Aim: to present the results achieved in a neonatal hearing screening program in Maceió. Materials and Methods: a retrospective analytical study was carried out in order to study the results from tests carried out from September 2003 to December 2006 in a private hospital of Maceió. Results: from a total of 2002 newborns, 1,626 fitted the inclusion criteria, 835 (51.4%) males. The hearing screening was considered appropriate in 1416 cases (87.1%), and the most frequently found age was between 16 and 30 days. Finally, 163 (10.0%) children presented risk indicators for hearing loss, and hyperbilirubinemia was the most common indicator. Conclusions: statistical results obtained from this hearing screening program show the importance of holding such programs. This study is important because it contributes to further regional or multinational studies.Since 1998, after we started the support group for neonatal hearing screening, many other hearing screening programs were held in Brazil. In Alagoas, the first program started in 2003, but none of its results were published. Hearing is paramount for human communication; therefore, childhood hearing loss can impair speech acquisition, emotional, educational and social development.  Communication skills is a distinctive trace of human existence, being one of the major factors associated with an individual’s well being. Within this context, hearing plays a fundamental role, since it is considered a cornerstone on top of which we build the complex human communication systemThe first two years of a child’s life have been considered a critical period for the apprehension and development of hearing and language skills. One of the main reasons for that is the fact that the central nervous system has a great plasticity when stimulated earlier on, especially before 6 months of age, increasing the number of nervous connections and, consequently, improving auditory pathway rehabilitationThe Brazilian Committee on Hearing Loss During Childhood - Comitê Brasileiro sobre Perdas Auditivas na Infância (CBPAI)The implementation of the hearing screening program is viable because of its low cost and the ease of execution under training and supervisionThe test for objective evaluation of the pre-neural peripheral hearing system, with major clinical application is the Otoacoustic Emissions Test (OAE). These are a type of acoustic energy, described by KEMPOAEs are the best screening procedure to detect hearing impairment early on, because it is a fast, non-invasive, easy to interpret and it is a high specificity and sensitivity test which reflects cochlear response without depending on central nervous system maturityWith the growing increase in the number of Universal Neonatal Hearing Screening Programs (UNHSP), there was a major concern in establishing principles and references to control the programs’ efficiency. Principle 1 stresses that every newborn must have access to hearing screening by physiological measure. Principles 2, 3 and 4 approach, diagnosis (before four months of age), intervention (before seven months of age), and auditory monitoring, respectively. Principle number 5 deals on the rights of children and families, guaranteed through informed choice, decision and consent. Principle # 6 makes sure the children’s family’s and test result information be kept confidential. The 7th principle establishes that information systems must be used in order to measure and report the impacts of neonatal hearing screening programs. And finally, principle 8 says that the neonatal hearing programs must provide data to monitor the quality, establish effective cost, mobilize and maintain support in the community, according to the legislation and regulationsSince 1998, after creating the neonatal hearing screening support group, many hearing screening programs were implemented in our countryWe carried out a retrospective, analytical and observational, cross-sectional study in which we analyzed the results of the tests carried out from September of 2003 through December of 2006, present in a data base from a private hospital in the city of Maceió-AL. This study was approved by the ethics committee of our institution under protocol number 666.In the sample we included the tests from newborns below 3 months of age and took off those non analyzable tests and/or out of the criteria pass/failure used by the Universal Neonatal Hearing Screening Support Group - Grupo de Apoio a Triagem Auditiva Neonatal Universal (GATANU)Hearing impairment risk indicators identified in our study were: family history of congenital hearing impairment; congenital infection ; consanguinity, skull-facial anomalies - including morphological alterations in the ear pinna and external auditory meatus; birth weight below 1,500 g or small for the gestational age (SGA); hyperbilirubinemia; ototoxic medication - including but not limited to aminoglycosides with or without diuretic agents; bacterial meningitis; Apgar score of 0-4 in the 1st minute or 0-6 in the 5th minute; mechanical ventilation for more than 5 days; syndromes associated with conductive or sensorineural hearing loss; neonates that spend more than 48 hours in the ICU and neonatal infectionsThe data collected were from newborns submitted to neonatal hearing screening by means of capturing otoacoustic emissions by transient stimulus, using the Capella OAE analyzer in order to analyze cochlear function integrity and to rule out the likelihood of the baby having sensorineural hearing loss. According to GATANU, we considered as a pass criterion, the presence of response above 6 dB in at least 3 frequency bands  with response reproducibility above 50%Research results were descriptively analyzed through the calculation of averages and proportions. We used the Z test for analysis of independent samples and the Odds Ratio in order to analyze the results obtained in respect of the hearing loss. The significance level used was of 5%.The data collected was stored and analyzed using Excel 2002® (Microsoft Corporation). We calculated the specific frequencies of variables and mean values; afterwards we created binomial tables with crossovers among the variables studied. The simple logistics regression test was used to compare the proportions, when we assessed whether or not there were associations between two or more variables. The significance level  was used to quantify the possibility of our figures present a random distribution. Values below 5% (p<0.05) were deemed significant. The Odds ratio was used in order to calculate the association strength between two variables, so as to quantify the possibility of a risk factor be associated with the event being studied, with a confidence interval of 95%. We also used the Z test to compare the results classified by age, gender and the incidence of risk factors associated with hearing impairment. In such a case, the 5% (or 0.05) significance level was accepted to reject the null hypothesis.From September of 2003 to December of 2006, we carried out NAS in 2002 newborns (NB) in a private hospital in the city of Maceió. Of these, 1,626 matched the inclusion criteria, 791 (48.6%) were girls and 835 (51.4%) were boys . Among bIn relation to the tests carried out by ear, we noticed that in the right ear there were 1,581 (48.62%) tests, while on the left ear they were 1,580 (48.59%), having that 91 (2.80%) ears were not assessed. 1,488 (91.64%) passed the NAS and 73 (4.49%) failed, on the left ear, 1,472 (90.59%) passed and 76 (4.67%) failed. When the Z test was used, p was equal to 0.2961 indicating a non-significant difference of results in relation to the ears.The most frequent age range at which the NB underwent NAS, as one can see in Of the 1,626 NB seen, 1,315 (80.9%) passed the test and 311 (19.1%) must be referred to a retest. Of these, 194 (11.9%) came and 117 (7.2%) did not come to the retest. With the retesting, we observed that in total, 1,416 (87.1%) NB passed the neonatal auditory screening, while 93 (5.7%) failed . Of the Our study picked up 163 (10.0%) NB with risk indicators for hearing impairment. Of the NB who failed NAS, 8.6% had some risk indicator for hearing impairment, while 91.4% did not. Of those who had RIHI, 42.9% used ototoxic medication for more than 5 days; 21.40% stayed in the incubator for more than 7 days; 14.30% had family history of congenital hearing impairment; 7.10% had hyperbilirubinemia with visible jaundice - not necessarily needing total blood transfusion; 7.10% used mechanical ventilation for more than 5 days and 7.10% remained in the neonatal ICU for more than 48 hours. Making up a mean value of 1.75 risk indicator per person.In the present investigation, NBs who had TOAE present were more often males and in the right ear; however, when we applied the Z statistical test, we perceived that these data were not significant. This finding does not match the ones in the literature we studiedNAS performance as soon as possible helps optimize intervention, contributing for the child’s development. On The present study showed that 87.1% passed NAS, 5.7% failed and 7.2% did not come for retest, despite being advised to. It important to stress that to “pass” the test means that at the time of the test, results matched TOAE responses, in other words, showed healthy outer hair cellsCBPAI4 states that retest indications should not go beyond 4%. The present investigation found an index of 19.1%, which is significant according to the Z statistical test (p=0.0001), inferring a high number of NB referred for a new assessment. This fact can be justified by the possible presence of noisy breathing, suction act or noise, factors which impair TOAE captureOf those NB who had RIHI, According to the literature, approximately 50% of the children diagnosed with hearing loss did not have risk indicators for itThis study disclosed the statistical results obtained from a neonatal hearing screening program carried out in the city of Maceió-AL, stressing the importance of implementing and maintaining such a program. Moreover, its publication contributes for multinational and/or regional programs.


