Disturbance in the protein landscape of cochlear perilymph in an Alzheimer's disease mouse model.

Hearing loss is a pivotal risk factor for dementia. It has recently emerged that a disruption in the intercommunication between the cochlea and brain is a key process in the initiation and progression of this disease. However, whether the cochlear properties can be influenced by pathological signals associated with dementia remains unclear. In this study, using a mouse model of Alzheimer's disease (AD), we investigated the impacts of the AD-like amyloid ß (Aß) pathology in the brain on the cochlea. Despite little detectable change in the age-related shift of the hearing threshold, we observed quantitative and qualitative alterations in the protein profile in perilymph, an extracellular fluid that fills the path of sound waves in the cochlea. Our findings highlight the potential contribution of Aß pathology in the brain to the disturbance of cochlear homeostasis.

The Breakdown of Blood-Labyrinth Barrier Makes it Easier for Drugs to Enter the Inner Ear.

PURPOSE: This study aimed to investigate dynamic change of permeability of blood-labyrinth barrier (BLB) after noise exposure and its effect on the drug delivery efficiency of systemic administration. METHODS: Gadopentetate dimeglumine (Gd-DTPA) and dexamethasone (DEX) were used as tracers, and magnetic resonance imaging (MRI) and immunofluorescence were used to observe the change of the BLB after strong noise exposure in guinea pigs. High-performance liquid chromatography-mass spectrometry (LC-MS) was used to observe the effect of the breakdown of BLB after noise exposure on the drug delivery efficiency of intravenous DEX. The guinea pigs were divided into 6 groups: normal group (N), 1, 3, 5, 8, and 12 days after noise exposure groups (P1, P3, P5, P8, P12), with 5 animals in each group. RESULTS: The BLB changes dynamically after noise exposure. Increased permeability of the blood-endolymph barrier, the endolymph-perilymph barrier, and the blood-nerve barrier was observed at days 1-3, 1-5, and 1-8, respectively, after noise exposure in guinea pigs. Higher drug concentration in the cochlear tissue was obtained by intravenous administration of DEX in guinea pigs during the time window of increased permeability of the BLB. CONCLUSION: After noise exposure, the increased BLB permeability makes it easier for drugs to enter the inner ear from blood. In guinea pigs, 1-8 days after strong noise exposure, the drug delivery efficiency of systemic administration increased. After 8 days, the efficiency gradually returned to normal level. 1-8 days after noise exposure may be the best intervention time for systemic administration. LEVEL OF EVIDENCE: NA Laryngoscope, 134:2377-2386, 2024.

Microneedles Facilitate Small-Volume Intracochlear Delivery Without Physiologic Injury in Guinea Pigs.

HYPOTHESIS: Microneedle-mediated intracochlear injection through the round window membrane (RWM) will facilitate intracochlear delivery, not affect hearing, and allow for full reconstitution of the RWM within 48 hours. BACKGROUND: We have developed polymeric microneedles that allow for in vivo perforation of the guinea pig RWM and aspiration of perilymph for diagnostic analysis, with full reconstitution of the RWM within 48 to 72 hours. In this study, we investigate the ability of microneedles to deliver precise volumes of therapeutics into the cochlea and assess the subsequent consequences on hearing. METHODS: Volumes of 1.0, 2.5, or 5.0 µL of artificial perilymph were injected into the cochlea at a rate of 1 µL/min. Compound action potential (CAP) and distortion product otoacoustic emission were performed to assess for hearing loss (HL), and confocal microscopy was used to evaluate the RWM for residual scarring or inflammation. To evaluate the distribution of agents within the cochlea after microneedle-mediated injection, 1.0 µL of FM 1-43 FX was injected into the cochlea, followed by whole mount cochlear dissection and confocal microscopy. RESULTS: Direct intracochlear injection of 1.0 µL of artificial perilymph in vivo , corresponding to about 20% of the scala tympani volume, was safe and did not result in HL. However, injection of 2.5 or 5.0 µL of artificial perilymph into the cochlea produced statistically significant high-frequency HL persisting 48 hours postperforation. Assessment of RWMs 48 hours after perforation revealed no inflammatory changes or residual scarring. FM 1-43 FX injection resulted in distribution of the agent predominantly in the basal and middle turns. CONCLUSION: Microneedle-mediated intracochlear delivery of small volumes relative to the volume of the scala tympani is feasible, safe, and does not cause HL in guinea pigs; however, injection of large volumes induces high-frequency HL. Injection of small volumes of a fluorescent agent across the RWM resulted in significant distribution within the basal turn, less distribution in the middle turn, and almost none in the apical turn. Microneedle-mediated intracochlear injection, along with our previously developed intracochlear aspiration, opens the pathway for precision inner ear medicine.

Anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the round window membrane.

BACKGROUND: We have developed 3D-printed microneedle technology for diagnostic aspiration of perilymph and intracochlear delivery of therapeutic agents. Single microneedle-mediated round window membrane (RWM) perforation does not cause hearing loss, heals within 48-72 h, and yields sufficient perilymph for proteomic analysis. In this study, we investigate the anatomic, physiologic, and proteomic consequences of repeated microneedle-mediated perforations of the same RWM at different timepoints. METHODS: 100-µm-diameter hollow microneedles were fabricated using two-photon polymerization (2PP) lithography. The tympanic bullae of Hartley guinea pigs (n = 8) were opened with adequate exposure of the RWM. Distortion product otoacoustic emissions (DPOAE) and compound action potential (CAP) were recorded to assess hearing. The hollow microneedle was introduced into the bulla and the RWM was perforated; 1 µL of perilymph was aspirated from the cochlea over the course of 45 s. 72 h later, the above procedure was repeated with aspiration of an additional 1 µL of perilymph. 72 h after the second perforation, RWMs were harvested for confocal imaging. Perilymph proteomic analysis was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Two perforations and aspirations were performed in 8 guinea pigs. In six, CAP, DPOAE, and proteomic analysis were obtained; in one, only CAP and DPOAE results were obtained; and in one, only proteomics results were obtained. Hearing tests demonstrated mild hearing loss at 1-4 kHz and 28 kHz, most consistent with conductive hearing loss. Confocal microscopy demonstrated complete healing of all perforations with full reconstitution of the RWM. Perilymph proteomic analysis identified 1855 proteins across 14 samples. The inner ear protein cochlin was observed in all samples, indicating successful aspiration of perilymph. Non-adjusted paired t-tests with p < 0.01 revealed significant changes in 13 of 1855 identified proteins (0.7%) between the first and second aspirations. CONCLUSIONS: We demonstrate that repeated microneedle perforation of the RWM is feasible, allows for complete healing of the RWM, and minimally changes the proteomic expression profile. Thus, microneedle-mediated repeated aspirations in a single animal can be used to monitor the response to inner ear treatments over time.

Intratympanic steroid treatment can reduce ROS and immune response in human perilymph investigated by in-depth proteome analysis.

Intratympanic (IT) steroid treatment is one of the most widely used and effective treatments for inner ear disorders such as sudden sensorineural hearing loss (SNHL). However, a clear mechanism of IT steroids in inner ear recovery has not yet been revealed. Therefore, we investigated proteome changes in extracted human perilymph after steroid treatment. In this study, we applied a tandem mass spectrometry (MS/MS)-based proteomics approach to discover global proteome changes by comparing human perilymph after steroid treatment with non-treated perilymph group. Using liquid chromatography-MS/MS analysis, we selected 156 differentially expressed proteins (DEPs) that were statistically significant according to Student's t-test. Functional annotation analysis showed that upregulated proteins after steroid treatment are related to apoptosis signaling, as well as reactive oxygen species (ROS) and immune responses. The protein-protein interaction (PPI) clusters the proteins associated with these processes and attempts to observe signaling circuitry, which mediates cellular response after IT steroid treatments. Moreover, we also considered the interactome analysis of DEPs and observed that those with high interaction scores were categorized as having equivalent molecular functions (MFs). Collectively, we suggest that DEPs and interacting proteins in human perilymph after steroid treatment would inhibit the apoptotic and adaptive immune processes that may lead to anti-inflammatory effects.

