However, controversial data have been reported. We present measures of the peripheral gustatory system activation in response to taste qualities by electrophysiological recordings from the tongue of 39 subjects classified for PROP taster status. The waveform of the potential variation evoked depended on the taste quality of the stimulus. Direct relationships between PROP sensitivity and electrophysiological responses to taste qualities were found. The largest and fastest responses were recorded in PROP super-tasters, who had the highest papilla density, whilst smaller and slower responses were found in medium tasters and non-tasters with lower papilla densities. The intensities perceived by subjects of the three taster groups correspond to their electrophysiological responses for all stimuli except NaCl. Our results show that each taste quality can generate its own electrophysiological fingerprint on the tongue and provide direct evidence of the relationship between general taste perception and PROP phenotype.Taste buds containing receptor cells that primarily detect one taste quality provide the basis for discrimination across taste qualities. The molecular receptor multiplicity and the interactions occurring between bud cells encode information about the chemical identity, nutritional value, and potential toxicity of stimuli before transmitting signals to the hindbrain. PROP (6- Taste buds are the peripheral organs of the gustatory system and are located on the surface of three different taste papillae, which are topographically arranged mostly on the tongue epithelium. These include the fungiform papillae on the anterior surface, foliate papillae on the lateral sides, and circumvallate papillae on the posterior part. Taste buds are clusters of 50–100 different types of columnar taste cells extending from the base to the top of the cluster, in which the mechanisms of taste reception and transduction are located .2+ [Taste bud cells are differentiated into three types according to their ultrastructure and function: type I, type II, and type III cells . Type I 2+ ,11, open2+ ,13,14. T2+ ,17,18,192+ imaging and electrophysiological experiments [The specific sensitivities of taste cells within taste buds provide the basis for discrimination across taste qualities . The seneriments ,21. Theseriments ,21,22 aneriments ,24,25,26eriments . The sigeriments . A multieriments ,29.n-propylthiouracil (PROP) has gained a consistent and appreciable consideration as a marker of general taste perception, dietary preferences, and habits that can impact on nutrition and health of individuals [rs1761667 SNP of the gene coding for the fatty acid scavenger receptor CD36 [In humans, taste perception varies greatly among individuals and is one of the most significant determinants influencing food preferences and therefore eating behavior, metabolism, and health ,30. Withividuals ,34,35,36ividuals , sour coividuals , umami tividuals , irritanividuals ,41, highividuals ,43,44, aividuals , and fruividuals ,47,48. Sividuals ,50, and ividuals ,52. Dataividuals  or healtividuals , body maividuals ,56, metaividuals ,58, smokividuals , consumpividuals ,65,66,67ividuals , and devividuals ,70,71, aividuals . Howeverividuals ,76,77,78ividuals ,80,81. Wividuals . Furthertor CD36 .The purpose of the present work is twofold. Firstly, we extend our previous results ,80,81 toSecondly, we verified the validity of using PROP tasting as a biomarker of general taste perception by comparing the electrophysiological response to each taste quality in subjects belonging to the three PROP taster categories , as an objective measure of their taste sensitivity for the different taste qualities. Since our previous studies showed robust relationships between the magnitude of electrophysiological taste responses and fungiform papillae densities, the latter measurements were also collected here.2. None had food allergies or were following a specific diet or taking medications that could interfere with taste sensitivity. Their taste function for the four basic tastes was screened by the taste strip test  to exclude any taste impairment. Subjects read and signed an informed consent form. This study was carried out in accordance with the latest revision of the Helsinki Declaration, and all procedures have been approved by the Ethical Committee of the University Hospital Company (AOU) of Cagliari, Italy. The trial was registered at ClinicalTrials.gov (identifier number is UNICADBSITB-1).Thirty-nine Caucasian non-smoking subjects  were recruited through usual procedures at the University of Cagliari. They were originally from Sardinia, Italy. No statistical methods were done to predetermine sample size. However the following guiding criteria were used: sample size was similar to the one already employed in electrophysiological experiments that assess the degree of activation of the receptors under study ,81; subjEach subject was tested on two consecutive days: on the first day, he/she was classified for PROP taster status. On the second day, he/she was tested for the electrophysiological responses to stimulations with six taste qualities and for density of fungiform papillae. All subjects had to abstain from drinking (except water), eating, using oral care products, or chewing gum for at least 2 h prior to testing. They had to be in the experiment room 15 min before the starting of the test (9.00 a.m.) in order to acclimate to the constant environmental conditions . Women were examined around the sixth day of the menstrual cycle to avoid changes of taste function resulting from the estrogen phase . StimuliSubjects were classified for their PROP taster status by two scaling methods. All were first assessed using the three-solution test according to Tepper et al. 2001 , which hDifferential electrophysiological recordings from the tongue of subjects were performed according to Sollai et al. ,81. BrieThe waveform of bio-potentials was analyzed by Clampfit 10.0 software . The voltage changes  in response to six taste qualities were obtained by subtracting the baseline value from the voltage at 0.1, 2.5, 5, 10, and 15 s from stimulation onset. The rate of potential variation (mV/s) was also calculated at the same time intervals.Taste stimulations were delivered by placing, for 15 s a paper disk (6 mm dia.) impregnated with 30 µL of taste solutions, or 30 µL of undiluted oleic acid, on the circular area of the tongue surface, which was left free by the hole of the second electrode. Sucrose (200 mM), NaCl (200 mM), citric acid (5.2 mM), caffeine (10 mM), and monosodium glutamate (MSG) (160 mM) solutions were used to represent the five primary taste qualities . The concentration for each stimulus was chosen based on preliminary tests. In order to verify that the potential variations recorded were certainly due to taste stimulations and not mechanical stimulations caused by placing the disks, subjects were also tested with dry paper disks.2 was calculated.The total number of fungiform papillae was measured in the same circular area of the tongue where the stimulations were performed during electrophysiological recordings according to Melis et al. 2013 . This arp values < 0.05 were considered significant.Repeated-measures ANOVA was used to analyze differences of mean values ± SEM of the bio-signal amplitude (mV) and potential variation rate (mV/s) evoked by the six taste qualities at 2.5, 5, 10, and 15 s after the application of stimulation. Data were also analyzed across PROP taster groups. Two-way ANOVA was used to compare differences in perceived intensity rating for the six taste qualities in super-tasters, medium tasters, and non-tasters. One-way ANOVA was used to compare differences in density of fungiform papillae according PROP taster status. Data were verified for the assumptions of normality, sphericity (when applicable), and homogeneity of variance. A Greenhouse–Geisser correction or Huynh–Feldt correction was applied whenever the sphericity assumption was violated. Post hoc comparisons were conducted with the Fisher’s least significant difference (LDS) test or Duncan’s test when the assumption of homogeneity of variance was violated. Statistical analyses were conducted using STATISTICA for WINDOWS . The electrophysiological recordings from the human tongue allowed the measurement of monophasic bioelectrical potential changes in response to taste stimulations, with respect to the baseline. NaCl stimulation evoked depolarizing monophasic potential changes, whereas all the other stimuli evoked hyperpolarizing monophasic potential changes. However, the waveform analysis of bioelectrical potentials allowed the observation that each stimulus determined a characteristic time course of the potential variation during stimulation. Examples of this are shown in 20,820 = 37.840; p < 0.0001 and rate: F20,820 = 5.5235; p < 0.0001). Post hoc comparison showed that all stimuli produced significant potential changes, with respect to baseline, after 2.5 s from stimulus application . The largest positive potential changes were recorded in response to citric acid and sucrose , the intermediate ones in response to caffeine and MSG , and the lowest in response to oleic acid ; negative potential changes were recorded in response to NaCl . Successively, the voltage variations elicited by citric acid decreased significantly at 10 s ; those in response to sucrose, caffeine, and MSG did not change during the stimulation time (p > 0.05); while that in response to oleic acid slowly continued to increase  to the end of stimulation. The depolarization in response to NaCl did not change during the stimulation time (p > 0.05).The mean values ± SEM of the potential amplitude (mV) and of potential change rate (mV/s) determined after 0.1, 2.5, 5, 10, and 15 s from the stimulus application in response to the six taste qualities are shown in p < 0.0001; Fisher LDS), then the values rapidly decreased across the time course of electrophysiological recordings, being halved at 2.5 s  and halved again at 5 s . Slower potential changes were determined at 0.1 s in response to sucrose , which significantly decreased only at 5 s . NaCl and caffeine produced intermediate values of potential change rate, which did not vary with time . The lowest values, unchanged over time, were observed with MSG and oleic acid .The quickest potential changes were recorded at 0.1 s in response to citric acid . After 2.5 s from stimulation, all stimuli evoked significant potential changes in all subjects , except for oleic acid in the non-taster group (p > 0.05). PROP super-tasters showed larger amplitudes than other PROP taster groups. Specifically, super-tasters