In the SSD group, the MAE on the intact ear side was worse than that of the acutely plugged controls, and it deteriorated with longer duration/younger age at SSD onset. On the impaired ear side, MAE improved with longer duration/younger age at SSD onset. Performance asymmetry across lateral hemifields decreased in the SSD group, and the maximum decrease was observed with the most extended duration/youngest age at SSD onset. The decreased functional asymmetry in patients with right SSD was related to greater cortical volumes in the right posterior superior temporal gyrus and the left planum temporale, which are typically involved in auditory spatial processing. The study results suggest that structural plasticity in the auditory cortex is related to behavioral changes in sound localization when utilizing monaural level cues in patients with SSD.Spatial hearing, which largely relies on binaural time/level cues, is a challenge for patients with asymmetric hearing. The degree of the deficit is largely variable, and better sound localization performance is frequently reported. Studies on the compensatory mechanism revealed that monaural level cues and monoaural spectral cues contribute to variable behavior in those patients who lack binaural spatial cues. However, changes in the monaural level cues have not yet been separately investigated. In this study, the use of the level cue in sound localization was measured using stimuli of 1 kHz at a fixed level in patients with single-sided deafness (SSD), the most severe form of asymmetric hearing. The mean absolute error (MAE) was calculated and related to the duration/age onset of SSD. To elucidate the biological correlate of this variable behavior, sound localization ability was compared with the cortical volume of the parcellated auditory cortex. In both SSD patients ( Unilateral loss of hearing deteriorates sound localization ability, which relies largely on binaural time and level differences in the sound source that reaches the two ears. Loss of binaural time/level cues results in poor localization performance, which is a significant behavioral deficit in patients with asymmetric hearing . HoweverAlthough adaptive changes in localization performance have been reported repeatedly, the neural correlates associated with functional changes have not yet been clearly addressed. Different auditory spatial cues are separately processed in the lower brainstem and are integrated in the higher subcortex or above. Across the auditory neural pathway, functional changes that improve localization performance occur at a higher level, where cue integration takes place and enables the reweighting of spatial cues. The auditory cortex plays a crucial role in auditory spectrotemporal analysis, and functional neuroimaging studies  and lesin = 23) to examine the behavioral change in auditory spatial ability in the SSD group. In the SSD group, clinical factors were analyzed to determine whether the duration of SSD/age at SSD onset was related to behavioral changes. To examine the biological substrate in the auditory cortex associated with behavioral adaptation, the GM volume was measured in the parcellated regions of the bilateral auditory cortices and compared to the changes in localization performance using monaural level cues in the SSD group.In the present study, sound localization ability using monaural level cues was evaluated in 26 patients with SSD and compared with those from acute plugged controls .Demographic and clinical information for all subjects is provided in Twenty-three healthy subjects  with normal hearing (PTA less than 25 dB HL in both ears) without a history of hearing problems served as the normal control (NC) group. The participants were matched to the SSD group in terms of the hearing threshold in the intact ear, sex, and age.All participants were right-handed. Subjects were excluded if they had any neurological/neuropsychiatric history or took any related medication. Medical history including diabetes, hypertension, and any condition that contraindicated MRI scanning were also exclusion criteria.This study protocol was approved by the Institutional Review Board of the Hallym University Sacred Heart Hospital  (IRB No. 2018-02-019-002).A sound localization test was performed using a DHA-8 apparatus  that assesses a person’s ability to identify the source location of a sound. Eight speakers were used in the experiment with two speakers in the front and rear of the subject (at 0° and 180°) and three additional speakers on each side of the individual, separated by 45°. All speakers were mounted at head level and labeled no. 1 to no. 8 .® software  was used to randomize trials and to record responses.As auditory stimuli, a 1 kHz warble tone at 40 dB SL was presented using GSI 61 Audiometry . The duration of the sound presentation was 3 s, with an approximate 5 s interstimulus interval. Five trials from each of eight loudspeakers were presented in random order, and a total of 40 trials were tested per subject. PresentationThe subjects were seated in the center of the sound booth and a chair facing the loudspeaker at 0° azimuth (front) and at 1 m from the speakers in any direction. Prior to the test, a training session composed of randomized trials of three presentations per speaker was conducted. Subjects answered the sound source by the number of speakers using their own voice. Participants were asked not to move their head in the course of the experiment. The response angle was averaged for five trials per speaker. To calculate the mean absolute error (MAE), the absolute difference between the response speaker and stimulus speaker was averaged for five trials per speaker, meaning smaller values indicated better sound localization. An asymmetry index (AI) was calculated to reflect performance discrepancies between the good- and poor-hearing sides. AI was defined as a relative difference in weighted angle differences between good- and poor-ear sides using the following equation:This index may span from −1 to 1. When this index approaches zero, it means that the sound localization performance is similar across the good- vs. poor-hearing ear sides. When the good-hearing side markedly outperformed the poor-hearing side, the AI was approximately 1.via the intact ear. In the NC group, a simulation experiment was conducted to compare the results with those of the SSD group. The right ear was covered with an earmuff and subjected to narrowband noise (centered at 1 kHz) for masking using an insert earphone , which prevented auditory stimuli from being processed by the covered right ear. The narrowband noise for the masking was provided through an audiometer at 20 dB SL for frequencies above 1 kHz .The SSD group was tested High-resolution 3D-T1 weighted images were obtained using a 3T MRI scanner  in both the SSD and NC groups. A 3D-gradient fast field echo sequence was applied with repetition time (TR) = 9.3 ms, echo time (TE) = 4.6 ms, field angle of field of view (FOV) = 230 × 230 mm, and matrix size = 230 × 200. The slice thickness was 1.0 mm, for a total of 160 slices without gaps.1) implemented in MATLAB version R2009a   with the Dartel toolbox running within SPM8  . First, 2 The ROIs encompassing auditory regions were the anterior part of the superior temporal gyrus (STG), posterior part of the STG, and PT. These ROIs were binarized and registered to the individual’s anatomical space. Additionally, a gray matter mask was applied to transformed ROIs to extract distinct anatomical indices. Finally, to correct for variation in brain size across the individuals, normalized cortical volume (nCV), which represents the GM volume of the predefined ROIs divided by whole-brain volume, was calculated for each ROI.Three regions of interest (ROIs) per hemisphere  were extracted from the Harvard-Oxford cortical and subcortical structural atlases using the FSL anatomy toolbox.t-test. Between the two SSD groups, the age at SSD onset, the duration of SSD, and PTA in the poor-hearing ear were compared using the Wilcoxon rank sum test. The PTA in the good-hearing ear was compared across the two SSD groups and the acutely plugged NC group (left ear) using the Kruskal–Wallis test.Statistical analyses were conducted with R using the ppcor, jmv, dunn.test, and WRS2 packages . A normapost hoc test with the p-value adjusted by the Benjamini–Hochberg method. Nonparametric partial correlation analyses were carried out between sound localization performances (MAE and AI) and clinical factors (age at SSD onset and duration of SSD), controlling for age.To analyze sound localization test results, response angles and MAE were analyzed to test the group differences and speaker directions effect (with interaction), using robust ANOVA for mixed design provided by WRS2 package. Nonparametric repeated-measures ANOVA tested the stimulation direction effect on the MAE across groups. The Wilcoxon rank sum test was used to compare the AI values and the MAE in each direction between the NC and SSD groups. For comparison across NC and SSD subgroups, the Kruskal–Wallis test was performed with Dunn’s When analyzing cortical structure, two SSD groups were analyzed separately, considering the innate structural/functional asymmetry of the brain. The nCV in each ROI was compared across groups using robust one-way ANOVA based on trimmed means. A nonparametric partial correlation was used to examine whether the nCV extracted from six ROIs was related to the age at onset of SSD, duration of SSD, and AI, controlling for age at the time of the experiment.F = 1.273, p = 0.290] and sex distribution  were not significantly different. PTA in the good (tested) ear was also not different across the three groups . Between the two SSD groups (SSD-R vs. SSD-L), there were no significant differences in PTA in the poor ear , duration of SSD (p = 0.716) or age at onset of SSD (p = 0.755).Across the three groups, age .In the NC group, PTA from both ears was not different . Therefore, all data reported here were standardized such that negative angular or stimulus values represent the impaired (plugged) ear side and positive values represent the intact (open) ear side. The pooled data from the SSD group were compared with those from the NC group (the right ear was covered and masked to simulate SSD-R).When the localization test results of the SSD-L group were flipped to match the good-/poor-hearing side with those of the SSD-R group, the response angle was different across stimulation directions, whereas no differences were found for groups. A mixed design ANOVA results showed no significant interaction between stimulation direction and group . When the SSD group was divided into three subgroups by the duration of SSD  = 19.3, p = 0.007; subgroup: F = 5.27, p = 0.078; stimulation direction × subgroup: F = 14.3, p = 0.012].The sound localization performance of all individuals in the NC and SSD groups is provided in n of SSD , stimulaBoth the NC and SSD groups showed a clear response bias toward the intact side. While 37.5% of the stimuli were provided on the good-hearing side (three out of eight speakers), 68.2 and 67.3% of responses were recorded on this hemifield in the plugged NC and SSD groups, respectively. However, in the impaired side of the SSD subgroup with a very long duration of SSD (43–46 years), bias toward the intact ear side was not apparent, of whom three out of five responded with the correct hemifield to stimuli from three speakers on the poor-hearing side  = 0, p = 0.967; stimulation direction: F = 41.3, p < 0.001; stimulation direction × group: F = 3.7, p = 0.028]. When comparing each direction separately, the simulated NC group showed significantly better performance  than the SSD group for the good-hearing