Extended life spans have led to an increase in the number of older people and an increase in the prevalence of disability among people older than 60 years of age.This study aims to investigate the association of sociodemographic variables and unhealthy behaviors with limitations in activities of daily living (ADL) among Thai older adults. The study also projects the number of older individuals likely to experience ADL limitations in the next 20 years.We performed sex-specific multinomial logistic regression analysis based on the 5th Thai National Health Examination Survey in 2014 to investigate the association between sociodemographic variables and health behaviors with ADL limitations among Thai older adults. Age- and sex-specific prevalence estimates of ADL limitations were obtained by applying the same models. These estimates were combined with population projections up to 2040 from the Office of the National Economic and Social Development Board, Thailand, to generate projections of older individuals with ADL limitations.Age and physical activity were significant factors for both sexes, with age positively associated with the level of ADL limitations and low physical activity associated with an increased relative probability of mild or moderate to severe ADL limitations compared to individuals with no ADL limitation (1.2-2.2 times). Other variables such as education, marital status, diabetes, hypertension, smoking, alcohol consumption, and having a fruit- and vegetable-based diet showed significant associations, but the results varied regarding sex and levels of ADL limitations. This study also projected the number of older adults with mild and moderate to severe ADL limitations over the next 20 years from 2020 to 2040, revealing an increase of 3.2 and 3.1 times, respectively, along with a significant increase in men compared to that in women.This study identified age and physical activity as significant factors associated with ADL limitations in older adults, while other factors showed varying associations. Over the next 2 decades, projections suggest a significant increase in the number of older adults with ADL limitations, particularly men. Our findings emphasize the importance of interventions to reduce ADL limitations, and health care providers should consider various factors impacting them. An aging society is characterized by a state where individuals aged 60 years and older constitute over 10% of the total population . The incUnhealthy behaviors such as low physical activity and having a high BMI, high cholesterol, and high blood sugar are known to increase the likelihood of disability among older adults -8. HandgGovernments require precise and prompt demographic information that is categorized by age, gender, and other pertinent factors to prepare for demographic changes and effectively implement policies and initiatives to address the effects and possibilities of an aging population. The United Nations suggests that governments be devoted to gathering, sharing, and evaluating demographic statistics, such as those pertaining to aging populations and older individuals. In Thailand, the studies on the correlation of social determinants of health and unhealthy behaviors with ADL limitations among older people are limited. Moreover, predictive values of personal health behavior and social determinants of health have seldom been applied to project the volume of older individuals with different ADL limitation levels. These estimates are crucial for driving health policy and addressing interventions that could decrease the number of disabilities among older individuals in the future ,13. TherOur study was based on two data sources: (1) the Thai NHES in 2014, and (2) population projections by 5-year age group and sex in Thailand.Data from the Thai NHES in 2014 were obtained . BrieflyADL limitations were assessed using the Barthel index —an ordinHypertension was defined as a diagnosis based on a systolic blood pressure of ≥140 mm Hg, diastolic blood pressure of ≥90 mm Hg from physical examinations, or self-reports of using antihypertensive medication.Diabetes mellitus was defined as a diagnosis based on a fasting plasma glucose level of ≥126 mg/dL from laboratory blood testing or self-reports of diagnosis from physicians, and currently receiving medical treatment.BMI was categorized as follows: underweight (<18.5 kg/m²), normal weight (18.5≤BMI<25 kg/m²), and overweight (≥25 kg/m²).Smoking status was categorized as follows: never smoked, current smokers (people who have smoked in the past 12 months), and former smokers (people who have smoked previously and not in the past 12 months).Alcohol consumption was defined as consuming at least 1 alcoholic drink (12.5 g/day) in the past 12 months.Physical activity level was categorized as high (≥150 minutes/week of combined intensity) and low (<150 minutes/week of combined intensity) in accordance with the global physical activity questionnaire of the World Health Organization .Fruit and vegetable consumption was categorized as high (≥5 portions/day) and low (<5 portions/day) .To project the number of older adults with ADL limitations in the future, we required population projections by age and sex. We used population projections from the National Economic and Social Development Board, which were based on population registration data from 2010 to 2015, collected by the Ministry of Interior and were generated using the cohort-component method. We selected the medium fertility assumption, which assumes that the fertility rate will decline from 1.62 in 2010 to 1.30 in 2040. The mortality assumption was based on life expectancy, which is projected to increase from 70.5 years to 76.8 years for men and from 77.8 years to 83.2 years for women between 2010 and 2040. The mortality pattern was created using a relational logit model. Lastly, a migration rate of 0 was used in the population projections.P<.05. To predict prevalence, we first selected potential covariate variables associated with ADL limitations. The results showed that age, sex, residential area, education, employment status, behavior risks, and health status were associated with ADL limitations . The NHES study was approved by the Ethical Review Committee for Research in Human Subjects, Ministry of Public Health, and all participants provided written informed consent.Overall, among 7306 respondents, the mean age in 2015 was 69.7 (SD 7.6) years, and the highest proportion of individuals (n=2493) were 34.1% in the age group of 60-64 years. Nearly equal proportions of men and women were observed . The prevalence rates of ADL limitations were 9.2% (n=672) for the mild level and 19.3% (n=1410) for the moderate to severe level.As shown in Furthermore, the proportion of individuals with no education attainment increased with the level of ADL limitation . The proportion of individuals with an abnormal BMI increased with the level of ADL limitation , particularly among underweight individuals. Additionally, one-third of older adults without ADL limitations were single, while the proportion of single individuals among those with ADL limitations was higher .The prevalence of hypertension tended to increase with the level of ADL limitation, but that of diabetes mellitus did not, the latter having been the highest in older adults with mild ADL limitations . The proportion of individuals with prior and current smoking experience decreased with an increase in the level of ADL limitation . The proportion of alcohol drinkers was higher among individuals with no ADL limitation  than among those with mild limitations  and those with moderate to severe limitations . Furthermore, the proportion of individuals with insufficient fruit and vegetable consumption was higher among those with mild ADL limitations  than in those with no limitation  and those with moderate to severe limitations , while that of individuals with low physical activity levels increased with the level of ADL limitations .Moderate to severe ADL limitations were significantly associated with all age groups among women and significantly associated with age groups of ≥70 years among men. Having hypertension had a significant association with moderate to severe ADL limitations among men but not among women. Having low physical activity levels exhibited a stronger significant association with having moderate to severe ADL limitations than with having high physical activity levels. Furthermore, the association between moderate to severe limitations and having a BMI in the underweight range was significant in both men and women.The overall population volume in 2015 was 4,304,000 for older men and 5,676,000 for older women. After adjusting for other variables in the model, the probability prediction of prevalence from postestimation regression was divided by age group and sex. The prevalence of mild functional ADL limitations was 7.1%  among older men and 10.5%  among older women, and for moderate to severe ADL limitations, it was 13.2%  among men and 20.6%  among women at the base year. The prevalence rates of functional ADL limitations increased by 23% among men and 8% among women every 5 years, in accordance with the rate of change in the prevalence of ADL limitations from the NHES from 2014 to 2019. Over 20 years, an increase in the predicted number of older individuals with ADL limitations was more likely among women than among men . Among men, the predicted number of people with mild ADL limitations in the population increased from 436,000 in 2020 to 1,866,000 in 2040. The number in the moderate to severe group also increased, from 826,000 to 3,414,000. Among women, the number of older people with mild ADL limitations showed a linear increasing pattern from 784,000 in 2020 to 2,040,000 in 2040, while that in the moderate to severe ADL limitations group increased from 1,542,000 to 3,993,000. The population estimates for both groups are presented in This study used data from a nationwide health survey among older individuals to investigate the association of sociodemographic variables and unhealthy behaviors with ADL limitations, and to estimate the predicted number of people living with ADL limitations from this association. The study identified age and physical activity as factors significantly associated with ADL limitations in older adults, while other factors showed varying associations. Over the next 2 decades, our projections suggest a significant increase in the number of older adults with ADL limitations, particularly men.This study found that ADL limitations remarkably increased with age among men and women. In addition, the estimated number of Thai older people with ADL limitations continuously increased over 20 years, and women accounted for a higher number of older people with ADL limitations than men. Consistent with several studies, our study found a significant association between ADL limitations and sociodemographic factors such as age, having chronic health conditions, and having unhealthy behaviors. It confirmed the profound effect of increasing age and chronic diseases on leading ADL limitations in older individuals ,10,12,23Our findings indicate that the prevalence of ADL limitations increased with age. Similarly, a previous study  reportedOur study used estimated marginal standardization from predictions of a fitted model ,22 at fiOur estimation of the average number of ADL limitations per year was 752,530 among men and 1,014,710 among women. Women were approximately 1-fold less likely to live with any ADL limitation than men. One explanation is that men exhibited a poorer health status and lifestyle and were more likely to be current alcohol consumers and smokers . FurtherOverall, the number of older Thai individuals will gradually increase over the next 20 years. This study found that the number of moderate to severe ADL limitations would reach 7.4 million in 2040, which is approximately 3-fold the current volume of individuals with moderate to severe ADL limitations. Our results project a larger number of individuals with mild ADL limitations than that suggested by Tantirat et al , who estOur findings suggest improvements in creating a friendly environment for older individuals, such as improving sidewalks and stairs to prevent falls, along with an increased number of health promotion and prevention programs to enhance overall physical activity, prevent chronic diseases and other limitations, and improve the quality of life of older Thai individuals. Longer healthy life expectancy would also reduce health care expenditure in older populations.The strength of this study lies in the use of a large cross-sectional national health survey that represents the general nationwide population. Furthermore, potential confounding factors were considered in the analyses. However, this study has limitations, as encountered in cross-sectional studies. First, this type of epidemiological study cannot establish causal relationships. Moreover, the direction of causality is indeterminate; ADL limitations and the presence of chronic disease could be reciprocally related to each other. Second, older individuals with severe disabilities or bedridden conditions might not have been included in the survey due to the data collection process, where respondents were invited to health care centers. Therefore, the prevalence of ADL limitations from these surveys might have been underestimated.In terms of methodology, this study encountered several limitations. We used a multinomial regression model to estimate the predicted probability of our outcome of interest. While certain demographic variables, such as marital status, residential area, and educational background, were considered in this model, it comprised a deterministic model that might not reflect the real situation . FurtherThe findings of this study highlight the vast number of older Thai individuals who will potentially experience ADL limitations in the next 20 years. This information could be used in policy making, particularly in health promotion planning. Consequently, relevant factors can be modified to reduce the number of people with disabilities in the future.


