Prevention of wound complications in salvage pharyngolaryngectomy by the use of well-vascularized flaps

Objective In an attempt to establish the mechanical relationships between the reticular lamina and tectorial membrane, we studied the morphological changes of the reticular lamina on a micrometer scale when an in vitro preparation of guinea pig cochlea with and without tectorial membrane was exposed to a potassium-rich medium.

Material and methods Using video-enhanced differential interference contrast microscopy, the radial displacement of the inner hair cells (IHCs) and outer hair cells (OHCs) in the reticular lamina was measured in real time after exposure to the potassium-rich medium for 3 min.

Results The amplitude of the displacement of the OHCs in preparations with an intact tectorial membrane was half of that observed in those in which the tectorial membrane had been removed. A similar displacement response was also observed for the IHCs, although it was smaller than that for the OHCs. There was no significant difference in the amplitude of the displacement among the three rows of OHCs.

Conclusions These results suggest that the structure linking the OHCs to the pillar cells is very elastic and that the movement of the OHCs in situ is weakly mechanically coupled to the IHCs. The tectorial membrane provides increased compliance in the motion of the IHCs and OHCs.     

The morphology and physiology of hair cells in organotypic cultures of the mouse cochlea

Organotypic explant cultures were prepared from the cochleas of 1 to 3 day post-natal mice and maintained in vitro for up to 5 days. The hair cells retain morphological integrity for the duration of the culture period although they exhibit embryological features such as a kinocilium and additional microvilli on their apical surfaces. The resting membrane potentials of mouse inner hair cells (IHCs) in vitro are similar to those of guinea-pig IHCs in vivo but the membrane potentials of outer hair cells (OHCs) in the mouse cochlea in vitro are less polarized than the resting membrane potentials of OHCs in the basal turn of the guinea-pig cochlea in vivo. The voltage responses of IHCs and OHCs to sinusoidal displacements of their stereocilia are similar to each other in waveform and dynamic range, although the responses of IHCs are larger than those of OHCs. The relationship between transducer conductance and stereocilia displacement in IHCs and OHCs is non-linear and largely accounts for the depolarizing asymmetry of the voltage response. The receptor potentials of IHCs and OHCs reverse close to 0 mV indicating that the transducer conductance is non-selective for cations. The voltage responses of IHCs and OHCs to intracellular current injection rectify when the membrane potentials are more depolarized than about -30 mV. This rectification is most pronounced in OHCs. OHCs also exhibit a time-dependent, voltage-sensitive conductance although they do not behave as electrical resonators.     

How are inner hair cells stimulated? Evidence for multiple mechanical drives

Recent studies indicate that the gap over outer hair cells (OHCs) between the reticular lamina (RL) and the tectorial membrane (TM) varies cyclically during low-frequency sounds. Variation in the RL-TM gap produces radial fluid flow in the gap that can drive inner hair cell (IHC) stereocilia. Analysis of RL-TM gap changes reveals three IHC drives in addition to classic SHEAR. For upward basilar-membrane (BM) motion, IHC stereocilia are deflected in the excitatory direction by SHEAR and OHC-MOTILITY, but in the inhibitory direction by TM-PUSH and CILIA-SLANT. Upward BM motion causes OHC somatic contraction which tilts the RL, compresses the RL-TM gap over IHCs and expands the RL-TM gap over OHCs, thereby producing an outward (away from the IHCs) radial fluid flow which is the OHC-MOTILITY drive. For upward BM motion, the force that moves the TM upward also compresses the RL-TM gap over OHCs causing inward radial flow past IHCs which is the TM-PUSH drive. Motions that produce large tilting of OHC stereocilia squeeze the supra-OHC RL-TM gap and caused inward radial flow past IHCs which is the CILIA-SLANT drive. Combinations of these drives explain: (1) the reversal at high sound levels of auditory nerve (AN) initial peak (ANIP) responses to clicks, and medial olivocochlear (MOC) inhibition of ANIP responses below, but not above, the ANIP reversal, (2) dips and phase reversals in AN responses to tones in cats and chinchillas, (3) hypersensitivity and phase reversals in tuning-curve tails after OHC ablation, and (4) MOC inhibition of tail-frequency AN responses. The OHC-MOTILITY drive provides another mechanism, in addition to BM motion amplification, that uses active processes to enhance the output of the cochlea. The ability of these IHC drives to explain previously anomalous data provides strong, although indirect, evidence that these drives are significant and presents a new view of how the cochlea works at frequencies below 3?kHz.     

