Characterising inner ear disorders represents a significant challenge due to a lack of reliable experimental procedures and identified biomarkers. It is also difficult to access the complex microenvironments of the inner ear and investigate specific pathological indicators through conventional techniques. Omics technologies have the potential to play a vital role in revolutionising the diagnosis of ear disorders by providing a comprehensive understanding of biological systems at various molecular levels. These approaches reveal valuable information about biomolecular signatures within the cochlear tissue or fluids such as the perilymphatic and endolymphatic fluid. Proteomics identifies changes in protein abundance, while metabolomics explores metabolic products and pathways, aiding the characterisation and early diagnosis of diseases. Although there are different methods for identifying and quantifying biomolecules, mass spectrometry, as part of proteomics and metabolomics analysis, could be utilised as an effective instrument for understanding different inner ear disorders. This study aims to review the literature on the application of proteomic and metabolomic approaches by specifically focusing on Meniere\'s disease, ototoxicity, noise-induced hearing loss, and vestibular schwannoma. Determining potential protein and metabolite biomarkers may be helpful for the diagnosis and treatment of inner ear problems.

Systemic drug administration provides convenience and non-invasive benefits for preventing and treating inner ear diseases. However, the blood-labyrinth barrier (BLB) restricts the transport of drugs to inner ear tissues. Ultrasound can stimulate specific areas and penetrate tissues, with the potential to overcome physiological barriers. We present a novel strategy based on low-pressure pulsed ultrasound assisted by microbubbles (USMB) to transiently open the BLB and deliver therapeutics into the inner ear. A pulsed ultrasound device with adjustable pressure was established; the generated ultrasound was transmitted through the external auditory canal into the guinea pig\'s inner ear. We observed that the application of microbubbles allowed the use of safe and efficient ultrasound conditions to penetrate the BLB. We found that USMB-mediated BLB opening seemed to be associated with a reduced expression of the tight junction proteins zonula occludens-1 and occludin. Following intravenous administration, hydrophilic dexamethasone sodium phosphate (DSP), hydrophobic curcumin (CUR), as well as drug-loaded nanoparticles (Fe3O4@CUR NPs) could be efficiently delivered into the inner ear. We observed better drug accumulation in the perilymph of the inner ear, resulting in less drug (cisplatin)-induced ototoxicity. Furthermore, physiological, hematological, and histological studies showed that the modulation of the BLB by low-pressure USMB was a safe process without significant adverse effects. We conclude that USMB could become a promising strategy for the systematic delivery of therapeutics in the treatment of inner ear diseases.

This study examined the association between hearing loss in sporadic vestibular schwannoma patients and the proteome of perilymph (PL), cerebrospinal fluid (CSF), and vestibular schwannoma. Intraoperative sampling of PL and of CSF, and biopsy of vestibular schwannoma tissue, was performed in 32, 32, and 20 patients with vestibular schwannoma, respectively. Perilymph and CSF in three patients with meningioma and normal hearing were also sampled. The proteomes were identified by liquid chromatography coupled to high-resolution tandem mass spectrometry. Preoperative hearing function of the patients was evaluated with pure tone audiometry, with mean values at frequencies of 500, 1000, 2000, and 4000 Hz (PTA4) in the tumor-affected ear used to delineate three hearing groups. Analysis of the PL samples revealed significant upregulation of complement factor H-related protein 2 (CFHR2) in patients with severe to profound hearing loss after false discovery rate correction. Pathway analysis of biofunctions revealed higher activation scores in the severe/profound hearing loss group of leukocyte migration, viral infection, and migration of cells in PL. Upregulation of CFHR2 and activation of these pathways indicate chronic inflammation in the cochlea of vestibular schwannoma patients with severe to profound hearing loss compared with patients with normal hearing or mild hearing loss.

