Hyperpolarization-Activated Current (I h) in Vestibular Calyx Terminals: Characterization and Role in Shaping Postsynaptic Events

Calyx afferent terminals engulf the basolateral region of type I vestibular hair cells, and synaptic transmission across the vestibular type I hair cell/calyx is not well understood. Calyces express several ionic conductances, which may shape postsynaptic potentials. These include previously described tetrodotoxin-sensitive inward Na⁺ currents, voltage-dependent outward K⁺ currents and a K(Ca) current. Here, we characterize an inwardly rectifying conductance in gerbil semicircular canal calyx terminals (postnatal days 3–45), sensitive to voltage and to cyclic nucleotides. Using whole-cell patch clamp, we recorded from isolated calyx terminals still attached to their type I hair cells. A slowly activating, noninactivating current (I_h) was seen with hyperpolarizing voltage steps negative to the resting potential. External Cs⁺ (1–5 mM) and ZD7288 (100 μM) blocked the inward current by 97 and 83 %, respectively, confirming that I_h was carried by hyperpolarization-activated, cyclic nucleotide gated channels. Mean half-activation voltage of I_h was −123 mV, which shifted to −114 mV in the presence of cAMP. Activation of I_h was well described with a third order exponential fit to the current (mean time constant of activation, τ, was 190 ms at −139 mV). Activation speeded up significantly (τ = 136 and 127 ms, respectively) when intracellular cAMP and cGMP were present, suggesting that in vivo I_h could be subject to efferent modulation via cyclic nucleotide-dependent mechanisms. In current clamp, hyperpolarizing current steps produced a time-dependent depolarizing sag followed by either a rebound afterdepolarization or an action potential. Spontaneous excitatory postsynaptic potentials (EPSPs) became larger and wider when I_h was blocked with ZD7288. In a three-dimensional mathematical model of the calyx terminal based on Hodgkin–Huxley type ionic conductances, removal of I_h similarly increased the EPSP, whereas cAMP slightly decreased simulated EPSP size and width.     

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.     

Directional Plasticity Rapidly Improves 3D Vestibulo-Ocular Reflex Alignment in Monkeys Using a Multichannel Vestibular Prosthesis

Bilateral loss of vestibular sensation can be disabling. We have shown that a multichannel vestibular prosthesis (MVP) can partly restore vestibular sensation as evidenced by improvements in the 3-dimensional angular vestibulo-ocular reflex (3D VOR). However, a key challenge is to minimize misalignment between the axes of eye and head rotation, which is apparently caused by current spread beyond each electrode’s targeted nerve branch. We recently reported that rodents wearing a MVP markedly improve 3D VOR alignment during the first week after MVP activation, probably through the same central nervous system adaptive mechanisms that mediate cross-axis adaptation over time in normal individuals wearing prisms that cause visual scene movement about an axis different than the axis of head rotation. We hypothesized that rhesus monkeys would exhibit similar improvements with continuous prosthetic stimulation over time. We created bilateral vestibular deficiency in four rhesus monkeys via intratympanic injection of gentamicin. A MVP was mounted to the cranium, and eye movements in response to whole-body passive rotation in darkness were measured repeatedly over 1 week of continuous head motion-modulated prosthetic electrical stimulation. 3D VOR responses to whole-body rotations about each semicircular canal axis were measured on days 1, 3, and 7 of chronic stimulation. Horizontal VOR gain during 1 Hz, 50 °/s peak whole-body rotations before the prosthesis was turned on was <0.1, which is profoundly below normal (0.94 ± 0.12). On stimulation day 1, VOR gain was 0.4–0.8, but the axis of observed eye movements aligned poorly with head rotation (misalignment range ∼30–40 °). Substantial improvement of axis misalignment was observed after 7 days of continuous motion-modulated prosthetic stimulation under normal diurnal lighting. Similar improvements were noted for all animals, all three axes of rotation tested, for all sinusoidal frequencies tested (0.05–5 Hz), and for high-acceleration transient rotations. VOR asymmetry changes did not reach statistical significance, although they did trend toward slight improvement over time. Prior studies had already shown that directional plasticity reduces misalignment when a subject with normal labyrinths views abnormal visual scene movement. Our results show that the converse is also true: individuals receiving misoriented vestibular sensation under normal viewing conditions rapidly adapt to restore a well-aligned 3D VOR. Considering the similarity of VOR physiology across primate species, similar effects are likely to occur in humans using a MVP to treat bilateral vestibular deficiency.     

