After insufficient recovery of the threshold with systemic therapy of ISSHL, intratympanic corticosteroid therapy appears to be associated with a significantly higher chance of an improved hearing threshold than no therapy or placebo. Both, hearing gain and final hearing threshold, however, appear to be independent from the onset of secondary therapy. Based on currently available data from clinical studies, no recommendation can be made with respect to the type of corticosteroid and specifics of the intratympanic application protocol.This article reviews recent aspects of diagnostics, differential diagnostics, and evidence in systemic and local therapy of idiopathic sudden sensorineural hearing loss (ISSHL). Since a number of disorders can be accompanied by sudden hearing loss, a meaningful and targeted diagnostic strategy is of utmost importance. An important differential diagnosis of sudden hearing loss are intralabyrinthine schwannomas (ILS). The incidence of ILS is probably significantly underestimated. This may be due to the lack of awareness or lack of explicit search for an intralabyrinthine tumor on MRI or an inappropriate MRI technique for the evaluation of sudden hearing loss . Therefore, the request to the radiologist should specifically include the question for (or exclusion of) an ILS. With special MRI techniques, it is possibly today to visualize an endolymphatic hydrops. The evidence in the therapy of ISSHL is – with respect to the quality and not quantity of studies – unsatisfying. The value of systemically (low dose) or intratympanically applied corticosteroids in the primary treatment of ISSHL is still unclear. In order to investigate the efficacy and safety of high dose corticosteroids as primary therapy for ISSHL, a national, multicenter, three-armed, randomized, triple-blind controlled clinical trial is currently performed in Germany ( According to the WHO, about 360 million people suffer from hearing impairment worldwide. In the European Union, the number is expected to amount to 434,000 people suffering from deafness and 44,000,000 people suffering from hearing impairment. A recent epidemiological investigation on the hearing status (HÖRSTAT) in Germany revealed a prevalence of hearing loss based on the WHO classification of about 16% ..7].First, causes of sudden hearing loss such as pathologies of the external auditory meatus and the middle ear as well as severe systemic diseases have to be excluded of which acute hearing loss is only an accompanying symptom, as for example cardiovascular emergencies (e.g. hypertensive crisis) or neurological emergencies (e.g. stroke).The identification of pathologies affecting the middle ear or the auditory meatus  is performed by ear microscopy and tuning fork tests.The current version of the German S1 guideline on ISSHL recommends the following elements as necessary diagnostics (translated from: ):“Intensive general and specific history takingENT-specific physical examinationBlood pressure measurementEar microscopyHearing tests Tympanometrypreliminary vestibular testing”Hereby, it appears to be reasonable to precisely define the term of “preliminary vestibular testing” in a revised version of the guideline.In the last version of the German S1 guideline on ISSHL, the following procedures are recommended as useful in individual cases . In this“Otoacoustic emissions (OAE)Auditory evoked brainstem potentials (ABR)Speech audiometryStapedius reflex measurementFunctional examination of the cervical spineLaboratory tests: blood glucose, CRP, procalcitonin, small blood count, differential blood count, creatinine, fibrinogen levelSerologic testing: borreliosis, syphilis, herpes simplex virus type 1, varicella zoster virus, CMV, HIVMRI: exclusion of a tumor of the cerebellopontine angle (hearing protection is recommended)CT scan: skull, temporal bone, cervical spineGlycerol test according to KlockhoffElectrocochleography: cochlear damage, exclusion of hydropsCERA: exclusion of psychogenic deafnessAuditory steady state responses (ASSR)Electronystagmography or video-oculographyDuplex sonographyTympanoscopyInterdisciplinary examinations ”When planning extended diagnostics, noise exposure of the inner ear  should be avoided, if possible, during the first days after acute hearing loss occurred. In cases of acute hearing loss in the context of an acute vestibular syndrome, i.e. if vertigo and/or nystagmus are observed, the following questions should always be clarified in the clinical examination to exclude a stroke (partly translated from ):Is a catch-up saccade observed in the clinical head impulse test for the horizontal vestibulo-ocular reflex (hVOR) and does it correspond to the affected side ?Are eye movement disorders and nystagmus observed?Is skew deviation observed?In 2009, Kattah et al.  introducBrandt et al.  recommenTesting for skew deviationDifferentiation of a peripheral spontaneous nystagmus and a central fixation nystagmus by means of fixation suppression and Frenzel gogglesExamination of a gaze-evoked nystagmus  in the opposite direction of the spontaneous nystagmusExamination of saccadic smooth pursuit Performance of a clinical head impulse test for the hVOR.Thus, the authors achieve also a high sensitivity and specificity in the differential diagnosis of stroke. If HINTS and/or the five procedures suggested by Brandt et al. do not indicate a central cause, a peripheral origin is very probable.Transient-evoked otoacoustic emissions and distortion product otoacoustic emissions are of general relevance for the evaluation of sensory hearing loss . The preTo assess the functional deficit after ISSHL, speech audiometry is more important than pure tone audiometry. Regarding speech audiometry, the Freiburg monosyllabic test in quiet , 17] at at17] atThe measurement of auditory evoked brainstem responses (ABR) provides information on the retrocochlear auditory pathways. Due to their direct neighborhood to the cochlear nerve in the internal auditory meatus, vestibular schwannomas (VS), also referred to as ”acoustic neuromas” (AN), that most frequently have their origin in the superior and inferior vestibular nerves, lead to an impairment of the conduction of action potentials in the auditory nerve. This results in a relatively or absolutely prolonged latency of the ABR waves JIII and JV generated in the brainstem during acoustic stimulation with click stimuli, when the two sides are compared. In order to exclude systematic errors, the latency difference of the waves JIII and JV is measured in relation to the latency of the wave JI generated in the cochlea.Since the late 1970ies, the measurement of the ABR has been established for the diagnosis of VS , 22]. F. F22]. FA meta-analysis evaluated 43 trials of 623 screened studies on the application of ABR for diagnosis of VS – from 1978 to 2009 . Howeverth patient with an actually present VS had the risk of a tumor that was not detected by ABR measurement. On the other hand, a false-positive rate of 18.0% shows that some patients would still undergo unnecessary MRI examination.These data, that are consistent over many trials, show that the ABR measurement has a high selectivity for the diagnosis and follow-up of VS. This also applies when a possible selection bias within the evaluated studies for large tumors and actual surgical intervention is considered. In practice, a false-negative rate of 14.2% means that with an incidence of VS in patients with asymmetric hearing loss of 1–7% , 0.55% oNonetheless, it must be mentioned that also patients that observe complete hearing recovery after ISSHL, should undergo MRI because even complete hearing recovery (spontaneous or for example after systemic corticosteroid administration) does not exclude vestibular schwannoma (acoustic neuroma) , 27]..27].Progress and development of electrophysiological diagnostics may further increase the sensitivity and specificity of VS diagnostics. For example, apart from measurements at supra-threshold levels, also registrations at 40 dB SL can be useful to differentiate cochlear and retrocochlear lesions. For patients with good hearing, those measurements could be the basis of intraoperative monitoring in cases of small VS in order to better evaluate possible level-related changes of the auditory nerve .Both methods, MRI and ABR, have their limitations and cannot be applied in all patients. On the one hand, the number of patients with contraindications or restrictions for MRI examinations continuously increases due to the high number of implants with limited MRI-compatibility. On the other hand, ABR measurements cannot or only to a limited extent be applied in patients with additional conductive hearing loss, because hereby the wave JI cannot be registered. MRI, however, provides information that go beyond the presence of VS which is not the case for ABR measurements. The question of cost efficiency will be answered also depending on different health systems in different countries. Both methods should be available as complementary procedures. This is useful for the preservation of the expertise and allows investigations of all patients despite possible, individually contraindications for one of the two methods.Specific laboratory examinations  may support the differential diagnostics of acute hearing loss. The existing evidence regarding the usefulness of laboratory examinations is based only on case series and small case control studies without sufficiently demonstrating their benefit. Routine examinations, however, are not recommended. In this context, “routine” refers to untargeted tests that are automatically performed or to test batteries without taking into account the specific characteristics of the patient and his concomitant diseases, additional symptoms, or anamnestic and geographic risk factors .Magnetic resonance imaging of the head and the temporal bone with contrast enhancement performed with special attention to the inner ear is generally indicated in all patients showing neuro-otological symptoms, in particular if unilateral and in every patient with acute idiopathic, sensorineural hearing loss. Usually, examination after an interval is sufficient. An immediate diagnostic is rarely indicated and should be performed only when a neurological emergency situation is expected. Even complete recovery of the hearing threshold after ISSHL does not exclude the cause of a pathology that may be diagnosed by MRI , 27]..27].In the context of an own investigation, 198 magnetic resonance images were carefully described that had been performed for diagnostics of patients with acute, sensorineural severe to profound hearing loss (>70 dB HL) or with acute deafness . The variety of pathological findings along or near the auditory pathways confirms the importance that not only vestibular schwannoma has to be excluded in the context of diagnostics of sudden hearing loss, but that the complete auditory pathway from the inner ear to the superior