Synchrotron Phase-Contrast Imaging and Cochlear Otosclerosis: A Case Report.

INTRODUCTION: Otosclerosis is a bone disorder affecting the labyrinthine capsule that leads to conductive and occasionally sensorineural hearing loss. The etiology of otosclerosis remains unknown; factors such as infection, hormones, inflammation, genetics, and autoimmunity have been discussed. Treatment consists primarily of surgical stapes replacement and cochlear implantation. High-resolution computed tomography is routinely used to visualize bone pathology. In the present study, we used synchrotron radiation phase-contrast imaging (SR-PCI) to examine otosclerosis plaques in a temporal bone for the first time. The primary aim was to study their three-dimensional (3D) outline, vascular interrelationships, and connections to the middle ear. METHODS: A donated ear from a patient with otosclerosis who had undergone partial stapedectomy with the insertion of a stapes wire prosthesis was investigated using SR-PCI and compared with a control ear. Otosclerotic lesions were 3D rendered using the composite with shading technique. Scalar opacity and color mapping were adjusted to display volume properties with the removal of bones to enhance surfaces. Vascular bone channels were segmented, and the communications between lesions and the middle ear were established. RESULTS: Fenestral, cochlear, meatal, and vestibular lesions were outlined three-dimensionally. Vascular bone channels were found to be frequently connected to the middle ear mucosa, perilabyrinthine air spaces, and facial nerve vessels. Round window lesions partly embedded the cochlear aqueduct which was pathologically narrowed, while the inferior cochlear vein was significantly dilated in its proximal part. CONCLUSION: Otosclerotic/otospongiotic lesions were imaged for the first time using SR-PCI and 3D rendering. The presence of shunts and abnormal vascular connections to the labyrinth appeared to result in hyper-vascularization, overloading the venous system, and leading to sensorineural hearing loss. We speculate about possible local treatments to alleviate the impact of such critical lesions on the labyrinthine microcirculation.

Immunohistochemistry Reveals TRPC Channels in the Human Hearing Organ-A Novel CT-Guided Approach to the Cochlea.

TRPC channels are critical players in cochlear hair cells and sensory neurons, as demonstrated in animal experiments. However, evidence for TRPC expression in the human cochlea is still lacking. This reflects the logistic and practical difficulties in obtaining human cochleae. The purpose of this study was to detect TRPC6, TRPC5 and TRPC3 in the human cochlea. Temporal bone pairs were excised from ten body donors, and the inner ear was first assessed based on computed tomography scans. Decalcification was then performed using 20% EDTA solutions. Immunohistochemistry with knockout-tested antibodies followed. The organ of Corti, the stria vascularis, the spiral lamina, spiral ganglion neurons and cochlear nerves were specifically stained. This unique report of TRPC channels in the human cochlea supports the hypothesis of the potentially critical role of TRPC channels in human cochlear health and disease which has been suggested in previous rodent experiments.

Micro-CT versus synchrotron radiation phase contrast imaging of human cochlea.

High-resolution images of the cochlea are used to develop atlases to extract anatomical features from low-resolution clinical computed tomography (CT) images. We compare visualization and contrast of conventional absorption-based micro-CT to synchrotron radiation phase contrast imaging (SR-PCI) images of whole unstained, nondecalcified human cochleae. Three cadaveric cochleae were imaged using SR-PCI and micro-CT. Images were visually compared and contrast-to-noise ratios (CNRs) were computed from n = 27 regions-of-interest (enclosing soft tissue) for quantitative comparisons. Three-dimensional (3D) models of cochlear internal structures were constructed from SR-PCI images using a semiautomatic segmentation method. SR-PCI images provided superior visualization of soft tissue microstructures over conventional micro-CT images. CNR improved from 7.5 ± 2.5 in micro-CT images to 18.0 ± 4.3 in SR-PCI images (p < 0.0001). The semiautomatic segmentations yielded accurate reconstructions of 3D models of the intracochlear anatomy. The improved visualization, contrast and modelling achieved using SR-PCI images are very promising for developing atlas-based segmentation methods for postoperative evaluation of cochlear implant surgery.

