The mesoporous structure of both powders was confirmed whereas the specific surface area of the MMO was enhanced by 69.9% to 36.86 m2 g−1 in the MMO/C powder with more electroactive sites. The higher electrical conductivity of the MMO/C electrode was proved using electrochemical impedance spectroscopy (EIS) results, with the MMO/C electrode achieving a specific capacitance of 571 F g−1 at 1 A g−1 current density, improved by more than 4.5 times that of the MMO. Furthermore, the rate performance and cycling stability of the MMO/C electrode reached 87% and 85.2%, respectively. Finally, a two-electrode energy storage device (MMO/C//AC) was assembled. It reveals a specific capacitance of 94.7 F g−1, a maximum energy density of 29.6 W h kg−1 at a power density of 660.1 W kg−1, and cycling performance of 84.3% after 2000 cycles. As a result, the resulting data demonstrate that the MMO/C electroactive material has promising abilities in capacitive energy storage systems.Recently, magnesium molybdate materials have attracted scientific attention for application in supercapacitor devices due to advantages like low synthesis cost and good redox reactions. Nevertheless, these materials endure low electrical conductivity leading to inferior electrochemical performance. To eliminate this drawback, we prepare a composite powder containing magnesium molybdate and functionalized carbon nanotubes (MMO/C) using a simple process to improve the supercapacitive properties. The results proved an electrostatic interaction between the two components of the composite powder, which contains 18–30 nm magnesium molybdate nanoparticles. A crystal model related to magnesium molybdate powder (MMO) was simulated, illustrating that MnO 4/MWCNT and MnMoO4 was conducted to assemble a high-performance supercapacitor device for future energy technologies.A comparative study on the supercapacitive properties of MnMoO These devices possess outstanding properties such as high specific capacitance, long cycling stability, good rate performance, fast charge/discharge capability, reliability, and low cost.3–6The rising demand for green energy and the worry about the fast diminishing of conventional fossil fuel has caused the research on energy storage to develop toward alternative energy sources.2,7 Even though pseudocapacitors prepare higher capacity and energy density, caused by faradaic fast redox reactions, these materials still suffer from low electrical conductivity. Hence, to employ the best benefits of the supercapacitive performances, applying a helpful technique for forming an efficient material is essential.There are two main charge storage mechanisms in supercapacitors: first, the electrical double layer mechanism related to the separation of charges at the electrode/electrolyte interface, and second, the faradaic redox reaction mechanism ascribed to electron and ion exchange on the noted interface. The former supercapacitors, usually made of carbon-based materials, are denoted as electrical double layer capacitors (EDLCs), and the latter, made of different transition metal oxides or conducting polymers, are known as pseudocapacitors.8 According to this, employing aqueous electrolytes provide better energy storage performances. On the other hand, the breakdown voltage of water in these electrolytes is around 1.2 V, and the potential window is restricted to lower values.9The supercapacitor electrode voltage can change depending on the electrolyte type. Accordingly, two common electrolyte types utilized in supercapacitors are organic and aqueous solutions. The organic electrolytes suffer from low ionic conductivity, inflammable nature, environmental incompatibility, overcharging during the process, and high prices. In contrast, an aqueous electrolyte yields more benefits than the organic one, like a more compatible nature, lower cost, higher ionic conductivity, and reduced heat generation.10 binary metal oxide materials have been considered an encouraging alternative due to their wide voltage windows, enhanced cyclability, remarkable electrical conductivity, and more electroactive sites. Additionally, among these oxides, magnesium molybdates can be assessed as a beneficial active material to raise electrochemical properties due to oxidation states coming from Mn ions and conductivity from Mo.11 This material suffers from two main drawbacks, low electrical conductivity and surface destruction during redox reactions. Employing carbon-based materials seems crucial to deal with the limitations.12,13According to the literature,14 Recent studies indicate that among these materials, multiwalled carbon nanotubes (MWCNTs), with their superior properties, are utilized to employ in EDLCs supercapacitors.15 Thus, using carbon nanotubes beside binary oxide materials, having high electrochemical properties and low cast, has encouraged the research community. For example, Ghosh et al.16 reported hydrothermally prepared MnMoO4/graphene composite electrode with 84% cycle life and 364 F g−1 capacitance at 2 A g−1 current density, whereas the produced pure MnMoO4 outputted 234 F g−1 at the same condition. This research states that the electrical conductivity of pure binary oxide and composite samples delivers 4.27 × 10−3 S cm−1 and 19.43 S cm−1 conductivities, respectively, justifying higher capacitance of the composite electrode. In another study investigated by Thangappan et al.17 the specific capacitance of MnMoO4/graphene nanocomposite improved by 49.7% to 302.08 F g−1 at a current density of 0.1 A g−1. Referring to this study, the chemical interaction of Mn2+ and MoO42− led to the formation of MnMoO4 crystalline nanoparticles, and at the same time, GO sheets are reduced using citric acid as the reducing agent attached to the binary oxide component. Thus, the presence of a large number of defects along with higher conductivity created by graphene prepared more active regions to enhance energy storage. Based on18 research, an electrospinning-carbonization-thermal annealing method was implemented to synthesize MnMoO4/carbon nanofibers composite. The specific capacitance of the electrode was obtained 389.7 F g−1 at 0.1 A g−1, and the cycle life results showed capacitance retention of 92.1% after 5000 cycles. Mu et al.19 reported hydrothermally treated hierarchical CNT/rGO on MnMoO4 nanosheets. The prepared device with 87.5% cycling stability demonstrated an energy and power density of 59.4 W h kg−1 and 1367.9 W kg−1, respectively.Carbonaceous materials possess high mechanical flexibility, large specific surface area, good aspect ratio, and excellent electrical conductivity.20,21 On the other hand, the mechanical mixing method has some benefits like simplicity, flexibility, and unit operation.22 As a result, introducing a MnMoO4/MWCNT electrode material with a simple technique could proficiently enhance the energy storage properties of the MnMoO4 with a simple synthesizing process enhancing its most common drawbacks.Co-precipitation is one of the simple and cost-effective approaches mainly employed caused by producing a uniform and high purity ceramic oxides along with ease of adjusting experimental parameters.−1 current density and by 582 at 10 mV s−1 scan rate. Furthermore, the cycle life and rate performance of the composite powder reached 85.2% and 87%, by 44.9% and 28% higher than the ones of the MMO powder, respectively.In this study, we have prepared simple co-precipitation and mixing approaches to achieve magnesium molybdate (MMO) and magnesium molybdate/multiwalled carbon nanotube (MMO/C) electroactive powders for being used as electrode materials to investigate their supercapacitive performance. Our studies clearly showed that by applying the MMO/C powder, the capacity of the MMO boosted from 125 to 571 at 1 A g2.3)2·4H2O (purity ≥ 97%), Na2(MoO4)·2H2O (purity ≥ 99.5%), and poly(vinyl pyrrolidone) (PVP) were purchased from Sigma-Aldrich. Nickel foam was prepared from LSS Company (Singapore). Poly(vinylidene fluoride) (PVDF), carbon black (CB), activated carbon (AC), and dimethylformamide (DMF) were procured from Sigma-Aldrich. The water utilized in all experimental sections was deionized (DI) with a conductivity of 0.1 μs cm−1.The as-obtained MWCNT (purity > 95%) was received from Cheap Tubes Inc. (USA). Mn(NO2.2.2(MoO4)·2H2O and 2.32 g Mn(NO3)2·4H2O were separately prepared in 100 mL DI-water and constantly mixed for about 30 min. At the same time, 0.02 g PVP was dissolved in 20 mL DI-water and added equally to the above solutions, 1 mL for each. Subsequently, the magnesium nitrate solution was dropwise added to the sodium molybdate under continuous stirring for up to 30 min. The obtained milky solution was separated and then rinsed well with ethanol and DI-water mixture several times via centrifugation under 1000 rpm for 15 min. Finally, the as-synthesized material was perfectly dried at 60 °C for 10 hours, named hydrated MnMoO4. To achieve the final product (MMO), the prepared precursor was calcined at 900 °C for 2 h.The MMO and MMO/C powders synthesis were conducted as follows: first, 2.25 g Na3 under ultrasonic conditions within 15 min and refluxed at 120 °C for 10 h. The mentioned powders were eventually washed and then dried under heating. For producing the composite powder (MMO/C), 120 mg of the MMO powder, synthesized in the first part, was mixed in 50 mL DI-water for 20 min. The noted solution was dropwise poured into 50 mL functionalized MWCNTs solution with a concentration of 2 mg mL−1 under stirring, and then the mixing was continued within 72 h at ambient temperature. The washing and drying processes were carried out the same way as in the first part.Second, the purchased MWCNTs were calcined at 380 °C for 2 h to eliminate the amorphous carbon materials. Thereafter, this powders were dispersed entirely in 7 M HNO2.3.−1 in the ambient atmosphere. In order to observe the surface morphology of both powders, a field emission scanning electron microscope (FESEM) with a MIRA3 TESCAN model was employed. The specific surface area of the materials was measured by utilizing the adsorption–desorption isotherms of nitrogen via the Brunauer–Emmett–Teller (BET), and pore size distribution was computed by the Barrett–Joyner–Halenda (BJH) method (Micrometrics TriStar II 3020). In addition, fourier transform infrared (FTIR) spectroscopy (by PerkinElmer–Frontier) was utilized to recognize the functional groups on the surface of materials.The crystallographic phase structure of the as-prepared materials was performed by X-ray diffraction (XRD) technique with Co-Kα radiation. The thermal characteristic of the MMO powder was appointed using an STA 503 BÄHR device at the range of 40–900 °C and a heating rate of 10 °C min2.4.2, denoted as working electrodes. The electrodes containing platinum and calomel were considered the counter and reference, respectively, in the three-electrode setup. The working electrode was prepared to produce the asymmetric two-electrode device, similar to what was mentioned, placed as the positive pole and AC as the negative. A piece of cellulose paper was positioned between two poles of the cell in order to separate them. The electrochemical features of the working electrodes were measured in a 2 M KOH aqueous solution in both systems. An OrigaFlex potentiostat workstation was implemented to measure the electrochemical characterizations using electrochemical impedance spectroscopy (EIS) (with a frequency range of 10 mHz to 100 kHz), cyclic voltammetry (CV), and galvanostatic charge–discharge (GCD) methods.The supercapacitive properties of the powders were investigated in both three- and two-electrode cell setups. The nickel foam was first washed with acetone for 15 min, and later a 2 M HCl solution under sonication was handled for 30 min to exclude debris and surface oxides. Finally, it was washed with acetone and ethanol. For assembling the three-electrode