Localization of histamine (H1, H2, H3 and H4) receptors in mouse inner ear

Conclusion The present findings show that all four types of histamine receptors (H1R, H2R, H3R, and H4R) are present in the inner ear, thus supporting the hypothesis that histamine plays a physiological role in the inner ear. Objective To analyse the presence of histamine receptors in the normal mouse inner ear. Methods CBA/J mice were used in this study. The localization of H1R, H2R, H3R, and H4R in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion, and endolymphatic sac, was studied by real-time PCR and immunohistochemistry. Results The mRNA for each receptor sub-type was detected in the inner ear. In the immunohistochemical study, the organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory epithelium, and endolymphatic sac cells showed an immunofluorescent reaction to all histamine receptors.     

Cell proliferation in the endolymphatic sac in situ during the immune response of inner ear

Background Normally, few immunocompetent cell are present in the endolymphatic sac (ES). During an active immune response in the inner ear, large amount of inflammatory cells, including immunocompetent cells, are seen in the ES. The current study aimed at assessing cellular proliferation within the ES during induced immune response in the inner ear. Methods Fifteen healthy, female SD rats were sensitized systemically with keyhole limpet hemocyanin (KLH), followed by local inoculation in the cochlea through basal turn fenestration with the same antigen. On Days 3, 7 and 14 following inoculation, the animal was sacrificed after intraperitoneal administration of 5-bromo-2'-deoxyuridine (BrdUrd), and the temporal bone harvested. Following decalcification, infiltration by BrdUrd- and IgG-positive cells in the ES was studied on frozen sections with H & E and immunohistochemical staining. Results During the secondary immune response in the inner ear against T-dependent antigens, there is increased cellular proliferation in the ES. The proliferated cells may differentiate into immunocompetent cells at the same location. Conclusions These findings indicate that the ES plays an important role in immune response of inner ear.     

Cell proliferation in endolymphatic sac during the immune response of inner ear]

OBJECTIVE: To study the cell proliferation in endolymphatic sac(ES) during the secondary immune response in inner ear. METHODS: Fifteen healthy, female SD rats were employed in the experiment. Sensitized systematically with keyhole limpet hemocyanin (KLH), the animals were inoculated with the same antigen through cochlea basal turn into the labyrinth. Administrated 5-bromo-2'-deoxyuridine (BrdUrd) intraperitoneally, the rats were sacrificed and the temporal bones were harvested at 3, 7, 14 day after labyrinth vaccination respectively. The frozen sections of the decalcified samples were dealt with H-E staining and immunohistochemical methods to investigate the cellular infiltration, BrdUrd and IgG positive cells in the ES. RESULTS: While the ES lumen and perisaccular region were infiltrated with mononuclear-phagocyte at the 3rd day post-vaccination, plasmocyte and lymphatic cells become the predominant infiltrating cells in the ES at the 7th day. The KLH in the ES lumen were phagocytized at the 3-7th day post-vaccination. S-phase cells and IgG positive cells in ES increased markedly in the 3rd day and 7-14 days post-vaccination respectively with similar localization. CONCLUSION: The activity of cell proliferation in the ES enhance and local proliferated cells may differentiate in situ into immunocompetent cells during the secondary immune response against TD antigen in the inner ear. This indicates that ES plays an important role in immune response of inner ear.     

Localization of melatonin and its receptors (melatonin 1a and 1b receptors) in the mouse inner ear

Abstract

Background: In the inner ear, evidence has been gathered indicating that melatonin plays important roles in inner ear physiology and pathophysiology. However, no attempt has been made previously to investigate the localization or expression of melatonin and its receptors in the whole inner ear.

Aims/objectives: To analyze the presence of melatonin and its receptors in the normal mouse inner ear.

Material and methods: C57BL6/J mice were used in this study. The localizations of melatonin, MT1a and MT1b in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion and endolymphatic sac (ES), were studied by immunohistochemistry.

Results: The organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory cells, vestibular dark and transitional cells, and ES epithelial cells showed an immunofluorescence reaction to melatonin, MT1a and MT1b.

Conclusion and significance: The present findings show that melatonin and its receptors (MT1a and MT1b) are present in the inner ear, thus supporting the hypothesis that melatonin plays a physiological role in the inner ear.     