showed larger signal amplitudes in response to NaCl and sucrose compared to those of other PROP taster groups at 2.5, 5, 10, and 15 s , as well as larger signal amplitudes in response to MGS and citric acid than those of other taster groups at 2.5 and 5 s . Super-tasters also showed larger responses to oleic acid than those of non-tasters at 10 and 15 s  and medium tasters showed higher values for oleic acid relative to non-tasters at 15 s . The amplitude of signals in response to caffeine was slightly higher in super-tasters than in other PROP taster groups but not in a significant way.The mean values ± SEM of the potential amplitude (mV) and of potential change rate (mV/s) determined after 0.1, 2.5, 5, 10, and 15 s from the application of stimulation with the six taste qualities according to PROP taster status are shown in 40,772 = 5.541; p < 0.00001). PROP super-tasters showed a quicker hyperpolarization with respect to the other taster groups, at 0.1 and 2.5 s, in response to sucrose and citric acid ; this same effect was only seen for caffeine at 0.1 .The mean values ± SEM of the potential change rate (mV/s) determined after 0.1, 2.5, 5, 10, and 15 s from the application of stimulation in response to the six taste qualities according to PROP taster status are shown in p ≤ 0.019; Fisher LDS, subsequent to two-way ANOVA), except for oleic acid for which the ratings of super-tasters were higher than those of non-tasters only . No significant difference related to PROP taster status was found for NaCl.The mean values ± SEM of the rating of the perceived intensity for the six taste qualities according to PROP taster status are shown in 2,33 = 13.105; p = 0.00006). PROP super-tasters had a higher density than medium tasters , who showed higher values than non-tasters .The mean values ± SEM of density of fungiform papillae determined in super-tasters, medium tasters, and non-tasters are shown in The first aim of this work was to characterize the electrophysiological responses evoked in taste buds on a localized area of the human tongue by stimulation with the six taste qualities, as a direct and quantitative measure of the degree of activation of the peripheral taste system generated by each taste stimulus. Interestingly, we found that each taste quality evoked a monophasic voltage change with a characteristic time course during stimulation. In fact, a comparative analysis of values of potential amplitude and of potential change rate, determined during stimulation, revealed that the waveform of the signal depends on the taste quality of stimulus. Although, the bioelectrical activity generated by all stimuli was represented by a monophasic potential change characterized by a fast-initial variation followed by a slow decline, NaCl was the only stimulus that evoked negative potential variations, while all other stimuli evoked positive potential changes, which nevertheless were differed from one another. +, by blocking a proton-sensitive K+ channel, and depolarize the cell membrane. On the other hand, the transduction mechanisms of sucrose, caffeine, MGS, or oleic acid are mediated by GPCRs, causing activation and diffusion of second messengers, which need more time to depolarize the cell. Differences in type of taste cells activated and in transduction mechanisms across taste qualities might explain these differences. Sweet, bitter, umami, and fat stimuli directly activate type II cells, which express specific receptors and, via ATP released during their tastant-induced stimulation, indirectly activate the adjacent type III cells, which instead directly respond to sour stimuli ,21. As m+ permeation through membrane ion channels [As noted above, the negative potentials we observed in response to NaCl stand in contrast to those of all other taste stimuli . Although the taste bud cells involved in the salty taste transduction have not been conclusively identified ,2, saltychannels . HoweverTaken together, these results indicate that each taste quality can generate a specific electrophysiological response in taste buds, and they strongly support other data showing that cell-to-cell signal interactions within taste buds, may represent a primary localization step where taste quality discrimination begins . Our results shed additional light on the validity of using the PROP phenotype as a biomarker of general taste perception. We found a direct relationship between PROP sensitivity and the electrophysiological response evoked by the six taste qualities in the buds of a localized area of human tongue. Specifically, the largest and quickest responses were recorded in PROP super-taster subjects, who had the highest density of fungiform papillae in the same area of the tongue where stimulations were applied during the recordings. Smaller and slower responses were observed in medium taster and non-tasters, who had lower densities of papillae in the same area of the tongue. The analysis of the time course of parameters defining the waveform of signals evoked by NaCl, sucrose, caffeine, MGS, and citric acid revealed that the super-taster phenotype, which was associated with a highest density of papillae, confers additional advantage already in the initial phase (2.5 s) of the response, which is certainly the most significant one to induce a behavioral response. Super-tasters showed a more prompt and intense response to sucrose and citric acid, a more intense one to NaCl and MGS, and a more rapid one to caffeine, as compared to medium tasters and non-tasters. Differently, the super-taster phenotype was important to elicit a large potential change in the last part of the response to oleic acid. The signal amplitude values slowly increased during stimulation in super-tasters, while no changes in the whole time course of recordings were found in medium tasters and non-tasters. This extended activation evoked by oleic acid in super-tasters, having a higher density of papillae, may reflect the high surface tension of the molecule that determines its slow diffusion toward the taste bud cells. These results strongly support previous psychophysical data showing a direct relationship between perception of a wide range of oral stimuli and PROP taster status ,46,47,48Importantly, the differences in the electrophysiological responses to sucrose, caffeine, MGS, oleic acid, and citric acid that we recorded in the PROP taster groups agree with the intensity ratings given by these subjects during oral stimulations, indicating that our bioelectrical measurements are consistent with common human psychophysical observations. It is noteworthy that the amplitude of electrophysiological response to NaCl by super-tasters was larger than those for medium tasters and non-tasters, however, the perceived intensities reported by subjects did not vary with PROP taster status. This is consistent with the psychophysical procedures for the classification of subjects by PROP taster status, which use NaCl as standard control showing that the rating of perceived intensity for this stimulus does not vary with the PROP phenotype of subjects . It is aThe findings of the present study were achieved thanks to a novel electrophysiological recording technique from the human tongue to directly and quantitatively measure the degree of activation of the peripheral gustatory system in response to taste stimuli ,81. ResuIn addition, our data provide the first direct and objective demonstration of the role of PROP phenotype in individual variability of general taste perception. They show that the influence of PROP status on the amplitude of electrophysiological response may reflect a summation effect associated with differences in papillae density, a well-known anatomical characteristic of the PROP phenotype ,94,95,96Finally, psychophysical measures have played a critical role in understanding human chemosensory experiences, by permitting the determination of taste responses at the CNS level, which can influence food choices and eating behaviors. Nevertheless, because taste intensity measures are subjective and sensitive to reporting bias, they are less useful for gaining insights into taste mechanisms, particularly those rapid responses that occur within the initial few seconds of oral exposure to a stimulus. The present work helps to fill gaps in knowledge of these processes by combining traditional behavioral methods with our electrophysiological recording method.


Purpose: The presence of endolymphatic hydrops (EH) in patients with intralabyrinthine schwannomas (ILSs) is poorly understood. This study aims to determine whether there is a correlation between endolymphatic hydrops and clinical presentations of ILS.Methods: Data from nine patients with ILSs were retrospectively reviewed between 2007 and 2020. Temporal bone MRI with intratympanic or intravenous injection of gadolinium was applied to detect ILSs and EH.Results: 3D real inversion recovery (IR) sequence MRI of the temporal bone confirmed ipsilateral EH in four patients (4/6). All four patients with EH on MRI presented with vertigo similar to Meniere's disease. Among these patients with EH, one patient with EH in the cochlea showed moderate sensorineural hearing loss, while three patients with EH in both the vestibule and cochlea showed profound hearing loss. MRI demonstrated a transmacular tumor (TMA) in one patient, intravestibular (IV) in four patients, and vestibulocochlear (VC) in four patients. Two IV cases showed moderated hearing loss, while the TMA and VC cases showed profound hearing loss. Transotic resection of the tumor was applied in five patients; translabyrinthine resection was applied in one patient; two patients were under observation; and one patient was given intratympanic injection of gentamicin (ITG). During follow-up, all of the treated patients reported relief of vertigo, and postoperative MRI was performed in two patients, which showed no tumor recurrence. The two patients under observation showed no deterioration of hearing loss or vertigo. One patient was lost to follow-up.Conclusion: EH concurrent with ILSs has been underestimated previously. With the extensive application of temporal bone MRI paradigms, such as 3D-real IR sequence MRI, more cases of potential EH in patients with ILS will be identified. The severity of hearing loss may be associated with the location of the tumor and the degree of EH. Intralabyrinthine schwannomas (ILSs) are rare benign tumors within the membranous labyrinth that are reported to arise from the distal branches of the cochlear, superior vestibular, or inferior vestibular nerves , 2. MayeUnilateral progressive hearing loss and recurrent vertigo are the most common