CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene ( CDKL5 , cause a range of phenotypes, including EIEE2 (MIM: 300672), a form of early infantile epileptic encephalopathy [CDKL5 mutations have also been described in patients diagnosed with West syndrome, Lennox-Gastaut syndrome and atypical forms of Rett syndrome (RTT) [CDKL5-related phenotypes result from loss of function mutations, but putative gain-of-function mutations [CDKL5 gene region, have also been identified [Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene, alopathy , 2, and MIM: 30062, a formme (RTT) , 5. In ame (RTT) –11. Studme (RTT) . It is wutations , as wellentified –15.CDKL5 is located on the X-chromosome (Xp22.13) and is subject to X-chromosome inactivation. All reported mutations thus far have dominant effects on phenotype and affected patients are almost always heterozygous females. Hemizygous males are known, however, and are invariably severely affected [affected . The cliaffected . IncidenCdkl5 knockout (KO) mouse models have been reported, with phenotypes including impaired motor control, altered behaviour, abnormal eye tracking, general hypoactivity and morphological features such as reduced dendritic arborisation of cortical neurons [Although the CDKL5 disorders are not congenital, the early postnatal onset of symptoms indicates that CDKL5 plays a crucial role in brain development. CDKL5 is also expressed within the mature adult nervous system , where i neurons , 23. HowCdkl5 transcripts are expressed widely throughout the body [The multiple cellular locations and potential functions of CDKL5 may, in part, be explained by its molecular heterogeneity. CDKL5 is known to exist as a number of isoforms resulting from alternative splicing, and several transcripts have been identified; however, nothing is known about their functional differences or developmental expression. the body , 25. Allthe body  located the body , 28. Oththe body . Loss-ofthe body , 29.Syngap gene, mutations in which also lead to EIEE [CDKL5 isoforms will provide information that is essential for the development of therapeutic avenues such as gene and protein replacement. A number of studies have investigated CDKL5 gene structure and transcript expression in different tissues [CDKL5 and mouse Cdkl5 using a combination of bioinformatic analyses and molecular methods, and to predict the protein isoforms translated from each transcript. The study has generated a new model of CDKL5 gene structure in both species and defines a range of previously uncharacterised transcript isoforms with unique developmental expression profiles. To accommodate these complexities in gene expression, we propose a new nomenclature system for CDKL5 gene products that encompasses both the range of transcript isoforms and the predicted suite of protein isoforms.Knowledge of gene transcript isoforms and their developmental expression profiles is essential for understanding their mechanistic roles in brain development. For example, different isoforms of the  to EIEE , have di to EIEE . Further tissues , 32, 33 The following RNA-seq datasets were analysed in this study: human brain GEO sample ID GSM1173806 ; human tSTAR --runMode genomeGenerate --genomeDir <species-index-dir> --genomeFastaFiles <fasta-files> --sjdbGTFfile <gtf-file><species-index-dir> is the directory into which the index files were written, <fasta-files> were FASTA-formatted files containing sequences from the primary assembly of the respective species' genome (Ensembl v80), and <gtf-file> was a GTF-formatted genome annotation file for the respective species . To map a particular set of RNA-seq reads to a species' genome, the following command line was run:where STAR --genomeDir <species-index-dir> --readFilesIn <read-files-1> <read-files-2> --outSAMstrandField intronMotif --twopassMode Basic --outSAMtype BAM SortedByCoordinate<species-index-dir> was the directory into which the species' index files were written, and <read-files-1> and <read-files-2> enumerated the FASTQ-formatted files containing paired-end RNA-seq reads for the dataset. STAR was run in two-pass mapping mode to give the most sensitive detection of reads mapping across novel splice junctions. RNA-seq output images were generated using the Integrative Genomics Viewer (IGV) and Sashimi plots [where mi plots –41.Human total RNA isolated from a range of adult tissues, including whole brain  and foetal brain  was obtained from Clontech , a widely-used commercial panel sourced with ethical approval based on consent; further approval from the local University of Glasgow ethics committee was not required. Mouse total RNA was isolated from tissues obtained from wild-type C57BL/6 mice using the RNeasy Lipid Tissue Kit (Qiagen), according to the manufacturer’s instructions. Animal samples were collected in accordance with the European Communities Council Directive (86/609/EEC) and with the terms of a project license under the UK Scientific Procedures Act (1986). The quality and quantity of isolated RNA was analysed using the RNA 6000 kit on a 2100 Bioanalyzer (Agilent), and using a Nanodrop 1000 spectrophotometer (Thermo Scientific).Total RNA was reverse transcribed using Superscript III (Life Technologies), according to the manufacturer’s protocol, in 20 μl reactions containing 200 ng of RNA template and 1 μM random hexamers. Reactions were then incubated with 2 units of RNaseH (Life Technologies) at 37°C for 20 min. End-point PCR was performed using Maxima Hot Start Green (Thermo Scientific), according to the manufacturer’s protocol, in 50 μl reactions containing 500 nM gene-specific primers, and products visualised on agarose gels. PCR was performed under the following cycling conditions: an initial denaturation at 95°C for 2 min, then 35 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 90 s, followed by a final extension of 72°C for 7 min. A list of primers used in the study is provided in th of the first-strand synthesis reaction and 300 nM gene-specific primers. PCR was performed under the following cycling conditions on an Mx3005P thermocycler (Agilent Technologies): an initial denaturation at 95°C for 30 s, then 40 cycles of 95°C for 10 s and 60°C for 60 s, followed by a dissociation curve. Appropriate controls were included as recommended by the MIQE guidelines [First-strand synthesis was performed using the qScript Flex kit (Quanta Bioscience), according to the manufacturer’s protocol, in 20 μl reactions containing 200 ng of total RNA template and 500 nM gene-specific primer or random hexamers. SYBR Green PCR reactions were carried out using the PerfeCTa SYBR kit (Quanta Bioscience), according to the manufacturer’s protocol, in 20 μl reactions using 1/20idelines . A list 3’-RACE was performed using the 3’-RACE System for Rapid Amplification of cDNA ends (Life Technologies), according to the manufacturer’s protocol, in 20 μl reactions containing 200 ng of total RNA template. 5’-RACE was performed using the 5’-RACE System for Rapid Amplification of cDNA ends, Version 2.0 (Life Technologies), according to the manufacturer’s protocol, in 25 μl reactions containing 500 ng of total RNA template and 2.5 pmol of gene-specific primer. Primary and nested PCR reactions were performed using Platinum Taq polymerase (Life Technologies), according to the manufacturer’s protocol, in 50 μl reactions containing 500 nM gene-specific primer. A list of primers used in the study is provided in CDKL5 transcripts, including the possibility of novel exons and splicing events, human tissue-specific RNA-seq datasets were analysed to identify all, including very rare, CDKL5 splicing events. Using sensitive alignment tools to detect reads mapping across all potential splice junctions we detected 27 discrete exons. In addition to confirming the locations of previously identified exon boundaries, we identified novel exons and cryptic splice sites  and was detected in all adult tissues tested, appearing especially abundant in CNS, kidney, testis, prostate gland, thymus and thyroid gland  constitute approximately 10% of CDKL5 transcripts expressed in whole brain, while those using the alternative splice site in exon 11 (hCDKL5_3 and hCDKL5_4) constitute less than 5% of transcripts in brain (hCDKL5_5 (previously known as CDKL5115) was only detected in testis amongst the adult tissues tested. These differences in relative abundance are consistent with a previous report [hCDKL5_5 differs from the other transcripts at the 3’ end by splicing from a cryptic