豚鼠耳蜗和内淋巴囊上皮钠通道的表达

目的 研究豚鼠耳蜗和内淋巴囊组织中上皮钠通道(epithelial sodium channel,ENaC)α、β、γ亚基的表达及其意义.方法 用兔抗大鼠ENaC α、β和γ亚基的多克隆抗体,采用免疫组化SP法观察豚鼠耳蜗和内淋巴囊组织中α、β、γ-ENaC的表达模式.另用α-ENaC cDNA质粒合成探针,原位杂交法检测豚鼠耳蜗和内淋巴囊组织中α-ENaC mRNA的表达.结果 免疫组化显示,在豚鼠耳蜗中,α-ENaC蛋白强烈表达于螺旋缘,而螺旋韧带、Corti器、Reissner膜等处的表达较弱;β-ENaC蛋白在螺旋韧带、螺旋缘和螺旋神经节、Corti器、Reissner膜等处的表达均呈弱阳性;γ-ENaC蛋白则在螺旋韧带的上半部、螺旋缘和螺旋神经节等处的表达呈强阳性,Corti器、Reissner膜等处也有阳性表达.三个亚基在血管纹中均呈阴性表达.在内淋巴囊中,α、β和γ亚基均较明显地表达于上皮细胞和上皮下纤维组织.原位杂交显示,α-ENaC mRNA除了表达于螺旋缘、螺旋韧带下部外,还表达于血管纹,而在内淋巴囊的上皮细胞和上皮下纤维组织也均呈阳性表达.结论 ENaC的各个亚基以不同的模式分布于豚鼠耳蜗和内淋巴囊的各个区域,形成功能性通道参与内淋巴的调节,从而保持内耳内环境的稳定.     

Localization of glucocorticoid receptors and glucocorticoid receptor mRNAs in the rat cochlea

The distribution of glucocorticoid (GR) receptor messenger RNAs (mRNAs) and GR receptors was studied by in situ hybridization histochemistry and immunocytochemistry, respectively. In situ hybridization histochemistry was performed with a biotin-labeled riboprobe complementary to rat GR receptor mRNA. GR receptor mRNAs were demonstrated in spiral ligament cells, spiral limbus cells, and spiral ganglion cells. GR receptor mRNAs were demonstrated neither in cells of the stria vascularis nor in cells of the organ of Corti region. With the use of a monoclonal and a polyclonal antibody, GR receptors were observed in the spiral ligament cells, stria vascularis cells, spiral limbus cells, and spiral ganglion cells by immunocytochemistry. Binding of anti-GR-receptor antibodies to a lesser extent was observed in the organ of Corti region; however, cellular distribution of the GR receptors could not be resolved with the applied techniques. These results suggest that the GR receptor is expressed differently in the heterogeneous cochlear tissues.     

Immunohistochemical localization of the epithelial sodium channel in the rat inner ear

Endolymph in membranous labyrinth is a K+-rich and Na+-poor fluid, and perilymph is conversely Na+-rich and K+-poor. Electrolyte transport between endolymph and perilymph is important for regulation of volume and osmotic pressure of the labyrinth. The epithelial sodium channel (ENaC) is a good candidate protein for Na+ transport in the tight epithelia, which has been well demonstrated in other tissues such as kidney, colon and lung. The purpose of the present study was to investigate the cellular localization of ENaC subunits in the rat inner ear immunohistochemically with the specific polyclonal rabbit antibodies against the rat alpha-, beta- and gamma-ENaC. All three subunits of ENaC were extensively labeled in the cochlea including the stria vascularis, spiral ligament, organ of Corti, spiral limbus, Reissner's membrane and spiral ganglion, and in the vestibule including the sensory epithelia and stroma cells of the macula utriculi, macula sacculi and ampullary crest. In conclusion, our results suggest that functional ENaC in the labyrinth may work in concert with other Na+ and K+ transport molecules to regulate endolymph and to maintain homeostasis in the inner ear.     

