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.

Evaluation of Levels of Triamcinolone Acetonide in Human Perilymph and Plasma After Intratympanic Application in Patients Receiving Cochlear Implants: A Randomized Clinical Trial.

Importance: The use of intratympanically applied steroids is of increasing interest. Consequently, research has focused on finding an ideal drug that diffuses through the round window membrane and can be retained in the perilymph. Objective: To compare levels of triamcinolone acetonide (TAC) in perilymph and plasma after intratympanic injection. Design, Setting, and Participants: This randomized clinical trial included 40 patients receiving cochlear implants at a single tertiary care center in Vienna, Austria. Patients were randomized to 1 of 4 treatment groups receiving 1 of 2 intratympanic doses of TAC (10 mg/mL or 40 mg/mL) at 1 of 2 approximate time points (24 hours or 1 hour) before sampling the perilymph. Inclusion was carried out between November 2017 and January 2020, and data were analyzed in December 2020. Interventions: All patients underwent intratympanic injection of TAC. During cochlear implantation, perilymph and plasma were sampled for further analysis. Main Outcomes and Measures: Levels of TAC measured in perilymph and plasma. Results: Among the 37 patients (median [range] age, 57 [26-88] years; 18 [49%] men) included in the analysis, TAC was present at a median (range) level of 796.0 (46.4-7706.7) ng/mL. In the majority of patients (n = 29; 78%), no drug was detectable in the plasma after intratympanic injection. Levels above the limit of detection were less than 2.5 ng/mL. The 1-factorial analysis of variance model showed lower TAC levels in the group that received TAC, 10 mg/mL, 24 hours before surgery (median, 271 ng/mL) compared with the group that received TAC, 10 mg/mL, 1 hour before surgery (median, 2877 ng/mL), as well as in comparison with the groups receiving TAC, 40 mg/mL, 24 hours before surgery (median, 2150 ng/mL) and 1 hour before surgery (median, 939 ng/mL). The 2-factorial analysis of variance model showed lower TAC levels in the group receiving TAC, 10 mg/mL, 24 hours before surgery than the group receiving TAC, 10 mg/mL, 1 hour before surgery, and higher TAC levels in the group receiving TAC, 40 mg/mL, 24 hours before surgery compared with the group receiving TAC, 10 mg/mL, 24 hours before surgery. Patients with thickening of the middle ear had statistically significantly higher plasma levels (median, 1.4 ng/mL vs 0 ng/mL) and lower perilymph levels (median, 213.1 ng/mL vs 904 ng/mL) than individuals with unremarkable middle ear mucosa. Conclusions and Relevance: In this randomized clinical trial, TAC was shown to be a promising drug for intratympanic therapies, with similar levels in perilymph 1 hour and 24 hours after injection (distinctly in the groups receiving the 40 mg/mL dose). There was also minimal dissemination to the plasma, especially in patients with unremarkable middle ear mucosa. Trial Registration: ClinicalTrials.gov Identifier: NCT03248856.

Endogenous α1-antitrypsin levels in the perilymphatic fluid correlates with severity of hearing loss.

OBJECTIVES: To determine the levels of endogenous α1-antitrypsin in the perilymph of patients undergoing cochlear implant (CI), and its reverse association with the severity of hearing loss. STUDY DESIGN: Retrospective study. SETTING: Tertiary care university hospital. PARTICIPANTS: The study includes 38 patients undergoing CI surgery, 11 patients diagnosed with congenital deafness and 27 non-congenital deafness, eight patients diagnosed with moderate hearing loss (N = 8; PTA = 70 dB), severe hearing loss (N = 11; PTA 70-90 dB) and profound hearing loss (N = 19; PTA > 90 dB). MAIN OUTCOME AND MEASURE: 1 to 12 µL perilymphatic fluids were collected by micropipette. α1-antitrypsin levels were determined, and current and historic audiological parameters were obtained. RESULTS: The congenital and non-congenital group exhibited AAT concentrations of 2.5 ± 1.9 × 106 LFQ and 3.2 ± 1.2 × 106 LFQ, respectively (mean ± SD; P = .38). Mean levels of α1-antitrypsin in the perilymph fluid within the moderate group was 3.64 × 106  ± 2.1 × 106 LFQ vs 3.5 × 106  ± 1.2 × 106 in severe hearing loss (P = .81) and 2.4 × 106  ± 1.1 × 106 LFQ in the profound hearings loss group (P = .06). The difference in levels of AAT in samples from the severe hearings loss group vs the profound hearings loss group reached statistical significance (P = .04). CONCLUSION: Insufficiency in α1-antitrypsin levels in the perilymph fluid of the inner ear appears to display a relationship with the severity of hearing loss. The prospect of introducing clinical-grade plasma-purified α1-antitrypsin directly onto the site of cochlear injury deserves thorough investigation.

