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.     

Mice lacking the B1 subunit of H+ -ATPase have normal hearing

Acid–base homeostasis of endolymph is thought to be essential for normal inner ear function. This assumption was supported by clinical data from individuals affected by autosomal recessive distal renal tubular acidosis with sensorineural hearing loss. This recessive syndrome was recently demonstrated to be due to mutations in the gene encoding the B1 subunit of H⁺-ATPase (ATP6B1). To examine the potential roles of H⁺-ATPase B1 subunit in inner ear development and function, we defined its spatial and temporal expression patterns in the developing mouse inner ear and examined the morphologic and physiologic effects of loss of its function. Standard in situ hybridization was used for the expression study with routine morphologic and physiologic assessments of hearing and balance in H⁺-ATPase B1 subunit (Atp6b1) null mutant mice. Atp6b1 mRNA was first detected at embryonic day 11.5 (E11.5) in the endolymphatic duct epithelia. From E16.5 onward, Atp6b1 was also observed in the presumptive interdental cell layer of the spiral limbus in the cochlea. Auditory brainstem response tests revealed normal hearing in mice lacking Atp6b1. The inner ears of these mice develop normally and show no overt morphological abnormalities. Our data demonstrate that Atp6b1 is not critical for normal inner ear development or normal inner ear function in mice and suggest that other proton-transporting mechanisms or pH buffering systems must allow the mouse inner ear to compensate for lack of normal Atp6b1 activity.     

Dynamic Expression of <Emphasis Type="Italic">Lgr5</Emphasis>, a Wnt Target Gene,in the Developing and Mature Mouse Cochlea

The Wnt signaling pathway is a recurring theme in tissue development and homeostasis. Its specific roles during inner ear development are just emerging, but few studies have characterized Wnt target genes. Lgr5, a member of the G protein-coupled receptor family, is a Wnt target in the gastrointestinal and integumentary systems. Although its function is unknown, its deficiency leads to perinatal lethality due to gastrointestinal distension. In this study, we used a knock-in reporter mouse to examine the spatiotemporal expression of Lgr5 in the cochlear duct during embryonic and postnatal periods. In the embryonic day 15.5 (E15.5) cochlear duct, Lgr5-EGFP is expressed in the floor epithelium and overlapped with the prosensory markers Sox2, Jagged1, and p27(Kip1). Nascent hair cells and supporting cells in the apical turn of the E18.5 cochlear duct express Lgr5-EGFP, which becomes downregulated in hair cells and subsets of supporting cells in more mature stages. In situ hybridization experiments validated the reporter expression, which gradually decreases until the second postnatal week. Only the third row of Deiters’ cells expresses Lgr5-EGFP in the mature organ of Corti. Normal cochlear development was observed in Lgr5^EGFP/EGFP and Lgr5^EGFP/+ mice, which exhibited normal auditory thresholds. The expression pattern of Lgr5 contrasts with another Wnt target gene, Axin2, a feedback inhibitor of the Wnt pathway. Robust Axin2 expression was found in cells surrounding the embryonic cochlear duct and becomes restricted to tympanic border cells below the basilar membrane in the postnatal cochlea. Both Lgr5 and Axin2 act as Wnt targets in the cochlea because purified Wnt3a promoted and Wnt antagonist suppressed their expression. Their differential expression among cell populations highlights the dynamic but complex distribution of Wnt-activated cells in and around the embryonic and postnatal cochlea.     

抗分泌因子与水通道蛋白1和2在大鼠内耳中的表达及其相互作用

目的研究抗分泌因子（antisecretory factor，AF）与水通道蛋白1、2（aquaporin-1，2，AQP1，2）在大鼠内耳中的表达及其相互作用。方法选取6只健康雄性SD大鼠，采用Envision二步法免疫组化技术，观察AF、AQP1和AQP2在大鼠内耳中的表达情况；选取20只健康雄性SD大鼠，分别取其前庭及耳蜗组织，运用免疫共沉淀结合蛋白质印记方法，用抗AQP1的单克隆抗体和抗AQP2多克隆抗体分别特异性地沉淀前庭和耳蜗组织中的蛋白抗原，用抗AF的特异性抗体检测沉淀物。结果AF在内耳分布广泛，耳蜗血管纹边缘细胞、螺旋韧带Ⅰ-Ⅴ型纤维细胞、前庭膜、基底膜、壶腹嵴等部位呈轻中度阳性反应，圆窗膜呈中强阳性反应，耳蜗螺旋神经节及前庭、耳蜗神经纤维均有阳性分布；AQP1主要分布于血管纹的中间细胞、螺旋韧带Ⅲ型纤维细胞、基底膜以及圆窗膜，染色强度为中重度；AQP2主要表达于螺旋韧带Ⅱ型、Ⅳ及Ⅴ型纤维细胞，呈中重度阳性染色反应，圆窗膜也有轻度表达。以AF特异性抗体分别检测AQP1及AQP2特异性抗体沉淀物中有清晰的阳性条带，相对分子质量约60000，与AF的相对分子质量吻合，而未加AQP1及AQP2特异性抗体的对照实验中无反应条带出现。结论AF、AQP1及AQP2在内耳组织中的分布有一定的规律，多位于与内淋巴关系密切的部位，提示三种蛋白可能参与内淋巴中水的调节。AF的分布区与AQP1及AQP2均有重叠，AQP1、AQP2与AF之间均存在相互结合作用。     

