Wideband frequency impedance for diagnosis of ossicular chain abnormality.

BACKGROUND: The accurate estimation of the ossicular chain abnormalities using existing functional examinations has been difficult. AIMS/OBJECTIVES: This study aimed to verify the accuracy of preoperative diagnosis of ossicular chain abnormalities using a wideband frequency impedance (WFI) meter, which can measure the dynamic characteristics of the middle ear. MATERIAL AND METHODS: Retrospective cohort study. Fourteen ears of patients with ossicular chain abnormalities that were definitively diagnosed surgically were included in this study. The following data were collected for each participant: sound pressure level (SPL) curve measured using the WFI meter and a sweep frequency impedance (SFI) meter, WFI measurements plotted on the resonance frequency (RF)-ΔSPL plane, distribution map of the dynamic characteristics of the middle ear, preoperative audiometry results, and the definitive surgical diagnosis. RESULTS: The SPL curve obtained using the WFI meter had lesser noise than that obtained using the SFI meter. The distribution map revealed that the ossicular chain separation range and ossicular chain fixation range were completely separated. The hearing data tended to be poor in cases with small ΔSPL. CONCLUSIONS AND SIGNIFICANCE: WFI can potentially enhance the accuracy of SFI. In addition, it can also be used for the classification of ossicular chain separation and fixation as well as the quantification of fixation in cases of ossicular chain anomalies that cannot be diagnosed using conventional tests.

Effects of salicylate derivatives on localization of p.H723R allele product of SLC26A4.

OBJECTIVE: Pendrin is a transmembrane protein encoded by the SLC26A4 gene that functions in maintaining ion concentrations in the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Variants in the SLC26A4 gene are responsible for sensorineural hearing loss. Although pendrin localizes to the plasma membrane, we previously identified that 8 missense allele products of SLC26A4 were retained in the intracellular region and lost their anion exchange function. We also found that 10 mM salicylate induced the translocation of 4 out of 8 allele products from the intracellular region to the plasma membrane and restored their anion exchanger activity. However, since 10 mM salicylate exhibits cytotoxicity, the use of chemical compounds with less cell toxicity is needed. In the present study, therefore, salicylate derivatives were used as the chemical compounds and their effects on the p.H723R allele products of SLC26A4 were investigated. METHODS: HEK293 cells were transfected with the cDNA of p.H723R. Cell proliferation, viability and toxicity assays were performed to investigate the response and health of cells in culture after treatment with four types of salicylate derivatives, i.e., 2-hydroxybenzyl alcohol, 2,3-dihydroxybenzoic acid, 2'-hydroxyacetophenone and methyl salicylate. The effects of these salicylate derivatives on the localization of the p.H723R were investigated by immunofluorescence microscopy. RESULTS: The application of 10 mM salicylate showed an increase in cell toxicity and decrease in cell viability, leading to a significant decrease in cell proliferation. In contrast, the application of 1 mM salicylate derivatives did not show any significant increase in cell toxicity and decrease in cell viability, corresponding to a logarithmic increase in cell concentration with an increase in culture time. Immunofluorescence experiments showed that the p.H723R retained in the endoplasmic reticulum (ER). Among the salicylate derivatives applied, 2-hydroxybenzyl alcohol induced the translocation of p.H723R from the ER to the plasma membrane 3 h after its application. CONCLUSION: The results obtained showed that 2-hydroxybenzyl alcohol restored the localization of the p.H723R allele products of SLC26A4 from the ER to the plasma membrane at a concentration of 1 mM by 3 h after its administration with less cytotoxicity than 10 mM salicylate.

Longitudinal changes in dynamic characteristics of neonatal external and middle ears.