CDKL5 gene mutations. We created a gene therapy vector that produces an Igk-TATk-CDKL5 fusion protein that can be secreted via constitutive secretory pathways and, due to the cell-penetration property of the TATk peptide, internalized by cells. We found that, although AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 vectors had similar brain infection efficiency, the AAVPHP.B_Igk-TATk-CDKL5 vector led to higher CDKL5 protein replacement due to secretion and penetration of the TATk-CDKL5 protein into the neighboring cells. Importantly, Cdkl5 KO mice treated with the AAVPHP.B_Igk-TATk-CDKL5 vector showed a behavioral and neuroanatomical improvement in comparison with vehicle or AAVPHP.B_CDKL5 vector-treated Cdkl5 KO mice. In conclusion, we provide the first evidence that a gene therapy based on a cross-correction approach is more effective at compensating Cdkl5-null brain defects than gene therapy based on the expression of the native CDKL5, opening avenues for the development of this innovative approach for other monogenic diseases.Although delivery of a wild-type copy of the mutated gene to cells represents the most effective approach for a monogenic disease, proof-of-concept studies highlight significant efficacy caveats for treatment of brain disorders. Herein, we develop a cross-correction-based strategy to enhance the efficiency of a gene therapy for CDKL5 deficiency disorder, a severe neurodevelopmental disorder caused by The online version contains supplementary material available at 10.1007/s13311-022-01295-8. CDKL5 gene, which lead to a lack of CDKL5 protein expression or function. CDD mainly affects girls and is characterized by early-onset epileptic seizures, hypotonia, intellectual disability, motor and visual impairment, and, in some cases, respiratory dysregulation [Cdkl5 knockout (KO) mice [CDKL5 (cyclin-dependent kinase-like 5) deficiency disorder (CDD) is a severe X-linked neurodevelopmental disease caused by mutations in the gulation –6. Althogulation , 8 suggegulation –11, implKO) mice –14, recaKO) mice , 15–17 aIn theory, for a monogenic disease such as CDD, the delivery of a wild-type copy of the mutated gene to cells which lack functional protein represents the most effective approach. Using adeno-associated virus (AAV), advancements in global central nervous system gene delivery have accelerated to the point that treatments for neurodevelopmental disorders, such as lysosomal storage disease, Rett syndrome, CDD, Fragile X, and autism seem within reach . HoweverWe recently developed an enzyme replacement therapy (ERT) based on a recombinant TATk-CDKL5 protein that, when systemically injected, was able to cross the blood–brain barrier and diffuse into brain cells due to the cell-penetration properties of the TATk peptide, retaining its native biological activity . DespiteCdkl5 KO mouse model to validate whether the Igk-TATk-CDKL5 approach, that enhances the biodistribution of the therapeutic CDKL5 kinase from genetically corrected cells to non-corrected cells via a cross-correction mechanism, significantly enhances therapeutic efficacy.Here, by combining the cell-penetrating property of our recently developed TATk-CDKL5 fusion construct with a secretory Igk-chain leader sequence and the advantages of a gene therapy approach, we have developed a new therapeutic approach that is able to overcome the limitations of both gene therapy and ERT for CDD. The idea behind it is that if the protein produced by the viral vector-infected cells can be secreted and enter into neighboring cells, this will amplify the effect of the gene therapy because, even if the transduced cells are low in number, they will become a “factory” for the production of the therapeutic protein, supplying therapeutic molecules to neighboring cells. In this scenario, the efficiency of gene delivery does not necessarily need to be high. This decreases the risk of insertional mutagenesis and toxic side effects connected with large vector doses while also providing the promise of a life-lasting treatment. We compared the effects of CDKL5 gene therapy with Igk-TATk-CDKL5 gene therapy in a 1 or hCDKL51 gene expression cassettes were subcloned in the backbone of pAAV-CBh-DIO-EGFP . The designed viral cassettes between the two AAV2 inverted terminal repeats (ITRs) contain a CBh promoter (0.8 kbp), the Igk-TATk-CDKL5 (3.1 kbp), or CDKL51 (2.9 kbp) open reading frame, followed by a WPRE and an SV40 polyadenylation signal (0.4 kbp). The TATk-CDKL5 and CDKL5 proteins were tagged with a haemagglutinin (HA)-tag. The AAV vector genomes containing the above-described expression cassettes were packaged in the AAVPHP.B capsid in HEK293 cells using an adenovirus-free triple transfection method . Igk-TATk sequence: MetETDTLLLWVLLLWVPGSTGDAAQPARRARRTKLAAYARKAARQARA.To express the Igk-TATk-CDKL5 and CDKL5 proteins by means of AAV vector-mediated gene delivery, the Igk-TATk-hCDKL52 at 37 °C. Total cell medium was replaced every 3 days and the cells were sub-cultured once they reached 90% confluence. Cells were transfected with designated plasmid DNA using Metafectene Easy Plus (Biontex). Forty-eight hours after transfection, cells were harvested, washed in PBS, and lysed for total protein extraction. Cell medium was also collected and 200 × concentrated as described previously [The HEK293T cell line was maintained in Dulbecco modified Eagle medium  supplemented with 10% heat-inactivated FBS (Gibco), 2 mM of glutamine (Gibco), and antibiotics , in a humidified atmosphere of 5% of COeviously . Cell exCdkl5 − /Y mice as previously described [l-lysine in 6-well plates and cultured in Neurobasal medium (Gibco) supplemented with B27 (Invitrogen) and glutamine (Gibco). Cells were maintained in vitro at 37 °C in a 5% CO2-humified incubator.Primary hippocampal neuronal cultures were prepared from 1-day-old (P1) wild-type and escribed . Briefly6) at day 2 in vitro (DIV), and fixed at DIV7 with 4% paraformaldehyde + 4% sucrose in 100 mM phosphate buffer pH 7.4. Fixed cells were stained with the primary and secondary antibodies listed in Supplementary Table Primary hippocampal neurons were infected with AAVPHP.B_Igk-TATk-CDKL5 and AAVPHP.B_CDKL5 MOI of 1006 at dayFor co-culture experiments, HEK293T cells were plated in a 6-well plate, while primary hippocampal neurons were plated on cover glasses. Twenty-four hours after plating, HEK293T cells were transfected with the AAV vector plasmid containing the Igk-TATk-CDKL5 or CDKL5 cassette. Twenty-four hours after transfection, the co-culture was prepared as follows: HEK293T cells were washed twice with fresh neuronal culture medium. The cover glasses with primary hippocampal neurons (DIV5) were transferred to the HEK293T 6-well plate in an elevated position with respect to the gel supports underneath. After 48 h of co-culturing, the cover glasses with neurons were removed; neurons were washed in PBS, fixed with 4% paraformaldehyde + 4% sucrose in 100 mM phosphate buffer pH 7.4 and processed for immunocytochemistry. The primary and secondary antibodies used are listed in Supplementary Table Cdkl5 − /Y strain in the C57BL/6 N background developed in [The mice used in this work derive from the loped in  and backloped in . Age-mat11 viral vector genomes were injected into 1-day post-gestation (P1) neonatal Cdkl5 − /Y mice via intracerebroventricular injection targeting the anterior horn of the lateral ventricle. Prior to the procedure, pups were incubated on ice for 1 min and subsequently injected using a 33-gauge needle . Injected neonates were subsequently returned to the dam. Mice were sacrificed 2 months (P60) post-injection.Neonatal injections were carried out as previously described . 1011 vi12 vg/mouse. When all the amount of the solution (200 µL) was infused, the infusion was stopped, and the catheter was gently pulled out. The threads were both tied to prevent bleeding. Permanent occlusion of one common carotid is a protocol largely used in mice to directly inject drugs into the brain with no long-term consequences for the animals. Indeed, the contralateral carotid and the Willis circle granted a physiological cerebral blood flow as previously documented [Surgery was performed under general anesthesia  with the mouse’s body temperature maintained at 37 °C by using a heating pad and intra-operative analgesia . All procedures were performed in sterile conditions. The tip of the catheter was flushed with sterile heparin. The isolation of the carotid artery was performed as previously described. Once isolated, two silk suture threads (Softsilk 5–0) were proximally and distally placed around the common carotid to create a free blood flow segment of artery in which to insert the catheter. The proximal thread  was permanently knotted and tied while the distal thread (near the bifurcation of the common carotid) was pulled by another operator to temporarily create an artery segment with no blood flow. With this approach, it was possible to cut the artery, avoiding any bleeding, and to insert the catheter for the infusions. The catheter was inserted between the suture threads, close to the proximal thread, through a hole made by a 90° bent needle 25 G)  that were tested with the following assays: marble burying, nesting test, hindlimb clasping, open field, Barnes maze. The second cohort consisted of 34 animals  that were tested with the following assays: marble burying, nesting test, hindlimb clasping, open field. The third cohort consisted of 19 animals  that were tested with the following assays: marble burying, nesting test, hindlimb clasping, open field, Barnes maze. The behavioral test was performed by operators who were blind to genotype and treatment. See Supplementary material for detailed behavioral methods.Behavioral tests were performed 2 months after the intracarotid infusion. The sequence of the tests was arranged to minimize the possibility of one test influencing the subsequent evaluation of the next test, and mice were allowed to recover for 2 days between different tests. 