Cochlear dimensions obtained in hemicochleae of four different strains of mice: CBA/CaJ, 129/CD1, 129/SvEv and C57BL/6J.

Because homologies between mice and human genomes are well established and hereditary abnormalities are similar in both, mice present a valuable animal model to study hereditary hearing disorders in humans. One of the manifestations of hereditary hearing disorders might be in the structure of cochlear elements, such as the gross morphology of the cochlea. Cochlear dimensions, however, are one factor that determines inner ear mechanics and thus hearing function. Therefore, gross cochlear dimension might be important when different strains of mice are compared regarding their hearing. Although several studies have examined mouse inner ear structures on a sub-cellular level, only few have studied cochlear gross morphology. Moreover, the sparse data available were acquired from fixed and dehydrated tissue. Dehydration, however, produces severe distortion of gel-like cochlear structures such as the tectorial membrane and the basilar membrane hyaline matrix. In this study, the hemicochlea technique, which allows fresh mouse cochlear material to be viewed from a radial perspective, was used to provide an itemized study of the dimensions of gross cochlear structures in four mouse strains (CBA/CaJ, 129/SvEv, 129/CD1 and C57BL/6J). Except for the CBA/CaJ, these strains are known to possess genes for age-related hearing loss. The measurements showed no major differences among the four strains. However, when compared with previous data, the thickness measures of the basilar membrane were up to 10 times larger. Such differences are likely to result from the different techniques used to process the material. The hemicochlea technique eliminates much of the distortion caused by dehydration, which was present in previous experiments.     

Energy Flux in the Cochlea: Evidence Against Power Amplification of the Traveling Wave

Traveling waves in the inner ear exhibit an amplitude peak that shifts with frequency. The peaking is commonly believed to rely on motile processes that amplify the wave by inserting energy. We recorded the vibrations at adjacent positions on the basilar membrane in sensitive gerbil cochleae and tested the putative power amplification in two ways. First, we determined the energy flux of the traveling wave at its peak and compared it to the acoustic power entering the ear, thereby obtaining the net cochlear power gain. For soft sounds, the energy flux at the peak was 1 ± 0.6 dB less than the middle ear input power. For more intense sounds, increasingly smaller fractions of the acoustic power actually reached the peak region. Thus, we found no net power amplification of soft sounds and a strong net attenuation of intense sounds. Second, we analyzed local wave propagation on the basilar membrane. We found that the waves slowed down abruptly when approaching their peak, causing an energy densification that quantitatively matched the amplitude peaking, similar to the growth of sea waves approaching the beach. Thus, we found no local power amplification of soft sounds and strong local attenuation of intense sounds. The most parsimonious interpretation of these findings is that cochlear sensitivity is not realized by amplifying acoustic energy, but by spatially focusing it, and that dynamic compression is realized by adjusting the amount of dissipation to sound intensity.     

In vivo micromechanical measurements of the organ of Corti in the basal cochlear turn

Cochlear mechanical measurements of organ of Corti motion are generally accomplished in the apical or basal turn as in vivo or in vitro studies. In the apex it is possible to observe and measure tectorial membrane vibration as well as vibrations of structures such as the reticular lamina or the basilar membrane (BM). However, compared to the basal turn, cochlear amplification and nonlinearity are not strong in the apex. Basal turn studies have typically been limited to point location measurements of the BM but improved technology for laser interferometry is now making possible the spatial mapping of BM motion. The 'complexity' of BM motion in the radial direction (particularly the phase variation) is important to new models of cochlear wave amplification. In future work it may be possible to learn about vibration of structures within the organ of Corti.     