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.

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.

Density difference between perilymph and endolymph: A new hypothesis for light cupula phenomenon.

Light cupula is an emerging concept accounting for positional nystagmus. It can be diagnosed when persistent geotropic direction-changing positional nystagmus (PG DCPN) is observed in a head-roll test. Although hypotheses explaining light cupula phenomenon such as "light debris", "lighter cupula", and "heavier endolymph" have been proposed, the mechanism underlying light cupula has not been clearly elucidated yet. In the present study, we proposed a new hypothesis accounting for light cupula, i.e., density difference between perilymph and endolymph could elicit characteristic PG DCPN in a head-roll test. We also discussed the mechanism how membranous canal containing endolymph became buoyant within the perilymphatic space under constant influence of gravity when the density of perilymph was higher than that of endolymph.

Development and performance of a biomimetic artificial perilymph for in vitro testing of medical devices.

OBJECTIVE: Cochlear implants interface with the fluid in the cochlea called perilymph. The volume of this fluid present in human and animal model cochlea is prohibitively low for isolation for in vitro studies. Thus, there is a need for an artificial perilymph that reflects the complexity of this fluid in terms of competitive protein adsorption. APPROACH: This study established a biomimetic artificial perilymph (BAP) comprising serum albumin, immunoglobulin G, transferrin, inter-alpha-trypsin inhibitor, apolipoprotein A1 and complement C3 to represent the major components of human perilymph. Adsorption of the BAP components to platinum was analysed. MAIN RESULTS: It was established that this six component BAP provided competitive and complex adsorption behaviours consistent with biologically derived complex fluids. Additionally, adsorption of the BAP components to platinum cochlear electrodes resulted in a change in polarisation impedance consistent with that observed for the cochlear device in vivo. SIGNIFICANCE: This study established a BAP fluid suitable for furthering the understanding of the implant environment for electroactive devices that interface with the biological environment.

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.

A Polymer-Based Extended Release System for Stable, Long-term Intracochlear Drug Delivery.

OBJECTIVE: Investigate a new polymer-based drug coating suitability for safe intracochlear delivery and ability to maintain long-term physiologically active levels of the corticosteroid fluticasone propionate. STUDY DESIGN: In vitro dissolution study to evaluate release profiles of polymer-coated drug particles and in vivo studies using a guinea pig model to measure perilymph drug concentrations at specific time points after implantation with polymer-coated drug particles and evaluate their effect on hearing function. METHODS: Polymer-coated fluticasone propionate (FP) particles were surgically implanted in guinea pigs through the round window membrane into the cochlear scala tympani. In the pilot study, pre- and post-op hearing thresholds were conducted on days 7, 14, and 42. In a second study, post-op hearing thresholds were conducted on days 90, 120, and 180. Perilymph drug concentrations were measured on the same time points. RESULTS: In 15 of 16 animals from day 7 through day 90, drug levels were within the targeted range, with no initial burst release detected. Drug was present in all animals on day 90 and was detected in some animals at 120 and 180 days. Hearing was tested and compared with non-implanted ears. Very good hearing preservation was observed in ears implanted with intracochlear particles when compared with contralateral ears. CONCLUSIONS: The polymer-based extended release system is effective in providing long-term, stable drug delivery for at least 90 days with good hearing outcomes. The results of this study support the potential for achieving long-term drug delivery with a single intracochlear administration.