system, the as-prepared electroactive materials were mixed with PVDF and CB powders in DMF solution, whereas their mass ratio was 80 : 10 : 10, respectively. After mixing, the acquired homogeneous paste was pressed onto the nickel foam with a dimension of 1 × 1 cm3.4 nanopowder was measured from 40 to 900 °C to obtain the optimal calcination temperature of the nanoparticles and the possible phase transformations during the heating. From 4 solid solution into MnMoO4 compound, similar to what was discussed elsewhere.23,24 Accordingly, the calcination temperature for producing MnMoO4 nanostructure was chosen at 900 °C in this study, as depicted in the experimental section.The simple schematic illustration of both MMO and MMO/C synthesis procedure is depicted in In order to confirm the successful synthesis and assess the phase composition of the prepared powders, the XRD analysis was utilized, as shown in θ ≈ 26. This peak corresponds to the (002) plane of MWCNTs and discloses the hexagonal graphite structure.25 Hence, the construction of the composite by coupling MnMoO4 nanoparticles with MWCNTs was evidently proved. Meanwhile, a comparative evaluation with the pure standard pattern represents that some XRD peaks of both MMO and MMO/C powders are more intense than usual, ascribed to those crystallographic plans with more X-ray exposure.26 The average grain size measurements obtained by the Scherrer equation from the XRD patterns were calculated to be 18.7 and 16.9 nm for the MMO and MMO/C powders, respectively. As one can see, although the mean crystallite size in the MMO/C is lower, there is no significant grain size difference between the powders.Compared to what was presented in the MMO pattern, the XRD pattern of the composite sample revealed a broader peak at around 2C2/m space group containing Mn and Mo atoms coordinated as MnO6 octahedra and MoO4 tetrahedra, respectively. 27 According to what was formerly concluded from the TG-DSC results, it is clear from the XRD patterns that the obtained calcination temperature, around 900 °C, is favorable for attaining the high purity MnMoO4 powder.Based on the XRD results, the MMO crystal delivers a monoclinic structure and For evaluating the morphologies of the powders, the FESEM technique was used. 28 leading to the path of ion penetration among nanoparticle voids facilitates, and also energy storage increases. In this way, the favorable benefits of each component collected to form the composite powder cause the supercapacitive performance to increase compared to the other powder, which will be discussed in the electrochemical part. However, the FESEM image of the MMO/C powder  shows th29 The strong bound at about 940 cm−1 is related to the stretching vibration mode of the Mo PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O functional group. Also, the peak at around 867 cm−1 corresponds to the characteristic bending mode of Mo–O–Mo. Finally, the strong bond at 718 cm−1 is attributed to the stretching vibration mode of Mo–O placed on the MoO4 tetrahedral structure discussed and modeled previously. For the composite sample, the number of characteristic peaks correlated to the carbonaceous material shown in −1 are ascribed to stretching vibration of CO and C–O bonds, and these functional groups are related to carboxylic acid (–COOH) groups30,31 due to the oxidation of a number of carbon atoms on the surface of MWCNTs. The peaks at 1523 and 3414 cm−1 are assigned to O–H in the –COOH groups. A strong bond at about 2886 cm−1 is related to the C–H stretching vibration of carbonyl groups.32The FTIR spectra were used for analyzing surface functional groups. −1 and three bonds with less intensity at 884, 828, and 359 cm−1. These peaks are three primary characteristic bands of the MMO.33Raman spectroscopy was collected to further study the structure of both powders. −1, it is clear that the value reaches 927 cm−1 in the composite one. Moreover, a similar behavior occurs for the other three peaks. The mentioned red-shift can be ascribed to an electrostatic interaction between magnesium molybdate and functional groups placed on the surface of MWCNTs during the preparation process. Owing to the positive effects of each composite component, the noted occurrence can increase the overall electrochemical properties of the composite powder.34 Besides, two additional typical bands are in the MMO/C sample, the D band at 1341 cm−1 and the G band at 1587 cm−1. It is known that the D band is related to crystal defects and disordered structure in carbon lattice, and the G band is attributed to the carbon atoms with sp2 hybridization.35 The ID/IG ratio reveals the degree of structural disorder in the composite powder. This ratio was calculated at about 1.14, suggesting defects of carbon structure in the MMO/C. The obtained value greater than one results in higher active sites for energy storage and more interactions in the electrode/electrolyte interface.36In the CNT-based composite, along with the red-shift compared to the MMO peaks, two extra peaks clearly appear. By carefully tracking the positions of the main peak of the MMO at 936 cmFinally, it is interesting to notice that the FTIR and Raman results strongly confirm the synthesis of both the composite and oxide powders.2 adsorption–desorption isotherms of the powders are illustrated in 2 g−1, and the pore volumes around 0.149 and 0.179 cm3 g−1, respectively. The more the specific surface area, the higher the specific capacitance will be, arising from the high surface area value of MWCNTs directly affecting it.The BET and BJH techniques were implemented to analyze the specific surface area and pore size distribution of the MMO and MMO/C powders. The N37 Accordingly, the penetration of electrolyte ions into the MMO/C mesopores, with smaller pore size, causes the quality of redox reactions, the charge transfer, and also supercapacitive performance to improve.Furthermore, the BJH curves of the powders  disclose−1 scan rates, respectively. It is evident that the potential windows of the electrodes are normally determined by increasing the potential until there is the occurrence of hydrogen evolution reaction (HER) on the negative side and oxygen evolution reaction (OER) on the positive side of the curve.38 The HER and OER denote the breakdown of aqueous electrolytes, which will be shown by a sharp increase in the current density. Thus, the best potential windows in the CV curves of the MMO and MMO/C samples were determined from 0 to 0.5 V and 0 to 0.55 V, respectively, as illustrated in The supercapacitive properties of the MMO and MMO/C powders are represented in 39 It is suggested that16,40 an electron transfer between Mn2+ and Mn3+ ions in the electrochemical double layer is responsible for redox activities leading to pseudocapacitance behavior.11 On the basis of 2+ to Mn3+, the peaks at about 325 mV in the MMO powder and at about 402 mV in the MMO/C powder are formed, whereas the scan rate value is 50 mV s−1. In the same scan rate, the reduction process arising from Mn3+ to Mn2+ is conducted at about 175 and 145 mV in the MMO and MMO/C samples, respectively. In this case, Mo role is to enhance the electrical conductivity of the active materials and cannot take part in the ion exchange phenomenon and eventually enhance the specific capacitance.41 Meanwhile, by increasing the scan rates in the CV curves of electrodes, the peaks at the oxidation and reduction branches of the curves shift toward the right and left, respectively. This case can be related to the more significant electron and ion transfer resistance at higher scan rates under the diffusion-controlled process.42,43A pair of redox peaks is observed by carefully assessing all CV curves, implying redox reactions happen during the cycles.−1 current densities show the maximum discharge time takes place at 1 A g−1 current density, as shown in 26 were obtained at 125 and 571 F g−1, respectively. With more calculations in the MMO/C electrode, the values at 3, 6, 9, and 12 A g−1 current densities achieve 541, 522, 509, and 499 F g−1 capacitance, respectively. Although the typical type of the curves does not vary notably, the area under any curves increases with declining the current density, occurring by increasing the specific capacitance. The IR-drop data of the MMO and MMO/C powders at 1, 3, 6, 9, and 12 A g−1 current densities were obtained from the GCD curves. The IR-drop is because of the electrodes’ internal resistance, the ionic conductivity of the electrolyte, and ionic movement of the electrodes.41 Our evaluations show the acquired IR-drops rise as the current densities do as well. The MMO and MMO/C IR-drops are in the range of 9.4 to 49.6 and 2.1 to 31.9 mV, respectively, demonstrating better conductivity of the MMO/C electrode during electrochemical assessments.The GCD curves related to both MMO and MMO/C electroactive materials at 1, 3, 6, 9, and 12 A g−1 at 1 A g−1 current density and 10 mV s−1 scan rate, respectively, indicating well-matched specific capacitances. Also the values were compared to the other literature studies. It is noteworthy that the capacitance can be affected by the initial materials, synthesis procedure, and the conditions of electrochemical measurements.A comparative assessment of the specific capacitances obtained from CV and GCD curves for the MMO/C electrode is exhibited in 34 Therefore, the lower the current densities were applied, the better the redox reactions were done throughout the surface.−1, more than 87% of the highest capacity at 1 A g−1 remains. In this condition, the corresponding value for the MMO sample is about 68%, by 22% lower. Hence, the rate performance of the MMO/C electrode is higher than the one of the MMO electrode. Evaluating these variations, the higher specific capacitance, by 357%, and rate performance, by 28%, of the composite sample are ascribed to its higher electrical conductivity and ion movement, resulting in a faster kinetic process due to the presence of conductive MWCNT networks.51 The specific capacitance of the electroactive materials can also be calculated using the CV data. The computed capacities of the MMO/C electrode at 10, 20, 35, and 50 mV s−1 scan rates are 582, 562, 488, and 376 F g−1, respectively. Nevertheless, the MMO electrode manifests the capacities of 119, 117, 98, and 93 F g−1 at the same scan rates. A complete evaluation of the CV curves of both electrodes indicates that as the scan rates decrease, so inversely do the specific capacitances, suggesting the mobility of the electrolyte ions increases while applying higher scan rates, causing the ionic exchange to decline.52 It is specified from the capacitances calculated from both GCD and CV curves that the MMO/C electrode reveals higher specific capacitance than that for the MMO.As it is apparent in the MMO/C sample, even if the current density reaches 12 A g−1. Altogether, the comparison of the overall capacitance retention of the MMO and composite electrodes indicates the former possesses 58.8% and the latter 85.2% of their initial capacitance values after 2000 cycles. Both electrodes reveal that almost over the first 180 cycles, a more noteworthy decrease in the specific capacitance takes place, according to the report.16 When cycle numbers increase from 180 to 700, the correlated capacitances decrease slower than the previous step, and up to 2000 cycles, it stays nearly constant. It seems that the degradation of the active materials due to internal stresses, along with their fast contraction and expansion during cycling, are the main reasons for these changes.48,53–55 As a consequence, the higher value of the cycle life obtained in the MMO/C electrode than that of the MMO arises from two main reasons: first, the higher electrical conductivity of MWCNTs applied throughout the electroactive material, which will prove further using the EIS results, and