Repair of surviving hair cells in the damaged mouse utricle.

Sensory hair cells (HCs) in the utricle are mechanoreceptors required to detect linear acceleration. After damage, the mammalian utricle partially restores the HC population and organ function, although regenerated HCs are primarily type II and immature. Whether native, surviving HCs can repair and contribute to this recovery is unclear. Here, we generated the Pou4f3DTR/+; Atoh1CreERTM/+; Rosa26RtdTomato/+ mouse to fate map HCs prior to ablation. After HC ablation, vestibular evoked potentials were abolished in all animals, with ∼57% later recovering responses. Relative to nonrecovery mice, recovery animals harbored more Atoh1-tdTomato+ surviving HCs. In both groups, surviving HCs displayed markers of both type I and type II subtypes and afferent synapses, despite distorted lamination and morphology. Surviving type II HCs remained innervated in both groups, whereas surviving type I HCs first lacked and later regained calyces in the recovery, but not the nonrecovery, group. Finally, surviving HCs initially displayed immature and subsequently mature-appearing bundles in the recovery group. These results demonstrate that surviving HCs are capable of self-repair and may contribute to the recovery of vestibular function.

Dynamic patterns of YAP1 expression and cellular localization in the developing and injured utricle.

The Hippo signaling pathway is a key regulator of tissue development and regeneration. Activation of the Hippo pathway leads to nuclear translocation of the YAP1 transcriptional coactivator, resulting in changes in gene expression and cell cycle entry. Recent studies have demonstrated the nuclear translocation of YAP1 during the development of the sensory organs of the inner ear, but the possible role of YAP1 in sensory regeneration of the inner ear is unclear. The present study characterized the cellular localization of YAP1 in the utricles of mice and chicks, both under normal conditions and after HC injury. During neonatal development, YAP1 expression was observed in the cytoplasm of supporting cells, and was transiently expressed in the cytoplasm of some differentiating hair cells. We also observed temporary nuclear translocation of YAP1 in supporting cells of the mouse utricle after short periods in organotypic culture. However, little or no nuclear translocation of YAP1 was observed in the utricles of neonatal or mature mice after ototoxic injury. In contrast, substantial YAP1 nuclear translocation was observed in the chicken utricle after streptomycin treatment in vitro and in vivo. Together, these data suggest that differences in YAP1 signaling may partially account for the differing regenerative abilities of the avian vs. mammalian inner ear.

Intense noise exposure alters peripheral vestibular structures and physiology.

The otolith organs play a critical role in detecting linear acceleration and gravity to control posture and balance. Some afferents that innervate these structures can be activated by sound and are at risk for noise overstimulation. A previous report demonstrated that noise exposure can abolish vestibular short-latency evoked potential (VsEP) responses and damage calyceal terminals. However, the stimuli that were used to elicit responses were weaker than those established in previous studies and may have been insufficient to elicit VsEP responses in noise-exposed animals. The goal of this study was to determine the effect of an established noise exposure paradigm on VsEP responses using large head-jerk stimuli to determine if noise induces a stimulus threshold shift and/or if large head-jerks are capable of evoking VsEP responses in noise-exposed rats. An additional goal is to relate these measurements to the number of calyceal terminals and hair cells present in noise-exposed vs. non-noise-exposed tissue. Exposure to intense continuous noise significantly reduced VsEP responses to large stimuli and abolished VsEP responses to small stimuli. This finding confirms that while measurable VsEP responses can be elicited from noise-lesioned rat sacculi, larger head-jerk stimuli are required, suggesting a shift in the minimum stimulus necessary to evoke the VsEP. Additionally, a reduction in labeled calyx-only afferent terminals was observed without a concomitant reduction in the overall number of calyces or hair cells. This finding supports a critical role of calretinin-expressing calyceal-only afferents in the generation of a VsEP response.NEW & NOTEWORTHY This study identifies a change in the minimum stimulus necessary to evoke vestibular short-latency evoked potential (VsEP) responses after noise-induced damage to the vestibular periphery and reduced numbers of calretinin-labeled calyx-only afferent terminals in the striolar region of the sacculus. These data suggest that a single intense noise exposure may impact synaptic function in calyx-only terminals in the striolar region of the sacculus. Reduced calretinin immunolabeling may provide insight into the mechanism underlying noise-induced changes in VsEP responses.

Lgr5+ cells regenerate hair cells via proliferation and direct transdifferentiation in damaged neonatal mouse utricle.