Elektromechanische Transduktion

BACKGROUND: The somatic electromotility of the outer hair cells can be induced by an extracellular electrical field. This enables us to investigate the electromechanically induced motion of the organ of Corti. METHODS: The electrically induced motion of the guinea-pig organ of Corti was measured with a laser Doppler vibrometer in three cochlear turns at ten radial positions on the reticular lamina (RL) and six on each of the upper and lower surfaces of the tectorial membrane (TM). RESULTS AND CONCLUSIONS: We found a complex vibration pattern of the RL and TM, leading to a stimulus synchronous modulation of the depth of the subtectorial space in the region of the inner hair cells (IHCs). This modulation causes radial fluid motion inside the space up to at least 3 kHz. This motion is capable of deflecting the IHC stereocilia and provides an amplification mechanism additional to that associated with basilar-membrane motion.     

Mechanical transduction in outer hair cells

The outer hair cells are responsible for the exquisite sensitivity, frequency selectivity and dynamic range of the cochlea. These cells are part of a mechanical feedback system involving the basilar membrane and tectorial membrane. Transverse displacement of the basilar membrane results in relative motion between the tectorial membrane and the reticular lamina, causing deflection of the stereocilia and modulation of the open probability of their transduction channels. The resulting current causes a change of membrane potential, which in turn produces mechanical force, that is fed back into the motion of the basilar membrane. Experiments were conducted to address mechanical transduction mechanisms in both the stereocilia and the basolateral cell membrane, as well as modes of coupling of the outer hair cell force to the organ of Corti.     

Single-neuron labeling and chronic cochlear pathology. III. Stereocilia damage and alterations of threshold tuning curves

Tuning curves were obtained from 100 to 150 auditory-nerve fibers spanning the range of characteristic frequencies (CFs) in each of eight cases of permanent noise-induced and three cases of permanent kanamycin-induced threshold shift. In each ear, from one to six neurons were intracellularly labeled with horseradish peroxidase. Locating the labeled terminals in plastic-embedded surface preparations of the cochlea enabled us to accurately correlate particular tuning-curve abnormalities with the condition of the sensory cells generating them. The correlations between structural and functional changes suggest that a normal tuning-curve tip requires that the stereocilia on both the IHCs and OHCs (especially those from the first row) be normal. Selective damage to the OHCs is associated with elevation of the tips and hypersensitivity of the tuning-curve tails. This tuning-curve pattern also originates from cochlear regions at the basal border of hair cell lesions where the local hair cells (and their stereocilia) appear completely normal at the light-microscopic level. Total destruction of the OHCs in a region in which the IHCs appear normal (as can happen in cases of kanamycin poisoning) is associated with bowl-shaped tuning curves which appear to lack a tip. Combined damage to the IHCs and OHCs (as typically happens in cases of acoustic trauma) is invariably associated with elevation of both tips and tails on the tuning curve. A framework for the interpretation of the results is suggested in which the activity of the OHCs is transmitted via the tectorial membrane to the tall row of stereocilia on the IHCs.     

A micromechanical model of the cochlea with radial movement of the tectorial membrane.

The function of the tectorial membrane in the cochlear micromechanics is uncertain. In modeling approaches some models have assumed it to be a resonator that participates in the sharp tuning mechanisms of the cochlea with its mass coupled to the ciliary stiffness of outer hair cells, being driven by the shear force between the reticular lamina and itself. This paper presents a different type of micromechanical model which assumes that the tectorial membrane is driven by a lymphatic fluid flow that can be shown to have a substantial radial component. It also assumes that the reticular lamina is relatively stiff and thereby restrains the top end of outer hair cells that exert a force to the basilar membrane via Deiters cells. When combined with a three-dimensional block model, it can simulate the sharp tuning mechanisms of the cochlea well.     

