Perilymph Samples Research Articles

Prestin in Human Perilymph, Cerebrospinal Fluid, and Blood as a Biomarker for Hearing Loss.

Determining the concentration of prestin in human blood, cerebrospinal fluid (CSF), and perilymph (PL), and evaluating its suitability as a clinical biomarker for sensori-neural hearing loss (SNHL). Human blood, CSF, and PL samples were intraoperatively collected from 42 patients with tumors of the internal auditory canal or with intracochlear tumors undergoing translabyrinthine or middle fossa tumor removal. Prestin concentration was measured using enzyme-linked immunosorbent assay and linear regression analyses were performed to investigate its associations with audiological as well as vestibular test results. Tertiary referral center. The median prestin concentration in blood samples of the 42 study participants (26 women, mean ± standard deviation age, 52.7 ± 12.5 years) was 1.32 (interquartile range, IQR, 0.71-1.99) ng/mL. CSF prestin levels were significantly higher with 4.73 (IQR, 2.45-14.03) ng/mL (P = .005). With 84.74 (IQR, 38.95-122.00) ng/mL, PL prestin concentration was significantly higher compared to blood (P = .01) and CSF (P = .03) levels. Linear regression analyses showed significant associations of CSF prestin concentration with preoperative hearing levels (pure-tone average and word recognition; P = .008, R2 = 0.1894; P = .03, R2 = 0.1857), but no correlations with blood or PL levels. This study's findings highlight the volatile nature of prestin levels and provide the first insights into this potential biomarker's concentrations in body fluids apart from blood. Future investigations should comprehensively assess human prestin levels with different etiologies of SNHL, prestin's natural homeostasis and systemic circulation, and its temporal dynamics after cochlear trauma. Finally, clinically approved detection kits for prestin are urgently required prior to considering a potential translational implementation of this diagnostic technique.

Hearing loss and its association with the proteome of perilymph, cerebrospinal fluid, and tumor tissue in patients with vestibular schwannoma

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.

Frequency of Herpes Simplex Virus Types 1 and 2 and Cytomegalovirus in Perilymph and Peripheral Blood Samples of Cochlear-Implanted Children Using the Polymerase Chain Reaction Method

Background and Aim: Inner ear infection with some viruses may be one of the possible causes of Sensorineural Hearing Loss (SNHL). This study aims to determine the frequency of Herpes Simplex Virus 1 and 2 (HSV-1, HSV-2) and Cytomegalovirus (CMV) in perilymph and peripheral blood samples of cochlear-implanted children. Methods: In this cross-sectional study, 30 children with severe-to-profound SNHL (aged 1.1–5 years) underwent cochlear implantation surgery. During surgery, their perilymph and peripheral blood samples were collected. The samples were analyzed separately for the presence of herpes HSV-1, HSV-2, and CMV by real-time Polymerase Chain Reaction (PCR) method. The load of IgG and IgM antibodies against these viruses was determined using the Enzyme-Linked Immunosorbent Assay (ELISA) method. Results: The frequency of CMV in perilymph samples was 16.7% (5 patients) and in peripheral blood samples was 3.3% (1 patient). The IgG antibody against CMV and HSV- 1 was positive in 80% and 46.7% of the patients, respectively. The IgM antibody against CMV was positive in 10%. The mean IgM serum antibody load against HSV-1, HSV-2, and CMV was 2.70, 1.70, and 5.47, respectively, and the mean IgG antibody load against these viruses was 56.07, 2.50, and 23.67, respectively. Conclusion: The IgG test is positive in cochlear-implanted children with CMV in their perilymph samples, and the CMV genome is not present in their peripheral blood. This may indicate the previous presence of this virus in the ear and its role in hearing loss. Keywords: Sensorineural hearing loss; perilymph fluid; cytomegalovirus; herpes simplex

Round Window Niche Veil is Visible on High-Resolution Computed Tomography and a Predictor of Local Drug Efficacy to Inner Ear.