Rapid Increase in Neural Conduction Time in the Adult Human Auditory Brainstem Following Sudden Unilateral Deafness

Individuals with sudden unilateral deafness offer a unique opportunity to study plasticity of the binaural auditory system in adult humans. Stimulation of the intact ear results in increased activity in the auditory cortex. However, there are no reports of changes at sub-cortical levels in humans. Therefore, the aim of the present study was to investigate changes in sub-cortical activity immediately before and after the onset of surgically induced unilateral deafness in adult humans. Click-evoked auditory brainstem responses (ABRs) to stimulation of the healthy ear were recorded from ten adults during the course of translabyrinthine surgery for the removal of a unilateral acoustic neuroma. This surgical technique always results in abrupt deafferentation of the affected ear. The results revealed a rapid (within minutes) reduction in latency of wave V (mean pre = 6.55 ms; mean post = 6.15 ms; p < 0.001). A latency reduction was also observed for wave III (mean pre = 4.40 ms; mean post = 4.13 ms; p < 0.001). These reductions in response latency are consistent with functional changes including disinhibition or/and more rapid intra-cellular signalling affecting binaurally sensitive neurons in the central auditory system. The results are highly relevant for improved understanding of putative physiological mechanisms underlying perceptual disorders such as tinnitus and hyperacusis.     

庆大霉素对离体培养小鼠前庭终器的损害

目的：观察并建立不同浓度庆大霉素损害新出生C 57小鼠前庭各终器的离体实验模型。方法：应用前庭终器分离取材技术、前庭器官离体培养技术和组织学检查技术观察不同浓度庆大霉素对新出生C 57小鼠前庭各终器的损害。结果：庆大霉素对离体培养前庭毛细胞的损害模式是随着剂量的增加而损害加重，庆大霉素对离体培养前庭毛细胞的破坏与其在体实验的耳毒性规律基本相似。结论：囊斑微纹区和壶腹嵴顶部的毛细胞比周边区的毛细胞更易遭受庆大霉素的破坏。     

Histopathology of the vestibular end organs after intratympanic gentamicin failure for Meniere's disease

CONCLUSION: To our knowledge, this is the first report of the histopathology of the vestibular end organs following intratympanic gentamicin for intractable Meniere's disease. There was relative sparing of the utricular macula, compared with the cristae ampullares. However, the utricular macula exhibited severe hair cell loss. Clinically, the patient has been free from vertigo spells for 3 years following labyrinthectomy. Objective: To describe the histopathology and morphometry of the vestibular end organs from a 59-year-old Meniere's patient who underwent transmastoid labyrinthectomy for recurrent vertigo after failed intratympanic gentamicin. MATERIALS AND METHODS: Light and transmission electron microscopy were utilized; with unbiased stereology-physical fractionator for type I, type II hair cell, and supporting cell counts. Comparison with end organ histopathology in a 56-year-old with Meniere's disease without gentamicin treatment was carried out. RESULTS: Histopathological analysis of the semicircular canal cristae ampullares showed severe atrophy of the neuroepithelium with undifferentiated cells, and fibrosis and edema of the stroma. The utricular macula had some remaining type I and type II vestibular hair cells, and nerve fibers and terminals within the underlying stroma. Morphometric measures were obtained from the utricular macula: 2000 type I and 500 type II hair cells, representing 7.3% of type I hair cells and 4.9% of type II hair cells compared with normative controls, and 24 000 supporting cells were obtained.     