Effect of inner and outer hair cell lesions on electrically evoked otoacoustic emissions

When the cochlea is stimulated by a sinusoidal current, the inner ear emits an acoustic signal at the stimulus frequency, termed the electrically evoked otoacoustic emission (EEOAE). Recent studies have found EEOAEs in birds lacking outer hair cells (OHCs), raising the possibility that other types of hair cells, including inner hair cells (IHCs), may generate EEOAEs. To determine the relative contribution of IHCs and OHCs to the generation of the EEOAE, we measured the amplitude of EEOAEs, distortion product otoacoustic emissions (DPOAEs), the cochlear microphonic (CM) and the compound action potential (CAP) in normal chinchillas and chinchillas with IHC lesions or IHC plus OHC lesions induced by carboplatin. Selective IHC loss had little or no effect on CM amplitude and caused a slight reduction in mean DPOAE amplitude. However, IHC loss resulted in a massive reduction in CAP amplitude. Importantly, selective IHC lesions did not reduce EEOAE amplitude, but instead, EEOAE amplitude increased at high frequencies. When both IHCs and OHCs were destroyed, the amplitude of the CM, DPOAE and EEOAE all decreased. The increase in EEOAE amplitude seen with IHC loss may be due to (1) loss of tonic efferent activity to the OHCs, (2) change in the mechanical properties of the cochlea or (3) elimination of EEOAEs produced by IHCs in phase opposition to those from OHCs.     

Age-related structural investigation of the Bronx waltzer mutant mouse cochlea: scanning and transmission electron microscopy

Cochleas of mice homozygous for the Bronx waltzer gene (symbol bv) were investigated using scanning (SEM) and transmission (TEM) electron microscopy. An age-related study was done from birth to postnatal day 100. With SEM, the arrangement of hair cells confirmed the unique feature of the bv/bv cochlea: the inner hair cells (IHCs) were either absent or abnormally haired but the outer hair cells (OHCs) appeared normal. No significant difference was observed with age. Using TEM, the remaining (20-25%) IHCs could be divided into two groups: normal-looking IHCs but with an abnormal synaptic pole, and abnormal, abortive-like IHCs. Very little if any sign of degeneration was observed whatever the age. OHCs displayed an almost normal cytology and pattern of innervation. The neurons of the spiral ganglion were very rare, even at birth. These findings suggest that the bv mutation should rather be classified in another group, than 'degenerative'. The persistence of normal structures at OHC level is discussed in light of the cochlear physiology of the bv/bv: it again raises the question of the real role of OHCs in the peripheral auditory mechanisms.     

Ultrastructural localization of calmodulin in gerbil cochlea by immunogold electron microscopy

Localization of calmodulin, a calcium binding protein, was identified in adult gerbil cochleas using paraffin section immunohistochemistry and immunogold electron microscopy with monoclonal antibody against bovine calmodulin. Immunoreactive calmodulin was abundant in inner hair cells (IHCs), outer hair cells (OHCs) and Boettcher cells of the cochleas. Other cell types containing calmodulin were marginal cells and basal cells of the stria vascularis, fibrocytes in the spiral ligament, spiral ganglion neurons and vascular smooth muscle cells. Immunogold labeling for calmodulin was observed in cuticular plate, stereocilia, and within cytoplasm of IHCs and OHCs. In OHCs the labeling was mostly observed in the region underlying lateral wall corresponding to subsurface cisterna. In IHCs the staining was diffuse in the cytoplasm and denser than that in OHCs. Boettcher cells showed dense staining along the microvillous projections facing to the intercellular spaces between Boettcher cells and Claudius cells and between the neighboring Boettcher cells. These distributions of calmodulin in the hair cells consist with the assumption that IHCs act as a true neurotransducer and OHCs as an active bi-directional mechanotransducer. The rich presence of calmodulin in Boettcher cells suggests that the cells may involve in mediating Ca(2+) regulation and play a distinctive active role in ion transport.     

