The blood labyrinthine barrier in the human normal and Meniere's disease macula utricle.

The ultrastructural organization of the blood labyrinthine barrier (BLB) was investigated in the human vestibular endorgan, the utricular macula, using postmortem specimens from individuals with documented normal auditory and vestibular function and surgical specimens from patients with intractable Meniere's disease. Transmission electron microscopic analysis of capillaries located in the normal human utricular stroma showed vascular endothelial cells with few pinocytotic vesicles, covered by a smooth and uniform basement membrane surrounded by pericyte processes. Meniere's disease specimens revealed differential ultrastructural pathological changes in the cellular elements of the microvasculature. With moderate degeneration of the BLB, there were numerous vesicles within the vascular endothelial cells (VECs), with increased numbers at the abluminal face, pericyte process detachment and disruption of the perivascular basement membrane surrounding the VECs. With severe degeneration of the BLB, there was severe vacuolization or frank apparent necrosis of VECs and loss of subcellular organelles. A higher severity of BLB degenerative changes was associated with a higher degree of basement membrane thickening and edematous changes within the vestibular stroma. This study presents the first ultrastructural analysis of the capillaries constituting the BLB in the human vestibular macula utricle from normal and Meniere's disease.

Deterioration of Vestibular Cells in Labyrinthitis.

OBJECTIVE: To quantitatively assess the effect of serous labyrinthitis, suppurative labyrinthitis, and labyrinthitis ossificans on vestibular hair cells, dark cells, and transitional cells. METHODS: We examined human temporal bone specimens with serous labyrinthitis, suppurative labyrinthitis, and labyrinthitis ossificans, then compared them with age-matched control groups without labyrinthitis. We evaluated the density of type I and II vestibular hair cells, dark cells, and transitional cells in the peripheral sensorial organs. RESULTS: The mean density of type I vestibular hair cells in the maculae of the saccule significantly differed between the serous labyrinthitis group and its control group. The loss of type I and II vestibular hair cells in all of the peripheral sensorial organs was significantly higher in the suppurative labyrinthitis group than in its control group. The mean density of dark cells in the lateral and posterior semicircular canals was significantly lower in the suppurative labyrinthitis group than in its control group. The mean density of type I and II vestibular hair cells, dark cells, and transitional cells was significantly lower in the labyrinthitis ossificans group than in its control group. CONCLUSION: The loss of vestibular hair cells and degenerative changes in dark cells and transitional cells could affect vestibular function in patients with labyrinthitis.

Variations in shape-sensitive restriction points mirror differences in the regeneration capacities of avian and mammalian ears.

When inner ear hair cells die, humans and other mammals experience permanent hearing and balance deficits, but non-mammalian vertebrates quickly recover these senses after epithelial supporting cells give rise to replacement hair cells. A postnatal decline in cellular plasticity appears to limit regeneration in mammalian balance organs, where declining proliferation responses are correlated with decreased spreading of supporting cells on artificial and native substrates. By culturing balance epithelia on substrates that differed in flexibility, we assessed spreading effects independent of age, showing a strong correlation between shape change and supporting cell proliferation. Then we made excision wounds in utricles cultured from young and old chickens and mice and compared quantified levels of spreading and proliferation. In utricles from young mice, and both young and old chickens, wounds re-epithelialized in <24 hours, while those in utricles from mature mice took three times longer. More cells changed shape in the fastest healing wounds, which accounted for some differences in the levels of proliferation, but inter-species and age-related differences in shape-sensitive restriction points, i.e., the cellular thresholds for shape changes that promote S-phase, were evident and may be particularly influential in the responses to hair cell losses in vivo.

Mefloquine damage vestibular hair cells in organotypic cultures.

Mefloquine is an effective and widely used anti-malarial drug; however, some clinical reports suggest that it can cause dizziness, balance, and vestibular disturbances. To determine if mefloquine might be toxic to the vestibular system, we applied mefloquine to organotypic cultures of the macula of the utricle from postnatal day 3 rats. The macula of the utricle was micro-dissected out as a flat surface preparation and cultured with 10, 50, 100, or 200 µM mefloquine for 24 h. Specimens were stained with TRITC-conjugated phalloidin to label the actin in hair cell stereocilia and TO-PRO-3 to visualize cell nuclei. Some utricles were also labeled with fluorogenic caspase-3, -8, or -9 indicators to evaluate the mechanism of programmed cell death. Mefloquine treatment caused a dose-dependent loss of utricular hair cells. Treatment with 10 µM caused a slight reduction, 50 µM caused a significant reduction, and 200 µM destroyed nearly all the hair cells. Hair cell nuclei in mefloquine-treated utricles were condensed and fragmented, morphological features of apoptosis. Mefloquine-treated utricles were positive for the extrinsic initiator caspase-8 and intrinsic initiator caspase-9 and downstream executioner caspase-3. These results indicate that mefloquine can induce significant hair cell degeneration in the postnatal rat utricle and that mefloquine-induced hair cell death is initiated by both caspase-8 and caspase-9.

