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.     

Biochemical studies on the embryonic development of the mammalian inner ear in organ culture

Summary Adenylate cyclase activity and phospholipid labeling were compared during embryonic development of the mouse inner ear in vivo and in vitro. Inner ears were explanted on the 16th gestational day and cultured in vitro for 3–12 days. The gestation time in vivo is 21 days.During the 1st week in vitro there is very little growth of the inner ear with regard to total protein content. In contrast, the labyrinth increases its protein content threefold during the corresponding period of time in vivo.The activity of adenylate cyclase develops parallel in vivo and in vitro until the 19th gestational day whereafter the specific activity of the enzyme in vitro surpasses that of the enzyme in vivo three- to fivefold suggesting a lack of control mechanisms in organ culture. Phospholipids are labeled by ³²P in an essentially similar quantitative relationship in vivo and in vitro, while some quantitative differences exist.According to the present study the usefulness of the organ culture for the investigation of inner ear development appears limited to a culture period corresponding to an age prior to birth.Presented at the 17th Workshop on Inner Ear Biology in Stockholm, June 23–25, 1980     

Organ culture of the crista ampullaris of the embryonic guinea pig

The embryonic inner ear of the guinea pig was cultured one to ten days in vitro. The explantation occurred on the 30th and 40th gestational days, respectively. Hair cells were found in various stages of cytodifferentiation. Both hair cells and supporting cells were ultrastructurally preserved at the end of the period in vitro. The gross morphologic features of the crista ampullaris were still immature. The embryonic inner ear of the guinea pig can be maintained in organ culture until at least as late as the 46th gestational day.     

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.     

Characteristic ionic composition of endolymph is maintained in cultured inner ear.

Inner ear anlagen from mouse were explanted on the 16th gestational day (gd) and cultured for 5 days, i.e. corresponding to the time of birth. By using energy dispersive X-ray technique an elemental composition characteristic for endolymph was found within the membranous labyrinth of the explants. The sodium to potassium ratio in the endolymphatic space of the cultured inner ears corresponded to endolymph of the 16th gd fetus in vivo. There was no difference in the endolymph compartment between the cochlear and vestibular halves of the in vitro specimens. Differences in Na to K ratio between endolymph of the inner ears and the surrounding medium were statistically significant. Thus, endolymph-regulating mechanisms are active also under organ culture conditions, although not fully optimal.     

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.     

Morphological and electrophysiological study of the inner ear and the central auditory pathways following whole body fetal irradiation

Pregnant CBA/CBA mice were whole body irradiated with 2 Gy on the 13th or 16th day of gestation, respectively. The exposed fetuses were raised to an age of 21 postnatal days. Auditory brainstem recordings of threshold levels showed a considerable elevation independent of if irradiation had been performed on either the 13th gestational day or the 16th gestational day. In exposed animals a latency difference occurs in the peaks that increases from peak 1 to peak 5, measuring in peak 5 up to 1.16 ms. Also the peak-to-peak length of waves 1-5 increases in irradiated animals. Scanning electron microscopy of the cochleae showed varying degrees of stereociliary derangement of both outer and inner hair cells, particularly in cochleae where irradiation had been performed on the 13th gestational day, but not loss of hair cells. Light microscopic analysis of auditory brainstem nuclei revealed normal conditions except that in inner ears exposed on the 16th gestational day the flocculus was fused to the lateral surface of the anterior ventral cochlear nucleus. It is concluded that the elevated threshold levels in irradiated animals are most likely due to pathological changes in the peripheral receptor organ whereas the increased latencies and the increased peak-to-peak length likewise reflect functional changes in the brainstem auditory nuclei.     

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.     

Formation of junctional complexes in otocysts developed in vitro. A freeze-fracture study

The thirteenth gestational day inner ear anlage (otocyst) was explanted to an in vitro system and cultured for 8 days, i.e. to a time corresponding to birth. The freeze fracturing technique was used to evaluate morphological differentiation, particularly as regards cell membrane specializations. The epithelial cells were found fully differentiated, e.g. there were regularly arranged stereocilia on the hair cells. The development of tight junctions and gap junctions followed the same pattern as in vivo, but tight junctions did not reach the same degree of regularity and maturation as they did in vivo. There were very few gap junctions in our in vitro specimens. Several tight junctions had an odd appearance, with loss of the normally punctate structure of the strands and areas with considerable thickening. It is possible that this specific morphology can be explained by differentiation in the in vitro system.     

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.

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.     

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.     

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     

Neurogene Stammzelltransplantation in die Kochlea

Background

Stem cell therapy is especially interesting for inner ear related diseases, since the hair cells are very sensitive and do not regenerate. Hair cell loss is therefore irreversible and is accompanied by hearing loss. In the last few years, different research groups have transplanted stem cells into the inner ear with promising results. In the presented study, our aim was to gain insight into how neuronal stem cells behave when they are transplanted, both in vitro and in vivo, into a damaged inner ear.