A newly identified surface coat on cochlear hair cells

Routine electron microscope methods do not well preserve or stain the surface coat or glycocalyx on cochlear hair cells. In other tissues, enhanced preservation and staining of these glycoconjugates was obtained following fixation with glutaraldehyde containing a cationic dye (e.g., Alcian blue and ruthenium red). When cochleas were fixed with glutaraldehyde containing Alcian blue, the endolymphatic surface of hair cells, but not the supporting cells, displayed an extensive (approximately 90 nm thick) surface coat. Alcian blue positive material was also observed in the tectorial and basilar membranes and in a portion of the spiral ligament. In addition, acellular bands of Alcian blue positive material were observed between the tectorial membrane and the reticular lamina or inner sulcus cells. Although the function of these cochlear glycoconjugates is not yet known, it is proposed that they serve to attach the tectorial membrane to the organ of Corti, and they are involved in stereocilia fusion following sound exposure and ototoxic drug administration.     

Structural relationships of the unfixed tectorial membrane

Although the tectorial membrane has a key role in the function of the organ of Corti, its structural relationship within the cochlear partition is still not fully characterised. Being an acellular structure, the tectorial membrane is not readily stained with dyes and is thus difficult to visualise. We present here detailed observations of the unfixed tectorial membrane in an in vitro preparation of the guinea pig cochlea using confocal microscopy. By perfusing the fluid compartments within the cochlear partition with fluorochrome-conjugated dextran solutions, the tectorial membrane stood out against the bright background. The tectorial membrane was seen as a relatively loose structure as indicated by the dextran molecules being able to diffuse within its entire volume. There were, however, regions showing much less staining, demonstrating a heterogeneous organisation of the membrane. Especially Hensen's stripe and regions facing the outer hair cell bundles appeared more condensed. Whereas no connections between Hensen's stripe and the inner hair cell bundles could be observed, there was clearly a contact zone between the stripe and the reticular lamina inside of the inner hair cell.     

THE COCHLEAR AMPLIFIER： IS IT HAIR BUNDLE MOTION OF OUTER HAIR CELLS？

Cochlear outer hair cells （OHCs） are involved in a mechanical feedback loop in which the fast somatic motility of OHCs is required for cochlear amplification. Alternatively, amplification is thought to arise from active hair bundle movements ob- served in non-mammalian hair cells. We measured the voltage-evoked hair bundle motions in the gerbil cochlea to determine if such movements are also present in mammalian OHCs. The OHCs displayed a large hair bundle movement that was not based on mechanotransducer channels but based on somatic motility. Significantly, bundle movements were able to generate radial motion of the rectorial membrane in situ. This result implies that the motility-associated hair bundle motion may be part of the cochlear amplifier.     

Tectorial membrane. II: Stiffness measurements in vivo

The tectorial membrane is assumed to play a crucial role in the stimulation of the cochlear hair cells and was thought for decades to serve as a stiff anchor for the tips of the hair-cell stereocilia, particularly those belonging to the OHCs. Yet, its stiffness has never been measured under conditions approximating its normal environment in live animals. We have developed a method for doing this. The tectorial membrane is approached through the lateral wall of scala media. The bony cochlear capsule is removed along scala media over somewhat less than 1/4 turn, and the underlying spiral ligament and stria vascularis are carefully reflected. With the help of a three axial hydraulic manipulator, a flexible micropipette filled with isotonic KCl is inserted into the tectorial membrane at one of two different angles and moved either transversally, away from the basilar membrane, or radially, toward or away from the modiolus. This causes the tectorial membrane to be deformed and the micropipette to bend. The micropipette stiffness is calibrated on an instrument of a new kind, so as to convert the bend into force. The calibration allows us to determine the point stiffness of the tectorial membrane from the amount of micropipette bend. The stiffness of the tectorial membrane per unit length has been calculated from the point stiffness with the help of the deformation pattern. Transversal and radial stiffness magnitudes have been determined in the second cochlear turn in Mongolian gerbils. Both are smaller by almost an order of magnitude than the corresponding aggregate stiffness of the OHC stereocilia. As a consequence, the tectorial membrane cannot act as a stiff anchor for the stereocilia but only as a mass load, except at relatively low sound frequencies where mass effects are negligible. This means that the classical model of shear motion between the tectorial membrane and the reticular lamina must be replaced.     