OBJECTIVE: Mesenchymal stem cells (MSCs) have the capability of providing ongoing paracrine support to degenerating tissues. Since MSCs can be extracted from a broad range of tissues, their specific surface marker profiles and growth factor secretions can be different. We hypothesized that MSCs derived from different sources might also have different neuroprotective potential.
OBJECTIVE: In this study, we extracted MSCs from rodent olfactory mucosa and compared their neuroprotective effects on auditory hair cell survival with MSCs extracted from rodent adipose tissue.
METHODS: Organ of Corti explants were dissected from 41 cochlea and incubated with olfactory mesenchymal stem cells (OMSCs) and adipose mesenchymal stem cells (AMSCs). After 72 hours, Corti explants were fixed, stained, and hair cells counted. Growth factor concentrations were determined in the supernatant and cell lysate using Enzyme-Linked Immunosorbent Assay (ELISA).
RESULTS: Co-culturing of organ of Corti explants with OMSCs resulted in a significant increase in inner and outer hair cell stereocilia survival, compared to control. Comparisons between both stem cell lines, showed that co-culturing with OMSCs resulted in superior inner and outer hair cell stereocilia survival rates over co-culturing with AMSCs. Assessment of growth factor secretions revealed that the OMSCs secrete significant amounts of insulin-like growth factor 1 (IGF-1). Co-culturing OMSCs with organ of Corti explants resulted in a 10-fold increase in IGF-1 level compared to control, and their secretion was 2 to 3 times higher compared to the AMSCs.
CONCLUSIONS: This study has shown that OMSCs may mitigate auditory hair cell stereocilia degeneration. Their neuroprotective effects may, at least partially, be ascribed to their enhanced IGF-1 secretory abilities compared to AMSCs.

UNASSIGNED: Age-related hearing difficulties have a complex etiology that includes degenerative processes in the sensory cochlea. The cochlea comprises the start of the afferent, ascending auditory pathway, but also receives efferent feedback innervation by two separate populations of brainstem neurons: the medial olivocochlear and lateral olivocochlear pathways, innervating the outer hair cells and auditory-nerve fibers synapsing on inner hair cells, respectively. Efferents are believed to improve hearing under difficult conditions, such as high background noise. Here, we compare olivocochlear efferent innervation density along the tonotopic axis in young-adult and aged gerbils (at ~50% of their maximum lifespan potential), a classic animal model for age-related hearing loss.
UNASSIGNED: Efferent synaptic terminals and sensory hair cells were labeled immunohistochemically with anti-synaptotagmin and anti-myosin VIIa, respectively. Numbers of hair cells, numbers of efferent terminals, and the efferent innervation area were quantified at seven tonotopic locations along the organ of Corti.
UNASSIGNED: The tonotopic distribution of olivocochlear innervation in the gerbil was similar to that previously shown for other species, with a slight apical cochlear bias in presumed lateral olivocochlear innervation (inner-hair-cell region), and a broad mid-cochlear peak for presumed medial olivocochlear innervation (outer-hair-cell region). We found significant, age-related declines in overall efferent innervation to both the inner-hair-cell and the outer-hair-cell region. However, when accounting for the age-related losses in efferent target structures, the innervation density of surviving elements proved unchanged in the inner-hair-cell region. For outer hair cells, a pronounced increase of orphaned outer hair cells, i.e., lacking efferent innervation, was observed. Surviving outer hair cells that were still efferently innervated retained a nearly normal innervation.
UNASSIGNED: A comparison across species suggests a basic aging scenario where outer hair cells, type-I afferents, and the efferents associated with them, steadily die away with advancing age, but leave the surviving cochlear circuitry largely intact until an advanced age, beyond 50% of a species\' maximum lifespan potential. In the outer-hair-cell region, MOC degeneration may precede outer-hair-cell death, leaving a putatively transient population of orphaned outer hair cells that are no longer under efferent control.

