Imaging the Aging Cochlea with Light-Sheet Fluorescence Microscopy.

Deafness is the most common sensory impairment, affecting approximately 5% or 430 million people worldwide as per the World Health Organization1. Aging or presbycusis is a primary cause of sensorineural hearing loss and is characterized by damage to hair cells, spiral ganglion neurons (SGNs), and the stria vascularis. These structures reside within the cochlea, which has a complex, spiral-shaped anatomy of membranous tissues suspended in fluid and surrounded by bone. These properties make it technically difficult to investigate and quantify histopathological changes. To address this need, we developed a light-sheet microscope (TSLIM) that can image and digitize the whole cochlea to facilitate the study of structure-function relationships in the inner ear. Well-aligned serial sections of the whole cochlea result in a stack of images for three-dimensional (3D) volume rendering and segmentation of individual structures for 3D visualization and quantitative analysis (i.e., length, width, surface, volume, and number). Cochleae require minimal processing steps (fixation, decalcification, dehydration, staining, and optical clearing), all of which are compatible with subsequent high-resolution imaging by scanning and transmission electron microscopy. Since all the tissues are present in the stacks, each structure can be assessed individually or relative to other structures. In addition, since imaging uses fluorescent probes, immunohistochemistry and ligand binding can be used to identify specific structures and their 3D volume or distribution within the cochlea. Here we used TSLIM to examine cochleae from aged mice to quantify the loss of hair cells and spiral ganglion neurons. In addition, advanced analyses (e.g., cluster analysis) were used to visualize local reductions of spiral ganglion neurons in Rosenthal's canal along its 3D volume. These approaches demonstrate TSLIM microscopy's ability to quantify structure-function relationships within and between cochleae.

Downregulation of Cav3.1 T-type Calcium Channel Expression in Age-related Hearing Loss Model.

OBJECTIVE: Age-related hearing loss (AHL), characterized by degeneration of cochlea structures, is the most common sensory disorder among the elderly worldwide. The calcium channel is considered to contribute to normal hearing. However, the role of the T-type voltage-activated calcium channel, Cav3.1, remains unclear in AHL. Here, we investigate the age-related change of Cav3.1 expression in the cochlea and D-gal-induced senescent HEI-OC1 cells. METHODS: Cochleae from C57BL/6 mice at 2 months and 12 months of age were assessed. Senescence in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells was induced by D-gal treatment. The immunofluorescence technique was employed to investigate the distribution of Cav3.1 in vivo and in vitro. Quantitative assessment was achieved by Western blotting and real-time PCR. RESULTS: In comparison with 2-month-old animals, 12-month old C57BL/6 mice exhibited great loss of hair cells and elevated auditory brainstem threshold. The Cav3.1 was located in hair cells, spiral ganglion cells, lateral walls, and the expression of Cav3.1 protein and mRNA decreased in the aged cochleae. D-gal-induced senescence assay confirmed the down-regulation of Cav3.1 expression in senescent HEI-OC1 cells. CONCLUSION: Our results show that age-related down-regulated expression of Cav3.1 in the cochleae is associated with AHL and may contribute to the pathogenesis of AHL.

Multiscale photonic imaging of the native and implanted cochlea.

The cochlea of our auditory system is an intricate structure deeply embedded in the temporal bone. Compared with other sensory organs such as the eye, the cochlea has remained poorly accessible for investigation, for example, by imaging. This limitation also concerns the further development of technology for restoring hearing in the case of cochlear dysfunction, which requires quantitative information on spatial dimensions and the sensorineural status of the cochlea. Here, we employed X-ray phase-contrast tomography and light-sheet fluorescence microscopy and their combination for multiscale and multimodal imaging of cochlear morphology in species that serve as established animal models for auditory research. We provide a systematic reference for morphological parameters relevant for cochlear implant development for rodent and nonhuman primate models. We simulate the spread of light from the emitters of the optical implants within the reconstructed nonhuman primate cochlea, which indicates a spatially narrow optogenetic excitation of spiral ganglion neurons.