Rapid analysis of neomycin in cochlear perilymph of guinea pigs using disposable SPE cartridges and high performance liquid chromatography-tandem mass spectrometry.

Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100 × 2.1 mm, 3 µm) at 40 °C in gradient mode with 0.2‰ (v/v) HFBA in water and 0.2‰ (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30 mL/min, with retention time of 3.50 and 3.62 min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1-115% of nominal values) and precise (CV% < 15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200 µg/mL, with LOD and LLOQ of 0.625 and 1.25 µg/mL in blank matrix. The matrix effect was corrected to (-0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and -20 °C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.

Validation of metabolomics analysis of human perilymph fluid using liquid chromatography-mass spectroscopy.

Although there is some data from animal studies, the metabolome of inner ear fluid in humans remains unknown. Characterization of the metabolome of the perilymph would allow for better understanding of its role in auditory function and for identification of biomarkers that might allow prediction of response to therapeutics. There is a major technical challenge due to the small sample of perilymph fluid available for analysis (sub-microliter). The objectives of this study were to develop and validate a methodology for analysis of perilymph metabolome using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Due to the low availability of perilymph fluid; a methodological study was first performed using low volumes (0.8 µL) of cerebrospinal fluid (CSF) and optimized the LC-HRMS parameters using targeted and non-targeted metabolomics approaches. We obtained excellent parameters of reproducibility for about 100 metabolites. This methodology was then used to analyze perilymph fluid using two complementary chromatographic supports: reverse phase (RP-C18) and hydrophilic interaction liquid chromatography (HILIC). Both methods were highly robust and showed their complementarity, thus reinforcing the interest to combine these chromatographic supports. A fingerprinting was obtained from 98 robust metabolites (analytical variability <30%), where amino acids (e.g., asparagine, valine, glutamine, alanine, etc.), carboxylic acids and derivatives (e.g., lactate, carnitine, trigonelline, creatinine, etc.) were observed as first-order signals. This work lays the foundations of a robust analytical workflow for the exploration of the perilymph metabolome dedicated to the research of biomarkers for the diagnosis/prognosis of auditory pathologies.

Permeation Enhancers for Intratympanically-applied Drugs Studied Using Fluorescent Dexamethasone as a Marker.

HYPOTHESIS: Entry of locally applied drugs into the inner ear can be enhanced by chemical manipulations. BACKGROUND: Perilymph drug concentrations achieved by intratympanic applications are well below the applied concentration due to limited entry through the round window (RW) membrane and stapes. Chemical manipulations to increase entry permeability could increase the effectiveness of drug therapy with local applications. METHODS: Dexamethasone-fluorescein (F-dex) was used as an entry marker. F-dex was applied to the RW niche of guinea pigs as a 20 µL bolus of 1 mM solution. After a 1 hour application, 10 samples of perilymph were collected sequentially from the lateral semicircular canal, allowing F-dex distribution throughout the perilymph to be quantified. Entry was also measured with the applied solution additionally containing dimethyl sulfoxide (DMSO), N-methylpyrrolidone (NMP), saponin, caprate, benzyl alcohol (BA) or poloxamer 407 (P407). Combinations of saponin or BA with P407 were also compared. RESULTS: In control experiments, F-dex entered the inner ear slowly at both the RW and stapes. The total F-dex recovered in all 10 samples from each animal averaged 2.1 pMoles for controls, 1.71 pMoles for 17% P407, 3.70 pMoles for caprate, 8.04 pMoles for DMSO, 16.32 pMoles for NMP, 31.0 pMoles for saponin, and 67.3 pMoles for 4% BA. Entry with DMSO, NMP, saponin and 4% BA were all significantly higher than the controls (one-way ANOVA). CONCLUSION: These studies confirm that entry of drugs into the ear can be markedly enhanced with the use of chemical permeation-enhancing agents.

Heat Shock Proteins in Human Perilymph: Implications for Cochlear Implantation.

