Epidermal Growth Factor Upregulates Production of Supernumerary Hair Cells in Neonatal Rat Organ of Corti Explants

The organ of Corti is highly ordered, with a single row of inner hair cells and three rows of outer hair cells. The number of hair cells produced was thought to be limited by the time of their terminal mitosis (i.e. E14 in the mouse). However, exogenous application of retinoic acid has been shown to stimulate the formation of supernumerary hair cells in organ of Corti explants from E13 to E16 mouse embryos. Using late embryonic and neonatal rat organ of Corti explants, we investigated the potential for production of supernumerary hair cells in more mature auditory sensory epithelia. When newborn rat organ of Corti explants were cultured under control conditions, an area of supernumerary hair cells was observed in a segment of organ of Corti that was at the junction between the basal and middle turns. In these areas of supernumerary hair cells the number of hair cells increased per unit of length, but remained constant per surface unit, further demonstrating the supernumerary character of this phenomenon. Organ of Corti explants treated with epidermal growth factor (EGF) showed a 50% increase in the length of the organ of Corti segment containing supernumerary hair cells. Upregulation of supernumerary hair cell formation by EGF was found to start and be maximal at birth (P0) and to disappear by 2 days after birth (P2). Treatment of EGF stimulated P0 explants with an antimitotic drug, cytosine arabinoside (ARAc), demonstrated that the production of supernumerary hair cells occurred independently of cell division.     

Supernumerary outer hair cells arise external to the last row of sensory cells in the organ of corti

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells.     

Identification of factors that maintain mammalian outer hair cells in adult organ of Corti explants

Both outer hair cells (OHCs) and inner hair cells (IHCs) survive and mature in 3 days old rat organ of Corti explants cultured for 1 month in a minimal essential medium. In contrast, under the same culture conditions, only IHCs survive in explants from adult guinea pig organ of Corti while many of the OHCs are lost within the first 48 h. Hair cell counts show OHCs loss to be greater in the lower portion (i.e. middle turn) of the cochlea than at the apex. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) indicates that there is DNA damage in adult OHCs within 8 h of explantation. Treatment of the adult organ of Corti explants with either actinomycin D (10⁻⁷ M) or cycloheximide (10⁻⁶ M) prevents most OHC losses. According to these results apoptosis may be the mechanism of OHC loss in adult organ of Corti explants. Stable membrane potentials recorded from the OHCs in both uncultured and actinomycin D-treated organ of Corti explants cultured for 72 h demonstrate the functional integrity of these hair cells. OHC losses in the adult guinea pig organ of Corti cultures can also be prevented by treatment with several of the growth factors tested, i.e. acidic fibroblast growth factor (aFGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), transforming growth factor-β1 (TGF-β1), and glial cell-derived neurotrophic factor (GDNF). The results of this study suggest that growth factor therapy may be applicable to the treatment of some hearing disorders.     

出生后大鼠听毛细胞纤毛发育过程

目的观察新生大鼠（P1、P7、P14、P21）耳蜗Corti器的形态结构及排列方式,以便了解听毛细胞纤毛发育的过程。方法取新乍SD大鼠四个阶段的耳蜗,采刖扫描电镜对出生后大鼠耳蜗Corti器形态结构进行系统观察。结果发现这四个阶段在体犬鼠耳蜗毛细胞从出生至毛细胞完全发育成熟,绝大部分都是以一排内毛细胞和三排外毛细胞的方式排列的,只有少部分在局部发现多出几个外毛细胞形成第四排。毛细胞的形态结构及排列方式随着时间的改变逐渐发育成熟。P14这个阶段是个重要的时期,Corti器表面柱细胞头板增宽,表面的微绒毛减少,Deiter细胞表面微绒毛也减少,动纤毛从底圄至顶圈逐渐退化已经基本结束,P14这个阶段Corti器各个部分发育已经完全成熟：结论出生后14天之内。大鼠听毛细胞静纤毛和动纤毛与听器其他细胞形态结构仍不同程度地处于由不成熟到成熟的逐渐发育过程,为研究大鼠出生后Corti器的发育、听觉功能的建立和完善提供了比较可靠的形态学证据。     

