The presence of laminin in the fetal human inner ear

Summary The expression of laminin was analyzed in the human fetal inner ear using immunohistochemical methods. In the 11-week-old human fetus, the presence of laminin was found in the basement membrane of the immature cochlea, endolymphatic sac and vestibular end organs. The reaction of the basement membrane of the endolymphatic sac was strong in the 15-week-old human fetus. A laminin reaction was seen in the cochlea, Reissner's membrane, epithelial cells of the limbus spiralis, the basilar membrane and the stria vascularis. In particular, the capillaries and basement membrane of the stria vascularis were strongly positive. These results suggest that laminin may be an essential component in the development of the inner ear and may possibly be related to filtration of the endolymph.     

Effect of furosemide on basal lamina anionic sites in guinea pig labyrinth

The authors studied the effects of acute furosemide administration on the basal lamina (BL) anionic sites in the stria vascularis, ampullar crista, and endolymphatic sac by using cationic polyethyleneimine (PEI). Furosemide was intravenously administered to albino guinea pigs with normal Preyer's reflexes. After 20 minutes, the bony labyrinth was removed and processed for histologic evaluation. Under a transmission electron microscope, a marked enlargement of the intercellular spaces was observed in the stria vascularis. The PEI distribution decreased significantly on the capillary BL in the stria vascularis and on the subepithelial BL in the sensory, transitional, and dark cell areas. However, no significant change was observed on the capillary BL or the subepithelial BL in the endolymphatic sac. These findings suggest that acute furosemide administration severely alters the distribution of the anionic sites in the strial capillary BL and in the subepithelial BL in the ampullar crista, but not in the capillary BL or the subepithelial BL of the endolymphatic sac.     

Ultrastructure of the stria vascularis and Reissner's membrane in experimental hydrops

A time-sequence study was made of the early ultrastructural changes of the stria vascularis and Reissner's membrane in the guinea pig after obliteration of the endolymphatic sac and duct. Pathological alterations of both the stria vascularis and Reissner's membrane were found to start in the apex of the cochlea. The morphological changes of the stria vascularis were characterized by an increase of vesicles in the marginal cells and by intercellular edema, followed by vacuolization and atrophy of marginal and intermediate cells. In Reissner's membrane extensive gaps in the mesothelial cell layer were observed together with intracellular pathology of the epithelial cells. The significance of these ultrastructural changes in the stria vascularis and Reissner's membrane with regard to the pathophysiology of the endolymphatic hydrops is discussed.     

水通道蛋白-1,3在豚鼠耳蜗及内淋巴囊的表达

目的:检测水通道蛋白(AQP)-1,3在豚鼠耳蜗及内淋巴囊中的表达情况。方法:运用免疫组织化学二步法及免疫荧光染色检测豚鼠耳蜗及内淋巴囊中AQP-1,3的表达。结果:AQP-1主要表达于耳蜗螺旋韧带基底部、Corti器基底膜及鼓阶内侧上皮面及内淋巴囊上皮细胞下的基质组织中;AQP-3主要表达于血管纹、螺旋韧带、Corti器、螺旋神经节细胞及内淋巴囊的上皮细胞及其下的基质成分中。结论:AQP-1,3在豚鼠耳蜗及内淋巴囊中存在广泛表达,但其功能仍未明确。     

豚鼠耳蜗和内淋巴囊水通道蛋白的表达

目的 研究豚鼠耳蜗和内淋巴囊组织中水通道蛋白(aquaporin，AQP)不同亚型的定位表达及其意义。方法 用兔抗大鼠AQP1、AQP2、AQP3、AQP4的多克隆抗体，采用免疫组化SP法分别检测相应AQP蛋白亚型在豚鼠耳蜗和内淋巴囊组织中的表达模式。结果 在耳蜗组织中，AQP1、4广泛分布于耳蜗的各个区域，如血管纹、螺旋韧带、Corti器、螺旋缘、螺旋神经节等，AQP3除了在血管纹表达呈弱阳性外，其余区域的表达与AQP1和AQP4相似，AQP2则仅表达于Reissner膜。在内淋巴囊组织中，AQP1、AQP3和AQP4在内淋巴囊上皮细胞和上皮下纤维组织均呈强阳性表达，只是AQP3的表达强度稍弱于AQP1和AQP4，而AQP2在内淋巴囊上皮细胞和上皮下组织均呈阴性表达。结论 水通道蛋白1、3、4以相似的方式广泛分布于豚鼠耳蜗和内淋巴囊组织中，而AQP2则仅表达于Reissner膜，提示不同亚型的AQP可能在不同区域协同作用参与内淋巴的调节，从而保持内耳内环境的稳定。     