Vascular Supply of the Human Spiral Ganglion: Novel Three-Dimensional Analysis Using Synchrotron Phase-Contrast Imaging and Histology.

Human spiral ganglion (HSG) cell bodies located in the bony cochlea depend on a rich vascular supply to maintain excitability. These neurons are targeted by cochlear implantation (CI) to treat deafness, and their viability is critical to ensure successful clinical outcomes. The blood supply of the HSG is difficult to study due to its helical structure and encasement in hard bone. The objective of this study was to present the first three-dimensional (3D) reconstruction and analysis of the HSG blood supply using synchrotron radiation phase-contrast imaging (SR-PCI) in combination with histological analyses of archival human cochlear sections. Twenty-six human temporal bones underwent SR-PCI. Data were processed using volume-rendering software, and a representative three-dimensional (3D) model was created to allow visualization of the vascular anatomy. Histologic analysis was used to verify the segmentations. Results revealed that the HSG is supplied by radial vascular twigs which are separate from the rest of the inner ear and encased in bone. Unlike with most organs, the arteries and veins in the human cochlea do not follow the same conduits. There is a dual venous outflow and a modiolar arterial supply. This organization may explain why the HSG may endure even in cases of advanced cochlear pathology.

Human Superior Olivary Nucleus Neuron Populations in Subjects With Normal Hearing and Presbycusis.

INTRODUCTION: Normative data on superior olivary nucleus neuron counts derived from human specimens are sparse, and little is known about their coherence with structure and function of the cochlea. The purpose of this study was to quantify the neuron populations of the divisions of the superior olivary nucleus in human subjects with normal hearing and presbycusis and investigate potential relationships between these findings and histopathology in the cochlea and hearing phenotype Methods: Histopathologic examination of temporal bone and brainstem specimens from 13 subjects having normal hearing or presbycusis was undertaken. The following was determined for each: number and density of superior olivary nucleus and cochlear nucleus neurons, inner and outer hair cell counts, spiral ganglion cell counts, and pure tone audiometry. RESULTS: The results demonstrate a significant relationship between cells within structures of the cochlear nucleus and the number of neurons of the medial superior olivary nucleus. No relationship between superior olivary nucleus neuron counts/density and cochlear histopathology or hearing phenotype was encountered. CONCLUSION: Normative data for superior olivary nucleus neuron populations are further established in the data presented in this study that includes subjects with normal hearing and also presbycusis.

The Human Cochlear Aqueduct and Accessory Canals: a Micro-CT Analysis Using a 3D Reconstruction Paradigm.

OBJECTIVE: We sought to study the anatomic variations of the cochlear aqueduct and its accessory canals in human temporal bones using micro-CT and a 3D reconstruction paradigm. More knowledge about the anatomic variations of these structures, particularly at the basal turn of the cochlea and round window niche, may be important to better preserve residual hearing as well as the neural supply during cochlear implant surgery. METHODS: An archival collection of 30 human temporal bones underwent micro-CT and 3D reconstruction. A surface enhancement paradigm was applied. The application displays reconstructed slices as a 3D object with realistic 3D visualization of scanned objects. Virtual sectioning or "cropping" of the petrous bone presented subsequent areas. Thereby, the bony canals could be followed from inside the basal turn of cochlea and middle ear to the jugular foramen. RESULTS: The cochlear aqueduct was always paralleled by an accessory canal containing the inferior cochlear vein. It ran from the basal turn of the cochlea and exited laterally in the jugular foramen. In 70% of the cases, a secondary accessory canal was observed and it derived mostly from a depression or infundibulum located in the floor of the round window niche. This canal also exited in the jugular foramen. The secondary accessory canal occasionally anastomosed with the primary accessory canal suggesting that it contains a vein that drains middle ear blood to the cranial sinus. CONCLUSION: Micro-CT with 3D surface reconstruction paradigm offers new possibilities to study the topographic anatomy of minor details in the human inner ear. The technique creates simulated transparent "castings" of the labyrinth with a coinciding surface view through enhancement of contrast between boundaries. Accessory canals that drain blood from the cochlea, spiral ganglion, and middle ear could be characterized three-dimensionally.