Electrical coupling differs in the in vitro and in vivo organ of Corti

Electrical communication between the supporting cells of the guinea pig organ of Corti was studied. For in vitro experiments, the inner ear was rapidly removed and placed in a heated perfusion chamber. Medium 199 was used. The bony cochlea and the lateral wall (spiral ligament and stria vascularis) were removed to expose the top two coils of the organ of Corti. In vivo experiments were performed upon anesthetized animals whose cochleas were exposed surgically. A tiny fenestra was made in the bony cochlea which permitted the passage of electrodes through the lateral wall and into the organ of Corti of the third turn. Coupling was assessed by impaling neighboring cells with 3 M KCl electrodes, and noting the spread of intracellularly injected current. Coupling ratios in the in vitro preparation were consistently greater than those obtained in vivo (0.58 +/- 0.17 vs. 0.104 +/- 0.064). Differences exist between the in vitro and in vivo preparations which might account for these results. In vivo the supporting cells are bathed in two different media, endolymph apically, and perilymph basally. Consequently, on their apical side the supporting cells are exposed to fluid high in K+, low in Ca2+ and at a potential of 80 mV, the endolymphatic potential. In vitro the cells are bathed on all sides in fluid similar to perilymph. Intermixing the fluids in an in vivo preparation, by tearing away the stria vascularis and Reissner's membrane, increases the magnitude of the coupling ratio (0.455 +/- 0.209). Thus the unique microenvironment of the inner ear maintains lower coupling ratios, and smaller space constants for the supporting cells.     

Cochleosaccular dysplasia: a morphometric and histopathologic study in a series of temporal bones.

OBJECTIVE: The objective of this study was to perform a morphometric analysis of a series of temporal bones with cochleosaccular dysplasia to clarify the extent of inner ear changes in this disease. STUDY DESIGN: This human temporal bone histopathologic study of a series of deaf-mute cases involves morphometric analysis, including stria vascularis and spiral ligament area measurements and spiral ganglion and hair cells counts. SUBJECTS: Thirteen temporal bones were selected from 35 with deaf mutism based on the histopathologic findings described by Scheibe. Twenty normal age-matched control subjects were used for comparisons. RESULTS: All temporal bones had the main histopathologic findings described by Scheibe, as well as severe affected stria vascularis. Seven temporal bones had cystic areas in the stria and three had concretions. Cross-sectional strial areas in temporal bones with cochleosaccular dysplasia were smaller than normal in all cochlear turns; however, no difference was found in spiral ligament cross-sectional areas. Reissner's membrane was hydropic in three temporal bones and the organ of Corti was absent in at least one cochlear turn in five. Concretions were present in the macula of seven temporal bones. Twelve temporal bones showed some level of spiral ganglion cell loss. No hair cells were observed in any temporal bone. A familial history of deafness was found in three cases. CONCLUSION: Pathologic findings were variable and limited to the saccule and scala media. The variation, perhaps, reflects the different etiologies involved in the origin of cochleosaccular dysplasia.     

Antigen Diffusion from the perilymphatic space of the cochlea

The diffusion pattern of horseradish peroxidase (HEP) injected into the scala tympani of the cochlear basal turn of guinea pigs was studied to test whether antigen presented in this manner can gain access to the endolymphatic sac. By two hours, HEP reaction product was found throughout the cochlea, with the greatest amounts in the spiral ligament, spiral lim-bus, basilar membrane, and organ of Corti. In several cochleas, very weak labeling was seen in the stria vascularis. HEP reaction product was maximal in the basal turn. By two hours, HEP reaction product was also observed in the endolymphatic sac lumen, epithelial cells, subepithelial tissue, and perisaccular connective tissue. It was more common in the proximal portion. At this time, macrophages within the lumen already appeared to have phagocytosed the HEP. By 72 hours after injection, the inner ear was cleared of HEP. The results of this study support the hypothesis that antigen in the scala tympani gains access to the endolymphatic sac lumen, where it may be presented by macrophages to the systemic immune system. Antigen most likely does not gain access to the endolymphatic space in the cochlea, but it gets to the endolymphatic sac through the perilymph and the Derisaccular tissue.     