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. Behavioral studies were carried out on the saline-injected Cdkl5 − /Y mice subjected to gene therapy with AAVPHP.B_Igk-TATk-CDKL5 or AAVPHP.B_CDKL5 vector was evaluated through an average behavioral score for each genotype and treatment in different tests. Mice were assessed on a 1–4 scale for each behavioral test: marble burying, nesting test, hindlimb clasping, open field (velocity and distance moved), and Barnes maze tests. For each test, the gap between the minimum and maximum values was then divided into four ranges. Scores of 1–4 for each quartile were assigned based on performance increase. We included the 5- to 15-min interval for the open field test (velocity and distance moved) in the global score evaluation.The overall behavioral improvement of Cdkl5 + /Y + vehicle n = 3, Cdkl5 − /Y + vehicle n = 3, Cdkl5 − /Y + AAVPHP.B_CDKL5 n = 4, and Cdkl5 − /Y + AAVPHP.B_Igk-TATk-CDKL5 n = 4), second cohort .At sacrifice, a total of 41 animals for the subsequent immunohistochemistry analyses and Golgi staining was randomly selected from two of the three independent animal cohorts; first cohort  , 28. Bri12 vg/mouse. Non-invasive transcranial IOS recordings were performed 45 days later under isoflurane anesthesia (0.5–1%), supplemented with an intraperitoneal injection of chlorprothixene hydrochloride (1.25 mg/kg), allowing us to use a lower level of isoflurane anesthesia during imaging to maintain a lightly anesthetized state. Images were obtained using an Olympus microscope (BX50WI). Red light illumination was provided by 8 red LEDs  attached to the objective  using a custom-made metal LED holder. Visual evoked responses were quantitatively measured as reported in [We measured cortical responses to visual stimulation by using intrinsic optical signal (IOS) imaging. The methods employed in  were useorted in .CDKL5 RNA was performed with the Base Scope® technology (Biotechne) following the manufacturer’s protocol using a 1ZZ probe designed on the CDKL5 exon 4. For double staining, the ISH was followed by immunohistochemistry for CDKL5 protein detection.Mice were perfused with 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4). Brains were collected and cut along the midline. Hemispheres were submerged in 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 24 h at 4 °C and then let sink in sucrose 15%, before being frozen at – 80 °C. The hemispheres were then cut with a cryostat into 15-µm-thick sagittal sections which were serially collected on glasses. The in situ hybridization (ISH) for the For immunohistochemistry, brain sections were incubated overnight at 4 °C with a primary anti-HA antibody  pixels within the area. Approximately 250 cells were analyzed from each slice. Distribution analyses were performed quantifying the percentage of cells expressing CDKL5 mRNA within different ranges of signal intensity.Hoechst and ISH images of the same cell were analyzed using NIS-Elements AR software . An area was traced around each cell comprising the nucleus (identified by Hoechst counterstaining); the intensity of ISH staining, corresponding with the CDKL5 mRNA and protein-positive cells, two images per section, from the cortex overlying field CA1 and the hindbrain (n = 3–4 sections) were acquired using an Eclipse TE 2000-S microscope equipped with a DS-Qi2 digital SLR camera (Nikon Instruments Inc.). All the positive cells present in the image were manually counted using the point tool of the Image Pro Plus software . The number of CDKL5 mRNA + cells was expressed as a percentage of the total number of cells, identified using Hoechst staining. The number of cross-corrected cells was estimated as the difference between the number of CDKL5 protein-positive cells and the number of CDKL5 mRNA-positive cells, and expressed as a percentage of the total number of infected cells (RNA + cells) or of the total amount of cells.For the quantification of n = 10–12 sections) from the hippocampal formation was incubated with the primary antibody. The primary and secondary antibodies used are listed in Supplementary Table Mice were sedated with isoflurane (2% pure oxygen) and sacrificed by cervical dislocation. The brains were quickly removed and cut along the midline. One hemisphere was fixed by immersion in a solution of 4% paraformaldehyde in 100 mM phosphate buffer (pH 7.4) for 48 h and subsequently stored in 20% sucrose for another 24 h, before being frozen in dry ice and kept at – 80 °C. The various brain regions of the other hemisphere was dissected, quickly frozen, and used for viral biodistribution analysis. The fixed hemispheres were then cut with a freezing microtome into 30-μm-thick coronal sections which were serially collected in a 96-well plate containing a solution consisting of 30% glycerol, 30% ethylene glycol, and 0.02% sodium azide in 1X PBS. One out of six free-floating sections . Quantification of Hoechst-positive nuclei and NeuN-positive cells was conducted in the field CA1 of the hippocampus while AIF-1-positive cells were measured and counted in the cortex overlying field CA1.2. Approximately 120 microglia cells were analyzed from each sample.For morphometric microglial cell analysis, starting from 20 × magnification images of AIF-1-stained slices, AIF-1-positive microglial cell body size was manually drawn using the Image Pro Plus (Media Cybernetics) measurement function and expressed in μm3.For the density of AIF-1-positive cells, Hoechst-positive nuclei, and NeuN-positive neurons, cells were manually counted using the point tool of the Image Pro Plus software (Media Cybernetics), and expressed as number of cells/mm2 were excluded from the evaluation. Three to four sections per animal were analyzed and the number of PSD-95 immunoreactive puncta was expressed per μm2.For quantification of PSD-95 immunoreactive puncta, images from the CA1 layer were acquired using a LEICA TCS SL confocal microscope . All images were acquired with the same gain and exposure time. To ensure that sampling was done at the same level of antibody penetration, all images were captured at a level corresponding to the mid-distance between the upper and lower focus of each section. A frame average process was used to improve image quality: an entire image frame was scanned multiple times and then the data for each point was averaged to reduce noise. Counting was manually carried out using Image Pro Plus software (Media Cybernetics) and fluorescence signals with an area lower than 0.15 µmHemispheres were Golgi-stained using the FD Rapid GolgiStain TM Kit (FD NeuroTechnologies) as previously described . DendritCdkl5 − /Y mice were homogenized in ice-cold RIPA buffer supplemented with 1 mM PMSF, and with 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich). Equivalent amounts of protein were subjected to electrophoresis on a 4–12% Mini-PROTEAN® TGXTM Gel (Bio-Rad) and transferred to a Hybond-ECL nitrocellulose membrane . The primary and secondary antibodies used are listed in Supplementary Table HEK293T cells transfected with plasmid DNA were lysates in Laemmli buffer supplemented with β-mercaptoethanol, sonicated, and boiled at 95 °C for 10 min. Brains of treated Animals were sedated with isoflurane (2% pure oxygen) and sacrificed for cervical dislocation. The brain was quickly removed and the various brain regions were dissected and stored at − 80 °C.®Tissue Kit (Macherey–Nagel) extraction kit and 100 ng of gDNA was used as a qPCR template. For the quantification of the number of viral copies, a portion of the viral promoter CBh was amplified . For data normalization, a portion of the mAgouti gene was also amplified . For data interpolation, the calibration curves for CBh and mAgouti were generated through the quantification of serial dilutions of the AAV vector plasmid and a plasmid containing Agouti fragment, respectively. A master calibration curve for CBh and mAgouti was calculated as the mean of several calibration curves obtained from multiple runs  [Genomic DNA (gDNA) was extracted from the brain region of interest with the NucleoSpinmAgouti) . Ct valuCdkl5 − /Y mice treated with vehicle, AAVPHP.B_CDKL5, or AAVPHP.B_Igk-TATk-CDKL5 with the GRS FullSample Purification Kit (GRISP) according to the manufacturer’s instruction. Isolated mRNA was subjected to a DNase I treatment (GRISP), and cDNA synthesis was achieved using iScript™ Advanced cDNA Synthesis Kit (Bio-Rad), according to the manufacturer’s instruction. Reverse transcriptase PCRs were performed using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) in iQ5 Real-Time PCR Detection System (Bio-Rad). A portion of the m-hCDKL5  sequence was amplified for the quantification of AAV vector genome transcripts. A portion of mGAPDH  sequence was amplified for data normalization. The differential folds of expression were calculated using the ΔΔCt method. Values were expressed as the fold increase in the CDKL5 expression in the cortex relative to that of the wild types.Total RNA was isolated from the brains of n indicates the number of mice. Statistical analysis was performed using GraphPad Prism software . All datasets were analyzed using the ROUT method (Q = 1%) for the identification of significant outliers and the Shapiro–Wilk test for normality testing. Datasets with normal distribution were analyzed for significance using Student’s t test or an ordinary one-way analysis of variance (ordinary one-way ANOVA). Post hoc multiple comparisons were carried out using the Fisher least significant difference (Fisher’s LSD) or a Tukey test. Datasets with non-parametric distribution were analyzed using the Kruskal–Wallis test. Post hoc multiple