The purpose of this study was to longitudinally assess the behavioral and electrophysiological hearing changes of a girl inserted in a CI program, who had bilateral profound sensorineural hearing loss and underwent surgery of cochlear implantation with electrode activation at 21 months of age. She was evaluated using the P1 component of Long Latency Auditory Evoked Potential (LLAEP); speech perception tests of the Glendonald Auditory Screening Procedure (GASP); Infant Toddler Meaningful Auditory Integration Scale (IT-MAIS); and Meaningful Use of Speech Scales (MUSS). The study was conducted prior to activation and after three, nine, and 18 months of cochlear implant activation. The results of the LLAEP were compared with data from a hearing child matched by gender and chronological age. The results of the LLAEP of the child with cochlear implant showed gradual decrease in latency of the P1 component after auditory stimulation (172 ms–134 ms). In the GASP, IT-MAIS, and MUSS, gradual development of listening skills and oral language was observed. The values of the LLAEP of the hearing child were expected for chronological age (132 ms–128 ms). The use of different clinical instruments allow a better understanding of the auditory habilitation and rehabilitation process via CI. The central auditory nervous system starts its development in intrauterine life; however, its maturation persists lifelong. The first phase of development is independent of external neurosensory stimulation. However, the second phase will only be effective from the sensory inputs that will organize and direct the process of connections of neural networks development . It is tIn cases where there is deprivation of sound stimulation, direct stimulation of the auditory nerve fibers through the cochlear implant (CI) has been an alternative for the CANS to receive the stimulation needed for the maturation process and, consequently, the development of auditory and oral language skills , 6. HencThus, there are outcome measures, standardized and validated for Brazilian Portuguese, that are able to assess the development of auditory and language skills of this population, providing important information that monitor the rehabilitation process. Concerning objective methods, electrophysiological assessment of hearing, through analysis of morphology and latency of Long Latency Auditory Evoked Potentials (LLAEP), has been a current method to verify the degree of development and the limits of central auditory plasticity. This can be done by monitoring the neurophysiological changes in the population with hearing loss after the onset of hearing intervention with the use of CI , 8.Considering that the development and organization of the central auditory pathways in children are directly related to an effective hearing experience, the P1 wave of LLAEP has been used as a biomarker to evaluate the maturation of the central auditory system in children. These measures, associated with behavioral assessment of auditory and language skills, can assist in verifying the effectiveness of auditory rehabilitation in children with a hearing aid (HA) and/or CI , 9–11.Several studies in the literature suggest that, especially in children implanted early, changes in synaptic connections and synchronization of neuronal transmission occur, reflecting a rapid decrease in latency and a concomitant increase in the amplitude of the LLAEP waves. Some authors observed that these children reached latency values of the P1 component expected for age after three months of implantation . Others Studies combining the results of LLAEP electrophysiological tests with behavioral assessments indicate that the decrease in latency of the P1 component is directly related to the improvement of communicative behaviors  , with imConsidering the variability of LLAEP results found in children with CI described in the literature and the scarcity of longitudinal studies on behavioral and electrophysiological changes with the use of the CI, the purpose of this study was to describe auditory maturation and changes on behavioral assessments of a child on early stages of stimulation with CI.This longitudinal case-control study was developed at the Audiology Sector of Department of Physical Therapy, Speech, Language and Hearing and Occupational Therapy and the Discipline/Department of Otolaryngology, Faculty of Medicine, University of São Paulo. The study was approved by the Ethics Committee of CAPPesq-HCFMUSP (process number 0319/11) and the procedures were performed when parents signed the informed consent.Data from electrophysiological and behavioral hearing assessments was collected at four time points: before CI activation and three, nine, and 18 months thereafter.LLAEP was recorded with a speech stimulus, syllable/ba/, presented in sound field with speakers positioned at an angle of 90° azimuth, 40 cm away from the side where the child uses the CI. The interstimulus interval was 416 ms and the intensity 70 dBHL, with presentation rate of 1.9 stimuli per second. Band pass filter from one to 30 Hz, gain of 100,000, averaging of 512 stimuli, and response analysis window from −100 ms prestimulus and 500 ms poststimulus were used. Two samples of each subject were collected to verify tracings reproducibility and to exclude doubts regarding waves/artefacts.Infant Toddler Meaningful Auditory Integration Scale (IT-MAIS): the adapted version to Brazilian Portuguese  was usedMeaningful Use of Speech Scales (MUSS) : the adaGlendonald Auditory Screening Procedure (GASP) : the adaThe following measures were used for behavioral assessment:The study was conducted with a female child whose diagnosis was performed at six months of age: bilateral profound sensorineural hearing loss of unknown etiology. Hearing aids were fitted bilaterally at one year of age, with limited results for the development of auditory and oral language skills.The child underwent CI implantation surgery in the right ear, with full insertion of electrodes. The electrodes were activated when the child was 24 months old, when she began speech and language therapy that focused on stimulation of hearing and oral language. After activation, she effectively used the CI, more than 8 hours per day.Before activation, the mean of 250 Hz and 500 Hz (85 dBHL) frequencies of her hearing thresholds in free field was below that of the other moments. After activation it was verified that the child answered more consistently to other frequencies . Her hearing thresholds after using the CI gradually decreased: with 3 months of use its mean was 60 dB and with 18 months of use its mean was 38 dBHL.From the perspective of the parents, the auditory behavior assessed by IT-MAIS increased from 32.5% at the first to 83% at the last assessment, and language skills, evaluated using the MUSS questionnaire, evolved from 17.5% to 55%, respectively.At the assessment of auditory skills preactivation, the child did not detect speech sounds; after 18 months of effective use of CI, she was able to detect all six Ling sounds, recognize her own name, and discriminate vowel sounds.Hearing thresholds obtained in the free field assessments in the four time points , questioRegarding the electrophysiological assessment of hearing, there was no response in the preactivation period. After three months of electrical stimulation via CI, the presence of the P1 component at 172 ms was observed. These values decreased over the 18 months of CI use, being observed at 141 ms after nine months and 134 ms after 18 months .The results of electrophysiological assessment were compared with the results of a hearing child matched by chronological age and gender, with no hearing or language impairments, whose latency values of the P1 component in the four assessments were, respectively, 132, 132, 130, and 128 ms .The auditory stimulation enables the nervous system to expand the synaptic connections (neuroplasticity), improving the efficacy of stimulus transmission through the central auditory pathways. This phenomenon is directly related to the development of auditory and oral language skills.A major difficulty in conducting studies with subjects who are CI users is the heterogeneity of this population, considering that many variables can influence the development of the auditory system and, consequently, the development of auditory and language skills. Among these variables are time of sensory deprivation, age at implantation, age of onset of hearing loss , etiology, speech and language therapy, and family motivation.For a better understanding of how the maturation of the auditory system occurs via electrical stimulation with the use of CI in children, a longitudinal study with the investigation of auditory changes found in electrophysiological and behavioral assessment of a child with a CI was carried out.In the initial electrophysiological evaluation of the current study, prior to CI activation, no response was observed. The trace consisted of several positive and negative waves arranged haphazardly, without a reproduction standard even after several recordings. This feature is reported in the literature as, demonstrating the presence of polyphasic waves, the central auditory pathways are abnormally organized front to sound deprivation , 20, 21.In the assessments subsequent to the CI activation, as the auditory experience provided by the effective use of this device increased, the electrophysiological tracings became more defined and latency values of the P1 component gradually decreased. This improvement in electrophysiological findings was possibly observed due to the fact that the peripheral auditory stimulation through the CI was able to provide improved functionality of synaptic connections, which gradually increased the speed of neural transmission and amplification of synaptic synchronization , 22.It was not the aim of this study to evaluate other components of the N1-P2-N2 complex. The literature reports that P1 is the component with the highest occurrence in young children and that the other components will define as maturation occurs; thus, the analysis of these components vary greatly at this age range, making it difficult to compare with what is expected for hearing children –26.When comparing the P1 component of electrophysiological evaluation through LLAEP of the child who is a CI user with that of the hearing child, it was observed that, after 18 months of CI use, the P1 latency (134 ms) component resembled the expected values for chronological age (128 ms). This confirms that, at this age range, the auditory system presents a high degree of plasticity and that the process of auditory rehabilitation was being effective.While there is no normative data regarding the P1 component latency in the literature, the data obtained in this study is similar to data from other authors. Some authors evaluated the P1 component latency in a group of children who received IC with up to three years and are five months old and observed average values of 378,18 ms at the time of activation and 137,5 ms after 12–18 months of CI use . Other aAccording to the literature, the auditory stimulation of deaf children before 36 months of age allows a very fast maturation of auditory system , 28, 29.After this critical period, synaptic connection abnormalities occur, resulting in an abnormal neuronal connectivity between neuronal cells, functional disintegration, and immaturity of auditory cortical areas; some auditory areas develop nonauditory functions  and abnormalities in the restructuring of cognitive functions occur .With respect to behavioral aspects, improvements were observed in hearing thresholds with the CI, which demonstrates that the electrical signal provided by the CI allowed access to the sounds of lower intensity . For theAccording to the estimated curve of normal development , the valThe results obtained in the electrophysiological and behavioral assessments demonstrated the effectiveness of the habilitation process of a child with a CI. Nevertheless, while the P1 component of the electrophysiological assessment showed rapid changes three months after the intervention and after nine months already approached the expected values for age (141 ms of the child with CI and 130 ms of the hearing child), the development of auditory and language skills required a longer follow-up for changes to be observed and measured. The presence of P1 wave was observed at the third month after activation, and an improvement on hearing and linguistic behavior after nine months after activation was noted .Whereas the observed changes in the electrophysiological assessment emerged shortly after the beginning of stimulation, this measure appeared to be a predictor of future behavioral auditory development, thus corroborating with the data presented in the literature that emphasizes the analysis of the P1 component as a biomarker of the development of the auditory system , 13, 32.The use of different clinical instruments allows a better understanding of the auditory habilitation and rehabilitation process via CI. The results of this study allowed the observation that the CI enabled the stimulation of auditory system structures in order to foster the development of auditory and oral language skills.Thus, the combined results of the electrophysiological and behavioral assessment analysis seems to be a proposal for monitoring the results obtained after the intervention via CI to provide information on the results of CI and to guide the therapeutic process , 33, 34.


