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.     

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.     

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.     

Alcohol and ultrastructural changes in the developing inner ear. An in vitro study

Inner ear explants from the CBA/CBA mouse were used in an organ culture system. The explants were cultured from the 16th gestational day until one day post partum. They were exposed to 1.5% or 3% ethanol in organ culture medium in order to determine any possible toxic effects upon the differentiating sensory structures of the sensory epithelium of the inner ear, that could be correlated to fetal alcohol syndrome. The higher concentration of ethanol caused a general and possibly unspecific destruction of the sensory epithelium, while the lower concentration caused characteristic changes including intracellular edema or vacuolization, especially confined to hair cells. Pathologic changes seemed dose-related but not time-related.     

A scanning electron microscopic study of vestibular organ malformation following prenatal gamma irradiation

Summary Pregnant CBA/CBA mice were exposed to 1 and 2 Gy whole-body gamma irradiation on the 13th and 16th gestational days, respectively. The litters were born on the 21st day of gestation and were tested for vestibular function at the age of 1 month. The animals were then sacrificed and their inner ears were analyzed by scanning electron microscopy. No disturbances of vestibular function were noted in the animals studied. However, the cristae ampullares showed severe malformations as regards their gross shape, with irregularities of their outer contours. Type I hair cells seemed to be more severely changed than Type II hair cells, with fusion of sensory hairs, giant hair formation and bulging of the cuticular plate. In certain sites the hair cells were totally missing. These derangements were usually located in the central areas of the cristae ampullares and in the striolar portion of the maculae utriculi. The morphological damage found showed a dose-dependent, time-related pattern.Supported by grants from the Swedish Medical Research Council (12X-0720) and from the Karolinska Institute     

Embryogenesis of the inner ear. I. Development and differentiation of the mammalian crista ampullaris in vivo and in vitro

The embryonic development of the crista ampullaris in the CBA/CBA mouse was followed comparatively in vivo and in vitro after explanation of the 13th gestational day otocyst. Special attention was paid to hair cell differentiation. In general the in vivo and the in vitro groups of inner ears showed a similar morphological maturation of hair cells on the ultrastructural level. The first sign of hair cell differentiation occurred on the 14th day in vivo and after 1 day in culture showing a thickening of microvilli presumably indicating the development of future sensory hairs. On the 16th day or after 3 days of in vitro culture the sensory hairs were regularely arranged with the kinocilium located at the cell periphery. Afferent nerve endings were identified on the 17th gestational day and some nerve terminals were suspected to be of efferent character appearing on the 18th day. On the day of birth there did not occur complete nerve chalices but a differentiation of hair cells into type I and II was indicated on the 19th gestational day by a slight difference in hair cell configuration and extension of innervation.     

Intermediate filament proteins in the embryonic inner ear of mice under normal conditions and after exposure to ototoxic drugs

The expression of the intermediate filament subclasses cytokeratins (CKs), vimentin, and neurofilament triplet proteins (NFs) was analysed in the embryonic inner ear of the CBA/CBA mouse, in vivo and in vitro, using well-defined monoclonal antibodies. Some of the cultured inner ears were exposed to 1-10 micrograms/ml of the ototoxic drugs gentamicin, ethacrynic acid or cisplatin. There was no difference in the expression of IF pattern in the cultured inner ears as compared with the in vivo developed labyrinths. Exposure to the ototoxic drugs did not affect the expression of CKs, vimentin, or NFs. CKs 8 and 18 were identified in all inner ear epithelia. In the mature hair cells, only the apical surfaces showed immunoreactivity to CKs. In addition, CKs 7 and 19 were visualized in the epithelia involved in maintaining endolymph homeostasis. The ganglion cells showed co-expression of CKs, vimentin and NFs, each having a characteristic localization in the cells.     