Super-resolution structured illumination fluorescence microscopy of the lateral wall of the cochlea: the Connexin26/30 proteins are separately expressed in man.

Globally 360 million people have disabling hearing loss and, of these, 32 million are children. Human hearing relies on 15,000 hair cells that transduce mechanical vibrations to electrical signals in the auditory nerve. The process is powered by the endo-cochlear potential, which is produced by a vascularized epithelium that actively transports ions in conjunction with a gap junction (GJ) system. This "battery" is located "off-site" in the lateral wall of the cochlea. The GJ syncytium contains the GJ protein genes beta 2 (GJB2/connexin26 (Cx26)) and 6 (GJB6/connexin30 (Cx30)), which are commonly involved in hereditary deafness. Because the molecular arrangement of these proteins is obscure, we analyze GJ protein expression (Cx26/30) in human cochleae by using super-resolution structured illumination microscopy. At this resolution, the Cx26 and Cx30 proteins were visible as separate plaques, rather than being co-localized in heterotypic channels, as previously suggested. The Cx26 and Cx30 proteins thus seem not to be co-expressed but to form closely associated assemblies of GJ plaques. These results could assist in the development of strategies to treat genetic hearing loss in the future.

Immuno-surveillance and protection of the human cochlea.

Background: Despite its location near infection-prone areas, the human inner ear demonstrates remarkable resilience. This suggests that there are inherent instruments deterring the invasion and spread of pathogens into the inner ear. Here, we combined high-resolution light microscopy, super-resolution immunohistochemistry (SR-SIM) and synchrotron phase contrast imaging (SR-PCI) to identify the protection and barrier systems in the various parts of the human inner ear, focusing on the lateral wall, spiral ganglion, and endolymphatic sac. Materials and methods: Light microscopy was conducted on mid-modiolar, semi-thin sections, after direct glutaraldehyde/osmium tetroxide fixation. The tonotopic locations were estimated using SR-PCI and 3D reconstruction in cadaveric specimens. The sections were analyzed for leucocyte and macrophage activity, and the results were correlated with immunohistochemistry using confocal microscopy and SR-SIM. Results: Light microscopy revealed unprecedented preservation of cell anatomy and several macrophage-like cells that were localized in the cochlea. Immunohistochemistry demonstrated IBA1 cells frequently co-expressing MHC II in the spiral ganglion, nerve fibers, lateral wall, spiral limbus, and tympanic covering layer at all cochlear turns as well as in the endolymphatic sac. RNAscope assays revealed extensive expression of fractalkine gene transcripts in type I spiral ganglion cells. CD4 and CD8 cells occasionally surrounded blood vessels in the modiolus and lateral wall. TMEM119 and P2Y12 were not expressed, indicating that the cells labeled with IBA1 were not microglia. The round window niche, compact basilar membrane, and secondary spiral lamina may form protective shields in the cochlear base. Discussion: The results suggest that the human cochlea is surveilled by dwelling and circulating immune cells. Resident and blood-borne macrophages may initiate protective immune responses via chemokine signaling in the lateral wall, spiral lamina, and spiral ganglion at different frequency locations. Synchrotron imaging revealed intriguing protective barriers in the base of the cochlea. The role of the endolymphatic sac in human inner ear innate and adaptive immunity is discussed.

The Spectral Extent of Phasic Suppression of Loudness and Distortion-Product Otoacoustic Emissions by Infrasound and Low-Frequency Tones.