To explore the potential role of retinaldehyde dehydrogenase (RALDH) enzymes and atRA in human postnatal ocular growth, RALDH activity, along with the distribution of RALDH1, RALDH2, and RALDH3 in the postnatal eye was determined.Retinaldehyde dehydrogenase 2 (RALDH2) has been implicated in regulating all-in vitro atRA synthesis assay together with HPLC quantification of synthesized atRA. Homogenates were compared by western blotting for RALDH1, RALDH2, and RALDH3 protein. Immunohistochemistry was used to determine RALDH1 and RALDH2 localization in posterior fundal layers of the human eye.Retina, retinal pigment epithelium (RPE), choroid, and sclera were isolated from donor human eyes. RALDH catalytic activity was measured in tissue homogenates using an In the postnatal human eye, RALDH catalytic activity was detected in the choroid (6.84 ± 1.20 pmol/hr/ug), RPE (5.46 ± 1.18 pmol/hr/ug), and retina (4.21 ± 1.55 pmol/hr/ug), indicating the presence of active RALDH enzymes in these tissues. RALDH2 was most abundant in the choroid and RPE, in moderate abundance in the retina, and in relatively low abundance in sclera. RALDH1 was most abundant in the choroid, in moderate abundance in the sclera, and substantially reduced in the retina and RPE. RALDH3 was undetectable in human ocular fundal tissues. In the choroid, RALDH1 and RALDH2 localized to slender cells in the stroma, some of which were closely associated with blood vessels.Results of this study demonstrated that: 1) Catalytically active RALDH is present in postnatal human retina, RPE, and choroid, 2) RALDH1 and RALDH2 isoforms are present in these ocular tissues, and 3) RALDH1 and RALDH2 are relatively abundant in the choroid and/or RPE. Taken together, these results suggest that RALDH1 and 2 may play a role in the regulation of postnatal ocular growth in humans through the synthesis of atRA. Postnatal ocular growth is regulated by a vision-dependent process that coordinates the growth of the eye such that the ocular axial length will align the retina with the focal plane to give clear, uncorrected vision . Interrutrans-retinoic acid (atRA) has been implicated as a key signaling molecule in the regulation of postnatal eye growth in chicks, guinea pigs, and primates  for 1 hr at RT with gentle rocking. Blots were then incubated with rabbit anti-human RALDH2 , rabbit anti-human RALDH1 , or rabbit anti-human RALDH3  overnight at 4°C (Table 1). Immunoblots were washed three times with PBS containing 0.05% Tween-20 followed by three washes with PBS. Immunoblots were then incubated with goat anti-rabbit IgG conjugated to alkaline phosphatase as a secondary labeling antibody . After incubation with the secondary antibody, blots were washed (as above), incubated in CDP-Star Ready-to-Use with Nitro-BlockII  for 5 min, and images were captured with a Chemigenius imager . For RALDH2 experiments, an identical blot of human samples was probed with rabbit anti-RPE65 [Protein concentrations of all tissue samples were determined by a Bradford assay . The protein concentration of each tissue sample was adjusted to a final concentration of 3.1 μg per 15 μL of dHti-RPE65   program. The faintest band on each image was assigned a value of 1 and all other bands were automatically assigned relative values to the faintest band. Automatic background correction was applied.Slides containing 5-micron thick paraffin sections of human posterior fundus containing the retina, RPE, choroid, and sclera were obtained from the National Disease Research Interchange . The donor was a 30 year old Caucasian male with a history of tobacco and marijuana use. Cause of death was not due to systemic disease.2O for 5 min, and transferred to a slide mailer (∼20 mL volume). Antigen retrieval was performed by incubating the slides in a 1% aqueous sodium borohydride solution for 2 min at room temperature, followed by washing with dH2O for 5 min (3X) and PBS for 5 min (3X). Following removal of the last wash, slides were incubated in sodium citrate  for 20 min in a 95°C water bath. The water bath was turned off, and the slide mailer remained in the bath for an additional 20 min. The mailer was removed from the water bath, allowed to cool to RT, and slides were washed with PBS for 5 min (3X). Slides were incubated in blocking buffer  for 1 hr at RT with rocking, and then incubated in blocking buffer containing rabbit anti-RALDH2 (1:300) or rabbit anti-RALDH1 (1:100) for 5 days at 4°C (Table 1). Slides were washed with PBS for 10 min (6X), incubated with goat anti-rabbit IgG conjugated to AlexaFluor 568  for 2 hrs at RT, and washed with PBS for 10 min (6X). Slides were then incubated with the nuclear stain, 4',6-diamidino-2-phenylindole  for 5 min and coverslipped using ProLong Gold Antifade reagent containing DAPI . For double labeling experiments, after incubation with anti-RALDH2, goat anti-rabbit IgG- AlexaFluor 568 conjugate, and subsequent washes, slides were incubated with mouse anti-α-smooth muscle actin  for an additional 5 days at 4°C. Slides were then washed (described above) and incubated with rabbit anti-mouse IgG conjugated to AlexaFlour 488  for 2 hrs. Slides were washed and mounted as described above and viewed using a Fluoview FV1000 confocal laser scanning microscope .Slides were deparaffinized in a xylene/ethanol series, washed three times in dHet. al., manuscript in preparation) before immunolabeling paraffin sections of human eyes.The following were used for negative controls: 1) primary antibody was omitted from the first incubation; 2) tissue sections were incubated in 10 μg/mL non-immune rabbit IgG  instead of the primary antibody. Additional preabsorption controls were performed in which the anti-RALDH2 antibody was incubated for 30 min at RT with 33.7 nM of purified recombinant chicken RALDH2 .trans-retinoic acid (atRA) from all-trans-retinaldehyde via RALDH enzymes [trans-retinaldehyde was measured in cytosol fractions of ocular tissues from each donor . Following 30 minutes of incubation of tissue cytosol fractions with all-trans-retinaldehyde in the presence of NAD, atRA could be detected by HPLC as a sharp peak eluting at approximately 12 minutes . This peak was not detected in samples incubated in the absence of tissue homogenate or in tissue homogenates incubated in the absence of NAD or all-trans-retinaldehyde, indicating all detectible atRA was synthesized by ocular tissues in vitro. All results were normalized to the total protein present in each cytosol fraction. RALDH activity could be detected in the retina, RPE, and choroid in all ages examined with activity ranging from 1.3–9.7 pmol/hr/μg total protein in the retina, 2.2–9.0 pmol/hr/μg total protein in the RPE, and 4.4–10.0 pmol/hr/μg total protein in the choroid . RALDH activity in the sclera was below the level of detection. It is interesting to note the relatively high RALDH activity in the retina of the 17 year old donor compared to the 16 year old donor. We speculate that postnatal differences in RALDH activity may underlie differences in ocular growth and refraction; however, since a detailed ocular history was not provided with the donor eyes, the interpretation of these results is limited. When averaged for all donors , RALDH activity was somewhat higher in the choroid , followed by the RPE , and retina , although differences were not statistically significant (p = .5385).Since results from experimental animal models have suggested thatpostnatal ocular tissues can synthesize all- enzymes , RALDH eFig. 2; see S1 Fig. for full gel). As expected, Coomassie Blue staining indicated distinct differences in protein profiles between cytosol fractions of each tissue type, but quantification of band intensities confirmed that similar amounts of total protein were loaded for each sample. In addition, minimal degradation was observed between donors based on the presence of distinct bands migrating between 10 and 200 kDa.In order to determine which RALDH isoforms could be contributing to the RALDH activity observed above, the presence of each RALDH isoform in postnatal human ocular tissues was examined. To ensure equal loading of protein (3 μg/lane), representative samples of cytosol from choroid, sclera, retina, and RPE were selected from each age group, separated by SDS-PAGE, and stained with Coomassie Blue . All results were normalized to total protein present in each cytosol fraction. In choroid samples, RALDH2 protein migrated as a closely spaced doublet with bands at ∼57 and 55 kDa. In retina and RPE samples, RALDH2 was present as a predominant band at 55 kDa together with a minor immunopositive band at ∼59 kDa . RALDH2 was also detected in the human sclera as a relatively faint band migrating at ∼55 kDa. It is suspected that the variations in size of the RALDH2 immunopositive bands observed within and among the tissues is due to various splice variants of the RALDH2 protein. Human RALDH2 has four known splice variants with molecular weights ranging from 46.1 to 56.7 kDa. As RALDH2 has recently been identified as synthesizing atRA in the postnatal chick choroid in response to visually guided eye growth , the pretrans-retinyl esters to 11-cis-retinol . Following immunoblotting with anti-RPE65, RPE65 could be readily detected in the RPE of every donor, with a singular band found at ∼65 kDa. To evaluate potential RPE contamination in the retina, choroid, and sclera, identical blots were probed using an antibody for RPE65, an isomerahydrlase specific to the RPE that converts all-Fig. 3B, 3C). Comparisons of relative RALDH2 abundance in human ocular tissue samples were reported as 1) “RALDH2 abundance by age” , where the integrated optical density (IOD) per 3 μg total protein for RALDH2 immunopositive bands was reported for each individual sample; and 2) “average RALDH2 abundance” , where values from Fig. 3B). When the absolute levels of RALDH2 , RALDH2 showed the same overall pattern of distribution across the tissues types; RALDH2  was highest in the RPE (17.86 ± 3.22), followed by the choroid (11.83 ± 3.55), retina (5.83 ± 1.44), and lowest in the sclera (3.11 ± 1.48). Significant differences in RALDH2 density were observed between the RPE and sclera  and between the RPE and retina .Relative RALDH2 abundance in human ocular tissue samples was determined by digital image analysis of band intensities on western blots in order to compare the relative tissue distribution of RALDH2 in the postnatal human eye , 3C. ComFig. 