Idiopathic sudden sensorineural hearing loss (ISSNHL) is characterized by abruptly appearing hearing loss, sometimes accompanied by vertigo. Vascular pathologies (e.g., cochlear ischemia, or cochlear infarction) are one of the most likely causes of ISSNHL. This review aims to present current understanding of inner ear anatomy, clinical features of ISSNHL, and its treatment strategies. The labyrinthine artery is the only end artery supplying blood to the inner ear, and it has three branches: the anterior vestibular artery, the main cochlear artery, and the vestibulo-cochlear artery (VCA). Occlusion of the VCA can be caused by a variety of factors. The VCA courses through a narrow bone canal. ISSNHL is usually diagnosed after excluding retrocochlear pathologies of sudden sensorineural hearing loss (SSNHL), such as vestibular schwannoma. Therefore, a head MRI or assessing auditory brainstem responses are recommended for patients with SSNHL. Severe SSNHL patients with high CHADS2 scores, an index of stroke risk, have a significantly lower rate of vestibular schwannoma than severe SSNHL patients with low CHADS2 scores, suggesting that severe ISSNHL in individuals at high risk of stroke is caused by vascular impairments. Intralabyrinthine hemorrhage causes SSNHL or vertigo, as in ISSNHL. The diagnosis of intralabyrinthine hemorrhage requires careful interpretation of MRI, and a small percentage of patients diagnosed with ISSNHL may in fact have intralabyrinthine hemorrhage. Many studies have reported an association between ISSNHL and atherosclerosis or cardiovascular risk factors (e.g., diabetes mellitus, hypertension, dyslipidemia and cardiovascular disease), and subsequent risk of stroke in patients with ISSNHL may be elevated compared to controls. Increased hearing level on the healthy ear side, high Framingham risk score, high neutrophil-to-lymphocyte ratio, high platelet-to-lymphocyte ratio, and severe white matter lesions may be poor prognostic factors for patients with ISSNHL. The association between thrombosis-related genes and susceptibility to ISSNHL has been reported in many studies (e.g., coagulation factor 2, coagulation factor 5, plasminogen activator inhibitor-1, platelet-associated genes, a homocysteine metabolism-related enzyme gene, endothelin-1, nitric oxide 3, phosphodiesterase 4D, complement factor H, and protein kinase C-eta). Treatment of ISSNHL with the aim of mitigating the vascular impairment in the inner ear includes systemically administered steroids, intratympanic steroid injections, hyperbaric oxygen therapy, prostaglandin E1, defibrinogenation therapy, and hydrogen inhalation therapy, but there is currently no evidence-based treatment for ISSNHL. Breakthroughs in the unequivocal diagnosis and treatment of ISSNHL due to vascular impairment are crucial to improve quality of life.

UNASSIGNED: Mutations in microRNA-96 (miR-96), a microRNA expressed within the hair cells (HCs) of the inner ear, result in progressive hearing loss in both mouse models and humans. In this study, we present the first HC-specific RNA-sequencing (RNA-seq) dataset from newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice.
UNASSIGNED: Bulk RNA-seq was performed on HCs of newborn Mir96Dmdo heterozygous, homozygous mutant, and wildtype mice. Differentially expressed gene analysis was conducted on Mir96Dmdo homozygous mutant HCs compared to wildtype littermate controls, followed by GO term and protein-protein interaction analysis on these differentially expressed genes.
UNASSIGNED: We identify 215 upregulated and 428 downregulated genes in the HCs of the Mir96Dmdo homozygous mutant mice compared to their wildtype littermate controls. Many of the significantly downregulated genes in Mir96Dmdo homozygous mutant HCs have established roles in HC development and/or known roles in deafness including Myo15a, Myo7a, Ush1c, Gfi1, and Ptprq and have enrichment in gene ontology (GO) terms with biological functions such as sensory perception of sound. Interestingly, upregulated genes in Mir96Dmdo homozygous mutants, including possible miR-96 direct targets, show higher wildtype expression in supporting cells compared to HCs.
UNASSIGNED: Our data further support a role for miR-96 in HC development, possibly as a repressor of supporting cell transcriptional programs in HCs. The HC-specific Mir96Dmdo RNA-seq data set generated from this manuscript are now publicly available in a dedicated profile in the gene expression analysis resource (gEAR-https://umgear.org/p?l=miR96).

