Autoimmune inner ear disease (AIED) is a rare clinical entity. Its pathogenicity, heterogenous clinical presentation in the context of secondary systemic autoimmune disease and optimal treatment avenues remain poorly understood. Vestibular impairment occurring in the context of AIED is rarely subject to detailed investigation given that the auditory symptoms and their responsiveness to immunosuppression are the focus of the few proposed diagnostic criteria for AIED. We present three cases of vestibulopathy occurring in the context of autoimmune inner ear conditions, including the first known report of autoimmune inner ear pathology arising with a temporal association to administration of the Pfizer-BioNTech SARS-CoV2 vaccination. We review the available literature pertinent to each case and summarise the key learning points, highlighting the variable presentation of vestibular impairment in AIED. Autoimmune inner ear disease (AIED) is a disabling but rare autoinflammatory condition of the inner ear, often leading to progressive and fluctuating, sensorineural hearing loss. It was first described in 1979 with a case series authored by McCabe et al. ,3,4). ThThe prevalence of AIED is estimated to be around 15 in 100,000 people   althoughThere are currently no universally accepted diagnostic criteria for AIED, although several definitions have been proposed:AIED is defined as progressive hearing loss (with or without vestibular dysfunction) arising secondary to autoimmune-driven inner ear pathology [athology . It is tathology ,3, althoathology ,3,6,7 whathology ,8,9. ThuPrimary AIED comprises isolated inner ear dysfunction.Secondary AIED describes inner ear dysfunction in the presence of systemic autoimmune disease , on Western blot analysis of inner ear proteins using patient serum . This anAIED research and clinical descriptions have largely focused on the hearing component of the inner ear. However, vestibular involvement is seen in approximately 50–60% of cases ,6,17. ThA 28-year-old male carpenter of white-British origin was referred to our tertiary Centre in December 2020, a year after developing sudden-onset spinning vertigo, imbalance and bilateral hearing loss. He had a 10-year history of ulcerative colitis which was in clinical remission on Azathioprine 200 mg daily. He also had long-standing classical migraine.He had a stormy clinical course over the first year; he was unable to work due to episodic vertigo, oscillopsia (due to primary gaze nystagmus) and increasing migraine frequency with new visual aura.The hearing loss was initially asymmetric, predominantly low-frequency and rapidly fluctuant over several weeks. When he first presented locally, he was treated with multiple short courses of Prednisolone whenever his hearing deteriorated, usually resulting in marked rapid improvement. Without a clear, defined local or national management pathway for AIED, serial audiograms were not routinely performed and his treatment with oral steroids was variable . Intratympanic steroid administration was attempted to avoid long-term oral steroid use but hearing recovery was not seen with three, separate, early attempts at this.Objective vestibular function tests conducted locally three months prior to referral to our Centre showed bilateral vestibular impairment. Contrast-enhanced magnetic resonance imaging (MRI) of the internal auditory meatus (IAM), in addition to a full auto-immune and vasculitic screen performed locally at the same time, were normal.In June 2021, 18 months after his initial onset of symptoms, repeat focused vestibular examination and investigations at our Centre confirmed persistent bilateral vestibulopathy. The clinical findings and objective audiovestibular function tests are summarised in Given the fluctuant, steroid-responsive hearing loss and vestibular involvement occurring in the context of pre-existing ulcerative colitis, a diagnosis of secondary AIED was made. He had a good initial response to high-dose oral prednisolone, although his hearing loss recurred on steroid withdrawal, requiring a maintenance dose of prednisolone 30 mg/day (administered with bone-protection and a proton pump inhibitor). Despite these measures, there was further deterioration of his baseline hearing over a two-year period. His ulcerative colitis remained quiescent during this time.Alongside the pharmacological management, he engaged in self-directed vestibular rehabilitation and a regular exercise program . This led to subjective improvement in his balance and oscillopsia.He was managed as part of a multidisciplinary, multi-site team with additional input from rheumatology, gastroenterology and ENT with regards to the most appropriate steroid-sparing maintenance agent. A preliminary decision was made to consider Methotrexate, but other options included anti-TNFα therapy, or a dose increase of Azathioprine, and a final decision is awaited.The subject in this case of AIED secondary to systemic ulcerative colitis presented with rapidly progressive, fluctuant, steroid-responsive hearing loss with episodic rotatory vertigo and imbalance secondary to bilateral vestibular impairment. His case demonstrates how central vestibular compensation occurs following a peripheral vestibular insult due to the plasticity of the vestibulo-ocular, vestibulo-spinal, vestibulo-collic and vestibulo-autonomic pathways, particularly in the young . The objWhilst the presence of an underlying autoimmune disorder supports AIED, a differential diagnosis would be bilateral Ménière’s disease, particularly as it is postulated that AIED and bilateral Ménière’s disease could be one single entity in continuum . The imaThis case also highlights the difficulties encountered by both the patient and treating clinicians when a clear management pathway for a rare disorder does not exist. This is of specific importance in AIED where funding issues for expensive biologics and disease-modifying antirheumatic drugs (DMARDs) can hinder management of a time-sensitive clinical condition.A 17-year-old man of Ghanaian heritage was referred to the neuro-otology clinic at our Centre in June 2019 with a four-month history of insidious right-sided hearing-loss and unsteadiness of gait, followed later by sudden complete loss of hearing on the left and rapid deterioration of his balance.The only significant past medical history was an isolated episode of bilateral uveitis at age four. This resolved at the time with Dexamethasone eye drops, which he continued long term with ophthalmology follow up.Upon presentation to us he had profound, bilateral, sensorineural hearing loss. He also had bilateral vestibular impairment and a global absence of peripheral reflexes. He had no recurrence of eye disease whilst taking corticosteroid eye drops. A summary of the investigations performed between June 2019 and June 2021 at our Centre is given in The hearing loss was initially treated with a left intra-tympanic steroid injection and oral Prednisolone 50 mg/day for six weeks. His hearing on the left improved significantly, particularly in the low frequencies.Post-contrast MRI IAMs  and braiA PET-CT showed widespread reactive lymphadenopathy. USS guided lymph node biopsy was not diagnostically helpful, although this was undertaken in the context of immunosuppression, secondary to steroid treatment. All other investigations were normal including nerve conduction studies, CSF analysis and an autoimmune screen.A diagnosis of atypical Cogan syndrome, a rare autoimmune condition affecting the eye and inner ear, was made. The Dexamethasone eye drops were discontinued. After completing six weeks of oral Prednisolone 50 mg/day, this was tapered down by 10 mg per month with a proton pump inhibitor and bone-protection. Long-term maintenance on a steroid-sparing agent, Mycophenolate Mofetil (MMF) 1 g twice daily, followed upon advice from the neuro-immunology team.His investigations and management followed multidisciplinary team advice with ENT, neuro-genetics, immunology, audiology and physiotherapy input. Although he saw significant improvement in his left-sided hearing loss and marginal improvement on the right, he went on to develop symptoms of visually induced vertigo, secondary to the underlying vestibulopathy. He was fitted with a left hearing aid and continues to benefit from vestibular rehabilitation.Although Cogan syndrome (CS) is a condition consisting of audiovestibular dysfunction, the focus of most reports on the subject is hearing loss, not specifically vestibular involvement, as with most other AIEDs. This is despite vestibular function being found to be abnormal in up to 90% of subjects with CS . This veIn the authors’ experience, most clinicians associate peripheral vestibular impairment with episodic vertigo but it is less well recognised that bilateral, sudden onset dysfunction can present with isolated marked imbalance, ataxia and oscillopsia in the absence of any reported dizziness or vertigo . PatientCS is an autoimmune vasculitis resulting in audiovestibular dysfunction and ocular inflammation, the latter most commonly manifesting as interstitial keratitis. Two distinct groups exist: those with typical or atypical disease. Typical CS occurs in young adults with flares of interstitial keratitis and Ménière’s disease-like vertiginous episodes resulting in progressive sensorineural hearing loss within approximately two years ,36. In tCS is rare, with just over 300 cases reported in the literature , althougFollowing the discovery of antibodies to inner ear and corneal antigens in patients suspected to have the condition, and given its favourable response to immunomodulatory drugs, CS was originally defined as systemic autoimmune disorder under the