Delayed hearing loss after cochlear implantation: Re-evaluating the role of hair cell degeneration.

Delayed loss of residual acoustic hearing after cochlear implantation is a common but poorly understood phenomenon due to the scarcity of relevant temporal bone tissues. Prior histopathological analysis of one case of post-implantation hearing loss suggested there were no interaural differences in hair cell or neural degeneration to explain the profound loss of low-frequency hearing on the implanted side (Quesnel et al., 2016) and attributed the threshold elevation to neo-ossification and fibrosis around the implant. Here we re-evaluated the histopathology in this case, applying immunostaining and improved microscopic techniques for differentiating surviving hair cells from supporting cells. The new analysis revealed dramatic interaural differences, with a > 80 % loss of inner hair cells in the cochlear apex on the implanted side, which can account for the post-implantation loss of residual hearing. Apical degeneration of the stria further contributed to threshold elevation on the implanted side. In contrast, spiral ganglion cell survival was reduced in the region of the electrode on the implanted side, but apical counts in the two ears were similar to that seen in age-matched unimplanted control ears. Almost none of the surviving auditory neurons retained peripheral axons throughout the basal half of the cochlea. Relevance to cochlear implant performance is discussed.

Neural Degeneration in Normal-Aging Human Cochleas: Machine-Learning Counts and 3D Mapping in Archival Sections.

Quantifying the survival patterns of spiral ganglion cells (SGCs), the cell bodies of auditory-nerve fibers, is critical to studies of sensorineural hearing loss, especially in human temporal bones. The classic method of manual counting is tedious, and, although stereology approaches can be faster, they can only be used to estimate total cell numbers per cochlea. Here, a machine-learning algorithm that automatically identifies, counts, and maps the SGCs in digitized images of semi-serial human temporal-bone sections not only speeds the analysis, with no loss of accuracy, but also allows 3D visualization of the SGCs and fine-grained mapping to cochlear frequency. Applying the algorithm to 62 normal-aging human ears shows significantly faster degeneration of SGCs in the basal than the apical half of the cochlea. Comparison to fiber counts in the same ears shows that the fraction of surviving SGCs lacking a peripheral axon steadily increases with age, reaching more than 50% in the apical cochlea and almost 66% in basal regions.

Predicting Atrophy of the Cochlear Stria Vascularis from the Shape of the Threshold Audiogram.

Several lines of evidence have suggested that steeply sloping audiometric losses are caused by hair cell degeneration, while flat audiometric losses are caused by strial atrophy, but this concept has never been rigorously tested in human specimens. Here, we systematically compare audiograms and cochlear histopathology in 160 human cases from the archival collection of celloidin-embedded temporal bones at the Massachusetts Eye and Ear. The dataset included 106 cases from a prior study of normal-aging ears, and an additional 54 cases selected by combing the database for flat audiograms. Audiogram shapes were classified algorithmically into five groups according to the relation between flatness (i.e., SD of hearing levels across all frequencies) and low-frequency pure-tone average (i.e., mean at 0.25, 0.5, and 1.0 kHz). Outer and inner hair cell losses, neural degeneration, and strial atrophy were all quantified as a function of cochlear location in each case. Results showed that strial atrophy was worse in the apical than the basal half of the cochlea and was worse in females than in males. The degree of strial atrophy was uncorrelated with audiogram flatness. Apical atrophy was correlated with low-frequency thresholds and basal atrophy with high-frequency thresholds, and the former correlation was higher. However, a multivariable regression with all histopathological measures as predictors and audiometric thresholds as the outcome showed that strial atrophy was a significant predictor of threshold shift only in the low-frequency region, and, even there, the contribution of outer hair cell damage was larger.SIGNIFICANCE STATEMENT Cochlear pathology can only be assessed postmortem; thus, human cochlear histopathology is critical to our understanding of the mechanisms of hearing loss. Dogma holds that relative damage to sensory cells, which transduce mechanical vibration into electrical signals, versus the stria vascularis, the cellular battery that powers transduction, can be inferred by the shape of the audiogram, that is, down-sloping (hair cell damage) versus flat (strial atrophy). Here we quantified hair cell and strial atrophy in 160 human specimens to show that it is the degree of low-frequency hearing loss, rather than the audiogram slope, that predicts strial atrophy. Results are critical to the design of clinical trials for hearing-loss therapeutics, as current drugs target only hair cell, not strial, regeneration.