temporal gyrus must be screened by means of magnetic resonance imaging. In this MRI study, 83 patients (41.9%) revealed pathological findings that were related to acute hearing loss. In the other 115 patients (58.1%) with sudden hearing loss, the MRIs were without pathological findings with respect to the auditory pathways. There was no correlation between the severity of sudden hearing loss and the probability of pathological MRI findings . In othePatients with sudden hearing loss, i.e. without any hint for a possible cause of the acute sensorineural hearing loss after history taking and examination, are not recommended to undergo immediate computed tomography for differential diagnosis because of the low significance, the costs, and radiation exposure . If a maTympanoscopy with sealing of the round window membrane  to close a “perilymph fistula” is often advocated as treatment for sudden hearing loss, especially for profound sudden hearing loss or acute deafness (anacusis without measurable hearing threshold) and vertigo. This practice is nearly exclusively based on case series which assumed, that the observed changes of hearing threshold were due to the intervention, although they were lacking comparison with an adequate control group , 34], , , 36], , . In thisEspecially in the context of clinical studies where the surgeons selects the intervention and also interprets the intraoperative findings, randomization (neither the patient nor the therapist/surgeon have an influence on the assignment to the treatment arm) and blinding at least of the endpoint measurements are crucial.In summary, the hypothesis of the efficacy of tympanoscopy with sealing of the round window membrane for treatment of idiopathic sudden hearing loss  can neither be confirmed nor rebutted on the basis of currently available data , 41]. . 41]. There is a high number of diseases that are characterized by the symptom of acute unilateral sensorineural hearing loss Table 3 .Therefore, it is important to look for anamnestic and clinical signs that make a definable cause of acute sensorineural hearing loss (hearing impairment) probable. Those are hints to other otological, or cardio-vascular, endocrinological, metabolic, (auto)immunological, neurological, and other diseases. Whenever a cause for the acute sensorineural hearing loss can be identified or is considered probable, it should not be termed “sudden (idiopathic) hearing loss”; e.g. acute sensorineural hearing loss due to an ipsilateral tumor of the cerebellopontine angle (vestibular schwannoma) should not be called “sudden (idiopathic) hearing loss”. The hearing loss (hearing impairment) should then be further examined and treated based on the guidelines of the identified disease or the “working diagnosis”.Such typical anamnestic data, symptoms, or hints might be for example: sudden bilateral hearing loss (which is rarely idiopathic), recurrent sudden or fluctuating hearing loss, isolated hearing loss in the lower frequencies, simultaneous, bilateral vestibular disorders with oscillopsia, accompanying focal neurological deficits such as pareses, dysarthria, ataxia, encephalopathy, headache, diplopia, vertical or gaze-evoked nystagmus and skew deviation, pathological findings of the central nervous system in the imaging, head trauma with timely relationship to the hearing loss, or ophthalmological symptoms.Bilateral hearing loss that appears suddenly or develops over a very short time has often vascular, metabolic, (auto)immunological, infectious, neoplastic, chemical toxic, or inflammatory causes, even if the bilateral occurrence is not pathognomonic for these causes. Also unilateral symptoms can be observed with those findings. Possible systemic causes for sudden bilateral sensorineural hearing loss must be clarified promptly .Fluctuating hearing loss or hearing loss in the low frequencies, often perceived (and treated) as recurrent sudden hearing loss, allow the assumption of hydropic ear disease of cochlear origin (see chapter on hydropic ear disease) Figure 2  or – if Some differential diagnoses (see Table 3 Vestibular schwannoma (acoustic neuroma), especially also with intralabyrinthine location, is an important and relatively frequent differential diagnosis of sudden hearing loss. In an own patient population, vestibular schwannoma was diagnosed in 8 of 198 (4%) of patients with sudden hearing loss by means of MRI. Although only patients with acute severe to profound hearing loss (mean hearing loss in the frequency ranges of 0.5–4 kHz >70 dB HL) or acute deafness were included in this study, a literature analysis did not show higher rates of vestibular schwannoma compared to patients with less severe idiopathic sudden hearing loss . The incidence of vestibular schwannomas detected by MRI in patients with ISSHL varies in the literature. The mean value of the studies displayed in Table 4 Even if a vestibular schwannoma is present, treatment as for ISSHL, e.g. with systemic corticosteroids, may lead to complete recovery of the hearing threshold after acute (and assumed idiopathic) sudden hearing loss , 27]. . 27]. Because of their location in the inner ear, intralabyrinthine schwannomas (ILS) appear to be a distinct entity and a rare but important differential diagnosis of ISSHL. Nearly all ILS cause an ipsilateral hearing loss as first symptom, which may be progressive, acute, or fluctuating, but usually of sensorineural nature . ILS canAccording to their location, ILS are classified into intracochlear, intravestibular, combined cochlea-vestibular, transmodioloar and transmacular , multilocular, and transotic tumors Figure 3  50], , The incidence of intralabyrinthine schwannomas is probably underestimated. If they are missed, this is often due to the small size at the time of first occurrence of symptoms , due to Requesting an MRI examination for sudden hearing loss should especially encompass the question to the radiologist of excluding an intralabyrinthine schwannoma. Cochlear implantation together with surgical removal of an ILS is an option for auditory rehabilitation in these cases. If ILS are diagnosed early, it might be a promising therapeutic approach in contrast to a wait-and-test-and-scan strategy. Radiotherapy is only indicated in very limited cases, e.g. for tumors with transmodiolar growth and extension into the internal auditory canal in older patients without vertigo , 58]..58].The large vestibular aqueduct syndrome (LVAS) (synonym: LEDS - large endolymphatic duct syndrome or large endolymphatic duct and sac) is observed as an isolated malformation or in combination with other malformations such as for example Mondini malformation, Branchio-Oto-Renal syndrome, or Pendred syndrome. Hearing loss is generally fluctuating and often progresses in stages. Thus, it may be confused with a recurrent (progressive) idiopathic sudden hearing loss. Acute deterioration of hearing is also typical for minor trauma of the head Figure 1 . Even ifHydropic ear disease comprises the spectrum of clinical manifestations of endolymphatic hydrops and must be differentiated from sudden hearing loss. The difference is made between the primary type  and the secondary type . Furthermore, symptoms may be of cochlear, vestibular, or cochlea-vestibular nature. Hence, Menière’s disease with the fully developed symptom triad corresponds to PHED of the cochlea-vestibular type while fluctuating low frequency hearing loss corresponds to PHED of the cochlear type. Also, the Menière-like clinical entity, for example of LVAS or “delayed endolymphatic hydrops” is described by this terminology (SHED). On the other hand, confusing terms such as “atypical Menière’s disease”, “monosymptomatic Menière’s disease”, “cochlear Menière’s disease”, “forme fruste” should be avoided , 46], , . With moThe labyrinthine artery supplying the inner ear is most frequently a branch of the anterior inferior cerebellar artery (AICA). It is the artery of the three cerebellar arteries with the smallest caliber and originates from the basilar artery. Its supply region encompasses a part of the anterior cerebellar hemispheres and the lateral pons. Arteriosclerosis, vascular dissection, or thrombosis may lead to infarctions in the supply area of the AICA. In most of the cases of AICA occlusion, the symptom complex may also include unilateral hearing loss and vestibular disorders  beside other symptoms like Horner’s syndrome, diplopia, facial paresis and dysesthesia, dysarthria, ataxia, nausea and vomiting, and contralateral reduction of pain and temperature sensation in different combinations. Those peripheral cochlea-vestibular disorders may also occur as isolated or prodromal symptoms . Some caA large number of different drugs have been suggested and used for treatment of ISSHL. An extensive overview is found for example in . In the The rational basis for the treatment of acute cochleo-vestibular disorders with corticosteroids is their effect on glucocorticoid and mineral receptors found in the inner ear . The proacute acoustic trauma , 69], , , 71], , ; amino-glycoside and cisplatin ototoxicity , 74]; ; 74]; pneumococcal meningitis ; autoimmune-associated hearing loss , 77]; ; 77]; insertion trauma in cochlear implantation (overview in ).The treatment of sudden hearing loss with systemic standard dose corticosteroid therapy has been investigated in numerous trials of different, mostly low level evidence and significant amount of bias (overview in ). On theThe rational basis for the treatment of idiopathic sudden sensorineural hearing loss (ISSHL) with systemic high-dose steroids are retrospective cohort studies. Alexiou et al. analyzed the audiograms of 603 patients with sudden hearing loss; 301 of them (from 1986 to 1991) did not receive steroids and 302 patients (from 1992 to 1998) were treated with high-dose intravenously administered steroids (prednisolone) . A treatHOch-DOsis-GlukoKORTikoidtherapie)”. Hopefully, the clinical insecurity of the value of high-dose corticosteroid therapy in routine practise will disappear  , , , 88]..88].Based on the current data and due to new knowledge from investigations of combined intratympanic corticosteroid therapy with hyperbaric oxygenation , 90], , , 92], a, a92], aThere are numerous suggestions and attempts of pharmacological therapy of sudden hearing loss based on the hypotheses of its etiology (an overview is found e.g. in ). In sumIn the last two decades, intratympanic therapy of inner ear diseases was placed more and more in the focus of research interests, in particular after the pioneering study by Parnes and co-authors (1999) . ReportsThe advantages of local versus systemic drug application are bypassing the blood-brain barrier; achieving higher drug levels in the inner ear; avoiding “first pass” effects;reduction of undesired systemic effects, and lower quantities of the drugs needed.For local drug application to the inner ear, the advantages are seen especially for i) drugs with a low therapeutic range; ii) drugs with large first-pass effects; iii) drugs with relevant undesired effects outside the ear, and iv) expensive drugs. These aspects apply for example for neurotransmitters and neurotransmitter antagonists, peptides, viral and non-viral gene transfer, and cell-based therapies , 95]. . 