OBJECTIVES: Neonates have smaller and less mature ears than adults. Developmental changes in structure and function continually occur after birth and may affect the diagnostic results obtained by audiometric assessment instrumentation, such as tympanometry and otoacoustic emission. In the present study, we investigated longitudinal changes in external and middle ear dynamic characteristics by performing sweep frequency impedance (SFI) tests. METHODS: SFI tests were longitudinally performed on healthy Japanese neonates (1 female and 1 male) from birth to 3 and 5 months, respectively. A sound of sweeping sinusoidal frequency, ranging from 0.1 kHz to 2 kHz, was presented to the ear canal at 50-daPa intervals of static pressure from +200 to -200 daPa. Test results were expressed a curve showing the sound pressure level (SPL) relative to probe tone frequency, called SPL curve. RESULTS: The first fluctuation in resonance frequency (RF1) and SPL (ΔSPL1), related to the external ear, showed significant developmental changes as chronological age increased; that is, RF1 and ΔSPL1 were respectively increased and decreased and thereafter became unmeasurable by 5 months of age. In contrast, the second fluctuation in resonance frequency (RF2) and SPL (ΔSPL2), related to the middle ear, did not show significant changes over the measurement period. CONCLUSIONS: The present results suggest that the dynamic characteristics of the external ear canal wall changed with increases in chronological age; the resonance of the wall at about 0.3 kHz at birth tended to increase to about 0.7 kHz and to be unmeasurable by 5 months of age, while those of the middle ear did not significantly changed. These results showing how neonatal-ear dynamics changes with chronological age may be an important key in further hearing research and the development of hearing devices and diagnostic tools suitable for neonates.

Predictive Accuracy of Sweep Frequency Impedance Technology in Identifying Conductive Conditions in Newborns.

BACKGROUND: Diagnosing conductive conditions in newborns is challenging for both audiologists and otolaryngologists. Although high-frequency tympanometry (HFT), acoustic stapedial reflex tests, and wideband absorbance measures are useful diagnostic tools, there is performance measure variability in their detection of middle ear conditions. Additional diagnostic sensitivity and specificity measures gained through new technology such as sweep frequency impedance (SFI) measures may assist in the diagnosis of middle ear dysfunction in newborns. PURPOSE: The purpose of this study was to determine the test performance of SFI to predict the status of the outer and middle ear in newborns against commonly used reference standards. RESEARCH DESIGN: Automated auditory brainstem response (AABR), HFT (1000 Hz), transient evoked otoacoustic emission (TEOAE), distortion product otoacoustic emission (DPOAE), and SFI tests were administered to the study sample. STUDY SAMPLE: A total of 188 neonates (98 males and 90 females) with a mean gestational age of 39.4 weeks were included in the sample. Mean age at the time of testing was 44.4 hr. DATA COLLECTION AND ANALYSIS: Diagnostic accuracy of SFI was assessed in terms of its ability to identify conductive conditions in neonates when compared with nine different reference standards (including four single tests [AABR, HFT, TEOAE, and DPOAE] and five test batteries [HFT + DPOAE, HFT + TEOAE, DPOAE + TEOAE, DPOAE + AABR, and TEOAE + AABR]), using receiver operating characteristic (ROC) analysis and traditional test performance measures such as sensitivity and specificity. RESULTS: The test performance of SFI against the test battery reference standard of HFT + DPOAE and single reference standard of HFT was high with an area under the ROC curve (AROC) of 0.87 and 0.82, respectively. Although the HFT + DPOAE test battery reference standard performed better than the HFT reference standard in predicting middle ear conductive conditions in neonates, the difference in AROC was not significant. Further analysis revealed that the highest sensitivity and specificity for SFI (86% and 88%, respectively) was obtained when compared with the reference standard of HFT + DPOAE. Among the four single reference standards, SFI had the highest sensitivity and specificity (76% and 88%, respectively) when compared against the HFT reference standard. CONCLUSIONS: The high test performance of SFI against the HFT and HFT + DPOAE reference standards indicates that the SFI measure has appropriate diagnostic accuracy in detection of conductive conditions in newborns. Hence, the SFI test could be used as adjunct tool to identify conductive conditions in universal newborn hearing screening programs, and can also be used in diagnostic follow-up assessments.

Sweep frequency impedance measures in young infants: developmental characteristics from birth to 6 months.

