Cytochemical localization of Na+/K+-ATPase activity in cochlear strial marginal cells after various catecholamine administrations

Sodium/potassium-activated adenosine triphosphatase (Na+/K+-ATPase) activity in the kidney and brain is high, and is regulated by catecholamines. Na+/K+-ATPase activity is also high in the basolateral infoldings of the strial marginal cells, where it aids in maintaining the characteristic electrolyte composition of the endolymph. To clarify the involvement of humoral control in strial function, particularly the role of catecholamines, the K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of strial marginal cells was investigated in guinea pigs using a cerium-based cytochemical method. The effects of reserpine, serotonin (5-HT), norepinephrine (NE), epinephrine (EP), both alone and in combination, were studied. High doses of reserpine cause depletion of sympathetic substances. Strial K+-NPPase activity was decreased after reserpine or dopamine treatment, and was increased after 5-HT, NE, and EP treatment. After reserpinization, repeated treatment with 5-HT, NE, or EP led to detectable strial enzyme activity. Thus, exogenous 5-HT, NE, and EP were able to restore strial K+-NPPase activity in the reserpine-treated animals. These results suggested that biogenic amines regulate strial K+-NPPase activity. Thus, the function of the stria vascularis may be regulated by the opposing actions of these catecholamines, and 5-HT.     

Cellular localization of facilitated glucose transporter 1 (GLUT-1) in the cochlear stria vascularis: its possible contribution to the transcellular glucose pathway

Immunoreactivity for the facilitated glucose transporter 1 (GLUT-1) has been found in the cochlear stria vascularis, but whether the strial marginal cells are immunopositive for GLUT-1 remains uncertain. To determine the cellular localization of GLUT-1 and to clarify the glucose pathway in the stria vascularis of rats and guinea pigs, immunohistochemistry was performed on sections, dissociated cells, and whole-tissue preparations. Immunoreactivity for GLUT-1 in sections was observed in the basal side of the strial tissue and in capillaries in both rats and guinea pigs. However, the distribution of the positive signals within the guinea pig strial tissue was more diffuse than that in rats. Immunostaining of dissociated guinea pig strial cells revealed GLUT-1 in the basal cells and capillary endothelial cells, but not in the marginal cells. These results indicated that GLUT-1 was not expressed in the marginal cells, and that another isoform of GLUT was probably expressed in these cells. Three-dimensional observation of whole-tissue preparations demonstrated that cytoplasmic prolongations from basal cells extended upward to the apical surface of the stria vascularis from rats and guinea pigs, and that the marginal cells were surrounded by these protrusions. We speculate that these upward extensions of basal cells have been interpreted as basal infoldings of marginal cells in previous reports from other groups. The three-dimensional relationship between marginal cells and basal cells might contribute to the transcellular glucose pathway from perilymph to intrastrial space. This study was supported by a grant-in-aid for scientific research (19570058) from The Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Another role for melanocytes: their importance for normal stria vascularis development in the mammalian inner ear

The stria vascularis of the mammalian cochlea is composed primarily of three types of cells. Marginal cells line the lumen of the cochlear duct and are of epithelial origin. Basal cells also form a continuous layer and they may be mesodermal or derived from the neural crest. Intermediate cells are melanocyte-like cells, presumably derived from the neural crest, and are scattered between the marginal and basal cell layers. The marginal cells form extensive interdigitations with the basal and intermediate cells in the normal adult stria. The stria also contains a rich supply of blood vessels. We investigated the role of melanocytes in the stria vascularis by studying its development in a mouse mutant, viable dominant spotting, which is known to have a primary neural crest defect leading to an absence of recognisable melanocytes in the skin. Melanocytes were not found in the stria of most of the mutants examined, and from about 6 days of age onwards a reduced amount of interdigitation amongst the cells of the stria was observed. These ultrastructural anomalies were associated with strial dysfunction. In the normal adult mammal, the stria produces an endocochlear potential (EP), a resting dc potential in the endolymph in the cochlear duct, which in mice is normally about +100 mV. In our control mice, EP rose to adult levels between 6 and 16 days after birth. In most of the mutants we studied, EP was close to zero at all ages from 6 to 20 days. Melanocyte-like cells appear to be vital for normal stria vascularis development and function. They may be necessary to facilitate the normal process of interdigitation between marginal and basal cell processes at a particular stage during development, and the lack of adequate interdigitation in the mutants may be the cause of their strial dysfunction. Alternatively, melanocytes may have some direct, essential role in the production of an EP by the stria. Melanocytes may be important both for normal strial development and for the production of the EP. We believe this is the clearest demonstration yet of a role for migratory melanocytes other than their role in pigmentation.     

