Myodes glareolus) from an experimental evolution model system: four lines selected for high swim-induced aerobic metabolism (A lines), which have also increased the basal, average daily, and maximum cold-induced metabolic rates, and four unselected control (C) lines. We measured the resting metabolic rate (RMR), evaporative water loss (EWL), and body temperature in 72 young adult (4 months) and 65 old (22 months) voles at seven ambient temperatures (13–32°C). The RMR was 6% higher in the A than in the C lines, but, regardless of the selection group or temperature, it did not change with age. However, EWL was 12% higher in the old voles. An increased EWL/RMR ratio implies either a compromised efficiency of oxygen extraction in the lungs or increased skin permeability. This effect was more profound in the A lines, which may indicate their increased vulnerability to aging. Body temperature did not differ between the selection and age groups below 32°C, but at 32°C it was markedly higher in the old A-line voles than in those from other groups. As expected, the thermogenic capacity, measured as the maximum cold-induced oxygen consumption, was decreased by about 13% in the old voles from both selection groups, but the performance of old A-line voles was the same as that of the young C-line ones. Thus, the selection for high aerobic exercise metabolism attenuated the adverse effects of aging on cold tolerance, but this advantage has been traded off by a compromised coping with hot conditions by aged voles.As with many physiological performance traits, the capacity of endotherms to thermoregulate declines with age. Aging compromises both the capacity to conserve or dissipate heat and the thermogenesis, which is fueled by aerobic metabolism. The rate of metabolism, however, not only determines thermogenic capacity but can also affect the process of aging. Therefore, we hypothesized that selection for an increased aerobic exercise metabolism, which has presumably been a crucial factor in the evolution of endothermic physiology in the mammalian and avian lineages, affects not only the thermoregulatory traits but also the age-related changes of these traits. Here, we test this hypothesis on bank voles ( Myodes glareolus, selected for the high rate of aerobic exercise metabolism , was higher in the A than in the C lines  and unselected control (C) lines , 2015. V C lines , althoug C lines . The sel C lines . Contrar C lines , the oxi C lines , 2015. I C lines . To summM. glareolus Schreber, 1780) from four replicate lines selected for the maximum swim-induced rate of aerobic metabolism (A lines: high aerobic metabolism) and four replicate lines of control, randomly bred voles (C lines: control). The selection criterion is the highest 1-min rate of oxygen consumption achieved during an 18-min swimming trial, adjusted for differences in body mass and other confounding factors, such as sex and measurement date. The swimming trial is performed at a water temperature of 38°C, which is close to the voles’ body temperature (Tb) to ensure that the increase of metabolic rate is due to exercise, not due to thermoregulation .This study was conducted on 72 young adult females from generation 27th and 65 old females from 24th generation. The age of the voles from the young group ranged from 93 to 161 days (mean ± SD = 114.4 ± 14.97 days), and voles from the old group were from 605 to 752 days (663.2 ± 22.1 days) old. Within the two age groups, the animals represented both selection directions and all replicate lines within the selection directions. We only used females because this work is a part of an ongoing project concerning limits to energy budgets during lactation cf. . The volTa = 13, 17, 23, 26, and 28°C) was started. The order of the measurements was randomized among individuals . Completing measurements on one block lasted 40 days. On the first day, we placed the voles in individual cages, and after 4 days thermosensitive data loggers were implanted to record the core body temperature. After a recovery period of 7–8 days, the resting metabolic rate (RMR) was measured at 20°C in order to familiarize all the voles with the respirometric measurements. After 3 days, a set of RMR measurements at five ambient temperatures . The loggers were programmed to record the via a nose cone for 7–25 min. Isoflurane  with oxygen was provided with a flow of 200 ml min–1  at 3% volume of breathing air  hing air . During ritoneum . The logritoneum . Post-suO2, in milliliters O2 per minute) in fasted animals. We recorded the body mass (MB) before and after each measurement (±0.1 g). We used two experimental setups for the RMR measurements: one for all Ta values ranging from 13 to 28°C and another for Ta = 32°C. In the first setup, the animals were acclimated for 2 h in room temperature (20°C) without food but with water provided ad libitum and then placed in the respirometric chambers for further measurements. The measurements were conducted at two time intervals: in the morning  and in the afternoon , each time for eight individuals.The RMR was measured as the minimum rate of oxygen consumption . The trial started at 28°C, then the temperature was gradually increased to 32°C (+1°C every 5 min) and then maintained constant for 2 h.The second setup was introduced because of a higher risk of mortality at 32°C . TherefoThe RMR was measured using an eight-channel open-flow respiratory system . We used black-colored glass chambers of about 800 ml fitted with wire baskets. To ensure that the voles could not exhale air directly into the outgoing air, the baskets with animals were suspended 3 cm below the ceiling of the chamber with the air inlet near the bottom and the outlet at the top of the chamber. We used paraffin oil at the bottom of each chamber to collect urine and feces; therefore, baskets were placed 4 cm above the chamber bottom . The cha–1 of fresh air dried with silica gel (Sigma-Aldrich) was regulated by a pump with an eight-channel mass flow controller . First, the water vapor pressure was measured in the subsamples of air flowing out of the chambers, then the air subsamples were dried by a small-volume magnesium perchlorate absorbent , and the oxygen and CO2 concentrations were measured with the integrated respirometry system . The air samples were drawn sequentially from an empty reference chamber and eight channels with animals. The channels were switched with a computer-controlled baselining unit and multiplexer . In each measurement cycle, lasting 19 min, the reference and the first active channel were sampled for 150 s, and each of the next seven active channels were sampled for 120 s, which ensured a complete washout of the system after switching channels. The time was longer for the reference and the first measurement channels because the change of the air composition after switching to those channels is larger than that in the case of the other channels (A flow rate of 500 ml minchannels . In the O2) and the rate of CO2 production (CO2), the last 20 s of the record before switching channels was used. The adequate baselines were computed by interpolating the oxygen concentration values (in percent) on the recorded baseline in the reference channel. The rate of oxygen consumption was computed using an appropriate respirometric equation .The data were recorded every second through an analog-to-digital interface UI2  with the ExpeData 1.9.17 v PRO data acquisition program. To calculate the rate of oxygen uptake (equation . BecauseTbrmr), the thermal conductance  was calculated for each ambient temperature as follows (CT = RMR/(Tbrmr - Ta).Based on the RMR and body temperature recorded at or near the time the RMR was recorded  and then the evaporative water loss   at the time when the RMR was measured and the mean barometric pressure calculated separately for each measurement were used for the calculation of the rate of the water vapor production ( minute) :i is the incoming air flow rate (500 ml min–1), FeH_2O the fraction of the water vapor in excurrent air (WVP/barometric pressure), and FiH_2O the fraction of the water vapor in incurrent air (WVP/barometric pressure).where FRW = J/s) according to Finally, the RMR and EWL measured together were converted to their energy equivalents  in completely soaked individuals placed in wet respirometric chambers (500 ml) maintained at +23°C in a temperature-controlled cabinet for up to 18 min (The thermogenic capacity was measured as the rate of oxygen consumption (o 18 min . Before o 18 min .–1 of fresh air dried with silica gel (Sigma-Aldrich) was regulated (±1%) by a mass flow controller . The subsample of excurrent air was pre-dried with an ND-2 gas drier (Sable Systems) and then by a small volume of a magnesium perchlorate absorbent . The oxygen and CO2 concentrations were measured in dried samples of ∼200 ml min–1 of air pull through the analyzers  by an ND-2 pump. The gas concentrations were recorded every 1 s through an analog-to-digital interface UI2 with the ExpeData 1.9.17 v PRO data acquisition program (Sable Systems Int.). The maximum thermogenic capacity was defined as the highest 1-min instantaneous rate of oxygen consumption . A pass of about 2,000 ml minsumption . The ratsumption . The effTbrmr, CT, EWL, and EWL/RMR at all ambient temperatures, we collected a total of 916 records of RMR (together with other variables) for all individuals. Among these records, 47 were excluded from the analyses due to death of animals , assumed bad condition of animals during measurements , or system errors (30 RMR records). The highest mortality was recorded at 32°C, where, despite the shorter trial time and real-time monitoring by video recordings, six individuals died (five from the A lines and one from the C lines). In addition, for the analyses of Tbrmr and CT, we excluded 11 missing records because of a malfunction of the data loggers and, for EWL and EWL/RMR analyses, of seven records with system errors. For the analyses of the thermogenic capacity, a total of 127 records were collected.For all analyses of RMR, 2 within the 20-s period from which the RMR was computed. When the animal is active, the variation is increased. Thus, the standard deviation (SD) of the 20 values can be used as an “activity index.” The distribution of the activity index showed that records with SD over 0.06 ml O2 min–1 are obvious outliers from the typical distribution, and the individuals had to be active when the minimum rate of oxygen was recorded (O2 second-by-second readings) has been included as an additional covariate in all analyses of RMR, Tbrmr, CT, EWL, and EWL/RMR.The size of the respirometric chambers was large enough to allow free movement, and many individuals showed long periods of intensive activity during the measurements carried out under conditions similar to those used in our experiment . To checrecorded . A totalTbrmr, CT, EWL, and EWL/RMR measured at 20 and 32°C and for O2cold, we used a cross-nested mixed analysis of covariance (ANCOVA) model [mixed procedure with the restricted maximum likelihood (REML) method and Satterthwaite option]. For the analyses of RMR, Tbrmr, CT, EWL, and EWL/RMR measured at 13, 17, 23, 26, and 28°C, we used the repeated-measures versions of the ANCOVA model. The results of the measurements at 20 and 32°C were not included in the repeated-measures models because these trials were always performed as the first and the last ones, respectively.All statistical analyses were performed in SAS v. 9.4 . For the analyses of MB, RMR, vs. old) and selection (A vs. C lines) as the main fixed categorical factors and the replicated lines nested within the selection groups as the random effect. Timing (“morning” and “afternoon”) and block (three groups) were included as fixed cofactors. In the analyses of RMR, CT, EWL, and EWL/RMR measured at 32°C, the models also included chambers as the fixed cofactor (two chambers), but in the analyses of these traits measured at other ambient