豚鼠耳蜗部分外支持细胞的分离法

目的 :探讨豚鼠耳蜗部分外支持细胞 (Deiter细胞、Hensen细胞和外柱细胞 )的分离方法 ,并建立细胞活性的鉴别标准。方法 :选取健康杂色豚鼠 5只 ,解剖出耳蜗基底膜 ,采用酶解加机械吹打法分离Deiter细胞、Hensen细胞和外柱细胞。结果 :可以获得较多数量、活性良好、长短不一的Deiter细胞和Hensen细胞 ,8h内细胞活性较好 ;但外柱细胞数量较少 ,存活时间较短。评价Deiter细胞、Hensen细胞和外柱细胞的活性标准 :①细胞膜无膨胀或扭曲 ;②细胞核无肿胀、移位 ;③在相差显微镜下细胞呈半透明状态 ,存在双折射现象 ;④细胞内无呈布朗运动的颗粒。结论 :熟悉耳蜗的解剖特性、分离出完整的基底膜 ,增加酶的浓度和加大机械吹打力度是获取活性良好的Deiter细胞、Hensen细胞和外柱细胞的关键 ;评价外毛细胞活性的 4项标准同样可用于判断Deiter细胞、Hensen细胞和外柱细胞的活性     

Displacement of the reticular lamina with and without the tectorial membrane in the guinea pig cochlea

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.     

Local mechanical properties of mouse outer hair cells: atomic force microscopic study

OBJECTIVES: Outer hair cells (OHCs) are capable of altering their cell length in response to changes in membrane potential. Due to this electromotility, OHCs probably subject the basilar membrane to force, resulting in cochlear amplification. To understand the mechanism of such amplification, knowledge of the mechanical properties of OHCs is required since the force produced by OHC electromotility is thought to depend on such properties. Various studies have been conducted to investigate the mechanical properties of guinea pig OHCs. With regard to mice, however, although various kinds of transgenic and knockout mice possess great potential as research models, the mechanical properties of mouse OHCs have not as yet been reported since the cells and/or tissues in the mouse hearing organ are relatively small and vulnerable to external stimuli, rendering sample preparation difficult. In this study, therefore, to establish indicators of the mechanical properties of OHCs in mice, such properties were measured by atomic force microscopy (AFM). METHODS: CBA/JNCrj strain male mice aged 10-12 weeks (25-30 g) were used. Cochleae were dissected out from the animal and both the basilar membrane and the organ of Corti were simultaneously unwrapped from the modiolus with forceps. Dissected coiled tissue was then incubated with an enzymatic digestion medium for 15 min. After digestion, OHCs were isolated by gently triturating the coiled tissue. Local mechanical properties of the OHCs were then measured by an indentation test using an AFM. RESULTS: Young's modulus and stiffness of the OHC in the apical turn of the mouse cochlea were 2.1+/-0.5 kPa and 4.4+/-1.2 mN/m, respectively. CONCLUSIONS: Young's modulus of the OHC in the apical turn of the cochlea in mice was roughly the same as that in the apical turn of the cochlea in guinea pigs; however, the stiffness of the former was about two times greater than that of the latter because the cell length of the former was shorter than that of the latter.     

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.     

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.     

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.     

Local mechanical properties of guinea pig outer hair cells measured by atomic force microscopy

In this study, mechanical properties of guinea pig outer hair cells (OHCs) were measured by atomic force microscopy (AFM). First, in order to confirm the availability of AFM for measurement of the mechanical properties of the OHC, Young's moduli of the OHCs measured in this study were converted into stiffnesses using a one-dimensional model of the cell and then compared with the values reported in the literature. Next, the difference in local mechanical properties of the OHC along the cell axis was measured. Finally, the relationship between Young's modulus in the middle region of the OHC and the cell length was evaluated. The results were as follows. (1) AFM is an adequate tool for the measurement of mechanical properties of the OHC. (2) Mechanical properties in the apical region of the OHC are a maximum of three times larger than those in the basal and middle regions of the cell. (3) Young's modulus in the middle region of a long OHC obtained from the apical turn of the cochlea and that of a short OHC obtained from the basal turn or the second turn are 2.0+/-0.81 kPa (n=10) and 3.7+/-0.96 kPa (n=10), respectively. In addition, it was found that Young's modulus decreases with an increase in the cell length.     