symptoms in patients with ILSs , 7. ThesEndolymphatic hydrops (EH) is considered a pathological hallmark of Meniere's disease. However, EH can also be detected in other inner ear diseases, such as delayed endolymphatic hydrops, endolymphatic sac tumors, extralabyrinthine and intralabyrinthine schwannomas –10. ShinIn this study, we retrospectively reviewed the medical records of nine patients with ILS and aimed to (1) demonstrate the occurrence of EH in ILSs; (2) reveal the correlation between clinical symptoms and imaging features of endolymphatic hydrops; and (3) discuss the diagnostic pitfalls of ILS.The clinical data of nine patients with ILS who were treated at the Otology & Skull Base Surgery Department, Fudan University, between 2007 and 2020, were retrospectively reviewed. We retrieved data on age, symptoms, pure tone test, imaging, and management.The diagnosis in six patients with ILS was confirmed by pathological examination postoperatively, while the other three patients who chose a wait-and-see strategy were diagnosed based on imaging. Six patients underwent both enhanced temporal bone MRI and temporal bone MRI with intratympanic or intravenous injection of gadolinium to detect EH. Patients 1, 2, and 3 only underwent enhanced temporal bone MRI, since MRI with intratympanic or intravenous injection of gadolinium was unavailable at that time in our hospital. According to the anatomic classification developed by Salzman et al. , ILSs inFor the intratympanic injection (IT) method, the patients received bilateral intratympanic injections of gadolinium diluted in saline (v/v 1:7) using a 22-gauge spinal needle and a 1-ml syringe. Gadopentetate dimeglumine was chosen in this research as the gadolinium contrast agent. Following the injections, the patients maintained an upright seated position for 30 min without speaking or swallowing. After 24 h, MRI scans were performed using a 3 T MR unit  with a 32-channel phased-array receive-only head coil. T2-space, 3D-real IR, and 3D-FLAIR sequence MRI images were collected. Briefly, the parameters for the 3D-real IR sequence were as follows: voxel size of 0.4 × 0.4 × 0.8 mm, scan time of 14 min, repetition time (TR) of 9,000 ms, echo time (TE) of 181 ms, inversion time (TI) of 1,730 ms, slice thickness of 0.80 mm, field of view (FOV) of 160 × 160 mm, and matrix size of 3,300 × 918. The parameters for the 3D-FLAIR sequence were as follows: voxel size of 0.7 × 0.7 × 0.6 mm, scan time of 6 min, TR of 6,000 ms, TE of 387 ms, TI of 2,100 ms, slice thickness of 0.60 mm, echo train length of 173, FOV of 220 × 220 mm, and matrix size of 1,701 × 810.For the intravenous (IV) method, the patients received an intravenous injection of a double dose (0.4 ml/kg body weight) of Gd-HP-DO3A; 4 h later, MRI was performed. All scans were performed on a 3 T MRI scanner  using a 32-channel phased array receive-only coil. T2-space and 3D real-IR sequence MRI scans were applied to collect images. The parameters for the 3D real-IR sequence were as follows: voxel size of 0.17 × 0.17 × 0.6 mm, scan time of 15 min and 20 s, repetition time of 6,000 ms, echo time of 181 ms, inversion time of 1,850 ms, slice thickness of 0.6 mm, field of view of 160 × 160 mm, and matrix size of 768 × 768.The clinical characteristics of the study cohort are summarized in Temporal bone MRI with enhancement was performed in nine patients, which revealed a hypointense filling defect within the labyrinthine on T2-weighted images, and enhanced T1-weighted images revealed a homogeneously enhanced mass within the labyrinthine in these patients . IntraopIn the present study, MRI demonstrated a transmacular tumor in one patient, intravestibular tumors in four patients, and vestibulocochlear tumors in four patients . The mea3D real inversion recovery (IR) sequence MRI of the temporal bone was applied in six patients to demonstrate endolymphatic hydrops, with the IV method applied in four patients and the IT method used in two patients. Four patients showed an intralabyrinthine hypointense mass concurrent with endolymphatic hydrops. Three patients presented with endolymphatic hydrops in both the cochlea and vestibule , and oneFive patients  with profound hearing loss underwent translabyrinthine resection of the tumor. Patient 2 with moderate hearing loss underwent surgery due to intractable vertigo. Patients 4 and 5 with serviceable hearing and tolerable vertigo chose conservative management. Intratympanic injection of low-dose gentamicin was used in patient 9 to relieve vertigo. No postoperative facial paresis, cerebrospinal fluid leakage, or other severe complications were encountered, except patient 7 who had delayed facial paresis 1 week after surgery and recovered completely in 1 month.Eight patients were followed up. One patient was lost to follow-up. The mean follow-up time was 48.6 months . During follow-up, five patients who underwent surgery reported no vertigo attacks, and two out of these five underwent MRI surveillance and did not show tumor recurrence . Two patILSs are rare disorders, but they may have been underestimated before. Van Abel et al. reported that the average delay between symptom onset and diagnosis of ILS was 7.0 ± 8.0 years . CurrentILS is also known as primary inner ear schwannoma, since this term differentiates ILS from extralabyrinthine schwannomas that involve the labyrinthine, thus more precisely describing this lesion . KennedySensorineural hearing loss is one of the most common symptoms of ILSs . It can Recurrent vertigo attacks, which mimic Meniere's disease, is another common symptom of ILSs, and combined with hearing loss and tinnitus, patients with ILS are prone to be misdiagnosed with MD at the early stage. Unfortunately, we still do not know if there are any typical differences in symptoms between patients with MD and patients with ILS, especially at the early stage. Though a differentiating clinical parameter between these two entities might be the extent of hearing loss. In ILS, frequently, total deafness occurs, whereas most MD cases end up with severe sensorineural hearing loss but not total deafness. A cohort study over a long period of time or a meta-analysis between the two groups could be meaningful to reveal the difference.Jerin et al. reported two cases of ILS presenting with clinical symptoms that were similar to patients suffering from delayed endolymphatic hydrops, but no EH was demonstrated on temporal bone MRI in the two cases. A few studies have reported recurrent vertigo attacks in patients with ILS, mimicking Meniere's disease or delayed endolymphatic hydrops. However, few studies have shown that EH can be identified in patients with ILSs –14. HomaIn the present study, the intralabyrinthine mass coexisting with EH was identified in four out of six patients using 3D real IR sequence MRI with injection of gadolinium. All four patients with EH on MRI presented with vertigo similar to Meniere's disease, so we assumed that EH is one typical imaging characteristic of ILSs, even in the early stage. It is hard to say whether EH or the intralabyrinthine mass is the first thing that can be detected on the MRI. Regarding the mechanism of EH in ILS, we speculated that the tumor may block the longitudinal flow of the endolymph, prevent the flow of the endolymph to the endolymphatic sac, or cause inflammation of the endolymph , 24.In patient 1 with positional vertigo, temporal bone MRI with intratympanic injection of gadolinium revealed that the tumor was located in the vestibular cavity and the fundus of the internal acoustic meatus, and no EH was detected, which is consistent with the presentation. Slattery et al. reported a high proportion of patients displaying positional vertigo and assumed that it may be attributed to the direct effect of the tumor exerted on the cochlear and vestibular end organs .In addition, the severity of hearing loss in the hydropic ears seems to be related to the site of hydrops, as hearing loss in patients with mild endolymphatic hydrops was moderate and profound in patients with both vestibular and cochlear hydrops. Further studies are needed to confirm the association between the symptoms and synchronous EH in ILSs.Inner ear tumors are easily missed due to their occult location and small size, especially on conventional MRI. From our experience, it is not difficult to identify tumors and EH in the labyrinth of patients with ILS after careful review of high-resolution MRI using the IT or IV method. According to a previous study, there is no difference in the detection rate of EH by the IT or IV method . The strSince vertigo is considered a risk factor for the growth of vestibular schwannomas , for patThe limitation of this study is that the number of cases is small; thus, statistical analysis between symptoms and imaging presentations cannot be performed.EH concurrent with ILSs has been historically underestimated. With the extensive application of clinical MRI paradigms, such as 3D real IR sequence MRI, more potential cases of EH presenting in patients with ILS will be identified. The severity of hearing loss may be related to the location of the tumor and the degree of EH.