umbrella of AIED. There is no diagnostic test or pathognomonic marker and the diagnosis is therefore a clinical one .Corticosteroids remain the initial treatment modality, with the primary aim of preventing progressive irreversible audiovestibular loss and ocular inflammation or complications from secondary vasculitis. In a study of 62 patients, 84% responded to oral steroids in isolation, 90% showed improvement with DMARD therapy and 100% responded to biologics . When a As with Case 1, we also highlight the importance of cross-specialty collaboration.A 46-year-old medical doctor of white-British origin had her first dose of the Pfizer-BioNTech SARS-CoV2 vaccination. That evening she experienced new-onset continuous, bilateral tinnitus. This continued and after two weeks a mild-to-moderate bilateral high frequency sensorineural hearing loss, which was worse on the left, was documented via her local ENT service. Apart from being atopic, she had no significant past medical history and she had not had clinical SARS-CoV2 infection itself.A course of high-dose oral Prednisolone  was commenced with a plan to reduce this slowly over a further ten days but on day 12 she developed right ear erythema, pain, swelling and vesicular lesions. There was no history of fever or a systemic inflammatory response. Unfortunately, a possible viral aetiology was not investigated and no anti-viral treatment was given. She was treated with almost three weeks of antibiotics  for suspected perichondritis. Complete resolution of the right ear symptoms was eventually seen.Following this episode, repeat audiometry showed improvement in the hearing thresholds on the on the left but deterioration on the right (the side with the perichondritis). This also improved over time. Then, one month after the original vaccination, she suddenly experienced marked unsteadiness and head movement-induced oscillopsia without vertigo. vHIT shortly after this showed reduced vestibulo-ocular reflex (VOR) gain and overt refixation saccades bilaterally, more so on the right. vHIT repeated three months later showed reduced gain on the right once again but also both covert and overt catch-up saccades. Saccades were also present on Suppression Head IMPulse (SHIMP) testing at that time, indicative of ongoing central compensation.Subsequent serial audiograms revealed bilateral high-frequency mild hearing loss; this was initially fluctuant on the right, but the thresholds became persistently raised over time. Additional investigations provided further evidence of right-sided audiovestibular dysfunction , absent vHIT repeated several weeks later eventually showed bilaterally reduced VOR gain suggestive of bilateral vestibular dysfunction. A summary of the audiovestibular and additional investigations undertaken for this patient is given in The peripheral vestibular impairment did not fluctuate on objective testing and she remained constantly unsteady, requiring walking aids. She reported a one-off brief episode of rotational vertigo whilst turning in bed and some visual motion sensitivity at work, particularly after prolonged computer screen use. With time, central compensation facilitated by vestibular rehabilitation took place and her balance improved both subjectively and objectively .This patient’s symptoms were extremely disabling, and she was concerned about the possibility of further vaccine doses resulting in progressive symptoms. Her age, occupation and the fact that she was a mother to two young children meant it was impossible for her to shield from symptomatic COVID-19 infection in the long term. Following discussion with the infectious diseases and virology teams, she received two further mRNA COVID-19 vaccination doses but from a different manufacturer, Moderna with Valaciclovir cover and serial audiovestibular testing. She had no further systemic or neurological symptoms and no adverse effects were seen apart from slight increased tinnitus perception.possibly triggered by the vaccine on a primed immune system. She continues to be closely monitored by the audiology, vestibular neurology, vestibular physiotherapy, rheumatology and infectious diseases teams.The clinical picture was suggestive of an autoimmune inner ear disease, In Case 3, the subject’s symptoms, including perichondritis, seemed to occur in a somewhat protracted, fluctuant manner following vaccination. However, although the symptom onset was temporarily associated with the first dose of the Pfizer BioNTech SARS-CoV2 vaccine, it is impossible to comment on true causality. As of 10th December 2021, 243 cases of ‘deafness’ following administration of the Pfizer vaccination have been reported via the UK MHRA Yellow Card reporting scheme, including 47 cases of sudden onset hearing loss. There have been 2011 reports of tinnitus and 1410 reports of vertigo .In this case, the initial evolution of right-ear perichondritis with vesicles, pinna inflammation and a limited response to antibiotics may have been suggestive of a viral aetiology, potentially VZV, particularly as some cases of herpes zoster or herpes simplex reactivation associated with Pfizer BioNTech vaccination , and folTo date, there is only one report of vestibular neuritis occurring following COVID-19 vaccination  and a haThe question remains as to whether vaccines or their adjuvants may trigger autoimmune disease. Such events can arise as a result of interaction between a susceptible, immunologically ‘primed’ subject in combination with reaction to a single or combination of vaccine components, in theory due to the concept of molecular mimicry . MoleculCase reports are emerging of SARS-CoV-2 vaccination being temporally associated with the onset of neurological autoimmune disease such as Guillain–Barre syndrome and Acute Disseminated Encephalomyelitis (ADEM) ,59,60. HThe three cases presented in this series highlight the variable presentation of vestibular involvement in autoimmune-related inner ear dysfunction.in the absence of vertigo or dizziness, which is crucial for non-vestibular specialists to appreciate to facilitate prompt referral to neuro-otology, diagnosis and treatment in this rare group of patients.The vestibulopathy and other baseline characteristics in these three cases are summarised in Vestibular involvement is seen in approximately 50–60% of cases of AIED ,6,17 andStandardised diagnostic criteria for AIED are needed, as is a platform to prospectively collect data across multiple sites. This would highlight gaps in our collective understanding of AIED, illustrate best practice, and allow the neuro-otology community to move towards creating a usable treatment guideline and undertake future prospective collaborative work to gain international expertise and standardisation. Given the potential overlap between bilateral Ménière’s disease and AIED, the newly formed UK Ménière’s Disease Registry  will likIntratympanic gadolinium three-dimensional MRI imaging has also been utilised to diagnose endolymphatic hydrops in AIED [Although no specific biomarkers exist in AIED, the newly developed MRI protocols involving delayed uptake of intravenous gadolinium show promising potential as a complementary diagnostic tool in AIED ,65. Wher in AIED . AlthougThe optimal treatment approach in AIED remains unclear at present. First-line treatment options for idiopathic sudden-onset sensorineural hearing loss include oral steroids, intra-tympanic steroid injections or a combination of both. Of these, the most effective treatment modality remains unclear but the three-arm UK STeroid Administration Routes For Idiopathic Sudden sensorineural Hearing (STARFISH) trial  due to cGiven that no diagnostic or treatment guideline is widely utilised at present, we propose that the next logical step would be to create a combined pathway to standardise data collection in AIED and develop a wider evidence base for management. We recommend that a full vestibular assessment  be undertaken in all patients suspected to have AIED.We believe that effective management of patients with such rare disorders requires close collaboration and inter-disciplinary advice across healthcare professionals  and specialties .The three cases discussed represent a wide spectrum of vestibular disease occurring in immune-mediated inner ear dysfunction, which has received little clinical focus in the literature to date.We present the first known case of autoimmune inner ear dysfunction following administration of the Pfizer BioNTech SARS-CoV2 vaccination, although further reports and research are required to prove causality over a mere temporal association. Autoinflammatory symptoms following COVID-19 infection have been noted and it can be hypothesized that, by the same mechanisms, vaccinations or their adjuvants may also trigger an autoimmune response, perhaps in a pre-primed subject. However, this appears to be an exceptionally rare occurrence and should not, based on the evidence available to date, preclude subjects even with pre-existing autoimmune disease from receiving vaccines, particularly in the context of an ongoing global pandemic. It remains to be seen whether further reports of either COVID-19 infection or vaccination triggering autoimmune cochleo-vestibular disease come to the fore.There is great potential for the nature of vestibulopathy in AIED, particularly in secondary AIED, to be characterised in further detail and for its optimal treatment to be understood. For this to take place, formal evidenced-based diagnostic criteria incorporating a vestibular assessment process should be agreed upon, which in turn requires a collaborative platform of audiovestibular, neurology and ENT professionals to gather data and compare outcomes from multiple sites.


