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.

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 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.

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.

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.

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

BACKGROUND: Due 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 FINDINGS: Sixteen 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. CONCLUSIONS: The 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.

Feasibility of microRNA profiling in human inner ear perilymph.

Hearing loss is common and caused by a wide range of molecular and cellular pathologies. Current diagnosis of hearing loss depends on a combination of physiologic testing, patient history, and in some cases genetic testing. Currently, no biopsy or equivalent procedure exists to diagnose inner ear disorders. MicroRNAs (miRNA) are short ribonucleic acids that regulate a variety of cellular processes. They have been found to be reliable markers for a variety of disease processes. In particular, a variety of miRNAs that are markers for neurodegenerative disease have been identified in cerebrospinal fluid. The aim of this study was to determine whether miRNAs could be identified in human perilymph potentially leading to the development of biomarkers for inner ear disease. Prospective sampling of human perilymph and its analysis were carried out. Patients undergoing surgery in which the inner ear is opened as part of the procedure (cochlear implantation, stapedectomy, labyrinthectomy) were recruited. A total of 2-5 µl of perilymph was collected and analyzed using Affymetrix GeneChip miRNA 4.0 microarrays. MiRNA common to all sampling approaches were selected. Analysis of miRNAs was carried out by evaluating miRNA targets in a cochlear transcriptome library using the Ingenuity Pathway Analysis software package. MiRNAs could be isolated from the perilymph of all patients. Evaluation of miRNAs shows the presence of miRNA populations that are predicted to interact with genes expressed in the inner ear. Additional analysis of miRNA populations shows that perilymph miRNAs could be linked to pathways associated with hearing disorders. Sampling of human perilymph is feasible and can potentially identify miRNAs associated with hearing disorders.

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.

Mannitol and the blood-labyrinth barrier.

BACKGROUND: Characterization of the blood labyrinth barrier (BLB) is extremely important to determine whether the BLB can be manipulated pharmacologically. However, experiments to investigate the BLB are technically difficult to perform. In this report, we demonstrated a unique method of controlling the BLB, and established the pharmacokinetics of gentamicin in perilymph, cerebrospinal fluid (CSF) and blood with and without mannitol. STUDY DESIGN: Controlled animal research project. METHODS: Permeability of the BLB and the blood brain barrier (BBB) to gentamicin with and without mannitol was studied by collecting 175 samples from 44 guinea pigs using concentrations relevant to human clinical situations. Samples were taken from two groups of 22 animals, with each animal undergoing sampling at a different time after administration of either 10 mg/ml gentamicin (4 mg/kg) (Gardena, CA) alone or gentamicin with 20% mannitol (250 mg/kg) (Mallinckrodt Inc., KY). The sample times varied from 0.5 to 17.5 h post-infusion. Samples were also taken from 4 animals as negative controls after administration of normal saline. Our goal was to simultaneously assess the pharmacokinetics of gentamicin in each of three different fluid samples in the same animal. Thus at the pre-determined post-infusion sampling time, each animal was sampled once for perilymph, CSF, and blood before being euthanized. Each animal contributed to a single time point on the subsequent pharmacokinetic curves with more than one animal per time point. RESULTS: Mannitol increased the rate of entry and egress of gentamicin through BLB significantly (p = 0.0044) but the effects on the BBB did not reach statistical significance (p = 0.581). Mannitol did not alter renal clearance of gentamicin from the blood (p = 0.433). The concentration of gentamicin in perilymph and CSF was always significantly lower than in blood. CONCLUSIONS: Mannitol administration transiently increases the permeability of the BLB. Potential clinical benefits may accrue from selected timing of administration of osmotic agents such as mannitol augmenting the rate of entry and egress of compounds such as gentamicin into and out of perilymph.

Pharmacokinetic Properties of Adenosine Amine Congener in Cochlear Perilymph after Systemic Administration.