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by The medical ethics committee of the Eye, Ear, Nose, and Throat Hospital of Fudan University, Shanghai, China. The patients/participants provided their written informed consent to participate in this study.YZ conceived the idea and drafted the manuscript, while FL collected the raw data and followed the patients. WW and CD generously provided the material and revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Multimorbidity presents a key challenge to healthcare systems globally. However, heterogeneity in the definition of multimorbidity and design of epidemiological studies results in difficulty in comparing multimorbidity studies. This scoping review aimed to describe multimorbidity prevalence in studies using large datasets and report the differences in multimorbidity definition and study design. Methods: We conducted a systematic search of MEDLINE, EMBASE, and CINAHL databases to identify large epidemiological studies on multimorbidity. We used the Preferred Reporting Items for Systematic Reviews and Meta-analysis Extension for Scoping Reviews (PRISMA-ScR) protocol for reporting the results. Results: Twenty articles were identified. We found two key definitions of multimorbidity: at least two (MM2+) or at least three (MM3+) chronic conditions. The prevalence of multimorbidity MM2+ ranged from 15.3% to 93.1%, and 11.8% to 89.7% in MM3+. The number of chronic conditions used by the articles ranged from 15 to 147, which were organized into 21 body system categories. There were seventeen cross-sectional studies and three retrospective cohort studies, and four diagnosis coding systems were used. Conclusions: We found a wide range in reported prevalence, definition, and conduct of multimorbidity studies. Obtaining consensus in these areas will facilitate better understanding of the magnitude and epidemiology of multimorbidity. Multimorbidity, the presence of multiple chronic conditions in an individual , challenWith the exception of relatively uncommon conditions, the majority of visits for the management of chronic conditions and any co-existing conditions are made to primary care physicians, not specialists. Additionally, specialists play a greater role in managing specific conditions of their expertise, but not the other comorbid conditions that the patient may have . Hence, The basic operational definition of multimorbidity includes the following parameters: the minimum number of chronic conditions to determine the presence of multimorbidity and the list of chronic conditions considered . Acute cIt is difficult to quantify the extent of the burden of multimorbidity, as the design of epidemiological multimorbidity studies varies greatly, and no consensus on the operational definition of multimorbidity exists . A systeLarger samples are able to provide a more precise estimate of multimorbidity . We defiSome challenges in using larger databases include the accuracy of the coding system used, and the reliability of coding as hospital records may be coded by non-physicians and may not reflect the actual codes from the physicians’ perspectives ,15. ThisTherefore, a scoping review was conducted on multimorbidity studies using large datasets. The main objective was to describe the range of prevalence of multimorbidity reported by these studies. The secondary objectives of the review were to identify and report the differences in the definitions of multimorbidity and the conduct of these studies.This scoping review was reported using Preferred Reporting Items for Systematic Reviews and Meta-analysis Extension for Scoping Reviews (PRISMA-ScR) protocol .Articles were included if they were (a) written in English, (b) involved human participants, (c) had a study population greater than 500,000, (d) included the primary healthcare or general population, and (e) used electronic databases. Articles were excluded if they (a) focused only on comorbidity, (b) used patient-reported data, (c) studied only inpatients, (d) included acute conditions in the list of conditions, (e) used less than 12 conditions to define multimorbidity, or (f) were qualitative, interventional studies, reviews, editorials, systematic reviews, or meta-analyses.The bibliographic databases of MEDLINE, EMBASE, and CINAHL were searched for all records from the inception date to 8 March 2020 to identify potentially relevant records. The search strategies were drafted and refined through team discussion amongst the authors. The final search strategies for the three databases can be found in The articles were screened by two independent reviewers (YPC and ESL) using Covidence. The reviewers sequentially evaluated the titles, abstracts, and then full text of all publications identified by our searches for potentially relevant articles. The reviewers resolved disagreements on article selection and data extraction by consensus and discussion with another reviewer (YX) if needed.A data-extraction form was jointly developed by two independent reviewers (YPC and ZSC) to determine the type of information to extract. The form captured the relevant information on multimorbidity prevalence, definitions of multimorbidity used , study settings , and conduct of the studies . The form also extracted information on the presence of any data governance or reliability standards of the electronic medical records used by the articles. The prevalence of multimorbidity was manually calculated from data provided in the articles if overall prevalence was not directly reported. In articles reporting prevalence estimates longitudinally over a period of time, we used prevalence estimates of the most recent data.The two reviewers independently extracted the data, discussed the inputs, and updated the data-extraction form after resolving any disagreements through discussion. Consensus was reached by involving a third reviewer (ESL) for unresolved items.The list of chronic conditions used by each article to define multimorbidity was compiled and organized into their respective categories based on the body systems. The organization of individual chronic condition into each category was conducted by two independent reviewers (YPC and ESL), and disagreements were resolved until consensus was reached via discussion with a third reviewer (YX). The organized conditions can be found in A total of 7235 records were obtained from the searches, and 42 full text articles were assessed for eligibility. Of these, 22 were excluded for various reasons as shown in The study characteristics and definitions of multimorbidity used by the 20 articles are presented in n = 9) [n = 7) [n = 4) [The majority of the included articles were from Europe (n = 9) ,29,30,31 [n = 7) ,32,33,34 [n = 4) ,35,36,37 [n = 4) ,33,34.n = 9) [n = 8) [n = 2) [There was significant variation in the age ranges of the study populations. Seven articles had no limits on age ,29,35,37n = 9) ,34,36,37 [n = 8) ,31,32,35 [n = 2) ,24. One  [n = 2) .The reported prevalence of multimorbidity was varied across the 20 articles. For MM2+ prevalence, 18 articles reported a prevalence ranging from 15.3% to 93.1%. For MM3+ prevalence, 12 articles reported a prevalence ranging from 11.8% to 89.7%. Nineteen articles reported crude prevalence rates, while one article [n = 17) used two chronic conditions and above to define the presence of multimorbidity. One article used at least three chronic conditions [Most of the articles  of the articles. “Genetic conditions” and “Others” were two categories that were only present in two articles [The number of chronic conditions used in defining multimorbidity varied greatly from 15 to 147. Six out of 21 categories were present in the list of chronic conditions reported by all 20 articles. These six categories were “Cardiovascular”, “Endocrine”, “Mental health”, “Musculoskeletal”, “Neurology”, and “Respiratory”. The presence of the other categories was more diverse, with nine categories present in less than half  to define the presence of multimorbidity. Only five articles provided the rationale for doing so: two were based on previous systematic reviews ,28, and The lists of chronic conditions used by the 20 articles ranged between 15 and 147 conditions. Most articles clustered around 15 to 30 conditions, which was partly due to the exclusion of articles with fewer than 12 conditions as recommended by Fortin et al. .The following six categories of conditions were included in all 20 articles: cardiovascular, endocrine, mental health, musculoskeletal, neurology, and respiratory. This suggests greater relevance of these categories in the primary care and general population, which is possibly due to prevalence of conditions, such as acute myocardial infarction (cardiovascular), diabetes mellitus (endocrine), or greater impact on patients’ lives, such as requiring lifelong medications. To derive the lists of conditions, most articles based their lists on previous studies, which was followed by clinical relevance of the conditions. Less commonly, government guidelines, indexes, or systematic reviews were used.All articles included mental health conditions, particularly depression, demonstrating that mental health conditions are significant health problems to consider in multimorbidity. Seven articles ,30,35,37While specialists may better provide care for patients with one dominant disease or closely related comorbidities, the management of physical–mental multimorbidity requires holistic care and delicate balance . For exaFour coding systems were used by the 20 articles: ICD-9-CM, ICD-10-CM, Read2, and ICPC-2. ICD-9-CM and ICD-10-CM were most commonly used by the 20 articles. However, the code accuracy, defined as the extent to which the ICD nosologic code reflects the underlying patient’s disease, is usually low . ICPC-2,n = 9) in this scoping review used prescription data as an additional means of diagnosing chronic conditions. Future studies may consider including prescription data or other means of confirming diagnoses in their diagnosis algorithms as well as standardizing them for large dataset multimorbidity studies to more accurately estimate multimorbidity prevalence.A lack of consensus on coding systems and diagnosis algorithms results in difficulty comparing among different multimorbidity studies. In studies using different coding systems, imperfect mapping of individual conditions onto a common coding system affects the accuracy of comparison studies. Differences in diagnosis algorithms also affect prevalence estimates of multimorbidity, as conditions may be under-reported if only diagnosis codes are used. Nearly half of the articles  did not mention any governance standards of the databases used as well. Indicating the presence of data governance is recommended to increase the reliability of multimorbidity studies using large databases.As such, data governance by a professional body is essential to ensure the reliability of large databases. However, data governance in healthcare is less mature compared to other industries, and no universal standard for healthcare data exists . Most ofOur scoping review has some limitations. A categorical approach to analyzing the chronic conditions rather than individual comparison was chosen for feasibility reasons. This made the identification of key chronic conditions difficult.In conclusion, our scoping review found a wide range in prevalence of multimorbidity as reported in studies using a large dataset, from 15.3% to 93.1% in MM2+ and 11.8% to 89.7% in MM3+. This is due to differences in both the definitions of multimorbidity and the conduct of the multimorbidity studies.Consensus is urgently needed to facilitate comparison across studies as well as ensure reproducibility. Additional research such as a qualitative study using the Delphi method  may be iMethods of diagnosing chronic conditions will also need to be standardized, harmonizing the current established coding systems and diagnosis algorithms. This is especially important as large datasets are reliant on multiple factors to ensure reliability, such as standards of governance of electronic medical records, accuracy of data coding, diagnosis codes, and algorithms used.