4). When RALDH2 was probed in the postnatal human eye with an anti-human RALDH2 antibody followed by a secondary antibody conjugated to AlexFlour 568 (red), RALDH2 could be detected in the retina, choroid, and RPE . Based on morphology and position in the retina, RALDH2 positive cells in the retina were speculated to be Müller cells and cone outer segments. No labeling of the retina or choroid was found in negative controls in which primary antibody was pre-incubated with recombinant RALDH2 prior to immunolabeling , when non-immune rabbit IgG was used in place of the primary antibody, or when sections were not exposed to primary antibody. However, some non-specific labelling was detected in the RPE and is most likely due to autofluorescence of RPE-associated lipofuscin [Immunohistochemistry was performed on sections of human posterior ocular tissues  in order to determine the cellular localization of RALDH2 and identify the cells responsible for atRA synthesis in these tissues , some of which were closely associated with the exterior of small blood vessels . In order to determine if the RALDH2-positive cells were vascular and/or extravascular smooth muscle, slides were double labeled with anti-RALDH2 together with anti-α-smooth muscle actin . RALDH2 did not co-localize with either non-vascular  or vascular  smooth muscle cells.In the choroid, RALDH2 was found in attenuated cells in the choroidal stroma . Following labeling with anti-RALDH1 antibodies, RALDH1 was detected faintly in the retina and RPE, moderately expressed in the sclera, and strongly expressed in the choroid at all age groups examined. RALDH1 protein migrated as a single band at ∼55 kDa in the retina and RPE and at ∼57 kDa in the choroid and sclera ( top panel). As RALDH1 has previously been shown to be abundantly expressed in the lens [S4 Fig. for full blot.) In addition, RALDH3 protein expression was evaluated in postnatal human ocular tissues by western blot using antibodies specific for the RALDH3 isoform. No immunopositive bands were detected in postnatal human ocular tissues by western blot ( bottom panel). Recombinant, purified human RALDH3 was employed as a positive control ( lane “R3”). Western blotting was employed to determine if the RALDH1 isoform was also present in postnatal human ocular tissues ; and 2) “average RALDH1 abundance” . RALDH1 appeared to be highest in relative abundance in the choroid, in moderate abundance in the sclera, and was present at relatively low levels in the retina and RPE . When the absolute levels of RALDH1 , average RALDH1  abundance was highest in the choroid (32.13 ± 4.07) with moderate levels in the retina (9.68 ± 1.70) and sclera (14.73 ± 3.63) and lowest levels in the RPE (2.39 ± 1.91). Significant differences in RALDH1 density were observed between the choroid and retina , the choroid and RPE , as well as the choroid and sclera .Similar to RALDH2 analyses, relative RALDH1 abundance in human ocular tissue samples was determined by digital image analysis of band intensities and was reported as 1) “RALDH1 abundance by age” ; and 2) Fig. 6). When sections of human fundus were probed with anti-human RALDH1 followed by anti-rabbit IgG conjugated to AlexFlour 568 (red), RALDH1 could be detected in the retina and choroid . Based on morphology and placement in the retina, RALDH1 immunopositive labeling was observed in rod outer segments and a subpopulation of cone outer segments. Additionally, punctate labelling could be detected throughout the inner plexiform layer and inner limiting membrane, possibly associated with Müller cells. No labeling of the retina or choroid was found in negative controls in which non-immune IgG was used as the primary antibody . Non-specific labelling was detected in the RPE and is most likely due to autofluorescence of RPE-associated lipofuscin (discussed above) [In order to determine the cellular localization of RALDH1, immunohistochemistry was performed on sections of human posterior ocular tissues  (d above) .Fig. 6C, 6D). Unlike RALDH2 labeling, RALDH1 labeling did not appear to associate with the exterior of blood vessels .In the choroid, strong RALDH1 labeling was found in extravascular cells throughout the choroidal stroma , which belong to the aldehyde dehydrogenase superfamily . Becauseal cells . Moreovetrans-retinaldehyde in the presence of NAD at rates ranging from 0.5–10.0 pmol/hr/μg total protein. Average RALDH catalytic activities were similar for choroid, retina, and RPE; 6.84 ± 1.20, 4.21 ± 1.55, and 5.46 ± 1.18 pmol/hr/μg total protein, respectively. Interestingly, the rates of RALDH catalytic activity measured from postnatal human ocular tissues are higher than those reported for the embryonic chick eye at 6 days of development, of which retina cytosol was reported to be the most abundant enzyme source  [in vivo. In vivo, however, RALDH activity is highly dependent on a number of factors including substrate and cofactor availability, the presence of cytosolic inhibitors, the rate of substrate turnover, and the presence of retinoid specific binding proteins, which may differ considerably from the assay conditions used in the present study. Therefore, extrapolation of RALDH activities determined in vitro from isolated human ocular tissues obtained postmortem to true RALDH activities in the postnatal human eye should only be done with caution.RALDH activity was detected in cytosol fractions of choroid, retina, and RPE of all donors. The choroid, retina, and RPE synthesized atRA from all-protein) . Althouget al. showed that in response to light, the postnatal murine RPE demonstrates RALDH activity and atRA synthesis above that of other ocular tissues [trans-retinaldehyde substrate from the photoreceptors following light-induced isomerization of 11-cis-retinaldehyde into the RPE, followed by oxidation of all-trans-retinaldehyde to atRA by RALDH. Although the specific RALDH isoform responsible for light-mediated atRA synthesis in the mouse RPE was not identified, our results suggest that RALDH2 is present in the human RPE where it may catalyze light-mediated atRA synthesis from all-trans-retinaldehyde most likely derived from the photoreceptors as a component of the light regulated retinoid fluxes between the retina and RPE [Based on the potential role of choroidal RALDH2 in modulating postnatal ocular growth, our initial studies examined relative RALDH2 abundance and distribution in the retina, RPE, choroid, and sclera of 5 human donor eyes, of ages 16–46 years. Variation in RALDH2 protein abundance was noted in ocular tissues between donors, either due to age-related or phenotypic differences between individual donors. However, when RALDH2 concentrations in each ocular tissue were averaged for all donors, significant differences in relative abundance could be detected. RALDH2 was greatest in relative abundance in the RPE of the postnatal human eye, with lesser amounts in the choroid, and lowest relative abundance in the sclera and the neural retina. These results are in contrast to those obtained from chick eyes, in which little to no RALDH2 was present in the postnatal chick RPE . However tissues . This li and RPE .In the choroid, RALDH2 was localized to a population of extravascular stromal cells in close association with vascular smooth muscle cells, as well as in attenuated cells throughout the choroidal stroma. In contrast to previous immunolocalization studies in chick , RALDH2 in vitro assay conditions may be beyond the limits of detection with this assay. In addition, it is possible that cytosolic inhibitors are present in the scleral lysates that inhibit RALDH activity in our in vitro assay.RALDH1 protein expression was also observed in the human choroid, comparable in abundance to that observed in the lens, with moderate expression in the sclera, and substantially lower expression in the retina and RPE. In the choroid, RALDH1 immunopositive cells were identified as attenuated cells in the stroma, similar in appearance to that of RALDH2 positive cells. RALDH3 was undetectable in postnatal human ocular tissues. Interestingly, in contrast to results obtained in humans, RALDH1 mRNA expression was undetectable in the chick choroid ; only RAtrans-retinaldehyde, produced by photoreceptors during the visual cycle, as previously described for the mouse eye [During embryogenesis, RALDH2, together with cytochrome CYP26A1, establish local atRA gradients that facilitate a number of developmental processes including neurogenesis, cardiogenesis, body axis extension, and limb, foregut, and eye development –35. The ouse eye . This liouse eye ,9. We thS1 FigS3 Fig.). C, choroid; S, sclera; R, retina; E, RPE.All data gathered from the 47 year old donor was excluded from analyses due to significant RPE65 contamination in the choroid of this donor (see (TIF)Click here for additional data file.S2 FigMolecular marker is shown in bright field, while the full western blot is shown as a chemi-luminescent image. C, choroid; S, sclera; R, retina; E, RPE.(TIF)Click here for additional data file.S3 FigMolecular marker is shown in bright field, while the full western blot is shown as a chemi-luminescent image. All data gathered from the 47 year old donor was excluded from analyses due to significant RPE65 contamination in the choroid of this donor. C, choroid; S, sclera; R, retina; E, RPE.(TIF)Click here for additional data file.S4 FigMolecular marker is shown in bright field, while the full western blot is shown as a chemi-luminescent image. L, lens; R, retina; E, RPE; C, choroid; S, sclera.(TIF)Click here for additional data file.S5 FigMolecular marker is shown in bright field, while the full western blot is shown as a chemi-luminescent image. L, lens; R, retina; E, RPE; C, choroid; S, sclera. R3, recombinant human RALDH3. Higher molecular weight bands in the “R3” lane are RALDH3 oligomers.(TIF)Click here for additional data file.