Noise-induced hearing loss (NIHL) is a global health problem affecting over 5% of the population worldwide. We have shown previously that acute noise-induced cochlear injury can be ameliorated by administration of drugs acting on adenosine receptors in the inner ear, and a selective A1 adenosine receptor agonist adenosine amine congener (ADAC) has emerged as a potentially effective treatment for cochlear injury and resulting hearing loss. This study investigated pharmacokinetic properties of ADAC in rat perilymph after systemic (intravenous) administration using a newly developed liquid chromatography-tandem mass spectrometry detection method. The method was developed and validated in accordance with the USA FDA guidelines including accuracy, precision, specificity, and linearity. Perilymph was sampled from the apical turn of the cochlea to prevent contamination with the cerebrospinal fluid. ADAC was detected in cochlear perilymph within two minutes following intravenous administration and remained in perilymph above its minimal effective concentration for at least two hours. The pharmacokinetic pattern of ADAC was significantly altered by exposure to noise, suggesting transient changes in permeability of the blood-labyrinth barrier and/or cochlear blood flow. This study supports ADAC development as a potential clinical otological treatment for acute sensorineural hearing loss caused by exposure to traumatic noise.

In vitro and in vivo pharmacokinetic study of a dexamethasone-releasing silicone for cochlear implants.

Cochlear implants have been widely used for patients with profound hearing loss and partial deafness. Residual low-frequency hearing, however, may deteriorate due to insertion trauma and tissue response around the electrode array. The present study investigated in vitro and in vivo release of dexamethasone from silicone used for cochlear implant electrode carriers. The in vitro experiment involved an apparatus simulating the inner ear fluid environment in humans. Release from two sizes of silicone films (200 µm × 1 mm × 10 mm and 500 µm × 1 mm × 10 mm), each loaded with 2 % dexamethasone, and was measured for 24 weeks. In the in vivo experiment, silicone rods loaded with 2 or 10 % dexamethasone, respectively, were implanted into the scala tympani of guinea pigs. Perilymph concentrations were measured during the first week after implantation. The results showed that dexamethasone was released from the silicone in a sustained manner. After a burst release, perilymph concentration was similar for silicone incorporated with 2 and 10 % dexamethasone, respectively. The similar pharmacokinetic profile was found in the in vitro experiment. The period of sustained drug delivery was maintained for 20 weeks in vitro and for 1 week in vivo. The results of the present study suggest that drugs like dexamethasone are released in a controlled manner from silicon electrode carriers of cochlear implants. Further studies will identify optimal release profiles for the use with cochlear implants to improve their safety and long-term performance.

Effects of a dexamethasone-releasing implant on cochleae: A functional, morphological and pharmacokinetic study.

AIM: This study evaluated the impact of a dexamethasone-releasing silicone implant on hearing function preservation, cochlear morphology and perilymph pharmacokinetics after cochlear implantation. METHODS: Guinea pigs were implanted unilaterally with silicone rods containing either 2% dexamethasone (DEXA group, n = 18) or no dexamethasone (control group, n = 17). Auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAEs) were measured preoperatively and over 6 months postoperatively. Cochlear histology using standard hematoxylin and eosin (H&E) staining and tumor necrosis factor (TNF)-alpha staining was performed 1 month postoperatively. Twenty-two guinea pigs were involved in the pharmacokinetic study, and real-time drug concentrations in perilymph were investigated using high-performance liquid chromatography (HPLC). The Mann-Whitney U test (1-tailed) was used for statistical analyses. RESULTS: ABR and DPOAE testing demonstrated decreased hearing function immediately postoperatively followed by a progressive hearing loss within the first day postoperatively. There was almost no observable hearing improvement in the control group from 1 week to 6 months postoperatively, but hearing levels in the DEXA group improved gradually from 1 week to 12 weeks. Hearing loss in the DEXA and control group was 5.0 ± 3.4 dB and 21.7 ± 5.3 dB, respectively at a 16-kHz stimulus frequency 6 months postoperatively. The difference in threshold shifts was present throughout all measured frequencies, and it was significant at 4-24 kHz. The morphological study revealed new fibrosis formation in the scala tympani, which encapsulated the implanted electrode. TNF-alpha positive staining in the cochleae of the DEXA group was less evident than the control group. The pharmacokinetic study revealed a peak perilymph concentration 30 min postoperatively and sustained dexamethasone release at least 1 week postoperatively. CONCLUSION: Cochlear implants that incorporate dexamethasone can release drug chronically in the inner ear and induce significant long-term recovery and preservation of auditory function after implantation.

