Assessment of the impact of noise over-exposure in stranded cetaceans is challenging, as the lesions that lead to hearing loss occur at the cellular level and inner ear cells are very sensitive to autolysis. Distinguishing ante-mortem pathology from post-mortem change has been a major constraint in diagnosing potential impact. Here, we outline a methodology applicable to the detection of noise-induced hearing loss in stranded cetaceans. Inner ears from two mass strandings of long-finned pilot whales in Scotland were processed for scanning electron microscopy observation. In one case, a juvenile animal, whose ears were fixed within 4 hours of death, revealed that many sensory cells at the apex of the cochlear spiral were missing. In this case, the absence of outer hair cells would be compatible with overexposure to underwater noise, affecting the region which transduces the lowest frequencies of the pilot whales hearing spectrum. Perfusion of cochlea with fixative greatly improved preservation and enabled diagnostic imaging of the organ of Corti, even 30 hours after death. This finding supports adopting a routine protocol to detect the pathological legacy of noise overexposure in mass stranded cetaceans as a key to understanding the complex processes and implications that lie behind such stranding events. There is an urgent need to develop methods for assessing the effects of underwater man-made noise on cetaceans. High intensity active sonar, and other loud noise sources, for example those from gas exploration, seismic surveys, etc., have the potential to cause lesions to exposed animals. Depending on the distance from the source, injuries can be lethal. As hearing is fundamental to cetaceans, changes to their hearing system may have a large impact on their ability to carry out vital activities. Deriving a forensic protocol to be able to detect if stranded cetaceans have suffered from noise-induced hearing loss is however key to understanding the physiological impact of anthropogenic noise on these species.Previous studies on terrestrial mammals using electron microscopy demonstrated structural alterations of the organ of Corti as a response to mechanical and metabolic fatigue of the sensory cells, in turn a result of high intensity and long duration sound exposure341The stereocilia of outer hair cells (OHCs) are in close contact with the tectorial membrane, which is depressed by the tips of the hairs. These depressions in the tectorial membrane are termed imprints. Experimental acoustic overstimulation in guinea pigs resulted in morphological changes in OHC stereocilia imprints on the undersurface of the tectorial membraneQuantifying the pathological changes in stranded marine mammals is very challenging due to the sensitivity of the organ of Corti epithelium to autolysis and the numerous potential causes that can lead to similar alterations.Hearing loss in cetaceans due to age9101113Long-term research has been undertaken to describe the ultrastructure of the normal organ of Corti in toothed whales with electron microscopyHere we present the methodology and findings from analysis of inner ears recovered from two mass stranding events (MSE’s) that occurred in Scotland; in September 2012 and in June 2015. In both cases, to assess potential acoustic causes and auditory pathology, inner ears from the freshest cases were analysed using scanning electron microscopy (SEM).Individual long-finned pilot whales involved in both MSE’s were necropsied as soon as possible after death. Both ear bones were dissected from the skull, removed and fixed in 10% neutral buffered formalin. Investigation into both MSE’s was coordinated by the Scottish Marine Animal Stranding Scheme (SMASS) with assistance provided from the Cetacean Stranding Investigation Programme, Sea Mammal Research Unit (SMRU) and Moredun Research Institute.Five teeth were collected from the mid-section of the left mandible of each sampled individual and preserved in 10% normal buffered formalin. Age was estimated for each individual using methods adapted from LockyerTable 1nd 2012, 21 long-finned pilot whales  live stranded and died in shallow rocky foreshore between Anstruther and Pittenweem, East Scotland (56.2147–2.7190). Gross necropsies were carried out on site on all 21 animals over the following 24–48 hours® , a rapid decalcifier based on hydrochloric acid . In total seven animals died or were euthanized by a trained marksman on welfare grounds, and a full necropsy was possible on six cases. One of the animals stranded was observed to be a pregnant female in significant distress and subsequent examination indicated a dystocia with a dead calf in utero. Recovery of carcasses for necropsy was logistically challenging. However, extraction and fixation of the ears from all cases were prioritized. Details for the cases are given in In contrast to the 2012 protocol, the cochlea were perfused with 10% neutral buffered formalin via the round and oval windows prior to the ears being immersed in the fixative. This followed the protocol implemented by Morell and AndréThe periotic bone surrounding the cochlea was decalcified using 14% ethylenediaminetetraacetic acid (EDTA) tetrasodium salt hydrate at pH 7.4 at room temperature . The 2015 samples were observed with an SEM at the University of British Columbia Bioimaging Facility, Canada (Hitachi S-4700).The decalcification of the periotic bone was stopped when the vestibular scala and the The same process was followed in all the samples except individual M280.2/12, which was discarded after observing macroscopic advanced autolysis.To map the findings of our analysis we divided the cochlea into several regions, with the base of the cochlea closest to the stapes and the apex or apical region at the tip of the spiral.A detailed post-mortem investigation was performed to assess likely factors causal or contributory to both MSE’s. Gross and histopathological investigation of all animals did not find any indication of specific disease processes although several animals involved in the 2012 MSE were in poor general health. Toxin burden from harmful algal blooms (HAB) and heavy metal analysis also did not find levels sufficiently elevated to explain the strandingpost-mortem (post-mortem (M280.1/12) was suitably well preserved; the organ of Corti was present throughout the cochlear spiral  did not show this pattern of missing OHCs at the apex . The senIn the case of noise-induced hearing loss, the sound sources responsible for such scars in individual M280.1/12 would mainly contain low frequency components. Studies of terrestrial mammals showed “the greatest hearing-loss occurs at a frequency about half an octave above the exposure tone”As expected and previously described in other odontocete speciesIn terms of the 2012 MSE, a scenario where some of the pod was acoustically compromised in their ability to communicate or navigate would offer one explanation for the stranding, but as only animal M280.1/12 was diagnostic along entire cochlear spiral, any causality on the mass stranding cannot be made. Inclusion of accurate inner ear perfusion in the 2015 samples yielded a considerable improvement in preservation of ultrastructural features of the inner ear sensory epithelium. Without perfusion, fixation of pilot whale ears after 18 hours post-mortem did not adequately preserve the organ of Corti cells for ultrastructural evaluation. However, the post-mortem interval of perfused inner ears can extend up to 24–30 hours in pilot whales with adequate retention of ultrastructural detail . ImportaAlthough many stranding networks are now implementing extraction and fixation of the inner ear in cetacean mass stranding necropsies, it is not yet common practice universally. The results presented here show that an appropriate protocol to recover and analyse ears from suitable candidates is available and may provide essential information to understand the complex processes involved in these acute events. This methodology can be applied to single strandings as well if the time from death to fixation is under 30 hours. Our results reinforce a recommendation presented at the International Whaling Commission workshop on cetacean stranding  that carcases are triaged for necropsy, with the tissues most sensitive to post-mortem decomposition, such as the ears, removed first.Overall, this demonstrates a method whereby ultrastructural changes in the ears of cetaceans can be accurately imaged in wild, dead stranded cetaceans. In turn this allows for quantification of any hearing impairment and, potentially, identification of the frequency range of noise overexposure responsible for ante-mortem hearing loss. This finding is of particular relevance to stranding network responders interested in collecting the ears for potential noise-induced hearing loss diagnosis. In turn this has important implications for policy making and ocean management, especially in those cases when overexposure to anthropogenic sounds may be linked to the cause of a stranding event.SEM was particularly useful for detecting lesions or abnormalities in the organ of Corti cuticular plate beyond the first μm from the apex, which can be associated with anthropogenic noise exposure.Perilymphatic perfusion of the cochlea greatly improved preservation of the inner ear ultrastructure, and extended considerably the diagnostic time window in which noise-induced hearing loss could be evaluated in pilot whales.Further research following the methodology presented here on normal apical morphology and the creation of cochlear frequency maps for pilot whales will be essential to distinguish between lesions and normal morphology. In cases of noise-induced hearing loss, these maps will ultimately provide information on the causal sources of such lesions.There is strong demand from the public to establish the reason for cetacean mass strandings. Thorough investigation relies on considering a large number of potential factors with the aim of establishing, when possible, the most probable cause. In many cases the information available may not permit definitive conclusions to be drawn and the significance of a factor or factors has to be interpreted with care so as not to speculate. The inner ear analysis of 2012 and 2015 mass stranding of pilot whales in Scotland revealed that:How to cite this article: Morell, M. et al. Implementation of a method to visualize noise-induced hearing loss in mass stranded cetaceans. Sci. Rep.7, 41848; doi: 10.1038/srep41848 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