水通道蛋白1在小鼠内淋巴囊的超微结构定位

目的研究水通道蛋白1（aquaporin1，AQP1）在小鼠内淋巴囊表达的精确定位，进一步探讨AQP1在内淋巴囊的作用。方法选取正常昆明小鼠30只，断头分离内耳内淋巴囊，应用冰冻切片免疫荧光染色法研究AQP1在内淋巴囊的组织分布情况，借助免疫电镜观察AQP1在内淋巴囊的超微结构定位。结果AQP1在内淋巴囊上皮下结缔组织层有表达。在纤维细胞胞膜特别是细胞突起的胞膜上可见大量胶体金颗粒标记，而上皮细胞的胞膜未见胶体金颗粒的标记。结论富含AQP1的内淋巴囊上皮下组织可能与内淋巴的吸收及平衡调节密切相关。     

Mechanisms of development of endolymphatic hydrops following secondary immune response in the endolymphatic sac of guinea pigs]

Previously the author reported the immediate development of endolymphatic hydrops (e. hydrops) following direct challenge of secondary antigen to the endolymphatic sac (ES) in guinea pigs, during the early phases of postchallenge, ranging from 1 to 5 weeks. The present study reports the results of specimens taken up to 10 weeks postchallenge, and correlation of e.hydrops to perilymph in antigen-specific antibody levels. From the present results, mechanisms of e.hydrops induced ES immune reaction are suggested as follows. 1) In the early stage of e.hydrops, an acute inflammatory reaction in the ES may produce endolymph by an increased vascular permeability of the inner ear and may impair endolymph absorption from the ES. 2) In the latter stage of e.hydrops, the moderate cellular infiltration in the ES may cause chronic impairment of endolymph absorption in the ES.     

The vein of the vestibular aqueduct with potential pathologic perspectives.

HYPOTHESIS: Pathologic changes around the vein of the vestibular aqueduct (VVA) may cause obstruction to the flow of blood toward the sigmoid sinus. Furthermore, a distal obstruction of this vessel may be responsible for a development of a retrograde flow of blood with concomitant drainage of endolymphatic sac (ES) substances to the inner ear. BACKGROUND: The VVA is responsible for the venous drainage of the vestibular apparatus and endolymphatic duct and ES. Previous studies have linked the VVA to Ménière's disease. The aim of the present article was a 3-dimensional perspective study of the VVA with its adjacent anatomic structures. METHODS: In 14 rats, the VVA was examined by 3-dimensional reconstruction of 2-microm serial sections, corrosion cast technique, and scanning electron microscopy. RESULTS: From the external aperture of the vestibular aqueduct, the VVA is interposed between the ES and the operculum. Three to 4 collecting venules from the ES drain into the VVA. The VVA merges at an oblique angle with the sigmoid sinus. CONCLUSION: The VVA courses near the ES, operculum, and sigmoid sinus and is potentially vulnerable to expanding structures in the cranial posterior fossa. The possible role of the VVA for the function of the ES under normal and pathologic conditions is discussed.     

细胞间粘附分子—1在内耳免疫反应中的表达

探讨细胞间占附分子－１在内耳免疫反应中的作用。方法 采用钥孔戚血蓝蛋白激发已全身致敏的２６只大鼠内耳，诱发其内耳免疫反应，然后通过免疫组化技术检测内耳扔ＩＣＡＭ－１的表达。结果 内耳激发后６小时，螺旋轴静脉及其回流小静脉即有ＩＣＡＭ－１表达，１２小时内淋巴囊及其下周区出现ＩＣＡＭ－１的表达，以２４－４８小时内各部位ＩＣＡＭ－１表达达最高峰。     

Immuno-injury to the inner ear auditory system following secondary endolymphatic sac immune response]

This study investigated immuno-injury to the inner ear auditory system following inner ear immune response in the guinea pig. The endolymphatic sac (ES) was directly challenged with keyhole limpet hemocyanin (KLH). Auditory brainstem response with click sound stimulation was assessed pre and post KLH challenge of the ES. In systematically presensitized animals, secondary KLH challenge to the ES produced significant elevation of the mean threshold level on day 3 and 17 as compared to that of pre challenge levels. However, no significant prolongation of the latency (N1-N3) was observed. On the other hand, neither phosphate buffered saline injection into the ES nor primary KLH challenges of the ES were capable of elevating the threshold level and changing the latency. These results indicate that elevation of threshold was apparently induced by a secondary immune response of the ES, not by nonspecific mechanical damage to the ES, the central auditory system or KLH toxicity to the inner ear, suggesting that local immune response of the ES is a possible pathogenesis of sensorineural hearing loss.     

Microorganism transport in the human endolymphatic duct.