Differential effects of AAV.BDNF and AAV.Ntf3 in the deafened adult guinea pig ear.

Cochlear hair cell loss results in secondary regression of peripheral auditory fibers (PAFs) and loss of spiral ganglion neurons (SGNs). The performance of cochlear implants (CI) in rehabilitating hearing depends on survival of SGNs. Here we compare the effects of adeno-associated virus vectors with neurotrophin gene inserts, AAV.BDNF and AAV.Ntf3, on guinea pig ears deafened systemically (kanamycin and furosemide) or locally (neomycin). AAV.BDNF or AAV.Ntf3 was delivered to the guinea pig cochlea one week following deafening and ears were assessed morphologically 3 months later. At that time, neurotrophins levels were not significantly elevated in the cochlear fluids, even though in vitro and shorter term in vivo experiments demonstrate robust elevation of neurotrophins with these viral vectors. Nevertheless, animals receiving these vectors exhibited considerable re-growth of PAFs in the basilar membrane area. In systemically deafened animals there was a negative correlation between the presence of differentiated supporting cells and PAFs, suggesting that supporting cells influence the outcome of neurotrophin over-expression aimed at enhancing the cochlear neural substrate. Counts of SGN in Rosenthal's canal indicate that BDNF was more effective than NT-3 in preserving SGNs. The results demonstrate that a transient elevation in neurotrophin levels can sustain the cochlear neural substrate in the long term.

Enhanced oval window and blocked round window passages for middle-inner ear transportation of gadolinium in guinea pigs with a perforated round window membrane.

To elucidate the communication between the middle and inner ear, and the fluid dynamics of the inner ear with the perilymphatic fistula (PLF) of the round window membrane (RWM). The PLF of the RWM was created in nine guinea pigs. Gadolinium diethylenetriamine pentaacetic acid bismethylamide (Gd-DTPA-BMA) was delivered into the middle ear and followed in the inner ear using a 4.7 Tesla MRI. Pressure was delivered to the external ear canal of PLF ear in an attempt to enhance the inner ear uptake of Gd-DTPA-BMA. The immediate loading of Gd-DTPA-BMA in the scala tympani of the basal turn was ablated by the outflow of perilymph through the leaking RWM while the oval window passage for Gd-DTPA-BMA was enhanced. There was more Gd-DTPA-BMA distribution in the scala tympani than in the scala vestibuli in the second turn of the PLF cochlea (within 20 min). Signal in the vestibulum and scala vestibuli of the basal turn and rest part of PLF cochlea was greater than that of the control cochlea with intact RWM within 30 min. Pressure applied to the external ear canal tended to enhance the loading of Gd-DTPA-BMA in the perilymphatic scalae of the PLF cochlea. The enhanced oval window passage of Gd-DTPA-BMA was proven by the distorted distribution in the inner ear with PLF. The radial communication of cochlear perilymph was supported by the Gd-DTPA-BMA gradient among the perilymphatic scalae. Applying positive pressure to the external ear canal caused backflow of perilymph into the cochlea which has a potential of transmitting microbes from the middle ear into the inner ear.

The mechanism underlying maintenance of the endocochlear potential by the K+ transport system in fibrocytes of the inner ear.