C. elegans CDKL-1, most closely related to CDKL1–4 and localized to neuronal cilia transition zones, modulates cilium length; this depends on its kinase activity and αJ helix-containing C terminus. Human CDKL5, linked to Rett syndrome, also localizes to cilia, and it impairs ciliogenesis when overexpressed. CDKL5 patient mutations modeled in CDKL-1 cause localization and/or cilium length defects. Together, our studies establish a disease model system suggesting cilium length defects as a pathomechanism for neurological disorders, including epilepsy.Various kinases, including a cyclin-dependent kinase (CDK) family member, regulate the growth and functions of primary cilia, which perform essential roles in signaling and development. Neurological disorders linked to CDK-Like (CDKL) proteins suggest that these underexplored kinases may have similar functions. Here, we present the crystal structures of human CDKL1, CDKL2, CDKL3, and CDKL5, revealing their evolutionary divergence from CDK and mitogen-activated protein kinases (MAPKs), including an unusual αJ helix important for CDKL2 and CDKL3 activity.   •CDKL2 and CDKL3 structures have distinct C-terminal αJ helix important for activity•C. elegans CDKL-1 localize to and have roles in ciliaHuman CDKL5 and •CDKL-1 cilia length control depends on kinase activity and C terminus with αJ helix•CDKL5 disease-linked mutations cause defects in CDKL-1 cilia length regulation C. elegans CDKL-1 in cilium length control, which is lost in a kinase-dead mutant and by introducing CDKL5 disease-linked mutations. The study suggests ciliary length impairment as a potential mechanism that contributes to human neurological disorders.Canning et al. reveal distinct structural features of CDKL kinases and a role for  Primary (non-motile) cilia are organelles found in most eukaryotic cells, including neurons, that perform essential roles in human sensory physiology, cell signaling, and development . They arThe length of cilia must be tightly regulated to ensure optimal functions in their given cell types . IFT plaChlamydomonas (Kinases implicated in cilium length control largely belong to two groups. One is the NIMA-related kinase (Nek) family, which includes mammalian Nek1 and Nek8, and CNK2, CNK4, and CNK11 from ydomonas . CMGC kiydomonas . Severalydomonas A also reydomonas . NotablyChlamydomonas CDKL5 orthologs regulate cilium length , which localizes to the ciliary TZ  motif  motif D. The tyF) motif D. SecondF) motif D. IntereF) motif . Hence, The CDKL structures all contain broad spectrum ATP-competitive inhibitors that bind to the kinase hinge region via 3 hydrogen bonds A–S3C. CDBy contrast, the CDKL1, CDKL3, and CDKL5 structures displayed characteristics of active kinases. Inhibitor DJM2005 showed a preference for CDKL1, and it formed an extra H-bond to the catalytic loop residue N131 C. Inhibi1 and αAct2). This forced the αC helix to swing outward, breaking the salt bridge between the catalytic lysine (K33) and glutamate . The CDKL1 structure showed an ∼11-Å shift in αC position that restored the salt bridge  proteins in yeast, and we performed e assays C. Proline assays C. These C. elegans, CDKL-1. This kinase is most closely related to mammalian CDKL1–4 and more distantly related to CDKL5  mutant used above contains a large out-of-frame deletion (850 bp) of exons 3–7 and is likely null, we also made another null (nx132) mutant using CRISPR-Cas9, which has a 5-bp deletion in the first coding exon, causing an early stop  animals. Whereas the median length of WT ADL cilia was 8.0 μm, cdkl-1 mutant cilia were 9.6 μm, or ∼20% longer , our results suggest that both CDKL5 and CDKL1–4 proteins may share functions in cilium length regulation. Vertebrates/mammals encode CDKL5 and CDKL1/2/3/4 proteins. This may indicate divergent functions for the different members, which may be cilium dependent and/or independent, including cell proliferation and tumorigenesis, neuronal differentiation, cognition, and learning  by converting a conserved lysine (K) in the ATP-binding site to arginine (R) (cdkl-1 (tm4182) null mutant, the kinase-dead mutant exhibited cilia ∼20% longer than WT  B and S5D than WT E. In add than WT F. Thus, C. elegans. An mNeonGreen-tagged CDKL-1A(ΔαJ) protein variant was expressed, and it was found to localize correctly to the TZ, suggesting that this region does not overtly affect protein stability  present in patients with epileptic encephalopathy, severe mental retardation, developmental delay, or spasms . The ress CDKL-1 D and S5Dlization B. Strikilization D. CDKL-1cdkl-1 mutant. Relative to WT CDKL-1, expression of each variant gave statistically longer cilia in almost all lines, with the phenotypic severity ranked L210P > G11R > P169L  also localize to, and control the length of, cilia (Chlamydomonas CDKL5 (LF5) localizes at the ciliary base and its disruption lengthens cilia  mutants, suggesting a link to length control. Specifically, CDKL5 is influenced by LF3, whose loss causes IFT protein accumulation at ciliary tips  are at the basal body  was cloned into the bacterial vector pNIC-CTHF. The CDKL2 , CDKL3 , and CDKL5  domains were cloned into the baculoviral vector pFB-LIC-Bse. More details are in the The CDKL1 kinase domain sequence  were determined empirically. See also the Kinase activity was profiled in 50 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgClcdkl-1A (Y42A5A.4A) translational fusions, all introns and exons of cdkl-1A, including its 1.8-kb 5′ UTR, were amplified from the genomic DNA, and they were fused to GFP or tdTomato with the unc-54 3′ UTR or cdkl-1 3′ UTR without any fluorescent proteins tags. The mutations found in CDKL5 human patients  or kinase-dead (CDKL5 K42R) mutations were introduced into the cdkl-1 gene by replacing the corresponding residues, and they were fused to tdTomato to assess localization. Plasmids encoding tdTomato-tagged or untagged CDKL-1A variants harboring CDKL5 mutations, and those encoding CDKL-1A(ΔαJ) (residues 1–286) with/without mNeonGreen, were prepared with the CloneJET PCR Cloning Kit.For the various All nematode strains  were maiADL cilia lengths  of L4 larvae were measured and plotted using Dot and Boxplots in R software. The distribution of each dataset was determined by Shapiro-Wilk test. The statistical significance  was calculated by the Dunn’s Kruskal-Wallis Multiple Comparisons with Holm-Sidak adjustment.A Gateway entry clone with the coding sequence of human CDKL5 (NM_003159.2) was obtained from the LTRI plasmid repository, and it was used to clone CDKL5 in fusion with GFP in the pcDNA5-FRT/TO-GFP vector. The GFP-CDKL5 fusion was subsequently subcloned into the lentiviral vector pHR-SIN-SFFV to generate the pHR-SIN-SFFV-GFP-CDKL5 plasmid.4 cells seeded in 12-well plates) were transfected with 40 nM  of 4 small interfering RNAs (siRNAs) targeting CDKL5 obtained from Dharmacon (ON-TARGET plus) using Lipofectamine RNAiMAX transfection reagent (Invitrogen). The Luciferase GL2 Duplex non-targeting siRNA from Dharmacon was used as a negative control. The cells were transfected and serum-starved for 72 hr to induce the formation of primary cilia. The p values are from two-tailed unpaired Student’s t tests.To silence CDKL5, hTERT RPE-1 and hTERT-RPE-1 GFP-CDKL5 cells (5 × 10


Hearing loss is a potential late effect after childhood cancer. Questionnaires are often used to assess hearing in large cohorts of childhood cancer survivors and it is important to know if they can provide valid measures of hearing loss. We therefore assessed agreement and validity of questionnaire-reported hearing in childhood cancer survivors using medical records as reference.In this validation study, we studied 361 survivors of childhood cancer from the Swiss Childhood Cancer Survivor Study (SCCSS) who had been diagnosed after 1989 and had been exposed to ototoxic cancer treatment. Questionnaire-reported hearing was compared to the information in medical records. Hearing loss was defined as ≥ grade 1 according to the SIOP Boston Ototoxicity Scale. We assessed agreement and validity of questionnaire-reported hearing overall and stratified by questionnaire respondents (survivor or parent), sociodemographic characteristics, time between follow-up and questionnaire and severity of hearing loss.Questionnaire reports agreed with medical records in 85% of respondents (kappa 0.62), normal hearing was correctly assessed in 92% of those with normal hearing (n = 249), and hearing loss was correctly assessed in 69% of those with hearing loss (n = 112). Sensitivity of the questionnaires was 92%, 74%, and 39% for assessment of severe, moderate and mild bilateral hearing loss; and 50%, 33% and 10% for severe, moderate and mild unilateral hearing loss, respectively. Results did not differ by sociodemographic characteristics of the respondents, and survivor- and parent-reports were equally valid.Questionnaires are a useful tool to assess hearing in large cohorts of childhood cancer survivors, but underestimate mild and unilateral hearing loss. Further research should investigate whether the addition of questions with higher sensitivity for mild degrees of hearing loss could improve the results. Hearing loss is a late effect of childhood cancer that is especially common after ototoxic treatments such as platinum chemotherapy, cranial radiation, and brain surgery –3. AudioPrevious studies of the validity of questionnaire-reported hearing have been conducted mainly in older populations –10. ChilWe therefore assessed validity and agreement of questionnaire-reported hearing in childhood cancer survivors using medical records as reference, and determined factors associated with agreement including the person answering the questionnaire (survivor or parent), sociodemographic characteristics, time between follow-up and questionnaire, and levels of hearing loss.The Swiss Childhood Cancer Survivor Study (SCCSS) is a population-based, long-term cohort study of all patients registered in the Swiss Childhood Cancer Registry (SCCR) who were diagnosed between 1976 and 2005 at age ≤ 20 years, and survived ≥5 years after initial diagnosis of cancer . The SCCWe included 2175 survivors in this validation study who were diagnosed in a Swiss specialized pediatric oncology (SPOG) clinic after 1989 because medical records of patients diagnosed earlier were rarely available . To deteBetween 2007 and 2013, we traced all addresses of survivors and sent them a long questionnaire. Parents of survivors ≤15 years old completed the questionnaire for their children. Survivors who were ≥16 completed questionnaires themselves. Nonresponders received a second copy of the questionnaire. If they again did not respond, we contacted them by phone. The main topics covered by the questionnaire were quality of life, somatic health, use of current medication and health services, psychological distress, health behaviors, and socioeconomic status. The detailed study design has been published elsewhere . The SCCIn 2015 we collected hearing tests, the corresponding audiologists´ reports, and oncological follow-up reports from the original medical records in the clinic archives of ear-nose-throat (ENT) departments and pediatric oncology clinics. When we found multiple reports or tests with hearing results that differed, we used the most recent result. We only considered reports or tests that were dated before the completion of the SCCSS questionnaire. The first author (A.W.) graded all hearing tests from these records for each ear separately and for frequencies up to 8 kHz according to the SIOP Boston Ototoxicity Scale  [15]. A.First, we determined prevalence, severity, and laterality of hearing loss assessed by questionnaire and by medical records, and compared differences in prevalence using McNemar statistics.Second, we assessed agreement between questionnaires and medical records. We calculated percent agreement  and Cohen´s kappa statistics . Kappa vThird, we evaluated validity of questionnaire-reported hearing. Information from medical records was treated as reference. We calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Sensitivity was the proportion of those with hearing loss as defined by the medical records whom the questionnaire classified correctly. Specificity was the proportion of those with normal hearing whom the questionnaire classified correctly. PPV was the proportion of those with questionnaire-reported hearing loss, in whom hearing loss was confirmed by medical records. NPV was the proportion of those with questionnaire-reported normal hearing, in whom normal hearing was confirmed by medical records. To investigate how validity might vary between subgroups, we also stratified measures of validity.Last, we assessed whether the questionnaire assessed hearing loss equally well for different severity levels of hearing loss. We calculated sensitivity and PPV stratified by severity level and laterality of hearing loss.We also performed a sensitivity analysis to compare results from all respondents (n = 361) to results from respondents for whom we found original hearing tests in medical records (n = 270) by excluding those for whom we did not find hearing tests in medical records and only used information from oncological follow-up (n = 91).We used Stata  for all analyses.Of the 2175 survivors diagnosed after 1989 in a SPOG clinic, 611 (28%) had received ototoxic cancer treatment. Among these, 222 did not reply to the questionnaire and 28 had missing medical records . This reFor 118 of the 361 respondents (33%) parents answered the questionnaire because they were younger than 16 (parent-reported). The 243 (67%) older respondents answered the questionnaire themselves   Tables. In this study, the first to comprehensively investigate agreement and validity of questionnaire-reported hearing in childhood cancer survivors, we found that questionnaires assessed hearing in childhood cancer survivors with acceptable validity. Medical records agreed with 85% of respondents’ questionnaires, which correctly assessed normal hearing in 92% of those with normal hearing and hearing loss in 69% of those with hearing loss. Results did not differ by questionnaire respondent (survivor- or parent-report) or sociodemographic characteristics. Questionnaires did underestimate mild or unilateral hearing loss.We assumed that medical records were complete and correct. However, we would have missed tests of survivors who had their hearing tested after oncological long-term follow-up at a private practice. We also used information on hearing from the oncological follow-up reports, when we found no hearing assessment in the medical records and assumed normal hearing when we found no information on hearing in those reports. This could have contribute to discrepancies in hearing loss between questionnaires and medical records, and may have increased the number of cases with false positive results. However, the sensitivity analysis suggested that this has not led to a relevant distortion of the results. Respondents with a long period between last follow-up and questionnaire might recall the doctor´s diagnosis less well, but validity decreased only in those with more than 10 years between follow-up and questionnaire. Only a minority of the respondents (11%) had such a long period. Finally, our results cannot be extrapolated to children who have not received ototoxic cancer treatment.Our study is strengthened by a study population that is large compared to those of previous validation studies of childhood cancer survivors –20. BecaThis is the first study to compare the questionnaire-reported prevalence of hearing loss with medical records in one group of childhood cancer survivors. The prevalence of hearing loss was slightly lower assessed by questionnaire than by medical records 27% vs. 31%). The prevalence of hearing loss is hard to compare in different study populations because of differences in diagnostic groups, cancer treatment, and type of auditory grading. In previous studies the prevalence of questionnaire-reported hearing loss in groups at risk of ototoxicity ranged from 16 to 31% ,21, and 1%. The pNo other study has investigated validity and agreement of questionnaire-reported hearing with medical records in childhood cancer survivors. Most publications on validity of questionnaire-reported hearing studied populations of older age using questions on subjective perception of hearing such as “Do you feel you have hearing loss?” ,9,25–29.We found very similar results for agreement and validity when either survivors or parents reported on hearing. Both survivors and parents recall hearing equally well. There is no other study investigating the difference between survivor- and parent-reported hearing.Validation studies on hearing in mainly elderly people reported that younger, male, and more highly educated respondents report hearing more accurately ,9,26,29.Not surprisingly, we found that survivors with mild and unilateral hearing loss are more likely to under-report hearing loss than survivors with severe hearing loss. This may have two causes. First, survivors may not have been aware of their mild hearing loss because their audiologist/clinician did not tell them about it. In 4% of cases, which we validated with the audiologist´s results of hearing tests, audiologists did not report on hearing loss though the audiogram showed limited results in the high frequencies. Second, unlike severe hearing loss unilateral or mild hearing loss may affect daily life little or not at all and fewer survivors may recall such a diagnosis, especially when it was some years ago. Several studies have also reported that the accuracy of questionnaire reports of mild hearing loss is low among the elderly ,9,27. ThStudies of hearing loss after treatment of childhood cancer ideally should collect data from medical records or, to obtain the most valid results, prospectively perform audiometry assessments. However, these approaches are often not feasible because they are expensive, and access to full medical records or additional audiometry assessments may be complicated. Many studies therefore assessed hearing loss by questionnaires, which inquire about doctor-diagnosed hearing problems. Reports of survivors and their parents appear equally valid.Questionnaires assess moderate or severe hearing loss well, but underestimate mild and unilateral hearing loss. Our questionnaire missed 90% of cases with mild unilateral and 67% of cases with moderate unilateral hearing loss. Particularly in the absence of well established questionnaires we suggest that researchers validate questionnaire-reported data in at least a subsample to avoid misclassification and reduce bias. Since level of hearing loss differentially affects the accuracy of hearing reports, the results observed for effects of ototoxic risk factors on hearing or the impact of hearing loss on an outcome could be biased. Instruments currently used in clinical assessment of hearing loss in childhood cancer patients such as the HUI and the HMS have questions that focus on difficulties following group conversations with or without a noisy environment. In a future project, we will investigate asking about this type of hearing difficulty to assess mild or unilateral hearing loss independent of clinicians who may not measure or report mild hearing loss.Questionnaires are a useful tool to assess hearing in childhood cancer survivors. However, they underestimate mild and unilateral hearing loss, so researchers should investigate whether inquiring about difficulty following conversation in a noisy environment can improve the assessment of this type of hearing loss in childhood cancer survivors.S1 Fig(TIF)Click here for additional data file.S2 FigAbbreviation: HL, hearing loss.* according to medical records.(TIF)Click here for additional data file.S3 FigAbbreviation: HL, hearing loss.*according to SIOP Boston Ototoxicity Scale.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Severe sepsis is known to result in various neurological long-term deficits in human. Recently, a link between severe, lethal sepsis and significant hearing loss with correlating histomorphological inner ear changes in mice (C57BL/6) was observed. However, if similar observations can be made in severe, non-lethal sepsis in mice is unclear. This study evaluates mice after severe, non-lethal sepsis for analogue functional and histomorphological alterations of the inner ear.A total of 63 C57BL/6 mice were included in the study. All underwent an initial hearing test with auditory brainstem response on day 1. In 35 mice sepsis was induced by cecal ligation and puncture (CLP), in 15 sham surgery was performed and 13 served as healthy control. A second hearing test was performed on day 7. All mice were sacrificed afterwards for further histomorphological evaluation of the inner ears. Immunohistochemical analysis with apoptotic markers Cleaved-caspase 3, BAX and BCL-2 were performed to identify structural inner ear damage.P<0.001). Positive immunostaining at different inner ear structures, like the spiral ligament or the supporting cells could be observed. The percentage of the immunostained positive area in the spiral ligament significantly correlated with the grade of hearing loss for BAX (P=0.027) and Cleaved-caspase 3 (P=0.024) but not for BCL 2 (P>0.05).Of all CLP mice, 21/35 (60.0%) died due to the induced sepsis. Of the surviving CLP mice, 14/35 (40.0%), post-treatment hearing thresholds differed significantly from the sham and control mice (The present data suggests that severe, non-lethal sepsis in mice results in significantly elevated hearing thresholds. A positive labelling for the pro-apoptotic markers BAX and Cleaved-caspase 3 suggested the induction of apoptosis in inner ear. Summary: This study shows that a mild sepsis leads to a significant hearing impairment due to apoptosis of different inner ear structures. Sepsis is a severe disease, which can result in multiple organ failure and death. Survivors suffer long-term sequelae. The responsible mechanisms for the chronic consequences of severe acute inflammation are not well understood .Hearing impairment is a serious complication associated with critical illness and goes along with a significant impact on the social life, causing feelings of loneliness, isolation and depression. Due to these implications hearing loss is one of the six most frequent diseases which impair the quality of life extensively .The causes of hearing loss in critical ill patients in the intensive care unit (ICU) range from mechanical or accidental trauma, administration of ototoxic medications, local or systemic infections, vascular and hematologic disorders, autoimmune diseases to environmental noise . Severe One possible influential factor resulting in hearing loss due to severe acute inflammation, like severe sepsis, has been reported by Schmutzhard and colleagues in a different setting. In an experimental murine malaria model, a significant hearing loss was observed. Furthermore, the histopathologic evaluation revealed apoptosis of fibrocytes of the spiral ligament. Malaria typical changes were not identified. The authors discussed systemic inflammatory response (SIRS) as possible cause for the observed hearing loss . ConsequThese observations were supported by two other experimental studies. Moon and colleagues observed a release of tumour necrosis factor-α from spiral ligament fibrocytes, if exposed to otitis media pathogens . FurtherTo detect apoptosis different markers were established the downregulation of the anti-apoptotic pathways can be demonstrated with decreased Bcl-2 labelling, which needs to be compared with the pro-apoptotic antagonist, the Bax protein. A Bax/Bcl-2 ratio favouring Bax expression with additional downstream Cleaved-caspase 3 (CC-3) upregulation is a solid sign for induced apoptosis .The link of hearing loss and severe, lethal experimental sepsis appears established. However, no such link has been made in a non-lethal experimental murine sepsis model yet. This study evaluates whether hearing impairment occurs in a non-lethal experimental sepsis and implies pathologic alterations of inner ear after survived disease.63 two-month-old C57BL/6 mice  were eligible for the study after the primary ABR measurement. Sepsis was induced in 35 mice with cecal ligation puncture, 15 mice underwent sham surgery  and 13 mice served as control . 