Acoustic overstimulation alters the morphology of the tectorial membrane

After a permanent threshold shift was induced by exposing guinea pigs to a 1 kHz pure tone at 105 dB(A) for 72 h, light microscopic observations of freshly dissected and stained tectorial membranes showed an increased waviness and clumping of the fibers of the middle zone. Hensen's stripe was not seen as a continuous dense structure running through the middle zone but was at times discontinuous and curved. As measured from cross-sections of the cochlea, the thickness of the tectorial membrane was decreased after acoustic overstimulation. The stereocilia of the inner and outer hair cells lie directly under the middle zone. Visual detection levels of threshold of tectorial membrane movement was determined by stimulating the marginal zone of the tectorial membrane of isolated cochlear coils by an oscillating water jet. After acoustic overstimulation the tectorial membrane became more compliant. The tectorial membrane abnormalities were restricted to the regions of the cochlea that demonstrated a 40–50 dB hearing loss.     

Measurement of Basilar Membrane Motion During Round Window Stimulation in Guinea Pigs

Driving the cochlea in reverse via the round window membrane (RWM) is an alternative treatment option for the hearing rehabilitation of a nonfunctional or malformed middle ear. However, cochlear stimulation from the RWM side is not a normal sound transmission pathway. The basilar membrane (BM) motion elicited by mechanical stimulation of the RWM is unknown. In this study, the BM movement at the basal turn was investigated in both reverse via RWM drive and acoustic stimulation in the ear canal or forward drive in postmortem isolated temporal bone preparations of guinea pigs. During reverse drive, a magnet-coil was coupled on RWM, and the BM vibration at the basal turn and the movement of the incus tip were measured with laser Doppler vibrometry. During forward drive, the vibration of the incus tip induced by sound pressure in the ear canal resulted in BM vibration and the BM movement at the same location as that in the reverse stimulation was measured. The displacement ratio of the BM to RWM in reverse drive and the ratio of the BM to incus in forward drive were compared. The results demonstrated that the BM response measured in both situations was similar in nature between forward and reverse drives. This study provides new knowledge for an understanding of BM movement induced by reverse drive via the RWM stimulation.     

Current Steering with Partial Tripolar Stimulation Mode in Cochlear Implants

The large spread of excitation is a major cause of poor spectral resolution for cochlear implant (CI) users. Partial tripolar (pTP) mode has been proposed to reduce current spread by returning an equally distributed fraction (0.5 × σ) of current to two flanking electrodes and the rest to an extra-cochlear ground. This study tested the efficacy of incorporating current steering into pTP mode to add spectral channels. Different proportions of current [α × σ and (1 − α) × σ] were returned to the basal and apical flanking electrodes respectively to shape the electric field. Loudness and pitch perception with α from 0 to 1 in steps of 0.1 was simulated with a computational model of CI stimulation and tested on the apical, middle, and basal electrodes of six CI subjects. The highest σ allowing for full loudness growth within the implant compliance limit was chosen for each main electrode. Pitch ranking was measured between pairs of loudness-balanced steered pTP stimuli with an α interval of 0.1 at the most comfortable level. Results demonstrated that steered pTP stimuli with α around 0.5 required more current to achieve equal loudness than those with α around 0 or 1, maybe due to more focused excitation patterns. Subjects usually perceived decreasing pitches as α increased from 0 to 1, somewhat consistent with the apical shift of the center of gravity of excitation pattern in the model. Pitch discrimination was not better with α around 0.5 than with α around 0 or 1, except for some subjects and electrodes. For three subjects with better pitch discrimination, about half of the pitch ranges of two adjacent main electrodes overlapped with each other in steered pTP mode. These results suggest that current steering with focused pTP mode may improve spectral resolution and pitch perception with CIs.     

Imprints of the inner sensory cell hairs on the human tectorial membrane

Summary Imprints indicating possible direct inner sensory cell hair contact with the tectorial membrane were observed in the cochlea of a 77-year-old woman under a scanning electron microscope (SEM). The imprints were seen in the lower and upper basal cochlear turns but not in the apical and middle turns. The small dot of imprints numbered from a few up to 12 and were arranged in various forms rather than straight lines. Contact between the tectorial membrane and inner and outer sensory cell hairs of the human cochlea was discussed from the SEM findings found in this case.     