Immunological identification of an inward rectifier K+ channel (Kir4.1) in the intermediate cell (melanocyte) of the cochlear stria vascularis of gerbils and rats

The cochlear stria vascularis produces the positive endocochlear potential (EP) and the endolymph. Both the EP and the endolymph are essential for the physiological function of hair cells. The intermediate cell is one of several cell types constituting the stria vascularis. It is known that inward rectifier K+ channels can play a constitutive role in the determination of the resting membrane potential. Localization of a member of the inward rectifier K+ channel family, Kir4.1, in the stria vascularis of gerbils and rats was investigated by immunological methods. A polyclonal antibody specific to the C-terminus of the rat Kir4.1 channel was raised in rabbits. Immunostaining of dissociated cells revealed that the Kir4.1 channel was localized to the intermediate cell, but not to the epithelial marginal cell. Subcellular localization of the Kir4.1 channel to the plasma membrane of the intermediate cell was confirmed by immunoelectron microscopy. Immunostaining of whole-tissue preparations revealed a network-like structure composed of intermediate cells. It seems likely that the Kir4.1 channel mediates the inwardly rectifying K+ current in the intermediate cell as shown previously by electrophysiological methods, and that this channel plays key roles in the production of the EP and K+ transport in the stria vascularis.     

Immunolocalization of P2Y4 and P2Y2 Purinergic Receptors in Strial Marginal Cells and Vestibular Dark Cells

K+ secretion by strial marginal cell and vestibular dark cell epithelia is regulated by UTP and ATP at both the apical and basolateral membranes, suggesting control by P2Y2 and/or P2Y4 purinergic receptors. Immunolocalization was used to determine the identity and distribution of these putative receptors. Membrane proteins from gerbil brain, gerbil vestibular labyrinth and gerbil stria vascularis were isolated and analyzed by Western blot. P2Y2 antibody stained one band at 42 kDa for each tissue, whereas P2Y4 antibody stained 3 bands on gerbil brain (75, 55 and 36 kDa), one band on gerbil stria vascularis (55 kDa) and two bands on vestibular labyrinth (42 and 56 kDa). All bands were absent when the antibodies were blocked with their respective antigenic peptide. P2Y4 was immunolocalized by fluorescence confocal microscopy to only the apical membrane of strial marginal cells and vestibular dark cells and was similar to apical immunostaining of KCNE1 in the same cells. By contrast, P2Y2 was observed on the basolateral but not the apical membrane of dark cells. Similarly, in the stria vascularis P2Y2 was observed in the basolateral region but not the apical membrane of marginal cells. Additional staining was observed in the spiral ligament underlying the stria vascularis. These findings identify the molecular bases of the regulation of K+ secretion by apical and basolateral UTP in the inner ear.     

Acute ischemia causes 'dark cell' change of strial marginal cells in gerbil cochlea

The cochlear stria vascularis produces the endolymph and generates the endocochlear DC potential, two indispensable ingredients of an auditory transduction process. The marginal cell, one of the several cell types constituting the stria vascularis, is called 'the dark cell' on the basis of its appearance by transmission electron microscopy (TEM). To clarify whether this commonly observed 'dark appearance' is a normal characteristic of marginal cells, as conjectured in the literature, or an experimental artifact, we developed an in vivo fixation method for minimizing ischemic tissue damages. While under sustained systemic circulation with oxygenated blood, the stria vascularis of gerbils was chemically fixed by perilymphatic perfusion with a fixative, and the stria vascularis was observed by TEM. In contrast to a number of previous reports, the cytoplasm of marginal cells was not dark, and quantitative analysis showed that the difference between the cytoplasmic electron density of marginal cells and that of intermediate cells (another type of strial cells) was not statistically significant. For comparison, the gerbils were allowed to undergo 3 min of ischemia following decapitation. Under these conditions, marginal cells showed typical 'dark appearance', as reported previously, and their cytoplasmic electron density was 1.7 times higher than that of the intermediate cells. In addition, the volume of mitochondria in marginal cells undergoing 3 min of ischemia was higher than that fixed in vivo. We therefore conclude that the widely recognized 'dark cell' appearance of marginal cells following conventional fixation procedures reflects cell injury due to ischemia, which is inherent in the standard fixation procedures, but can be avoided by our fixation protocol here introduced.     