Immunohistochemical localization and mRNA expression of aquaporins in the macula utriculi of patients with Meniere's disease and acoustic neuroma.

Meniere's disease is nearly invariably associated with endolymphatic hydrops (the net accumulation of water in the inner ear endolymphatic space). Vestibular maculae utriculi were acquired from patients undergoing surgery for Meniere's disease and acoustic neuroma and from autopsy (subjects with normal hearing and balance). Quantitative immunostaining was conducted with antibodies against aquaporins (AQPs) 1, 4, and 6, Na(+)K(+)ATPase, Na(+)K(+)2Cl co-transporter (NKCC1), and alpha-syntrophin. mRNA was extracted from the surgically acquired utricles from subjects with Meniere's disease and acoustic neuroma to conduct quantitative real-time reverse transcription with polymerase chain reaction for AQP1, AQP4, and AQP6. AQP1 immunoreactivity (-IR) was located in blood vessels and fibrocytes in the underlying stroma, without any apparent alteration in Meniere's specimens when compared with acoustic neuroma and autopsy specimens. AQP4-IR localized to the epithelial basolateral supporting cells in Meniere's disease, acoustic neuroma, and autopsy. In specimens from subjects with Meniere's disease, AQP4-IR was significantly decreased compared with autopsy and acoustic neuroma specimens. AQP6-IR occurred in the sub-apical vestibular supporting cells in acoustic neuroma and autopsy samples. However, in Meniere's disease specimens, AQP6-IR was significantly increased and diffusely redistributed throughout the supporting cell cytoplasm. Na(+)K(+)ATPase, NKCC1, and alpha-syntrophin were expressed within sensory epithelia and were unaltered in Meniere's disease specimens. Expression of AQP1, AQP4, or AQP6 mRNA did not differ in vestibular endorgans from patients with Meniere's disease. Changes in AQP4 (decreased) and AQP6 (increased) expression in Meniere's disease specimens suggest that the supporting cell might be a cellular target.

Quantitative cellular level analysis of mitochondrial DNA 3243A > G mutations in individual tissues from the archival temporal bones of a MELAS patient.

CONCLUSION: We could represent the first quantitative analysis of the mutation rate at the cellular level in human inner ear of a patient with MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) by combining laser capture microdissection (LCM) and quantitative real time PCR. OBJECTIVES: We previously reported combining LCM and PCR to isolate mtDNA from the cells of specific tissues within a human archival celloidin-embedded temporal bone section without known otological history. Using this method, we quantitatively analyzed the rate of mtDNA 3243A > G mutation in the inner ear of a MELAS patient, and examined the correlation of the mutation rate at the cellular level and their histopathological condition. METHODS: We extracted each inner ear organs using LCM from temporal bone sections of a MELAS patient, and studied the mutation rate, which was calculated as the ratio of the amount of mutant mtDNA to the total mtDNA. RESULTS: We found that the mtDNA mutation rate was high in spiral ganglion cells and the saccular macula, but was comparatively low in hair cells of the organ of Corti, the stria vascularis and the facial nerve. With the exception of the stria vascularis, there was a good correlation between the mutation rate and the histological findings.

Damage and functional recovery of the Atlantic cod (Gadus morhua) inner ear hair cells following local injection of gentamicin.

This study addresses the ultrastructural and functional damage and subsequent recovery of the inner ear in the Atlantic cod following intrasaccular gentamicin injection. Inner ear damage was assessed using SEM and measurements of AEP following 250-Hz pure-tone stimuli. Data from gentamicin-treated fish were compared with control (no injection) and sham (injection of saline) fish. Control fish had normal response thresholds associated with well-developed hair cell bundles in their macula sacculi. Sham fish had higher response thresholds compared with control fish during the first week post-intervention, but response thresholds were subsequently normal. Treated fish displayed significant inner ear damage associated with an increased average AEP threshold on the third day following treatment. Thereafter, inner ear tissue displayed signs of progressive regeneration until it was comparable to controls from the 14th day. Response thresholds were similar to those of control fish from the 17th day following treatment. These observations suggest that the macula sacculi of Atlantic cod can regenerate towards a near-complete functional and ultrastructural recovery within 17-21 days following ototoxic gentamicin treatment.