Incomplete Partition Type II Cochlear Malformations: Delineating the Three-Dimensional Structure from Digitized Human Histopathological Specimens.

HYPOTHESIS: There are clinically relevant differences in scalae anatomy and spiral ganglion neuron (SGN) quantity between incomplete partition type II (IP-II) and normal cochleae. BACKGROUND: IP-II is a commonly implanted cochlear malformation. Detailed knowledge of intracochlear three-dimensional (3D) morphology may assist with cochlear implant (CI) electrode selection/design and enable optimization of audiologic programming based on SGN maps. METHODS: IP-II (n = 11) human temporal bone histological specimens were identified from the National Institute on Deafness and Other Communication Disorders National Temporal Bone Registry and digitized. The cochlear duct, scalae, and surgically relevant anatomy were reconstructed in 3D. A machine learning algorithm was applied to map the location and number of SGNs. RESULTS: 3D scalae morphology of the basal turn was normal. Scala tympani (ST) remained isolated for 540 degrees before fusing with scala vestibuli. Mean ST volume reduced below 1 mm 2 after the first 340 degrees. Scala media was a distinct endolymphatic compartment throughout; mean ± standard deviation cochlear duct length was 28 ± 3 mm. SGNs were reduced compared with age-matched norms (mean, 48%; range, 5-90%). In some cases, SGNs failed to ascend Rosenthal's canal, remaining in an abnormal basalward modiolar location. Two forms of IP-II were seen: type A and type B. A majority (98-100%) of SGNs were located in the basal modiolus in type B IP-II, compared with 76 to 85% in type A. CONCLUSION: Hallmark features of IP-II cochleae include the following: 1) fusion of the ST and scala vestibuli at a mean of 540 degrees, 2) highly variable and overall reduced SGN quantity compared with normative controls, and 3) abnormal SGN distribution with cell bodies failing to ascend Rosenthal's canal.

Supporting-cell vs. hair-cell survival in the human cochlea: Implications for regenerative therapies.

Animal studies have shown that the supporting-cells surviving in the organ of Corti after cochlear insult can be transdifferentiated into hair cells as a treatment for sensorineural hearing loss. Clinical trials of small-molecule therapeutics have been undertaken, but little is known about how to predict the pattern and degree of supporting-cell survival based on audiogram, hearing loss etiology or any other metric obtainable pre-mortem. To address this, we systematically assessed supporting-cell and hair cell survival, as a function of cochlear location in 274 temporal bone cases from the archives at the Massachusetts Eye and Ear and compared the histopathology with the audiograms and hearing-loss etiologies. Results showed that supporting-cell survival was always significantly greater in the apical half than the basal half of the cochlea, that inner pillars were more robust than outer pillars or Deiters' cells, and that total replacement of all supporting cells with a flat epithelium was rare outside of the extreme basal 20% of the cochlea. Supporting cell survival in the basal half of the cochlea was better correlated with the slope of the audiogram than with the mean high-frequency threshold per se: i.e. survival was better with flatter audiograms than with steeply down-sloping audiograms. Cochlear regions with extensive hair cell loss and exceptional supporting cell survival were most common in cases with hearing loss due to ototoxic drugs. Such cases also tended to have less pathology in other functionally critical structures, i.e. spiral ganglion neurons and the stria vascularis.

Supporting-cell vs. hair-cell survival in the human cochlea: Implications for regenerative therapies.