Laying hens may peck each other, painfully pulling out feathers and damaging flesh. There are many potential reasons and no obvious solutions, so the sharp tip of the beak is often carefully removed after hatching to limit the damage inflicted. Scientist have found it is useful to provide hens with lots of other things to forage in and peck at. Our study compared the effect of providing pans containing an abrasive material designed to attract pecking behaviour to eight treatment flocks of young hens, with eight flocks acting as controls. Each group had four beak-trimmed and four intact-beak flocks paired by farm. The pans significantly reduced the side and top beak lengths in samples of birds measured at 6–7 weeks and 10–11 weeks of age, and beak growth appeared to be linear. Thus, provision of abrasive material in pans effectively blunted beaks of younger birds. By the end of rear (14–15 weeks), birds seemed to lose interest in pecking at the pans, so there was no difference between treatment and control flocks, indicating scope for redesign to retain their interest.p < 0.005). There was no effect on feather pecking or plumage cover. Mean side-beak length and mean top-beak lengths were shorter in treatment flocks at 6–7 weeks and 10–11 weeks (p < 0.001). Intact-beak treatment flocks had shorter mean side-beak length at 10–11 weeks (p < 0.001) and at 14–15 weeks (p < 0.05) and mean top-beak length at 6–7 weeks (p < 0.05) and at 10–11 weeks (p < 0.05). BT treatment flocks had shorter side-beak and top-beak lengths at 6–7 weeks and at 10–11 weeks (p < 0.001). Beak lengths showed linear growth, with individual bird variation indicating a potential for genetic selection. The study demonstrated that abrasive material can reduce beak length in pullets.Injurious pecking, commonly controlled by beak trimming (BT), is a widespread issue in laying hens associated with thwarted foraging. This controlled study compared the effect in intact and beak-trimmed pullets of providing pecking pans to eight treatment flocks from six weeks of age. Flocks (mean size 6843) comprised eight British Blacktail, six Lohmann Brown and two Bovans Brown. All young birds (6–7 weeks) pecked more frequently at the pecking pans (mean 40.4) than older pullets  ( Injurious pecking (IP) is an umbrella term for inter-bird pecking behaviours including severe feather pecking (SFP), vent pecking (VP), cannibalistic pecking (CP) and toe pecking (TP) . The forA further form of inter-bird pecking is gentle feather pecking (GFP), which comprises repeated pecks at the edges of feathers—often aimed at the tip of the tail and sometimes other areas. GFP results in minor damage of little welfare concern . The occBeak trimming (BT) remains one of the most effective management tools to reduce the impact of IP ,13,14, aThere are two methods: the hot blade technique (HB), still widely used in some countries, and infra-red beak trimming (IRBT), which has replaced HB trimming of day-old chicks in the UK and in Europe ,19.Hot-blade trimming involves the removal of up to one third of the maxilla (upper) and (lower) mandible ,17, whicIRBT is recognised as less damaging with fewer adverse consequences, suggesting a low risk of long-term pain . HoweverThere is increasing awareness of the impact of the environment and management during rear on the development of IP in the subsequent laying period ,3. Young2 to 18.1 birds/m2, a significant reduction in GFP and SFP was seen. On average, the highest rate of enrichment pecking per bird per 3 min was observed for lucerne bales, followed by pecking stones and pecking blocks, but, again, the separate effect of the pecking stones could not be evaluated [A complementary approach with a different focus is to try to maintain birds with “naturally” blunted beaks, which may be achieved by providing hard materials as pecking substrates. Commercially available durable pecking blocks and stones have two potential functions, first, by reducing or blunting beaks to avoid beak trimming and, second, by providing a type of enrichment. Some studies have examined the effects of providing hard materials as part of a package of changes. For example, Pettersson et al.  includedvaluated ,32.p = 0.070), but there were no effects on other beak measures and no treatment effects of enrichment or location on any measure. However, pecks at the extra enrichment generally decreased over time. In other studies, pecking stones have been more successful as beak blunting tools; for example, in turkeys after six months of access to pecking stones, the beaks of about 80% of treatment birds resembled those of beak trimmed conspecifics [Whilst the studies mentioned above have included hard pecking materials, they have not examined whether these materials are effective in blunting birds’ beaks via abrasion. Abrasion could be an alternative to BT in laying hens, but this has only been examined in a handful of studies. Fiks-van Niekerk and Elson  placed apecifics .In recent years, there has been a shift in the technique used to measure hen beak characteristics. Previous work in other avian species has revealed diversity in beak length, depth and strength in Darwin’s finches  using lan = 8), Lohmann Brown (LB) (n = 6) and Bovans Brown (BB) (n = 2). Flock size ranged from 3300 to 11,000 and the average flock size was 6843  at day old at the hatchery and the other half were intact-beak flocks . Strains were British Blacktail (BBT)  to give a representative sample. A 2-min habituation period allowing the birds to become accustomed to the presence of the observer was used and observations were taken at a 1 m distance to minimise bird disturbance. The number of birds in the observation area was counted before and after the observation to calculate the average number present during each observation period. Observations included all bouts of gentle feather pecking (GFP), where a bout was defined as continuous pecking until another behaviour was performed or the behaviour stopped for a 5 s gap [Observations of feather pecking behaviour were recorded at each age  between the hours of 08.30 a.m. and 12.00 noon and generally matched for time of day across all visits. Five-minute observations of behaviour were recorded in 2 m 5 s gap . All ind2 traumatized)). Birds that were selected for beak measurements  were further inspected for plumage condition using the same five-point scale (Plumage condition was assessed at the end of the rearing period (bird age at 14/15 weeks). This time point was selected as young chicks and pullets go through many moults throughout rear. Twelve birds per area  were visually scored during the morning behaviour observations in nine areas of the house on the litter area. Birds were not handled in order to minimise flock disturbance. Pullets were randomly selected by counting three birds to the right of the first bird focused upon. Feather cover was assessed and scored for five areas of the body—neck, back, tail, rump and wings—using a five-point scale and photographs for reference, a method similar to Bright and others (2006)  was selected from different areas of the rearing house for beak measurement. Birds were randomly selected using the method described above. Birds were caught in the afternoon so the flock behaviour during observations would not be influenced by catching and handling of the sampled birds. Birds were gently restrained by one person and wrapped in a disposable cloth to restrain their wings so they could be easily handled. The top of the right leg of each bird was marked with a black dot with a permanent marker pen to ensure that these birds would not be recaptured throughout the study. Beak length was measured with vernier callipers using methodology slightly adjusted from that of Van de Weerd (2005)  see Tab. During The data collected from the study were evaluated for significant relationships using statistical tests in IBM SPSS version 26. General linear modelling (GLM) was used, with boot-strapping procedures where appropriate to account for variable data distributions, to examine effects of treatment (pecking pans provided or not), beak status (intact or trimmed) and bird age at , treated as a repeated measure. A flock was taken to be the independent unit for analysis for measures of bird pecking behaviour towards pans or other birds and for plumage condition, as these measures would be highly influenced by the behaviour of other birds in each flock. Pecking activity directed towards pecking pans was analysed for treatment flocks with the model including beak status and bird age. Bird pecking behaviour at the pans was corrected for the length of time for each focal bird and is presented as a pecking rate (pecks/bird/min).GLM repeated measures analyses were used for analysis of feather pecking (SFP and GFP) in both treatment and control flocks, with models including beak status and bird age. For each flock, a mean value of the nine observations of feather pecking behaviour was calculated for each visit: visit 1 at 6–7 weeks, visit 2 at 10–11 weeks and visit 3 at 14–15 weeks. Plumage condition on non-handled birds was assessed only at the end of the rearing period, and so a simple model examined the effect of treatment and beak status. Comparable simple statistical tests were performed on the 45 birds/flock which were plumage scored during handling for beak measurements.Analysis of bird beak characteristics was performed at the individual bird level using data for side length and top of beak for each of the 45 birds sampled at each age . Normality tests showed that none of the data were normally distributed even after transformation, therefore, non-parametric statistical analyses were used. Mann–Whitney U tests were used to compare side-beak length and top-beak length between intact and IRBT birds across age , and patterns of beak growth were plotted for each breed. Further Mann–Whitney U tests were conducted to explore the effect of pecking pan provision on side-beak and top-beak length. A Bonferroni adjustment was used to reduce the possibility of incorrectly rejecting the null hypothesis (Type 1 error) because independent tests were being performed simultaneously on a single dataset (15 tests 0.05/15 = 0.003).p = 0.003 (p = 0.27).There was a reduction in pecking pan activity (peck rate) with age for the eight flocks with access to the pecking pans, with young birds at 6–7 weeks pecking more often (mean 40.4) compared to 10–11 weeks (mean 26.0) and 14–15 weeks (mean 23.32), pecks/bird/min: F = 25.51; df = 1,6;  = 0.003 . Howeverp = 0.08). SFP