The results were also statistically significant for preservation of postoperative hearing with pre- and postoperative GR 1–3 (p = 0.024). There were no differences in tumor sizes between the treatment and the control groups in men, whereas statistically significant larger tumors were observed in the female treatment group compared with the female control group. Prophylactic nimodipine is safe, and an effect for hearing preservation in 40 men with preoperative hearing ability of GR 1–4 was shown in this retrospective investigation. The imbalance in tumor size with larger tumors in females of the treatment group may falsely suggest a gender-related effect. Further investigations are recommended to clarify whether gender has impact on nimodipine’s efficacy.A 2016 published randomized multicenter phase III trial of prophylactic nimodipine treatment in vestibular schwannoma surgery showed only a tendency for higher hearing preservation rates in the treatment group. Gender was not included in statistical analysis at that time. A retrospective analysis of the trial considering gender, preoperative hearing, and nimodipine treatment was performed. The treatment group received parenteral nimodipine from the day before surgery until the seventh postoperative day. The control group was not treated prophylactically. Cochlear nerve function was determined by pure-tone audiometry with speech discrimination preoperatively, during in-patient care, and 1 year after surgery and classified according to the Gardner-Robertson grading scale (GR). Logistic regression analysis showed a statistically significant effect for higher hearing preservation rates (pre- and postoperative GR 1–4) in 40 men comparing the treatment ( There are ongoing discussions about the best management of sporadic vestibular schwannomas (VS) particularly regarding hearing preservation. Unilateral hearing loss is associated with severe deterioration of quality of life with impaired speech recognition in noise, lack of directionality of sound, and daily fatigue . In prinExcluding the gender-specific characteristics, the previous studies suggest a neuroprotective beneficial effect of the prophylactic nimodipine administration in VS and maxillofacial surgeries , 25. In Considering these divergent findings, an additional, retrospective analysis of the 2016 published multicenter, randomized phase III trial investigating gender-related differences in hearing preservation rates after VS surgery with prophylactic nimodipine treatment was performed .Regarding the detailed description of applied methods, the authors refer to the published paper on the phase III trial  . The mosThe prospective, open-label, 2-arm, randomized, and multicenter study was conducted in compliance with the principles of the Declaration of Helsinki and Good Clinical Practice guidelines and approved by the German Competent Authority. The study protocol was approved by the Ethics Committee and all local review boards of the participating institutions. All patients granted informed consent prior to inclusion. No changes to methods were performed after the trial was started.Adults from 18 years of age with an indication for VS surgery were included. Reasons for exclusion were contraindications against nimodipine, surgery for recurrent VS, pregnancy and lactation period, neurofibromatosis type 2, inoperability, preoperative facial nerve function grade VI according to the House-Brackmann (HB) grading scale , and parVS resections were performed by a retrosigmoid approach with neurophysiological monitoring . Nimodipine  was started the day before surgery and was continued until the seventh postoperative day. Individual dosage adjustments of nimodipine are described in the previous publication of the trial .Since there was a trend only for better hearing preservation rates  , only heEffect-measure statistics were applied to evaluate risk differences. Results are expressed as probability to retain hearing. We used generalized linear models (GLM) with the binomial distribution and identity link. Due to the low number of patients with tumor sizes of Koos 1 and 4, the analysis was performed in a subgroup of patients with tumor sizes of Koos 2 and 3 .In addition, hypothesis testing was conducted using chi-square and Student’s n = 21; female: n = 27) or to the control group  , and 4 patients had to be excluded . The primary endpoint of the underlying multicenter study  was “facThe authors refer to the published paper on the phase III trial .p = 0.028). As shown in Table The treatment and the control groups of both males and females were comparable in age, extent of resection, and preoperative cranial nerve function. There was no significant difference regarding tumor sizes between the treatment and the control group in men. In contrast, tumors were significantly larger in the female treatment group as compared with control group (p = 0.084). Adding a gender-treatment interaction to this model resulted in interaction contrast for hearing preservation RD of IC = 0.31 . Risk differences of hearing preservation with and without treatment were RD = 0.36  for male and RD = 0.05  for female patients.Effect-measure statistics evaluated risk differences of hearing preservation 1 year after surgery. A first GLM with terms for treatment, gender, tumor size, and gender-tumor size interactions yielded a risk difference of RD = 0.19  compared with the male control (n = 19) group , but not in females comparing 27 women in both groups (p = 0.077). Preoperative hearing of GR 1–2 was documented in 28 men (15 in the treatment and 13 in the control group). However, postoperative hearing ability of GR 1–2 was preserved in only five men (three patients in the treatment group). A robust statistical evaluation in men with preoperative GR 1–2 was therefore not possible.Logistic regression analysis similarly showed an effect for higher hearing preservation rates (pre- and postoperative GR 1–4 and GR 1–3) in the male treatment .Further subanalysis regarding tumor sizes showed statistically significant higher hearing preservation rates in males with Koos 2 tumors (p = 0.709). However, all females with Koos 4 tumors of the control group became deaf, whereas in two of eight (25%), hearing was preserved in the female therapy group. Concerning Koos 2 tumors of females, no statistical evaluation is reasonable since 12 of 15 Koos 2 tumors were randomized to the control group .The GLM with terms for treatment, gender, tumor size, and gender-tumor size interactions yielded a risk difference of RD = 0.02 , HB II in 10% of the males and in 15% of the females, HB III in 12% of the males and in 14% of the females, HB IV in 2% of the males and in 1% of the females, and HB V in 2% of the males and in 1% of the females.Logistic regression analysis showed no gender-related effect and no interaction between nimodipine and gender regarding preservation of facial nerve function. Facial nerve function 1 year after surgery was HB I in 74% of the males and in 69% of the females  3A4 have been reported. CYP3A4 is responsible for the metabolism of nimodipine , it may , the antiapoptotic effect of nimodipine pretreatment is associated with the activation of AKT and CREB by phosphorylation [Without considering tumor size and drug metabolism, the molecular mechanisms of this putative gender effect are unclear. In vitrorylation . Pan et rylation . In summNimodipine’s efficacy was predominantly observed in men with larger differences in patients with Koos 2 tumors. This might be interpreted as a gender-related nimodipine effect; however, statistical analysis did not reveal significant differences. The lack of a treatment effect in women could thus be mainly due to the imbalance of tumor sizes. Since hearing preservation rates are strongly influenced by tumor sizes, nimodipine is obviously more useful in tumor sizes with higher chances for postoperative hearing. This finding was limited to postoperative hearing classes GR 1–4 and GR 1–3, but not to GR 1–2. Due to the small sample size, further studies are needed to clarify if postoperative useful hearing (GR 1-2) also benefits from nimodipine treatment. For Koos 2 tumors in women, a robust statistical evaluation was not possible due to strongly unbalanced distribution with only 3 women in the treatment group.A benefit from nimodipine for facial nerve outcome was not observed. Considering that in both the female and the male groups’ facial nerve function was excellent or good (HB 1–2) 1 year after surgery in 84%, a potential effect of nimodipine may not be detectable. In contrast to the cochlear nerve, the facial nerve has the potential for regeneration, which may influence the analysis of nimodipine’s efficacy.In the presented retrospective analysis, prophylactic nimodipine was safe and preserved hearing predominantly in men with Koos 2 tumors but also in the male overall group. Whether the perioperative administration of nimodipine has also beneficial effects in women has to be clarified by further investigations. A possible gender effect should be investigated in further studies.


