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.     

Ultrahistochemical demonstration of glucose-6-phosphatase in hair cells of the guinea pig organ of corti (author's transl)]

This study is concerned with the ultrahistochemical demonstration of glucose-6-phosphatase activity and its localization in inner and outer hair cells of the guinea pig organ of Corti. The enzyme activity has been demonstrated by a method according to Hugon et al. (1970). In hair cells of the organ of Corti a characteristic distribution pattern of reaction products has been registered. Subsurface cisterns and the Hensen's bodies of outer hair cells were heavily loaded with reaction products. In addition, the endoplasmic reticulum and the nuclear membrane as well as postsynaptic cisterns were rich in precipitates. With regard to their morphologic pecularities the inner hair cells show a more homogeneous distribution of enzyme activity. The findings corroborate the former assumption of a genetic relationship of either subsurface cisterns and Hensen's body to the endoplasmic reticulum of outer hair cells. Furthermore, the high glucose-6-phosphatase activity of both subsurface cisterns and Hensen's bodies are considered indicative of their participitation in the energy metabolism of outer hair cells. Referring to biochemical studies of Thalmann and associates (1973), the narrow spatial relationship of glucose-6-phosphatase positive ER membranes to mitochondria presumably represents a morphologic correlation of aerobic and anaerobic metabolic pathways in the guinea pig 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.     

Glutamate-like Immunoreactivity During Hair Cell Recovery After Gentamicin Exposure in the Chinchilla Vestibular Sensory Periphery

Objective: Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista ampullaris after gentamicin ototoxicity. Study Design: In five groups of three animals each, ototoxicity was produced by placing gentamicin (50 μg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1, 2, 4, 8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed. Methods: For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immunoreaction was detected using the avidinbiotiny-lated-complex technique and diaminobenzidine was the chromogen. Results: Normal sensory epithelia demonstrated type I and type II hair cells with moderate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-like immunoreactivity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type II hair cells surviving ototoxic treatment (15%–18%) contained moderate glutamate-like immunoreactivity, supporting cells showed no immunoreactivity, and nerve terminals and fibers displayed strong immunoreactivity. At 4 and 8 weeks after treatment, recovered hair cells (80%) had greater glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level. Conclusion: Glutamate may be used as an indicator of hair cell differentiation and as an index of the molecular recovery of hair cells after ototoxicity.     

supporting cells-a new area in cochlear physiology study

For many years, studies about the cochlea have been mainly focused on sensory cells, i.e. the inner hair cell (IHC) and outer hair cell (OHC), and the neuron system. Supporting cells, such as Hensen's cells and Deiters' cells are less studied. Their physiological functions and other characteris-tics are not well documented. Nowadays, supporting cells are a new world attracting to scientists" inter-ests. The scope of this review is to detail the biological properties of the supporting cells, mainly Hensen's cells and Deiters' cells in the cochlea. Studies on this subject will be helpful in understanding physiology of the cochlea, and hopefully provide new approaches in treating diseases of inner ear.     

Cochlear de-efferentation and impulse noise-induced acoustic trauma in the chinchilla

The olivocochlear bundle (OCB) has been shown to protect the ear from acoustic trauma induced by continuous noise or tones. The present study examines the OCB's role in the ear's response to impulse noise (150 dB pSPL, 100 impulses, 50 s total exposure duration). Successful section of the OCB was achieved through a posterior parafloccular fossa approach for the right ears of six out of 15 adult chinchillas. The left ears from the same animals served as efferent-innervated controls. Measurements of inferior colliculus evoked potentials (ICPs) showed that the de-efferented ears incurred similar temporary and permanent threshold shifts as the control ears. Twenty days after noise exposure, depressed ICP amplitudes had virtually recovered to pre-values in the control ears whereas those in the de-efferented ears remained significantly depressed. Greater loss of inner hair cells was seen in the de-efferented ears than in the control ears. Both control and de-efferented ears incurred large loss of outer hair cells, with no statistically significant differences between groups. The current data are intriguing, yielding tentative evidence to suggest that inner hair cells of de-efferented ears are more susceptible to impulse noise than those in efferented control ears. In contrast, outer hair cell vulnerability to impulse noise appears to be unaffected by de-efferentation.     