appeared to increase linearly with bird age . There were no age or treatment effects on GFP behaviour. The frequency of GFP was 2.58 (se 0.52) for beak trimmed birds and 1.12 (se 0.48) for intact beak birds .No significant treatment differences in scan observations of SFP were found in birds with or without access to the pecking pans, nor were there beak status effects. For SFP, overall mean pecks/5 min scan were 0.241 (0.016–0.465) with the provision of pans versus a mean of 0.526 (0.285–0.767) with no pans   . The pat< 0.001) . A linea< 0.001) . There wWhen individual birds from all treatment flocks were considered together, irrespective of beak status, mean side-beak length and mean top-beak length were significantly shorter with a pecking pan present at 6–7 weeks and 10–11 weeks . No signp < 0.001) and at 14–15 weeks  and on mean top-beak length at 6–7 weeks  and at 10–11 weeks . For IRBT birds, there was a significant shortening effect of pecking pans on mean side-beak length at 6–7 weeks  and at 10–11 weeks , with similar effects on mean top-beak length at 6–7 weeks  and 10–11 weeks .Intact and IRBT birds were next considered separately. For intact birds, there was a significant shortening effect of pecking pans on mean side-beak length at 10–11 weeks . However, relatively low levels of IP were observed and, in general, the flocks had good plumage cover and quality.It was hypothesized that treatment flocks provided with pecking pans would show reduced levels of feather pecking, yet no differences were observed, and neither were there any significant effects of beak treatment, strain or age. This finding contrasts with other studies, which show that providing some form of enrichment reduces IP during the rearing and laying period ,31,32. HFew studies have considered whether pecking enrichments that supply hard substrates can reduce beak sharpness in laying hens, with only three previous studies on birds during the rearing stage. Our study measured beaks at the individual bird level and confirmed, as expected, intact beaks were longer than trimmed beaks. Beak length generally increased with age, except in BB birds, where growth between age 6 and 7 weeks and 10 and 11 weeks was relatively low, followed by a greater increase between 10 and 11 weeks and 14 and 15 weeks of age. Variation between individual birds in beak length tended to decrease with age. Pecking pans had a significant shortening effect on side-beak length and top-beak length at 6–7 weeks of age and at 10–11 weeks. Finding significantly shorter beaks at 6–7 weeks was unexpected, as the pans had been placed just the day before observations commenced. This could therefore be due to a random effect of uncontrolled differences between housing or flocks. However, it is also possible that the pecking pans had a very rapid effect, shortening the birds’ beaks dramatically in just a few hours or days. In support, other authors  found thAt 10–11 weeks, both mean side-beak length and mean top-beak length were significantly shorter in the birds provided with a pecking pan when birds of all beak status were considered together, and separately for both intact and IRBT birds. By 14–15 weeks, there was no significant difference in the two measurements when all birds were considered together, but the side-beak length of the intact birds remained shorter at this final visit when pans were provided. One explanation for the reduced effect of pans at the older age is that the birds’ beaks are continually growing and becoming stronger, so blunting may not have been achieved as easily as when the chicks were younger, having much softer beaks, or, more likely, blunting needs to occur continuously in growing birds and the use of the pans had substantially declined.This study has highlighted factors that are likely to be important when considering a long-lasting enrichment to enhance bird welfare. It is the first to investigate, on multiple rearing farms, the potential benefit of blunting the bird’s beak naturally through normal levels of wear. This was achieved by provision of pecking pans containing an abrasive material and provided as an intended environmental enrichment. Evaluation of the results and methodology has given rise to questions to be addressed for future work based upon the improvements to the design and presentation of the pecking pan enrichment device and to some of the methods developed in this study. Despite some limitations in methodology, this study demonstrates that pecking pans for chicks/pullets may provide an environmental enrichment, with the potential added benefit of blunting the bird’s beak naturally through normal level of wear with a very quick onset of effect at 6–7 weeks and continuing to 10–11 weeks of age for all birds, and through to 14–15 weeks for birds with intact beaks. It remains to be determined whether the effect could be extended by presenting the abrasive material differently so that it remained clean and accessible. Furthermore, these preliminary results indicate genotypic variation in beak characteristics, which gives scope for selecting for genotypes with blunter, less damaging beaks.


Sensorineural hearing loss is a well-known side effect of cisplatin (CDDP). There is limited research on the effect of dosing, infusion times, and schedules of cisplatin administration and their impact on hearing loss.A retrospective review of 993 pediatric patients’ medical and audiological charts from August 1990 to March 2015 was conducted using stringent inclusion criteria to characterize patients with hearing loss. 248 of these patients received CDDP. Of these, 216 patients had sufficient CDDP infusion data to assess for sensorineural hearing loss attributable to CDDP and its associated risk factors. Chart reviews were performed to extract clinical data including CDDP dosing information. Demographic and clinical characteristics were summarized by descriptive statistics, and univariate and multivariate logistic regressions were performed to examine the relationship between hearing loss and specific parameters of cisplatin administration . Stepwise variable selection procedure was performed in the multivariate model building to extract the best subset of risk factors for the prediction of hearing loss and worsening ototoxicity grade using an established ototoxicity grading scale from the International Society of Pediatric Oncology (SIOP).p-value=0.0086], radiation to any part of the body, , amount infused per dose (mg/m2) , and cumulative cisplatin exposure (mg/m 2)  were associated with hearing loss. Similar associations were also found between these risk factors and worsening SIOP grade.A total of 153 patients with complete medical and audiologic data were evaluable for analysis. Hearing loss was identified in 72.6% of the patients. Multivariate analysis revealed that age [OR=0.90 (0.84-0.97), In one of the largest studies examining the influence of CDDP dosing and schedules on hearing loss, we found the amount of CDDP infused per dose is a significant risk factor. Considerations in designing regimens that reduce the amount of CDDP infused per dose may reduce the risk of hearing loss. Randomized prospective trials are needed. Sensorineural hearing loss (SNHL) is a well-known complication from the administration of cisplatin (CDDP) –6. EvideThis study, approved by the Institutional Review Board of the Washington University School of Medicine Human Research Protection Office, was a retrospective chart review of medical record data existing at the initiation of our study. Audiology charts of pediatric oncology patients at St. Louis Children’s Hospital treated from August 1, 1990 through March 31, 2015 were reviewed. From this cohort, patients were assessed for treatment containing CDDP.Inclusion criteria into our current study required prior CDDP treatment. Patients whose chemotherapy treatment did not include CDDP were excluded. Evaluable patients had either completed their CDDP therapy or had documented CDDP hearing loss from current therapy and were still being treated at the cutoff for study entry in March 2015. Data was not collected for patients still undergoing treatment who had normal hearing at the cutoff point of the study. No patients received oto-protectants. The medical records of these patients were reviewed to extract the following variables of interest: gender, birthdate, date of diagnosis, race, ethnicity, diagnosis, CDDP dosage information , presence of carboplatin, radiation exposure to any part of body, radiation exposure to head, date when all therapy ended, date of last CDDP administration, living status, date of most recent audiogram, presence of hearing loss based on the worse ear, and right/left ear toxicity grades according to the International Society of Pediatric Oncology (SIOP)  at our institution is defined as thresholds of ≤ 30 dBnHL from 0.5 kHz -1 kHz and ≤ 20 dBnHL at 2 kHz - 8 kHz, using both earphone and inserts as transducers.All audiograms included in our analysis were of good to fair reliability, as determined by the testing audiologist. Soundfield and ABR testing was allowed for baseline testing. All patients were required to have a normal baseline audiogram with a subsequent ear specific behavioral audiogram, testing out to 6000 and/or 8000 Hz; baseline audiograms obtained All evaluable baseline hearing tests required at least 2 thresholds from 1 kHz - 4 kHz. If any frequency was outside the defined normal range, from 1kHz - 4kHz, the audiogram for that ear was not evaluable. Ears with conductive hearing loss ≥ 1000 Hz at baseline were excluded. Patients were included in our analysis if they presented with normal hearing in at least one ear at baseline. In this case, audiologic data was only collected on the single, normal hearing ear. Finally, our study allowed the baseline audiogram to be absent if a subsequent audiogram documented normal hearing.All audiograms evaluated as the “most recent audiogram” were behavioral hearing tests; soundfield alone and ABR testing were not permissible. All sensorineural hearing loss ≤ 4 kHz, were confirmed by bone conduction thresholds. Bone is not tested above 4 kHz at our institution. If the hearing loss was observed at ≥ 6 kHz, documentation of a normal middle ear status through static admittance of ≥ 0.3 mmho, large ear canal volume consistent with patent tubes, and/or a normal otologic exam by an otolaryngologist was needed to confirm sensorineural nature of the hearing loss. In the current study, the presence of normal bone conduction thresholds and/or a normal hearing