comparisons were carried out using Dunn’s multiple comparison test. For the open field and the learning phase of the Barnes maze, statistical analysis was performed using a repeated measure two-way analysis of variance (RM two-way ANOVA). A probability level of P < 0.05 was considered to be statistically significant. A descriptive statistic of the treatment factor is given in the Supplementary Table Results are presented as mean ±  standard error of the mean (± SE), and To express the Igk-TATk-CDKL5 and CDKL5 proteins by means of AAV vector-mediated gene delivery, an Igk-TATk-CDKL5 or a CDKL5 gene expression cassette  was inseCdkl5 KO (− /Y) mice that had undergone intraventricular injection with AAVPHP.B_Igk-TATk-CDKL5 vector at the neonatal stage and that were sacrificed 2 months after the injection. By combining fluorescent in situ hybridization (ISH) and immunohistochemical staining to simultaneously visualize CDKL5 mRNA and protein, respectively, we found CDKL5 protein replacement in brain cells that did not show ISH staining  mice were used as controls for behavioral tests. Importantly, no changes in terms of body weight, sleep pattern, microglial cell number, or cell survival were observed in vector-treated Cdkl5 − /Y mice compared to age-matched vehicle-treated mice  Cdkl5 − /Y mice is associated with autistic-like (ASD-like) phenotypes, analyzed through home-cage social behaviors (marble burying and nest building ability) [Cdkl5 − /Y mice buried a significantly lower number of marbles and showed a reduced nest building ability compared to wild-type (+ /Y) mice  . Cdkl5 −ice Fig. a, b. Sixice Fig. a. Similator Fig. b.Fig. 3ECdkl5 − /Y mice [Cdkl5 − /Y mice showed a higher clasping score  imaging, a method that allows us to monitor the visually evoked responses in the same animal at different time points . We founion Fig. h, the viice Fig. h.Cdkl5 − /Y mice [Cdkl5 − /Y mice exhibit a deficit in dendritic spine structure and stabilization [Cdkl5 − /Y mice treated with AAVPHP.B_Igk-TATk-CDKL5 vector, the length of both apical and basal dendrites was recovered compared to vehicle-treated and AAVPHP.B_CDKL5-treated Cdkl5 − /Y mice  as a model of genetic brain disorder, our study provides novel evidence that a gene therapy approach based on a vector expressing a therapeutic protein fused to an Igk-TATk polypeptide provides increased protein biodistribution and therapeutic efficacy compared to the same vector devoid of the fusion peptide.Cdkl5 − /Y mice to be more suitable as an in vivo disease model than Cdkl5 + / − mice. Moreover, the age of the mice  and the route of administration  was chosen to avoid a massive viral infection that would target a high number of cells, potentially hindering, in part, the assessment of effectiveness of the cross-correction mechanism.Although most CDD patients are females who are heterozygous for CDKL5 deficiency due to random X-chromosome inactivation, hemizygous males have also been reported. The testing of therapies in all genotypes would, of course, be of interest. However, in this proof-of-concept study of gene therapy based on a cross correction mechanism, to avoid the mosaicism interaction that would confound the comparative analysis, we considered Cdkl5 − /Y mice treated with AAVPHP.B_Igk-TATk-CDKL5 vector underwent a higher neurodevelopmental and behavioral improvement than mice treated with AAVPHP.B_CDKL5 vector. Importantly, no toxic effects, including immunogenicity problems related to the secreted TATk-CDKL5 protein, were observed in AAVPHP.B_Igk-TATk-CDKL5-treated mice, suggesting the safety of this approach. However, a comprehensive toxicology study needs to be carried out to confirm the safety of this novel gene therapy approach. These promising results suggest that a gene therapy with the Igk-TATk-CDKL5 transgene may be an effective approach to treat CDD. Even if we detected around 6% of cross-corrected cells in the brain of AAVPHP.B_Igk-TATk-CDKL5-treated Cdkl5 − /Y mice, we believe that the total number of cross-corrected cells was underestimated due to technical detection problems, mainly concerning the difficulty in detecting cells that received a small amount of protein through a cross-correction mechanism. The number is likely to be higher, however, as supported by the many improved outcomes.We demonstrated that Cdkl5 − /Y mice did not exhibit improvement in behavior in comparison with vehicle-treated mice. The poor therapeutic effect of gene therapy with only CDKL5 has previously been reported [Cdkl5 − /Y mouse, such as innate behaviors and motor performance, and to ameliorate visual function. Albeit in a more marginal way, hippocampal-dependent learning was improved. The lesser therapeutic effect may be attributed to the lower number of viral copies reaching the hippocampus compared to other brain regions. The correlation between levels of CDKL5 re-expression and effectiveness of the gene therapy was confirmed by the finding that in the hindbrain, where the CDKL5 endogenous levels are lower than in the rest of the brain [Cdkl5 − /Y mice, treated with either AAVPHP.B_CDKL5 or AAVPHP.B_Igk-TATk-CDKL5 vectors, could then explain the normalization of the breathing pattern during REM sleep.Following AAVPHP.B_CDKL5 vector delivery, reported  and was reported . It is nhe brain , treatmeCdkl5 − /Y mice, we found that the impaired dendritic and synaptic development was restored, as was neuronal survival in the hippocampus. By contrast, the lack of behavioral improvement in Cdkl5 − /Y mice treated with AAVPHP.B_CDKL5 may be accounted for by the reduced effect of treatment on neuronal survival and dendritic development. Similarly, microglia over-activation, a recently described alteration in the brains of Cdkl5 − /Y mice [Cdkl5 − /Y mice does not induce the full recovery of hippocampus-dependent cognitive abilities may be attributable to the complex and intricate in vivo brain function which goes beyond structural restorations.As an anatomical substrate of the ameliorated behavioral performance, in AAVPHP.B_Igk-TATk-CDKL5-treated  /Y mice , is inhiCDKL5 mRNA is mainly expressed in the cerebral cortex and in the hippocampus. This different distribution could explain the modest therapeutic efficacy shown by the CDKL5 gene therapy. Moreover, we observed unexpected inconsistency between the expression of the viral mRNA and the low CDKL5 protein expression, suggesting a relatively low half-life or reduced translation of the delivered CDKL5. The hypothesis of CDKL5 instability is in agreement with a recent finding showing that the viral MeCP2 mRNA was not actively translated by ribosomes, underscoring the importance of complex endogenous regulatory elements for MeCP2 protein expression [CDKL5 mRNA levels suggest a high CDKL5 re-expression in the brain, i.e., in the hindbrain, the CDKL5 protein level was much lower compared to that found in wild-type mice, indicating that CDKL5 protein levels correlate poorly with viral mRNA levels. Future studies are needed to characterize regulatory elements of the CDKL5 mRNA and to design a CDKL5 transgene cassette that would be useful to obtain higher CDKL5 protein levels. However, it was demonstrated that even a modest 5–10% re-expression of MeCP2 has a promising therapeutic effect on the Rett Syndrome (RTT) phenotype in a mouse model of RTT [Cdkl5 − /Y mice are sufficient to ameliorate CDD phenotypes if supported by an increased biodistribution due to the properties of the Igk-TATk fusion protein.Interestingly, despite finding a broad distribution of the delivered cassettes over the different brain areas of the viral-injected mice, the viral transcript biodistribution differs to that of the wild-type mice, in which pression . Similarl of RTT . Althougl of RTT , that thl of RTT . Here, wRegardless of the encouraging results in the mouse model, much more needs to be investigated in view of the therapeutic potential of such treatment for CDD patients. First of all, a study on heterozygous females is necessary to verify whether CDKL5 protein production/cross-correction in wild-type cells may lead to an abnormal phenotype. Moreover, a long-term behavioral assessment needs to be performed to demonstrate the efficacy and persistence of transduction over a long time period.Cdkl5 − /Y mice support the amplified therapeutic effect of the secretable TATk-CDKL5 protein compared to CDKL5 alone. Thus, we believe that this study has provided the first proof-of-principle that an innovative gene therapy approach based on the unique advantages of the Igk-TATk fusion peptide is more efficient in improving neurodevelopmental and behavioral impairments in a mouse model of CDD. Such promising results imply that this approach may become a powerful tool for the cure of CDD, and could open avenues to the development of gene therapy for other monogenic diseases based on the unique and compelling properties of the Igk-TATk-fusion protein approach.The improved behavioral performances and neuroanatomical deficits observed in AAVPHP.B_Igk-TATk-CDKL5-treated Supplementary file1 (PDF 508 KB)Supplementary file2 (PDF 508 KB)Supplementary file3 (PDF 508 KB)Supplementary file4 (PDF 533 KB)Supplementary file5 (PDF 508 KB)Supplementary file6 (PDF 508 KB)Supplementary file7 (PDF 508 KB)Supplementary file8 (PDF 534 KB)Supplementary file9 (PDF 508 KB)Supplementary file10 (PDF 508 KB)Supplementary file11 (DOCX 4804 KB)Supplementary file12 (PDF 508 KB)Supplementary file13 (PDF 508 KB)Supplementary file14 (PDF 551 KB)Supplementary file15 (PDF 517 KB)Supplementary file16 (DOCX 39 KB)Supplementary file17 (DOCX 43 KB)Supplementary file18 (PDF 526 KB)Supplementary file19 (PDF 508 KB)Supplementary file20 (PDF 508 KB)Supplementary file21 (PDF 508 KB)Supplementary file22 (PDF 508 KB)Supplementary file23 (PDF 508 KB)Below is the link to the electronic supplementary material.