Synthetic Biology relies on the concept that biological systems are organized into modular components, that can be easily assembled, tested, and modified to generate biological machineries .Synthetic biology explores the directed metabolic alterations to produce high-value products transforming microorganisms into chemical factories. Lignocellulosic biomass has been suggested as an alternative to fossil fuels being a promising renewable resource from an economic and environmental perspective . This strain showed an improved ability to metabolize pyrolyzed organic matter (PyOM), a relative recalcitrant subproduct resulting from the combustion of vegetal organic matter during wildfires. Using high resolution transcriptomic analysis, the authors of the manuscript investigated the response of P. domesticum to PyOM observing an induction pattern of genes related to stress tolerance, in addition to metabolism and mineralization of aromatic compounds. The mineralization of PyOM by the dominant early-successional fungus P. domesticum is likely to have broad impacts on post-fire succession and recovery in soil microbial communities .Another paper by Stecker et al., describes the functional characterization of the L-proline biosynthetic route by osmotolerant strains of Bacillus subtilis. A genomic analysis of osmotolerant strains determined the presence of mutations affecting either the AhrC transcriptional regulator or its operator positioned in front of the argCJBD-carAB-argF L-ornithine/L-citrulline/L-arginine biosynthetic operon. These mutations, together with regulatory mutations affecting rocR-rocDEF expression, were responsible for the repurposing of three different routes: L-arginine biosynthesis, RocD-dependent degradation for L-ornithine, and the last step in L-proline biosynthesis. The authors concluded that these genetic adaptations are a demonstration of the genetic plasticity and metabolic flexibility of B. subtilis to counteract the changing environmental conditions .Additionally, environmental stress due to abiotic factors such as osmolarity impose considerable energetic and growth constrains on bacterial cells in their growing habitats , quantifying the physiological responses  and the expression of genes related with fatty acid biosynthesis. They concluded that the treatment of the cells at 5°C increases DHA biosynthesis. Moreover, this metabolic increment was accompanied by a transcriptional induction of the metabolic enzymes involved in fatty acid biosynthesis, determined by qPCR .Two additional papers reported studies on secondary metabolites with potential interest in the areas of human health, specifically nutrition and therapeutics. In the nutritional field, long chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) are essential fatty acids for human health with fish oil being its current main source. Due to DHA high demand for pharmaceutical and nutraceutical purposes, microorganisms have been described as an emerging alternative source of this omega-3 fatty acid. Zhang et al. describes the molecular basis for the transcriptional regulation of the biosynthesis of the antibiotics 2,4-diacetyphloroglucinol  and pyoluteorin by Pseudomonas protegens FD6. In this bacterial strain, the sigma factor RpoS is an essential regulator of cell cycle. RpoS negative mutants showed an impairment of biofilm formation, swimming motility, swarming motility, and resistance to chemical stress, but an increase in the antibiotic biosynthesis. The authors concluded that RpoS negatively controlled 2,4-DAPG biosynthesis and transcription of the 2,4-DAPG operon phlACBD by directly interacting with phlG and phlA promoters. Moreover, RpoS also inhibited the production of pyoluteorin by a transcriptional repression mechanism of its biosynthetic operon pltLABCDEFG exerted by a direct binding to the promoter regions of pltR, pltL and pltF genes .Antibiotics are one of the most relevant products of secondary metabolism from microorganisms, showing a wide chemical diversity and action mechanisms. The production of an antibiotic is tightly regulated, and the study of the molecular details of this regulation has become an essential topic to develop improved strains with enhanced biosynthetic potential (Zhang et al., In summary, despite the multiple biological functions that secondary metabolites could have, they are very relevant to understand the biological response to stress conditions, opening a new avenue of opportunities for synthetic biology applications.AL and FE: conceptualization, idea, writing, organization, and writing the manuscript. All authors contributed to the article and approved the submitted version.


Natural hearing which means hearing naturally like normal people is critical for patients with hearing loss to participate in life. Cochlear implants have enabled numerous severe hearing loss patients to hear voice functionally, while cochlear implant users can hardly distinguish different tones or appreciate music subject to the absence of rate coding and insufficient frequency channels. Here a bioinspired soft elastic metamaterial that reproduces the shape and key functions of the human cochlea is reported. Inspired by human cochlea, the metamaterials are designed to possess graded microstructures with high effective refractive index distributed on a spiral shape to implement position‐related frequency demultiplexing, passive sound enhancements of 10 times, and high‐speed parallel processing of 168‐channel sound/piezoelectric signals. Besides, it is demonstrated that natural hearing artificial cochlea has fine frequency resolution up to 30 Hz, a wide audible range from 150–12 000 Hz, and a considerable output voltage that can activate the auditory pathway in mice. This work blazes a promising trail for reconstruction of natural hearing in patients with severe hearing loss. A bioinspired soft elastic metamaterial with a size equivalent to the current cochlear implant that can be seen as a prototype of passive artificial cochlea with a frequency resolution of 30 Hz is proposed. This work will inspire future work in the fabrication of totally implantable passive artificial cochlea that enables the users to hear naturally. The deficiency of hair cells is the main reason for severe or profound hearing loss.In the natural hearing process of normal people Figure 1a, exterFigure 1 Howe Hearing naturally like normal people is a long‐term wish of patients with severe or profound hearing loss, but has not been implemented.Cochlear implants (CI), which have been commercialized for nearly 40 years, brought functional hearing to numerous hearing loss patients by generating electric currents to directly stimulate the auditory nerve. The route of CI is to stimulate auditory nerves with implanted current sources and electrodes while ignoring the users’ original auditory path. CI users usually have adequate communication abilities in a relatively quiet environment but can hardly distinguish different tones, recognize speech signals from noises, or appreciate music mainly subject to the absence of rate coding and insufficient frequency resolution.3, 4 Nevertheless, either increasing frequency channels or providing variable‐frequency stimuli will rapidly increase both the power consumption and the size of the CI. Therefore, the average frequency resolution of current CI is about 300 Hz in 0–8 kHz, while the frequency variation between two pitches in music can be less than 30 Hz. As for other aspects of CI, totally implantable cochlear implants were investigated by implanting the external devices of CI into human body to avoid discrimination. Besides, artificial basilar membranes or hair cells were explored to obtain a passive sound‐electric response to simplify the structure of CI. But good pitch‐place mapping with high frequency resolution has not yet been achieved. Acoustic metamaterial is one of the prominent technologies for manipulating sound waves at will. Previous works about acoustic metamaterials with effective refractive index  demonstrated pitch‐place mapped acoustic concentrators at relatively high frequencies  with tens of centimeters in size. If the working frequency comes to hundreds of hertz, the sample size will be several meters. Several previous studies on metamaterials have explored cochlea‐inspired acoustic sensors or cochlea‐like vibration modes in rigid resonators. However, few have demonstrated hearing reconstruction, acoustic‐electric transversion, or biomedical engineering applications. Acoustic metamaterials give promising opportunities for implementing pitch‐place mapping and sound‐electric conversion in cochlea but are limited to the bulky and rigid body.To improve the auditory performance of CI, efforts were made by increasing frequency channels and adding partial temporal fine structure into the stimulating signal.6 NeveHere we report a bioinspired soft elastic metamaterial (BSEM) that reproduces the shape and function of the human cochlea. BSEM keeps nearly the same auditory path as normal people and reproduces the key functions of human cochlea, as shown in Figure 2 enabling fine place‐pitch mapping and high‐speed parallel processing of electric signals. Moreover, the whole process is analog, which means the electric signals converted by BSEM can synchronize the change of external sound (complete rate coding) without extra delay compared to the natural hearing process. The side view of a completed BSEM is exhibited in Figure −3, 1.69 MPa  = 1.8 × 1.09θ mm and r2(θ) = r1(θ) + d(θ) + w(θ), (6.01π < θ < 9.91π). The branches are formed by cutting minimal cuboids from the ensemble spiral tapes. The length, width, and angular position of cut cuboids are l(n) = 2.4(168−N)7 × 10−17 mm + 0.27 mm, w(N) = 0.1 × 1.0067169−N mm, θ(N) = 0.255π[(169−N)0.64−1], , respectively. The bottom margin d and height of BSEM are 2.1 mm and 2.0 cm, respectively.The core of our method for rehabilitating natural hearing is the bioinspired metamaterials, a kind of soft elastic metamaterials25 witL = 142.8 mm  along the propagation direction. At the same time, the wavelength gradually decreases and the wave pressure field is progressively growing, which renders a spatial compression of the acoustic wave as well as the amplitude enhancement in the high refractive index region. Besides, the effective refractive index for different frequencies will reach a maximum value at different locations  of 8 channels  in BSEM at a series of tone‐burst excitations . The results show good pitch discrimination and considerable frequency resolution (≈30 Hz) of BSEM. We also tested the performance of BSEM at a broad frequency range. The Vpp of 8 channels  in BSEM at a series of tone‐burst excitations  are illustrated in Figure After fabricating the BSEM sample according to the optimized structure, a biomimetic measuring system was built to evaluate the performance of BSEM /Vunstructured(f), V and f for voltage and frequency respectively) to characterize the frequency response of each channel. V (f) is obtained by Fourier transform of the output voltage of samples under sweep excitation. Figure In order to evaluate the voltage enhancement effect of metamaterials, we prepared a sample without microstructure. Eight  of 168 channels were picked and analyzed  and intensities (80–90 dB) converted by BSEM could lead to ABR in mice. For comparison, the results of normal mice and deaf mice  under acoustic stimulations are illustrated in Figure  were also undergoing irreversible changes. The mean peak‐to‐peak values (recorded 10 times) shown in Figure In order to verify the biological effect of the electrical signal generated by BSEM, we stimulated auditory nerves in the cochlea of Kunming mice by inserting electrodes of BSEM and recorded their auditory brainstem response (ABR) that is often used to study and detect the functional state of the auditory system in basic and clinical practice.29 ParThe above results indicate that the BSEM possesses a broad audible range, more than 150 frequency channels, a similar analog sound sensing mechanism to natural hearing, and good robustness. Besides, BSEM has the advantages of convenience  and safety , which is hard to achieve for current electronic CI. Immunity to electromagnetic interferences and MRI‐compatibility are verified in Figures 3−3. Air is sealed inside the matrix to keep body fluid out. We conducted simulations to predict the propagation of sound waves through small BSEM  have shown the potential for manufacturing such complex microstructures. We will attempt to manufacture small BSEM based on these technical means in future work.In this work, we provided a proof of concept for natural hearing artificial cochlea. The main results in the manuscript are obtained from a 168‐channels BSEM with the size of 5 cm × 5 cm  in the simulation. The linear elastic model (the isotropic damping is set to be 0.01) is utilized to simulate elastic waves propagating under different conditions. The simulation model and boundary condition settings are shown in Figure COMSOL Multiphysics 4.2a was used to conduct the finite element analysis for simulating the elastic waves propagating in SBMC. The type of the mesh element is free triangular and the maximum size is 0.3 mm . 