95]. In the last years, nearly 200 articles with case reports, case series without control groups, retrospective cohort studies, and also about 20 randomized controlled trials (RCTs) on therapy of sudden hearing loss with intratympanic glucocorticoid application were published. Despite the large number of publications on this topic, only relatively uncertain conclusions regarding the efficacy of intratympanic corticosteroids for sudden hearing loss may be drawn on the basis of the current data. Even carefully described patient cohorts with improvements of hearing threshold do not allow conclusions regarding the efficacy without providing data from a control group that underwent either no or another treatment . The detailed analysis of the about 20 RCTs , reveals that the quality of most of these RTCs is disappointing and the risk of bias is high. Various systematic review articles and meta-analyses dealt with the different therapeutic strategies of intratympanic corticosteroid application for sudden hearing loss , 102], , 102], , 104], , 104], , 106], , 106], , 108]. . 108]. BRegarding the primary therapy of sudden hearing loss, systemic (low-/medium-dose) and intratympanic glucocorticoid therapy seem to be equally effective  , 103], , 103], . StudiesIn cases of insufficient recovery of hearing after systemic therapy, current data suggest that intratympanic glucocorticoid therapy as secondary treatment is associated with a significantly higher probability of an improved hearing threshold , 103], , 103], , 106]..106].Primary combined therapy (intratympanic and systemic therapy) seems to be superior to primary systemic glucocorticoid therapy. However, the effect is clearly lower than the one for intratympanic secondary (reserve) therapy and the risk of bias of those trials is very high . In a meSummarizing the results of randomized as well as non-randomized trials and comparing intratympanic, systemic, and combined therapy, no significant difference was found between the different treatment strategies, neither regarding primary nor secondary therapy Figure 5 . In addiFor secondary therapy, hearing improvement seems to be independent from treatment onset (2–4 weeks after sudden hearing loss or 4–6 weeks afterwards) . If the final absolute hearing threshold is chosen as an outcome parameter – a parameter, which seems to be of higher importance for the patients than the change of hearing threshold, the relevance of which depends from the initial hearing loss – the final pure tone hearing threshold seems to be completely independent from the onset of treatment. This applies to primary as well as secondary therapy, at least for the available data with treatment onset within approximately 2 months after ISSHL Figure 6  107], , Regarding primary intratympanic, primary combined, and secondary intratympanic therapy of sudden hearing loss with glucocorticoids, no correlation of individual parameters of the application protocol could be observed so far  , 111]. . 111]. MThe staged approach of treating sudden hearing loss currently applied in the author’s department is presented in Figure 7 As primary therapy, systemic high-dose prednisolone administration is performed for 5 days (prednisolone i.v. 250 mg/d). Only in cases of contraindication for systemic high-dose prednisolone therapy , a primary intratympanic therapy is performed. In very rare cases of suspected rupture of the round window membrane, e.g. in the context of barotrauma or mechanical trauma, tympanoscopy with exploration and sealing of the window is performed with triamcinolone acetonide soaked connective tissue.As secondary therapy, “blind” (i.e. without inspection of the round window niche) transtympanic injection of dexamethasone-phosphate solution  is applied. After injection, the patients are asked to lay on the opposite side for 30 minutes.® sponges soaked with triamcinolone acetonide are inserted into the niches of the round and oval windows  will probably play a major role in the future. They allow targeted and controlled release of substances over a predefined time period outside or inside the cochlea .http://hodokort-studie.hno.org/) , the audiogram of the contralateral side, or the age- and gender-specific normal audiogram (DIN ISO 7029). As frequency range for the main outcome parameter, the 3 frequencies are considered that are most affected by the hearing loss, as already suggested in other studies , 124]. . 124]. FAnother problem of clinical studies on sudden hearing loss is that for mild and moderate hearing losses, improvements of hearing threshold achieved with or without treatment cannot be statistically differentiated. Hence, only the inclusion of patients with a certain minimum hearing loss is currently recommended for clinical studies on ISSHL , 120], , 120], , 127]..127].Up to now, there is no international consensus in terms of measuring treatment success. The pure tone hearing threshold, however, is currently the internationally most standardized parameter. In most of the trials on sudden hearing loss or other inner ear diseases, the change of hearing threshold is reported. Even current high-quality randomized clinical trials on sudden hearing loss apply this parameter. Speech understanding (percentage of the correctly understood monosyllables of the Freiburg test at 65 and 80 dB SPL), but especially speech understanding in noise even better reflect how a patient may communicate in daily life. Nevertheless, it is not easy to compare the results of speech audiometries of different languages. In addition, they are influenced by other parameters such as speech competence and cognitive factors.There are different possibilities to classify improvement of hearing based on the changes of pure tone or speech reception threshold. Internationally, however, no consensus could be found on when hearing improvement can be considered as helpful or meaningful. For example, hearing aids are used for a broad range of hearing thresholds. Furthermore, a current American guideline emphasized that defined grades of improvement may be associated with different levels of benefit for the individual patient .Specific patient-related and patient reported outcome parameters on the quality of life such as for example the patient’s self-assessment of the general health-related quality of life  and the assessment of the subjective hearing handicap (score of the HHIE questionnaire) or other standardized and validated QoL instruments should be involved more frequently in the future.diagnostics. Patients with acute unilateral cochlea-vestibular symptoms should undergo appropriate MRI examination  to assess peripheral (inner ear) and retro-cochlear/central causes according to the recommendations of the German Radiological Society . Requesting an MRI examination to clarify the causes of sudden hearing loss, it is recommened to ask explicitly for the exclusion of an intralabyrinthine schwannoma. New developments of imaging diagnostics improve the possibilities of differential diagnostics, e.g. the diagnosis of endolymphatic hydrops.Even from a prognostic point of view, sudden hearing loss is not an emergency that has to be treated immediately . This retherapy of sudden hearing loss is very unsatisfactory – regarding the quality, not the quantity of trials. There is an existing rational basis for the treatment of acute cochlea-vestibular disorders with corticosteroids. The value of systemically applied corticosteroids in the initial treatment of sudden hearing loss  remains unclear. Regarding primary therapy of sudden hearing loss, systemic corticosteroids (low/medium dose) and intratympanic corticosteroids seem to be equally effective . Up to now there is no sufficient evidence for the efficacy of primary systemic high-dose glucocorticoid therapy for sudden hearing loss as recommended in the current German AWMF guideline. To answer this question, a multicenter clinical trial entitled “Efficacy and safety of high dose glucocorticosteroid treatment for idiopathic sudden sensorineural hearing loss – a three-armed, randomized, triple-blind, multicenter trial (HODOKORT)” is performed supported by the German Study Center of Otolaryngology, Head & Neck Surgery (http://hodokort-studie.hno.org/). The study situation with respect to the Primary combined therapy (intratympanic and low-/medium dose systemic application) seems to be superior to primary low-dose systemic glucocorticoid therapy. However, the effect is clearly lower compared to intratympanic secondary (reserve) therapy (see below) and the risk of bias in these trials is very high. Data on the efficacy of a primary combined therapy compared to primary systemic high-dose prednisolone therapy are not available.The tendency of increased hearing improvement after early onset of primary therapy of sudden hearing loss (intratympanic or combined) can be considered a sham effect because this is most likely to be due to spontaneous recovery.secondary therapy is associated with a significantly higher probability of an improved hearing. Hereby, the hearing improvement seems to be independent from the onset of therapy (2–4 weeks after sudden hearing loss or 4–6 weeks afterwards). Therefore, the necessity of early onset of intratympanic secondary therapy is not supported on the basis of the currently available data. In case of insufficient recovery of hearing after systemic therapy of sudden hearing loss, current data suggest that intratympanic glucocorticoid therapy as In the context of primary intratympanic, primary combined , and secondary intratympanic therapy of sudden hearing loss with glucocorticoids, the therapeutic success does not seem to depend on individual parameters of the application protocol  so that currently no recommendation for a specific therapy protocol of intratympanic therapy can be given.This work was partially done within the project KS2013-190 supported by the Federal Ministry of Education and Science in Germany . The author is head of the scientific advisory board of AudioCure Pharma GmbH, Berlin, Germany. The authors institution receives research grant support from MedEl, Austria and MedEl, Germany. The author received honorary for lectures in this topic from the ENT-Physician’s organisation in Germany and Infectopharm, Heppenheim, Germany. The author received travel support, e.g. for lectures from Cochlear Deutschland GmbH & Co. KG. The author received a major research grant from the Federal Ministry of Education and Science in Germany (BMBF: KS2013-190). The author also received honorary for lectures or session moderations not related to this topic by Merck Serono, Darmstadt, Germany.


Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker’s yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA. In sexually reproducing organisms, crossing over between homologous chromosomes in meiosis creates physical linkages required to segregate chromosomes into haploid gametes. In baker’s yeast, which utilizes meiotic recombination pathways conserved in mice and humans, the majority of meiotic crossovers are initiated through the formation of a branched DNA intermediate, which is stabilized by the Msh4-Msh5 complex. This DNA intermediate is further processed to form a structure (a double Holliday junction), which requires the endonuclease activity of the Mlh1-Mlh3 DNA mismatch repair factor to be resolved exclusively into a crossover product. Current meiotic recombination models invoke the use of structure-specific enzymes that symmetrically cleave single Holliday junctions. In this study, we provide evidence that the yeast Mlh1-Mlh3 complex is unlikely to act as a structure-specific enzyme. Furthermore, we showed that Mlh1-Mlh3’s endonuclease activity is dependent upon its ability to form a polymer on DNA and suggest that it is capable of cleaving DNA that is captured in an active complex. Together, our biochemical observations support a novel model involving regulated polymerization of Mlh1-Mlh3 for its cleavage function, potentially in meiotic crossovers or in mismatch repair. Escherichia coli, DNA mismatches are initially recognized by MutS, which recruits MutL in an ATP-dependent reaction to activate MutH. MutH functions as a latent endonuclease that nicks the newly replicated and undermethylated DNA strand up to several kilobases away from the mismatch. The resultant nick, 5′ or 3′ to the mismatch, directs excision and resynthesis steps that remove the misincorporated DNA –44Sf9 ced ~50 nM for loop mismatch; Kd ~200 nM for homoduplex) ) or to the primer complementary to the opposing strand (primer B [AO3518]). Template annealed to the radiolabeled primer was then isolated using S-300 HR spin columns (GE) and incubated with 2.5 mM dNTPs and 2,000 U of T4 DNA polymerase (NEB). Primer extension products were analyzed by 15%, 8 M urea PAGE and phosphorimaging. Radiolabeled oligonucleotide AO3535 was used as a marker for migration of a 60-mer primer extension product.pUC18 plasmid was either nicked with 2, 40 μg/mL BSA, and 0.1 mM DTT. Upon the addition of Mlh1-Mlh3, reactions were incubated for 10 min at 30°C. The reaction was then filtered through KOH-treated nitrocellulose filters using a Hoefer FH225V filtration device for approximately 1 min. Filters were subsequently analyzed by scintillation counting.DNA filter binding assays were performed essentially as described . Briefly-/Å2. Movies were collected with a defocus range of –1 to –2.5 microns and a nominal magnification of 11,500x, which produced images with a calibrated pixel size of 3.15Å. The 30 frames in each move were aligned using the program alignframesleastsquares_list [https://sites.google.com/site/rubinsteingroup/home). These programs perform whole frame alignment of the movies using previously published motion correction algorithms [Mixtures of Mlh1-Mlh3 alone or in combination with either circular or linear DNA were applied at the concentrations stated in the Results onto EM grids freshly coated with a continuous layer of amorphous carbon. Grids were floated on a 5 μL drop of the diluted assembly reaction for 2 min immediately after a glow discharge treatment of 5 mA for 15 s. Excess of sample was blotted with filter paper and the grids were stained with 1% uranyl acetate for 1 min. Grids were loaded in a room-temperature holder and introduced into a FEI Tecnai F20 electron microscope operated at 200 kV and equipped with a Gatan K2 Summit direct detector camera. This detector was used in counting movie mode with five electrons per pixel per second for 15-s exposures and 0.5 s per frame. This method produced movies consisting of 30 frames with an exposure rate of ~1 eres_list  and averres_list . These pgorithms . A denoiBsaHI and BsrGI at 37°C for 60 min. The digested sample was then resolved by 4%, 8 M urea PAGE and phosphorimaged.For experiments in ScaI, HindIII, or used as a substrate in an Mlh1-Mlh3 endonuclease reaction containing 300 nM Mlh1-Mlh3 in quintuplicate. The linearized product was then gel isolated from these reactions and replicates were combined. Where + T4 polymerase is indicated, in a 10 μL reaction, ~1 μg of linearized DNA was combined with 100 μM dNTPs in NEB buffer 2.1. One unit of T4 DNA polymerase (NEB) was added to the reaction. The reaction was incubated for 15 min at 12°C followed by a heat inactivation step at 75°C for 20 min. An 8 μL portion of the reaction was then added to a 10 μL reaction containing the supplied buffer and 400 units of T4 DNA ligase (NEB) where indicated. The reaction was incubated at room temperature for 2 h followed by heat inactivation per the manufacturer’s instructions.The closed circular form of pEAE99 was initially gel isolated and treated with For all plotted data, individual data points from each trial as well as means and standard deviations are found in S1 FigLeft, SDS-PAGE analysis of treatment of 1.2 μM Mlh1-Mlh3 with 200 U lambda (λ) protein phosphatase (NEB) protein phosphatase. Where - Mlh1-Mlh3 is indicated, a mock treatment was performed omitting Mlh1-Mlh3 and combining with DNA in the endonuclease reaction to assess any background nicking from increasing amounts of the phosphatase treatment conditions. Where - phosphatase is indicated, Mlh1-Mlh3 was subjected to treatment with the phosphatase buffer, but MnCl2 and phosphatase were omitted. + phosphatase is the full reaction including phosphatase and Mlh1-Mlh3. (B) Average of quantification for four separate experiments, errors bars represent standard deviation. (C) Agarose gel analyzing nicking on 2.7 kb circular substrate following treatment of 1.2 μM Mlh1-Mlh3 with 50 U CDK1-cyclinB (NEB) (see (A) NEB) see . Where -NEB) see . Where -(TIFF)Click here for additional data file.S2 FigHindIII linearized pUC18 (red) in the presence of 1 mM MnSO4. (B) Quantification of data in A; fraction nicked defined as fraction of substrate lost plotted against yeast Mlh1-Mlh3 concentration. (C) Native agarose gel electrophoresis analysis of yeast Mlh1-Mlh3 (150 nM) endonuclease activity on circular substrate ranging from 2.7 kb to 12 kb. The concentration of nucleotide in each reaction is 15 μM. Lane 1 contains 2-log DNA ladder (NEB). Where + Mn2+is indicated, 1 mM MnSO4 was added to the reaction. (D) Quantification of nicking in lanes 4, 7, 10, and 13 in C averaged from two separate experiments. Error bars indicate standard deviation.(A) Denaturing agarose analysis of yeast Mlh1-Mlh3 nicking on circular pUC18 (2.7 kb) (black) or (TIFF)Click here for additional data file.S3 FigHindIII, followed by treatment with T4 DNA ligase (NEB). Ligated product and supercoiled pUC18 were resolved in one-half of an agarose gel not containing ethidium bromide. In the other half of the gel, Nt.BstNBI-digested pUC18 and supercoiled pUC18 were resolved as markers. After running, the gel was cut and the half containing the markers was stained with ethidium bromide. The relaxed circlular and supercoiled circluar substrates were extracted from the unstained half using the stained half as a guide. Endonuclease reactions were assembled and carried out as described in the Materials and Methods using either supercoiled or relaxed circular DNA as a substrate. Left, where + Mlh1-Mlh3 is indicated, reactions contain 300 nM Mlh1-Mlh3. In lanes 4–7 and 10–13, Mlh1-Mlh3 is 50, 150, 300, and 500 nM, respectively. Right, quantification of agarose gel.Relaxed circular pUC18 plasmid was prepared by linearization with (TIFF)Click here for additional data file.S4 FigBsaHI and BsrGI (2.3 kb fragment) for analysis by denaturing PAGE. (C) Left, total nicking on circular substrate measured by agarose gel analysis. Right, 8 M urea PAGE analysis of Mlh1-Mlh3 nicking ± Msh2-Msh3. Amount of radioactive probe was quantified and the fraction of substrate nicked was calculated as amount of radioactivity lost compared to the negative controls . < indicates that the amount of substrate lost was less than 3%. Approximate position of 150 nucleotide migration is indicated.(A) Endonuclease activity performed with 25 nM wild-type or D523N Mlh1-Mlh3 in the presence of 60 nM Msh2-Msh3 and/or 0.5 mM ATP where indicated. (B) Schematic describing mapping assay. Radiolabeled substrate combined with 40 nM Msh2-Msh3 and 20 nM Mlh1-Mlh3. After endonuclease activity was stopped, substrate was cleaved with (TIFF)Click here for additional data file.S5 Fig2+ unless otherwise indicated. (A) 15 μM total nucleotide in each reaction combined with 150 nM human MLH1-PMS2 in the presence of RFC/PCNA with ATP. Experiment is otherwise identical to that performed in Materials and Methods. 1.4 kb circular substrate was generated by re-ligating the ~1400 bp BssSαI fragment of pUC18. 15 kb circular substrate is plasmid pEAM58 amplified and mini-prepped from DH5α competent cells by standard methods. Reactions were combined as described in A and in Materials and Methods and analyzed by agarose gel. (F) Average of quantification for four separate experiments from E, errors bars represent standard deviation. (G) hMLH1-PMS2 (300 nM) does not create linear product on 2.7 kb closed or nicked circular substrate. Reaction was performed as described for All reactions contain 1 μM yeast RFC and yeast PCNA, 0.5 mM ATP, and 1 mM Mn(TIFF)Click here for additional data file.S6 Fig(A-B) Experiment performed identical to that in (TIFF)Click here for additional data file.S7 FigEcoRI(E111Q) is a variant of EcoRI that binds to, but does not cleave, the EcoRI recognition sequence [EcoRI site) in the Mlh1-Mlh3 endonuclease reaction buffer and incubated for 30 min at 37°C. Binding was confirmed by gel shift. EcoRI(E111Q)-bound substrate or substrate without EcoRI(E111Q) was then combined in an endonuclease reaction  with increasing amounts of Mlh1-Mlh3. The reaction was combined, allowed to proceed, and stopped as described in the Materials and Methods. Reactions were analyzed by agarose gel and quantified.sequence . In a 5 (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