Perilymph metabolomic and proteomic MALDI-ToF profiling with porous silicon chips: A proof-of-concept study.

INTRODUCTION: Sensorineural hearing losses (SNHLs) are a significant public health issue, and the hearing loss field is desperately in need of effective therapy. Pathophysiological mechanisms are not yet clearly understood in the absence of validated methods to assess the inner ear content. Proteomic and metabolomic analysis of perilymph is opening new research perspectives for SNHLs. We aimed to demonstrate the feasibility of an innovative mass spectrometry (MS) strategy using porous silicon chips (PSCs) to investigate the low molecular weight (LMW) protein and metabolite content of human perilymph. Our second objective was to stratify perilymph samples according to their MS profiles and compare these results with clinical data. MATERIAL AND METHODS: Perilymph samples obtained during cochlear implant surgery from patients with SNHLs were retrieved from a validated biobank. To focus on LMW entities, we used a PSC enrichment protocol before MALDI-ToF MS analysis. PSCs were used as a LMW molecular preanalytical stabilizer and amplifier. Patients' clinical data and SNHL characteristics were retrieved retrospectively from medical charts. RESULTS: We successfully acquired and compared 59 exploitable MS profiles out of 71 perilymph samples. There was a good correlation between duplicates. Comparing both ears from the same patient, we found good reproducibility even when there was a one-year interval between samplings. We identified three distinct groups when comparing the samples' metabolomic profiles and four homogeneous groups comparing their LMW proteome profiles. Clinical data analysis suggested that some groups shared clinical or preanalytical characteristics. CONCLUSION: This proof-of-concept study confirms that LMW proteome and metabolome content of perilymph can be analyzed with PSCs. Based on protein profiles, we managed to stratify perilymp samples according to their molecular composition. These results must be confirmed with a larger population, and sampling methods require improvement, but this approach seems promising. In the future, this approach may pave the way for companion test strategies to precisely diagnose and define potential molecular targets for audioprotective therapies.

Triamcinolone acetonide can be detected in cerebrospinal fluid after intratympanic injection.

Intratympanically applied treatments are of increasing interest to the otologic community to treat sudden sensorineural hearing loss or vestibular disorders but also to deliver gene therapy agents, or biologics to the inner ear. Further diversion from the middle ear and perilymph to blood circulation and cerebrospinal fluid via the cochlear aqueduct are one of the limiting factors and so far not understood well enough. In this study, intratympanically applied triamcinolone acetonide was determined in cerebrospinal fluid. Additionally, perilymph was sampled through the round window membrane as well as at the lateral semicircular canal to determine drug levels. Of the twenty-one included patients, triamcinolone acetonide was quantifiable in cerebrospinal fluid in 43% at very low levels (range 0 ng/ml-6.2 ng/ml) which did not correlate with perilymph levels. Drug levels at the two different perilymph sampling sites were within a range of 13.5 ng/ml to 1180.0 ng/ml. Results suggest an equal distribution of triamcinolone acetonide to semicircular canals, which might support the use of triamcinolone acetonide as a treatment option for vestibular pathologies such as Menièrés disease. On the other hand, the distribution to cerebrospinal fluid might be limiting current approaches in gene therapy where a central distribution is unwanted.

Evaluating Neurotrophin Signaling Using MicroRNA Perilymph Profiling in Cochlear Implant Patients With and Without Residual Hearing.

