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Objective: The German waltzing guinea pig is a special strain of animal with a recessively inherited inner ear defect, resulting in deafness and a severe vestibular dysfunction. The hearing loss in the cochlea of the German strain is a result of a collapse of the Reissner membrane and the absence of scala media. The vestibular organ has not yet been described., Materials and Methods: German waltzing guinea pigs (homozygote and heterozygote) of different ages ranging from embryologic age 25 days to adulthood were investigated. The living animals were tested with four different vestibular tests, and the fetuses were controlled according to breeding. The morphology of the vestibular parts (ampulla, saccule, and utricle) was observed by using the light and transmission electron microscopy., Results: Collapse of the membranous labyrinth was found already at embryologic age 50 days and progressed over time. Vestibular dysfunction was noted already from birth., Conclusions: Vestibular atelectasis has been shown to have the same morphology as the reported vestibular dysfunction in the German waltzing guinea pig. Owing to this similarity, this animal can be a good model for vestibular research.

Mix-mice, a new strain of mice with inner ear dysfunction, and their littermates were used in the present study in order to investigate vestibular morphology, visualised by light microscopy (LM) and transmission electron microscopy (TEM). Contrary to the mix-mice, their littermates showed less stained nerve chalices and it was possible to detect that hair cells showed surface herniations and so-called 'blebs' in the apical portion of the epithelium. Sensory hairs showed a disarrayed pattern. The mix-mice had severe microscopical abnormalities: collapse of the membranous cell layer, cavities inside the neuroepithelium, severe loss of hair cells, herniations of the few remaining hair cells and increased supporting cells were evident. In the utricle and saccule, hair cells were missing and the epithelial surface was covered by a single layer of smooth, flattened epithelial cells of hitherto unknown origin., (Copyright 2003 S. Karger AG, Basel)

The DBA/2 strain of mice have genetically induced vestibular dysfunction that presents grossly at an early age as circling behavior and abnormal righting reflexes. The vestibular morphology of this strain has not previously been examined. DBA/2 mice of different ages that showed circling behavior were sacrificed and then had their inner ears immediately removed and fixed in glutaraldehyde. The specimens were prepared for light, transmission and scanning electron microscopy. Additional specimens at 10 months of age were fixed with paraformaldehyde for immunohistochemical investigation and labelling of alpha-actinin. Non-circling litter mates served as controls. The morphology and immunohistochemistry of the vestibular end-organs were evaluated as a function of age and circling behavior. The sensory epithelium of the ampulla and utricle in the circling mice showed softening of the cuticle, hair cell cytoplasmic herniation, expelled cellular debris, fused stereocilia and giant hair cells that progressively increased in severity with age. The non-circling litter mates showed similar but less severe pathology of the vestibular sensory epithelium. The immunohistochemical analysis showed no differences at a magnification of 400 x.

The murine endolymphatic sac (ELS) can survive for several weeks in tissue culture. The epithelial cells mature in vitro and demonstrate functional activity under these artificial conditions. The goal of the present study was to investigate fluid and particle transport between the ELS cells, the ELS lumen, and the surrounding tissue culture medium. A series of tracers, including anti-mouse IgG-labelled colloidal gold, horseradish peroxidase (HRP), and ruthenium red (RR) were used to elucidate different aspects of ELS transport. Results indicated that both light and dark cells of the ELS took up HRP from both the luminal and the basal side of the epithelial cells, forming coated vesicles. Only luminal uptake occurred when the HRP was injected into the lumen, and only basal uptake was observed when the HRP was placed in the culture media. HRP introduced into the tissue culture media never entered the lumen of the ELS. Anti-mouse IgG labelled with gold showed no uptake at all from either side of the epithelial cells. Gold could be found only in membrane-bound vesicles in the cytoplasm of reticuloendothelial (RES) cells in the ELS lumen and lateral intercellular spaces (LIS) of the sac. This suggests that there are no IgG sites in sac cells and that the circulating RES cells seen in the lumen of the ELS come from the surrounding connective tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

A recent report from our laboratory describes techniques for growing fetal murine endolymphatic sac (ELS) in vitro in tissue culture. The purpose of the present study is twofold: first, to determine whether the in vitro endolymphatic cells function in their artificial environment; and second, to begin to understand the nature of luminal cell function in vitro when separated from subepithelial connective tissue and blood. The endolymphatic sac was dissected from 18th gestational day fetal mouse otocysts and grown in DME tissue culture media for 7 days. Light microscopic sections of the endolymphatic sac were stained with Periodic Acid Schiff (PAS) reagent, alcian blue, Verhoff's elastin stain, and van Gieson's collagen stain to reveal deposits of glycogen as well as mucopolysaccharides, elastic fibers, and collagen. Controls for glycogen staining were prepared using the amylase enzyme. For electron microscopical evaluation, 'en bloc' staining was used, to confirm the location of cellular glycogen. Results indicate that in vitro luminal cells of the murine ELS are viable and show signs of functional activity with the markers used. The luminal substance and apical cytoplasm shows distinct purple metachromasia with toluidine blue and PAS-positive staining that disappears with amylase digestion. The ELS cells and luminal substance were negative for alcian blue at pH 1.0 and pH 2.5. These findings are similar to those seen in in vivo murine control sac specimens and demonstrate the ability of cultured sac cells to store glycogen and produce complex carbohydrates. The similarity in staining between the luminal substance and the cytoplasm of sac cells in all in vitro specimens suggests some secretory function by sac cells.