Is the older ear more susceptible to noise damage?

Eight chinchillas aged 8.9 to 12.8 years were used to examine the effect of noise on the aging ear. The left malleus/incus complex was removed to produce a 50-dB conductive hearing loss which protected those ears from noise damage. The animals were then exposed for 36 days to an octave band of noise with a center frequency of 0.5 kHz and a sound pressure level of 95 dB. After 1 hour (n=2) or 1 month (n=6) of recovery, their cochleas were prepared for microscopic examination. The percentages of missing inner hair cells (IHCs) were 7.4 ± 6.0% and 7.8 ± 5.1% for their protected and exposed ears, respectively. Outer hair cell (OHC) loss was 12.8 ± 8.7% and 20.6 ± 7.8% for their protected and exposed ears, respectively. A paired sample Student's t test revealed that OHC loss was significantly greater (P=.003) in the older-exposed compared to the older-protected ears whereas IHC loss was not significantly different. For younger-exposed ears (i.e., 1 to 3 years), the percentages of missing IHCs and OHCs averaged 2.6 ± 2.0% and 12.3 ± 4.6%, respectively. When the aging-related cell loss was subtracted from total loss in the younger-and older-exposed ears, the noise-induced loss of sensory cells in the older ears was not significantly different from that in the younger ears. Therefore, it is concluded that older chinchilla ears are not more susceptible to noise damage than younger ears.     

耳蜗毛细胞和螺旋神经节及其神经纤维的联合定量观察

目的：将耳蜗基底膜取材技术与耳蜗切片技术相结合以观察同一耳蜗中几个主要结构在病理发展中的相互关系。方法：耳蜗铺片用于定量观察毛细胞，骨性螺旋板切片用于定量观察缰孔内的神经纤维，中轴切片用于定量观察螺旋神经节。结果：将卡铂耳中毒灰鼠的耳蜗标本与正常灰鼠进行比较，显示内毛细胞，神经纤维和螺旋神经节的损失百分比基本一致。结论：卡铂对内毛细胞，神经纤维和螺旋神经节都有破坏作用，至于在卡铂病变早期，究竟哪一     

Pronounced infracuticular endocytosis in mammalian outer hair cells.

Endocytosis in cochlear hair cells was investigated by staining with the vital fluorescent dye FM 1-43, that partitions reversibly into membranes and is trapped in vesicles during endocytosis. The temporal development and spatial distribution of FM 1-43 induced fluorescence was investigated using confocal laser-scanning microscopy. FM 1-43 rapidly and intensely stained cochlear hair cells, leaving the supporting cells unstained. For short application (0.2-30 s), only the infracuticular region of outer hair cells (OHCs) was labeled, whereas for long application (30-60 s), the OHCs were also labeled in the infranuclear zone and along a central strand extending from the infracuticular zone down to the nucleus, as well as along the entire cell membrane. Except for the cell membrane, the infracuticular zone, directly below the cuticular plate, showed the most rapid and intense staining, and in most cases staining was spherically shaped with a diameter of 3-7 microm. Localization and size of this infracuticular staining coincided with Hensen's body, a specialized variant of the endoplasmic reticulum. In contrast to the OHCs, apical fluorescence of inner hair cells presented a homogeneous distribution. When OHCs were incubated in FM 1-43 for longer than 1 min, many points of contact between the central strand, the infracuticular zone and the lateral cell membrane were observed. Since Hensen's bodies are a specialty of OHCs and the fluorescent staining pattern of these cells was unique, it is proposed that Hensen's body is involved in the turnover of OHC-specific proteins, such as those involved in the molecular machinery of the motor action of the plasma membrane.     