正常豚鼠内耳水通道蛋白的表达及意义

目的：检测正常豚鼠内耳组织中水通道蛋白（aquaporins,AQPs）的表达,探讨其在内耳液体平衡中的意义.方法：用免疫组织化学方法,以兔抗大鼠AQP0、1、2、3、5、7、8的多克隆抗体,检测正常豚鼠内耳组织中水通道蛋白亚型0、1、2、3、5、7、8的表达.结果：水通道蛋白亚型0、1、2、3、5、7、8在豚鼠内耳有不同程度、不同模式的表达,其中AQP0仅在血管纹上皮细胞、螺旋神经节细胞有较弱的表达,AQP1的分布见于包绕骨迷路、内淋巴囊、内淋巴管的纤维细胞,基底膜鼓阶面细胞、螺旋韧带纤维细胞、螺旋缘纤维细胞、Corti器、内外螺旋沟、血管纹、椭圆囊壁、球囊壁、螺旋神经节细胞等.AQP2表达在血管纹、Corti器、螺旋神经节细胞和内淋巴囊中.AQP3、7、8的分布类似,在螺旋神经节和包绕膜迷路的组织中均有表达,其中Corti器、内外螺旋沟、血管纹、螺旋神经节表达较强,在螺旋韧带、螺旋缘纤维细胞表达较弱.AQP5则在Corti器、内外螺旋沟、螺旋神经节细胞表达较强,在螺旋韧带纤维细胞表达稍弱.结论：在正常豚鼠内耳中,尤其是膜迷路中有多种水通道蛋白亚型,以不同的方式表达,他们可能在维持膜迷路液体平衡中起着协同作用.     

水通道蛋白-2在大鼠内耳的定位及其意义

目的：探讨水通道蛋白-2(Aqp-2)在大鼠内耳中的定位，分析其在内耳水代谢中的作用机制。方法：使用正常成年SD大鼠15只，制备颞骨组织标本，采用SP免疫组织化学技术，检测Aqp-2蛋白在大鼠内耳的表达情况。结果：Aqp-2在大鼠的内淋巴囊、血管纹、螺旋神经节有较强表达，在Corti器、基底膜、螺旋缘的前庭唇和鼓唇、盖膜、螺旋凸也有表达。结论：Aqp-2主要分布在与内淋巴代谢有关的结构(内淋巴囊和血管纹)，它在Corti器的表达为膜迷路积水时伴有的听力下降提供了又一解释；Aqp-2在螺旋神经节的表达，提示它可能与正常听觉的维持有关。     

Presence of plasma membrane-bound Ca(2+)-ATPase in the secretory epithelia of the inner ear.

The Ca2+ concentration of the endolymph is low, around 0.023 mM. Yet, because of the positive endocohlear potential, Ca2+ must be actively transported into the endolymphatic space. The mechanisms responsible for the active Ca2+ transport into the endolymph are not known. In this study, the presence of plasma membrane-bound Ca(2+)-ATPase (PMCA ATPase) in the endolymph-producing, secretory epithelia of the inner ear from guinea pig was investigated with immunoblotting and immunohistochemistry. The antibody used was a monoclonal antibody which recognizes an epitope shared by all four known isoforms of PMCA ATPase. With immunoblotting, a band corresponding to PMCA ATPase was found in the stria vascularis, the ampullary tissue, the utricle and the endolymphatic sac in assays from at least three different batches of tissue. With immunohistochemistry, a strong positive staining reaction for PMCA ATPase could be seen in the stria vascularis and the dark cells of the ampullary tissue and the utricle. The epithelial cells in the endolymphatic sac showed a moderate positive staining reaction. Accordingly, in this study the presence of PMCA ATPase was shown in all the endolymph-producing, secretory epithelia of the inner ear. These results indicate that PMCA ATPase plays a role in the regulation of the Ca2+ concentration in the endolymph.     