The development of perisaccular blood vessels is described during embryology and the postnatal period of the mouse. Primitive sinusoidal vessels already appear at the early otocyst stage as the future endolymphatic sac is formed. Before birth the vasculature attains a more mature appearance with tubular, somewhat fully developed blood vessels. At this stage a primitive basement membrane is also formed. Soon after birth the blood vessels appear mature with developed fenestrations and micropores, giving them an appearance comparable to blood vessels in other fluid transporting organs.

Numerous studies have attempted to elucidate the function of the mammalian endolymphatic sac (ELS). All of these studies have been performed on in vivo specimens and are thus influenced by humoral and tissue factors extraneous to the sac. In contrast, an in vitro model would provide an opportunity to study ELS cells in a carefully controlled environment. This report presents our experience with tissue culturing the murine endolymphatic sac removed from 16 and 18 gestational day fetuses. Light (LM) and transmission electron microscopical (TEM) evaluations of the developing endolymphatic sac were performed over periods of one, four, and seven days in tissue culture. In order to confirm growth and maturation, three-dimensional reconstructions from serial sections of the cultured ELS were made and compared with published accounts of in vivo murine ELS development for equivalent periods of time. Both whole and dissected otocysts were grown in tissue culture and compared with one another. Two different tissue culture medias were investigated, each with and without the addition of collagenase, used to soften the dense fibrous capsule of the otocyst and thus facilitate dissection and histological preparation. The impact of collagenase and the tissue culture medias on endolymphatic sac growth were studied. Results demonstrated that murine ELS cells were able to differentiate and mature in tissue culture, as confirmed by LM, TEM, and three-dimensional reconstructions. After an initial delay, in vitro maturation of cells in tissue culture paralleled normal in vivo growth and in some specimens appeared to show accelerated maturation. This in vitro model should prove useful in efforts to define ELS function and in providing a technique for tissue culturing human ELS from normal and diseased ears.

The well-known toxicity pattern of aminoglycoside antibiotics has been used with the aim to produce waste products in the inner ear. The endolymphatic sac was studied after daily intraperitoneal injection of 75 mg/kg kanamycin for 10 and 20 days to see whether or not the endolymphatic sac showed signs of increased phagocytic activity. The epithelium of the endolymphatic sac was reduced in height, the cells were swollen and extremely thin. The dark epithelial cells were outnumbering the light cells. The subepithelial tissue showed an edematous swelling containing few blood vessels, collagen fibrils and fibroblasts with an empty appearance. There were few wide lateral intercellular spaces and there were intraluminal free floating cells. There were, in all, no clear morphological signs of increased phagocytic activity in the epithelium of the endolymphatic sac.

Radioactively labeled sulphur was injected into 12 mice of the NMRI strain 20 days after pretreatment with kanamycin. The animals were decapitated after intervals ranging from 2 minutes to 24 hours after injection. After a routine autoradiographic procedure, darkly stained silver grains were detected in the endolymphatic sac (ES) and its surroundings. One hour after injection, the grains were found in the surrounding blood vessels. Eleven hours later, maximal uptake was seen in the epithelial cells of the ES in the kanamycin-treated animals. Twenty-four hours after injection, a faint S35 uptake in the ES could still be detected. An increase in the ES activity, indicated by an increased number of free-floating cells and the secretion of a sulphur-containing intraluminal substance, did not occur. The spreading pattern of sulphur, as shown in this investigation, does not support the theory that waste products from the inner ear, transported to the ES by the longitudinal flow, produces an increased activity in the sac after long pretreatment with kanamycin. In 12 control animals, no difference in the distribution of labeling was observed, but there was an additional uptake in the free intraluminal cells which was not seen in the kanamycin group.

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.

Pregnant CBA/CBA mice were exposed to 0.5, 1 and 2 Grey (Gy), (1 Gy = 100 rad) in single doses with whole body gamma-irradiation on the 12th, 13th and 16th gestational days, respectively. The animals were tested at an age of one month for vestibular and cochlear function. Thereafter the inner ears were analyzed with scanning electron microscopy. A morphological analysis with cytocochleograms was performed. Morphological changes in the vestibular part showed gross malformations in the cristae ampullares. Hair cells of type I seemed to be more severely changed than hair cells type II. The macula utriculi also showed malformations of the otoconia. All these changes were more pronounced when the irradiation was given early during pregnancy and with the highest doses used, except the otoconia which were more injured when irradiated day 16 of gestation. No disturbances of the equilibrium reflexes were noted. In the cochlea a dose-dependent, time-related damage pattern was demonstrated with pathological changes of outer (OHC) and inner (IHC) hair cells. When tested electrophysiologically for auditory function with auditory brainstem recordings (ABR), elevated thresholds were revealed different in shape depending on when during pregnancy irradiation took place. A good correlation existed between the morphological changes as seen in the cytocochleograms and the functional changes documented with the ABR.

Pregnant mice were whole-body irradiated on the 12th, 13th and 16th gestational day with single doses of 0.5, 1, 2, 3, 4, 10 and 20 Gy, using a 60Co source. No surviving litters occurred in groups exposed to more than 2 Gy. Below this dose, irradiation can cause malformation of the cristae ampullares and the maculae. Malformations were more severe and more frequent in fetuses exposed on the 12th than on the 13th gestational day, indicating that, prior to or the earlier during morphogenesis irradiation takes place, the more vulnerable is the developing tissue. Inner ears exposed to irradiation on the 16th gestational day did not reveal severe malformations.