Sensorineural hearing loss is the most common sensory deficit. The etiologies of sensorineural hearing loss have been described and can be congenital or acquired. For congenital non-syndromic hearing loss, mutations that are related to sites of cochlear damage have been discovered . For cytomegalovirus infection or auditory neuropathies, mechanisms are also well known and well researched. Although the etiologies of sensorineural hearing loss may be evident for some patients, the damaged sites and pathological mechanisms remain unclear for patients with progressive post-lingual hearing loss. Metabolomics is an emerging technique in which all metabolites present in a sample at a given time are analyzed, reflecting a physiological state. The objective of this study was to review the literature on the use of metabolomics in hearing loss. The findings of this review suggest that metabolomic studies may help to develop objective tests for diagnosis and personalized treatment. SNHL is a chronic disease that can affect oral language development [Sensorineural hearing loss (SNHL) is the most common sensory deficit and a major public health problem . It is eSNHL may result from damage between the cochlea and the auditory cortex. It can be attributed to endocochlear lesions, neural impairment , or central hearing loss. Endocochlear damage is thought to be the leading cause of SNHL.SNHL can be congenital (present at birth) or acquired. Congenital SNHL may be due to hereditary or non-hereditary causes. Genetic SNHL (approximately 33% of cases) is classically divided into syndromic forms, such as Waardenburg’s syndrome and Jervell and Lange-Nielsen syndrome, and non-syndromic forms, e.g., autosomal dominant, autosomal recessive, X-linked, and mitochondrial. Genetic analysis has allowed for a better understanding of the pathophysiological mechanisms of SNHL. For example, some genes can affect inner ear homeostasis  or mechano-electrical transduction  coding for potassium channels). Non-hereditary causes include prenatal infections , prematurity, anoxia, preeclampsia, maternal diabetes, and drug or alcohol use during pregnancy.The major causes of acquired SNHL are aging, ototoxic drugs, genetic mutations, sudden hearing loss, and external factors  ,3. Howev2+ in hair cells, resulting in mitochondrial perturbation, apoptosis, and hair cell death. (3) The involvement of mitogen-activated protein kinase (MAPK), which is associated with signaling proteins that help to activate stress-activated protein kinase, a key mediator of oxidative stress and inflammation related to cell death. (4) Programmed hair cell death via the intracellular or extracellular pathway (caspase). (5) Infection and/or inflammation that induces apoptosis via an innate immunity-based response, promoting pathogen death. While many pathways leading to ciliated cell loss and nerve cell loss have been described and appear to be interrelated, there are currently no available diagnostic tools with which to characterize the site of injury in the cochlea and the intracellular mechanisms of SNHL [Five main pathways have been described for the cellular mechanisms involved in SNHL ,3: (1) Taspase).  InfectioThe cellular mechanisms involved in hearing loss can be intricately linked. Currently, there is no tool available with which to characterize the pathophysiological mechanisms in SNHL of an undetermined etiology, or to identify the lesion sites involved. This makes an accurate etiological diagnosis challenging, as no specific biomarker has been identified with which to characterize SNHL in terms of its origin, severity, and evolution. Identifying biomarkers linked to the metabolic mechanisms involved could allow for better characterization of SNHL, better prediction of the evolution of SNHL, and the personalized management of SNHL. Modifications to metabolic pathways can be investigated using metabolomic analysis, an emerging technique that can be used to identify metabolic biomarkers via the analysis of individual metabolites present in an organic sample. The “metabolome” refers to all metabolites of biological origin below 1500 daltons  , and proMetabolomic research appears to be a promising tool with which to study SNHL. Metabolomic analysis may provide additional data regarding the known etiologies of deafness and help to better categorize SNHL when etiology remains unknown. The objectives of this review are to alert readers to the growing interest in metabolomic approaches for the study of inner ear pathologies, to provide innovative scientific findings from metabolomics studies, and to discuss new experimental designs with which to better understand progressive hearing loss.This systematic review was written in accordance with the Cochrane Handbook for Systematic Reviews of Interventions Version 5.2.0 (June 2017) and followed the PRISMA  method . The PROhttp://www.isrctn.com; accessed on 12 September 2021), https://www.clinicaltrials.gov/ (accessed on 12 September 2021), and Scientific Electronic Library Online (SciELO). The search began in November 2020 and was completed in August 2021.This systematic literature review was conducted by searching medical literature analysis and retrieval system online , Excerpta Medical dataBASE , Cochrane Central Register of Controlled Trials , International Standard Randomised Controlled Trial Number (ISRCTN) registry  thesaurus was used to search the MEDLINE database. The search terms used were “Hearing loss” and “Metabolomic”, “Deafness” and “Metabolomic”, and “Metabolomic” and “Inner Ear”. The Cochrane Central Register of Controlled Trials (CENTRAL) was searched for “Hearing loss” and “Metabolomic”, “Deafness” and “Metabolomic”, and “Metabolomic” and “Inner Ear”.The study selection was carried out according to the “Participants, Interventions, Comparisons, Outcomes” (PICO) characteristics. Biomarker studies in patients with SNHL or animal models of deafness based on a metabolomic approach were included. The eligibility criteria were English language studies dealing with metabolomic analysis related to hearing loss, regardless of the fluid that was analyzed. All articles without metabolomic analysis or without the analysis of hearing loss were excluded.Two authors independently collected papers based on titles, abstracts, and keywords. The research was conducted according to the exclusion and eligibility criteria defined above. The data extraction method was blinded and double-reviewed. In the case of disagreement, the two authors met to reach a consensus, and the final adjudication was based on the expert opinion of the senior author.Here, the methodologies of the studies are described, focusing on the analytical technique used, the types of samples studied, and the types of experiments performed. The main results concerning the metabolites found and the metabolic pathways are also presented.In the included studies, different types of samples were subjected to metabolomic analysis. Perilymph was used in six of thirteen studies; perilymph has the advantage of being closer to metabolic mechanisms because hair cell bodies are immersed in this fluid ,14 The vBlood plasma was collected in three studies to investigate cisplatin-induced changes at the systemic level , the metUrine, composed of the final products from metabolic breakdown, was analyzed in two studies ,23. The In another study, rat brain tissue was subjected to metabolomic analysis to better understand metabolic changes at the cortical level in the genesis of tinnitus induced by acoustic trauma .One study investigated dexamethasone-induced changes in a ciliated cell-like HEI-OC1 cell line . This waDifferent types of samples are associated with different functions and purposes. Perilymph was the most commonly used fluid in the studies presented, given the hypothesis that some metabolomic changes could be observed in perilymph following induced SNHL. Perilymph is located in the inner ear, close to pathophysiological events; however, its volume is small, and collection requires invasive sampling. Perilymph samples may be contaminated, and volumes may be insufficient . For theThe analytical methods used to perform the metabolomic analyses were NMR spectrometry in one study , GCMS inLCMS is the most commonly used approach in different studies. No consensus exists on the analytical method that is most appropriate in this context; the choice depends on the volume of the sample inherent to its nature and the targeted metabolic pattern. Consequently, the exploration of the perilymph by LCMS seems to be the most informative strategy with which to analyze the pathophysiology of hearing lossFour studies investigated metabolomic changes in animals following noise exposure.One study performed a metabolomic analysis of the entire cochlea located in the temporal bone of mice after noise-induced trauma . The aimIn one study, metabolic brain changes were investigated in rats following sound exposure with a sound level of 110 dB at 16 kHz for 1 h . Auditor2 gas [2 gas, four to H2 gas alone, and two were not exposed. The results showed an increase in eight metabolites  and a decrease in stachydrine and homostachydrine in the noise-exposed group, compared to the H2-exposed group. Stachydrine and homostachydrine decreased in the noise-exposed group alone, with similar levels observed in the noise-exposed with H2 gas, H2 gas alone, and unexposed groups. Acyl-carnitines were increased in the noise-exposed group compared to the noise-exposed group with H2 gas, and were involved in oxidative stress. Audiometric results were significantly improved in guinea pigs exposed to noise combined with H2 gas compared to noise alone. These two articles showed metabolic differences following noise exposure in guinea pig perilymph; however, the differences did not allow for any conclusions.Two studies focused on noise-induced changes in the metabolomic analysis of perilymph ,17. One 2 gas inhalation on cisplatin-induced ototoxicity in 34 guinea pigs via a morphological, immunohistochemical, and metabolomic analysis of the perilymph [2 gas inhalation showed an otoprotective effect against cisplatin-induced toxicity at the auditory level, as well as immunohistochemically. Subsequently, another study examined metabolomic changes in guinea pig blood serum after an intravenous injection of cisplatin or NaCl [l-acetylcarnitine, ceramide, and cysteinylserine.Metabolomic analysis following cisplatin-induced ototoxicity has been the subject of two studies ,20. One erilymph . The autThe literature review found five out of thirteen articles on metabolomic analysis and SNHL in humans.One study performed a metabolomic analysis of urine to look for a correlation between presbycusis and renal dysfunction as defined by traditional Chinese medicine. The authors found differences in 23 metabolites between subjects with presbycusis and those with normal hearing. After analysis by the Kyoto Encyclopedia of Genes and Genomes (KEGG) software, these metabolites were found to be involved in the metabolism of glutathione, amino acids, glucose, N-methyl-D-aspartate (NMDA), and gamma-aminobutyric acid (GABA) receptors . AnotherThe identification of specific biomarkers of hearing impairment in urine may help to better understand the pathophysiological mechanisms and to classify the etiologies of sudden or mitochondrial deafness.One article performed a feasibility study for the metabolomic analysis of a small amount of human perilymph (<1 μL) collected during cochlear implantation using LCMS . Ninety-One study performed an exome and plasma metabolome analysis of a child with mitochondrial kidney disease (RMND1 mutation) and deafness. Ceramide accumulation was observed, which provided a better understanding of the pathophysiological mechanism involved . AnotherOne study investigated dexamethasone-induced changes in a ciliated cell-like cell line at the metabolic level under two growth conditions . They weOf the seven papers, two investigated perilymph, two urine, two serum, and one HEI-OC1 cells in vitro. With the exception of two papers ,22, the Most of the included metabolomic studies investigated noise-induced hearing loss ,19,22,242 gas [Metabolomic changes following cisplatin-induced ototoxicity were investigated in two studies on guinea pigs using perilymph analyzed by LCMS with the addition of H2 gas  and bloo2 gas . ChangesIn another study, sudden hearing loss was explored by the metabolomic analysis of urine using NMR spectrometry . The resIn two studies with humans, the metabolomic analysis of perilymph was performed by LCMS for cochlear implant candidates and the perilymph was sampled during surgery ,15. Too Another research axis using metabolomic analysis could be Ménière’s disease. Previous studies have identified endolymphatic hydrops in these patients. However, the pathophysiological mechanisms are not yet fully understood, although genetic, vascular, immunological origin, the aberrant expression of aquaporins, or a combination of causes have been proposed . SeveralMetabolomic analysis is an innovative and recent technique. The analysis approach depends on the type and volume of the sample, and the type of metabolites sought. Due to inter-individual variability and the role of environmental conditions , it is nAmong the different types of samples analyzed in the included studies, perilymph is likely to be in the closest proximity to pathophysiological events; hair cell bodies are bathed in perilymph. Metabolomic analysis has shown a difference in metabolites between blood and perilymph in the baseline state . There iCurrently, there are no comparative metabolomic studies on the different fluids that can be sampled to determine which fluid is most promising. In the future, it would be interesting to define the best sample to use in terms of ease of collection, sensitivity, and specificity in the context of SNHL.The evolution of analytical techniques has allowed for the characterization of a larger number of metabolites. In 2015, 77 metabolites were found in perilymph with GCMS ; in 2019In the future, it will be necessary to conduct complementary studies in animal models to discover metabolic profiles linked with hearing loss phenotypes. For example, noise-induced hearing loss can be explored by comparing normal perilymph to perilymph following noise-induced hearing loss; differences in metabolic profiles may explain the pathophysiology.Sampling the perilymph from a large cohort of patients might allow for the identification of biomarkers that could explain the pathophysiological mechanisms associated with SNHL or be related to the duration and etiology of SNHL. For example, studies of perilymph during cochlear implantation have provided interesting information on the etiologies of SNHL and on the duration of hearing loss, allowing for improved management of SNHL ,28. FurtTo date, there are few studies regarding metabolomic analysis and hearing loss, and even fewer on the metabolomic analysis of perilymph. Furthermore, the metabolomic analysis of perilymph in normal hearing humans is not ethically feasible due to the induction of cophosis during sampling. This could be overcome by proposing a metabolomic study of perilymph in organ donors. The studies conducted to date in humans have all been carried out on patients receiving cochlear implantation for profound deafness; however, the etiology and duration of the deafness are not always easy to define. In addition, most of the experimental studies have been performed on small animals . It would be interesting to study the perilymph in larger mammals, such as sheep or dogs, which are physiologically and anatomically similar to humans ,33. For The current methods for the exploration of deafness with subjective and objective audiometry do not allow for the precise identification of damaged sites within the cochlea for patients where the etiology of SNHL is unknown. The purpose of this review was to assist investigators and Ear, Nose and Throat (ENT) surgeons to better understand the value of metabolomic analysis for patients with SNHL. Further research is needed to define normal and pathological states using metabolomic analysis. This will help to define biomarkers linked with etiology, which has great clinical relevance. Metabolomic analysis will also increase our understanding of the pathways involved and foster the development of new therapies. One of the challenges faced in these future studies will be to sample the perilymph without adverse effects.


