Effect of noise exposure on blood-labyrinth barrier in guinea pigs

The influence of noise exposure on the endothelial transport system in the cochlea was investigated using cationic polyethyleneimine (PEI), since systemically administered PEI passes through the capillary endothelial cell and attaches to basal lamina (BL) anionic sites in the cochlea. Under general anesthesia, all guinea pigs were administered an intravenous injection of 0.5% PEI. Thirty minutes later, five animals were exposed to noise (10 kHz, broad band noise, 105 dB SPL) for 30 min, via speakers inserted into the external auditory canal. The remaining five animals (controls) were left without noise exposure for 1 h following PEI injection. All guinea pigs were then immediately sacrificed, and the bony labyrinths were removed. PEI distribution on the BL was assessed in the stria vascularis, spiral ligament, basilar membrane, spiral limbus and Reissner's membrane throughout the cochlea with transmission electron microscopy. Compared to control animals, PEI distribution in the noise-exposed animals was significantly increased in the strial vessels of the basal and second turns and in Reissner's membrane of all turns. In the spiral ligament, basilar membrane and spiral limbus, no significant difference in PEI distribution was observed between the control and noise-exposed animals. These findings indicate that noise exposure increases macromolecular transport in the stria vascularis but not in the spiral ligament, spiral limbus and basilar membrane and that systemically administered macromolecules are more readily transported to Reissner's membrane by noise exposure.     

Importance of type IV collagen, laminin, and heparan sulfate proteoglycan in the regulation of labyrinthine fluid in the rat cochlear duct

The distribution of major components of the basement membrane, such as type IV collagen, laminin, and heparan sulfate proteoglycan (HSPG), was investigated in the rat cochlear duct. Immunofluorescence demonstrated that type IV collagen, laminin and HSPG were distributed along capillaries in the cochlear duct, including the stria vascularis, spiral ligament, spiral prominence and spiral limbus. Additionally, type IV collagen, laminin and HSPG were found to be distributed from the basement membrane of Reissner’s membrane to that of the spiral prominence in a linear pattern. The scala media was surrounded by these basement membrane components, demarcating endolymph from perilymph, along epithelial cells except at the stria vascularis. These findings suggest that type IV collagen, laminin and HSPG create the anatomical separation between endolymph and perilymph, thus indicating that they may be involved in the regulation of fluid transport between the endolymph and perilymph. Received: 3 December 1997 / Accepted: 12 February 1998     

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.     

Ultrastructure and immunohistochemical identification of the extracellular matrix of the chinchilla cochlea

The molecular composition and three-dimensional organization of the extracellular matrix (ECM) was studied by immunofluorescent microscopy, transmission and scanning electron microscopy in three connective tissue structures of the cochlea: the spiral limbus, basilar membrane and spiral ligament. Type II collagen, fibronectin, tenascin, chondroitin sulfate proteoglycans, alphav and beta1 integrins were immunolocalized in the ECM of these connective tissue structures. Electron micrographs showed a continuum of cross-striated collagen fibrils having a similar diameter and axial periodicity that spread from the spiral limbus via the basilar membrane and into the spiral ligament. Some of collagen fibrils were aggregated laterally into bundles. Bundle images, and their digital Fourier transformations, showed a major 67-nm axial D-repeat characteristic for collagen fibrils. Transmission electron microscopy showed numerous proteoglycans associated with the collagen fibrils. The spiral limbus, basilar membrane and spiral ligament demonstrated regional differences in molecular composition and structural organization of their ECM. The glycoproteins fibronectin, tenascin and alphav integrin were immunolocalized mainly in the basilar membrane. Collagen fibrils of the spiral limbus and spiral ligament did not appear to be strongly oriented. However, most of the collagen fibrils in the basilar membrane were arranged into radially directed bundles. Collagen fibrils in the basilar membrane were also surrounded by a homogeneous matrix, which was immunoreactive to fibronectin and tenascin antibodies. A more complete understanding of the composition and structural organization of the ECM in these connective tissue structures in the cochlea provides a foundation upon which micromechanical models of cochlear function can be constructed.     

