla of the emu was investigated quantitatively with light and scanning electron microscopical techniques. The emu is a member of the Paleognathae, a group of flightless birds that represent the most primitive living avian species. The comparison of the emu papilla with that of other, more advanced birds provides insights into the evolution of the avian papilla. The morphology of the emu papilla is that of an unspecialised bird, but shows the full range of features previously shown to be typical for the avian basilar papilla. For example, the orientation of the hair cells' sensitive axes varied in characteristic fashion both along and across the papilla. Many of the quantitative details correlate well with the representation of predominantly low frequencies along the papilla. The most distinctive features were an unusually high density of hair cells and an unusual tallness of the hair-cell bodies. This suggests that the evolution of morphologically very short hair cells, which are a hallmark of avian papillae, is a recent development in evolution. The small degree of differentiation in hair-cell size contrasts with the observation that a significant number of hair cells in the emu lack afferent innervation. It is therefore suggested that the development of functionally different hair-cell types in birds preceded the differentiation into morphologically tall and short hair cells.

Postnatal maturation of the organ of Corti in gerbils: morphology and physiological responses.

The organ of Corti, the sensory epithelium of hearing in mammals, matures postnatally in the gerbil. Quantitative analyses of the postnatal development of the organ of Corti, including supporting cells and the basilar membrane, were carried out. The morphological study confirmed that maturation of the sensory cells proceeds with a base-to-apex gradient, with the outer hair cells appearing to mature before the inner hair cells. Maturation of the supporting cells and the basilar membrane commenced first in the middle turn. Expansion of the second row of Deiters' cells began at 6 days after birth in the middle turn, before enlargement of the pillar cell heads at 8 days postnatally. Pillar cell head enlargement continued until 20 days postnatally in the middle turn. The tunnel of Corti and spaces of Nuel appeared first in the middle turn between 8 and 10 days postnatally. The maturation of the basilar membrane involved the thickening of the central hyaline layer and a reduction in the epithelial cells on the tympanic aspect. This process continued until about 20 days after birth. The cochlear microphonic potential, whole nerve action potential, and stimulus frequency otoacoustic emissions were recorded from 12 days after birth onward and related to changes in organ of Corti morphology. The results show that changes in the accessory structures continue throughout the period of onset and development of cochlear responses between 12 and 20 days after birth, and may therefore influence the micromechanical responses of the organ of Corti to acoustic stimuli during this period.