N = 1103). Under statistical control for demographics and in agreement with pre-registered hypotheses, lower neuroticism and higher extraversion independently explained superior self-reported noise resistance, speech-hearing ability and acceptable background noise levels. Surprisingly, objective speech-in-noise recognition instead increased with higher levels of neuroticism. In turn, the bias in subjectively overrating one's own hearing in noise decreases with higher neuroticism but increases with higher extraversion. Of benefit to currently underspecified frameworks of hearing in noise and tailored audiological treatments, these results show that personality explains inter-individual differences in coping with acoustic noise, which is a ubiquitous source of distraction and a health hazard.Acoustic noise is pervasive in human environments. Some individuals are more tolerant to noise than others. We demonstrate the explanatory potential of Big-5 personality traits neuroticism  and extraversion  on subjective self-report and objective psycho-acoustic metrics of hearing in noise in two samples (total  Differeosf.io/fgyj9), lower neuroticism and higher extraversion were associated with better subjective hearing in noise. To our surprise, these relationships reversed for objective hearing in noise. In other words, we found underrated (i.e. lower subjective than objective) hearing in noise with higher neuroticism and overrated hearing in noise with higher extraversion. We focused particularly on these two personality dimensions since both have been consistently related to subjective hearing in noise in the literature .The goal of the present study was to establish robust estimates of the association of personality traits neuroticism and extraversion with recognized subjective and objective measures of hearing in noise in a large sample, under statistical control for demographic factors and other personality traits. In agreement with pre-registered hypotheses (. 2N = 1103 participants (a). We tested the hypotheses that lower neuroticism and higher extraversion are both associated with better subjective and objective hearing in noise.We conducted an online study to assess the explanatory power of Big-5 personality traits  for established, subjective and objective measures of hearing in noise in a sample of icipants a. We tes. 2.1Neuroticism = 0.73, CAExtraversion = 0.8, CAOpenness = 0.67, CAAgreeableness = 0.56, CAConscientiousness = 0.6). As common and as expected, the Big-5 personality dimensions showed small-to-moderate pairwise correlations  but were sufficiently independent to investigate their individual associations with noise tolerance.In agreement with previous research , a shortWNSS = 0.88), (ii) speech-hearing ability using the speech, spatial and qualities of hearing scale  and (iii) the self-adjusted, maximally acceptable noise level (ANL) while listening to speech. Additionally, a psycho-acoustic test quantified objective hearing in noise as (iv) the noise level at which listeners correctly reported 50% of presented digit triplets . Small pairwise correlations between these tests  confirmed that they assess partly independent facets of hearing in noise.Separate facets of hearing in noise were measured using four tests. Subjective self-report measures of hearing in noise were assessed using (i) noise resistance on the Weinstein noise sensitivity scale  but with higher extraversion . To estimate the size of these effects, we report the common language (CL) effect size, which here indicates the following: for a randomly sampled person with above-average self-reported noise resistance (WNSS), the probability for an above-average neuroticism score is about 41%, but about 56% for an above-average extraversion score.In agreement with our hypotheses and with previous evidence from smaller samples e.g. –16), biv, biv16])r = −0.174; p < 0.001; CL = 0.444; ANL: r = −0.153; p < 0.001; CL = 0.451) and to higher extraversion . Interestingly, and against what we had hypothesized, this pattern of results showed a tendency to reverse for objective hearing in noise in the digit-triplet-test (DTT): Correct report of digit triplets at higher background noise levels was associated with higher neuroticism . However, extraversion was not significantly associated with DTT performance .Beyond previous research, we found that better self-reported speech-hearing ability (SSQ) and higher self-adjusted acceptable levels of background noise (ANL) were related to lower neuroticism , sound presentation (speakers versus headphones) and self-reported hearing loss . Goodnesβ = −0.279; p < 0.001; CL = 0.416), speech-hearing ability  and the acceptable noise level , but positively predicted objective performance of speech-in-noise recognition . Conversely, extraversion positively predicted noise resistance , speech-hearing ability  and the acceptable noise level , but was not significantly related to objective hearing in noise in the digit-triplet-test .Overall, results of multiple regression analyses largely converged with bivariate correlations: under statistical control for other predictors, neuroticism negatively predicted noise resistance , we estimated the replicability of obtained results by division of the total sample into the two cohorts of participants . figure 2a, top right). However, random sub-sampling from the total sample (10 000 sub-samples of Nsub-sample = 550) revealed that this relation was somewhat overestimated in cohort one (only 3.28% of sub-samples resulted in a βsub-sample ≥ βcohort 1) and underestimated in cohort two (only 2.43% of sub-samples resulted in a βsub-sample ≤ βcohort 2). Most importantly, across all sub-samples, the relation of neuroticism and objective hearing in noise (DTT) was significantly positive .Following our pre-registered analysis plan  versus an objective test (DTT) of hearing in noise .a), by extraversion (b) and—for comparison—by age (c). Individuals expressing better subjective than objective hearing in noise can be considered to overrate their hearing in noise, while the opposite (underrated hearing in noise) holds for individuals expressing better objective than subjective hearing in noise.Notably, higher neuroticism predicted lower scores of subjective, but higher scores of objective hearing in noise. The reverse pattern was observed for extraversion. Compared with surrogate data with permuted personality scores, neuroticism and extraversion significantly displaced participants from an equilibrium of subjective and objective hearing in noise. That is, higher scores on neuroticism were accompanied by underrated own hearing in noise. Instead, higher scores on extraversion were associated with overrated hearing in noise. By contrast to personality, higher age reduced objective hearing in noise but left the subjective hearing in noise largely unaffected.extraversionz-score − neuroticismz-score explained inter-individual differences in subjective hearing in noise . This effect corresponds to a considerable displacement along the vertical axis in figure 3d. Although smaller in effect size, the extraversion–neuroticism difference score also explained objective hearing in noise , corresponding to a small displacement of participants along the horizontal axis in figure 3d. In other words, relatively higher extraversion and lower neuroticism explain the tendency to subjectively overrate one's objective hearing in noise.For statistical analysis, we employed a new joint extraversion–neuroticism predictor in additional multiple regression analyses: the normalized difference e shows median splits along both personality dimensions that divide participants with scores below the median (−) from participants with scores above the median (+).To further illustrate this dissection along the personality dimensions neuroticism (N) and extraversion (E), figure 3β = 0.116; p < 0.001; CL = 0.538) but with lower openness  and conscientiousness scores . Furthermore, better self-reported speech-hearing ability in the SSQ was associated with higher openness  and conscientiousness scores .Beyond personality traits neuroticism and extraversion, . 3N = 1103; 18–74 years) and employed statistical control of demographics and other personality traits. Higher neuroticism and lower extraversion were associated with substantially reduced subjective hearing in noise but with somewhat increased objective hearing in noise. These relations were largely orthogonal to the effect of older age, which primarily explained decreasing objective hearing in noise.Humans differ considerably in their tolerance of acoustic noise in the environment. Here, we investigated the association of Big-5 personality traits neuroticism and extraversion with subjective and objective measures of hearing in noise. We used an online experiment to recruit a large, age-varying sample of participants (. 3.1Our results replicate well-established associations of neuroticism and extraversion with subjective (i.e. self-reported) noise tolerance on the WNSS . Our effThe relationship of personality and scores on the WNSS are unsurprising, given that this scale was designed to assess affective reactions to noise  and thatA major goal of the present study was to test whether neuroticism and extraversion would also explain scores on hearing in noise tests used in audiological screening and hearing aid fitting. Our results clearly showed that this was the case: Higher neuroticism and lower extraversion independently explained lower self-reported speech-hearing ability  and lowThe difficulty of speech comprehension in noise is a common complaint, which has primarily been associated with older age, hearing loss  and lowe. 3.2The DTT is a widely used audiological screening tool available in many languages . It is aIn the present study, better objective hearing in noise was to large extents explainable by younger age and use of headphones (versus tabletop or in-built loudspeakers). Opposite to our hypotheses, neuroticism exhibited a small positive association with objective hearing in noise under statistical control for demographic information and sound presentation as well as other personality traits: if a person sampled at random scored above average on neuroticism, the probability of their objective hearing in noise being also above average is about 52%. It is obvious that the association of DTT scores with neuroticism was considerably smaller than their association with age, which is known to covary with hearing thresholds. Since hearing sensitivity (i.e. audiometry) was not included in the present study, we cannot differentiate whether higher