Expression of aquaporins in the cochlea and endolymphatic sac of guinea pig

It has been shown that the aquaporin (AQP) family forms membrane pores selectively permeable for water and some small solutes such as glycerol and urea, and thus plays important roles in regulating the fluid in many organs involved in fluid transport such as kidney, lung and brain. The aims of the present study were to investigate the cellular localization and its significance of aquaporins (AQPs) in various subregions of the cochlea and endolymphatic sac of guinea pig. The expression patterns of AQP1, 2, 3 and 4 were immunolabeled with the specific polyclonal rabbit antibodies against the rat AQP1, 2, 3 and 4. Our immunohistochemical examination showed that in the cochlea, AQP1, 3 and 4 were widely distributed in various locations including stria vascularis, spiral ligament, the organ of Corti and spiral ganglion in the similar patterns except that AQP3 in the stria vascularis was lightly weaker than AQP1 and AQP4. AQP2 was labeled only in Reissner's membrane. In the endolymphatic sac, AQP1, AQP3 and AQP4 were strongly expressed in the epithelial cells and subepithelial cells similarly with the exception that AQP3 was lightly weaker than AQP1 and AQP4. No AQP2 immunoreactivity was detected in the endolymphatic sac. Theses results suggest that different members of the AQP family in the labyrinth may work in concert to regulate endolymph and to maintain homeostasis in the inner ear.     

Ultrastructure of the endolymphatic sac in two-phase endolymphatic hydrops in the guinea pig.

Two-phase endolymphatic hydrops is a subtle experimental model for Meniere's disease. Chronic dysfunction of the endolymphatic sac, induced by dissection of the most distal part without causing damage to the intermediate part, is combined with increased endolymph production induced by administration of aldosterone which stimulates the N/K-ATPase in the stria vascularis. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea pig cochleas: controls: non-operated aldosterone-treated cochleas; operated (dissection of the endolymphatic sac) cochleas; operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone treatment had no visible effect. Dissected ears revealed severe deviations. The epithelium of the intermediate sac was low, showed dilated lateral intercellular spaces indicating elevated fluid transport and displayed serious degenerative processes. Distally, the endolymphatic sac was completely blocked by newly formed bone. Additional aldosterone treatment had no cumulative effect on the dissected ears.     

Ultrastructure of the Endolymphatic Sac in Two-phase Endolymphatic Hydrops in the Guinea Pig

Two-phase endolymphatic hydrops is a subtle experimental model for Meniere's disease. Chronic dysfunction of the endolymphatic sac, induced by dissection of the most distal part without causing damage to the intermediate part, is combined with increased endolymph production induced by administration of aldosterone which stimulates the Na/K-ATPase in the stria vascularis. A transmission electron microscopic study was performed on the endolymphatic sacs of four groups of guinea pig cochleas: controls; non-operated aldosterone-treated cochleas; operated (dissection of the endolymphatic sac) cochleas; operated and aldosterone-treated cochleas. Light and electron microscopy showed a normal morphology in the controls. Aldosterone treatment had no visible effect. Dissected ears revealed severe deviations. The epithelium of the intermediate sac was low, showed dilated lateral intercellular spaces indicating elevated fluid transport and displayed serious degenerative processes. Distally, the endolymphatic sac was completely blocked by newly formed bone. Additional aldosterone treatment had no cumulative effect on the dissected ears.     

Cytokeratin diversity in epithelia of the human inner ear

The expression of cytokeratins (Cks) nos. 5, 7, the combination 5 and 8, 8, 10, 17, 18 and 19 was analysed in the inner ear of 14-24-week-old human fetuses, using 10 different well characterized monoclonal antibodies (mAbs). A complex pattern of Ck immunoreactivity was found. Cks 8 and 18 were identified in both cochlear and vestibular hair cells as well as in all other epithelia. Epithelia involved in fluid regulation (stria vascularis, Reissner's membrane and dark cell epithelium) revealed the same pattern of Ck immunoreactivity in spite of morphological differences in cell configuration. A specific Ck, no. 10 as detected with the mAbs RKSE 60, was found in the endolymphatic duct and sac. The expression of Cks in the human inner ear is in principle similar to that found in the labyrinth of other animal species, for instance in the mouse. However, the human inner ear had a more complex pattern than any inner ear so far known in animal species.     