We investigated the effect of a biasing tone close to 5, 15, or 30 Hz on the response to higher-frequency probe tones, behaviorally, and by measuring distortion-product otoacoustic emissions (DPOAEs). The amplitude of the biasing tone was adjusted for criterion suppression of cubic DPOAE elicited by probe tones presented between 0.7 and 8 kHz, or criterion loudness suppression of a train of tone-pip probes in the range 0.125-8 kHz. For DPOAEs, the biasing-tone level for criterion suppression increased with probe-tone frequency by 8-9 dB/octave, consistent with an apex-to-base gradient of biasing-tone-induced basilar membrane displacement, as we verified by computational simulation. In contrast, the biasing-tone level for criterion loudness suppression increased with probe frequency by only 1-3 dB/octave, reminiscent of previously published data on low-side suppression of auditory nerve responses to characteristic frequency tones. These slopes were independent of biasing-tone frequency, but the biasing-tone sensation level required for criterion suppression was ~ 10 dB lower for the two infrasound biasing tones than for the 30-Hz biasing tone. On average, biasing-tone sensation levels as low as 5 dB were sufficient to modulate the perception of higher frequency sounds. Our results are relevant for recent debates on perceptual effects of environmental noise with very low-frequency content and might offer insight into the mechanism underlying low-side suppression.

Two Photon Fluorescence Microscopy of the Unstained Human Cochlea Reveals Organ of Corti Cytoarchitecture.

Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, and it typically originates from the cochlea. Methods to visualize intracochlear cells in living people are currently lacking, limiting not only diagnostics but also therapies for SNHL. Two-photon fluorescence microscopy (TPFM) is a high-resolution optical imaging technique. Here we demonstrate that TPFM enables visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified adult human cochlea.

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.

Finite element analysis and three-dimensional reconstruction of tonotopically aligned human auditory fiber pathways: A computational environment for modeling electrical stimulation by a cochlear implant based on micro-CT.

The application of cochlear implants can be studied with computational models. The electrical potential distribution induced by an implanted device is evaluated with a volume conductor model, which is used as input for neuron models to simulate the reaction of cochlear neurons to micro-stimulation. In order to reliably predict the complex excitation profiles it is vital to consider an accurate representation of the human cochlea geometry including detailed three-dimensional pathways of auditory neurons reaching from the organ of Corti through the cochlea-volume. In this study, high-resolution micro-CT imaging (Δx = Δy = Δz = 3 µm) was used to reconstruct the pathways of 30 tonotopically organized nerve fiber bundles, distributed over eight octaves (11500-40 Hz). Results of the computational framework predict: (i) the peripheral process is most sensitive to cathodic stimulation (CAT), (ii) in many cases CAT elicits spikes in the peripheral terminal at threshold but with larger stimuli there is a second spike initiation site within the peripheral process, (iii) anodic stimuli (ANO) can excite the central process even at threshold, (iv) the recruitment of fibers by electrodes located in the narrowing middle- and apical turn is complex and impedes focal excitation of low frequency fibers, (v) degenerated cells which lost the peripheral process are more sensitive to CAT when their somata are totally covered with 2 membranes of a glial cell but they become ANO sensitive when the myelin covering is reduced.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea.

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCII and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy (SR-SIM) were used in the study. CD4+ and CD8+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons, CD4+ and CD8+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus, macrophages, CD4+ and CD8+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4+ and CD8+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Real-time intracochlear imaging of automated cochlear implant insertions in whole decalcified cadaver cochleas using ultrasound.

OBJECTIVES: This study aimed to determine the feasibility of combining high-frequency ultrasound imaging, automated insertion, and force sensing to yield more information about cochlear implant insertion dynamics. METHODS: An apparatus was developed combining these aspects along with software to control implant and imaging probe positions. Decalcified unfixed human cochleas were implanted at various speeds, insertion sites, and implant models while imaging near the implant tip throughout insertion and recording force data from the cochlea mounting stage. Ultrasound video data were also captured. RESULTS: The basilar membrane (BM) was frequently penetrated by the implant in either the mid-basal or lower middle turn. Measurements were also performed of apical BM motion in response to upstream implant movement at varying insertion speeds. Increasing insertion speed resulted in greater BM displacement. DISCUSSION: Multiple insertions per cochlea increase the volume of data per specimen while also reducing variability due to differences between cochleas. However, to image inside the cochlea with ultrasound, the bone had to be decalcified, which likely had a significant effect upon the response of tissue to contact by the implant. As calcified bone strongly reflects ultrasound, we also found ultrasound imaging to be an excellent method for easily assessing bone decalcification progress. CONCLUSION: This technique may be very useful for some studies, although the confounding effects of bone decalcification may make results of other studies too difficult to generalize. The approach could be adapted to other real-time imaging modalities, such as optical coherence tomography.