Animal studies have shown that the supporting-cells surviving in the organ of Corti after cochlear insult can be transdifferentiated into hair cells as a treatment for sensorineural hearing loss. Clinical trials of small-molecule therapeutics have been undertaken, but little is known about how to predict the pattern and degree of supporting-cell survival based on audiogram, hearing loss etiology or any other metric obtainable pre-mortem. To address this, we systematically assessed supporting-cell and hair cell survival, as a function of cochlear location in 274 temporal bone cases from the archives at the Massachusetts Eye and Ear and compared the histopathology with the audiograms and hearing-loss etiologies. Results showed that supporting-cell survival was always significantly greater in the apical half than the basal half of the cochlea, that inner pillars were more robust than outer pillars or Deiters' cells, and that total replacement of all supporting cells with a flat epithelium was rare outside of the extreme basal 20% of the cochlea. Supporting cell survival in the basal half of the cochlea was better correlated with the slope of the audiogram than with the mean high-frequency threshold per se: i.e. survival was better with flatter audiograms than with steeply down-sloping audiograms. Cochlear regions with extensive hair cell loss and exceptional supporting cell survival were most common in cases with hearing loss due to ototoxic drugs. Such cases also tended to have less pathology in other functionally critical structures, i.e. spiral ganglion neurons and the stria vascularis. Highlights: Supporting cell survival was systematically assessed in 274 human cochleasSupporting cell survival was better with flat than with down-sloping audiogramsSupporting cell survival was most robust when hearing loss was from ototoxic drugsOtotoxic cases also showed less pathology in other critical cochlear structuresThe data can inform clinical trials for regeneration via supporting cell conversion.

Computed Tomography Density as a Bio-marker for Histologic Grade of Otosclerosis: A Human Temporal Bone Pathology Study.

HYPOTHESIS: Computed tomography (CT) density measurement can be used to objectively distinguish otosclerosis from normal bone and to determine histologic grades of otosclerosis. BACKGROUND: Otosclerosis can be seen on CT as subtle radiolucent areas. An objective radiologic measurement that corresponds to known otosclerosis pathology may improve diagnostic accuracy, and could be used as a radiologic biomarker for otosclerosis grade. METHODS: A blinded, randomized evaluation of both histologic grade on histopathology slides and CT density measurement was performed on 78 human temporal bone specimens (31 with otosclerosis and 47 controls) that had undergone high-resolution multi-detector CT before histologic processing. Assessments were performed at 11 regions of interest (ROIs) in the otic capsule for each specimen. RESULTS: The CT density measurement mean (Hounsfield Units) ± standard deviation for all ROIs (Nos. 1-9) was 2245 ± 854 for grade 0 (no otosclerosis, n = 711), 1896 ± 317 for grade 1 (inactive otosclerosis, n = 109), and 1632 ± 255 for grades 2 and 3 combined (mixed/active otosclerosis, n 35). There was a strong inverse correlation of CT density to histologic grade at ROIs Nos. 1-5 (ANOVA, p < 0.0001). The inter-rater reliability for CT density was very good (correlation coefficient 0.87, p < 0.05). ROC curves suggested a cut-off of 2,150HU to distinguish otosclerosis from normal bone, and 1,811HU to distinguish low grade from mixed/high grade otosclerosis. CONCLUSIONS: In human temporal bone specimens, CT density may be used to distinguish normal bone from bone involved by otosclerosis. A higher histologic grade (i.e., indicating a more active otosclerotic focus) correlated with lower density.

Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina.

Hearing loss in patients with vestibular schwannoma (VS) is commonly attributed to mechanical compression of the auditory nerve, though recent studies suggest that this retrocochlear pathology may be augmented by cochlear damage. Although VS-associated loss of inner hair cells, outer hair cells, and spiral ganglion cells has been reported, it is unclear to what extent auditory-nerve peripheral axons are damaged in VS patients. Understanding the degree of damage VSs cause to auditory nerve fibers (ANFs) is important for accurately modeling clinical outcomes of cochlear implantation, which is a therapeutic option to rehabilitate hearing in VS-affected ears. A retrospective analysis of human temporal-bone histopathology was performed on archival specimens from the Massachusetts Eye and Ear collection. Seven patients met our inclusion criteria based on the presence of sporadic, unilateral, untreated VS. Tangential sections of five cochlear regions were stained with hematoxylin and eosin, and adjacent sections were stained to visualize myelinated ANFs and efferent fibers. Following confocal microscopy, peripheral axons of ANFs within the osseous spiral lamina were quantified manually, where feasible, and with a "pixel counting" method, applicable to all sections. ANF density was substantially reduced on the VS side compared to the unaffected contralateral side. In the upper basal turn, a significant difference between the VS side and unaffected contralateral side was found using both counting methods, corresponding to the region tuned to 2000 Hz. Even spiral ganglion cells (SGCs) contralateral to VS were affected by the tumor as the majority of contralateral SGC counts were below average for age. This observation provides histological insight into the clinical observation that unilateral vestibular schwannomas pose a long-term risk of progression of hearing loss in the contralateral ear as well. Our pixel counting method for ANF quantification in the osseous spiral lamina is applicable to other pathologies involving sensorineural hearing loss. Future research is needed to classify ANFs into morphological categories, accurately predict their electrical properties, and use this knowledge to inform optimal cochlear implant programming strategies.