temperatures, the models included chambers as the random effect (eight chambers). For the analysis of O2cold, timing was not included, but analyzer was an additional cofactor (two respirometric systems). In all models, we also included interactions between the age and selection factors and random interactions between age and line nested within selection. In all models, the mean body mass measured before the RMR measurements and the activity index were included as covariates. To test the assumption of the homogeneity of body mass slopes, all initial, full ANCOVA models included interactions between the main factors (and their interactions) and the initial body mass. These interactions were not significant; therefore, we removed them from the final models. The repeated-measures extension of the above models was used to perform the analyses of RMR, Tbrmr, CT, EWL, and EWL/RMR for combined results from measurements at other ambient temperatures. These models included a fixed repeated-measures factor for Ta (order of the measurements treated as a grouping factor) and the interactions of Ta with age, selection, and age × selection interaction.All models included age  number of an animal was included as a random effect. For the RMR analysis, we used a model with an “unstructured” type of residual (co)variance matrix and, for the other response variables, the “compound symmetry” type of residual (co)variance matrix. For the RMR, we performed additional analysis with The results of all analyses are expressed as adjusted least squares mean (LSM) ± SD. All presented LSMs were calculated for fixed body mass = 25 g and activity index = 0.03. Before estimating the final models, we analyzed studentized residuals and removed from the final analysis observations with residuals higher than 3.5 and lower than -3.5 as the outliers .CT estimated for the lowest measured temperature (13°C) for the Selection × Age subgroups and the respective O2cold values, the lower lethal temperature , i.e., the minimum temperature at which the animals can retain homeothermy, was estimated from the linear equation of the thermoregulatory model  or between the morning and afternoon trials . The mean mass was higher in old voles (LSM ± SE = 28.5 ± 0.89 g) than in young ones  and tended to be higher in voles from the A lines (27.6 ± 1.14 g) than in those from the C lines .The body mass of the voles, measured before the first respirometric trial, ranged from 15.9 to 37.5 g (mean ± SD = 25.6 ± 5.35 g). The body mass did not differ between the three experimental blocks (p < 0.05), and the RMR estimated for the modal value of SD = 0.02 was 3–5% higher in comparison to that estimated for SD = 0.05  at all Ta values below 32°C. At 32°C, this RMR difference was 7% for the A lines and 8% for the C lines. The effect of the activity index was not significant for Tbrmr at ambient temperatures below 32°C (p > 0.1), but at 32°C the body temperature increased with the activity index (p = 0.04). The activity index did not affect the results of thermal conductance (p > 0.15). The EWL increased with increasing activity index at all temperatures except at 20°C .In all analyses, the RMR increased with increasing activity index (2 min–1 (mean ± SD = 1.5 ± 0.25 ml O2 min–1) and increased with body mass  and varied among the three experimental blocks . Analysis of covariance showed that the mass-adjusted RMR did not differ between age groups  or between selection directions . The interaction between these factors was also not significant  revealed that the measurement order affected the results , although no systematic trend was observed , and that the RMR decreased with increasing Ta from 1.86 ± 0.02 ml O2 min–1 at 13°C to 1.09 ± 0.02 ml O2 min–1 at 28°C . The mass-corrected RMR was, on average, 6% higher in the A lines than in the C lines  and did not differ between the two age groups . The analysis with Ta treated as a quantitative predictor gave practically the same results concerning the main factors. The resting metabolic rate decreased with increasing Ta . The slope did not differ between selection directions or age groups either . The RMR was higher by 0.09 ± 0.02 ml O2 min–1 in the A than in the C lines , but it did not differ between the two age groups , and the interaction between selection direction and age was not significant either . The mass-adjusted RMR was 16% higher in the A than in the C lines , but the age effect  and the interaction between the factors were not significant , the RMR ranged from 0.53 to 1.67 ml OTbrmr), measured at the ambient temperature (Ta) of 20°C, ranged from 36.3 to 39.4°C (mean ± SD = 38.0 ± 0.6°C). The body temperature decreased with body mass . The ANCOVA-adjusted least squares means did not differ between age groups  or selection directions .Body temperature at the time when the RMR was recorded  showed a significant effect of the measurement order  and no significant effect of the timing . The body temperature decreased with increasing ambient temperatures, from 38.1 ± 0.06°C at 13°C to 37.7 ± 0.06°C at 28°C , selection , or their interaction . Interactions between Ta and age or selection were not significant either .The repeated-measures analysis applied to the combined data of Tbrmr was markedly higher than those at temperatures of 28 and 26°C  or between age groups . However, the Tbrmr was higher in the A than in the C lines , and the interaction between age and selection direction was also significant  than the young voles , the and 26°C . Unlike CT) at 20°C ranged from 0.06 to 0.12 ml O2 min–1°C–1 (mean ± SD = 0.08 ± 0.01 ml O2 min–1°C–1) and increased with body mass , but the CT differed among the three experimental blocks . Analysis of covariance revealed that the mass-adjusted CT did not differ between age groups  or between selection directions , while in the old group the effect of selection was not detectable , but the interaction between these factors was not significant  showed a significant effect of the measurement order , but no systematic trend was observed. Thermal conductance increased with increasing Ta, from 0.073 ± 0.002 ml O2 min–1°C–1 at 13°C to 0.112 ± 0.002 ml O2 min–1°C–1 at 28°C  and was also higher in the old than in the young voles , but differed among the three experimental blocks . The mass-adjusted CT did not differ significantly between age groups , but it was higher in the A than in the C lines . However, the interaction between age and selection was also significant because in the C lines old individuals tended to have a higher CT, whereas in the A lines the pattern was the opposite , but not in the old group  and did not change with body mass . The mass-adjusted EWL was 26% higher in the old (2.50 ± 0.10 mg H2O min–1) than in the young voles . The interaction between age and selection was also significant, and the EWL was much higher in the A than in the C lines in the old group , but not in the young group . In addition, the difference in the EWL between age groups was more remarkable in the A lines  than in the C lines  showed no effect of the measurement order , but the EWL decreased with increasing Ta, from 2.47 ± 0.10 mg H2O min–1 at 13°C to 1.99 ± 0.10 mg H2O min–1 at 28°C  than in the young groups  and was higher in the A lines (2.34 ± 0.10 mg H2O min–1) than in the C lines . However, neither the interaction between age and selection  nor the interactions of these factors with Ta were significant . Unlike in lower temperatures, at 32°C, the mass-adjusted EWL did not differ significantly between age groups , but it was higher in the A lines (1.96 ± 0.07 mg H2O min–1) than in the C lines . The interaction between age and selection was not significant . The EWL/RMR was 24% higher in the old (0.20 ± 0.008) than in the young voles . The interaction between age and selection was significant, and the EWL/RMR was higher in the A than in the C lines in the old group , but not in the young group . In addition, the difference in the EWL/RMR ratio between age groups was more profound in the A lines  than in the C lines  at 20°C ranged from 0.09 to 0.35 (mean ± SD = 0.18 ± 0.055) and decreased with body mass  showed no effect of the measurement order , but EWL/RMR increased with increasing Ta, from 0.17 ± 0.006 at 13°C to 0.22 ± 0.006 at 28°C . However, neither the interaction between age and selection  nor the interactions of these factors with Ta were significant . Unlike in lower temperatures, at 32°C, the EWL/RMR did not differ significantly between age groups  and between selection directions . The interaction between age and selection was not significant  ranged from 2.88 to 7.10 ml O2 min–1 (mean ± SD = 4.86 ± 0.93 ml O2 min–1), and it increased with body mass . The mean values obtained by the two respirometric systems differed by 4% , but, as the animals from all groups were randomly assigned to the two systems, the difference had no bearing on the conclusions. The mass-adjusted O2cold was 13% lower in the old (4.55 ± 0.07 ml O2 min–1) than in the young voles . However, the interaction between these factors was not significant  calculated for a 25-g vole was lower in the A than in the C lines and higher in the old than in the young voles, but the difference between age groups was similar in the A and C lines  ranging from 13 to 28°C, designed to analyze the relationship between the traits and ambient temperature and to detect differences between the experimental groups in the lower critical temperature . Finally, to compare the performance under extreme thermal conditions, we measured the thermogenic capacity (the maximum cold-induced metabolic rate) and the RMR (and the associated traits) at 32°C, i.e., at the temperature above the expected UTC. However, it has turned out that the RMR decreased steadily with increasing ambient temperatures and was lower at 32°C than at 28°C, with no evidence of the thermoneutral zone  in the range of temperatures from about 26 to 31°C and that the trials near and above the upper critical temperature  are associated with an increased risk of mortality . We alsoral zone . This inral zone . Thus, tTa range of 13–28°C, did not differ between the selection and age groups , measured simultaneously with the RMR, did not differ significantly between the experimental groups, but it increased nearly linearly with decreasing Ta  calculated separately for each individual and Ta, revealed that the characteristics of heat exchange were affected both by the selection and aging  revealed a more interesting pattern . The EWLTa analyzed, Tb was higher than that at 28°C  is higher in voles from the selected lines than in those from the control lines  theory provides a conceptual perspective on the evolution of life histories in relation to maximum capacity to dissipate body heat in endotherms . Our finAll datasets presented in this study are included in the article/Supplementary Material.The animal study was reviewed and approved by II Local Bioethical Committee in Kraków, Poland.ES and MG conceived the experiment. MG performed the experiment and carried out all the measurements. MG, PK, and ES analyzed the data. MG and PK wrote the manuscript with the assistance of ES and UB. All authors commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Newborn hearing screening (NHS) can reduce the economic and social burden of hearing impairment. To track the progress of the goal set by the National Program of Action for Child Development (NPACD) and to estimate the detection rates of hearing impairment, the present study examined NHS coverage in 2008–2010 and 2016 and the detection of hearing impairment across China in 2016.Licensed medical institutions across China were surveyed in 2012 and 2018 by the National Center for Birth Defects Monitoring of China to collect data for the 2008–2010 period and for 2016 on live births, initial screening rates , secondary screening rates , and rates of hearing impairment diagnosis among infants who were referred in the secondary screening. To