Col11a1 and Col11a2 mRNA expression in the developing mouse cochlea: implications for the correlation of hearing loss phenotype with mutant type XI collagen genotype

OBJECTIVE: Mutations in the fibrillar collagen genes COL11A1 and COL11A2 can cause sensorineural hearing loss associated with Stickler syndrome. There is a correlation of hearing loss severity, onset, progression and affected frequencies with the underlying mutated collagen gene. We sought to determine whether differences in spatial or temporal expression of these genes underlie this correlation, and to identify the cochlear cell populations expressing these genes and the structures likely to be affected by mutations. MATERIALS AND METHODS: We used in situ hybridization analysis of C57BL/6J mouse temporal bones. RESULTS: Similar, diffuse expression of Col11a1 and Col11a2 mRNA was first observed in the cochlear duct at embryonic Day 15.5, with increasingly focal hybridization being noted at postnatal Days 1 and 5 in the greater epithelial ridge and lateral wall of the cochlea. The greater epithelial ridge appeared to be the main, if not only, source of mRNA encoding Col11a1 and Col11a2 in the tectorial membrane. At postnatal Day 13, Col11a1 and Col11a2 expression became more focal and co-localized in the inner sulcus, Claudius' cells and cells of Boettcher. CONCLUSIONS: We did not observe spatial or temporal differences in mRNA expression that could account for the auditory phenotype genotype correlation. The expression patterns suggest essential roles for Col11a1 and Col11a2 in the basilar or tectorial membranes.     

Cochlear abnormalities in insulin-like growth factor-1 mouse mutants

Insulin-like growth factor 1 (IGF-1) modulates inner ear cell proliferation, differentiation and survival in culture. Its function in human hearing was first evidenced by a report of a boy with a homozygous deletion of the Igf-1 gene, who showed severe sensorineural deafness [Woods et al., New Engl. J. Med. 335 (1996) 1363–1367]. To better understand the in vivo role of IGF-1 during inner ear differentiation and maturation, we studied the cochleae of Igf-1 gene knockout mice by performing morphometric stereological analyses, immunohistochemistry and electron microscopy on postnatal days 5 (P5), P8 and P20. At P20, but not at P5, the volumes of the cochlea and cochlear ganglion were significantly reduced in mutant mice, although the reduction was less severe than whole body dwarfism. A significant decrease in the number and average size of auditory neurons was also evident at P20. IGF-1-deficient cochlear neurons showed increased apoptosis, along with altered expression of neurofilament 200 kDa and vimentin. The eighth nerve, the cochlear ganglion and the fibers innervating the sensory cells of the organ of Corti of the P20 mouse mutants presented increased expression of vimentin, whereas the expression of neurofilament was decreased. In addition, the myelin sheath was severely affected in ganglion neurons. In conclusion, IGF-1 deficit in mice severely affects postnatal survival, differentiation and maturation of the cochlear ganglion cells.     

Delta/Notch-Like EGF-Related Receptor (DNER) is Expressed in Hair Cells and Neurons in the Developing and Adult Mouse Inner Ear