Enhanced Vestibulo-ocular Reflex to Electrical Vestibular Stimulation in Meniere’s Disease

Meniere’s disease is characterized by sporadic episodes of vertigo, nystagmus, fluctuating sensorineural hearing loss, tinnitus and aural pressure. Since Meniere’s disease can affect different regions of the vestibular labyrinth, we investigated if electrical vestibular stimulation (EVS) which excites the entire vestibular labyrinth may be useful to reveal patchy endorgan pathology. We recorded three-dimensional electrically evoked vestibulo-ocular reflex (eVOR) to transient EVS using bilateral, bipolar 100-ms current steps at intensities of 0.9, 2.5, 5.0, 7.5 and 10.0 mA with dual-search coils in 12 unilateral Meniere’s patients. Their results were compared to 17 normal subjects. Normal eVOR had tonic and phasic spatiotemporal properties best described by the torsional component, which was four times larger than horizontal and vertical components. At EVS onset and offset of 8.9 ms latency, there were phasic eVOR initiation (M = 1,267 °/s²) and cessation (M = −1,675 °/s²) acceleration pulses, whereas during the constant portion of the EVS, there was a maintained tonic eVOR (M = 9.1 °/s) at 10 mA. However in Meniere’s disease, whilst latency of EVS onset and offset was normal at 9.0 ms, phasic eVOR initiation (M = 1,720 °/s²) and cessation (M = −2,523 °/s²) were enlarged at 10 mA. The initiation profile was a bimodal response, whilst the cessation profile frequently did not return to baseline. The tonic eVOR (M = 20.5 °/s) exhibited a ramped enhancement of about twice normal at 10 mA. Tonic eVOR enhancement was present for EVS >0.9 mA and disproportionately enhanced the torsional, vertical and horizontal components. These eVOR abnormalities may be a diagnostic indicator of Meniere’s disease and may explain the vertigo attacks in the presence of declining mechanically evoked vestibular responses.     

Characterization and Inflammatory Response of Perivascular-Resident Macrophage-Like Melanocytes in the Vestibular System

A large number of perivascular cells expressing both macrophage and melanocyte characteristics (named perivascular-resident macrophage-like melanocytes, PVM/Ms), previously found in the intra-strial fluid–blood barrier, are also found in the blood–labyrinth barrier area of the vestibular system in normal adult cochlea, including in the three ampullae of the semicircular canals (posterior, superior, and horizontal), utricle, and saccule. The cells were identified as PVM/Ms, positive for the macrophage and melanocyte marker proteins F4/80 and GSTα4. Similar to PVM/Ms present in the stria vascularis, the PVM/Ms in the vestibular system are closely associated with microvessels and structurally intertwined with endothelial cells and pericytes, with a density in normal (unstimulated) utricle of 225 ± 43/mm²; saccule 191 ± 25/mm²; horizontal ampullae 212 ± 36/mm²; anterior ampullae 238 ± 36/mm²; and posterior ampullae 223 ± 64/mm². Injection of bacterial lipopolysaccharide into the middle ear through the tympanic membrane causes the PVM/Ms to activate and arrange in an irregular pattern along capillary walls in all regions within a 48-h period. The inflammatory response significantly increases vascular permeability and leakage. The results underscore the morphological complexity of the blood barrier in the vestibular system, with its surrounding basal lamina, pericytes, as well as second line of defense in PVM/Ms. PVM/Ms may be important to maintain blood barrier integrity and initiating local inflammatory response in the vestibular system.     