To determine the morphologies and effect of the round window niche veil (RWNV) on local drug delivery efficacy and develop diagnostic criteria on high-resolution computed tomography (HRCT). Patients diagnosed with otosclerosis, bilateral profound sensorineural hearing loss or vestibular schwannoma were enrolled from 2019 to 2022, receiving temporal bone HRCT scanning, and anatomic variations of RWMV were summarized intraoperative. For patients with vestibular schwannoma, 1 mL of dexamethasone solution (4 mg/mL) was administered via facial recess during operation, and samples of perilymph were collected to analyze. The diagnostic criteria of RWNV on HRCT were developed and verified. A total of 85 patients were enrolled. RWNV was observed in 54 cases intraoperatively with an incidence of 63.5% (95% CI, 52.9%-73.0%). The median perilymph concentrations were 4.86-fold higher in the group without RWNV than with RWNV (p < 0.0001). RWNV could be visualized on HRCT with a window width of 3500-4500 HU and a window level of 300-500 HU. The characteristic features were as follows: (1) a thin soft tissue shadow could be seen at the entrance of the round window niche (RWN); (2) it was visible in at least 2 consecutive layers along the upper margin of RWN from top to bottom; (3) it was discontinuous with the adjacent bone margin. The sensitivity and specificity of the diagnostic criteria were 77.8% and 93.6%, respectively. RWNV could reduce local dexamethasone diffusion efficacy to the inner ear, which could be diagnosed on HRCT and used as a predictor of local drug delivery efficacy to the inner ear. 3 Laryngoscope, 134:1396-1402, 2024.

Investigation of inner ear drug delivery with a cochlear catheter in piglets as a representative model for human cochlear pharmacokinetics.

Hearing impairment is the most common sensory disorder in humans, and yet hardly any medications are licensed for the treatment of inner ear pathologies. Intricate pharmacokinetic examinations to better understand drug distribution within this complex organ could facilitate the development of novel therapeutics. For such translational research projects, animal models are indispensable, but differences in inner ear dimensions and other anatomical features complicate the transfer of experimental results to the clinic. The gap between rodents and humans may be bridged using larger animal models such as non-human primates. However, their use is challenging and impeded by administrative, regulatory, and financial hurdles. Other large animal models with more human-like inner ear dimensions are scarce. In this study, we analyzed the inner ears of piglets as a potential representative model for the human inner ear and established a surgical approach for intracochlear drug application and subsequent apical sampling. Further, controlled intracochlear delivery of fluorescein isothiocyanate-dextran (FITC-d) was carried out after the insertion of a novel, clinically applicable CE-marked cochlear catheter through the round window membrane. Two, six, and 24hours after a single injection with this device, the intracochlear FITC-d distribution was determined in sequential perilymph samples. The fluorometrically assessed concentrations two hours after injection were compared to the FITC-d content in control groups, which either had been injected with a simple needle puncture through the round window membrane or the cochlear catheter in combination with a stapes vent hole. Our findings demonstrate not only significantly increased apical FITC-d concentrations when using the cochlear catheter but also higher total concentrations in all perilymph samples. Additionally, the concentration decreased after six and 24hours and showed a more homogenous distribution compared to shorter observation times.

Sonoporation of the Round Window Membrane on a Sheep Model: A Safety Study.

Sonoporation using microbubble-assisted ultrasound increases the permeability of a biological barrier to therapeutic molecules. Application of this method to the round window membrane could improve the delivery of therapeutics to the inner ear. The aim of this study was to assess the safety of sonoporation of the round window membrane in a sheep model. To achieve this objective, we assessed auditory function and cochlear heating, and analysed the metabolomics profiles of perilymph collected after sonoporation, comparing them with those of the control ear in the same animal. Six normal-hearing ewes were studied, with one sonoporation ear and one control ear for each. A mastoidectomy was performed on both ears. On the sonoporation side, Vevo MicroMarker® microbubbles (MBs; VisualSonics-Fujifilm, Amsterdam, The Netherlands) at a concentration of 2 × 108 MB/mL were locally injected into the middle ear and exposed to 1.1 MHz sinusoidal ultrasonic waves at 0.3 MPa negative peak pressure with 40% duty cycle and 100 μs interpulse period for 1 min; this was repeated three times with 1 min between applications. The sonoporation protocol did not induce any hearing impairment or toxic overheating compared with the control condition. The metabolomic analysis did not reveal any significant metabolic difference between perilymph samples from the sonoporation and control ears. The results suggest that sonoporation of the round window membrane does not cause damage to the inner ear in a sheep model.