Automated analysis of human cochlea shape variability from segmented µCT images.

The aim of this study is to define an automated and reproducible framework for cochlear anatomical analysis from high-resolution segmented images and to provide a comprehensive and objective shape variability study suitable for cochlear implant design and surgery planning. For the scala tympani (ST), the scala vestibuli (SV) and the whole cochlea, the variability of the arc lengths and the radial and longitudinal components of the lateral, central and modiolar paths are studied. The robustness of the automated cochlear coordinate system estimation is validated with synthetic and real data. Cochlear cross-sections are statistically analyzed using area, height and width measurements. The cross-section tilt angle is objectively measured and this data documents a significant feature for occurrence of surgical trauma.

The 1.06 frequency ratio in the cochlea: evidence and outlook for a natural musical semitone.

A frequency ratio of about 1.06 often appears in cochlear mechanics, and the question naturally arises, why? The ratio is close to that of the semitone (1.059) in music, giving reason to think that this aspect of musical perception might have a cochlear basis. Here, data on synchronised spontaneous otoacoustic emissions is presented, and a clustering of ratios between 1.05 and 1.07 is found with a peak at 1.063 ± 0.005. These findings reinforce what has been found from previous sources, which are reviewed and placed alongside the present work. The review establishes that a peak in the vicinity of 1.06 has often been found in human cochlear data. Several possible cochlear models for explaining the findings are described. Irrespective of which model is selected, the fact remains that the cochlea itself appears to be the origin of a ratio remarkably close to an equal-tempered musical semitone, and this close coincidence leads to the suggestion that the inner ear may play a role in constructing a natural theory of music. The outlook for such an enterprise is surveyed.

Supernumerary human hair cells-signs of regeneration or impaired development? A field emission scanning electron microscopy study.

BACKGROUND: Current attempts to regenerate cochlear sensorineural structures motivate further inspection of the human organ of hearing. Here, we analyzed the supernumerary inner hair cell (sIHC), a possible sign of regeneration and cell replacement. METHODS: Human cochleae were studied using field emission scanning electron microscopy (FESEM; maximum resolution 2 nm) obtained from individuals aged 44, 48, and 58 years with normal sensorineural pure-tone average (PTA) thresholds (PTA <20 dB). The wasted tissue was harvested during trans-cochlear approaches and immediately fixed for ultrastructural analysis. RESULTS: All specimens exhibited sIHCs at all turns except at the extreme lower basal turn. In one specimen, it was possible to image and count the inner hair cells (IHCs) along the cochlea representing the 0.2 kHz-8 kHz region according to the Greenwood place/frequency scale. In a region with 2,321 IHCs, there were 120 scattered one-cell losses or 'gaps' (5%). Forty-two sIHCs were present facing the modiolus. Thirty-eight percent of the sIHCs were located near a 'gap' in the IHC row (±6 IHCs). CONCLUSIONS: The prevalence of ectopic inner hair cells was higher than expected. The morphology and placement could reflect a certain ongoing regeneration. Further molecular studies are needed to verify if the regenerative capacity of the human auditory periphery might have been underestimated.

The pre- and post-somatic segments of the human type I spiral ganglion neurons--structural and functional considerations related to cochlear implantation.

