Tumor Necrosis Factor-alpha-mutant Mice Exhibit High Frequency Hearing Loss

Exogenous tumor necrosis factor-alpha (TNF-α) plays a role in auditory hair cell death by altering the expression of apoptosis-related genes in response to noxious stimuli. Little is known, however, about the function of TNF-α in normal hair cell physiology. We, therefore, investigated the cochlear morphology and auditory function of TNF-α-deficient mice. Auditory evoked brainstem response showed significantly higher thresholds, especially at higher frequencies, in 1-month-old TNF-α^−/− mice as compared to TNF-α^+/− and wild type (WT); hearing loss did not progress further from 1 to 4 months of age. There was no difference in the gross morphology of the organ of Corti, lateral wall, and spiral ganglion cells in TNF-α^−/− mice compared to WT mice at 4 months of age, nor were there differences in the anatomy of the auditory ossicles. Outer hair cells were completely intact in surface preparations of the organ of Corti of TNF-α^−/− mice, and synaptic ribbon counts of TNF-α^−/− and WT mice at 4 months of age were similar. Reduced amplitudes of distortion product otoacoustic emissions, however, indicated dysfunction of outer hair cells in TNF-α^−/− mice. Scanning electron microscopy revealed that stereocilia were sporadically absent in the basal turn and distorted in the middle turn. In summary, our results demonstrate that TNF-α-mutant mice exhibit early hearing loss, especially at higher frequencies, and that loss or malformation of the stereocilia of outer hair cells appears to be a contributing factor.     

Preliminary Characterization of Voltage-Activated Whole-Cell Currents in Developing Human Vestibular Hair Cells and Calyx Afferent Terminals

We present preliminary functional data from human vestibular hair cells and primary afferent calyx terminals during fetal development. Whole-cell recordings were obtained from hair cells or calyx terminals in semi-intact cristae prepared from human fetuses aged between 11 and 18 weeks gestation (WG). During early fetal development (11–14 WG), hair cells expressed whole-cell conductances that were qualitatively similar but quantitatively smaller than those observed previously in mature rodent type II hair cells. As development progressed (15–18 WG), peak outward conductances increased in putative type II hair cells but did not reach amplitudes observed in adult human hair cells. Type I hair cells express a specific low-voltage activating conductance, G _K,L. A similar current was first observed at 15 WG but remained relatively small, even at 18 WG. The presence of a “collapsing” tail current indicates a maturing type I hair cell phenotype and suggests the presence of a surrounding calyx afferent terminal. We were also able to record from calyx afferent terminals in 15–18 WG cristae. In voltage clamp, these terminals exhibited fast inactivating inward as well as slower outward conductances, and in current clamp, discharged a single action potential during depolarizing steps. Together, these data suggest the major functional characteristics of type I and type II hair cells and calyx terminals are present by 18 WG. Our study also describes a new preparation for the functional investigation of key events that occur during maturation of human vestibular organs.     

Current Steering with Partial Tripolar Stimulation Mode in Cochlear Implants

The large spread of excitation is a major cause of poor spectral resolution for cochlear implant (CI) users. Partial tripolar (pTP) mode has been proposed to reduce current spread by returning an equally distributed fraction (0.5 × σ) of current to two flanking electrodes and the rest to an extra-cochlear ground. This study tested the efficacy of incorporating current steering into pTP mode to add spectral channels. Different proportions of current [α × σ and (1 − α) × σ] were returned to the basal and apical flanking electrodes respectively to shape the electric field. Loudness and pitch perception with α from 0 to 1 in steps of 0.1 was simulated with a computational model of CI stimulation and tested on the apical, middle, and basal electrodes of six CI subjects. The highest σ allowing for full loudness growth within the implant compliance limit was chosen for each main electrode. Pitch ranking was measured between pairs of loudness-balanced steered pTP stimuli with an α interval of 0.1 at the most comfortable level. Results demonstrated that steered pTP stimuli with α around 0.5 required more current to achieve equal loudness than those with α around 0 or 1, maybe due to more focused excitation patterns. Subjects usually perceived decreasing pitches as α increased from 0 to 1, somewhat consistent with the apical shift of the center of gravity of excitation pattern in the model. Pitch discrimination was not better with α around 0.5 than with α around 0 or 1, except for some subjects and electrodes. For three subjects with better pitch discrimination, about half of the pitch ranges of two adjacent main electrodes overlapped with each other in steered pTP mode. These results suggest that current steering with focused pTP mode may improve spectral resolution and pitch perception with CIs.     