Proteoglycan arrays in the cochlear basement membrane

Indirect immunofluorescence and transmission electron microscopy were used to investigate the composition and assembly of proteoglycans in the basement membranes of the spiral limbus, basilar membrane, spiral ligament, Reissner's membrane, myelinated nerve fibers, and blood capillaries of the spiral ligament and stria vascularis in the chinchilla cochlea. Four types of basement membrane components: laminin, entactin/nidogen, type IV collagen and heparan sulfate proteoglycans were immunolocalized in all basement membranes in association with heparan sulfate proteoglycans. beta 1 and alpha 1 integrin subunits were also detected along these basement membranes. The concentration of the basement membrane-associated proteins and integrin subunits differed according to the adjacent cell type. Electron microscopy showed that all basement membranes, with exception of those of stria vascularis, consist of two layers: lamina lucida and lamina densa. In the stria vascularis only a homogeneous lamina densa was observed. Cuprolinic blue treatment revealed heterogeneity in the ultrastructure and arrangement of proteoglycans in the cochlear basement membranes. Proteoglycans of the subepithelial basement membrane in the spiral limbus and spiral ligament formed quasi-regular, linear arrays within the lamina lucida, or were located at both sides of the lamina densa in the basilar membrane and Reissner's membrane. In the basement membranes of nerve fibers, and capillaries in the spiral ligament and stria vascularis, proteoglycans were scattered throughout these basement membranes, but showed different concentration and ultrastructural appearance, which may be related to different filtration and mechanical properties. In the basilar membrane, PGs were located above and below the lamina densa. An additional layer of PGs below the lamina densa may function as increased mechanical support of organ of Corti by its interaction with underlying fibrillar collagen layer. In the stria vascularis capillaries, PGs were stained considerably less with Cuprolinic blue and were scattered through the lamina densa of the basement membrane compared to capillaries of spiral ligament. This observation is compatible with a higher permeability of the strial capillaries.     

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。     

Immunohistochemical detection of platinated DNA in the cochlea of cisplatin-treated guinea pigs

Cisplatin-induced ototoxicity is correlated with functional and morphological changes in the organ of Corti, the stria vascularis and the spiral ganglion. However, the cochlear sites of cisplatin uptake and accumulation have not been properly identified. Therefore, we have developed an immunohistochemical method to, indirectly, detect cisplatin in semithin cryosections of the guinea pig cochlea (basal turn) using an antiserum containing antibodies against cisplatin-DNA adducts. Platinated DNA was present in the nuclei of most cells in the organ of Corti and the lateral wall after cisplatin administration. Nuclear immunostaining was most pronounced in the outer hair cells, the marginal cells and the spiral ligament fibrocytes. This study is the first to demonstrate the presence of cisplatin in histological sections of the cochlea.     

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.     

Fast alterations of vascular endothelial growth factor (VEGF) expression and that of its receptors (Flt-1, Flk-1 and Neuropilin) in the cochlea of guinea pigs after moderate noise exposure

Vascular endothelial growth factor (VEGF) is a vascular permeability regulating, proangiogenic factor with neuroprotective properties. Its expression in the inner ear has been demonstrated, but little is known concerning its subcellular distribution or potential involvement in sound perception and adaptation to noise. Therefore, we determined the expression patterns and levels of VEGF and the three VEGF-receptors FLK, FLT and Neuropilin in the cochlea of guinea pigs, and examined the alterations occurring after noise exposure. After 70 dB exposure, VEGF expression was found to be reduced in all cell types of the organ of Corti, in the stria vascularis and in spiral ganglion cells. Additional down-regulation was observed in the spiral ligament and in interdental cells after 90 dB. In contrast, VEGF showed an in tendency increased level after both intensities in nerve fibers of the osseous spiral lamina. Expression of FLT was affected similarly, showing down-regulation after 70 and 90 dB on spiral ganglion cells, the nerve fibers of the osseous spiral lamina and on Deiters cells. Additionally, down-regulation was observed in the remaining cell types of the organ of Corti, the stria vascularis, the spiral ligament and the interdental cells. The Neuropilin levels remained unchanged by our experiments; apart from the blood vessel endothelium, there was no detectable expression in any of the cell types investigated. The FLK expression pattern was likewise unaffected by exposure to 70 or 90 dB, with the notable exception of an increased level occurring in Schwann cells after 90 dB. We postulate that modulation of VEGF and its receptors may be part of a neuroprotective mechanism in response to noise.     