Prevalence of Macrophages Within the Cochlear Vessels Following Cochlear Implantation in the Human: An Immunohistopathological Study Using Anti-Iba1 Antibody.

HYPOTHESIS: The prevalence of monocyte-derived macrophages within cochlear vessels may increase following cochlear implantation. BACKGROUND: Recently, we reported an increase in the number of ionized calcium-binding adaptor molecule 1 (Iba1)-positive macrophages in selected cochlear sites such as the osseous spiral lamina and Rosenthal's canal following cochlear implantation. Activation of the immune system induces the recruitment of monocyte-derived macrophages. The prevalence of monocyte-derived macrophages within cochlear vessels may increase following cochlear implantation. However, the delivery system of macrophages to the human cochlea is incompletely understood. METHODS: The prevalence of macrophages and monocytes within cochlear blood vessels in 10 human subjects who had undergone unilateral cochlear implantation was studied by light microscopy using anti-Iba1 immunostaining. The densities of Iba1-positve monocytes per area of lumen of cochlear vessels in the sections near the round window in implanted ears were compared with the contralateral unimplanted ears. The correlation between the densities of Iba1-positve monocytes and the duration (months after the cochlear implantation) was also evaluated. RESULTS: The prevalence of Iba1-positive macrophages/monocytes in vessels near the round window in implanted ears (mean 26%, median 21%) was greater than in opposite unimplanted ears (mean 5.2%, median 2.5%: p < 0.01). The density of Iba1-positive monocytes in implanted ears (mean 32, median 16 cells/105 µm2) tended to be greater than that in unimplanted ears (mean 6.6, median 0.93 cells/105 µm2: p = 0.08). The density of Iba1-positive monocytes was significantly correlated with duration of implantation but not in the unimplanted ears. CONCLUSION: An increase in prevalence of Iba1-positive macrophages/monocytes within cochlear blood vessels after cochlear implantation was demonstrated. These findings suggest a delivery system of Iba1-positive macrophages through cochlear vessels in human that is ongoing for long duration.

Primary Neural Degeneration in Noise-Exposed Human Cochleas: Correlations with Outer Hair Cell Loss and Word-Discrimination Scores.

Animal studies suggest that cochlear nerve degeneration precedes sensory cell degeneration in both noise-induced hearing loss (NIHL) and age-related hearing loss (ARHL), producing a hearing impairment that is not reflected in audiometric thresholds. Here, we investigated the histopathology of human ARHL and NIHL by comparing loss of auditory nerve fibers (ANFs), cochlear hair cells and the stria vascularis in a group of 52 cases with noise-exposure history against an age-matched control group. Although strial atrophy increased with age, there was no effect of noise history. Outer hair cell (OHC) loss also increased with age throughout the cochlea but was unaffected by noise history in the low-frequency region (<2 kHz), while greatly exacerbated at high frequencies (≥2 kHz). Inner hair cell (IHC) loss was primarily seen at high frequencies but was unaffected by noise at either low or high frequencies. ANF loss was substantial at all cochlear frequencies and was exacerbated by noise throughout. According to a multivariable regression model, this loss of neural channels contributes to poor word discrimination among those with similar audiometric threshold losses. The histopathological patterns observed also suggest that, whereas the low-frequency OHC loss may be an unavoidable consequence of aging, the high-frequency loss, which produces the classic down-sloping audiogram of ARHL, may be partially because of avoidable ear abuse, even among those without a documented history of acoustic overexposure.SIGNIFICANCE STATEMENT As regenerative therapeutics in sensorineural hearing loss enter clinical trials, it becomes critical to infer which cochlear pathologies are present in addition to hair cell loss. Here, by analyzing human autopsy material, we show that acoustic injury accelerates age-related primary neural degeneration, but not strial degeneration, neither of which can be inferred from audiometric thresholds. It exacerbates outer hair cell (OHC) loss only in the high-frequency half of the cochlea, suggesting that the apical loss is age-related, whereas the basal loss is partially noise induced, and therefore avoidable. Statistical analysis suggests that neural loss helps explain differences in word-recognition ability among individuals with similar audiometric thresholds. The surprising correlation between neural loss and OHC loss in the cochlea's speech region also implicates neural loss in the well-known decline in word scores as thresholds deteriorate with age.