splice site in the newly-named exon 19 to exons 20, 21 and 22  predicts that all of these different 5’ ends would utilise the same ATG start codon in exon 2, and therefore the different TSSs reflect differences in 5’-untranslated region (UTR) composition only (hCDKL5_1 –hCDKL5_5) but was never used in combination with exons 1, 1a or 1b. Alternative splicing and read-through events (such as from exon 1c to 1d in some isoforms) result in a complex set of CDKL5 5’-UTRs, described in Analysis of the 5’ region of the gene from RNA-seq and 5’-Rapid Amplification of cDNA Ends (5’-RACE) data revealed multiple transcription start sites (TSSs) . This inion only . Exons 1CDKL5 transcript 3’-UTRs have never been characterised in detail before. Previous northern blot data [CDKL5 transcripts may be extremely large, potentially bigger than 10 kb. Here, we analysed 3’-UTRs using RNA-seq datasets and 3’-RACE to search for potential polyadenylation sites within the predicted UTR regions encoded by exons 19 and 22. We identified two separate canonical polyadenylation signals (AATAAA) in exon 19 used in the hCDKL5_1, _2, _3 and _4 transcripts . In the case of the hCDKL5_5 transcript, the composition of the C-terminal region of the protein and the sequence of the 3’-UTR is entirely different to that of the other isoforms. 3’-RACE identified a non-canonical polyadenylation signal (AATTAA) used by this isoform downstream of the stop codon in exon 22  revealed that the cryptic splice site consensus sequence is present in all mammals with Cdkl5 sequences in Genbank except mouse and a few other rodent species (not shown).New RT-PCR assays capable of differentiating between  tissues . mCdkl5_us study . We alsodatasets . No mousfference . BLAST amCdkl5_6, _7 and _8). Bioinformatic analysis of RNA-seq datasets indicated that three exons lie downstream of exon 19 , indicating usage of exon 1 by transcript isoforms mCdkl5_1 and _2  downstream of the stop codon in exon 22 , within the proposed system. The gene symbol is followed by an underscore and a number, which will differentiate transcripts with different coding sequences. The system is based on orthology, so the same number in different species indicates an orthologous coding sequence; for example, hCDKL5_1 comprises exonic sequences that seem to be entirely orthologous to those found in mCdkl5_1. The proteins made from each transcript will be named according to the same convention i.e. transcript hCDKL5_1 encodes protein isoform hCDKL5_1.Based on the work described above, a new nomenclature system for human sed here . For eacCDKL5 isoforms expressed in the brain, most transcripts splice from exon 1 directly to exon 2 . RT-PCR and qRT-PCR analysis showed that mCdkl5_1 and mCdkl5_2 are expressed throughout embryonic and early postnatal development, increasing in levels and peaking within the first few weeks after birth, with mCdkl5_2 being expressed at lower levels than mCdkl5_1  is the predominant brain isoform of CDKL5. hCDKL5_1 is a 9.7 kb transcript comprised of 18 exons and a large 6.6 kb 3’-UTR. However, we now demonstrate considerably greater diversity in human and mouse CDLK5 transcripts than previously realised. This diversity arises from alternative promoter and first exon usage and from alternative splicing, and these patterns are in many cases species-specific.CDKL5 disorder is a rare, debilitating form of early infantile epileptic encephalopathy and severe intellectual disability caused by a range of of CDKL5 , 32, 33 CDKL5 coding region is orthologous and well-conserved between human and mouse  is the most abundant transcript isoform in the CNS for both mouse and human [CDKL5/Cdkl5 transcripts were expressed in human and mouse brains, and that a different, smaller transcript is expressed in human and mouse testes [CDKL5 UTRs confirms that, in adults, large transcripts of ~9.7 and ~12.8 kb in human, and ~9.7 kb in mouse, are predominantly expressed in the CNS; and that transcripts of ~3.4 kb in human and ~3.2 kb in mouse are almost exclusively expressed in the testis.Our results confirm that nd human . This ise testes . HoweverhCDKL5115+ex.16b and hCDKL5-ex.19m predicted by previous studies [hCDKL5_2 and hCDKL5_4 and its inclusion adds 41 amino acids to the protein. This exon is extremely well conserved between human and mouse, but we can find no evidence of it containing known functional elements. Use of the cryptic splice site in exon 11 (hCDKL5_3 and hCDKL5_4) results in the loss of 17 amino acids, a region that contains a putative nuclear localisation signal [CDKL5 transcripts expressed in whole brain  in the major brain isoforms may suggest additional, previously unrecognised, modes of regulation of ynthesis . It is ps region . HoweverCDKL5/Cdkl5 expression reveals a dynamic regulation of individual transcript levels during neurodevelopment. Indeed we find that transcripts mCdkl5_8 and hCDKL5_5, previously believed to be testis-specific, are expressed in the brain during development. This finding is of particular significance since mutations in exons 20–22 have hitherto been classified as being non-pathogenic [mCdkl5_1 increases from E17-P24, whereas mCdkl5_8 shows a gradual decrease over the same period. The detection of different transcript isoforms is suggestive of complex developmental regulation of CDKL5 expression patterns and levels during pre- and postnatal development. However, the functional significance of different CDKL5 transcript isoforms and whether there are levels of redundancy across CDKL5 transcripts remains to be assessed.Our analysis of thogenic . More gethogenic . Our resCDKL5 and mouse Cdkl5 transcript isoforms a new standardised nomenclature system is proposed. In addition to the former names described in CDKL5 terminology. The new proposed nomenclature system retains flexibility and takes into account the complexity of transcript isoforms characterised in this study, providing a reference for future work on this gene.To accompany the analysis of human CDKL5 transcripts highlighted in this study needs to be taken into account when developing gene-based, protein-based or pharmacotherapies for CDKL5 disorders. The data may also aid the development of isoform-specific antibodies. The complexity and dynamic regulation of CDKL5 may also be important when considering the time-course of CDKL5 disorder pathophysiology and potential time-points for therapeutic intervention. The detailed characterisation of CDKL5/Cdkl5 should not only inform the design of CDKL5 mutation screening assays but, importantly, also provide a valuable platform for fundamental research into the biology of CDKL5.Overall, the complexity of S1 FighCDKL5_1 will also amplify hCDKL5_5, if hCDKL5_5 is expressed in that tissue. As hCDKL5_5 is expressed primarily in the adult testis, the assay is therefore quite specific for hCDKL5_1. An alternative primer pair that amplifies hCDKL5_1 + _3 simultaneously was tested in the panel of adult tissues and a similar pattern of expression observed.PCR Primer pairs specific for each isoform in (A) human and (B) mouse are listed, and the diagrams indicate the coverage of the resultant amplicons. (C) The primer pair designed to amplify (TIF)Click here for additional data file.S2 FigCDKL5 transcript isoforms in a panel of adult tissues. GAPDH was used as a loading control.RT-PCR analysis of (TIF)Click here for additional data file.S3 FigCDKL5 and Cdkl5 transcripts were gel-purified and sequenced (the 285 bp band in the left gel image). A low abundance product confirming the use of an alternative splice site in exon 11 in CDKL5 transcripts was gel-purified and sequenced (the 146 bp band in the right gel image).  Sequence of PCR amplicons in (C). Primers are indicated by red arrows. The 3’ end of exon 11 in human and mouse, showing the presence of a cryptic splice site in human and its absence in mouse due to a single nucleotide difference (highlighted in red).RNA-seq data from brain (red) and testis (blue) datasets show reads mapping to (A) exons 16, 17 and 18 in human and mouse and (B) exons 10, 11 and 12 in human. In both diagrams the number on the y-axis indicates maximum read count and the dotted lines between splice donor and acceptor sites indicate the number of reads that map to that exon-exon junction. All reads contributing to these data span a maximum of two exons. (C) RT-PCR of total RNA isolated from brain tissue in human and mouse. Products confirming the presence of exon 17 in (TIF)Click here for additional data file.