Reticular lamina vibrations in the apical turn of a living guinea pig cochlea.

The reticular lamina of the apical turn of a living guinea pig cochlea was viewed through the intact Reissner's membrane using a slit confocal microscope. Vibrations were measured at selected identified locations with a confocal heterodyne interferometer, in response to tones applied with an acoustic transducer coupled to the ear canal. The position coordinates of each location were recorded. Mechanical tuning curves were measured along a radial track at Hensen's cells, outer hair cells, inner hair cells and at the osseous spiral lamina, over a frequency range of 3 kHz, using five sound pressure levels (100, 90, 80, 70 and 60 dB SPL). The carrier to noise ratio obtained throughout the experiments was high. The response shape at any measuring location was not found to change appreciably with signal level. The response shape also did not change significantly with the radial position on the reticular lamina. However, the response magnitude increased progressively from the inner hair cell to the Hensen's cell. The observed linearity of response at the fundamental frequency is explained by the presence of negative feed back in the apical turn of the cochlea.     

Inner ear damage induced by Mycobacterium fortuitum

Summary We used electron microscopy to investigate Mycobacterium fortuitum -induced changes in the inner ears of mice. We found that the inner and outer hair cells had degenerated and disappeared in the organ of Corti. Changes in the lower turn of the cochlea were more severe than those of the upper turn while the changes of the outer hair cells were more severe than those of the inner hair cells. Disappearance, fusion and ballooning of the sensory hairs were observed in the vestibular organs. The bacterial extract also induced inner ear damage which was similar to that caused by live M. fortuitum.     

Potentiation of inner ear damage following electron beam irradiation with CDDP administration

The study was designed to examine the combined ototoxic effect of CDDP (Cisplatin, Cis-diammine dichloroplatinum) and electron beam irradiation, using guinea pigs. One group received physiological saline solution of 4 ml/kg/day, and another group received CDDP of 2 mg/kg/day for five days. And following the injection of saline or CDDP, the electron beam of 14Gy/day was applied to the both groups to the right ear for five days. Animals were sacrificed after 21 days, and temporal bones of these animals were removed for the inner ear histopathology. Temporal bones were classified into four groups (control, electron beam irradiation, CDDP administration, and combined administration group), and the inner ears were observed by the surface preparation technique with a phase contrast microscope, and by scanning and transmission electron microscopy, and by temporal bone study of serial sectioned slides. The main pathologic findings of the inner ear are as follows: Electron beam irradiation group showed no hair cell damage. CDDP group induced slight damage to the outer hair cells. Combined administration group (CDDP + electron beam irradiation) showed severe outer hair cell damage. Stria vascularis was degenerated moderately in the combined administration group and slightly in some animals of electron beam irradiation group. And Reissner's membrane and Hensen's cells, were damaged in the basal turn of cochlea of the combined administration group. All other cochlear structures (spiral ganglion, spiral lamina, basilar membrane, spiral prominence, tectorial membrane, blood vessels of the cochlea) and vestibular organs were lack of significant changes in all groups by a light microscopic observation. This study was clarified that combined administration of CDDP and electron beam irradiation showed severe ototoxic potentiation. Therefore, it is important that we must pay attention to the inner ear damage caused by combined therapy of CDDP and electron beam irradiation involving inner ear for the head and neck tumor.     

Mechanical properties of sensory and supporting cells in the organ of Corti of the guinea pig cochlea--study by atomic force microscopy