evaluation at both baseline and at the most recent hearing test superseded abnormal tympanometric measures. The patient was determined to have hearing loss if the most recent audiogram showed change from normal baseline and a SIOP grade of greater than 0 in the worse evaluable ear.Each evaluable ear was assigned a SIOP grade relative to the bone conduction thresholds of the most recent audiogram. The SIOP grading scale has been suggested as the superior grading scale in regards to classifying ototoxic hearing loss , 18. AudStudy data were collected and managed using REDCap electronic data capture tools hosted at Washington University School of Medicine , 20. REDAll statistical analyses were carried out using SAS version 9.4 . Demographic and clinical characteristics were summarized by descriptive statistics, i.e., median and IQR (interquartile range) for continuous variables; and count and percentage for categorical variables. Group differences were examined by Kruskal-Wallis test for continuous variables and Fisher’s exact test (if cell count less than 5) or Chi-square test for categorical variables. Univariate and multivariate logistic regression were used to examine the relationship between hearing loss/SIOP grade and various risk factors, including sex, race, diagnosis, carboplatin use, radiation, radiation to the head, age, total number of doses, the amount of CDDP infused per dose, number of doses/cycle, number of cycles, prescribed dose time, and cumulative CDDP dose. A stepwise variable selection procedure was performed in the multivariate model building for the outcome measure, hearing loss. A significance level of 0.3 was required to allow a risk factor to enter or stay in a model during the variable selection process. To make the results comparable, the final multivariate risk model of hearing loss that obtained during the stepwise selection was used as the multivariate risk model for the outcome measure, SIOP grade.There were 993 patients in the entire cohort. After assessing their treatment, 248 patients were included in the study who had a prior history of CDDP exposure Figure 12, ranging from 90 to 1000 mg/m 2. The mean amount per dose was 74.1 mg/m 2, ranging from 20 to 150 mg/m 2. Patients received an average of 6.9 doses (range=1-30) and 3.7 cycles (range=1-8) with an average of 2.1 dose per cycle (range= 1-9). The average prescribed dose time was 4.4 hours, (range= 1-8).Analysis of evaluable patients revealed a difference in hearing loss frequency linked to age (p=0.0003), diagnosis (p= 0.0013), radiation to any part of the body (p=0.010), radiation to the head (p=0.012), amount of CDDP per dose (p=0.019), and number of doses per cycle (p=0.048) Table 1.Univariate and multivariate multinomial logistic regression analyses were also completed to determine associated risk factors with SIOP grade Table 3.th-75th percentile = 300-480 mg/m 2). Thus, cumulative dosing may have been obscured as a risk factor due to the minimal difference in cumulative dosing between those with hearing loss and those without (mean = 400 mg/m2vs. 383.5 mg/m2).Increasing our understanding of the parameters of CDDP infusion effects on hearing loss can lead to strategies that may reduce the risk. This study has demonstrated the amount of CDDP infused per dose was strongly associated with an increased risk of hearing loss; it is also associated with more severe hearing loss as reflected by a worsening SIOP grade. Other parameters such as number of doses, number of doses per cycle, number of cycles, and infusion times failed to reach significance using multivariate analysis. Cumulative CDDP was also associated with hearing loss, to a lesser degree. Prior studies have repeatedly identified cumulative incidence as a risk factor for hearing loss , 11, 21.Our analysis also revealed age was strongly associated with a risk of hearing loss with the youngest patients at greatest risk. Radiation to any part of the body was also shown to be a risk factor, with radiation directly to the head failing to achieve significance in multivariate analysis. Our previous investigations had established radiation to locations other than the head still place patients at risk for hearing loss . RadiatiThis report supports a recent study published which also identified the amount per dose as a risk factor for hearing loss . Our stuOur audiometric data is representative of a purely pediatric population; our mean age is older at 8.3 years. Obtaining reliable data in a young, ill child is fraught with difficulties, leading to audiograms that often fail to meet our stringent criteria for study eligibility. Many audiograms in our youngest subjects were not included in this study driving up the average age of the cohort. Finally, we included hearing loss at all levels of severity, while the recent publication only examined patients with higher SIOP grades. We had previously shown hearing loss continues to decline long after therapy is completed. We also have demonstrated that the patients with the longest follow-up had the highest likelihood of diagnosed hearing loss. Our mean time in years from the last CDDP administration to the last audiogram was 3.73 years while the previous study reported a mean of 1.5 years from the end of treatment. The increased prevalence of hearing loss as patients are followed longer and the use of the better verses worse ear may explain the difference in hearing loss observed by those investigators (43.8%) compared to our study (72.6%) .This latThis study was limited by the large fraction of patients (39%) excluded for analysis due to missing infusion or audiology data. .Ineligible patients were younger, more frequently treated with carboplatin, and were less likely to be treated with radiation. We suspect that these findings could be attributed to the differences in the number of neuroblastoma patients in the ineligible cohort . Neuroblastoma patients are typically younger, may receive carboplatin, and may receive less radiation due to their young age. Given the difficulties in obtaining audiograms in young patients, it is possible that young neuroblastoma patients were disproportionately excluded due to insufficient audiologic dataThe study was a cross sectional analysis with no specific time point that could be used for hearing testing that could encompass the entire cohort. Audiograms were not obtained after each CDDP infusion so we could not assess any variables associated with a specific infusion encounter. However, despite these limitations and given the paucity of studies examining this subject, our audiologic monitoring program provided evidence that calls for new approaches for CDDP administration.In order to increase the number of evaluable patients, we included patients that demonstrated SNHL, even if they had not completed therapy. However, we did not include patients undergoing therapy if they had not demonstrated hearing loss at the end of the study period. Given that our patients were carefully screened for SNHL, which we would expect to be irreversible, we were not concerned that patients who had established SNHL would subsequently convert to the “no hearing loss” cohort with time. In contrast, there are patients who may lose hearing with subsequent cycles; this was our justification for not including those patients receiving therapy in the no hearing loss group. Furthermore, the patients with hearing loss on therapy were not included in the SIOP analysis, as it is possible the SIOP grade could shift, even while on therapy. We therefore were able to add additional patients to enhance our sample size, without including those subjects that would compromise the integrity of the cohort.This study also collected patients over a period of almost thirty years. Although there have been advances in this field, there are no significant changes in audiology testing that would have influenced the data. However our monitoring of patients became more robust in recent years, where we now test at risk patients at regular and set intervals. Thus, the results may have been influence by the frequency of testing rather than differences in testing methods. We addressed this issue by examining the year of diagnosis (based on the date of diagnosis) as a continuous variable. The year of date of diagnosis ranges from 1983 to 2015 (median: 2006). The median diagnosis year of patients with hearing loss is 2005 ranging from 1983 to 2015; while the median diagnosis year of non-hearing loss group is 2007 ranging from 1996 to 2013. There is no significant relationship between hearing loss and the year of diagnosis to the date of the last audiogram, . Subgroup analysis comparing 2010-2015 to 1990-1995 also shows the year of diagnosis has no significant relationship with hearing loss (p = 0.16).Although we examined specific infusion parameters, we were unable to assess how patients may vary in CDDP exposure based on varying pharmacokinetics. Such variations could lead to variances in area underneath the curve (AUC) that could account for different levels of toxicity. Calvert established a formula to establish more consistent dosing and AUC attainment for carboplatin . UnfortuDespite these limitations and given the paucity of studies examining this subject, our audiologic monitoring program provided evidence that calls for new approaches for CDDP administration.This project is one of the largest audiologic studies examining the influence of CDDP dosing and schedules on hearing loss. We demonstrated the amount of CDDP infused per dose is a significant risk factor. These findings support an observation in a previously reported clinical trial by the Children’s Oncology Group where the two cohorts only varied with the amount of CDDP per dose with worse ototoxicity observed in the group assigned the higher dose . Such doThe original contributions presented in the study are included in the supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Institutional Review Board of the Washington University School of Medicine Human Research Protection Office. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.Design of the trial – RH, SH, Execution of the trial – RH, SH, JH, KS, Data collection – SH, MC, AS, Data analysis – NW, Writing Manuscript – RH, SH, MC, AS, NW. All authors contributed to the article and approved the submitted version.Data analysis was provided by the Biostatistics Shared Resource, of The Siteman Cancer Center and is supported in part by an NCI Cancer Center Support Grant #P30 CA091842, Eberlein, PI.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.