Proteome profile of patients with excellent and poor speech intelligibility after cochlear implantation: Can perilymph proteins predict performance?

Modern proteomic analysis and reliable surgical access to gain liquid inner ear biopsies have enabled in depth molecular characterization of the cochlea microenvironment. In order to clarify whether the protein composition of the perilymph can provide new insights into individual hearing performance after cochlear implantation (CI), computational analysis in correlation to clinical performance after CI were performed based on the proteome profile derived from perilymph samples (liquid biopsies). Perilymph samples from cochlear implant recipients have been analyzed by mass spectrometry (MS). The proteins were identified using the shot-gun proteomics method and quantified and analyzed using Max Quant, Perseus and IPA software. A total of 75 perilymph samples from 68 (adults and children) patients were included in the analysis. Speech perception data one year after implantation were available for 45 patients and these were used for subsequent analysis. According to their hearing performance, patients with excellent (n = 22) and poor (n = 14) performance one year after CI were identified and used for further analysis. The protein composition and statistically significant differences in the two groups were detected by relative quantification of the perilymph proteins. With this procedure, a selection of 287 proteins were identified in at least eight samples in both groups. In the perilymph of the patients with excellent and poor performance, five and six significantly elevated proteins were identified respectively. These proteins seem to be involved in different immunological processes in excellent and poor performer. Further analysis on the role of specific proteins as predictors for poor or excellent performance among CI recipients are mandatory. Combinatory analysis of molecular inner ear profiles and clinical performance data using bioinformatics analysis may open up new possibilities for patient stratification. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

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

IntroductionSensorineural 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 methodsPerilymph 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. ResultsWe 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. ConclusionThis 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.

Isolation of sensory hair cell specific exosomes in human perilymph

Evaluation of hearing loss patients using clinical audiometry has been unable to give a definitive cellular or molecular diagnosis, hampering the development of treatments of sensorineural hearing loss. However, biopsy of inner ear tissue without losing residual hearing function for pathologic diagnosis is extremely challenging. In a clinical setting, perilymph can be accessed, potentially allowing the development of fluid based diagnostic tests. Recent approaches to improving inner ear diagnostics have been focusing on the evaluation of the proteomic or miRNA profiles of perilymph. Inspired by recent characterization and classification of many neurodegenerative diseases using exosomes which not only are produced in locally in diseased tissue but are transported beyond the blood brain barrier, we demonstrate the isolation of human inner ear specific exosomes using a novel ultrasensitive immunomagnetic nano pom-poms capture-release approach. Using perilymph samples harvested from surgical procedures, we were able to isolate exosomes from sensorineural hearing loss patients in only 2–5 μL of perilymph. By isolating sensory hair cell derived exosomes through their expression level of myosin VIIa, we for the first-time sample material from hair cells in the living human inner ear. This work sets up the first demonstration of immunomagnetic capture-release nano pom-pom isolated exosomes for liquid biopsy diagnosis of sensorineural hearing loss. With the ability to isolate exosomes derived from different cell types for molecular characterization, this method also can be developed for analyzing exosomal biomarkers from more accessible patient tissue fluids such as plasma.

Impact of Systemic versus Intratympanic Dexamethasone Administration on the Perilymph Proteome.