Mammalian hearing is refined by amplification of the motion of the cochlear partition. To understand the cochlear amplification, mechanical models of the cochlea have been used. When the dynamic behavior of the cochlea is analyzed by a model, elastic properties of the cells in the organ of Corti must be determined in advance. Recently, elastic properties of outer hair cells (OHCs) and pillar cells have been elucidated. However, those of other cells have not yet been clarified. Therefore, in this study, using an atomic force microscope (AFM), elastic properties of Hensen's cells, Deiters' cells and inner hair cells (IHCs) in the apical turn and those in the basal and second turns were estimated. As a result, slopes indicative of cell elastic properties were (8.9 +/- 5.8) x 10(3) m(-1) for Hensen's cells (n = 30), (5.5 +/- 5.3) x 10(3) m(-1) for Deiters' cells (n = 20) and (3.8 +/- 2.6) x 10(3) m(-1) for IHCs (n = 20), and Young's modulus were 0.69 +/- 0.45 kPa for Hensen's cells and 0.29 +/- 0.20 kPa for IHCs. There was no significant difference between elastic properties of each type of cell in the apical turn and those in the basal and second turns. However, it was found that there is a significant difference between Young's moduli of cells estimated in this study and those of the OHCs and pillar cells reported previously.     

Guinea pig tectorial membrane profile in an in vitro cochlear preparation

The guinea pig cochlea was examined under high-magnification light microscopy in an in vitro preparation. After extraction of the otic capsule, the bulla was opened widely and a small hole made into the fourth turn of the scala vestibuli. The organ of Corti was visualized under artificial endolymph at 600 X magnification. Added 1-micron titanium dioxide particles settled on the upper surface of the transparent tectorial membrane. Particle positions showed that much of this upper surface lay in a flat sheet that extended centrifugally almost to the Hensen's cells, giving the impression it was attached there. The sheet extended at least to the level of the inner hair cells, where a tectorial membrane thickness of about 40 micron was reached. Titanium dioxide particles were seen regularly in immediate proximity to the hair cell cilia, indicating that scala media is continuous with the subtectorial space. Upon mechanical manipulation, Hensen's cells proved to be extremely cohesive and elastic. It is suggested that hair cell stereocilia provide major mechanical connections for the tectorial membrane.     

Cytomegalovirus-induced pathology in human temporal bones with congenital and acquired infection

ObjectivePublications on histopathology of human temporal bones with cytomegalovirus (CMV) infection are limited. We aim to determine histopathology of the inner ears and the middle ears in human temporal bones with congenital and acquired CMV infections.MethodsTemporal bones from 2 infants with congenital and 2 adults with acquired CMV infection were evaluated by light microscopy.ResultsTwo infants with congenital CMV infection showed striking pathological changes in the inner ear. There was a hypervascularization of the stria vascularis in the cochlea of the first infant, but no obvious loss of outer and inner hair cells was seen in the organ of Corti. However, cytomegalic cells and a loss of outer hair cells were found in the cochlea of the second infant. The vestibular organs of both infants showed cytomegalic cells, mostly located on dark cells. There was a loss of type I and type II hair cells in the macula of the saccule and utricle. Loss of hair cells and degeneration of nerve fibers was also seen in the semicircular canals. Both infants with congenital infection showed abundant inflammatory cells and fibrous structures in the middle ear cavity. No evidence of cytomegalic cells and hair cell loss was found in the cochlea or vestibular labyrinth in acquired CMV infection.ConclusionsIn two infants with congenital CMV infection, the cochlea, vestibule, and middle ear were highly affected. Temporal bones of adult donors with acquired viral infection showed histological findings similar to donors of the same age without ear disease.     

The inner ear and the in vitro system.

The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.     

Degeneration of the organ of Corti following intravenous administration of cisplatin.

The development of acute morphological changes in the cochlea was studied in guinea pigs given one intravenous high-dose injection of cisplatin. In the light microscope three major stages of degeneration in the organ of Corti could be recognized: 1) an initial swelling of the Hensen's cells and protrusion of the Deiters' cells into the space of Nuel enclosing the outer hair cells, 2) a gradual degeneration of the outer hair cells together with a vacuolization in the region of the base of the inner hair cells, 3) a collapse of the Reissner's membrane and the entire organ of Corti with different degrees of damage to the inner hair cells. Sporadic bulging of the marginal cells of the stria vascularis into the endolymphatic space could be observed 4 days after injection.     