不同种属实验动物耳蜗毛细胞年龄相关性缺失的不同模式

目的 展示自然衰老和耳聋相关基因遗传缺陷之间耳蜗毛细胞缺失的不同模式。方法 用不同龄的长尾猴、南美栗鼠、豚鼠、Sprague-Dawley 大鼠、CBA/CaJ 小鼠、C57BL/6J 小鼠、A/J小鼠、DBA/2J 小鼠和侏儒灰色突变纯合子 (dwg/dwg) 小鼠作为受试对象。所有测试动物的耳蜗基底膜都被制作成平坦的耳蜗基底膜铺片。沿着耳蜗基底膜的全长,基底膜上所有的内外毛细胞都被完整计数,毛细胞的计数结果被输入到耳蜗图软件并自动生成每组实验条件的平均耳蜗图。结果 在天然衰老的动物中,耳蜗毛细胞的缺失总是发生在老年阶段。与此不同的是,在耳聋相关基因缺陷的动物中,耳蜗毛细胞的缺失却是发生在青年阶段甚至幼年阶段。发生在天然老化动物的耳蜗毛细胞缺失总是呈均匀分布或从耳蜗的顶回向底回扩展。 但是,发生在具有耳聋相关基因遗传缺陷动物的耳蜗毛细胞缺失却通常表现为从耳蜗的底回向顶回扩展。结论 本实验观察结果表明,发生在天然衰老的不具备耳聋相关基因缺陷动物身上的年龄相关性耳蜗毛细胞缺失反映的是真正由衰老引起的耳蜗退化性病变,而发生在伴有耳聋相关基因遗传缺陷的年幼动物身上的年龄相关性耳蜗毛细胞缺失可能与耳聋相关基因的遗传缺陷有关。     

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.     

Representation of acoustic signals in the human cochlea in presence of a cochlear implant electrode

BACKGROUND: In subjects with remaining low frequency hearing, combined electric-acoustic stimulation (EAS) of the auditory system is a new therapeutic perspective. Intracochlear introduction of a cochlear implant electrode, however, may alter the biomechanical properties of the inner ear and thus affect perception of acoustic stimuli. STUDY DESIGN: Based on histological observations of morphologic changes after cochlear implantation in cadaveric and post mortem studies the effects of basilar membrane (BM) stiffening in the ascending basal and middle turns of the cochlea due to close contact of the BM with the electrode were simulated in a 3D-computational finite element model of the inner ear. To verify our simulated results, pre- and postoperative pure-tone audiograms of 13 subjects with substantial residual hearing, who underwent cochlear implantation, were evaluated. RESULTS: In the scenario of partial BM-fixation, acoustic energy of middle (2 kHz) and high (6 kHz) frequency was focused basally and apically to the fixed section, increasing BM displacement amplitudes up to 6 dB at a stimulation level of 94 dB (SPL). Lower frequencies were not affected by fixation in the basal and middle turn of the cochlea. In implanted subjects, a small but significant decrease of thresholds was observed at 1.5 kHz, a place in tonotopy adjacent to the tip region of the implanted electrode. CONCLUSION: Our model suggests that stiffening of the basilar membrane adjacent to an implanted electrode into the basal and middle cochlear turn did not affect BM movement in the low frequency area. Focussing of acoustic energy may increase perception in regions adjacent to the fixed section. Observations in implanted subjects were concordant with our model predictions. High frequencies, however, should not be amplified in patients using EAS to avoid disturbances in discrimination due to tonotopically incorrect frequency representation.     