21/35 (60.0%) of the CLP mice died due to the induced sepsis.At day one an initial objective hearing test was performed with auditory evoked brainstem responses. The mean pure tone average in the sepsis group was 40.9 dB (SD5.4), in the sham group 42.8 dB (SD4.3) and in the control group 40.8 dB (SD6.5) A.Fig. 1At day 7 the mean hearing thresholds of surviving mice in the sepsis group was 48.0 dB (SD7.6), in the sham group 42.6 dB (SD6.5) and in the control group 41.0 dB (SD6.7) B.The pre- and post-treatment average hearing threshold and the mean hearing loss in sepsis mice at different frequencies of 4, 8, 16 and 32 KHz were shown in P<0.001). While in the sepsis group an average hearing loss of 9.8 dB was found, no change of hearing thresholds could be seen in the sham and control mice  and in low hearing loss (10.09), but only a slight reactivity in sham mice (2.87).The staining of BCL-2 in the spiral ligament was highest in sepsis mice with low hearing loss (4.02). A lower reactivity in sepsis mice with high hearing loss (2.29) and only a minimal reactivity in the sham mice (1.21) was observed.P=0.027) and CC-3 (P=0.024), but no statistically significant correlation could be found for Bcl2 (P=0.222)  .Fig. 5.A previous publication has demonstrated that experimental sepsis can lead to a significant hearing impairment in mortally sick mice. In this murine model an apoptosis in the supporting cells of the organ of Corti and a disturbance of the synapses of the inner hair cells could be described . A critiTherefore, the question arises if the observed functional and morphologic impairment of the inner ear develops in animals surviving sepsis.In the current study experimental sepsis has been induced with cecal ligation puncture. Rittirsch et al. defined two grades of sepsis in CLP technique: a ligation of the cecum at the middle containing 50% of the cecum results in a survival rate of 40%, this has been referred to as a mid-grade sepsis. In contrast, if 75% of the cecum is ligated a mortality of 100% can be expected and has been defined as high-grade sepsis . In thisIn the initial objective hearing test a nearly similar mean hearing threshold at an average of 41.5 dB in all three groups could be observed, which is considered a regular hearing threshold for C57BL/6 mice .All surviving mice were scheduled for a second ABR measurement on day 7. In the surviving sepsis mice the mean hearing threshold increased significantly for 9.8 dB. In contrast the hearing thresholds of the sham and control group remained stable with a mean difference of only ±0.2 dB. These results confirm prior findings, describing an average hearing loss of 8.1 dB in lethal septic mice and no change in the control and sham group .In a malaria mouse model an apoptosis in the fibrocytes of the spiral ligament and a breakdown of the blood-labyrinth barriers as pathological mechanisms of the hearing loss could be observed . In a prP=0.027) and CC-3 (P=0.024) in sepsis mice with a high hearing loss could be found. Animals of the sham group did not show any reaction or only a very mild staining in the supporting cells of the cochlea.The histomorphologic work-up for the surviving mice revealed a good structure preservation. Furthermore the immunolabelling for the pro-apoptotic markers BAX and CC-3 was positive in the Deiter’s cells and inner hair cells as well as in the spiral ligament. For this the percentage of positive immunoreactive area in the spiral ligament was compared in mice with and without sepsis. A significant activity of BAX  were used. An initial objective hearing test was performed with ABR. Animals with thresholds below 40 dB at 16 kHz were eligible. Sepsis was induced in 35 mice with cecal ligation puncture; 15 mice underwent sham surgery  and 13 mice served as control . The course of sepsis was monitored for signs of prostration, lowered temperature and weight loss at 12-hour intervals. On day 7 in all surviving mice a second ABR measurement was performed. Deeply anesthetised the animals were sacrificed afterwards. Within 7 min after death the inner ears were prepared and fixed for further morphologic and immunohistochemical examination.®, Senden-Bösensell, Germany) (67.5 mg/kg body weight), xylazine hydrochloride  (5.4 mg/kg body weight) and atropine sulfate  (0.085 mg/kg). After immobilizing the animal, the fur of the belly was trimmed and disinfected with 90% alcohol. A median incision was done on the linea alba. The cecum was mobilized and ligated with a 4.0 Vicryl suture  in the middle of the cecum in order to induce an anticipated mortality of 60% , xylazine hydrochloride (5.4 mg/kg body weight) and atropine sulphate (0.085 mg/kg). Mice were kept on a pre-warmed (37.0°C) gel pad, which provides a constant body temperature during measurements.Auditory nerve responds were measured with a custom made portable system controlled by the software Audiology Lab  in a custom-made electrically shielded sound attenuated chamber. Potentials were recorded via three subcutaneous needle-electrodes. Tone bursts of 4, 8, 16 and 32 kHz were used as stimuli. Tone bursts were produced by two different transducers. Starting with 0 dB the stimuli increased by 5 dB up to 90 dB. Hearing thresholds were determined as the minimum stimulation level that produced a clearly recognizable potential. After amplification and band pass filtering , electrical signals were averaged over 128 repetitions of stimulus pairs. The ABR results were evaluated by two independent Otologists in a blinded manner.®Plus, Menzel-Gläser, Braunschweig, Germany), air-dried for 1 h and stored at −20°C.Immediately after sacrifice, the cochleae were harvested and dissected within 7 min. The round and the oval windows were opened and 4% formaldehyde in phosphate buffered saline (PBS), pH 7.4, freshly prepared from paraformaldehyde was perfused into the fluid spaces. Subsequently inner ears were immersed in the fixative. Decalcification was performed with an 8-hour exposure of tissue in 20% EDTA  in PBS, pH 7.4, at 37°C. For immunohistochemical examination, cryoembedded serial sections (10 μm) were cut on a cryostat (LEICA CM 3050), mounted on glass slides .Slides were immunostained with an automated system (Ventana Roche Discovery Classic) according to the DAB-MAP discovery research standard procedure to guarantee reproducible results. Slides were rehydrated in distilled water and exposed to primary antibodies without any antigen unmasking strategies. Antibodies raised against BAX , Cleaved caspase-3  and BCL-2 , raised against a peptide mapping at the N-terminus of human Bcl-2 were incubated for 60 min and detected with biotinylated secondary antibodies  visualized by a peroxidase-antiperoxidase system developed with Diaminobenzidin intensified with copper sulfate. Blocking of endogenous peroxidase was accomplished by competitive blocking with an excess of peroxidase. For control purposes, representative sections were processed in the same way by substituting primary antibodies with isotype matching immunoglobulins . Slides were counterstained with haematoxylin, dehydrated in ascending grades of isopropanol cleared in xylene and mounted with EntellanTable S1.Antibodies utilised in the study for immunohistochemical analyses are summarized in ® GmbH, Vienna, Austria) and evaluated with the analysis software HistoQuest . The system consisted of a Zeiss Imager Z2 microscope equipped with a 3 megapixel area scan colour camera Pixelink PL-623 CF. The motorised stage from Maerzhauser had an 8-slice insert easing the batch scan. The white light was delivered by a VISLED lamp based on LED technology which ensured a stable reproducible colour temperature over the entire light intensity spectrum. The image acquisition phase was done with a Zeiss 20x, 0.6 apochromat objective. Every scanning project acquired a non-saturated white image for shading compensation. The sections were investigated and the spiral ligament was manually selected and marked for analysis using regions of interest (ROI). The computer analysis was done using HistoQuest 3.5 cytometry software calculating the mean optical density of the immunohistochemical signal across the total ROI area. The software splits the colour into marker-specific channels using an integrated colour separation method termed single reference shade. This approach can reliably separate Haematoxylin and colorimetric DAB developed antibody immunoreactivity. A manual background threshold was set once interactively to extract positive areas and used throughout all slides processed.The immunohistochemical slides were digitised and analysed with TissueFAXS  was calculated as the mean of the left and right ear ABR-thresholds for the frequencies 4, 8, 16 and 32 kHz for each animal. For descriptive statistics whisker plots were used to demonstrate the cumulative PTA score. To test treatment effects (CLP versus sham or control), one-way analysis of covariance was used with post-treatment PTA as the outcome variable adjusted for pre-treatment PTA as a covariate, with a ® Excel® containing all relevant data: total analysed area, optical density/µm² and segment positive area µm² of each domain.The measurement of the immunohistochemical slides were exported from the validated analysed projects to Microsoft® Excel®.Statistical analysis was performed in SPSS 21  using the data from exported Microsoft


Chronic tinnitus is a prevalent hearing disorder, and yet no successful treatments or objective diagnostic tests are currently available. The aim of this study was to investigate the relationship between the presence of tinnitus and the strength of the middle-ear-muscle reflex (MEMR) in humans with normal and near-normal hearing. Clicks were used as test stimuli to obtain a wideband measure of the effect of reflex activation on ear-canal sound pressure. The reflex was elicited using a contralateral broadband noise. The results show that the reflex strength is significantly reduced in individuals with noise-induced continuous tinnitus and normal or near-normal audiometric thresholds compared with no-tinnitus controls. Due to a shallower growth of the reflex strength in the tinnitus group, the difference between the two groups increased with increasing elicitor level. No significant difference in the effect of tinnitus on the strength of the middle-ear muscle reflex was found between males and females. The weaker reflex could not be accounted for by differences in audiometric hearing thresholds between the tinnitus and control groups. Similarity between our findings in humans and the findings of a reduced middle-ear muscle reflex in noise-exposed animals suggests that noise-induced tinnitus in individuals with clinically normal hearing may be a consequence of cochlear synaptopathy, a loss of synaptic connections between inner hair cells (IHCs) in the cochlea and auditory-nerve (AN) fibers that has been termed hidden hearing loss. Chronic tinnitus is a prevalent condition that in some cases can lead to debilitating consequences. It may also indicate some damage to the inner ear, termed cochlear synaptopathy, even in cases of clinically normal hearing. However, there are currently no objective diagnostic tests for either tinnitus or cochlear synaptopathy in humans. This study compares the strength of the middle-ear muscle reflex in people with clinically normal or near-normal hearing but suffering from noise-induced tinnitus to that in an age-matched control group without tinnitus. The results show that the tinnitus group had greatly reduced reflex strength. The outcomes are consistent with recent results obtained in mice and suggest that it may be possible to diagnose tinnitus and cochlear synaptopathy in humans.Tinnitus, the chronic perception of sound in the absence of an acoustic source, affects ∼10-15% of the adult human population worldwide . Most inThe aim of this study was to investigate the relationship between tinnitus and the strength of the middle-ear-muscle reflex (MEMR) in humans with normal and near-normal audiometric thresholds. A wideband measure of the MEMR was used to probe the reflex strength because this measure is more sensitive to changes in middle-ear impedance than the standard clinical measure, which uses a low-frequency probe tone . There aFor the study, we recruited 18 individuals with tinnitus  and 18 individuals with no tinnitus . The mean age of the individuals with tinnitus was 46 years (range: 25–63 years) and the mean age of the individuals with no tinnitus was 43 years (range: 27–62 years). Individuals with tinnitus were either employees of the University of Minnesota or were acquaintances of the employees and were recruited because they had complained about experiencing continuous tinnitus. Individuals for the control group were recruited via flyers and personal contacts and were selected to approximately