Embryogenesis of the inner ear. II. The late differentiation of the mammalian crista ampullaris in vivo and in vitro

The embryonic development of the crista ampullaris of the CBA/CBA mouse was followed both in organ culture of explanted inner ears of the 16th gestational day and in vivo from the 16th gestational day until the 21st day, an age corresponding to birth. Cytodifferentiation of the sensory epithelium of the crista ampullaris occurs during this period. At partus, there is a rather mature crista with well developed hair cells and 1-2 layers of supporting cells. Innervation and differentiation into type I and type II hair cells have started prior to partus but occur mainly post partum. The in vitro development followed that of the in vivo but with a slight delay, especially concerning the later stages of the in vitro development. At the time corresponding to partus, differentiation of hair cells is almost identical in the two groups but innervation is delayed in the in vitro group of inner ears. Because of the very high reproducibility/stability in vitro and morphologic maturation of both hair cells and gross structure of the crista ampullaris, organ culture of the 16th gestational day inner ear explant is a suitable model in the study of the late embryonic development under normal and pathological conditions.     

Low-dose gamma irradiation effects on labyrinthine development in vitro

The 13th and 16th gestational day inner ear anlagen, respectively, were exposed after explantation to an organ culture system to low-dose gamma irradiation with a 2 Gy single dose. The explants were thereafter cultured in vitro for 8 vs. 5 days to an age corresponding to birth in vivo. The explants were analysed with regard to gross morphology and at the light and electron microscopic levels. The 13th gestational day inner ear anlage showed malformations of the gross shape. The gross morphology of the 16th gestational day inner ear explant was unaffected. Irradiated specimens showed a delayed development in general as compared with controls. A defective cytodifferentiation of hair cells was observed at the ultrastructural level. Sensory hair fusion occurred, the sensory hair rootlets were poorly developed as also was the cuticle. Nerve terminals were not identified. The observations in the present in vitro study are in agreement with corresponding earlier published in vivo investigations. The organ culture model can thus be used for irradiation induced selective effects on labyrinthine development.     

CBA小鼠内耳感觉上皮的参考数据

目的测量成年CBA小鼠耳蜗和前庭的有关数据,为内耳感受器定量分析提供参考依据。方法将6只出生后3个月左右的成年CBA小鼠(12耳)的耳蜗和前庭各终器取出并制备成全耳蜗基底膜铺片和全前庭终器铺片。测量全耳蜗基底膜的总长度,以及基底膜和Corti器在耳蜗不同部位的宽度。观察并记录全耳蜗毛细胞的总数和基底膜上不同区间的毛细胞密度。观察并记录椭圆囊斑和球囊斑毛细胞的总数及其微纹区和周边区的毛细胞密度,记录壶腹嵴上的毛细胞密度。结果正常成年CBA小鼠的耳蜗基底膜长度为(5.76±0.196)毫米(平均数±标准差,下同),基底膜的宽度在耳蜗底部距起始端约1.5毫米处为(339.1±9.87)微米,在耳蜗中部距起始端约3毫米处为(304.5±11.82)微米,在耳蜗顶部距起始端约5毫米处为(300.1±7.22)微米,说明小鼠耳蜗基底膜的宽度从底回到顶回逐渐变窄。Corti器的宽度在耳蜗底部距起始端1.5毫米处为(37.80±2.24)微米,在耳蜗中部距起始端约3毫米处为(45.00±2.67)微米,在耳蜗顶部距起始端约5毫米处为(52.20±3.16)微米,可见Corti器的宽度从底回到顶回逐渐变宽。CBA小鼠全耳蜗毛细胞的总数为(3116.41±151.91)个,其中内毛细胞总数为(680.67±17.50)个,而外毛细胞的总数是(2435.8±143.46)个。耳蜗内、外毛细胞的平均密度为(541.1±9.36)个/毫米,其中内毛细胞的平均密度为(118.3±2.68)个/毫米,外毛细胞的平均密度为(422.8±11.87)个/毫米,耳蜗毛细胞在耳蜗基底膜上不同区间的毛细胞密度无明显差异(P>0.05)。小鼠椭圆囊斑上的毛细胞总数为(3300±177.51)个,球囊斑上的毛细胞总数为(3045±361.57)个。前庭球囊斑和椭圆囊斑微纹区和周边区的毛细胞密度基本相同(P>0.05),两个囊斑微纹区的平均毛细胞密度为(40.2±6.59)个/0.0025mm2,两个囊斑周边区的平均毛细胞密度为(53.2±7.18)个/0.0025mm2,可见囊斑微纹区的毛细胞密度低于周边区。尽管小鼠上半规管和后半规管壶腹嵴的毛细胞区被位于嵴中央的上皮细胞分隔为两个区域,但三个壶腹嵴上的毛细胞密度基本相同,其平均密度为(44.7±7.15)个/0.0025mm2。结论本实验所得CBA小鼠耳蜗和前庭测量数据,为进一步定量观察CBA小鼠内耳病理学改变提供了重要的参考依据。     

The inner ear and the in vitro system.