Tps establish close contacts  with other neighbouring cells . The ELISA testing of the supernatant of primary culture of TCs indicated that the concentrations of VEGF and EGF increased progressively. In conclusion, our study shows the existence of typical TCs at the level of oesophagus  and suggests their possible role in tissue repair.Telocytes (TCs), a new type of interstitial cells, were identified in many different organs and tissues of mammalians and humans. In this study, we show the presence, in human oesophagus, of cells having the typical features of TCs in lamina propria of the mucosa, as well as in muscular layers. We used transmission electron microscopy (TEM), immunohistochemistry (IHC) and primary cell culture. Human oesophageal TCs present a small cell body with 2–3 very long Telopodes (Tps). Tps consist of an alternation of thin segments ( Telocytes (TCs) were recently described as a new type of interstitial cells by Popescu The ultrastructure of the oesophageal wall  was previously described, with special reference to Interstitial Cells of Cajal (ICCs) Considering previously described close spatial relations of TCs with (new-formed) blood vessels in normal tissue i) patients who suffered primary oesophagus cancer were confirmed histologically; (ii ) patients underwent curative surgery, but did not receive any pre-operative treatment; and (iii ) adjacent oesophagus tissues samples, used as control, were confirmed histologically as the normal. The study was approved by the Ethic Committee of Fudan University, Zhongshan Hospital. All collections of human tissues were performed after obtaining informed consent from the patients.Patient selected for this study fulfilled the following criteria: (i ) histology and immunochemical staining; (ii ) electronic microscopy; (iii ) fresh tissues for isolation and primary cell culture of TCs.Human oesophagus samples were obtained from patients undergoing thoraco-abdominal incision radical esophagectomy (six patients) or Ivor-Lewis esophagectomy (four patients) for neoplastic oesophagus diseases. Normal human oesophagus samples were collected from the corresponding normal tissues adjacent to resection margins from patients who had no anti-cancer treatment before tumour resection. All samples were examined and confirmed by pathologist. In all, 10 primary oesophagus tissue samples for study were obtained. Human adjacent normal oesophagus tissue samples underwent protocols for  embedded in paraffin. The primary antibodies were used as follows: vimentin, rabbit polyclonal , 1:100; CD34, rat monoclonal , 1:100. Negative controls were performed by omitting the primary antibody. Tissue sections were examined and photographed under an Olympus light microscope (BX51) equipped with a digital camera . The images were digitally acquired by using the software ImagePro-Express.Oesophageal samples from each patient were prepared for primary cell culture and vital staining as previously described 2 plastic culture flasks with primary TCs at 24 and 48 hrs, separately  and stored at −20°C. Blank medium hatched in the same atmosphere of cell culturing was collected simultaneously as control (n = 10). The concentrations of VEGF and EGF in the supernatant were detected by the method of ELISA with corresponding ELISA kit .The supernatant was collected in 25 cmi) very long; (ii) beads-on-a-string appearance: alternation of thin segments (podomers) – less than 0.2 μm – and dilated segments (podoms) 2+ uptake/releasing units’ composed of endoplasmic reticulum, mitochondria and caveolae , as for TEM. Thus, Figures Under light microscopy, vimentin-positive cells were seen within human oesophageal submucosa Fig. A. PositiTelocytes were successfully maintained in primary culture and could easily be identified before reaching confluence. Starting with the 3rd day in culture, TCs appeared with characteristic long, moniliform processes . Also, cell culture obtained from submucosa of human oesophagus shows a major population of interstitial cells with morphology typical of TCs, as it was stained for Giemsa .Immunohistochemistry labelling for vimentin and CD34 demonstrates that in the submucosa of oesophagus resides a population of vimentin/CD34-positive cells having evocative morphology for TCs. The positive expression for these markers is present at the level of cell body and also their prolongations, and can discriminate TCs from other interstitial cells , previous published data showed that the detected levels of IL-6 and VEGF in TCs culture increased with passage number (to 964.47 pg/ml for IL-6 and 262.20 pg/ml for VEGF) In this study, by ELISA testing, we showed that, In conclusion, our study clearly shows the existence of TCs within interstitium of mucosa, submucosa and muscular layer of oesophagus and suggests possible role(s) of TCs in pathogenesis of several oesophageal disorders that involve (neo)angiogenesis and/or cell differentiation.


Ganoderma stipitatum and Lentinus swartzii, two autochthonous Colombian forest white rot fungi (WRF), previously identified as high enzyme producers, were used to remove BPA and TCS from aqueous solutions. A Box-Behnken factorial design showed that pH, temperature, and duration of treatment were significant model terms for the elimination of BPA and TCS. Our results demonstrated that these EDCs were extensively removed from 5 mg L−1 solutions after a contact time of 6 hours. Ninety-four percent of TCS and 97.8% of BPA were removed with the enzyme solution from G. stipitatum; 83.2% of TCS and 88.2% of BPA were removed with the L. swartzii enzyme solution. After a 6-hour treatment with enzymes from G. stipitatum and L. swartzii, up to 90% of the estrogenic activity of BPA was lost, as shown by the yeast estrogen screen assay. 2,2-Azino-bis-(3-ethylthiazoline-6-sulfonate) (ABTS) was used as a mediator (laccase/mediator system) and significantly improved the laccase catalyzed elimination of BPA and TCS. The elimination of BPA in the absence of a mediator resulted in production of oligomers of molecular weights of 454, 680, and 906 amu as determined by mass spectra analysis. The elimination of TCS in the same conditions produced dimers, trimers, and tetramers of molecular weights of 574, 859, and 1146 amu. Ecotoxicological studies using Daphnia pulex to determine lethal concentration (LC50) showed an important reduction of the toxicity of BPA and TCS solutions after enzymatic treatments. Use of laccases emerges thus as a key alternative in the development of innovative wastewater treatment technologies. Moreover, the exploitation of local biodiversity appears as a potentially promising approach for identifying new efficient strains for biotechnological applications.Bisphenol A (BPA) and triclosan (TCS) are known or suspected potential endocrine disrupting chemicals (EDCs) which may pose a risk to human health and have an environmental impact. Enzyme preparations containing mainly laccases, obtained from  Bisphenol A (BPA) and triclosan (TCS) are present in a great number of products of daily use. Residues of these products are found in many environmental matrices such as rivers and wastewater treatment plant effluents , 2. BPA Oryzias latipes exposed to TCS −, [2M-H]−, [3M-H]−, and [4M-H]− of BPA. The molecular weights of the BPA dimer, trimer and tetramer detected are, respectively, 454, 680, and 906 amu. m/z values of 287, 573, 859, 1145, and 1431 represent, respectively, [M-H]−, [2M-H]−, [3M-H]−, [4M-H]−, and [5M-H]− of TCS. The molecular weights of these compounds are, respectively, 288, 574, 860, 1146, and 1432 amu. Coriolopsis polyzona laccase were the first to demonstrate that enzymatic treatment of nonylphenol (NP), bisphenol A (BPA), and triclosan (TCS) produced high molecular weight metabolites through a radical polymerization mechanism of NP, BPA, and TCS. This result was confirmed for TCS by Murugesan et al. -biphenyl-2,2′-diol [Huang and Weber  have shon et al.  with G. ,2′-diol . Huang a,2′-diol  also pro,2′-diol  have sho,2′-diol  identifi,2′-diol , 22, or ,2′-diol ). L. swartzii was the most efficient for elimination of BPA, whereas laccase of G. stipitatum was the most effective for the removal of TCS. This is most probably due to different kinetic behavior  as a function of strain and substrate that may strongly affect the extent of reaction and the metabolites produced [According to our results, the panel of reaction products associated with the elimination of BPA and TCS by the enzyme solution produced by the WRF tested appeared to be strain- and micropollutant-dependent. For example, laccase of produced . −1BPA solution. Upon contacting this solution for 3.5 hours, with 250 U L−1 laccase from L. swartzii, there was a decrease of up to 70% in estrogenic activity and a 50% decrease with an equivalent treatment with laccase from G. stipitatum. Finally, after a 6 h treatment, estrogenicity loss of up to 90% of the BPA solution was observed for the laccases from both strains.Although the elimination of BPA and TCS was studied using the Box-Behnken factorial design, it was of great concern to address the elimination of the estrogenic activity associated with these compounds. To determine the estrogenic activity of the treated solutions, we used the well-established YES assay . TCS was−1 BPA solution was almost entirely removed after a laccase treatment of only 6 hours. The loss of estrogenic activity of BPA solution is apparently due to structural modification of the parent compound [It had been previously observed that the estrogenic activity of a 0.88 mM solution of BPA was almost entirely removed by extending the laccase treatment to 12 h . In our compound  as conficompound , 46. Thecompound . Further D. pulex acute toxicity assay. Dilution factors used  to determine the acute toxicity represent the proportion of the BPA or TCS solution added to the culture media. The smaller the LC50 value, the more toxic is the tested solution.The toxicities of the aqueous samples were measured using the microcrustacean50 determined for the solutions containing the initial 5 mg L−1 concentration of the micropollutants and for the treated solutions using 250 U L−1 of laccase activity from the different WRF strains at a pH of 5 and a temperature of 60°C. The initial BPA solution presented a LC50 value of 10% while the TCS solution had a LC50 < 12.5%. For both contaminants, the best toxicity reductions were obtained with the enzyme solution from the WRF strain G. stipitatum. In the analysis performed on samples from the BPA solution treated with that enzymatic solution, there was no observation of D. pulex death after the 48 hours incubation period at any of the dilutions tested . It can also be concluded that the chemicals formed by the oxidative action of laccase presented a lower acute toxicity than the parent compounds. In this paper, it was shown that among two newly described WRF strains producing laccases , the mosThe biotreatment of wastewater using laccase-containing solutions secreted by WRF seems therefore to be an attractive solution for the elimination of micropollutants, including the known and suspected endocrine disruptors BPA and TCS. Laccases, which are regarded as environmentally friendly and relatively low-cost catalysts, may emerge as a key alternative in the development of new tertiary or polishing wastewater treatment technologies. The present contribution shows also that the exploitation of local biodiversity appears as potentially promising for identifying new efficient strains for biotechnological applications.


The prevalence of hearing loss (presbycusis) is 35–50% in those aged 65 years or older; consequently, hearing assistant devices become more and more important . CochleaIt has motivated intense investigation on developing stem cell therapy as a new therapeutic strategy, for example, through the transplantation of stem cells into the inner ear for hearing restoration . H.-C. CRecent advances in hearing preservation studies have introduced new concepts and technologies to be applied in CI , 5. To dThe benefits of residual hearing preservation in cochlear implant recipients have promoted the development of atraumatic surgeries. The surgeons prefer round window approach to preserve low frequency hearing . The incA bone-anchored hearing aid (BAHA) or bone-anchored hearing device is a type of hearing aid based on bone conduction , 8. TheyThe diagnostic value of high resolution computed tomography (HRCT) and magnetic resonance imaging (MRI) before CI is very high . M. BusiLimiting the assessment of CI performance strictly to speech perception improvement does not properly evaluate the characteristics of the prosthesis-neural interface. Electrophysiological testing should provide a more accurate proxy of the interaction between the electrodes of the CI and the auditory neurons. F. Venail et al. modeled the activation of auditory neurons in CI recipients. Distribution of Neural Responses Telemetry residues could provide a proxy of auditory neurons functioning in implanted cochleas.The outcome of CI varies over a wide range among pediatric patients . M. ParkAccording to H.-S. Hsieh et al., implanted children tend to write stories that are shorter, worse organized, and without a plot, while formulating morphosyntactically correct sentences. Special attention is required on their auditory and language performances, which could be the underlying causes of the written language problems.In this special issue, we collected both basic and clinical original research articles stimulating the continuing efforts to understand the cochlear implant technology, the development of strategies to treat deafness, and the evaluation of outcomes. It is our wish to increase interest in CI and its related science research with this special issue and further accelerate the development of novel therapies for hearing impairment.