There are indications that endolymphatic sac (ES) may be an immunologically active part of the inner ear. So far, no microorganisms or foreign substances have been localized in this area under 'normal' conditions. Only a limited number of human specimens, including the entire endolymphatic duct (ED) and ES, have been collected and analyzed from cadavers or surgical biopsy specimens. In this study, 6 human ED and ES collected from cadavers and at surgery were analyzed by light and electron microscopy. This was done in order to investigate if microorganisms may normally be drained at this route into the ES. Some microorganisms (Mycoplasma pneumoniae) were found in the lumen and subepithelial tissue of 1 human ED. These observations suggest that microorganisms may also be locally processed and disposed at the level of the ED. These results add further evidence as to the immunodefensive role of the human ES.     

内淋巴囊的脉管分布

目的：通过对豚鼠内淋巴囊表面的脉管及其粘膜下层结构的研究,了解其对内耳功能的影响,方法：用碳素黑水加１０％甲醛进行左心室灌注,将豚鼠的颞骨标本用冬青油透明并进行显微检查及透射电镜检查。结果：内淋巴囊表面有非常丰富的血管网,而且与乙状窦有很密切的联系,内淋巴囊周围被毛细血管,微静脉,静脉和个别微动脉形成的一个细密的环状血管网所包绕。毛细血管周围有丰富的淋巴窦、毛细血管与淋巴窦构成一个完整、细密的网状     

Functional morphology of the human endolymphatic sac. A review.

Modern immunohistochemical methods allow a functional characterization of the human endolymphatic sac (ES) and its associated cell populations. The currently available immunohistochemical data on the extraosseous part of the human ES support the assumption that the epithelium is metabolically active and capable of both secretion and absorption. The reactivity of some epithelial cells with antibodies against neuroendocrine antigens implies a paracrine activity of the human ES. Further results provide evidence for a possible role of the human ES in inner ear immune defense and indicate a putative functional relationship of the human ES to the common mucosa-associated immune system.     

Anatomic distribution and localization of immunocompetent cells in normal mouse endolymphatic sac

The distribution and anatomic localization of immunocompetent cells in normal mouse endolymphatic sac (ES) were examined by immunohistochemical methods. Antibodies against T cells (anti-Thy-1, -Lyt-1, -Lyt-2), macrophage (anti-Mac-1) and immunoglobulins (anti-IgM, -IgG, -IgA) were employed in an indirect technique utilizing strept-avidin-biotin complex. In the normal mouse ES, each of these cells could be detected by a difference in its frequency and distribution within and around the ES. Thy-1 positive (Thy-1+) cells are the most predominant in this tissue and can be seen throughout the ES. Lyt-1+ cells were found within the ES epithelium and in the perisaccular region, whereas Lyt-2+ cells were rarely present. Mac-1+ cells were present primarily in the lumen of the distal portion of the ES. IgM-bearing (IgM+) cells were seen in the subepithelial region, IgG+ cells were occasionally detected in the lumen and only a few IgA+ cells were present in the perisaccular region. This study revealed that the ES has the necessary immunological components for antigen processing and the generation of local immune responses within the inner ear.     

Time-dependent alterations of 3-fucosyl-N-acetyl-lactosamine (CD15) expression in the endolymphatic sac of adult guinea pigs after glycerol administration

The present study examined the effect of glycerol administration on 3-fucosyl-N-acetyl-lactosamine (CD15) epitope expression in the endolymphatic sac (ES) of the guinea pig's inner ear. Adult guinea pigs were injected intravenously with glycerol (2 g/kg body wt.). CD15 expression was studied at 80 min up to 5 h after treatment. In untreated animals single cells and cell groups in the ES expressing CD15 epitope intra- and intercellularly were identified by immunohistochemistry to be mainly in the epithelial layer of the rugosal and distal part of the sac. Glycerol administration modulated the expression of CD15 epitope. In the epithelial layer, expression decreased and was nearly depleted after 3 h. After 4 h of glycerol administration, CD15 expression reappeared and reached the comparable level of controls. The numbers of CD15-positive cells in the lumen of the ES increased steadily and arrived at their the highest levels in 2-h specimens. The localization of CD15-epitope expression and its modulation after glycerol administration within the ES implies that this molecule may play a role in re-establishing the sac's normal function. In addition, we speculate that CD15 may be associated with processes of an immune response in the inner ear.     

内淋巴囊同种抗原免疫致自身免疫性Mnière病的实验研究

采用同种粗制内耳抗原（CIEAg）在已致敏的豚鼠内淋巴囊（ES）局部免疫，诱发出明显的前庭和听觉功能障碍，以及与Meniere病极为相似的内耳膜迷路水肿改变。从而证明ES局部同种内耳抗原免疫是一种有效的造成Meniere病的方法，揭示部分Meniere病的发病机制可能与自身免疫因素有关。     

Immunohistochemical characterization of the human endolymphatic sac and its associated cell populations.