168 shims with a thickness of 125 µm were inserted into the reserved gap in molds. The matrix materials  were poured into the molds after the release agent  was added to the inner surface of the mold. After being placed at 23 °C for 48 h, the samples were formed and taken out. Then the shims were replaced by piezoelectric flakes with the same thickness and similar density as well as modulus. Copper twisted pairs were welded to the electrodes of piezoelectric flakes. More details are shown in Figure All the testing platforms were basically constructed with an optical bench, acoustic sponges, vibration exciters, amplifiers, and an 8‐channel data acquisition system . Detailed measure setups for different experiments are shown in figures in main text and Supporting Information.Isolated hind limbs were obtained from frogs. Ringer's solution was added to isolated hind limbs to keep their physiological activity. In experiments, electrodes of 8 channels were attached to sciatic nerves in hind limbs for electrical stimulation.−1, 1 wt%) intraperitoneal injection. In brief, the hair on the top of the head and around one ear was cut, the skin on the head was cut with a scalpel to make a small incision and the local anesthetic procaine was dropped for pain relief, and the skull was exposed to fix the small flat nail  for fixing the cochlear stimulation electrode. Referring to previous relevant research reports, the skin around one auricle was cut to expose the milky white translucent auditory bulla. A small hole was drilled in the auditory bulla with a probe, and part of the bone wall of the auditory bulla was peeled off along the hole with sharp‐nosed forceps to enlarge the opening for insertion of the stimulating electrode. Under a stereomicroscope, a small hole about 30–50 µm in diameter was drilled in the cochlear bone wall adjacent to the round or cochlear widow with a fine probe for inserting a thin insulted tungsten wire electrode for stimulating the cochlea, as shown in Figure The animal experiments were approved by Institutional Animal Care and Use Committee, Huazhong University of Science and Technology with the IACUC number being 2714. A total of 31 healthy and normal‐hearing adult Kunming mice   were used in the experiment. General surgery was performed after anesthesia with Nembutal sodium (45 mg kgnjection.36 In H. T. and J. Z. are inventors of a patent application (CN patent application: 2022109096269) that covers the mechanism and the design of BSEM.Supporting InformationClick here for additional data file.Supplemental Movie 1Click here for additional data file.Supplemental Movie 2Click here for additional data file.Supplemental Movie 3Click here for additional data file.Supplemental Movie 4Click here for additional data file.Supplemental Movie 5Click here for additional data file.Supplemental Movie 6Click here for additional data file.Supplemental Movie 7Click here for additional data file.Supplemental Movie 8Click here for additional data file.Supplemental Movie 9Click here for additional data file.Supplemental Movie 10Click here for additional data file.Supplemental Video 11Click here for additional data file.


The most common sensorineural disorder in humans is hearing impairment and approximately 60% of prelingual hearing disorders are genetic. Especially parents with a congenital deaf child want to know as early as possible whether their second born child has the same genetic defect or not. The aim of this study is to demonstrate that postnatal genetic umbilical cord analysis is both the earliest detection possibility and sufficient.We included first born children with severe hearing impairment that underwent cochlear implantation. All included patients were analyzed genetically and exhibited mutations of either DFNB1 loci or SLC26A4 gene. Additionally, the umbilical cord of the sibling underwent genetic analysis to detect hereditary genetic mutations as early as possible.49 newborn children out of 22 families were included in this study. Genetic analysis revealed clinical relevant mutations in all first born children and in four siblings via umbilical cord analysis. All patients who have been diagnosed with a relevant genetic mutation that caused severe hearing impairment underwent hearing rehabilitation via cochlear implant surgery.This study demonstrates the sufficient and early as possible detection of known genetically hearing disorders via umbilical cord analysis. In case of a known familial genetic hearing disorder, it is advisable to analyze newborn siblings for the corresponding genetic defect as soon as possible, to be able to plan and initiate clinical care for the patient as early as possible. It is also extremely important for the parents to obtain clear information about the auditory status of the newborn. Hearing impairment is the most common sensorineural disorder in humans. Approximately 1–3/1000 newborns are affected by profound hearing impairment to deafness at birth or in the first years of life. The same number of children become deaf before reaching adulthood. The causes of severe hearing impairment are very heterogeneous. Based on the form of the physiological defect, hearing disorders are classified as conductive hearing loss, sensorineural hearing loss, or a combination of both. Reasons for hearing disorders may include environmental factors or viral infections, strong noise sources, ototoxic substances, and genetic causes. Hearing disorders can occur anytime in development and show a progressive course over several years from mild hearing loss to deafness .Approximately ~ 60% of all prelingual hearing disorders are genetic. Inherited hearing disorders are divided into syndromic or non-syndromic; non-syndromic hearing disorders (NSHL) occur in isolation, whereas syndromic hearing disorders (SHL) are associated with additional disorders. Nearly 70% of cases of inherited hearing loss are non-syndromic and predominantly due to sensorineural causes. Of these, about 80% of the cases follow an autosomal recessive (DFNB) and 18% an autosomal dominant (DFNA) mode of inheritance; about 2% are x-linked (DFNX) or mitochondrial linked (MT). A total of 193 gene loci have been described to date, for which 143 genes have been identified so far, and at least 50 genes are still unknown , localized in chromosomal segment 13q11-12, is caused by mutations in the gene GJB2 [MIM 121011] , which eHalf of all cases of nonsyndromic recessive hearing impairment are due to alterations in this gene locus. This results in a prevalence rate of these heterozygous gene defects, at the DFNB1 gene locus of approximately 1/60 in the general population .Pendred syndrome is one of the most common forms of genetic syndromic hearing disorders with about 1–8/100,000 cases. It is inherited in an autosomal recessive manner, clinically characterized by a sensorineural hearing disorder associated with thyroid dysfunction, which, however, occurs only at the beginning or in the middle of the second decade of life. Furthermore, a dilated aqueductus vestibuli (EVA = enlarged vestibular aqueduct) or Mondini dysplasia is present . Until nOur experience has shown that parents want to be informed as soon as possible whether their newborn child has the same genetic defect as their child with the genetical disease . PreviouWe performed a retrospective analysis of second and third born siblings of genetically positive tested hearing impaired first born children out of 22 families at a quaternary university hospital and associated cochlear implant center, included between 2002 and 2022. All included first-born patients showed a severe hearing impairment in the first two years of life, so that a cochlear implant treatment was necessary.We performed a genetic analysis of the gene locus DFNB1 in which the genes GJB2 (connexin-26) and GJB6 (connexin-30) are arranged in tandem orientation. In patients who had enlarged vestibular aqueduct on preliminary CI examination by high-resolution thin-slice CT, sequence analysis of the gene SLC26A4 (Pendrin) was performed as an alternative. Primers, PCR, and sequencing conditions were selected according to procedures described before , 17.Immediately after birth of a sibling, a part of the umbilical cord (10–250 mg) was sent sterile to the molecular genetic laboratory of the University ENT Clinic Freiburg and promptly analyzed for the already known genetic defect of the sibling.This study took place in the Department of Otorhinolaryngology, Head and Neck Surgery at the Implant Center of the University Hospital Freiburg. The study was approved by the Hospital’s Ethics Committee  according to the Declaration of Helsinki  ). Informed consent was obtained from all subjects and/or their legal guardian(s). The data that support the findings of this study are available from the department of otorhinolaryngology  but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are, however, available from the authors upon reasonable request and with permission of the Medical Center Freiburg and the local Ethics Committee of the Freiburg University.Bilateral severe hearing loss was diagnosed in 22 first born children in the first months of life. Inner ear malformations could be excluded in these cases by high-resolution computed tomography (CT) of the temporal bone. Two mutations were identified in 16 of the patients being homozygous carriers of the c.35delG mutation in the GJB2 gene . Hearing loss was progressive in these patients. Two different mutations were identified in each of these three patients. The newborn sibling had no mutation in one case. In three cases a heterozygous mutation could be found.In the case of a proven genetic cause of hearing impairment, the affected families are interested in finding out soon after delivery to what extent their next born child is also affected by hearing impairment. The considerable emotional stress of the parents must also be taken into account in this context .Practical experience shows newborn hearing screening (NHS) with OAE (otoacoustic emissions) or ABR (auditory brainstem response) is not always reliable. Especially in cases of progressive hearing loss, as is typical for Pendred syndrome, an unremarkable finding in newborn hearing screening can lead to a delay in diagnosis and thus treatment , 20.In contrast, our results from eight cases Table  clearly These analyses can be performed within 2–3 days. Only small amounts of the umbilical cord tissue are needed, 10 mg is approximately equivalent to the head of a pin. Ideally, the collection and shipment of umbilical cord tissue or blood is done in collaboration with the obstetrics department and no additional blood test is required.On the one hand, mutation analysis of the umbilical cord tissue can rule out the disease and spare both the children and their parents other expensive and time-consuming diagnostic procedures. Alternatively, prenatal diagnosis can be applied to know the hearing status of the second child earlier. However, unlike umbilical cord diagnosis, this method poses a risk to both mother and child. A diagnosis one week after birth is early enough for hearing rehabilitation if necessary. If the condition is disclosed, all necessary steps can be taken to efficiently support speech development in the newborn. Furthermore, it is possible for the parents to be clear at an early stage that the hearing impairment is present or not.In case of a known familial genetic hearing disorder, it is reasonable and easy to analyze newborn siblings for the corresponding genetic defect by umbilical cord analysis as soon as possible, to be able to plan and initiate clinical care for the patient as early as possible. It is also extremely important for the parents to obtain clear information about the health of the newborn.