Age-Related Hearing Loss Is Dominated by Damage to Inner Ear Sensory Cells, Not the Cellular Battery That Powers Them.

Age-related hearing loss arises from irreversible damage in the inner ear, where sound is transduced into electrical signals. Prior human studies suggested that sensory-cell loss is rarely the cause; correspondingly, animal work has implicated the stria vascularis, the cellular "battery" driving the amplification of sound by hair cell "motors." Here, quantitative microscopic analysis of hair cells, auditory nerve fibers, and strial tissues in 120 human inner ears obtained at autopsy, most of whom had recent audiograms in their medical records, shows that the degree of hearing loss is well predicted from the amount of hair cell loss and that inclusion of strial damage does not improve the prediction. Although many aging ears showed significant strial degeneration throughout the cochlea, our statistical models suggest that, by the time strial tissues are lost, hair cell death is so extensive that the loss of battery is no longer important to pure-tone thresholds and that audiogram slope is not diagnostic for strial degeneration. These data comprise the first quantitative survey of hair cell death in normal-aging human cochleas, and reveal unexpectedly severe hair cell loss in low-frequency cochlear regions, and dramatically greater loss in high-frequency regions than seen in any aging animal model. Comparison of normal-aging ears to an age-matched group with acoustic-overexposure history suggests that a lifetime of acoustic overexposure is to blame.SIGNIFICANCE STATEMENT This report upends dogma about the causes of age-related hearing loss. Our analysis of over 120 autopsy specimens shows that inner-ear sensory cell loss can largely explain the audiometric patterns in aging, with minimal contribution from the stria vascularis, the "battery" that powers the inner ear, previously viewed as the major locus of age-related hearing dysfunction. Predicting inner ear damage from the audiogram is critical, now that clinical trials of therapeutics designed to regrow hair cells are underway. Our data also show that hair cell degeneration in aging humans is dramatically worse than that in aging animals, suggesting that the high-frequency hearing losses that define human presbycusis reflect avoidable contributions of chronic ear abuse to which aging animals are not exposed.

Anatomy of the Human Osseous Spiral Lamina and Cochlear Partition Bridge: Relevance for Cochlear Partition Motion.

The classic view of cochlear partition (CP) motion, generalized to be for all mammals, was derived from basal-turn measurements in laboratory animals. Recently, we reported motion of the human CP in the cochlear base that differs substantially from the classic view. We described a human soft tissue "bridge" (non-existent in the classic view) between the osseous spiral lamina (OSL) and basilar membrane (BM), and showed how OSL and bridge move in response to sound. Here, we detail relevant human anatomy to better understand the relationship between form and function. The bridge and BM have similar widths that increase linearly from base to apex, whereas the OSL width decreases from base to apex, leading to an approximately constant total CP width throughout the cochlea. The bony three-dimensional OSL microstructure, reconstructed from unconventionally thin, 2-µm histological sections, revealed thin, radially wide OSL plates with pores that vary in size, extent, and distribution with cochlear location. Polarized light microscopy revealed collagen fibers in the BM that spread out medially through the bridge to connect to the OSL. The long width and porosity of the OSL may explain its considerable bending flexibility. The similarity of BM and bridge widths along the cochlea, both containing continuous collagen fibers, may make them a functional unit and allow maximum CP motion near the bridge-BM boundary, as recently described. These anatomical findings may help us better understand the motion of the structures surrounding the organ of Corti and how they shape the input to the cochlear sensory mechanism.