胚胎小鼠耳蜗感觉上皮的形态发生

目的了解胚胎小鼠耳蜗感觉上皮发育的形态学特征及规律。方法利用光镜、扫描电镜、透射电镜观察从胚胎第8天到刚出生期间C57BL/6小鼠耳蜗感觉上皮发育过程中的形态变化。结果C57BL/6小鼠胚胎第10.5天耳蜗开始发育,感觉上皮由高柱状上皮细胞组成,到胚胎第14天才开始分化为原始的毛细胞;胚胎第16天耳蜗底转Corti器原基形成,毛细胞和支持细胞初具雏形;到出生时,耳蜗形态基本成熟,但底转Corti器仅趋向成熟,蜗尖才形成Corti器原基。结论小鼠耳蜗感觉上皮发育的形态变化在胚胎第13天到出生时最明显,感觉细胞都来源于一种高柱状上皮细胞,到胚胎第14天开始分化为毛细胞;而Corti器的分化成熟是从底转向顶转进行的,出生时尚未完全发育成熟。     

Vector-mediated delivery of bcl-2 prevents degeneration of auditory hair cells and neurons after injury

OBJECTIVE: To test the hypothesis that bcl-2 prevents oxidative stress-induced apoptosis of auditory sensory cells in explants of the organ of Corti and dissociated cell cultures of the spiral ganglion. METHODS: Organ of Corti explants and dissociated spiral ganglion cell cultures obtained from 3-day-old (P3) rats or adult spiral ganglion cell cultures from 28-day-old (P28) rats were transduced with vectors containing a human bcl-2 gene. Cultures were then exposed to neomycin, cisplatin or subjected to withdrawal of neurotrophin supplementation. Outcome measures included hair cell and neuron counts, mitochondrial membrane potential and a histological measure of apoptosis. RESULTS: Expression of bcl-2 in the organ of Corti explants and neuronal cell cultures provided a significant level of protection against cell death. Bcl-2 expression in the organ of Corti explants also protected mitochondria from loss of membrane potential and blocked an early step in the commitment of hair cells to apoptosis. CONCLUSION: Expression of bcl-2 in cochlear tissues protects sensory cells from a variety of insults that have been demonstrated to damage the inner ear.     

Triamcinolone acetonide protects auditory hair cells from 4-hydroxy-2,3-nonenal (HNE) ototoxicity in vitro

CONCLUSION: Triamcinolone acetonide crystalline suspension (e.g. Volon A) was not ototoxic to the auditory hair cells present within organ of Corti explants and protected them from an ototoxic molecule, i.e. 4-hydroxy-2,3-nonenal (HNE), that is produced within the organ of Corti as a result of oxidative stress-induced damage. OBJECTIVES: To test the corticosteroid, triamcinolone acetonide, for ototoxicity and otoprotective capacity in organ of Corti explants. MATERIALS AND METHODS: Organ of Corti explants excised from 4-day-old rats were the test system, HNE was the ototoxin challenge. Hair cell integrity counts were performed with fluorescent microscopy on fixed explants stained with FITC-labeled phalloidin. Statistical significance was set at p<0.05. RESULTS: Triamcinolone acetonide did not affect hair cell integrity in the organ of Corti explants and it provided a high level of protection of hair cells against the ototoxic effects of a damaging level of HNE as determined by hair cell density counts.     

Triamcinolone acetonide protects auditory hair cells from 4-hydroxy-2,3-nonenal (HNE) ototoxicity in vitro

Abstract

Conclusion. Triamcinolone acetonide crystalline suspension (e.g. Volon A®) was not ototoxic to the auditory hair cells present within organ of Corti explants and protected them from an ototoxic molecule, i.e. 4-hydroxy-2,3-nonenal (HNE), that is produced within the organ of Corti as a result of oxidative stress-induced damage. Objectives. To test the corticosteroid, triamcinolone acetonide, for ototoxicity and otoprotective capacity in organ of Corti explants. Materials and methods. Organ of Corti explants excised from 4-day-old rats were the test system, HNE was the ototoxin challenge. Hair cell integrity counts were performed with fluorescent microscopy on fixed explants stained with FITC-labeled phalloidin. Statistical significance was set atp <0.05. Results. Triamcinolone acetonide did not affect hair cell integrity in the organ of Corti explants and it provided a high level of protection of hair cells against the ototoxic effects of a damaging level of HNE as determined by hair cell density counts.     

细胞增殖及凋亡与耳蜗感觉上皮的分化

目的观察耳蜗发育中细胞增殖、凋亡的变化规律，探讨它们与耳蜗感觉上皮分化的关系。方法利用透射电镜观察从胚胎第8天到刚出生的C57BL/6小鼠耳蜗感觉上皮的增殖、凋亡及分化的变化。结果胚胎第10天，耳蜗才开始发育；到胚胎第11天耳蜗感觉上皮出现核分裂像并逐渐增多，胚胎第14天时已开始减少；细胞凋亡现象出现在胚胎第13天，从胚胎第14天到胚胎第15天细胞凋亡最明显，后逐渐减少；同时在胚胎第14天毛细胞开始分化形成，到胚胎第16天，Corti器原基形成时，毛细胞和支持细胞形态趋向成熟，但到出生时，Corti器尚未发育成熟。结论细胞增殖和凋亡是耳蜗发育中的必然现象，细胞增殖、凋亡及毛细胞的分化成熟相继发生但又互相重叠；细胞增殖与凋亡的动态平衡在耳蜗感觉上皮分化成熟中起着重要作用。     