Influence of acrylamide monomer addition to the acrylic denture-base resins on mechanical and physical properties

The aim of the study was to evaluate the effect of adding acrylamide monomer （AAm） on the characterization, flexural strength, flexural modulus and thermal degradation temperature of poly（methyl methacrylate） （PMMA） denture-base resins. Specimens （n= 10） were fabricated from a conventional heat-activated QC-20 （Qc-） and a microwave heat-activated Acron MC （Ac-） PMMA resins. Powder/ liquid ratio followed the manufacturer＇s instructions for the control groups （Qc-c and Ac-c） and for the copolymer groups, the resins were prepared with 5% （-5）, 10% （- 10）, 15% （- 15） and 20% （-20） acrylamide contents, according to the molecular weight ratio, respectively. The flexural strength and flexural modulus were measured by a three-point bending test. The data obtained were statistically analyzed by Kruskal-Wallis test （a=O.05） to determine significant differences between the groups, The chemical structures of the resins were characterized by the nuclear magnetic resonance spectroscopy. Thermal stabilities were determined by thermogravimetric analysis （TGA） with a heating rate of 10 ~C.min-1 from 35 ~C to 600 ~C. Control groups from both acrylic resins showed the lowest flexural strength values. Qc-15 showed significant increase in the flexural strength when compared to Qc-c （P〈O.01）. Ac-10 and Ac-15 showed significance when compared to Ac-c （P〈O.01）. Acrylamide incorporation increased the elastic modulus in Qc-10, Qc-15 and Qc-20 when compared to Qc-c （P〈0.01）. Also significant increase was observed in Ac-10, Ac-15 and Ac-20 copolymer groups when compared to Ac-c （P〈0.01）. According to the 1H-nuclear magnetic resonance （NMR） results, acrylamide copolymerization was confirmed in the experimental groups. TGA results showed that the thermal stability of PMMA is increased by the insertion of AAm.     

cAMP mediates transepithelial K+ and Na+ transport in a strial marginal cell line

Because cytoplasmic cAMP has been reported to be the secondary messenger mediating K⁺ transport in marginal cells of freshly isolated stria vascularis, the possible role of cAMP in ion transport processes of an immortalized marginal cell line (MCPV-8) showing evidence of K⁺ and Na⁺ reabsorption was evaluated in this study. Confluent MCPV-8 monolayers were mounted into Ussing chambers and perfused on both sides with perilymph-like Ringer's solution. Transepithelial short-circuit current (I_SC), resistance (R_T) and open-circuit voltage (V_T) were measured using voltage clamp technique. The following results were obtained. (1) Addition of forskolin (10⁻⁴ M) to the basolateral perfusate increased I_SC to 311±42%; no significant change in R_T was observed. Addition of BaCl₂ (2 mM) to the apical perfusate at the maximal response of forskolin blocked 50–60% of I_SC and subsequent addition of amiloride (10⁻⁵ M) to the apical perfusate further blocked I_SC to a value close to 0. (2) To evaluate the effect of cellular cAMP on Ba²⁺-sensitive K⁺ current, amiloride-sensitive Na⁺ current was blocked first by addition of amiloride (10⁻⁵ M) to the apical perfusate; subsequent addition of 3-isobutyl-1-methylxanthine (IBMX, 1 mM) or N⁶,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate (dbcAMP, 1 mM) to the basolateral perfusate increased I_SC to 175±13% and 411±32%, respectively. The stimulated I_SC was blocked to close to 0 by addition of BaCl₂ (2 mM) to the apical perfusate. N²,2′-O-Dibutyrylguanosine 3′,5′-cyclic monophosphate (dbcGMP, 1 mM) had no effect on I_SC. (3) To assess the effect of cellular cAMP on amiloride-sensitive Na⁺ current, Ba²⁺-sensitive K⁺ current was blocked in advance by addition of BaCl₂ to the apical perfusate; subsequent addition of IBMX or dbcAMP to the basolateral perfusate increased I_SC to 219±21% and 388±39%, respectively. The stimulated I_SC was blocked to close to 0 by addition of amiloride to the apical perfusate. dbcGMP had no effect on I_SC. Hence, these results suggest that cellular cAMP is the secondary messenger that mediates the transepithelial transport of both K⁺ and Na⁺ in MCPV-8 monolayers.     