An anionic charge barrier in the guinea pig cochlea

Summary A charge barrier has been found in the the glomerular basement membrane of the kidney and plays an important role in the filtration of solutes. In the present study, we used electron microscopy to localize anionic sites of a similar charge barrier in the guinea pig cochlea. Polyethyleneimine (PEI) was used as a cationic marker to detect anionic sites. Our results showed a localization of PEI with regular interspaces, indicating the anionic sites to the charge in the capillary basement membrane of the stria vascularis and the spiral ligament, and in the basal lamina of Reissner's membrane and the spiral prominence. This charge barrier, as well as structural size barrier, may play an important role in the maintenance of normal inner ear functions.     

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.     

Age-related morphological changes in the basement membrane in the stria vascularis of C57BL/6 mice

Basement membrane anionic sites (BMAS) are involved in the selective transport of electrically charged macromolecules in cochlear capillaries. Using cationic polyethyleneimine (PEI), we examined age-related changes in BMAS in the cochleae of C57BL/6 mice. The mice were grouped according to age as follows: 3 days, 4 weeks, 8 weeks, 6 months, and 12 months. In the right bony labyrinths, widths of the stria vascularis were measured in paraffin-embedded sections using light microscopy. The left bony labyrinths were immersed in a 0.5 % cationic PEI solution and embedded in epoxy resin. Ultrathin sections of the left cochlea were examined using transmission electron microscopy. A significant difference in stria vascularis width was observed between the 4-week-old and 12-month-old mice. The PEI distribution in the capillary and epithelial basement membranes (BMs) of the cochlea was observed. In all animals, PEI particles were evenly distributed in the capillary BM of the spiral ligament and in the subepithelial BM of Reissner’s membrane. In the stria vascularis, PEI particles were evenly distributed in the capillary BM in 3-day-old mice. In 4- and 8-week-old mice, PEI particle sizes were markedly lower than those observed in 3-day-old mice. In 6- and 12-month-old mice, PEI particles were hardly detected in the strial capillary BM. In the strial capillary BM in these mice, the laminae rarae externa and interna disappeared, but the lamina densa became larger. We speculated that age-related changes of strial capillary BMAS may affect electrically charged macromolecule transport systems in the stria vascularis of C57BL/6 mice.     

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.     

溶酶体神经氨酸酶基因缺乏小鼠听觉和耳形态学改变初步研究

目的研究溶酶体神经氨酸酶基因（Neul）敲除小鼠听功能和耳形态学改变，探讨唾液酸沉积症听力损害的病理生理机制。方法应用听性脑干反应测试和常规颞骨连续切片观察3周、2个月和4个月龄的Neul敲除纯合子（Neul-／-）和野生型（Neul＋／＋）小鼠听阈和光镜下外耳、中耳及内耳形态。结果3周龄的Neul-／-小鼠，短声和短音8、16及32kHz听阈（声压级）较Neul＋／＋提高50—55dB；2个月和4个月龄小鼠听阈提高60—68dB。Neul-／-小鼠3周龄即有明显的中耳和内耳改变，特别是2个月和4个月龄有显著的外耳道堵塞和严重中耳炎，听小骨和耳蜗骨壁细胞、血管纹边缘层和中间层细胞、耳蜗螺旋神经节细胞、螺旋缘纤维细胞、前庭膜、基底膜及沿前庭阶外淋巴隙的间皮细胞明显囊泡化，但Corti器细胞正常。前庭神经节细胞、壶腹嵴及球囊毛细胞和支持细胞也呈现明显囊泡化。结论溶酶体神经氨酸酶的缺乏可导致较严重的听力损害和耳形态改变；外耳道阻塞或中耳炎和听骨改变可能引起传导性聋；耳蜗螺旋神经元、血管纹、螺旋缘、前庭膜和基底膜等细胞的溶酶体储积可能导致感音神经性聋。     