HYPOTHESIS: MicroRNAs predicted to regulate neurotrophin signaling can be found in human perilymph. BACKGROUND: Animal and human temporal bone studies suggest that spiral ganglion health can affect cochlear implant (CI) outcomes. Neurotrophins have been identified as a key factor in the maintenance of spiral ganglion health. Changes in miRNAs may regulate neurotrophin signaling and may reflect neurotrophin expression levels. METHODS: Perilymph sampling was carried out in 18 patients undergoing cochlear implantation or stapedotomy. Expression of miRNAs in perilymph was evaluated using an Agilent miRNA gene chip. Using ingenuity pathway analysis (IPA) software, miRNAs targeting neurotrophin signaling pathway genes present in a cochlear cDNA library were annotated. Expression levels of miRNAs in perilymph were correlated to the patients' preoperative pure-tone average. RESULTS: Expression of mRNAs coding for neurotrophins and their receptors were identified in tissue obtained from normal human cochlea during skull base surgery. We identified miRNAs predicted to regulate these signaling cascades, including miR-1207-5p, miR-4651, miR-103-3p, miR-100-5p, miR-221-3p, miR-200-3p. There was a correlation between poor preoperative hearing and lower expression of miR-1207 (predicted to regulate NTR3) and miR-4651 (predicted to regulate NTR2). Additionally, miR-3960, miR-4481, and miR-675 showed significant differences in expression level when comparing mild and profound hearing loss patients. CONCLUSIONS: Expression of some miRNAs that are predicted to regulate neurotrophin signaling in the perilymph of cochlear implant patients vary with the patient's level of residual hearing. These miRNAs may serve as biomarkers for changes in neurotrophin signaling.

MicroRNA Profiling in the Perilymph of Cochlear Implant Patients: Identifying Markers that Correlate to Audiological Outcomes.

HYPOTHESIS: MicroRNA (miRNA) expression profiles from human perilymph correlate to post cochlear implantation (CI) hearing outcomes. BACKGROUND: The high inter-individual variability in speech perception among cochlear implant recipients is still poorly understood. MiRNA expression in perilymph can be used to characterize the molecular processes underlying inner ear disease and to predict performance with a cochlear implant. METHODS: Perilymph collected during CI from 17 patients was analyzed using microarrays. MiRNAs were identified and multivariable analysis using consonant-nucleus-consonant testing at 6 and 18 months post implant activation was performed. Variables analyzed included age, gender, preoperative pure tone average (PTA), and preoperative speech discrimination (word recognition [WR]). Gene ontology analysis was performed to identify potential functional implications of changes in the identified miRNAs. RESULTS: Distinct miRNA profiles correlated to preoperative PTA and WR. Patients classified as poor performers showed downregulation of six miRNAs that potentially regulate pathways related to neuronal function and cell survival. CONCLUSION: Individual miRNA profiles can be identified in microvolumes of perilymph. Distinct non-coding RNA expression profiles correlate to preoperative hearing and postoperative cochlear implant outcomes.

MicroRNA Profiling as a Methodology to Diagnose Ménière's Disease: Potential Application of Machine Learning.

OBJECTIVE: Diagnosis and treatment of Ménière's disease remains a significant challenge because of our inability to understand what is occurring on a molecular level. MicroRNA (miRNA) perilymph profiling is a safe methodology and may serve as a "liquid biopsy" equivalent. We used machine learning (ML) to evaluate miRNA expression profiles of various inner ear pathologies to predict diagnosis of Ménière's disease. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary academic hospital. SUBJECTS AND METHODS: Perilymph was collected during labyrinthectomy (Ménière's disease, n = 5), stapedotomy (otosclerosis, n = 5), and cochlear implantation (sensorineural hearing loss [SNHL], n = 9). miRNA was isolated and analyzed with the Affymetrix miRNA 4.0 array. Various ML classification models were evaluated with an 80/20 train/test split and cross-validation. Permutation feature importance was performed to understand miRNAs that were critical to the classification models. RESULTS: In terms of miRNA profiles for conductive hearing loss versus Ménière's, 4 models were able to differentiate and identify the 2 disease classes with 100% accuracy. The top-performing models used the same miRNAs in their decision classification model but with different weighted values. All candidate models for SNHL versus Ménière's performed significantly worse, with the best models achieving 66% accuracy. Ménière's models showed unique features distinct from SNHL. CONCLUSIONS: We can use ML to build Ménière's-specific prediction models using miRNA profile alone. However, ML models were less accurate in predicting SNHL from Ménière's, likely from overlap of miRNA biomarkers. The power of this technique is that it identifies biomarkers without knowledge of the pathophysiology, potentially leading to identification of novel biomarkers and diagnostic tests.