Although research on hearing loss, including the identification of causative genes, has become increasingly active, the pathogenic mechanism of hearing loss remains unclear. One of the reasons for this is that the structure of the inner ear of mice, which is commonly used as a genetically modified animal model, is too small and complex, making it difficult to accurately capture abnormalities and dynamic changes in vivo. Especially, Reissner\'s membrane is a very important structure that separates the perilymph and endolymph of the inner ear. This malformation or damage induces abnormalities in hearing and balance. Until now, imaging analyses, such as magnetic resonance imaging (MRI) and computed tomography, are performed to investigate the inner ear structure in vivo; however, it has been difficult to analyze the small inner ear structure of mice owing to resolution. Therefore, there is an urgent need to develop an image analysis method that can accurately capture the structure of the inner ear of mice including Reissner\'s membrane, both dynamically and statically. This study aimed to investigate whether it is possible to accurately capture the structure (e.g., Reissner\'s membrane) and abnormalities of the inner ear of mice using an 11.7 T MRI. By combining two types of MRI methods, in vivo and ex vivo, we succeeded for the first time in capturing the fine structure of the normal mouse inner ear, such as the Reissner\'s membrane, and inflammatory lesions of otitis media mouse models in detail and accurately. In the future, we believe that understanding the state of Reissner\'s membrane during living conditions will greatly contribute to the development of research on inner ear issues, such as hearing loss.

Calvarial bone marrow has been found to be central in the brain immune response, being connected to the dura through channels which allow leukocyte trafficking. Temporal bone marrow is thought to play important roles in relation to the inner ear, but is still largely uncharacterized, given this bone complex anatomy. We characterized the geometry and connectivity of rat temporal bone marrow using lightsheet imaging of cleared samples and microCT. Bone marrow was identified in cleared tissue by cellular content (and in particular by the presence of megakaryocytes); since air-filled cavities are absent in rodents, marrow clusters could be recognized in microCT scans by their geometry. In cleared petrosal bone, autofluorescence allowed delineation of the otic capsule layers. Within the endochondral layer, bone marrow was observed in association to the cochlear base and vestibule, and to the cochlear apex. Cochlear apex endochondral marrow (CAEM) was a separated cluster from the remaining endochondral marrow, which was therefore defined as \"vestibular endochondral marrow\" (VEM). A much larger marrow island (petrosal non-endochondral marrow, PNEM) extended outside the otic capsule surrounding semicircular canal arms. PNEM was mainly connected to the dura, through bone channels similar to those of calvarial bone, and only a few channels were directed toward the canal periosteum. On the contrary, endochondral bone marrow was well connected to the labyrinth through vascular loops (directed to the spiral ligament for CAEM and to the bony labyrinth periosteum for VEM), and to dural sinuses. In addition, CAEM was also connected to the tensor tympani fossa of the middle ear and VEM to the endolymphatic sac. Endochondral marrow was made up of small lobules connected to each other and to other structures by channels lined by elongated macrophages, whereas PNEM displayed larger lobules connected by channels with a sparse macrophage population. Our data suggest that the rat inner ear is surrounded by bone marrow at the junctions with middle ear and brain, most likely with \"customs\" role, restricting pathogen spread; a second marrow network with different structural features is found within the endochondral bone layer of the otic capsule and may play different functional roles.

The planar polarized organization of hair cells in the vestibular maculae is unique because these sensory organs contain two groups of cells with oppositely oriented stereociliary bundles that meet at a line of polarity reversal (LPR). EMX2 is a transcription factor expressed by one hair cell group that reverses the orientation of their bundles, thereby forming the LPR. We generated Emx2-CreERt2 transgenic mice for genetic lineage tracing and demonstrate Emx2 expression before hair cell specification when the nascent utricle and saccule constitute a continuous prosensory domain. Precursors labeled by Emx2-CreERt2 at this stage give rise to hair cells located along one side of the LPR in the mature utricle or saccule, indicating that this boundary is first established in the prosensory domain. Consistent with this, Emx2-CreERt2 lineage tracing in Dreher mutants, where the utricle and saccule fail to segregate, labels a continuous field of cells along one side of a fused utriculo-saccular-cochlear organ. These observations reveal that LPR positioning is pre-determined in the developing prosensory domain, and that EMX2 expression defines lineages of hair cells with oppositely oriented stereociliary bundles.