side only . In the NC group, MAE values were significantly different across source directions ; the MAE for the good-hearing side was significantly better than that for the poor (p = 0.001) and front (p < 0.001) directions but was not better than that for the back direction (p = 0.234), for which the MAE was also smaller (better) than that for the poor-hearing (p = 0.036) and front (p < 0.001) directions. When MAE values were further analyzed across the NC group and three SSD subgroups with different durations of SSD  = 43.7, p < 0.001] and interacted with the group , but the group effect was not significant . In the post hoc comparison, the group difference was significant only in the good-hearing direction , with a significantly smaller MAE in the NC group over two SSD subgroups with over 10 years of SSD . Similar to the NC group, the SSD <7years group showed the best MAE on the good-hearing side compared with all three other directions , but in the other two subgroups with over 10 years of SSD, MAE values on the good-hearing side were not significantly superior to the MAE values on the poor-hearing side and back (p > 0.05)  and compared between groups . When coide only . On the s of SSD , MAE val > 0.05) .p = 0.046), meaning that localization performance between the good- and poor-hearing sides was more symmetric in the SSD group than in the acutely plugged controls. When further compared between the NC group and SSD subgroups, AI values differed significantly across groups , with significantly smaller AI values in the SSD43–46years group than in the NC group (p = 0.042) and SSD <7years group (p = 0.033).The AI values were calculated in each group to represent the difference in sound localization performance between good- and poor-hearing sides . AI valup > 0.05), MAE values from the poor-hearing side showed a significant correlation with both factors . On the good-hearing side, a negative trend showing worse performance with SSD onset at a younger age was found (p = 0.124). As expected with the strong negative correlation between onset age and duration of SSD, a reverse relationship was found with the duration of SSD. When the duration of SSD increased, localization performance improved on the poor-hearing side , and tended to deteriorate on the good-hearing side . As a result, the difference in sound localization ability between the good- and poor-hearing sides was the largest when the duration of SSD was the shortest (and the age onset of SSD was the latest), and the performance asymmetry between the good- and poor-hearing sides decreased as the duration of SSD increased (and with a younger age of SSD onset). This change in performance asymmetry was confirmed with the analysis of AI in this group. The AI value was smallest when the onset age of SSD was the youngest . In both the SSD-R and SSD-L groups, nCV in all six ROIs did not reveal a significant relationship with either the onset age of SSD or duration of SSD . In the SSD-R group, greater volumes in the right posterior STG  and the left PT  were associated with smaller AI values  In the SSD group, localization ability using level cues on the intact ear side was worse than that in the acutely plugged controls  and tendFor binaural listeners, interaural time and level differences are the two most reliable sources for azimuthal localization, and monaural spectral cues play an auxiliary role, such as discriminating sources in the cone of confusion . The monOn the intact ear side, both the SSD and simulated NC groups showed clear response bias toward the intact (open) ear side , but theTo pursue the biological substrate that grounds the above observed behavioral changes, the cortical structure of the parcellated auditory cortex was compared to the behavioral performance in the SSD group. The AI was used as a composite index representing behavioral changes across impaired and intact sides. Right and left SSDs were separately analyzed considering the functional/structural asymmetry of the auditory cortex. As a result, the GM volume in the right posterior STG and the left PT were strongly related to the AI in the right SSD patients; a greater volume in those areas was related to more symmetric localization performance between the good-/poor-hearing sides.via the PT and dorsally to the inferior parietal lobule, premotor cortex, and dorsolateral prefrontal cortex, the latter of which is involved in top-down modulation to the primary and nonprimary auditory regions (Auditory spatial processing is mediated by the dorsal stream of the cortical auditory pathway. From the primary auditory cortex, the dorsal stream is directed posteriorly  regions . The pos regions .Compared to bilateral deafness, SSD is a more recent focus of dedicated research, and less is known about neuroplasticity following unilateral hearing loss . Althougvia the intact left ear. Accordingly, the relatively preserved sensory input might incur more extensive structural changes in the auditory cortex in this group.This study found cortical structural changes in relation to auditory spatial behavior in the SSD-R group only. Ear-specific neuroplasticity in patients with asymmetric hearing has been reported, yet the findings are controversial. Recent neuroimaging studies have shown a clear impact of the deafness side, with a greater degree of neural plasticity for right-sided hearing loss . On the The behavioral changes in auditory spatial ability related to the experience of asymmetric hearing have motivated the development of sound localization training programs and devices for patients with asymmetric hearing loss . A recenIn SSD patients, sound localization ability changes in relation to the onset/duration of SSD and prolonged experience/earlier onset of SSD were related to a more symmetric performance of using the monaural level cue across the intact and impaired sides. A significant structural correlation of the posterior auditory region to the adaptive behavioral change in sound localization suggests that neuroplasticity occurs in the cortical areas for auditory spatial processing in subjects who suffer from deficits in function due to asymmetric auditory input.The raw data supporting the conclusions of this article is available from the corresponding author on reasonable request.The studies involving human participants were reviewed and approved by the Institutional Review Board of the Hallym University Sacred Heart Hospital. The patients/participants provided their written informed consent to participate in this study.JHK and H-JL contributed to the conception and design of the study. JHK and LS collected and analyzed the data. H-JL and JB contributed to the final version of the manuscript. All authors discussed the result and contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


The cumulative knowledge of retina development has been instrumental in the generation of retinal organoid systems from pluripotent stem cells; and these three-dimensional organoid models, in turn, have provided unprecedented opportunities for retinal research and translational applications, including the ability to model disease in a human setting and to apply these models to the development and validation of therapeutic drugs. In this review article, we examine how retinal organoids can also contribute to our understanding of retinal developmental mechanisms, how this knowledge can be applied to modeling developmental abnormalities, and highlight some of the avenues that remain to be explored. The vertebrate retina is an extension of the central nervous system composed of seven main types of neurons and glia specialized for visual function. Its delicate and complex organization arises during embryonic development through tightly spatiotemporally regulated mechanisms that are highly conserved among vertebrates .The retina originates from the ventral diencephalon, where a group of cells begins to co-express a set of transcription factors including Pax6, Rax, Six3, Six6, and Lhx2, and becomes specified as the eye field  retinas or retinal “organoids” from embryonic and induced pluripotent stem cells (ESC and iPSC) from different species. But how does this happen? What can we learn about retinal development by studying organoids? And can we harness the potential of organoids to gain a better understanding of congenital retinal abnormalities? Rather than compiling a comprehensive account of the literature on these topics, we highlight some key studies that illustrate the contributions of retinal organoids to answering these critical questions and propose avenues for further exploration.Considering its complexity, it is extraordinary that this process could be reproduced Retinal organoids mimic the cellular composition and histoarchitecture of the native retina, including the differentiation of all major cell types organized in the characteristic trilaminar structure. Moreover, they are capable of achieving an advanced level of maturation, including the ability to respond to light stimulation and form functional synapses  surface, with interkinetic nuclear migration displacing nuclei radially to their final location  signaling from the surface ectoderm is necessary to specify the presumptive neural retina territory by upregulating the transcription factor Vsx2, which is involved in a cross-repressive loop with MITF. In mouse and chick, removal of the surface ectoderm at the optic vesicle stage results in failure to specify a neural retina, leading to the development of microphthalmic pigmented vesicles, which can be rescued by exogenous FGF administration  gene that causes familial forms of glaucoma, with organoids derived from control subjects, and found that RGCs in OPTN mutant organoids displayed a significant increase in caspase-3 activation. Further, treatment of OPTN RGCs with BDNF or PEDF caused a reduction in caspase-3 activation, highlighting the utility of this model as a tool for pharmacological development , which was significantly upregulated in this model, led to apoptosis in cancerous organoids, which could be relevant as a potential therapeutic agent (Liu et al., Finally, retinal organoids have also been used to model retinoblastoma, the most prevalent intraocular malignancy in children, which has a developmental origin (Liu et al., Despite these and other encouraging results, the potential of organoids to study congenital retinal defects remains largely untapped. For instance, these models could be used to elucidate the mechanisms that lead to the retinal phenotype observed in conditions like Down syndrome, where progress has been slow due in part to the limitations of animal models in recapitulating human pathophysiology; and they could also contribute to our understanding of how viruses, toxins and other environmental exposures affect the human retina during embryonic development, as it has been described for human brain organoids. For example, infection with Zika virus, which causes fetal microcephaly, has been modeled in forebrain organoids from hiPSCs (Garcez et al., The cumulative knowledge of retina development has been instrumental in the generation of retinal organoid systems. The time is now ripe for retinal organoids to inform our understanding of retina development. This deeper understanding, combined with the advantages of retinal organoids as culture models that allow tight control of experimental manipulations and the possibility to model disease in a human setting, offer unique opportunities to gain insights into the pathophysiology of congenital retinal abnormalities for the development of potential therapeutic approaches.MV and AV wrote the initial manuscript draft. YP and CS prepared the illustrations. 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.