One of the goals of a Newborn Hearing Screening Program (NHSP) is to have a low retesting rate.To investigate the association between the retesting rate and the bath of newborn babies. Study design: cross-sectional contemporary cohort.Transient otoacoustic emissions (TOE) results have been compared to the information received from the nurse's aide who bathed the babies on the day of the test (373 newborns), and the time interval between the TOE study and the last bath (350 newborns).Significant statistical differences were found in relation to the percentage of retesting when babies were bathed by certain nurse's aides. On the other hand, the percentage of retesting decreased significantly when the time interval between the last bath and the TOE study was longer than 7 hours and 50 minutes.Moisture of the external acoustic meatus, caused by inappropriate protection against water during bath, and the short interval between the bath and the ETOE study, could be considered as possible factors causing retesting in NHSP programs. Hearing deficiency is a serious problem in public health. However, since it is not evident in the first months of life, this deficiency may have a late diagnosis, and as a consequence, it may cause damages to speech development and to the overall development of the child.American Academy of PediatricsThe rate of children born alive reported in Datasus website indicates the birth of 3,026,548 babies in 2004 in BrazilAccording to the Joint Committee of Infant HearingDespite the importance of early intervention for the development of those children with hearing deficiency, the average age at diagnosis of hearing deficiency in Brazil is late. Nóbrega7, evaluated 442 subjects with hearing deficiency from 1990 to 1994, and from 1994 to 2000, revealed that even with the increased information available for the population and a higher level of medical knowledge on target exams, considering the second period studied, this average age did not show significant statistic changes, remaining at around two years old. In our clinical practice, we see that hearing alterations are commonly identified by speech delay generally at 18 to 24 months old.In order to prevent this situation, UNHS, that is to say, to screen the hearing of all babies born at maternity hospitals is being done in some Brazilian maternity hospitals, as it is done in the United States and Europe.The importance of programs for early identification of hearing deficiency, in which all newborn babies must be screened, is stressed by the widely disseminated data that 50% of patients with hearing difficulties do not show risk factors for hearing deficiencyHowever, it is noted that despite UNHS being one of the most important tools in early identification programs for hearing disorders, its functionality depends directly on the criteria used to do it.The National Institutes of Health, in 1993In the last survey done by the Newborn Hearing Screening Supporting Group in 2005, it was seen that there were 237 places in 22 states developing NHS Programs with an objective methodology 11 in Brazil. At the International Conference Beyond Newborn Hearing Screening, that took place in Como, Italy in 2004, Chapchap and Ribeiro reported that out of the 192 places that at the time had NHS Programs in Brazil, 61% used transient evoked otoacoustic emissions (TEOAE), 24% evoked otoacoustic emissions- distortion product and 15% Brain Stem Hearing PotentialWith objective exams, NHS Programs became feasible. However, there are difficulties. In the study of Evoked Otoacoustic Emissions for example, some factors may distort the result, such as professional training when placing the probe; excessive noise during the exam; baby´s condition at the moment of the exam, that is to say, if he/she is asleep or awake, hearing screening test within the first 24 hours of life, a period that increases the possibility of vernix in the external acoustic meatusDespite confirmation of above mentioned factors, there is not enough literature regarding the difficulties found with Evoked Otoacoustic Emissions specifications at NHS. A difficulty that was mentioned more frequently is the presence of vernix. Chang et al.In order to establish the final result, without interference from the possible presence of vernix, re-testing is normally indicated no longer than 30 days of life. However, we also observed in our practice that the required time for re-test can cause anxiety in the family as a consequence and it can also create a lasting doubt for speech therapists, due to the failure of patients to come back to be re-tested. Thus, the recommendation resulting from this statement is that we should try to achieve a reduction of re-tests.Vernix is defined as a lipid rich substance that covers the fetus skin and it is also present in the newborn skinSince vernix is a fatty substance which does not mix up with water, it is believed that there may be a greater difficulty of water absorption when there is vernix. It is supposed that this may explain our practical observation that after bath, it is often more difficult to capture TEOAEs, possibly due to humidity in the external acoustic meatus.Another issue that called our attention in our practice was that when we did the same amount of exams in alternate days, at the same maternity hospital, the number of re-tests showed a sensitive alteration, for example, on one day, in 10 screenings, not even one re-test whereas on the following day, with the same amount of exams, there were three or four re-tests.-re-testing rate could vary depending on the person responsible for the baby´s bath, due to the way and/or the experience of the assistant nurse who allows water into the external acoustic meatus during bath;-the re-testing rate could vary depending on the time passed between the last bath and the Evoked Otoacoustic Emissions test, because with moist external acoustic meatus, the failure in the exam seemed to be even more frequent, mainly with the presence of vernix.The following hypothesis resulted from our practical observation:Taking into account these hypothesis and considering the importance of trying to reduce the number of re-tests on the EOE study at NHS, this research aimed to investigate the relationship between re-testing rate and the newborn bath, at a NHS program, considering the professional responsible for the bath and the time passed between the TEOAE study and the last bath.This research was approved by the Research Ethics Committee at the University where we did the study, authorization no 017/2004. Following current standards on ethics on research with human beings, all participants  of this study agreed on taking part and they received information on the project and signed an Informed Consent.Data were gathered at a maternity hospital in Curitiba, where there is a Hearing Deficiency Early Identification Program. This Program started in 1996 and its methodology uses TEOAE in newborn children. All screenings are done within the first 24 hours or more of life, preferably closer to the hospital discharge (approximately 48 hours old), in a big room with the baby in the adequate state and it is done by speech therapists with a large experience on NHS. Thus, the possible re-tests resulting from the lack of training of the professional in placing the probe, excessive noise during the exam, an inadequate state of the baby at the moment of the test and hearing screening before the baby is 24 hours old were avoided.After the test, when the baby does not pass the exam, a new test is scheduled in 30 days. If TEOAE are still altered, the baby is sent for a complete audiological evaluation for diagnosis confirmation.In the daily routine of maternity hospitals, when babies are born they are kept in a warmed crib for about two hours and then they received the first bath. Assistant nurses are responsible for baths and they use a special technique to place the fingers pressing the tragus to prevent water from coming in.We started gathering data for this study in March 2004 and we finished in June the same year. Despite the fact hearing screening is optional, we screened 130 babies per month for each 200 born babies on average, making up a total of 538 babies on NHS during the period of the study. However, the study of the causes of this research was different according to the variable we studied, taking into account the amount of information that can be obtained regarding each baby.The first analysis done tried to check the correlation between the assistant nurse who gave the bath just before the TEOAE test and the of re-tests. On the second analysis, we looked at a possible correlation between re-testing rate and the time span between the last bath and the TEOAE test.Data related to the assistant nurse responsible for the last bath before the test and to the time span between the bath and the TEOAE study were gathered with the help from the team of assistant nurses at the nursery, who filled in a form to record all this information .AppendixConsidering that not all assistant nurses filled in all necessary data for all babies, the number of babies we studied was different according to the variable studied, that is to say, 373 babies for the variable of the assistant nurse who gave the last bath and 350 babies for the variable of the time span between the last bath and the TEOAE. Thus, sample inclusion criteria were to have forms completely filled out by the nursing team and the lack of risk factors for hearing lossFor TEOAE test, we used the ILO Echocheck equipment by Otodynamics, and as stimulus we used a click in an intensity of 83 dBNPS, and the frequency range analyzed was between 1,600 and 3,600 Hz. For the normality standard the equipment allows us to choose the minimum values of the signal/noise ratio, as well as the response level of TEOAEs. In this study, the values adjusted as normality criteria for the presence of TEOAE  were the following: TEOAE amplitude and the signal/noise ratio as the same or above 6 dBNPS, according to what Román et al suggested.The data obtained from the forms were analyzed together with TEOAE records.Proportion Test was used in the statistic analysis with a significance level of 5%.Taking into account that some assistant nurses only gave a few baths, we choose to do a statistical analysis including only those who gave at least 10 baths. Therefore, we used the Proportion Test with the aim of checking if the differences in percentages of re-tests obtained by these nursing assistants differed significantly. Considering a 5% significance level, we saw a statistical significant difference between the re-test percentages obtained by assistant nurses G and M , G and N  and M and Z , suggesting that some assistant nurses may have produced more re-tests than others.Another interesting data that may be obtained from looking at It can be observed that 41 out of the 350 babies who had the TEOAE test done, had to be