However, there was no hearing restoration or intact hair cell in the D7 group. GV1001 protected against cochlear hair cell damage, and furthermore, delayed administration of GV1001 up to 3 days rescued hair cell damage and hearing loss in KM/furosemide-induced deaf mouse model.The cell-penetrating peptide GV1001 has been investigated as an anticancer agent and recently demonstrated anti-oxidant and anti-inflammatory effects. It has shown a protective effect on a kanamycin (KM)-induced ototoxicity mouse model. In the present study, we administered GV1001 at different time points after inducing hair cell damage, and examined if it rescues hair cell loss and restores hearing. A deaf mouse model was created by intraperitoneal injection of KM and furosemide. First, to test the early temporal change of hearing and extent of hair cell damage after KM and furosemide injection, hearing and outer hair cells (OHCs) morphology were evaluated on day 1, day 2 and day 3 after injection. In the second experiment, following KM and furosemide injection, GV1001, dexamethasone, or saline were given for three consecutive days at different time points: D0 group , D1 group , D3 group  and D7 group . The hearing thresholds were measured at 8, 16, and 32 kHz before ototoxic insult, and 7 days and 14 days after KM and furosemide injection. After 14 days, each turn of the cochlea was imaged to evaluate OHCs damage. GV1001-treated mice showed significantly less hearing loss and OHCs damage than the saline control group in the D0, D1 and D3 groups ( Aminoglycoside antibiotics are used in the treatment of gram-negative bacterial infections, multi-drug resistant tuberculosis, and other hospital acquired serious infections. Dose-limiting side effects include cochlear and/or vestibular toxicity and nephrotoxicity. Aminoglycosides result in hair cell death by either caspase-dependent or -independent mechanisms  derived from the active site of human telomerase reverse transcriptase (hTERT), has been investigated as an anticancer agent. GV1001 has been used against advanced pancreatic cancer, melanoma, non-small cell lung cancer, advanced hepatocellular carcinoma, cutaneous T-cell lymphoma and B-cell chronic lymphocytic leukemia. As an anticancer agent, GV1001 binds multiple human leukocyte antigen (HLA) class II molecules and elicits combined CD4 and CD8 T-cell responses , Day-2 (N = 3) and Day-3 (N = 3). After injection of KM and furosemide on day 0, hearing loss and cochlear hair cell damage were evaluated on day 1, day 2 and day 3, respectively (Supplementary file S1).A deaf mouse model  was created by intraperitoneal injection of KM (1000 mg/kg) followed by furosemide (100 mg/kg) within 30 min. The study protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (BA-1504-174-017). In Experiment 1, to assess the initial temporal change of hearing and the extent of hair cell damage in this deaf mouse model, total nine mice were divided into three groups: Day-1 (N = 40), dexamethasone (N = 40) and saline (N = 40). GV1001 , dexamethasone (15 mg/kg), or saline was subcutaneously administered for three consecutive days after the injection of KM and furosemide. To compare the rescue effect of GV1001 on different time points, each group was divided into four subgroups according to the time points of GV1001, dexamethasone, and saline treatment: D0 group , D1 group , D3 group , and D7 group .In Experiment 2, to test the rescue effect of GV1001, total 120 mice were divided into the following three treatment groups: GV1001 (All of the mice underwent an auditory brainstem response (ABR) test  under intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) after inhalation of isoflurane. During the ABR test, a heating pad was applied to maintain body temperature. Tone burst  stimuli at 8, 16 and 32 kHz were delivered to the external auditory meatus through plastic earphones connected to an EC1 electrostatic speaker. Subdermal needle electrodes were applied behind the ipsilateral mastoid (reference electrode), behind the contralateral mastoid (active electrode), and on the vertex (ground electrode).The evoked responses were amplified, and 1024 sweeps were averaged in real time. To acquire auditory thresholds, the sound intensity of the tone burst stimuli was lowered by 10 dB intervals from 90 dB SPL. The auditory threshold was defined as the lowest sound intensity at which the most robust and stable component was evoked around 4 ms , and examined using confocal microscopy  under uniform 63× magnification for all imaging analyses.Under anesthesia, venous blood was obtained before cardiac perfusion with PBS, followed by 4% paraformaldehyde (pH 7.4), and the cochlea and kidney were immersion-fixed  was evaluated for the presence or absence of hair cells. OHCs were considered missing if there was a gap in the normal hair cell array or if there were no apparent stereocilia or cuticular plates. The number of morphologically intact OHCs was counted manually and averaged in five different fields. The percentage of morphologically intact OHCs, defined as “intact OHCs rate”, was calculated at each cochlear turn (apex/middle/base).® Critical Care Xpress . The operating range for BUN was 6–102 mg/dL; the normal range of BUN in mice is 13–35 mg/dL  values prior to trans-cardiac perfusion with saline and 4% paraformaldehyde fixative (pH 7.4). The BUN values were measured using Stat ProfileRenal tubules were harvested and imaged by optical microscopy after hematoxylin and eosin (H&E) staining. The morphology was quantified as 0 , 1 , 2 , 3 , and 4  and STATA v. 14.0 for Windows software . The Kruskal-Wallis H test with Bonferroni correction were performed to compare continuous variables among the three groups and four subgroups: From the Experiment 1 designed to assess the initial temporal change of hearing and the extent of hair cell damage Figure , the heap < 0.0167).In the Experiment 2 designed to test the rescue effect of GV1001 Figure , the heap = 0.007), 16 kHz (p = 0.006), and 32 kHz (p = 0.001) at 1 week, and 8 kHz (p = 0.013), 16 kHz (p = 0.008), and 32 kHz (p = 0.003) at 2 weeks. In the D1 group, the significant difference between the GV1001 and saline control group showed at one points: 32 kHz (p = 0.014) at 2 weeks. In the D3 group, the significant difference between the GV1001 and saline control group showed at three points: 8 kHz (p = 0.006) at 1 week, and 8 kHz (p = 0.005) and 16 kHz (p = 0.001) at 2 weeks. On the other hand, a statistically significant difference to the saline control group, in the hearing thresholds of the dexamethasone group, was seen at two points in the only D0 group: 8 kHz (p = 0.007) and 32 kHz (p = 0.012) at 2 weeks. There was no statistically significant difference in the hearing thresholds between the GV1001 and dexamethasone group. In the D7 group, the hearing thresholds of all three groups were increased to nearly 100 dB SPL (complete hearing loss).Difference in the hearing thresholds between the GV1001 and saline control group was statistically significant at all six points in the D0 group: 8 kHz , 16 kHz (p = 0.001), and 32 kHz (p = 0.001) at 1 week, and 16 kHz (p = 0.002) at 2 weeks, were significantly different. In comparison between D1 and D7 group, hearing thresholds at three points, 32 kHz (p = 0.003) at baseline, and 8 kHz (p = 0.004) and 32 kHz (p = 0.003) at 2 weeks, showed statistically significant difference. Moreover, in comparison between D3 and D7 group, hearing thresholds at one points, 8 kHz (p = 0.007) at 2 weeks, showed statistically significant difference. However, we found no significant difference in the hearing thresholds at any treatment time points among the D0, D1 and D3 groups (Supplementary file S4).In the GV1001 group, when compared the hearing thresholds among the D0, D1, D3 and D7 groups, the hearing thresholds of the D0, D1 and D3 groups were found to be statistical significantly different vs. that of the D7 group (p < 0.0167). In the D0 group , middle (p = 0.002), and base (p = 0.003). In the D1 group  and basal turn (p = 0.007). In the D3 group . On the other hand, dexamethasone-treated mice showed a statistically significant intact OHCs morphology than the saline group at each cochlear turn only in the D0 group: apex (p = 0.010), middle (p = 0.009), and base (p = 0.002). However, the OHCs and hearing could not be rescued by dexamethasone or GV1001 when treated 7 days after injection of KM and furosemide : apex (p = 0.005), middle (p = 0.003), and base  mg/dL, and these values were within the normal range for BUN values in mice (8–33 mg/dL). Moreover, the renal tubules of all GV1001, dexamethasone, and saline treated mice showed normal morphology in the H&E staining. Thus, the administration of 10 mg/kg GV1001 was safe in mice and did not cause nephrotoxicity (Supplementary file S4).In the present study, cell penetrating peptide GV1001 was tested if it rescues hair cell loss and restores hearing after inducing hair cell damage using KM/furosemide-induced deaf mouse model. We also tested if there is a therapeutic window to reverse hair cell damage by investigating agents at different time points of D0, D1, D3 and