Intratympanic application of poloxamer 407 hydrogels results in sustained N-acetylcysteine delivery to the inner ear.

N-acetylcysteine is a thiol-containing antioxidant, which has shown otoprotective effects in in vitro as well as in vivo models of cisplatin-induced hearing loss. Systemic administration of antioxidants, however, is associated with the major potential drawback of interference with the tumoricidal effect of cisplatin. This therapeutic limitation can be overcome by local intratympanic injection of the antioxidant N-acetylcysteine, which results in very restricted systemic uptake of the drug, whilst intracochlear drug levels are substantially higher. Furthermore, osmolality and pH properties of formulations for intratympanic injection need to be controlled, as they impact the fraction of drug crossing the barriers of the inner ear and could potentially damage middle and inner ear structures. This study focused on (i) the evaluation of concentration-time profiles of N-acetylcysteine in perilymph, cerebrospinal fluid and plasma after intratympanic administration, (ii) the influence of the dosage form, i.e. a thermoreversible poloxamer 407 hydrogel versus a solution, on N-acetylcysteine pharmacokinetics, and (iii) the development of a pH- and osmolality-adjusted formulation for intratympanic N-acetylcysteine delivery. 49 female albino guinea pigs were randomized into two treatment groups, receiving either a single intratympanic injection of a 4% N-acetylcysteine poloxamer 407 hydrogel or a 4% N-acetylcysteine solution. 8 animals served as untreated controls. N-acetylcysteine levels in perilymph, cerebrospinal fluid and plasma were monitored over a period of 24 h. Samples were taken at 1, 3, 6, 12 and 24 h (poloxamer 407 hydrogel group) and 1, 6 and 24 h (solution group) post injection, and analysed by high performance liquid chromatography-tandem mass spectrometry. Intratympanic application of the 4% N-acetylcysteine poloxamer 407 hydrogel resulted in a 4-fold larger perilymph area under the concentration-time curve (0-24 h) than topical administration of the equally concentrated N-acetylcysteine solution but in similar plasma N-acetylcysteine levels. N-acetylcysteine concentrations in the cerebrospinal fluid were below the level of detection (5 ng/ml) in both treatment groups. N-acetylcysteine-containing formulations applied to the middle ear were isohydric and osmolality was reduced by up to 200 mosmol/kg compared to equally concentrated formulations used in previous studies. In summary, we were able to demonstrate that the intratympanic injection of thermoreversible poloxamer 407 hydrogels increases and sustains N-acetylcysteine delivery to the inner ear. Given the low plasma N-acetylcysteine levels after topical application and the physiological pH and osmolality of the hydrogel, the risk of compromising the antineoplastic effects of cisplatin therapy and of local side effects is minimal.

A possible mechanism of the formation of endolymphatic hydrops and its associated inner ear disorders.

The pathology of Meniere's disease (MD) is well established to be endolymphatic hydrops. However, the mechanism underlying deafness and vertigo of MD or idiopathic endolymphatic hydrops is still unknown. In order to evaluate the pathogenesis of deafness and vertigo in MD, it seems to be rational to investigate the interrelationship between hydrops and inner ear disorders using animals with experimentally-induced endolymphatic hydrops. In spite of intense efforts by many researchers, the mechanism of vertiginous attack has been unexplained, because animals with experimental hydrops usually did not show vertiginous attack. Recently, there are two reports to succeed to evoke vertiginous attack in animals with experimental hydrops. In the present paper were first surveyed past proposals about underlying mechanism of the development of hydrops and inner ear disorders associated with hydrops, and were discussed the pathogenetic mechanism of vertiginous attack in hydrops. In conclusion, abrupt development of hydrops was thought to play a pivotal role in the onset of vertiginous seizure.