calculate universal newborn hearing screening (UNHS) coverage, the number of newborns who received NHS within 4 weeks after birth was divided by the number of live births. The detection rate of hearing impairment was calculated by combining referral rates on primary and secondary screening with the rate of diagnosis.National UNHS coverage increased from 29.9% in 2008 to 86.5% in 2016, with different regions showing different increases. During this period, the number of provinces with UNHS coverage over 90.0% increased from 2 to 17, with UNHS coverage in 2016 being substantially higher in eastern provinces (93.1%) than in western provinces (79.4%). In 2016, the detection rate of hearing impairment across the country was 0.23% (95% CI 0.15–0.25%), and it varied from 0.17% in western provinces to 0.22% in central provinces and 0.28% in eastern provinces. The lowest rate was 0.02% in Heilongjiang Province and the highest rate was 0.63% in Hainan Province.National UNHS coverage increased substantially from 2008 to 2016, although provinces and regions still showed differences. The detection rate of infant hearing impairment in China is comparable to that in other countries. A national individual-level information system is urgently needed in China to facilitate the integration of screening, diagnosis and treatment of infant hearing impairment, which may also lead to a more accurate estimate of the detection rate. Hearing impairment in children is a serious obstacle to their development and education: it has been associated with delayed development of speech and language and cognitive skills, as well as with slow learning and difficulty in school . AccordiNewborn hearing screening (NHS) can effectively enable the diagnosis of hearing impairment and intervention during the first 6 months, ensuring better outcomes for children , 4, 5. SIn China, NHS was first adopted for high-risk neonates in Beijing in 1989, and UNHS was initiated in May 1996 in a few provinces . In 1999With the aims of tracking the progress of the goal set by the NPACD and the estimation of detection rates of hearing impairment, the National Center for Birth Defects Monitoring of China (NCBDMC) conducted two surveys authorized by the National Health Commission of China (NHCC) in 2012 and 2018. Therefore, the baseline of the coverage of UNHS in 2008–2010 and its mid-term status were obtained, while the national and subnational detection rates of hearing impairment were estimated based on the survey in 2018.This study used a cross-sectional survey to collect the tabulated data on NHS and diagnosis, which were implemented in 2012 and 2018, respectively, by the medical institution. The data in the 2012 survey covered the period from 2008 to 2010, and the data in the 2018 survey covered the year 2016.The two surveys were legal and mandatory data collections with the permission of the NHCC, and all the original data were tabulated data that did not involve individual information. The publication of the study has received permission from the NHCC. It meets the conditions of ethical exoneration according to the Council for International Organizations of Medical Sciences .Regulation of Neonatal Disease Screening promulgated by NHCC in 2009 ,At the same time, several Chinese provinces, such as Tibet and Guizhou, show quite low UNHS coverage, akin to that of several developing countries . This loWe report detection rates of neonatal hearing impairment at the provincial and national levels in China. Since UNHS coverage has reached 86.5%, our detection rates may be reasonable estimates of the actual incidence of newborn hearing impairment. We measured a national detection rate of 0.23% 95% CI 0.15–0.26%) in 2016. According to the data we acquired from the survey conducted in 2018, only 72.7% of neonates who were referred in the primary screening underwent secondary screening (secondary screening rate), and only 53.9% of neonates who were referred in the secondary screening were referred to medical institutions for diagnosis . Given the lack of a national, individual-level information system, we had to collect all our data from licensed medical institutions providing diagnosis, which means that we missed patients who were diagnosed at unlicensed places. Therefore, our estimated referral rate may underestimate the true rate. Nevertheless, our estimate falls within the global incidence of 0.05–0.5% among neonates and infants 5–0.26% i–33.Several limitations of the study should be noted. First, although several methods of data quality control had been carried out, the UNHS coverage was estimated based on the tabulated data rather than individual-level data, which may affect the accuracy of the data. Therefore, we compared some provincial screening rates with those of previous studies, such as the rates in Guangdong , Hubei , and HunNational UNHS coverage in China increased substantially from 2008 to 2016, which substantially contributed to the achievement of goals set by the NPACD, but geographic disparities remain. The detection rate of infant hearing impairment in China appears to be similar to that in many other countries. A national individual-level information system is needed to help integrate screening, diagnosis and treatment in China, which may also help determine a more accurate detection rate.Additional file 1. The process of newborn hearing screening specified in national technical guidelines for screening of neonatal diseases.Additional file 2. Coverage area of medical institutions that participated in the two surveys. *NHS, newborn hearing screening.Additional file 3. Detection rate of newborn hearing impairment in 2016 in China, by province and region. *The data of cases diagnosed as hearing impairment in the table could not totally represent the actual data of cases diagnosed in 2016, as the data of cases diagnosed as hearing impairment in our study were collected from licensed medical institutions providing diagnostic/treatment. a Referral rate of primary screening was estimated from the formula: number of referral in primary screening / NHS number *100. b Referral rate of secondary screening was estimated from the formula: number of referral in secondary screening / number of secondary screens *100. c Diagnostic rate of hearing impairment was estimated from the formula: number of referral in secondary screening / number of cases diagnosed with hearing impairment * 100. d Detection rate of hearing impairment was estimated from the formula:  *  * (Diagnostic rate of hearing impairment / 100) *100. e The 95% confidence interval for this detection rate was 0.15 to 0.26%, which was estimated as described in Methods. f Chongqing and Tibet were not included in the analysis.


NXNL1) gene which interrupts the metabolic and redox signaling between rods and cones. After providing scientific evidence supporting this mechanism, we propose a way to restore this lost signaling and prevent the cone vision loss in animal models of RP. We also explain how we could restore this signaling to prevent cone vision loss in animal models of the disease and how we plan to apply this therapeutic strategy by the administration of both products of NXNL1 encoding the trophic factor RdCVF and the thioredoxin enzyme RdCVFL using an adeno-associated viral vector. We describe in detail all the steps of this translational program, from the design of the drug, its production, biological validation, and analytical and preclinical qualification required for a future clinical trial that would, if successful, provide a treatment for this incurable disease.The loss of cone photoreceptor function in retinitis pigmentosa (RP) severely impacts the central and daily vision and quality of life of patients affected by this disease. The loss of cones follows the degeneration of rods, in a manner independent of the causing mutations in numerous genes associated with RP. We have explored this phenomenon and proposed that the loss of rods triggers a reduction in the expression of rod-derived cone viability factor (RdCVF) encoded by the nucleoredoxin-like 1 ( RPE65-related form of Leber congenital amaurosis (LCA), a severe blinding disease, by a recombinant adeno-associated viral (AAV) vector that corrects the genetic deficit in those patients  prepared with the same protocol as negative control [An important step in the transfer of the treatment of RP using the combination of RdCVF and RdCVFL is to provide a proof-of-concept in an animal mouse model of the disease using the pilot material of the clinical-grade AAV. We can use a mouse model of recessive RP, and the NL1 gene ,111. Thisitivity . The effy assays ,111,137.y assays . In thisby light . To avoiby light . The mosby light . It is nby light ,111. It  control ). Each m control ,169.NXNL1 gene are expressed in the retina of patients prior to the degeneration of photoreceptors, an immune response against the transgene products is not expected. Those studies are done according to regulatory guidelines [11 vg/mL were injected in the subretinal space in the tapetal region, the area centralis, of the fundus of the dog retina. By estimating the size of that bleb using the human retina and the known distance separating the optic nerve head and the fovea it is possible to calculate the volume to be injected.After the completion of that study, the clinical grade AAV is produced and used for toxicological and biodistribution studies under good laboratory practices (GLP) conditions. Since the products of the idelines ,171,172 idelines . Only laidelines ,174. Theidelines . Lateralidelines . Two hunIn order to liberate clinical batches of the drug, they must be qualified using a potency assay that demonstrates that the drug is biologically active. In a research laboratory this is performed using primary cells and animal models that cannot be used within an industrial process. At the early clinical stages, the expression of RdCVF and RdCVFL mRNA can be measured using quantitative RT-PCR and the proteins by sandwich ELISA, but both requires the transduction of an immortalized cell line, cells that can be handled in a confined environment. Since RdCVFL is under control of a cone opsin promoter, only retinoblastoma cell lines that originate from cone precursors are expressing this transgene after their transduction with the AAV expressing RdCVF and RdCVFL . At morehttps://sph.uth.edu/retnet/) may result in an enormous phenotype heterogeneity that will mask any possible benefit for vision after statistical analysis. Since we have demonstrated the positive effects of cone vision of a rodent models of recessive and dominant RP, patients carrying a recessive mutation of the PDE6B gene  and those with dominant mutations in the RHO gene  are better suited for this phase of the project [rd10 mouse in the preclinical study to which must be applied a coefficient calculated by the difference in the number of cones in the mouse and human retina: 113,000 cones in the mouse and 6,400,000 cones in the human retina [One of the main aspect of a trial for RP treatment is to recruit a population of patients with a homogenous secondary degeneration of cones ,183,184. project . The coh project , in a mon retina ,185. A ln retina ,187.The absence of adverse effects and the benefit of the treatment will be assessed by comparing the rate of loss of cone vision of the treated eyes to untreated eyes. The time necessary in the trial to see efficacy may be of several years by measuring visual acuity using kinetic perimetry but other parameters, such as an autofluorescent ring, may evolve faster ,189. ThiNXNL1 gene provides the rational for a therapy aimed at preventing or delaying the loss of central vision in all genetic forms of RP.The dominance of rods in the retina of most mammals is the result of an evolutionary constraint known as nocturnal bottleneck . The reaThis section is not mandatory, but may be added if there are patents resulting from the work reported in this manuscript.