Development of endolymph during maturation of the mammalian inner ear

Summary The development of the elemental composition in the endolymphatic space was investigated during embryologic and early post natal maturation of the CBA/CBA mouse. At birth the elemental distribution was similar in the endo- and perilymphatic spaces. Mature composition of endolymph was reached 6–8 days post partum. The maturation of endolymph corresponded well in time with the morphological maturation of the stria vascularis.Supported by grants from the Swedish Medical Research Council (12X-720), The Swedisch Society of Medical Sciences and funds from Tysta Skolan     

Organ culture of the postnatal mouse crista ampullaris part I

Summary The 2 day post partum mouse Crista ampullaris was cultured in vitro for 7 days. The differentiation into secretory and sensory areas was preserved. The ultrastructure of the dark cells of the secretory region was well maintained. The hair cell morphologically was considerably changed though the hair cells themselves survived 7 days in culture.Supported by grants from Karolinska Institutet, and Swedish Medical Research Council (Grant No. 12X-720)     

Vestibular hair cell pathology in the Shaker-2 mouse

Summary The circling-waltzing behaviour of the Shaker-2 mouse is suggested, at least in part, to be of peripheral origin. In this hereditary inner ear disease, degeneration of hair cells type I has been observed showing specific pathologic features: rod-shaped inclusion bodies and sensory hair fusion. Later, the hair cells type I are expelled into the endolymphatic space. A large number of sensory cells type II are morphologically normal. The failure of earlier investigators to demonstrate pathological changes in the sensory epithelia of this animal is likely to be due to the use of light microscopical methods only.Supported by grants from the Torsten and Ragnar Söderberg's Stiftelse, funds from Tysta Skolan and the Swedish Medical Research Council (12X-00720)     

Junctional complexes of the in vitro developed inner ear. A freeze fracture study

The freeze fracture technique was used to study intercellular junctions of inner ear anlages developed in vitro. The 16th gestational day inner ear from the CBA/CBA mouse was cultured for 5 days whereafter the specimens were analyzed. Inner ears developed in vivo were used as controls. A considerable variation in the maturation of the tight junctional complexes occurred in both the vestibular and cochlear parts of the labyrinth. The sequential maturation of tight junctions and gap junctions showed the same structural features in the in vivo and the in vitro developed inner ears, although it seemed that the in vivo developed inner ears showed a slightly more overall mature morphology of tight junctions.     

A scanning electron microscopic study of vestibular organ malformation following prenatal gamma irradiation

Pregnant CBA/CBA mice were exposed to 1 and 2 Gy whole-body gamma irradiation on the 13th and 16th gestational days, respectively. The litters were born on the 21st day of gestation and were tested for vestibular function at the age of 1 month. The animals were then sacrificed and their inner ears were analyzed by scanning electron microscopy. No disturbances of vestibular function were noted in the animals studied. However, the cristae ampullares showed severe malformations as regards their gross shape, with irregularities of their outer contours. Type I hair cells seemed to be more severely changed than Type II hair cells, with fusion of sensory hairs, giant hair formation and bulging of the cuticular plate. In certain sites the hair cells were totally missing. These derangements were usually located in the central areas of the cristae ampullares and in the striolar portion of the maculae utriculi. The morphological damage found showed a dose-dependent, time-related pattern.     

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker ?1 and Shaker ?2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique.Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlea during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker ?1 and Shaker ?2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.     

Organ culture of the postnatal mouse Crista ampullaris. Part I

The 2 day post partum mouse Crista ampullaris was cultured "in vitro" for 7 days. The differentiation into secretory and sensory areas was preserved. The ultrastructure of the dark cells of the secretory region was well maintained. The hair cell morphologically was considerably changed though the hair cells themselves survived 7 days in culture.     