Glucocorticoids are the first-line treatment for sensorineural hearing loss, but little is known about the mechanism of their protective effect or the impact of route of administration. The recent development of hollow microneedles enables safe and reliable sampling of perilymph for proteomic analysis. Using these microneedles, we investigate the effect of intratympanic (IT) versus intraperitoneal (IP) dexamethasone administration on guinea pig perilymph proteome. Guinea pigs were treated with IT dexamethasone (n = 6), IP dexamethasone (n = 8), or untreated for control (n = 8) 6 h prior to aspiration. The round window membrane (RWM) was accessed via a postauricular approach, and hollow microneedles were used to perforate the RWM and aspirate 1 μL of perilymph. Perilymph samples were analyzed by liquid chromatography-mass spectrometry-based label-free quantitative proteomics. Mass spectrometry raw data files have been deposited in an international public repository (MassIVE proteomics repository at https://massive.ucsd.edu/) under data set # MSV000086887. In the 22 samples of perilymph analyzed, 632 proteins were detected, including the inner ear protein cochlin, a perilymph marker. Of these, 14 proteins were modulated by IP, and three proteins were modulated by IT dexamethasone. In both IP and IT dexamethasone groups, VGF nerve growth factor inducible was significantly upregulated compared to control. The remaining adjusted proteins modulate neurons, inflammation, or protein synthesis. Proteome analysis facilitated by the use of hollow microneedles shows that route of dexamethasone administration impacts changes seen in perilymph proteome. Compared to IT administration, the IP route was associated with greater changes in protein expression, including proteins involved in neuroprotection, inflammatory pathway, and protein synthesis. Our findings show that microneedles can mediate safe and effective intracochlear sampling and hold promise for inner ear diagnostics.

Automated Sequential Analysis of Hydrophilic and Lipophilic Fractions of Biological Samples: Increasing Single-Injection Chemical Coverage in Untargeted Metabolomics.

In order to increase metabolite coverage in LC–MS-based untargeted metabolomics, HILIC- and RPLC-mode separations are often combined. Unfortunately, these two techniques pose opposite requirements on sample composition, necessitating either dual sample preparations, increasing needed sample volume, or manipulation of the samples after the first analysis, potentially leading to loss of analytes. When sample material is precious, the number of analyses that can be performed is limited. To that end, an automated single-injection LC–MS method for sequential analysis of both the hydrophilic and lipophilic fractions of biological samples is described. Early eluting compounds in a HILIC separation are collected on a trap column and subsequently analyzed in the RPLC mode. The instrument configuration, composed of commercially available components, allows easy modulation of the dilution ratio of the collected effluent, with sufficient dilution to obtain peak compression in the RPLC column. Furthermore, the method is validated and shown to be fit for purpose for application in untargeted metabolomics. Repeatability in both retention times and peak areas was excellent across over 140 injections of protein-precipitated blood plasma. Finally, the method has been applied to the analysis of real perilymph samples collected in a guinea pig model. The QC sample injections clustered tightly in the PCA scores plot and showed a high repeatability in both retention times and peak areas for selected compounds.

Sheep as a large animal model for cochlear implantation

IntroductionIn surgical training, large animal models are more suitable as their anatomy is more similar to humans. In otology, there have been relatively few studies about large animal models for surgical training. ObjectiveIn this study, we aimed to do a neuroradiologic evaluation and surgical insertion of a cochlear implant electrode array on a sheep head model. MethodsTwenty cadaveric sheep heads were studied. A computed tomography scan and neuroradiologic evaluation was performed on each head, obtaining measurements of the inner ear for each sheep. Sheep measurements were compared to those from temporal bone computed tomography scans from 20 female humans. Surgical procedures were first trained with 13 of the sheep temporal bones, after which cochlear implantation was performed on the remaining 7 temporal bones. The position of the inserted electrode array insertion was confirmed by computed tomography scan after the procedure. ResultsNeuroradiologic evaluation showed that, relative to the 20 female humans, the mean ratio for sheep was 0.60 for volume of cochlea, 0.70 for height of cochlea, 0.73 for length of cochlea; ratios for other metrics were >0.80. For the surgical training, the round window was found in all 20 sheep temporal bones. Computed tomography scans confirmed that electrode insertions were fully complete; the mean value of electrode array insertion was 18.3 mm. ConclusionThe neuroradiologic and surgical training data suggest that the sheep is a realistic animal model to train cochlear implant surgery and collection of perilymph samples, but less so for surgical training of mastoidectomy due to pneumatization of the mastoid.