Danio rerio) is an important model organism in hearing research. However, data on the hearing sensitivity of zebrafish vary across different reports. In the present study, the hearing sensitivity of zebrafish was examined by analysing the auditory evoked potentials (AEPs) over a range of total lengths (TLs) from 12 to 46 mm. Morphological changes in the hair cells (HCs) of the saccule (the main auditory end organ) and their synapses with primary auditory neurons were investigated. The AEPs were detected up to a much higher frequency limit (12 kHz) than previously reported. No significant difference in the frequency response range was observed across the TL range examined. However, the AEP thresholds demonstrated both developmental improvement and age-related loss of hearing sensitivity. The changes in hearing sensitivity were roughly consistent with the morphological changes in the saccule including (1) the number and density of HCs, (2) the organization of stereocilia, and (3) the quantity of a main ribbon protein, Ribeye b. The results of this study established a clear baseline for the hearing ability of zebrafish and revealed that the changes in the saccule contribute to the observed changes in TL (age)-related hearing sensitivity.Zebrafish ( Danio rerio), which has become an important vertebrate model organism, is a potentially powerful tool for evaluating hearing genetics, the mechanisms involved in hair cell (HC) regeneration and medical screening for the treatment of auditory dysfunction12The zebrafish  in two studies10et al. reported higher thresholds than those obtained from their AEP data 1012et al. focused on zebrafish from only 28 to 36 mm TL. More recently, two studies investigated hearing ability in larval zebrafish (before 7 dpf) by observing microphonic potentialIn 1993, Platt71011121314In summary, to date, there is no clear, accepted baseline AEP profile for zebrafish hearing according to their ontogenetic development, although such data are available for many other species1617181920212223Because AEPs are a sensitive and widely used measure of fish hearing252627Due to the role of the Weberian apparatus in the hearing of “hearing specialists”, most researchers consider the saccule as the major auditory organ, although there are data indicating a role of the lagena in hearing29We examined the hearing ability of zebrafish using frequency-specific AEPs. To establish the reliable recording of auditory responses, we used various tests to confirm that the waveforms were auditory responses, not artefacts see . The firF4,368 = 54.64, P < 0.001). Post hoc pairwise tests showed significant differences between these groups, as summarized in We measured AEP thresholds across 11 frequencies between 100 Hz and 8 kHz in five TL groups .To observe developmental changes, we labelled saccular HCs with Phalloidin and counted the number of HCs across 7 TL groups . In the youngest group , each fish saccule had an average of 400 ± 26 (mean ± SEM) HCs . This nuTo examine region-related differences in HC morphology, we divided the entire saccular epithelium into two portions (anterior and posterior) and 9 regions, as labelled in 2 from 9 regions, as labelled in F4,16 = 18.68, P < 0.001). The results of the post hoc pairwise comparisons are described in P < 0.001) for the marginal regions  but not (P > 0.05) for the central regions . This finding was confirmed by Tukey’s post hoc tests. The asterisks in To investigate developmental changes in HC density, we counted Phalloidin-labelled HCs in unit areas of 1,000 μmP = 0.58 for height and P = 0.99 for diameter), but they showed significant differences between regions according to both height  and diameter . Similar to the reported findings in goldfishTo further examine the density and the changes in the morphology of HCs in the saccule, semi-thin sections of the saccular epithelium along the rostral-caudal axis were collected from 12–15 mm and 32–37 mm TL fish (n = 3 for each TL group). Although no TL (age)-related effect was observed for either height or diameter, the structure of the saccular epithelium, especially the architecture of HC arrangement, appeared to change with TL (age) in addition to the changes in HC number. The degeneration of HC bundles was observed in fish with a TL > 32 mm , largely due to the loss of stereocilia. ribeye a and b. Ribeye b is expressed most strongly in the inner earThere are two ribeye genes expressed in the synaptic ribbons of zebrafish sensory cells: F3, 36 = 101.04, P < 0.0001; region: F2, 36 = 329.25, P < 0.0001) and the interaction between the two factors  were significant. Tukey’s post hoc tests revealed region-specific differences between the TL groups (To quantify the TL (age)-related changes in Ribeye b expression, we compared the average fluorescence intensity of this protein in a fixed area of the saccular epithelium across the 4 TL groups  based on two-way ANOVA, which also demonstrated an effect of TL . Post hoc pairwise comparisons showed significant differences in ribbon diameters between the 12–15 and 32–37 mm TL groups at both the 60% and 80% distances  was used to observe ribbon morphology. The ribbon diameters of the saccular HCs were examined across 4 locations  and 3 TL groups . For each region in each TL group, the ribbons were measured from multiple HCs that were collected from at least 3 fish. Similar to immunohistological observation, this experiment revealed that the ribbon diameter appeared to increase along the rostral-caudal axis : HCs in In the present study, we examined the hearing ability changes in zebrafish with a TL ranging from 12 to 46 mm (corresponding to an age from 40 dpf to 20 months post-fertilization) and assessed the age-related morphological variations in saccular HCs. The major findings are summarized as follows: 1, the hearing sensitivity, but not the hearing frequency range, of zebrafish changes with TL (or age) ; 2, the Ontogenetic changes in hearing sensitivity have been examined behaviourally and/or eletrophysiologically in a few fish species. However, the overall outcomes of these previous reports present a complicated and conflicted picture102426333435361124263435112638et al., used AEP recordings to observe the changes in hearing ability that occur with size/age10In zebrafish, only two studies, by Higgs In addition to the AEP technique, various methods have been used to evaluate hearing abilities in zebrafish121440Trichopsis vittataSynodontis schoutedeniLophiobagrus cyclurusAge/size-related hearing threshold changes similar to those found in the present study have been observed in other hearing specialist fish species using the AEP technique. These species include the labyrinth fish S.schoutedeniL.cyclurus10The available data do not present a clear picture regarding the age/size-related changes in the hearing frequency range. In the present study, we did not detect a significant change in the hearing frequency range between juvenile and adult fish. This result is in agreement with the observations mentioned above by Zeddies and Fay, who studied startle responseIt has been widely accepted that the Weberian ossicles and the swim bladder are responsible for the high sound sensitivity of fish species containing this apparatus relative to other fish speciesL.cyclurus, the expansion of the hearing frequency range with changing hearing thresholds was identifiedS. schoutedeniThe effect of the Weberian ossicles and the swim bladder on the hearing frequency range of zebrafish is debatable. In a study by HiggsOther age/size-related changes observed in this study included changes in HC number and density and in the size of the sensory epithelium  and 4A, et al.Porichthys notatus, saccular-specific HC additions were concurrent with the seasonal changes in reproductive female auditory sensitivity.Fish, different from most other vertebrates, continue to accumulate HCs in their inner ear after hatching46434445101446Based on our data, the AEP threshold continually decreased with TL up to 32–37 mm ; this paS. schoutedeni. In addition, our data suggest that in addition to the role of Weberian ossicles, changes in HC number, Ribeye b protein expression, and stereocilia abundance each contribute to hearing sensitivity changes over time.We used AEPs to study zebrafish hearing during development and analysed both hearing sensitivity and hearing frequency range over a large TL range. The overall pattern of the hearing sensitivity of zebrafish observed in this study present a pattern similar to that of the squeaker catfish All animal experiments were performed according to the guidelines and the approval of the Committee of Experimental Animal Service, Shanghai, China.AB line zebrafish were bred and reared in filtered aquaria at 28 ± 1 °C in our fish colony. The fish were maintained under a 12:12 light:dark cycle and were fed three times per day.The AEP recordings were conducted in a rectangular tank (100 cm × 42 cm × 50 cm) that contained two chambers, including a small chamber housing a grounded speaker that was isolated by magnetic shielding. The border between the two chambers was made acoustically transparent by mesh holes that were present in the area facing the speaker diaphragm. The holes were covered with a thin plastic membrane to physically separate the water between the two chambers. The tank was placed in a soundproof chamber see .Zebrafish were temporarily anesthetized and immobilized in 0.01% tricaine methanesulphonate . Then, they were mounted and pinned on a 10 cm dish padded with silica gel. The dish containing the fish was fixed on a plastic strip 5 cm below the surface of the water and 25 cm away from the underwater speaker. The temperature of the water was 25 ± 1 °C. Three electrodes , in which the signals were amplified by 20-fold, filtered between 0.1 and 1 kHz, and digitized. The output of this pre-amplifier was sent to a real-time processor . The signal processing by all TDT hardware used for sound delivery (RP2.1) and AEP acquisition (RA16PA and BA) was controlled by a PC using BioSigRP software. At each frequency, the AEP was recorded in a descending sequence of SPLs in 5 dB steps from 160 dBw to the hearing response threshold, which was defined as the lowest level at which a visible and repeatable AEP wave was observed in two averaged trials. In each trial, the responses were averaged over 1,000 sweeps.Briefly, fish heads were fixed with 4% paraformaldehyde at 4 °C overnight. After rinsing the tissue three times with 0.25% PBST, the sensory epithelia of the saccules were dissected according to a previous report2 regions of interest were manually selected, as marked by white dotted rectangles in  using a 20 × or 100 × oil immersion objective lens. DAPI (405 nm), Alexa 488 (488 nm) or DyLight 549 was visualized via blue-violet diode, Argon-ion and Green HeNe excitation, respectively. Z-stack images (0.3 μm apart over 5–10 μm) were transformed into TIFF images, and the mean fluorescence intensity of Ribeye b immunolabelling was determined using ZEN 2011 software (Carl Zeiss) with background correction. Three 500 μmngles in . The meaTEM of ribbon synapses was performed according to routine procedurespost hoc test was used for pairwise comparisons between TL groups or regions when significant main effects were found. Two-way ANOVAs were used to evaluate HC height and diameter, fluorescence intensity of Ribeye b immunolabelling, and ribbon diameter considering the factors TL and region, followed by Tukey’s post hoc tests when significant main effects were found.The data were analysed using GraphPad Prism (GraphPad Software Inc.) and SPSS software (SPSS Inc.). Mean hearing thresholds were determined for each group and at each frequency, and audiograms and histograms were drawn using GraphPad Prism 5. We used ANOVA to determine whether the average HC density varied significantly according to TL or region. Tukey’s How to cite this article: Wang, J. et al. Ontogenetic development of the auditory sensory organ in zebrafish (Danio rerio): changes in hearing sensitivity and related morphology. Sci. Rep.5, 15943; doi: 10.1038/srep15943 (2015).