Assessing fractional hair cell survival in archival human temporal bones.

OBJECTIVES/HYPOTHESIS: Histopathological analysis of hair cell survival in human temporal bone sections has historically been binarized such that each hair cell row is rated as either present or absent, thereby greatly underestimating the amount of hair cell loss. Here, we describe and validate a technique to reliably assess fractional hair cell survival in archival sections stained with hematoxylin and eosin (H&E) using high-resolution light microscopy and optical sectioning. STUDY DESIGN: Technique validation. METHODS: Hair cell counts in archival temporal bone slide sets were performed by several observers using either differential interference contrast (DIC) or confocal microscopy of the endogenous eosin fluorescence in hair cells. As a further cross-check, additional decelloidinized sections were immunostained with hair cell markers myosin VI and VIIa. RESULTS: Cuticular plates and stereocilia bundles are routinely resolvable in DIC imaging of archival H&E-stained human material using standard research-grade microscopes, allowing highly accurate counts of fractional hair cell survival that are reproducible across observer and can be verified by confocal microscopy. CONCLUSIONS: Reanalysis of cases from the classic temporal bone literature on presbycusis suggests that, contrary to prior reports, differences in audiometric patterns may be well explained by the patterns of hair cell loss. LEVEL OF EVIDENCE: NA Laryngoscope, 130:487-495, 2020.

The Distribution and Prevalence of Macrophages in the Cochlea Following Cochlear Implantation in the Human: An Immunohistochemical Study Using Anti-Iba1 Antibody.

HYPOTHESIS: Cochlear implantation may cause an increase in the number of macrophages in the human cochlea similar to previous findings in the vestibular endorgans. BACKGROUND: Macrophages play a key role in both an inflammatory response and homeostatic maintenance. Recently, an increase in the prevalence of macrophages was demonstrated in the human vestibular endorgans after implantation. However, the prevalence of macrophages in the cochlea after implantation is unclear. The aim of this study was to compare the distribution and prevalence of macrophages in implanted human cochleae and the contralateral unimplanted ears. METHODS: The prevalence of macrophages in the cochlea in 10 human subjects who had undergone unilateral cochlear implantation was studied by light microscopy using anti-Iba1 immunostaining. The densities of macrophages in the osseous spiral lamina (OSL) and Rosenthal's canal (RC) in implanted cochleae were compared with the contralateral unimplanted ears. The distribution of macrophage morphology (amoeboid, transitional, and ramified) was also compared. RESULTS: There were activated and phagocytosing macrophages within the fibrotic sheath surrounding the electrode track and within fibrous tissue with lymphocytic infiltration in implanted ears. The densities of macrophages in OSL and RC in implanted ears were significantly greater than in unimplanted ears in some areas. There was also a difference in the prevalence of macrophage phenotype between the OSL and RC. CONCLUSION: An increase in the density of macrophages in the cochlea after cochlear implantation was demonstrated. Both phagocytosis and anti-inflammatory activity of macrophages were suggested by the distribution and prevalence of macrophages in the implanted cochlea.

Density of Macrophages Immunostained With Anti-iba1 Antibody in the Vestibular Endorgans After Cochlear Implantation in the Human.