Recruitment of endogenous progenitors is critical during tissue repair. The inner ear utricle requires mechanosensory hair cells (HCs) to detect linear acceleration. After damage, non-mammalian utricles regenerate HCs via both proliferation and direct transdifferentiation. In adult mammals, limited transdifferentiation from unidentified progenitors occurs to regenerate extrastriolar Type II HCs. Here we show that HC damage in neonatal mouse utricle activates the Wnt target gene Lgr5 in striolar supporting cells. Lineage tracing and time-lapse microscopy reveal that Lgr5+ cells transdifferentiate into HC-like cells in vitro. In contrast to adults, HC ablation in neonatal utricles in vivo recruits Lgr5+ cells to regenerate striolar HCs through mitotic and transdifferentiation pathways. Both Type I and II HCs are regenerated, and regenerated HCs display stereocilia and synapses. Lastly, stabilized ß-catenin in Lgr5+ cells enhances mitotic activity and HC regeneration. Thus Lgr5 marks Wnt-regulated, damage-activated HC progenitors and may help uncover factors driving mammalian HC regeneration.

Vestibular evoked myogenic potentials in children after cochlear implantation.

OBJECTIVE: The aim of this study was to report the effect of unilateral cochlear implantation to vestibular system using vestibular evoked myogenic potentials (VEMPs) by air-conduction in a sample of children aged less than 5 years. MATERIALS: This study consisted of 10 children (6 boys and 4 girls), who underwent cochlear implantation surgery at our clinic, and 8 normal hearing children (5 boys and 3 girls) matched for age. The VEMPs were performed before, 10 days, and 6 months after surgery. Both the implanted and unimplanted ears of each child were evaluated, with the cochlear implant both off and on. RESULTS: Preoperatively, six (60%) children had abnormal VEMPs responses on both ears. In the postoperative sessions, no child showed any VEMPs response on the implanted side. The VEMPs were not recorded on the unimplanted side either, except for one case. At 6 months, the VEMPs response on the unimplanted side of three children became normal when the cochlear implant was on, and in two children with the device off. CONCLUSION: In the postoperative 6-month-period, the disappearance of VEMPs suggests that the saccule of children can be extensively damaged following cochlear implantation. A recovery of VEMPs can take place on the unimplanted side, with the cochlear implant both on and off. Despite this saccular injury, the absence of clinical signs in children could be explained by their ability to effectively compensate for such vestibular deficits.

Inhibition of Notch activity promotes nonmitotic regeneration of hair cells in the adult mouse utricles.

The capacity of adult mammals to regenerate sensory hair cells is not well defined. To explore early steps in this process, we examined reactivation of a transiently expressed developmental gene, Atoh1, in adult mouse utricles after neomycin-induced hair cell death in culture. Using an adenoviral reporter for Atoh1 enhancer, we found that Atoh1 transcription is activated in some hair cell progenitors (supporting cells) 3 d after neomycin treatment. By 18 d after neomycin, the number of cells with Atoh1 transcriptional activity increased significantly, but few cells acquired hair cell features (i.e., accumulated ATOH1 or myosin VIIa protein or developed stereocilia). Treatment with DAPT, an inhibitor of γ-secretase, reduced notch pathway activity, enhanced Atoh1 transcriptional activity, and dramatically increased the number of Atoh1-expressing cells with hair cell features, but only in the striolar/juxtastriolar region. Similar effects were seen with TAPI-1, an inhibitor of another enzyme required for notch activity (TACE). Division of supporting cells was rare in any control or DAPT-treated utricles. This study shows that mature mammals have a natural capacity to initiate vestibular hair cell regeneration and suggests that regional notch activity is a significant inhibitor of direct transdifferentiation of supporting cells into hair cells following damage.

The Effect of CO(2) and KTP laser on the cat saccule and utricle.

OBJECTIVES/HYPOTHESIS: To assess the potential carbon dioxide (CO(2)) and potassium-titanyl-phosphate (KTP) laser-related trauma to the saccule and utricle in a cat model. STUDY DESIGN: Basic science experiment utilizing cat model. METHODS: Twelve adult male cats were divided into two groups-CO(2) and KTP-to assess the potential saccule and/or utricle trauma with direct discharge of laser energy into the vestibule after the stapes was removed. Both groups were subdivided to assess the effects with acute sacrifice and three-month survival. Bone conduction auditory brain-stem response thresholds were used to monitor auditory function. Clinical observation was used to monitor vestibular function. The temporal bones were harvested, processed, and stained with hematoxylin and eosin (H&E) in all animals with the uninvolved side serving as the control. RESULTS: None of the animals demonstrated changes in bone conduction auditory brain-stem responses. None of the animals in the survival group demonstrated clinical vestibular dysfunction. Saccular and utricular wall rupture was observed in all animals sacrificed acutely. None of the saccular and utricular wall ruptures were of a size and location that could be attributed to laser trauma, and none of the saccular and utricular wall ruptures were associated with neuroepithelial trauma. CONCLUSIONS: There is no evidence of a difference between the CO(2) and KTP laser in potential laser-related trauma. Using bone-conducting auditory brain-stem response threshold and clinical monitoring of vestibular function, there was no evidence of clinical auditory or vestibular dysfunction. The histologic evidence of saccular and utricular wall rupture is more consistent with stapes extraction trauma than laser-related trauma.

Shape change controls supporting cell proliferation in lesioned mammalian balance epithelium.