D7. The KM/furosemide-induced ototoxicity mouse model was ideal for this experiment since substantial hearing loss was documented on the next day of KM and furosemide injection, and complete loss of OHCs was seen on the second day of injection. GV1001-treated mice showed significantly less hearing loss and less hair cell damage than the saline control group in the D0, D1 and D3 subgroups. Therefore, delayed administration of GV1001 up to 3 days could rescue hearing loss and hair cell damage, while hearing loss was irreversible if it was given 7 days after ototoxic insult in KM/furosemide-induced deaf mouse model.The potential otoprotective effects of several antioxidants have been tested in animal models. Iron chelators such as desferoxamine and dihydroxybenzoate  with dexamethasone (15 mg/kg) and saline by measuring the hearing thresholds and OHCs morphology. When compared with saline, the rescue effect of GV1001 was proven in all D0, D1 and D3 groups. As shown in Figures A previous study investigated the effect of different dexamethasone treatment times on OHCs survival following ototoxic insult with KM and furosemide. Mice pre-treated with dexamethasone (prior to 1 h of the insult) showed a statistically significant improvement in intact OHCs counts compared with the controls, but the mice subjected to dexamethasone post-treatment  showed highly variable OHCs counts (Fernandes and Lin, The preventive effect of GV1001 pre-treatment against cochlear hearing loss and hair cell damage in a KM-induced deaf mouse model was verified in our previous study. As shown in Figure In conclusion, we demonstrated that the cell-penetrating peptide GV1001 rescues hair cell damage and restores hearing in a KM/furosemide-induced deaf mouse model. Given that the rescue effect of GV1001 could be related to its anti-inflammatory, anti-oxidant, and anti-apoptotic activities, we predict that GV1001 could be useful not only in aminoglycoside-induced hearing loss, but also in acute cochlear hearing loss due to other causes. Because SNHL is caused by an imbalance in redox homeostasis and subsequent increase in ROS followed by the apoptotic pathway, GV1001 may have a potential clinical role in restoring hearing in cases of acute SNHL.SHK analyzed data and wrote the manuscript. GJ performed the animal experiments and wrote the manuscript. SK provided technical assistance and wrote the manuscript. JWK designed the study and revised the manuscript. All authors read and approved the final 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.


Auditory neuropathy is a significant and understudied cause of human hearing loss, diagnosed in patients who demonstrate abnormal function of the cochlear nerve despite typical function of sensory cells. Because the human inner ear cannot be visualized during life, histopathological analysis of autopsy specimens is critical to understanding the cellular mechanisms underlying this pathology. Here we present statistical models of severe primary neuronal degeneration and its relationship to pure tone audiometric thresholds and word recognition scores in comparison to age-matched control patients, spanning every decade of life. Analysis of 30 ears from 23 patients shows that severe neuronal loss correlates with elevated audiometric thresholds and poor word recognition. For each ten percent increase in total neuronal loss, average thresholds across patients at each audiometric test frequency increase by 6.0 dB hearing level (HL). As neuronal loss increases, threshold elevation proceeds more rapidly in low audiometric test frequencies than in high frequencies. Pure tone average closely agrees with word recognition scores in the case of severe neural pathology. Histopathologic study of the human inner ear continues to emphasize the need for non- or minimally invasive clinical tools capable of establishing cellular-level diagnoses. Damage to the auditory nerve (AN), via demyelination or loss of synapses, axons, or neuronal cell bodies in the spiral ganglion, can result in imprecise temporal coding and a disruption in firing synchrony, yielding a faulty representation of the input signal3. In Starr’s patients, (1) the auditory brainstem response (ABR), an electrophysiological measurement consisting of several peaks, the first of which reflects the summed activity of the AN, was absent or severely distorted; (2) auditory brainstem reflexes, such as the stapedius muscle reflex, were absent; and (3) speech intelligibility scores were disproportionately poorer than expected based on audiometric threshold measurements, though (4) typical measures of outer hair cell (OHC) function, such as otoacoustic emissions and cochlear microphonic potentials, remained within normal limits3. These criteria remain the diagnostic hallmarks of auditory neuropathy, though further evaluation via electrocochleography and advanced tests of neural function can refine a diagnosis4.In 1996, Arnold Starr and colleagues coined the term “auditory neuropathy” as a means of classifying patients who demonstrated aberrant responses to assessments of AN function despite normal responses to assessments of sensory cell function5, and among healthy infants, routine screening identifies AN dysfunction in 1 in every 70002. This phenotype has been documented to accompany an array of medical conditions, ranging from developmental dystrophies and genetic mutations6 to acquired neuropathies, such as those induced by neoplasm, infection, exposure to noise or drugs, or the deleterious effects of age8.Recent studies suggest that auditory neuropathy is a more prevalent form of hearing loss than initially assumed. Among preterm infants, 1 in every 423 graduates of the newborn intensive care unit meets Starr’s criteria for auditory neuropathy9. Studies of human patients with auditory neuropathy demonstrate that pure tone audiometric thresholds associated with this phenotype can vary from near-normal to highly elevated4. Such variation is likely due to the diverse etiology associated with this phenotype. For example, though the synapse between the spiral ganglion neuron (SGN) and inner hair cell (IHC) is the most vulnerable element in the inner ear, human and animal studies historically suggest that damage to the AN and its terminals may not be reflected in traditional measurements of hearing thresholds13. Indeed, since the 1950s, diffuse damage to the AN without damage to hair cells has been assumed to have little effect on pure tone thresholds14.In adults, the prevalence of auditory neuropathy is likely underestimated, because the most widely used diagnostic tool in audiology, the pure tone audiogram, is not capable of predicting cell-type specific damage in the inner ear8. To date, five independent studies of temporal bone histopathology in specific forms of hereditary AN dysfunction have been undertaken, all of which involved single patients or small cohorts (n < 5)18. The consistent finding among these studies was a severe loss of SGNs despite the presence of sensory cell populations within normal limits for age. To place results like these in the context of normal presbycusis, the expected age-related decline in SGNs over the human lifespan, Makary et al. charted SGN cell counts from 100 individuals with no known cochlear pathology, generating baseline values for primary neuronal loss against which pathologic degeneration can be compared19.Because there is currently no way to biopsy or visualize cellular-level structures of the inner ear in living humans, histopathological study of human temporal bones has formed the basis of scientific understanding for numerous auditory pathologies. In 2001, Nadol reviewed primary cochlear neuronal degeneration in individual temporal bones associated with genetic, toxic, immunologic, degenerative, idiopathic, and infectious causes; however, no attempt at collective quantitative analysis was madeTo date, there has been no histological study of pathologic neural degeneration spanning various etiologies. Here, we present hypothesis-driven statistical models of neuronal loss in severe neural degeneration and its relationship to pure tone audiometric thresholds and word recognition scores among 30 ears from 23 patients, spanning every decade of life. Understanding the natural progression of auditory neural degeneration holds important implications for patient counseling and will prove integral for the development of future therapies, as no existing treatment can ameliorate this phenotype.Representative cochlear photomicrographs demonstrate the abnormal histopathology characteristic of primary neural degeneration. Sectioning the human cochlea parallel to the modiolus, or conical central axis, reveals Rosenthal’s canal, a spiraling bony channel within the two and a half turns of the cochlea which houses SGN cell bodies. Myelinated peripheral axons fan out from SGNs in this canal, reaching through the osseous spiral lamina to innervate hair cells embedded in the organ of Corti, while central axons project from Rosenthal’s canal to the cochlear nucleus in the brainstem. In humans with no known cochlear pathology, like the 71-year-old male presented in Fig. 20. Here, SGN cell bodies in 51 ears from 34 patients identified as candidates for inclusion in this study were quantified and compared with the age-based mean for normal SGN cell death expected for decade of life, published by Makary et al.19 . Given only ten observations, hair cell loss could not be reliably controlled for in the existing model. However, nontrivial threshold elevation in Segment I, even in the case of neuronal loss no different than is typical for age, suggests the contribution of other cell types to the observed high-frequency hearing