An LCMS-based untargeted metabolomics protocol for cochlear perilymph: highlighting metabolic effects of hydrogen gas on the inner ear of noise exposed Guinea pigs.

INTRODUCTION: Noise-induced hearing loss (NIHL) is an increasing problem in society and accounts for a third of all cases of acquired hearing loss. NIHL is caused by formation of reactive oxygen species (ROS) in the cochlea causing oxidative stress. Hydrogen gas (H2) can alleviate the damage caused by oxidative stress and can be easily administered through inhalation. OBJECTIVES: To present a protocol for untargeted metabolomics of guinea pig perilymph and investigate the effect of H2 administration on the perilymph metabolome of noise exposed guinea pigs. METHODS: The left ear of guinea pigs were exposed to hazardous impulse noise only (Noise, n = 10), noise and H2 (Noise + H2, n = 10), only H2 (H2, n = 4), or untreated (Control, n = 2). Scala tympani perilymph was sampled from the cochlea of both ears. The polar component of the perilymph metabolome was analyzed using a HILIC-UHPLC-Q-TOF-MS-based untargeted metabolomics protocol. Multivariate data analysis (MVDA) was performed separately for the exposed- and unexposed ear. RESULTS: MVDA allowed separation of groups Noise and Noise + H2 in both the exposed and unexposed ear and yielded 15 metabolites with differentiating relative abundances. Seven were found in both exposed and unexposed ear data and included two osmoprotectants. Eight metabolites were unique to the unexposed ear and included a number of short-chain acylcarnitines. CONCLUSIONS: A HILIC-UHPLC-Q-TOF-MS-based protocol for untargeted metabolomics of perilymph is presented and shown to be fit-for-purpose. We found a clear difference in the perilymph metabolome of noise exposed guinea pigs with and without H2 treatment.

Utility of Perilymph microRNA Sampling for Identification of Active Gene Expression Pathways in Otosclerosis.

HYPOTHESIS: Profiling of microRNA (miRNA) within perilymph samples collected at the time of stapedectomy can be used to identify active gene expression pathways in otosclerosis as compared with controls. BACKGROUND: miRNAs are small non-coding RNAs that effect gene expression by post-transcription regulation and silencing. Perilymph sampling allows for a novel way to collect material actively involved in the disease process. METHODS: Perilymph was collected at time of stapedectomy, underwent a microarray analysis, and significantly expressed miRNAs were correlated to known bone morphology pathways using a cochlear transcriptome library. To determine miRNA related specifically to otosclerosis, cochlear implant controls were used for statistical analysis. RESULTS: A total of 321 significantly expressed miRNAs were identified within the four otosclerosis perilymph samples. miRNAs associated with 23 genes involved in bone morphology pathways were significantly expressed. A significant difference in the otosclerotic samples as compared with control was noted in miRNA expression regulating HMGA2, ITGB3, SMO, CCND1, TP53, TP63, and RBL2 gene pathways. No significant difference was noted in miRNAs expression associated with ACE, RELN, COL1A1, and COL1A2 genes which were previously correlated with otosclerosis. CONCLUSIONS: Perilymph miRNA profiling obtained at the time of stapedectomy consistently identifies differentially expressed genes compared with controls. Perilymph miRNA sampling with cochlear transcriptome library cross-referencing can be successfully used to identify active gene expression pathways in otosclerosis.

Proteome of normal human perilymph and perilymph from people with disabling vertigo.