The Notch signaling pathway is known to play important roles in inner ear development. Previous studies have shown that the Notch1 receptor and ligands in the Delta and Jagged families are important for cellular differentiation and patterning of the organ of Corti. Delta/notch-like epidermal growth factor (EGF)-related receptor (DNER) is a novel Notch ligand expressed in developing and adult CNS neurons known to promote maturation of glia through activation of Notch. Here we use in situ hybridization and an antibody against DNER to carry out expression studies of the mouse cochlea and vestibule. We find that DNER is expressed in spiral ganglion neuron cell bodies and peripheral processes during embryonic development of the cochlea and expression in these cells is maintained in adults. DNER becomes strongly expressed in auditory hair cells during postnatal maturation in the mouse cochlea and immunoreactivity for this protein is strong in hair cells and afferent and efferent peripheral nerve endings in the adult organ of Corti. In the vestibular system, we find that DNER is expressed in hair cells and vestibular ganglion neurons during development and in adults. To investigate whether DNER plays a functional role in the inner ear, perhaps similar to its described role in glial maturation, we examined cochleae of DNER−/− mice using immunohistochemical markers of mature glia and supporting cells as well as neurons and hair cells. We found no defects in expression of markers of supporting cells and glia or myelin, and no abnormalities in hair cells or neurons, suggesting that DNER plays a redundant role with other Notch ligands in cochlear development.     

Expression patterns of aquaporins in the inner ear: evidence for concerted actions of multiple types of aquaporins to facilitate water transport in the cochlea

Water transport between the perilymph and endolymph is important in regulations of volume and osmotic pressure of the inner ear labyrinth. It is now known that expression of water channels (aquaporins or AQPs) in the cell membrane dramatically increases the ability of water to cross epithelial cells. The aims of the current study were to investigate the cellular localization of AQPs by immunolabeling, and to study the developmental expression and relative abundance of various subtypes of AQPs. We report here that AQP3, AQP7 and AQP9 were expressed in the inner ear. Specific subtypes of AQPs were found in discrete regions expressed by both epithelial cells and fibrocytes in cochlear and vestibular organs. Semi-quantitative measurements showed that AQP4 and AQP1 were the two most abundantly expressed AQP subtypes in the inner ear, and their expressions were dramatically upregulated during development. These data showed a highly localized and largely non-overlapping distribution pattern for different subtypes of AQPs in the inner ear, suggesting the existence of regional subtype-specific water transport pathways, and global regulation of water transport in the inner ear may require concerted actions of multiple types of AQPs.     

水通道蛋白1、2、5在膜迷路积水豚鼠耳蜗中的分布及表达研究

目的观察水通道蛋白(aquaporin,AQ)1、2、5(AQP1、AQP2、AQP5)三种蛋白在膜迷路积水豚鼠耳蜗的表达及分布情况,探讨其在内淋巴积液中可能的发生机制。方法选择健康红目豚鼠60只,随机分为空白组(30只)和模型组(30只),模型组给予醋酸去氨加压素6μg·kg-1·d-1腹腔注射10 d,空白组以1 ml生理盐水腹腔注射10 d;常规饲养7天后取出耳蜗,通过免疫组化、Western-blot方法观察AQP1、AQP2、AQP5蛋白在豚鼠耳蜗的分布及表达。结果免疫组化示空白组、模型组豚鼠耳蜗组织中AQP1表达的光密度值分别为0.134±0.021、0.273±0.053;AQP2表达分别为0.089±0.013、0.261±0.100;AQP5表达分别为0.264±0.065、0.151±0.098;与空白组比较,模型组AQP1、AQP2蛋白表达明显上调(P<0.01),AQP5蛋白表达明显下调(P<0.05)。Western-blot示空白组、模型组豚鼠耳蜗组织中AQP1表达分别为0.214±0.034、0.313±0.032;AQP2表达分别为0.283±0.050、0.544±0.042;AQP5表达分别为0.291±0.041、0.199±0.033;与空白组比较,模型组AQP1、AQP2蛋白表达上调(P<0.01),AQP5蛋白表达明显下调(P<0.05)。结论AQP1、AQP2、AQP5蛋白均可能参与了豚鼠耳蜗膜迷路积水的发生。     

共同腔畸形人工耳蜗手术适应证及手术方法的探讨

目的：探讨共同腔畸形人工耳蜗手术适应证以及人工耳蜗电极植入人路的选择。方法：在对重度或全聋患者进行人工耳蜗植入术前影像掌检查中,发现了6例耳蜗、前庭、外半规管呈共同腔畸形,其中5例有残留听力,1例未查到残留听力。结果：6例影像学检查呈共同腔畸形患者中,对5例有残留听力患者进行了人工耳蜗植入,其中3例选择了常规入路植入电极,2例选择了经乳突侧入路植入电极,术后均建立了人工耳蜗的听觉反应。1例因未查到残留听力,放弃了人工耳蜗手术治疗。结论：有残留听力的共同腔畸形患者,如果能够接受术后听觉言语识别效果差的事实,可以进行人工耳蜗手术。无残留听力或无法了解到有听觉反应的共同腔畸形患者,在现有技术条件下应放弃人工耳蜗植入手术。     