Impaired stria vascularis integrity upon loss of E-cadherin in basal cells

In the cochlea, sensory transduction depends on the endocochlear potential (EP) and the unique composition of the endolymph, both of which are maintained by a highly specialized epithelium at the cochlear lateral wall, the stria vascularis. The generation of the EP by the stria vascularis, in turn, relies on the insulation of an intrastrial extracellular compartment by epithelial basal cells. Despite the physiological importance of basal cells, their cellular origin and the molecular pathways that lead to their differentiation are unclear. Here, we show by genetic lineage tracing in the mouse that basal cells exclusively derive from the otic mesenchyme. Conditional deletion of E-cadherin in the otic mesenchyme and its descendants does not abrogate the transition from mesenchymal precursors to epithelial basal cells. Rather, dedifferentiation of intermediate cells, altered morphology of basal and marginal cells and hearing impairment due to decreased EP in E-cadherin mutant mice demonstrate an essential role of E-cadherin in terminal basal cell differentiation and their interaction with other strial cell types to establish and maintain the functional architecture of the stria vascularis.     

Hyperpigmentation of Chinchilla Stria Vascularis Following Acoustic Trauma

This report describes morphological alterations of the chinchilla stria vascularis seen 30 days after exposure to impulse noise. The observed changes included a dramatic increase in strial melanin content which occurred in 7 of 36 animals exposed to electronically synthesized impulses presented in various temporal patterns at either 135 or 150 dB peak SPL. In these animals, densely pigmented areas of stria 1.5 to 3 mm in length were found in the basal cochlear turn. Light and electron microscopic study revealed that these areas contained large numbers of melanin granules situated primarily in pale-staining cells of the middle layer of the stria. Unlike the pigment granules present in normal chinchilla stria, the melanosomes found in the noise-exposed material clearly showed ultrastructural features characteristic of eumelanin. Melanin granules were also observed in marginal and basal cells of the noise-exposed stria. In some cases, pigment granules which had apparently been expelled from the marginal cells were present in the endolymphatic space beneath Reissner's membrane and on the strial surface. These findings support the view that the melanin-bearing cells of the inner ear are capable of markedly increased activity in response to stressful conditions.     

Hyperpigmentation of chinchilla stria vascularis following acoustic trauma.

This report describes morphological alterations of the chinchilla stria vascularis seen 30 days after exposure to impulse noise. The observed changes included a dramatic increase in strial melanin content which occurred in 7 of 36 animals exposed to electronically synthesized impluses presented in various temporal patterns at either 135 or 150 dB peak SPL. In these animals, densely pigmented areas of stria 1.5 to 3 mm in length were found in the basal cochlear turn. Light and electron microscopic study revealed that these areas contained large numbers of melanin granules situated primarily in pale-staining cells of the middle layer of the stria. Unlike the pigment granules present in normal chinchilla stria, the melanosomes found in the noise-exposed material clearly showed ultrastructural features characteristic of eumelanin. Melanin granules were also observed in marginal and basal cells of the noise-exposed stria. In some cases, pigment granules which had apparently been expelled from the marginal cells were present in the endolymphatic space beneath Reissner's membrane and on the strial surface. These findings support the view that the melanin-bearing cells of the inner ear are capable of markedly increased activity in response to stressful conditions.     