The acoustic signal is crucial for animals to obtain information from the surrounding environment. Like other sensory modalities, the central auditory system undergoes adaptive changes  during the developmental stage as well as other stages of life. Owing to its plasticity, auditory centers may be susceptible to various factors, such as medical intervention, variation in ambient acoustic signals and lesion of the peripheral hearing organ. There are critical periods during which auditory centers are vulnerable to abnormal experiences. Particularly in the early postnatal development period, aural inputs are essential for functional maturity of auditory centers. An aural deprivation model, which can be achieved by attenuating or blocking the peripheral acoustic afferent input to the auditory center, is ideal for investigating plastic changes of auditory centers. Generally, auditory plasticity includes structural and functional changes, some of which can be irreversible. Aural deprivation can distort tonotopic maps, disrupt the binaural integration, reorganize the neural network and change the synaptic transmission in the primary auditory cortex or at lower levels of the auditory system. The regulation of specific gene expression and the modified signal pathway may be the deep molecular mechanism of these plastic changes. By studying this model, researchers may explore the pathogenesis of hearing loss and reveal plastic changes of the auditory cortex, facilitating the therapeutic advancement in patients with severe hearing loss. After summarizing developmental features of auditory centers in auditory deprived animals and discussing changes of central auditory remodeling in hearing loss patients, we aim at stressing the significant of an early and well-designed auditory training program for the hearing rehabilitation. The sense of hearing is a major pathway by which humans use to recognize environment. Acoustic cues assist humans to engage in neural activities integrated from multiple brain stations, which make constant adjustment of their behavior, such as learning, communication and exercise. Hearing impairment directly affects the recognition of language and social competency among humans. Hearing relies on the signal transmission from the auditory organ to the central auditory system. The central auditory system consists of cochlear nucleus, superior olive nucleus, lateral lemniscus, inferior colliculus, medial geniculation body and auditory cortex. Earlier studies suggested connections between different stations of the auditory system which are structurally hard-wired. This view was however challenged by recent research upon discovering the plasticity of sensory systems  9 in rats, and detection of the auditory brainstem response (ABR) on P12 demonstrated the auditory function. The hearing threshold detected on P22 was close to that in adulthood  showed a reduced volume of the cochlear nucleus after aural deprivation. Later studies revealed that the degree of apoptosis after aural deprivation was associated with the developmental stage of the affected mice. A comparison of aural deprivation in P5–7 and P21 mice showed that the P5–7 mice displayed earlier neuronal apoptosis in the cochlear nucleus than the P21 mice , which could regulate neural excitability and improve the time analytical precision of the signal input from both ears. Through whole-cell patch-clamp analysis, the results demonstrated that cochlear resection prior to the development of hearing increased the Ih in the LSO and reduced the Ih in the medial nucleus of the trapezoid body (MNTB), resulting in the formation of a depolarized resting membrane potential in the LSO and decreasing the neuronal input resistance.Many studies have reported that aural deprivation can delay inhibitory synaptic neuronal development in the lateral superior olivary nucleus (LSO) in rats, thereby blocking inhibitory synapse formation and the associated maturation of the axonal and dendritic morphology  were used to promote GABAergic neurotransmission and to restore the reduced strength of inhibitory postsynaptic currents caused by auditory damage. However, the GABAB receptor agonist could not achieve this goal, suggesting the activation of GABAA receptors may be necessary for promoting the maturation of central inhibitory transmission  as well as the flip subunit of the AMPA receptor was up-regulated  glutamate receptors following aural deprivation induced by amikacin treatment during development, and the result demonstrated that decreased mRNA expression of the NR1 (NMDA receptor 1), NR2a and NR2b subunits of NMDA receptors as well as the flop subunit of the AMAP receptor in the bilateral cochlear nucleus. However, the expression of GABARegarding neurotransmitters, earlier researchers believed that cochlear damage would inevitably cause a series of complex changes, including the release, binding, and reuptake of glutamatergic, glycinergic, and GABA energy pathway neurotransmitters  as a tracer for monitoring during a 48-h period following unilateral conductive hearing loss and observed a significant down-regulation of protein synthesis in bilateral medial superior olive nuclei. However, the protein synthesis of bilateral medial superior olive nuclei and the ipsilateral trapezoid body was increased 6 h after the operation. Wang and Rubel  and c-fos, among the immediate early genes, and activity-regulated cytoskeleton associated protein (Arc), synaptogyrin 1 gene (Syngr 1), and brain-derived neurotropic factor (Bdnf), among neural plasticity genes, in the aural deprivation group was first reduced and then increased from the 2nd to the 4th week after the operation. The expression of the gamma-aminobutyric acid A receptor, alpha 5 (Gabra5), cholinergic receptor nicotinic beta polypeptide 3 (Chrnb3), and cholinergic receptor nicotinic epsilon (Chrne) genes, which are associated with neurotransmission, was reduced in the 12th week after the operation , but not in primary visual cortex (V1); and the cross modal TC-plasticity was effectively recruited in V1 when aural deprivation in adult mice. According to the result, researchers suggested that multimodal training paradigms may benefit individuals with auditory processing disorders , functional magnetic resonance imaging (fMRI), and evoked potentials have been widely used to allow imaging, becoming important techniques for studying development and determining plasticity changes in the auditory center. Through the detection of central metabolism and electrical activities, these methods allow non-invasive imaging analyses of patients’ brains. A previous study demonstrated that the glucose metabolism of the local cerebral cortex of humans less than 16 years of age first increases and then declines as a positive waveform. Glucose metabolism reaches its peak at 6 years old. This change is consistent with the timing of cortical synaptic maturation during development Chugani, . Hsu et In clinical practice, researchers have demonstrated that the duration of aural deprivation affects functional recovery after interventions in hearing-impaired individuals. Patients with binaural hearing impairment who only wore one hearing aid showed a worse outcome of unaided ear than those wearing no hearing aids in both ears  to predict the prognosis of hearing-impaired children before the cochlear implantation. The results demonstrated that children in whom cochlear implantation was delayed and initially relied on sign language communication displayed overexpression of SEP in the region of the left temporal cortex, suggesting the presence of cross-modal reorganization (Charroó-Ruíz et al., Similar to other sensory modalities, the peripheral hearing input is essential for the development and functional maturation of the auditory center. Long-term blockade of peripheral signal or an asymmetric input throughout life may induce structural and functional reorganization of the auditory center. These effects may be due to changes in a large number of molecules, proteins, genes, and signaling pathways. During the critical period of auditory development, the auditory experience, environmental experience, and different damaging factors may directly or indirectly cause different degrees of central adaptive changes. Some changes may remain to adulthood in individuals and affect their auditory skill acquisition. In the future, research of aural deprivation-induced plastic changes may continuously increase at different levels in the aforementioned areas. Molecular mechanisms corresponding to the observed morphological changes, correlations between different functional changes, the synaptogenesis, the degeneration of nerve fibers, the reconstruction of neural circuits, neurotransmitter regulation and the activation of many proteinases will become hot research topics. With the increasingly attention of translational medicine, research findings involving aural deprivation and central plasticity may affect clinical auditory rehabilitation strategies. That reversing the reorganization of the auditory center caused by aural deprivation will allow patients with hearing impairment to benefit from early interventions and proper auditory rehabilitation trainings, although this is still a challenge for both neuroscientists and clinical audiologists.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There are errors in the Author Contributions. The correct contributions are: Conceived and designed the experiments: SP, AM. Performed the experiments: SP, SD, NJ, JJ, PTC. Analyzed the data: SP, AM, NC. Contributed reagents/materials/analysis tools: AM, AMR, NC. Wrote the paper: SD, SP, NJ, AM


Immune-mediated pathogenesis has been suggested for idiopathic sensorineural hearing loss (iSNHL). Recent studies have investigated the relationship between iSNHL and autoantibodies against inner ear antigens, but specific tests are not available.To assess the positivity of autoantibodies against inner ear in a series of pediatric patients with idiopathic sensorineural hearing loss.We conducted a prospective, non-interventional observational study in a series of pediatric patients affected with iSNHL. Autoantibodies against inner ear , previously described in the serum of patients with Cogan's syndrome, were detected in our populations. The characteristics of children who resulted positive were also evaluated to verify if clinical data, disease progression and response to treatment could confirm an immune-mediated pathogenesis.Eleven patients were enrolled and 9 of them resulted positive for the inner ear antibodies detected. Non-organ specific autoantibodies were present in 5 children out of 9. An immune-mediated condition was diagnosed in 2 cases and minor immune manifestations were found in other 2 patients. In 5 cases hearing loss remained stable with no therapy, otherwise 4 children developed hearing loss progression. Two subjects were treated with steroids and methotrexate achieving hearing improvement. Another subject started methotrexate treatment showing hearing loss stabilization.Most of clinical characteristics and comorbidities described, added to immunologic tests results and to treatment efficacy, suggest an immune-mediated pathogenesis of hearing loss. Inner ear autoantibodies are not able to identify children affected with autoimmune sensorineural hearing loss but, integrated with clinical data, they can represent a diagnostic aid for the physician. Large prospective studies are needed to investigate usefulness, diagnostic and prognostic role of these autoantibodies.None declared.