The vast majority of hearing loss, the most common sensory impairment, and vertigo, which commonly causes falls, both reflect underlying dysfunction of inner ear cells. Perilymph sampling can thus provide molecular cues to hearing and balance disorders. While such "liquid biopsy" of the inner ear is not yet in routine clinical practice, previous studies have uncovered alterations in perilymph in patients with certain types of hearing loss. However, the proteome of perilymph from patients with intact hearing has been unknown. Furthermore, no complete characterization of perilymph from patients with vestibular dysfunction has been reported. Here, using liquid-chromatography with tandem mass spectrometry, we analyzed samples of normal perilymph collected from three patients with skull base meningiomas and intact hearing. We identified 228 proteins that were common across the samples, establishing a greatly expanded proteome of the previously inferred normal human perilymph. Further comparison to perilymph obtained from three patients with vestibular dysfunction with drop attacks due to Meniere's disease showed 38 proteins with significantly differential abundance. The abundance of four protein candidates with previously unknown roles in inner ear biology was validated in murine cochleae by immunohistochemistry and in situ hybridization: AACT, HGFAC, EFEMP1, and TGFBI. Together, these results motivate future work in characterizing the normal human perilymph and identifying biomarkers of inner ear disease.

Multivariate discrimination of heat shock proteins using a fiber optic Raman setup for in situ analysis of human perilymph.

Raman spectroscopy has proven to be an effective tool for molecular analysis in different applications. In clinical diagnostics, its application has enabled nondestructive investigation of biological tissues and liquids. The human perilymph, for example, is an inner ear liquid, essential for the hearing sensation. The composition of this liquid is correlated with pathophysiological parameters and was analyzed by extraction and mass spectrometry so far. In this work, we present a fiber optic probe setup for the Raman spectroscopic sampling of inner ear proteins in solution. Multivariate data analysis is applied for the discrimination of individual proteins (heat shock proteins) linked to a specific type of hearing impairment. This proof-of-principle is a first step toward a system for sensitive and continuous in vivo perilymph investigation in the future.

Using Machine Learning to Predict Sensorineural Hearing Loss Based on Perilymph Micro RNA Expression Profile.

Hearing loss (HL) is the most common neurodegenerative disease worldwide. Despite its prevalence, clinical testing does not yield a cell or molecular based identification of the underlying etiology of hearing loss making development of pharmacological or molecular treatments challenging. A key to improving the diagnosis of inner ear disorders is the development of reliable biomarkers for different inner ear diseases. Analysis of microRNAs (miRNA) in tissue and body fluid samples has gained significant momentum as a diagnostic tool for a wide variety of diseases. In previous work, we have shown that miRNA profiling in inner ear perilymph is feasible and may demonstrate distinctive miRNA expression profiles unique to different diseases. A first step in developing miRNAs as biomarkers for inner ear disease is linking patterns of miRNA expression in perilymph to clinically available metrics. Using machine learning (ML), we demonstrate we can build disease specific algorithms that predict the presence of sensorineural hearing loss using only miRNA expression profiles. This methodology not only affords the opportunity to understand what is occurring on a molecular level, but may offer an approach to diagnosing patients with active inner ear disease.

Is oval window transport a royal gate for nanoparticle delivery to vestibule in the inner ear?