While endovenous thermal ablation (ETA) become first choice of treatment for varicose veins, overuse of ETA for the inappropriate indication is growing problem. ETA is performed not only on varicose cases without symptom but also non diseased cases with segmental reflux of saphenous veins or no reflux. Indications of ETA was demonstrated in “the Clinical Practice Guidelines for ETA for Varicose Veins 2019” by Japanese Society of Phlebology. Purpose of this supplement is description of basics of correct indication for ETA. We also demonstrate the typical case of overuse of ETA for wrong indication.  However, while several patients have benefited from ETA, it is performed for partial valvular insufficiency of the saphenous vein without stasis symptoms and even for normal veins at some medical institutions. In addition, simultaneous treatment of both lower limbs at a higher frequency than that expected epidemiologically  and planned repetitive procedures on the same lower limb are performed at these institutions. Amid such situations, the Japanese Committee for Endovascular Treatment for Varicose Veins has alerted implementing surgeons and supervisory doctors to perform appropriate treatment.Since its approval for national health insurance coverage in 2011, the use of endovenous thermal ablation (ETA) for varicose veins of the lower extremities has become widespread due to its minimal invasiveness, currently accounting for approximately 90% of surgical procedures for lower extremity varicose veins in Japan. describing appropriate ETA indications. These indications, including the appropriate ranges of the venous diameter and valvular insufficiency, as defined in the previous edition, were considered self-explanatory with the widespread use of ETA and were not specified in the 2019 guidelines. However, the number of improperly treated cases has increased as mentioned above, with inexperienced doctors getting involved in the treatment. Therefore, an addendum describing more basic surgical indications was preferred by many clinicians and insurance certification committee doctors. Under the circumstances described above, the addendum to the guidelines was created to present the basic surgical indications and the details of inappropriate treatment cases, to perform appropriate ETA. Additionally, the indications for cyanoacrylate venous closure, approved in December 2019, are similar to those of ETA, with similar cases not indicated for both modalities.In 2019, the Japanese Society of Phlebology published the “2019 Guidelines for Endovenous Thermal Ablation for Lower Extremity Varicose Veins” (ETA Guidelines), consisting of recommendations based on the common clinical questions on ETA selected by the guideline committee and evidence,The addendum to the guidelines recommends not performing ETA for the cases listed in  The presence or absence of valvular insufficiency should always be evaluated using ultrasonography, and is identified if the reflux time measured by pulsed Doppler through blood flow induction in a standing or sitting position exceeds 0.5 s. The previous edition of the ETA guidelines recommended measuring venous diameters and indicating ETA if the mean diameter of the saphenous vein was 4 mm or greater. However, the recommended saphenous vein diameter was not stated in the 2019 guidelines as it varies between GSV and SSV and depends on the physique; i.e., it can only be an indicator for the blood reflux volume. If the diameter is 4 mm or smaller, symptoms are often mild even with reflux. Therefore, to determine the indication for ETA, thorough consideration and detailed examination is necessary. Also, if there is no valvular insufficiency in the saphenous vein, regardless of the diameter, it is not primary varicose vein. Therefore, even if venous dilatation with a diameter of 4 mm or greater exists, or spider or reticular veins and edema classified into C1 and C3 of the Clinical Etiological Anatomical Pathophysiological Classification (CEAP) exist, there is no indication for ETA if valvular insufficiency in the saphenous vein is absent.According to the guidelines, ETA is indicated for symptomatic primary varicose veins of the lower extremities, and the treatment target veins include the great saphenous vein (GSV), small saphenous vein (SSV), and accessory saphenous vein. According to the mechanism of development, varicose veins of the lower extremities are classified into primary, secondary, and congenital. Primary varicose veins of the lower extremities are defined as those developing due to valvular insufficiency of the superficial venous system, mostly the saphenous vein.Performing ETA on the saphenous vein without valvular insufficiency would cause unnecessary invasion, leading to the loss of an alternative graft option for future cardiac or lower limb bypass surgery. Therefore, “patients should not be harmed,” as such an act is against the “medical ethics” and must be strictly avoided. Partial valvular insufficiency does not cause symptoms, and although blood reflux worsens over time, all patients do not develop chronic venous insufficiency. To date, clinical studies of ETA for lower extremity varicose veins have been conducted in patients with symptomatic lower extremity varicose veins, including mostly axial reflux cases associated with extensive saphenous vein valvular insufficiency ranging from the sapheno-femoral junction (SFJ) to below the knee  or pudendal varicose vein serves as a reflux source and causes valvular insufficiency of the GSV without SFJ insufficiency. These cases are indications for ETA  and pulmonary thromboembolism (PTE). However, while the prevalence of varicose veins of the lower extremities is high, the incidence of PTE is low. A recent report analyzing health insurance data showed that the incidence of DVT is 5.3 times and that of PTE 1.7 times higher in patients who were diagnosed with varicose veins of the lower extremities.A typical case of inappropriate treatment is shown below. This case has been reconstructed as a representative case based on the one that was provided and reported to the Japanese Society of Phlebology. It has been modified for protection of personal information and does not refer to a specific individual patient or medical institution.The patient was a woman in her 50s who visited a medical institution with a chief complaint of varicose veins, fatigability, and edema of the right lower limb. Ultrasonography of the lower extremity veins revealed valvular insufficiency in the right SSV and femoral vein with left distal GSV, and right SSV dilatation with a diameter of 6.8×6.8 mm, diagnosed as an indication for ETA on the right SSV (Although we were aware of a small number of doctors providing improper treatment, we believed that the approval of the health insurance coverage of ETA would eliminate inappropriate treatment in ETA. However, recently improper treatment has been provided more systemically, on a larger scale. Even though the majority of doctors adhere to the guidelines and sincerely provide proper medical care to their patients, a small number provide improper treatment due to ignorance or financial motivation, affecting the budget of healthy health care services under public health insurance. We prepared this ETA guideline addendum to improve the situation. This article concludes with the Hippocratic Oath related to the ethics of medicine.“I will apply dietetic and lifestyle measures for the benefit of the sick to the best of my ability and judgment and will keep them from harm and injustice.”


Based on the antigenic distances, subgroup II and III strains resulted in two clear antigenic clusters 2 and 3, separated from the vaccine strain F98, cluster 1. New antigenic properties of subgroup III viruses were associated with 11 amino acid changes in the HA protein, suggesting antigenic drift in H9N2 viruses. Our phylogenetic and antigenic analyses of the H9N2 strains circulating in local markets in Southern China provide new insights on the antigenic diversification of H9N2 viruses.The H9N2 subtype avian influenza virus (AIV) has become endemic in poultry globally; however due to its low pathogenicity, it is not under primary surveillance and control in many countries. Recent reports of human infection caused by H9N2 AIV has increased public concern. This study investigated the genetic and antigenic characteristics of H9N2 AIV isolated from local markets in nine provinces in Southern China from 2013 to 2018. We detected an increasing annual isolation rate of H9N2 AIV. Phylogenetic analyses of hemagglutinin (HA) genes suggests that isolated strains were rooted in BJ94 lineage but have evolved into new subgroups (II and III), which derived from subgroup I. The estimated substitution rate of the subgroup III strains was 6.23 × 10IMPORTANCE The H9N2 low pathogenicity avian influenza (LPAI) virus has become endemic in poultry globally. In several Asian countries, vaccination against H9N2 avian influenza virus (AIV) was approved to reduce economic losses in the poultry industry. However, surveillance programs initiated after the introduction of vaccination identified the persistence of H9N2 AIV in poultry . Recent reports of human infection caused by H9N2 AIV has increased public concern. Surveillance of H9N2 circulating in poultry in the fields or markets was essential to update the vaccination strategies. This study investigated the genetic and antigenic characteristics of H9N2 AIVs isolated from local markets in nine provinces in Southern China from 2013 to 2018. The discovery of mutations in the hemagglutinin (HA) gene that result in antigenic changes provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry. Currently, H9N2 avian influenza virus (AIV) is one of the predominant subtypes (together with H5 and H7 subtype AIVs) circulating in both wild birds and poultry , 2. Ever5–Surveillance of H9N2 AIV is carried out in many countries, especially China and other Asian countries in which live poultry markets (LPMs) are potential areas for human-poultry transmission or reassortment . GeneticIn addition to surveillance programs, some countries have also implemented poultry vaccination to prevent H9N2 AIV from becoming endemic –18. Afte–Since 2003, monitoring programs of LPAI H9N2 in Southern China were conducted by the Research Team of the Etiologic Ecology of Animal Influenza and Avian Emerging Viral Disease in Shanghai Veterinary Research Institute (SHVRI). Swab