CLRN1), leading to combined progressive hearing loss and retinal degeneration. The cellular distribution of CLRN1 in the retina remains uncertain, either because its expression levels are low or because its epitopes are masked. Indeed, in the adult mouse retina, Clrn1 mRNA is developmentally downregulated, detectable only by RT‐PCR. In this study we used the highly sensitive RNAscope in situ hybridization assay and single‐cell RNA‐sequencing techniques to investigate the distribution of Clrn1 and CLRN1 in mouse and human retina, respectively. We found that Clrn1 transcripts in mouse tissue are localized to the inner retina during postnatal development and in adult stages. The pattern of Clrn1 mRNA cellular expression is similar in both mouse and human adult retina, with CLRN1 transcripts being localized in Müller glia, and not photoreceptors. We generated a novel knock‐in mouse with a hemagglutinin (HA) epitope‐tagged CLRN1 and showed that CLRN1 is expressed continuously at the protein level in the retina. Following enzymatic deglycosylation and immunoblotting analysis, we detected a single CLRN1‐specific protein band in homogenates of mouse and human retina, consistent in size with the main CLRN1 isoform. Taken together, our results implicate Müller glia in USH3 pathology, placing this cell type to the center of future mechanistic and therapeutic studies to prevent vision loss in this disease. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Usher syndrome type 3 (USH3) is an autosomal recessively inherited disorder caused by mutations in the gene clarin‐1 ( CLRN1), whose main isoform encodes a 232‐amino acid protein predicted to have four transmembrane domains, and a single glycosylation site in the first extracellular loop In vitro biochemical assays suggest that CLRN1 functions as a molecular scaffold, recruiting proteins involved in cell adhesion at distinct plasma membrane regions and playing a role in organizing the actin cytoskeleton Clrn1 knockout (KO) and N48K knock‐in mice display poorly developed and disorganized F‐actin‐rich stereocilia at a young age, and are profoundly deaf by postnatal day 21 (P21) Usher syndrome (USH) represents a group of autosomal recessive disorders, characterized by combined vision and hearing loss, and in some cases vestibular dysfunction Clrn1 mRNA in the mouse retina was shown to have the highest expression in the early postnatal retina, and was detected exclusively in the inner nuclear layer (INL) by in situ hybridization Clrn1 mRNA was detectable only by RT‐PCR and remained confined to the inner retina. During postnatal development, Clrn1 transcripts in the INL were found to co‐localize with Müller cell‐specific markers, suggesting that in the retina, Clrn1 was expressed primarily in Müller glia cells. Another group reported that CLRN1 protein was expressed in mouse photoreceptors, in synaptic and connecting cilium regions in situ, leading some investigators to suggest that its levels are very low, or its epitopes are masked The function of CLRN1 in the retina is currently unknown, primarily due to the lack of appropriate USH3 animal models and a major gap in our knowledge regarding its cellular localization. Three previous studies focusing on localizing CLRN1 in the retina have yielded conflicting results Clrn1 mRNA is developmentally downregulated in mouse retina and the documented repeated failed attempts to reliably detect retinal CLRN1 protein, raises a number of fundamental questions. Is the cellular pattern of Clrn1 mRNA expression in the mouse retina different from human? Is CLRN1 protein in mouse retina present only transiently during development, or does it exhibit continuous expression into adulthood? Do human and mouse retinas express distinct CLRN1 isoforms at the protein level? The answers to these questions are critical for designing adeno‐associated virus (AAV)‐based treatment strategies for therapeutic interventions to prevent vision loss in USH3 patients and may also provide clues for understanding the differences in the ocular phenotype between mouse models and human USH3. Therefore, in the current study we examined the pattern of CLRN1 expression in mouse and human retina by using a combination of novel tools, including the highly sensitive RNAscope ISH technique for visualizing Clrn1 transcripts in tissue sections and single‐cell RNA sequencing (scRNA‐seq) analysis to identify the specific cell types in which Clrn1 mRNA is expressed. Interestingly, our findings reveal that Clrn1 transcripts in both mouse and human adult retinas are concentrated in the INL, being specifically expressed in Müller glia, and not in photoreceptor cells. In addition, to overcome the existing challenges in reliably detecting CLRN1 protein expression, we generated and characterized a novel knock‐in mouse with an N‐terminal hemagglutinin (HA) epitope‐tagged endogenous CLRN1. We found that although Clrn1 mRNA is developmentally downregulated, CLRN1 protein is expressed continuously during adulthood in the mouse. Following enzymatic deglycosylation, a single CLRN1 protein product was detected by immunoblotting in both mouse and human retinal extracts, consistent in size with the main CLRN1 isoform. This study shifts our understanding of the pathological mechanisms in USH3 in a novel direction, one that requires the study of glia–neuron interactions in the human retina, instead of focusing exclusively on photoreceptor neurons. Our results provide important fundamental knowledge for developing therapeutic approaches to prevent vision loss in USH3, and for future studies aimed at uncovering the mechanisms by which CLRN1 mutations lead to photoreceptor degeneration.The phenotypic discrepancy between the mouse and human USH3 retinal phenotype, combined with the knowledge that http://www.arvo.org/About/policies/statement-for-the-use-of-animals-in-ophthalmic-and-vision-research/). Wild‐type C57BL/6J mice served as controls and were purchased from the Jackson Laboratory . The N‐HA‐tagged Clrn1 knock‐in mouse contains a 30‐nucleotide sequence (TAC CCA TAC GAT GTT CCA GAT TAC GCT GAG) at the 5′‐end of Clrn1 (NM_153385.3), immediately after the ATG start codon. This mouse was generated at the Jackson Laboratory by using the CRISPR/Cas9 technology in the C57BL/6J strain as described in supplementary material, Supplementary materials and methods.All animal procedures, maintenance, and handling were approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research (In situ hybridization (ISH) was performed on formalin‐fixed, paraffin embedded sections as directed by the manufacturer . We used RNAscope LS 2.5 Probe – Mm‐Clrn1  to detect mouse Clrn1 and RNAscope® 2.5 LS Probe – Hs‐CLRN1  to detect human CLRN1. The Clrn1 mouse probe was designed to span exons 1, 3, and 4 of the main Clrn1 isoform, and included the region comprising nucleotides 106–1146 of mouse Clrn1 (NM_153385.2). The human probe included the region 325–2352 of human CLRN1 (NM_174878.2). Both probes were designed to detect all validated mRNA isoforms. Positive control sections detected reference transcripts encoding RNA polymerase II subunit A . Negative control sections were probed for bacterial dihydrodipicolinate reductase mRNA (dapB). Detailed information on the ISH procedure can be found in supplementary material, Supplementary materials and methods.Clrn1/CLRN1 transcripts were analyzed in mouse, non‐human primate (NHP), and human retinas. The data for NHP were a reanalysis from a previously reported database CLRN1 gene expression data in known cell types Details on processing human and mouse retina samples are described in supplementary material, Supplementary materials and methods. A rabbit monoclonal anti‐HA antibody  was used to detect the N‐HA‐tagged CLRN1 protein; an anti‐CLRN1 rabbit polyclonal antibody  was used to detect human CLRN1; and mouse monoclonal anti‐alpha tubulin  served as a loading control antibody.Different tissue preparation methods and antibodies were used for CLRN1‐HA detection, as described in supplementary material, Supplementary materials and methods.post hoc comparisons or by a two‐sided Student's t‐test . Graphs show mean ± SEM; P values less than 0.05 were considered statistically significant.GraphPad Prism version 8  was used for statistical analysis. Differences between groups were analyzed either by one‐way analysis of variance (ANOVA) with Tukey's Clrn1 transcripts in the intact retinal tissue, we used RNAscope chromogenic ISH on sections of mouse and human retinas Clrn1 mRNA expression in the mouse retina during postnatal development and adult stages, either under standard bright‐field or by fluorescence microscopy  used as a negative control produced no signal  used as a positive control generated an abundant signal in all the layers of the retina, as expected  and outer limiting membrane (OLM) that may result from Müller cell processes extending into those regions We examined a Figure . Transcra Figure . In addiClrn1 mRNA, we used single‐cell RNA‐seq (scRNA‐seq) transcriptomic analysis on retinas from mice, macaque NHP, and humans. In order to identify and cluster retinal cell types, we used the t‐distributed stochastic neighbor embedding (t‐SNE) principal component method in Seurat to visualize cells that are plotted in clusters based on highly variant gene expression Clrn1. In all three species, Clrn1‐expressing cells co‐localized with Müller cells , encoding a 232‐amino acid protein. By using the F1 primer against exon 1, we amplified two bands, which sequencing identified as the main isoform 2 and the shorter isoform 3, containing exons 1 and 4, and encoding a 172‐amino acid CLRN1 protein. This result is consistent with previous studies, showing that these two transcripts are primarily amplified from retinal cDNA using primers that span all four Clrn1 exons To overcome challenges in detecting CLRN1 protein in the retina, we generated and characterized an epitope‐tagged Clrn1 KO and N48K knock‐in mice are deaf by P21 but display normal retinal function and morphology throughout their lifetime when raised on a C57BL/6J background. Therefore, any potential detrimental effects of the HA tag on CLRN1 protein function will most likely impact the hearing and hair cell morphology at early stages during development. Consequently, we first tested if the presence of the HA tag results in the loss of hearing function in homozygous HA‐tagged Clrn1 knock‐in mice. We performed acoustic brainstem response (ABR) testing of broadband clicks stimuli on N‐HA Clrn1 knock‐in mice at P30 compared with age‐matched C57BL/6J controls. We did not observe any significant differences in hearing between the two groups  and outer hair cells (OHCs), with no IHC or OHC loss at any locations along the cochlear turns . We did not observe any significant changes in structure of the retina at 2, 4 or 12 months of age , a protein essential for the integrity of adherens junctions and Müller glia–photoreceptor interactions, are associated with a wide spectrum of retinal dystrophies, including Leber congenital amaurosis (LCA) and early‐onset recessive retinitis pigmentosa (RP) CLRN1 mRNA expression in different species, and provide a firm answer to the essential question regarding the cellular source of CLRN1 in the adult human retina. These results are relevant for future studies addressing the biological significance of CLRN1 protein expression in Müller glia and its impact on photoreceptor cells, with the ultimate goal of developing safe therapeutic strategies to prevent vision loss in the USH syndrome.In summary, by combining the scRNA‐seq data analysis with a highly sensitive ISH assay, we were able to directly examine the pattern of AD and LX wrote the manuscript, and conceived and designed the study. LX, SNB, CPS, FD, OA and YW carried out experiments, generated figures, and analyzed data. ESL, KAM, WWH, WCS, JTH, SB, JDA and AD interpreted data and revised the manuscript. All the authors agreed with the submission in its final form.Supplementary materials and methodsClick here for additional data file.Figure S1. Detection of Clrn1 mRNA in the mouse retinas during postnatal development and throughout adulthoodFigure S2.Clrn1 mRNA localization in albino A/J mice (P40)Figure S3. Dual RNAscope Clrn1 ISH and glutamine synthetase (GS) immunohistochemistry in mouse retina (P8)Figure S4. Detection of CLRN1 transcripts in the human retinaFigure S5. Single‐cell RNAseq analysis of Clrn1 expression in mice, NHP, and human retinaFigure S6. Characterization of the N‐terminal HA‐epitope‐tagged Clrn1 knock‐in mouse modelFigure S7. Detection of the endogenous HA‐tagged CLRN1 protein in the HA‐tagged Clrn1 knock‐in mice during postnatal development and adulthoodFigure S8. Immunofluorescence analysis of N‐HA‐Clrn1 knock‐in retinasFigure S9. Detection of recombinant HA‐tagged CLRN1 protein in HEK293 cells and mouse retina by immunohistochemistryClick here for additional data file.