Drug delivery to the inner ear by nanomedicine strategies has emerged as an effective therapeutic approach for the management of inner ear diseases including hearing and balance disorders. It is well accepted that substance enters the perilymph from the middle ear through the round window membrane (RWM), but the passage through the oval window (OW) has long been neglected. Up to now, researchers still know little about the pathway via which nanoparticles (NPs) enter the inner ear or how they reach the inner ear following local applications. Herein, we engineered fluorescence traceable chitosan (CS) NPs, investigated the NP distribution within cochlear and vestibular organs, and assessed the availability of RWM and OW pathways to NP transport. Intriguingly, there were high levels of CS NPs in vestibular hair cells, dark cells and supporting cells, but negligible ones in cochlear hair cells and epithelial cells after intratympanic administration. However, the NPs were visualized in two cell models, L929 and HEI-OC1 cell lines, and in the hair cells of cochlear explants after co-incubation in vitro. These combined studies implied that CS NPs might enter the vestibule directly through the OW and then preferentially accumulated in the cells of vestibular organs. Thus, in vivo studies were carried out and clearly revealed that CS NPs entered the inner ear through both the RWM and OW, but the latter played a governing role in delivering NPs to the vestibule with vivid fluorescence signals in the thin bone of the stapes footplate. Overall, these findings firstly suggested that the OW, as a royal gate, afforded a convenient access to facilitate CS NPs transport into inner ear, casting a new light on future clinical applications of NPs in the effective treatment of vestibular disorders by minimizing the risk of hearing loss associated with cochlear hair cell pathology.

Adjuvant agents enhance round window membrane permeability to dexamethasone and modulate basal to apical cochlear gradients.

Glucocorticoids have direct anti-inflammatory, anti-oxidant and anti-apoptotic effects on cochlear hair cells. Cochlear glucocorticoid therapy has gained particular attention for its ability to enhance the protection of residual hearing following hearing preservation cochlear implantation. Local drug delivery methods achieve high drug concentrations within the inner ear fluids but are reliant upon diffusion across the round window membrane. Diffusion has been shown to demonstrate large individual variability. This study explores the role of "adjuvant agents", which when administered with glucocorticoids, enhance inner ear absorption and distribution. Guinea pig cochleae were administered either dexamethasone alone or in combination with hyaluronic acid, histamine, or combination histamine and hyaluronic acid, targeted at the round window membrane. Control subjects received saline. Perilymph was sampled from the cochlear apex, and basal to apical dexamethasone concentrations recorded with mass spectroscopy. Cochleae were harvested, and immunohistochemistry employed to explore dexamethasone tissue penetration and distribution. Basal to apical gradients were observed along the scala tympani, with higher dexamethasone concentrations observed at the cochlear base. Gradients were more pronounced and uniform when administered on a hyaluronic acid sponge, while histamine increased absolute concentrations reaching the inner ear. Tissue penetration correlated with perilymph concentration. Our results demonstrate that adjuvant agents can be employed to enhance dexamethasone absorption and distribution in the inner ear, thus proposing therapeutic strategies that may enhance steroid facilitated hearing protection.

Intracochlear drug delivery: Fluorescent tracer evaluation for quantification of distribution in the cochlear partition.

Measurement of drug distribution in the inner ear has important roles in the design of local delivery methods, such as direct, intracochlear delivery, and in assessment of emerging drug candidates in preclinical animal models. Sampling methods have been used in the past to measure drug concentrations in the cochlear fluids, but these methods provide no direct information about drug distribution in the cochlear tissues. In this work, we evaluated four fluorescent markers that simulate drug distribution in the organ of Corti after intracochlear delivery to the cochlea's scala tympani compartment. Our hypothesis is that ultimately, a cocktail comprising several fluorescent drug surrogates or fluorescently-tagged drugs, each with differing distribution, spreading, and clearance behavior, can be used to evaluate both transient and cumulative drug distributions associated with different delivery techniques. In this study, FITC-dextran, Qtracker™ 655, gentamicin Texas-Red, and FM 1-43 FX were each evaluated as candidate markers by direct intracochlear infusion into guinea-pig cochleae. Distribution of the markers was measured using fluorescence confocal microscopy imaging of cochlear whole mount dissections from animals sacrificed 3 h after the tracer-infusion. For all four tracers, strong fluorescence was observed in the tissue sections near the base, but only Qtracker™-655, gentamicin Texas-Red (GTTR) and FM 1-43 FX exhibited any specificity in labelling of the sensory hair cells. Therefore, these substances represent leading candidates for the quantification drug distribution achieved by different delivery approaches to the scala tympani.