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 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.     

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.     

Effect of impulse noise exposure on the endocochlear potentials]

Effects of impulse noise exposure (25 impulses, peak level 165 dB SPL) on endocochlear potentials (EP) and CAP threshold were investigated in guinea pigs. Eight hours after exposure, EP recorded from the second turn of the cochlea was very low (19.0 +/- 1.7 mV) and the negative EP resulted from prolonged anoxia was markedly diminished (-EPmax = -6.4 +/- 1.3 mV). The return rate of EP after reventilation was also reduced. The positive EP returned to normal values 7 days after exposure but the negative EP and the CAP threshold did not. The results suggest that the stria vascularis was also damaged together with the organ of Corti, the degree of damage seemed to be heavier in the latter if only complete recovery of positive EP is taken into consideration. The stria vascularis was also not completely restored when examining the return rate of EP after reventilation. Mechanisms underlying the changes in EP and hearing sensitivity after noise exposure are discussed.     

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Student's t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Student's t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Student's t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.     

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.     

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.     

Viral labyrinthitis: early pathology in the human

The histologic findings in the temporal bones of three patients who died from viral encephalopathy are presented. Pathology was restricted to the scala media, vestibular labyrinth, and internal auditory canal and was considered to be expressions of viral labyrinthitis. The changes were different degrees of degeneration of the organ of Corti, early encapsulation of the tectorial membrane, degeneration of the stria vascularis, and round cell infiltration of the modiolus and contents of the internal auditory canal. A new finding in the organ of Corti and early stages of cystic degeneration of the stria vascularis are documented. In all cases, the saccule was degenerated with sloughing of the otolithic membrane and vestibular labyrinth was involved in varying degrees.     

Dystroglycan expression in the developing and senescent gerbil cochlea

Dystroglycan (DG) forms part of a cell surface laminin receptor complex and is believed to play a critical role in the assembly and homeostasis of basement membranes (BM). The receptor complex is made up of alpha- and beta-DG subunits and is found in muscle, epithelial and nerve tissue. In the cochlea, DG may be involved in the abnormal accumulation of laminin seen in the thickened BM of strial capillaries with age. This excess deposition of laminin is thought to lead to capillary necrosis and contribute to degeneration of the stria vascularis (SV). Here we assessed the presence and distribution of DG in the developing, mature and senescent gerbil cochlea in order to ascertain whether altered patterns of expression are a factor in age-related pathology. Western blots of proteins isolated from the entire cochlea demonstrated the presence of the alpha-DG subunit. mRNA encoding DG was identified in microdissected specimens of the lateral wall and the combined organ of Corti/modiolus by RT-PCR analysis. Immunohistochemical experiments localized alpha-DG in epithelial BMs and regions of epithelial cell-cell contact with no intervening BM in the developing and mature cochlea. Immunoreactive alpha-DG was present in the BM underlying strial capillaries and in vessels of the central portion of the auditory nerve, but was not detected in any other vessels in the cochlea. Age-related changes in alpha-DG expression were observed only in the SV where a marked decrease in alpha-DG immunoreactivity was seen in the BM of strial capillaries as well as throughout the SV. The results demonstrate the selective expression of alpha-DG in both BM and non-BM sites in the mature cochlea and suggests its involvement in both developmental and aging processes.     

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.     

Localization of Inducible Heat Shock Protein mRNA in the Guinea Pig Cochlea with a Nonradioactive In Situ Hybridization Technique

Cell types of the guinea pig cochlea that contain mRNA of the inducible heat shock protein (HSP72) were determined with an in situ hybridization technique, using a biotinylated oligonucleotide probe. Staining was present in the spiral limbus, spiral prominence, stria vascularis, and organ of Corti (supporting, pillar, outer hair, and interdental cells). In sections digested with pepsin, only spiral ganglion cells stained. The pattern of staining was similar in normal and heat-stressed animals. Therefore, HSP72 mRNA is present in many guinea pig cochlear cell types, some of which have not previously been shown to contain HSP72 protein. Differences in HSP72 mRNA and protein staining may be attributable to stress or processing techniques, but they may also suggest mechanisms unique to guinea pigs and primates, who normally express the inducible form of HSP.     

水杨酸钠作用后大鼠耳蜗热休克蛋白27表达的研究

目的　研究水杨酸钠作用后大鼠耳蜗热休克蛋白 2 7(heatshockprotein2 7,HSP2 7)的表达特点 ,探讨HSP2 7在水杨酸钠耳毒性中的作用及意义。方法　用免疫组化SP法结合图像分析技术检测水杨酸钠注射后大鼠耳蜗HSP2 7的表达。结果　HSP2 7在正常及水杨酸钠作用后大鼠耳蜗各回均有不同程度的染色 ,主要阳性部位是Corti器、螺旋神经节、螺旋韧带和血管纹。但水杨酸钠作用后HSP2 7在耳蜗各阳性部位的表达均明显增强 ,其中以Corti器的表达更明显 ,P值均小于 0 .0 1。结论　HSP2 7在大鼠耳蜗组织表达有选择性 ,水杨酸钠能够明显诱导HSP2 7在大鼠耳蜗中的表达 ,HSP2 7可能对耳蜗形态结构损害后的修复起作用 ,且与耳鸣的产生发展可能有关。     

Expression of vascular endothelial growth factor and its receptors in the cochlea of various experimental animals

CONCLUSION: The results of this study indicate that vascular endothelial growth factor (VEGF) may be an important regulator of the vascular network of the inner ear and suggest that the VEGF signalling pathway may play a role in pathophysiologic conditions. OBJECTIVE: In order to clarify the role of vascular growth factor in the modulation of the vascular network of the cochlea, we studied the expression of VEGF and its receptors-fms-like tyrosine kinase (Flt-1) and foetal liver kinase (Flk-1)-in the inner ear of 3-month-old rodents of different species: C57BL/6J mice, Wistar albino rats and Hartley albino guinea pigs. MATERIAL AND METHODS: Qualitative immunohistochemical studies were performed by using specific antibodies to VEGF and its receptors on paraffin sections of the cochlea. The expression levels of VEGF and its receptors were quantified by means of Western blot analysis of cochlea protein extracts. RESULTS: We demonstrated that VEGF and its receptors are expressed in the cochlea and described their distribution in the inner ear. In particular, VEGF and Flt-1 are present at the level of the modiolus, spiral ganglion, spiral ligament, basilar membrane, supporting cells, outer and inner hair cells and stria vascularis. Flk-1 was less strongly expressed in the cochlea and was not detected in the organ of Corti.