HYPOTHESIS: Cochlear implantation may result in an increase in the density of macrophages in vestibular endorgans in the human. BACKGROUND: Vestibular symptoms are a common complication of cochlear implantation. In a previous study, we demonstrated histological evidence of a foreign-body response caused by silicon and platinum in the human cochlea following cochlear implantation. The objective of the current study was to seek evidence of a possible immune response in vestibular endorgans after cochlear implantation. METHODS: The density of macrophages immunostained with anti-Iba1 antibody in the vestibular endorgans (lateral and posterior semicircular canals, utricle and saccule) in 10 human subjects who had undergone unilateral cochlear implantation was studied by light microscopy. The densities of macrophages in the neuroepithelium, subepithelial stroma, and among dendritic processes in the mid-stromal zone in four vestibular endorgans in the implanted and the opposite unimplanted ears were compared. The distributions of macrophage morphology (amoeboid, transitional and ramified) were also compared. RESULTS: The densities of macrophages in implanted ears in four vestibular endorgans were significantly greater than that in opposite unimplanted ears except in the subepithelial zone of the utricle and posterior semicircular canal. In contrast to the neuroepithelium, the subepithelial distribution of amoeboid macrophages in implanted ears was significantly less than in unimplanted ears. CONCLUSION: An increase in the density of macrophages in four vestibular endorgans after implantation was demonstrated. The transition among phenotype of macrophages suggested possible migration of amoeboid macrophages from the subepithelial stroma into the neuroepithelium.

Mechanical Compression of Coverslipped Tissue Sections During Heat-induced Antigen Retrieval Prevents Section Detachment and Preserves Tissue Morphology.

Heat-induced antigen retrieval (HIAR) is routinely employed on aldehyde-fixed tissue sections to enhance the reactivity of antibodies that exhibit weak or no specific interactions with tissue antigens when applied in conventional immunohistochemical protocols. A major drawback of HIAR protocols is, however, the heat-induced detachment of sections from the microscope slide with resultant impaired tissue morphology or loss of the section. We developed a method in which tissue sections mounted on glass slides are temporally coverslipped, and a clamp is used to compress the sections on the microscope slide during HIAR treatment. This "pressurized coverslipping" during HIAR was tested on various formalin-fixed tissues (murine kidneys and temporal bones, human tonsils and temporal bones) that were embedded in paraffin or celloidin. The method reliably kept the sections adherent to the slide, preserved the tissue morphology, and effectively retrieved tissue antigens for improved results in immunohistochemical labeling, even for exceptionally delicate, large, and poorly adhering sections, that is, decalcified human temporal bone sections. In summary, we present a simple method for improved slide adherence and morphological preservation of tissue sections during HIAR treatment that can be combined with all HIAR protocols and that requires only basic lab equipment.

Inner ear pathologies impair sodium-regulated ion transport in Meniere's disease.

Meniere's disease (MD), a syndromal inner ear disease, is commonly associated with a pathological accumulation of endolymphatic fluid in the inner ear, termed "idiopathic" endolymphatic hydrops (iEH). Although numerous precipitating/exacerbating factors have been proposed for MD, its etiology remains elusive. Here, using immunohistochemistry and in situ protein-protein interaction detection assays, we demonstrate mineralocorticoid-controlled sodium transport mechanisms in the epithelium of the extraosseous portion of the endolymphatic sac (eES) in the murine and human inner ears. Histological analysis of the eES in an extensive series of human temporal bones consistently revealed pathological changes in the eES in cases with iEH and a clinical history of MD, but no such changes were found in cases with "secondary" EH due to other otological diseases or in healthy controls. Notably, two etiologically different pathologies-degeneration and developmental hypoplasia-that selectively affect the eES in MD were distinguished. Clinical records from MD cases with degenerative and hypoplastic eES pathology revealed distinct intergroup differences in clinical disease presentation. Overall, we have identified for the first time two inner ear pathologies that are consistently present in MD and can be directly linked to the pathogenesis of EH, and which potentially affect the phenotypical presentation of MD.

Focal Degeneration of Vestibular Neuroepithelium in the Cristae Ampullares of Three Human Subjects.