To describe and study the population statistics, hearing phenotype, and pathological changes of a porcine congenital single-sided deafness (CSSD) pedigree.Click auditory brainstem response (ABR), full-frequency ABR, and distortion product otoacoustic emission (DPOAE) were used to assess the hearing phenotype of the strain. Tympanogram was used to assess the middle ear function since birth. Celloidin embedding–hematoxylin–eosin (CE-HE) stain and scanning electron microscopy (SEM) were used to study the pathological changes of cochlear microstructures. Chi-square analysis was used to analyze the relation between hearing loss and other phenotypes.P = 0.0841 > 0.05) and gender (P = 0.4621 > 0.05) by chi-square analysis. The deaf side of CSSD offspring in the fifth generation had no relation with that of their maternal parent (P = 0.2387 > 0.05). All individuals in this strain exhibited congenital severe to profound sensorineural hearing loss with no malformation and dysfunction of the middle ear. The good hearing ear of CSSD stayed stable over age. The deaf side of CSSD and BHL presented cochlear and saccular degeneration, and the hair cell exhibited malformation since birth and degenerated from the apex to base turn through time. The pathology in BHL cochlea progressed more rapidly than CSSD and till P30, the hair cell had been totally gone. The stria vascularis (SV) was normal since birth and degenerated through time and finally exhibited disorganization of three layers of cells.The mating mood of CSSD with CSSD was most efficient in breeding-targeted CSSD phenotype (47.62%), and the prevalence of CSSD reached 46.67% till the fifth generation, where 42.22% were bilateral hearing loss (BHL) and 9.00% were normal hearing (NH) individuals. Hearing loss was proved to have no relation with coat color (This inbred porcine strain exhibited high and stable prevalence of CSSD, which highly resembled human non-syndromic CSSD disease. This porcine model could be used to further explore the etiology of CSSD and serve as an ideal tool for the studies of the effects of single-sided hearing deprivation on neural, cognitive, and behavioral developments and the benefits brought by CI in CSSD individuals. Single-sided deafness (SSD) is defined as profound sensorineural hearing loss in one ear with normal hearing on the opposite side. Congenital SSD (CSSD) often refers to those who did not pass the newborn hearing screening and then was diagnosed with unilateral hearing loss at birth. The incidence of sensorineural hearing loss is estimated to be 1.86 per 1,000 newborns; among them, 30–40% are unilateral , but theKIT or KITLG. KIT-KITLG signaling pathway and MITF were suggested to mutually interact in the migration process of melanocyte from the neural crest to stria vascularis (SV). The imbalanced migration and distribution of melanocytes in stria vascularis might be cause by laterality of hearing  in neuroscientific studies. The mechanism and the onset of neural reorganization might differ for CSSD and ASSD, which should be considered . Since cPAX3 and KIT , which would influence the differentiation and migration of melanocytes in cochlea during embryogenesis  were feline homolog of the human Waardenburg syndrome  because  and KIT . Andrej  and KIT , which w and KIT  with pre and KIT . Most st and KIT . Hearing and KIT . Howeverogenesis . People Although the above animal breeds exhibited high prevalence of deafness, however, few hereditary components were verified, and nearly all breeds mentioned above mimicked the phenotypes of human Waardenburg syndrome, and the prevalence of CSSD was casual. In our study, we describe a naturally occurring inbred CSSD porcine pedigree with high and stable prevalence of CSSD. Auditory physiology and pathological presentations of different hearing phenotypes in the pedigree were uniform and detailly described.All animals of this inbred Bama Miniature pig pedigree in this paper were provided by the Laboratory Animal Science Center of College of Basic Medicine in the Army Military Medical University . All animals were raised in a standard pathogen-free (SPF) condition. Animals younger than P30 were raised in the Lab Animal Science Center in Chongqing; animals older than P30 were delivered to the Lab Animal Center of PLA General Hospital and being raised in the same condition.In all experiments, animals were anesthetized with 1.5–5% Isoflurane in 3:3 mixture of oxygen and air by inhalation machine for animal use . Animals were put on a heating pad to maintain body temperature.Since the full-frequency auditory brainstem response (ABR) measurement would take about 2 h, pigs under P30 could not stand long-time anesthesia. Therefore, for the pigs younger than P30, only stimulus click  was used to diagnose if they are deaf or not, and hearing tests were taken in a small sound-proof booth for animals. Insert ear plug was put into the ear and sealed the external ear canal. Ground electrode was put at the apex nasi; reference electrodes were put in the ipsilateral earlobes of the tested ear, and recording electrode was inserted into the skin of the calvaria along the centerline. The click ABR tests only took about 10 min each pig.Tucker Davis Technology RZ6 (TDT RZ6) was used to apply the full-frequency—from 1 to 32 kHz—ABR tests in pigs over P30. Electrodes were put in the same position as described above. This part of hearing tests was conducted in a standard soundproof booth. A loudspeaker (MF1 2356) was put in the external ear canal meatus, and the untested ear was masked by a calibrated 60 dB SPL white noise by an inserted earphone.Titan  was used to do the tympanometry, which reflected the function of the middle ear, and parameters like middle ear compliance and volume were obtained. Distortion product otoacoustic emission (DPOAE) from 500 to 10 kHz was measured by Titan, which reflected the function of outer hair cells (OHCs). Tympanogram and DPOAE were tested at P1 and P30.Celloidin embedding–hematoxylin–eosin (CE-HE) stained cochlear section and scanning electron microscopy (SEM) samples were made following the methods described in our previous studies. Animals were sacrificed in accordance with the Care and Use of the of Laboratory Animals. Cochleae were extracted from the temporal bone within 10 min and being postfixed in 4% paraformaldehyde (CE-HE) or 2.5% glutaraldehyde (SEM) at 4°C overnight. After being washed in 1% phosphate-buffered saline (PBS) three times, each time 10 min, the cochleae were shifted into 10% ethylenediaminetetraacetic acid (EDTA) solution for decalcification at room temperature (RT) on a shaker for 2 weeks.2. Fixed sections were then coated using a sputter coater and examined under a scanning electron microscope (Helios Nanolab 600i).For the HE staining, the cochleae were dehydrated using graded ethanol , and each grade would take 2 days. Then, the cochleae were transferred into graded celloidin , and each grade would take 7 days at RT. The cochleae were embedded in 15% celloidin in a glass dish for 1–2 months until solidification. Then, the samples were put into 75% ethanol for 2–5 days. The samples were sectioned (15 μm each slice) using a freezing microtome (Leica CM1900) and stained with hematoxylin and eosin. The images were visualized and captured using a Leica DMI3000 microscope. For the SEM, decalcified cochleae were postfixed for 2 h in 1% osmium at RT, dehydrated in graded ethanol , treated with 2% tannin acid for 2 h at RT, rinsed in 0.24 M phosphate buffer (pH 7.4) for 2 h at RT, and dried in a critical point dryer  using liquid COSpiral ganglion cells were counted, using the CE-HE specimen under a Leica DMI3000 light microscope. Two cochleae of each hearing phenotype were counted; 12 serial sections, 15 μm each specimen, containing modiolus were counted, and for each specimen, apical, middle, and basal turn ganglion cells were counted, respectively. The mean values of the above 12 serial sections at each turn were used as the ganglion cell counting number for each turn. Only the cells with clear nucleus were counted .t-test was used to analyze the statistical difference of tympanogram parameters and ganglion cell counting number of each group compared to the normal group, and P < 0.05 was considered as statistical different.Prism GraphPad 8.4.0 was used to do statistical analysis and draw graphs. Chi-squared test was used to analyze the possible hereditary mode. Unpaired All experiments and procedures in this paper were conducted under the guidelines of the Care and Use of the Laboratory Animals and approved by both the Animal Ethics Committee of Army Military Medical University and PLA General Hospital.P = 0.0841 > 0.05). Since we also observed the pigmentation of iris color in some pigs within the pedigree, we listed the iris color change in different phenotypes .In the whole pedigree, female individuals amounted to 16 (55.17%) and male individuals 13 (44.83%). The ratio of female CSSD to male CSSD was 1.23:1, close to 1. In the whole pedigree, L-CSSDs amounted to 14 (48.28%), and R-CSSD totaled 15 (51.72%) with a ratio of 1:1.07. Among female CSSDs, the proportion of L-CSSD (9/56.25%) to R-CSSD (7/43.75%) was 1.28:1. Among the male CSSDs, the proportion of L-CSSD (5/38.46%) to R-CSSD (8/71.54%) was 1:1.6 . As showIn the fifth generation, there were 45 siblings with 21 (46.67%) CSSD individuals, 19 (42.22%) BHL individuals, 4 (9.00%) NH individuals, and 1 (2.11%) unknow phenotype (died at P1 because of diarrhea before hearing test) . In the P = 0.2387 > 0.05).Next, we explored the relation of deaf laterality between the offspring and their parents. The paternal pig of the fifth generation was the same R-CSSD male pig; the maternal pigs included both L-CSSD and R-CSSD individuals. Under the mating strategy of Male R-CSSD with female L-CSSD, two (25.00%) of the offspring were R-CSSDs and six (75.00%) were L-CSSDs, and under the strategy of male R-CSSD mating with female R-CSSD, eight (61.64%) of the offspring were R-CSSDs and five (48.36%) were L-CSSDs . As showbilateral hearing losses and the deaf ear of CSSD individuals presented congenital profound sensorineural hearing loss since P1 through all frequencies. NHs and the normal side of CSSD showed normal hearing thresholds since P1 through all frequencies . The heaThe volume and the