re-tested, that is to say 11.71% of them. Re-test results were as follows: 38 normal tests (92%) and three altered tests (8%). The three babies with altered re-tests were sent for diagnosis and they had normal results. As it can be seen on From the results obtained and showed on One of the main concerns of professionals working with hearing deficiency early detection programs is related to re-testing ratesThe consequences of these re-tests can be many: anxiety of parents related to a possible confirmation of hearing deficiency, the baby does not show up for re-test and the consequent “loss” of the child for diagnosis, increased demand on the service flowchart of hospital/maternity/clinic, increased cost-effectiveness of the hearing screening program, among others.Therefore, this study tried to investigate some factors that may contribute to a very important cause in the production of re-tests: moisture in the baby's external acoustic meatus at the hearing screening.Among these factors, we can mention the work done by assistant nurses who bathe the baby on the day of the exam. We can state that some of them had re-testing percentages significantly different form others. It is believed that this confirmation may result from the more or less skills of these professionals to protect the baby's ear to avoid the water going into the ear at bath time. Furthermore, we also noticed that those assistant nurses who did not sent babies to be re-tested were the most experienced professionals of the sector, according to the information obtained from the head nurse. Thus, the findings obtained confirmed the hypothesis that re-testing rates can be influenced by the experience the assistant nurse has in bathing babies, avoiding or not water from coming into the external acoustic meatus.Another result that confirms the hypothesis that water inside the babies’ external acoustic meatus may have caused the re-testing, has to do with the time span between the bath at the day of the exam and the TEOAE test. The results obtained on Therefore, the results obtained on the analysis of the two variables  suggest that humidity on external acoustic meatus may be increasing the re-testing percentage of TEOAEs, considering that the variable experience of examiner, noise level at the room, exam made with less than 24 hours and the state of the baby at the moment of the exam were not considered for the methodology used.It is believed that the persistent moisture at the external acoustic meatus may result from the presence of vernix, considering that it works as a hydrophobic barrierWe think it is important to point out that a possible cause for re-testing seen at this study was not found in any other study we reviewed. According to Maxon et al.Thus, the results obtained in this study suggest that we need to include specific guidance for nursing teams and parents into the protocols developed on NHS programs. These guidelines should include being extremely careful to avoid water coming into the baby's ears during bath. Besides, the results also suggest that the TEOAE test should be made with the highest possible time interval after the newborn's bath.It is believed that if this care is taken, re-testing percentage may be considerably reduced and therefore it would be possible to reduce the cost-effectiveness of the hearing screening program, as well as the anxiety experienced by parents when receiving an altered result.From this point of view, we stress that the success of an early identification program of hearing loss is related to the possibility of reducing false-positive results caused by the conditions of the external ear, among other factors.From the results obtained, we can conclude that moisture in the external acoustic meatus of babies, caused by a bath time too close to the study of otoacoustic emissions and by an inefficient protection of the external acoustic meatus regarding water penetrating the the ear at bath time, can be considered a possible factor that generates re-testing on NHS programs that use TEOAE as a hearing screening procedure.The confirmation that the baby's bath at the day of the exam may influence the result of the TEOAE test, offers a very relevant information for NHS programs that have as one of their goals low re-testing percentages.


This study investigates the effect of hearing aid use on the peripheral auditorypathways in children with sensorineural hearing loss prior to cochlearimplantation, as revealed by the electrically evoked auditory brainstem response(EABR). Forty children with hearing aids were recruited. Half of them had normalinner ear structures and the other half had inner ear malformations (IEMs). TheEABR was evoked by electrically stimulating the round window niche (RWN) andround window membrane (RWM) during the cochlear implantation operation. Theonset age of hearing aid use was significantly correlated with the peaklatencies, but not amplitudes, of the wave III (eIII) and wave V (eV). HigherEABR thresholds were found for RWN stimulation than for RWM stimulation and inthe children with IEMs than in those without IEMs. Our study providesneurophysiological evidence that earlier use of hearing aids may amelioratephysiological functions of the peripheral auditory pathway in children with andwithout IEMs. The EABR evoked by the electrical stimulation at RWM is moresensitive compared with that at RWN for evaluating functions of the auditoryconduction pathway. The development of the auditory system depends to a large extent on sufficientauditory input during the sensitive period, i.e., a period in which the auditorysystem requires external stimulation to develop skills for auditory processing.Auditory deprivation during the sensitive period will adversely affect many aspectsof cortical maturation, hinder the development of auditory pathways, and result indecreased behavioral performance and deficits of spoken language acquisition . In the The effect of hearing aid use on functions of the auditory pathway has been assessedby using behavioral tests and cortical evoked responses. Relevant evidencedemonstrates that hearing aids can help patients with hearing loss improve theirspeech performance and reduce auditory processing effort . ImagingThe electrically evoked auditory brainstem response (EABR) is an objective andeffective method to evaluate functions of the auditory conduction pathway up to thelevel of the brainstem . It can In this study, we investigated the effect of hearing aid use on physiologicalfunctions of the auditory pathway in hearing-impaired children with and withoutinner ear malformations (IEMs) by using the EABR. The EABR was evoked by electricalstimulation at RWN and round window membrane (RWM) during the operation to implantthe CI. We hypothesized that hearing aid use prior to CI activation could promotethe development of the auditory brainstem pathway. Therefore, we predicted thatchildren with earlier or longer use of hearing aids would show lower EABR thresholdsand/or shorter eIII and eV latencies. Exploratory analyses further compared the EABRthresholds and latencies for two stimulation sites (RWN and RWM) in two groups(children with and without IEMs).Forty children : 5.09  ±  3.79years old) with sensorineural hearing loss who received their first CI in ourhospital from September 2018 to June 2020 were included in this study. Thesechildren were right-handed according to an assessment with the EdinburghHandedness Inventory . They stμm. A stainless steel needle electrode was used as thereference electrode and was placed in the coarse protuberance of the occipitalbone at the operation side. The EABR output signal was filtered online with aband-pass of 0.1–3 kHz and was averaged from 512 sweeps at each stimulus levelwith a time window of 15 ms. The electrical pulse was the alternating wave with100-μs duration and was delivered at a rate of 21 Hz.Electrode impedances were less than 3 kΩ.The EABR was recorded by Neuro-Audio NET1.0.103.3. .The recording electrode, the ground electrode and the reference electrode werebody surface button electrodes and were placed in the middle of the forehead,between the eyebrows, and about 1 cm in front of the tragus of the operativeear, respectively. The electrical stimulation was generated from an EMG externalelectric stimulator . The facial nerve stimulationprobe  was selected as the stimulation positiveelectrode. Its surface was coated with Parylene insulating coating except forthe exposed tail end of the electrode and the diameter was 500All surgical procedures were performed via a mastoidectomy. Posterior tympanotomywas performed through the facial recess. Meticulous hemostasis could be achievedby using diamond burs. After the RWN was exposed, we injected patients withmuscle relaxant cis-atracurium at 0.5 mg/kg according to the body weight toreduce the interference of muscle activity from EABR signals. During the firstEABR recording, the stimulation probe was placed on the surface of the RWN.Then, a diamond bur was used to remove the RWN and maximally expose the RWM. Weperformed the second EABR recording by placing the stimulation probe on thesurface of the RWM. The initial electrical stimulation intensity for the EABRwas 2.0 mA. To assess the EABR threshold, namely the minimum stimulationintensity eliciting eIII or eV, we increased or decreased the stimulationintensity in a first step of 0.5 mA followed by a smaller step of 0.1 mA untilthe eIII or eV appeared or disappeared. The maximum stimulation intensity was3.0 mA. The EABR waveform for each stimulation intensity was averaged by 512epochs and detected visually. Each test of the EABR for the RWN or RWMstimulation lasted 3–5 min.r  =  0.989; RWM:r  =  0.996), eIII latencies  and eVlatencies marked by the first observer showed high correlations with the second. Twocriteria for identifying the eIII and eV were that the two components should bereproducible at least at two different stimulation intensities and thedifference in peak latencies of each component should be ≤0.3 ms betweenadjacent stimulation intensities. One child without IEMs and one with IEMsshowed no robust eIII and eV, and these two children were excluded from furtheranalysis. SPSS Statistics V.24  was used for statisticalanalysis. The eIII and eV latencies for each individual were determined at astimulation intensity of 2.0 mA when the EABR threshold was ≤2.0 mA or at thestimulation intensity of the EABR threshold when the threshold was >2.0 mA.Differences in the EABR thresholds and latencies between the two stimulationmethods and the two groups were analyzed by using a two-way analysis of variance(ANOVA) with stimulation method (RWN and RWM) as the within-subjects factor andgroup (children with and without IEMs) as the between-subjects factor. TheGreenhouse–Geisser adjustment was applied when the variance sphericityassumption was not satisfied. A Pearson correlation test was used to assess thecorrelations between the onset