samples from domestic poultry  were collected annually in the local markets in Southern China. The most recent samples collected from 2013 to 2018 showed an increasing annual isolation rate of H9N2 AIV in Southern China. The phylogenetic and antigenic analyses of the H9N2 isolates were combined to identify a common clustering change. The most recent isolates clustered together but were far from the vaccine strains. Isolated strains forming a novel antigenic group in antigenic assays also clustered in a new subgroup in the phylogenetic tree. The evolution rate of strains in the new antigenic group was estimated to be higher than the average rate of H9N2 in China. Moreover, amino acid changes on HA protein indicating potential antigenic sites provide new evidence for the molecular evolution of H9N2.To monitor the prevalence of H9N2 AIV in Southern China, we collected oropharynx and cloaca samples of poultry from LPMs in nine cities of different provinces in Southern China. From 2013 to 2018, a total of 13,981 samples were collected from chickens , ducks , geese (81 samples), and pigeons (32 samples) in different LPMs . H4, H6,To analyze the genetic evolution of the HA gene of the H9N2 isolates, 113 representative isolates were selected to perform HA gene sequencing and further analysis based on geographical and host differences. These included 21 viruses isolated in 2013, 42 in 2014, 23 in 2015, 7 in 2016, 11 in 2017, and 9 in 2018. Online HA sequences were used for the phylogenetic tree, including 4 in 2013, 3 in 2014, 7 in 2015, 11 in 2016, 8 in 2017, and 9 in 2018. Phylogenetic analysis showed that the HA genes of the isolates from 2013 to 2018 were all distributed in subgroup II or subgroup III, except for A/chicken/Guangdong/D2703/2013 and A/chicken/Fujian/F1132/2015 and the commercial vaccine strain A/chicken/Shanghai/F/1998 (F/98), which were colocated in subgroup I . Among tIn addition, a new branch of evolution, subgroup III, began to appear after 2013, including 14 viruses  isolated in 2013, 28  in 2014, 19  in 2015, 6  in 2016, 7  in 2017, and 9  in 2018.To estimate the time when subgroup I, subgroup II, and subgroup III viruses started to appear, the most recent common ancestor (tMRCA) for these time points was estimated based on the HA genes of all H9N2 isolates in the time-scaled phylogenic tree . Subgroup I lineage viruses first emerged in August 1992, with a 95% highest posterior density (HPD) interval from September 1991 to May 1993. The tMRCA of subgroup II viruses was September 1996 (July 1995 to November 1996). The most recently isolated H9N2 strains from 2018 were in subgroup III, of which the tMRCA was estimated as September 2010 .To monitor the antigenic map of the H9N2 isolated viruses, 27 representative isolates were selected for crossing hemagglutination inhibition (HI) tests, including three viruses isolated in 2013, two in 2014, two in 2015, seven in 2016, seven in 2017, and six in 2018 . A vacci−3  substitutions/site/year over a 25-year period , with only 18.75% of the average evolution rate of the HA gene of H9N2 AIVs. In contrast, the HA genes of antigenic cluster 3 viruses (subgroup III) evolved most rapidly with 6.23 × 10−3 substitutions/site/year, 1.5-fold faster than that of the average substitution rate of HA genes for H9N2 viruses  of the viral effective population size . The popThe homology of HA protein from different antigenic clusters was analyzed to assess the potential amino acid residues resulting in antigen drift. Eleven amino acid residues were conserved in vaccine cluster viruses  but mutated into the same amino acid residues both in cluster 2 and 3 viruses, including M96L, T205A, D208E, Q226L, N273K, S274R, N275S, V276T, R285K, P306S, and A325S (H3 numbering), indicating that these residues in HA may contribute to the antigen change between cluster 1 and clusters 2/3 ; Fig. 4aInvestigation of the key amino acids characterizing antigenic properties of strains in cluster 2 showed 10 mutated amino acid residues in the HA protein between cluster 2 viruses and cluster 3 viruses, including N/H57Q, G81E, S/N133D, K137T, D145G, Q156G, N159G, A160D/N/E, V/A190T, and T192R ; Fig. 4bTo gain information on the evolution of potential antigenic amino acid residues on HA from H9N2 over time in nature, we analyzed HA proteins of H9N2 isolates and other viruses submitted to NCBI. Of the 11 potential antigenic amino acids characteristic of cluster 1, 10 were dominant patterns in China, including M96L, T205A, D208E, Q226L, N273K, N275R, V276T, R285K, P306S, and A325S in HA protein (H3 numbering). Currently, S274R is the predominant amino acid for H9N2 HA, mixed with some S274 . MoreoveThe H9N2 virus is a predominant subtype of AIV circulating in Asia and other countries all over the world –33. H9N2–35–Our surveys of LPMs conducted in China from 2013 to 2018 demonstrate that H9N2 AIVs are still prevalent, as described previously . An incrTogether with the high isolation rate and long-term endemic circulation, H9N2 AIVs in mainland China are undergoing a rapid antigenic drift. The hypothesis that vaccination triggered rapid evolution leading to antigenic drift was reported in Korea only 4 years after the national vaccination program was applied in poultry in 2007 . Similar−3 substitutions/site/year. This substitution rate increased almost 1.5-fold (6.23 × 10−3 substitutions/site/year) when antigenic differences were involved.Phylogenic analysis of 113 H9N2 strains isolated from LPMs in Southern China from 2013 to 2018 provided sufficient circulating and evolutionary information of H9N2 in local poultry. Consistent with previous studies indicating G9 (A/chicken/Hong Kong/G9/1997) lineage (h9.4.2)  as the d−3 substitutions/site/year)  using the sequences from the influenza database has been estimated. These rates are similar to the estimations using the same method for H3 (HA1 domain) in humans (5.7 × 10te/year) . For betWe found 21 amino acid mutations on H9 antigenic sites (in region 66 to 334 on the H9 protein sequence) specific to strains in antigenic cluster 2, different from those in cluster 1 as well as three vaccine strains. Referring to the global online database of AIV H9N2 in Nextstrain, all these mutations evolved after 2013 and became dominant in Southern China. This explains why the isolation ratios in LPMs from 2013 to 2018 did not decrease even though vaccines had been used long term in poultry, with reports that the H9N2 virus continues to circulate in vaccinated chickens , 46.Structural analyses of the HA1 protein to predict key potential antigenic sites dominating the subgroup of current H9N2 isolates from field markets in Southern China were performed. The four surface locations  at the top of the HA head also show high entropy in real-time tracing . This inIn summary, our study shows that the currently circulating H9N2 viruses form a new antigenic group to those from vaccine strains and are also clearly divided into new subgroups in phylogenic trees. The declining diversity together with the high substitution rate of H9N2 strains in the new antigenic group suggest that vaccination triggers the evolution of H9N2 into new antigenic groups. Our study provides a baseline reference for evolutionary studies of H9N2 viruses and vaccination strategies in poultry.All animal experiments were conducted in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China and the Netherlands. The protocols (SHVRI-SZ-20190730-01 and SHVRI-SZ-20190906-02) used in the study were approved by the Animal Care and Use Committee of Shanghai Veterinary Research Institute (SHVRI).TTCTAACCGAGGTCGAAAC-3′ (M-229U) and 5′-AAGCGTCTACGCTGCAGTCC-3′ (M-229L). Hemagglutination inhibition (HI) assays were then used as antiserums specific for H9 subtype influenza viruses.Between 2013 and 2018, a total of 13,981 oropharyngeal swabs and cloaca swabs were collected from chickens, ducks, geese, and pigeons in local markets from nine provinces in China . Swab samples were inoculated in the allantoic cavity of 10-day-old specific-pathogen-free (SPF) chicken egg embryos. The harvested allantoic fluids were tested by hemagglutination assay. Total viral RNAs of HA-positive allantoic fluids were extracted to detect influenza viruses by amplifying the M gene by PCR. The primers used for this detection were 5′-The viral RNA was extracted, and reverse transcription was performed with the Uni12 primer as previously described , 50. ComFJ190132, EPI_ISL_260531, AF156378, CY005632, EPI_ISL_146723, and KF188265) were identified and removed from the data set. We then constructed time-scaled phylogenies using a Bayesian Markov chain Monte Carlo (MCMC) framework implemented in BEAST (v1.10.2) .To generate a time scale tree of the whole lineage containing the H9N2 strains isolated from this lab , we inclAccording to the phylogenetic tree of the HA gene, 27 H9N2 viruses were selected from the isolated strains from 2013 to 2018; six stored virus strains from previous research  were selected for the antigenic analysis. Briefly, specific antisera with inactivated virus isolates were produced in SPF Leghorn chickens with adjuvant Montanide ISA 71VG (SEPPIC). Four-week-old SPF Leghorn chickens were vaccinated with a total of 1.5 mL by intramuscular injection. After 3 weeks, sera were collected from vaccinated chickens. The hemagglutination inhibition (HI) assay was conducted to test cross-reactivity between virus and antisera as described previously by the World Organization for Animal Health (OIE) . First, 1JSD)  position of the identified molecular changes on the sites of the HA protein, the HA1 structure of H9 was simulated. A/swine/Hong Kong/9/98 (H9N2) (1JSD)  was used1JSD) .https://nextstrain.org)  . To zoomhttps://platform.gisaid.org/epi3/cfrontend#80368) with isolate IDs from EPI_ISL_462482 to EPI_ISL_462520. These HA sequences were submitted to the GenBank database (OK563122 to OK563235).All the nucleotide sequences of HA and NA genes from H9N2 isolated in Southern China from 2013 to 2018 were submitted in the GISAID database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) hosted by NCBI. Details are at https://doi.org/10.1371/journal.pcbi.1007189.s012 (The G9 lineage strains recovered from the data set in reference 189.s012 .https://nextstrain.org/flu/avian/h9n2/ha) consisted of 1,092 of 1,332 genomes sampled between January 1976 and June 2019. The data used in this research were filtered to avian  from Africa (115), China (358), Europe (55), South Asia (126), Southeast Asia (93), and West Asia (395).Data and analysis from Nextstrain (