Gentamicin induced nitric oxide-related oxidative damages on vestibular afferents in the guinea pig

Gentamicin is a well-known ototoxic aminoglycoside. However, the mechanism underlying this ototoxicity remains unclear. One of the mechanisms which may be responsible for this ototoxicity is excitotoxic damage to hair cells. The overstimulation of the N-methyl-d-aspartate (NMDA) receptors increases the production of nitric oxide (NO), which induces oxidative stress on hair cells. In order to determine the mechanism underlying this excitotoxicity, we treated guinea pigs with gentamicin by placing gentamicin (0.5 mg) pellets into a round window niche. After the sacrifice of the animals, which occurred at 3, 7 and 14 days after the treatment, the numbers of hair cells in the animals were counted with a scanning electron microscope. We then performed immunostaining using neuronal nitric oxide synthase (nNOS), inducible NOS (iNOS) and nitrotyrosine antibodies. The number of hair cells in the animals was found to decrease significantly after 7 days. nNOS and iNOS expression levels were observed to have increased 3 days after treatment. Nitrotyrosine was expressed primarily at the calyceal afferents of the type I hair cells 3 days after treatment. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining revealed positive hair cells 3 days after treatment. Our results suggest that inner ear treatment with gentamicin may upregulate nNOS and iNOS to induce oxidative stress in the calyceal afferents of type I hair cells, via nitric oxide overproduction.     

Unilateral Adaptation of the Human Angular Vestibulo-Ocular Reflex

A recent study showed that the angular vestibulo-ocular reflex (VOR) can be better adaptively increased using an incremental retinal image velocity error signal compared with a conventional constant large velocity-gain demand (×2). This finding has important implications for vestibular rehabilitation that seeks to improve the VOR response after injury. However, a large portion of vestibular patients have unilateral vestibular hypofunction, and training that raises their VOR response during rotations to both the ipsilesional and contralesional side is not usually ideal. We sought to determine if the vestibular response to one side could selectively be increased without affecting the contralateral response. We tested nine subjects with normal vestibular function. Using the scleral search coil and head impulse techniques, we measured the active and passive VOR gain (eye velocity / head velocity) before and after unilateral incremental VOR adaptation training, consisting of self-generated (active) head impulses, which lasted ∼15 min. The head impulses consisted of rapid, horizontal head rotations with peak-amplitude 15 ^o, peak-velocity 150 ^o/s and peak-acceleration 3,000 ^o/s². The VOR gain towards the adapting side increased after training from 0.92 ± 0.18 to 1.11 ± 0.22 (+22.7 ± 20.2 %) during active head impulses and from 0.91 ± 0.15 to 1.01 ± 0.17 (+11.3 ± 7.5 %) during passive head impulses. During active impulses, the VOR gain towards the non-adapting side also increased by ∼8 %, though this increase was ∼70 % less than to the adapting side. A similar increase did not occur during passive impulses. This study shows that unilateral vestibular adaptation is possible in humans with a normal VOR; unilateral incremental VOR adaptation may have a role in vestibular rehabilitation. The increase in passive VOR gain after active head impulse adaptation suggests that the training effect is robust.     

Glutamate-like Immunoreactivity During Hair Cell Recovery After Gentamicin Exposure in the Chinchilla Vestibular Sensory Periphery

Objective: Determine the expression of glutamate by immunohistochemistry in normal and recovering vestibular hair cells in the chinchilla crista ampullaris after gentamicin ototoxicity. Study Design: In five groups of three animals each, ototoxicity was produced by placing gentamicin (50 μg)-impregnated Gelfoam pellets within the perilymphatic space of the superior semicircular canal. Animals were sacrificed at 1, 2, 4, 8, and 16 weeks after treatment. A group of normal (n=3) animals was also processed. Methods: For the detection of glutamate the inner ears of these animals were dissected, and the horizontal cristae ampullaris embedded in plastic. Two-micron-thick tissue sections were obtained and incubated with monoclonal antibodies against glutamate. The immunoreaction was detected using the avidinbiotiny-lated-complex technique and diaminobenzidine was the chromogen. Results: Normal sensory epithelia demonstrated type I and type II hair cells with moderate glutamate-like immunoreactivity. Supporting cells demonstrated no glutamate-like immunoreactivity. Afferent nerve fibers and calyxes surrounding type I hair cells demonstrated strong glutamate-like immunoreactivity. At 1 and 2 weeks after treatment the few type II hair cells surviving ototoxic treatment (15%–18%) contained moderate glutamate-like immunoreactivity, supporting cells showed no immunoreactivity, and nerve terminals and fibers displayed strong immunoreactivity. At 4 and 8 weeks after treatment, recovered hair cells (80%) had greater glutamate-like immunoreactivity when compared with normal hair cells, supporting cells displayed no glutamate-like immunoreactivity, and afferent fibers contained strong glutamate-like immunoreactivity. At 16 weeks, glutamate-like immunoreactivity in hair cells returned to normal level. Conclusion: Glutamate may be used as an indicator of hair cell differentiation and as an index of the molecular recovery of hair cells after ototoxicity.     