新霉素损伤后小鼠耳蜗毛细胞的改变[论著]

目的　观察新霉素对小鼠听功能及耳蜗毛细胞形态学变化的影响。方法　选取C57BL/6小鼠16只在出生后第8天开始连续10 d皮下注射硫酸新霉素，对照组（10只）皮下注射等量生理盐水。停药后1、4、8、12 w进行听性脑干反应（ABR）检测。每次ABR检测后，新霉素组随机取3只小鼠，对照组随机取2只小鼠，处死后，取耳蜗进行冰冻切片，用免疫荧光方法观察耳蜗Corti器及毛细形态学变化。结果　新霉素可造成小鼠耳蜗毛细胞严重损伤，且小鼠ARB阈值显著增高，不可恢复；新霉素对耳蜗Corti器毛细胞的损伤顶圈最轻，中圈次之，底圈最重，且随着时间推移Corti器形态结构破坏逐渐加重且不可自我修复。结论　硫酸新霉素造成小鼠Corit器毛细胞的损伤是不可逆的。     

Transplantation of neural stem cells into explants of rat inner ear

Damage and loss of hair cells in the inner ear is the most frequent cause of hearing loss and balance disorders. Mammalian hair cells do not regenerate in the conventional ways. To regenerate the hair cell in the mammalian inner ear we transplanted neural stem cells into explants of rat inner ear. The stem cells integrated successfully into the sensory epithelium of the vestibular organs, but not into the organ of Corti. This method is useful to investigate efficient ways to transplant stem cells into the inner ear.     

Comparative anatomy of the cochlea and auditory nerve in mammals

The numbers and structure of hair cells; afferent, efferent, and reciprocal synapses as seen at the base of hair cells; innervation patterns of first order cochlear neurons; and number and morphology of spiral ganglion cells will be discussed and compared in the guinea pig, rat, cat, monkey and man. Despite many similarities both in the organ of Corti and the spiral ganglion in these species, there are a number of differences which may have important physiologic implications. In the organ of Corti, the major differences among species are the length and width of the basilar membrane, the number of inner and outer hair cells, and the length of hairs on both inner and outer hair cells. Significant differences in the innervation pattern of the inner hair cell among these species include the number of afferent nerve terminals per inner hair cell, the degree of branching of afferent fibers, and the number of synapses per afferent nerve terminal. Among outer hair cells, the number of afferent nerve terminals per outer hair cell, presence or absence of a pre-synaptic body, presence or absence of reciprocal synapses, the number of efferent terminals per outer hair cell, and the presence of dendodendritic synapses in outer spiral bundles may be differences important physiologically. In the spiral ganglion, there are significant differences in the number of spiral ganglion cells, the number of cochlear nerve fibers, the percentage of spiral ganglion cells which are myelinated, and the presence of synapses on spiral ganglion cells.     

重组杆状病毒载体介导新生大鼠耳蜗Corti器体外模型基因转染研究

目的建立新生大鼠耳蜗Corti器体外培养模型,探讨重组杆状病毒作为内耳基因转染载体的可行性及其转染特点。方法应用Bac-to-Bac杆状病毒表达系统制备重组杆状病毒Bac-GFP,建立新生大鼠耳蜗Corti器体外模型,加入携带报告基因的病毒悬液,观察外源基因表达情况。结果耳蜗Corti器体外培养48小时后,培养组织生长良好。Bac-GFP联合丁酸钠可以高效转染毛细胞和螺旋神经节细胞,同时并没有改变Corti器的正常结构。结论新生大鼠离体内耳组织转基因培养法是内耳基因治疗实验研究的一种可行方法。杆状病毒可以在体外高效、安全、快速的转染毛细胞和螺旋神经节细胞,有希望成为内耳基因治疗的新型载体。     

Dissecting the molecular basis of organ of Corti development: Where are we now?