Ultrastructural correlates of selective outer hair cell destruction following kanamycin intoxication in the chinchilla

Kanamycin ototoxicity, combined with behavioral audiometry to evaluate threshold shifts, was used to destroy outer hair cells (OHCs) in the basal cochlea of the chincilla while leaving the inner hair cell (IHC) population largely intact. After survival times of four weeks to one year, transmission electron microscopy was employed to determine the condition of surviving hair cells and neural elements. Throughout the region of OHC loss, IHCs and their innervation were normal in appearance if their adjacent supporting cells were undamaged. When IHC supporting cells, specifically the inner pillar cells, were damaged or absent, damage to IHCs was commonly obsereved. Such supporting cell-related damage included extrusion of the cuticular plate from the surface of the reticular lamina, encapsulation and/or fusion of stereocilla, and gross distortion of hair cell shape. When the outer supporting cells of the organ of Corti were undamaged following OHC loss, outer spiral fibers were found to have survived in near-normal numbers in the region from 0.5–1.0 mm basalmost surviving OHC, but suffered progressive attrition toward the basal end of the cochlea. It is concluded that kanamycin-induced OHC loss can occur without concommitant IHC damage or outer spinal fiber loss.     

Ultrastructure of the normal human organ of corti. new anatomical findings in surgical specimens

CONCLUSION: A thorough scanning electron microscopy (SEM) investigation of immediately fixed human adult cochleae obtained during surgery for petro-clival meningiomas conveyed new information about morphology. OBJECTIVE: To investigate the ultrastructure of human adult cochleae using SEM. MATERIAL AND METHODS: Two human cochleae were decalcified, fixed with glutaraldehyde and osmium and prepared for SEM. RESULTS: The excellently preserved morphology showed the pathways of nerve fibres through the organ of Corti. Undulating lateral cell membranes of Hensen and Claudius cells created an enlarged surface that may be important for homoeostasis. The distal attachment of the tectorial membrane to the reticular lamina was present in the shape of a marginal net, which was extended through marginal pillars. Stereocilia imprints extended as far as the distal end of the marginal pillars. The presence of an irregularly distributed fourth row of outer hair cells attached to the marginal pillars raises questions about differences in the excitation of the last row of outer hair cells as a result of differences in the composition of the tectorial membrane.     

Nitric oxide distribution and production in the guinea pig cochlea

Production sites and distribution of nitric oxide (NO) were detected in cochlear lateral wall tissue, the organ of Corti and in isolated outer hair cells (OHCs) from the guinea pig using the fluorescent dye, 4,5-diaminofluorescein diacetate. Fluorescent signal, indicating the presence of NO, was found in the afferent nerves and their putative endings near inner hair cells (IHCs) and putative efferent nerve endings near OHCs, the IHCs and OHCs, the endothelial cells of blood vessels of the spiral ligament, the stria vascularis, and the spiral blood vessels of the basilar membrane. An increased NO signal was observed following exposure to the substrate for NO, L-arginine, while exposure to NO synthase inhibitors resulted in a decrease in NO signal. Observation of OHCs at the subcellular level revealed differentially strong fluorescent signals at the locations of cuticular plate, the subcuticular plate region, the infranuclear region, and the region adjacent to the lateral wall. The findings indicate the presence of NO in the cochlea and suggest that NO may play an important role in both regulating vascular tone and mediating neurotransmission in guinea pig cochlea.     