Extended exposure to high-speed handpieces and other noise-intensive devices might put dentists at risk for possible hearing impairment. The aim of this study was to determine the hearing ability of dentists and other scientists for comparison.U-test.After approval by the ethics committee, 115 subjects  of both genders were enrolled in the study. Exclusion criteria were colds, ear-blockages or abnormal hearing-thresholds. An audiometric determination  was performed in the frequency range of 125Hz to 8 kHz for both ears. Anamnestic data and number of years in the profession were assessed using a questionnaire. Differences between groups were analyzed with the Mann–Whitney-Data from 53 dentists and 55 other academic professionals  with a mean age of 51.7 ± 9.6 years and similar gender distributions in both groups were analyzed. The audiometric tests for the right and left air conduction showed that the hearing of dentists tended to be slightly more impaired than in the control subjects. For the frequencies 3 kHz and 4 kHz these differences were statistically significant for both ears. In contrast, no significant differences were found in this range for bone conduction.Hearing impairment in dentists was slightly higher than in controls. Although other factors like environmental noise exposure were comparable for both groups, occupational exposure to high-speed handpieces and other noisy devices can be an additional burden for the hearing. All sounds, regarded as pleasant or unpleasant depending on the subjective experience, stimulate hearing in humans. The respective vibrational energy of the sound reflecting surfaces is captured as sound pressure through the ear canal at the ear drum. Noise is defined to consist mostly of undesirable tones or sounds, where these limits have to be regarded as very individual, because often certain frequencies are perceived subjectively by different individuals as sound or noise. Exposure to prolonged irregularly composed frequency ranges and raised noise levels are perceived as uncomfortable or even painful, and can cause numerous physical and psychological disorders or can even induce hearing loss-3. AmongIn general, acute damage to the hearing can be caused by constant exposure to sound pressure levels exceeding 85 dB, and by a sound pressure level exceeding 120 dB already after a few seconds. Also, extremely high sound pressure levels like from blasts or explosions in close proximity to the ear normally lead to permanent damage.In their daily profession especially dentists and dental personnel are exposed to a noise level of different frequency ranges due to the use of high speed handpieces, various instruments and ultrasound devices-14. The The impairment by dental noises always depends on the frequency intensity, the daily intervals of the noise exposure, the daily treatment time and the years in the profession, the individual sensitivity and the distance from the respective instruments and devices. In numerous studies the impairment through noise was tested in dentists and the dental personnel, and possible consequences were assessed-18. WithA total of 115 subjects of both genders could be recruited for the present cross-sectional study to assess the hearing of dentists and other academic professionals. The subjects were alerted to participation in the free-of-charge study on hearing partly through flyers and partly by means of person-to-person communication. The project was introduced during the annual board meeting of the professional representation of the dentists from the state of Rhineland-Palatinate (Germany). All participants of the study lived and worked in the metropolitan Rhine-Main region, Germany, have worked in their profession for at least 10 years, and comprised an age range of 38 to 73 years. After the approval by the ethics commission of the professional representation of the dentists from the state of Rhineland-Palatinate (Nr. 837.439.11(7981)), a survey was conducted among dentists and other academic professionals (control subjects), comprising physicians, mathematicians, computer scientists, biologists and chemists, using a questionnaire, which provided, in addition to general anamnestic data, information about earlier or acute damage to the hearing or other ear disorders as well as about the years in the profession. Prior to an assessment of the hearing all subjects received detailed information about the audiometric tests, and only after having obtained a written consent from them, the subjects were enrolled in the study. All data and findings were evaluated anonymously.By means of an extensive questionnaire, inclusion and exclusion criteria were determined for the participation in this hearing assessment. Among the inclusion criteria were for the dentists at least ten years working as dental practitioner; in addition, in all subjects no impaired hearing, no general disorders of the ear or the sound pathway as well as no hearing damages like tinnitus or acoustic hallucinations could be present and the subjects were to be free from common colds.After filling in the questionnaires, all subjects participated in an audiometric test for both ears, using both air and bone conduction. The tests for the determination of the subjective hearing took place in shielded rooms. By means of the audiometer  of the Institute of Occupational, Social and Environmental Medicine of the Johannes Gutenberg University Mainz, the hearing for the frequency range of 125 Hz to 8 kHz was assessed in the dental practitioners and the non-dental control subjects.U-Test (for the continuous variables age and intensities in dB). A significance level of p < 0.05 was chosen.The statistical analyses of the data were performed using the program STATA/IC 12.1  in cooperation with the Institute of Occupational, Social and Environmental Medicine. Absolute and relative frequencies were given for the categorical variable gender, and means and standard deviations for the continuous variables age and intensities in dB. Differences in the variables between the two groups were analyzed using the Chi square-test  and the Mann–Whitney After the checking of all questionnaires and audiometric tests, the data from a total of 75 men and 33 women  could be evaluated. Subjects with prior ear disorders or presently suffering from hearing loss and those with incomplete or missing data were excluded from the study. In Table U-Test). However, nearly identical residual hearing was observed for dentists and control subjects for bone conduction of the left and right ear, representing the integrity of the inner ear, and the values were no longer statistically significant.When the results of the audiometric tests were analyzed using a direct comparison of the dental practitioners with the academics not from the dental profession, it became apparent that as a rule the hearing impairment was only slightly more pronounced in the dentists than in the control subjects. At frequencies of 3 kHz  and 4 kHz  these slight differences were marginally statistically significant for air conduction of the left as well as the right ear . Bali et al. could shIn the present investigation a special emphasis was based on the fact that the study was conducted in a very densely populated area in Germany, which is located in close vicinity to one of the largest European airports as well as an extensive industrial area and road network.Therefore, for the hearing assessment, it has to be considered in particular that environmental influences make up a significant part of the possible noise exposure, because the subjects of the study have been located in this area for at least 10 years. In addition, the dental treatment devices are subject to constant further developments, so that occupational damages to the hearing loss recede more and more into the background.It can be concluded from the results of this study that hearing impairment in dentists was slightly higher than in the control subjects, but besides occupational exposure to high-speed handpieces and other noisy devices the additional noise exposure in modern industrialized nations also has to be considered. Especially important in this context is environmental noise pollution in densely populated urban areas.The authors declare that they have no competing interests.TGW, LS and DW carried out the study. AC and VE performed the statistical analysis. BW, TGW, VE, SL and AC conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Objectives. We describe an unusual case of surgical management of congenital mixed hearing loss in a female patient with inner ear malformation. This report outlines the role of temporal bone imaging and previous surgical history in evaluating a patient's risk of perilymph gusher during stapes surgery. Methods. A 68-year-old female patient with a history of profound bilateral mixed hearing loss due to ossicular and cochlear malformation presented to our otology clinic. She had undergone multiple unsuccessful previous ear surgeries. Computed tomography revealed bilateral inner ear malformations. She elected to proceed with revision stapedectomy. Results. The patient received modest benefit to hearing, and no operative complications occurred. Conclusions. Although stapedectomy has been shown to improve hearing in patients with stapes fixation, there is risk of perilymph gusher in patients with inner ear abnormalities. Evaluation and counseling of the risk of gusher during stapes surgery should be done on a case-by-case basis. Stapes fixation is a relatively common cause of conductive hearing loss, but it rarely presents with concomitant inner ear malformation, comprising a special challenge for the otologist. The combination of these two conditions has been well described previously in male patients with X-linked mixed hearing loss . In thisA 68-year-old female patient with a history of congenital hearing loss presented to the otology clinic to discuss management of her hearing loss. She had previously been diagnosed with ossicular and cochlear malformation but had no significant family history of hearing loss. She had undergone several surgeries in the right ear between the ages of 4 and 36 years to improve her hearing. Previous surgeons had attempted this by stapedectomy and implantation of various stapes prostheses in the patient's right ear, and she reported immediate hearing improvement after the most recent surgery. However, several hours after that surgery she