Vestibular evoked myogenic potentials are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs

This study applied the vestibular evoked myogenic potential (VEMP) test to guinea pigs coupled with electronic microscopic examination to determine whether VEMPs are dependent on type I or II hair cell activity of the saccular macula. An amount of 0.05 ml of gentamicin (40 mg/ml) was injected directly overlaying, but not through, the round window membrane of the left ear in guinea pigs.One week after surgery, auditory brainstem response test revealed normal responses in 12 animals (80%), and elevated thresholds in 3 animals (20%). The VEMP test using click stimulation showed absent responses in all 15 animals (100%). Another 6 gentamicin-treated animals underwent the VEMP test using galvanic stimulation and all 6 also displayed absent responses. Ultrathin sections of the saccular macula in the gentamicin-treated ears displayed morphologic alterations in type I or II hair cells, including shrinkage and/or vacuolization in the cytoplasm, increased electron density of the cytoplasm and nuclear chromatin, and cellular lucency. However, extrusion degeneration was rare and only present in type II hair cells. Quantitative analysis demonstrated that the histological density of intact type I hair cells was 1.1 +/- 1.2/4000 microm(2) in the gentamicin-treated ears, showing significantly less than that in control ears (4.5 +/- 1.8/4000 microm(2)). However, no significant difference was observed in the densities of intact type II hair cells and supporting cells between treated and control ears. Furthermore, the calyx terminals surrounding the damaged type I hair cells were swollen and disrupted, while the button afferents contacting the damaged type II hair cells were not obviously deformed. Based on the above results, we therefore conclude that VEMPs are heavily dependent on type I hair cell activity of the saccular macula in guinea pigs.     

Protective effects of alpha-tocopherol against gentamicin-induced Oto-vestibulo toxicity: an experimental study

OBJECTIVE: Free radicals are involved in gentamicin ototoxicity and vestibular dysfunction and it has been demonstrated that free radical scavengers, such as alpha-tocopherol, are able to inactive free radicals, attenuating tissue damage This study was designed to investigate the possible protective effects of alpha-tocopherol against gentamicin-induced oto-vestibulo toxicity. MATERIAL AND METHODS: Adult albino guinea pigs were divided into four groups and were treated for 2 weeks as follows: Group A, controls; Group B, gentamicin plus corn oil; Group C, gentamicin only; and Group D, gentamicin plus alpha-tocopherol. To evaluate vestibular function, the animals underwent sinusoidal oscillations in the dark about their vertical and longitudinal axes to evoke horizontal and vertical vestibulo-ocular reflexes (VORs), respectively. Electrocochleographic recordings were performed using an implanted round window electrode. The compound action potentials (CAPs) at 2, 4, 8 and 16 kHz were measured every 5 days Morphological changes were analysed by means of scanning electron microscopy. RESULTS: Gentamicin induced a consistent reduction in VOR responses and a progressive high-frequency hearing loss of 50-60 dB sound pressure level. Alpha-Tocopherol co-therapy slowed the progression of hearing loss and significantly attenuated the final threshold shifts The impairment of vestibular function was reduced, as evidenced by an increased VOR gain. The massive loss of outer hair cells in the cochlear basal turn and of cristae ampullaris stereocilia in gentamicin-treated animals was not observed in the cochlea of animals protected with alpha-tocopherol. CONCLUSION: This study supports the hypothesis that alpha-tocopherol interferes with gentamicin-induced free radical formation, and suggests that this drug may be useful in preventing aminoglycoside oto-vestibulo toxicity.     