Ultrastructural changes in otoconia of osteoporotic rats.

The etiology of benign paroxysmal positional vertigo (BPPV) remains obscure in many cases and women are affected more often than men. A recent prospective study, performed in women >50 years of age suffering from recurrent BPPV, showed associated osteopenia or osteoporosis in a large percentage of these patients. These results suggested the possible relationship between recurrent BPPV and a decreased fixation of calcium in bone in women >50 years. To test this hypothesis, an experimental study was performed in adult female rats. Utricular otoconia of female rats in which osteopenia/osteoporosis was induced by bilateral ovariectomy (OVX) were compared to those of sham-operated adult females rats (SHAM), as control group. FIRST STUDY: The morphology of theutricles of OVX and SHAM rats was analyzed with scanning electron microscopy. In osteopenic/osteoporotic rats, the density of otoconia (i.e. the number of otoconia per unit area) was decreased (p = 0.036)and their size was increased (p = 0.036) compared to the control group. SECOND STUDY: To test the role of calcium turnover in such morphological changes, utricular otoconia of 2 other groups of OVX and SHAM rats, previously injected with calcein subcutaneously, were examined by conventional and epifluorescence microscopy. In epifluorescence microscopy, labeling with calcein showed no significant fluorescence in either group. This finding was interpreted as a lack of external calcium turnover into otoconia of adult female rats. The ultrastructural modifications of otoconia in osteopenic/osteoporotic female adult rats as well as the role of estrogenic receptors in the inner ear are discussed. The possible pathophysiological mechanisms which support the relationship between recurrent BPPV in women and the disturbance of the calcium metabolism of osteopenia/osteoporosis are debated.

A place principle for vertigo.

OBJECTIVE: To provide a road map of the vestibular labyrinth and its innervation leading to a place principle for different forms of vertigo. METHOD: The literature describing the anatomy and physiology of the vestibular system was reviewed. RESULTS: Different forms of vertigo may be determined by the type of sense organ, type of ganglion cell and location in the vestibular nerve. CONCLUSION: Partial lesions (viral) of the vestibular ganglion are manifested as various forms of vertigo.

Training and aging modulate the loss-of-balance phenotype observed in a new ENU-induced allele of Otopetrin1.

BACKGROUND INFORMATION: The sensing of head movement in mammals depends upon the vestibular endorgan of the inner ear, a complex structure made up of the semicircular canals and otoliths. Due to the similarity between the human and mouse vestibular apparatus, the analysis of mutant mouse is a valuable strategy aiming to identify genes involved in the control of balance and movement. RESULTS: In the course of a genome-wide chemical-mutagenesis programme, we isolated a recessive mutation, named ied (inner ear defect), which induced a severe loss-of-balance. A detailed phenotypic analysis of the mutant mice demonstrates that the balance impairment does not affect the motor activity and can be rescued, in part, by training, despite a complete agenesis of otoconia in the utricule and the saccule of the inner ear. Molecular characterization of the ied mutation revealed a transversion that affects the splicing of the second exon of the Otopetrin1 gene located on mouse chromosome 5. The consequence of such a mutation leads to a disruption of the transcription of the gene. CONCLUSIONS: The identification of the ied knock-down allele strengthens the role of the Otopetrin1 in the sensing of balance. Moreover, the rescue of the ied mutant phenotype in specific behavioural tasks confirmed that other sensory inputs or neural plasticity can compensate, to some extent, for the loss-of-balance. In the future, the ied mutant mice might be helpful to study the genetic control of the compensation strategies developed by organisms to counteract balance defects.

Histology of the utricle in kinetotically swimming fish: a parabolic aircraft flight study.

OBJECTIVES: Humans taking part in parabolic aircraft flights (PAFs) may suffer from space motion sickness, which is a form of kinetosis. As it has been repeatedly shown that some fish in a given batch also reveal kinetotic behaviour (especially so-called spinning movements and looping responses) during PAFs, and as a result of the homology of the vestibular apparatus of all vertebrates, fish can be used as model systems to investigate the origin of susceptibility to motion sickness. Therefore. we were prompted to examine the utricular maculae, which are responsible for the internalization of gravity in teleosteans of fish swimming kinetotically in microgravity (microg) in comparison with those of animals from the same batch who swam normally. MATERIAL AND METHODS: Larval cichlid fish (Oreochromis mossambicus) were subjected to PAFs. Post-flight, animals which had behaved normally or kinetotically during the microg phases were examined histologically The sizes of the inner ear utricular maculae as well as the numbers of sensory and supporting cells were determined. RESULTS: The total numbers of both sensory and supporting cells of the utricular maculae did not differ between kinetotic and normally swimming fish. Cell density (number of sensory and supporting cells/100 microm2) was, however, reduced in kinetotic animals (p < 0.0001), which seemed to be due to the presence of malformed epithelial cells of increased size in the kinetotic specimens. CONCLUSION: These results indicate that susceptibility to kinetosis may originate from genetically predisposed malformed sensory epithelia.