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.     

K+ Currents in Isolated Vestibular Afferent Calyx Terminals

Vestibular hair cells transduce mechanical displacements of their hair bundles into an electrical receptor potential which modulates transmitter release and subsequent action potential firing in afferent neurons. To probe ionic mechanisms underlying sensory coding in vestibular calyces, we used the whole-cell patch-clamp technique to record action potentials and K⁺ currents from afferent calyx terminals isolated from the semicircular canals of Mongolian gerbils. Calyx terminals showed minimal current at the mean zero-current potential (−60 mV), but two types of outward K⁺ currents were identified at potentials above −50 mV. The first current was a rapidly activating and inactivating K⁺ current that was blocked by 4-aminopyridine (4-AP, 2.5 mM) and BDS-I (up to 250 nM). The time constant for activation of this current decreased with membrane depolarization to a minimum value of ∼1 ms. The 4-AP-sensitive current showed steady-state inactivation with a half-inactivation of approximately −70 mV. A second, more slowly activating current (activation time constant was 8.5 ± 0.7 ms at −8 mV) was sensitive to TEA (30 mM). The TEA-sensitive current also showed steady-state inactivation with a half-inactivation of −95.4 ± 1.4 mV, following 500-ms duration conditioning pulses. A combination of 4-AP and TEA blocked ∼90% of the total outward current. In current clamp, single Na⁺-dependent action potentials were evoked following hyperpolarization to potentials more negative than the resting potential. 4-AP application increased action potential width, whereas TEA both increased the width and greatly reduced repolarization of the action potential.     

Chlorogenic acid alters the voltage-gated potassium channel currents of trigeminal ganglion neurons

Chlorogenic acid（5-caffeoylquinic acid, CGA） is a phenolic compound that is found ubiquitously in plants, fruits and vegetables and is formed via the esterification of caffeic acid and quinic acid. In addition to its notable biological functions against cardiovascular diseases, type-2 diabetes and inflammatory conditions, CGA was recently hypothesized to be an alternative for the treatment of neurological diseases such as Alzheimer＇s disease and neuropathic pain disorders. However, its mechanism of action is unclear.Voltage-gated potassium channel（Kv） is a crucial factor in the electro-physiological processes of sensory neurons. Kv has also been identified as a potential therapeutic target for inflammation and neuropathic pain disorders. In this study, we analysed the effects of CGA on the two main subtypes of Kv in trigeminal ganglion neurons, namely, the IK,Aand IK,Vchannels. Trigeminal ganglion（TRG）neurons were acutely disassociated from the rat TRG, and two different doses of CGA（0.2 and 1 mmol·L21） were applied to the cells.Whole-cell patch-clamp recordings were performed to observe alterations in the activation and inactivation properties of the IK,Aand IK,Vchannels. The results demonstrated that 0.2 mmol·L21CGA decreased the peak current density of IK,A. Both 0.2 mmol·L21and1 mmol·L21CGA also caused a significant reduction in the activation and inactivation thresholds of IK,Aand IK,V. CGA exhibited a strong effect on the activation and inactivation velocities of IK,Aand IK,V. These findings provide novel evidence explaining the biological effects of CGA, especially regarding its neurological effects.     

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.     