Pathohistological study on active endolymphatic hydrops in guinea pig--with stress stimulations

Recently Meniere's disease is believed to show a pathological features of endolymphatic hydrops, but the etiology of this disease has not yet been ascertained. Many investigators tried to create animal models with endolymphatic hydrops by obliterating endolymphatic sac and duct. However, these methods have not been adequate to explain the mechanism of development of Meniere's disease, because it gives mechanical damage to destroy endolymphatic sac and duct. On the other hand, Meniere's disease is thought to develop even as a result of stress. The present paper reported the conduction of an acute experiment, creating active endolymphatic hydrops by stress load, to study the occurrence rate of endolymphatic hydrops and the pathological findings of inner ears by a light microscope. This was followed by a light and electron microscopic study on the changes in permeability of the cochlear lateral wall with horseradish peroxidase and a study of the development mechanism of endolymphatic hydrops. Results obtained were as follows: 1. Although it was impossible to cause endolymphatic hydrops with a single stimulation, four kinds of stress stimulations could cause endolymphatic hydrops with the rate of 37.8%. 2. Judging from the different pathological findings between the group with four kinds of stress stimulations and the untreated control group, vessel permeability in the stria vascularis was highly changeable, while that in the spiral ligament was not. 3. It was found out that increased permeability of the stria vascularis vessels was by increased pinocytotic vesicular transport and through tight junctions from vessel lumen to basal lamina and that it was presumably only by increased pinocytotic vesicular transport beyond basal lamina. 4. It was assumed that increased permeability of the stria vascularis vessels was one of the causes of endolymphatic hydrops which were believed to be pathological features of Meniere's disease.     

Detection of immunoglobulin in the inner ear tissue by PAP method

The distribution of immunoglobulins IgG, IgA and IgM in the inner ear tissue from a patient who died of lung bleeding followed after sepsis was studied, and also the normal guinea pig inner ears and the inner ear disorders induced by Kanamycin injection were studied for the distribution of IgG. The temporal bones were fixed in formaldehyde, decalcified in EDTA and embedded in paraffin. The PAP method was used for the demonstration of the immunoglobulins. In both the human inner ear tissue and the normal control inner ear tissue of the guinea pigs deposits of IgG were found in the sensory organs and the endolymphatic sac, however, in the stria vascularis was slight. The severe damaged inner ears induced by Kanamycin the remarkable decreased deposits of IgG were found in the cochlea, but in the endolymphatic sac the remarkable increased deposits of IgG were found. No IgA and IgM were found in the human inner ear tissue.     

Expression of caspase-activated deoxyribonuclease (CAD) and caspase 3 (CPP32) in the hydropic cochlea of guinea pigs – second report

Apoptosis was induced in the cochlea by the injection of keyhole limpet hemocyanin (KLH) into the endolymphatic sac of guinea pigs and immunohistochemically examined. Keyhole limpet hemocyanin was injected into the right endolymphatic sac. The temporal bones were fixed via cardiac infusion of fixative and immunohistochemically stained for caspase-activated deoxyribonuclease or caspase 3. Endolymphatic hydrops became evident in the cochlea 1 day after the injection of keyhole limpet hemocyanin (n=6). The temporal bones in the control group did not show any caspase-activated deoxyribonuclease or caspase 3 immunoreactivity (n=6). Immunoreactivity for caspase 3 was detected in the supporting cells of the organ of Corti, the stria vascularis and the spiral ganglion cells. Caspase-activated deoxyribonuclease was also detected in the same areas. These findings suggest that apoptosis is involved in the pathogenesis of endolymphatic hydrops. This phenomenon could lead to cochlear dysfunction, as seen in endolymphatic hydrops.     

Expression pattern of adenylyl cyclase isoforms in the inner ear of the rat by RT-PCR and immunochemical localization of calcineurin in the organ of Corti.