Gentamicin tympanoclysis: effects on the labyrinthine sensory cells.

OBJECTIVES: The objective of the study was to determine whether a selective vestibular hair cell toxicity with sparing of the cochlear hair cells could be achieved by infusing different concentrations of gentamicin into the middle ears of adult cats. STUDY DESIGN: Prospective experimental animal study treating only the left ear of each cat, the right ear serving as individual control. METHODS: Gentamicin solution at concentrations of either 30 or 3 mg/mL was infused daily into the left middle ear of adult cats until overt ataxia occurred. After 1 month or 6 months, each cat was killed and its temporal bones prepared for optical microscopy. RESULTS: Animals treated with 30 mg/mL gentamicin until ataxic required a median of five daily doses. These animals had clear-cut cochlear basal turn hair cell losses accompanying toxic lesions in the utricle and cristae. In contrast, animals treated with 3 mg/mL gentamicin until ataxic required an average of 19 daily doses. These animals had lesions restricted to the utricle and cristae with sparing of the cochlea hair cells. Animals that failed to develop ataxia manifested neither lesions of the cochlear nor vestibular hair cells. CONCLUSION: Gentamicin tympanoclysis in the cat animal model, using a dilute solution and continued once daily until clinical ataxia occurs, is capable of producing selective vestibular hair cell toxicity while sparing cochlea hair cells.

Hyper-vision in a patient with central and paracentral vision loss reflects cortical reorganization.

SM, a 21-year-old female, presents an extensive central scotoma (30 deg) with dense absolute scotoma (visual acuity = 10/100) in the macular area (10 deg) due to Stargardt's disease. We provide behavioral evidence of cortical plastic reorganization since the patient could perform several visual tasks with her poor-vision eyes better than controls, although high spatial frequency sensitivity and visual acuity are severely impaired. Between 2.5-deg and 12-deg eccentricities, SM presented (1) normal acuity for crowded letters, provided stimulus size is above acuity thresholds for single letters; (2) a two-fold sensitivity increase (d-prime) with respect to controls in a simple search task; and (3) largely above-threshold performance in a lexical decision task carried out randomly by controls. SM's hyper-vision may reflect a long-term sensory gain specific for unimpaired low spatial-frequency mechanisms, which may result from modifications in response properties due to practice-dependent changes in excitatory/inhibitory intracortical connections.

Study of the gerbil utricular macula following treatment with gentamicin, by use of bromodeoxyuridine and calmodulin immunohistochemical labelling.

Effects of ototoxic drugs on the gerbil vestibular sensory epithelium were probed by use of immunocytochemical labelling with antibodies to both a mitogenic marker (bromodeoxyuridine) and a hair cell specific protein (calmodulin). Nine animals had gentamicin administered once daily for 5 days, as a transtympanic injection into the right middle ear. They additionally were given a daily intraperitoneal injection of bromodeoxyuridine, starting on the same day as the gentamicin injection and continuing until the day of sacrifice. Nine other animals, serving as controls for bromodeoxyuridine incorporation, received only the intraperitoneal injections of bromodeoxyuridine. The inner ears from three gerbils were obtained at 1, 2 or 4 weeks following the last gentamicin injection and utricles from the injected ears were processed for immunohistochemical analysis. In specimens where gentamicin was administered, we found evidence of bromodeoxyuridine incorporation in 17 cells (10 single cells and 7 pairs of cells) in a total of 216 sections taken from the central regions of the 9 utricles. However, in control specimens, no bromodeoxyuridine labelling was found in any cells of the 216 sections examined. Of 10 single cells labelled with bromodeoxyuridine, two cells in the hair cell layer were labelled with antibodies against calmodulin. One had a faint labelling in the nucleus and the other in the stereocilia, but not in the cell bodies. Of 7 pairs of cells, two pairs with nuclei localized in the hair cell layer had faint labelling for calmodulin in the nuclei, but no labelling in any other part of the cell. The other 13 cells labelled with antibodies to bromodeoxyuridine were not labelled with antibodies to calmodulin. Our results suggest that the bromodeoxyuridine-labelled cells could not be positively identified as hair cells based on immunohistochemical labelling for calmodulin.

Ultrastructural findings of the macula utriculi in a case of a petrous apex cholesteatoma: a comparison with findings in a patient with an acoustic neuroma.