Age related hearing loss (presbycusis) is a natural process represented by elevated auditory thresholds and decreased speech intelligibility, especially in noisy conditions. Tinnitus is a phantom sound that also potentially leads to cortical changes, with its highest occurrence coinciding with the clinical onset of presbycusis. The aim of our project was to identify age, hearing loss and tinnitus related structural changes, within the auditory system and associated structures. Groups of subjects with presbycusis and tinnitus (22 subjects), with only presbycusis (24 subjects), young tinnitus patients with normal hearing (10 subjects) and young controls (17 subjects), underwent an audiological examination to characterize hearing loss and tinnitus. In addition, MRI  scans were used to identify changes in the cortical and subcortical structures. The following areas of the brain were analyzed: Heschl gyrus (HG), planum temporale (PT), primary visual cortex (V1), gyrus parahippocampus (PH), anterior insula (Ins), amygdala (Amg), and hippocampus (HP). A statistical analysis was performed in R framework using linear mixed-effects models with explanatory variables: age, tinnitus, laterality and hearing. In all of the cortical structures, the gray matter thickness decreased significantly with aging without having an effect on laterality (differences between the left and right hemispheres). The decrease in the gray matter thickness was faster in the HG, PT and Ins in comparison with the PH and V1. Aging did not influence the surface of the cortical areas, however there were differences between the surface size of the reported regions in the left and right hemispheres. Hearing loss caused only a borderline decrease of the cortical surface in the HG. Tinnitus was accompanied by a borderline decrease of the Ins surface and led to an increase in the volume of Amy and HP. In summary, aging is accompanied by a decrease in the cortical gray matter thickness; hearing loss only has a limited effect on the structure of the investigated cortical areas and tinnitus causes structural changes which are predominantly within the limbic system and insula, with the structure of the auditory system only being minimally affected. Hearing deterioration can begin as early as the age of 30, however it starts to manifest itself as high frequency hearing loss and worsened speech perception at around the age of 60  related to aging, presence of age-related sensorineural hearing loss and its degree (presbycusis), presence of tinnitus and its specific characteristics  within different cortical regions; the Freesurfer analytic tool was used for cortical reconstruction. MR morphometry enables the detection of changes in the cortical surface, thickness, and overall volume.Seventy-three participants  were examined in this study. Based on their age, hearing thresholds and presence of tinnitus, six different groups were formed: young controls (Y) with physiological hearing thresholds  and no tinnitus (NT) ; a group of aged participants (O) with normal hearing for their age group (mild degree of presbycusis) represented by a physiological age-related decline of the auditory thresholds and no tinnitus ; a group of aged participants with hearing loss  having significant elevation of the auditory thresholds compared to O-NH-NT and no tinnitus ; a young group with normal hearing, an analog to Y-NH-NT group but with present tinnitus ; an analog group to O-NH-NT but with tinnitus ; and analog group to O-HL-NT but with tinnitus . The age, hearing threshold, and presence of tinnitus specificity of each group enabled differentiation between the possible independent effects of each factor.Audiograms of all participants showed only minimal asymmetry (up to 2 dB) and therefore data from both ears were pooled together. The audiograms were then compared with average audiograms for the specific age group according to our previously published data , Y-NH-T , O-NH-NT , O-NH-T , O-HL-NT , O-HL-T .All of the examined participants declined any previous otologic surgery: vestibular lesion, chronic exposure to loud noise, severe head trauma, lesion of the facial nerve, disorder of the cervical spine or had self-reported central nervous system disorder. None of the participants were musical professionals, but several in the elderly group played musical instruments sporadically (not more than once a month). An otoscopic examination, with removal of the cerumen and confirmation of an intact tympanic membrane, was performed on all of the participants. The examination procedures were approved by the Ethics Committee of the University Hospital Motol, in Prague. All participants signed written informed consent.The presence of tinnitus was self-reported by participants. To establish the degree of tinnitus discomfort, participants completed the Tinnitus Handicap Inventory (THI) questionnaire  and tinnitus laterality . The duration of tinnitus ranged from 1.5 to 8 years, with a median of 3 years. Fourteen participants suffered from unilateral tinnitus and seventeen participants suffered from bilateral/intracranial tinnitus.Pure tone audiometry was measured over an extended frequency range from 125 Hz to 16 kHz . Hearing thresholds were measured separately for each ear with a resolution of 2 dB.All acoustic stimuli were delivered separately to each ear. An acoustic signal was delivered via Sennheiser HDA 200 high-frequency audiometric headphones connected to a custom-made audiometric apparatus, based on a high-quality audio device (RME Fireface), complemented by a custom-made programmable attenuator. The apparatus provided a digital-to-analog conversion and attenuation/amplification of the measurement signals, communication between the experimenter and the examined subject, and an acquisition of the subject's responses using a comfortable interface with backlit buttons (Arturia BeatStep). The apparatus was controlled by a custom-made software package built in the Matlab environment, which provided all the necessary functions including the generation and/or playback of digital measurement signals, acquisition of the subjects' responses, and basic data evaluation. The equipment was calibrated according to ISO 389-5, ISO 389-8, ISO 8253-3, and IEC 60645-3 standards using the Brüel & Kjær 4153 Artificial Ear.3, phase oversampling 40%, without slice oversampling, bandwidth 290 Hz/pixel, echo spacing 6.3 ms, without parallel imaging, prescan normalize, eliptical filter, acquisition time 9:26. Additional T2 3D structural image using 3D turbo-spin echo with variable flip angle (SPACE) was acquired with the parameters: TR/TE = 3,200/453 ms, 1.7 averages, turbo factor 167, slice turbo factor 2, slice partial Fourier 7/8, matrix 256 × 256 × 208, voxel size 0.86 × 0.86 × 0.86 mm3, phase oversampling 20%, without slice oversampling, bandwidth 751 Hz/pixel, echo spacing 3.5 ms, with parallel imaging iPAT GRAPPA, acceleration factor 2, reference lines 24, weak raw filter, acquisition time 8:18 was measured. The T2 image was used for bias field correction and refinement of the pial surface position in subsequent HCP pipelines/FreeSurfer data processing. Both sequences were acquired in a sagittal orientation.All MR measurements were performed using a Siemens Trio 3T MR system and a 12-channel head coil. T1 3D structural image was acquired by using the magnetization prepared rapid acquisition gradient echo (MPRAGE) sequence with the parameters (TI—inversion time) TI/TR/TE = 900/2,100/2.63 ms, flip angle 10°, 1 average, phase partial Fourier 6/8, matrix 256 × 256 × 256, voxel size 0.86 × 0.86 × 0.86 mmStructural images were preprocessed by “PreFreeSurferPipeline.sh” script as part of HCP pipelines framework , the atlas provided by the Martinos Center for Biomedical Imaging and the Institute of Neurosciences and Biophysics was used . Although there is also a subdivision to substructures available, for simplicity we only used volumes of the whole structures in our study. To increase robustness of the results, we used both T1 (using only T1 images) and T1T2 (using jointly T1 and T2 images) modes of segmentation.Estimated intracranial volume (eTIV), which was used as a covariate in the analyses of area and volume, was derived from the determinant of the transform matrix used to align structural image with an atlas , age related hearing loss (presbycusis) presence/absence (PRESB), ROI laterality (LAT), age, sex, eTIV, anatomical parcel, segmentation method  were modeled as fixed effects. The inter-individual variability  was modeled as a random effect. Several analyses were conducted with differing involvement of the above listed fixed effects:M1: LAT + REGION + AGE + sex and interactions LAT:REGION + LAT:AGE + REGION:sex + REGION:AGE + LAT:REGION:AGE to compare degree of aging between selected regions and influence of laterality in all groups. The aim of M1 is to identify the effect of aging, its gradient (change over time), laterality (left vs. right hemisphere) and its interactions on the studied regions. The dependent variable is cortical thickness.M2: for each region a separate model was fitted with effects:TIN + PRESB + LAT + AGE + sex (+ eTIV) to test the effect of aging, age related hearing loss, tinnitus and laterality in all groups. The aim of M2 is to identify effects of aging, hearing loss, tinnitus and laterality (difference between the specific region in the left and right hemisphere) on the thickness and surface area (both were modeled as dependent variables) of each region.M3: for each region a separate model was fitted with effects:TIN + PRESB + LAT + AGE + sex (+ eTIV). To address the observed robust effect of aging on data from all subjects that could potentially cover non-age related changes, only data from groups O-NH-NT, O-NH-T, O-HL-NT, and O-HL-T were used for this analysis to test the effect of tinnitus and hearing. The aim of the M3 is to decrease the robust effect of aging and potentially identify effects of hearing loss and tinnitus that could be covered by impact of aging. Therefore, only aged groups were included in this model.M4: for each region a separate model was fitted with effects:TIN_LATERALITY + LAT + AGE + sex (+ eTIV) and interaction TIN_LATERALITY: LAT to test the effect of tinnitus laterality in tinnitus subjects from O-NH-T and O-HL-T groups. The aim of M4 is to decrease the robust effect of aging and potentially identify the effect of tinnitus laterality . Therefore, only aged tinnitus groups were included in this model (O-NH-T and O-HL-T).M5: volume of hippocampus and amygdala was fitted with effects:TIN + PRESB + LAT + AGE + sex + eTIV + method to test the effect of age, laterality, tinnitus, and age related hearing loss on hippocampal/amygdalar volume in all groups.M6: in regions with a significant effect of tinnitus, the effect of tinnitus duration and severity (THI index) on a dependent variable was tested: area of planum temporale with effects TIN_LENGTH or THI + LAT + AGE + sex + eTIV in O-NH-T and O-HL-T groups. Hippocampal and amygdalar volumes were computed using T1 method with effects TIN_LENGTH or THI + LAT + AGE + sex + eTIV in Y-NH-T, O-NH-T, and O-HL-T groups.In models with the area and volume estimated, eTIV was added as a nuisance covariate due to its effect on brain surface area. Also, sex was included as a nuisance covariate in accord with the common practice in most morphometric studies.p-value of Shapiro-Wilk test of the residuals of a particular model. A common practice in statistical modeling is to follow the principle of parsimony and select the minimal model, i.e., retain only the terms which contribute significantly to the explanation of the variance in the data. The model simplification by using a combination of the Akaike information criterion and elimination of the non-significant terms, was therefore performed. For clarity, the structure of the full and simplified models is listed in the The statistical modeling was performed as follows: First, the Box-Cox transformation of the dependent variable to optimize normality of the model residuals was done. The search for an optimal lambda for transformation was done by iterative evaluation of the p = 0.05 for each test controls the probability of false detection of each effect under the validity of the null hypothesis at 5%. However, given the multitude of hypotheses as well as the multitude of related tests for each of the hypotheses, the overall probability of spurious detection of at least one effect using the uncorrected significance threshold in the case of the validity of all null hypotheses (no effects) is