Most studies concerning adenylyl cyclases in the inner ear were carried out before the advent of molecular biology. In a PCR approach using cDNAs of six inner ear tissues (stria vascularis, endolymphatic sac, organ of Corti, vestibulum, cochlear and vestibular nerve) we found tissue specific expression of adenylyl cyclase isoforms. Adenylyl cyclases types 2 and 4 are predominant in the fluid controlling tissues, i.e. in the stria vascularis and endolymphatic sac. In the organ of Corti and vestibulum the Ca2+-modulated isoforms types 1, 6 and 9 were expressed. The regulation of adenylyl cyclase 9, which is the major isoform expressed in the organ of Corti, proceeds via the Ca2+-activated protein phosphatase 2B (calcineurin, PPP3). PCR with specific primers for calcineurin demonstrated its abundant expression in the organ of Corti. Using a monoclonal antibody we localized calcineurin immunochemically to the cochlear nerve, the nerve fibers and the inner hair cells. In the cochlear and vestibular nerves a characteristic neuronal expression pattern of adenylyl cyclase isoforms was observed, i.e. adenylyl cyclases types 2, 3 and 8. The functional consequences of the adenylyl cyclase expression pattern in the inner ear are discussed in conjunction with its unique sensory performance.     

Prenatal low-dose gamma irradiation of the inner ear induces changes in the expression of intermediate filaments

The expression of intermediate filaments (1F) was analysed in the inner ear in normally developed adult CBA/CBA mice and in mice of the same age which had been gamma irradiated in utero with a low dose 1-2 Gy single exposure. Well characterized monoclonal antibodies (mAbs) against all classes of intermediate filament proteins (cytokeratins-Cks, vimentin, neurofilaments, desmin and glial fibrillar acidic protein) were used. With the exception of neurofilament proteins, the expression of intermediate filament proteins was the same in adult normal and irradiated inner ears, irrespective of gestational age at exposure. A complex Ck pattern occurred in the various cell types comprising the membranous labyrinth. In spite of the differences in cell shape and internal organization of organelles, epithelia actively involved in inner ear fluid homeostasis (stria vascularis, dark cell epithelium, endolymphatic duct and sac) revealed, according to our mAbs, the same expression of Cks, except for the mouse counterpart of human Ck 7, which was found exclusively in the stria vascularis and the endolymphatic duct and sac. The pattern of intermediate filament composition in the labyrinth was the same in the mouse as in man. Irradiation on gestational days 12 or 13 (the otocyst stage)--but not at more advanced embryonic age--induced immunoreactivity for neurofilament proteins in vestibular hair cells (HC) and to a minor extent also in cochlear HC. No such positivity was found in the control material.     

The histologic study of the inner ear in guinea pigs on anaphylatoxin

Complement is known to relate to many inflammatory reactions. C4a, C3a and C5a, known as anaphylatoxins, are known to cause strong inflammatory reactions. In this study, the role of anaphylatoxins on the pathogenesis in the cochlea was examined. On hundred forty six male Harley guinea pigs, weighing about 350 grs, all susceptible to preyer's reflex, were used in this study. Anaphylatoxins were made from guinea pig serum treated with zymosan, and inoculated into the carotid artery of the guinea pigs. Parts of these animals were sacrificed and examined at ten minutes, one day, two days, three days, seven days, ten days and fifteen days after injection of anaphylatoxins. Pathological changes in inner ears were observed by light microscopy. After 10 minutes, inner ears were found morphologically normal. After one day, inner ears were found to be almost morphologically normal but the stria vascularis was observed with cystic formation. After two days, cystic formations in the stria vascularis were enlarged and Reissner's membranes were collapsed in some other animals. After three days, the stria vascularis in the various cochlear turns except in the basal turn, were extremely atrophied, some cochlear nerves showed degeneration and some cochlea showed endolymphatic hydrops. After seven days, ten days and fifteen days, the morphological changes showed atrophy in the stria vascularis similar to the results observed on the third days. Atrophy in the stria vascularis was improved gradually with time, but the degeneration of the cochlear nerve was not improved. Opinions have been divided on the cause of inner ear disease including Meniere's disease. Many authors have reported that infectious diseases, for example mumps, measles and cytomegalovirus infection, have caused human sensorineural hearing loss. These diseases have been reported to result in atrophy in the stria vascularis, degeneration of the cochlear nerve and some other pathological changes. In this study, it was clearly observed that the atrophy of the stria vascularis, the endolymphatic hydrops and other morphological changes were caused by introduction of anaphylatoxins. These results were similar to the pathological changes observed in inner ear diseases in human beings. Therefore, inflammatory substances, including anaphylatoxins, were closely related to the cause of inner ear diseases. The animal model used in this report is considered to be important for elucidating the pathogenesis of inner ear diseases.