Effects of lithium on endolymph homeostasis and experimentally induced endolymphatic hydrops

There is evidence to suggest that water homeostasis in the inner ear is regulated via the vasopressin (VP)-aquaporin 2 (AQP2) system in the same fashion as in the kidney. The VP-AQP2 system in the kidney is well known to be inhibited by lithium, resulting in polyuria due to a decrease in reabsorption of water in the collecting duct of the kidney. Therefore, lithium is also likely to inhibit the VP-AQP2 system in the inner ear, and consequently exert some influence on inner ear fluid homeostasis. In this study, we investigated the effects of lithium on AQP2 expression in the rat inner ear, and on the cochlear fluid volume in hydropic ears of guinea pigs. A quantitative PCR study revealed that lithium reduced AQP2 mRNA expression in the cochlea and endolymphatic sac. Lithium application also decreased the immunoreactivity of AQP2 in the cochlea and endolymphatic sac. In a morphological study, lithium intake significantly reduced endolymphatic hydrops dose-dependently. These results indicate that lithium acts on the VP-AQP2 system in the inner ear, consequently producing a dehydratic effect on the endolymphatic compartment.     

内耳结构异常的语前聋人工耳蜗植入儿童听觉语言康复效果评价

目的评价不同类型内耳结构异常的语前聋儿童人工耳蜗植入术后听觉语言康复效果。方法选取人工耳蜗植入儿童10例(12耳),其中内耳结构发育正常的3例,前庭导水管扩大3例,Mondini畸形1例,蜗孔狭窄1例,内听道狭窄听神经纤细2例,分别采用电刺激诱发听神经复合动作电位(electrically evoked compound action potential,eCAP)、助听听阈、听障儿童听觉能力、语言能力评估标准及方法,从听神经客观电生理水平、听察觉阈、听觉辨识与理解能力以及语言能力4方面评价患儿的康复效果。结果①客观电生理:单纯前庭导水管扩大患者听神经对电刺激敏感度与内耳发育正常者相似,Mondini畸形患者比内耳发育正常者略高,蜗孔狭窄及听神经纤细患者听神经敏感度较差;②听察觉阈:与内耳结构正常及轻度畸形(Mondini畸形)患者相比,蜗孔狭窄及听神经纤细患者听察觉阈偏高,高频比低频阈值低;③听觉辨识与理解能力:在安静环境近距离交谈情境下,单纯前庭导水管扩大、Mondini畸形及蜗孔狭窄患者对熟悉语词和短句的辨识与理解能力与内耳发育正常者无差别,听神经纤细患者得分较低;④语言能力:单纯前庭导水管扩大、Mondini畸形患者与内耳发育正常者无差别,蜗孔狭窄患者的语言交往与表达能力落后于内耳发育正常者,听神经纤细患者在语法、理解、交往、表达4个维度均落后于内耳发育正常者。结论语前聋儿童人工耳蜗植入术后听觉语言康复效果需要使用多种方法综合评价。Mondini畸形患者给予足够电刺激量后,其听觉语言能力可达到内耳发育正常者水平。蜗孔狭窄、听神经纤细等严重内耳畸形患者,人工耳蜗对其听觉语言能力发展有一定帮助,家长需要建立合理期望值并坚持长期康复。     

Trophic effects of insulin-like growth factor-I (IGF-I) in the inner ear

Insulin-like growth factors (IGFs) have a pivotal role during nervous system development and in its functional maintenance. IGF-I and its high affinity receptor (IGF1R) are expressed in the developing inner ear and in the postnatal cochlear and vestibular ganglia. We recently showed that trophic support by IGF-I is essential for the early neurogenesis of the chick cochleovestibular ganglion (CVG). In the chicken embryo otic vesicle, IGF-I regulates developmental death dynamics by regulating the activity and/or levels of key intracellular molecules, including lipid and protein kinases such as ceramide kinase, Akt and Jun N-terminal kinase (JNK). Mice lacking IGF-I lose many auditory neurons and present increased auditory thresholds at early postnatal ages. Neuronal loss associated to IGF-I deficiency is caused by apoptosis of the auditory neurons, which presented abnormally increased levels of activated caspase-3. It is worth noting that in man, homozygous deletion of the IGF-1 gene causes sensory-neural deafness. IGF-I is thus necessary for normal development and maintenance of the inner ear. The trophic actions of IGF-I in the inner ear suggest that this factor may have therapeutic potential for the treatment of hearing loss.