match the ages of the participants in the tinnitus group. Hearing sensitivity was tested using a calibrated audiometer Madsen Conera (GN Otometrics). A method of limits was used to measure hearing thresholds at audiometric frequencies from 250 Hz to 8 kHz in one-octave steps. Each tone was first presented at an audible level between 30 and 45 dB sound pressure level (SPL). The listeners signaled that they heard the tone via a button press. The level of the tone was decreased by 10 dB after each response coinciding with the tone, and increased by 5 dB after the lack of response (the tone not heard). The lowest level that was consistently heard for three repeated tone presentations was taken as hearing threshold. Levels below 0 dB HL were not tested. The wideband measure of MEMR effects was obtained using clicks with a flat spectrum between 250 Hz and 12 kHz as the probe stimulus and wideband noise bursts as the reflex elicitors. The clicks were generated using a 60th-order recursive exponential filter . In eachDuring the sound presentation and recording, the subjects were seated comfortably in a reclining chair in a double-walled sound-insulating booth. The subjects were asked to stay relaxed and as still as possible during the recordings. The entire test took ∼12–14 min per subject.The stimuli were generated on a PC in Matlab (MathWorks) with a LynxTwo-B sound card (Lynx Studio Technology) using a sampling rate of 48 kHz. The stimuli were presented to the listeners using ER10X probes (Etymotic Research) with the ear tips chosen to best fit the subject’s ear. The ear canal sound pressure was recorded from the ear with the clicks via ER10X microphone and was digitized using the LynxTwo-B sound card before being stored for offline analysis.Recorded waveforms were screened visually for artifacts. On the detection of artifacts, a test was repeated, and the clean recordings were analyzed separately for each elicitor level. The artifacts were detected for only two out of 36 subjects and were due to inadvertent removal of the probe from the ear.To obtain the average baseline (pre-elicitor) and comparison (during-elicitor) click pressure for each elicitor-level condition, the ear-canal sound pressure wave form during nine clicks preceding the onset of the elicitor and nine clicks during the second half of the elicitor was Fourier transformed. The complex-valued frequency-domain representations for the pre-elicitor and during-elicitor clicks were averaged separately across all 30 trials. Only the clicks during the second half of the elicitor were analyzed to allow for sufficient buildup of the MEMR strength . The mea2. In addition, the size of the effect of tinnitus on MEMR strength for the highest elicitor level used (88 dB SPL) was calculated in terms of Cohen’s d  and sex as the between-subjects effects, and age and the average of hearing thresholds at 4 and 8 kHz as covariates. Greenhouse-Geisser corrections were used when Mauchly’s test revealed a violation of sphericity assumption. The effect size for the presence of tinnitus after accounting for effects of age and hearing sensitivity in the ANOVA was assessed by calculating the value of ηohen’s d .Relative changes in ear-canal sound pressure across frequency elicited by the contralateral noise were averaged separately for the individuals in the control group A and theThe values of MEMR strength calculated from the changes in ear-canal sound pressure for each elicitor level, are shown in F = 2.24, p = 0.01), as expected based on existing animal data  = 3.38, p = 0.08), indicating that elevated thresholds in some of the individuals with tinnitus cannot fully account for the weaker MEMR strength in this study. The effect of sex also was not significant  = 0.02, p = 0.88) and there was no significant interaction between sex and hearing status  = 0.50, p = 0.48). Even with the effects of age and high-frequency hearing loss accounted for, there remained a highly significant effect of tinnitus  = 31.76, p < 0.001, η2 = 0.47) indicating significantly weaker MEMR activation in this group of subjects. The within-subjects effect of level was not significant  = 1.92, p = 0.09) but there was a highly significant interaction between level and presence of tinnitus  = 19.45, p < 0.001) reflecting the slower growth of the MEMR strength with increasing elicitor level in the tinnitus group. No other interactions were significant.The ANOVA showed that the effect of age was significant  the effect size estimated by a Cohen’s s “huge” , meaningThe main finding from this study was a significantly weaker MEMR in humans with tinnitus related to excessive noise exposure but with clinically normal or near-normal hearing than in age-matched humans with similar audiometric hearing but without tinnitus. A recent study in mice found a significantly weaker MEMR in noise-exposed mice with cochlear synaptopathy confirmed by postmortem confocal analysis . Thus, tNevertheless, there is growing converging evidence from humans and animals suggesting that tinnitus in the absence of audiometric hearing loss is triggered by a diffuse loss of synaptic IHC/AN connections. In mice, eliminating over 95% of synapses with afferent AN fibers via ouabain treatment has been shown to trigger progressive neural gain at the level of the inferior colliculus and the auditory cortex  that couEvidence from humans who have normal hearing but experience chronic tinnitus is based mainly on ABRs which show a reduction in the amplitude of wave I representing the integrity of AN responses in the absence of changes to the amplitude of wave V originating from the brainstem . Small iGiven the large size of the effect of tinnitus on MEMR strength after accounting for age and differences in hearing sensitivity at 4 and 8 kHz, and the likely relationship between tinnitus and cochlear synaptopathy, the wideband MEMR measure appears to be a highly promising test for detecting synaptopathy in humans. As shown by individual data in In this study, the test population was limited to individuals with normal and near-normal hearing because of the possible complex relationship between hearing loss and the MEMR. Even for carefully matched audiograms, hearing loss may be due to different combinations of outer-hair cell and IHC damage in different individuals. Disentangling these contributions in humans is time consuming and often unreliable . In mildWe did not measure hearing thresholds for frequencies extending beyond the audiometric range in this study. Elevated hearing thresholds in some of our tinnitus subjects did not account for the sizeable difference in MEMR strength between the two groups. It is possible that the tinnitus group had impaired hearing sensitivity at very high frequencies (>8 kHz) compared to the group without tinnitus. Wideband noises are more effective elicitors of the MEMR that pure tones or narrowband noises , but unfStudies of cochlear synaptopathy in animals have used otoacoustic emissions to evaluate the health of the cochlear responses . In humaIn summary, the MEMR measurement presented here provides a promising objective diagnostic test for tinnitus in humans with no or mild clinical hearing loss. Based on earlier animal studies , we surm


The TCDI technique based on two-step heating is capable of not only stably capturing the Ag ionic ink, but also reducing the porosity of thermally decomposed Ag nanoparticle structures by eliminating large amounts of organic complexes. The porosity reduction of metal grid TCs on a glass substrate leads to a significant decrease of the sheet resistance from 21.5 to 5.5 Ω sq−1 with an optical transmittance of 91% at λ = 550 nm. The low-porosity metal grid TCs are effectively embedded to uniform, thin and transparent polymer films with negligible resistance changes from the glass substrate having strong interfacial fracture energy (~8.2 J m−2). Finally, as the porosity decreases, the flexible metal grid TCs show a significantly enhanced electromechanical durability under bending stresses. Organic light‐emitting diodes based on the flexible metal grid TCs as anode electrodes are demonstrated.Next-generation transparent conductors (TCs) require excellent electromechanical durability under mechanical deformations as well as high electrical conductivity and transparency. Here we introduce a method for the fabrication of highly conductive, low-porosity, flexible metal grid TCs  Indium tin oxide (ITO)-based films have been the industrial standard for a long time due to their low electrical sheet resistance (Rs) and high optical transmittance (T); however, ITO-based films include several drawbacks, including inherent brittleness and the need for expensive sputtering processes2. Alternative materials to ITO-based films, including conducting polymers3, graphene4, carbon nanotubes5, random metal nanowire networks10, and regular metal grids11, have been reported for use in flexible optoelectronic devices. Among these materials, regular metal grids offer many advantages, such as facile control over their grid width and spacing, scalability to large-area application and low junction resistance13.Transparent conductors (TCs) are indispensable in a variety of optoelectronic devices, including organic light-emitting diodes (OLEDs), organic solar cells, and touch screen panels16. This method facilitates the fabrication of metal grid structures with the relatively high aspect ratio (=height/width), while they should be sintered at low temperature (<150 °C) due to thermal degradation of the polymer substrate; (2) metal NP structures on a glass substrate are fabricated using a variety of wet deposition methods, such as direct imprinting (DI)18, gravure printing19, electrohydrodynamic jet printing21 and inkjet printing22. Although this method is not suitable for fabricating the high aspect ratio structures of metal NPs, the metal grid TCs on the glass substrate can be sintered at high-temperatures (>200 °C) to improve the electrical conductivity24. After high-temperature sintering process, the metal grid TCs are transferred from the glass substrate to the polymer substrate for the fabrication of flexible metal grid TCs.Cost-effective and solution-processed metal grid TCs on flexible substrates have been reported using two different fabrication schemes: (1) grid-patterned cavity is formed into a polymer substrate using hot embossing process and then is filled with metal nanoparticle (NP) ink26. Recently, our group suggested a reservoir-assisted DI method based on an Ag ionic ink, which has several advantages such as higher metal content and less aggregation than colloidal metal NPs, for the fabrication of high-performance flexible metal grid TCs18. However, the Ag NP structures fabricated by the thermal decomposition of Ag ionic ink include numerous micro/nanoscale pores due to the elimination of organic complexes28. Unfortunately, those pores can deteriorate electrical and mechanical properties of the flexible metal NP structures33. First, the formation of many pores precludes the generation of highly conductive metal grid TCs. Second, since high-temperature sintering process (~300 °C) induces strong interfacial fracture energy (IFE) between the metal and the substrate, micro/nanoscale pores can cause serious damage to metal NP structures during the transfer process. Finally, as the cracks are initiated and propagated near numerous pores under mechanical deformations, the flexible metal grid TCs may be easily damaged by static or dynamic bending stresses. The formation of micro/nanoscale pores inside the metal NP structures should be minimized in the development of flexible optoelectronic applications.Among the wet deposition methods, the DI of colloidal metal NPs can directly produce micro/nanoscale metal structures at low-costs and in a high-throughput manner without expensive etching steps and metal evaporationρm) and Rs of the metal grid TCs at a fixed transmittance (T550nm) at 550 