Hearing impairment, the third largest health burden worldwide, currently lacks definitive treatments or preventive drugs. This study compared the effects of hydrophilic and lipophilic statin on hearing loss using a common database model. This retrospective multicenter study was conducted in three hospitals in South Korea . We enrolled patients with hyperlipidemia with an initial hearing loss diagnosis. Data were collected during January 1, 2022–December 31, 2021 using the Observational Health Data Science and Informatics open-source software and Common Data Model database. The primary outcome was the occurrence of first-time hearing loss following a hyperlipidemia diagnosis, as documented in the Common Data Model cohort database. The measures of interest were hearing loss risk between hydrophilic and lipophilic statin use. Variables were compared using propensity score matching, Cox proportional regression, and meta-analysis. Among 37,322 patients with hyperlipidemia, 13,751 (7669 men and 6082 women) and 23,631  were treated with hydrophilic and lipophilic statins, respectively. After propensity score matching, according to the Kaplan–Meier curve, hearing loss risk did not significantly differ among the hospitals. The hazard ratio (HR) of the male patients from Anam , 0.05–1.51]), Guro , and Ansan  hospitals were analyzed using Cox proportional regression. Overall effect size  was estimated using meta-analysis, which indicated that hearing loss risk among hydrophilic statin users was less than that among lipophilic statin users and was statistically significant. Men in the hydrophilic statin group had a lower risk of hearing impairment than those in the lipophilic statin group. For decades, efforts to develop medical interventions for preventing or mitigating hearing loss have been ongoing. Several drugs and approaches have been studied in vitro, in animals, and in clinical trials. While some have looked promising, to date, no drug has been approved by the U.S. Food and Drug Administration (FDA) for treating hearing loss5.Hearing impairment is a pervasive disorder, and its incidence increases with age and is the third largest health burden globally8. Statins, which are commonly used to treat cardiovascular and neurological disorders, may also be effective for treating hearing impairment9. Statins are a class of drugs that are commonly used for the treating hyperlipidemia and for preventing heart attack and stroke. Statins are classified into two categories: hydrophilic and lipophilic. Lipophilic statins, which have a high permeability to adipose tissue, have been shown to attenuate adipocyte maturation. In contrast, hydrophilic statins do not have this effect10. Hydrophilic statins have been found to lower serum adiponectin and C-reactive protein levels. Some previous studies have suggested the potential of hydrophilic statins to yield a more pronounced impact on the occurrence of hearing loss when contrasted with their lipophilic counterparts10. Other studies have investigated the association between statin intake and the risk of hearing loss and found that the association may be influenced by sex12.Cardiovascular damage and neurodegeneration are known risk factors for hearing loss13.The Common Data Model (CDM) cohort study involved a correlation analysis of specific topics through a model that defines different large-scale clinical data in each hospital with the same standards of structure and meaning. This approach allows for easy gathering and utilization of data, making it popular in multicenter researchIn this multicenter study, we compared the impact of hydrophilic and lipophilic statins on hearing loss by utilizing a common database model. Additionally, we specifically examined the sex-related effect of statins on hearing loss.14 open-source software and the CDM database15. The OHDSI network is an international collaboration that aims to develop a data-sharing system16 by applying open-source data analytics to a large number of health databases17.This multicenter retrospective cohort study was conducted at three hospitals  from January 1, 2002 to December 31, 2021. It used the Observational Health Data Sciences and Informatics (OHDSI)Patients (aged 40–80 years) with hyperlipidemia, identified using the International Classification of Diseases, Tenth Revision, Clinical Modification (ICD-10-CM) diagnosis code E78.2, were selected. These were identified using a concept ID code in the CDM database and their details, including hearing loss, are provided in Table 18 and platinum compounds20, as these may impact hyperlipidemia regardless of the type of statins used.The primary outcome was the first occurrence of hearing loss followed by a diagnosis of hyperlipidemia. Hearing loss includes sensorineural hearing loss, sensory hearing loss, neural hearing loss, noise-induced hearing loss, and sudden hearing loss. The classification and identification were based on the systematic nomenclature of medicine (SNOMED), which is a standardized vocabulary for diagnostic codes. To eliminate confounding factors while setting covariates, we excluded patients taking aminoglycosidesProportion is the proportion of participants receiving the treatment.To control the selection bias, we used propensity score (PS) matching to balance the covariates between the hydrophilic statin and lipophilic statin groups. However, in this study, the preference score was used after suitable adjustments. The equation of the preference score is as follows:2 value test) were used to test for heterogeneity in the meta-analysis21. All statistical analyses were conducted using R 4.1.3 (http://www.R-project.org) and the OHDSI Cohort Method R package22. Meta-analyses of the random-effects model were performed using meta for the R package.We performed a matched group analysis using 1:1 propensity matching with a 0.25 caliper. Additionally, we created a table for baseline characteristics and applied Cox proportional regression to calculate the hazard ratio (HR), which was used to investigate the association between statin intake and hearing loss. The Kaplan–Meier cumulative survival plot was used to compare the difference before and after PS matching for each hospital. The HR was calculated using Cox proportional regression and the overall correlations were evaluated using a meta-analysis. Statistical approaches , and the requirement for informed consent was waived by the ethics committee along with the Institutional Review Board of Korea University Anam, Guro, and Ansan hospitals. All methods were performed in accordance with the relevant guidelines and regulations.This study included 37,322 patients with hyperlipidemia from three hospitals. Among these participants, 13,751 (7669 men and 6082 women) and 23,631  patients used hydrophilic and lipophilic statins, respectively. Figure Table The aggregated baseline characteristics of hydrophilic and lipophilic statin users from each hospital before and after PS matching are described in Table 23. The X-coordinates of each graph show the absolute value of the standardized difference of the mean before PS matching, and the Y-coordinates represent the value after PS matching. Each dot represents a covariate and perfect balance after matching, according to most dots in the Y-coordinate located below 0.1. Among these patients, the covariates were well-balanced in each data source. The types of covariates included demographics , conditions in the prior 30 days and 365 days, and aggregation of the condition in the SNOMED CT. Aggregation of drug is ingredient and Anatomical Therapeutic Chemical Classification System (ATC class). The results of the preference distribution into a PS adjustment for each hospital by sex are shown in Fig. Figure Figure Nevertheless, the summary of the HRs from the three hospitals demonstrates interesting results. In Fig. However, the HR of the male patient groups from the Anam , Guro , and Ansan  hospitals still indicates that the risk of hearing loss in hydrophilic statin users was less compared to that in lipophilic statin users. However, the overall effect size  was statistically significant.In addition to the overall effect size , the HR of female patient groups from the Anam , Guro , and Ansan  hospitals showed a higher risk of hearing loss in hydrophilic statin users than that in lipophilic statin users. Although these results differ from the previous ones, they were not statistically significant.Figure 24.Statins belong to a class of drugs that are widely used for treating hyperlipidemia and preventing heart attacks and strokes. They work by inhibiting HMG-CoA reductase to varying degrees. Drugs classified as statins share common mechanisms of action while displaying distinct molecular structures and varying degrees of lipophilicity or hydrophilicity. The variation in their chemical structure contributes to factors such as enzyme binding affinity, drug efficacy, tissue selectivity, bioavailability, degradation mechanism, absorption mechanism, and elimination half-life26. Studies on statins and sensorineural hearing loss have generally found a reduced risk of hearing impairment29. In a study using national health screening data, the odds ratio of hearing impairment was lower in the statin group, with a more significant effect observed in the hydrophilic statin group9.Previous research on the association between statin use and sudden sensorineural hearing loss has yielded conflicting results13. Therefore, we conducted the first large-scale CDM cohort study to investigate the effect of hydrophilic and lipophilic statins on hearing loss in patients with hyperlipidemia. Our study found that hydrophilic statins had a protective effect on hearing impairment compared to that of lipophilic statins in men. Homogenization was conducted because this study included a large patient group from three hospitals, and the patients were divided into two groups: hydrophilic and lipophilic statin users. Moreover, the variations of the patient group were corrected through covariate balancing and PS matching.Since statins are not clinically used to treat hearing impairment, it is difficult to conduct a meta-analysis using large-scale data. However, CDM models enable accurate analysis by creating uniformity in large-scale data and allowing reproducibility. The effectiveness of CDM has been demonstrated in animal experiments and retrospective studies in patients. The CDM cohort study is a correlation analysis of specific topics using a model that standardizes large clinical data from each hospital in terms of structure and meaning. Organizing CDMs preserve the original data from a source and allows maximum adaptability. Fully organized data models are easy to utilize because all rough codes are mapped to a medical composition. The adaptive rules system expands a database of easily adaptable, reusable measures to maintain adaptability, facilitate analysis, and ensure study-specific transparency9. The protective effect of statins may be stronger in men due to a higher rate of risk factors for cardiovascular diseases such as obesity, alcohol use, or smoking30. Additionally, the more careful approach to health of women may cause increased suspicions regarding the need for statins or more distresses about their potential side effects. Therefore, men have taken statins for a relatively longer duration than have women31. Hydrophilic statins may have fewer side effects compared to those of lipophilic statins due to their lower tissue uptake and reduced dependence on cytochrome P450 metabolism24. In addition, a previous study that included patients with atherosclerosis reported superior anti-inflammatory effects (with higher levels of adiponectin and lower levels of C-reactive protein) of hydrophilic statins compared to the effects of lipophilic statins32.In this study, the protective effect of hydrophilic statins over lipophilic statins against hearing impairment was only significant in males, which is consistent with the results of previous studies  that have also shown that the protective effect of statins is more prominent in older and male populations more susceptible to cardiovascular eventsThis study has several limitations. First, because this was a code-based study, we might have determined hearing loss only in the context of codes. Conducting a thorough qualitative analysis would have been ideal. Furthermore, we did not conduct an in-depth analysis of the correlation between the duration of statin administration and the degree of hearing loss. Moreover, we did not consider the degree of hyperlipidemia related to coding. Future studies should consider these limitations, in addition to including a control group (with no statin intake).In conclusion, our large-scale, multicenter study provides evidence supporting the protective effect of hydrophilic statins on hearing impairment. In particular, our findings indicate that men in the hydrophilic statin group exhibited a lower risk of hearing impairment than did those in the lipophilic statin group. This research expands our understanding of the differential impacts of hydrophilic and lipophilic statins and highlights their potential in mitigating hearing loss in patients with hyperlipidemia.Supplementary Information 1.