Methods

Neuronal stem cells from E9.5 day old mouse embryos were collected and infected with an adenoviral vector encoding green fluorescent protein (GFP). GFP+ cells were then transplanted into a damaged organ of Corti in vitro or into a damaged mouse inner ear in vivo.

Results

We were able to detect GFP+ cells close to the organ of Corti in vitro and in the organ of Corti in vivo. The GFP+ cells do not seem to be randomly distributed in either the in vitro or in vivo situation. Most interestingly, GFP+ cells could be detected close to places where hair cells had been lost in vivo.

Conclusion

Neuronal stem cells are interesting candidates to replace lost hair cells. However, a great deal of research is still needed before they can enter clinical trials.     

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     

Developmental stage-dependent pattern of inner ear expression of intermediate filaments

The expression of vimentin, cytokeratins (CKs) and neurofilament (NF) proteins was analysed (using monoclonal antibodies) in the mouse inner ear at the otocyst stage (13th gestational day), when organogenesis was largely completed (16th gestational day) and at birth (21st gestational day). Co-expression of vimentin and CKs occurred at the otocyst stage. On the 16th gestational day, most epithelial cells lacked immunoreactivity for vimentin and considerable variation in CK positivity was found between different regions of the epithelial lining. At birth, CK positivity was lacking in the developing organ of Corti but was present in other types of epithelium lining the scala media. In the vestibular half of the labyrinth, positivity for CKs was found at the apical surfaces of both sensory cells and supporting cells and in epithelia lining the membranous labyrinth. Vimentin positivity occurred in the greater epithelial ridge of the differentiating organ of Corti. Even at this stage the statoacoustic ganglion comprised two subpopulations of ganglion cells: those staining for NF proteins and those lacking this immunoreactivity. Thus, as the inner ear matures, a pattern of cytoskeletal reorganization occurs that is dependent on developmental stage.     

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.     

An in vitro rapid evaluation of drug-induced ototoxicity and of reductive effect of calcium on aminoglycoside ototoxicity using organ culture

The ototoxicity of aminoglycoside and anticancer platinum drug was analysed using an organ culture system. The effect of calcium antagonism on aminoglycoside ototoxicity was investigated by the same system. The inner ears of mice, 16-day embryos, were cultured for 5 days with or without gentamicin (GM) or kanamycin (KM) or streptomycin (SM), or ribostamycin (RSM), including 1, 10, 100, or 1000 micrograms/ml respectively. The 21st gestational day-inner ear was cultured in vitro during 4 days with or without Cisplatin (CDDP) or platinum analog DWA2114R (DWA), including 0.1, 1, or 10 micrograms/ml, respectively. The 16th gestational day-inner ear was cultured in vitro for 5 days with 10 micrograms/ml KM, adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium. The damages of crista ampullaris and macula utriculi of cultured inner ear were estimated according to the ototoxicity score based on morphological changes by a light microscopic observation of serial sections of the materials. We defined four grades for the damages according to the following criteria; grade 1: damage of apical surface of the hair cells, grade 2: the existence of debris in the endolymph space, grade 3: disappearance of the hair cells, grade 4: degeneration of the supporting cells. Using this system following results were obtained: 1) the effect of aminoglycoside was dose dependent, 2) the order of ototoxicity was following; GM greater than KM greater than SM greater than RSM, 3) the drug concentration of 1000 micrograms/ml is sufficient to study its ototoxic potential in this system, 4) the effect of both CDDP and DWA was obvious at a concentration of 0.1 microgram/ml, 5) DWA showed almost the same ototoxicity as CDDP at the same concentration, 6) Adding 5 mM Ca2+ or 10 mM Ca2+ to the culture medium, the ototoxic damage induced by 10 micrograms/ml of KM was not noticed. A protective effect of Ca2+ against KM ototoxicity was observed in vitro. This organ culture and ototoxicity score system can serve as a useful and adequate model system for evaluating the ototoxicity.     

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。     

Microanalytic and light microscopic studies on the developing otic capsule

The structure of the middle layer of the bone of the otic capsule is histologically unique: it is of endochondral origin, has no Haversian canals and the ossification develops from several centres which fuse to form the hardest bone in the body. Our study has been concentrated on the development of the otic capsule in the CBA/CBA mouse, followed from the 13th gestational day to early postnatal age. In the 14th gestational day inner ear, a condensation of mesenchyme is detected around the membranous labyrinth. A cartilaginous capsule is present on the 15th-16th gestational day. Prior to birth, ossification centres occur close to the stapedial footplate. Serial cryosectioning of the newborn inner ear reveals very few regions containing high levels of calcium (microprobe analysis) although by light microscopy, several ossification centres can be identified.