OBJECTIVE: International Journal of Audiology To study the developmental characteristics of sweep frequency impedance (SFI) measures in healthy infants from birth to 6 months. DESIGN: All infants were assessed using high-frequency tympanometry (HFT), distortion product otoacoustic emission (DPOAE) and SFI tests. SFI measures consisted of measurement of resonance frequency (RF) and mobility (ΔSPL) of the outer and middle ear. A mixed model analysis of variance was applied to the SFI data to examine the effect of age on RF and ΔSPL. STUDY SAMPLE: Study included 117 ears from 83 infants of different age groups from birth to 6 months. RESULTS: The mean RF of the outer ear increased from 279 Hz at birth to 545 Hz at 4 months, whereas mean ΔSPL of the outer ear decreased from 7.9 dB at birth to 3.7 dB at 4 months of age. In contrast, the mean RF and ΔSPL of the middle ear did not change significantly with age up to 6 months. CONCLUSIONS: Developmental characteristics should be considered when evaluating the function of the outer and middle ear of young infants (≤6 months) using the SFI. The preliminary normative SFI data established in this study may be used to assist with the evaluation.

Effects of ear canal static pressure on the dynamic behaviour of outer and middle ear in newborns.

OBJECTIVE: The present study investigated the effect of ear canal pressure on the dynamic behaviour of the outer and middle ear in newborns with and without a conductive condition using the sweep frequency impedance (SFI) technology. METHODS: A test battery consisting of automated auditory brainstem response (AABR), transient evoked otoacoustic emission (TEOAE) and 1000-Hz tympanometry (HFT) was performed on 122 ears of 86 healthy newborns and 10 ears of 10 newborns with a conductive condition (failed TEOAE and HFT). The dynamic behaviour of the outer and middle ear, when the pressure applied to the ear canal was varied from 200 to -200daPa, was evaluated in terms of the sound pressure level (SPL) in the ear canal, resonance frequency (RF) and displacement (ΔSPL). RESULTS: Application of either a positive or negative static pressure to the ear canal of healthy newborns increased the resonance frequency of the outer (RF1) and middle ear (RF2), but decreased the displacements of the outer (ΔSPL1) and middle ear (ΔSPL2). Positive static pressures resulted in lower SPL while negative static pressures resulted in higher SPL than that at ambient pressure (0daPa). At -200daPa, more than 90% of ears showed signs of collapsed ear canal. The dynamic behaviour under various positive and negative static pressures for newborn ears with a conductive condition indicated similar pattern of SPL, RF1 and ΔSPL1 responses for the outer ear as per healthy ears, but abnormal responses for the middle ear. CONCLUSIONS: While both positive and negative pressures applied to the ear canal have the same effect of stiffening the outer and middle ear, negative pressure of up to -200daPa resulted in more than 90% of ears with a collapsed ear canal. The results of the present study do not only offer useful clinical information for differentiating healthy ears from ears with a conductive condition, but also provide information on the maturation aspects of the outer and middle ear in newborns.

All Three Rows of Outer Hair Cells Are Required for Cochlear Amplification.

In the mammalian auditory system, the three rows of outer hair cells (OHCs) located in the cochlea are thought to increase the displacement amplitude of the organ of Corti. This cochlear amplification is thought to contribute to the high sensitivity, wide dynamic range, and sharp frequency selectivity of the hearing system. Recent studies have shown that traumatic stimuli, such as noise exposure and ototoxic acid, cause functional loss of OHCs in one, two, or all three rows. However, the degree of decrease in cochlear amplification caused by such functional losses remains unclear. In the present study, a finite element model of a cross section of the gerbil cochlea was constructed. Then, to determine effects of the functional losses of OHCs on the cochlear amplification, changes in the displacement amplitude of the basilar membrane (BM) due to the functional losses of OHCs were calculated. Results showed that the displacement amplitude of the BM decreases significantly when a single row of OHCs lost its function, suggesting that all three rows of OHCs are required for cochlear amplification.

Sweep frequency impedance measures in Australian Aboriginal and Caucasian neonates.