Dogs and cats have differences in vitamin D metabolism compared to other mammalian species, as they are unable to perform vitamin D cutaneous synthesis through sun exposure. Therefore, they are dependent on the dietary intake of this nutrient. The classic functions of vitamin D are to stimulate intestinal calcium and phosphate absorption, renal calcium and phosphate reabsorption and regulate bone mineral metabolism. Thus, it is an important nutrient for calcium and phosphorus homeostasis. This review highlights the evidence of the direct and indirect actions of vitamin D on bone mineral metabolism, the consequences of nutritional imbalances of this nutrient in small animals, as well as differences in vitamin D metabolism between different size dogs. It is worth mentioning that renal synthesis is largely responsible for circulating calcitriol. However, it has been demonstrated in humans that several other tissues express 1α-hydroxylase, including the breast, parathyroid gland, colon, prostate, beta-pancreatic cells, placenta, brain, immune cells, endothelial cells and keratinocytes [Dogs convert ergocalciferol to calcidiol as well as cholecalciferol . Howeverinocytes .Although calcitriol is the active metabolite, vitamin D status is assessed by measuring circulating calcidiol concentrations as it has a long biological half-life, ranging from 10 days to three weeks, whereas the biological half-life of calcitriol varies from three to six hours . BesidesCalcitriol synthesis regulation is influenced by several factors, such as plasma calcium and phosphate concentrations, parathyroid hormone (PTH), fibroblast growth factor FGF-23), klotho gene and calcitonin , klotho ,53,54,553 3D3) which, in turn, undergoes an oxidation process giving rise to 1α,25-dihydroxyvitamin D3 -26,23-lactone.The 24-hydroxylase enzyme is responsible for vitamin D catabolism. This mitochondrial enzyme is found mainly in proximal renal tubules but it is also expressed in intestine, keratinocytes, fibroblasts, lymphocytes and macrophages . The 24-3 2D3) and 1α,24,25-trihydroxyvitamin D3 3D3), respectively. The 1α,24,25(OH)3D3 still undergoes an oxidation process, forming calcitroic acid, a metabolite excreted through bile [2D3 is believed to be related to the bone mineralization process [Through a second metabolic pathway, 24-hydroxylase promotes hydroxylation on carbon 24 of calcidiol and calcitriol, resulting in the formation of 24,25–dihydroxyvitamin Dugh bile ,61,62. T process .Calcitriol stimulates 24-hydroxylase activity, playing a protective role against hypercalcemia . In situ2D3. However giant breed dogs had lower 24,25(OH)2D concentrations, with values which were about 10 times lower than small breed dogs.It is known that dogs of giant breeds are prone to develop skeletal disorders when compared to dogs of small breeds fed with low or excessive calcium diets . Among o2D concentrations and lower plasma 24,25(OH)2D concentrations compared to miniature poodle puppies. According to Hazewinkel and Tryfonidou [2D concentrations may be related to disturbances in endochondral ossification during the rapid growth phase observed in large and giant breed dogs. Osteochondrosis is a disorder of maturation that results in a delay of cartilage mineralization. It is known that both 24,25(OH)2D and 1,25(OH)2D play an important role in differentiating cartilage cells and matrix mineralization [2D and 1,25(OH)2D is of major importance [In another study, carried out by Tryfonidou et al. , Great Dyfonidou , the lowlization ,69. In oportance . Therefo2D concentrations is not yet fully elucidated, but it is believed that this can be explained by higher circulating growth factor concentrations that these animals present, such as growth hormone (GH) and insulin-like growth factor I (IGF-I) [2D concentrations has been reported after GH therapy in children with GH deficiency [2D synthesis in mammalian renal cells under conditions of hypophosphatemia. In addition, an association has been demonstrated between high plasma GH and IGF-I concentrations and low plasma 24,25(OH)2D concentrations [The reason why giant breed dogs have lower plasma 24,25(OH) (IGF-I) . Even in (IGF-I) , higher ficiency , normal ficiency  and eldeficiency , and Conficiency  observedtrations , as welltrations , which mBrumbaugh and Haussler  first deThe VDR can also show nongenomic responses, which occurs when calcitriol interacts with VDR located in sphingolipid and cholesterol rich cell membranes . In respRegarding vitamin D functions related to bone metabolism, the target organs of calcitriol are intestine, kidneys, bone tissue and parathyroid gland ,7.2+–ATPase (PMCA 1b) and/or a Na-Ca exchanger [In the intestine, calcitriol acts on both transcellular and passive paracellular calcium transport ,9. Transxchanger , which rxchanger ,93. The xchanger ,95.Paracellular transport occurs mainly in the most distal regions of the intestine, but it can happen over the entire length of the intestine . This paIn addition to calcium absorption, in the intestine, vitamin D also acts on phosphate absorption. Calcitriol stimulates phosphate absorption via the Naþ phosphate transporter (NaPi-IIb) pathway so that calcitriol increases Na-Pi transporter expression, which corresponds to a family of SLC34 solute carriers (SLC34A2) .Calcitriol stimulates renal calcium reabsorption so that calcium transport through renal epithelial cells is similar to calcium transport through intestinal epithelial cells. In the apical cell membrane of the distal convoluted tubules and collecting ducts there is an epithelial calcium channel (TRPV5) that is responsible for transporting calcium into the cells. Calcium transport occurs through renal tubular cells through the calbindin-D28K protein, and in the basolateral cell membrane there are calcium transporters PMCA1b and NCX responsible for transporting calcium into the bloodstream . In addiIt is known that calcitriol receptor mRNA has high expression in the parathyroid gland ,108. AftIn humans, the treatment with calcitriol in CKD patients promotes beneficial effects on the kidneys unrelated to PTH and mineral metabolism, including activity of renin-angiotensin-aldosterone system suppression, reduction of proteinuria, reduction of podocyte loss associated with glomerular hypertrophy and antiproliferative and antifibrotic effects ,116,117.The mechanisms by which vitamin D decreases PTH synthesis are well described, so that it suppresses the parathyroid gland cells growth gland by decreasing growth factors  and through the increase in inhibitors of cell growth (cyclin-dependent kinase inhibitors p21 and p27) . In addiIn bone tissue, under conditions of hypocalcemia, calcitriol works in conjunction with PTH to mobilize calcium from bones for circulation to increase circulating ionized calcium concentrations ,120. Thi2D-mediated signaling in the growth of endochondral bone [In the past, it was believed that calcitriol influenced bone growth and mineralization only indirectly, that is, by increasing the calcium and phosphorus concentrations in the circulation through stimulating intestinal absorption and renal reabsorption of these minerals . Later, ral bone .As previously discussed, in negative calcium balance, VDR signaling is directed towards maintaining circulating calcium concentrations, that is, there is an increase in resorption and a decrease in bone mineralization . In contMinimum and maximum recommendations of cholecalciferol for dogs and cats according to National Research Council (NRC) , EuropeaFew studies have investigated vitamin D requirements for dogs and cats, and all of these studies were performed in puppies and kittens since the demand for skeletal calcification is higher at this life stage and growing animals are more sensitive to vitamin D deficient diets. As there are no studies that investigated vitamin D requirements for adult dogs and cats, as well as breeding females, the recommendations are based on studies of growing animals.−1  for growing puppies consuming a diet with 9.0 g of calcium and 7.5 g of phosphorus per kg. However, it was only in 1944 that the first vitamin D recommendation for dogs was proposed by Michaud and Elvehjem [−1·d−1. Finally, Tryfonidou [−1 to Great Dane puppies and did not observe any adverse bone disease.A pioneering study carried out in 1918 by Mellanby  observedElvehjem , in whicyfonidou  providedRegarding cats, few studies have evaluated different concentrations of vitamin D in feline nutrition. The first study was published by Gershoff et al.  in which−1 of diet). In this study, the authors provided low cholecalciferol concentration diets to the kittens’ mothers during pregnancy and lactation, thus inducing a reduction in the vitamin D kitten reserves. The kittens were fed from nine to 22 weeks of age with the diets and it was noticed that the plasma 25(OH)D concentrations grew linearly with dietary intake of cholecalciferol. In addition, animals fed 0.0 and 125 IU cholecalciferol kg−1 of diet, consumed the food until 34 weeks of age. In this part of the study, the authors observed a sharp drop in the plasma 25(OH)D concentrations of the animals that consumed the food without the addition of vitamin D. However, the kittens that consumed the food with 125 IU cholecalciferol kg−1 of diet had higher plasma 25(OH)D concentrations at 22 and 34 weeks than at nine weeks of age, which indicates that the inclusion of 125 IU cholecalciferol kg−1 of diet would be suitable for kittens. However, the authors recommended a dose of 250 IU cholecalciferol kg−1 of diet to maintain a safety margin for both kittens, pregnant and lactating cats.Morris et al.  evaluate−1 of purified diet, with calcium concentrations ranging from 3.8 to 8.1 g/kg of diet and constant Ca:P ratio. In this study, the authors also observed plasma 25(OH)D concentrations within the normal range.Morris and Earle  publisheVitamin D deficiency can be caused by abnormal vitamin D metabolism related to congenital abnormalities due to genetic mutations ,138, as As previously discussed, vitamin D requirements are not well established for dogs and cats, which are dependent on vitamin D dietary intake. Vitamin D deficiency results in hypocalcemia and hypophosphatemia, which can result in clinical manifestation of bone disease characterized by impaired bone mineralization associated either with rickets or osteomalacia ,16. RickIn humans, nutritional rickets continues to be reemerging as a disease problem across the world ,141. HumVitamin D-dependent rickets type I (VDDR-I), also referred to as vitamin D 1α-hydroxylase deficiency, is an autosomal recessive disorder caused by a deficiency of the renal enzyme 1α-hydroxylase due to a defect in the gene encoding it. Affected animals have normal serum calcidiol concentrations and low calcitriol concentrations ,147. VitA case of VDDR-I in a cat was characterized by hypocalcemia, hyperphosphatemia and skeletal abnormalities such as abnormal bone calcification, delayed teething and hyperparathyroidism . The autVDDR-II has been reported in dogs and cats and is a disease characterized by hypocalcemia, secondary hyperparathyroidism, low bone mineralization, rickets and, in some cases, alopecia ,145. TheOsteomalacia, another metabolic bone disease, rarely causes clinical signs in dogs or cats. However, the inability to detect bone pain and the insensitivity of radiographs of domestic animals, suggest that this condition may be underdiagnosed. In humans, the incidence of this disease remains largely underestimated across the globe as well . OsteomaNutritional secondary hyperparathyroidism (NSHP) is a common