second, lower surface destruction coming from better stress distribution on the overall surface of the active material during cycling.Rct) is ascribed to charge exchange resistance throughout the electrode/electrolyte interface, and the latter, named Warburg impedance (Zw), is related to the kinetics of hydroxide ions on the active material surface. Additionally, the value of x-intercept (Rs) is attributed to the internal resistance of the powders as the electroactive materials.56–60The EIS technique was used to evaluate further the electrochemical properties of both electroactive materials. Rs values of the electrode containing the MMO and MMO/C powders are 2.1 and 1.8 Ω, respectively, demonstrating that the MMO/C reveals higher conductivity and improved mobility of ions.61 Meanwhile, the radius of the semicircle region related to the MMO/C electrode is smaller, and as a result, its Rct value is lower than that of the MMO, which manifests better ionic exchange on the electrode/electrolyte interface. Besides, the lower Warburg impedance of the MMO/C electrode, at the lower frequency part, shows more excellent hydroxide kinetics for diffusion on the surface of the active material.47,62,63 Altogether, the EIS results imply that the MMO/C material contains a good electrostatic connection of the MMO particles and MWCNTs and shows higher ion exchange, electron transfer, and ionic diffusion kinetics at the electrode/electrolyte interface than those of the MMO due to the presence of MWCNTs.It is apparent in the EIS curves that the −1 scan rate. Our investigations revealed that the most favorable voltage window is in the 0–1.5 V range, as observed in 64 that a greater operating voltage, in our case 1.7 V, brings about a higher current density due to water splitting and electrolyte decomposition throughout the electrode surface.The MMO/C and AC were applied as the positive and negative electrodes, respectively, while assembled in a two-electrode system (MMO/C//AC). To find the best operating voltage of the device, the CV measurements were conducted at various voltage windows and 50 mV s−1 at 1 A g−1 current density via these curves. As one can recognize, similar to what was stated in the three-electrode system, the rate performance curve in the device was obtained using GCD plots, as shown in −1, the device discloses a great rate performance, demonstrating rapid electron transfer and good ion penetration coming from its good electrical conductivity.65−1 within 2000 cycles. It is recognizable that the cyclical behavior of two- and three-electrode systems are similar. According to the cyclic life results of the device, about 84.3% of its initial specific capacitance remains after 2000 cycles, which reveals its remarkable stability.41 The energy density values were calculated to be around 29.6, 28.2, 26.6, 24.5, 22.1, and 20 W h kg−1 at the power density values of 660.1, 1298.1, 3447.8, 6784.6, 14 184.6, and 34 951.8 W kg−1, respectively. On the basis of obtained results, a maximum energy density of 29.6 W h kg−1 at a power density of 660.1 W kg−1 was computed.Finally, we studied the supercapacitive and characteristic properties of the MMO and MMO/C electrode materials under a comparative study. The data obtained from the asymmetric two-electrode MMO/C//AC device demonstrate excellent electrochemical abilities to implement in high-performance supercapacitor applications.4.−1 at 1 A g−1 current density and from 119 to 582 F g−1 at 10 mV s−1 scan rate under identical measurement conditions. Thanks to its electrochemical performance, the MMO/C active material was selected to be evaluated in an assembled device. The outstanding supercapacitance properties of the MMO/C material are related to its higher electrical conductivity, more electroactive sites, and better ion exchange. The resulting data implied that owing to the easy experimental procedure and promising supercapacitive abilities, such a composite powder could be considered a good choice for electronic devices.In summary, a simple co-precipitation method was implemented for the synthesis of the MMO powder, and then this material was electrostatically interacted with functionalized multiwalled carbon nanotubes to prepare the MMO/C composite. Both powders were further characterized, and their supercapacitive performances were compared in a three-electrode system. The MMO/C electroactive material maintains a high capacity retention of 85.2% after 2000 cycles and retains the rate performance of 87%. By preparing the composite electrode, the specific capacitance of the MMO was successfully enhanced from 125 to 571 F gThere are no conflicts to declare.


The vertebrate retina contains a large number of different types of neurons that can be distinguished by their morphological properties. Assuming that no location should be without a contribution from the circuitry and function linked to a specific type of neuron, it is expected that the dendritic trees of neurons belonging to a type will cover the retina in a regular manner. Thus, for most types of neurons, the contribution to visual processing is thought to be independent of the exact location of individual neurons across the retina. Here, we have investigated the distribution of AII amacrine cells in rat retina. The AII is a multifunctional amacrine cell found in mammals and involved in synaptic microcircuits that contribute to visual processing under both scotopic and photopic conditions. Previous investigations have suggested that AIIs are regularly distributed, with a nearest-neighbor distance regularity index of ~4. It has been argued, however, that this presumed regularity results from treating somas as points, without taking into account their actual spatial extent which constrains the location of other cells of the same type. When we simulated random distributions of cell bodies with size and density similar to real AIIs, we confirmed that the simulated distributions could not be distinguished from the distributions observed experimentally for AIIs in different regions and eccentricities of the retina. The developmental mechanisms that generate the observed distributions of AIIs remain to be investigated. After removing the eyes, the retinas were dissected out (in HEPES-buffered extracellular solution) and used for immunolabeling.The use of animals in this study was carried out under the approval of and in accordance with the regulations of the Animal Laboratory Facility at the Faculty of Medicine at the University of Bergen . Albino rats  had m phosphate buffer  were added from above and allowed to soak through. This procedure was repeated two to three times, after which the filter paper with the attached retina was transferred to a larger volume of 4% paraformaldehyde in 0.1 m PB and fixed for 30 min at room temperature. The retina was then washed six times (10 min each) in 0.01 m phosphate-buffered saline  and incubated overnight at 4°C in antibody incubation solution consisting of PBS with 5% normal goat serum (Sigma-Aldrich), 0.5% Triton X-100 (Sigma-Aldrich), and 0.05% NaN3. The retina was then incubated for four nights (at 4°C) with primary antibody  in antibody incubation solution (with 0.2% Triton X-100). Both retinas were also immunolabeled with an antibody against ankyrin-G, but this was reported in Liu et al.  to place a spot of permanent ink (xylene free) on the dorsal part of the eye. After the eye had been enucleated and opened, a small incision was made in the dorsal part of the retina, choroid and sclera before dissecting the retina from the eye cup. To flatten the retina, we made four radial incisions from the periphery almost to the center and transferred the retina onto the nongridded surface of nitrocellulose filter paper . Together with the attached retina, the filter paper was placed on a piece of folded tissue paper (Kimwipe). A few drops of HEPES-buffered extracellular solution were added from above and allowed to soak through. For fixation, a few drops of 4% paraformaldehyde in 0.1 u et al.  and willThe primary antibody against parvalbumin was a polyclonal antibody raised in guinea pig against amino acids 1–133 . The antibody has been validated by the supplier, as indicated in the supplier’s data sheet which showed a band of the respective molecular weight of the protein detected, as revealed by Western blot.μm2) by sequential scanning (between lines) of the different channels. For each frame (corresponding to an acquisition channel), each line was scanned two times and accumulated to increase the signal-to-noise ratio (SNR). The confocal pinhole was set to 0.5 Airy units . To obtain well-sampled image stacks that could be processed with deconvolution (see section “Image deconvolution”), images were sampled at a rate slightly higher than the ideal Nyquist rate. The Nyquist sampling distance in the lateral direction was calculated asλex is the wavelength of the excitation light, λem is the wavelength of the emission light, n is the lens medium refractive index (1.45 for the glycerol used for immersion), and α is the half-aperture angle of the objective . The XY pixel size was ~46 nm and the focal plane interval was ~150 nm, sufficient to satisfy Nyquist rate sampling according to the stated equations.For imaging the immunolabeled wholemount preparations, we used a TCS SP8 confocal microscope (Leica) equipped with a ×63 glycerol immersion objective  and HyD detectors. The image stacks were acquired at 8‑bit resolution and the acquisition was controlled by LAS X software (Leica). We used the “white light” laser and acousto-optic filters to obtain an excitation laser line of 488 nm (for Alexa 488) and the emission bandwidth was set to 500–550 nm. The laser intensity for the acquisition channel was adjusted to maximize the dynamic range, with minimal saturation at the highest intensities. We used the Navigator module of the Leica LAS X software to acquire a total of 16 image stacks arranged roughly along two orthogonally oriented lines across the retina (see section “Results”). Each high-resolution image stack was acquired as a series of optical slices .In addition to the high-resolution image stacks, we also used the Navigator module of the Leica LAS X software to acquire a number of low-resolution images that tiled the entire retina . Each low-resolution image was acquired at a single focal plane  and decrease axial and lateral blurring, we digitally deconvolved each image stack with Huygens . Huygens reassigned out-of-focus light with a theoretically calculated point spread function, using the classic maximum likelihood estimation (CMLE) deconvolution algorithm. For each image stack, we estimated an optimal value for the user-selectable SNR parameter by repeating the deconvolution for several values of the SNR while keeping all other parameters and settings constant  and manually traced the contour of the cell body. In addition to AII amacrine cells, antibodies against parvalbumin also label a population of non-AII, widefield amacrine cells in this location of the rat retina. To distinguish between AII amacrines and the widefield amacrines, we used three morphological criteria  to calculate the number of nearest neighbors for each cell, corresponding to the number of edges in each Dirichlet domain.After we had segmented all cell bodies of immunolabeled AII amacrine cells in an image stack, we used Neurolucida 360 to automatically position a marker within each cell body contour. For each cell, the marker was located at the XYZ coordinate of the center-of-mass (centroid) of the corresponding contour. The segmentation results were processed with built-in analysis functions of Neurolucida Explorer . IGOR Pro  was used to compute the Dirichlet (Voronoi) domains (including their areas) and the nearest-neighbor distances for the population of XY coordinates corresponding to the centroids of the AII amacrine cell bodies. To compute the nearest-neighbor distances, we only took into account the XY coordinates. The regularity index  of the stack. Unfortunately, this will underestimate the spatial density. To compensate for this, we also counted immunolabeled cell bodies intersected by the top or left edge of the image frame, as long as they were considered to unequivocally belong to AII amacrine cells. In a few cases where we were uncertain if cell bodies belonged to AII amacrines or not, we included every other such cell in the total count of AIIs.