OBJECTIVE: Despite high prevalence of otitis media in Aboriginal children, the acoustic-mechanical properties of their outer and middle ear during the neonatal period remain obscured. The objective of this study was to compare the acoustic-mechanical properties of outer and middle ear using Sweep Frequency Impedance (SFI) measures between Australian Aboriginal and Caucasian neonates. METHODS: SFI data from 40 ears of 24 Aboriginal neonates (16 males, 8 females) with mean gestational age of 39.57 weeks (SD = 1.25) and 160 ears of 119 Caucasian neonates (57 males, 62 females) with mean gestational age of 39.28 weeks (SD = 1.25) serving as controls were analysed. SFI data in terms of resonance frequency (RF) and mobility of the outer and middle ear (ΔSPL) were collected from neonates who passed a test battery that included automated auditory brainstem response, distortion product otoacoustic emissions test and 1000-Hz tympanometry. SFI data were analysed using descriptive statistics and analysis of variance. RESULTS: There was no significant difference in mean gestational age, age of testing and birth weight between the Aboriginal and Caucasian neonates. The mean resonance frequencies for the outer ear (mean RF1 = 264.9 Hz, SD = 58.6 Hz) and middle ear (mean RF2 = 1144 Hz, SD = 228.8 Hz) for Aboriginal neonates were significantly lower than that of Caucasian neonates (mean RF1 = 295.3 Hz, SD = 78.4 Hz and mean RF2 = 1241.8 Hz, SD = 216.6 Hz). However, no significant difference in the mobility of outer ear (ΔSPL1) and middle ear (ΔSPL2) between the two groups was found. Middle ear resonance was absent in 22.5% (9 ears) of Aboriginal ears but present in all Caucasian ears. CONCLUSIONS: This study provided evidence that despite passing the test battery, Aboriginal neonates had significantly lower resonance frequencies of the outer and middle ear than Caucasian neonates. Furthermore, 22.5% of Aboriginal neonates showed no middle ear resonance, indicating the possibility of subtle middle ear issues not detected by the test battery. Reasons for the different acoustic-mechanical properties between the two ethnic groups remain unclear and require further investigation.

Normative sweep frequency impedance measures in healthy neonates.

BACKGROUND: Diagnosing middle ear disorders in neonates is a challenging task for both audiologists and otolaryngologists. Although high-frequency (1000 Hz) tympanometry and acoustic stapedial reflex tests are useful in diagnosing middle ear problems in this age group, they do not provide information about the dynamics of the middle ear in terms of its resonance frequency (RF) and mobility. The sweep frequency impedance (SFI) test can provide this information, which may assist in the diagnosis of middle ear dysfunction in neonates. PURPOSE: This study aimed to investigate the feasibility of testing neonates using the SFI technique, establish normative SFI data for RF and mobility of the middle ear in terms of changes in sound pressure level (∆SPL in dB), and describe the dynamics of the middle ear in healthy Australian neonates. STUDY SAMPLE: A prospective sample of 100 neonates (58 males, 42 females) with a mean gestational age of 39.3 wk (SD = 1.3 wk; range = 38-42 wk), who passed all three tests, namely, automated auditory brainstem response, transient evoked otoacoustic emissions, and 1000 Hz tympanometry, were included in this study. DATA COLLECTION AND ANALYSIS: A SFI research prototype was used to collect the data. First, the SPL in the ear canal was measured as a probe-tone frequency was swept from 100-2000 Hz with the ear canal static pressure held constant at 200 daPa. Then, this measurement was repeated with the static pressure reduced in 50 daPa steps to -200 daPa. Additional measurement was also performed at the static pressure, where the peak of the 1000 Hz tympanogram occurred. A graph showing the variation of SPL against frequency at all static pressures was plotted. From this graph, the RF and ∆SPL at tympanometric peak pressure (TPP) were determined. Descriptive statistics and an analysis of variance (ANOVA) were applied to the RF and ∆SPL data with gender and ear as independent variables. RESULTS: The results showed two resonance regions of the outer/middle ear with the high RF (mean = 1236 Hz; 90% range: 830-1518 Hz) being approximately equal to four times that of the low RF (mean = 287 Hz; 90% range = 209-420 Hz). The low RF was more easily identifiable than the high RF. The ∆SPL at the low RF (mean = 8.2 dB; 90% range = 3.4-13 dB) was greater than that at the high RF (mean = 5.0 dB; 90% range = 1.5-8.1 dB). There were no significant differences or interactions between genders and ears. CONCLUSION: The study showed that the SFI is a feasible test of middle ear function in neonates. The SFI results revealed two regions of resonance with the lower resonance (287 Hz) possibly related to the movements of the outer ear canal wall and higher resonance (1236 Hz) related to the resonance of the middle ear. The normative data developed in this study will be useful in evaluating outer and middle ear function in neonates.