The incomplete partition type III is a severe cochlear malformation present in X-linked deafness. It is a rare, non-syndromic cause of severe to profound mixed hearing loss, often progressive. The complete absence of bony modiolus and the wide communication between the cochlea and the internal auditory canal make cochlear implantation challenging, with still no consensus on the management of these patients. To the best of our knowledge, no results have ever been published in the literature on the treatment of these patients with hybrid stimulation (bone and air). We present three cases in which this hybrid stimulation gave better audiological results then air stimulation alone. A literature review on audiological results of the current treatment options in children affected by IPIII malformation was conducted independently by two researchers. Ethical considerations on the treatment of these patients were conducted by the Bioethics department of the University of Insubria. In two of the patients, the bone–air stimulation, associated with prosthetic–cognitive rehabilitation, meant that surgery was avoided, obtaining similar communication performances of those present in the literature. We believe that, when the bone threshold appears partially preserved, a stimulation through the bone or hybrid modality, such as the Varese B.A.S. stimulation, should be attempted. X-linked deafness is a rare condition caused by mutations in POU3F4, a gene related to brain and cochlear development. The mutation results in a severe cochlear malformation classified as incomplete partition type III (IPIII), which consists of an absent bony modiolus and no separation between the base of the cochlea and the Internal Auditory Canal (IAC). The bony interscalar septa and the external dimension of the cochlea are preserved . Males pIn the case of moderate to severe hearing loss, a traditional hearing aid may be sufficient to ensure adequate communication, however the majority of IPIII patients present with severe to profound mixed hearing loss ,3. The cCochlear implantation is possible in IPIII, albeit burdened with significant complications, especially due to the wide communication between the cochlea and the IAC. A severe gusher is practically unavoidable, with a combined difficulty in performing a correct placement of the array in a malformed cavity, and an increased risk of meningitis due to the ample cerebrospinal fluid (CSF) leak or the necessity of a lumbar drainage ,6. MoreoOn the other hand, the worsening in the auditory performances over time could destine the child to a life of sound deprivation.Bone conduction stimulation is an alternative to air stimulation that propagates sound directly to the inner ear through skull bone vibrations. In patients with a preserved hearing bone threshold, this method can be utilized to restore hearing discrimination even if the air threshold is compromised. This is possible using bone conduction hearing devices that do not always necessitate surgery, which would be a simple procedure. Patients with IPIII malformation and severe to profound mixed hearing loss are nowadays given the only treatment option of cochlear implantation.To the best of our knowledge, no results have ever been published in the literature on the treatment of these patients with hybrid stimulation (bone and air).We present three cases of children with IPIII malformation treated with hybrid bone–air stimulation (B.A.S.) and the Varese B.A.S. audiological features and results are carefully described. A literature review was conducted on the clinical and audiological outcomes of all the known treatments available for patients with IPIII malformation. In light of the data that emerged, an analysis of the ethical implications of the therapeutic options available for these patients was performed, with consideration of the risks and potential benefits of each intervention.A retrospective review of all pediatric patients with IPIII malformation treated in the referral center SSD of Audiovestibology Varese from 2001 to 2022 was conducted. Inclusion criteria were: the presence of mixed hearing loss, a bone hearing threshold of at least 60 dB HL and the radiological evidence of IPIII malformation.t-test with GraphPad Software  with a significance value of p < 0.05. Informed consent was obtained from all subjects involved in the study.Audiological data  in noise), anamnestic information and follow-up data were collected. The audiological results obtained with air, bone and hybrid stimulation were statistically analyzed. The graphical representation and the statistical analysis were performed using Student’s ® SKY Ultra Power. The devices were adjusted according to the patient’s audiometric threshold. The DSL® 5 pediatric prescription formula, with adaptive microphones and feedback suppression systems, was used as an amplification target. The target gain subjectively did not guarantee sufficient loudness, so it was increased based on the patient’s responses. A bone conduction device coupled with a soft band was used to stimulate the bone component. To guarantee the best gain and maximum power output, the BAHA® 5 Superpower was chosen: which, in addition to being one of the most powerful bone conduction devices, is the only one equipped with the EveryWear™ standard. The transducer was placed on the contralateral mastoid through a connecting cable. The BAHA® was adjusted based on the audiometric threshold detected through the worn device . The proprietary cochlear BAHA prescription formula was used. The daily program was set as follows: microphone set in adaptive mode, active feedback management  and position compensation set to “on”. In addition, the gain was increased to satisfy the loudness required by the patient. Despite the critical gain measurement, the new volume increase required a review of the feedback.Both devices using either the airway or bone route are acceptable amplification options for patients with conductive or mixed hearing loss, whereas behind the ear hearing aids (BTEs) are usually the first choice for patients with sensorineural hearing loss. In the Varese B.A.S., a BTE hearing aid and a bone conduction device are employed simultaneously in each patient. A powerful BTE hearing aid was used to treat the aerial component of hearing loss, namely PhonakSpeech reception thresholds (SRTs) in noise were measured using an adaptive testing procedure, the simplified Italian matrix test (SiIMax). The simplified version uses a shortened version of the original word matrix, forming a three-word sentence with an unpredictable meaning, and is specially designed for pediatric patients or adults with a reduced word span. Measurements were conducted during the follow-up visits in several sessions which allowed the patient to become familiar with the test. All measurements were conducted in a silent booth with the patient one meter away from a single loudspeaker. The signal was kept constant at 65 dB SPL and the noise varied from an initial signal to noise ratio (SNR) of 0 from the same front source. Both OlSa-noise and the International Speech Test Signal (ISTS), with different masking modulation effects, were used as competition to the signal. The participants conducted the tests with the various configurations of the devices as shown later. In our department, the highest accepted value was +20 S/R, beyond this limit the results were considered not significant.A literature review on audiological results of the current treatment options in children affected by IPIII malformation was conducted independently by two researchers. The results were analyzed by the Bioethics department of the University of Insubria and ethical considerations were conducted on the treatment of these patients.Among 1004 pediatric patients followed because of hearing loss or with risk factors for hearing loss in the Audiovestibology Unit of Varese in 2022, five patients had the radiological diagnosis of IPIII malformation. All of the patients had mixed hearing loss. Two patients were excluded from the study due to a bone threshold above the value of 60 dB HL.p = 0.0126).Three patients were included in the study and their cases are shown below. Audiological results are shown in He was born preterm (35 w) from a pregnancy complicated by maternal hypothyroidism. The birth weight was 3100 g with Apgar 8–10. He presented with neonatal jaundice (bilirubin 21 mg/dL) treated with prolonged phototherapy (10 days). The outcome of the neonatal auditory screening was not available. The mother had undergone stapes surgery in another department for mixed hearing loss with an intraoperative finding of a severe gusher which required the suspension of the surgery.The first evaluation of the child at our Audiovestibology Unit was made at the age of 1 year with diagnosis of moderate to severe bilateral mixed hearing loss requiring a hearing aid and adequate speech therapy training. He underwent CT (Computed Tomography) scan and MRI (Magnetic Resonance Imaging) with a finding of bilateral IPIII malformation.The child grew up following our prosthetic-rehabilitation program achieving the normal stages of language development. The hearing threshold worsened, up to the age of five y.o. when the air prosthesis was no longer adequate, and it was indicated he should receive cochlear implantation. A severe gusher occurred during surgery and the procedure, even with the agreement of the parents, was suspended. Fortunately, the threshold was preserved, and the child continued to use his hearing aid. The parents have always refused further attempts to place a cochlear implant.At school, the child used a wireless remote microphone with good academic performances, until the age of nine, when the child started complaining of difficulty in understanding the teacher and a deflated mood. Although the air thresholds were low, the bone thresholds were partially preserved and to the suggestion of a BAHA softband in association with his air stimulation was made. Audiological results are shown in After three years of follow-up, the child presents with slightly worsened left air and bone thresholds, however, he continues to constantly wear the double prosthesis with benefit with acceptable communicative and scholastic results. Communication was evaluated on the Categories of Auditory Performance (CAP) scale (Score = 4) and he reached score 5 according to the Speech Intelligibility Rating (SIR).He was full-term born from a regular pregnancy. There were no maternal infections and the birth weight was 3170 g. There were no perinatal complications. He had no family history of hearing loss. He was referred to our department at the age of 13 months, after failing the newborn auditory screening. He was diagnosed with moderate to severe bilateral sensorineural hearing loss and inadequate communicative performances. It was suggested that the child received an immediate hearing aid fitting and speech rehabilitation and then he underwent a CT scan and MRI with a finding of bilateral IPIII malformation. The child was later diagnosed with mild cognitive retardation; however, he attended school with a support teacher and benefited from wireless remote microphone. At the age of 6 y.o., the hearing thresholds worsened to a profound mixed hearing loss and, with a partially preserved bone threshold, a BAHA