Purification of a low-molecular-weight excitatory substance from the inner ears of goldfish

The neurotransmitter released by the hair cell has not been identified; little is known about other neuroactive substances that may be important in hair-cell organ function. To identify neuroactive substances in hair cell tissue, we have examined substances in extracts of the inner ears of goldfish that can excite afferent fibers innervating hair cells. The extracts contain an unidentified low-molecular-weight (LMW) excitatory substance that is a candidate to be the hair-cell neurotransmitter. The LMW excitatory substance has been highly purified by a sequential combination of (1) treatment with cation-exchange resin; (2) gel-permeation chromatography; (3) gradient-elution cation-exchange chromatography; (4) isocratic-elution cation-exchange chromatography; and (5) high-performance anion-exchange chromatography. Based upon its separation behavior in these purification steps, the LMW excitatory substance may be a small, zwitterionic compound with titratable anionic and cationic groups.     

Age-Related Primary Cochlear Neuronal Degeneration in Human Temporal Bones

In cases of acquired sensorineural hearing loss, death of cochlear neurons is thought to arise largely as a result of sensory-cell loss. However, recent studies of acoustic overexposure report massive degeneration of the cochlear nerve despite complete hair cell survival (Kujawa and Liberman, J Neurosci 29:14077–14085, 2009). To assess the primary loss of spiral ganglion cells (SGCs) in human ears, neuronal counts were performed in 100 temporal bones from 100 individuals, aged newborn to 100 years, selected to include only cases with a normal population of inner and outer hair cells. Ganglion cell counts declined at a mean rate of 100 cells per year of life. There were no significant gender or inter-aural differences, and a slight increase in degeneration in the basal turn re upper turns was not statistically significant. The age-related decline in SGCs was significantly less than that in prior studies that included ears with hair cell loss (Otte et al., Laryngoscope 88:1231–1246, 1978), but significantly more than for analogous data on vestibular ganglion cells in cases without vestibular hair cell loss (Velazquez-Villasenor et al., Ann Otol Rhinol Laryngol Suppl 181:14–19, 2000). The age-related decline in SGC counts may contribute to the well-known decline in hearing-in-noise performance, and the data will help in interpretation of histopathological findings from temporal bones with known otologic disease.     

Ionic coupling among cells in the organ of Corti.

Gap junctions have been demonstrated morphologically among the supporting cells of the mammalian organ of Corti but, in contradistinction to reptiles, evidence for their existence between the supporting cells and hair cells is equivocal. The literature is ambiguous with respect to electrical coupling and dye coupling among the supporting cells, and no coupling of either kind has been demonstrated for the hair cells. We found strong coupling of both kinds among the supporting cells in the cochleas of live Mongolian gerbils and a less stable coupling between the supporting cells and the outer hair cells. The electrical coupling was established by recording alternating receptor potentials in the hair cells and following their decrement in the population of Hensen's cells; the dye coupling, by injecting Lucifer yellow electrophoretically into the hair cells or the supporting cells and investigating its spread to the neighboring cells. The electrical recordings were made by means of microelectrodes filled with either 1.5 or 3 M KCl or 1 M LiCl with 6% Lucifer yellow, the latter used for dye injection. The electrode resistances ranged from about 20 to 60 M omega in the first instance, and from about 50 to 110 M omega, in the second. The electrodes were inserted into the organ of Corti through scala media according to the method of Dallos, Santos-Sacchi and Flock (1982) modified by us. The alternating potential in Hensen's cells was usually larger than in the outer tunnel of Corti and remained practically constant up to the outer margin of the Hensen's-cell population. Its phase was the same as in the outer hair cells. When the dye was injected into a Hensen's cell, it always spread to neighboring Hensen's cells and often to Deiter's cells. Dye injected into outer hair cells (identified according to anatomical and physiological criteria) also spread to Deiter's and Hensen's cells and, usually, to other outer hair cells. Stained cells were identified in surface preparations and, on two occasions, in serial sections from plastic embedded cochleas.