vomited and the hearing improvement ceased. Previous surgical records could not be obtained. At the time of the initial visit, she used behind-the-ear hearing aids bilaterally with poor benefit.A previously obtained audiogram revealed a profound mixed hearing loss bilaterally , with air-conduction pure-tone averages  of 108 dB HL in the right ear and 95 dB HL in the left ear . Word reThe patient had never undergone otologic imaging. Because of the patient's unusual story and multiple previous surgeries, a noncontrast temporal bone computed tomography (CT) scan was obtained. Results revealed multiple bilateral middle ear and cochlear malformations. The ossicles demonstrated abnormal morphology and appeared to be fused bilaterally. There were only 1.5 turns to both cochleae instead of the expected 2.5 turns. The vestibules were enlarged bilaterally. Incomplete separation of the basal turns of both cochleae from the internal auditory canals (IACs) was seen . A metalAfter thorough discussion of risks, benefits, and alternatives of surgery, the patient elected to undergo a right revision stapedectomy under general anesthetic. During surgery, the incus and malleus were identified as partially fused and malformed. Two prostheses from previous surgeries were found unattached to any ossicles and were removed. A vestibulotomy was made with a KTP laser anterior to an opening from a previous operation that, on review of the CT, appeared to open into a superior extension of a hypotympanic air cell. On creating the new vestibulotomy, a pulsatile but low-flow perilymph leak was noted, which was slowed by placing a tragal perichondrium graft over the vestibulotomy. A new titanium total ossicular chain prosthesis was then placed onto the graft at the site of the vestibulotomy, which resulted in cessation of the perilymph leak. Perichondrium and tragal cartilage were used to support the tympanic membrane over the prosthesis, and absorbable gelatin foam pieces were used to pack the middle ear and stabilize the prosthesis. The external auditory canal was then filled with antibacterial ointment and a cotton ball was placed, concluding the procedure.Three weeks after surgery, the patient returned for a postoperative visit. She was doing well and reported minimal dizziness postoperatively, improved hearing in the operated ear, and no evidence of cerebrospinal otorrhea or rhinorrhea. Three months postoperatively, she was doing well with subjective improvement in hearing and a well-healed ear. Postoperative audiogram showed a persistent right-sided mixed hearing loss, with a modestly improved unaided pure-tone average of 90 dB HL and an aided pure-tone average of 45 dB HL, although her word recognition remained at 20% unaided. Subjectively, she was happy with her outcome and improvement using her hearing aid. We discussed the possibility of cochlear implantation in the future, but she was content with her current status using hearing aids.The patient presented in this report was likely diagnosed with congenital stapes fixation as a child and had undergone several stapedectomy attempts in the right ear to correct it. Congenital stapes fixation presents with a conductive hearing loss early in childhood. Juvenile otosclerosis is another entity that may cause a progressive fixation of the stapes, leading to a conductive hearing loss in the pediatric patient. For these patients, however, onset of hearing loss is typically after puberty. Stapedectomy has been proven to be an effective treatment for congenital stapes fixation , 3. A reA major concern, for which extensive preoperative counseling and discussion was held with the patient, was the potential for a perilymph gusher  with her inner ear malformation identified on CT. Perilymph gusher is a rare complication of stapes surgery but may have a tremendous adverse effect on the patient's hearing . PatientPerilymph gushers are common in individuals with X-linked mixed hearing loss. This is a well-known condition with many similarities to the case presented here. Patients present with severe mixed hearing loss in childhood due to congenital stapes fixation and inner ear malformation. A characteristic temporal bone CT will reveal bulbous IACs with only partial or no separation of the IAC from the cochlea, giving the patients a very high risk of perilymph gusher . This co


The evolution of cooperation remains a central issue in socio-biology with the fundamental problem of how individuals minimize the risks of being short-changed (‘cheated’) should their behavioural investment in another not be returned. Economic decisions that individuals make during interactions may depend upon the presence of potential partners nearby, which offers co operators a temptation to defect from the current partner. The parcelling model posits that donors subdivide services into parcels to force cooperation, and that this is contingent on opportunities for defection; that is, the presence of bystanders. Here we test this model and the effect of bystander presence using grooming interactions of wild chimpanzees. We found that with more bystanders, initiators gave less grooming at the beginning of the bout and were more likely to abandon a grooming bout, while bouts were less likely to be reciprocated. We also found that the groomer’s initial investment was not higher among frequent groomers or stronger reciprocators, suggesting that contrary to current assumptions, grooming decisions are not based on trust, or bonds, within dyads. Our work highlights the importance of considering immediate social context and the influence of bystanders for understanding the evolution of the behavioural strategies that produce cooperation. Gallus gallus domesticusPan troglodytesAstatotilapia burtoniProcambarus clarkij 4Betta splendensPongo spp.In group-living species, many social interactions occur in the presence of others. The influence of such bystanders has attracted increasing attention, both in their role as an audience that influences performance Macaca fascicularis) respond to local market conditions in their grooming of females, a result interpretable as a bystander effect. Unlike parcelling, however, BMT predicts only the total amount of investment as a function of market conditions, and does not predict the internal structuring of interactions. Thus while both parcelling and BMT predict a bystander effect, the division of behaviour into parcels is not predicted, or accounted for, by BMT.Biological markets theory10Social grooming exchanges provide an excellent system within which to explore models of social cooperation19202122232425262829Pan troglodytes) of the M-group community of the Mahale Mountains National Park (Tanzania). We focus on adult males, as they are more gregarious than adult females and exhibit higher rates of grooming3434Here we test the effect of bystander presence on grooming interactions among wild chimpanzees  bystanders we expect: less investment (i.e. less grooming effort: prediction 1), as well as fewer (prediction 2) and smaller (prediction 3) parcels (episodes of grooming), from an initiator during the initial phase of the grooming bout, and a higher probability of unidirectional bouts (prediction 4). As episodes of grooming can vary in duration, such that a given amount of grooming (grooming effort) can in principle be provided by multiple shorter episodes or fewer longer episodes, we also tested the relationships between grooming effort, number and length of episodes.We recognise that rank differences within a dyad can influence economic decision-making if subordinates trade grooming for rank-related or rank-restricted commodities2025202542Parcel size may be contingent also on the expectation by the donor of receiving grooming from the recipient: the more likely it is that the recipient will reciprocate grooming, the bigger the donor’s parcel ought to be. Despite a growing set of findings supporting a view that primate grooming exchanges are driven by immediate economic considerations18192021242526274445463248502122et al.Papio ursinus). Fruteau et al.Cercocebus atys) and vervet monkeys (Chlorocebus aethiops), but in the opposite direction: dyads that groomed more frequently tended to give shorter first episodes than did infrequent groomers. The relationship between initial investment and the degree of reciprocity has not been investigated and we provide the first test of this among wild chimpanzees. Under the relationship model, we predict that groomer’s initial investment should be greater among frequent groomers (prediction 5) and/or strong reciprocators (prediction 6) than among infrequent groomers and weak reciprocators, respectively.Few studies have tested this prediction. Barrett Across 774 bouts, we recorded 33 hours of grooming in the initial phase with a mean (±SD) per bout duration of 153.6 ± 179.9 s (median = 95.5 s): 139.9 ± 174 s (median = 81.5 s) during unidirectional bouts and 197.5 ± 191.6 s (median = 133.5 s) during bidirectional bouts. The initial phase of unidirectional bouts  consisted on average of 1.9 ± 1.6 episodes , of 72.6 ± 73.4 s (median = 51.8 s) in duration. The initial phase of bi-directional bouts consisted on average of 2.2 ± 1.7 episodes , of 96.3 ± 91.4 s (median = 74 s) in duration. Both the number of episodes and mean episode length predicted grooming effort in the initial phase . Number and length of episodes were not collinear , and one did not predict the other ; chimpanzees appeared to vary their grooming effort by altering either the number or length of episodes.Only 4% of grooming bouts were interrupted; this was less likely to occur with more male bystanders , although the number of male bystanders did not depend on the context during which the grooming interaction occurred .The number of male bystanders (i.e. those within 10 m) significantly predicted both groomer’s initial investment  and rank distance  predicted the occurrence of bidirectional bouts : groominfrequent = 12, Ninfrequent = 32, W = 209, p = 0.664). We also found no support for prediction 6: strong reciprocators did not invest significantly more in the initial phase of a bout  than weak reciprocators .We classified 12 dyads as frequent groomers, while 9 dyads had a grooming reciprocity index greater than 0.8. Six dyads were both frequent groomers and strong reciprocators. We found no support for prediction 5: groomer’s initial investment was not significantly longer among frequent groomers  than