compliance of middle ear showed that there were no statistical differences among each group of the CSSD pedigree . This reIn the CE-HE images of NHs and the normal sides of CSSDs, three rows of OHCs and one row of inner hair cells (IHCs) could be identified, and supporting cells like Dieter cells, Hansen cell, and inner and outer pillar cells closely and regularly arranged . In the In the BHLs, HC damage progressed the same trajectory as the CSSD but was more severe and rapid compared to the deaf sides of CSSDs. At P1, hair bundles of IHCs at the apex turn were fused; OHCs were disorganized with fused bundles . IHCs ofThe ganglion cells counting number of BHL, deaf side (DS) of CSSD, and normal side (NS) of CSSD showed significant decrease compared to the NH and that of the DS of CSSD showed significant reduction compared to the NS of CSSD; about 76, 77.4, and 66.7% cells survived for BHL, DS of CSSD, and NS of CSSD, respectively . For theIn NHs, the stria vascularis was composed of three layers of cells: marginal cells, intermediate cells, and basal cells. Marginal cells (star) were tightly attached with each other by gap junction. Basal cells (triangle) were interconnected to separate stria vascularis (SV) from spiral ligament. Intermediate cells (circle) were in the middle layer in which melanin spots could be identified. At P30, the SV of NHs and both sides of CSSDs were of normal structure, while in BHL, SV was disorganized with only two layers of cells survived .The structure of SV degenerated through age. In DS of CSSD cochleae, the structure of SV remained normal till P154. At P180, the SV was dominated by marginal and basal cells; the intermediate cells declined . In BHL Sensorineural hearing loss is often induced by loss of HCs and SGNs in the inner ear cochlea . HC tranIn this paper, we studied the auditory physiology and pathology of BHL, NH, and CSSD individuals. All the deaf cochlear showed cochlear–saccular degeneration, also known as Scheibe dysplasia. For DS of CSSD and BHL, bundles of hair cells were fused since birth, and hair cells degenerated from the apex to the base. In the CSSDs, sporadic hair cells could be identified at P80–P154; basal membrane was replaced by the epithelial cells. BHL showed the same but more severe and rapid pathological process; till P30, hair cells and supporting cells could not be identified, and the Reissner’s membrane tensely attached to the basal membrane. Only sporadic pillar cells survived. The ipsilateral saccule exhibited degenerated hair cells since P1. Scheibe dysplasia had been proven to occur in many animals, like deaf white cats, Dalmatian dogs, waltzing guinea pigs, and mice . It was For the degeneration of the ganglion, we did not have enough animals at different ages, so we chose pigs of P30 to analyze the pathology of ganglion cells. The pathology changes in each hearing phenotype were uniform in this pedigree. Significant reduction in ganglion cells of BHL, DS of CSSD, and NS of CSSD could be observed. For BHL, the ganglion cell counting number at each turn decreased proportionately. For CSSDs, the most severe cell loss occurred in the apex turn; we should also pay attention to the NS of CSSD; the total ganglion cell number and that of each turn showed a significant reduction compared to NHs. This might indicate that the NS of CSSD might be vulnerable to factors that harm hearing like noise exposure and ototoxic drugs. This coincided with the reports of the vulnerability of the good hearing ear of CSSD humans.P = 0.19 > 0.05), 40.77 ± 7.70 (P < 0.0001), 36.00 ± 6.06 (P < 0.0001), respectively, and the counting number at apex turn showed no significant differences with the NH. Meanwhile, the organ of Corti was flattened, and supporting cells vanished at the apex and mid turn at P1 (P < 0.001), 37.10 ± 7.16 (P < 0.001), and 31.30 ± 7.14 (P < 0.001), while the organ of Corti showed the same but much milder pathological changes. These results indicated that the pathological process started from embryogenesis period because the degeneration of supporting cells occurring after the acquired hearing loss would trigger the neural degeneration at least 2 weeks away , broad availability, large litter size, and lower ethical concern because they are of a kind of food source instead of companion animals like cats and dogs . Pigs arAll the relevant data is contained in this article. The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.The animal study was reviewed and approved by The Ethics Committee of Chinese PLA General Hospital. Written informed consent was obtained from the owners for the participation of their animals in this study.WR, CX, and F-JZ mainly conducted the experiment. YW, T-TL, C-HL, X-YZ and L-LW were mainly in charge of breeding and maintaining the pedigree. YZ mainly conducted the SEM and TEM studies and illustrated the relative results. HZ, W-WG, PJ, and S-MY designed the study, analyzed the data, and illustrated the pathology results of the pedigree. WR finally contributed to the writing of the article, figures, and tables. 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.


For patients with severe and profound hearing loss, communication is significantly more difficult (50.0% and 45.0% respectively) when the interlocutor wears a face mask. There is a significant correlation between the degree of hearing loss and the way in which communication is made more difficult when the interlocutor wears a face mask (p < 0.001). A statistically significant difference was determined between the degrees of hearing loss in all measured subscales: stress (p = 0.024), anxiety (p = 0.026) and depression (p = 0.016). Conclusions: We have determined that face masks used during the COVID-19 pandemic significantly hamper communication among the study groups (p = 0.007) and there is a significant correlation between the degree of sensorineural hearing loss and the presence of symptoms in all three DASS-21 subscales, meaning that the symptoms of stress, anxiety and depression were more intense in severe and profound hearing loss.Background. The incidence of hearing loss is constantly increasing and according to the World Health Organization, by 2050, 900 million people will suffer from hearing loss. The main Objective of the study was to determine the differences between the severity of the symptoms of stress, anxiety and depression in participants with varying degrees of sensorineural hearing loss during the COVID-19 pandemic. An additional aim was to examine the extent and manner in which protective face masks impact the communication of people with hearing loss. Matrials and Methods: A cross-sectional study was conducted, which included 160 patients (81 men and 79 women) with bilateral sensorineural hearing loss. The patients’ age range was 50 to 80 years. Depending on the degree of hearing loss or pure-tone threshold, the participants were divided into four groups: mild hearing loss, moderate hearing loss, severe hearing loss and profound hearing loss. The research used the Depression, Anxiety and Stress Scale (DASS-21) and a questionnaire in which the participants reported whether surgical face masks  worn by speakers makes communication difficult, to what extent and in what way. Results: The average age of the patients was 67.97 ± 8.16. A significant correlation was found between the degree of hearing loss and communication difficulties caused by the use of protective face masks ( The incidence of hearing loss is constantly increasing and, according to the World Health Organization (WHO), hearing loss had been determined in 466 million people worldwide until 2018. It has been estimated that by 2030, 630 million people worldwide will suffer from hearing loss and by 2050 the number will reach 900 million  in patieThe COVID-19 pandemic itself affects mental health and the development of the symptoms of stress, anxiety, depression, insomnia, anger and fear, all of which can even lead to suicide . Fear anThe main aim of the study was to determine the differences in the severity of the symptoms of stress, anxiety and depression in participants with varying degrees of sensorineural hearing loss during the COVID-19 pandemic.The additional aim was to determine how the degree of hearing loss affected communication while people were wearing a face mask.The study was designed as a cross-sectional survey which included 160 patients (81 men and 79 women) with bilateral sensorineural hearing loss, aged 50 to 80 years. The study was conducted at the Otorhinolaryngology Clinic (ENT) of the University Clinical Center (UCC) Nis in the period from March to May 2021 during the COVID-19 pandemic.Inclusion criteria were: bilateral sensorineural hearing loss of patients aged 50 to 80.Exclusion criteria were: conductive and mixed hearing loss, congenital hearing anomalies, Meniere’s disease, posterior cranial fossa tumor, acoustic neurinoma, glomus tumor, tinnitus, incorrigible vision loss, psychiatric disorders, neurological disorders caused by alcohol and psychoactive substance abuse, patients with severe heart, kidney, lung and malignant diseases.The research was approved by the Ethics Committee of the University Clinical Center Nis .Data were collected using semi structured questionnaire specially created for this investigation. The questionnaire consisting of 18 questions and with two distinct sections.A Likert scale is a psychometric scale commonly used to represent participants opinions and attitudes to a topic or subject matter. The four point Likert scale was used in this study. Patients assessed each item on the Likert scale from zero-0  to 3 (it concerns me most of the time) in relation to how often they felt that way during the last week.The scores on each subscale multiplied by two, so the results ranged from 0 to 42, where higher scores indicate higher levels of anxiety, depression and stress.The participants stated whether and to what extent  they experienced hearing difficulties in communication with an interlocutor wearing a surgical face mask  as well as how communication is most impeded .After signing an informed consent, the participants filled in the questionnaire with the help of an examiner.Depression, Anxiety and Stress Scale (DASS-21) was used to assess the emotional state . The DASThe DASS-21 is based on a dimensional rather