nm were represented as a function of the organic complex contents and geometrical calculation. The effect of porosity on the transfer process of metal grid TCs to the polymer film was explored at different sintering temperatures. In addition, the effect of porosity on an electromechanical durability of the flexible metal grid TCs was examined under the static and dynamic bending stresses. Finally, the utility of flexible metal grid TCs as anode electrodes was demonstrated by fabricating of OLEDs.In order to resolve this problem, we introduce a novel temperature-controlled direct imprinting (TCDI) process of Ag ionic ink based on two-step heating for the generation of highly conductive, low-porosity, flexible metal grid TCs. This TCDI technique led to stably capturing the Ag ionic ink and reducing the porosity of thermally decomposed Ag NP structures. The electrical resistivity (P = 120 kPa) and low temperature; (ii) the first heating step was performed at an evaporation temperature (TE) of 50 °C over 5 min to stably capture the Ag ionic ink inside the grid-patterned cavity. It should be noted that when the Ag ionic ink is initially heated at an increased TE, large amounts of the filled ink leave from the grid-patterned cavity through a liquid film between the mold and the substrate due to carbon dioxide generation during the thermal decomposition of Ag ions34; (iii) for a liquid film with negligible thickness (hf ≈ 0), the second heating step was carried out at decomposition temperature (TD) = 90 °C to eliminate organic complexes derived from the thermal decomposition of the Ag ions and to improve their thermal decomposition rate; (iv) after complete solvent evaporation, the grid-patterned mold was carefully removed from the substrate. The Ag NP structures were thermally treated at sintering temperature (TS) = 300 °C for 10 min. Figure TD was increased from 50 °C to 90 °C, the micro/nanoscale pores in the metal grids were significantly reduced. This micro/nanoscale pores may be mainly generated by the elimination of organic complexes during the thermal decomposition process. Amounts of organic complexes inside the Ag NP structures were compared using thermogravimetric analysis (TGA) and derivative thermogravimetric (DTG) analysis of Ag NP-organic complex powders, respectively. Figure TD = 50 and 90 °C, respectively. The Ag NP-organic complex powders dried at the lower TD showed the larger weight loss and the faster decomposition rate in the temperature range of 100–200 °C. The Ag NP-organic complex powders dried at TD = 50 and 90 °C showed a weight loss of 21.24 and 6.17%, respectively. These results indicated that the metal grids generated using the DI of Ag ionic ink may include a greater fraction of the organic complex by weight, about 15% more, than those obtained using the TCDI of Ag ionic ink. Figure TD. As TD increased from 50 to 100 °C, the organic complex contents of the powder decreased. The inset shows photographic images of the metal-organic complex powders. The Ag NP-complex powders dried at TD = 50 °C showed dark black due to high organic complex contents (left), while those obtained at TD = 90 °C showed bright silver due to relatively low organic complex contents (right).The TCDI of Ag ionic ink, which involves the two-step heating, is schematically illustrated in Fig. Rs and T550nm for the metal grid TCs generated using the TCDI of Ag ionic ink. As TD increased from 50 to 90 °C, Rs of the metal grid TCs decreased significantly, holding T550nm over 91.2%; however, the value of Rs for the metal grid TCs fabricated at TD = 100 °C increased and fluctuated as solvent evaporation and thermal decomposition occurred near the boiling point of the base-solvent . Figure TD. The spectral transmittance of metal grid TCs fabricated at TD = 100 °C did significantly decrease from 350 to 500 nm. Also, figure of merit (FoM), which generally predicts the performances of TCs, was compared for the metal grid TCs generated at different values of TD in Figure TD increased to 90 °C, the FoM of the metal grid TCs was increased from 250 to 572. The value of ρm of thermally reduced Ag NP line structures fabricated at different values of TD is compared in Fig. ρm could be expressed using the following relationship:ρo is the electrical resistivity of the thermally reduced metal structures without organic complex contents, wp is the weight percent of organic complex and kp is a correction factor from experimental data. The correction factor was determined to be kp = 0.0727. The value of ρo (6.5287 × 10−6 Ω cm) was 4 times larger than that of the bulk Ag (1.59 × 10−6 Ω cm).Figure Rs of the metal grid TCs was calculated based on the structural size and organic complex weight. The equation used to predict Rs was constructed by modifying the expression for Rs obtained using a square wire network, as suggested by Van de Groep et al.13. The cross-section of the Ag NP structures was regarded as trapezoidal, and Rs was predicted using the following equation:hm is the thickness of the metal grid line, d1 is the bottom width and d2 is the top width of the metal grid line, and s is the spacing between the metal grid lines. The value of km depends on a difference between the predicted shape of the Ag NP structures and their actual shape. The mean values of d1, d2, hm, sm for the metal grid line were 8 μm, 6 μm, 2 μm and 257 μm, respectively. The correction factor, extracted from experimental results, was determined to be km = 3.74. Figure Rs of the metal grid TCs fabricated at different values of TD could be suitably fit to the theoretical values.The r) due to a decrease of the nominal bending strain35. The Norland Optical Adhesive 81 (NOA 81), which shows a high optical transparency over a wide spectral range with a good mechanical flexibility and a strong adhesion force to metal structures, is used as the polymer film. The NOA 81 solution was poured onto the metal grids-patterned glass substrate, and then was covered by the glass substrate. After the UV exposure, the metal grids embedded within NOA 81 film were detached from two glass substrates.The metal grid TCs on the glass substrate should be transferred onto the polymer substrate for use in flexible optoelectronic devices. A transfer process of metal grid TCs provided the flexibility and significantly reduced the surface roughness that can cause optoelectronic device failure. Figure TS. The higher TS induces the more increase of the IFE between the metal and the glass substrate, which causes serious damage to the flexible metal grid TCs during the transfer process. Especially, the metal structures with many pores are prone to be broken due to the increase of the IFE. In Figure TS. In Figure TS over the range 200–300 °C. Although the glass substrate was treated by the fluorine, the IFE between the Ag NP film and the glass substrate depended strongly on TS. The Ag NP films sintered at TS = 300 °C, in particular, displayed 3-fold larger IFE (~8.2 J m−2) than those sintered at TS = 200 °C (~2.7 J m−2). Figure R/Ro) and Rs of the flexible metal grid TCs after the transfer process of the metal grid TCs sintered TS = 200–300 °C. As TS increased, ∆R/Ro of the flexible metal grid TCs based on the DI of Ag ionic ink (TD = 50 °C) significantly increased from 33% to ~104% due to an increase of the IFE. On the other hand, the flexible metal grid TCs obtained using the TCDI of Ag ionic ink (TD = 90 °C) displayed a small value of ∆R/Ro (~6%). Although the IFE increased, the porosity reduction led to preventing a significant increase in ∆R/Ro from serious mechanical damage. The Rs of the flexible metal grid TCs fabricated at TD = 50 and 90 °C showed 5.5 and 52.5 Ω sq−1, respectively.The effect of the porosity reduction on the transfer process of metal grid TCs was explored by measuring the resistance changes of metal grid TCs sintered at different values of T and Rs for the flexible metal grid TCs with the values obtained from a commercially available ITO-coated polyethylene terephthalate (PET) film. Here, it should be noted that the transmittance spectra of the bare NOA 81 film, the flexible metal grid TCs, and the ITO-coated PET film include the transmittance through the substrate. The flexible metal grid TCs performed better (Rs of 5.5 Ω sq−1 and T550nm of 81.47%) than the ITO-coated PET film (Rs of 15.0 Ω sq−1 and T550nm of 78.35%). The T of the ITO-coated PET film fluctuated over the wavelength range of 350–800 nm, whereas the corresponding values of the flexible metal grid TCs remained constant.Figure 36. As the number of pores in the metal grid structures decreased, the mechanical durability of the flexible metal grid TCs can be improved by inhibiting pore-induced crack initiation and propagation under bending stresses33. This resulted in reducing the electrical resistance changes of the metal grid structures under bending stresses. Figure R/Ro obtained from the flexible metal grid TCs fabricated using TCDI and DI of Ag ionic ink under a static bending test. The value of ∆R/Ro of the ITO-coated PET film under a static bending test was evaluated as a reference. As the flexible metal grid TCs were bent up to r = 1 mm, the values of ΔR/Ro increased for these two flexible metal grid TCs (Ro = 6.3 and 21.0 Ω sq−1) by a factor of 2.2 and 113.3 respectively. The R/Ro of the flexible metal grid TCs fabricated using the DI of Ag ionic ink significantly increased at r = 1 mm due to serious damage. Figure R/Ro obtained from the flexible metal grid TCs fabricated using TCDI and DI of Ag ionic ink under a dynamic bending test with r = 7.5 mm. The value of ∆R/Ro of the ITO-coated PET film under a dynamic bending test was evaluated as a reference. After the only several bending cycles, the resistance of the ITO-coated PET film significantly increased to a few tens of kΩ due to formation of numerous and visible cracks. After 1000 cycles of repeated bending/relaxation, ∆R/Ro obtained from the flexible metal grid TCs (Ro = 7.4 and 43.5 Ω sq−1) increased by a factor of 1.5 and 86.4, respectively. When TD increased, the porosity reduction of the flexible metal grids led to decreasing ∆R/Ro under static and dynamic bending stresses . The low-porosity, flexible metal grid TCs fabricated using the TCDI of Ag ionic ink showed an excellent electromechanical durability under static and dynamic bending tests, indicating their great potential for use in various flexible optoelectronic devices.The electromechanical durability of the flexible metal grid TCs under static and dynamic bending stresses is an important factor to be considered in flexible optoelectronic devicesRs of 5.5 Ω sq−1 and T550nm of 81.47%. Poly polystyrene sulfonate (PEDOT:PSS) and MoO3 layers were used on the top of IZO films for efficient hole injection. The 4,4′-Bis(N- carbazolyl)-1,1′-biphenyl (CBP) layer doped with green phosphorescent emitters of bis(2-(2-pyridinyl-N)phenyl-C)(acetylacetonate) iridium (III) (Ir(ppy)2acac, 7 wt%) was used as an emitting layer. The CBP and 2,2′,2″--tris(1-phenyl-1-H-benzimidazole) (TPBi) layers were used as hole- and electron- transport layers, respectively. Figure ηEQE) and power efficiency (ηPE) of the devices. Therefore, the OLEDs based on the flexible metal grid TCs show the comparable values of ηEQE and ηPE with those of ITO-based OLEDs, as shown in Fig. ηEQE of 22.0% and ηPE of 61.8 lm W−1 at 111.6 cd m−2, while ITO-based OLEDs show ηEQE of 21.5% and ηPE of 60.6 lm W−1 at 116.2 cd m−2, respectively.Figure via the TCDI of Ag ionic ink. As TD increased during the thermal decomposition of Ag ionic ink, the porosity of metal grid structures was significantly reduced due to the elimination of large amounts of organic complexes. Also, both ρm and Rs of metal grid TCs were estimated based on the weight percent of organic complex and geometrical calculations. The porosity reduction led to improving the optoelectrical properties of metal grid TCs (Rs < 6 Ω sq−1 at T550nm = 91%) and preventing serious