μm) and fitted each histogram with a Gaussian function (IGOR Pro):y0 is the baseline (fixed at 0), A is the amplitude, and w (width) is equal to s.d. From the number of AIIs in an image stack, we also calculated the expected probability density function for a randomly distributed population with the same spatial density , where the integral is equal to 1, with the total number of points (n) in a given region before the corresponding function was compared with a given observed distribution:n/A and A is the area of each acquired region (246.03 × 246.03 μm2).For the AII amacrines in a given image stack, we generated histograms of nearest-neighbor distance distributions (bin width set to 1 μm2). Each cell body was represented as a circle and the location was represented by the XY coordinates of the center of the circle. When a cell was added, its location was determined by generating two random numbers (for the X and Y center coordinates) with equal probability within the limits of the side length of a stack. The center location was also constrained by the location and size of the other cell bodies to prevent overlap. For each simulation cycle of adding a cell body, the diameter was randomly selected from a Gaussian distribution with the same mean and s.d. as observed for the segmented AII amacrine cells in a given image stack. For a segmented cell body, the apparent diameter was calculated as the diameter of a circle with the same area.To compare the distribution of nearest-neighbor distances observed for a given region with that for random distributions, we performed simulations (IGOR Pro). For each simulation run, we generated a random distribution of the observed number of cells within an image region (XY) of the same size as used during acquisition (246.03 × 246.03 s.d. (n = number of cells or points).For data analysis and visualization, we used Huygens, Neurolucida Explorer, IGOR Pro, MATLAB, and Excel. Density recovery profiles (DRP), based on two-dimensional point autocorrelograms, were generated (IGOR Pro) as described by Rodieck . ExperimIn this study, we immunolabeled two wholemount retinas  for parvalbumin, a calcium-binding protein that serves as a marker for AII amacrine cells in rat retina  to center (Region #4), as illustrated in l retina . For Retl retina .μm2 (n = 4005 cells) versus 59.2 ± 6.3 μm2 . The eccentricity-dependence of cell density was paralleled by similar (but opposite) relationships for Dirichlet domain area and nearest-neighbor distance. Specifically, the Dirichlet domain area and the nearest-neighbor distance increased from the center toward the periphery, concomitant with the corresponding decrease in density (s.d.) for both Dirichlet domain area and nearest-neighbor distribution, with the former preferred by some authors because it takes into account a cell’s relation to all its neighbors, not only its nearest  in Retina-1 (n = 16 image stacks) in Retina-2  distributed and that this pertains to all regions and eccentricities of the retina  and 4. Hμm. The corresponding histogram of nearest-neighbor distances was well fitted by a Gaussian function and appeared distinctly different from the expected random distribution  generated nearest-neighbor distance histograms that were well fitted by Gaussian functions and appeared markedly different from the expected random distributions for the identical spatial densities. The results for an individual image stack in one of the wholemounts are illustrated in We next performed identical simulations where we set the average cell body size to zero , with markedly different results. The results for a single simulation run are illustrated in The results presented in μm and multiplied the average histograms by 4 to compare with the expected random distributions. μm). It was readily apparent, however, that for both density and diameter, the values had to be reduced to much lower levels than observed for the real AIIs for the frequency histograms to match the corresponding expected random distributions. In both cases, the 50% reductions were markedly different, whereas the 25% reductions were very similar  cell diameter to 75, 50, or 25% of the values observed for a given stack. For a given simulation condition, we repeated the simulation 500 times and calculated the average frequency distribution. To generate histograms with higher resolution, we set the bin width to 0.25  similar  and 6B.Fμm). Also in this case, however, it was readily apparent that the values both for density and diameter had to be markedly reduced relative to the values for the real AIIs before the distributions became similar to the expected random distribution  and the average apparent diameter was relatively high . A given distribution is then compared with the expected probability density function for a random (Poisson) distribution of points of the same density. This kind of analysis has been performed for many different types of neurons in several different species. When the distribution of AII amacrine cells was analyzed in retina of cat, rabbit, rat, macaque, mouse, and bat, it was in every case concluded that the observed mosaic displayed relatively high regularity, with a regularity index of approximately 4–5.5  and lowest  density within a given quadrant was 1.5 and almost 3, respectively. For the ventronasal retina, there was much less of a center-periphery gradient. The center-periphery gradient of the dorsotemporal retina is much more pronounced than what was observed for mouse retina by Pérez de Sevilla Müller et al. . Althougnd Reese  for a nu2) is somewhat lower than the peak densities reported for the two retinas analyzed by Wässle et al. . As suggested in a previous study from our laboratory (Liu et al., 2. The soma size was not reported, but from their figure the average diameter can be estimated as ~8.7 μm. Accordingly, simulations of random soma distributions with density and size matched to the data of Wässle et al. (The peak density observed for the two retinas in our study (~5200 and ~4900 cells/mme et al.  would bee et al. .For the two retinas analyzed in our study, we calculated a regularity index for the distribution of AIIs in each field, both for the nearest-neighbor distance distribution and for the Dirichlet domain area distribution. It has been argued that because the nearest-neighbor distance distribution only takes into account the relation between an individual neuron and one of its neighbors, the Dirichlet domain area is a more general measure which takes into account the relation to all the neighbors (reviewed by Reese, In addition to nearest-neighbor distance distribution, other, more complicated methods have been developed to analyze whether a mosaic displays regular or random distribution. The method known as the DRP Rodieck,  is basedThe most important question, however, is whether our results for the distribution of AII amacrines in rat retina differ in any substantial way from the corresponding results for AIIs in mouse retina (Keeley & Reese, The fact that the real distributions of AII amacrine cells cannot be discriminated from simulated random distributions matched for density and constrained by soma size does not by itself mean that the distribution is inherently random. Arguably, the important point is how the final distribution is generated during development of the retina. With the constraint that the AII cell bodies are essentially confined to a single tier of the inner nuclear layer, the real distributions could potentially be generated by a series of mechanisms without an intrinsic ability to establish regularity. For the relatively high density of the population of AII amacrine cells, there is no clear need for a mechanism to explicitly establish regularity in the distribution. Future work will be required to decipher the actual mechanisms involved during development (cf. Reese & Keeley,


Eight implanted children showed residual hearing below 2000 Hz worse than 100 dB nHL and 2 children showed pantonal residual hearing worse than 100 dB nHL (p > 0.05). Conclusion: The audiological evaluation of infants with a comprehensive protocol is highly reliable. RW-ECoG provided a better definition of hearing thresholds, while E-ABR added useful information in cases of auditory nerve deficiency.Background: An electrophysiological investigation with auditory brainstem response (ABR), round window electrocochleography (RW-ECoG), and electrical-ABR (E-ABR) was performed in children with suspected hearing loss with the purpose of early diagnosis and treatment. The effectiveness of the electrophysiological measures as diagnostic tools was assessed in this study. Methods: In this retrospective case series with chart review, 790 children below 3 years of age with suspected profound hearing loss were tested with impedance audiometry and underwent electrophysiological investigation . All implanted cases underwent pure-tone audiometry (PTA) of the non-implanted ear at least 5 years after surgery for a long-term assessment of the reliability of the protocol. Results: Two hundred and fourteen children showed bilateral severe-to-profound hearing loss. In 56 children with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 70 dB nHL. In the 21 infants with bilateral profound sensorineural hearing loss receiving a unilateral cochlear implant, no statistically significant differences were found in auditory thresholds in the non-implanted ear between electrophysiological measures and PTA at the last follow-up ( Infant hearing loss is associated with multiple adverse sequelae, including poor speech and language acquisition, decreased global quality of life, and increased societal cost for the care of deaf people. Cochlear implantation (CI) is one of the most common procedures to address this problem, together with auditory brainstem implantation (ABI), with steadily improving outcomes. Yet, despite the success of cochlear implantation, several important questions remain; specifically, the early identification of which infants would benefit from cochlear implantation ,10,11,12The primary aims of electrophysiological investigation in children with suspected hearing loss are: (1) the determination of hearing levels, and subsequently, in combination with radiological investigations, (2) the determination of candidacy for CI or ABI. Several electrophysiological tests have been proposed ,21,22,23Since 1998, we have been developing a comprehensive electrophysiologic testing protocol. In addition to auditory brainstem responses (ABR), round window electrocochleography (RW-ECoG) and round window electrical auditory brainstem responses (RW-EABR) have been applied in a standardized manner. RW-ECoG, a near-field cochlear potential, has the advantage over ABR of better defining hearing thresholds and residual hearing levels. RW-EABR can provide important information regarding the excitability of the auditory nerve, the site of the hearing loss, and therefore the candidacy for CI or ABI.The aim of the present study is to evaluate this auditory electrophysiological testing protocol in deaf children for the selection of candidates for auditory implantation by reviewing the long-term follow-up of the non-implanted ear.Since 1998, we have used an electrophysiological testing protocol for determining hearing thresholds in children suspected of having profound hearing