it was among infrequent groomers  initiators invested less in their grooming bouts and provided both less grooming and fewer parcels; (2) bouts were less likely to be reciprocated; and (3) initiators were more likely to abandon the grooming if the recipient did not reciprocate. These results suggest that male chimpanzees make economic decisions on how much to invest in grooming interactions that are based not only on whether the partner reciprocates, but also on whether there are other potential social partners in close proximity. This is a key aspect of Connor’s parcelling model122024402352personal observations, and similarly, in Japanese macaques (Macaca fuscata) groomers are more likely to receive aggression from recipients in post-grooming contexts than during controlsContrary to our expectations, we found evidence that dominance rank influenced episode length: initiators gave shorter episodes when they groomed high-ranking males, regardless of rank distance. Given that high-ranking individuals were more likely to terminate boutset al.1819202124252627444546We found no support for predictions derived from the relationship model of primate grooming interactions. There was no influence of grooming frequency on the grooming invested in the initial phase, and we found initiators in dyads with a history of grooming reciprocity, who should under the relationship model trust their partner and expect to receive grooming in return, were no different in their initial investment to those without such a history. Thus the grooming relationship  of a dyad appeared irrelevant to the behaviour of an individual initiating a grooming bout, and we have no evidence that individuals who had received high levels of reciprocity in the past from their current partner trusted that they would do so again. This finding calls into question the concept that grooming builds trust between individuals. Our findings echo the results of Machanda We have presented evidence elsewhere as to the importance of market forces on the grooming decisions of male chimpanzees1011Here, we focused on the initial phase of grooming bouts and showed that this is particularly important for partner-choice decisions, with the presence of bystanders having no effect on later phases of bidirectional bouts. If we consider bystanders to represent a local market of alternative suppliers of grooming, then the question is why we need to consider parcelling at all as an explanation ? The obvious answer is that chimpanzees deliver their grooming effort in discrete episodes within bouts – they parcel their grooming – an effect that is not predicted  by biological market theory. Furthermore, individuals continue to parcel their grooming throughout bidirectional boutsOverall, our study shows that male chimpanzees modify the amount of grooming investment in the initial phase of the bout in relation to the number of male bystanders: when there are more males in close proximity, initiators tend to give less grooming, with fewer grooming episodes, and are more likely to interrupt a bout in the absence of immediate reciprocation, making unidirectional bouts more likely to occur. Our results show that the number of bystanders present during a grooming interactions can affect parcel size, at least in terms of the amount of grooming invested, as predicted by Connor’s model and highlight the importance of considering the presence of other individuals during cooperative interactions, and the distinction between local and global markets, as a way towards a better understanding of the evolved behavioural strategies that generate cooperative interactions.>16 years old) and 23 adult females (>14 years old)We studied the grooming behaviour of adult male chimpanzees of M-group community from the Mahale Mountains National Park, Tanzania , between February and November 2011. This community has been continuously studied for over 30 yearsWe collected data through long-day focal follows on eight adult males. Data on grooming and agonistic interactions were collected using all-occurrence samplingWe defined grooming as the visual examination, searching and manipulation of the skin and hair with one or both hands, with the occasional use of the lower lip to part the hair. When a male initiated a grooming bout, we recorded: (1) the identity of the individual who approached the partner either to give or receive grooming, together with the time of the approach; (2) the identity of other males within 10 m (whom we labelled bystanders); (3) the changes in the identity of groomer and recipient, including the initiator; (4) the end time of the grooming bout, and the identity of the individual who terminated the bout. A grooming bout was considered to have ended when neither individual groomed for at least 30s24We examined grooming bouts as pairwise (dyadic) interactions. Bouts were classified as either bidirectional when both individuals contributed some groomingTo determine social dominance rank we used the outcome of agonistic interactions to calculate dominance rank order using Elo-ratings2517lmer function in the package ‘lme4’ (in R 3.1.2) to perform a Linear Mixed Model (LMM) analysis. We set grooming effort (i.e. duration) as the dependent variable, while the number and mean duration of initial episodes were entered as independent fixed factors. We ran a similar model to assess whether mean episode length predicted the number of episodes. We used the drop1 function in R to compute a likelihood ratio test and thereby assess whether the independent factor(s) exerted a significant effect on the dependent variable.We tested whether groomer’s investment in the initial phase was predicted by the number and mean length of episodes in this phase, using the glmer function in the package ‘lme4’. In one model we included information on whether or not the bout was interrupted as binomial dependent variable, with the number of bystanders set as independent factor. In a separate model, we considered the number of male bystanders as a dependent variable entered as count data with Poisson distribution, while the context was included as an independent factor.To assess whether the number of male bystanders significantly affected the likelihood that a grooming bout was interrupted, and depended on the context of the grooming interaction, we ran two different Generalized Linear Mixed Model (GLMM) analyses, using the lmer command and the drop1 function in R 3.1.2. The initiator’s initial investment and the mean length of episodes were entered as continuous dependent variables in separate models, while in each model the number of male bystanders, rank difference between partners, the context during which the grooming interaction occurred and a binary variable describing whether the bout was interrupted were set as fixed factors. To determine whether the initial phase was distinct from the remainder of bi-directional bouts in its sensitivity to bystander effects , we investigated whether the remaining investment provided by an initiator was influenced by the number of bystanders and/or the rank distance between partners. To this end, we used an LMM analysis with the total grooming effort in each bout, minus that in the respective initial phase, as the dependent variable and with the number of bystanders and rank distance between partners as fixed factors.To test whether groomers’ initial investment (prediction 1), and the mean length of grooming episodes (prediction 3) were affected by the number of male bystanders and rank difference between partners, we ran two Linear Mixed Model analyses with the glmmadmb function in R 3.1.2. In this model, the number of episodes was entered as count data, with the number of male bystanders, rank difference, context and whether the bout was interrupted included as fixed factors, as above. We used two Generalized Linear Mixed Models with binomial distributions (logistic GLMM) to test (a) whether reciprocation was less likely with more bystanders , and (b) whether the initiator or the recipient were more likely to terminate a unidirectional bout in the presence of bystanders, again using the glmmadmb function. In the first of these models, we set grooming pattern  as the dependent variable; in the second, the identity of the individual who terminated the bout . The number of bystanders, rank difference, context and whether the bout was interrupted were entered as fixed factors in both models.We used a Generalized Linear Mixed Model with negative binomial distribution to assess the effect of bystander and dominance rank on the number of grooming episodes given by initiators (prediction 2) in the initial phase of the bout, using the In all these models (LMM and GLMM), the identity of the dyad and the day on which each bouts occurred  were included as random factors (intercepts) with nested structure (within-day bouts nested within dyads) in order to control for the different contribution that each dyad gave to the data set, and for the non-independency of the grooming bouts that were exchanged within a day.et al.24To test predictions 5 and 6, we categorised dyads on the basis of the frequency of grooming exchanged and the strength of grooming reciprocity. To identify the dyads that exchanged grooming time more frequently, we calculated the time spent grooming (grooming effort) for each dyad as a proportion of the total observation time for both members of that dyad. We ranked dyads on this proportion from the highest to lowest and, following Koski gAb is the grooming that individual A directed towards B, gBa is the grooming that B directed towards A and gAb + gBa is the total grooming exchanged between A and B. This index can range between 0 (no reciprocity) and 1 (complete reciprocity). Dyads with an RI equal or greater than 0.8 were defined as strongly reciprocatingin which We used Mann-Whitney U tests to compare the groomer’s initial investment across dyads. As initial grooming investments in different bouts are potentially not independent when they occur within the same dyad, we calculated mean groomer’s initial investment for each dyad and compared these values between dyads that were frequent and infrequent groomers, and between the strongly reciprocating and other dyads.How to cite this article: Kaburu, S. S. K. and Newton-Fisher, N. E. Bystanders, parcelling, and an absence of trust in the grooming interactions of wild male chimpanzees. Sci. Rep.6, 20634; doi: 10.1038/srep20634 (2016).


