鼠内耳内淋巴系统的免疫组化特点

目的 众所周知,内耳有两套淋巴系统:外淋巴与内淋巴.外淋巴本质与脑脊液相似,支持膜迷路.内淋巴成分与外淋巴不同、充满膜迷路,且参与内耳免疫应答.本研究旨在使用多种针对淋巴管及血管的特异性抗体来研究内耳淋巴系统的特征,试图探寻内耳淋巴系统与外周淋巴系统、血管系统有何异同.方法 我们采用多重免疫方法,检测小鼠内淋巴系统中CD31,tomato lectin和podoplanin,LYVE-1,LA102的表达.结果 显示内淋巴系统不显示血管标记(CD31和tomato lectin),而血管纹的内皮细胞则与其它普通血管内皮细胞有相同的免疫表型.内淋巴系统表达部分淋巴标记(podoplanin).结论 这些数据提示与血管系统相比,内淋巴系统可能从结构及功能上更类似于体内其它部位的淋巴系统.     

Intermediate filaments in the newborn inner ear of the mouse

The presence of intermediate filaments in the inner ear of the newborn mouse was analyzed with immunofluorescence techniques using antibodies against the five classes of intermediate filaments: cytokeratins, vimentin, desmin, neurofilaments and glial fibrillary acid protein (GFA). Neurofilaments were found in all nerve fibers from the ganglion cell to the hair cell. In the vestibular ganglion two subpopulations of ganglion cells were identified: a minor part staining intensively with neurofilament and the major part of cells lacking this immunofluorescence. Vimentin occurred in a number of supporting structures in the membranous labyrinth, but not in vestibular or cochlear ganglion cells. Cytokeratins, desmin or GFA were not identified in the inner ear.     

Developmental expression of aquaporin 2 in the mouse inner ear

OBJECTIVES: The maintenance of endolymph homeostasis is critical for the inner ear to perform its functions of hearing and maintaining balance. The identification and cloning of aquaporins (a family of water channel proteins) has allowed the study of a novel cellular mechanism potentially involved in endolymph homeostasis. The objective of the present study was to define the developmental temporal and spatial expression pattern of aquaporin 2 (Aqp2) in the developing mouse inner ear. STUDY DESIGN: A systematic immunohistochemical study of Aqp2 protein expression was performed on embryonic mouse inner ears ranging from embryonic day 10 (otocyst stage) to embryonic day 18 (just before birth). METHODS: Serial cryosections of embryonic mouse inner ears were used for immunohistochemical experiments. A rabbit polyclonal antisera raised against a synthetic Aqp2 peptide was used with a standard nickel intensified 3,3-diaminobenzidine reaction protocol for immunolocalization of Aqp2 in tissue sections. RESULTS: Aquaporin 2 is expressed diffusely in the early otocyst, then becomes progressively restricted as the inner ear matures. During early cochlear duct formation (embryonic days 12 and 13), expression of Aqp2 is homogeneous; later, it becomes restricted to specific regions of the endolymphatic compartment (embryonic days 15 and 18). Similar restriction of expression patterns could be noted for the vestibular structures. Endolymphatic duct and sac and stria vascularis expression of Aqp2 was noted to occur fairly late during development but demonstrated a distinct pattern of immunolabeling. CONCLUSIONS: Aquaporin 2 shows an early and specific pattern of expression in the developing mouse inner ear, suggesting a significant role for this water channel protein in the development of endolymph homeostasis and meriting further functional studies of Aqp2 in the inner ear.     

Inner ear fine structures of the hamster in frozen sections used for immunohistochemical assays for inner ear diseases

Summary Frozen sections of the inner ear of the hamster enable detailed investigations of the fine structures in immunofluorescence assays. At high magnification single mitochondria can be identified by their reactions with an antiserum containing antibodies against mitochondria. In the positive reaction with an antiserum against nuclei, the typical green fluorescence is restricted to the nuclei, which are mostly separated by the surrounding cytoplasm. The method of immunohistochemical assay using frozen sections from the non-decalcified inner ear is very time-consuming and cannot be recommended for the routine diagnosis of inner ear diseases, although it may be useful in research and for studying critical clinical cases. Offprint requests to: N. R. Wei     

Expression of glycoconjugates in the mouse inner ear after prenatal irradiation

Summary Irradiation of the murine fetal inner ear is known to produce damage both to the vestibular and cochlear parts in the adult mouse. Fluorescein-labelled lectins were used to reveal possible differences in the glycoconjugate content between normal and irradiated inner ears. In the vestibular part, the otoconia showed the highest uptake of labelled sugars. This uptake was weaker after irradiation when compared to non-irradiated specimens. The type I hair cells in the ampulla and in the utricle showed a weaker uptake, but no labelling was demonstrated in the type II hair cells compared to the non-irradiated controls. In the cochlear part of the inner ear almost no uptake of fluorescent-binding lectins could be demonstrated in the irradiated groups except for in the tectorial membrane. In the endolymphatic sac no uptake was shown after prenatal irradiation. These findings are discussed and correlated to the already known damage of the inner ear following prenatal irradiation.     