The spiral ganglion and cochlear nuclei of deafness mice

Deafness mice (dn/dn) never develop hearing, but, except for the associated physical defects, have no other known abnormalities. For this study, cochleas and brains of five adult homozygotes (dn/dn) and five adult heterozygotes (+/dn), matched by weight and sex, were prepared for serial section light microscopy. In the homozygotes, the organ of Corti was totally degenerated basally, gradually improving toward the apex where supporting cells, border cells and pillar cells were present; however, the stria vascularis was dystrophic in the apex. The saccular macula was atrophied or dystrophic in seven of the ten homozygote ears. The homozygotes had only 23% of the number of spiral ganglion cells found in the heterozygotes, but they appeared robust. In six of the ears of each group there was clumping of apical spiral ganglion neurons. In the homozygotes, the volume of Rosenthal's canal was 121%, that of the dorsal nuclei was 90%, and that of the ventral cochlear nuclei was 63% of the comparable volumes in heterozygotes. The globular cells of the homozygote ventral cochlear nucleus were 72% of the size of those of heterozygotes. These quantitative morphological abnormalities of the homozygote spiral ganglion and cochlear nuclei may result from organ of Corti atrophy.     

Age-related changes in the murine cochlear lateral wall

Cochleas from C57BL/6 mice were investigated electrophysiologically and histochemically to evaluate the pathology of presbycusis. The average auditory brainstem response thresholds from 6-week-old mice were significantly lower than those of 6-month-old mice and those of 1-year-old mice. Histologic observation revealed changes in the cochlea after age 6 months. Conventional hematoxylin and eosin (H&E) staining showed disorganization of the organ of Corti, a decrease in the number of spiral ganglion cells, and atrophy of the stria vascularis. Although H&E staining and type II collagen immunolabeling did not show obvious changes in the spiral ligament (SL), the density of connexin 26 staining was reduced in this region. Sodium-potassium-adenosinetriphosphatase immunolabeling was increased in the SL, whereas its average density was not significantly altered in the stria vascularis. These results suggest that the SL could be among the regions responsible for cochlear malfunction with aging.     

Application of argon laser to the inner ear

The effects of argon laser irradiation on the inner ear were studied morphologically. The stria vascularis showed focal coagulation, vesicle formation, perforation and atrophy after irradiation through the otic capsule. These changes vary according to the power and duration of irradiation. Atrophy of the stria vascularis can be produced without injuring the organ of Corti, by suitable application of the laser beam. The round window membrane transmits the argon laser. Tissues in the hook region and a part of the lower basal turn demonstrated changes of varying degree. Loss of the nerve fibres was observed in the osseous spiral lamina, which was fractured and dislocated. Irradiation through the stapedectomized oval window destroyed the macula sacculi without rupturing the saccular wall.     

大鼠耳蜗组织中Nedd4及SGK1的表达

目的 研究大鼠耳蜗组织中神经前体细胞表达-发育性下调基因亚型(neural precursor celI-ex-pressed,developmentally downregulated isoforms,Nedd4)及血清糖皮质激素诱导激酶1(serum glucocorticoid-in-ducible kinase1,SGK1)蛋白分子的表达.方法 选取健康Wistar大鼠8只.用特异性多克隆兔抗鼠Nedd4-1/2、Nedd4-2及SGK1抗体,采用免疫组化法研究大鼠耳蜗组织中Nedd4-1/2、Nedd4-2及SGK1蛋白的表达模式.结果 Nedd4-1/2、Nedd4-2及SGK1蛋白广泛表达于大鼠耳蜗组织的各个区域中,包括血管纹、螺旋韧带、Corti器、螺旋缘、螺旋神经节、前庭膜等处.结论 在耳蜗组织中,存在一个由SGK1、Nedd4和上皮钠通道(ENaC)组成的Na+转运系统,它们协同作用转运Na+,从而保持内耳内环境的稳定.     