Previous studies have indicated that extended exposure to a high level of sound might increase the risk of hearing loss among professional symphony orchestra musicians. One of the major problems associated with musicians’ hearing loss is difficulty in estimating its risk simply on the basis of the physical amount of exposure, i.e. the exposure level and duration. The aim of this study was to examine whether the measurement of the medial olivocochlear reflex (MOCR), which is assumed to protect the cochlear from acoustic damage, could enable us to assess the risk of hearing loss among musicians. To test this, we compared the MOCR strength and the hearing deterioration caused by one-hour instrument practice. The participants in the study were music university students who are majoring in the violin, whose left ear is exposed to intense violin sounds (broadband sounds containing a significant number of high-frequency components) during their regular instrument practice. Audiogram and click-evoked otoacoustic emissions (CEOAEs) were measured before and after a one-hour violin practice. There was a larger exposure to the left ear than to the right ear, and we observed a left-ear specific temporary threshold shift (TTS) after the violin practice. Left-ear CEOAEs decreased proportionally to the TTS. The exposure level, however, could not entirely explain the inter-individual variation in the TTS and the decrease in CEOAE. On the other hand, the MOCR strength could predict the size of the TTS and CEOAE decrease. Our findings imply that, among other factors, the MOCR is a promising measure for assessing the risk of hearing loss among musicians. Excessive noise exposure can cause temporary hearing deterioration as well as permanent hearing damage to the cochlear, which is known as noise induced hearing loss (NIHL). Previous studies have indicated that professional symphony orchestra musicians are exposed to a high level of sound, comparable to noise in industrial settings (between 79 and 98 dBA ), and arAt present, the risk factors for musicians’ hearing loss have not been sufficiently identified. A considerable number of studies have assessed the risk of NIHL among musicians by sound exposure. The degree of risk varies with environmental factors such as instrument type and position in the orchestra. Nevertheless, the variation of PTS has only partially been explained by the amount of exposure: Some studies reported that groups with a higher exposure level tend to show larger PTSs , 6], wh, wh6], wAlthough the environmental factors involved in musicians’ hearing loss have been intensively investigated, as mentioned above, very little attention has been focused on the inter-individual variation of susceptibility to NIHL in musicians. Previous studies on occupational hearing loss have revealed that the amount of hearing loss depends on the susceptibility to NIHL, which varies substantially between individuals, as well as on the amount of exposure ; individTo identify factors underling the variability of susceptibility, previous studies have examined various biological factors such as eye color, gender, age, and middle-ear muscle reflex, and environmental factors such as exposure to drugs or chemicals . Among tBoth types of MOCR have been implicated in protecting the ear from acoustic damage, although their usefulness in natural environment is under debate : Severalhumans is still equivocal. It was only recent that Wolpert et al. , m, mhumanst et al. , discotht et al. , and occt et al. —failed tWe hypothesized that MOCR measurement could be applied to musicians to assess their risk of NIHL, because musicians’ MOCRs are stronger than non-musicians , 32] an an32] anTo test this hypothesis, we examined whether MOCR strength can predict the hearing deterioration caused by short-duration instrument practice. The temporary hearing deterioration was quantified by measuring audiograms and click-evoked otoacoustic emissions (CEOAEs) before and after a one-hour instrument practice. OAEs can exhibit an observable change before changes in conventional audiometry associated with noise exposure  and may Sixteen audiometrically normal college-age violinists participated in the experiment. They were students of Kyoto City University of Arts, majoring in violin. None reported a clinical history of hearing disorders. Three participants  whose CEOAE signal-to-noise ratio was too low (< 2 dB) were not included in the post-experiment statistical analysis relevant to the OAE and the MOCR data. Their distributions of their age, gender and violin-playing experience are summarized in For CEOAE and MOCR measurement, stimuli were digitally synthesized with sampling rates of 96 kHz and converted to analog signals using an Edirol UA-101 (24 bits). The analog signals were amplified by a headphone buffer and presented through Etymotic Research ER-4s earphones connected to an ER-10B low-noise microphone system. Ear-canal sound pressure was recorded with an Etymotic Research ER-10B low-noise microphone system inserted in each ear. Sounds for eliciting the ipsilateral and contralateral MOCR were delivered by ER-4s earphones. Prior to the measurement, the outputs from the ER-4s were calibrated using a DB2012 accessory  of a Bruel and Kjaer Type 4257 ear simulator (IEC 711).Sounds during the practice were recorded with custom-made equipment comprising two microphones (SOUNDMAN OKM-II) and headbands. This equipment allowed us to place two small microphones 1–2 cm away from either ear. Recording of the practice session were made by directing the line output of the microphones to a TASCAM DR-05 linear PCM recorder with a 44.1-kHz sampling rate and 16-bit resolution. Prior to the measurement, the frequency characteristics of the microphones were calibrated with Bruel and Kjaer BK4192 microphones. All measurements and violin practices were conducted in a quiet classroom or conference room at Kyoto City University of Arts.Hearing thresholds were measured with an audiometer (RION Type AA-58) and expressed as hearing level (in dB HL). The thresholds were measured at five frequencies  with 5-dB intensity resolutions using the audiometer’s search algorithm. The order of the right- and left-ear measurement was randomized for each participant.We measured both the ipsilateral and contralateral MOCRs as CEOAE suppression induced by noise presented to the ipsilateral and contralateral ear, respectively. The amount of suppression, typically ~ 4 dB, was assumed to be related to the MOCR strength .In the measurement of the contralateral MOCR, a continuous noise was presented to the contralateral (opposite) ear during the CEOAE recording . The noiIn the measurement of the ipsilateral MOCR, we applied the forward suppression paradigm , in whicOne set of MOCR measurements was composed of two blocks of contralateral-MOCR measurement and ten blocks of ipsilateral MOCR measurement. The order of the blocks in one measurement set was randomized. This MOCR measurement set was repeated three times for both ears alternately (the order of left- and right-ear MOCR measurements was randomized for each participant). Thus, a total of six and 30 blocks of contralateral and ipsilateral MOCR measurements, respectively, were performed. Accordingly, for a given ear, each participant was presented with a total of 1800 clicks without noise, 1500 clicks with the contralateral noise, and 1500 clicks with the ipsilateral noise.withc, Pwithi and Pwithout (in Pa). Contralateral and ipsilateral MOCR strength was defined as 20log10(Pwithc/Pwithout) and 20log10(Pwithi /Pwithout), respectively. To specify the frequency region where noise exposure has the largest effect, CEOAEs for the without-noise condition were analyzed in three frequency bands centered at 1, 2, and 4 kHz using a one-octave-wide band pass filter. The pressure level at each frequency (in dB SPL) was defined as an RMS value in the 8–18 ms region of the band-passed waveform. In the following, we refer to those CEOAEs in the without-noise condition as “CEOAE”.Recorded waveforms were averaged for the without-noise, with-ipsilateral-noise, and with-contralateral-noise conditions. MOCR was computed by using the level of CEOAEs that were band-pass filtered between 1 and 3 kHz, in which the largest MOCR-related CEOAE suppression was observed . The preAudiograms, CEOAEs, and MOCR strength were obtained just before and immediately after the one-hour