小鼠内耳水通道蛋白的定位及其意义

目的检测水通道蛋白(waterchannelprotein;aquaporin,Aqp)亚型在小鼠内耳不同部位的分布和各亚型所在的部位。方法使用成年小鼠30只,经活体灌注,切取双侧颞骨,按石蜡包埋技术处理和切片。使用免疫组织化学方法标记确认小鼠内耳组织中水通道蛋白亚型1、3、4、5、7、9分布情况。结果Aqp-1为1∶200和Aqp-3、7、9为1∶100的一抗浓度可以看到它们在内耳不同部位稳定、清晰的染色反应,但Aqp-4和5使用1∶50甚至1∶30也未观察到染色。Aqp-1在圆窗膜、螺旋韧带、内淋巴囊和内淋巴管、椭圆囊和球囊以及内耳血管壁等处被标记;Aqp-3在螺旋韧带、前庭唇、内、外螺旋沟、基底膜和基底膜嵴、内淋巴囊和内淋巴管、膜半规管和椭圆囊、球囊斑等处显示荧光染色。Aqp-7在血管纹、基底膜、前庭膜、椭圆囊和球囊及其囊斑有反应染色,而Aqp-9在螺旋缘、前庭唇、内、外螺旋沟、前庭膜、膜半规管和球囊及其囊斑有较强染色。结论水通道蛋白亚型1、3、7、9广泛分布于小鼠内耳不同组织中,其分布部位和反应强弱存在差异,主要存在与内淋巴密切相关的组织结构中。     

A morphometric study of the pallid mutant mouse inner ear

Mice homozygous for the mutant gene pallid (pa/pa) often lack otoconia in some or all of their maculae and are used to study the influences of gravity receptor hypostimulation on vestibular-related behaviors. Since the value of this animal model is based on the assumption that the vestibular sensorineural elements are normal, a morphometric analysis was done on the inner ear of these otoconia-deficient mice to see whether sensori-neural structures are also affected by the pallid gene. In pallid mice lacking all otoconia, the sensory epithelia of the utricle, saccule, and semicircular canal cristae were the same size as in their heterozygous (pa/+) controls. Although the superior and inferior divisions of the vestibular ganglion of the pallid mice were smaller than normal, the first-order neurons within these divisions were normal in size, number, and density. However, the superior divisions in both groups had larger neurons than did the inferior divisions. Within the pallid cochlea, first-order auditory neurons within the spiral ganglion were smaller than normal, but the scala media was larger. Since the significant vestibular influences of the pallid gene are limited primarily to the otoconia, behavioral abnormalities reported for these otoconia-deficient mice are apparently due only to gravity receptor hypostimulation.     

A new immunohistochemical method for the detection of gentamicin in inner ear fluid compartments

A new method was developed for frozen section detection of antigens that natively occur in the cochlear peri- and endolymph. A combination of immunohistochemistry and immunoblot assay enabled topological and quantitative detection of small and hydrophilic molecules (such as the aminoglycoside antibiotics) in frozen sections of the inner ear compartments (scala tympani, scala vestibuli and cochlear duct). A selective localization is possible in the peri- and endolymphatic region of each coil of the cochlea. During sectioning of the cochlea, a small piece of a nitrocellulose membrane is placed to the surface of the intersection and briefly warmed. The sections are cut, simultaneously attached to a nitrocellulose membrane on which the aminoglycoside antibiotics remain adsorbed without any fixation procedure. Using this method, immunoincubation to detect gentamicin was performed in a way usually done in western blot analysis. Results with two different enzyme reactions with the enzyme conjugated to a second antibody (i.e., dye as substrate and the chemiluminescence detection system) are presented and compared. This histoimmunoblot assay provides a general non-radioactive and sensitive immunohistochemical tool for the localization of compounds occurring in extracellular body fluid compartments. For inner ear research this method now enables the investigation of the penetration and distribution of therapeutics in peri-and endolymphatic sites and can even be applied to separately quantifying concentrations of a substance in different coils of the same cochlear section.     

Spatiotemporal expression of otogelin in the developing and adult mouse inner ear

Using a PCR-based subtractive method on cDNA from 2-day-old mouse cochlea, we identified a gene encoding otogelin, Otog, an inner ear specific glycoprotein expressed in all acellular structures. Here, we provide evidence that otogelin is detected as early as embryonic day 10 in the otic vesicle. At this stage, otogelin is detected in the epithelial cells which do not overlap with the myosin VIIA-expressing cells, namely the precursors of the hair cells, thus arguing for an early commitment of the two cell populations. Analysis of otogelin spatiotemporal cell distribution allows a molecular tracing for the contribution of the cochlear and vestibular inner ear supporting cells to the formation of the acellular structures. Throughout embryonic and adult life, the expression of the otogelin gene as monitored by LacZ inserted into Otog, and the abundance of the protein are greater in the vestibule than in the cochlea. In adult, otogelin is still produced by the vestibular supporting cells, which argues for a continuous process of otogelin renewal in the otoconial membranes and cupulae. In contrast, in the tectorial membrane, otogelin should be a long-lasting protein since both the otogelin gene and protein were almost undetectable in adult cochlear cells. The data are consistent with the requirement for otogelin in the attachment of the otoconial membranes and cupulae to their corresponding sensory epithelia as revealed in Otog -/- mice.     