BACKGROUND: We report a unique pattern of focal degeneration of the neuroepithelium of cristae ampullares, thick subepithelial extracellular deposits, and neural degeneration in three humans. OBJECTIVE: To characterize the pattern of vestibular degeneration and measure the thickness of subepithelial deposits in these three cases and controls. METHODS: The subepithelial deposits of vestibular end organs in three subject cases and controls were studied using hematoxylin and eosin, periotic acid-Schiff, Gomori trichrome staining, and immunostaining for antineurofilament, antimyosin VIIa, and anticollagen 4a1. The thickness of deposit as measured by light microscopy was compared with that of control groups (age-matched controls, patients with unilateral Menière's disease, vestibular neuritis, cupulolithiasis, severe nonfocal degeneration of the vestibular neuroepithelium, and Alport syndrome). The correlation of thickness of deposits with age from 0 to 100 years was also investigated. RESULTS: Focal loss of hair cells in the neuroepithelium, thick subepithelial deposits, and degeneration of subepithelial dendrites and Scarpa's ganglion were found in all three cristae of three subject cases. Immunostaining demonstrated a decrease of afferent neural fibers in the cristae and focal fragmentation of the basement membrane adjacent to the deposits. The thickness of the subepithelial deposits in three cristae of three subject cases was significantly greater than that of all controls. In the three cristae of normal controls, the thickness of deposits demonstrated a positive correlation with age. CONCLUSION: Although both age and degeneration of the vestibular neuroepithelium may be associated with the thickness of the subepithelial deposits, in this unique pattern of degeneration, the thickness of the subepithelial deposits was significantly greater than that in all controls.

Preservation of Cells of the Organ of Corti and Innervating Dendritic Processes Following Cochlear Implantation in the Human: An Immunohistochemical Study.

HYPOTHESIS: This study evaluates the degree of preservation of hair cells, supporting cells, and innervating dendritic processes after cochlear implantation in the human using immunohistochemical methods. BACKGROUND: Surgical insertion of a cochlear implant electrode induces various pathologic changes within the cochlea including insertional trauma, foreign body response, inflammation, fibrosis, and neo-osteogenesis. These changes may result in loss of residual acoustic hearing, adversely affecting the use of hybrid implants, and may result in loss of putative precursor cells, limiting the success of future regenerative protocols. METHODS: Twenty-eight celloidin-embedded temporal bones from 14 patients with bilateral severe to profound sensorineural hearing loss and unilateral cochlear implants were studied. Two sections including the modiolus or basal turn from each temporal bone were stained using antineurofilament, antimyosin-VIIa, and antitubulin antibodies in both the implanted and unimplanted ears. RESULTS: Inner and outer hair cells: Immunoreactivity was reduced throughout the implanted cochlea and in the unimplanted cochlea with the exception of the apical turn.Dendritic processes in the osseous spiral lamina: Immunoreactivity was significantly less along the electrode of the implanted cochlea than in the other segments.Inner and outer pillars, inner and outer spiral bundles, and Deiters' cells: Immunoreactivity was similar in the implanted and unimplanted cochleae. CONCLUSION: Insertion of a cochlear implant electrode may significantly affect the inner and outer hair cells both along and apical to the electrode, and dendritic processes in the osseous spiral lamina along the electrode. There was less effect on pillar cells, Deiters' cells, and spiral bundles.

Foreign Body Response to Silicone in Cochlear Implant Electrodes in the Human.

HYPOTHESIS: Silicone as part of a cochlear implant electrode may be responsible for a foreign body response in the human. BACKGROUND: Clinical evidence of a foreign body response to a cochlear implant has been reported. In a previous study, particulate material found within the fibrous sheath and within macrophages surrounding a cochlear implant has been identified as being consistent with platinum. However, to date, there has been no histologic evidence of a role for silicone in this cellular immune response. METHODS: A total of 44 temporal bone specimens from 36 patients were reviewed by light microscopy for evidence of presumed platinum and/or silicone foreign bodies in an extracellular or intracellular location. Identification of cell type involved in phagocytosis of foreign body material was accomplished using CD163 immunostaining. The identity and source of the foreign body material was confirmed using energy-dispersive X-ray spectroscopy and scanning electron microscopy. RESULTS: Evidence for both platinum and silicone was found in all 44 specimens. In three patients, anti-CD 163 immunostaining demonstrated phagocytized platinum and silicone foreign bodies. In five specimens, energy-dispersive X-ray spectroscopy demonstrated that the birefringent foreign bodies were consistent with silicone. Scanning electron microscopy of two electrodes removed from temporal bones demonstrated small cracks, fragmentation, and small circular defects in the silicone carrier. CONCLUSION: Histologic evidence of a foreign body response to the presence of platinum and silicone in a cochlear implant has been demonstrated and may be responsible for some reported delayed failures or extrusion.