loss. This protocol was adopted in a tertiary referral center after newborn hearing screening test failure (otoacoustic emission and/or ABR during sleep). The protocol involves a series of tests including ABR, RW-ECoG, and RW-EABR performed under general anesthesia in a single recording session. The aim of the testing protocol is to exclude possible residual functional hearing, determine the site of the hearing loss, and subsequently, in association with imaging studies, select candidates for CI or ABI. If the electrophysiological data indicate that a child is a suitable candidate for CI or ABI, he or she additionally undergoes computed tomography (CT) and magnetic resonance imaging (MRI) to confirm the diagnosis and evaluate for inner ear malformations. All subjects underwent genetic, pediatric, and neuropsychiatric evaluation, in order to understand if any associated disabilities  or genetic factors for hearing loss were present. A flow chart for the testing protocol is shown in With our testing protocol, we identified 21 infants who were diagnosed with bilateral profound sensorineural hearing loss before 6 months of age between 2002 and 2009 and were fitted with a unilateral cochlear implant  before the age of 1 year. This group of implanted infants was used to test the efficacy of the protocol below 1 year of age through long-term audiological follow-up. All children were implanted by the senior author (VC). All patients underwent pure-tone audiometry (PTA) and speech perception testing in quiet (mono/disyllabic word lists) of the non-implanted ear at least 5 years after implantation. n = 6).Exclusion criteria were children who were unable to complete PTA testing due to cognitive disabilities or had developmental delay; diagnosed with auditory neuropathy; whose parents did not consent to CI or ABI even after diagnosis . For ABR testing, needle electrodes were placed subcutaneously with the standard vertex to pre-tragus montage. Electrode impedance was smaller than 3 kΩ. Electrodes were connected to an evoked potential system and responses were band-pass filtered between 100 and 3000 Hz. Clicks of 100 μs duration, produced by a 100 μs electrical square wave, were presented via TDH-39 headphones at a rate of 21 per second with alternating polarity. Click signals were calibrated in dB nHL. At each presentation, the number of sweeps was set at 1024. Data were collected in time windows of 10–15 ms. The threshold was defined as the minimum stimulating level where an ABR response was both identifiable and repeatable. Starting at 120 dB nHL, clicks were presented twice for each sound intensity level in order to ensure reproducible responses. The level of stimulation was decreased in 20 dB nHL steps until no response was present. Thereafter, ABR was repeated using 10 dB steps and then 5 dB steps until the threshold was established. Round Window Electrocochleography (RW-ECoG). RW-ECoG was performed using the cotton-wick electrode over the round window niche, placed as described above . The eleRound window electrical auditory brainstem responses (RW-EABR). After being used as a recording electrode for RW-ECoG, the same cotton-wick electrode placed on the round window niche was used to deliver electrical stimuli to test the viability of the cochlear nerve fibers. To minimize stimulus artifacts, biphasic rectangular pulses were delivered by a constant current stimulator to the cotton-wick electrode. The stimulating electrode was referenced to a sub-dermal needle electrode placed in the ipsilateral pre-tragus area. The electrical stimulus was a biphasic pulse of 100 μs per phase and its intensity ranged from 1 to 1.5 mA. EABR were recorded using a contralateral electrode montage with the positive electrode inserted at the vertex, the negative electrode at the contralateral pre-tragus area, and the ground at the forehead. Each recorded waveform consisted of 1024 samples of EEG activity over a 10 ms time base, and the results were band-pass filtered between 1 and 2500 Hz. The EABR threshold was defined as the minimum current level required to evoke an identifiable wave V. The best threshold identified with ABR or ECoG testing was used to define the preoperative electrophysiological threshold for the frequency of the stimulus used. Before undergoing implantation, children underwent a trial with hearing aids, vibrotactile devices, or both with no improvement.p < 0.05. Statistical analysis was performed using the paired T-test and Mann–Whitney test as appropriate (we compared the implanted ear with the non-implanted ear). Statistical significance was set at A total number of 790 subjects underwent the diagnostic protocol, including 401 males and 389 females. The median age at the time of diagnosis was 13.4 ± 9.6 months.Out of the 790 tested children, 214 (27%) showed bilateral severe-to-profound hearing loss. Among these children, 13 (6.1%) also had a diagnosis of auditory neuropathy  ,25,26. OElectrophysiological thresholds obtained with ABR and RW-ECoG tests are presented in In 56 subjects (6.8%) with either ABR thresholds between 70 and 90 dB nHL or no response, RW-ECoG showed thresholds below 75 dB nHL  due to either the testing protocol or general anesthesia were identified. Test protocol duration was assessed in a subset of recording sessions (163 subjects). Test protocol duration for ABR is related to the number of intensity levels tested and ranged from 4 min to 15 min (mean 9 ± 4 min). The RW-ECoG portion, including myringotomy and placement of the electrode, lasted between 8 min and 20 min (mean 14 ± 5 min). RW-EABR testing lasted between 4 and 11 min (mean 8 ± 3 min). Total electrophysiological protocol duration ranged from 28 to 65 min (mean 41 ± 13 min).To further assess the efficacy of our protocol, hearing thresholds obtained at initial diagnosis by electrophysiological methods were compared to PTA at least 5 years after the implantation. Twenty-one infants diagnosed with bilateral profound sensorineural hearing loss before 6 months of age and fitted with a unilateral cochlear implant before 12 months underwent pure-tone audiometry (PTA) of the non-implanted ear at least 5 years after the implantation. Demographics and clinical data of these cases, including the etiology of hearing loss if known, are presented in None of the implanted children had functional hearing in the non-operated ear, defined as at least one threshold better than or equal to 75 dB nHL for 250, 500, or 1000 Hz. In 11 children (52.5%), PTA confirmed the previous diagnosis of profound hearing loss (no responses up to 120 dB nHL), with no differences between pre- and post-operative PTA in the non-operated ear.p > 0.05) with residual hearing below 2 kHz (0.5 and 1 kHz) (Eight children (38%) had hearing worse than 100 dB nHL at frequencies below 2000 Hz and two children (9.5%) showed a flat residual hearing worse than 100 dB nHL. No statistically significant differences in the paired T-test and Mann–Whitney test were identified between auditory thresholds of the non-implanted ear between the results at diagnosis (115.8 ± 4.9 dB nHL) and at the last follow-up (113 ± 6.2 dB nHL) among the 8 subjects (d 1 kHz) .Residual hearing was not detectable on the implanted side in any children. Speech discrimination scores at the last follow-up with the cochlear implant off were 0%.The mean speech perception outcome at the last follow-up for the 21 children implanted (implant ON) was 85.2 ± 10.7. As a result of the critical importance of auditory input early in life for speech and language development and the ability to intervene with hearing devices , many countries have adopted mandatory newborn hearing screening. Children deprived of auditory information in the first 3–5 years of life will have difficulty in oral communication even if their hearing is restored because their brain mechanisms for developing auditory pattern recognition will have passed the critical period of brain plasticity ,28.For many years, it was also thought that RW-ECoG was not reliable enough to predict hearing status and thus that the risks of the procedure  did not justify the limited benefit. However, as the present results show, RW-ECoG does have the reliability to diagnose severe and profound hearing loss in infants. This objective electrophysiological procedure, combined with RW-EABR, can be used to establish candidacy for CI or ABI in infants. RW-ECoG represents cochlear microphonics and summation potentials and is related to hair cell presence and health. RW-EABR, in contrast, is an evoked neural potential and indicates the presence and health of the auditory nerve. Recording these potentials from the round window provides a clear signal that has high reliability, selectivity, and specificity for hearing diagnosis. Children with damaged hair cells but an intact auditory nerve can benefit from a CI, while children with no response on either measure require an ABI because this indicates the absence of an auditory nerve, so a CI would be of no benefit. This lack of auditory nerve can be cross-checked with MRI results. Buchmann et al. (2011)  showed tFurthermore, RW-ECoG demonstrated moderate hearing loss (<70 dB nHL) in 53/790 (6.8%) subjects with an ABR threshold between 70 and 90 dB nHL or no response. All these children were referred to the audiologist for hearing aid evaluation. Conversely, extratympanic ECoG seems less effective ,30. RW-EThe combination of the presented extensive electrophysiological and radiological (CT and MRI) protocols is helpful in improving the efficacy of diagnosis, despite being mildly invasive. The extra information that RW-ECoG provided can be fundamental in view of a better threshold definition as well as to rule out auditory neuropathy. In cases of cochlear nerve hypoplasia, RW-EABR could add important information regarding the candidacy between CI and ABI.The audiological evaluation of infants with a comprehensive protocol seems to be highly effective in subjects with profound hearing loss who are candidates for cochlear implantation below 1 year of age and even before 6 months. Residual hearing was detected in half of the 21 subjects who underwent CI, but no subjects showed serviceable residual hearing for hearing aid fitting.Our results agree with Vlastarakos et al., who underline the safety and efficacy of the diagnosis and, eventually, CI in infants ,13. RegaThe major limitations of the present study are the small number of children below 1 year of age implanted and that the non-implanted ear has been considered as the control group since they had substantially the same hearing threshold. Finally, as in all studies concerning cochlear implants, the heterogeneity of the population and multiple covariates must be taken into consideration.In our experience, RW-ECoG and RW-EABR are at present the most reliable electrophysiologic techniques for establishing hearing thresholds and the site of lesion in infants with severe to profound hearing loss, furnishing useful indications for candidacy to CI or ABI in a quick, inexpensive, cost-effective, and reproducible way. The entirety of the procedure lasts on average 40′, a duration that may be achieved thanks to general anesthesia. The important information obtained from RW-ECoG and RW-EABR can avoid placing an unnecessary CI as well as placing a cochlear implant in a “dead” cochlea unsuitable for electrical stimulation.A comprehensive electrophysiological evaluation of infants is highly reliable in identifying children with profound hearing loss who may be candidates for CI or ABI. Severe-to-profound loss was detected in half of the examined cohort, and some had minor residual hearing, but none showed residual hearing sufficient for hearing aid use. We propose RW-ECoG as a reliable method for diagnosing and defining hearing loss and for determining whether a hearing aid, CI, or ABI is the most appropriate management approach.