The responses of cochlear hair cells to tonic displacements of the sensory hair bundle

Hair bundle displacements and receptor potentials were recorded from outer hair cells (OHCs) in organotypic cultures of the mouse cochlea during force steps applied to the bundles with a silica probe of known stiffness. The receptor potentials of some OHCs adapt for excitatory displacements and the time constants of receptor potential adaptation and hair bundle force relaxation for excitatory displacements are very similar. Thus in these OHCs, the receptor potentials correspond to the applied force for excitatory displacements. For inhibitory displacements, the receptor potentials correspond to hair bundle displacement. Some OHC receptor potentials are nonadapting and follow displacement in both the excitatory and inhibitory directions. The hair bundles of nonadapting OHCs are less stiff than those of adapting OHCs and nonadapting OHCs are an order of magnitude less sensitive to hair bundle displacement than adapting OHCs. In response to a combination of excitatory, tonic, hair bundle displacement and current injection, the receptor potentials of nonadapting OHCs decline as the membrane potential is made more positive and reverse near 0 mV. When the receptor potentials of adapting OHCs measured during current injection are compensated for constant input resistance and driving voltage across the transducer conductance, the receptor potential amplitude at the offset of the step displacement is independent of the level and polarity of the injected current. Before adaptation, at the onset of the step displacement of the hair bundle, the amplitude of the receptor potential increases as the injected current becomes more positive. For adapting OHCs, the receptor potential amplitude is a linear function of excitatory bundle displacement for amplitudes less than 50 nm. With negative, but not positive, current injection the receptor potentials at the onset of the displacement tend to saturate and the slope of the function decreases. This voltage dependent control of OHC transducer operating range is proposed to have a role in regulating the sensitivity of the cochlea.     

Distribution of F-actin and fodrin in the hair cells of the guinea pig cochlea as revealed by confocal fluorescence microscopy.

We double-stained paraformaldehyde fixed guinea pig cochleas with rhodaminated phalloidin to detect F-actin and with a monoclonal antibody against non-erythroid spectrin (fodrin). The hair cells were studied in surface specimens of the organ of Corti with confocal fluorescence microscopy. In serial optical sections, phalloidin stained the stereocilia, cuticular plate, and a circumferential ring beneath it in the inner and outer hair cells (IHCs and OHCs). The cytoplasm of the IHCs and the OHCs was unlabelled, but the infracuticular network of the OHCs in the upper turns showed a strong reaction. The lateral plasma membrane was unreactive with phalloidin in the IHCs and OHCs, except in the basal turn, where a moderate reaction, probably representing actin of Deiter's cups, was seen along the lateral walls of the basal pole of the OHCs. Fodrin was similarly seen in the cuticular plate, in a circumferential ring beneath it, and in the infracuticular network of the apical OHCs. The most interesting finding was the fodrin-specific distinct labelling of the lateral cell surface in the OHCs of the basal cochlear turn. This staining diminished towards the apex and was practically absent in the OHCs located above the level of 15 mm from the round window. The lateral cell surface of IHCs showed moderate fodrin labelling in all cochlear turns. This staining was much weaker than that seen in the basal OHCs. Fodrin labelling revealed deformation from the regular cylindrical shape in midportion of the OHC bodies in the basal turn of the cochlea.     

Development of outward potassium currents in inner and outer hair cells from the embryonic mouse cochlea

We recorded K(+) currents in inner (IHCs) and outer (OHCs) hair cells from mice at embryonic days 16 and 18 and on the day of birth (PO) to characterize their early physiological differentiation. In both cell types, outward currents increased in size during late embryonic development, in cells situated in both the apical and basal coils of the cochlea. Currents increased up to six-fold, with current density increasing four-fold. Currents in basal cells were generally larger than in the apex, and currents in IHCs were larger than in OHCs at any given stage. In OHCs, they were initially non-inactivating but gained the partial inactivation characteristic of the K(+) current of neonatal mouse cochlear hair cells, I(K,neo), by day 18 in the base and by P0 in the apex. In IHCs, there was little change, other than in amplitude, with partial inactivation already evident in the base by embryonic day 16. These results suggest that changes in the channel complement of OHCs occur within a few days of terminal mitosis, whereas in IHCs any such development would occur earlier. The progressive development of K(+) currents correlates with a developmental delay of around 2 days from the base to the apex of the cochlea.