Role of class D L-type Ca2+ channels for cochlear morphology

Voltage-gated Ca(2+) channels formed by subunits (class D Ca(2+) channels) tightly regulate neurotransmitter release from cochlear inner hair cells (IHCs) by controlling the majority of depolarisation-induced Ca(2+) entry. We have recently shown that the absence of these channels can cause deafness and degeneration of outer hair cells (OHCs) and IHCs in alpha1D-deficient mice (alpha1D(-/-)) (Platzer et al., 2000. Cell 102, 89-97). We investigated the time-dependent patterns of degeneration during postnatal development in the alpha1D(-/-) mouse cochlea using light and electron microscopy. At postnatal day 3 (P3), electron microscopy revealed no morphological aberrations in sensory cells, in afferent as well as in efferent nerve endings. But at P7 we observed a beginning degeneration of afferent nerve fibres by electron microscopy. By P15, we found a loss of OHCs in apical turns but electron microscopy revealed no ultrastructural changes in IHCs and efferent axons as compared to C57 black control animals (C57BL). We demonstrated by serial ultrathin sectioning of 15 days old alpha1D(-/-) mice that intact efferent nerve fibres formed direct contacts with IHCs as the degeneration of afferent nerve fibres progressed. We also saw a notable degeneration of spiral ganglion cells at P15. By 8 months, nearly all spiral ganglion and sensory cells of the organ of Corti were absent. Random ultrathin sectioning gave the impression that synaptic bodies abundant in wild-type animals were absent in nearly all alpha1D(-/-) mice investigated. We conclude that besides presumably reduced synaptic bodies the absence of class D L-type Ca(2+) channels does not prevent morphological development of the cochlea until P3 but may cause cochlear degeneration thereafter. The observed pattern of degeneration involves afferent nerve fibres (P7) followed by cell bodies in the spiral ganglion (P15), OHCs (P15) and IHCs (after P15).     

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.     

Heterogeneity of phospholipase C in the cochlea of the guinea pig

In the present study, we investigated the presence of phosphatidylinositol-specific phospholipase C (PLC) isoforms in the cochlea of the guinea pig using Western blot analysis and immunocytochemistry. By Western blotting, PLCbeta1 and delta1 were expressed in the cochlear sensory epithelia (CSE) and PLCbeta1, gamma1 and delta1 were expressed in the cochlear lateral wall. By immunocytochemistry of the CSE, PLCbeta1-like immunoreactivity was mainly expressed in the outer hair cells (OHCs), but not in the inner hair cells (IHCs). PLCgamma1 and delta1 were expressed neither in the OHCs nor in the IHCs. In the cochlear lateral wall, PLCbeta1, delta1 and gamma1 were expressed in the stria vascularis and the spiral ligament. In addition, PLCbeta1, delta1 and gamma1 were also present in type I spiral ganglion cells. Based on these results, we discussed the function of these PLC isoforms in the cochlea.     

Inner hair cells isolation and morphological observation]

OBJECTIVE: To set up an effective technique for isolating the single inner hair cells (IHCs) and observe morphological features to distinguish IHCs from other hair cells (OHCs) in vitro. METHODS: Surface preparations contained rows of OHCs and IHCs were prepared from guinea pigs. The cluster and single IHCs were separated microsurgically combination with an enzymatic digestion. The clusters of IHCs were separated using two fine electrodes and transferred by a gentle suction using a glass micropipette. RESULTS: On the average, one guinea pig cochlear yielded approximately 30 to 50 viable solitary IHCs. The cell body of the IHCs was pear or flask shape. The nucleus was located in the central of the cell body, and the cytoplasm was filled with the structured and scattered rough granules. The stereocilia were seen in the most of IHCs (93 out of 98). The stereocilia were aligned, almost lineally or in a "C" shape, in one side on the surface of the cuticular plate. The tight neck and the angle between the cuticular plate and the axis of the cell were observed in some IHCs but not obvious in most cases, which might be due to the orientation of the cells. The length of isolated IHCs was ranged from 13 to 31 microns and with an average of (22.45 +/- 4.14) micron (mean +/- s, n = 98). The diameter of IHCs was ranged from 7 to 15 microns and with an average of (11.95 +/- 1.59) micron. The length of stereocilia was ranged from 2 to 7.5 microns and with an average of (5.21 +/- 1.00) micron. CONCLUSION: Unambiguous solitary IHCs were successfully harvested from guinea pig cochlea using a microsurgical technique. The tight neck and the angle between the cuticular plate and the axis of the IHCs have been considered as an important landmark to distinguish the IHCs from OHCs.     