damage (∆R/Ro < 6%) from the strong IFE between the metal and the substrate during the transfer process. In addition, we verified that the porosity reduction resulted in significantly enhancing an electromechanical durability of the flexible metal grid TCs under static and dynamic bending stresses. The OLED based on flexible metal grid TCs as anode electrodes was demonstrated. The uniformity, reliability and scalability of the metal grid TCs should be further improved by the combination of uniform pressing-stable detaching system and the exact control of fluidic properties (viscosity and surface tension) before this technology can be commercialized. We believe that this strategy can provide a useful approach to enhance optoelectrical properties and an electromechanical durability of solution-processed metallic TCs for the development of next-generation flexible optoelectronic devices.In summary, we demonstrated the fabrication of highly conductive, low-porosity, flexible metal grid TCs 34.The Ag ionic ink  consisted of Ag ions , a base solvent (methanol and toluene) and additives. Prior to the TCDI of Ag ionic ink, methanol was evaporated at 100 °C for 10 min to improve the concentrated ink filling. The Ag alkyl carbamate complexes were decomposed to Ag NPs, carbon dioxide, and the corresponding alkyl amines by heating above 50 °C for a few minutesw) of 15 μm and a spacing (s) of 250 μm by calculating the geometrical shadow zone, T = s2/(s + w)2. The value of a cavity height was limited to 7.5 μm to prevent the destruction of Ag NP structures during the detachment of PDMS mold. However, the value of T550nm measured from the metal grid TCs was higher than that obtained using the geometrical calculation of grid-patterned mold due to mold deformation .The PDMS solution  was generated by mixing the silicon elastomer kit and a curing agent (10:1), and this mixture was poured onto a SU-8 master. After PDMS curing at 100 °C for 1 hour, a PDMS mold was carefully released from the SU-8 master. In the PDMS mold, the grid-patterned cavity was designed with a width 37. At 30 kPa, most of the ink was not captured inside the grid-patterned mold due to a large value of hf. The ink was effectively captured inside the mold cavity over 100 kPa. The pressure was optimized at 120 kPa by comparing Rs and T550nm of the metal grid TCs. Also, the relatively uniform P based on horizontal levels led to decreasing both Rs and resistance fluctuations of the metal grids.The effect of All types of glass substrate, including soldalime glass and borosilicate glass, were treated using 1 H, 1 H, 2 H, 2H-perfluorooctyl-trichlorosilane  for 3 min in a vacuum chamber. After the sintering process, a metal grid-patterned glass substrate  was re-treated with the fluorinated silane. The NOA 81 solution was poured onto the metal grid-patterned glass substrate, and it was covered by a borosilicate glass substrate. After UV exposure, the borosilicate glass substrate was detached from the NOA film, and then the metal grid-embedded within NOA 81 film (its thickness of 150 μm) was detached from the sodalime glass substrate.−1, AMG Inc., Korea) as a reference after O2 plasma treatment . The samples with PEDOT:PSS were dried to remove any residual solvent at 100 °C for 10 min on the hot plate. Finally, the PEDOT:PSS-coated samples were loaded into a thermal evaporator for deposition of organic, metal oxide layers, and metal electrodes under high vacuum (3 × 10−6 Torr) conditions. The stacked multilayers of OLEDs were composed of MoO3 (10 nm)/CBP (20 nm)/CBP doped with 7 wt% of Ir(ppy)2acac (20 nm)/TPBi (55 nm)/LiF (1 nm)/Al (100 nm).The IZO film (~50 nm) was deposited on the flexible metal grid TCs using an RF sputtering system (RF power: 120 W). The PEDOT:PSS  layers were spin-coated on the top of the IZO films. PEDOT:PSS was coated also on pre-coated ITO glass substrates  specimens were fabricated with the size of 37.5 mm × 9 mm. The specimens were sandwiched by an additional glass beam using an epoxy . The final structure of the DCB test specimen was the glass substrate/epoxy/Ag/glass substrate. The connection parts with the DCB test machine and aluminum loading tabs were attached to the specimens with the selective epoxy  and the specimens were cured at 120 °C for 1 h in a convection oven.Rs of the metal grid TCs was measured using the two-terminal method and four-point probe method 38. Two electrodes between the metal grids, separated by a square area (25 mm2), were fabricated using conductive pens .The images of the metal grid TCs were measured using field-emission SEM . Cross-sectional images of the metal grid TCs were measured using FIB-SEM , and the surface roughness of the flexible metal grid TCs was measured using AFM . The transmittance spectra were measured using a UV-VIS-NIR spectrophotometer . The Supplementary Information


The purpose of this study was to investigate hearing threshold changes of workers with unilateral conductive hearing loss who were exposed to workplace noise for 8-years.t-test was conducted to compare the hearing threshold changes over time between conductive hearing loss (CHL) ear and sensorineural hearing loss (SNHL) ear.Among 1819 workers at a shipyard in Ulsan, 78 subjects with an air-bone gap ≥10 dBHL in unilateral ears were selected. Factors that could affect hearing were acquired from questionnaires, physical examinations, and biochemistry examinations. Paired p < 0.05). The threshold change at 4 kHz was 3.2 dBHL higher in SNHL ears which was statistically significant (p < 0.05). When workers were exposed to noise levels of 85 dBA and above, threshold change at 4 kHz was 5.6 dBHL higher in SNHL ears which was statistically significant (p < 0.05). Among workers aged below 50, the threshold change values were lower in low-frequency (0.5–2 kHz) in SNHL ears, with a small range of changes, whereas in high-frequency (3–6 kHz), the range of changes was greater SNHL ears (p < 0.05). Among workers aged 50 and above, SNHL ears showed a wider range of changes in both high- and low-frequency areas (p < 0.05).The study included male subjects aged 48.7 ± 2.9, having worked for 29.8 ± 2.7 years. Hearing thresholds increased significantly in CHL ears and SNHL ears at all frequencies (0.5–6 kHz) during follow-up period (At high-frequencies, particularly at 4 kHz, the range of hearing threshold changes was lower in ears with conductive hearing loss than in contralateral ears. This is suggested as a protective effect against noise exposure. Hearing loss is one of the most frequent chronic sensorineural injuries. In 2013, The National Institute on Deafness and Other Communication Disorders reported the following statistics about hearing loss in adults: adults aged 45–54 showed a hearing loss level of 2 %, those aged 55–64 showed 8.5 %, 65–74 of 25 %, and 75 and above showed almost 50 % hearing loss [According to “Workers’ periodic health examination”, a report produced by the Ministry of Employment and Labor in Korea in 2013, 25,891 workplaces had noisy working environment, and accordingly, 564,663 workers underwent hearing examination [An expected consequence of conductive hearing loss in these workers is a decrease in the degree of noise-related damage in the inner ears as a result of reduced sound energy transfer in the air conduction process. However, a few studies have investigated this. While a study on the protective effects of conductive hearing loss against excessive noise has been reported , the stuIn this study, we conducted an 8-year follow-up investigation on workers having unilateral conductive hearing loss (CHL), and compared the results with hearing changes in the contralateral ears. Until now, studies that observe long-term follow-up of conductive hearing loss have rarely been reported. Moreover, this study is the first of its kind to be conducted in South Korea. In addition to noise, this study analyzes the relation between hearing loss and other factors previously reported to affect hearing, including age , 7, smokA total of 13,139 male workers aged 20–62 at a shipyard in Ulsan underwent special health examination for noise exposure, in 2006. Of these, 1819 workers completed audiometry examinations including bone conduction examination and tympanometry. These 1819 workers were selected as our initial subjects. There is controversy in definition of conductive hearing loss. Chung had defined as 15 dB or more air-bone gap for at least one frequency [During the health examination procedures, subjects were asked to complete self-reported questionnaires. All information concerning age, working period, working department, history of ear disease, and trauma history of head was collected from these questionnaires. All damaged eardrum diagnoses were obtained from the otoscopy and tympanometry results. In order to investigate additional factors that may affect hearing, data regarding body mass index (BMI), blood pressure, fasting blood sugar (FBS), and total cholesterol were collected.Levels of noise in the work environment were measured in accordance with the Ministry of Employment and Labor Notification No. 2009-78 . Regarding noise-exposure levels, the subjects’ mean noise levels were calculated for each working department and process by using the regional and individual measurement results obtained in the first year of the research (2006). A noise dose badge , audio dosimeter , two noise logging dosimeter  were used as the noise-level measuring devices, and the devices were adjusted with a sound calibrator before and after the use.Audiometry was conducted by experts trained through the quality assurance program for audiometry by the Korea Occupational Safety and Health Agency (KOSHA). The Hughson-Westlake procedure was used to detect air conduction thresholds at 0.5, 1, 2, 3, 4, and 6 kHz and to detect bone conduction thresholds at 0.5, 1, 2, 3, and 4 kHz. An AC40 diagnostic audiometer  with TDH-39P headphones attached was used for this procedure. An AT235 impedance audiometer  was used for tympanometry, and SDW-2000  was used for soundproof booths. Audiometry and tympanometry were adjusted annually in accordance with the KOSHA guidelines, and the soundproof booths used were in accordance with American National Standards Institute (ANSI) S3.1-1999 .t-test was conducted 1) to compare the differences between the air conduction threshold changes for the sensorineural hearing loss ears (ΔS) and those for the conductive hearing loss ears (ΔC), 2) to compare the hearing threshold changes between mean of low-frequency  and that of high-frequency .Air conduction threshold changes were investigated for the 8-year follow-up period (2006–2013) at each frequency for all SNHL and CHL ears. A paired t-test was used to compare mean of difference (2013-2006) between hearing threshold of SNHL ear and that of CHL ear, on air and bone conduction, according to average work environment noise level and age. SPSS 21.0  was used for data analysis. When the p-value was lower than 0.05, we considered it statistically significant.All subjects were subsequently divided into two groups based on average work environment noise level (85 dBA) and age (50 and above). A paired 2 and systolic blood pressure was 125.9 ± 12.5 mmHg. FBS and total cholesterol levels were 102.8 ± 17.7 mg/dL and 189.3 ± 31.2 mg/dL, respectively. Five subjects showed an FBS level above 126 mg/dL and seven subjects showed a total cholesterol level higher than 230 mg/dL. Among the 78 subjects, there were 14 smokers and 64 non-smokers. The mean noise level of their work environment was 83.5 ± 5.0 dBA, and 25 subjects were exposed to a noise level of 85 dBA or higher (Table The mean age of subjects at the start of the study was 48.7 ± 2.9 years (though 90 % subjects were aged 45 and above) and the mean length of their working period was 29.8 ± 2.7 years. BMI was 23.3 ± 1.8 kg/mThe mean thresholds of air and bone conduction of SNHL and CHL ears increased over time at all