Growth inhibitory response and ultrastructural modification of oral-associated candidal reference strains （ATCC） by Piper betle L. extract

Candida species have been associated with the emergence of strains resistant to selected antifungal agents. Plant products have been used traditionally as alternative medicine to ease mucosal fungal infections. This study aimed to investigate the effects of Piper betle extract on the growth profile and the ultrastructure of commonly isolated oral candidal cells. The major component of Po betle was identified using liquid chromatography-mass spectrophotometry （LC-MS/MS）. Seven ATCC control strains of Candida species were cultured in yeast peptone dextrose broth under four different growth environments： （i） in the absence of P. betle extract; and in the presence of P. beUeextract at respective concentrations of （ii） 1 mg.mL-1; （iii） 3 mg.mL-1; and （iv） 6 mg.mL- 1 The growth inhibitory responses of the candidal cells were determined based on changes in the specific growth rates （μ）. Scanning electron microscopy （SEM） was used to observe any ultrastructural alterations in the candida colonies. LC-MS/MS was performed to validate the presence of bioactive compounds in the extract. Following treatment, it was observed that the p-values of the treated cells were significantly different than those of the untreated cells （P〈0.05）, indicating the fungistatic properties of the P. beUe extract. The candidal population was also reduced from an average of 13.44× 10^6 to 1.78×10^6 viable cell counts （CFU）.mL-1, SEM examination exhibited physical damage and considerable morphological alterations of the treated cells. The compound profile from LC-MS/MS indicated the presence of hydroxybenzoic acid, chavibetol and hydroxychavicol in P. betle extract. The effects of P. betle on candida cells could potentiate its antifungal activity.     

Experimental Measurement of Utricle System Dynamic Response to Inertial Stimulus

The membranous utricle sac of the red-eared turtle was mounted in a piezoelectric actuated platform mounted on the stage of a light microscope. The piezoelectric actuator oscillated the base of the neuroepithelium along a linear axis. Displacements were in the plane of the utricle and consisted of a linear sinusoidal-sweep signal starting at 0 and increasing to 500 Hz over 5 s. This inertial stimulus caused measurable shear displacement of the otoconial layer’s dorsal surface, resulting in shear deformation of the gelatinous and column filament layers. Displacements of the otoconial layer and a reference point on the neuroepithelium were filmed at 2,000 frames/s with a high-speed video camera during oscillations. Image registration was performed on the video to track displacements with a resolution better than 15 nm. The displacement waveforms were then matched to a linear second-order model of the dynamic system. The model match identified two system mechanical parameters—the natural circular frequency ω_n and the damping ratio ζ—that characterized the utricle dynamic response. The median values found for the medial-lateral axis on 20 utricles with 95 % confidence intervals in parenthesis were as follows: ω_n = 374 (353, 396) Hz and ζ = 0.50 (0.47, 0.53). The anterior-posterior axis values were not significantly different: ω_n = 409 (390, 430) Hz and ζ = 0.53 (0.48, 0.57). The results have two relevant and significant dynamic system findings: (1) a higher than expected natural frequency and (2) significant under damping. Previous to this study, utricular systems were treated as overdamped and with natural frequencies much lower that measured here. Both of these system performance findings result in excellent utricle time response to acceleration stimuli and a broad frequency bandwidth up to 100 Hz. This study is the first to establish the upper end of this mechanical system frequency response of the utricle in any animal.     

I h and HCN Channels in Murine Spiral Ganglion Neurons: Tonotopic Variation,Local Heterogeneity,and Kinetic Model