The role of preexisting sensorineural hearing impairment on the risk for sudden sensorineural hearing loss (SSHL) is still unclear. In this study, we aimed to assess the risk of SSHL in patients with common preexisting sensorineural hearing impairment using population-based data.A population-based case-control study design was used to analyze claims data between January 2001 and December 2011 obtained from the Taiwan National Health Insurance Research Database. The cases consisted of 514 patients with SSHL and the controls were frequency matched to 2,570 cases by sex, 10-year age group, and year of index date. Common sensorineural hearing impairments were retrospectively assessed in the cases and controls. Associations between sensorineural hearing impairment and risk of SSHL were evaluated using unconditional univariate and multivariate logistic regression analyses.p < 0.001). Other comorbidities including hypertension, diabetes mellitus, and hyperlipidemia also showed significant associations with SSHL. Similar results were obtained when the association between SSHL and sensorineural hearing impairment was adjusted with either all the covariates  or with only those selected using a backward elimination procedure .The mean age for the 3,084 study subjects was 53.1 years . Of the 514 cases, 49 (9.5%) had sensorineural hearing impairment while only 44 (1.7%) of the 2,570 controls had the same condition. Univariate logistic regression analyses indicated that preexisting sensorineural hearing impairment was significantly associated with SSHL (odds ratio, OR = 6.05, Results from this population-based case-control study revealed that common sensorineural hearing impairment might be a novel risk factor for SSHL. Sudden sensorineural hearing loss (SSHL) is defined as a loss of 30 dB or more in three contiguous frequencies over less than three days. It can be an isolated symptom or a presenting symptom of systemic diseases. The etiology and pathogenesis remain largely unknown ,2. A sysThis study was approved by the institutional review board of the Dalin Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taiwan (No. B10202022). Since the NHIRD files contain only de-identified secondary data, the review board waived the requirement for obtaining informed consent from the patients.The data for this nationwide population-based case-control study were obtained from the Longitudinal Health Insurance Database 2000 (LHID 2000), which is a subset database of the Taiwan National Health Insurance Research Database (NHIRD). The LHID 2000 contains medical services utilization information since 1996 for a randomly selected sample of one million beneficiaries registered in 2000, representing approximately 5% of Taiwan’s population. The Taiwan National Health Insurance is a universal single-payer compulsory health insurance program that instituted in March 1995 and it has a coverage of 99.9% of the Taiwan's population .th Revision, Clinical Modification [ICD-9-CM] code 388.2), (2) the two diagnoses must occurred within 90 days or less, and (3) the diagnoses must be made by an otolaryngologist. Five controls per case were randomly selected from the LHID 2000, frequency matched by sex, age group , and year of index date. Patients with conductive hearing loss (ICD-9-CM codes 389.0x), Ménière's disease (ICD-9-CM code 386.0), or vestibular schwannoma (ICD-9-CM code 225.1) were excluded from the study  patients who received at least two diagnoses of SSHL , DM (ICD-9-CM code 250.xx), coronary artery disease (CAD) or myocardial infarction (MI) , CKD (ICD-9-CM codes 585.xx and 586.xx), hyperlipidemia (ICD-9-CM codes 272.xx), and obesity  were also assessed.Urbanization of levels of the residence of cases and controls were constructed according to a published categorization scheme, which is based on a combination of population density, percentage of residents with college level or higher education, percentage of residents 65 years and older, percentage of residents who were agriculture workers, and the number of physicians per 100,000 people . In addip-value of the likelihood ratio test in the multivariate logistic regression analysis. All analyses were performed using IBM SPSS Statistics software package, version 22.0 . A p < 0.05 was considered statistically significant.Distributions of age groups, sex, urbanization level of patient's residence, and tertile of insured amount between cases and controls were evaluated using Chi-square test. Unconditional univariate logistic regression analyses were used to evaluate the risks of SSHL associated with sensorineural hearing impairment and various comorbidities. In addition, two multivariate logistic regression models including different comorbidities were used to assess the independent association between SSHL and sensorineural hearing impairment. In Model 1, ten covariates including age, sex, urbanization level of patient's residence, tertile of insured amount, hypertension, DM, CAD or MI, CKD, hyperlipidemia, and obesity were included. Model 2 included only covariates that were obtained based on a backward elimination procedure using the p < 0.001). Other comorbidities including hypertension, DM, and hyperlipidemia also showed significant associations with SSHL.p < 0.001) or with only hypertension and hyperlipidemia that were selected using a backward elimination procedure during the model development .This nationwide, population-based case-control study in Taiwan showed that patients with preexisting sensorineural hearing impairment had an increased risk of SSHL. The association remained significant after adjusting a number of comorbidities. This finding suggests that common sensorineural hearing impairment may share the common etiologies and/or pathophysiological changes with SSHL, which can make the inner ear more susceptible to occurrence of SSHL. Conversely, our finding does not support the notion that hypoxia preconditioning or noise preconditioning can protect patients from subsequent SSHL.Previous research indicated that common sensorineural hearing impairment could result from aging, noise exposure, ototoxic drug exposure, obesity, hypertension, DM, dyslipidemia, CKD, or vertebrobasilar insufficiency , 16, 17.Inner ear with preexisting sensorineural hearing impairment can have a poorer functional reserve due to angiopathy, neuropathy, lower antioxidative enzyme activities and/or higher oxidative stress , 19. TheIn this study, we did not included patients with conductive hearing impairments because these diseases were not supposed to be pathophysiologically associated with SSHL. In addition, we also did not included Ménière's disease and vestibular schwannoma because these conditions were associated with recurrent SSHL . TherefoIn conclusion, our study showed that preexisting sensorineural hearing impairment might be a novel risk factor for SSHL. To minimize the risk of SSHL, clinicians should pay close attention to the presence of common sensorineural hearing impairment in their patients and to advise them to avoid noise and ototoxic medications.


In most of the surviving patients after a lightning strike, audiovestibular abnormalities have been reported. The most frequently reported type of abnormalities is a tympanic membrane perforation with hearing loss and external ear canal burn. However a sensor neural hearing loss and mixed type hearing loss can also occur, but these occur rarely. A nineteen-year-old female patient had, after a lightning strike, serious burns on the left ear, behind the ear, and on the chest and neck. She also had in her left ear 108 dB hearing loss with irregular central perforation and in her right ear 52 dB sensorineural hearing loss. There was no hearing loss before the strike. A hearing aid was recommended for the right ear and good care and follow-up were recommended for the left ear. A lightning strike can cause serious audiological damage. Therefore, it is necessary to make a careful audiovestibular evaluation of the patients. Although there exist rarely healed cases from sensorineural hearing loss after lightning strike in literature, in our case hearing loss occurred bilaterally and then it healed unilaterally. This condition is quite rare in literature. Lightning is a phenomenon in which an electric discharge of 40,000 to 50,000 A and temperature of 20,000°C are produced between the earth and the clouds, and it can cause important injury to humans . Most ofLightning strike has been reported as a sporadic cause of middle and inner ear damage . OtologiThe pathophysiology of sensorineural hearing loss is described by several authors. This pathology may be attributed to vascular and structural changes. Firstly, hypoxia theory is defined as hypoxia in cochlear system secondary to the cardiorespiratory arrest . SecondlExternal ear burns may occur , 12. AltA nineteen-year-old female patient, after being struck by lightning, came to the emergency room with burns on her body and somnolence. To observe the possible cardiac risks and for the treatment of burns on the body, the patient was treated in intensive care for eight days, and she applied to our otolaryngology clinic because of hearing loss ten days after lightning strike. It is written in her medical history that she had no hearing loss before the lightning strike and she cannot remember the case. During the physical inspection, right external auditory canal and tympanic membrane and the left external ear canal are examined as a normal. On the left tympanic membrane, about 5 × 4 mm perforation was determined . Walk, RInjuries from lightning may occur in four methods . A direcIn most of the surviving patients after a lightning strike, audiovestibular abnormalities have been reported. The most frequently reported type of abnormalities is a tympanic membrane perforation with hearing loss and external ear canal burn. However a sensor neural hearing loss and mixed type hearing loss can also occur, but these occur rarely . Wright It is very difficult that tympanic membrane, middle ear, and labyrinth are affected by the current direction. It is because the impedance of these ways is higher than skin or soft tissue . In our Variable factors affect the formation of inner ear lesions due to lightning strike. These are blast injury and the exposure of the direct cochlea throughout the electric current. Also the vessel rupture, which causes bleeding in the inner ear and endothelial damage, which causes occlusion by the small vessels, can be accused in this process as a result of long acting common vasospasm and sympathetic instability after a lightning strike. However, these mechanisms are estimates and may vary depending on the case. There are only a few temporal bone studies made on this topic. Particularly by the temporal bone studies of Youngs and friends on a patient who had bilateral sensor neural hearing loss and died because of transverse mellitus after a lightning strike, the absence of Corti member, rupture, and collapse by Reissner's membrane, strial degeneration, and reduction in the spiral ganglion cell populations were determined .We thought that the reason for the hearing loss in our patient can be blast injuries or developed vasomotor change. The convalescence on the right ear of our patient can be explained with a long acting common vasospasm after lightening and improvement of endothelial damage, which causes occlusion in small vessels as a result of sympathetic instability and provision of revascularization. In our opinion, affection of the left side dominant hearing loss can result from the striking of the lightning to the left side of the body.In the first-degree burns in auricle and external auditory canal daily dressing with cold water and analgesics is sufficient. In the second-degree burns, the burned area shall be cleaned with cold water and soap and then dead tissue shall be removed. The auricle shall be covered with antibiotic ointment and dressed with mastoid. There are full-thickness skin damage and probably tissue loss by third-degree burns so that cartilage can be seen. Skin grafts or local flaps are necessary for these patients. Oral and topical antibiotics should be used against secondary wound infections . In our Various animal experiments are done to explain the central and cochleovestibular pathology occurring due to lightning strike. Low voltage and alternating current have been used in animal models. However, these studies using animal models were insufficient to explain the size of the pathology as a result of lightning strikes, because the current shape and power of lightning strikes are variable .In patients exposed to lightning strike, prognosis depends systemic effects, including primarily central nervous system. Renal failure due to muscle necrosis, myocardial dysfunction, and neurological disorders can occur after a lightning strike. The presence of developing neurological deficits is a serious condition and the most common of them is the loss of consciousness and confusion and these occur in 74–80% of all patients . In our Tympanic membrane perforation and transmission type hearing loss due to lightning strike can heal by self-improvement of tympanic membrane or tympanoplasty. In the treatment of patients with tympanic membrane perforation due to lightning strike, except in cases with suspected fistula, spontaneous healing may be delayed due to local vascular structure and environmental damage. Therefore it is useful to wait up to six months for the tympanoplasty. If perforations do not heal within six months, the tympanoplasty is indicated . In the Lightning strike can cause very different effects on auditory system. This may show different shapes like a simple change in hearing threshold up to total loss of hearing. It has to be considered that an audiovestibular pathology might occur the patients admitted to the emergency department after being struck by lightning and these patients have to be examined more carefully because of this reason. The convalescence on the right ear of our patient can be explained with a long acting common vasospasm after lightening and improvement of endothelial damage, which causes occlusion in small vessels as a result of sympathetic instability and provision of revascularization. But we think we need further investigations to clarify this situation.