neuroticism relates to lower (i.e. better) hearing thresholds or to better hearing in noise, which are both captured by the DTT.Together, the differential relations of neuroticism with subjective versus objective hearing in noise have important implications for audiological rehabilitation: it might be instructive to imagine an audiologist who encounters over a few months' time or so 100 customers with above-average neuroticism. Based on the present results, the audiologist would expect 63 of them to also be of sub-average self-reported hearing in noise (and not 50)—but should not be surprised if 53 of them in fact perform above average in an objective hearing in noise test . Note thDiverse associations between neuroticism and extraversion on the one hand and sensory processing on the other have been reported in the literature. For instance, lower extraversion correlates with lower (i.e. better) auditory thresholds ,48 and u. 3.3High levels of neuroticism have traditionally been considered a predisposition for negative affect –54, whicIn theory, individuals with poorer subjective hearing in noise might choose better hardware for sound presentation, which in turn might improve objective measures of hearing in noise. This should result in a negative correlation of subjective and objective hearing in noise, which was not the case in the present data. Furthermore, our statistical models did control for quality of sound presentation to some extent (headphones versus in-built or tabletop speakers). However, a caveat of the present online study was the unfeasibility to control the level of ambient noise settings in which individual participants performed the study.We consider three views that explain the neuroticism-objective hearing in noise relationship. First, the view that unstable introverts  are characterized by higher levels of neural excitation and lower inhibition –59 and sThird, and more promisingly, individuals with higher neuroticism might allocate more cognitive resources , which eexpended listening effort or listening engagement [β = 0.099; p = 0.031) but not of experienced mental demand . These results are consistent with the view of higher expended listening effort being associated with higher levels of neuroticism.In the context of the hearing, the allocation of cognitive resources is referred to as gagement –70. In tper se, but rather that they allocate more cognitive resources, which eventually results in lower speech reception thresholds in the DTT.Reminiscent of trait neuroticism, the listening effort construct has been related to both affect and cognition . RecentlOf note, objective hearing in noise was significantly associated with only one of the three subjective hearing in noise tests (SSQ). This agrees with and extends the literature on discrepancies between subjective and objective hearing ability ,19,20. HAs a direct consequence of the differential relation of personality with subjective versus objective hearing in noise, higher neuroticism associates with a tendency to underrate one's own hearing in noise, whereas the opposite holds for higher extraversion. Put differently, subjective and objective hearing in noise tests are of limited validity: the former might be sensitive to affect and the latter to the allocation of cognitive resources (i.e. listening effort), both of which vary as a function of neuroticism. Future research should (i) explore to what degree the link between neuroticism and performance in objective hearing in noise tasks is mediated by listening effort, (ii) test whether the observed associations with personality generalize to non-auditory sensory processing sensitivity  and . Participants completed all tests of the experiment in the browser. After piloting the functionality of the online study, all procedures were pre-registered with the Open Science Framework before data collection started (osf.io/fgyj9).The present experiment was implemented using the online platform To make sure that a participant's computer was playing audio files at an appropriate sound intensity, each participant was presented with a spoken German digit triplet and had to select the corresponding triplet among six options in the beginning of the online study. The study continued only in case this task was successfully accomplished.. 5.2Participants were recruited via crowdsourcing. The study was only available to participants who, according to their registration with the crowdsourcing platform, were German. They provided informed consent and were financially compensated by €3.60 if they completed all tests of the study. Experimental procedures were approved by the ethics committee of the University of Lübeck.N = 500. To be able to replicate the obtained results, the planned sample size in cohort two was also at least N = 500. Participants who completed at least the demographic questionnaire and one additional test were included in the final sample, which were Ncohort 1 = 554 recorded in December 2019 and January 2020, and Ncohort 2 = 549 recorded in April and May 2020, resulting in a complete sample of Ntotal = 1103 .Data were collected in two cohorts. The planned sample size in cohort one was at least  —Demographic information. Participants provided information on their gender , age, number of siblings, education , musicality , knowledge of existing hearing loss (yes/no), use of hearing prostheses (no/hearing aid/cochlear implant/other) and the type of sound presentation of their computer . Of note, no additional direct measures of hearing sensitivity  were in —Personality questionnaire. Big-5 personality dimensions  were assessed using the short BFI-S questionnaire [ionnaire ,81, comp —Noise resistance. Noise resistance was assessed using a German version [ version  of the W version , compose —Speech-hearing ability. Six items of the speech, spatial and qualities questionnaire  were us —ANL test. We implemented an adapted version of the ANL test [Eine kurze Geschichte der Menschheit; Yuval Noah Harari) narrated by a male voice, which was embedded in 10-talker babble noise .ANL test . Particited from ). Using  —DTT. To obtain an objective measure of speech-in-noise reception, we used an adapted version of the DTT [50). To this end, the test started at an SNR of 0 dB. For every subsequent trial, the SNR was raised by 2 dB in case of an incorrect response on the previous trial and decreased by 2 dB in case of a correct response on the previous trial. Participants completed 24 trials. The SRT50 was calculated as the average SNR across the last 6 trials. Participants completed two runs of the DTT, with mean SNR values of MDTT1 = −11.03 dB and MDTT2 = −11.42 dB. the DTT . On everTo better agree with the assumption of normality, obtained scores (DTTraw) were shifted into the range of positive values and log-transformed using the formula: DTT = log10 (DTTraw + 16) for further statistical analyses. Since previous research has shown that performance in the DTT reaches stable performance levels in the second run of the test [Every participant completed the questionnaire on demographic information first, followed by the remaining five tasks  in randomized order to avoid order effects.the test , we usedAfter the DTT, participants in cohort two completed two short questionnaires to assess some aspects related to listening effort. Two questions  were in. 5.3http://www.jamovi.org) are available online (osf.io/fgyj9). Further analysis scripts and raw data are available from the corresponding author upon request.A table including pre-processed data and an accessible script for most important data analyses in the open access software Jamovi .. 5.4fitlm function). Standardized coefficients were obtained by z-transforming all variables in the regression model. The 95% CIs for all effects (i.e. standardized regression coefficients) can be found in the online data (osf.io/fgyj9).We used two-sided statistical test. Planned statistical analyses were supposed to test the relation of personality dimensions and different facets of hearing in noise. To this end, bivariate Pearson correlations and multiple linear regression analyses were performed as implemented in Jamovi (v. 1.1.9) and in Matlab ; using tp-values for the number of performed tests but instead tested the replicability of all effects by splitting up the dataset into the two cohorts. Bivariate Pearson correlations among predictors in regression models were small to moderate  and the variance inflation factors of regression models were small , such that multicollinearity was not an issue.The major statistical analysis comprised four multiple regression models . To contMissing values on individual tests were not imputed. Thus, any case (participant) with a missing value was discarded from the respective correlation analysis or regression model. The number of observations for the four multiple regression models  were: WNSS , SSQ , ANL  and DTT .N = 550, sampled at random without replacement) from the whole sample of participants, followed by the calculation of a multiple linear regression to predict DTT performance. Standardized coefficients for the relation of neuroticism and DTT performance were extracted (βsub-sample) and corresponding bounds of the 95% CI were estimated by the 2.5th and 97.5th percentiles of the distribution of βsub-sample.To estimate the internal consistency of the relation of neuroticism and performance in the DTT , we used a sub-sampling approach. We drew 10 000 sub-samples  effect size , which wz-transform of hearing in noise test scores , personality dimensions neuroticism and extraversion, and age. For purpose of visualization (a–c), we generated scatterplots of subjective hearing in noise  as a function of objective hearing in noise (DTT), followed by colour-coding of individual participants' data points by neuroticism, extraversion and age. For statistical analysis, we permuted neuroticism, extraversion and age across participants (10 000 permutations), followed by calculation of the weighted centre of gravity (COG) of data in the objective-versus-subjective hearing in noise space. For instance, the x- and y-coordinates of the neuroticism-weighted COG of objective (DTT) versus subjective (WNSS) hearing in noise was calculated according toFurthermore, we performed unplanned analyses to statistically test the relation of personality with diverging scores on subjective versus objective hearing in noise. To this end, we calculated normalized scores by lization a–c, we gerror_ellipse for Matlab [Elliptical 95% CIs of surrogate COGs were calculated using the function r Matlab .Click here for additional data file.