OBJECTIVE: Biomarkers reflecting the etiology and pathophysiology of inner ear diseases are limited. Evaluation of proteins in the perilymph may improve our understanding of inner ear disease. Heat shock proteins (HSP) belong to a superfamily of stress proteins and promote refolding of denatured proteins. The aim of the study was to analyze HSP in human perilymph and to identify possible correlation with audiological and etiologic data. METHODS: Sampling of the perilymph was performed during cochlear implantation and vestibular schwannoma removal. Individual proteins were identified by a shot-gun proteomics approach by orbitrap mass spectrometry. Expression of HSP genes was determined in human cochlear tissue that was obtained during transcochlear surgeries. RESULTS: Ten subgroups of HSP were identified in human perilymph samples. Increased levels of HSP were detected in a higher percentage in the perilymph of patients with residual hearing when compared with patients with no residual hearing in cochlear implantation. In patients with complete preservation of residual hearing, HSP 90 is identified in a lower percentage whereas HSP 70 1A/1B and 6 was identified in all the samples. Constitutive expression of HSP family members was verified in normal cochlear tissue. CONCLUSION: The 10 HSP variants are not identified in all the perilymph samples, but in a higher proportion in patients with residual hearing compared with patients with no residual hearing. In-depth proteome analysis of perilymph samples in correlation to patients' audiogram data shows an increased concentration of HSP in patients with residual hearing. An increase in specific HSP in patients with loss of residual hearing after cochlear implantation was not observed.

Cannula-based drug delivery to the guinea pig round window causes a lasting hearing loss that may be temporarily mitigated by BDNF.

Sustained local delivery of drugs to the inner ear may be required for future regenerative and protective strategies. The round window is surgically accessible and a promising delivery route. To be viable, a delivery system should not cause hearing loss. This study determined the effect on hearing of placing a drug-delivery microcatheter on to the round window, and delivering either artificial perilymph (AP) or brain-derived neurotrophic factor (BDNF) via this catheter with a mini-osmotic pump. Auditory brainstem responses (ABRs) were monitored for 4 months after surgery, while the AP or BDNF was administered for the first month. The presence of the microcatheter - whether dry or when delivering AP or BDNF for 4 weeks - was associated with an increase in ABR thresholds of up to 15 dB, 16 weeks after implantation. This threshold shift was, in part, delayed by the delivery of BDNF. We conclude that the chronic presence of a microcatheter in the round window niche causes hearing loss, and that this is exacerbated by delivery of AP, and ameliorated temporarily by delivery of BDNF.

Proteome Analysis of Human Perilymph Using an Intraoperative Sampling Method.

The knowledge about the etiology and pathophysiology of sensorineural hearing loss (SNHL) is still very limited. This study aims at the improvement of understanding different types of SNHL by proteome analysis of human perilymph. Sampling of perilymph was established during inner ear surgeries (cochlear implantation, vestibular schwannoma surgeries), and safety of the sampling method was determined by checking hearing threshold with pure-tone audiometry postoperatively. An in-depth shot-gun proteomics approach was performed to identify cochlear proteins and the individual proteome in perilymph of patients. This method enables the identification and quantification of protein composition of perilymph. The proteome of 41 collected perilymph samples with volumes of 1-12 µL was analyzed by data-dependent acquisition, resulting in overall 878 detected protein groups. At least 203 protein groups were solely identified in perilymph, not in reference samples (serum, cerebrospinal fluid), displaying a specific protein pattern for perilymph. Samples were grouped by patient's age and surgery type, leading to the identification of some proteins specific to particular subgroups. Proteins with different abundances between different sample groups were subjected to classification by gene ontology annotations. The identified proteins might serve as biomarkers to develop tools for noninvasive inner ear diagnostics and to elucidate molecular profiles of SNHL.

Enhanced local bioavailability of single or compound drugs delivery to the inner ear through application of PLGA nanoparticles via round window administration.