内耳免疫反应诱导Fas和FasL表达与凋亡的关系

目的研究内耳免疫反应过程中是否存在细胞凋亡,以及细胞凋亡是否与Fas和FasL信号转导有关.方法选用雌性白色豚鼠16只,随机分为实验组和对照组各8只,以钥孔虫戚血蓝蛋白(keyhole limpet hemocyanin,KLH)全身免疫后,实验组以相同抗原进行内耳免疫,对照组内耳注射等量的磷酸盐缓冲生理盐水(phosphate buffered saline,PBS),在内耳免疫5d后处死动物,取内耳免疫侧耳蜗做石蜡切片.通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(terminal-deoxynucleotidyl transferase mediated nick end labeling,TUNEL)检测内耳凋亡细胞,免疫组化检测内耳Fas和FasL的表达.结果实验组豚鼠内耳Corti器毛细胞,血管纹的缘细胞和螺旋神经节细胞存在TUNEL染色阳性细胞,而对照组动物切片仅在支持细胞、血管纹和螺旋神经节细胞中发现极少数TUNEL染色阳性细胞.免疫组化染色实验组Corti器、螺旋神经节细胞、血管纹和螺旋韧带Fas和FasL蛋白表达阳性,而对照组只有螺旋神经节细胞和血管纹有较弱的Fas蛋白表达,FasL蛋白表达阴性.结论内耳免疫反应可诱导细胞凋亡的发生,Fas-FasL途径是参与此过程重要的信号转导途径之一.     

Isolation,cultivation, and characterization of primary bovine cochlear pericytes: A new in vitro model of stria vascularis

The study of strial pericytes has gained great interest as they are pivotal for the physiology of stria vascularis. To provide an easily accessible in vitro model, here we described a growth medium-based approach to obtain and cultivate primary bovine cochlear pericytes (BCP) from the stria vascularis of explanted bovine cochleae. We obtained high-quality pericytes in 8–10 days with a > 90% purity after the second passage. Immunocytochemical analysis showed a homogeneous population of cells expressing typical pericyte markers, such as neural/glial antigen 2 (NG2), platelet-derived growth factor receptorβ (PDGFRβ), α-smooth muscle actin (α-SMA), and negative for the endothelial marker von Willebrand factor. When challenged with tumor necrosis factor or lipopolysaccharide, BCP changed their shape, similarly to human retinal pericytes (HRPC). The sensitivity of BCP to ototoxic drugs was evaluated by challenging with cisplatin or gentamicin for 48 hr. Compared to human retinal endothelial cells and HRPC, cell viability of BCP was significantly lower ( p < 0.05) after the treatment with gentamicin or cisplatin. These data indicate that our protocol provides a simple and reliable method to obtain highly pure strial BCP. Furthermore, BCP are suitable to assess the safety profile of molecules which supposedly exert ototoxic activity, and may represent a valid alternative to in vivo tests.     

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current “two-cell model” of the stria vascularis.     

Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique

We have made several improvements in the method of fixation of the inner ear and the enzyme-histochemical technique for carbonic anhydrase (CA) detection. The results confirmed that CA is localized in the hair cells of the organ of Corti, Deiters' cells or nerve endings, inner pillar cells, Boettcher's cells, stria vascularis, spiral ligament, spiral limbus, and spiral ganglion cells. These results generally agree with previous histochemical observations but showed some differences. Our method preserved tissue morphology and showed more detailed localization of CA activity in the inner ear. In particular, the marginal zone of stria vascularis and the epithelial cells of spiral prominence, facing the endolymph, showed no CA activity, while the suprastrial region of the spiral ligament and the supralimbal region of the spiral limbus, juxtaposed to the perilymph, showed CA activity. In outer hair cells, the cuticular plate, which faces the endolymph showed CA activity, but the lateral membrane, which faces the perilymph showed no CA activity. In contrast, the inner hair cell cytoplasm showed diffuse CA activity. These results will be useful in considering ion exchange between endolymph and its adjacent cells, and between perilymph and its adjacent structures.     

A Study of the Histologic and Enzyme Histochemical Changes in the Cochlea of Guinea Pigs After Non-Focused Ultrasound Irradiation

We evaluated the histologic and histochemical (succinate dehydrogenase, SDH) changes in cochleas of guinea pigs after non-focused ultrasound (NFU) irradiation. For this purpose, the cochleas of 50 guinea pigs (both ears in Groups 1–10) were irradiated at 2.5–8.0 MHz NFU for 6 h and the histologic/histochemical changes were determined. Our data show that after NFU irradiation for 6 h, no histological changes were observed in the cochleas by silver staining and scanning electron microscopic examination; however, the SDH activity of the basilar membranes and stria vascularises at the corresponding position decreased significantly. The SDH values of basilar membranes and stria vascularis were higher at 8 h than at 30 min after the NFU irradiation. SDH activity of the outer hair cells presented more declination than that of the inner hair cells. It was, therefore, concluded that a certain dosage of NFU irradiation at various frequencies could lead to metabolic changes in the basilar membrane and stria vascularis at different areas of cochlea. Moreover, these changes were found to be reversible or partially reversible. These changes also suggest that the cochlear hair cells located at different areas might be related to ultrasonic perception.     