far higher than 5%. Therefore, the p-values corrected for multiple comparisons by Holm method  the thresholds were slightly elevated compared with both young groups, specifically at frequencies above 4 kHz, where the audiograms exceeded 20 dB HL that is considered clinically physiologic. However, these thresholds are considered normal for the age group >60 years of age, according to Jilek et al. . Both grBased on our previous results on structural changes of the gray matter in patients with a different degree of presbycusis (mild vs. expressed), that showed only a minimal effect of hearing loss on thickness of the auditory cortex and examined auditory related regions . No significant difference was found in the decline of the thickness between the left and right hemispheres, suggesting that the cortical gray matter thinning is symmetrical in all regions and aging had no effect on the laterality .The morphometric analysis of the cortical thickness within all selected regions pooled into one statistical model (M1), showed the interaction between the age and region . Post-hop < 0.0001, corrected p < 0.0001, PT p < 0.0001, corrected p < 0.0001, V1 p < 0.0001, corrected p = 0.0002, Ins p < 0.0001, corrected p < 0.0001, PH p = 0.002, corrected p = 0.05).The effects of aging, tinnitus, hearing and laterality on the cortical thickness and surface were also evaluated for each region separately (M2). The effect of age on the cortical thickness was significant in all of the regions  with a greater thickness in the right hemisphere.The effect of laterality on cortical thickness was present at borderline level only in the PH . The effect of laterality was present in all of the examined regions . Hearing loss (in our case presence of expressed presbycusis) showed only minimal effect on surface of HG . We observed a borderline decrease of the insular surface due to the tinnitus   . The surp = 0.002, corrected p = 0.04), PT , V1 , PH , Ins . The significant effect of aging on cortical surface was present in the PT  and borderline effect in PH .Due to the strong effect of aging on our data, only data from the age coherent groups  were used to detect the possible effects of hearing loss and tinnitus (M3). In the age coherent groups, the effect of aging on cortical thickness was significant in the HG   (The hearing loss (comparison of the effect of mild vs. expressed presbycusis) had no significant effect in the examined areas with a borderline effect in the case of HG where the surface was found to decrease (p = 0.1) . Tinnitup = 0.1) .The effect of tinnitus laterality (M4) was also considered and unilateral vs. bilateral (capitis) tinnitus was examined (in groups O-NH-NT and O-HL-T). Laterality of tinnitus had no effect on the cortical thickness or surface in any of the examined regions.p = 0.009, corrected p = 0.1 . Laterality showed significantly greater volumes in the right amygdala  as well as the right hippocampus .Hearing loss had no significant influence on the volume of either the amygdala or hippocampus (M5). However, tinnitus caused a borderline effect of the volume increase in both regions . The duStatistical significance of results is summarized in Age-related hearing loss and tinnitus are the two most common disorders affecting the auditory system. In principle both pathologies can also potentially influence non-auditory brain structures, especially if they appear at the same time. In our project we have identified several age, hearing loss and tinnitus related pathologies that alter the cortical gray matter in various regions. Age-related cortical atrophy was present in all of the examined regions, however the extent of it differed. Hearing loss and tinnitus were studied among age matched groups while controlled for the effect of age. The effect of hearing loss (degree of presbycusis) showed only a borderline effect on the surface of the AC structures, whereas the main effect of tinnitus led to an increase of the volume of structures of the limbic system . The effect of aging was not significantly region-specific and did not alter the left-right hemisphere relationship.The input criteria for the selection of volunteers were based on the goal to assemble age balanced groups: with normal hearing with and without tinnitus, with a physiologic age related decline with and without tinnitus, and elderly groups with expressed presbycusis (elevation of hearing thresholds exceeding physiologic aging) with and without tinnitus. The hearing thresholds criteria to distinguish between the mild and expressed presbycusis were based on our previous studies  and presence/absence of tinnitus, is what distinguishes this study from previous ones.The most dominant finding in our study was the effect of aging on the cortical gray matter. Aging essentially, leads to multiple changes of gray matter and its effect differs among the cortical regions. Cortical atrophy caused by gray matter loss is linear in relation to aging  is very common in the brain regarding functional specificities, as well as the difference in volume. A typical example is the AC, where the left hemisphere is focused on the temporal parameters of sound and speech processing, whereas the right hemisphere is focused on the processing of spectral parameters and music  on the gray and white matter of the auditory cortex and pathway  related morphometric changes among our volunteers, such changes were reported by several previous studies specifically in the insula and bilaterally in superior and middle temporal cortices .We are aware of the potential limitations of our study. Firstly, coherency of the subject subgroups is important. Although we have initially established a strict criteria and patients/volunteers were strictly selected, it is possible to be even more precise. However, the strictness of criteria dramatically decreases the number of subjects involved and therefore the statistical significance of data. Furthermore, even in cases when tinnitus is accompanied by physiological audiometric results, more detailed examination shows the presence of hearing pathology, therefore it is illusive to recruit subjects with tinnitus and normal hearing. Another factor is the limitation of the MRI examination and its precision. In contrast to human MRI studies, a histopathologic post-mortem examination of animal brain tissue shows morphometric changes due to hearing loss, and also the presence of tinnitus Syka, . CurrentOur study revealed age related structural changes within the several cortical regions involved in the auditory processing and tinnitus network. Regarding the effect of tinnitus, our results show a trend specifically within the limbic structures. The structure of the auditory system is dominantly affected by aging and additionally we were also able to identify borderline hearing loss related changes.A lack of significant changes caused by tinnitus and hearing loss in the auditory cortex can be attributed to several factors, such as the limitations of MRI resolution, signal to noise ratio, strictness of the inclusion criteria, number of participants. The strong prevalence of age effect shows that the coherence of subject subgroups is essential for the credible detection of tinnitus and hearing loss related changes in potential future studies.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the University Hospital Motol, in Prague. The patients/participants provided their written informed consent to participate in this study.OP: manuscript preparation, examination, auditory examination, statistical analysis, and work load cca 25%. AŠ: MR examination, statistical analysis, manuscript preparation, and work load cca 20%. JT: MR examination, statistical analysis, and work load cca 15%. VS: auditory examination, database administration, and work load cca 13%. DK: auditory examination, database administration, and work load cca 12%. JSB: auditory examination, database administration, and work load cca 5%. JS: manuscript preparation, project overview, and work load 10%. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In human study, we used a Cox regression hazard model to indicate that a significantly lower percentage individuals developed SNHL compared with and without DXM use . After adjustment for age and other variables [adjusted hazard ratio: 0.725 ], this study also demonstrated that DXM use appeared to reduce the risk of developing SNHL. This animal study demonstrated that DXM significantly attenuated noise-induced hearing loss. In human study, DXM use may have a protective effect against SNHL.The effect of dextromethorphan (DXM) use in sensorineural hearing loss (SNHL) has not been fully examined. We conducted an animal model and nationwide retrospective matched-cohort study to explore the association between DXM use and SNHL. Eight-week-old CBA/CaJ hearing loss was induced by a white noise 118 dB sound pressure level for 3 h. DXM (30 mg/kg) was administered intraperitoneally for 5 days and boost once round window DXM socking. In population-based study, we examined the medical records over 40 years old in Taiwan’s National Health Insurance Research Database between 2000 and 2015 to establish retrospective matched-cohort to explore the correlation between DXM use and SNHL. Using click auditory brainstem response (ABR), hearing threshold was measured as 48.6 ± 2.9 dB in control mice compared with 42.6 ± 7.0 dB in DXM mice, which differed significantly ( Hearing loss is a growing and alarmingly high burden in the world reporting from the Global Burden of Disease Studies and the third leading cause of years lived with disability ,2. SensoN-methyl-d-aspartate (NMDA) receptor activation and related auditory nerve excitation -ADAM/micro-PET and showed DXM prevent hearing loss and preserved brain serotonin transporter function [Given that NMDA receptor antagonists tend to have negative central nervous system side effects, such as hallucinations or anesthesia, their clinical potential has been limited . In the function . This effunction .Based on aforementioned results, DXM has the potential to be a promising therapeutic intervention, we directly explored the association between DXM use and the risk of SNHL using Taiwan’s NHIRD database. After adjusting variables by multiple Cox regression analysis, the use of DXM significantly reduced the risk of SNHL compared with matched-cohort control group. DXM use is frequently prescribed for patients when they might catch cold in Taiwan. We used cumulative DDD (cDDD) of DXM to analyze the association between DXM use and risk of SNHL. The result revealed longer duration of DXM use (larger amount of cDDD) had more significantly decreased the risk of SNHL. Herein, we further provided the evidence to support the finding that the use of DXM has a strong impact in preventing SNHL.The mechanism for the preventive effect of DXM from hearing loss may be through the inhibition of glutamate-induced excitotoxicity at the highly active synapse due to NMDA receptor activation, dendritic swelling, and the production of reactive oxygen species ,29. GlutIn recent years, a hidden hearing loss theory has advocated that synaptic loss is the primary pathology even with only temporary threshold shifts, and that this synaptic loss is independent from both IHC and SGN loss ,30. CochOur study had some limitations. In animals, it was not clear which approach  resulted in the therapeutic effect because we combined both approaches and we did not assess the immunohistochemistry staining for synaptic ribbon. In humans, we were unable to reduce non DXM contributing factors in the retrospective-matched cohort study. This is because: (1) we cannot assess a patient’s history and cause of SNHL according to the diagnosis by using the ICD-9-CM code (389.1x), (2) the severity of hearing threshold of individuals as determined by audiometry was not available in the current NHIRD database, and (3) the timing of DXM application was inaccessible as it was not prescribed for treatment of SNHL. All of these factors may have contributed to the otoprotective effect of DXM in this study. Future clinical trials would have to sufficiently reduce non DXM contributing factors to confirm our findings.Our study demonstrated that DXM significantly attenuated noise-induced hearing loss and may have a protective effect against SNHL. The effect may be through synaptic regulation. Therefore, to develop a novel effect of pharmaceutical medicine to prevent cochlear synaptopathy is practical to the hearing preservation program. DXM can be an alternative medication applying in future clinical trials for SNHL.