In‐depth proteome of perilymph in guinea pig model

The vast majority of sensorineural hearing loss is caused by impairment of the inner ear cells. Proteomic analysis of perilymph may therefore improve our understanding of inner ear diseases and hearing loss. However, the investigation of the human perilymph proteome was limited due to technical difficulties in perilymph sampling. The guinea pig (Cavia porcellus) is frequently used as an experimental model in preclinical hearing research. In this study, we analyzed samples of perilymph collected from 12 guinea pigs to overcome limited experimental information regarding its proteome. We identified a total of 1413 proteins, establishing a greatly expanded proteome of the previously inferred guinea pig perilymph. This provides a comprehensive proteomic resource for the research community, which will facilitate future molecular-phenotypic studies using the guinea pig as an experimental model of relevance to human inner ear biology.

Novel 3D-printed hollow microneedles facilitate safe, reliable, and informative sampling of perilymph from guinea pigs

BackgroundInner ear diagnostics is limited by the inability to atraumatically obtain samples of inner ear fluid. The round window membrane (RWM) is an attractive portal for accessing perilymph samples as it has been shown to heal within one week after the introduction of microperforations. A 1 µL volume of perilymph is adequate for proteome analysis, yet the total volume of perilymph within the scala tympani of the guinea pig is limited to less than 5 µL. This study investigates the safety and reliability of a novel hollow microneedle device to aspirate perilymph samples adequate for proteomic analysis. MethodsThe guinea pig RWM was accessed via a postauricular surgical approach. 3D-printed hollow microneedles with an outer diameter of 100 µm and an inner diameter of 35 µm were used to perforate the RWM and aspirate 1 µL of perilymph. Two perilymph samples were analyzed by liquid chromatography-mass spectrometry-based quantitative proteomics as part of a preliminary study. Hearing was assessed before and after aspiration using compound action potential (CAP) and distortion product otoacoustic emissions (DPOAE). RWMs were harvested 72 h after aspiration and evaluated for healing using confocal microscopy. ResultsThere was no permanent damage to hearing at 72 h after perforation as assessed by CAP (n = 7) and DPOAE (n = 8), and all perforations healed completely within 72 h (n = 8). In the two samples of perilymph analyzed, 620 proteins were detected, including the inner ear protein cochlin, widely recognized as a perilymph marker. ConclusionHollow microneedles can facilitate aspiration of perilymph across the RWM at a quality and volume adequate for proteomic analysis without causing permanent anatomic or physiologic dysfunction. Microneedles can mediate safe and effective intracochlear sampling and show great promise for inner ear diagnostics.

Relationship between Metabolomics Profile of Perilymph in Cochlear-Implanted Patients and Duration of Hearing Loss.

Perilymph metabolomic analysis is an emerging innovative strategy to improve our knowledge of physiopathology in sensorineural hearing loss. This study aims to develop a metabolomic profile of human perilymph with which to evaluate the relationship between metabolome and the duration of hearing loss. Inclusion criteria were eligibility for cochlear implantation and easy access to the round window during surgery; patients with residual acoustic hearing in the ear to be implanted were excluded. Human perilymph was sampled from 19 subjects during cochlear implantation surgery. The perilymph analysis was performed by Liquid Chromatography−High-Resolution Mass and data were analyzed by supervised multivariate analysis based on Partial Least-Squares Discriminant Analysis and univariate analysis. Samples were grouped according to their median duration of hearing loss. We included the age of patients as a covariate in our models. Statistical analysis and pathways evaluation were performed using Metaboanalyst. Nineteen samples of human perilymph were analyzed, and a total of 106 different metabolites were identified. Metabolomic profiles were significantly different for subjects with ≤12 or >12 years of hearing loss, highlighting the following discriminant compounds: N-acetylneuraminate, glutaric acid, cystine, 2-methylpropanoate, butanoate and xanthine. As expected, the age of patients was also one of the main discriminant parameters. Metabolic signatures were observed for duration of hearing loss. These findings are promising steps towards illuminating the pathophysiological pathways associated with etiologies of sensorineural hearing loss, and hold open the possibilities of further explorations into the mechanisms of sensorineural hearing loss using metabolomic analysis.