than a categorical conception of psychological disorders, and scores emphasize the degree to which someone is experiencing symptoms rather than having diagnostic cutoff points. DASS-21 is a standardized scale for self-assessment of three unpleasant emotional states and it contains three subscales each with seven items.The first scale is about depression: dysphoria, hopelessness, devaluation of life, self-deprecation, lack of interest/involvement, anhedonia and inertia. . The second scale is about anxiety: autonomic arousal, skeletal muscle effects, situational anxiety and subjective experience of anxious affect. . The third scale is about stress: levels of chronic nonspecific arousal, difficulty relaxing, nervous arousal, and being easily upset/agitated, irritable/over-reactive and impatient. . Score are presented as a total score and a score for the three subscales [The diagnostic procedure included an audiological examination where the hearing threshold was determined by liminal tonal audiometry on a Diagnostic Audiometer AD 629  in a soundproof room with pure tones at 128 Hz to 8 kHz frequencies. Hearing loss is defined by hearing the threshold in dB in the frequency range of 0.5 to 4 kHz. The degree of hearing loss was determined based on the assessment by the World Health Organization (WHO) where the following applied: 0–25 dB—normal hearing, 26–40 dB—mild hearing loss, 41–60 dB moderate, 61–80 dB severe, and >80 dB profound hearing loss [p < 0.05.The analysis of the obtained data was done using the SPSS 16.0 software package for Windows . The obtained data are presented as arithmetic mean and standard deviation (SD). For the purposes of analysis, the data were also presented as absolute and relative numbers. The chi-squared test was used to analyze and test category features. The nonparametric Kruskal–Wallis H test was used to examine the differences in DASS-21 symptoms intensity among groups of patients with varying degrees of sensorineural hearing loss. The null hypothesis was tested with a significance threshold of p = 0.965) and age (p = 0.491). Protective face masks used in the COVID-19 pandemic affected communication differently among the study groups and the differences were statistically significant (p = 0.007). The least affected was the Mild HL group (77.5%) whereas for the other groups, the difficulty was more even (90.9–97.5%). It was recorded that the patients’ ability to lip read differed statistically significantly between the examined groups (p = 0.424).The study included 160 patients, average age 67.97 ± 8.16 years . The groups, which were formed in relation to the degree of hearing loss, were equalized according to gender (d groups . Only twp = 0.024), anxiety subscale (p = 0.026) and depression subscale (p = 0.016).p < 0.001). When the interlocutor wears a face mask, communication is significantly more difficult for patients with SHL and PHL  (A significant correlation was determined between the degree of hearing loss and difficulties in communication occurring due to the use of a protective face mask (ctively) .p < 0.001). It was found that in addition to hearing less sound and not understanding what was said, a major subjective problem for patients with SHL and PHL was the inability to read from the lips, 32.5% to 37.5%  may be one of the ways to improve verbal communication during the pandemic due to better auditory perception in people with and without hearing loss . StudiesIn the prevention of future pandemics, it is necessary to understand the importance of protective face masks and to find new options and strategies for fighting against this and other potential viral infections. In patients with hearing loss, when the speaker is wearing a mask, it is necessary to enable communication in such way that the speaker first attracts the listener’s attention, that the lighting is good, that they speak intelligibly and slowly and that they use gestures when they talk. It is also necessary to use hearing amplifiers (which can be adjusted to higher volume levels), cochlear implants, wireless systems and mobile phone applications . It is rOne of the limitations of our research is a small sample of respondents. Also, it does not take into account whether the participants or their families suffered from COVID-19, which may impact their mental status.Based on the presented results, it can be concluded that there are differences in the severity of the symptoms of stress, anxiety and depression among participants with varying degrees of sensorineural hearing loss, where those with SHL and PHL reported more intense symptoms of stress, anxiety and depression. There is a correlation between the degree of hearing impairment of participants and disordered communication resulting from the use of a face masks during the COVID-19 pandemic.


In this study, we confirmed the changes in hearing function and inner ear structure over a long period of time in DBA/2 mice, a laboratory animal model suitable for studying hearing loss. We believe that our study is the first to report findings regarding hearing function and structural changes in DBA/2 mice aged ≥34 weeks. These results are of significance for researchers and the scientific community using laboratory animal models.DBA/2 mice are a well-known animal model for hearing loss developed due to intrinsic properties of these animals. However, results on the phenotype of hearing loss in DBA/2 mice have been mainly reported at an early stage in mice aged ≤7 weeks. Instead, the present study evaluated the hearing ability at 5, 13, and 34 weeks of age using DBA/2korl mice. Auditory brainstem response test was performed at 8–32 KHz at 5, 13, and 34 weeks of age, and hearing loss was confirmed to be induced in a time-dependent manner. In addition, histopathological evaluation at the same age confirmed the morphological damage of the cochlea. The findings presented herein are the results of the long-term observation of the phenotype of hearing loss in DBA/2 mice and can be useful in studies related to aging-dependent hearing loss. With the development of modern civilization, individuals are at an increased risk of exposure to excessive noise. Moreover, with the increase in the number of aging individuals, senile hearing loss is emerging as a major social and environmental concern . As repoStudies using laboratory animals have great merits in that the research environment can be controlled . In addiIn this study, DBA/2 mice, which are frequently used in hearing-loss-related research studies ,9, were Male DBA/2korl mice were kindly provided by the Department of Laboratory Animal Resources at the National Institute of Food and Drug Safety Evaluation . The mice had access to a standard irradiated chow diet  and sterilized water ad libitum. In this study, the animal facility conditions were maintained under a strict light cycle , 23 ± 2 °C, and 50 ± 10% relative humidity under specific pathogen-free conditions. To evaluate hearing loss in the DBA/2korl mice, we used 37 male mice for ABR test and 15 male mice for histopathological analysis. ABR was recorded and histopathological analysis was conducted at 5, 13, and 34 weeks of age. The animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of The Laboratory Animal Center of the DGMIF  and were in accordance with their guidelines.Auditory functions were assessed by recording ABRs using an ABR workstation , Inc., Alachua, FL, USA), as described previously . All tesAfter recording the ABRs, the mice were perfused with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and their inner ears were isolated. For preparing paraffin sections, the inner ears were fixed with 4% PFA in PBS for 24 h at 4 °C and then decalcified with 10% ethylenediaminetetraacetic acid in PBS for 24 h at 4 °C. The specimens were dehydrated using a graded ethanol series, permeabilized with xylene, and embedded in paraffin. The paraffin-embedded inner ears were serially sectioned to obtain 6-μm-thick sections using a microtome , mounted on Superfrost Plus microscope slides , and then subjected to H&E staining.t-test and Excel  and are presented as mean ± standard error in graphical plots. Statistically significant differences are indicated using asterisks. p values of <0.0001 were considered statistically significant.The results were analyzed using Student’s In the present study, four stimuli  were delivered at 5, 13, and 34 weeks of age in the mice, according to the average hearing thresholds A–D. At 5To observe the structural changes in the cochlea of the inner ear with increasing age in DBA/2korl mice, a histopathological analysis was performed using H&E staining at 5, 13, and 34 weeks of age A–C. The DBA/2 mice, an inbred mouse strain, have been widely used as disease models for research related to hearing loss and neuronal degeneration ,11. DBA/In the present study, the ABR test revealed that the hearing thresholds shifted to higher frequencies in elderly DBA/2korl mice. In the organ of Corti in the cochlea, damage such as hair cell loss was observed from 13 weeks of age. These hair cells are extremely fragile and cannot regenerate after being damaged . In the ahlcdh23 allele causes age-related hearing loss [Our study confirmed that this change in hearing function is senile hearing loss, which occurs rapidly in the high-frequency region with age. Taken together, the results obtained here should be useful for senile deafness-related research, understanding the phenotype of laboratory animals, and evaluating the efficacy of new drugs. Of the several mouse models associated with the age-related hearing loss mouse model, the most widely used mouse strain is probably C57BL/6. However, as shown in the results of the previous study , C57BL/6ing loss . These f