The increase of storage modulus (G’) of MGC-g-CD-ic-TCS after visible light irradiation for 200 s indicated its hydrogelation. The swollen hydrogel in aqueous solution resulted in two release behaviors of an initial burst and sustained release. Importantly, in vitro and in vivo results indicated that MGC-g-CD-ic-TCS inhibited bacterial infection and improved wound healing, suggesting its high potential application as an antibacterial tissue bio-adhesive.Accelerating wound healing with minimized bacterial infection has become a topic of interest in the development of the new generation of tissue bio-adhesives. In this study, we fabricated a hydrogel system (MGC-g-CD-ic-TCS) consisting of triclosan (TCS)-complexed beta-cyclodextrin (β-CD)-conjugated methacrylated glycol chitosan (MGC) as an antibacterial tissue adhesive. Proton nuclear magnetic resonance ( Self-healing tissue adhesives have been regarded as attractive sealants for wound closure in surgery and trauma handling because they perform several important functions including promoting wound healing acceleration by preferably binding to tissues, as well as sealing leaks and stopping tissue adhesion . HoweverHydrogels are promising candidates as self-healing tissue adhesives, since damaged wounds can be regenerated by their hydrophilic and crosslinked structures, such as soft tissues, and high permeability to oxygen, nutrients, and water-soluble metabolites . In addiAmong the basic materials for hydrogels, water-soluble chitosan derivatives are coming into the spotlight as self-healing tissue adhesives ,9. These1H NMR) and differential scanning calorimetry (DSC) analyses. The MGC bio-adhesive was prepared using a blue visible light irradiation with riboflavin as a photoinitiator, which were characterized using rheology, in vitro drug release, in vitro cytotoxicity, and antibacterial activity assays. The tissue adhesion properties of MGC-g-CD-ic-TCS were compared with those of commercially used adhesives such as fibrin glue and cyanoacrylate. In addition, the self-healing capacity of MGC-g-CD-ic-TCS was compared with that of untreated incision, suture, cyanoacrylate, and MGC hydrogel.In this work, we prepared a self-healing hydrogel tissue bio-adhesive (MGC-g-CD-ic-TCS) with antibacterial activity based on visible light-cured methacrylated glycol chitosan (MGC) and succinyl-beta-cyclodextrin/triclosan (β-CD-ic-TCS) for wound closure. The efficacy was evaluated using a rat model of skin incision . A metha1H NMR analysis in D2O  of the methyl group was calculated in consideration of the integration ratio between the glucopyranosyl ring peak area of GC at 3.23–4.21 ppm and the -CH3 peak area of the methacrylic group at 1.89 ppm, resulting in a methacrylate of 70%. MGC-g-CD produced new peaks at 2.42 ppm and 2.59 ppm, and 5.00 ppm, which were assigned for -CH2CH2- of the succinyl group and H-1 of the glucopyranosyl group, respectively [2- peak area of succinyl group at 2.34 ppm was 24%. Inclusion complex formation between MGC-g-CD and TCS made the appearance of new peaks possible, because 1H NMR analysis of poorly water-soluble TCS is impossible in D2O. The peaks of complexed TCS were observed at 6.45 ppm, 6.64 ppm, 6.88 ppm, 6.96 ppm, 7.30 ppm, and 7.56 ppm. In comparison with the -CH2CH2- peak of the succinyl group, the inclusion complex percentage of TCS was almost 100%.Conjugation of succinyl-β-CD to the MGC backbone and the inclusion complex between MGC-g-CD and TCS were confirmed by s in D2O . We prevsing D2O ,12,13,14ectively . In addiTo further investigate the inclusion complex formation between MGC-g-CD and TCS, DSC was employed and compared with the results of MGC-g-CD and TCS A. TCS poThe storage modulus of MGC-g-CD-ic-TCS hydrogel precursor solution before and after visible light irradiation for 120 s was monitored from 0 Hz to 10 Hz at 37 °C B. VisiblThe cell proliferation test of TCS, MGC and MGC-g-CD-ic-TCS was performed at 37 °C for 7 days using a cell counting kit-8 (CCK-8) assay . A graduEscherichia coli and Staphylococcus aureus  for evaluating the tissue adhesive strength and the result was compared to those of fibrin glue, cyanoacrylate, and MGC, as shown in For improving the bioavailability of TCS in wound healing, its water solubility and release behavior must be advanced. β-CD-grafted MGC-based hydrogel can meet the characteristics by inclusion of complex formation between the ring molecule and the drug as shown in To further investigate the histological changes in the incisions of all groups, hematoxylin and eosin (H&E) and Masson’s trichrome (MT) staining were performed at days 7 and 14, respectively . From thCollagen deposition in the incisions was evaluated by MT staining B. Compar2, respectively, and the numbers of mannose receptor-positive cells were 280, 550, 260, 500, and 600 cells/mm2, respectively. At day 14, the M1/M2-related cells increased in the five groups, which exhibited 28, 18, 32, 20, and 19 cells/mm2 in the case of iNOS-positive cells, and 310, 650, 290, 750, and 820 cells/mm2 in the case of mannose receptor-positive cells. To further investigate the effect of M1 and M2 macrophages on wound healing, M1, M2, and M1/M2 ratio were calculated  was used. MGC-g-CD was purified using a dialysis membrane tube . Mouse fibroblast L-929 cell line was supplied by the Korean Cell Line Bank . Chemicals were used as received.GC  and glycidyl methacrylate (GM) were purchased from Sigma-Aldrich  for MGC preparation. β-CD-COOH  was used for MGC-g-CD preparation. Riboflavin 5′-monophosphate sodium salt  was used as a photoinitiator. 4--4-methyl-morphoninium chloride (DMT-MM) as a condensing agent was supplied by Wako Pure Chemical Industries . TCS obtained from Sigma-Aldrich  was used as an antibacterial agent. As a cyanoacrylate adhesive, Liquiband® was covered to the entrance. The mixture was continuously stirred until acetone was perfectly evaporated and precipitate was eliminated by centrifugation at 1200 rpm. After lyophilization of the supernatant, MGC-g-CD-ic-TCS was characterized by analyses of 1H NMR, DSC, and storage/loss moduli. The 1H NMR analysis was performed using D2O. The DSC  was employed for analyzing the thermal behavior of MGC-g-CD-ic-TCS, which was monitored from 50 °C to 170 °C at 10 °C/min by heating the hydrogel (50 mg) onto an aluminum pan. The storage/loss moduli of MGC-g-CD-ic-TCS were measured using an AR 2000ex rheometer  at 37 °C from 0 Hz to 10 Hz.MGC was prepared according to procedures from our previous studies ,12,13,142, light-emitting diode curing light, Foshan Keyuan Medical Equipment Co., Ltd., Foshan, China). The behavior was calculated by the ratio of swollen weight to initial weight at each time interval.The hydrogel was prepared on a clean bench. Aqueous MGC-g-CD-ic-TCS solution was filtered using a 0.22 µm syringe filter for the removal of microorganisms, and then lyophilized. To a solution of MGC-g-CD-ic-TCS (200 mg) in DPBS (3 mL), aqueous riboflavin  was added, and the sample was vortexed gently. MGC-g-CD-ic-TCS was stabilized in a refrigerator (4 °C) for stabilization before use. For hydrogelation, the hydrogel precursor solution was irradiated for 200 s using a blue light  at a wavelength of 282 nm.The release test was carried out according to a previously reported method . A speci4 cells/well) on the hydrogels, they were incubated for 1, 3, 5, and 7 days in an incubator set at 37 °C and 5% CO2. In the case of TCS, the cells (1 × 104 cells/well) were attached on the wells of 96-well plate for 3 h and TCS-containing media was treated. At each time interval, CCK-8 (100 µL) was added in wells filled with the hydrogels and incubated for additional 2 hours. The optical density of supernatants extracted from CCK-8-treated wells was measured at 450 nm using a microplate reader .L-929 cell line was cultured with Eagle’s Minimal Essential Medium supplemented with FBS (5%), penicillin (100 units/mL) and streptomycin (100 µg/mL). For this experiment, cells cultured for 10 passages were used. The cell proliferation assay was performed on two hydrogel samples: TCS (TCS: 1.8 µg), MGC, and MGC-g-CD-ic-TCS (TCS: 1.8 µg). The hydrogel precursor solutions were immersed in a 96-well plate and photocured using a visible light irradiation for 200 s. After seeding the cells . Prior to the test, the fat layer of the skin, which was used for this study, was removed. MGC-g-CD-ic-TCS hydrogel precursor solution was applied on the surface of the cleaned skin with 10 × 10 mmEscherichia coli ATCC25922 (E. coli) and Staphylococcus aureus ATCC6538 (S. aureus) were used for evaluating the antibacterial activity of MGC and MGC-g-CD-ic-TCS (TCS: 1.8 µg) hydrogels. It was estimated by monitoring the optical density of the two bacteria at 600 nm. E. coli or S. aureus  were inoculated in fresh nutrient LB broth medium containing the two hydrogels (60 mg/L per each hydrogel) in flasks. LB medium alone was used as a negative control. Afterward, the flasks were incubated at 37 °C with a continuous stirring at 150 rpm. The optical density was measured for 48 h at 1-h intervals.n = 5). A constant length of incision was produced on the back of each rat and the incision depth was made including the dermis. A specific volume (150 µL) of cyanoacrylate, MGC, and MGC-g-CD-ic-TCS (TCS: 1.8 µg). were used for three groups, respectively. Other two groups include incision + no treatment and incision + suture. After treating MGC and MGC-g-CD-ic-TCS hydrogel precursor solutions into the incisions, visible light  was irradiated for 120 s. At the predetermined time intervals, the mice of each group were anesthetized, the skins including the incisions were removed, and the mice were sacrificed. All skin tissue samples were used for histological evaluations.This study was approved by the Institutional Animal Care and Use Committee of Chonbuk National University . Male Sprague Dawley rats  was used and randomized into five groups on each time interval . As a control, the same thickness of paraffin slide using normal skin tissue was made. The tissue sections were treated with H&E and MT staining . For immunostaining, deparaffinized tissue sections were treated using a microwave antigen-retrieval procedure with 0.01 M sodium citrate buffer. After blocking, the sections incubated with antibodies against iNOS  and mannose receptor (Abcam). For visualization, an HRP/DAB detection IHC kit (Abcam) was used according to the manufacturer’s instructions. These sections were observed using a slide scanner  and a panoramic viewer  program.p < 0.05 was considered statistically significant.All quantitative data were expressed as the mean ± standard deviation. Statistical analysis was performed with one-way analysis of variance (ANOVA) using SPSS software . A value of * 1H NMR and DSC analyses. Due to the migration of water molecules into its 3-D structure, the hydrogel was swollen, and this influenced the release behavior of TCS. MGC-g-CD-ic-TCS exhibited better tissue bonding strength than commercially used fibrin glue. In addition, the hydrogel showed high antibacterial activity against E. coli and S. aureus because of the cocktail effect of MGC and TCS. More importantly, MGC-g-CD-ic-TCS was associated with accelerated wound healing in vivo, suggesting its great potential as a tissue bio-adhesive.Here, we designed and prepared a MGC-g-CD-ic-TCS hydrogel system as an antibacterial tissue bio-adhesive. Inclusion complexes between MGC-g-CD and TCS were confirmed by


Introduction: A Tarlov cyst (TC) is a perineural cyst filled with cerebrospinal fluid that originates from the dorsal ganglion or the spinal posterior nerve root. TCs are usually asymptomatic and incidentally found in the sacral region. Endopelvic extension of TCs is uncommon and can be misdiagnosed as an adnexal mass on gynecological ultrasound imaging.Methods: We performed a search for all clinical studies of TCs that mimicked adnexal masses that had been published through October 12, 2020. We placed no restrictions on language or year of publication in our search, and we performed searches with the following keywords: perineural cyst, Tarlov cyst, sclerotherapy, management, and prognosis. We included all misdiagnosed cases or cases considered as adnexal masses on pelvic sonography.Results: We identified 21 cases of TCs mimicking adnexal masses and conducted a comprehensive analysis of these 21 cases to assess the epidemiology, symptoms, initial diagnoses, provisional ultrasound diagnoses, confirmative modalities, sizes, locations, treatments, and outcomes. The 21 cases included 16 symptomatic cases (76%) and 5 cases with incidental findings (24%), and the average patient age was 41.3 years. The initial diagnosis was performed with ultrasonography in all cases. The most frequent misdiagnosis was unspecified adnexal mass. Confirmative diagnostic modalities were MRI only (67%), CT only (5%), and both MRI and CT (28%). Treatments were surgery (33%), conservative treatment (19%), percutaneous intervention (5%), and alcohol sclerotherapy (5%). In two symptomatic cases misdiagnosed as pelvic masses, cystectomy was performed and leakage of cerebrospinal fluid occurred, necessitating repair of the leak. In one of the asymptomatic patients, cauda equina syndrome occurred after alcohol sclerotherapy for misdiagnosed TC. However, the patient improved with no neurologic deficit after 18 months of conservative treatment.Conclusion: The possibility of large TCs should be considered when assessing adnexal masses in sonography. Since TCs can masquerade as pelvic masses, they should be considered if the mass appears tubular/cystic or multilocular/multiseptate, does not move with respiration, and originates from the sacrum in sonography with or without neurologic symptoms. Accurate diagnosis can prevent medical mismanagement and reduce patient discomfort. Tarlov cysts (TCs) were first described by Dr. Tarlov in 1938 . TCs areWe report a case in which a TC was misdiagnosed as an adnexal cyst in a 38-year-old woman . She had visited a local gynecology hospital for a routine health checkup without any symptoms, and pelvic ultrasonography showed a left 6.51 × 4.96 cm cystic mass . The lesWritten informed consent was obtained from the patient for the publication of any potentially identifiable images or data included in this article.ClinicalTrials.gov) through October 12, 2020. We placed no restrictions on language or year of publication in our search, and we performed searches using the following keywords: Tarlov cyst, perineural cyst, adnexal mass, sclerotherapy, management, and prognosis.We reviewed all papers that reported TCs mimicking adnexal masses, with the papers identified by searching MEDLINE, EMBASE, SCOPUS, Web of Science, the Cochrane Central Register of Controlled Trials, the World Health Organization International Clinical Trials Registry Platform, and the clinical trials registry and database of the U.S. National Institutes of Health . All patients were female, and 16 were symptomatic (76%). Pelvic pain  , 12 and The initial diagnostic modality was ultrasonography in all cases. The most common incorrect diagnosis or initial diagnosis was an unspecified adnexal mass , 6, 14. The patients were treatments with surgery 7 cases, 33%) , 13, 15,3% , 13, Five patients were asymptomatic. Among them, one underwent unnecessary surgery , one th, 6, 9 diThe types of surgery were laminectomy in three cases , 10, 11,Among the three symptomatic patients who underwent laminectomy performed on symptomatic patients, two showed pain relief , 11 and In one case , an asymIn the present case, the clinician performed alcohol sclerotherapy after a misdiagnosing a TC as a simple ovarian cyst. Alcohol sclerotherapy is a transvaginal ultrasound-guided aspiration and ethanol injection technique that is presently used as an alternative therapeutic modality for simple ovarian cysts or endometriomas , 17. TheOur literature review outlines the importance of familiarizing gynecologists and obstetricians to TCs, which are cystic masses distinct from functional cysts, endometriomas, teratoma, hydrosalpinges, and peritoneal inclusion cysts with similar characteristics . TCs shoThe size of the TCs that were mistaken for adnexal masses in the literature review was 5 cm or more (mean size: 6.9 cm), while the median age of the population was 41 years. Accordingly, clinicians should consider the possibility of TCs when treating middle-aged women with adnexal cysts that are 5 cm or greater in size.Since TCs are extraperitoneal cysts, they do not show mobility during respiration.Since TCs are perineural cysts originating from the lumbosacral nerve root, identification of a connection to the posterior pelvic wall is important, i.e., TCs are located posteriorly on the sacrum.In contrast to other adnexal cysts, TCs are separated from reproductive organs such as the ovary and salpinx.Patients presenting with a symptomatic endopelvic TC may show low back pain, sciatica, leg weakness, and other neurologic deficits, so these findings may also serve as a point of differentiation.These differentiating points are summarized in A comprehensive understanding of TCs, including the points of differentiation, begins with a thorough understanding of anatomy. TCs are perineural cysts of the sacral area that arise between the perineurium and endoneurium. In 1938, Dr. Tarlov first reported a TC of the filum terminale as an incidental autopsy finding, and the lesion was classified as a type II meningeal cyst by Nabors et al. . TCs areThe pathogenesis of these cysts is unclear, although various hypotheses have been proposed to explain the formation of the slit valve mechanism that allows CSF to pass into the cysts . There iCommon clinical presentations include low back pain, sacrococcygeal pain, leg weakness or pain, sciatica, perianal pain, neurogenic claudication, bowel and bladder dysfunction, and sexual disturbances. TCs can also cause unusual clinical symptoms  if the cysts are in the presacral region . MRI, CTAsymptomatic TCs generally do not require treatment . ConservOur study had a few limitations. The primary limitation was the lack of cases, with only 21 relevant cases identified in the literature search. Although misdiagnosed TCs for adnexal masses are not common, we performed a search for all clinical studies of TCs that mimicked adnexal masses. Secondly, the data were obtained retrospectively. Thus, additional research is needed to prevent misdiagnosis and enable more accurate diagnosis and treatment in the future.Our data indicates the importance of considering the possibility of a large TC when assessing adnexal masses on sonography. Since TCs can masquerade as pelvic masses, if the masses appear tubular/cystic or multilocular/multiseptate, do not move with respiration, and originate from the sacrum in sonography with or without neurologic symptoms, TC should be considered. Our case is the first to report chemical CES caused by alcohol sclerotherapy for a TC that was incorrectly diagnosed as an adnexal mass. However, the patient recovered almost completely without significant complications after 18 months. Accurate diagnosis can prevent incorrect medical management and reduce patient discomfort.The original contributions presented in the study are included in the article/supplementary materials, further inquiries can be directed to the corresponding author/s.Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SK and TK: data analysis, data interpretation, literature search, and writing of the manuscript. HL and JP: conceptualization, methodology, and writing of the manuscript. KN: supervision, project administration, and review of the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.