consequence of a nutritional deficiency in pets consuming unbalanced diets ,151,152.Moreover, when low vitamin D dietary concentrations are available, there is a decrease in calcium absorption, which may result in hypocalcemia ,155. LowFew studies have reported NSHP in dogs and cats. Usually, the development of this change has been associated with the consumption of full meat-based diets by young animals due to the high calcium demand for bone growth ,152,157.Morphological changes in bones due to calcium mobilization makes the bones poriferous and prone to fracture. Mature bone replacement by fibrous tissue can lead the animal to develop fibrous osteodystrophy, which affects especially the jaw bones ,151,152.The treatment of animals with NSHP includes a complete and balanced diet, but sometimes it is necessary for the addition of calcium supplements. Cage rest is also important for the first few weeks of treatment in order to decrease the risk of bone fractures. The prognosis for noncomplicated cases is good and usually dietary correction normalizes body levels of calcium, phosphorus and PTH. On the other hand, complicated cases evolving spinal fractures, or more severe bony changes, support a bad prognosis ,157.Signs of acute vitamin D toxicosis are related to the development of hypercalcemia when serum total calcium concentrations exceed 18 mg/dL, and serum ionized calcium concentrations are greater than 1.40 mmol/L. Clinical signs include polydipsia and polyuria due to difficulty in concentrating urine and stimulation of the thirst center, anorexia, vomiting and constipation as a consequence of decreased excitability of gastrointestinal smooth muscle and, finally, decreased neuromuscular excitability may cause weakness, depression, muscle twitching and seizures.On the other hand, smaller but chronic intake of higher quantities of vitamin D (exceeding by 100 times de NRC recommended) are known as a cause of osteochondrosis and retarded bone remodeling in growing giant breed puppies like Great Danes, who do not have tight control of mineral deposition in large bones compared to smaller breeds ,36,37.Until now, cases of hypervitaminosis D are apparently rare and experimentally-induced, since the inclusion of this nutrient is controlled during pet food fabrication, although, in 2005, Mellanby et al.  reportedIn both cases, dogs presented at the veterinary hospital with pyrexia, inappetence, polydipsia, polyuria, lethargy, a stiff gait, marked ionized calcium and total hypercalcemia. Palpation of both carpi demonstrated marked soft tissue swelling in one of them and flexion of both carpi caused considerable discomfort to the dog whose radiographs of the left and right carpi, elbows and stifles revealed multiple joint effusions. The consumption of the same diet indicated nutritional involvement. The dog from case one was treated only with intravenous saline solution for 24 h and sent home. The owners considered the dog to be generally free of clinical signs by day 180. On the other hand, the dog from case two received home oral steroid treatment associated with antibiotics and the owners reported that its condition was much improved and exercising normally by day 16.In both cases, the food with vitamin D excess was withdrawn, ionized calcium was close or within the reference range by the second week after the first visit to the hospital. However, 180 days after, plasma concentrations of 25(OH)D were above the reference range, possibly due to its long half-life (29 days approximately), and may be why hypercalcemia signs persist for a while even when vitamin D was promptly taken out from diet .As the etiology of vitamin D toxicosis is related to hypercalcemia, methods that inhibit bone resorption are the best alternative for treatment. In this scenario, intravenous injections of bisphosphonates are the best alternative, already described for humans and dogs ,159. It The causes of hypervitaminosis D are not always nutritional, and the most common one is cholecalciferol rodenticide described in dogs ,26,27,28The nonclassical actions of vitamin D were not a focus of this review. However it is important to remember that vitamin D is also related to numerous functions not associated with bone metabolism ,44. FromIn small animals has been demonstrated an association between low vitamin D circulating concentrations and diseases not associated with bone metabolism, such as: cancer ,163,164,In addition, in-vitro antineoplastic effects of calcitriol have been shown in various types of canine tumors, including osteosarcoma , squamouIt is not yet clear whether low vitamin D status is the cause or consequence of the diseases mentioned above, with evidence supporting both hypotheses .It is concluded that dogs and cats have different vitamin D metabolisms compared to other mammal species. Little is known about the vitamin D requirements for dogs and cats, especially concerning adult animals and females in the reproductive phase. New trends in dog and cat nutrition, such as a homemade diet, may increase the risk of developing vitamin D deficiency. Vitamin D poisoning is not common in dogs and cats, but it can occur as a result of errors in the formulation of commercial foods, accidental consumption of rodenticides, plants containing calcitriol glycosides and topical ointments based on vitamin D analogues.


Background: Coronavirus disease-19 (Covid-19) is one of the most devastating epidemics in the 21st century, which has caused considerable damage to the physical and mental health of human beings. Despite a few regions like China having controlled the epidemic trends, most countries are still under siege of COVID-19. As the emphasis on cleaning and hygiene has been increasing, the problems related to obsessive-compulsive disorder (OCD) may appear.Objective: This study was designed to investigate the prevalence of OCD in the urban population in Wuhan during the stage of regular epidemic control and prevention. Meanwhile, characteristics and risk factors for OCD were also explored.Method: Five-hundred and seventy residents in urban areas of Wuhan were recruited using the snowball sampling method to complete questionnaires and an online interview from July 9 to July 19, 2020. Collected information encompassed socio-demographics, Yale-Brown Obsessive-Compulsive Scale (Y-BOCS) scores, Social Support Rating Scale (SSRS) scores and Pittsburgh Sleep Quality Index(PSQI) values.Results: Three months after lifting the quarantine in Wuhan, the prevalence of OCD was 17.93%. About 89% of OCD patients had both obsessions and compulsions, while 8% had only obsessions and 3% had only compulsions. Top 3 common dimensions of obsessions were miscellaneous (84.0%), aggressive (76.6%), and contamination (48.9%), and of compulsions were miscellaneous (64%), checking (51.7%), and cleaning/washing/repeating (31.5%). The unmarried were more vulnerable to OCD than the married . Students had 2.103 times the risk of developing OCD than health care workers (p < 0.05). Those with positive family history of OCD and other mental disorders  and presence of psychiatric comorbidity  were also at higher risk. Each level increase in sleep latency increased the risk of OCD to 1.646 times (p < 0.05).Conclusion: In the background of regular epidemic control, the prevalence of OCD was high, and the symptoms were widely distributed. Obsessions often accompanied compulsions. Being single and a student, positive family history of OCD and other mental disorders, presence of psychiatric comorbidity, and longer sleep latency were predictors of OCD. Early recognition and detection of these issues may help to intervene in OCD. The new coronavirus disease COVID-19), which was first detected in December 2019, was declared as a public health emergency of international concern (PHEIC) on January 30, 2020  and repetitive stereotyped behaviors (compulsions), is a common chronic mental disease, which is often under-recognized . The estSocial support and sleep quality have been linked to mental health in many previous studies; enough social support and good sleep quality could ensure a better mood , 16. HowCurrently, most countries are still under the siege of Covid-19. The occurrence of mental problems may be delayed, and these problems can persist for a long time. Therefore, the mental health effects of the pandemic need to be investigated. Would people in reopened areas suffer from OCD in the background of regular epidemic prevention and control?Thereby, we investigated urban residents in Wuhan, aiming at collecting concrete clues on OCD and its risk factors, which might, in turn, assist in providing valuable reference for other countries as well as handling this issue instantaneously and potentially. The hypotheses for this study were the following.Hypothesis 1: the prevalence of OCD in the regular epidemic stage is higher than what it used to be pre-pandemic.Hypothesis 2: social support and sleep quality may help to predict OCD diagnosis in this background.People from central areas of Wuhan, China, were recruited online through “Wenjuanxin” and “WeChat” using the snowball sampling method from July 9 to July 19, 2020, around 3 months since the quarantine had been lifted.Inclusion criteria were: (1) a resident of a central urban area in Wuhan, (2) aged 15 years or above, and (3) ability to understand the contents of questionnaires.People were excluded if they: (1) could not meet the inclusion criteria, (2) spent <2 min or over 1 h for filling questionnaires, (3) stumbled upon “trap questions,” or (4) dropped out.Eleven participants spent over 1 h for filling questionnaires, 17 failed in “trap questions,” and 1 quit midway. Thus, 29 invalid questionnaires were eliminated, and 541 samples were included in the analysis; the valid response rate was 94.91%.All respondents participated voluntarily under the premise of written informed consent and could quit at any time. Ethical approval was obtained from Renmin Hospital of Wuhan University.Several socio-demographic characteristics, such as sex, age, income, marital status, educational level, and number of family members, were included in the questionnaire .In particular, information on family history of mental disorders or comorbid mental disorders was acquired by items in the questionnaire saying “Have you ever been diagnosed with a mental illness like schizophrenia, depressive disorder, maniac disorder, bipolar disorder, anxiety disorders, post-traumatic stress disorder, Tourette syndrome in the hospital and remained uncured,” “Do you have family members diagnosed with OCD or mental disorders like above?” We also reconfirmed this information orally in a brief online interview. Respondents who reported the presence of an additional mental disorder as well as the context where the diagnosis was given, plus the duration of the disorder, were confirmed as participants with psychiatric comorbidity. The same criteria were applied to ascertain a positive family history for a mental disorder.The widely used Y-BOCS consists of a checklist for symptoms (58 items) and a scale for severity . There is a moderate correlation in consistency and discrepancy between self-reported and clinician-rated Y-BOCS scores and patients tend to rate symptoms lower than clinicians from experience , 20. GivA 10-min oral online interview was conducted for all participants through the “Wechat” app  for rendering explanations of purpose of this research, reconfirmation of participation, as well as interpretation of colloquial definition of obsessions and compulsions, thereby aiming to minimize the confusion to a maximum level.The Chinese version