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

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. 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. 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. 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. 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.

Defining the Inflammatory Microenvironment in the Human Cochlea by Perilymph Analysis: Toward Liquid Biopsy of the Cochlea.

The molecular pathomechanisms in the majority of patients suffering from acute or progressive sensorineural hearing loss cannot be determined yet. The size and the complex architecture of the cochlea make biopsy and in-depth histological analyses impossible without severe damage of the organ. Thus, histopathology correlated to inner disease is only possible after death. The establishment of a technique for perilymph sampling during cochlear implantation may enable a liquid biopsy and characterization of the cochlear microenvironment. Inflammatory processes may not only participate in disease onset and progression in the inner ear, but may also control performance of the implant. However, little is known about cytokines and chemokines in the human inner ear as predictive markers for cochlear implant performance. First attempts to use multiplex protein arrays for inflammatory markers were successful for the identification of cytokines, chemokines, and endothelial markers present in the human perilymph. Moreover, unsupervised cluster and principal component analyses were used to group patients by lead cytokines and to correlate certain proteins to clinical data. Endothelial and epithelial factors were detected at higher concentrations than typical pro-inflammatory cytokines such as TNF-a or IL-6. Significant differences in VEGF family members have been observed comparing patients with deafness to patients with residual hearing with significantly reduced VEGF-D levels in patients with deafness. In addition, there is a trend toward higher IGFBP-1 levels in these patients. Hence, endothelial and epithelial factors in combination with cytokines may present robust biomarker candidates and will be investigated in future studies in more detail. Thus, multiplex protein arrays are feasible in very small perilymph samples allowing a qualitative and quantitative analysis of inflammatory markers. More results are required to advance this method for elucidating the development and course of specific inner ear diseases or for perioperative characterization of cochlear implant patients.

Detection of BDNF-Related Proteins in Human Perilymph in Patients With Hearing Loss.

The outcome of cochlear implantation depends on multiple variables including the underlying health of the cochlea. Brain derived neurotrophic factor (BDNF) has been shown to support spiral ganglion neurons and to improve implant function in animal models. Whether endogenous BDNF or BDNF-regulated proteins can be used as biomarkers to predict cochlear health and implant outcome has not been investigated yet. Gene expression of BDNF and downstream signaling molecules were identified in tissue of human cochleae obtained from normal hearing patients (n = 3) during skull base surgeries. Based on the gene expression data, bioinformatic analysis was utilized to predict the regulation of proteins by BDNF. The presence of proteins corresponding to these genes was investigated in perilymph (n = 41) obtained from hearing-impaired patients (n = 38) during cochlear implantation or skull base surgery for removal of vestibular schwannoma by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS). Analyzed by mass spectrometry were 41 perilymph samples despite three patients undergoing bilateral cochlear implantation. These particular BDNF regulated proteins were not detectable in any of the perilymph samples. Subsequently, targeted analysis of the perilymph proteome data with Ingenuity Pathway Analysis (IPA) identified further proteins in human perilymph that could be regulated by BDNF. These BDNF regulated proteins were correlated to the presence of residual hearing (RH) prior to implantation and to the performance data with the cochlear implant after 1 year. There was overall a decreased level of expression of BDNF-regulated proteins in profoundly hearing-impaired patients compared to patients with some RH. Phospholipid transfer protein was positively correlated to the preoperative hearing level of the patients. Our data show that combination of gene expression arrays and bioinformatic analysis can aid in the prediction of downstream signaling proteins related to the BDNF pathway. Proteomic analysis of perilymph may help to identify the presence or absence of these molecules in the diseased organ. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