loss.Segment I corresponds with the cochlear base, a region particularly vulnerable to myriad forms of damage throughout the lifetimeIn 30 ears from 23 patients with severe neural degeneration, a linear mixed effects regression model was used to correlate audiometric thresholds at each of six individual audiometric test frequencies with total neuronal loss as a percentage of the mean expected for age Fig. . For eacA significant increase in hearing thresholds was observed as neuronal loss increased over all six audiometric test frequencies (p < 0.001). As observed in Fig. Word recognition scores, reported as a percentage of correctly repeated words after being read a word list in quiet, were available for 15 of 30 ears of interest (12 patients). Word recognition among these patients declined significantly as total neuronal loss increased Fig. . Linear 25. In fact, on a clinical audiogram, normal hearing thresholds at frequencies up to and including 2 kHz are covered by the belly of a down-sloping area colloquially referred to as the “speech banana”27. Among low-frequency observations, mean hearing thresholds increase by 6.8 dB HL per 10% total neuronal loss, while among high-frequency observations, mean hearing thresholds increase by 4.8 dB HL per 10% total neuronal loss. The difference in rate of threshold increase between low-frequency and high-frequency observations is statistically significant (p = 0.007). As in Fig. To further explore the observation that elevated thresholds at high frequencies may reflect the involvement of factors outside neuronal pathology Fig. , thresho28. A linear mixed effects regression model was used to describe the relationship between pure tone average and total neuronal loss as a percentage of the age-based mean in normal presbycusis , defined as the average of the two or three lowest thresholds at audiometric test frequencies between 0.5–2 kHz, is used in the otology clinic as a reliable approximation of speech reception and speech detection thresholdsWe present the largest quantitative study to date of severe neural degeneration in the human inner ear. As cellular structures within the cochlea cannot be biopsied or visualized during life, approaching auditory neuropathy from a histopathological perspective provides important evidence for the cellular substrates of clinical assessments. Our model suggests that mean hearing thresholds in ears with severe primary neural degeneration increase by 6.0 dB HL per 10% total neuronal loss Fig. . This is11. Schuknecht and Woellner, who pierced the auditory nerves of cats with needles to damage up to 50% of AN fibers, observed only mild effects of this trauma on a behavioral audiogram29. More recently, Lobarinas et al. used carboplatin to induce large amounts of IHC death in trained chinchillas, observing aberrant responses in the behavioral audiogram only when IHC loss exceeded 80%30. However, significant threshold elevation is known to accompany multiple forms of auditory neuropathy regardless of hair cell loss in humans, including cochlear nerve hypoplasia, a diagnosis of gradable severity thought to represent most cases of unilateral auditory neuropathy in humans7.However, though the positive relationship between these variables is clear, a simple linear increase in hearing thresholds is not well explained by animal models of excitotoxic, physical, or drug-induced neuropathy. For example, after noise exposure in mice, Kujawa and Liberman observed a temporary elevation of audiometric thresholds followed by a return to normal values, despite widespread synaptopathy31. Though the defining characteristic of this patient population is severe neural degeneration, diseases thought to be primarily neurodegenerative often affect the function of and communication between neighboring supporting or interconnected cells that form corresponding parts of a less-than-functional system32.Importantly, despite the fact that the hair cells of patients presented in this study are morphologically intact and present in numbers normal for age, no evidence exists as to whether or not these cells were functional in life. It is well known that functional and chemical changes can long precede morphologic changes in the cochlea7. A classic complaint from such a patient is that they “can hear, but cannot understand,” and this claim is documented in the written medical records of at least two patients included in this study. For these patients, perceiving speech in noisy environments can be a nearly insurmountable task, perhaps due to loss of SGNs with low spontaneous firing rates and high sound thresholds, which confer important dynamic range to the response patterns of the auditory nerve and are selectively lost as a result of traumatic noise exposure33. In our model, the slope of the regression line representing decline in word recognition with increasing neuronal loss is −0.68  were documented to have a population of IHCs and OHCs normal for age as verified by light microscopy, and a qualitatively abnormal population of spiral ganglion neurons, which we subsequently quantified. Cytocochleograms were available for ten of 30 patients.All human temporal bones analyzed for this study were stored in the archival collection at Massachusetts Eye and Ear and handled according to protocols approved by the associated Human Studies Committee. Informed consent regarding participation in the National Temporal Bone Registry was obtained from all individuals prior to death. Sample collection and human specimen storage was conducted in accordance with Massachusetts Eye and Ear protocols and the Helsinki Declaration. Temporal bones were fixed in 10% neutral buffered formalin or Heidenhain Susa solution, decalcified in ethylenediaminetetraacetic acid, embedded in celloidin, and serially sectioned (20 μm) according to published protocols43. In every tenth section of each cochlear turn, SGNs in which a nucleus was visible were manually counted. Total neuronal counts per cochlea were then estimated by multiplying SGN counts by 10 to account for intervening sections, and by 0.91 to correct for dual-counting of nuclei spanning section boundaries, in accordance with the most recent recommendations44. Totals were compared to published reference values for total cochlear neuronal populations observed in age-matched patients with typical age-related cochlear neuronal degeneration19. Bones with total neuronal counts more than one standard deviation below the expected mean for normal, age-related cochlear neuronal degeneration (30 ears from 23 patients) were included in subsequent linear regression models. Otologic diagnoses recorded for these patients varied  was used. Word recognition scores, recorded as a percentage of correctly repeated words after being read a word list in quiet, were available for 15 of 30 ears (12 patients) of interest.The most recent clinical audiogram recorded prior to the death of each patient, comprising pure tone detection thresholds at 250, 500, 1000, 2000, 4000, and 8000 Hz, was included in regression models. Pure tone average (PTA), defined at our institution as the average of the two lowest pure tone thresholds at test frequencies between 0.5 and 2 kHz19, in total and after dividing the cochlear spiral into four segments of relatively equal length. All models included controls for linear age, sex, and time interval between each patient’s most recent audiogram and date of death. For omnibus tests of differences in slope, conditional F-tests with Kenward-Rogers correction for degrees of freedom were performed. Computations were carried out and graphs generated in R45. For all analyses, p < 0.05 was considered statistically significant.Linear mixed effects regression models were used to estimate the relationship between audiometric test frequencies and neuronal damage as a percentage of the published age-based meanThe temporal bones and patient records analyzed in the current study are publicly available to trained researchers and can be accessed through the National Temporal Bone, Hearing, and Balance Pathology Resource Registry at Massachusetts Eye and Ear, established by the National Institute on Deafness and Other Communication Disorders (NIDCD) of the National Institutes of Health (NIH).Supplementary PDF File