Expression of basement membrane type IV collagen chains during postnatal development in the murine cochlea

An immunofluorescence study was performed to examine the temporal and spatial patterns of expression for the different type IV collagen chains during postnatal cochlear development. At birth, the classical chains (4A1 and 4A2) were widely expressed, while the novel chains (4A3, 4A4, and 4A5) were completely absent. Activation of the novel chains was observed at 4 days of age, with intense, widely distributed immunostaining suggesting that most of the cells in the cochlea express the novel chains at this developmental stage. From day 8 through day 14, developmental inactivation of the novel chains results in a reduction of generalized immunoreactivity with a concomitant elevation of specific staining in the membranous structures bounding the interdental cells of the spiral limbus, the inner sulcus, the basilar membrane, and in a fibrous bed of staining radiating from the spiral prominence into the region of the spiral ligament which corresponds to the location of the root cell processes. This pattern of intense immunostaining for the novel chains persists through adulthood. The classical chains are expressed in these same anatomical regions only transiently (from day 6 to day 10), after which a gradual developmental inactivation leads to the adult expression pattern where classical collagen chains are found primarily in the perineurium, in the membranes surrounding the spiral ganglion cell bodies, and in the vascular basement membranes of the spiral ligament and the stria vascularis. The complex developmental pattern of expression for the type IV collagen chains in the murine cochlea is similar to that observed in the murine kidney, which is the other major site for basement membrane pathology in Alport syndrome.     

Effect of cisplatin on the negative charge barrier in strial vessels of the guinea pig A transmission electron microscopic study using polyethyleneimine molecules

The anionic sites on the basement membrane in the cochlea are believed to act as a charge barrier. Using polyethyleneimine (PEI) as a cationic tracer, we examined the effects of cisplatin (CDDP) on anionic sites in the basement membrane in the cochlea. Eight Hartley-strain guinea pigs were separated into control (buffer administration) and CDDP groups. Ultrathin sections of the basal and third turns of the cochlear ducts were examined using a transmission electron microscope. A marked decrease of PEI distribution was noted in the strial vessels of the CDDP group compared to the control group. However, the two groups showed no signigicant differences in PEI particles on the basement membranes in the basilar and Reissner's membranes, or in the basal and third turns. These findings suggest that the charge barrier in the stria vascularis may be easily inured by the administration of CDDP.     

Long-term ototoxic effects of neomycin applied topically in the middle ear: a morphological study in the guinea pig

Neomycin was instilled daily, uni- or bilaterally, into the middle ear of guinea pigs for three months. The cochleae were examined, by light and electron microscopy, six months after the end of treatment. The organs of Corti of the treated ears were completely destroyed, and in the most advanced lesions, were substituted by a single layer of very thin flat cells. In the spiral ganglion only some glial cells and a few neurons could be observed. All surviving neurons were myelinated, and their ultrastructure was greatly altered, with disorganization of the rough endoplasmic reticulum and ribosomes. The basilar membrane almost disappeared, losing it amorphous and filamentous components. The spiral limbus and the stria vascularis were atrophic and were also covered, in the final stages, by flat elongated cells. In view of its morphological characteristics, this epithelium may arise from the displacement of the interdental cells and perhaps from the cochlear surface of Reissner's membrane.     