superpower softband was prescribed in addition to his air stimulation. Audiological results are shown in He was full-term born from a regular pregnancy. There were no known maternal infections during pregnancy. The birth weight was 3000 g with Apgar 9–10. No perinatal complications occurred. He had no family history of deafness. He presented with language delay. The parents’ first doubts about hearing emerged at the age of two. The diagnosis of deafness was performed in another department with the first use of a hearing aid at the age of four with unsatisfactory communication results. He underwent a CT scan and MRI with a finding of bilateral IPIII malformation. A cochlear implant was proposed which the parents refused. At the age of 5 y.o., the patient was brought to our department, with a diagnosis of mild to severe bilateral mixed hearing loss with indications for a prosthetic upgrade, rehabilitation management and use of a wireless remote microphone. After the initial benefit, the thresholds gradually worsened and the possibility of performing a cochlear implant was re-evaluated at the age of 8. Given the high surgical risks, in agreement with the parents, a bone conduction device coupled with a soft band was fitted in association with his air stimulation with a consistent benefit from the double stimulation. The audiological results are shown in A total of 178 abstracts published after 2009 were selected on the basis of the following keywords “IP III malformation”, “X-Linked deafness”. Titles and abstract were screened and 29 articles were fully reviewed. The final analysis was conducted on 17 papers ,20,21,22Incomplete partition type III is a severe cochlear malformation with an estimated incidence of 0.9–2% of all inner ear malformations ,23. In oTraditionally, these patients are treated with hearing aids until the progressive worsening of the acoustic threshold leads to cochlear implantation, an operation burdened by high surgical risks, especially when performed on very young children.Moreover, the literature outcomes of cochlear implantation in IPIII malformation are very discordant, when and if adequately described. The audiological tests performed are different and often not comparable.Smeds et al.  describeOther authors reported the audiological outcomes varying from sound detection to a CNC word recognition score of 100% passing through a series of different audiological tests that make it difficult to compare the results of the various studies ,20,21,22The most used tool to measure the CI-related discrimination abilities in the literature, of patients with IPIII, are the CAP (Categories of Auditory Performance) and the SIR (Speech Intelligibility Rating) scales. On average, the results reveal recognition of some speech sounds without lip reading. However, the rehabilitation method used is never described.According to the aforementioned tests, our patients benefited from hybrid bone and air stimulation and they achieved speech production equal to those of normal hearing children of the same age.-early and correct audiological diagnosis;-prosthetic–cognitive rehabilitation in order to correct the sensory impairment and the consequent communicative handicap. With this kind of rehabilitation, described by S. Burdo ,26, it iHowever, the audiological result alone is not enough to justify the communication results of these patients. Once the acoustic perception is guaranteed, all patients have to undergo a period of rehabilitation aimed at developing communication abilities. In our department, all deaf patients, regardless of the etiology of deafness, undergo an initial prosthetic-rehabilitation method, as described in other articles . A transIn our experience, with the limits of the cognitive variables peculiar to each case, as well as the family adherence to the rehabilitative method, the residual cerebral plasticity and the auditory memory at the time of diagnosis, patients are given the opportunity to achieve a complete scholastic integration with comparable communicative performances to those of normal hearing.Criteria for ethical issues concerning patients being treated with B.A.S. involve various aspects.Decision-making in children is a very complex problem because there are essentially two apparently conflicting requirements. The first is related to the need to perform the effective intervention in time to guarantee the recovery of perceptive and linguistic abilities; the second is related to the need to avoid creating functional damage to the child. Parents have the burden of a choice which on the one hand contemplates a surgical intervention with high intraoperative risks and no guarantees of success and on the other an acoustic stimulation which allows the maintenance of effective communication performances associated with a close audiological and pedagogical follow-up.Parents not only have an accountability to ensure an acceptable quality of life for their child, but also to promote the development of their future autonomy and self-fulfillment.From a careful analysis of the audiological results published in the literature and those achieved by the patients involved in the study, we believe that the application of double air and bone acoustic stimulation is proportionate  and ethiIf any critical audiological and pedagogical issues are identified during the follow-up, the most appropriate therapeutic procedure is re-discussed with the parents.IPIII malformation is a severe condition related to hearing loss that often makes pediatric patients eligible for cochlear implantation. When the bone thresholds are partially preserved, a stimulation through the bone or hybrid modality, such as the Varese B.A.S. stimulation, should be attempted. In our experience, this type of stimulation, associated with a correct rehabilitation, can avoid subjecting young children to a surgery burdened by potential serious complications, and to obtain similar communication performances at the same time. However, further studies with a larger sample size are needed to confirm the statements reported.


Neurons that transmit information from the retina to other parts of the brain are more affected by anesthesia than previously thought. Related research article Boissonnet T, Tripodi M, Asari H. 2023. Awake responses suggest inefficient dense coding in the mouse retina. eLife12:e78005. doi: 10.7554/eLife.78005.To better understand how animals sense their environment, studies measuring brain activity need to include neural recordings in awake behaving animals . HoweverExperiments in awake animals capture more natural behavior, while those conducted in anesthetized animals or acute brain slices allow sensory stimuli to be controlled more tightly. The latter can also identify more mechanistic details of the brain structures involved in sensory processing. Thus far, it has been widely assumed that neurons behave similarly enough across these different conditions so that insights from one type of experiment can be translated to another.However, the effects of anesthesia (and brain slicing) on various parts of the central nervous system have been a topic of considerable attention . For exaBoissonnet et al. recorded and compared how the output neurons of the retina  responded to light in awake animals, in anesthetized animals and in experimentally isolated retinas. Activity levels of the ganglion cells in the isolated retinas were recorded through spike recordings. For recordings in living animals, Boissonnet et al. inserted electrical probes directly into the optic tract, the nerve bundle that relays visual information to the brain. Boissonnet et al. then applied full-field light modulation to stimulate the entire retina with light of equal intensity to avoid the effects of eye movements, and also accounted for pupil constriction. This allowed them to give the retina nearly identical stimuli across the three conditions.The study revealed that ganglion cells in awake animals were able to respond to substantially higher temporal frequencies of light stimuli than ganglion cells in an animal under anesthesia or ganglion cells in isolated retinas. Isolated retinas also showed markedly lower spiking activity .As with many innovative approaches, the study of Boissonnet et al. raises at least as many new questions as it answers. Contrary to previous hypotheses, anesthesia appears to have profound effects even at the early stages of visual processing within the retina. From a mechanistic perspective, this is, perhaps, not terribly surprising. Both anesthetics used in the study are known to interact with a set of inhibitory receptors for the neurotransmitter GABA (γ-aminobutyric acid), and they are prevalent throughout the retina .Still, the magnitude of the effect – the anesthetized preparation was substantially slower than even the isolated retina – is an important point to consider when interpreting recordings in the visual system of awake and anesthetized animals. Interestingly, a higher firing activity and faster response dynamics in awake animals have also been found in the parts of the brain that the axons of the retinal ganglion primarily project onto (that is, the dorsal lateral geniculate nucleus and the superior colliculus; While this work is a critical first step, there is still a long way to go before it is possible to measure the response of retinal ganglion cells in a natural context. Boisonnet et al. did not measure spatial or movement-related responses. Instead they ensured that the stimuli were spatially uniform, and the animals were head-fixed to minimize their eye movement. Future research could build on this method to further refine the technique and ensure the behavioral contexts of the animal match how retinal ganglion cells work in natural conditions. It will also be interesting to see if other neurons in early sensory systems, particularly those in the peripheral nervous system, like the nose and ear, have comparable results to the retina, which is part of the central nervous system . For now