age and duration of hearing aid use and the EABRthreshold, eIII latency, and eV latency for all participants. Thep values were corrected for multiple comparisons using thefalse discovery rate (FDR). All values in this study are expressed asmean  ±  SD.The eIII and eV were marked by two observers blinded to the information of eachchild. The EABR thresholds   =  13.380, p < 0.001]and group .There was no significant interaction between these two factors. The EABRthresholds were significantly higher for RWN stimulation than for RWMstimulation and in children with IEMs than in those without IEMs  < 0.001, p  =  0.996; eV:F < 0.001, p  =  0.995], group orsignificant interaction between these two factors   and eV latencies but not with theEABR thresholds  , eIII latency, or eV latency  . No sign  0.864) .In this study, we examined the effect of hearing aid use on the peripheral auditorypathway in children with sensorineural hearing loss by recording the intraoperativeEABRs. We found positive correlations between the onset age of hearing aid use andthe peak latencies of the eIII and eV. Furthermore, higher EABR thresholds werefound for RWN stimulation than for RWM stimulation and in children with IEMs than inthose without IEMs. Our neurophysiological results suggest that earlier use ofhearing aids can ameliorate physiological functions of the peripheral auditorypathway in children with sensorineural hearing loss with and without IEMs.Furthermore, the EABR evoked by the electrical stimulation at RWM is more sensitivecompared with that at RWN for evaluating functions of the auditory conductionpathway.The EABR eIII and eV are less affected by electrical artifacts compared with eI andeII and are therefore usually used as neurophysiological indicators in implantedindividuals. The two later components (eIII and eV) indicate that the signal hasreached the brainstem at a higher level along the auditory pathway (the superiorolivary nucleus and the inferior colliculus). We previously reported that the EABRresponse rates were 80% and 55% for patients with no IEMs and those with Mondinimalformation, respectively . The higPrevious electrophysiological studies have shown the positive effect of an early CIon the development of the central auditory system . Long-teIt is suggested that a hearing aid is fitted as early as possible for the developmentof the auditory system, which is also supported by our finding that the onset age ofhearing aid use was positively correlated with latencies of eIII and eV. However, inthis study, children with hearing loss received hearing aids relatively late (at amean age of 2.3 years old). A late intervention with a hearing aid has also beenfound in other studies . This caThe EABR can be evoked via intracochlear CI stimulation  or by exIn conclusion, our EABR findings demonstrate that earlier use of hearing aids canameliorate physiological functions of the peripheral auditory pathway in pediatricCI candidates. Early hearing aid fitting can promote the development of the auditorysystem during the sensitive periods, providing the neural basis for speechrehabilitation after a CI is received. Our neurophysiological evidence also suggeststhat the EABR evoked by the electrical stimulation at RWM may be more sensitive andeffective for evaluating auditory conduction functions than stimulation at RWN.


Perpetuated exposure to higher decibels is one of the leading causes of noise-induced hearing loss (NIHL). The occurrence of NIHL has likewise been expanding in kids and adolescents. NIHL in youngsters and adolescents is connected with personal music players, which need earphones or headphones, and have powerful sound tones. The effects of such leisure activities and their relation to NIHL remains poorly studied. Various studies have been conducted to establish the degree of correlation between audio device usage and hearing disablement; recent studies show more agreeable data about the same. Therefore, the use of earphones and their effects on hearing needs to be well assessed, considering the high prevalence of the problem and the relative lack of literature for the same.The intent of the following review is to gather information regarding the manifestations of impairment in hearing due to audio devices with the objective to assess the correlation between hearing impairment and the use of audio devices. Also, guidelines established by Central Pollution Control Board regarding noise pollution need to be revised as per the present-day setting. The implementation of these guidelines is the responsibility of the companies that design and produce different types of audio devices. These devices should be made ear-safe according to standard safety guidelines related to hearing impairment from loud noise. This review has taken into consideration 23 authentic studies from all around the globe that state a positive or negative result after studying the correlation between NIHL and long-term noise exposure. The findings of this narrative review indicate that leisure activities involving the use of audio devices pose a risk of NIHL, and a need for further research in this domain is seen, given the ubiquitous and ever-increasing nature of their consumption. Noise-induced hearing loss (NIHL) is an impairment in hearing due to perpetuated exposure to loud sounds. This is classified as sensorineural hearing loss, which usually presents with bilateral, irreversible, and progresses with continual exposure to higher decibels. It is a general medical condition that can restrict regular day-to-day activities. The threat of disablement in hearing because of prolonged exposure to noise is present in about 12% of the population worldwide, which is also among the most preventable causes .The Global Burden of Disease Study  found thStudies carried out in the period between 1985-1990A pilot study was made by Lees et al. in 1985 to assess the prevalence of high-frequency hearing loss due to non-occupational noise exposure in 60 randomly selected students. The audiometric assessment was performed on 30 males and 30 females between the ages of 16-25 years. Using a 10 dB attenuation of hearing at one test frequency as a positive result, 40% of the subjects had evidence of noise-induced hearing loss. The majority of positive cases manifest hearing impairment at 6000 Hz and not the classically accepted 4000 Hz frequency. A highly significant correlation was found between leisure noise exposure and positive test results .Lee et al. in 1985 conducted another study. Sixteen individuals participated in this pilot study in which they listened to earphones at their habitual maximum volume for three hours. A total of seven participants showed notable transient shifts, which returned to normal within a day. Above mentioned facts indicated that recreational sound often caused transient shifts, and rules for auditory public sustainability must be laid down .In 1985, Catalano et al. carried out a study involving three well-known testing models: Bruel and Kjaer sound level meter, Octave band filter, and Artificial ear. Researchers provided around 200 students with a questionnaire, and audiometric testing (frequencies used: 250-8000) was carried out. Nearly 31% of all radio users surpassed the maximum permissible noise dose considering the Occupational Safety and Health Administration (OSHA) criteria. Fifty per cent of the "at risk" group went above and beyond the risk threshold .In 1990, about 500 youngsters (15-24 years) in Hong Kong were interviewed by Wong et al. about their audio device usage frequency. More than 80% of them reported using audio devices for the last three years averaging about four and a half hours per week. The audiometric and otoscopic examination did not suggest a strong correlation between personal cassette player (PCP) users and NIHL, as most of them used these devices at safe levels .Studies carried out in the period between 1991-1995Hetu and Fortin, in 1995, took into consideration highly amplified music as a potential risk of hearing damage. Intense pulsations (rate 2 Hz), a limited dynamic range, and a sloping spectrum with the maximal energy in the 1/3-octave centred at 63 Hz were found to be the characteristics of party music. Persistent threshold shift seemed extremely unlikely for people who routinely listened to loud music, even though temporary threshold shift is anticipated for various durations of exposure to this type of sound .Studies carried out in the period between 1996-2000In 1999, according to Zenner's analysis of clinical statistics, it was found that one in ten teenagers suffered from NIHL caused by leisure-time noise .Studies carried out in the period between 2001-2005In 2002, Mazlan et al. conducted an analysis on 136 Client Service Representatives from a Malaysian telecommunication company who utilised audio headsets during their office hours. The objective of this study was to quantify the frequency of auditory canal infection and numerous ear, nose, and throat infections connected to it. Using the Amplaid 309 Clinical Audiometer, their hearing limits also were determined. In the subjects, we did not discover any common disease of the external ear canal. In four cases, the centre ear condition persisted, and in four patients, the wax was impacted. Twenty-five participants were examined for hearing impedance (21.2 per cent). However, because the use of earphones did not indicatively affect the high frequencies, there was little correlation between hearing disability and sound exposure .Studies carried out in the period between 2006-2010In a study conducted in China by Peng et al. in 2007, hearing impairment was discovered in 14% of the ears of 120 youngsters who used personal listening devices (PLD). These 120 PLD users, alongside 30 youngsters with normal hearing, were subjected to audiometry of both 'conventional' and 'extended high-frequency' types. The obtained results indicated that prolonged use of PLD contributed to hearing damage. The research also demonstrates that extended high-frequency audiometry is a reliable technique for identifying hearing loss brought on by noise at the initial stages [In an article published in 2007, Vogel et al. carried out a qualitative survey of focus-group discussions with adolescents aged 12 to 18 years from two large secondary schools  for pre-vocational and pre-university education. The semi-structured question route was theoretically drafted keeping in mind the protection motivation theory. The majority of young people revealed that they frequently listened to their MP3 players at the highest possible volume, particularly male students and students from pre-professional schools. Most young people claimed that they would not identify any limitations on their propensities for being open to music. They came to the conclusion that interventions should concentrate on educating young people about loud music's potential dangers and how to protect themselves, with