Quantitative evaluation of pinocytosis of capillaries of the stria vascularis under normal and experimental conditions

In order to examine evidence of high activity of pinocytosis in capillaries of the stria vascularis, quantitative morphometry of pinocytotic vesicles was carried out ultrastructurally by the horseradish peroxidase (HRP) tracer method. In guinea pigs, there was a significant difference between the stria vascularis and spiral ligament in the number of vesicles per micron 2 (p less than 0.05) and labelled vesicles per micron 2 (p less than 0.001). In normotensive control rats, the number of labelled vesicles per micron 2 in capillaries of the stria vascularis was significantly greater (p less than 0.001) than that of the spiral ligament. Results of acute hypertensive and acute hypotensive experiments both indicated that enhanced permeable capillaries of the stria vascularis showed no significant increase in the number of pinocytotic vesicles per micron 2, but that they showed a more significant increase in the number of labelled vesicles per micron 2 than non-enhanced permeable capillaries of the stria vascularis (p less than 0.01) and capillaries of the spiral ligament (p less than 0.001). These findings provide ultrastructural confirmation of our previous studies (4, 8, 9) that pinocytosis contributes to the high permeability of capillaries of the stria vascularis under normal and experimental acute hypertensive and acute hypotensive conditions.     

Quantitative evaluation of pinocytosis of capillaries of the stria vascularis under normal and experimental conditions

In order to examine evidence of high activity of pinocytosis in capillaries of the stria vascularis, quantitative morphometry of pinocytotic vesicles was carried out ultrastructur-ally by the horseradish peroxidase (HRP) tracer method. In guinea pigs, there was a significant difference between the stria vascularis and spiral ligament in the number of vesicles per urn(2) (p<0.05) and labelled vesicles per urn(2) (p<0.001). In normotensive control rats, the number of labelled vesicles per u,m(2) in capillaries of the stria vascularis was significantly greater (p<0.001) than that of the spiral ligament. Results of acute hypertensive and acute hypotensive experiments both indicated that enhanced permeable capillaries of the stria vascularis showed no significant increase in the number of pinocytotic vesicles per urn(2), but that they showed a more significant increase in the number of labelled vesicles per um(2) than non-enhanced permeable capillaries of the stria vascularis (p<0.01) and capillaries of the spiral ligament (p<0.001). These findings provide ultrastruc-tural confirmation of our previous studies (4, 8, 9) that pinocytosis contributes to the high permeability of capillaries of the stria vascularis under normal and experimental acute hypertensive and acute hypotensive conditions.     

Immunohistochemical localization of fibronectin in the chinchilla cochlea

The purpose of this study was to better understand the molecular composition of the cochlea. Fibronectin (FN), a well characterized adhesive glycoprotein, was localized by immunofluorescence microscopy in fresh and fixed cochlear tissues, and in fixed kidney tissue, using a polyclonal, affinity-purified, rabbit, anti-fibronectin antibody and a secondary antibody coupled to FITC. The FN antibody was free from cross-reactivity with other known basement membrane and cell matrix molecules. FN reactivity in the cochlea was most intense in the basilar membrane, latero-basal borders of Boettcher's cells, otic capsule, endothelial basement membranes (particularly those of the stria vascularis), and as a diffuse, fan-shaped network radiating into the spiral ligament. Little FN labelling was present in the epithelial basement membranes. Negative control tissue showed no immunoreactivity; whereas, positive kidney control tissue showed appropriate FN immunoreactivity in the mesangium of the glomerulus. The most significant finding of this study was that FN is a major component of the basilar membrane and its distribution appears to correspond to the amorphous ground substance. FN was not localized in the organ of Corti or at the tips of the hair-cell stereocilia.     

Scanning Electron Microscopic Examination of the Extracellular Matrix in the Decellularized Mouse and Human Cochlea