This review summarizes recent progress in our understanding of the molecular basis of cochlear duct growth, specification of the organ of Corti, and differentiation of the different types of hair cells. Studies of multiple mutations suggest that developing hair cells are involved in stretching the organ of Corti through convergent extension movements. However, Atoh1 null mutants have only undifferentiated and dying organ of Corti precursors but show a near normal extension of the cochlear duct, implying that organ of Corti precursor cells can equally drive this process. Some factors influence cochlear duct growth by regulating the cell cycle and proliferation. Shortened cell cycle and premature cell cycle exit can lead to a shorter organ of Corti with multiple rows of hair cells (e.g., Foxg1 null mice). Other genes affect the initial formation of a cochlear duct with or without affecting the organ of Corti. Such observations are consistent with evolutionary data that suggest some developmental uncoupling of cochlear duct from organ of Corti formation. Positioning the organ of Corti requires multiple genes expressed in the organ of Corti and the flanking region. Several candidate factors have emerged but how they cooperate to specify the organ of Corti and the topology of hair cells remains unclear. Atoh1 is required for differentiation of all hair cells, but regulation of inner versus outer hair cell differentiation is still unidentified. In summary, the emerging molecular complexity of organ of Corti development demands further study before a rational approach towards regeneration of unique types of hair cells in specific positions is possible.     

Tubulin expression in the developing and adult gerbil organ of Corti

In the late stages of inner ear development, the relatively undifferentiated cells of Kollicker's organ are transformed into the elaborately specialized cell types of the organ of Corti. Microtubules are prominent features of adult cells in the organ of Corti, particularly supporting cells. To test the possible role of microtubules in organ of Corti development, the microtubule organization in the organ of Corti has been examined using indirect immunofluorescence to beta-tubulin in the developing gerbil cochlea. Tubulin first appears at post-natal day 0 (P0) as filamentous asters in inner hair cells and by P2, asters are also seen in outer hair cells. Tubulin appears at P3 in inner pillar cells in a tooth crown-like figure. By P6, tubulin expression is also evident in outer pillar cells and by P9, it is seen in Deiters cells. Elaboration of microtubules in pillar cells was observed to proceed from the reticular lamina towards the basilar membrane. The pattern of tubulin expression in the apical organ of Corti lags the base by about 3 days until P6, but by P9, apical and basal organ of Corti appear substantially the same.     

重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜的实验分析

目的观察重组腺病毒Ad-GFP转染新生小鼠离体耳蜗基底膜培养组织的情况。方法取新生1～5 d小鼠的耳蜗基底膜,离体培养1 d后,加入重组腺病毒Ad-GFP继续培养1 d,在荧光显微镜及Confocal显微镜下观察病毒对离体耳蜗基底膜的转染情况。结果耳蜗基底膜离体培养1 d后,在显微镜下观察,可见基底膜贴壁生长良好,外周有新生上皮细胞和成纤维细胞长出;高倍显微镜下可见耳蜗内外毛细胞和支持细胞等结构。离体耳蜗基底膜培养组织加入重组腺病毒Ad-GFP继续培养1 d后,可见重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜及其外周长出的新生上皮细胞和成纤维细胞;在新生小鼠离体耳蜗基底膜上,不仅大上皮嵴细胞区域、小上皮嵴细胞区域的细胞能被高效转染,毛细胞也能被高效转染。结论重组腺病毒Ad-GFP能高效转染新生小鼠离体耳蜗基底膜培养组织。     

Postnatal development of the organ of Corti in cats: a light microscopic morphometric study

This study quantitatively characterizes the development of the major morphological features of the organ of Corti during the first 2 weeks postnatal, the period when the cat auditory system makes the transition from being essentially non-functional to having nearly adult-like responses. Four groups of kittens (n=3) were studied at one day postnatal (P1), P5, P10, P15, and compared to adults. Measurements were made of the organ of Corti at 3 cochlear locations: 20%, 60% and 85% of basilar membrane length from the base – cochlear locations which in the adult correspond to best frequencies of ≈20 kHz, 2 kHz and 500 Hz, respectively. In addition, measurements of basilar membrane length and opening of the tunnel of Corti were made in 20 cochlear specimens from kittens aged P0–P6. Results indicate that: (i) at P0 the basilar membrane has attained adult length, and the tunnel of Corti is open over approximately the basal one-half of the cochlea; (ii) the initial opening of the tunnel of Corti occurs at a site about 4 mm from the cochlear base (best frequency of ≈25 kHz in the adult cochlea); (iii) the thickness of the tympanic cell layer decreases markedly at the basal 20-kHz location; (iv) the areas of the tunnel of Corti and space of Nuel and the angulation of the inner hair cells (IHC) relative to the basilar membrane all show marked postnatal increases at both the middle and apical locations; (v) IHC are nearly adult-like in length and shape at birth, whereas the OHC (at 2-kHz and 500-Hz locations) undergo marked postnatal changes; (vi) disappearance of the marginal pillars and maturation of the supporting cells are not yet complete by P15.