Dynamic characteristics of the middle ear in neonates.

OBJECTIVE: Early diagnosis and treatment of hearing disorders in neonates is highly effective for realization of linguistic competence and intellectual development. To objectively and quickly evaluate the dynamic characteristics of the middle ear, a sweep frequency impedance (SFI) meter was developed, which allowed the diagnosis of middle-ear dysfunctions in adults and children. However, this SFI meter was not applicable to neonates since the size of the measurement probe was too large. In the present study, therefore, the SFI meter was improved, i.e., the diameter of the probe was reduced to that of the neonatal external ear canal. By using this newly designed SFI meter, SFI tests were performed in healthy neonates. METHODS: A sound of the sweeping sinusoidal frequency between 0.1 kHz and 2.0 kHz in 0.02-kHz step intervals is presented to the ear canal by an SFI probe while the static pressure of the ear canal is kept constant. During this procedure, the sound pressure level (SPL) is measured. The measurements are performed at 50-daPa intervals of static pressure from 200 daPa to -200 daPa. RESULTS: Measurements were conducted in 10 ears of 9 neonates. The SPL showed two variations at 0.26 ± 0.03 kHz and 1.13 ± 0.12 kHz. Since the SPL is known to show a variation at frequencies from 1.0 kHz to 1.6 kHz due to the resonance of the middle ear in adults and children with normal hearing, the second variation is probably related to such resonance in neonates. The measurement of gel models, which mimics the neonatal external ear canal, showed a variation in SPL at around 0.5 kHz. This implies that the source of the first variation may possibly be related to the resonance of the external ear canal wall. CONCLUSIONS: SFI tests revealed that there were two variations in the SPL curve in neonates, one at 0.26 ± 0.03 kHz and the other at 1.13 ± 0.12 kHz, the former and the latter being possibly related to the resonance of the external ear canal wall and that of the middle ear, respectively. This result suggests that the dynamic characteristics of the middle ear in neonates are different from those in adults.

Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear.

Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A(665) and A(666)) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A(665) and A(666) shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.

Salicylate restores transport function and anion exchanger activity of missense pendrin mutations.

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.

Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer.

Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti-prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring-like with a diameter of about 11 nm.

Salicylate-induced translocation of prestin having mutation in the GTSRH sequence to the plasma membrane.

Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin-transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.

Mutation-induced reinforcement of prestin-expressing cells.

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.

Atomic force microscopy in studies of the cochlea.

The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. The origin of this cochlear amplification is the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. The gene of this protein has been identified and termed prestin. We, herein, present a method for observation by atomic force microscopy (AFM) of prestin expressed in the Chinese hamster ovary (CHO) cell plasma membrane. To obtain a stable sample for AFM imaging in liquid, we used as an example in the protocol provide here, CHO cells transfected with prestin or FLAG-tagged prestin, and untransfected CHO cells. The cells attached to a substrate were subjected to ultrasonic waves generated from a sonicator probe so that the inside-out plasma membranes remained on the substrate. Prestin was immunostained with mouse anti-FLAG primary antibody and FITC-conjugated goat anti-mouse IgG secondary antibody. The lipid of the plasma membrane was labeled with fluorescence probes. The cytoplasmic faces of the cells were then observed in liquid by the tapping mode of AFM at low and high magnifications. More particle-like structures 8-12 nm in diameter were observed in the plasma membranes of the prestin-transfected CHO cells than in those of the untransfected CHO cells. Since the difference between these two types of cells is due to the existence of prestin, such particle-like structures in the prestin-transfected CHO cells are possibly constituted by prestin.

Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots.

Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.

Effects of heat stress on filamentous actin and prestin of outer hair cells in mice.