of SSRS designed by Shuiyuan Xiao was used to evaluate the type and levels of social support received from others. The questionnaire consists of 3 aspects, namely, subjective social support, objective social support, and the availability of social support, with a total point ranging from 7 to 56. The more the points you score, the more the social support you have .The modified PSQI with 4 dimensions, such as sleep satisfaction, sleep disturbance, sleep latency, and sleep duration, was applied to appraise sleep quality. Each dimension is scored between 0 and 3, with a total point ranging from 0 to 21, and the more the points you score, the poorer the quality of your sleep .T-test, while non-normally distributed variables were processed using non-parametric tests. A p < 0.05 indicated a significant difference. Besides, all significant factors in univariate analysis as well as those believed to be relevant variables were introduced in a multi-factorial logistic regression stepwise equation  for a deeper insight into relatively independent risk factors of OCD; p < 0.05 indicated significance.SPSS 24.0 software  was used for statistical analyses. The dependent variable in the current study, OCD or non-OCD, was categorical, while independent variables consisted of both categorical and quantitative ones. Thereby, comparisons of group differences in categorical data were performed by the chi square test. Quantitative variables with normal distribution were processed using the Five-hundred and seventy residents from all 7 central urban areas in Wuhan participated in the research, 29 among whom were excluded due to invalid response to the questionnaire. Thus, 541 appropriate respondents were included; 57.5% of them were females, and the rest were males. Most respondents were young (86.7%). About 7.6% of respondents admitted to having co-morbidity with other mental diseases, and 5.2% claimed to have family members with mental disorders. Complete baseline information is shown in n = 8) or compulsions (n = 3) presenting alone were rare.In total, 97 respondents were confirmed to have OCD according to Y-BOCS, among whom 86 had both obsessions and compulsions; obsessions . Further, the univariate analysis indicated that the prevalence of OCD diagnosis differed depending on some sociodemographic variables such as marital status, occupation, and employment status (p < 0.05). Moreover, the prevalence in respondents who were asymptomatic cases, with comorbid mental disorders, family history of OCD or other mental disorders, sleep disorders, or poor social support levels turned out higher than that in those without these factors (p < 0.05).Altogether, 97 respondents met the criterion for OCD diagnosis, so the prevalence of OCD in the background of regular epidemic prevention and control was 17.93%. The prevalence of OCD increased as age decreased, with the highest being 22.66% in the young group aged between 15 and 24 years (p < 0.05). Students were at 2.169 times the risk of having an OCD diagnosis compared to that of health care workers (HCWs). The prevalence of OCD in people with comorbid mental disorders or a positive family history of OCD or other mental disorders was much higher than that in those without other mental disorders (p < 0.05). Notably, sleep latency, which was one of the assessments for sleep quality in the current research, turned out to be an independent predictor for OCD; each unit increase in sleep latency was associated with 0.646 times higher risk for developing OCD (p < 0.05).Significant variables from To the best of our knowledge, this is the first study on the prevalence of OCD and possible influencing factors among central urban residents in Wuhan in the background of regular epidemic control and prevention. As known to all of us, Wuhan, one of the first areas that were heavily thrashed by COVID-19, has achieved great success in the battle against this pandemic through hard work and generous support from all circles. New cases have not been observed since March 18, 2020, and the lockdown policy was removed on April 8, 2020, under the premise of the mitigated situation.Notwithstanding, something worth much attention is the fact that people from this area might still suffer from certain mental disorders in the stage of regular epidemic prevention. As observed in this study, despite being a relatively secure area compared with many other countries where the pandemic progressed, 3 months after reopening, people in Wuhan were still affected by OCD with a prevalence rate of 17.93%. Occupation, marital status, comorbid mental disorders, family history and sleep latency were associated with OCD.p < 0.05), indicating that students were the vulnerable ones. Indeed, students, under great pressure and with dubiously-oriented coping skills were often prone to mental disorders . A family history of OCD also increased the risk of developing OCD with an odds ratio of 2.497. Previously, both twin studies and genome-wide association reports indicated the heritability of OCD ; future research should address this limitation. Finally, it remains to be seen to what extent and how people from other parts of the world experience OCD in this special situation.The present cross-sectional study conducted among urban people in Wuhan indicated that OCD, with wide-ranging symptomatic dimensions, was very pervasive in the stage of regular epidemic control. In addition, it was observed that obsessions and compulsions occurred independently. Being single or a student, family history of OCD, comorbid status, and longer sleep latency appear to be potential predictors of OCD in this situation; therefore, more attention should be paid to these factors, allowing for early detection and intervention in OCD.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Clinical Research Ethics Committee of Renmin Hospital of Wuhan University. Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.YZ, LX, and HW: design and concept. GW: supervision and management. YZ and LX: draft of manuscript. YZ and YX: processing, analysis of statistics, collection, acquisition, and verification of data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we address this issue by conducting nine different variations of an N-Back task manipulating stimulus type and task structure. Furthermore, we explored the effect of the pre-processing method used and differences in the laboratory environment. Results reveal significant differences in behavioral and electrophysiological signatures in response to N-Back stimulus type, task structure, pre-processing method, and laboratory environment. In conclusion, we suggest that experimental factors, analysis pipeline, and laboratory differences, which are often ignored in the literature, need to be accounted for when interpreting findings and making comparisons across studies.The N-Back, a common working memory (WM) updating task, is increasingly used in basic and applied psychological research. As such, an increasing number of electroencephalogram (EEG) studies have sought to identify the electrophysiological signatures of N-Back task performance. However,  Working memory (WM), defined as a limited capacity system responsible for temporary storage and manipulation of relevant information  vs. verbal (for instance when the presented stimulus is word or syllable) stimuli. This indicates that stimulus and load factors play a significant role in modulating P2, N2, and P3 components  and  but also in terms of different data pre-processing pipelines and laboratory effects, such as differences in room setup, computer testing stations, as well as electroencephalogram (EEG) hardware and software. While this is true of numerous areas of ERP research, the N-Back is particularly notorious in how it varies across studies , USA. The purpose of this study was to explore the potential factors that affect ERP morphology and behavioral signatures of the N-Back task and to replicate experimental procedures described in two published datasets collected in different labs (Datasets II and III). Dataset II was collected at KU Leuven, Belgium , undergraduate students, were recruited from UCR. The experimental protocol was approved by the Institutional Review Board of UCR and all participants gave their informed consent before the experiment. They received course credit and a payment of $10 for participating in two sessions.task structures (see below) with three stimulus types: words , pictures , and colors . Task structures differed in terms of stimulus duration, ISI, response contingency, and feedback  and incorrect responses (red circle around the stimulus). For task 1 and task 2 no response was allowed during stimulus presentation.N = 16) in each variant. Each session consisted of 11 blocks presented in the following order: 1-back practice block, 2-back practice block, four 2-back test blocks, 3-back practice block, and four 3-back experimental blocks. Instructions were provided before each new N-level and 15-s breaks were given between blocks. Practice blocks consisted of sixteen trials during which the participant performed task 3 with Color stimuli whereas experimental blocks consisted of N + 40 trials .Each participant performed four out of the nine N-Back variations across two different sessions conducted on different days, where the same difficulty levels were administered each day, for a total of approximately 90 min per session. This ensured that all combinations of conditions existed in a within-subject design, even though not all participants completed every condition. The assignment of each participant to each N-Back variant was done randomly based on the subject number to ensure an equal number of participants  and Psychtoolbox Version 3.0.8 were used to present the task and generate the stimuli  operating at a sampling rate of 2,048 Hz. Active Two system stored the EEG signal with no high-pass filter and low-pass filtered only by the anti-aliasing filter. Thirty-two active Ag/AgCl electrodes placed according to the 10/20 system Jasper,  at O1, Ovia advertisements and flyersTwenty-three healthy adults  were recruited task structure similar to task 2 of Dataset I, mentioned above, where each stimulus was presented for 1,000 ms, followed by an ISI of 2,000 ms. The stimuli were generated using MATLAB 2007b  and Psychtoolbox Version 3.0.8  at a sampling rate of 2,000 Hz, using 32 Ag/AgCl electrodes placed at O1, Oz, O2, PO3, P8, P4, Pz, P3, P7, TP9, CP5, CP1, CP2, CP6, TP10, T7, C3, Cz, C4, T8, FC6, FC2, FC1, FC5, F3, Fz, F4, AF3, AF4, Fp1, Fp2. The reference was placed at AFz and the ground at CPz. Also, four external electrodes around the eyes were used for EOG recording following the instructions of Croft and Barry .Seventy-two healthy adults were recruited from the University of Maribor, Slovenia  and displayed on a CRT monitor. Participants had to respond to both targets and non-targets. Two types of stimuli were used: two-letter words and colors  with sintered electrodes. The EEG data were recorded using a SynAmps RT system and had a band-pass filter of 0.15–100.0 Hz. The 19 EEG traces were sampled online at 1,000 Hz. Vertical eye movements were recorded using two external electrodes placed above and below the left eye and a ground electrode was applied to the forehead. Two ear lobe references (A1 and A2) were used for online referencing, followed by common average re-referencing.Two pre-processing pipelines were used to analyze Dataset I: pipeline I and pipeline II. For ERP comparison across the three datasets, only pipeline I was used. The pipelines were chosen as they represented different, but standard