Atherosclerosis is reported to be a risk factor for the severity of idiopathic sudden sensorineural hearing loss (ISSNHL). We evaluated the hypothesis that atherosclerosis affects the hearing thresholds of both the affected and healthy sides of ISSNHL patients. We conducted multivariate analyses on retrospectively collected data of patients with ISSNHL (N = 762) to evaluate the relationship between known factors linked to atherosclerosis and hearing thresholds on affected and healthy sides and whether these factors are prognostic for hearing recovery. Older ages, vertigo or dizziness, diabetes mellitus, and congestive heart failure were significantly related to higher hearing thresholds on the affected side. Older ages, male, and vascular disease were significantly related to higher hearing thresholds on the healthy side. Vertigo or dizziness, severe hearing loss and hearing loss at high frequencies on the affected side, higher hearing thresholds on the healthy side, regular anticoagulant medication, and delayed steroid treatment were significantly related to lack of recovery. Since several atherosclerosis-related factors are associated with higher hearing thresholds on both affected and healthy sides in ISSNHL and higher hearing thresholds on the healthy side predict poorer prognosis, diagnosis, and predicting prognosis of ISSNHL may benefit from rigorous evaluation of patients’ cardiovascular comorbidities and hearing levels on both the healthy and affected sides. The pathogenesis of ISSNHL also remains unclear, with some reports suggesting it may involve viral infections2, autoimmunity3, circulatory disturbances4, among others. Although the etiology and pathogenesis of ISSNHL have been studied for decades, major advances in these areas have been elusive. This gap has hindered diagnosis, selection of appropriate preventive measures, and testing and development of therapeutic strategies.The etiology of idiopathic sudden sensorineural hearing loss (ISSNHL) remains unknown, more than half of patients don’t completely recover1. Indeed, hearing thresholds on the patient’s unaffected side, or healthy side, are typically ignored. This constraint means that the hearing loss of ISSNHL is treated as a single disease, even though it arises from various pathologies. Supplemental Table 1, which is consistent with clinical practice guideline by American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS)5.One reason for this lack of major progress may be that the diagnostic criteria for ISSNHL depend on the mode of onset and the form of the audiogram of the affected sideHow do currently used criteria affect the evaluation of difficult-to-diagnose hearing disorders? Take, for example, the two hypothetical cases shown in Fig. 6, but also to a variety of medical conditions and factors related to a patient’s background, such as whether the patient has atherosclerosis and is subject to certain genetic factors8. Therefore, we hypothesized that the audiogram on the healthy side of patients with ISSNHL could be a useful additional measure to aid in the diagnosis and prognosis of ISSNHL, which is currently not done. With this approach, it may be possible to distinguish the two patterns of hearing loss shown in Fig. In the present study, we focused our study on audiograms of the healthy side in patients with ISSNHL. The pattern of the audiogram of the patient’s healthy side may reflect normal changes related not only to aging9. Although the study had few subjects, older patients with normal hearing on the healthy side had a better chance for recovery than those with hearing loss on the healthy side. These findings and ideas prompted us to study the influence of various factors on hearing level not only on the affected side of ISSNHL patients but also on the healthy side, as well as to determine whether they might be prognostic factors.There are some suggestions in the literature that analyzing the audiogram on the healthy side in patients with ISSNHL may be beneficial. One report suggested that the pattern of hearing levels on the healthy side are predictive of a good/poor prognosis in older patients with ISSNHL18. We hypothesized that certain atherosclerosis factors affect hearing levels on both the affected and healthy sides in ISSNHL patients and have the potential to provide an additional measure to classify ISSNHL patients. Specifically, patients with ISSNHL who also have atherosclerosis, are more likely to be severely affected, and because atherosclerosis likely affects both sides equally, age-related hearing loss on the healthy side may be more advanced. Thus, in patients with ISSNHL who also have factors linked to the development of atherosclerosis, the severity on the affected side and elevated PTA thresholds on the healthy side may occur simultaneously.We were particularly interested in evaluating the influence of known factors linked to atherosclerosis on ISSNHL occurrence and severity, because these factors are reported to also present a risk for triggering ISSNHL, and they can impact both severity of ISSNHL and the progression of age-related hearing lossThe aim of the present study was to evaluate possible associations between particular patient variables—mainly atherosclerosis factors—and hearing levels (PTA) on both the affected and healthy sides in patients with ISSNHL. Multivariate analysis allowed us to determine the association between hearing levels on the healthy side and prognosis of ISSNHL (recovered/not recovered), controlling for a number of patient variables.19. For the present study, we used the same dataset but excluded patients who had vestibular schwannoma, which was verified by head MRI. We also included additional data from the included patients’ charts for analysis in the present study. Any patients diagnosed with bilateral simultaneous onset of ISSNHL were also excluded.We conducted a retrospective study by analyzing data from medical charts of ISSNHL patients treated previously at six tertiary-level hospitals in Japan. These study subjects and some of their data were described fully in our previous report19 comprised audiometric, demographic, and basic medical data gathered from the medical records of 916 SSNHL patients that sought treatment between 2012 and 2020. For the included patients in the present study, we analyzed additional data from their charts about hearing prognosis (recovery/no recovery) and audiometric data from their healthy side. The diagnosis of ISSNHL was done using the criteria of the Sudden Deafness Research Committee of the Ministry of Health, Labour and Welfare (MHLW), Japan (2015) , hypertension (HTN), hyperlipidemia (HLD), atrial fibrillation (Afib), stroke or transient ischemic attack (TIA), venous thromboembolism (VTE), congestive heart failure (CHF), and any indicators of vascular disease 19. Additional data we collected from the patients’ charts for the present study were current or past smoking status (yes/no); hearing thresholds  at low frequencies ; hearing thresholds (dB) at middle frequencies ; hearing thresholds (dB) at high frequencies ; PTA on the healthy side; regular antiplatelet medication at onset of hearing loss (yes/no); regular anticoagulant medication at onset of hearing loss (yes/no), systemic steroid therapy, initial or salvage intratympanic steroid therapy; latency (days) to begin steroid treatment, prostaglandin E1 treatment, and whether they received hyperbaric oxygen therapy. We also gathered from the patients’ charts whether they recovered (yes/no) from their hearing loss related to ISSNHL. Some patients never received steroid treatment, systemic steroid therapy, or intratympanic steroid therapy. For those patients, they were manually assigned to the group who began steroid treatment 8 or more days after ISSNHL onset.We obtained data from the patients’ charts about their age, sex, affected side regarding ISSNHL (left ear [L] or right ear [R]), pure tone audiogram (PTA) on the affected side assessed at standard testing frequencies (below), presence of vertigo or dizziness (V/D) symptoms, and selected comorbidities250 + PTA500 + PTA1000 + PTA2000 + PTA4000)/5. The severity of hearing loss on the affected side was graded according to the following MHLW criteria, the Japanese guidelines for ISSNHL: Grade 1: PTA < 40 dB; Grade 2: 40 dB ≤ PTA < 60 dB; Grade 3: 60 dB ≤ PTA < 90 dB; and Grade 4: 90 dB ≤ PTA1. The severity of hearing loss on the healthy side was graded according to the following criteria: Grade 0: PTA < 25 dB; Grade 1: 25 dB ≤ PTA < 40 dB; Grade 2: 40 dB ≤ PTA < 60 dB; Grade 3: 60 dB ≤ PTA < 90 dB; and Grade 4: 90 dB ≤ PTA. Hearing loss at low, middle, and high frequencies was also estimated. Hearing loss at low, middle, and high frequencies was defined as a hearing loss of ≥ 30 dB at two or more of the three frequencies .To get a clearer picture of the patients’ hearing level, we calculated the arithmetic mean of individual PTA data obtained at five frequencies  as follows: mean PTA =  in patients with ISSNHL, we conducted multivariate regression analyses. Hearing levels on affected side and healthy side (dB thresholds) were analyzed in separate models. We carried out a three-step analysis. First, we selected independent variables related to hearing levels on each side based on previous literature. The selected independent variables on affected side included potential factors related to the severity of ISSNHL: patient age, DM, HTN, HLD, current or past smoking status; cardiac disease ; and presence of V/D symptoms2) in PTA (dependent variable) explained by certain clinical characteristics (independent variables). The independent variables were patient age, sex, affected ear (L/R), presence of V/D symptoms, current or past smoking status, and patient comorbidities . If a variable had a significant P-value of less than 0.20 for the outcome, it was included in the final multivariate model.Second, we performed separate univariate regression analyses for the affected and healthy sides. The single outcome we were interested in for each side was what was the percentage of variance  and the various independent variables. The independent variables selected in the first and second steps were incorporated into the final model. We performed normal log-transformation on the dependent variable (PTA) when residuals in the model were non-normal.22.To determine possible associations between hearing levels on the patients’ healthy side and prognosis for recovering from ISSNHL, we carried out a two-step statistical analysis. First, we used Fisher’s exact test to evaluate the relationship between recovery of hearing loss (yes/no) and other relevant data we collected. Recovery was defined as a study subject’s audiogram having one of the following two patterns: (1) the arithmetic mean of thresholds obtained at five frequencies  on the affected side was 20 dB or less; (2) If there was hearing loss on the healthy side and the hearing level on the healthy side was determined to be stable, the arithmetic mean of thresholds at five frequencies  on the affected side recovered to within 10 dB of the healthy side’s arithmetic mean of the thresholds at the five frequencies. These definitions were determined with reference to the Japanese and AAO-HNS guidelines which described the definition of recovery using the hearing level on the healthy side37. These variables were patient age, DM, HTN, HLD; use of systemic steroid therapy, intratympanic steroid therapy , latency to begin steroid treatment, use of prostaglandin E1, use of hyperbaric oxygen therapy; severity of hearing loss on the affected side, pattern of audiogram , severity of hearing loss on the healthy side; and presence of V/D symptoms. We also included as independent variables all variables from the univariate analyses that had a P-value of less than 0.20.For the second step of our analysis of prognostic factors, we conducted a multivariate regression analysis. The final multivariate model was developed by fitting a multivariate logistic regression model using recovery/no recovery of hearing loss as the dependent variable and the independent variables for factors previously reported as prognostic factors reported for ISSNHL recovery30.The cutoff values for MHLW Grade level of the affected side and Grade level of the healthy side were determined appropriately based on the distribution of Grades. The cutoff values for patient age and latency (days) to begin steroid treatment were based on values reported in previous studies38. Using a