Dexamethasone and Dexamethasone Phosphate Entry into Perilymph Compared for Middle Ear Applications in Guinea Pigs

Dexamethasone phosphate is widely used for intratympanic therapy in humans. We assessed the pharmacokinetics of dexamethasone entry into perilymph when administered as a dexamethasone phosphate solution or as a micronized dexamethasone suspension, with and without inclusion of poloxamer gel in the medium. After a 1-h application to guinea pigs, 10 independent samples of perilymph were collected from the lateral semicircular canal of each animal, allowing entry at the round window and stapes to be independently assessed. Both forms of dexamethasone entered the perilymph predominantly at the round window (73%), with a lower proportion entering at the stapes (22%). When normalized by applied concentration, dexamethasone phosphate was found to enter perilymph far more slowly than dexamethasone, in accordance with its calculated lipid solubility and polar surface area properties. Dexamethasone phosphate therefore has a problematic combination of kinetic properties when used for local therapy of the ear. It is relatively impermeable and enters perilymph only slowly from the middle ear. It is then metabolized in the ear to dexamethasone, which is more permeable through tissue boundaries and is rapidly lost from perilymph. Understanding the influence of molecular properties on the distribution of drugs in perilymph provides a new level of understanding which may help optimize drug therapies of the ear.

The proteome of perilymph in patients with vestibular schwannoma. A possibility to identify biomarkers for tumor associated hearing loss?

BackgroundDue to the surrounding bone, the human inner ear is relatively inaccessible and difficult to reach for cellular and molecular analyses. However, these types of investigations are needed to better understand the etiology, pathophysiology and progression of several inner ear disorders. Moreover, the fluid from the inner ear cannot be sampled for micro-chemical analyses from healthy individuals in vivo. Therefore, in the present paper, we studied patients with vestibular schwannoma (VS) undergoing trans-labyrinthine surgery (TLS). Our primary aim was to identify perilymph proteins in patients with VS on an individual level. Our second aim was to investigate the proteins identified at a functional level and our final aim was to search for biological markers for tumor-associated hearing loss and tumor diameter.Methods and findingsSixteen patients underwent TLS for sporadic VS. Perilymph was aspirated through the round window before opening the labyrinth. One sample was contaminated and excluded resulting in 15 usable samples. Perilymph samples were analyzed with an online tandem LTQ-Orbitrap mass spectrometer. Data were analyzed with MaxQuant software to identify the total number of proteins and to quantify proteins in individual samples. Protein function was analyzed using the PANTHER Overrepresentation tool. Associations between perilymph protein content, clinical parameters, tumor-associated hearing loss and tumor diameter were assessed using Random Forest and Boruta. In total, 314 proteins were identified; 60 in all 15 patients and 130 proteins only once in 15 patients. Ninety-one proteins were detected in at least 12 out of 15 patients. Random Forest followed by Boruta analysis confirmed that alpha-2-HS-glycoprotein (P02765) was an independent variable for tumor-associated hearing loss. In addition, functional analysis showed that numerous processes were significantly increased in the perilymph. The top three enriched biological processes were: 1) secondary metabolic processes; 2) complement activation and 3) cell recognition.ConclusionsThe proteome of perilymph in patients with vestibular schwannoma has an inter-individual stable section. However, even in a cohort with homogenous disease, the variation between individuals represented the majority of the detected proteins. Alpha-2-HS-glycoprotein, P02765, was shown to be an independent variable for tumor-associated hearing loss, a finding that needs to be verified in other studies. In pathway analysis perilymph had highly enriched functions, particularly in terms of increased immune and metabolic processes.