approaches to ERP analysis . The method was developed as it accounts for eye artifacts using an automatic procedure .Pipeline II was conducted by using MATLAB R2016a . The data were resampled to 1,000 Hz and filtered in the 0.1–30 Hz range using a zero-phase 4th-order Butterworth filter. All electrodes were re-referenced offline to the average of the two mastoid signals , and ERP and behavioral data comparisons respectively. We note that this p-value is monotonically relatable to other measures of reliability, such as differences in signal to noise ratio (SNR).To assess the effect of N-Back task variations on behavioral responses  and ERP morphology , a positive deflection with a peak around 100 ms after stimulus presentation. It is distributed over the lateral occipital electrodes and reflects the early sensory processing of visual stimuli. P1 latency depends on stimulus contrast, such as luminance or SNR, while its amplitude is modulated by attention , a negative deflection that peaks around 100–200 ms after stimulus onset. It has a distribution over the entire scalp, but it peaks earlier over the frontal regions of the scalp. It has been shown that its amplitude is modulated by attention. Larger amplitude is associated with attended stimuli, while smaller is associated with increasing stimulus presentation frequency , a positive deflection with a peak of around 150–275 ms after stimulus presentation. It is distributed over the fronto-central and parieto-occipital regions of the scalp, but its maximal is over the frontal area. It is elicited by visual stimuli and modulated by attention , a negative deflection detected around 200–350 ms after stimulus onset. It is distributed over the frontal regions of the scalp and posterior regions in visual attention tasks , a positive deflection with a peak occurring around 250–600 ms after stimulus onset. It shows a stronger distribution over the centro-parietal electrodes on the scalp. Its amplitude becomes larger with infrequent target stimuli and decreases with habituation and task difficulty. Its latency is modulated by the difficulty in discriminating the target stimulus Picton, .N400 (N4), a negative deflection detected between 400–600 ms after stimulus onset. It is typically stronger over centro-parietal regions of the scalp and reflects brain response to semantically meaningful stimuli that can include visual and auditory words, sounds, pictures, and faces , a positive deflection, with a peak occurring around 500–1,000 ms after stimulus onset. It is most prominent for posterior scalp channels. The PLC amplitude is modulated by stimulus repetition: suppressed for stimuli that have been already presented, and generally larger for new stimuli , in both long- and short-term memory paradigms , we performed a nonparametric permutation-based analysis on behavioral and electrophysiological data.To investigate the effect of experimental features . On the other hand, results revealed a robust overall effect of task structure , and for task 3 vs. task 2 for words (p < 0.001) and colors (p < 0.012) but only a trend for pictures (p = 0.067). However, there was no statistically significant difference between task 1 and 2 . These results show that while there is a highly significant effect of tasks, especially task 3 vs. the others, that the choice of stimulus has a lesser effect on task performance.While ture see , showingstimulus type and task structure. This can be seen in p-value of less than or equal to 0.05 for at least 12 consecutive bins with Δt of 1/512 Hz.Overall, ERP morphologies changed substantially both as a function of task 1, ERP morphologies differed more frequently for pictures compared to colors and words, as seen in pictures vs. words differed more frequently in the N1, N2 and P2 components in channels Fz, Cz, and Pz (the latter only for P2), pictures vs. colors showed differences in the N2, P2, and P3 components in channels Fz and Cz. Additionally, words vs. colors showed differences in the P2 component in channels Fz, Cz, and Pz.For task 2, we found that ERP morphologies differed more frequently for colors compared to pictures and words , the main effect of the load is shown in p < 0.05) for all the components mentioned in this paper except for P1 . Likewise, we also observed differences in the ERPs as a function of metrics of performance (p < 0.001) for all the components, except for P1. However, again, this effect was largely independent of the task, and stimulus types (see stimulus and task conditions).To understand how other factors may have influenced the ERPs, we also examined the effects of memory load and performance on ERP waveforms see . Concernformance ; hits . Moreover, the word N-Back variation with task structure 1 showed significant differences in P3 components between the two pipelines. For task structure 2 and words, Cz showed a significant difference in N2 and P3 components. For task structure 2 and pictures, Fz revealed significant differences in PLC and Cz in P3 and PLC. For task structure 2 and color stimulus, Fz showed significant differences in P3, N4, and PLC signatures and Cz in P3 and PLC. For task structure 3 and words, Fz showed significant differences in N2, P3, N4, and PLC components. Further, Cz showed differences for N2 and P3 and Pz for PLC. For task structure 3 and pictures, Fz and Cz showed a significant difference in PLC. Finally, for task structure 3 and colors, Fz showed significant differences in P3, N4, and PLC components and Cz showed a significant difference in PLC.We next examined the extent to which differences in analysis pipelines used across labs resulted in changes in estimated ERP morphologies. Interestingly, early ERP components are relatively preserved across the pipelines, but that later ERP components showed significant differences between pipeline I and pipeline II see . FurtherBecause Pipelines I and II differ in several ways ranging from analysis toolbox, eye artifacts removal to reference electrodes, etc., there are too many candidate parameters to be causally related to a specific difference in an ERP component. Nevertheless, these results are interesting as they highlight how the use of different pre-processing pipelines commonly used in the EEG literature can affect ERP morphology at an aggregate level, and in particular, the choice of the pipeline can impact the extent to which one correctly/incorrectly determines differences between conditions. While it would be interesting to unveil possible causal relations between these differences in the pipeline, the goal of the present study is to illuminate the impacts of common methodological differences between studies rather than to fully explain such differences, which would require a larger study. Furthermore, considering the few existing studies in the literature  as used in Dataset I (UCR) and Dataset II (KU Leuven), as well as task 1  which was used in Dataset I (UCR) and Dataset III (UM). We did not compare Datasets II and III as the stimuli were different: pictures vs. words respectively, whereas Dataset I included both words and pictures and could therefore be compared to both datasets. We note, that while this analysis is far from comprehensive and it would be ideal to collect data on identical procedures across the labs, however, this is at least illuminative of other, unexplained, variance that can be expected from different labs conducting similar research but not coordinating on the exact details of the studies, which is typical of the extant literature.Another potential aspect of variation is the experimental location resulting in behavioral and ERP morphology differences. Specifically, we refer to different laboratories to explore differences in several characteristics such as lab settings, stimuli, tasks, subject pools, subject instructions, processing pipelines, and so on. Using pipeline I, we compared task 2 showed significantly higher accuracy in Dataset II compared to Dataset I  were found in P1, N1, P2, N2, and P3 components, in channels Fz and Cz.Behavioral results for task 1, higher accuracy was observed in Dataset III compared to Dataset I , revealing higher accuracy for task 3 compared to the other two, perhaps due to having the longest stimulus duration  thereby supporting the process for the encoding of information that is facilitated when stimulus duration is longer. Indeed, Kunimi (stimulus type and observed better performance for pictures of objects compared to words and colors. In contrast, Nystrom et al. (Given the fact that most meta-analyses overlook differences in the N-Back task adopted in each study (Glahn et al., , Kunimi  showed t, Kunimi  showed tm et al.  reportedtask structure and stimulus type is their impact on ERP morphology. To highlight this variance, we examined differences in several ERP components, named N1, P2, N2, and P3 for both factors, as previous studies suggested ERP component modulation in response to WM experimental features, particularly for stimulus type, and observed their spatial distribution. Mecklinger and Pfeifer (Another important aspect when considering the following factors such as  Pfeifer  reported Pfeifer  showed v Pfeifer  observedstimulus type and task structure, we suggest that different experimental laboratories and pre-processing procedures might also affect the accuracy and ERP morphology. Seemingly arbitrary procedures are employed by different laboratories, in terms of environment and equipment, as well as data pre-processed and analyzed by different pipelines, which have been shown to produce different findings (Busch et al., In addition to Our study presents several limitations. We considered only accuracy during N-Back performance, because the three Datasets and the related tasks had different response requirements, and so it would have been very complex to compare them. As Dataset I utilized a mixed within/between design, individual differences might have affected ERP signatures attributed to laboratory effects. Indeed, a recent review paper highlighted the variety of features that may impact N-Back performance, including both task and individual features (Pergher et al., stimulus type, task structure, and laboratory and processing pipeline give rise to differences in behavioral and physiological outcomes. While future research is needed to help us understand the mechanisms that underly these observed differences, the present work can help readers appreciate effect sizes to be expected related to the many variations considered here. We note that while, in general, it is well acknowledged any difference between studies can have an impact, the significance of these variations in the case of the N-Back has been largely overlooked, thus limiting understanding of their role in affecting accuracy and ERP morphology and of potentially important information related to the mechanisms that regulate WM processes. We suggest that for the field to move forward, experimental features, analysis pipeline, and laboratory differences need to be taken into consideration when interpreting findings and making comparisons across studies.In conclusion, the present data sets help clarify the extent to which common N-Back task variations in terms of The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Institutional Review Board of the University of California Riverside. The patients/participants provided their written informed consent to participate in this study.VP, MS, and AS conceived the presented idea and developed the theory. MS, VP, and AP carried out the experiment. MS and VP performed the computations. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.