Patients suffering from conductive or mixed hearing loss may benefit from bone-conduction hearing systems (BAHS). The amount of amplification provided by the hearing system is selected based on the individual's sensorineural frequency-specific threshold. With patients who are not able to provide thresholds behaviorally, such as young children, objective methods are required to estimate the unaided and aided hearing threshold and thus the success of the hearing system fitting. In a prospective study with ten adult Baha softband users, aided and unaided frequency-specific thresholds were estimated. Aided thresholds to tone bursts via Baha stimulation were obtained behaviorally and electrophysiologically using cortical auditory evoked potentials (CAEPs) and were compared to pure-tone thresholds using routine clinical audiometry. For all stimulation frequencies, the frequency-specific electrophysiological and behavioral hearing thresholds measured with Baha stimulation were highly correlated and not different. Increased thresholds were observed only with the 0.5 kHz Baha stimulation as compared to the pure-tone audiogram. Objective measurement of frequency-specific hearing thresholds with CAEPs is applicable to BAHS users. Frequency-specific estimation of pure-tone hearing thresholds is used as behavioral measure in clinical routine . As subjCAEPs can also be reliably recorded in individuals when the acoustic stimulation is processed through a hearing aid , 14. In Measuring hearing thresholds with Baha users objectively, the recording of auditory evoked potentials using Baha as stimulator was recently shown in normal hearing adults . The behIn this study, we measure the aided frequency-specific pure-tone hearing thresholds objectively by CAEP recordings with patients showing a sensorineural or mixed hearing loss. Therefore, electrophysiological thresholds were estimated with three audiometric frequencies and compared to the behaviorally measured aided and unaided thresholds.M = 62 years; three females, seven males) participated in the study. According to the WHO classification [Ten adults aging between 16 and 81 years  and bone conduction (BC-PTA) using narrow band noise as masker if applicable. An SD50 audiometer  with a TDH39 headphone  for air-conduction stimulation and a KH70 transducer  for bone-conduction stimulation were used which are established in the clinical routine. The better hearing side was determined by averaging the 0.5, 1, and 2 kHz bone-conduction thresholds .For the bone-conduction hearing systems (BAHS) portion of the experiment tone bursts with frequencies of 0.5 kHz, 1 kHz, and 2 kHz were generated by the ESTIM2 signal generator and amplifier  which also recorded the EEG. The stimulus length was 500 ms containing 15 ms of rise and fall time and its repetition rate was 0.5 Hz. The stimuli were delivered electrically to the external audio port of the Baha Intenso  whose amplification was not fitted to the individual hearing threshold. The vibrator was connected to the mastoid of the better hearing side using the Baha softband . The stimulation level was set by changing the amplitude level of the electrical signal provided by the signal generator. The program switch of the Baha Intenso was set to “external,” and the gain and tone controls were adjusted to its middle position which provided a linear I/O function of the Baha up to 60 dB SPL and a gain ranging from 55 dB (0.5 kHz and 2 kHz) to 60 dB (1 kHz). The volume switch of the Baha was adjusted to a value of “2.” The calibration of the stimuli was done subjectively. Therefore, the bone-conduction hearing thresholds for the tone bursts were determined behaviorally for the frequencies of 0.5 kHz, 1 kHz, and 2 kHz using the Baha as stimulator.μV and the A/D rate was 400 Hz. The EEG data were filtered with a 15 Hz low pass. Prior to the averaging, the EEG was online epoched whereas whereas epochs with amplitudes reaching more than 95% of the maximal amplification range were rejected. The sound intensity level was initially set to 70 dB nHL and then reduced in steps of 5 dB until the individual electrophysiological threshold was estimated. At least, 90 artifact-free epochs were collected for every sound intensity level and averaged. The P1-N1-P2 waveforms and the lowest sound intensity level where a P1-N1-P2 waveform could be observed (CAEP threshold) were determined by visual inspection of the averaged waveforms by two audiologists. At every stimulation level, the recording was repeated. The P1 response was defined as the most prominent positive peak in the latency range of 0–80 ms; the N1 response was defined as the most prominent negative peak in the latency range of 50–150 ms; the P2 response was defined as the most prominent positive peak in the latency range of 100–200 ms. The absence of the response was stated if there was no reproducible P1, N1, and P2 responses in both recordings. For all conditions, the subjects were comfortably seated in a sound attenuated booth. The total duration of the experiment including breaks was about 1.5 hours.The electrophysiological estimation of the thresholds was done with the same setup. For the two-channel EEG recording Ag/AgCl electrodes were connected to the electrodes sites F3 (inverting) and the left mastoid (noninverting) and to F4 (inverting) and the right mastoid (noninverting). The gain was set to 100 α was set to 95% for all comparisons. Regression analysis was done between the behavioral and electrophysiological thresholds for all frequencies. The electrophysiological hearing thresholds and the behavioral pure-tone bone-conduction threshold measured with the routine clinical audiometry were compared descriptively.The individual Baha hearing thresholds were compared by a two-way ANOVA for repeated measures using the factors frequency  and method . The assumption of sphericity for the repeated measures variables was tested with the Mauchly test. Greenhouse-Geisser corrections for violations of sphericity were applied when necessary. Post hoc comparisons were made with Fisher's least significant difference test (LSD). M: 22.5 dB) for the 0.5 kHz stimulation, from 0 to 25 dB (M: 11.1 dB) for the 1 kHz stimulation, and from 5 to 20 dB (M: 16.5 dB) for the 2 kHz stimulation.F = 23.3, P < 0.01). No difference between the frequencies or interaction of the frequency and the method was observed. The mean difference between the behavioral and electrophysiological Baha thresholds  was 4.4 dB (SD: 0.9 dB) which is below the resolution of the threshold determination (5 dB). A regression analysis showed a significant linear regression between the electrophysiological and the behavioral thresholds for 0.5 kHz , 1 kHz , and 2 kHz .In this study, we measured CAEPs when auditory stimulation was applied via a Baha transducer. With this, electrophysiological pure-tone thresholds were measured frequency-specifically for a group of subjects with different behavioral bone-conduction thresholds. For all stimulation frequencies used, clear P1-N1-P2 waveforms could be discerned. The latencies and amplitudes are comparable to those evoked with headphone stimulation , 7. The CAEP thresholds would be helpful to control the fitting of the Baha processor objectively. As in this study the Baha processor was not fitted to the individuals' PTA, the electrophysiological thresholds cannot be compared to the behavioral bone-conduction thresholds. For that reason, the behavioral pure-tone thresholds were also measured by Baha stimulation using the same acoustical stimulation setup as used for the CAEP recording. For all stimulation frequencies used, the electrophysiological and behavioral pure-tone thresholds show a good correlation with almost no behavioral-electrophysiological offset as described by previous measurements –26. In pRecording CAEPs using a Baha as transducer could interfere with its unknown signal processing of the stimulus and artifacts in the EEG recording caused by the electromagnetic transducer as described by Rahne et al.  for audiObviously, the electrophysiological threshold did not coincide with the bone-conduction threshold measured behaviorally with the routine clinical audiometry. This was expected because the Baha was temporarily fitted to the hearing impaired subject's skin by a softband. It is known that threshold shifts of 5 to 20 dB occur if transcutaneous and percutaneous coupling of Baha devices are compared , 29. Tha


Understanding the logic behind how a fruit fly's brain tells it to groom its body parts in a stereotyped order might help us understand other behaviours that also involve a series of actions. Related research article Seeds AM, Ravbar P, Chung P, Hampel S, Midgley Jr FM, Mensh BD, and Simpson JH. A suppression hierarchy among competing motor programs drives sequential grooming in Drosophila. eLife3:e02951. doi: 10.7554/eLife.02951Image Fruit flies groom themselves to remove dirt and dust from their bodiesWhen performing kata, a skilled martial artist flows through a sequence of individual movements—attacks and blocks, turns and jumps—to defeat a series of imaginary opponents. Of course, such action sequences are not restricted to either karate or humans. Instead, serial behaviours are widespread throughout the animal kingdom, with examples including navigation, courtship and communication. However, it is not clear how a nervous system is able to generate sequences of actions.eLife, Andrew Seeds and co-workers—who are all at the Janelia Farm Research Campus—have taken a novel approach to investigate serial behaviours by using the powerful genetic tools that are available in the fruit fly Drosophila  suppress lower priority behaviours (such as abdomen cleaning), but not vice versa. Thus, these modules interact in a ‘suppression hierarchy’.The results indicate competitive queuing, and Seeds et al. used a simulation to demonstrate that such a model is sufficient to reproduce sequential grooming behaviour . In a diSeeds et al. suggest two possible ways of implementing hierarchical suppression. A module could be high priority either because it is more sensitive to dust or because it inhibits the downstream, ‘low priority’, modules. However, further study is needed to determine which is the case for grooming behaviour in fruit flies. Furthermore, the cells and circuits that implement grooming behaviour also remain to be identified.This study provides causal evidence that modules being activated in parallel combined with hierarchical suppression can produce serial behaviour. More broadly, it also illustrates the usefulness of studying model organisms, as it is remarkable that the same algorithm that is thought to underpin complex, learned behaviours unique to humans (such as typing and speech) also produces a widespread behaviour that is innate in fruit flies (i.e. grooming). Perhaps this algorithm is evolutionarily ancient and has been elaborated in humans, in which case, understanding computation in fruit flies can indeed teach us about karate.