POU3F4 gene. We present two siblings diagnosed with the condition and their long-term follow-ups. They both presented audiovestibular symptoms and showed progressive mixed losses and bilateral vestibular weakness. They were treated with cochlear implant, digital amplification and with vestibular rehabilitation. Significant others around them were involved in their journey with the medical team, and in both, a very favourable outcome was achieved. This is the first time that we have reported evolving audiovestibular function with vestibular quantification in X-linked gusher disorder and emphasize on the multidisciplinary holistic approach to manage these children effectively.Conductive hearing losses are typically present in disorders of the external/middle ear. However, there is a rare group of inner ear conditions called third windows that can also generate a conductive hearing loss. This is due to an abnormal connection between the middle and the inner ear or between the inner ear and the cranial cavity. X-linked gusher disorder is an extremely rare congenital inner ear dysplastic syndrome with an abnormal connection due to a characteristic incomplete cochlear partition type III and an incomplete internal auditory meatus fundus. The disorder is inherited in an X-linked fashion due to the mutation of the  X-linked gusher disease is a rare genetic disorder that presents a conductive element in hearing loss measured by pure tone audiometry. First identified by Nance in 1971, this entity was considered a congenital stapedial fixation disorder, where during surgery, a perilymphatic gusher was identified . SubsequPOU3F4 gene. The POU family of genes is a family of eukaryotic transcription factors regulating neuroendocrine function, of which the POU3F4 gene is a key factor for mesenchymal integrity, spiral ganglion functioning, spiral ligament structure and stria vascularis activities [The clue that the disorder is genetically inherited was suggested by observing the disorder in males. The gene responsible for the disorder was identified in 1995  as the Ptivities . The muttivities .X-linked gusher disorder mostly, if not always, occurs in males given its mode of inheritance. However, females may be obligatory carriers presenting a milder phenotype ,8. ThereThe hearing loss encountered is heterogenous, ranging from moderate to severe and profound conductive to sensory cochlear hearing loss, but the mixed variety is the most common ,13. The In this study, we present two siblings who were diagnosed with the condition and underwent full behavioural/objective audiological and objective vestibular quantification. These siblings were reported earlier as part of a cohort  with sumPOU3F4 gene.Two children out of a total of fifty-three third-window disorders in a tertiary paediatric vestibular centre in the UK over a period of 3.5 years were diagnosed with X-linked gusher disorder, comprising 3.7% of all third-window disorders. Both children were diagnosed with detailed anamnesis, exclusion of other conditions and imaging studies (HRCT/T2 drive MRI). Both would show mutations in the Pure tone audiometry following British Society of Audiology standards, tympanometry and acoustic reflexes, transient otoacoustic emissions were performed to quantify hearing with an Aurical Audiometer, Otometrics Zodiac and Otodynamics equipment, respectively.Videonystagmography (VNG) with and without optic fixation , vestibulospinal test battery (VST) with and without foam cushion , video head impulse test (vHIT), suppression head impulse test (SHIMP), cervical vestibular-evoked myogenic potential test (cVEMP) and measurement of the subjective visual vertical (SVV) were utilised to quantify the vestibular system. Our own laboratory norms were used when analysing vHIT, SHIMP and cVEMP results . The ICS Impulse 2015 and 2019 versions and Neurosoft 2019 software were used for VNG/vHIT/SHIMP and VEMPs, respectively. The first author performed all vestibular tests except the VEMPS, which was performed by one of the co-authors, and consistency was maintained.We did not perform ocular vestibular-evoked myogenic potentials (oVEMP), as paediatric norms are hardly available, and we are in the process of gathering our own norms. These tests were repeated several times as part of audiovestibular surveillance. All children also underwent neurological, paediatric, cardiological and development assessment as mandated in investigating children with suspected vestibular disorders. The families, the sensory services, the school and significant others were all involved in the journey of the children effectively.Informed signed consent was obtained from both children and their legal guardian for reporting their conditions to scientific journals. Ethical Committee approval was not required as the study was not classed as research but as a case series with less than 3 subjects.This child did not undergo the universal newborn hearing screening as it was in its infancy in the country of study. He was referred due to delayed speech and motor development at the age of 2 years and 3 months and a severe mixed hearing loss was observed in both ears. His tympanometry was bilaterally normal as was stapedial reflex on the right only and his transient otoacoustic emissions were absent bilaterally. He was fitted immediately with digital amplification and monitored twice every year. The hearing loss progressed over the next years, especially on the left ear, becoming profound , which nHRCT and T2 drive MRI demonstrated a bulbous end of IAM with dilatation, absent cochlear modiolus with intact septa, incomplete cochlear partition type III and incomplete IAM fundus gene  that conHe perceived balance problems and was apprehensive in playground activities when he was a toddler. He was fond of dancing but was unable to indulge in the twists/turns that dancing demanded. He was diagnosed with bilateral vestibular dysfunction 12 years after diagnosis, showing an initial six semicircular canal involvement in the vHIT, absent cVEMP on the left with low thresholds on the right, with abnormal VST but normal SHIMP, SVV and VNG . There wSibling B failed his newborn hearing screening and showed a moderate mixed hearing loss on both sides. He was fitted at the age of 6 weeks and was regularly monitored. There was a mild speech delay and he received speech and language therapy. His hearing loss also showed progression and fluctuation and became severe over the years . His tymPOU3F4 mutation by genetic typing.HRCT and T2 drive MRI was identical to his brother , confirmHis motor skills were delayed, and like his brother, he was also apprehensive about playground activities. He was diagnosed with bilateral vestibular weakness 10 years after diagnosis  involvinPOU3F4 gene with 51 different loci identified [X-linked gusher disorder is diagnosed by a characteristic phenotype of congenital progressive mixed hearing loss with normal middle ear function and by a pathognomonic HRCT demonstratable cochlear dysplasia. The disorder is due to a mutation in the entified . Our sibPOU3F4 gene in the animal model participates in the remodeling of the otic capsule [The typical feature of an X-linked gusher disorder in HRCT is an incomplete cochlear partition type III. The  capsule . A breac capsule . Althoug capsule . In X-li capsule . These fOther cochlear structural abnormalities defined in X-linked gusher disorder are an enlarged superior vestibular nerve canal, enlarged labyrinthine facial nerve canal, enlarged singular nerve canal, vestibule with cystic appearance, semicircular canal with cystic appearance, dysplasia of oval window, dysplasia of round window and abnormal stapes .Unlike HRCT, MRI observations in the condition have hardly been reported. T2 MRI in X-linked gusher disorder shows a bulbous internal auditory canal, an incomplete separation of the basal turn of the cochlea with the fundus and a fluid-filled cochlear cavity without the modiolus or the spiral lamina . An MRI A typical feature observed in X-linked gusher disorder is a conductive element to the hearing loss that it presents, which was universally observed in all case reports ,20,21,22The third-window effect was later detailed by Merchant et al. in 2008 . They atOther typical third-window symptoms are conductive dysacusis, autophony, Hennebert’s and Tullio’s phenomenon, disequilibrium, dizziness/vertigo, tinnitus/pulsatile tinnitus, misophonia and gaze-induced tinnitus ,14. It iFor a child with a third-window disorder, their cognitive reaction, emotional reaction, effect on schoolwork and social life and on overall development should be factored in. Once our siblings were made aware of the pathology, they reconciled to their conditions very well to do well in life and develop a positive insight for the future. The observation that characteristic third-window symptoms may be absent in children suggests that the possibility of a third window in a child is not just based on anamnesis but rather on a more holistic and an emerging clinical scenario. Indeed, this holistic approach is quite essential to investigate and then confirm a third window for effective management, avoiding missing the diagnosis.The mixed hearing loss encountered in X-linked gusher disorder is progressive ,22. ThisThe entire publication spectrum regarding X-linked gusher disorder has investigated the hearing loss and the radiological phenotype in some detail but all but one made no mention of a vestibular phenotype. The first author reported vestibular findings in their study on rare third-window disorders in children with the two cases reported in this study but did not elaborate on the evolution of vestibular function or a SHIMP test . The sibThe hallmark of diagnosing a third window is a characteristic vestibular-evoked myogenic test (VEMP) that assesses saccular  and utricular (ocular oVEMP) function. Both these tests show elevated amplitudes and lowered thresholds in mobile third windows due to hyperstimulation of the otolith sensors because of the third-window effect on the acoustic energy transmission . StudiesThe video head impulse test (vHIT) has revolutionized vestibular diagnostics in recent times . NormatiThe suppression head impulse test (SHIMP) is a new test in the vestibular test battery that is useful for assessing vestibular compensation and is used as an adjunct to the vHIT . This haWe followed-up with these children for a period of seventeen years, and the older child has transitioned to the adult services formally, while the younger one is still under follow-up. They were fitted early with hearing aids. They underwent regular monitoring of hearing loss and digital amplification adjusted accordingly given the progressive nature of their hearing losses. The older sibling was implanted later on one ear and did not want a second implant while the younger one is doing very well with bilateral hearing aids. The outcome was development of normal speech and communication skills that are reported after early intervention for hearing loss in a child . HoweverSimilarly, a vestibular weakness is required to be addressed early. Both the siblings underwent a course of vestibular rehabilitation and recovered well with their balance. They received situational counselling as to how to avoid provocation that could lead to vestibular decompensation. They could indulge in playground activities and one child resumed his hobby of dancing.The limitation of this study is its small number, which of course leaves questions for generalisation. However, we believe that these two cases provide a rather important insight into this very complex disorder. It might be difficult to launch dedicated controlled and blind studies given the rarity of the condition, and thus, snapshots like this yield valuable information. We recommend scrupulous follow-ups and a holistic and cognitive approach  that is crucial to manage a child.X-linked gusher disorder is a rare genetic disorder in children that manifests with an audiovestibular phenotype. Hearing loss is usually a mixed one due to a third-window effect as a result of characteristic bony deformities. Vestibular function hitherto unreported may be weak and can be quantified by objective vestibular tests. The condition is progressive and evolves over a period of time where hearing loss progresses, and vestibular weakness may undergo partial compensation, rendering children symptomatic. Early audiovestibular intervention is pivotal with multidisciplinary engagement in a holistic way involving significant others in the care. This achieves a maximal favourable outcome underpinning the importance of regular follow-ups for these children.