In this paper, the potential of poly(D,L-lactide-co-glycolide acid) (PLGA) nanoparticles (NPs) for carrying single or compound drugs traversing the round window membrane (RWM) was examined after the round window (RW) administration of different NPs to guinea pigs. First, coumarin-6 was incorporated into PLGA NPs as a fluorescent probe to investigate its ability to cross the RWM. Then, PLGA NPs with salvianolic acid B (Sal B), tanshinone IIA (TS IIA), and total panax notoginsenoside (PNS) including notoginsenoside R1 (R1), ginsenoside Rg1 (Rg1), and ginsenoside Rb1 (Rb1) were developed to evaluate whether NPs loaded with compound drugs would pass through the RWM and improve the local bioavailability of these agents. PLGA NPs loaded with single or compound drugs were prepared by the emulsification solvent evaporation method, and their particle size distribution, particle morphology, and encapsulation efficiency were characterized. In vitro release study showed sustained-release profiles of Sal B, TS IIA, and PNS from the NPs. The pharmacokinetic results showed that NPs applied to the RWM significantly improved drug distribution within the inner ear. The AUC0-t of coumarin-6 in the perilymph (PL) following RW administration of NPs was 4.7-fold higher than that of coumarin-6 solution, and the Cmax was 10.9-fold higher. Furthermore, the AUC(0-t) of R1, Rg1, and Rb1 were 4.0-, 3.1-, and 7.1-fold greater, respectively, after the application of NPs compared to the compound solution, and the Cmax were, respectively, 14.4-, 10.0-, and 16.7-fold higher. These findings suggest that PLGA NPs with unique properties at the nanoscale dimensions have a powerful ability to transport single or compound drugs into the PL through the RWM and remarkably enhance the local bioavailability of the encapsulated drugs in the inner ear. The use of PLGA NPs as nanoscale delivery vehicles to carry drugs across the RWM may be a promising strategy for the treatment of inner ear diseases.

Beta-trace protein in pediatric otitis media with effusion.

OBJECTIVES: The middle ear cleft connects by membranes to the inner ear and the subarachnoid space particularly in infants. In order to gain more insight about the permeability between the two compartments we quantified the concentration of beta-trace protein - a highly specific marker for CSF and perilymph but not for serum and mucosal effusion - in middle ear secretions from children with otitis media with effusion. METHODS: One-hundred and three patients were included and 93 samples from secretory otitis media were collected during myringotomy or explorative tympanotomy. Thirty-eight patients of 103 had to be excluded (36.9%). Of the 93 collected samples from 65 subjects, 82 viscous samples were pre-diluted 1:1 with tyloxapol. In spite of the attempt to pre-dilute the viscous samples, 30 glue-like samples of 93 were not applicable for nephelometry. The final analysis was made on 63 samples of 52 subjects (median age 3 years) which were quantified for beta-trace protein using immunonephelometry. RESULTS: In 3/63 samples the beta-trace protein values were below the detection range (<0.2 mg/L) and in 1/63 it was beyond with 18.3mg/L. The median beta-trace protein value for 59 samples within the measuring range was 2.4 mg/L, range 0.2-14.2. CONCLUSION: In pediatric middle ear effusions, the beta-trace protein concentration was found to have a high range compared to other body fluids from other studies. In other studies, the values for serum (0.59 mg/L) or mucosal secretion (0.003-0.12 mg/L) were lower and the values in CSF (18.4 mg/L) or perilymphatic fluid (23.5 mg/L) were highest. This finding might indicate a weak barrier between the cerebrospinal fluid space or inner ear fluid compartments on the one side and the tympanic cavity on the other side given the condition of otitis media with effusion. The detection of beta-trace protein might be important to assess the risk of impending complications.

A novel aerosol-mediated drug delivery system for inner ear therapy: intratympanic aerosol methylprednisolone can attenuate acoustic trauma.