Mature mammals are uniquely vulnerable to permanent auditory and vestibular deficits, because the cell proliferation that produces replacement hair cells in other vertebrates is limited in mammals. To investigate the cellular mechanisms responsible for that difference, we created excision lesions in the sensory epithelium of embryonic and 2-week-old mouse utricles. Lesions in embryonic utricles closed in <24 h via localized expansion of supporting cells, which then reentered the cell cycle. Pharmacological treatments combined with time-lapse microscopy demonstrated that the healing depended on Rho-mediated contraction of an actin ring at the leading edge of the lesion. In contrast, lesions in utricles from 2-week-old and older mice remained open even after 48 h. Supporting cells in those utricles remained compact and columnar and had significantly stouter cortical actin belts than those in embryonic sensory epithelia. This suggests that cytoskeletal changes may underlie the age-related loss of proliferation in mammalian ears by limiting the capacity for mature supporting cells to change shape. In mature utricles, exogenous stimulation with lysophosphatidic acid overcame this maturational block and induced closure of lesions, promoting supporting cell expansion and subsequent proliferation. After lysophosphatidic acid treatment, 85% of the mature supporting cells that had spread to a planar area >300 microm2 entered S-phase, whereas only 10% of those cells that had a planar area <100 microm2 entered S-phase. Together, these results indicate that cellular shape change can overcome the normal postnatal cessation of supporting cell proliferation that appears to limit regeneration in mammalian vestibular epithelia.

Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity.

Calpains, a family of calcium-activated proteases that breakdown proteins, kinases, phosphatases and transcription factors, can promote cell death. Since leupeptin, a calpain inhibitor, protected against hair cell loss from acoustic overstimulation, we hypothesized that it might protect cochlear and vestibular hair cells against gentamicin (GM) ototoxicity. To test this hypothesis, mouse organotypic cultures from the cochlea, maculae of the utricle and the crista of the semicircular canal (P1-P3) were treated with different doses of GM (0.1-3 mM) alone or in the presence of leupeptin (0.1-3 mM). The percentage of outer hair cells (OHCs) and inner hair cells (IHCs) decreased with increasing doses of GM between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced GM-induced damage to IHCs and OHCs; this protective effect was dose-dependent. GM also significantly reduced hair cell density in the crista and utricle in a dose-dependent manner between 0.1 and 3 mM. The addition of 1 mM of leupeptin significantly reduced hair cell loss in the crista and utricle for GM concentrations between 0.1 and 3 mM. These results suggest that one of the early steps in GM ototoxicity may involve calcium-activated proteases that lead to the demise of cochlear and vestibular hair cells.

An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles.

Sensory epithelia from normal rat utricles and those cultured with and without neomycin treatment were assayed for the presence of growth factor receptor mRNAs by RT-PCR (reverse transcriptase-polymerase chain reaction). Both undamaged and damaged utricles showed mRNA for Insulin receptor, IGF-I receptor, FGF receptor 1, EGF receptor, and PDGF alpha receptor. Neomycin-damaged sensory epithelia showed less PDGF alpha receptor mRNA than undamaged epithelia, suggesting that this message by expressed at higher copy levels in hair cells than in supporting cells. Consistent with that hypothesis, immunohistochemistry revealed much stronger PDGF alpha receptor staining in the hair cells than in the supporting cells. Preliminary evidence suggests that IGF-I receptor message also may be lowered in neomycin-damaged epithelia.

[Histopathology of nonacoustic labyrinth following head injury in guinea pigs].

The histopathological changes in nonacoustic labyrinth induced by experimental head injury were studied in 11 guinea pigs. The animals were divided into two groups. The first group were killed after the pain sense recovered and the second group were allowed to survive for 15 days. The temporal bones were serially sectioned and observed under light microscope. The pathological findings of the vestibular organs included arrangement disturbance, lytic, exfoliate and vacuolization of the sensory epithelia, massive spherical bodies in the region of cilium. The otolithic membranes were exfoliated in the utricular and saccular maculae. There was the otolith by the ductus reuniens separated from the saccular maculae in one ear. There were basophilic staining homogenous deposit on the cristae ampullaris. These findings showed that impairments of vestibulae following head injury were obvious. The secondary impairments, cupulolithiasis and obstruction of the ductus reuniens, from the utricular and saccular maculae were one of the pathologic changes in hearing loss and vertigo following head injury.

Impulse noise induced damage in the vestibular end organs of the guinea pig. A light microscopic study.

Guinea pigs were exposed to the impulse noise from 90-300 rifle shots (peak 158 dB SPL, maximal energy content at 1.1 kHz). This exposure induced severe cochlear damage. The vestibular end organs also showed damage of varying degree. The ampullary cristae were most severely damaged, but changes were also seen in the utricular and saccular maculae. The changes appear to be primarily mechanical and to result from the effect of the acoustic pressure wave on the vestibular labyrinth.