Electrophysiological diagnosis of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) may be challenging. Thus, with the aim ofproviding some practical advice in electrophysiological approach to a patient with suspected CIDP, we analyzed electrophysiological data from 499 patients enrolled inthe Italian CIDP Database.We calculated the rate of each demyelinating feature, the rate of demyelinating features per nerve, the diagnostic rate for upper andlower limb nerves, and, using a ROC curve analysis, the diagnostic accuracy of each couple of nerves and each demyelinating feature, for every CIDP subtype.Moreover, we compared the electrophysiological data of definite and probable CIDP patients with those of possible and not-fulfilling CIDP patients, and by a logisticregression analysis, we estimated the odds ratio (OR) to make an electrophysiological diagnosis of definite or probable CIDP.The ulnar nerve had the highestrate of demyelinating features and, when tested bilaterally, had the highest diagnostic accuracy except for DADS in which peroneal nerves were the most informative.In possible and not-fulfilling CIDP patients, a lower number of nerves and proximal temporal dispersion (TD) measurements had been performed compared to definiteand probable CIDP patients. Importantly, OR for each tested motor nerve and each TD measurement was 1.59 and 1.33, respectively.Our findingsdemonstrated that the diagnosis of CIDP may be missed due to inadequate or incomplete electrophysiological examination or interpretation. At the same time, thesedata taken together could be useful to draw a thoughtful electrophysiological approach to patients suspected of CIDP.The online version contains supplementary material available at 10.1007/s10072-021-05321-z. Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a chronic and disabling disease with a range prevalence of 0.8–8.9 cases per 100,000 and incidence of 0.2–1.6/100,000 .Diagnosis of CIDP is made by demonstrating peripheral nerve demyelination, commonly by electrophysiological testing. Hence, several sets of electrophysiological criteria for diagnosis of CIDP have been proposed, and the most widely accepted criteria are those recommended by the European Federation of Neurological Societies and Peripheral Nerve Society (EFNS/PNS). Based on the number and electrophysiological features , motor nerve conduction velocity (MNCV) slowing, temporal dispersion (TD), probable or definite conduction block (CB), and absent or prolonged F-wave), three diagnostic electrophysiological classes are identifiable: definite, probable, and possible CIDP –7.The diagnosis may be easy when demyelinating signs are widespread, while it may be challenging when demyelination is limited to few or specific nerves or nervous tracts  –10.Accordingly, in clinical practice, neurophysiological examination in search of demyelinating signs may be time-consuming and eventually not decisive, so making tricky the diagnosis and delaying the start of a proper treatment.Thus, having the opportunity to access the Italian CIDP Database , 4, we hMoreover, we analyzed the electrophysiological data from patients who, although diagnosed as having CIDP based on clinical and supportive features, did not fulfill the electrophysiological criteria for demyelination.This was a retrospective multicenter cohort study in a large sample of CIDP patients implementing a web database  to collect demographical, clinical, and electrophysiological data from patients diagnosed and followed by 22 centers throughout Italy with expertise on CIDP.The ethical committee of each participating center approved the study and all the patients gave written informed consent.Clinical and neurophysiological data were obtained by an experienced neurologist with a neuromuscular subspecialty. Verification of the diagnostic data for all of the enrolled patients was centralized in the database coordinator center (Humanitas). Data monitoring included diagnosis revision, suspect double entries, missing data, and plausibility checks. Patients with an alternative diagnosis for the neuropathy, increased titers of anti-myelin-associated glycoprotein IgM antibodies, or without available nerve conduction studies were excluded .For the current study, all electrophysiological data  were further evaluated independently and after discussed all together by three expert neurophysiologists  in the center of Naples. Based on EFNS/PNS criteria for demyelination, patients were classified into four diagnostic categories: “definite,” “probable,” “possible,” and “not-fulfilling” electrophysiological diagnosis of CIDP. Afterward, the electrophysiological results were discussed with the coordinating center  and combined with clinical and laboratory data. The neurophysiological study was contemporaneous with clinical classification of CIDP.We calculated in CIDP patients  the rate of each demyelinating feature, the rate of demyelinating features per nerve , and the diagnostic rate as established by EFNS/PNS for upper and lower limb nerves. To exclude median neuropathy at the wrist , distal motor latency in the median nerve was not considered in our analysis when associated with slowing of sensory nerve conduction velocities or absence of sensory action potential.We ran a receiver operating characteristic (ROC) curve analysis to measure the diagnostic accuracy for each possible couple of nerves and for each demyelinating feature for CIDP diagnosis . For ROC curve analysis, we used as diagnostic gold standard the diagnosis of CIDP based on the combination of clinical features, neurophysiology data, and supportive criteria.We also ran ROC curve analysis separately for typical CIDP and for each atypical CIDP subtype , distal acquired demyelinating symmetric (DADS), pure sensory ,and pure motor). Values for the area under the curve (AUC) obtained from ROC curve analysis above 0.8 were considered excellent .Moreover, we compared the electrophysiological approach  among the patients with definite and probable CIDP and those with possible CIDP and not fulfilling electrophysiological CIDP criteria. We decided to analyze together possible and not-fulfilling CIDP patients as “possible” category is a very poorly specific category .Thus, through a logistic regression analysis, we estimated the odds ratio (OR) of differences in electrophysiological approach applied to patients with definite and probable CIDP with respect to that applied to possible and not-fulfilling groups and then we used a multivariable logistic regression analysis for estimating the independent weight of differences emerged from the univariate logistic regression analysis.Lastly, we evaluated the percentage of diagnosed patients (definite and probable) according to the number of tested nerves  and the number of proximal temporal dispersion measurements. After finding that the percentage of CIDP diagnosis progressively increased up to six tested nerves while after (more than six nerves) the diagnostic rate remained unchanged, the value of 6 (nerves) was set as a cut-off and as a reference value for a regression analysis.Each regression analysis was controlled for age and sex, and odds ratios and 95% confidence intervals were reported.The study was approved by the ethical committee of each participating center.Written informed consent was obtained from all participants at enrollment.Any data not published within this article is fully available previous anonymization by request from a qualified investigator.We analyzed data from 499 (out of 545) patients included consecutively into the Italian CIDP Database from January 2015 to January 2019. Forty-four patients were excluded from the analysis for the presence of a different cause for neuropathy or unavailable neurophysiological data . Two patFirst, we divided our dataset according to electrophysiological data: 352/499 patients 70.6%) were classified as definite CIDP, 10/499 (2%) as probable CIDP, 57/499 (11.4%) as possible CIDP, and 80/499 (16%) as CIDP not fulfilling the EFNS/PNS electrophysiological criteria [% were clFor patients that did not meet EFNS/PNS electrophysiological criteria for demyelination, the diagnosis of CIDP was discussed among the neurophysiologist panel, the database coordinator center, and the physician who had enrolled the patient. Therefore, these patients were considered affected by CIDP because, though they did not have nerve conduction abnormalities , MNCV was reduced in 285/419 (68%), proximal TD was present in 183/419 (43.7%), probable and definite CBs were observed in 46/419 (11%) and 134/419 (32%), respectively, and F-wave was absent or prolonged in 145/419 (34.6%) patients. Prolonged distal compound muscle action potential (CMAP) duration was excluded from this analysis because of heterogeneity of data.The highest rate of demyelinating features was observed in the ulnar nerve . The frequency for each demyelinating feature per nerve  is reported in Table Overall, at least one nerve with demyelinating features was observed in 65.7% of patients considering only the upper limbs, and in 64.5% of patients considering only the lower limbs. However, the analysis per subtypes showed that the rate of demyelinating features was comparable between upper and lower limbs for typical and sensory forms while it was higher in upper than lower limbs in MADSAM (70.6 vs 58.9%) and motor (64.7% vs 35.3%) subtypes and it was higher in lower limbs in DADS phenotype (73.8% vs 40.5%) Table .Table 2The ROC curve analysis for nerves showed the highest diagnostic accuracy for ulnar nerves and the lowest for tibial nerves Table  in the oIn typical CIDP, as well in MADSAM, sensory, and motor subtypes, the ROC curve analysis showed the highest diagnostic accuracy for ulnar nerve evaluated bilaterally, followed by the combination of ulnar and peroneal nerves, independently from the side . An excellent AUC was reported for the combination