Development of the Gerbil Inner Ear Observed in the Hemicochlea

A frequency-dependent change in hearing sensitivity occurs during maturation in the basal gerbil cochlea. This change takes place during the first week after the onset of hearing. It has been argued that the mass of a given cochlear segment decreases during development and thus increases the best frequency. Changes in mass during cochlear maturation have been estimated previously by measuring the changes in cochlear dimensions. Fixed, dehydrated, embedded, or sputter-coated tissues were used in such work. However, dehydration of the tissue, a part of most histological techniques, results in severe distortion of some aspects of cochlear morphology. The present experiments, using a novel preparation, the hemicochlea, show that hydrated structures, such as the tectorial membrane and the basilar membrane hyaline matrix, are up to 100% larger than estimated previous studies. Therefore, the hemicochlea was used to study the development of cochlear morphology in the gerbil between the day of birth and postnatal day 19. We used no protocols that would have resulted in severe distortion of cochlear elements. Consequently, a detailed study of cochlear morphology yields several measures that differ from previously published data. Our experiments confirm growth patterns of the cochlea that include a period of remarkably rapid change between postnatal day 6 and 8. The accelerated growth starts in the middle of the cochlea and progresses toward the base and the apex. In particular, the increase in height of Deiters' cells dominated the change, "pushing" the tectorial membrane toward scala vestibuli. This resulted in a shape change of the tectorial membrane and the organ of Corti. The tectorial membrane was properly extended above the outer hair cells by postnatal day 12. This time coincides with the onset of hearing. The basilar membrane hyaline matrix increased in thickness, whereas the multilayered tympanic cover layer cells decreased to a single band of cells by postnatal day 19. Before and after the period of rapid growth, the observed gross morphological changes are rather small. It is unlikely that dimensional changes of cochlear structures between postnatal days 12 and 19 contribute significantly in the remapping of the frequency-place code in the base of the cochlea. Instead, structural changes affecting the stiffness of the cochlear partition might be responsible for the shift in best frequency.     

Characterization of the Sheep Round Window Membrane

Intratympanic injection is a clinically used approach to locally deliver therapeutic molecules to the inner ear. Drug diffusion, at least in part, is presumed to occur through the round window membrane (RWM), one of the two openings to the inner ear. Previous studies in human temporal bones have identified a three-layered structure of the RWM with a thickness of 70–100 μm. This is considerably thicker than the RWM in rodents, which are mostly used to model RWM permeability and assess drug uptake. The sheep has been suggested as a large animal model for inner ear research given the similarities in structure and frequency range for hearing. Here, we report the structure of the sheep RWM. The RWM is anchored within the round window niche (average vertical diameter of 2.1 ± 0.3 mm and horizontal diameter of 2.3 ± 0.4 mm) and has a curvature that leans towards the scala tympani. The centre of the RWM is the thinnest (55–71 μm), with increasing thickness towards the edges (< 171 μm), where the RWM forms tight attachments to the surrounding bony niche. The layered RWM structure, including an outer epithelial layer, middle connective tissue and inner epithelial layer, was identified with cellular features such as wavy fibre bundles, melanocytes and blood vessels. An attached “meshwork structure” which extends over the cochlear aqueduct was seen, as in humans. The striking anatomical similarities between sheep and human RWM suggest that sheep may be evaluated as a more appropriate system to predict RWM permeability and drug delivery in humans than rodent models.     

Developmental Changes of Mechanics Measured in the Gerbil Cochlea

This report describes stiffness and best frequency measurements obtained in vitro from the basilar membrane of the gerbil cochlea at the onset of hearing, during hearing maturation, and after hearing has matured. Our stiffness data constitute the first direct experimental evidence of developmental stiffness changes in the basal and middle turns. Stiffness changes by a factor of 5.5 in the basal turn between postnatal day 11 and adult, and the difference from adult is statistically significant for all ages measured up to postnatal day 16. For the middle turn, stiffness changes by a factor of 1.6 between postnatal day 11 and adult. Whereas for postnatal day 12 and beyond there is no statistically significant difference from adult, our data suggest that there may be a significant difference of stiffness between day 11 and adult in the middle turn. For the basal turn, our motion measurements confirm a passive component to the developmental best frequency shift. For the middle turn, changes in best frequency are not statistically significant. Best frequency was determined by stimulating the tissue at audio frequencies with a glass paddle and measuring motion with a computer-based imaging system. Tissue stiffness was measured with a piezoelectric-based sensor system. Tissue stiffness changes have previously been postulated to contribute to the best frequency shift observed in the cochlear base. Incorporating our data into a simple spring-mass resonance model demonstrates that our experimentally measured stiffness change can account for the change of best frequency. These results suggest that a stiffness change is, in fact, a critical component of the best frequency shift observed in the basal turn of the gerbil cochlea after the onset of hearing.     