Lawsonia intracellularis (L. intracellularis) and is endemic in pig herds worldwide. However, a L. intracellularis-specific monoclonal antibody plays an important role in the evaluation of L. intracellularis infection in vitro. Therefore, the objective of this study was to produce and identify the characteristics of a new monoclonal antibody against the outer membrane protein (Omp2) of L. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC). The results indicated that three highly specific monoclonal antibodies against the Omp2 protein  of L. intracellularis were obtained by using purified Omp2 as an immunogen, the titers of ascitic fluids of 4D9, 3G2, and 7G5 cells were 1:2,048,000, 1:512,000, and 1:256,000, respectively. IFA analysis showed that the 4D9, 3G2, and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs or closely related to L. intracellularis, such as Desulfovibrio, Bilophila wadsworthia (B. wadsworthia), Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), and Brachyspira hyodysenteriae (B. hyodysenteriae). IFA and IHC results indicated that the monoclonal antibodies can be successfully used as primary antibodies to detect L. intracellularis in infected cells and in the crypt of the ileum from infected tissues of PE. Our findings suggested that the new monoclonal antibody specific against L. intracellularis will be useful for the evaluation of L. intracellularis infection in vivo and in vitro.Proliferative enteropathy (PE) is an infectious enteric disease caused by  Lawsonia intracellularis  is an infectious enteric disease characterized by thickening of the ileal wall as a result of enterocyte proliferation associated with the presence of llularis , 2. L. iprimates –6. Porciprimates , 8. Prolprimates , 8.L. intracellularis has been widely spread in more than 20 countries, such as China, Canada, Brazil, Finland, France, South Africa, Greece, and India . A higher rate of seroprevalence was found in fattening pigs, sows, and boars than in preweaning piglets and weaners  (2), 8.8% carbon dioxide (CO2), and 83.2% nitrogen (N2), which is time consuming  presents many advantages such as repeatable, sensitive, and less time consuming  , and immn (FISH) , these mn (FISH) , 22. Moronsuming . Therefoonsuming . FurtherL. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC). Our findings may lay the basis for evaluating the monolayer of McCoy, IEC-18, IPEC-J2, and many other cell lines and tissues infected with L. intracellularis.Therefore, the objectives of the present study was to produce and identify the characteristics of a new monoclonal antibody against outer membrane protein (Omp2) of L. intracellularis strain  was purchased from Boehringer Ingelheim Vetmedica, Germany, and Escherichia coli (CAU0751), Salmonella choleraesuis (CVCC2139), and Salmonella typhimurium (CVCC2220) were purchased from the China Veterinary Culture Collection Center (CVCC). Desulfovibrio (ATCC 27774) and Bilophila wadsworthia (ATCC 49260) were purchased from the American Type Culture Collection (ATCC). Brachyspira hyodysenteriae is courtesy of the Shanghai Animal Disease Prevention and Control Center. The pGex-6p-1 (+) vector was purchased from Novagen, USA. The myeloma cell line SP2/0 (stored in our laboratory) and rat small intestine cells  were cultured in DMEM/high glucose  in a humidified 5% CO2 atmosphere at 37°C. All the cell culture media were supplemented with 10% fetal bovine serum . L. intracellularis was grown as described (L. intracellularis and incubated in an atmosphere of 83.2% nitrogen (N2), 8.8% carbon dioxide (CO2), and 8% oxygen (O2) for 7 days at 37°C.The oli CAU071, SalmonOmp2) gene sequences of L. intracellularis were selected and analyzed. The partial Omp2 sequence GXNN strain was synthesized according to the 103–993 bp regions from L. intracellularis (no. EU621796.1) by Sangon Biotech Co., Ltd.  using restriction enzymes . The synthetic Omp2 gene was cloned into the prokaryotic expression vector pGex-6p-1 (+). The positive recombinant plasmids were identified by enzyme digestion and sequencing and then transformed into E. coli Rosetta (DE3) competent cells. The expression of the recombinant Omp2 protein was induced with 0.3 mmol/L isopropyl-β-D-thiogalactopyranoside  at 16°C for 18 h, and the protein was examined by SDS-PAGE. The recombinant protein was also detected by SDS-PAGE after purification with a high-affinity GSTRAP HP column  according to the instructions of the manufacturer, and the concentration was determined with a NANODROP 2000 Spectrophotometer . In addition, Western blotting was performed to evaluate the reactivity of the purified Omp2 protein. The same method was described in the reference with some modifications  for 1 h at 37°C. After three washes with TBST, the membrane was incubated with goat anti-pig IgG H&L (HRP) secondary antibody for 1 h at 37°C . After three washes with TBST, the target protein bands were detected using 3,3-diaminobenzidine tetrahydrochloride (DAB), and the reaction was stopped by rinsing with distilled water and drying the membrane.The outer membrane protein ; and all experiments were performed in accordance with the relevant guidelines and regulations. The purified Omp2 protein was used to immunize 6-week-old BALB/c mice . The mice were immunized with Freund's adjuvant  three times. Three days after the final immunization, the mice with the highest antibody titers were euthanized, and the spleen cells were harvested. Subsequently, splenocytes from immunized mice were fused with sp2/0 murine myeloma cells by using 50% polyethylene glycol  according to a previously described method . The fus2SO4. The OD450nm was measured in a micro titer plate reader (Bio-Rad Benchmark Plus). The cutoff value was determined when the OD450nm ratio (OD450nm of purified antibody/OD450nm of unimmunized rabbit serum) was above 2.1. The antibody titer was defined as the highest dilution that yielded a net OD450nm value greater than the calculated cutoff.The antibody titers of ascitic fluid were further determined by ELISA, as previously described . BrieflyThe expressed Omp2 proteins were subjected to gel electrophoresis on 12% SDS-PAGE after denaturation with 2 × SDS loading buffer at 100°C for 3~5 min. The protein bands were transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TBST overnight at 4°C. After washing three times in TBST, the membranes were incubated with mAbs against Omp2 protein, positive control  or negative control  at 37°C for 1 h. After washing with TBST, the membranes were immersed in goat anti-mouse HRP-conjugated polyclonal serum at 37°C for 1 h. After washing in the same way, the membranes were immersed in DAB, and the reaction was stopped by rinsing with distilled water and drying the membrane.5L. intracellularis, 1 × 105 CFU of Desulfovibrio, B. wadsworthia, S. choleraesuis, S. typhimurium, E. coli, and B. hyodysenteriae were tested using a bacterial suspension to infect IEC-18 cells. The cell cultures were detected after being incubated at 37°C in 8.0% O2, 8.8% CO2, and 83.2% N2 in a humidified incubator for 3 h, the rest of the experiment steps were the same as before.The cross-reactivity of the mAbs was detected by IFA. Briefly, about 1 × 105L. intracellularis at a multiplicity of infection (MOI) of 100 for 3 h and 7 days in a tri-gas incubator with 83.2% N2, 8.8% CO2, and 8% O2 at 37°C  according to methods described in the literature  was used to perform IF and IHC to stain L. intracellularis bacteria according to the previously reported methods  staining and immunocytochemistry (IHC) in histological fragments of lesions suggestive of PPE. Briefly, infected  and normal intestinal samples were obtained from commercial pigs in a slaughterhouse in Jiangsu Province, China. Subsequently, the samples of ileum were fixed by immersion in 10% neutral-buffered formalin.  methods , 30. UniOmp2 gene was correctly inserted into the pGex-6p-1 vector according to nucleotide sequencing and restriction analysis (AM180252.1) is 99.1%. Compared with LI0902 of PHE/MN1-00, the 175th , 223th (Glu to Gly), and 254th (Leu to Ser) amino acids generated mutation amino acids from the GXNN strain. Subsequently, the recombinant Omp2 protein was successfully expressed in BL21 (DE3) cells after it was induced by IPTG. The expressed recombinant GST-Omp2 protein was ~58.6 kDa and present in the supernatant of bacterial lysates  before it was used as a coating antigen in the ELISA for screening (data not shown). Finally, three hybridoma cell lines named 4D9, 3G2, and 7G5 were acquired by subcloning and screening. The titers of ascitic fluids were measured by indirect ELISA, and the titers of 4D9, 3G2, and 7G5 were 1:2,048,000, 1:512,000 and 1:256,000, respectively .The results of subtype identification showed that the heavy chains of the three mAbs  all belonged to IgG1, and the light chains were of the kappa type .Western blotting analysis showed that all the mAbs and the positive control (the mouse serum against Omp2 protein) could react with the Omp2 protein , lane 5.Desulfovibrio, B. wadsworthia, S. choleraesuis, S. typhimurium, E. coli, and B. hyodysenteriae. The results showed that 4D9, 3G2, and 7G5 did not have cross-reactions with enteric bacteria commonly found in the ileum of pigs or closely related to L. intracellularis . Our findings indicated that the L. intracellularis in the monolayer of eukaryotic cells could be detected at 3 h post infection  gene is LI0902 of L. intracellularis. The homology of amino acid sequence between Omp2 and LI0902 is 99.1%. Watson et al. used LC-ESI-MS/MS to analyze the two isolates of L. intracellularis from heavily infected epithelial cell cultures, and 19 proteins containing LI0902 were identified, and the function of Omp2 of L. intracellularis is thought to mediate peptidoglycan recognition and play an important role in protein–protein interactions via the vWF domain. In addition, the recombinant Omp2 protein could be recognized by sera from all but one of the infected groups and was not recognized by sera from any of the uninfected animals  can be found in the article/supplementary material.The animal study was reviewed and approved by the Ethical Committee of the Faculty of Veterinary Science of Nanjing Agricultural University.NX designed and drafted the work and wrote the manuscript. NX, JL, ML, YH, and HL performed the experiments, analyzed the data, and interpreted the results. HF helped conceive the project and edit the manuscript. All authors contributed to the article and approved the submitted version.This study was supported by the National Key Research and Development Program of China (2017YFD0500203), the Jiangsu Agricultural Science and Technology Innovation Fund (CX(19)2020), the Industry-University Cooperation Program of Jiangsu Province (NBG0060265-1), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).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.