The effect of noise on deoxyglucose uptake into inner ear tissues of the mouse

Summary The uptake of deoxyglucose into inner ear tissues was studied in the mouse. Sixty minutes after a single i.v. injection of 5 mCi 2-deoxy-D-[³H]glucose/kg body weight, stria vascularis/spiral ligament and organ of Corti as well as other body tissues were dissected and analyzed for radioactivity. Uptake into inner ear tissues was three to five times lower than into brain or heart. The ratio of deoxyglucose-6-phosphate to deoxyglucose was 6040 and the compounds were eliminated from the inner ear with a half life of approximately 60 min. Exposure to 100 dB of white noise during the radioactive pulse decreased uptake of deoxyglucose into both stria vascularis/spiral ligament and organ of Corti by 50%.This work was supported by grant NS 12881 from the National Institutes of Health and Program Project grant NS 05785 and training grant NS 07106     

小鼠耳蜗胚胎发育过程中Smad4基因的表达

目的探讨小鼠内耳胚胎发育演变过程，检测Smad4基凶在小鼠耳蜗中的表达情况。方法选用耳廓反应灵敏、健康的C57BL／6小鼠作为种鼠交配，用观察阴栓方法获得适龄胎鼠，≤17天取胚胎头，≥18天在显微镜下取耳蜗，胚胎头水平冰冻切片，耳蜗平行于蜗轴冰冻切片，HE染色方法观察小鼠耳蜗发育形态演变过程，免疫组织化学方法检测Smad4蛋白在小鼠胚胎10～20天耳蜗巾的表达情况。结果胚胎10天，听泡发育．胚胎12天听泡下部有蜗管始基形成并开始发育．胚胎18天，蜗管发育2圈，形成了可以辨认的内、外毛细胞．血管纹开始分化。Smad4从胚胎15天才开始表达，最初表达部位为耳蜗底转将发育成蜗轴以及柱状上皮分化为感觉细胞及支持细胞的区域，但在胚胎早期表达较弱，后期在耳蜗内广泛表达，在螺旋器、血管纹、前庭膜均有表达，且表达逐渐增强，而在蜗轴部位的表达逐渐减弱。结论Smad4在小鼠内耳分化期有阳性表达，且表达具有明显的阶段性和区域性，说明其参与内耳听觉器官的发育过程并且可能存耳蜗的形成过程中起着重要的作用。     

The orientation and its significance of water channel protein in the mouse inner ear]

OBJECTIVE: To examine the distribution of various subtypes of water channel protein (aquaporin, Aqp) in various structures of the mouse inner ear. METHODS: Thirty mice with white color were used in this study and were cardiacally perfused. The temporal bones were taken out and were processed and sectioned by paraffin-embedded technique. The sections were labeled with fluorescent antibody by immunohistochemical method. The distribution of Aqp1, 3, 4, 5, 7, 9 were confirmed in the inner ear of mouse. RESULTS: The constant and clear fluorescent reaction could be observed in the inner ear tissue with the first antibody concentration of 1:200 for Aqp-1 and 1:100 for Aqp-3, 7, 9. But the reaction of Aqp-4 and 5 could not be found with the concentration of 1:50, even 1:30. Aqp-1 was labeled in the round window membrane, spiral ligament, endolymphatic sac and duct, utricle and saccule and the wall of inner ear blood vessel; Aqp-3 in the spiral limb, vestibular lip, internal and external sulci, basilar membrane and basilar membrane crest, endolymphatic sac and duct, membranous semicircular canal and utricle and saccular macula; Aqp-7 in the stria vascularis, basilar membrane, Reissner's membrane, utricle and saccule and their maculae; Aqp-9 in the spiral limb, vestibular lip internal and external sulci, Reissner's membrane, membranous semicircular canal, saccule and its macula. CONCLUSION: Aqp-1, 3, 7, 9 were extensively distributed in various tissues of mouse inner ear. Their distribution sites and reaction degree were different, and mainly located in the structures related to the endolymph.     

A ferritin-containing cell type in the stria vascularis of the mouse inner ear

This report describes a new cell type within the stria vascularis of the mouse inner ear. The cell is similar ultrastructurally to the classically described intermediate cell. However, it can be distinguished by the presence of dense vacuoles, presumably lysosomes, within which can be visualized electron dense particles resembling ferritin molecules. In addition, the ferritin-like particles are present throughout the cytoplasm and occasionally within the endoplasmic reticulum. These cells characteristically abut capillary basal lamina. Electron probe analysis of the dense vacuoles revealed the presence of iron. It is suggested that these cells may sequester iron released from dying erythrocytes in the strial capillary system, whereupon the iron is conserved through ferritin synthesis.