Measurement for Detection of Incomplete Partition Type II Anomalies on MR Imaging.

BACKGROUND AND PURPOSE: Incomplete partition type II of the cochlea, commonly coexisting with an enlarged vestibular aqueduct, can be a challenging diagnosis on MR imaging due to the presence of a dysplastic spiral lamina-basilar membrane neural complex, which can resemble the normal interscalar septum. The purpose of this study was to determine a reproducible, quantitative cochlear measurement to assess incomplete partition type II anomalies in patients with enlarged vestibular aqueducts using normal-hearing ears as a control population. MATERIALS AND METHODS: Retrospective analysis of 27 patients with enlarged vestibular aqueducts (54 ears) and 28 patients (33 ears) with normal audiographic findings who underwent MR imaging was performed. Using reformatted images from a cisternographic 3D MR imaging produced in a plane parallel to the lateral semicircular canal, we measured the distance (distance X) between the osseous spiral lamina-basilar membrane complex of the upper basal turn and the first linear signal void anterior to the basilar membrane. RESULTS: The means of distance X in patients with normal hearing and prospectively diagnosed incomplete partition type II were, respectively, 0.93 ± 0.075 mm (range, 0.8-1.1 mm) and 1.55 ± 0.25 mm (range, 1-2.1 mm; P < .001). Using 3 SDs above the mean of patients with normal hearing (1.2 mm) as a cutoff for normal, we diagnosed 21/27 patients as having abnormal cochleas; 4/21 were diagnosed retrospectively. This finding indicated that almost 20% of patients were underdiagnosed. Interobserver agreement with 1.2 mm as a cutoff between normal and abnormal produced a κ score of 0.715 (good). CONCLUSIONS: Incomplete partition type II anomalies on MR imaging can be subtle. A reproducible distance X of ≥1.2 mm is considered abnormal and may help to prospectively diagnose incomplete partition type II anomalies.

Novel Role of the Mitochondrial Protein Fus1 in Protection from Premature Hearing Loss via Regulation of Oxidative Stress and Nutrient and Energy Sensing Pathways in the Inner Ear.

AIMS: Acquired hearing loss is a worldwide epidemic that affects all ages. It is multifactorial in etiology with poorly characterized molecular mechanisms. Mitochondria are critical components in hearing. Here, we aimed to identify the mechanisms of mitochondria-dependent hearing loss using Fus1 KO mice, our novel model of mitochondrial dysfunction/oxidative stress. RESULTS: Using auditory brainstem responses (ABRs), we characterized the Fus1 KO mouse as a novel, clinically relevant model of age-related hearing loss (ARHL) of metabolic etiology. We demonstrated early decline of the endocochlear potential (EP) that may occur due to severe mitochondrial and vascular pathologies in the Fus1 KO cochlear stria vascularis. We showed that pathological alterations in antioxidant (AO) and nutrient and energy sensing pathways (mTOR and PTEN/AKT) occur in cochleae of young Fus1 KO mice before major hearing loss. Importantly, short-term AO treatment corrected pathological molecular changes, while longer AO treatment restored EP, improved ABR parameters, restored mitochondrial structure, and delayed the development of hearing loss in the aging mouse. INNOVATION: Currently, no molecular mechanisms linked to metabolic ARHL have been identified. We established pathological and molecular mechanisms that link the disease to mitochondrial dysfunction and oxidative stress. CONCLUSION: Since chronic mitochondrial dysfunction is common in many patients, it could lead to developing hearing loss that can be alleviated/rescued by AO treatment. Our study creates a framework for clinical trials and introduces the Fus1 KO model as a powerful platform for developing novel therapeutic strategies to prevent/delay hearing loss associated with mitochondrial dysfunction. Antioxid. Redox Signal. 27, 489-509.

Coding deficits in hidden hearing loss induced by noise: the nature and impacts.