Background: Zebrafish (Danio rerio) have been established in recent years as a model organism to study Diabetic Retinopathy (DR). Loss of endothelial cells and pericytes is an early hallmark sign of developing DR in the mammalian retina. However, morphology, numbers, ratios, and distributions of different vascular cells in the retinal compartment in zebrafish have not yet been analyzed and compared with the mammalian retina.Methods: The retinal trypsin digest protocol was established on the zebrafish retina. Cell types were identified using the Tg(nflk:EGFP)-reporter line. Cells were quantified using quantitative morphometry.Results: Vascular cells in the zebrafish retina have distinct morphologies and locations. Nuclei of vascular mural cells appear as long and flat nuclei located near the vessel wall. Round nuclei within the vessel walls can be identified as endothelial cells. The vessel diameter decreases from central to peripheral parts of the retina. Additionally, the numbers of vascular cells decrease from central to peripheral parts of the retina.Discussion: The retinal trypsin digest protocol, which can be applied to the zebrafish retina, provides novel insights into the zebrafish retinal vascular architecture. Quantification of the different cell types shows that, in comparison to the mammalian retina, zebrafish have higher numbers of mural cells and an increased mural cell to endothelial cell ratio. This protocol enables to quantify mural cell and endothelial cell numbers, is easily adaptable to different transgenic and mutant zebrafish lines and will enable investigators to compare novel models on a single cell level. Multiple ocular pathologies are associated with changes in the retinal vasculature. Diabetic retinopathy is one of the leading causes of vision impairment or blindness globally  and one Improved understanding of the cellular mechanisms of diabetic retinopathy has shown that disruption of the neurovascular unit is critically involved in the pathogenesis . The terEarly structural changes in diabetic retinopathy are the loss of pericytes and consecutively the loss of endothelial cells, leading to vasoregression and, when progressing, decreased blood flow in the retina . In the Danio rerio) have come into focus as a convenient animal model to study diabetic complications (Tg(fli1a:EGFP) reporter line • Conical tubes • Cover slips • Culture dishes • Dumont Tweezers No. 5 (neoLab)• Feather disposable scalpel No. 10 (Feather)• Leica TCS SP5 Confocal Microscope (Leica)• Microscissors Vannas 2.5 mm (Fine Science Tools)• Microscope slides (76 × 26 mm) (neoLab)• Olympus BX51 Microscope (Olympus)• Safe-lock tubes • Syringes • XC10 camera (Olympus)Tg(nflk:EGFP) (Tg(nflk:EGFP) transgenic line, GFP is expressed in nuclei of endothelial cells under the control of the flk1/vegfr2 promotor, which is activated early during the development of endothelial cells  in the morning and fish flake food in the afternoon. For these experiments, zebrafish from the Tg(nflk:EGFP) line were used at >24 months post fertilization (mpf). The zebrafish were sacrificed through hypothermal shock in 2°C–4°C cold water (Zebrafish embryos of the lk:EGFP)  line weral cells . Zebrafial cells  and wereld water .The eyes were carefully extracted from the head at the optic nerve and fixed in 4% formalin at room temperature for 48 h. Retina dissection was performed as previously described by our laboratory . The preTo identify endothelial cells in the retinal vasculature, confocal images of the air-dryed digest preparations before staining with Mayer’s hemalum solution (Millipore) were acquired using a confocal microscope (DM6000 B) with a scanner (TCS SP5 DS). Images of distinct areas were taken with a ×20 water immersion objective, 400 Hz bidirectional, 1,024 × 1,024 pixels and z-stack steps of 1.5 µm thickness.To properly visualize the different cell types in the retina, nuclei staining with undiluted Mayer’s hemalum solution (Millipore) was performed. After air-drying the preparations, the slides were shortly placed in aqua bidest and then moved to fresh undiluted Mayer’s hemalum solution for 7 min. Afterwards, they were placed in lukewarm tap-water for 2 min and then moved shortly to 70% and then 80% ethanol before being put in 96% and then 99.8% ethanol for 5 min each. After the last change of ethanol, the slides were placed in two changes of xylene and kept there for 5 min each before the slides were covered with cover slips using DPX mounting medium (Thermo Fisher Scientific). Hemalum stained samples can be kept at room temperature for approximately 2 weeks without significant loss of quality.2 of capillary area).To determine the numbers and morphologies of retinal vascular mural cells and endothelial cells, the retinal digest preparations were analyzed using the image analyzing system Cell-F . To establish the number of endothelial cells and vascular mural cells in the retina of healthy adult zebrafish images of all areas of the retina were taken with an Olympus BX51 microscope  under ×200 magnification. Images were taken in all density areas of the retina  and in central, intermediate, and peripheral parts of the retina . Nuclei p-value of p < 0.05 was considered as significant.Results are expressed as mean ± SD. One-way ANOVA followed by appropriate multiple comparison tests was used to analyze statistical significance between different groups. Statistical analyses were performed using GraphPad Prism software. A Experimental procedures on zebrafish were approved by Medical Faculty Mannheim (License no. I-21/18). All experiments were carried out in accordance with the approved guidelines.The application of the retinal digest protocol on mouse retinas has provided very valuable information about pathological retinal cellular changes under diabetic conditions for decades . Since n2, which is now provided manually.Some technical challenges arose during the adaptation of the retinal trypsin digest protocol to the zebrafish retina. The retinal vasculature in zebrafish is located on top of the inner limiting membrane (ILM) and the vessels are tightly connected to the ILM. While the ILM is removed during the digestion process in the mammalian retina, removal of the ILM in zebrafish led to removal of the vasculature. Therefore, this experimental step for the zebrafish retina digestion was skipped. Since the remaining zebrafish ILM consists mainly of collagen and showed a strong background color after hemalum stain, we also needed to adapt the quantification of the capillary area and number of cells per mm2.The intermediate part of the zebrafish retina was the most reliably quantifiable part . In the Tg(nflk:EGFP) zebrafish line in which the nuclei of endothelial cells express endothelial green fluorescence protein (EGFP). Retinae of Tg(nflk:EGFP) zebrafish were processed using the digest protocol described above. Prior to staining, confocal images were taken, and matched with digest preparations after staining. Respective images were compared in specific locations allowing for the unequivocal identification of endothelial cells as the third cell type . We defined the main vessels before they divide as vessels in the “central” part of the retina, the vessels between the first division point and the second division point as vessels in the “intermediate” part of the retina and the vessels which were connected to the IOC as vessels in the “peripheral” part of the retina. Furthermore, the zebrafish retina has areas with differing vascularity due to an uneven pattern of intervascular distance in the periphery of the retina. There is one high density, one low density and two medium density areas. Highest and lowest vascularity are located opposite each other .2. To establish the unit cells/mm2 we counted the cells over 200 µm of vessel length and measured the vessel diameter to calculate the area.To ensure comparability between zebrafish and mammalian retinal vascular cell numbers it was necessary to calculate the number of endothelial cells and vMCs per mmWe found that the vessel diameter decreased from central to peripheral parts of the retina. In the central part of the retina, vessels had a diameter of on average 15.13 ± 1.96 µm, which decreased to 12.22 ± 1.61 µm in the intermediate area and to 9.99 ± 1.12 µm in the peripheral area. If compared to the mammalian retina, only the vessels in the periphery of the retina would be qualified as capillaries, while the vessels in the intermediate and central parts of the retina would be arterioles .Furthermore, we found that the vessel diameters varied between the different density areas in the intermediate part of the retina while they remained comparable in the central and peripheral part of the retina .2 to ensure comparability.In conclusion, we established a method to count the different cell types. We discovered that due to the variation in vessel diameter between different density groups in the intermediate part of the retina it is essential to quantify cells in the zebrafish retina as cell numbers per mmTo conclusively analyse the retinal vascular architecture, we quantified vascular cell numbers in all areas of the retina of adult zebrafish.2 vascular area decreased significantly from central to peripheral parts of the retina . This variability in cell densities of the different parts of the retina requires careful pre-test definition of which areas are compared  of the retina  rather than capillaries, as shown before .It has been described that vascular mural cells change their phenotype and marker expression dependent on which vessel type they are associated with . Typicalpdx1−/− zebrafish at 5 days post fertilization. This phenotype could be rescued through antihyperglycemic and antiangiogenic pharmacological interventions, indicating that the mechanism may be observed and studied in zebrafish models of DR at an early stage (Apart from vSMC loss, persistent dilation of retinal arterioles has been also observed in diabetes, which may lead to increased retinal blood flow and may represent arteriolar dysfunction . Interesly stage .pdx1−/− model of DR it was shown that mutants expressed less Transgelin1 than controls from the age of 3 mpf onwards (Furthermore, in the  onwards . Transge onwards , furtherAnother striking feature of the adult zebrafish retina is the different vessel densities according to retinal area. The reasons for this observation remain unknown. In zebrafish larvae, photoreceptor distribution is unisotropic, which may cause different vascular densities, but in adult zebrafish retinae, photoreceptors are organized in a highly regular array . Thus, iPerforming the retinal trypsin digest method on the zebrafish retina may help researchers analyze the role of arterioles and vSMC in the pathogenesis of diabetic retinopathy.Another key difference that needs to be considered when applying the retinal trypsin digest protocol to the zebrafish retina is the anatomy. In mammals, the retinal vascular network is the result of coupled hyaloid regression and retinal angiogenesis leading to two vascular plexuses, the choroid and the intraretinal vasculature . In zebraldh2.1−/− retinae proving the usefulness of the protocol (In conclusion, we have further characterized the zebrafish retinal vasculature using the retinal trypsin digest protocol and we provide a novel statistical quantification of the vasculature and its cell types. We found that the different vascular cell types can be identified through morphology and location and by applying the quantification protocol, we report novel vascular features within the physiological retina. In addition, quantification of the cell numbers showed that zebrafish have an increased number of vascular mural cells in comparison to the mammalian retina and an increased mural cell to endothelial cell ratio. We have recently successfully applied the protocol showing an increased vessel diameter and reduced mural cell coverage in adult protocol . Thus, t