To determine whether hearing loss is associated with incident physician‐diagnosed dementia in a representative sample.Retrospective cohort study.English Longitudinal Study of Ageing.Adults aged 50 and older.Cross‐sectional associations between self‐reported  and objective hearing measures  and dementia were examined using multinomial‐logistic regression. The longitudinal association between self‐reported hearing at Wave 2 (2004/05) and cumulative physician‐diagnosed dementia up to Wave 7 (2014/15) was modelled using Cox proportional hazards regression.OR = 1.6, 95% CI = 1.1–2.4 moderate hearing; OR = 2.6, 95% CI = 1.7–3.9 poor hearing, objective: OR = 1.6, 95% CI = 1.0–2.8 moderate hearing; OR = 4.4, 95% CI = 1.9–9.9 poor hearing). Longitudinally, the hazard of developing dementia was 1.4 (95% CI = 1.0–1.9) times as high in individuals who reported moderate hearing and 1.6 (95% CI = 1.1–2.0) times as high in those who reported poor hearing.After adjustment for potential confounders, in cross‐sectional analysis, participants who had self‐reported or objective moderate and poor hearing were more likely to have a dementia diagnosis than those with normal hearing (self‐reported: odds ratio Older adults with hearing loss are at greater risk of dementia than those with normal hearing. These findings are consistent with the rationale that correction of hearing loss could help delay the onset of dementia, or that hearing loss itself could serve as a risk indicator for cognitive decline. The global estimate of the number of individuals living with dementia was 46.8 million in 2015, with approximately 800,000 residing in the United Kingdom and 676,000 living in England.A number of modifiable risk factors for dementia have been identified, including social interactions, physical activity, and type 2 diabetes mellitus.Previous longitudinal epidemiological studies conducted in the United States and Wales have provided evidence that hearing loss is independently associated with dementia,Data were used from a cohort of men and women aged 50 and older from the English Longitudinal Study of Ageing (ELSA),A three‐way assessment protocol was used to define dementia as described previously.Participants were asked to rate their hearing from 1 (excellent) to 5 (poor) in Waves 1 to 7.A hearing screening device  was used to obtain objective hearing scores for participants at Wave 7 (2014/15). This device has been validated and was used in the Health Survey for England in 2014.Age was classified into four categories . Economic status was defined using quintiles of non‐pension wealth  as calculated by the Institute for Fiscal Studies.a priori that age, sex, ethnicity, wealth, education, and hearing aid use were possible confounders.The sociodemographic and clinical risk profiles were summarized according to self‐reported  and objective  hearing categories. Chi‐square tests were performed to ascertain whether there were significant differences in the distribution of sociodemographic and clinical categories between hearing groups. For the cross‐sectional analyses of self‐reported and objective hearing impairment, odds ratios and 95% confidence intervals of diagnosed dementia at Wave 7 (2014/15) were calculated, with normal hearing as the reference group. Separate analyses were conducted on self‐reported and objective hearing impairment. On the basis of the existing literature, it was decided Cox proportional hazards regression was used to model the association between self‐reported hearing  and cumulative diagnosed dementia (July 2005 to June 2015). The Health Survey for England has calculated cross‐sectional and longitudinal weighting to adjust for non‐response bias.Individuals who had been diagnosed with dementia in Wave 2 (2004/05) were excluded. Time to dementia was measured in years from the beginning of Wave 2 (2004/05). Date of dementia diagnosis was used if known; if not known, the midpoint date between waves of data collection was used. Individuals who were known to have died or left the study were censored. Mortality data for ELSA were available up to February 2013. If an individual dropped out of the study between waves, the last interview date was used for the censor date. The Schoenfeld residual test was used to test the proportional hazards assumption of the models.To examine whether the self‐report and objective measures had independent effects on risk of dementia, both measures were included in one model. Individuals who wore hearing aids were excluded from the sensitivity analysis because the self‐report measure was based on hearing aid use, and the objective measure was not. Finally, to avoid misinterpreting the results of communication difficulties as a sign of cognitive impairment, a sensitivity analysis that omitted individuals who fit the IQCODE cut‐off criteria but did not have a physician diagnosis of dementia were included.All data were analyzed using STATA version 14 .Ninety‐five percent  of participants in Wave 7 rated their hearing, with 23.1%  reporting poor and 34.7%  moderate hearing difficulties. Self‐reported hearing difficulties were associated with older age, male sex, lower wealth and education, hearing aid use, history of stroke, and diabetes or hypertension  of the participants had a hearing screening test in Wave 7 Table. Fewer iDementia was associated with worse self‐reported and objective hearing Table . After aOf the 8,780 core members in Wave 2, 22% n = 1,933) reported poor hearing, and 31.6%  moderate hearing difficulties. Self‐reported hearing difficulty was associated with older age, male sex, lower wealth and education, hearing aid use, hypertension, and history of stroke (Table 3 reporteThere were 269 incident cases of diagnosed dementia between Wave 2 and the end of Wave 7 (June 2015). During the mean follow‐up of 11 years, individuals in the moderate (hazard ratio (HR) = 1.39, 95% CI = 1.01–1.92) and poor  hearing groups were at greater risk of developing dementia than the normal hearing group. Older age and diabetes mellitus also emerged as significant independent risk factors Table .κ = 0.262, 95% CI = 0.257–0.269). The association between objective hearing and physician‐diagnosed dementia remained significant for the poor hearing group when including self‐reported hearing in the model. In contrast, the association between self‐reported hearing difficulties weakened and became non‐significant. The associations did not change when individuals who used a hearing aid were excluded. When individuals with an IQCODE greater than 3.5 only were omitted from the model, the odds ratios were smaller but remained significant.There was fair agreement between the objective and self‐reported hearing measures . Longitudinal analysis over an 11‐year period showed that the incidence was 39% higher in individuals with moderate self‐reported hearing and 57% higher in those with poor self‐reported hearing than in those with normal hearing after adjusting for multiple covariates.The Health and Retirement Study, which has a profile similar to that of ELSA, also examined self‐reported hearing and dementia cross‐sectionally and found that 44% of participants who reported fair to poor hearing had probable dementia.The current study built on previous longitudinal studies conducted in the United States and Wales that found that individuals with moderate and severe hearing loss were at greater risk of developing dementia.The previous studies of objective hearing loss used a pure tone audiometry test, whereas the current study used self‐reported hearing measures in the longitudinal analyses.A unique strength of using ELSA is that it involves a large national sample of resident English men and women aged 50 and older. The dataset includes repeated measures of chronic conditions, so it was possible to capture cumulative physician‐diagnosed dementia cases and analyze time to dementia diagnosis. The dataset also had measures of self‐reported and objective hearing measures at Wave 7 that could be compared in the presence of other potential covariate measures.There are also several limitations to these analyses. First, there were fewer individuals with dementia in this study than in population estimates,Second, only self‐reported hearing measures were available in ELSA for longitudinal analysis; objective measures would be desirable. Comparison of subjective and objective measures of hearing loss was also challenging, because only self‐reported hearing was based on hearing aid use, although the two measures had a significant positive association. In addition, use of a hearing aid was included as a confounder in the analysis.Finally, categorizations of subjective and objective hearing measures do not match directly because they are based on rather different criteria.Although longitudinal associations between self‐reported hearing loss and future dementia were identified, it is not certain that the associations are causal. Major confounders such as age, education, and cardiovascular risk factors were adjusted for statistically, but there could be residual confounding by unmeasured variables. In addition, dementia develops over many years, so pathological processes may have been initiated even before baseline measures of hearing. Nevertheless, there are three possible mechanisms by which hearing loss could cause dementia; cognitive burden, changes in brain structure and function, and social isolation. First, cognitive burden or the “effortfulness hypothesis” has been demonstrated in older adults (66–81).Second, lack of sensory input and difficulty processing may lead to changes in brain structure and function.Finally, lack of interaction and intellectual stimulation has been associated with dementia in prospective studies.Is hearing loss an indicator of early stages of dementia and a preventable risk factor, or is dementia an indicator of hearing loss? There are opposing arguments regarding the direction of the association between hearing and dementia, but either pathway could have major public health implications.In the United Kingdom, approximately 4 million people with hearing impairment delay seeking medical help.Hearing aids could help with treatment of hearing loss and possibly decrease social isolation, although they need to be acceptable and effective. The quality of hearing aids seems to have improved, and 70% of older adults reported being fairly satisfied with their hearing aids in the Health Survey for England.This study supports previous literature that has found that older adults with hearing loss have a higher rate of developing dementia. It also found that hearing aid use had a protective effect in cross‐sectional analysis. The findings suggest that treatment of hearing loss with hearing aids could help delay the onset of dementia. The public health implications are substantial because more than 3 million U.K. adults aged 50 and older have hearing loss. Further studies are needed to confirm the possible biological and social mechanisms involved, and a large prospective study is needed to examine treatment of hearing loss.Table S1. Descriptive statistics of dementia and objective hearing testClick here for additional data file.