dataset that excluded patients with any missing baseline characteristics data, we calculated the probability of missing values in the outcome variable using logistic regression that included variables in baseline characteristics. We used an IPW-weighted multivariate logistic regression model to determine the ISSNHL prognostic factors and to compute odds ratios for the lack of ISSNHL recovery outcome.Since some patients lacked complete data and were excluded from the complete-case analysis, we conducted a sensitivity analysis to evaluate the stability of the prognosis results for recovering from ISSNHL. One straightforward approach for dealing with missing data is to analyze only cases that have no missing data. However, this method can induce bias. Inverse probability weighting (IPW) is a commonly used method to correct this bias. We used IPW to evaluate selection bias due to missing outcome data (recovery/no recovery from ISSNHL)39. EZR is based on R and R commander and is available at http://www.jichi.ac.jp/saitama-sct/SaitamaHP.files/statmed.html. Population means, standard deviations (SD), and counts were also calculated with EZR. For all tests, a P-value of < 0.05 was defined as statistically significant. For multivariate analyses, we used the variance inflation factor (VIF) to check for the presence of multicollinearity in the independent variables. A VIF cutoff of less than 5 was adopted. The VIF reflects the degree to which linear dependencies among the independent variables is inflated due to collinearity. It is generally accepted that a VIF value > 10 is unacceptable for multiple regression40.For all analyses, we used EZR  statistical software19, details of institutional approvals are described in that publication19. Briefly, the typical requirement to obtain written informed consent was waived by the institutional review boards because of the retrospective design. Participant data were anonymized, and all efforts were made to protect participant data. We carried out the present study in accordance with the guidelines of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement41.As the current study used the same dataset as that reported in our previous studyA total of 863 patients with ISSNHL met the inclusion criteria. However, 101 of the 863 patients had missing data and were excluded from the complete-case analysis Fig. . Thus, 72 = 0.145), while on the affected side, V/D symptoms accounted for the most variance (r2 = 0.050). For both the affected and healthy side, PTA was significantly associated with patient age, presence of V/D symptoms, DM, HTN, and vascular disease. Other factors linked with atherosclerosis showed different patterns in their association with PTA on the affected and healthy sides in patients with ISSNHL. Stroke or TIA, CHF, and VTE were significantly associated with PTA only on the affected side. Sex of the patient and Afib were significantly associated with the PTA only on the healthy side. HLD and Afib were the only factors in our univariate model that were not significantly associated with the PTA on the affected side, which previously had been reported to be closely associated with the occurrence and severity of ISSNHL12.Several factors previously linked to atherosclerosis predicted severity of hearing loss (higher PTA) in patients diagnosed with ISSNHL. Table Table Table Supplemental Table Supplemental Table 4, intralabyrinthine hemorrhage42, viral infection2, and autoimmunity3 are reported to contribute to this frightening condition. The diagnostic criteria for ISSNHL are constructed based on the sudden onset of hearing loss and the hearing pattern on the affected side, as revealed in the patient’s PTA. These currently used criteria make it difficult to clearly identify the pathogenesis of ISSNHL, as a variety of pathologies may lead to ISSNHL. This deficiency, in turn, makes it difficult to develop an appropriate treatment.The pathogenesis of ISSNHL is unknown, although circulatory disturbances44. Aging, DM, smoking, and a history of heart disease are reported to be factors in the severity of ISSNHL13. Aging, DM, and smoking are also factors in the development of atherosclerosis, which is in itself a risk factor for the development and severity of heart disease45.Focusing on the pathogenesis of circulatory disturbances, atherosclerosis factors that are reported to be associated with ISSNHL include DM, HTN, dyslipidemia (DLP), and cardiovascular disease (CVD)In the present study, we evaluated factors related to circulatory disturbances and whether they predict the severity and prognosis of ISSNHL. Specifically, we focused on factors linked to the development of atherosclerosis and whether these are associated with hearing thresholds on the healthy side of patients with ISSNHL. These factors are not currently included in the diagnostic criteria for ISSNHL. We also examined whether factors linked to atherosclerosis are predictive of ISSNHL recovery. Specifically, we focused on the relationship between hearing thresholds on the affected and healthy sides in ISSNHL and prognostic factors for ISSNHL recovery.12.We had two main findings. First, we documented significant associations between atherosclerosis factors and higher thresholds of PTA on the patients’ healthy side as well as on their affected side. Our study provided evidence that older ages, V/D, DM, and CHF are associated with higher thresholds of PTA on the patients’ affected side. Previous studies have reported that V/D, cardiac disease, atherosclerosis factors such as older age and DM are related to the severity of ISSNHL46. In the present study, older ages, being male, and vascular disease were associated with higher PTA thresholds on the healthy side. To the best of our knowledge, there have been no reports examining the patient factors that influence hearing in the healthy ear in patients diagnosed with ISSNHL.We also found evidence that the ISSNHL patients’ healthy sides also show impairments in hearing. Although there are no reports on risk factors associated with an increase in hearing thresholds on the healthy side of patients with ISSNHL, the primary risk factors are those related to aging. There are a number of reported risk factors for age-related hearing loss, including advancing age, being male, smoking, alcohol use, DM, HTN, heart disease, noise exposure, and genetic predisposition27. In addition to factors linked to atherosclerosis, other medical conditions and hearing parameters are reported to be associated with ISSNHL. The severity of hearing loss on the affected side , advanced age, V/D, a flat or “down-sloping” audiogram, and latency from onset of hearing loss to start of treatment appear to be associated with prognosis and recovery rate of ISSNHL30. There are also reports of positive therapeutic effects of various treatments. For example, systemic steroid therapy, intratympanic steroid therapy , prostaglandin E1 administration, and hyperbaric oxygen therapy are reported to have beneficial effects on ISSNHL, but none of these have been sufficiently evaluated in terms of how they impact recovery from ISSNHL37. In the present study, poor prognostic factors include presence of V/D symptoms, having an initial MHLW Grade 4 hearing loss in the affected ear, higher PTA thresholds at high frequencies in the affected ear, delaying steroid treatment 8 or more days after onset of hearing loss, initial Grade 2 or higher hearing loss in the healthy ear, and regular anticoagulant medication at onset of hearing loss. Some of these negative factors have been reported in the past. V/D, severe hearing loss in the affected ear, and delayed steroid treatment are reported to be poor prognostic factors for ISSNHL30. Higher PTA thresholds at high frequencies in the affected ear might have the same significance as that of flat or “downsloping” hearing audiograms, which have been reported to be poor prognostic factors28. To the best of our knowledge, there have been no reports indicating that higher PTA thresholds on the healthy side and regular anticoagulant medication at onset of hearing loss are poor prognostic factors for ISSNHL. However, the relationship between PTA on the healthy side and prognosis of ISSNHL, as well as the relationship between atherosclerosis factors and PTA on the healthy side, require further investigation. Although regular anticoagulant medication at onset of hearing loss might be a risk factor for intralabyrinthine infarction or hemorrhage, more rigorous investigation is needed, one that combines imaging of the inner ear and audiometric assessment in patients with ISSNHL.Our second main finding related to factors that affect the chances for hearing recovery in ISSNHL patients. Factors linked to atherosclerosis are reported to be prognostic factors for whether patients recover from ISSNHL. Specifically, the comorbidities of DM, HTN, DLP, metabolic syndrome, and Framingham CVD risk score lower the rate of ISSNHL recovery and lead to poorer prognosisContrary to our expectations, HTN in the present study was a favorable prognostic factor for ISSNHL. Since the HTN variable in the present study was retrospective data gathered through chart review, we know that most of the study patients were treated properly with an antihypertensive drug. Therefore, lacking data about accurate blood pressure measurements at the point of ISSNHL development may have confounded our results.The present study has two strengths. Because we were able to analyze data from a relatively large number of patients from six different hospitals, we were able to confidently carry out multivariate regression analyses. This first strength allowed us to comprehensively examine PTA on both the affected and healthy sides of patients with ISSNHL and rigorously evaluate several factors linked to atherosclerosis that may be predictors of ISSNHL and prognostic factors for recovery. The second strength was that we evaluated not only prognostic factors’ possible relationship to PTA on the affected side but also to PTA on the healthy side, which has not been reported as a prognostic factor for ISSNHL. With further study, our results might point to new advice about predicting prognosis of ISSNHL by assessing PTA on the healthy side.The present study also has three possible limitations. First, the study may be influenced by selection bias related to the data coming from retrospective chart review. However, there were few missing outcome values in the prognostic factors analysis, and the results of IPW-weighted analysis were similar to those of the complete-case analysis. Moreover, the cases were consecutive cases arriving at the six hospitals. Therefore, we place high confidence in the reliability of the results.Another cautionary note is that we mainly examined known factors linked to atherosclerosis in the analysis of hearing on the healthy side. Therefore, other factors that are known to affect hearing on the healthy side, such as noise exposure, alcohol consumption, and genetic predisposition, were not investigated.Third, only a small number of patients received salvage treatment such as hyperbaric oxygen therapy or intratympanic steroid therapy in the present study. Therefore, insufficient adjustment for salvage treatment might have affected the analysis of hearing recovery. In Japan, intratympanic steroid therapy is rarely selected due to medical insurance coverage. In addition, hyperbaric oxygen therapy is not common in Japan because few medical facilities have the equipment for hyperbaric oxygen therapy.In conclusion, certain atherosclerosis factors appear to be associated with higher PTA hearing thresholds not only on the patients’ affected side in ISSNHL but also with higher hearing thresholds on their healthy side. Higher hearing thresholds of 40 dB or higher on the healthy side were associated with poor prognosis for recovery from ISSNHL. Thus, a clearer picture of a patient’s ISSNHL onset and chances for recovery may emerge from a more rigorous evaluation of hearing levels on the unaffected, or healthy side, in addition to that done on the affected side.In the near future, we will conduct prospective studies of ISSNHL and the impact of atherosclerosis factors, focusing on hearing on the healthy side. In addition, it will be informative to determine prospectively whether intralabyrinthine infarction or hemorrhage is present on MRI in patients with ISSNHL who were taking regular anticoagulant medication at the time of sudden hearing loss onset.Supplementary Tables.