The endocochlear potential (EP) of +80 mV in the scala media, which is indispensable for audition, is controlled by K+ transport across the lateral cochlear wall. This wall includes two epithelial barriers, the syncytium and the marginal cells. The former contains multiple cell types, such as fibrocytes, which are exposed to perilymph on their basolateral surfaces. The apical surfaces of the marginal cells face endolymph. Between the two barriers lies the intrastrial space (IS), an extracellular space with a low K+ concentration ([K+]) and a potential similar to the EP. This intrastrial potential (ISP) dominates the EP and represents the sum of the diffusion potential elicited by a large K+ gradient across the apical surface of the syncytium and the syncytium's potential, which is slightly positive relative to perilymph. Although a K+ transport system in fibrocytes seems to contribute to the EP, the mechanism remains uncertain. We examined the electrochemical properties of the lateral wall of guinea pigs with electrodes sensitive to potential and K+ while perfusing into the perilymph of the scala tympani blockers of Na+,K+-ATPase, the K+ pump thought to be essential to the system. Inhibiting Na+,K+-ATPase barely affected [K+] in the IS but greatly decreased [K+] within the syncytium, reducing the K+ gradient across its apical surface. The treatment hyperpolarized the syncytium only moderately. Consequently, both the ISP and the EP declined. Fibrocytes evidently use the Na+,K+-ATPase to achieve local K+ transport, maintaining the syncytium's high [K+] that is crucial for the K+ diffusion underlying the positive ISP.

Cochlear pharmacokinetics of cisplatin: an in vivo study in the guinea pig.

OBJECTIVES/HYPOTHESIS: Cisplatin produces toxic lesions to outer hair cells (OHCs) in the cochlear base but not in the apex. The objective of this study was to compare the pharmacokinetic profile of cisplatin in scala tympani (ST) perilymph in the cochlear base and apex, respectively. STUDY DESIGN: In vivo animal study. METHODS: Forty-seven guinea pigs were given an intravenous bolus injection of an ototoxic dose of cisplatin. Ten to 240 minutes after cisplatin was given, blood, cerebrospinal fluid (CSF), and ST perilymph were aspirated within the same target time. ST perilymph was aspirated from the basal turn and from the apex of the cochlea by two different sampling techniques. Liquid chromatography with postcolumn derivatization was used for quantitative determination of the parent drug. RESULTS: Ten minutes after administration, the concentration of cisplatin in ST perilymph was 4-fold higher in the basal turn of the cochlea than in the apex. At 30 minutes, the drug concentrations did not differ. At 60 minutes, the level of cisplatin in ST perilymph and blood UF was equivalent. The perilymph-blood ratio increased thereafter with time. CONCLUSION: The pharmacokinetic findings of an early high concentration of cisplatin in the base of the cochlea and delayed elimination of cisplatin from ST perilymph compared to blood might correlate to the cisplatin-induced loss of OHCs in the base of the cochlea.

Pulmonary delivery of d-methionine is associated with an increase in ALCAR and glutathione in cochlear fluids.

In animals, hearing loss resulting from cochlear mechanosensory cell damage can be mitigated by antioxidants such as d-methionine (d-met) and acetyl-l-carnitine (ALCAR). The systemic routes of administration of these compounds, that must of necessity transit trough the cochlear fluids, may affect the antioxidant levels in the cochlea and the resulting oto-protective effect. In this study, we analyzed the pharmacokinetics of [(14)C]d-met in the cochlea and four other tissues after intratracheal (IT), intranasal (IN), and oral by gavage (OG) administration and compared it to intravenous administration (IV). We then analyzed the effect of these four routes on the antioxidant content of the cochlear fluids after d-met or ALCAR administration, by liquid chromatography/mass spectrometry. Our results showed that the concentration of methionine and ALCAR in cochlear fluids significantly increased after their respective systemic administration. Interestingly, d-met administration also contributed to an increase of ALCAR. Our results also showed that the delivery routes differently affected the bioavailability of administered [(14)C]d-met as well as the concentrations of methionine, ALCAR and the ratio of oxidized to reduced glutathione. Overall, pulmonary delivery via IT administration achieved high concentrations of methionine, ALCAR, and oxidative-related metabolites in cochlear fluids, in some cases surpassing IV administration, while IN route appeared to be the least efficacious. To our knowledge, this is the first report of the direct measurements of antioxidant levels in cochlear fluids after their systemic administration. This report also demonstrates the validity of the pulmonary administration of antioxidants and highlights the different contributions of d-met and ALCAR allowing to further investigate their impact on oxidative stress in the cochlear microenvironment.