Hidden hearing refers to the functional deficits in hearing without deterioration in hearing sensitivity. This concept is proposed based upon recent finding of massive noise-induced damage on ribbon synapse between inner hair cells (IHCs) and spiral ganglion neurons (SGNs) in the cochlea without significant permanent threshold shifts (PTS). Presumably, such damage may cause coding deficits in auditory nerve fibers (ANFs). However, such deficits had not been detailed except that a selective loss of ANFs with low spontaneous rate (SR) was reported. In the present study, we investigated the dynamic changes of ribbon synapses and the coding function of ANF single units in one month after a brief noise exposure that caused a massive damage of ribbon synapses but no PTS. The synapse count and functional response measures indicates a large portion of the disrupted synapses were re-connected. This is consistent with the fact that the change of SR distribution due to the initial loss of low SR units is recovered quickly. However, ANF coding deficits were developed later with the re-establishment of the synapses. The deficits were found in both intensity and temporal processing, revealing the nature of synaptopathy in hidden hearing loss.

Visualization of Reissner membrane and the spiral ganglion in human fetal cochlea by micro-computed tomography.

PURPOSE: Although visualization of fine structures in the cochlea such as Reissner membrane (vestibular membrane) is important for elucidation of the mechanism and the establishment of therapy for inner ear diseases, they cannot be visualized by even the most advanced high-resolution medical computed tomography (CT) and magnetic resonance imaging. Visualization of Reissner membrane in dissected animals by micro-magnetic resonance imaging has been reported, but bone could not be visualized. We attempted to visualize human fetal Reissner membrane and the spiral ganglion by micro-focus x-ray CT (micro-CT), which has a spatial resolution several hundred times greater than the conventional medical CT. MATERIALS AND METHODS: Serial tomograms of a dissected pyramis, including the cochlea of human fetuses (stillborn specimens), were obtained by micro-CT, and 3-dimensional reconstruction was performed by a volume-rendering method. RESULTS: Clear tomograms (theoretical spatial resolution, 12.2 x 12.2 microm; slice thickness 77.5 microm) and 3-dimensional reconstructed images (theoretical spatial resolution, 6.8 x 6.8 microm; slice thickness, 40.0 microm) of Reissner membrane and the spiral ganglion with a bony labyrinth (cochlear bone) were successfully obtained for the first time. The thickness of Reissner membrane obtained by the tomogram was 12 microm, which corresponds to the optical macroscopic value from resin-embedded histologic sections. CONCLUSIONS: This study showed that micro-CT enables us to visualize the internal fine structure of the human cochlea. As the success rate of the visualization of Reissner membrane is not high, it is necessary to improve the image quality and contrast resolution of micro-CT to enable stable visualization of fine structures. The development of imaging equipment such as micro-CT for medical use should play an important role in the elucidation of the mechanism and the establishment of therapy for inner ear diseases.

Assessment of intracochlear electrode position and correlation with behavioural thresholds in CII and 90K cochlear implants.

CONCLUSION: The image quality of 64-MDCT provided excellent definition of the fine osseous structures and individual electrode contacts. Evaluation of electrode distances revealed a more focused stimulation for the Helix contacts, with better optimization of pulse width and frequency of stimulation. OBJECTIVES: A multi-slice CT scan was performed postoperatively to evaluate electrode distance from the modiolus and variability of fitting parameters (M level) for two different types of cochlear implant electrode carriers, CII and 90K implants with 1J and Helix electrode carriers. MATERIALS AND METHODS: The electrode's position in different cochlear implant (CI) electrodes, Advanced Bionics 90K 1J and Helix, was assessed postoperatively in 20 adult patients by means of a 64-MDCT scanner. Axial, coronal, and oblique 0.3 mm multiplanar reconstructions (MPRs) were obtained and datasets were analyzed to assess the intracochlear position and distance from the surface of the electrodes to the bony edge of the modiolus. Patients' fitting characteristics were gathered at the time the CT was performed and correlated to intracochlear measurements. RESULTS: Determination of contact distances confirmed smaller average values for the Helix at the apex and medial segments. Helix electrodes were closer to the modiolus in all segments. Likewise, M level determination showed lower values for the Helix carrier, confirming a more focused stimulation and better optimization of pulse width and frequency of stimulation.