a particular focus on pre-professional school students. There may be a need for MP3 player volume-level guidelines in addition to customised MP3 player modifications and hearing instruction for children .Vogel et al., in 2009, focused on 1687 young people (12-to 19-year old). Of all members, 90% detailed paying attention to music via headphones on portable audio devices; about 29% were arranged as audience members in danger of hearing misfortune because of assessed openness of 89 dBA for more than one hour out of every day. Compared with listeners not at risk for hearing loss, at-risk listeners were more likely not to live with both guardians, to experience satisfaction of listening to higher decibels, to give an account of increased propensity strength connected with risky MP3 listening, and were not much inclined to be roused to safeguard their ears. The most reliable indicator of high-risk listening behaviour was propensity strength, suggesting that it might be challenging to change the young generation's intentional behaviour and that a variety of different approaches might be necessary to prevent music-related hearing impairment ,23.In 2009, Mostafapour et al. conducted a potential ear testing among a group of students aged above 18 years who presented with a history of exposure to various sources of higher decibels of leisure noise. Volunteered students were subjected to typical auditory examinations. In the study, it was identified that the presence or absence of particular notches in audiograms could not be linked to a particular noise-pollution source, and a low degree of correlation was found between PLD users and NIHL. But this could not indicate a total disregard for the correlation between NIHL and prolonged use of audio devices in larger, more exposed populations .In 2010, McNeill et al. evaluated the possible threat to hearing incidental to the usage of handy audio devices. A 49-item survey measuring client listening preferences and emotional aspects of hearing wellness was completed by 28 college freshmen . In a variety of study halls with foundation noise levels between about 40 and 55 dBA, sound level estimations of members' self-recognised common and "considering the worst possible scenario" volume levels were made. The term of usage was about two hours each day for six and a half days. The interquartile ranges (IQR) at average and 'worst case' volume settings were IQR=12 and IQR=9, respectively. Nineteen students who participated in the survey said they occasionally experienced a potential disturbance's adverse side effects, which led to hearing loss. Between those who had experienced tinnitus and those who had not, there were stark differences in MP3 client listening styles . Table 1Studies carried out in the period between 2011-2015In 2011, Schlauch and Carney conducted a study to calculate the percentage of false-positive results for guidelines proposed to detect early NIHL by studying audiograms for typical NIHL-associated notches. Audiograms of children belonging to the age group of 6-11 years showed no significant findings. Inadmissible high false-positive values were obtained after applying the pass-fail criteria to the data collected for measuring hearing acuity. Getting rid of systematic calibration errors, repeating and averaging threshold measurements, and utilising headsets with lower variability for 6.0 and 8.0 kHz, two frequencies vital for distinguishing noise notches, all have the potential to increase audiometric precision .Another study in 2011, conducted by Punch et al., was aimed at assessing PLDs and their impact on hearing. It was found that young adults and teenagers seem to view the dangers of PLD use to ears somewhat differently. Therefore, messages intended to propose actions they may take to avoid or minimise these dangers must be targeted in order to have the best results .In a cross-sectional study in November 2011 by Ansari et al., a 20-item questionnaire based on headphone usage was given to 2,400 random high school attendees. The results displayed that about 45% of the involved population had a record of hearing issues . About 37% of the respondents confessed to listening to music non-stop, and about 50% stated that volume was kept at higher than usual levels. This included use of various kinds of audio devices, and only about 16% of students were not using any kind of music device. This study observed that the depicted hearing habits of students were pretty risky .A 2013 article by Skånland investigated the use of music players and their role as affect regulators. This article stated that music, if integrated into one's life on a daily basis, would help us to regulate our emotions and achieve desirable outcomes in most situations. With newer portable technology such as MP3 players, it becomes easier to access music. This is an excellent tactic, as any hearing damage caused would be insignificant when compared to detrimental side effects of other risky regulation tactics such as alcoholism and smoking .In 2013 itself, Sulaiman et al. assessed early hearing impacts connected with PLD utilisation in 35 youthful grown-up PLD clients and their age-and sex-matched controls using a mix of typical and extended high-frequency audiometry along with Transient-Evoked Otoacoustic Emission (TEOAE) and Distortion Product of Otoacoustic Emission (DPOAE) estimations. Increased values of the mean hearing threshold at prolonged high frequencies in PLD clients, along with decreased amplitudes of TEOAE and DPOAE, were observed, which could be inferred as a leading road to hearing impairment .In 2014, Naik et al. conducted a study amongst 1000 students from a reported institute. Four groups were made, Group A involving 250 understudies who had a propensity for listening to music using earphones for no less than two hours daily, Group B including the same number of understudies who listen to headphones for under one hour each day and Group C including 250 understudies who once in a while use headphones but do use speakers often and lastly Group D involving the rest 250 understudies who are not accustomed to listening music via headphones. All four groups were exposed to pure tone audiometry. About 5%- 10% of understudies from Group A and 1%- 5% from Group B depicted high-frequency hearing loss. No significant hearing disablement was observed in Group C and D. This suggested a strong correlation between the duration of hearing and NIHL .In 2015, Keppler et al. evaluated the effects of leisure-noise exposure on hearing function in about 160 young adults. Weekly and lifetime equivalent noise exposure calculations were the basis of the division of participants into low, intermediate, and highly exposed categories. Between the three groups, there were no appreciable changes in hearing thresholds, TEOAE amplitudes, or DPOAE amplitudes. More than 85% of subjects complained of temporary post-exposure tinnitus. No significant difference between the participants of the three groups was appreciated in the short duration .Studies carried out in the period between 2016-2020In 2019, Zia et al. directed a detailed observational review at the Outpatient Department of a renowned hospital in Karachi, Pakistan. Data in regards to the earphone, use of Q-tips and swimming and their manifestations as ear disease were studied. Injury to the auditory canal, tingling sensations, ear discharge, tinnitus, and hearing disablement were some of the occurrences as per the data gathered through a pre-organised survey. About 56% of the population under consideration used headphones, and a majority of them presented with infection of the ear, itching, and hearing damage .Byeon, in 2020, led an observational review. The study was based on South Korean youths who were habitual of using headphones in public places with a loud environment. This study assessed around 530 subjects (12-19 years) who also were a part of a national survey in the past. Teenagers who were presented to high commotion levels through earphones in an uproarious climate, along with teenagers whose headphone utility extended 80 mins each day, had about 20%-25% predominance of possible hearing loss over others. This indicated that these students were 4.5 times more prone to hearing damage than others. The researcher noticed that longitudinal examinations are expected to give proof of causality between headphone use and hearing loss .Studies carried out post-2020In a 2021 review by Pienkowsk, the researchers address modern-day fun, which includes party clubs, sports stadiums and concerts, as "leisure noise". NIHL is one the most common preventable manifestations of this leisure noise, tinnitus and hyperacusis being the others. A much more comprehensive range of settings should implement noise limitations, and public health initiatives should place more emphasis on promoting aural conservation through education .In 2022, AlQahtani et al. played out a cross-sectional review to concentrate on the mindfulness of NIHL and hearing misfortune. The goal of the evaluation was to determine how much awareness there is regarding the connection between using headphones and NIHL. The poll included about 40 questions, which were divided into as many as six categories. The review incorporated a total of 1086 members. Inspected populace's age went from 18 to 55 years. Hearing issues were persistent in people who were exposed to noisy work environments and were considerably greater when listening sessions lasted more than five hours per day. The age of the subject, loudness levels he/she is exposed to, and weekly session length were some of the intangibles taken into consideration. A low degree of public knowledge about hearing loss due to bad hearing habits was reported . Table 2The term NIHL literally means any degree of hearing disablement caused due to prolonged noise exposure. With the advent of smartphones and various applications such as YouTube and other social media sites and video streaming platforms, leisure time activity and usage of earphones, headphones have become almost ubiquitous amongst the youth of developed and developing nations. Not only that, but high-quality headphones are also used during gaming, which involves the simulation of loud and sudden noises, such as those of gunshots, blasts, screams, and car crashes, delivered to the ear at close proximity, which are usually reasons for premature hearing loss.Therefore, the use of earphones and their effects on hearing needs to be assessed, given the high prevalence of the problem and the relative paucity of literature on the same. After taking into consideration 23 authentic studies regarding the correlation between NIHL and sound exposure, the findings of this narrative review indicate that leisure activities involving the use of audio devices pose a threat of NIHL, and further research is needed in this particular area, given the ubiquitous and ever-increasing nature of their consumption.