Chemical composition of Galla chinensis extract and the effect of its main component(s) on the prevention of enamel demineralization in vitro

To determine the chemical composition of Galla chinensis extract(GCE) by several analysis techniques and to compare the efficacy of GCE and its main component(s) in inhibition of enamel demineralization,for the development of future anticaries agents,main organic composition of GCE was qualitatively determined by liquid chromatography-time of flight-mass spectrometry(LC-TOF-MS) and quantified by high-performance liquid chromatography-diode array detector(HPLC-DAD).Inorganic ions were tested by inductively coupled plasma-atomic emission spectroscopy and F was especially measured by ion chromatography.Then,bovine enamel blocks were randomly divided into four treatment groups and were subjected to a pH-cycling regime for 12 times.Each cycle included 5-min applications with one of four treatments:4 g?L 21 GCE solution,4 g?L 21 gallic acid(GA) solution,1 g?L 21 NaF solution(positive control),deionized water(DDW,negative control),and then 60-min application in pH 5.0 acidic buffer and 5-min application in neutral buffer.Acidic buffers were retained for calcium analysis.The main organic composition of GCE were GA and its isomer,and,to a lesser extent,small molecule gallotannins.The content of GA in GCE was 71.3%60.2%(w/w).Inorganic ions were present in various amounts,of which Ca was(13662.82) mg?g 21,and Zn was(6.860.1) mg?g 21.No F was detected in GCE.In pH cycling,GA showed an effect similar to GCE in inhibiting enamel demineralization(P.0.05).GA was found to be the main effective,demineralization inhibiting component of GCE and could be a promising agent for the development of anticaries agents.     

Distribution of efferent cholinergic terminals and α-bungarotoxin binding to putative nicotinic acetylcholine receptors in the human vestibular end-organs

Although acetylcholine (ACh) has been identified as the primary neurotransmitter of the efferent vestibular system in most animals studied, no direct evidence exists that ACh is the efferent neurotransmitter of the human vestibular system. Choline acetyltransferase immunohistochemistry (ChATi), acetylcholinesterase (AChE) histochemistry, and α-bungarotoxin binding were used in human vestibular end-organs to address this question. ChATi and AChE activity was found in numerous bouton-type terminals contacting the basal area of type II vestibular hair cells and the afferent chalices surrounding type I hair cells; α-bungarotoxin binding suggested the presence of nicotinic acetylcholine receptors on type II vestibular hair cells and on the afferent chalices surrounding type I hair cells. This study provides evidence that the human efferent vestibular axons and terminals are cholinergic and that the receptors receiving this innervation may be nicotinic. Laryngoscope, 105:1167-1172, 1995     

Gentamicin uptake in the chinchilla inner ear

Studies of transtympanic gentamicin have focused on clinical use and outcomes. This study presents evidence of bilateral uptake and retention of gentamicin in certain inner ear cells and structures following transtympanic gentamicin application. Middle ear application of gentamicin was performed by either minipump (Alza model, 2002) or transtympanic injection in a chinchilla model. Histological sections of decalcified temporal bones were stained to identify the distribution of gentamicin. Using both anti-gentamicin immunohistochemistry and autoradiography of tracer amounts of tritiated gentamicin, Scarpa’s and spiral ganglion cells, stria vascularis, and vestibular dark cells of the injected ear were found to have higher levels of gentamicin and retain it within cell bodies while staining levels fell to background levels in the rest of the injected ear over the course of 14 days. There was no evidence of an apical to basal gradient of anti-gentamicin staining within the spiral ganglion. Contralateral inner ear cells showed light anti-gentamicin staining. Cell bodies in the ipsilateral dorsal cochlear nucleus bordering the cochlear aqueduct (CA) showed a lateral to medial gradient of gentamicin staining, suggesting the CA as a potential site of transfer of gentamicin to the contralateral ear. Direct effects of aminoglycosides on ganglion cells may have implications on both the success of cochlear implantation in patients deafened following systemic aminoglycoside therapy and on the advisability of clinical practices of transtympanic gentamicin therapy and ototopic aminoglycoside treatment.     