frequencies during the study period. The reported values of the air-bone gap in the CHL ears at the initial stage of the study remained at a similar level during the follow-up period Fig. .Fig. 2HeIn low-frequency areas, the observed hearing threshold changes were higher 8.7–11.2 dBHL) in CHL ears (compared to 7.9–10.3 dBHL in SNHL ears). In contrast, in high-frequency areas, hearing threshold changes were higher (4.7–9.4 dBHL) in SNHL ears (compared to 4.0–7.8 dBHL in CHL ears). On comparing hearing threshold changes between SNHL and CHL ears at each frequency, significantly 3.2 dBHL higher hearing threshold changes were detected at 4 kHz in SNHL ears than in CHL ears. However, significant differences were not detected at other frequencies (Table  dBHL in p < 0.05). In subjects experiencing noise levels of 85 dBA and above, the mean hearing threshold ranges increased by 6.9 dBHL in SNHL ears and by 7.7 dBHL in CHL ears in low-frequency areas, and increased by 5.8 dBHL in SNHL ears and by 3.2 dBHL in CHL ears in high-frequency areas (p < 0.05). Thus, hearing changes were more pronounced in low-frequency areas than in high-frequency areas , and mean hearing threshold changes were investigated for each of the SNHL and CHL ears at each frequency. The range of changes was observed to be higher in CHL ears than in SNHL ears in low-frequency areas. Conversely, in high-frequency areas, the range was found to be higher in SNHL ears than in CHL ears. In subjects experiencing noise levels below 85 dBA, the mean hearing threshold ranges increased by 10.5 dBHL in SNHL ears and by 11.3 dBHL in CHL ears in the low-frequency areas. These ranges increased by 8.5 dBHL in SNHL ears and by 7 dBHL in CHL ears in the high-frequency areas , whereas the ranges increased by 8.3 dBHL in SNHL ears and by 6.8 dBHL in CHL ears in high-frequency areas (p < 0.05). In subjects aged 50 and above, the mean hearing threshold ranges increased by 10.5 dBHL in SNHL ears and by 9.0 dBHL in CHL ears in low-frequency areas (p < 0.05), whereas the ranges increased by 6.9 dBHL in SNHL ears and by 4.6 dBHL in CHL ears in high-frequency areas (p < 0.05). Hearing threshold changes were observed to be higher in low-frequency areas than in high-frequency areas in both age groups. However, in subjects aged 50 and above, the range was higher in SNHL ears than in CHL ears in both low- and high-frequency areas. In contrast, in subjects aged below 50, the range of changes was observed to be higher in CHL ears than in SNHL ears in low-frequency areas, and it was higher in SNHL ears than in CHL ears in high-frequency areas . In subjects aged below 50, the mean hearing threshold ranges increased by 8.4 dBHL in SNHL ears and by 11.1 dBHL in CHL ears in low-frequency areas  in SNHL ears than in CHL ears, and the threshold of bone conduction was observed to be significantly lower by 4.0 dBHL) in SNHL ears than in CHL ears. In subjects aged 50 and above, the threshold changes of air conduction in SNHL ears at 4 kHz was observed to be significantly higher (by 4.1 dBHL) than that in CHL ears . Workplaces where the noise level at the work environment exceeds the permissible level (or where NIHL has previously occurred) are placed under constant supervision, and the employers are advised to implement a hearing-conservation program. However, because of the unwelcome economic burden of noise reduction placed on employers and the lukewarm responses towards these measures shown by workers themselves, NIHL comprises the majority of cases of occupational diseases reported since the 1990s, and this trend is expected to continue.In this study, the hearing ability of workers with unilateral conductive hearing loss was analyzed, and the results compared with those of contralateral ears to investigate how existing conductive hearing loss affects the progress of NIHL. Hearing has been reported to deteriorate with age . During Regarding hearing threshold changes of air and bone conduction at each frequency, the air-bone gaps were not observed in SNHL ears for most frequencies during the follow-up period; however, in CHL ears, the air-bone gaps observed in the early period of study, and persisted during the follow-up period, without much change. This indicates the air-bone gap induced by conductive hearing loss consistently affects the subjects’ hearing threshold level without any improvement over time. In calculating the changes in SNHL and CHL ears at each frequency and comparing the differences, the results showed that the changes in SNHL ears were significantly greater than the changes in CHL ears (by 3.2 dBHL at 4 kHz). Thus, when conductive hearing loss is a precondition, hearing deterioration induced by noise exposure is comparatively small.In the two noise-level groups (below 85 dBA vs. 85 dBA and above), hearing ability was observed to gradually decrease over time in both CHL and SNHL ears. However, when considering the differences in threshold changes between SNHL and CHL ears at each frequency, significant differences were observed in threshold changes of bone conduction at 0.5 kHz, at noise levels below 85 dBA. At noise levels of 85 dBA and above, as shown in the previous result, threshold changes of air conduction were significantly greater at 4 kHz in SNHL ears than in CHL ears. These results indicate that, at 4 kHz, the threshold changes of air conduction were smaller in CHL ears than in SNHL ears. In other words, conductive hearing loss is associated with a decrease in noise-induced injuries. Our interpretation of this result is that, in agreement with Chung’s study , CHL earWhen looking at hearing threshold changes in subjects categorized in accordance with age (below 50 vs. 50 and above), hearing deteriorated in both ears in the two groups over time. When comparing difference of the changes in SNHL and CHL ears at each frequency, the air and bone conduction threshold changes at 1 kHz were significantly greater in CHL ears than in SNHL ears in subjects aged below 50. This result indicates that in relatively younger subjects, hearing deterioration induced by conductive hearing loss continues in low-frequency over time. On the other hand, in the subjects aged 50 and above, significant differences were not detected at 1 kHz, and threshold changes of air conduction in SNHL ears at 4 kHz were significantly greater than that in CHL ears. This result suggested that in relatively older subjects, hearing deterioration induced by noise exposure can be accelerated in high-frequency over time. In other words, hearing in the ears without conductive hearing loss gets affected by noise exposure to a greater extent in the younger age group and to a smaller extent in the higher age group. The reasons for this can be found in aging-induced overall hearing damage. In particular, in cases involving relatively older subjects, high-frequency hearing loss were observed in the early stage of the study, thus, this ceiling effect might have caused the relatively lower changes observed during the follow-up period. Gates et al. investigated the correlation between age and noise-induced injury with 203 adults included in the Framingham Heart Study cohort (aged 58–80) as the subjects. The subjects were categorized into three groups according to the audiogram results obtained in the early stage of the study. The threshold increasing patterns in high-frequency  were then evaluated, and the changes in the patterns were confirmed after the follow-up period . The resThe C5-dip is observed in NIHL demonstrates a remarkable increase of the hearing threshold in the audiogram at 4 kHz compared to other frequencies. This study shows that hearing threshold changes were observed to be significant greater in SNHL ears than in CHL ears at around 4 kHz, and in accordance with noise levels, these differences continued only when exposed to noise levels of 85 dBA and above. This indicates that hearing threshold changes are greater in SNHL ears than in CHL ears at 4 kHz, that is, CHL ears are affected lesser by noise exposure than SNHL ears are. Thus, it can be concluded that a protective effect against noise exists in CHL ears in comparison with in SNHL ears.In cases involving middle ear diseases, such as chronic otitis media, a decrease in eardrum elasticity or an eardrum perforation, accompanied in part by loss of the ossicles, there is a decrease in sound energy transfer through the eardrum and ossicles to the inner ear. The cochlea resonates at frequency-specific locations, that is high-frequency sounds with short wavelengths are received at the base and low-frequency sounds with long wavelengths are received near to the apex. The precise mechanism of occurrence of NIHL is unknown. Pathologic lesions have been observed in the outer and inner hair cells of the inner ear in previous studies on humans and animals, and our understanding of the occurrence of noise-induced deformation of inner ear organs is based on these findings –22. NoisThe effects of reported influencing variables on hearing threshold changes were analyzed by multiple linear regression tests, although it is not shown in the table. Except for CHL ears at 2 kHz, a significant positive association with FBS was detected in low-frequency areas. Systolic blood pressure showed significant negative association in SNHL ears at 1 kHz. In terms of the noise level, a significant negative association was detected in SNHL ears at 0.5 and 1 kHz, also at 3 and 4 kHz in CHL ears.The strengths of this study are as follows: This study conducted an 8-year follow-up for the first time in South Korea on noise exposure effects with workers who have unilateral conductive hearing loss as the subjects in comparison with hearing changes in contralateral ears. Previous studies that have investigated a long-term follow-up in this topic are rare overseas as well. Furthermore, hearing thresholds of individuals on both sides were compared and analyzed, minimizing the effects induced by individual characteristics. Several previous studies have compared hearing thresholds of individuals on both sides on identical individuals as subjects, but the measured frequency ranges have been limited. Moreover, they have been mostly cross-sectional studies, thereby limited in confirming causal relationships , 15.The limitations of this study are as follows: First, the subjects were selected using initial audiometry results because the confirmation of the onset of conductive hearing loss is impossible. Second, most of the subjects (over 90 %) were aged 45 and above, with over 46 % at 50 and above, and thus, age-induced hearing loss could not be excluded. Third, the subjects’ working periods were mostly over 25 years, and thus in most cases susceptibility to noise is assumed to have decreased. Moreover, there was no noticeable tendency for the susceptibility to change during the study period. The protective effect of conductive hearing loss under noise exposure was observed at some frequencies even though the evidence was not conclusive. In the future, additional follow-up studies are required to examine the early effects of noise exposure on relatively young male and female workers with working periods of less than 20 years, or to observe changes in workers’ hearing level to noise exposure immediately after their otologic surgery.During the 8-year follow-up period, this study investigated 78 workers with unilateral conductive hearing loss who have been exposed to workplace noise. Hearing threshold changes in the ears with conductive hearing loss was significantly smaller than that in the contralateral ears at 4 kHz, though statistically significant differences were not found in other frequency area. From the results of this study, we suggest that protective effects are against noise in workers with CHL. Further studies are needed to investigate early effects of noise exposure on relatively young male and female workers with working periods of less than 5 years to complement this study.