电码针对庆大霉素耳中毒的防治作用及其机制的研究

目的：观察电码针对豚鼠庆大霉素（GE）耳毒性的防治作用,方法：测定脑干听觉诱发电位（BAEP）和用组织化学方法测定耳蝇毛细胞及血管纹的琥珀酸脱氢酶（SDH）。结果：电针能降低CE引起的BAEP反应阈的上升幅度,缩小BAEP波峰潜伏期及波峰间期的延长;能保护毛细胞及耳蜗血管纹细胞线粒体呼吸酶的活性。结论：电针能降低GE5的耳毒性,保护毛细胞及耳蜗血管纹细胞线粒体酶的活性。保证这些细胞能量代谢,维持细胞所需要能量的各种功能的活动。减少细胞的损伤,可能有是电针防治GE耳毒性的机制之一。     

Independence of the Endovestibular Potential in Homeotherms

The endolymphatic potential was recorded from various vestibular parts of the labyrinth from which the cochlea (in the case of guinea pigs) or the cochlea, lagena, and sacculus (in the case of pigeons) had been removed. This endovestibular potential of the isolated vestibule declined during anoxia and recovered after anoxia in the same manner as the endovestibular potential of the intact labyrinth. Its non-anoxic level was the same as in the intact labyrinth; i.e., +5 to +8 mv in the pigeon and +2 to +5 mv in the guinea pig. It is, therefore, concluded that the endovestibular potential is independent of the cochlea, stria vascularis, and endocochlear potential.     

微波对豚鼠耳蜗微循环的影响

本文对７４只豚鼠,通过颈静脉注入大剂量的速尿,建立了豚鼠急性耳蜗微循环障碍动物模型。利用动态观测手段中的激光多普勒测试技术及静态观察方法的螺旋韧带血管纹红细胞计数技术,探讨了微波对成年豚鼠耳蜗微循环的保护作用。为保护和改善动物的听力水平提供更多的资料。     

线粒体毒素诱导突发性耳聋模型血管纹损伤机制的研究

目的研究线粒体毒素诱导豚鼠突发性耳聋模型血管纹损伤的机制。方法20只杂色豚鼠随机分为3-硝基丙酸（3-NP）组和磷酸盐缓冲液（PBS）对照组,分别经圆窗膜给予0.3 mmol/L 3-NP或PBS 10min。分别检测两组豚鼠的听性脑干反应测听（ABR）,血管纹丙二醛（MDA）、超氧化物歧化酶（SOD）及边缘细胞超微结构变化。结果与PBS对照组相比较,3-NP组ABR阈移增大、幅值减小;血管纹MDA含量增高（P〈0.05）,SOD活性降低（P〈0.05）;边缘细胞超微结构改变,尤以线粒体肿胀,空泡变最为显著。结论线粒体毒素通过氧化应激反应损伤突发性耳聋豚鼠的血管纹。     

Sound needs sound melanocytes to be heard

Intermediate cells in the stria vascularis of the mammalian cochlea are melanocytes, which contain melanin pigments and are capable of synthesizing melanin. These melanocytes are required for normal development of the cochlea, as evidenced by studies of mutant mice with congenital melanocyte anomalies. Melanocytes are also needed for developed cochleae to function normally, as evidenced by studies of mutant mice with late-onset melanocyte anomaly and humans with acquired melanocyte anomaly. Melanin, per se, does not seem to be essential for normal hearing function, but it may protect against traumata to the cochlea, e.g., noise and ototoxic aminoglycosides. Recent electrophysiological studies have revealed that strial intermediate cells are provided with specific ionic channels, such as inwardly rectifying K+ channels (Kir4.1) and voltage-dependent outwardly rectifying K+ channels. These channels may play central roles in strial function and thus in normal hearing.