Human auditory nerve afferents consist of two separate systems; one is represented by the large type I cells innervating the inner hair cells and the other one by the small type II cells innervating the outer hair cells. Type I spiral ganglion neurons (SGNs) constitute 96% of the afferent nerve population and, in contrast to other mammals, their soma and pre- and post-somatic segments are unmyelinated. Type II nerve soma and fibers are unmyelinated. Histopathology and clinical experience imply that human SGNs can persist electrically excitable without dendrites, thus lacking connection to the organ of Corti. The biological background to this phenomenon remains elusive. We analyzed the pre- and post-somatic segments of the type I human SGNs using immunohistochemistry and transmission electron microscopy (TEM) in normal and pathological conditions. These segments were found surrounded by non-myelinated Schwann cells (NMSCs) showing strong intracellular expression of laminin-ß2/collagen IV. These cells also bordered the perikaryal entry zone and disclosed surface rugosities outlined by a folded basement membrane (BM) expressing laminin-ß2 and collagen IV. It is presumed that human large SGNs are demarcated by three cell categories: (a) myelinated Schwann cells, (b) NMSCs and (c) satellite glial cells (SGCs). Their BMs express laminin-ß2/collagen IV and reaches the BM of the sensory epithelium at the habenula perforata. We speculate that the NMSCs protect SGNs from further degeneration following dendrite loss. It may give further explanation why SGNs can persist as electrically excitable monopolar cells even after long-time deafness, a blessing for the deaf treated with cochlear implantation.

Immunohistological analysis of neurturin and its receptors in human cochlea.

OBJECTIVE: Difficulties in obtaining properly preserved human cochlea have been a major obstacle to in vitro study of this deeply located and hard bone-fortressed hearing organ. Our study aimed at investigating GDNF family ligands (GFLs) and their receptors in the human cochleae that were surgically obtained during a transcochlear approach dealing with life-threatening, intra-cranial meningiomas. METHODS: The specimens were properly fixed with 4% paraformaldehyde in the operating room. By using immunohistochemical techniques, distribution of GDNF, Neurturin (NTN, one member of GFLs), as well as cRet, GFRα-1 and GFRα-2 receptors in the human cochleae was investigated. Five cochleae from five adult patients were processed for the study. The patients had normal hearing threshold before operation. RESULTS: cRet receptor immunoreactivity was seen in the spiral ganglion neurons, mainly inside the cell bodies but rarely in the nerve fibers and not in the organ of Corti. Immunolabeling for GFRα-1 and GFRα-2 receptors was identified mainly in the cell bodies of the spiral neurons than in the nerve fibers. In the organ of Corti, GFRα-1 immunostaining could be demonstrated in the Deiters' cells, Hensen cells, inner pillar cells, and weakly in the inner hair cells but not in the outer hair cells; no structures in the organ of Corti were labeled with GFRα-2 receptor antibody. NTN immunostaining was found in the supporting cells of organ of Corti, including Deiters' cells, Hensen cells as well as Claudius' cells. In the spiral ganglia, NTN immunostaining was seen in both the cell bodies and the nerve fibers of neurons. GDNF immunoreactivity was not revealed in human cochlea. CONCLUSION: Surgically obtained human cochleae were properly fixed and underwent immunohistochemical investigation of neurotrophic elements. NTN and its receptors discovered in current study can be responsible for the unique neuronal survival properties in human spiral ganglion (hSG); a prerequisite for the function of cochlear implants.

Laser-doppler evaluation of the human tympanic membrane by measuring blood flow, volume, and velocity.

From a hospital setting, a sample base of 50 people with normal hearing, was used to study the otoacoustic phenomenon of the ear. Human volunteers were taken into an acoustic room and allowed to rest for 5 minutes. Each volunteer inserted a probe as close to the tympanic membrane as possible. The objective of the study was to determine the pattern of distribution of blood flow, volume, and velocity of erythrocytes circulating in blood vessels of the tympanic membrane. This was achieved by recording numerical values for the flow, volume, and velocity of erythrocytes using the laser-Doppler flowmeter. Analysis of the results showed two distinct relationships, a negative exponential curve in the blood flow for the right and left ear, and a Gaussian distribution for the velocity and volume of erythrocytes for both ears.