The use of monoclonal and polyclonal antibodies as specific markers for the localization of tissue constituents in situ allows the characterization of cells according to their state of differentiation and the detection of cellular antigens related to the function of cells and tissues. Our studies focus on the immunohistochemical characterization of the human endolymphatic sac (ES) and its associated cell populations. A panel of 37 monoclonal and polyclonal antibodies were used on frozen sections and fixed material from 64 ES of 32 persons without any clinically known inner ear disorders. The ES were removed at the time of autopsy; case histories were available. The results of our studies on the extraosseous part of the human ES support the assumption that the epithelium is metabolically active and capable of both secretion and absorption. The reactivity of the epithelial cells with antibodies against neuron specific enolase, chromogranin and somatostatin, respectively, implies a paracrine activity of the ES. Further results obtained with antibodies specific for cells of the immune system indicate a possible role of the human ES in the inner ear immune defence and a functional relationship of the ES to the common mucosa-associated immune system.     

内淋巴囊的外源性抗原特异性免疫应答

目的　研究内淋巴囊对于外源性胸腺依赖性抗原的特异性免疫应答。方法　用SD大鼠 32只 ,以抗原全身免疫后 ,经耳蜗底周向外淋巴腔注入相同抗原 ,分别于此后 1、3、7、14天处死动物取颞骨作组织学处理。然后应用免疫组化等技术 ,观察内淋巴囊的细胞浸润 ,免疫细胞增殖及其对抗原的吞噬清除作用。结果　内耳抗原接种后第 1、3天 ,内淋巴囊出现单核吞噬细胞浸润 ,第 7天出现浆细胞和淋巴细胞浸润 ;内耳抗原接种后第 3、7天 ,内淋巴囊的IgG阳性细胞增多 ,同时抗原被捕捉、递呈和吞噬。结论　内淋巴囊对外源性抗原可产生特异性免疫应答 ,是内耳局部免疫应答的重要场所。     

The mRNA of claudins is expressed in the endolymphatic sac epithelia

Objective

Claudins are a family of membrane proteins which localize to tight junctions (TJs). Recent studies have shown that claudins can form pores for ions in the TJs and regulate the permeability of epithelial paracellular ion transport. The endolymphatic sac (ES) is a part of the inner ear, absorbing the endolymphatic fluid. ES dysfunction may result in endolymphatic hydrops. In this study, we focused on the paracellular transport and examined claudin mRNA expression in the ES epithelia.

Materials and methods

Total RNA was isolated from whole ES epithelia of rats by laser capture microdissection. RT-PCR was used to evaluate the expression of claudins. The expression of each claudin mRNA in the epithelial cells of rat ES was confirmed by in situ hybridization.

Results

RT-PCR indicated the expression of cldn2, cldn4, cldn6, cldn7, cldn9, cldn11, cldn12, and cldn14. The expression of these claudin mRNAs in the epithelial cells of rat ES was confirmed by in situ hybridization.

Conclusion

We demonstrated mRNA expression of multiple claudins in the rat ES epithelia. These results in the ES epithelia were consistent with a role of claudins in paracellular ion transport.     

细胞间粘附分子-1在内耳免疫反应中的表达

目的探讨细胞间粘附分子１（ｉｎｔｅｒｃｅｌｕｌａｒａｄｈｅｓｉｏｎｍｏｌｅｃｕｌｅ１，ＩＣＡＭ１）在内耳免疫反应中的作用。方法采用钥孔血蓝蛋白激发已全身致敏的２６只大鼠内耳，诱发其内耳免疫反应，然后通过免疫组化技术检测内耳的ＩＣＡＭ１的表达。结果内耳激发后６小时，螺旋轴静脉及其回流小静脉即有ＩＣＡＭ１表达，１２小时内淋巴囊及其囊周区出现ＩＣＡＭ１表达，在２４～４８小时内耳各部位ＩＣＡＭ１表达达最高峰。７２小时各种炎性细胞浸润到内耳的各个部位。随后ＩＣＡＭ１表达逐渐减弱，２８天完全消失。结论内耳免疫反应时，ＩＣＡＭ１在炎性细胞从循环系统进入内耳的过程中发挥着重要作用，调控ＩＣＡＭ１表达的细胞因子也可能还来自内淋巴囊以外的其他细胞。