Effect of colouring green stage zirconia on the adhesion of veneering ceramics with different thermal expansion coefficients

This study evaluated the adhesion of zirconia core ceramics with their corresponding veneering ceramics, having different thermal expansion coefficients （TECs）, when zirconia ceramics were coloured at green stage. Zirconia blocks （N=240; 6 mm x 7 mm x 7 mm） were manufactured from two materials namely, ICE Zirconia （Group 1） and Prettau Zirconia （Group 2）. In their green stage, they were randomly divided into two groups. Half of the specimens were coloured with colouring liquid （shade A2）, Three different veneering ceramics with different TEC （ICE Ceramic, GC Initial Zr and IPS e.max Ceram） were fired on both coloured and non-coloured zirconia cores. Specimens of high noble alloys （Esteticor Plus） veneered with ceramic （VM 13） （n= 16） acted as the control group. Core-veneer interface of the specimens were subjected to shear force in the Universal Testing Machine （0.5 mm-min-1）. Neither the zirconia core material （P=0.318） nor colouring （P=0.188） significantly affected the results （three-way analysis of variance, Tukey＇s test）. But the results were significantly affected by the veneering ceramic （P=0.000）. Control group exhibited significantly higher mean bond strength values （45.7__.8） MPa than all other tested groups （（27.1__.4.1）-（39.7__.4.7） and （27.4__.5.6）-（35.9___4.7） MPa with and without colouring, respectively） （P~0.001）. While in zirconia-veneer test groups, predominantly mixed type of failures were observed with the veneering ceramic covering ~ 1/3 of the substrate surface, in the metal-ceramic group, veneering ceramic was left adhered 1/3 of the metal surface. Colouring zirconia did not impair adhesion of veneering ceramic, but veneering ceramic had a significant influence on the core-veneer adhesion. Metal-ceramic adhesion was more reliable than all zirconia-veneer ceramics tested.     

A Genome-Wide Association Study of Chronic Otitis Media with Effusion and Recurrent Otitis Media Identifies a Novel Susceptibility Locus on Chromosome 2

Chronic otitis media with effusion (COME) and recurrent otitis media (ROM) have been shown to be heritable, but candidate gene and linkage studies to date have been equivocal. Our aim was to identify genetic susceptibility factors using a genome-wide association study (GWAS). We genotyped 602 subjects from 143 families with 373 COME/ROM subjects using the Illumina Human CNV370-Duo DNA Bead Chip (324,748 SNPs). We carried out the GWAS scan and imputed SNPs at the regions with the most significant associations. Replication genotyping in an independent family-based sample was conducted for 53 SNPs: the 41 most significant SNPs with P < 10⁻⁴ and 12 imputed SNPs with P < 10⁻⁴ on chromosome 15 (near the strongest signal). We replicated the association of rs10497394 (GWAS discovery P = 1.30 × 10⁻⁵) on chromosome 2 in the independent otitis media population (P = 4.7 × 10⁻⁵; meta-analysis P = 1.52 × 10⁻⁸). Three additional SNPs had replication P values < 0.10. Two were on chromosome 15q26.1 including rs1110060, the strongest association with COME/ROM in the primary GWAS (P = 3.4 ×10⁻⁷) in KIF7 intron 7 (P = 0.072), and rs10775247, a non-synonymous SNP in TICRR exon 2 (P = 0.075). The third SNP rs386057 was on chromosome 5 in TPPP intron 1 (P = 0.045). We have performed the first GWAS of COME/ROM and have identified a SNP rs10497394 on chromosome 2 is significantly associated with COME/ROM susceptibility. This SNP is within a 537 kb intergenic region, bordered by CDCA7 and SP3. The genomic and functional significance of this newly identified locus in COME/ROM pathogenesis requires additional investigation.     