of ulnar and peroneal nerves, taken together (AUC: 0.95). Detailed data for each subtype are reported in Supplementary Table The ROC curve analysis for demyelinating features showed the highest diagnostic accuracy for MNCV slowing (AUC: 0.8401) Fig. .Fig. 2RThe patients with a diagnosis of definite and probable CIDP with respect to those with possible CIDP or not fulfilling electrophysiological CIDP criteria had a higher median value (6 vs 4) of tested motor nerves as a whole and also for upper (3 vs 2) and lower limbs (3 vs 2) considered separately.In the entire cohort of patients, the median number of tested motor nerves was 6 , 2 in the upper limbs  and 3 in the lower limbs .In definite CIDP patients, the median number of tested motor nerves was 6 , 3 in the upper limbs  and 3 in the lower limbs .In probable CIDP patients, the median number of tested motor nerves was 6 , 3 in the upper limbs  and 3 in the lower limbs .In possible CIDP patients, the median number of tested motor nerves was 4 , 2 in the upper limbs  and in 2 in the lower limbs .In CIDP patients not fulfilling the diagnostic criteria, the median number of tested motor nerves was 4 , 2 in the upper limbs (mean 1.9 SD 1.2) and 2 in the lower limbs .z: 7.2; p<0.00) meaning that for each nerve added to neurophysiological evaluation there was an additional 60% probability to make an electrophysiological diagnosis of definite or probable CIDP  than lower limbs .The univariate logistic regression analysis showed an odds ratio (OR) of 1.59 .In possible CIDP patients as well in those not fulfilling CIDP electrophysiological criteria, proximal TD was calculated less times compared to definite and probable CIDP patients ; z: 6.52; p<0.00), meaning that for each TD measurement, there was an additional 33% probability to reach an electrophysiological diagnosis of definite or probable CIDP .The logistic regression analysis demonstrated an OR of 1.33  and showed the best diagnostic accuracy (AUC: 0.8401).The most informative nerve was the ulnar nerve, which had the highest rate of demyelinating features , as well as ulnar nerve tested bilaterally, which showed the highest diagnostic accuracy in CIDP population as a whole (AUC: 0.84) and in each CIDP category except for DADS, in which peroneal nerves followed by tibial nerves were the most informative nerves.Lower rate of demyelinating features as well as lower diagnostic accuracy (AUC: 0.78) of median with nerve respect to ulnar nerve could depend on the exclusion of prolonged DML from our analysis as a possible sign of carpal tunnel syndrome, when coupled with a consistent sensory conduction study. This certainly reduces the diagnostic potential of the median nerve and it should be properly considered when performing the electrophysiological exam.On the other hand, the fact that the lower limb nerves had the most diagnostic accuracy in DADS patients is certainly consistent with a length-dependent neuropathy such as DADS neuropathy.When facing a suspected CIDP patient, it is well known that if electrophysiological criteria for demyelination are not satisfied, the diagnosis as well as the access to treatment may be delayed or denied. Thus, in order not to miss early diagnosis of CIDP, several diagnostic criteria have been proposed and the most widely accepted criteria are those recommended by EFNS/PNS which appear to have the best combination of sensitivity 73%) and specificity (90%) for the diagnosis of CIDP –7. Impor3% and spAlthough expected, our analysis certainly demonstrated that the diagnosis of CIDP may be missed due to inadequate or incomplete electrophysiological examination or interpretation. Indeed, in possible and not-fulfilling CIDP patients, a lower number of motor nerves  and of TD  measurements had been performed compared to definite and probable CIDP patients.Consistently, the calculation of odds ratio revealed that each nerve added to examination increased the probability (OR: 1.59) to reach an electrophysiological diagnosis of definite and probable CIDP, especially when nerves of the upper limbs were tested (OR: 1.97). However, the investigation of more than 6 nerves did not improve further the diagnostic rate of CIDP.In addition, for each proximal TD measurement, there was an additional 33% probability to reach an electrophysiological diagnosis of definite and probable CIDP, regardless of the number of tested motor nerves.These data taken together could be useful to draw a thoughtful electrophysiological approach to patients suspected of CIDP. The joint task force of EFNS/PNS guidelines reports to test at first four nerves  on one side and, if criteria are not fulfilled, to study the same nerves at the other side.Our findings suggest to start electrophysiological examination by testing ulnar and peroneal nerves bilaterally; afterward, taking into account the cut-off of six nerves, adding tibial nerves for DADS phenotype and median nerves for all other CIDP subtypes  could improve the electrophysiological diagnostic chance. Importantly, electrophysiological examination should consider all nerve conduction demyelinating findings including the evaluation of temporal dispersion.However, distal CMAP amplitudes (<1mV) and especially from peroneal and tibial nerves may be too low for properly interpreting MNCV slowing as demyelinating , 21. MorLastly, we are absolutely aware that distal CMAP duration can be very useful for CIDP diagnosis, but unfortunately, we could not consider it, as, given the retrospective nature of this study, filter settings to adjust the cut-off values were not available , 24.ESM 1(DOCX 19 kb)


CDKL5) gene cause a rare neurodevelopmental disorder characterized by early-onset seizures and severe cognitive, motor, and visual impairments. To date there are no therapies for CDKL5 deficiency disorder (CDD). In view of the severity of the neurological phenotype of CDD patients it is widely assumed that CDKL5 may influence the activity of a variety of cellular pathways, suggesting that an approach aimed at targeting multiple cellular pathways simultaneously might be more effective for CDD. Previous findings showed that a single-target therapy aimed at normalizing impaired GSK-3β or histone deacetylase (HDAC) activity improved neurodevelopmental and cognitive alterations in a mouse model of CDD. Here we tested the ability of a first-in-class GSK-3β/HDAC dual inhibitor, Compound 11 (C11), to rescue CDD-related phenotypes. We found that C11, through inhibition of GSK-3β and HDAC6 activity, not only restored maturation, but also significantly improved survival of both human CDKL5-deficient cells and hippocampal neurons from Cdkl5 KO mice. Importantly, in vivo treatment with C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice, suggesting that dual GSK-3β/HDAC6 inhibitor therapy may have a wider therapeutic benefit in CDD patients.Mutations in the X-linked cyclin-dependent kinase-like 5 ( CDKL5 deficiency disorder (CDD) is a complex and severe neurodevelopmental disorder caused by mutations of the CDKL5 gene [CDKL5 is located on the X chromosome, the prevalence of CDD among women is four times higher than in men. Genetic mutations of the CDKL5 gene cause absence of a functional CDKL5 protein, a serine/threonine kinase that is highly expressed in the brain and, in particular, in neurons [KL5 gene , for whiKL5 gene ,2,3,4. I neurons ,7.Cdkl5 deficiency in mice impairs spine maturation and dendritic arborization of hippocampal and cortical neurons [CDKL5 deletion in human neuroblastoma cells induces an increase in cell death and in DNA damage-associated biomarkers [Cdkl5 was shown to decrease neuronal survival in different brain regions such as the hippocampus, cortex, and basal ganglia in Cdkl5 knockout (KO) mice, paralleled by increased neuronal senescence [In vitro and in vivo models of CDD have helped to provide important insights into the mechanism of CDKL5 functions in neuronal development. CDKL5 has been found to regulate neuronal migration, axon outgrowth, dendritic morphogenesis, and synapse development in cultured rodent neurons as a model system ,10,11,12 neurons ,16,17,18 neurons ,20,21. Comarkers . During nescence .Cdkl5 KO mice [Cdkl5, the AKT-GSK-3β signaling pathway is particularly interesting due to its pivotal role in brain development and function [Cdkl5-deficient brains. Importantly, pharmacological inhibition of GSK-3β activity rescues dendritic morphogenesis and synapse development in the Cdkl5 KO mouse [Cdkl5 KO mice [Cdkl5 KO mice [Cdkl5 KO mice [In view of the variety of cellular processes regulated by protein kinases and of the severity of the neurological phenotype of CDD patients, it is widely assumed that CDKL5 may have a very complex role in neurons, influencing the activity of a variety of intracellular pathways. Indeed, through a kinome profiling study, Wang and colleagues demonstrated that several signaling transduction pathways involved in neuronal and synaptic plasticity are disrupted in the forebrain of  KO mice . Among tfunction ,25. IncrKO mouse ,27. Simi KO mice . However KO mice , suggest KO mice , suggest11 (C11) [Combinatorial therapies have recently become one of the most successful drug development strategies for complex diseases. For instance, it was reported that combined inhibition of GSK-3β and HDACs induces synergistic effects compared to the single target drug, with a potential improved therapeutic efficacy . Due to 11 (C11) , to resc11 (C11) was selected for its high dual activity against GSK-3β and histone deacetylase 6 (HDAC6) [C11 on GSK-3β and HDAC6, we treated a recently generated human neuronal cell model of CDKL5 deficiency, the CDKL5 knockout (KO) SH-SY5Y neuroblastoma cell line , and, as a control for selective GSK-3β inhibition, with NP-12 .Compound  (HDAC6) , which o (HDAC6) ,31. In oDKL5-KO; ), with CCDKL5-KO compared with parental SH-SY5Y cells , the phosphorylation of