Decellularized tissues have been used to investigate the extracellular matrix (ECM) in a number of different tissues and species. Santi and Johnson JARO 14:3-15 (2013) first described the decellularized inner ear in the mouse, rat, and human using scanning thin-sheet laser imaging microscopy (sTSLIM). The purpose of the present investigation is to examine decellularized cochleas in the mouse and human at higher resolution using scanning electron microscopy (SEM). Fresh cochleas were harvested and decellularized using detergent extraction methods. Following decellularization, the ECM of the bone, basilar membrane, spiral limbus, and ligament remained, and all of the cells were removed from the cochlea. A number of similarities and differences in the ECM of the mouse and human were observed. A novel, spirally directed structure was present on the basilar membrane and is located at the border between Hensen and Boettcher cells. These septa-like structures formed a single row in the mouse and multiple rows in the human. The basal lamina of the stria vascularis capillaries was present and appeared thicker in the human compared with the mouse. In the mouse, numerous openings beneath the spiral prominence that previously housed the root processes of the external sulcus cells were observed but in the human there was only a single row of openings. These and other anatomical differences in the ECM between the mouse and human may reflect functional differences and/or be due to aging; however, decellularized cochleas provide a new way to examine the cochlear ECM and reveal new observations.     

Fine structure histopathology of labyrinthitis ossificans in the gerbil model

Labyrinthitis ossificans (LO) is the pathological deposition of new bone within the lumen of the cochlea and labyrinth. This process occurs most commonly as a result of infection or inflammation affecting the otic capsule. Trauma and vascular compromise can also lead to neo-ossification within the otic capsule. The mechanism that regulates this process remains unestablished. This study details the end-stage histopathology in high-resolution plastic thin sections. Twenty Mongolian gerbils were infected by intrathecal injection of Streptococcus pneumoniae type 3 followed by subcutaneous penicillin G procaine (8 days) and were painlessly sacrificed 3 months later. The cochleas were serially divided and sectioned for light and electron microscopy. Sixteen of 20 animals (27 of 40 cochleas) demonstrated LO. Cochlear damage was most extensive in the vestibule and basal turn and decreased toward the apex, which often appeared normal. The histopathologic findings consisted of 1) new bone, calcospherites, osteoid, and fibrosis without dense connective tissue or osteoblasts extending from the endosteal wall into the lumen of the vestibule and scala tympani; 2) areas of dense connective tissue and osteoid enclosed by epithelial cells conjoined with the organ of Corti, stria vascularis, spiral ligament, and vestibular (Reissner's) membrane; and 3) partial to complete loss of the organ of Corti, spiral ligament cell bodies, stria vascularis, and spiral ganglion cells. Osteoblastic activity was not demonstrated in end-stage ossification in LO in the gerbil model. Neo-ossification appears to occur by calcospherite deposition along collagen-like fibrils within osteoid. The destruction of the organ of Corti, spiral ganglion cells, stria vascularis, and cells of Reissner's membrane and the spiral ligament occurs even in the absence of ossification of the cochlear duct.     

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。     

Changes in the capillaries of the stria vascularis after hemorrhagic shock

As a result of severe hemorrhagic shock in guinea pigs, the blood flow in the capillaries of the stria vascularis was blocked, but that of the spiral ligament was unaffected. It is therefore postulated that the thick layer of basal cells and fibrocytes around the stria vascularis and the capillaries leaving the stria vascularis plays an important role in the formation of blood sludging.     

Ultrastructural study of the effect of acute hypertension on the stria vascularis and spiral ligament

The effect of acute hypertension, induced in rats by intravenous injection of methoxamine chloride (Mexan), on the stria vascularis and spiral ligament was studied electronmicroscopically with the tracer method of horseradish peroxidase (HRP). Considerable extravasation of HRP occurred in the stria vascularis, due to the increased vesicular transport. The leaked HRP spread into intercellular spaces, but was prevented from spreading towards the endolymph by zonulae occludentes between marginal cells and towards the perilymph by zonulae occludentes between basal cells. The reaction product was occasionally found between basal cells. No leakage of HRP from capillaries was observed in the spiral ligament, although some labelled micropinocytotic vesicles were present in the endothelium. It is suggested that, under acute hypertensive conditions, areas of zonulae occludentes bordering the stria vascularis play an important role as a barrier to HRP, whereas capillaries in the spiral ligament themselves act as a barrier to it.