One of the major contributors to the response profile of neurons in the auditory pathways is the I _h current. Its properties such as magnitude, activation, and kinetics not only vary among different types of neurons (Banks et al., J Neurophysiol 70:1420–1432, 1993; Fu et al., J Neurophysiol 78:2235–2245, 1997; Bal and Oertel, J Neurophysiol 84:806–817, 2000; Cao and Oertel, J Neurophysiol 94:821–832, 2005; Rodrigues and Oertel, J Neurophysiol 95:76–87, 2006; Yi et al., J Neurophysiol 103:2532–2543, 2010), but they also display notable diversity in a single population of spiral ganglion neurons (Mo and Davis, J Neurophysiol 78:3019–3027, 1997), the first neural element in the auditory periphery. In this study, we found from somatic recordings that part of the heterogeneity can be attributed to variation along the tonotopic axis because I _h in the apical neurons have more positive half-activation voltage levels than basal neurons. Even within a single cochlear region, however, I _h current properties are not uniform. To account for this heterogeneity, we provide immunocytochemical evidence for variance in the intracellular density of the hyperpolarization-activated cyclic nucleotide-gated channel α-subunit 1 (HCN1), which mediates I _h current. We also observed different combinations of HCN1 and HCN4 α-subunits from cell to cell. Lastly, based on the physiological data, we performed kinetic analysis for the I _h current and generated a mathematical model to better understand varied I _h on spiral ganglion function. Regardless of whether I _h currents are recorded at the nerve terminals (Yi et al., J Neurophysiol 103:2532-2543, 2010) or at the somata of spiral ganglion neurons, they have comparable mean half-activation voltage and induce similar resting membrane potential changes, and thus our model may also provide insights into the impact of I _h on synaptic physiology.     

The growth of Staphylococcus aureus and Escherichia coli in low-direct current electric fields

Electrical potentials up to 800 mV can be observed between different metallic dental restorations. These potentials produce fields in the mouth that may interfere with microbial communities. The present study focuses on the impact of different electric field strengths （EFS） on the growth of Staphylococcus aureus （ATCC 25923） and Escherichia coil （ATCC 25922） in vitro. Cultures of S. aureus and E. coil in fluid and gel medium were exposed to different EFS. Effects were determined by calculation of viable counts and measurement of inhibition zones. In gel medium, anodic inhibition zones for S. aureuswere larger than those for E. coliat all field strength levels. In fluid medium, the maximum decrease in the viable count of S. aureus cells was at 10 V.m-1. Field-treated S. aureus cells presented ruptured cell walls and disintegrated cytoplasm. Conclusively, S. aureus is more sensitive to increasing electric field strength than E. coll.     

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.     

On the High Frequency Transfer of Mechanical Stimuli from the Surface of the Head to the Macular Neuroepithelium of the Mouse

Vestibular macular sensors are activated by a shearing motion between the otoconial membrane and underlying receptor epithelium. Shearing motion and sensory activation in response to an externally induced head motion do not occur instantaneously. The mechanically reactive elastic and inertial properties of the intervening tissue introduce temporal constraints on the transfer of the stimulus to sensors. Treating the otoconial sensory apparatus as an overdamped second-order mechanical system, we measured the governing long time constant (Τ_L) for stimulus transfer from the head surface to epithelium. This provided the basis to estimate the corresponding upper cutoff for the frequency response curve for mouse otoconial organs. A velocity step excitation was used as the forcing function. Hypothetically, the onset of the mechanical response to a step excitation follows an exponential rise having the form Vel_shear = U(1-e^−t/TL), where U is the applied shearing velocity step amplitude. The response time of the otoconial apparatus was estimated based on the activation threshold of macular neural responses to step stimuli having durations between 0.1 and 2.0 ms. Twenty adult C57BL/6 J mice were evaluated. Animals were anesthetized. The head was secured to a shaker platform using a non-invasive head clip or implanted skull screws. The shaker was driven to produce a theoretical forcing step velocity excitation at the otoconial organ. Vestibular sensory evoked potentials (VsEPs) were recorded to measure the threshold for macular neural activation. The duration of the applied step motion was reduced systematically from 2 to 0.1 ms and response threshold determined for each duration (nine durations). Hypothetically, the threshold of activation will increase according to the decrease in velocity transfer occurring at shorter step durations. The relationship between neural threshold and stimulus step duration was characterized. Activation threshold increased exponentially as velocity step duration decreased below 1.0 ms. The time constants associated with the exponential curve were Τ_L = 0.50 ms for the head clip coupling and T_L = 0.79 ms for skull screw preparation. These corresponded to upper −3 dB frequency cutoff points of approximately 318 and 201 Hz, respectively. T_L ranged from 224 to 379 across individual animals using the head clip coupling. The findings were consistent with a second-order mass-spring mechanical system. Threshold data were also fitted to underdamped models post hoc. The underdamped fits suggested natural resonance frequencies on the order of 278 to 448 Hz as well as the idea that macular systems in mammals are less damped than generally acknowledged. Although estimated indirectly, it is argued that these time constants reflect largely if not entirely the mechanics of transfer to the sensory apparatus. The estimated governing time constant of 0.50 ms for composite data predicts high frequency cutoffs of at least 318 Hz for the intact otoconial apparatus of the mouse.     