which is regulated by GSK-3β [CDKL5-KO cells. Treatment with C11 recovered CRMP2 phosphorylation in SH-CDKL5-KO cells (CDKL5-KO cells (GSK-3β is a constitutively active serine/threonine kinase that is predominantly modulated by inhibitory serine-9 (Ser9) phosphorylation . Consist5Y cells A,B. SH-C5Y cells A,B. Simirylation A,B. As ay GSK-3β . In lineKO cells C,D. As eKO cells C,D.C11 on HDAC6, we investigated acetylated tubulin levels in untreated and treated SH-CDKL5-KO cells. Interestingly, we found that SH-CDKL5-KO cells showed decreased levels of acetylated tubulin compared with parental SH-SY5Y cells , to compare drug efficacy we used C11 at a concentration that was 10 times higher (10 µM) than that of NP12 (1 µM). We found that treatment with C11 restored neuritic length in SH-CDKL5-KO clones  for 5 days, a considerable proportion of cells differentiated to a more neuronal phenotype by extending neuritic processes . As preval cells . Since C= 2.7 µM ; NP12: IO clones A,D. Afteal cells A,D, confCDKL5-KO clones, we evaluated the percentage of mitotic and pyknotic nuclei visualized with Hoechst staining. As previously reported [CDKL5-KO showed a reduced number of mitotic cells  stress. SH-CDKL5-KO cells were more sensitive to treatment with H2O2 than were parental SH-SY5Y cells, showing an increased number of pyknotic nuclei and γH2AX levels  neurons  cultures , neuron treatment with survival .Cdkl5 −/Y brain. Since to date there are no studies on the action of C11 in vivo, a pilot study was performed to examine the effect of an acute treatment with increasing C11 doses  on GSK-3β inhibition. We found a dose response increase of phosphorylated GSK-3β levels in both the hippocampus and cortex of Cdkl5 −/Y mice  daily for 15 days with C11 (50 mg/kg), the lower dose that was shown to improve or even restore phosphorylated GSK-3β (P-GSK-3β) to control levels , such as clasping and hand-sucking [Common features of -sucking .Cdkl5 −/Y mice spent significantly more time hanging onto the bar than did control (+/Y) mice  mice C. Treatm−/Y mice C, suggesCdkl5 KO mice exhibited a hind-limb clasping behavior [C11 on motor stereotypies, we evaluated the hind-limb clasping time during the two minutes’ testing period  test in order to explore hippocampus-dependent spatial memory. As previously reported [Cdkl5 −/Y mice showed a clear learning deficit over the 2–4 days of the trial  mice  mice A–C. Treac spines A,C and tc spines B,C in CdC11-induced restoration of spine maturation was accompanied by a restoration of synaptic connectivity, we evaluated the number of immunoreactive puncta for PSD-95 in the hippocampal of Cdkl5 −/Y mice. As previously reported [Cdkl5 −/Y mice, the number of postsynaptic terminals was lower in comparison with that of Cdkl5 +/Y mice  mice  mice A–C, confsurvival A–C. Othe−/Y mice , assesseC11, in addition to its dual GSK-3β/HDAC6 inhibitory action, decreases neuroinflammation by modulating the microglial phenotypic switch from the M1 (pro-inflammatory) to the M2 (anti-inflammatory) phenotype [Cdkl5 KO mouse [Cdkl5 −/Y mice compared to their wild-type counterparts  mice  mice D,E.CDKL5 failure, alterations in multiple complex molecular networks are involved in the mechanisms that underlie the CDD-related phenotype. The complexity of this disease might limit the efficacy of commonly used single molecular drug therapeutics. Therefore, the severity of the neurological phenotype of CDD patients could be better tackled using a multi-target approach that is able to simultaneously modulate multiple targets involved in the onset of the disease. Our study provides novel evidence that, in in vitro experimental models of CDD, the GSK-3β/HDAC6 dual inhibitor, C11 [C11 restored synapse development, neuronal survival, and microglia over-activation, and improved motor and cognitive abilities of Cdkl5 KO mice. Overall, our data suggest that a GSK-3β/HDAC6 dual inhibitor therapy may engender a more effective strategy with which to achieve therapeutic benefits in CDD patients.It is likely that, due to tor, C11 , is moreC11 in CDD we selected, as an in vitro model, a recently generated human cellular model of CDKL5 deficiency that exhibited alterations in the GSK-3β signal pathway [CDKL5 deficiency in these cells causes decreased acetylation of a well-known substrate of HDAC6 deacetylase activity, αtubulin [CDKL5 deficient cells obtained with the single GSK-3β inhibitor, NP12.To validate the effect of  pathway  as well αtubulin . The decαtubulin . Among tαtubulin . The linC11 improved or even restored neuronal maturation and connectivity in in vitro and in vivo models of CDD. In both human CDKL5 deficient cells and hippocampal neurons from Cdkl5 KO mice a complete restoration of the extension of the neuritic processes was obtained when cells were treated with either C11 or NP12. Similarly, the effects of in vivo treatment with C11 or NP12 were comparable in terms of dendritic spines and PSD-95 positive puncta in CDKL5 KO mice  ,49,50. Tthose of ).C11 treatment was much more effective at recovering neuronal survival than treatment with NP12 in both in vitro and in vivo experimental models of CDD, suggesting that the C11 pro-survival effect is conveyed to HDAC6 inhibition. In support of this, our findings are in line with a previous study showing that inhibition of HDAC6 activity in neurons promotes protection against oxidative stress-induced death [CDKL5 KO cells, our findings showing that treatment with C11 selectively increases αtubulin acetylation, without altering histone H3 acetylation, strongly confirm the higher specificity of C11 for HDAC6.Interestingly, we showed here that ed death . There ied death ,52,53. Ced death . HDAC6, ed death . Importaed death ; specified death . HDAC6 ied death . A furthed death ,59. WhilC11 might contribute to restoring neuronal survival and connectivity in the hippocampus of Cdkl5 KO mice. The anti-inflammatory effect exerted by C11 may depend on both of its inhibitory activities. GSK-3 inhibitors lead to a reduction in microglia activity [Evidence that microglia-mediated inflammation contributes to the neuropathology of CDD has recently been reported . Chronicactivity ,63 via iactivity . The mecactivity ,66, and activity ,68.Cdkl5 KO mice [CDKL5-null neurons. The importance of the use of an inhibitor with large selectivity for HDAC6 such as C11 is that it can overcome all of the neuronal toxicity associated with the use of pan-HDAC inhibitors, suggesting that its specific inhibition may eliminate a range of untoward effects seen with the clinical application of pan-HDAC inhibitors in cancer [C11 improved motor and cognitive behavior in Cdkl5 KO mice suggest that dual GSK-3β/HDAC6 inhibitors might be an effective treatment option for CDD. Since previous findings showed that a pharmacological intervention aimed at normalizing impaired GSK-3β activity elicits different age-dependent outcomes in Cdkl5 KO mice [C11 double inhibitor is more efficient at recovering brain abnormalities in CDD at different developmental ages than a therapy with a selective GSK-3β or HDAC6 inhibitor. Future studies may clarify this issue.The search for better results in clinical practices, because of the inefficacy of some treatments based on single drugs, has encouraged the adoption of polypharmacology as a new therapeutical strategy. This multi-target approach could be: association of drugs, combination of drugs, or a single drug with multiple ligands. However, the deleterious effects caused by drug–drug interactions and toxicity of combination therapy have reinforced the emergence of novel strategies in drug discovery. The most recent approach is now considering single drugs that concomitantly recognize more than one molecular target. Although GSK-3β and HDAC inhibitors have been previously tested in  KO mice ,26,27, tn cancer . This isn cancer . Our fin11 (C11) was synthetized as previously reported [Compound reported .CDKL5 knockout (KO) SH-SY5Y neuroblastoma cell line  and the DKL5-KO; ), were m5 cells per well in culture medium supplemented with 10% FBS. The day after, cells were exposed to C11 , NP-12 , or vehicle (0.1% DMSO) for 24 h.Cells were plated onto poly-D-lysine-coated slides in a 6-well plate at a density of 2.5 × 105 cells per well in culture medium supplemented with 10% FBS. The day after, cells were exposed to H2O2  and C11 (10 μM) or NP12 (1 μM) for 24 h. The following day, cells were fixed in a 4% paraformaldehyde 4% glucose solution at 37 °C for 30 min.Cells were plated onto poly-D-lysine-coated slides in a 6-well plate at a density of 2.5 × 105 cells per well in culture medium supplemented with 10% FBS. Twenty-four hours after cell plating, retinoic acid  10 mM in ethanol was added to the medium at 10 μM final concentration each day for 5 days. Cells were co-treated with C11 (10 μM), NP12 (1 μM), or vehicle (0.1% DMSO) every 2 days.For differentiation analyses, cells were plated onto poly-D-lysine-coated slides in a 6-well plate at a density of 1 × 10Phase contrast images of cells were taken with an Eclipse TE 2000-S microscope  equipped with a DS-Qi2 digital SLR camera . Images were taken from random microscopic fields. Neurite outgrowth was measured using the image analysis system Image Pro Plus . Only cells with neurites longer than one cell body diameter were considered as neurite-bearing cells. In each experiment, a total of 900 cells was analyzed. All experiments were performed at least three times. The total length of neurites was divided by the total number of cells counted in the areas.Nuclei were stained with Hoechst 33342 . Apoptotic cell death was assessed by manually counting the number of pyknotic nuclei and apoptotic bodies and is expressed as a percentage of the total number of cells. The number of mitotic cells was assessed by manually counting the cells in prophase (chromosomes are condensed and visible), metaphase (chromosomes are lined up at the metaphase plate), and anaphase/thelophase (chromosomes are pulled toward and arrive at the opposite poles) and expressed as a percentage of the total number of cells.Cdkl5 knockout (KO) strain in the C57BL/6N background developed in [Cdkl5 KO mice (Cdkl5 −/Y) and age-matched controls , when possible littermates, were used for all experiments and genotyped as previously described [The mice used in this work derive from the loped in  and backescribed . The dayCdkl5 KO mice as described [5 cells were plated on coverslips coated with poly-L-lysine in 6-well plates and cultured in Neurobasal medium supplemented with B27  and glutamine . Cells were maintained in vitro at 37 °C in a 5% CO2-humified incubator and fixed for immunostaining or western blot analysis on day 10 after plating (DIV10). Treatment was added to the conditioned medium and replenished every second day until DIV10. Cell cultures were fixed in a 4% paraformaldehyde 4% glucose solution at 37 °C for 30 min and processed for immunocytochemistry analysis.Primary hippocampal neuronal cultures were prepared from 1-day-old (P1) wild-type (WT) and escribed . BrieflyTo evaluate neuritic outgrowth and connectivity, fixed cells were stained with the following primary antibodies: a rabbit polyclonal anti-MAP2  and a mouse monoclonal anti-PSD-95  antibody. Detection was performed using a FITC-conjugated anti-rabbit IgG  and a Cy3-conjugated anti-mouse IgG  antibody, respectively. Nuclei were counterstained with Hoechst-33342  and fluorescent images were acquired using a Nikon Eclipse Te600 microscope equipped with a Nikon Digital Camera DXM1200 ATI system . The neuritic length of MAP2-positive neurons was measured and quantified by tracing along each neuronal projection using the image analysis system Image Pro Plus  as previously described . The degIn order to assess survival of differentiated hippocampal neurons in culture, the number of MAP2-positive neurons was counted and expressed as a percentage of the total number of cells in culture evaluated through Hoechst staining.C11 was dissolved in a vehicle solution of 5.5% dimethyl sulfoxide (DMSO) in water.Cdkl5 +/Y mice were treated with vehicle (5.5% of DMSO in water) and two-month-old Cdkl5 −/Y mice were treated with vehicle or C11  administered intraperitoneally. Animals were sacrificed after 4 h.Two-month-old Cdkl5 KO mice [Cdkl5 +/Y and Cdkl5 −/Y male mice were randomly assigned to the experimental conditions and, starting from postnatal day 30 (P30), were treated with vehicle (5.5% of DMSO in water) or C11 (50 mg/kg) administered via a daily intraperitoneal injection for 15 consecutive days. Body weight of the mice was monitored daily.Since in the mammalian brain, the dramatic rearrangements in structure and function that characterize childhood/adolescence result in a critical and sensitive period of brain development and suggest that early interventions would provide potential clinical benefits, we decided to chronically treat juvenile  KO mice . Cdkl5 +Cdkl5 +/Y and Cdkl5 −/Y male mice were treated with vehicle  or NP-12 , administered via subcutaneous injection every other day for 20 days. The dose of NP12 was chosen based on [Brain sections that were processed for Hoechst staining, and used to evaluate hippocampal neuron survival, derived from animals used in . Brieflybased on .Cdkl5 +/Y n = 1, Cdkl5 −/Y n = 8, and Cdkl5 −/Y + C11n = 6) that were tested with the following assays: clasping, catalepsy, and Morris water maze. The second cohort consisted of 18 animals  that were tested with the following assays: clasping, catalepsy, and Morris Water Maze. The third cohort consisted of 11 animals (Cdkl5 +/Y n = 5 and Cdkl5 −/Y n = 6) that were tested with the following assays: clasping and catalepsy.A total of 44 animals, from 9 litters, were used for behavioral studies. The sequence of the tests was arranged to minimize the effect of one test influencing subsequent evaluation of the next test, and mice were allowed to recover for 1–2 days between different tests. All behavioral studies and analyses were performed blind to genotype. Mice were allowed to habituate to the testing room for at least 1 h before the test, and testing was always performed at the same time of day. Three independent test cohorts were used. The first test cohort consisted of 15 animals  were excluded from the analysis.Hippocampal-dependent spatial learning and memory was assessed using the Morris Water Maze (MWM) as previously described . Mouse bThe bar was set at a height of 6 cm. Mice were gently positioned, by placing both forelimbs on the bar and their hindlimbs on the floor. The time needed for the mice to remove both paws from the bar was measured using a stopwatch.Animals were suspended by their tail for 2 min and hind-limb clasping was assessed independently by two operators from video recordings. A clasping event is defined as the retraction of limbs into the body and toward the midline. The time spent hind-limb clasping was expressed as a percentage.CDKL5-KO cell lines were lysates in ice-cold RIPA buffer  supplemented with 1mM PMSF, and with 1% protease and phosphatase inhibitor cocktail . Total protein from the hippocampus and cortex of 2-month-old Cdkl5 −/Y and Cdkl5 +/Y male mice were homogenized in ice-cold RIPA buffer supplemented with 1mM PMSF, and with 1% protease and phosphatase inhibitor cocktail . Protein concentration for both cell and tissue extracts was determined using the Bradford method [® TGXTM Gel  and transferred to a Hybond-ECL nitrocellulose membrane . For acetylated tubulin detection, 10 μg of proteins were loaded on the gel. The primary and secondary antibodies used are listed in TM Software . Images acquired with exposition times that generated protein signals out of a linear range were not considered for the quantification.Total proteins from SH-SY5Y and SH-d method . EquivalFor histological and immunohistochemistry analysis, animals were euthanized with isoflurane (2% in pure oxygen) and sacrificed through cervical dislocation. Brains were quickly removed and cut along the midline. Right hemispheres were fixed via immersion in 4% paraformaldehyde in 100 mM phosphate buffer, pH 7.4, stored in fixative for 48 h, kept in 20% sucrose for an additional 24 h, and then frozen with cold ice. Subsequently, hemispheres were cut with a freezing microtome into 30 μm thick coronal sections that were serially collected in anti-freeze solution  and processed for immunohistochemistry procedures as described below. Left hemispheres were Golgi-stained as previously described . All ste3. Starting from 20X magnification images of AIF-1-stained hippocampal slices, AIF-1 positive microglial cell body size was manually drawn using the Image Pro Plus measurement function, and expressed in µm2.One out of every 8 serial brain sections from the hippocampal formation was incubated with one of the following primary antibodies: mouse monoclonal anti-NeuN antibody  or rabbit polyclonal anti-AIF-1 antibody . Sections were then incubated for 2 h at room temperature with a Cy3-conjugated anti-mouse secondary antibody  or with a Cy3-conjugated anti-rabbit secondary antibody . Nuclei were counterstained with Hoechst-33342 . Fluorescent images were acquired using a Nikon Eclipse TE600 microscope equipped with a Nikon Digital Camera DXM1200 ATI System . The number of Hoechst-positive and NeuN-positive cells were manually counted using the Image Pro Plus software  and established as cells/mmOne out of every 6 free-floating sections from the hippocampal formation was incubated with a rabbit polyclonal anti-PSD-95 antibody  and with a CY3-conjugated anti-rabbit IgG secondary antibody . For quantification of PSD-95 immunoreactive puncta, images from the stratum oriens of the hippocampus were acquired using a LEICA TCS SL confocal microscope . Three to four sections per animal were analyzed and puncta counts were performed on a single plan, 1024 × 1024 pixel images. Counting was carried out using Image Pro Plus software .In Golgi-stained 100-μm-thick sections, spines of CA1 pyramidal neurons were counted using a 100× oil immersion objective lens. Dendritic spine density was measured by manually counting the number of dendritic spines on basal dendrites of CA1 pyramidal neurons. In each mouse, 15 dendritic segments (segment length: 10–30 μm) from each zone were analyzed and the linear spine density was calculated by dividing the total number of counted spines by the length of the sampled dendritic segment. The total number of spines was expressed per 10 μm. Based on their morphology, dendritic spines can be divided into five different classes which fall into two categories which also reflect their state of maturation . The total number of spines was expressed per μm and the number of spines belonging to each class was counted and expressed as a percentage.p < 0.05 was considered to be statistically significant.Data from single animals represented the unity of analysis. Results are presented as mean ± standard error (SE). Statistical analysis was performed using GraphPad Prism . All datasets were analyzed using the ROUT method (Q = 1%) to identify significant outliers and the D’Agostino–Pearson omnibus test for normality. Datasets with normal distribution were analyzed for significance using Student’s t-test or a one-way analysis of variance (ANOVA) with genotype and treatment as factors. Post hoc multiple comparisons were carried out using the Fisher least significant difference (Fisher LSD) test. For the learning phase of the MWM test, statistical analysis was performed using a repeated three-way mixed ANOVA with genotype and treatment as grouping factors and day as a repeated measure. For categorical data, that is, percentages of spines, we used a chi-squared test. Animals identified as outliers by the use of Grubb’s test were excluded from the analyses . A proba