Histopathologic Changes of the Inner ear in Rhesus Monkeys After Intratympanic Gentamicin Injection and Vestibular Prosthesis Electrode Array Implantation

Bilateral vestibular deficiency (BVD) due to gentamicin ototoxicity can significantly impact quality of life and result in large socioeconomic burdens. Restoring sensation of head rotation using an implantable multichannel vestibular prosthesis (MVP) is a promising treatment approach that has been tested in animals and humans. However, uncertainty remains regarding the histopathologic effects of gentamicin ototoxicity alone or in combination with electrode implantation. Understanding these histological changes is important because selective MVP-driven stimulation of semicircular canals (SCCs) depends on persistence of primary afferent innervation in each SCC crista despite both the primary cause of BVD (e.g., ototoxic injury) and surgical trauma associated with MVP implantation. Retraction of primary afferents out of the cristae and back toward Scarpa’s ganglion would render spatially selective stimulation difficult to achieve and could limit utility of an MVP that relies on electrodes implanted in the lumen of each ampulla. We investigated histopathologic changes of the inner ear associated with intratympanic gentamicin (ITG) injection and/or MVP electrode array implantation in 11 temporal bones from six rhesus macaque monkeys. Hematoxylin and eosin-stained 10-μm temporal bone sections were examined under light microscopy for four treatment groups: normal (three ears), ITG-only (two ears), MVP-only (two ears), and ITG + MVP (four ears). We estimated vestibular hair cell (HC) surface densities for each sensory neuroepithelium and compared findings across end organs and treatment groups. In ITG-only, MVP-only, and ITG + MVP ears, we observed decreased but persistent ampullary nerve fibers of SCC cristae despite ITG treatment and/or MVP electrode implantation. ITG-only and ITG + MVP ears exhibited neuroepithelial thinning and loss of type I HCs in the cristae but little effect on the maculae. MVP-only and ITG + MVP ears exhibited no signs of trauma to the cochlea or otolith end organs except in a single case of saccular injury due to over-insertion of the posterior SCC electrode. While implanted electrodes reached to within 50–760 μm of the target cristae and were usually ensheathed in a thin fibrotic capsule, dense fibrotic reaction and osteoneogenesis were each observed in only one of six electrode tracts examined. Consistent with physiologic studies that have demonstrated directionally appropriate vestibulo-ocular reflex responses to MVP electrical stimulation years after implantation in these animals, histologic findings in the present study indicate that although intralabyrinthine MVP implantation causes some inner ear trauma, it can be accomplished without destroying the distal afferent fibers an MVP is designed to excite.     

Inhibition of caspases alleviates gentamicin-induced cochlear damage in guinea pigs

The efficacy of caspase inhibitors for protecting the cochlea was evaluated in an in vivo study using guinea pigs, as the animal model system. Gentamicin (12 mg/ml) was delivered via an osmotic pump into the cochlear perilymphatic space of guinea pigs at 0.5 microl/h for 14 days. Additional animals were given either z-Val-Ala-Asp (Ome)-fluoromethyl ketone (z-VAD-FMK) or z-Leu-Glu-His-Asp-FMK (z-LEHD-FMK), a general caspase inhibitor and a caspase 9 inhibitor, respectively, in addition to gentamicin. The elevation in auditory brain stem response thresholds, at 4, 7, and 14 days following gentamicin administration, were decreased in animals that received both z-VAD-FMK and z-LEHD-FMK. Cochlear sensory hair cells survived in greater numbers in animals that received caspase inhibitors in addition to gentamicin, whereas sensory hair cells in animals that received gentamicin only were severely damaged. These results suggest that auditory cell death induced by gentamicin is closely related to the activation of caspases in vivo.