Detection and localization of heat shock protein 70 in the normal guinea pig cochlea

Normal guinea pig cochlear sections were treated with an antibody against heat shock protein (HSP70). HSP70-like immunoreactivity was observed in Claudius' cells and in the interdental cells of the spiral limbus. In the organ of Corti, immunoreactivity was confined to pillar cells, as well as Hensen's and Deiters' cells.     

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.     

顺铂中毒后豚鼠耳蜗电位变化的特征及形态学实验观察

目的 观察顺铂对耳蜗微音电位（ＣＭ）、总和电位（ＳＰ）及复合动作电位（ＣＡＰ）的影响及毛细胞形态学改变。方法 用人工外淋巴液和顺铂灌流豚鼠耳蜗，分别记录耳蜗第三回中阶的ＣＭ、－ＳＰ及ＣＡＰ，琥珀酸脱氢酶染色观察毛细胞的数量变化，透射电镜观察外毛细胞结构。结果 顺铂灌流１ｈ：≤６０ｄＢ ＳＰＬ声强级刺激时ＣＭ、－ＳＰ和ＣＡＰ幅度均较灌流顺铂前略下降，≥７０ｄＢ ＳＰＬ声强级刺激时幅度比灌流顺铂前明显     

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.     

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.     

不同龄CBA/J 小鼠耳蜗毛细胞的自然衰退

目的 为了观察CBA/J小鼠耳蜗毛细胞的自然退化现象，方法 本文采用了耳蜗铺片技术配合耳蜗毛细胞图直观方法对出生后不同月龄小鼠内外毛细胞作了形态学定量分析。结果 从出生6个月开始，随着月龄增加，CBA/J小鼠的内外毛细胞缺失从底回逐渐向顶回发展，以外毛细胞为最明显。结论 本实验证实了CBA/J小鼠耳蜗毛细胞存在自然退化现象，并有一定的规律。为临床研究耳蜗遗传性疾病提出 可靠的对照依据。     

Salicylate-induced morphological changes of isolated inner hair cells and outer hair cells from guinea-pig cochlea

Objectives

The aim of this study is to investigate the salicylate-induced morphological changes of cochlear inner hair cells (IHCs) and outer hair cells (OHCs).

Methods

IHCs and OHCs were acutely isolated from the guinea-pig cochlea. Cells were observed under the inverted microscope and 10 mmol/L sodium salicylate solutions or 0.01 mmol/L dexamethasone-plus-salicylate solutions were applied. The cell length or the ratio between the length and width was the indices of the morphological changes in cells.

Results

Isolated IHCs did not demonstrate any significant changes in sodium salicylate solutions in 20 min and in 40 min, whereas OHCs were shortened by the 10 mmol/L sodium salicylate to 83% in 20 min and 75% in 40 min. There were no significant differences between in the dexamethasone-plus-salicylate solutions and in the control solutions after 20 min and 40 min both in IHCs and OHCs.

Conclusions

Although salicylate affected the isolated OHCs from guinea-pig cochlea, IHCs were not changed morphologically by sodium salicylate applications. Dexamethasone inhibited the salicylate-induced morphological changes of OHCs.     

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.     

ATP and Nitric Oxide Modulate Intracellular Calcium in Isolated Pillar Cells of the Guinea Pig Cochlea

Supporting cells in the mammalian cochlea have recently received attention as potential targets of neurotransmitters, neuromodulators, and neurohumoral agents. Calcium homeostasis in Deiters' and Hensen's cells, for example, is regulated by ATP and nitric oxide. We studied the intracellular calcium concentration [Ca2+]i in isolated pillar cells of the guinea pig cochlea in response to extracellular ATP and nitric oxide using the fluorescent indicator fluo-3. [Ca2+]i increased rapidly and significantly throughout the pillar cell in response to a bolus of ATP or 2-methylthio ATP while a, b-methylene ATP was ineffective. The response to ATP was inhibited by suramin and Cibacron Blue but not by pyridoxal phosphate 6-azopheny1-2',4'-disulfonic acid. This pharmacological profile is consistent with a [Ca2+]i increase largely mediated by P2Y receptors. In Ca2+-free medium supplemented with EGTA, the response to extracellular ATP was reduced by 33%, suggesting a contribution of calcium influx to the overall effect. The ATP-induced increase of [Ca2+]i was attenuated by NO donors (sodium nitroprusside or diethylamine NONOate), and this attenuation was reversed by KT5823, an antagonist to protein kinase G. The results indicate the involvement of purinergic mechanisms and the nitric oxide/cyclic GMP/protein kinase G pathway in the regulation of [Ca2+]i in cochlear pillar cells.