Lesions to cochlear inner hair cells induced by noise

Summary A total of 28 un-anesthetized rabbits of the small chinchilla strain were unilaterally exposed to noise (2–7 kHz, 135 dB SPL in the ear canal). After a follow-up time ranging from 15 minutes to 10 months, the ears were perfused with glutaraldehyde and prepared for analysis by secondary emission electron microscopy and or transmission electron microscopy. The typical finding was a fusion and clumping of inner hair cell (IHC) sensory hairs. In two of the animals, no loss of outer hair cells (OHC) was observed; in several of the others, only a small local loss of OHC was observed in the 2 and 4 kHz regions in spite of extensive IHC abnormality. A frequency map of the rabbit cochlea was obtained by pure tone lesions to OHC. The extent of IHC abnormalities corresponds to the 1–16 kHz region. The findings may provide a basis for the study of the functional relationship between the IHC and OHC.Supported by grants from the Swedish Medical Research Council (14X-04958-05B, 12X-03156-09C), Stifteisen Tysta Skolan, K A Wallenberg Foundation, The Swedish Work Environment Fund (79/80) and The Karolinska Institute fundsPresented at the 17th Workshop on Inner Ear Biology in Stockholm, June 23–25, 1980     

Cytoskeletal identification of intermediate filaments in the inner ear of the jerker mouse mutant

The expression of the five main groups of intermediate filaments (IF) and their subgroups, especially cytokeratins, was analysed in cryosections of the labyrinth of the jerker mouse mutant and compared with normal CBA/CBA controls. Fourteen different well characterized monoclonal antibodies were used. In principle the same pattern of IF was found in the two mouse strains. IF were not found in hair cells of the mouse inner ear, which may reflect a disparity as compared with human fetal hair cells. Chain-specific (epitope-specific) differences were visualized in cytokeratin no. 8 with regard to cells in epithelia involved in the homeostasis of inner ear fluids. Differences in immuno-staining occurred between adjacent cells in the same epithelium, for instance in the semicircular canals.     

Acoustic reflex after experimental lesions to inner and outer hair cells

Chronic effects of noise or kanamycin on the acoustic intra-aural reflex in nonanesthetized rabbits were studied. The intra-aural reflex activity was simultaneously recorded in both ears upon alternate stimulation of the left and the right side. The inner ears were analyzed in scanning and transmission electronmicroscopy. Exposure to bandlimited high-level noise was found to induce extensive deformations in the sensory hairs of the inner hair cells without loss of outer hair cells. Kanamycin caused a degeneration of outer hair cells in the basal 1–2 turns without affecting the morphology of the inner hair cells. The morphological changes, as a function of the location on the basilar membrane, were compared to physiological changes, as a function of frequency. In the noise-exposed animals the threshold of the intra-aural reflex was found to be raised in a frequency range corresponding to the extent of abnormal sensory hairs of the inner hair cells. In the kanamycin-treated animals reflex changes correlated to the loss of outer hair cells. The observations were interpreted as indicating that both types of hair cells cooperate in the activation of the intra-aural reflex in rabbits.     

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。     

Deafness in an old English sheepdog

Summary The histopathological features of the inner ears in a case of deafness in an old English sheepdog reveal a cochleo-saccular (Scheibe) type of degeneration. However, also the hair cells in the vestibular part of the labyrinth were reduced in number though there appeared no clinical signs or symptoms of vestibular dysfunction. The present case is the so far only known old English sheepdog with a hearing loss reported in Sweden.Supported by grants from Karolinska Institutet and the Swedish Medical Research Council (B77-12X-00720-12B)     

Embryonic Inner Ear Cells Reaggregate into Specific Patterns In Vitro

The sensory epithelia of the mammalian inner ear consist of a highly precise pattern of sensory hair cells and supporting cells. The mechanisms regulating this patterning are only beginning to be determined. The present study describes a method for culturing dissociated embryonic inner ear cells and the resulting patterning that occurs in these cultures. The results indicate that developing inner ear cells aggregate into precise patterns on a two-dimensional substrate, suggesting that intrinsic patterning mechanisms remain active in vitro. Using antibodies and scanning electron microscopy to detect hair cells and nonsensory cells, it was determined that only a subset of aggregates contained sensory hair cells. The hair cells were organized into specific patterns and surrounded by supporting cells, similar to the in vivo pattern. Additionally, hair cells increased their immunoreactivity and number of stereocilia over time, suggesting that hair cells continue to mature in vitro. Thus, the study reveals that the cells of the developing inner ear provide the necessary signals that direct sensory hair cells and supporting cells to reassociate into very precise patterns in vitro and that these patterns are reminiscent of the patterning that occurs in vivo.