The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.     

豚鼠内耳椭圆囊斑体外原代培养

目的探讨豚鼠内耳椭圆囊斑体外正常培养状态及其在体外用氨基糖苷类抗生素庆大霉素后的毒性损伤特征,以期建立前庭外周器官体外实验模型。方法豚鼠被随机分成三组:①正常未培养对照组;②正常原代培养组,利用体外组织培养方法对豚鼠内耳椭圆囊斑进行正常原代培养;③庆大霉素损伤原代培养组。正常原代培养组和庆大霉素损伤原代培养组在培养期间每天用相差显微镜观察生长晕,正常培养组在培养的第5天和第10天固定用石蜡包埋切片光镜观察。庆大霉素组在48小时用树脂包埋半薄切片观察。结果正常原代培养组在培养10天过程中,用相差显微镜观察椭圆囊斑在培养第1～4天无明显生长晕,在培养第5天明显从椭圆囊边缘长出。随培养时间延长,生长晕不断扩大,以成纤维细胞为主。庆大霉素损伤原代培养组在培养2天内每天用相差显微镜观察椭圆囊斑未见明显生长晕。正常原代培养至第5和第10天,石蜡包埋切片显示支持细胞和毛细胞存活。庆大霉素组在48小时,用树酯包埋半薄切片观察,感觉上皮层的毛细胞溶解破坏,支持细胞存活。结论椭圆囊斑器官体外培养方法可行,并培养成功,为今后进一步研究椭圆囊斑提供了实验模型。     

The inner ear and the in vitro system

Summary The embryologic labyrinthine development of the CBA/CBA mouse occurs parallell in vivo and in vitro. Regarding post partum inner ears, either as cultured otocysts passing a corresponding time in vitro or inner ear explants of newborn/mature animals, the extracorporal system becomes unable to maintain specialized hair cell structures for more than a few days. The sensory cells themselves, however, survive for considerably longer time. Vestibular hair cells show sensory hair fusion. Cochlear hair cells loose their surface structures but the sensory hair rootlets penetrating into the cuticle are preserved. Post partum inner ears from the guinea pig reacted in a similar way in vitro as did labyrinths from the CBA/CBA mouse.Supported by grants from Karolinska Institutet, The Swedish Medical Research Council (grant no 12X-720) and The Swedish Society of Medical Sciences     

Tissue culture of middle ear epithelium of the guinea pig--differences of the cellular growth activity in the middle ear cavity using collagen gel culture method

Primary tissue culture system from the middle ear and the tracheal epithelium of the guinea pig was established using collagen gel method. The cultured epithelium was studied by phase-contrast microscopy, light and scanning electron microscopy. The outgrowth area of the epithelium was quantitatively measured for period of up to ten days. In the middle ear cavity, the mucosal explants were sampled from various sites in order to compare their differential and proliferative activities. The mucosal explants attached on collagen substrates were composed of ciliated, non-ciliated, goblet and basal cells. This basic structure was similar to the natural middle ear epithelium. The ciliated cells showed well organized cilia. Most of the outgrowth cells devoid of fibroblastic cells in the monolayer were polygonal shaped with numerous microvilli. The morphology of the outgrowth cells changed from columnar or cuboidal to squamous shapes in the area away from the explants. There was a correlation between the distribution of the ciliated cells in the outgrowths and these in the explants. The explants with columnar or cuboidal ciliated epithelia sampled from the opening of the eustachian tube or its neighborhood formed more than eightfold outgrowth sheets in vitro. This is comparable to tracheal epithelia. On the other hand, the explants with simple squamous epithelia sampled from the area distal to the eustachian tube showed about fourfold proliferative activity. We concluded that this culture system would be useful for the study of cellular multiplication and differentiation mechanisms of the respiratory tract epithelium.     

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.     

Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants

A method for long term culture of utricular macula explants is demonstrated to be stable and reproducible over a period of 28 days in vitro (DIV). This culture system for four-day-old rat utricular maculae is potentially suitable for studies of hair cell loss, repair and regeneration processes as they occur in post-natal mammalian inner ear sensory epithelia. The cellular events that occur within utricular macula hair cell epithelia during 28 days of culture are documented from serial sections. Vestibular hair cells (HCs) and supporting cells (SCs) were systematically counted using light microscopy (LM) and the assistance of morphometric computer software. Ultrastructural observations were made with transmission electron microscopy (TEM) for describing the changes in the fine detailed morphological characteristics that occurred in the explants related to time in vitro. After 2 DIV the density of HCs was 77%, at 21 DIV it was 69%, and at 28 DIV it was 52% of HCs present at explantation. Between 2 DIV and 28 DIV there was a 1.7% decrease of the vestibular macula HC density per DIV. The corresponding decrease of SC density within the utricular explants was less than 1% per DIV. The overall morphology of the epithelia, i.e. relationship of HCs to SCs, was well preserved during the first two weeks in culture. After this time a slight deterioration of the epithelia was observed and although type I and type II HCs were identified by TEM observations, these two HC types could no longer be distinguished from one another by LM observations. In preparations cultured for 21 DIV, SC nuclei were located more apical and further away from the basal membrane compared to their position in macula explants fixed immediately after dissection. The loss of cells that occurred was probably due to expulsion from the apical (i.e. luminal) surface of the sensory epithelia, but no lesions of the apical lining or ruptures of the basal membrane were observed. There were no significant changes in the volume of the vestibular HC comprising macular epithelium during the observation period of 28 DIV.     

Organ culture system for inner ears

This paper reports an organ culture system using 2-hydroxyethylmethacrylate (HEMA) hydrogel as a substrate to study the development of otocysts and statoacoustic ganglions of the mouse embryos in vitro. Twelfth and thirteenth gestation-day otocysts and statoacoustic ganglions with otic sensory epithelium and/or rhombencephalon developed well on the HEMA hydrogel. Normal morphogenesis of the inner ears and cytodifferentiation of their sensory epithelia were noted. Neurons of the statoacoustic ganglions differentiated well with outgrowth of nerve fibers. The new organ culture system can be used for cultivating otocysts extracorporeally, facilitating investigation of the effects of various biologically active extracellular proteins, glycoproteins and glycosaminoglycans on statoacoustic ganglion-target tissue explants.     

Extracorporeal preservation. Organ culture of the post-natal mammalian inner ear.

Postnatal (newborn and mature) inner ear organs from CBA/CBA mouse and guinea pig were analysed concerning hair cell survival in an in vitro system. After only a few days in the artificial surroundings a transformation of hair cell characteristics occurred, in the form of either loss of sensory hairs (cochlear hair cells) or hair fusion (vestibular hair cells), although the hair cell itselt survived for a considerably longer time. Intracellular myelin figures became evident after 2--3 days in culture. However, a considerable individual variation among hair cells was observed concerning the ultrastructure of the cell at this stag in vitro. Completely normal hair cells could in rare cases be recognised after 1 week in organ culture (cochlear inner hair cells of the CBA/CBA mouse). Loss of the surface structures of the hair cells is likely to constitute an irreversible transformation not in agreement with the true hair cell characteristics.     

Comparative ototoxic potential of hyaluronic acid and methylcellulose.

In experimental animal studies, exogenous hyaluronan (HA) has been shown to exert beneficial effects on the healing of tympanic membrane perforation. As any other exogenous substance, HA may prove potentially toxic, by filling the middle ear cavity, to the sensory cells of the organ of Corti. Electrophysiological (ABR) and morphological studies were carried out in the rat to examine the auditory function and the structure of the sensory epithelium. Rats received either HA or hydroxy-propyl-methyl-cellulose by trans-tympanic injection (middle ear cavity was completely filled up) and were compared to untreated, age- and weight-matched rats. In both treated groups ABR revealed transitory, mild conduction hearing loss, in particular for high frequencies, until day 7 postinjection. This loss recovered completely within the 15th day. Morphologically, no significant degenerative/necrotic lesions were observed in the organ of Corti, from both treated groups.     

Hair cell differentiation following tissue interactions for induction of otocyst morphogenesis

The 12th-12.5th gestational day inner ear otocyst from the CBA/CBA mouse was explanted to organ culture with and without surrounding mesenchyme. One group of otocysts from which the mesenchyme had been removed was cultured in conditioned medium (i.e., medium in which mesenchyme alone had been cultured but had been removed prior to explantation of the "stripped" otocyst, without adjacent mesenchyme). Morphogenesis was good in organ cultures with preserved mesenchyme, acceptable in "stripped" otocysts cultured in conditioned medium, but very poor (or even lacking) in specimens deprived of mesenchyme and cultured in normal medium. In the two latter groups, only a small number of hair cells were identified. Although morphogenesis can be induced in specimens initially deprived of adjacent mesenchyme, a normal tissue relationship seems essential for cytodifferentiation of a normal number of hair cells.     