Immunohistochemical detection of vascular endothelial growth factor (VEGF) and VEGF receptors Flt-1 and KDR/Flk-1 in the cochlea of guinea pigs

Vascular endothelial growth factor (VEGF) is known as an endothelial cell-specific mitogen. There are no reports concerning the presence of VEGF in the inner ear. To gain information, immunohistochemical analysis using specific antibodies to VEGF and to both known VEGF receptors Flt-1 and KDR/Flk-1 was performed on paraffin-sectioned temporal bones from five guinea pigs. Immunoreactivity of VEGF, Flt-1 and KDR/Flk-1 was detectable in spiral ganglion cells. VEGF could also be found in the endothelium of blood vessels, in the spiral ligament and in the organ of Corti. Flt-1 was found in the limbus epithelium, in all supporting cells of the organ of Corti, in Claudius cells, cells of the sulcus and in the spiral ligament. Flk-1 could be detected in some supporting cells of the organ of Corti (inner pillar cells and Deiters' cells). Immunoreactivity to Flk-1 was also found in endothelium of blood vessels and in the spiral ligament. Hair cells showed VEGF immunostaining, but did not contain staining to Flt-1 nor Flk-1. In the stria vascularis any immunoreactivity to all used VEGF and VEGF receptor antibodies could not be detected. The findings were supported by Western blot analysis on inner ear tissues and ovaries from guinea pigs. We may conclude that the growth factor VEGF and both receptors participate in cochlear physiology.     

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.     

Experimental mumps labyrinthitis in monkeys (Macaca irus)--immunohistochemical and ultrastructural studies

Three monkeys (Macaca irus) were inoculated with mumps virus into unilateral cochleas and their inner ear were examined by immunofluorescent microscopy and transmission electronmicroscopy. The temporal bones were removed after survival period of 14 days when serological tests disclosed elevation of anti-mumps antibody titers. Immunofluorescent microscopy revealed that the viral antigen was positive in the stria vascularis. The ultrastructural study revealed that the pathologic changes in the cochleas were marked in the organ of Corti and stria vascularis. The outer hair cells were more susceptible to the infection than the inner hair cells. In the stria vascularis, both marginal and intermediate cells were affected. It was possible to find some of marginal cells in the basal turn shedding a large number of mature virions into the endolymph. These pathologic changes observed in the cochleas of the monkeys were similar to those previously revealed in the guinea pig cochleas and thus were considered as the specific features of acute mumps labyrinthitis.     

Localization of the NO/cGMP-pathway in the cochlea of guinea pigs.

The presence of nitric oxide synthase (NOS) in substructures of the cochlea of guinea pigs is an issue of current focus. Moreover, information concerning the localization of cells effected by the NO/cGMP-pathway are rare. Paraffin sections of guinea pig cochlea were incubated with specific antibodies to the three known NOS isoforms, soluble guanylyl cyclase (sGC) and cyclic guanosine-monophosphate (cGMP), the second messenger system of NO. While detection of inducible iNOS failed in all cochlear structures, expression of endothelial eNOS was found in the spiral ligament, in the stria vascularis, in cells of the organ of Corti, in nerve fibers and in some perikaryia of the spiral ganglion. The cochlear nerve showed an accentuated affinity for immunostaining in distal, basal segments of the cochlea. Neuronal bNOS was found predominantly in the endosteum of the modiolus and cochlea and was less intensively present in all perikaryia of the spiral ganglion and in the spiral ligament. Supporting cells of the organ of Corti and cells in the limbus spiralis displayed only modest immunostaining, while bNOS was not found in outer and inner hair cells. NOS detection was accompanied by immunoreactivity to sGC and to cGMP. The presence of NOS and its second messenger system gives evidence for a possible involvement in neurotransmission, regulation of the cochlear amplifier and in homeostasis.