The loss of auditory sensory hair cells (HCs) is the most common cause of sensorineural hearing loss (SNHL). As the main sound transmission structure in the cochlea, it is necessary to maintain the normal shape and survival of HCs. In this review, we described and summarized the signaling pathways that regulate the development and survival of auditory HCs in SNHL. The role of the mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channels, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant. The molecular interactions of these signaling pathways play an important role in the survival of HCs, which may provide a theoretical basis and possible therapeutic interventions for the treatment of hearing loss. Globally, hearing loss is the most common sensory disorder, and its severity ranges from mild hearing impairment to total deafness. More than 500 million people are affected by this health problem, and 1 in every 500 newborns worldwide is deaf , 3. ThesHair cells (HCs) and spiral ganglion neurons (SGNs) are the main structures of sound transmission in the cochlea –11. The Several signaling cascades are activated following injury to the cochlea. These pathways can be proinflammatory, prodeath, and even prosurvival. The signaling cascades that occur at the cellular and molecular levels are highly complex and intertwined . HoweverAt present, there are an increasing number of studies that have focused on cellular molecular pathways that could represent potential checkpoints in the mechanism of hearing loss and HC damage. As research into the necrobiology of the inner ear progresses, the pathways specific to auditory HC death will become better defined. Among the identified pathways, mitogen-activated protein kinase (MAPK), phosphoinositide-3 kinase/protein kinase B (PI3K/Akt), Notch/Wnt/Atoh1, calcium channel, and oxidative stress/reactive oxygen species (ROS) signaling pathways are the most relevant –29. ImpoTo summarize the research regarding hearing loss based on cellular molecular pathways in recent years and to guide hearing recovery and the prevention of hearing loss, this paper reviews the current research on the biology of HC damage. The various cellular molecular pathways are discussed systematically. The potential of damage and survival mechanisms as targets for pharmacological intervention to prevent or ameliorate hearing loss is reviewed, and conclusions are drawn in the last section.Metabolomic and bioinformatic analyses have indicated that MAPK signaling is the major pathway in various types of hearing loss , 31. In The MAPK/ERK pathway is reportedly associated with cell proliferation, differentiation, migration, senescence, and apoptosis. It consists of a series of proteins including Ras, Raf, MEK, and ERK. In many cases, ERK1/2 activation is thought to promote cell proliferation and survival . Studiesα, FasL, Bak, Bim, and Bax) and blocking transcription of antiapoptotic genes (such as Bcl-2) [JNKs, also known as stress-activated protein kinases, phosphorylate the transcription factor, c-Jun. JNK has three isoforms: JNK1, JNK2, and JNK3. The MAPK/JNK signal transduction pathway is activated in response to the exposure of cells to environmental stress and contributes to an apoptotic response by stressed cells that have been damaged by ROS . Studiess Bcl-2) –47. Takeα-) induced ototoxicity [P38 is another downstream protein of the MAPK pathway and is involved in many cellular processes, including inflammation, cell cycle regulation, and apoptosis . It can toxicity –59.In conclusion, different MAPK kinases have different effects on HCs. In the future, treatments and drugs based on the MAPK pathway may play a significant role in HC regeneration and protection from ototoxic drug–induced hearing loss.The gradual loss of HCs leads to hearing loss, and thus identifying specific signaling pathways that promote HC survival is an effective therapy. Among the identified signaling pathways, PI3K is a powerful candidate for improving the survival of HCs. In recent decades, intensive investigations have been carried out towards PI3K pathway activation for the treatment of aminoglycoside-induced –62, suddα-induced apoptosis in vitro by activation of PI3K/Akt and NF-κB signaling [Ototoxic drugs are the main environmental factors of hearing loss –73. Howeignaling . Zhang eignaling .Sudden sensorineural hearing loss (SSNHL) is a rapid and unexplained SNHL that occurs within 72 hours, with hearing loss ≥ 20 dB in at least 2 adjacent frequencies , 77. By Increased PI3K/Akt signaling has a protective effect on hearing loss induced by ototoxicity or adverse environmental factors. Understanding the mechanisms of PI3K/Akt signaling may provide therapeutic ideas for combating hearing loss and identifying new disease strategies.β, PAX, Notch, Wnt, NFκB, insulin/IGF1, and AP-1 [The human cochlea has about 16,000 sensory HCs, which are necessary for normal hearing . In contand AP-1 . Among tand AP-1 –94. Simuand AP-1 –100.Atoh1 is one of the most important transcription factors involved in the development of the inner ear . PreviouNotch signaling plays a key role in Atoh1 regulation and inner ear development. Atoh1-dependent cell development support and sensory patch patterning are currently believed to be dependent on Notch signaling-mediated lateral inhibition . Cheng eDuring the development of the cochlea, the Notch signaling pathway interacts with the FGF signaling pathway to control the expression of Atoh1 . Doetzlhβ-catenin signaling has been confirmed to be upstream of both Atoh1 and SOX2 during the development of cochlear HCs and retina cells [β-catenin signaling can promote HC regeneration by increasing cell proliferation and Atoh1 expression. The Wnt pathway also interacts with the Notch pathway. Studies have demonstrated that Wnt activation followed by Notch inhibition significantly promotes the transformation of supporting cells into HCs. Lineage tracing has shown that new HCs, predominantly OHCs, arise from inner pillar and third-row Deiter's cells [Wnt/na cells , 112. Wn's cells , 114. In's cells . Thus, Wγ-secretase inhibitor LY411575 on the Notch signaling pathway results in the formation of new hair cells in the outer hair cell region and a mild reduction in noise-induced auditory brainstem response (ABR) threshold shifts; although, the mice were still functionally deaf [Based on the importance of the Notch/Wnt/Atoh1 pathway, the researchers provide novel therapeutic strategies. Pan et al. reported that genetically engineered Atoh1 knockout mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy . Mizutarlly deaf . In addilly deaf , 118.In summary, Notch interacts with the FGF to inhibit the expression of Wnt and further suppress the downstream molecule Atoh1, thereby preventing the differentiation of supporting cells into HCs  97, 115115, 119.2+) is critical for cell survival. On this basis, transport channels, ligand-gated channels, and voltage-gated calcium channels (VGCCs) are located on the plasma membrane of HCs and facilitate Ca2+ entry into HCs [α1G, α1H, and α1I [Acquired hearing loss, including noise-induced hearing loss, age-related deafness, and ototoxicity-induced hearing loss, has complex mechanisms for each disease. Despite the complexity of these mechanisms, numerous studies have shown that apoptosis of inner ear structures is a common theme among many types of acquired hearing loss –123. Calinto HCs . Also, w and α1I . In the  and α1I .α1H T-type calcium channel subunit comprising one or more Cav3 calcium channel types in the cochlea [2+ influx that activates proapoptotic signaling cascades in HCs. There is evidence that the Ca2+-sensitive neuropeptide, somatostatin (SST), can antagonize aminoglycoside-induced cell death. SST analogs have the same effect [Therapeutic agents aimed at preventing apoptosis are a concept central to many therapies that target acquired hearing loss. Research is currently underway to evaluate potential therapeutic targets within the peripheral auditory system and apoptotic pathways. Numerous studies have shown that calcium channel blockers (CCBs) can effectively prevent damage to cochlear cells. More recent in vivo research has provided evidence to suggest that systemic and intratympanic (direct) application of CCBs can prevent hearing loss in cisplatin- and noise-induced ototoxic models . For exa cochlea . In addie effect .In view of the apoptotic effect of calcium channels in cochlea HCs, these channels may become a potential pharmacological target for clinical interventions in the future. Pharmacological inhibition of CCBs may represent a promising approach to the treatment of auditory impairment of various etiologies.2O2), and singlet oxygen, are mainly generated by the mitochondria in most mammalian cells [ROS-induced oxidative stress has been reported to play a key role in several systems and in cochlear damage –136. Numan cells , 144. Inan cells . This evan cells , 147.Many drugs, including cisplatin, gentamicin, and neomycin, are ototoxic and may cause irreversible apoptosis of cochlear HCs. These can increase the levels of ROS, which ultimately leads to cell injury and mitochondrial dysfunction. As for the important role of ROS in cell apoptosis, considerable research has been focused on inhibiting ROS production. For example, Guo et al. reported that FSK significantly reduced cisplatin-induced ototoxicity in both HEI-OC1 cells and cochlear explant cultures by inhibiting the mitochondrial apoptotic pathway and ROS production . Shin etIncreased oxidative stress and ROS play an important role in the initiation and progression of hearing loss induced by diverse ototoxic agents or adverse health conditions. Understanding the mechanisms of oxidative stress and ROS signaling may provide therapeutic options for combating hearing loss and identifying new treatment strategies.Numerous types of inner ear damage can cause harm to auditory HCs and ultimately result in hearing loss. Multiple signaling pathways are involved in ototoxicity, noise, aging, and traumatic stress events. The most well studied molecular mechanisms behind cell death in auditory HCs are the MAPK, PI3K/Akt, Notch/Wnt/Atoh1, calcium channel, and oxidative stress/ROS signaling pathways. In this review, we provide a schematic showing how the listed pathways interact within the hair cells , 150–152