This study examines the effects of TCS on thyroid hormone secretion and the early development of zebrafish. The fertilized zebrafish eggs were exposed to TCS at 0 (control), 3, 30, 100, 300, and 900 ng/mL, and the hatching rate and the larvae mortality were inspected within the first 14 days. The total triiodothyronine (TT3), total thyroxine (TT4), free triiodothyronine (FT3), and free thyroxine (FT4) were measured at 7, 14, and 120 days post-fertilization (dpf). The histopathological examinations of thyroid follicles were conducted at 120 dpf. TCS exposure at 30-300 ng/mL reduced the hatching rate of larvae to 34.5% to 28.2 % in the first 48 hours and 93.8 .7 % to 86.8 % at 72 h. Extremely high TCS exposure (900 ng/mL) strongly inhibited the hatching rate, and all the larvae died within 1 day. Exposure to TCS from 3 to 300 ng/mL reduced the thyroid hormones production. The mean TT3 and FT3 levels of zebrafish decreased in 300 ng/mL TCS at 14 dpf , and the FT4 decreased at 120 dpf ( 0.09 ± 0.04 vs. 0.20 ± 0.14 pg/mg). At 120 dpf , in the 300 ng/mL TCS exposure group, the nuclear area and the height of thyroid follicular epithelial cells became greater, and the follicle cell layer got thicker. This happened along with follicle hyperplasia, nuclear hypertrophy, and angiogenesis in the thyroid. Our study demonstrated that early life exposure to high TCS levels reduces the rate and speed of embryos hatching, and induces the histopathological change of thyroid follicle, and decreases the TT3, FT3, and FT4 production in zebrafish.Triclosan (TCS) is an antimicrobial chemical widely used in personal care products. Most of the TCS component is discharged and enters the aquatic ecosystem after usage. TCS has a similar structure as thyroid hormones that are synthesized by thyroid follicular epithelial cells, thus TCS has a potential endocrine disrupting effect. It is still not clear how the different levels of the environmental TCS would affect early development  Thyroid hormones are synthesized and secreted by the thyroid follicular epithelial cells, and are critical for fetal growth and neuro-development in early life , 2. The 4) and triiodothyronine (T3) levels  levels , 27 at t) levels , 29. Mor) levels , but fewZebrafish has been a good model for studies of thyroid function, since the development of its thyroid system is comparable to human . None haTCS  phenol, ≥ 97.0 % purity (HPLC), CAS no. 3380-34-5) and dimethyl sulfoxide  were purchased from Sigma-Aldrich .Zebrafish strains of AB wild-type line were used in the study. The male and female zebrafish at 5-months-old were acclimated in tanks containing dechlorinated tap water for 4 weeks before mating under a photoperiod of 14:10 h light/dark cycle. The fish were fed with brine shrimp twice per day.Fertilized eggs were collected within 30 min after natural mating and unfertilized eggs were discarded. Fertilized eggs were randomly assigned to 0 (0.01 % DMSO as solvent control), 3, 30, 100, 300, and 900 ng/mL TCS (600 eggs per group) and raised up to 7 and 14 days at 28.0 ± 0.5°C under 14:10 h light/dark photoperiod cycle . These TThere were 200 fertilized eggs with a density of one embryo per 2 mL medium put into a culture dish until 72 h post-fertilization (hpf). The larvae were transferred into glass tanks with a density of one larva per 60 mL medium until 14 days post-fertilization (dpf) . After 1The hatching rate was calculated as the number of larvae hatched during the first 3 days divided by the total number of fertilized eggs. The death of larvae was inspected and recorded twice per day for first 14 days, and the dead larvae were removed from dishes/tanks. The mortality was calculated as the number of dead larvae divided by the total number of hatched larvae.At 7, 14, and 120 dpf, the zebrafish were euthanized by immersing in ice-cold water for 40 min , 38, and3), free thyroxine (FT4), total triiodothyronine (TT3), and total thyroxine (TT4) levels were measured by ELISA   to normalize thyroid hormone concentration  . The larntration . The limThe histological analysis of the thyroid gland was conducted in zebrafish at 120 dpf. The heads of zebrafish were fixed in 10% neutral formalin  for at least 12 h, and transferred to 70% ethanol, all at room temperature. Each zebrafish head was placed in processing cassettes, dehydrated through a serial alcohol gradient, and embedded in the wax blocks.Serial transverse cross-sections were cut using a microtome 5 μm and dewaxed in xylene, rehydrated through decreasing concentrations of ethanol, and washed in PBS. The sections were then stained with hematoxylin for 4−5 min and with eosin for 1−2 min . Thyroid follicles were located according to their being dispersed among the afferent branchial arterioles  in the subpharyngeal region . StainedPhotographs of each follicle were taken at the largest follicle diameter  with an Olympus digital camera . The height of follicular epithelial cells and the long and short diameters of their cell nuclei were quantitatively analyzed. The nuclear size was calculated as the long diameter × short diameter × π/4 . At leasP value < 0.05. All analyses were performed using the SAS 9.3 software .The ANOVA F-test was used to compare the differences in thyroid hormone levels, the height of thyroid follicle epithelial cells and nuclear size, and chi square test was used to compare the difference in hatching rate and mortality of larvae among TCS exposure groups. We used linear regression models to evaluate the associations of TCS exposures with thyroid hormones and thyroid follicle histopathological changes. The level of significance was two-sided P < 0.01. All the hatched larvae presented a well-developed head, body, and tail within 72 h. At 48 hpf, the hatching rate was 42% in the control group, and 47.8 % in 3 ng/ml TCS exposure group . But with the increase of TCS exposure concentrations , the hatching rate significantly dropped from 34.5 % to 4.3 % in 30 - 900 ng/mL TCS exposure . The TT3, TT4, and FT3 levels were comparable among the five TCS exposure groups. After adjusted for sex, the FT4 levels were still lower in TCS 30, 100, and 300 ng/mL groups than that in the control group . TCS exposure induced follicle hyperplasia  and hypertrophy (enlarged follicular cell size and nuclear size), shown as increased nuclear area and cell height of thyroid follicle epithelial cells  of thyroid follicular cell turned larger in the thyroid gland of adult zebrafish at 120 dpf.In this study, we examined the disrupting effects of early exposure to TCS on the thyroid hormones in larvae and adult zebrafish, and the pathological changes of thyroid follicles. We found that the hatching rate of zebrafish embryos reduced whin 48 hpf with high TCS exposure, while the hatching rate became similar as the control when exposing to TCS at 0 - 300 ng/mL. TCS exposure did not change the mortality of larvae at 0 - 300 ng/mL, either. But in the extremely high TCS exposure (900 ng/mL), both the hatching rate and the survival of larvae were severely reduced. As to the effects of TCS on thyroid hormones, the levels of TTZebrafish is an ideal animal model for study of chemical pollutants in water and thyroid hormone-disruption, partly due to its high (71 %) genetic similarity to humans . We set 4 and T3 levels at 9 days post-hatching (dph) and 12 dph, and increased T4 levels at 15 dph in Cyprinodon variegatus. Pinto et al. (via diet) induced a reduction in circulating thyroid hormones, hyperplasia of follicles and the thyrocyte height. In our experiments, the TT4 and FT4 levels tended to be decreased with higher TCS exposure after 7 days, although they were not significant. The obvious thyroid disrupting effect by TCS shown at 14 dpf, and the FT4 decreased significantly with the increasing TCS exposure concentration both at 14 and 120 dpf. The impact of TCS exposure on thyroid hormone levels (TT3 and FT3) at 14 dpf provides the causal support for the findings in the human population  based on the environmental TCS levels, including wildlife and general population TCS exposure levels in a natural environment. The 3-100 ng/mL TCS is the range of urinary TCS concentrations in pregnant women , and 3-34 and FT4, but no change was found in the levels of blood thyroid stimulating hormone (TSH)  , 62–64.  in mice , 65, or s laevis .4 metabolism and decreased T4 bioavailability by increasing the hepatic enzymes activity of glucuronyltransferase and pentoxyresorufin-O-deethylase (PROD) in the liver  axis or due to the increasing thyroid hormones clearance. Some mechanisms have been studied but the data were controversial. TCS exposure may promote  or inhibhe liver –69, or bhe liver , 61. It he liver . Therefo3 level in Chinese newborns  in 30, 100, and 300 ng/mL TCS exposure. This is the first study to explore the thyroid disturbed effect by long-term TCS exposure starting from the early life in zebrafish. It has been reported that the formation, growth, and differentiation of thyroid follicles in zebrafish appear to be independent of TSH (Our study found that zebrafish exposed with TCS for 120 days (from embryo to adult phase) caused thyroid histopathology changes in 3, 30, 100, and 300 ng/mL TCS exposure, and disturbed the thyroid hormone level  levels in the zebrafish larvae and the FT4 level in adult zebrafish (even adjusted for age). Moreover, TCS exposure affected the structure of thyroid follicles by increasing nuclear area and epithelial cell height of zebrafish thyroid follicles. Considering the reduced level of thyroid hormones which are crucial for neurodevelopment (We found that exposure to TCS delayed the hatching of zebrafish embryos, decreased the thyroid hormone (FTelopment , our stuThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving animals were reviewed and approved by the Ethics Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.FO conceptualized the study. FO, NT, and PF implemented the study, FO and NT interpretated data and drafted the manuscript. FO, NT, PF, WYW, WJW, and XY contributed to acquisition and analysis of research data. LC had intensively revised the manuscript. All authors have reviewed and approved the manuscript as submitted.This study was supported by grants from the National Natural Science Foundation of China , Shanghai Municipal Education Commission - Gaofeng Clinical Medicine Grant [grant number 20152518], and in part by the Collaborative Innovation Program of Shanghai Municipal Health Commission [grant number 2020CXJQ01].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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.