We developed a novel aerosol-mediated drug delivery system for inner ear therapy by using a silicon-based multiple-Fourier horn nozzle. Intratympanic aerosol (ITA) methylprednisolone (MP) delivery can protect hearing after acoustic trauma. The highest concentration of MP (38.9 ± 5.47 ppm) appeared at 2 h and declined rapidly within 10 h. The concentrations of MP remained at a relatively low level for more than 10 h. Compared to the baseline, the auditory brainstem response (ABR) thresholds shifted markedly at 1 h after noise exposure in all groups (p < 0.05). From the cochleograms, it can be noted that the main lesions encompassed the 2-20 kHz frequency range. Significant differences ( ) were observed for the range between 5 and 8 kHz in the cell loss of outer hair cells (OHCs). The losses for IHCs were lower than for OHCs. The MP movement in the middle ear was simulated by a convection diffusion equation with a relaxation time. The relaxation time was 0.5 h, and the concentration threshold of MP on the round window membrane (RWM) in the middle ear (C T) was 8900 ppm. Using the unit hydrograph (UH) method, we obtained a proper boundary concentration on the RWM at the cochlea, which resulted in a well-fit concentration. Finally, a linking mechanism between the middle ear and the cochlea was established by the RWM. The adjustable permeability and concentration threshold provide the flexibility to match the peak times and peak values of the concentration on the RWM in the middle ear and the cochlea.

Proteome of human perilymph.

Current diagnostic tools limit a clinician's ability to discriminate between many possible causes of sensorineural hearing loss. This constraint leads to the frequent diagnosis of the idiopathic condition, leaving patients without a clear prognosis and only general treatment options. As a first step toward developing new diagnostic tools and improving patient care, we report the first use of liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to map the proteome of human perilymph. Using LC-MS/MS, we analyzed four samples, two collected from patients with vestibular schwannoma (VS) and two from patients undergoing cochlear implantation (CI). For each cohort, one sample contained pooled specimens collected from five patients and the second contained a specimen obtained from a single patient. Of the 271 proteins identified with high confidence among the samples, 71 proteins were common in every sample and used to conservatively define the proteome of human perilymph. Comparison to human cerebrospinal fluid and blood plasma, as well as murine perilymph, showed significant similarity in protein content across fluids; however, a quantitative comparison was not possible. Fifteen candidate biomarkers of VS were identified by comparing VS and CI samples. This list will be used in future investigations targeted at discriminating between VS tumors associated with good versus poor hearing.

Intratympanic versus intravenous delivery of dexamethasone and dexamethasone sodium phosphate to cochlear perilymph.

OBJECTIVE: To measure dexamethasone concentrations in the plasma and perilymph of the human ear after intravenous (IV) and intratympanic (IT) administration and to compare these with previous studies with methylprednisolone. METHODS: Patients were administered dexamethasone by the IT or IV routes approximately 0.5 to 2 hours before cochlear implantation. The IT dose of 1.6 to 7.2 mg (0.4-1.8 ml of a 4 mg/ml solution) of dexamethasone sodium phosphate was administered by injection into the middle ear cavity through the external auditory canal via a 27-gauge needle passed through a small anterosuperior myringotomy. The IV dose of dexamethasone sodium phosphate was 0.17 mg/kg given as a single injection for 30 seconds. A sample of perilymph (approximately 20 µl) was collected using a needle passed through the round window, and blood was sampled simultaneously. Concentrations of free dexamethasone and dexamethasone sodium phosphate were measured using a validated liquid chromatography-tandem mass spectrometry method. RESULTS: In the 22 patients studied, 22 perilymph samples and 19 plasma samples were available and suitable for measurement. The median perilymph concentration of dexamethasone after IV injection of 0.17 mg/kg was 0.016 mg/L (n = 9; range, 0.008-0.17), and 1.4 mg/L (n = 13; range, 0.1-16.3) after IT administration of approximately 4 mg. Perilymph concentrations were approximately 88-fold higher after IT compared with IV administration (p = 0.0004) or approximately 260 fold after correction for dosage. The median plasma concentration of dexamethasone after IV injection was 0.12 mg/L (n = 7; range, 0.07-0.14) and 0.003 mg/L (n = 12; range, <0.0005-0.005) after IT injection. Plasma concentrations were approximately 40-fold lower (p = 0.0005) or approximately 13-fold lower after dose correction. Concentrations of dexamethasone sodium phosphate were more variable and were even higher in perilymph and lower in plasma. CONCLUSION: Administration of dexamethasone IT results in much higher perilymph concentrations and much lower plasma concentrations compared with IV administration.