BackgroundAutoimmune inner ear disease (AIED) represents less than 1% of all cases of sensorineural hearing loss (SNHL) but its frequency may be underestimated due to lack of specific clinical and laboratory criteria. AIED can be associated with a systemic autoimmune disease (SAID) in 15%-30% of the cases. The objective of the present study was to characterize the clinical and prognostic factors of a cohort of patients with AIED.Materials and methodsThe authors conducted a retrospective descriptive analysis of a cohort of patients with AIED referred from the otorhinolaryngology department to a systemic immune-mediated diseases unit between March 2013 and November 2020. A consecutive sample of 39 patients with suspected AIED was referred. SNHL was defined as a fall of the hearing threshold of at least 30 decibels in three consecutive frequencies. Eight patients were excluded for not meeting the audiometric criteria or having confounding factors. The remaining 31 patients were included with a total of 50 affected ears. To classify the intensity of hearing loss, an arithmetic mean of pure tone was calculated. Normal hearing or mild hearing loss at the last pure tone audiometry of the follow-up were an indicator of good prognosis and were considered the outcome of interest.ResultsThirty-two percent of the patients had an associated SAID. There were no differences regarding demographic and clinical characteristics when comparing patients with AIED alone and patients with AIED and a SAID, except for the positivity of antinuclear antibodies (ANA). ANA positivity was superior in patients with the association of AIED and a SAID when compared with patients with AIED alone . The SAID was diagnosed after the AIED in 70% of the patients, in which diagnosis of the SAID occurred a median of 4,2 (IQR 0.8-5.1) years after the diagnosis of the AIED. Normal audiometric evaluation or a mild hearing loss was achieved in 31% of the ears at the last audiometric evaluation. A normal audiometry or a mild hearing loss at the time of diagnosis was independently associated with a better outcome . Bilateral hearing loss was independently associated with a worse outcome . The use of systemic corticosteroids (p=0.941), transtympanic corticosteroids (p=0.700) and non-steroid immunomodulator drugs (p=0.986) did not affect prognosis. The presence of a SIAD did not affect the prognosis (p=0.986).ConclusionsIn this cohort, SAID was present in one-third of the patients with AIED. A good prognosis was achieved in one-third of the patients. A normal audiometry or mild disease at presentation was associated with a good outcome, whilst bilateral involvement was associated with a bad one. Association of a SAID did not seem to influence the hearing-related prognosis. Positivity of ANA antibodies may justify performing a complementary investigation to determine the presence of a SAID. Sensorineural hearing loss (SNHL) has been defined as a fall of at least 30 dB of the hearing threshold in at least three consecutive frequencies . It has AIED usually presents as a subacute, rapidly progressive, often fluctuating, bilateral and asymmetrical SNHL -4. AlmosThe pathophysiology of AIED is not entirely understood. The concept of the inner ear as an immune-privileged site has been refuted . AntibodDiagnosis is based on the clinical presentation as there are no diagnostic criteria or pathognomonic laboratory tests. Essentially, AIED is a diagnosis of exclusion, suspected in case of a documented progressive SNHL, when other etiologic causes have been ruled out. Meniere’s disease should be considered in the presence of vestibular symptoms and retro-cochlear pathology should be excluded by computed tomography or magnetic resonance imaging .AIED can be classified as primary, when the inner ear is the only organ affected, or secondary when, in 15-30% of cases, it develops in association with a systemic autoimmune disease (SAID) such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), granulomatosis with polyangiitis, Vogt-Koyanagi-Harada disease or antiphospholipid syndrome (APS) .The recommended treatment is systemic corticosteroids, with transtympanic infusion being preferred in cases of failure, incomplete response or contraindication to systemic therapy . HoweverThe authors conducted a retrospective descriptive analysis of a cohort of patients with SHNL referred from the otorhinolaryngology department to a systemic immune-mediated diseases unit between March 2013 and November 2020 for a suspected AIED. Diagnosis of SNHL was based on clinical presentation and the presence of a fall of the hearing threshold of at least 30 decibels (dB) in three consecutive frequencies. The diagnosis of associated SAID was applied when appropriated and was based on clinical and serologic criteria according to the respective EULAR recommendations. Data were collected from hospital records and handled in an anonymous and population-based fashion. Patient’s audiometric evaluations were performed with a standard audiometer . To classify the intensity of hearing loss, an arithmetic mean of pure tone was calculated. The arithmetic mean of frequencies 250 Hz, 500 Hz, 1 KHz and 2 KHz was obtained when low and medium frequencies were affected; 1, 2, 3, 4, 6 and 8 KHz when medium and high frequencies were affected; 3, 4, 6 and 8 KHz when only high frequencies were affected and of all eight frequencies when low, medium and high frequencies were affected together. In cases of deep loss, when the hearing threshold was not detected, the maximum response audiometer of 120 dB was considered. The intensity of hearing loss was classified as mild when the arithmetic mean of pure tone was between 26 and 40 dB, moderate when between 41 and 70 dB, severe if between 71 and 90 dB and profound if above 90 dB. The first and last pure tone audiometry available were used. In cases of bilateral involvement but with only one ear affected at first presentation, the contralateral one was considered normal. Anti-HSP70 antibodies were determined by an immunoenzymatic assay. Antinuclear antibodies (ANA) determination was performed by indirect immunofluorescence followed by immunoblot. The definition of positivity was expressed as a titre of at least 1:160. Anti-neutrophil cytoplasmic antibodies (ANCA) were determined by indirect immunofluorescence followed by enzyme-linked immunosorbent assay (ELISA).Categorical data were presented as counts and percentages and were analyzed with chi-square test and Fisher’s exact test, as appropriate. Normality was tested with Kolmogorov-Smirnov test. The skewed distributions were described with medians and interquartile ranges (IQR) and were compared by the Mann-Whitney. Normal distributions were described with means and standard deviations and were compared with the use of student’s t-test. For the comparison of more than two variable groups chi-square test or ANOVA analysis were used accordingly. For the analysis of prognosis, an analysis of each ear affected by AIED was performed. In order to assess prognostic predictors, variables were selected for a logistic regression analysis, assessed by the estimated odds ratio (OR) with 95% confidence interval (CI). Variables yielding p values of less than or equal to 0.20 in the univariate analyses were entered into a multiple logistic regression model. Normal hearing or mild hearing loss at the last pure tone audiometry of the follow-up were an indicator of good prognosis and were considered the outcome of interest. The two-sided alpha level was set at 0.05.PopulationBetween March 2013 and November 2020, 39 patients with suspected AIED were referred to the systemic immune-mediated diseases unit. Of these, eight were excluded. Three had vestibular symptoms and hearing loss complaints without meeting audiometric criteria for SNHL. Five were considered to have confounding factors for the purpose of the present study: one had history of otosclerosis and used bilateral hearing aids; one had a history of chronic otitis media; one had had labyrinthitis following trans-tympanic steroid infusion; one suffered from hearing impairment since childhood; one had an erratic follow-up with presentation, evolution and therapy difficult to understand from hospital records. The remaining 31 patients fulfilled the criteria for this study.Patients’ characteristics are summarized in Table Systemic autoimmune disease and autoimmune inner ear diseaseTen patients (32%) with AIED also had a SAID. Four patients had SLE, three had systemic sclerosis (SS), two RA and one APS. In two patients the diagnosis of SAID occurred four and 20 months before hearing loss, respectively. In one patient the diagnosis was simultaneous. In seven patients (70%) the diagnosis of the SAID occurred a median of 4.2 (0.8-5.1) years after the diagnosis of AIED. When compared with patients with AIED alone, patients with the association of AIED and SAID had similar age and sex predominance Table . There wPrognosis and predictors of good outcomeThe intensity of hearing loss in the final audiometric evaluation during follow-up was classified as mild, moderate, severe or profound according to the arithmetic mean of the pure tones obtained. Good outcome was considered when the final audiometry was normal or the intensity of hearing loss was mild. A total of 50 ears were affected. At the time of the analysis the final audiometry was unavailable in three patients with bilateral affection. Therefore, the final analysis included 42 ears.At the time of presentation, 20% of the ears had normal audiometry or a mild hearing loss, 51% had moderate and the remainder 29% had severe or profound hearing loss. At the time of the last evaluation, 31% of the ears had a normal audiometric evaluation or a mild hearing loss, 38% had moderate and 31% had severe or profound hearing loss , none seemed to have influenced prognosis. In 1997 Rauch et al. . DespiteAs a retrospective analysis, this study has many limitations. It did not follow a specific treatment and diagnostic protocol and last pure tone audiograms were performed at different disease stages. Speech discrimination testing and vestibular symptoms and tinnitus were not used to define prognosis. Finally, hearing threshold fluctuations, frequent in AIED, may have also influenced the overall prognosis.In this cohort, SAID was present in one-third of the patients with suspected AIED. A normal audiometry or mild disease at presentation was associated with a good outcome, whilst bilateral involvement was associated with a bad one. Association of a SAID did not seem to influence the hearing-related prognosis. Determination of ANA may have a role in patients with suspected AIED to justify performing a complementary investigation to determine the presence of a SAID. It is important to achieve a future consensus on the definition of hearing improvement that considers the morbidity caused by remaining hearing loss, even if some hearing recovery is achieved with therapy.