Synaptogenesis in co-cultured inner ear explants which share a single statoacoustic ganglion

Co-cultured otic explants which shared a single statoacoustic ganglion were grown 'in vitro' to the equivalent of gestation day 21 and then processed either for light microscopic or ultrastructural study. Only vestibular sensory epithelium was studied. Nerve fibres from the statoacoustic ganglion (SAG) of the (+) SAG otic explant were attracted to the sites of differentiating vestibular sensory epithelium in the (-) SAG otic explant. Synaptic contact between vestibular sensory hair cells and statoacoustic ganglion neurites was observed in both the (-) SAG and (+) SAG otic explants.     

Effect of vitamin A on the growth and differentiation of rat external auditory canal epithelium in organ culture.

Intact rat external ear canal explants were maintained either in retinoid-deficient or retinoid-supplemented medium for 7, 10, 14, and 21 days. The morphology of the external ear canal epidermis was well maintained, including the presence of sebaceous glands even after 21 days in culture with retinoid-deficient medium. However, after culturing in retinoid-supplemented medium, the external ear canal epithelium showed both a loss of keratohyalin granules and the formation of keratin. Extensive microvilli formation occurred, even though the desquamation process continued from the superficial layer after 7, 10, and 14 days in culture with retinoid-enriched medium. After 21 days in culture with retinoid-supplemented medium, the ear canal epithelium contained well-developed Golgi apparatus and secretory granules as well. It was concluded that the ear canal epithelium was transformed into a secretory-like mucosal epithelium by retinoid supplementation.     

Interleukin-1 facilitates airway epithelial migration in response to injury

OBJECTIVE: To test the hypothesis that interleukin (IL)-1 plays a permissive role in respiratory epithelial cell migration and proliferation. STUDY DESIGN: Primary cultures of porcine respiratory epithelial cells or tracheal organ explants were cultured in the presence or absence of function-blocking antibodies to IL-1. Areas of epithelial cell outgrowth were determined in control and antibody-treated organ explants daily for 4 days. At intervals, cultured cells were collected for cell counting and viability determination. Time course and dose-response curves were constructed for control and antibody-treated groups. RESULTS: Interleukin-1 secretion into culture supernatants increased sharply from days 3 to 7. Outgrowths from tracheal explants were reduced by greater than 60% by single antibody treatment, and by over 90% by treatment with antibodies to both IL-1alpha and -1beta by day 4 of culture. Function-blocking antibodies to IL-1 significantly reduced cell number by day 7 of culture. CONCLUSIONS: Interleukin-1 is produced by respiratory epithelial cells in culture during log phase growth and plays a permissive role in cell migration and proliferation.     

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.     

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.     

Determinants of ganglion-receptor cell interaction during development of the inner ear. A heterochronic ganglia study

It has been suggested that inner ear sensory receptors produce attractant fields that guide neurite outgrowth from statoacoustic ganglion (SAG) neurons to appropriate target sites within the developing labyrinth. This experiment tested the temporal limitations of SAG neurons in their ability to respond to these attractant fields. Statoacoustic ganglia were excised from 12, 13, 14 and 15 gestation day (GD) mouse embryos. This temporal series of SAG was implanted into aganglionic 12 GD otocysts. All cultures were grown for 7 days in vitro, then fixed and processed for nerve fiber staining. Specimens were evaluated for the presence of neurites associated with the inner ear sensory receptors that developed within the otic explants. All of the implanted heterochronic ganglia (i.e. 13, 14 or 15 GD) as well as the homochronic (i.e. 12 GD) ganglion controls extended neurites to sensory epithelium of both vestibular and auditory character. Neurites made contact with the base of hair cells in all of the sensory structures. These findings demonstrate that SAG neurons are capable of extending processes in response to otic attractant fields for an extended period during the embryonic development of this ganglion. This observation supports the hypothesis that the onset and duration of receptor generated attractant fields may act as a controlling factor in establishing patterns of innervation within the developing inner ear.     

Evolution of Laryngeal Cancer Surgery

Conclusion. Olfactory epithelium explant culture represents a model that will be useful in studying the calcium signal transduction of olfactory sensory neurons (OSNs). Objectives. The aim of this study was to test the feasibility and efficacy of olfactory epithelium explant culture in the study of calcium signl transduction. Materials and methods. Using explant culture, devoid of any enzyme use, a mixed cell type culture system from newborn murine olfactory epithelium was achieved. Morphological and immunochemical criteria were employed to identify the cultured cells. Calcium imaging, using the fluorescent calcium indicator fura-2, was used to monitor changes in the intracellular calcium concentration of candidate OSNs following exposure to forskolin, a cAMP pathway stimulant. Results. The putative OSNs synthesized both neuronal proteins and olfactory-specific markers. The cultured neurons originated from the ‘precursors’ present in the olfactory epithelium explants, became fully developed, and could survive for at least 9 days in vitro. Upon stimulation, the intracellular calcium concentration of the OSNs increased from 58.5±12.8 nM (n=20) to 577.2±57.6 nM (n=10).