The morphological characteristics of the vestibular sensory cells of the macula utriculi obtained during surgery in a patient with a petrous apex cholesteatoma were examined using scanning and transmission electron microscopy. Findings were compared to cells studied in a patient with acoustic neuroma. Scanning electron microscopy showed that compared to the apparently normal cells in the acoustic neuroma case, most sensory cells in the cholesteatoma case had large cuticular plates, irregular locations of cilia and no clear polarizations. Supporting cells showed profuse short microvilli on the whole surface. With transmission electron photomicrographs, type I hair cells were not seen and certain morphological changes were observed in type-II-like cells and supporting cells. We presume that the degenerative changes in the vestibular epithelia were due to circulatory disturbances and/or direct pressure applied to the vestibular nerve at the internal auditory canal, with subsequent involvement of the macula utriculi.

Vestibular morphology in relation to age and circling behavior.

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.

Argon laser irradiation of the otolithic organ.

An argon laser was used to irradiate the otolithic organs of guinea pigs and cynomolgus monkeys. After stapedectomy, the argon laser (1.5 W x 0.5 sec/shot) irradiated the utricle or saccule without touching the sensory organs. The stapes was replaced over the oval window after irradiation. The animals used for acute observation were killed immediately for morphologic studies; those used for long-term observation were kept alive for 2, 4, or 10 weeks. Acute observation revealed that sensory and supporting cells were elevated from the basement membrane only in the irradiated area. No rupture of the membranous labyrinth was observed. Long-term observation revealed that the otolith of the macula utriculi had disappeared in 2-week specimens. The entire macula utricili had disappeared in 10-week specimens. No morphologic changes were observed in cochlea, semicircular canals, or membranous labyrinth. The saccule showed similar changes.

Morphological changes in the vestibular epithelia and ganglion induced by ototoxic drug.

The morphological changes of the vestibular sensory epithelia and the vestibular ganglions induced by Gentamicin(GM) were investigated using scanning electron microscope, transmission electron microscope and light microscope. The guinea pigs were injected with a single application of 4 mg (0.1ml) of GM into the middle ear through the tympanic membrane. The vestibular organs and the ganglions were observed up to 6 months after the treatment. Four days after the injection, fused, ballooned and missing cilia were observed in the vestibular sensory epithelia. These changes progressed and extended toward the periphery of the crista and the macula. The changes of the vestibular ganglions were first observed one month after the treatment. The degenerative process started from destruction of the mitochondrial cristae and vacuolization of the cytoplasm in the Schwann cell. The next step of the change was dissociation of the myelin sheath around the ganglion cell. The cytoplasmic organelles in the ganglion cell gradually deteriorated. At the later stage, the myelin sheath around the ganglion cell disappeared and the number of the cell reduced. Furthermore, the myelin sheath of the nerve fiber was dissociated. In this study the signs of the vestibular ganglion damage were later than that of the vestibular organ. However, we thought the changes in the ganglion are probably due to direct influence of GM, since the degeneration was found to develop in a relatively short period.

Effect of macular ablation on frequency and latency of motion-induced emesis in the squirrel monkey.

Three previously motion-emetic sensitive squirrel monkeys were rendered refractory to a standard motion-emetic regimen by a two-stage utriculosacculectomy procedure which preserved the cristae ampullares of semicircular canals. Three non-operated control squirrel monkeys tested on the same motion-emetic regimen time schedule as the operated animals remained motion-emetic sensitive with regard to incidence, frequency, and latency of motion-induced emetic responses. Following a sham surgical procedure (stapedectomy) performed on two of the latter animals and one additional new animal, the emetic incidence decreased from 100% to 89%, but the frequency and latency were not altered significantly.

Sensorineural and vascular changes in an ear with acoustic neurinoma.

The temporal bones from a 55-year-old woman with deafness and dizzy spells caused by a right-sided acoustic neurinoma were examined by the techniques of microdissection, surface preparations, and celloidin sections. Sensorineural degeneration was present in the basal end of the right cochlea but was not severe enough to explain the deafness. The compression of the cochlear nerve by the tumor was postulated to have caused deafness by injury to neurons adjacent and central to the tumor. There was profound degeneration of the vestibular nerve and sensory cells. A gelatinous material occluded the scala vestibuli, and the vestibular fluid spaces contained an amorphous proteinaceous substance. Fewer erythrocytes were seen in the vessels of the right cochlea, but there was no atrophy of capillaries to indicate long-term reduction of circulation. Vasculoneogenesis of venous vessels had occurred in the scala tympani, probably as a result of venous stasis.