Prostaglandin E2 receptor EP4 is involved in inflammation and related tumorigenesis in the colorectum. This study aimed to investigate the chemopreventive ability of RQ-15986, a selective EP4 antagonist, in colitis-related colorectal tumorigenesis. Male Kyoto APC delta rats, which have APC mutations, were treated with azoxymethane and dextran sulfate sodium and subsequently administered RQ-15986 for eight weeks. At the end of the experiment, the development of colorectal tumor was significantly inhibited in the RQ-15986-treated group. The cell proliferation of the crypts and tumors in the colorectum was decreased following RQ-15986 treatment. RQ-15986 also suppressed the expression of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-18, and monocyte chemotactic protein-1, in the colon mucosa. In addition, the expression levels of indoleamine 2,3-dioxygenase, which is involved in immune tolerance, were decreased in the colorectal epithelium and tumors of the RQ-15986-treated group. These findings indicate that RQ-15986 inhibits colitis-associated colorectal tumorigenesis by attenuating inflammation, suppressing cell proliferation, and modulating the expression of indoleamine 2,3-dioxygenase. Targeting prostaglandin E2/EP4 signaling might be a useful strategy for chemoprevention of inflammation-related colorectal cancer. Colorectal cancer (CRC) is one of the most serious health concerns owing to its high morbidity and mortality. Because the prevalence of CRC has increased gradually worldwide , there iProstaglandins are inflammatory mediators generated from arachidonic acids by the enzyme cyclooxygenase-2 (COX-2). Among the prostaglandins, prostaglandin E2 (PGE2) is a key COX-2 product that regulates various inflammatory processes . PGE2 exIn addition to colorectal inflammation, PGE2/EP4 signaling is profoundly associated with colorectal tumorigenesis . In humaRQ-15986, a selective EP4 antagonist that was originally reported to improve signs and symptoms of osteoarthritis and rheumatoid arthritis , is expeThe purpose of this study was to clarify the potential chemopreventive ability of a selective EP4 antagonist in colitis-related colorectal tumorigenesis. For this purpose, we used a colitis-related CRC model of Kyoto adenomatous polyposis coli (APC) delta (KAD) rats with an Apc mutation, which mimics human inflammation-related colon carcinogenesis well ,20. In tAzoxymethane (AOM) was obtained from Sigma Chemical Co. . DSS was purchased from MP Biomedicals, LLC . RQ-15986  and IDO in the colorectal mucosa was performed using the labeled streptavidin-biotin method  with primary anti-PCNA antibody  and anti-IDO antibody . The positive cell rates of immunohistochemistry for PCNA or IDO were evaluated based on previous methods ,21.Foxp3), Ido1, Ifng, Il6, Il18, monocyte chemotactic protein-1 (Mcp1), Tnfa, and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) genes were designed using the software available at the Roche Universal Probe Library Assay Design Center . The sequences for these primers are shown in Foxp3, Ido1, Ifng, Il6, Il18, Mcp1, and Tnfa were normalized to the expression level of Gapdh.Total RNA was isolated from the scraped colon mucosa of experimental rats using the RNeasy Mini Kit . cDNA was synthesized from 0.2 μg of total RNA using High Capacity cDNA Reverse Transcription Kit . Quantitative real-time reverse transcription-PCR (RT-PCR) analysis was performed using LightCycler Nano  with LightCycler 480 SYBR Green I Master Mix (Roche Diagnostics). The specific primers used for amplifying forkhead box protein-3  were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum (Sigma) in an incubator at 37 °C under a humidified atmosphere containing 5% CO2. The cells were preincubated in the absence or presence of RQ-15986 at various concentrations  for 2 h and then stimulated with interferon (IFN)-γ (10 ng/mL) for 24 h. IFN-γ was used for induction of IDO1. Cell extracts were prepared and RT-PCR analysis for P < 0.05 was considered significant.All data are expressed as mean ± SD. The differences between the groups were analyzed using two-way ANOVA. For significant differences indicated by ANOVA, Tukey–Kramer multiple comparison test was performed. Fisher’s exact test was used to compare the incidence of intestinal tumors. A value of P < 0.01). During the course of the experiment, administration of RQ-15986 did not cause any clinical symptoms. No pathological alterations that would suggest toxicity of RQ-15986 in the liver, kidney, and spleen of rats were observed , indicating that RQ-15986 suppressed colitis-related colorectal carcinogenesis.Colorectal tumors were observed endoscopically 3 weeks after the end of DSS administration, and they increased over time in the colons of rats that received AOM and DSS . At sacrThe effects of RQ-15986 on cell proliferation in the colonic mucosa and CRC tissues of the experimental rats were evaluated through immunohistochemical staining for PCNA. Both in the non-lesional crypts and CRC tissues, administration of RQ-15986 markedly decreased the PCNA-labeling indices . TherefoIfng, Il6, Il18, Mcp1, and Tnfa were markedly elevated in the colonic mucosa of AOM/DSS-treated rats compared to those in untreated rats. In AOM/DSS-treated rats, RQ-15986 administration significantly decreased the expression levels of these mRNAs. These findings indicate that RQ-15986 attenuated colorectal inflammation associated with tumorigenesis.The effects of RQ-15986 on the mRNA levels of pro-inflammatory cytokines in the colonic mucosa were examined. As shown in Ido1 mRNA in the colonic mucosa were significantly elevated in AOM/DSS-treated rats, but this effect was suppressed upon RQ-15986 administration Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Cranial nerve involvement is not commonly encountered in chronic inflammatory demyelinating polyradiculoneuropathy (CIDP); this is especially true for involvement of the hypoglossal nerve. Neither Beevor's sign nor its inverted form has previously been described in CIDP.A 28-year-old man presented with distal-predominant limb weakness and numbness at the age of 18. A diagnosis of CIDP was made, which was confirmed by electrodiagnostic evidence of demyelination. He responded well to intravenous immunoglobulin and glucocorticoid treatment and achieved remission for 5 years. However, the same symptoms relapsed at the age of 28 and lasted for 10 months. On examination, in addition to limb sensory impairment and muscle weakness, mild bilateral facial paresis, tongue atrophy and fasciculations, and inverted Beevor's sign were also observed. A brief literature review of cranial nerve involvements in CIDP and Beevor's sign or its inverted form were also performed.Cranial nerves may be affected in patients with CIDP. Facial palsy is most frequently present, while hypoglossal nerve involvement is rare. Inverted Beevor's sign can appear in CIDP patients.The online version contains supplementary material available at 10.1186/s12883-021-02287-5. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare, acquired, immune-mediated neuropathy that affects the peripheral nerves and nerve roots. It is characterized by a relapsing–remitting or progressive course and evidence of demyelination . TypicalA 28-year-old man presented with a relapsing–remitting course of distal-predominant limb weakness and numbness for 10 years. Ten years earlier, he had gradually progressive weakness and numbness in the distal limbs. He was unable to write, button his clothes, or open jars, but had no difficulty in climbing stairs or rising from a seated position. He was treated with intravenous dexamethasone and subsequent oral prednisolone for 1 month in another hospital, although the specific dosage was unknown. His symptoms disappeared after treatment. However, 7 years ago, the same symptoms relapsed and were gradually progressive. He presented at our hospital because of persistence of the aforementioned symptoms. He was treated with intravenous immunoglobulin at 0.4 g/kg/day for 5 days, and intravenous methylprednisolone at 500 mg per day for 3 days, followed by 240 mg per day for 1 day and 120 mg per day for 1 day. Subsequently, he received 55 mg of oral prednisolone for 1 month, after which it was gradually tapered off. He also took cyclosporine at 50 mg twice per day. In addition, he was subjected to another two 5-day courses of intravenous immunoglobulin, at 6 years ago and 5 years ago. He began to gradually recover after treatment and achieved remission, and he stopped all treatment 5 years ago. However, his symptoms relapsed 10 months ago, with gradually progressive asymmetrical weakness and numbness in his distal arms and legs. His left upper limb was most affected. He had no known comorbid conditions and no relatives with a similar condition.On neurological examination, the patient had mild bilateral facial paresis, asymmetric atrophy of the tongue muscle, and tongue fasciculations Fig. . Tendon Additional file 1: Video 1.Additional file 2: Video 2.Laboratory evaluations, including a complete blood count, erythrocyte sedimentation rate, C-reactive protein, antinuclear antibodies, anti-neutrophil cytoplasmic antibody, fasting serum glucose, thyroid function studies, serum and urine protein electrophoresis and immunofixation, and angiotensin-converting enzyme, were all unremarkable. Anti-ganglioside antibodies, myelin-associated glycoprotein, neurofascin 155 and 186, and contactin 1 were all negative. In addition, cerebrospinal fluid analysis revealed a normal protein content, cell count, and glucose level. Electrodiagnostic testing revealed severe, diffuse, asymmetric sensorimotor polyneuropathy that fulfilled the criteria for primary demyelination in CIDP. This included prolonged distal motor latencies, delay and disappearance of F waves, and conduction block in the radial and ulnar nerves Table . MagnetiThe patient was diagnosed with CIDP, and was again treated with intravenous immunoglobulin at 0.4 g/kg/day for 5 days, intravenous methylprednisolone at 500 mg per day for 3 days, and then oral prednisolone at 60 mg/day for 1 month before it was gradually tapered off. The patient was followed up by telephone. After 3 months of treatment, his numbness was completely relieved, his limb strength was recovered about 70%, he had no difficulty in daily life, and he returned to work.In the current report, we present a case with chronically recurrent limb weakness and sensory dysfunction as well as cranial nerve involvement. The patient’s tendon reflexes were absent in all extremities. His nerve conduction velocity study results met the electrodiagnostic criteria for CIDP, and he responded well to immunomodulatory treatment. Other causes were excluded by extensive clinical and laboratory evaluations. According to the European Federation of Neurological Societies and the Peripheral Nerve Society criteria, the diagnosis of the present patient was definite CIDP . FurtherOne notable feature in the present case was cranial nerve involvement, which included the facial and hypoglossal nerves. Cranial neuropathy is clinically uncommon in patients with CIDP, although it has been described in several case series or reports, including in cranial nerves II to X, and XII , 3, 8–11TCAP mutations [Another notable feature in the present case was the presence of an inverted Beevor's sign, which has not been reported previously in CIDP. Beevor's sign, which is an upward movement of the umbilicus with truncal flexion from a supine position, derives its name from Dr. Charles Edward Beevor. It first appeared in his 1898 textbook "Diseases of the Nervous System: A Handbook for Students and Practitioners", and was used to indicate a spinal cord lesion between the levels of T10 and T12 , 5, 13. utations , sporadiutations , 21, amyutations , and acuutations . Less frutations , 24 and utations . TherefoIn conclusion, we described a rare case of CIDP presenting with cranial nerve involvement and inverted Beevor's sign. Cranial nerves may be affected in patients with CIDP. Facial palsy is most frequently present, while hypoglossal nerve involvement is rarely reported. CIDP should be considered in patients who present with cranial nerve involvement. To our knowledge, this is the first report of the inverted Beevor's sign in CIDP. Neurologists should therefore pay attention to the movement of the umbilicus on neck flexion during neurological examinations in neuromuscular disorders.Additional file 3: Table 1. Muscle strength evaluation with Medical Research Council scale. Table 2. The nerve ultrasonography of the patient. Figure 1. The magnetic resonance imaging of the lumbosacral plexus of the patient.