High-Frequency Force Generation in the Constrained Cochlear Outer Hair Cell: A Model Study

Cochlear outer hair cell (OHC) electromotility is believed to be responsible for the sensitivity and frequency selectivity of the mammalian hearing process. Its contribution to hearing is better understood by examining the force generated by the OHC as a feedback to vibration of the basilar membrane (BM). In this study, we examine the effects of the constraints imposed on the OHC and of the surrounding fluids on the cell's high-frequency active force generated under in vitro and in vivo conditions. The OHC is modeled as a viscoelastic and piezoelectric cylindrical shell coupled with viscous intracellular and extracellular fluids, and the constraint is represented by a spring with adjustable stiffness. The solution is obtained in the form of a Fourier series. The model results are consistent with previously reported experiments under both low- and high-frequency conditions. We find that constrained OHCs achieve a much higher corner frequency than free OHCs, depending on the stiffness of the constraint. We analyze cases in which the stiffness of the constraint is similar to that of the BM, reticular lamina, and tectorial membrane, and find that the force per unit transmembrane potential generated by the OHC can be constant up to several tens of kHz. This model, describing the OHC as a local amplifier, can be incorporated into a global cochlear model that considers cochlear hydrodynamics and frequency modulation of the receptor potential, as well as the graded BM stiffness and OHC length.     

Secretion of a new basal layer of tectorial membrane following gentamicin-induced hair cell loss

Scanning electron microscopy (SEM) and video-enhanced DIC light microscopy were used to assess morphological changes in the chick tectorial membrane (TM) following gentamicin-induced hair cell loss. Gentamicin was administered (100 mg/kg/day for 3 days) and isolated and in-situ TMs were examined in both fixed and unfixed preparations at days 5 and 10 after the initial injection. Although this protocol induced hair cell damage extending up to 75% of the length of the basilar papilla, there was no apparent damage to the TM itself. However, the ejection of damaged hair cells appeared to sever the filamentous attachments between the TM and the apical surface of the basilar papilla. In SEM preparations this detachment caused the TM to shrink back toward the superior edge. Interestingly, despite the lack of TM damage, gentamicin treatment did reveal the secretion of a new basal layer of TM. Secretion of this new basal layer had begun by day 5 and it was well organized by day 10. This new layer formed attachments to both the recovering basilar papilla and the overlying original TM, a step thought to be necessary for the restoration of auditory function in the regenerating cochlea.     

Characterization and Inflammatory Response of Perivascular-Resident Macrophage-Like Melanocytes in the Vestibular System

A large number of perivascular cells expressing both macrophage and melanocyte characteristics (named perivascular-resident macrophage-like melanocytes, PVM/Ms), previously found in the intra-strial fluid–blood barrier, are also found in the blood–labyrinth barrier area of the vestibular system in normal adult cochlea, including in the three ampullae of the semicircular canals (posterior, superior, and horizontal), utricle, and saccule. The cells were identified as PVM/Ms, positive for the macrophage and melanocyte marker proteins F4/80 and GSTα4. Similar to PVM/Ms present in the stria vascularis, the PVM/Ms in the vestibular system are closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, with a density in normal (unstimulated) utricle of 225 ± 43/mm²; saccule 191 ± 25/mm²; horizontal ampullae 212 ± 36/mm²; anterior ampullae 238 ± 36/mm²; and posterior ampullae 223 ± 64/mm². Injection of bacterial lipopolysaccharide into the middle ear through the tympanic membrane causes the PVM/Ms to activate and arrange in an irregular pattern along capillary walls in all regions within a 48-h period. The inflammatory response significantly increases vascular permeability and leakage. The results underscore the morphological complexity of the blood barrier in the vestibular system, with its surrounding basal lamina, pericytes, as well as second line of defense in PVM/Ms. PVM/Ms may be important to maintain blood barrier integrity and initiating local inflammatory response in the vestibular system.     

Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma

Damage to the tectorial membrane caused by acoustic trauma was examined with scanning and transmission electron microscopy immediately after exposure and at selected time points over a 10 day recovery period. At 0 h of recovery the structure of the tectorial membrane overlying the region of hair cell damage was severely disrupted and connections between the membrane and the basilar papilla were lost. By 24 h of recovery, regeneration of the tectorial membrane was evident in the secretion of new matrix materials by the supporting cells of the basilar papilla. By 10 days of recovery a new honeycomb-like matrix had replaced the segment of damaged tectorial membrane, re-established connections with hair cell stereocilia and become fused with adjacent regions of undamaged tectorial membrane. However, the regenerated segment included only the honeycomb-like structure of the lower layer of the normal tectorial membrane. The laterally-oriented fibers which form the upper layer of the membrane were not regenerated over the damaged region. These findings indicate that the tectorial membrane is regenerated in parallel with the hair cells during recovery from acoustic trauma but the full extent of this recovery and its effect on cochlear function are not yet clear.     

Divergent Aging Characteristics in CBA/J and CBA/CaJ Mouse Cochleae

Two inbred mouse strains, CBA/J and CBA/CaJ, have been used nearly interchangeably as ‘good hearing’ standards for research in hearing and deafness. We recently reported, however, that these two strains diverge after 1 year of age, such that CBA/CaJ mice show more rapid elevation of compound action potential (CAP) thresholds at high frequencies (Ohlemiller, Brain Res. 1277: 70–83, 2009). One contributor is progressive decline in endocochlear potential (EP) that appears only in CBA/CaJ. Here, we explore the cellular bases of threshold and EP disparities in old CBA/J and CBA/CaJ mice. Among the major findings, both strains exhibit a characteristic age (∼18 months in CBA/J and 24 months in CBA/CaJ) when females overtake males in sensitivity decline. Strain differences in progression of hearing loss are not due to greater hair cell loss in CBA/CaJ, but instead appear to reflect greater neuronal loss, plus more pronounced changes in the lateral wall, leading to EP decline. While both male and female CBA/CaJ show these pathologies, they are more pronounced in females. A novel feature that differed sharply by strain was moderate loss of outer sulcus cells (or ‘root’ cells) in spiral ligament of the upper basal turn in old CBA/CaJ mice, giving rise to deep indentations and void spaces in the ligament. We conclude that CBA/CaJ mice differ both quantitatively and qualitatively from CBA/J in age-related cochlear pathology, and model different types of presbycusis.     

小鼠出生后早期耳蜗柯蒂氏器巨噬细胞形态的变化

目的 揭示小鼠出生后早期耳蜗柯蒂氏器是否存在巨噬细胞及柯蒂氏器巨噬细胞形态和分布的变化.方法 选1～4周龄的C57BL/6J小鼠,解剖取耳蜗基底膜.CD45抗体(一种全白细胞标记物)染色耳蜗基底膜,F4/80(巨噬细胞专有蛋白标记物)确认巨噬细胞,碘化丙锭标记细胞核,荧光显微镜下观察CD45染色阳性细胞的形态和分布变化...     

Location of small cochlear lesions by phase contrast microscopy prior to thin sectioning

A new technique for the study of the inner ear is described. Cochleae are fixed, dehydrated and infiltrated with plastic while their bony walls are intact except for small holes at the third turn and near the round window. After the plastic polymerizes, the cochlear bone is removed so that half turns of cochlear duct can be separated from the modiolus. These specimens are re-embedded in a thin layer of plastic after trimming so that their basilar membrane sides lie close to the surface of the plastic layer. Using phase contrast microscopy, the entire organ of Corti is examined by focusing through the surface of the plastic and through the specimens from the basilar membrane up to the tectorial membrane. Segments of organ of Corti containing small lesions are divided in such a way that cross-sections of those areas for study by light and electron microscopy are obtained readily using an ultrotome.