When the ear is exposed to traumatic loud noise, outer hair cells (OHCs) are damaged and thus permanent hearing loss occurs. Recently, prior conditioning with heat stress has been reported to protect OHCs from traumatic noise exposure by increasing the stiffness of the OHC soma and has also been reported to enhance distortion product otoacoustic emissions [DPOAEs; Murakoshi, M., Yoshida, N., Kitsunai, Y., Iida, K., Kumano, S., Suzuki, T., Kobayashi, T., Wada, H., 2006. Effects of heat stress on Young's modulus of outer hair cells in mice. Brain Res. 1107, 121-130]. In the present study, to further investigate the heat stress-induced protective mechanism of hearing and such stress-induced DPOAE enhancement mechanism, the amount of filamentous actin (F-actin), which is concerned with cell stiffness, and the amount of prestin, which is concerned with the generation of DPOAEs, were examined in OHCs, with and without heat stress. Heat stress was found to increase the amount of F-actin 6-24 h after heat stress. The greatest increase in the amount of F-actin was observed at the cuticular plate where F-actin anchors the roots of the stereocilia to the cell body. Based on this result, the part of the stereocilia most reinforced and protected by heat stress was concluded to be the roots of the stereocilia. In contrast with F-actin, heat stress did not affect the amount of prestin.

Effects of heat stress on Young's modulus of outer hair cells in mice.

Intense sound exposure causes permanent hearing loss due to hair cell and cochlear damage. Prior conditioning with sublethal stressors, such as nontraumatic sound, heat stress and restraint protects the ear from acoustic injury. However, the mechanisms underlying conditioning-related cochlear protection remain unknown. In this paper, Young's modulus and the amount of filamentous actin (F-actin) of outer hair cells (OHCs) with/without heat stress were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. Conditioning with heat stress resulted in a statistically significant increase in Young's modulus of OHCs at 3-6 h after application, and such modulus then began to decrease by 12 h and returned to pre-conditioning level at 48 h after heat stress. The amount of F-actin began to increase by 3 h after heat stress and peaked at 12 h. It then began to decrease by 24 h and returned to the pre-conditioning level by 48-96 h after heat stress. These time courses are consistent with a previous report in which heat stress was shown to suppress permanent threshold shift (PTS). In addition, distortion product otoacoustic emissions (DPOAEs) were confirmed to be enhanced by heat stress. These results suggest that conditioning with heat stress structurally modifies OHCs so that they become stiffer due to an increase in the amount of F-actin. As a consequence, OHCs possibly experience less strain when they are exposed to loud noise, resulting in protection of mammalian hearing from traumatic noise exposure.

Imaging by atomic force microscopy of the plasma membrane of prestin-transfected Chinese hamster ovary cells.

The high sensitivity of mammalian hearing is achieved by amplification of the motion of the cochlear partition. This cochlear amplification is thought to be generated by the elongation and contraction of outer hair cells (OHCs) in response to acoustical stimulation. This motility is made possible by a membrane protein embedded in the lateral membrane of OHCs. Although a fructose transporter, GLUT-5, was initially proposed to be this protein, a later study identified the gene of the motor protein distributed throughout the OHC plasma membrane. This protein has been named "prestin." However, although previous morphological studies by electron microscopy and atomic force microscopy (AFM) found the lateral wall of OHCs to be covered with 10-nm particles, believed to be motor proteins, it is unknown whether such particles consist only of prestin or are a complex of GLUT-5 and prestin molecules. To determine if the 10-nm particles are indeed constituted only of prestin, plasma membranes of prestin-transfected and untransfected Chinese hamster ovary (CHO) cells, which do not express GLUT-5, were observed by AFM. First, the cells attached to a substrate were sonicated so that only the plasma membrane remained on the substrate. The cytoplasmic face of the cell was observed by the tapping mode of the AFM in liquid. As a result, particle-like structures were recognized on the plasma membranes of both the prestin-transfected and untransfected CHO cells. Comparison of the difference in the frequency distribution of these structures between those two cells showed approximately 75% of the particle-like structures with a diameter of 8-12 nm in the prestin-transfected CHO cells to be possibly constituted only by prestin molecules. Our data suggest that the densely packed 10-nm particles observed on the OHC lateral wall are likely to be constituted only of prestin molecules.