Hearing loss can lead to a number of disabilities, subsequently reducing the quality of life. In general, hearing thresholds of adolescents are better than adults and the elderly. However, occasionally, adolescents acquire hearing loss for a number of reasons. In this study, our goal was to estimate the prevalence of hearing loss in the Korean population and to investigate the factors related to hearing thresholds in adolescents.A cross-sectional study was conducted using data from the Korean National Health and Nutrition Examination Survey (KNHANES) between 2010 and 2012. We enrolled a total of 1,658 participants, ages 13 to 18 years. We investigated the prevalence of hearing loss and the factors associated with hearing thresholds at various frequencies .Weighted prevalence of unilateral and bilateral hearing loss in Korean adolescents was 2.2% and 0.4%, respectively. Weighted prevalence of hearing thresholds ≥ 20 dB at speech and high frequencies were 3.1% and 5.0%, respectively, for unilateral hearing loss and 0.7% and 1.9%, respectively, for bilateral. Age group, tympanometric data, and household income were significantly related to unilateral or bilateral hearing thresholds ≥ 20 dB at speech frequencies. Earphone use in noisy places was associated with bilateral hearing thresholds ≥ 20 dB at high frequencies.The prevalence of hearing loss in Korean adolescents was 2.6% using the general standard threshold associated with hearing loss. However, the prevalence of hearing thresholds ≥ 20 dB for speech and high frequencies was much higher. The results from this study provide an estimate of hearing loss in adolescents and suggest the need for early detection and hearing preservation programs among adolescents. Hearing loss is currently one of the most prevalent, chronic conditions. Recent estimates by the World Health Organization report that 360 million individuals, 5.3% of the global population, suffer from permanent disabling hearing loss . There aIndependent of the cause of hearing loss, minimal and unilateral permanent damage may result in poorer educational test performance, higher incidence of failed grades, and greater dysfunction in areas such as behavior and self-esteem , 10. EarIn this study, we aimed to determine the prevalence of hearing loss in South Korean adolescents and to analyze the risk factors associated with hearing loss using data from the 2010–2012 Korean National Health and Nutrition Examination Survey (KNHANES).The institutional review board at the Korean Centers for Disease Control and Prevention (KCDC) approved the protocol used in this study and obtained written consent from the parents, next of kin, or guardians on behalf of the adolescents enrolled in our study.This study is based on data from the 2010–2012 KNHANES, a cross-sectional and nationally representative survey conducted by the KCDC. KNHANES has been conducted periodically since 1998 to assess the health and nutritional status of the non-institutionalized population of Korea. Participants were selected using a proportional, allocation systematic sampling with multi-stage stratification.In total, 1,658 patients  ages 13–18 years (junior and high school students) were enrolled in this study. Applying the recommended weighted values from the KNHANES, the total effective frequencies analyzed were 4,047,854 .Participant demographics, including age group , sex, residence type (apartment or non-apartment), town , household monthly income , and noise exposure history were categorized as personal risk factors. Noise exposure history was surveyed in detail and was classified into the following four categories: 1) earphone use in noisy places; 2) occupational noise, which includes at least 3 mo of work in an excessively noisy environment ;, 3) non-occupational noise, including exposure to loud noise  for >5 h per week; and, 4) momentary noise, which included exposure to brief loud sounds . In addition, we sought tympanometric findings likely to affect the hearing threshold (otitis media or drum perforation). Abnormal tympanometry was defined as (1) no peak; (2) a peak pressure below minus 100 daPa; or, (3) a peak pressure above plus 100 daPa.An audiometric examination was administered to all participants. Air-conduction pure-tone thresholds were obtained in a soundproof booth using an automatic audiometer . Supra-auricular headphones were used and the otolaryngologist provided basic instructions to the participants regarding the automated hearing test. Automated testing was programmed according to a modified Hughson-Westlake procedure using a single pure tone for 1–2 sec. The lowest level at which the subject responded to 50% of the pure tone was set as the subject’s threshold. The automated hearing test had good reliability and validity, comparable to the manual pure-tone audio test , 13. ParWe investigated hearing loss prevalence using the following criteria. Hearing loss was defined as a pure tone average (PTA) of the following frequencies: 0.5, 1, 2, and 3 KHz at a threshold of ≥ 26 dB HL. Moderate to profound hearing loss was defined as a pure-tone hearing threshold level of ≥ 41 dB. For early detection of hearing loss in adolescents, we investigated the prevalence of adolescents with hearing thresholds ≥ 20 dB HL in speech  and high frequencies . Lastly, we investigated the factors associated with hearing thresholds ≥ 20 dB HL.KNHANES participants were not randomly sampled. The survey was designed using a complex, stratified, multistage probability-sampling model. To obtain representative prevalence information from the data, it was necessary to consider the power of each participant as representative of the Korean population (sample weight). Survey sample weights were calculated by considering the sampling rate, response rate, and age/sex proportion of the reference population.P-value < 0.05 indicated statistical significance.We performed cross-tabulation analysis using a complex sampling method. Two-tailed analyses were conducted using SPSS , and a Of the 1,658 participants, 1,534  completed a bilateral audiometric examination. Overall, the weighted prevalence of unilateral and bilateral hearing loss among Korean adolescents was 2.2%  and 0.4% , respectively. The prevalence of unilateral and bilateral moderate to profound hearing loss was 1.0%  and 0.3% , respectively .Weighted prevalence of adolescents with unilateral and bilateral hearing thresholds ≥ 20 dB at speech frequencies was 3.1%  and 0.7% , respectively. The prevalence of unilateral and bilateral hearing thresholds ≥ 20 dB at high frequencies was 5.0%  and 1.9% , respectively .P = 0.018, P = 0.044, respectively). Furthermore, a household income of $2,000 or less was associated with a bilateral hearing threshold ≥ 20 dB (P = 0.011)  and abnormal tympanometric results were associated with a unilateral hearing threshold ≥ 20 dB (= 0.011) .For participants with hearing thresholds ≥ 20 dB at high frequencies, no factor was associated with a unilateral hearing threshold ≥ 20 dB. Only earphone use in noisy places was associated with a bilateral hearing threshold ≥ 20 dB .In this study, we demonstrated that the weighted prevalence of hearing loss in Korean adolescents is 2.6% . The prevalence of the Korean population ≥ 19 years is 20.5% . AlthougIn general, the standard for conventional hearing loss is ≥ 26 dB at average threshold during pure-tone audiometry. However, normal hearing in children is defined as a hearing threshold > 20 dB . TherefoPreviously, a nationally representative study was published reporting demographic and audiometric results for adolescents residing in the United States. In this study, they reported that the prevalence of any hearing loss increased significantly from 14.9% between 1988–1994 to 19.5% during 2005–2006 ,18. HoweWe also evaluated associations between demographic and other factors, and hearing thresholds ≥ 20 dB at speech and high frequencies. Aging is a well-known risk factor for hearing loss but we found that being of high-school age was associated with a unilateral hearing threshold ≥ 20 dB at speech frequencies. This was not attributable to aging; all subjects were young. Also, a relatively low household monthly income  was associated with a bilateral hearing threshold ≥ 20 dB at speech frequencies.No type of noise exposure was associated with unilateral or bilateral hearing thresholds ≥ 20 dB at speech frequencies. Of the types of noise investigated, earphone use in noisy places was associated with a bilateral hearing threshold ≥ 20 dB at high frequencies. It has been reported that portable music players can have deleterious effects on hearing thresholds in adolescents . Noise eBut, using of TDH-style earphone, calibrated in a IEC303 couple, collapsing ear canal, and the presence of ear wax might result in an elevation of high-frequency thresholds ,23, and Occupational or non-occupational noise was not associated with a unilateral or bilateral hearing threshold ≥ 20 dB at speech and high frequencies. In fact, most adolescents of the present study were students, and very few were exposed to occupational noise (they did not work). Neither non-occupational noise nor momentary noise were associated with hearing loss.In adolescents, any association between hearing thresholds and noise remains controversial. Dehnert et al. and Williams et al. failed to find any relationship between leisure noise and hearing thresholds , 8. HoweAnother report suggested that while some leisure-noise is hazardous level, the relationship between noise and hearing loss is yet to be determined . All of Tympanometry abnormalities are associated with unilateral hearing thresholds ≥ 20 dB at speech. Tympanometry abnormalities include ear drum perforation, otitis media, impacted cerumen, or eustachian tube dysfunction. All participants with tympanometry abnormalities have the possibility of acquiring conductive hearing loss. Hearing loss due to tympanometry abnormalities can sometimes be recovered, but hearing loss due to noise exposure is usually permanent. Moreover, adolescents with hearing loss can be asymptomatic, preventing early detection in these individuals. In particular, high frequency hearing loss is not noticed until it progresses to the point of affecting speech and communication. It is thought that for hearing preservation in adolescents, early detection of hearing loss is important and hearing tests using a standard hearing threshold ≥ 20 dB should be administered.One limitation was that KNHANES did not obtain data at 8 KHz, thus we analyzed threshold data ≤ 6 KHz. Data at a higher frequency may provide more detailed information on hearing thresholds.This study estimated the prevalence of hearing loss in adolescents and the results suggest the need for early detection, as well as hearing preservation programs, among adolescents.