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.     

Decreased Immunoreactivities and Functions of the Chloride Transporters, KCC2 and NKCC1, in the Lateral Superior Olive Neurons of Circling Mice

Objectives

We tested the possibility of differential expression and function of the potassium-chloride (KCC2) and sodium-potassium-2 chloride (NKCC1) co-transporters in the lateral superior olive (LSO) of heterozygous (+/cir) or homozygous (cir/cir) mice.

Methods

Mice pups aged from postnatal (P) day 9 to 16 were used. Tails from mice were cut for DNA typing. For Immunohistochemical analysis, rabbit polyclonal anti-KCC2 or rabbit polyclonal anti-NKCC1 was used and the density of immunolabelings was evaluated using the NIH image program. For functional analysis, whole cell voltage clamp technique was used in brain stem slices and the changes of reversal potentials were evaluated at various membrane potentials.

Results

Immunohistochemical analysis revealed both KCC2 and NKCC1 immunoreactivities were more prominent in heterozygous (+/cir) than homozygous (cir/cir) mice on P day 16. In P9-P12 heterozygous (+/cir) mice, the reversal potential (E_gly) of glycine-induced currents was shifted to a more negative potential by 50 µM bumetanide, a known NKCC1 blocker, and the negatively shifted E_gly was restored by additional application of 1 mM furosemide, a KCC2 blocker (-58.9±2.6 mV to -66.0±1.5 mV [bumetanide], -66.0±1.5 mV to -59.8±2.8 mV [furosemide+bumetanide], n=11). However, only bumetanide was weakly, but significantly effective (-60.1±2.9 mV to -62.7±2.6 mV [bumetanide], -62.7±2.6 mV to -62.1±2.5 mV [furosemide+bumetanide], n=7) in P9-P12 homozygous (cir/cir) mice.

Conclusion

The less prominent immunoreactivities and weak or absent responses to bumetanide or furosemide suggest impaired function or delayed development of both transporters in homozygous (cir/cir) mice.     

Hearing Loss is an Early Consequence of Npc1 Gene Deletion in the Mouse Model of Niemann–Pick Disease,Type C

Niemann–Pick disease, type C1 (NPC1) is a rare lysosomal lipidosis that is most often the result of biallelic mutations in NPC1, and is characterized by a fatal neurological degeneration. The pathophysiology is complex, and the natural history of the disease is poorly understood. Recent findings from patients with NPC1 and hearing loss suggest that multiple steps along the auditory pathway are affected. The current study was undertaken to determine the auditory phenotype in the Npc1^nih mutant mouse model, to extend analyses to histologic evaluation of the inner ear, and to compare our findings to those reported from human patients. Auditory testing revealed a progressive high-frequency hearing loss in Npc1^−/− mice that is present as early as postnatal day 20 (P20), well before the onset of overt neurological symptoms, with evidence of abnormalities involving the cochlea, auditory nerve, and brainstem auditory centers. Distortion product otoacoustic emission amplitude and auditory brainstem response latency data provided evidence for a disruption in maturational development of the auditory system in Npc1^−/− mice. Anatomical study demonstrated accumulation of lysosomes in neurons, hair cells, and supporting cells of the inner ear in P30 Npc1^−/− mice, as well as increased numbers of inclusion bodies, myelin figures, and swollen nerve endings in older (P50–P70) mutant animals. These findings add unique perspective to the pathophysiology of NPC disease and suggest that hearing loss is an early and sensitive marker of disease progression.