instrument practice session. To observe the recovery from the temporary deterioration, we also conducted the same measurement 60 minutes after the end of the violin practice. The order of hearing thresholds and CEOEAs and MOCR strength measurement was randomized for each participant. For a given participant, the order was identical for the pre- and post-exposure measurements. The measurement of hearing thresholds took approximately five minutes; that of CEOAEs and MOCR strength took approximately 15 minutes.Participants were instructed to play the violin as in their usual solitary practice. Throughout the instrument practice session, participants played the violin almost continuously, except for short breaks to prepare scores for a next piece. The sounds to which they were exposed during the one-hour practice were recorded by small microphones placed 1–2 cm away from either ear. The average exposure level throughout the one-hour of violin playing was computed with A-weighted root-mean-square (in dBA), 1/3 octave band and octave band sound levels (in dB SPL) with center frequencies between 250 and 10000 Hz.The effect of the exposure during the practice on CEOAE and audiogram was assessed by a two-way split-plot analysis of variance (ANOVA) with condition (before and after the practice) and frequency as factors. Subsequent post-hoc pairwise comparisons were performed to separately test significance of differences between the data before- and immediately after the practice and between the data before and 1h after the practice. Relationships among changes in CEOAE or audiogram with exposure level and the MOCR strength were assessed by using Pearson’s product-moment correlation coefficient. For all analyses, normality of distribution and equality of variance were ascertained by a one-sample Kolmogorov—Smirnov test and Levene’s tests, respectively. Statistical significance was set at a probability value of p = 0.05 except for the post-hoc pairwise comparisons in which p-values were adjusted by Bonferroni correction.T15 = 8.4, p<0.001). The exposure level to the left ear was larger than to the right ear in the middle- to high-frequency region (> 500 Hz)  and significant interactions of laterality and frequency . Significant simple main effects were found at frequencies above 500 Hz .During the violin practice, participants played the violin almost continuously . A-weigh 500 Hz) : A two-wF1, 15 = 0.43, p = 0.52) or interaction of laterality and frequency . There was also no significant difference in CEOAE between the right and left ear .All participants showed normal audiograms (HL < = 25 dB HL) before the violin practice and no significant difference between the right and left ear: A two-way split-plot ANOVA with laterality and frequency as factors shows no significant main effect  and left ear . Mean ipsilateral MOCRs were -2.0 dB (SD 1.7) for the left ear and -2.2.dB (SD 1.9) for the right ear. Mean contralateral MOCRs were -2.5 dB (SD 1.6) for the left ear and -2.6 dB (SD 1.6) for the right ear. There was no significant difference between the right and left ear for the contralateral MOCR  and ipsilateral MOCR .CEOAE level was significantly smaller in the with-contralateral-noise and with-ipsilateral-noise condition than the without-noise condition both for the right  and a significant interaction of condition and frequency . A simple main effect was observed at 4 kHz in audiograms . An ANOVA as above for the right ear showed no significant main effect  or interaction . The right-ear hearing thresholds straight after the practice were lower in 1–2 kHz than before practice, but those differences were not statistically significant.The short-duration instrument practice caused a left-ear and 4-kHz specific TTS : For lefF1, 12 = 20.4, p<0.001) and a significant interaction of condition and frequency . An ANOVA as above for the right ear showed no significant main effect  or a significant interaction . A simple main effect was observed at 4 kHz in the left and right ear . The TTS at 4 kHz and the CEOAE decrease at 4 kHz were significantly correlated  and the CEOAE decrease at 4 kHz ; the data for the left ear were distributed around the right top, and the data for the right ear were distributed around the left bottom on the regression line derived from the exposure level and the size of hearing deterioration  and CEOAE decrease  only for the left ear.The larger TTS and the greater CEOAE decrease in the left ear are likely to reflect the larger exposure level in the left ear than in the right ear : When thioration . NeverthR12 = 0.71, p = 0.0064; R12 = 0.66, p = 0.014; R12 = 0.21, p = 0.49 for audiogram; R12 = 0.18, p = 0.56 for CEOAE). Contralateral MOCR strength was not significantly correlated with the TTS or the CEOAE decrease in the right and left ear .For the left ear, ipsilateral MOCR strength was negatively correlated with the TTS  and no significant interaction of condition and frequency  in the left ear. An ANOVA as above showed no significant main effect or interaction in the right ear .To observe the recovery from the temporary deterioration, we also conducted the same measurement 60 minutes after the end of the violin practice and found that the left-ear hearing thresholds were lower (better audibility) than before the practice : A two-w and a significant interaction . The simple main effect at 4 kHz was significant . For the right ear, there was no significant difference before and 60 min after the practice: An ANOVA as above showed no significant main effect  or significant interaction . There was no simple main effect .On the other hand, a significant CEOAE decrease persisted even 60 minutes after the practice for the left ear : An ANOVIn this study, we showed that MOCR strength could predict hearing deterioration caused by a short-duration instrument practice. To our knowledge, this is the first study to report an association between MOCR strength and the risk of hearing loss among musicians.There was no significant difference between the left and right ear for audiograms and CEOAE before the violin practice. In contrast, some studies have reported that professional violinists tend to have left-ear-specific hearing loss in audiograms , 6]. Pr. Pr6]. PAlthough we should be cautious when comparing our results with previous studies because of several methodological differences , 31], w, w31], wAn instrument practice as short as one-hour caused a 4-kHz and left-ear specific TTS, which is similar to a typical pattern of the PTS among professional violinists , 5], , [CEOAE decreased at 4 kHz also for right ears. CEOAE might be sensitive to a slight functional change of the right ear that could not be revealed in the conventional audiometry. It has been shown that CEOAE can exhibit an observable change before conventional audiometry changes .Interestingly, the left-ear hearing thresholds one hour after a violin practice were even lower than those before it. Considering that a significant CEOAE decrease persisted even 60 minutes after the practice, this enhanced hearing sensitivity may originate in functional changes in the retrocochlear auditory pathway. Indeed, several animal studies observed enhancement of neural responses after acoustic exposure: Salvi et al.  measuredMOCR strength was negatively correlated with the TTS and the CEOAE decrease caused by a short-duration instrument practice; the participants who had strong efferent activity tended to show a small TTS and small OAE decrease. This result is consistent with studies of NIHL reporting that individuals with a stronger MOCR show a smaller TTS   is larger than non-musicians’ and what produces the inter-individual variation of MOCR strength among musicians. Perrot and Collet  suggesteA short-duration instrument practice causes a temporary hearing deterioration among violinists. The larger exposure level in the left ear could account for the more pronounced hearing deterioration in the left ear than in the right ear, but it could not entirely explain the inter-individual variation of the hearing deterioration. On the other hand, MOCR strength assessed by CEOAE suppression could predict the size of the hearing deterioration moderately well. Our findings imply that the exposure level depending on the proximity of the instrument can partly determine the risk of hearing loss and that MOCR measurement is promising for assessing the risk of hearing loss among musicians.