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.

The role of monocytes and macrophages in the dynamic permeability of the blood-perilymph barrier.

The blood-perilymph barrier serves a critical role by separating the components of blood from inner ear fluids, limiting traffic of cells, proteins and other solutes into the labyrinth, and allowing gas (O2-CO2) exchange. Inflammation produces changes in the blood-perilymph barrier resulting in increased vascular permeability. It is commonly thought that compromise of the blood-inner ear barrier would lead to hearing impairment through loss of the endocochlear potential (EP). In fact, the effect of increasing cochlear vascular permeability on hearing function and EP is poorly understood. We used a novel method to measure the integrity of the blood-perilymph barrier and demonstrated the effects of barrier compromise on ABR threshold and EP. We also investigated the contribution of CX3CR1 cochlear macrophages and CCR2 inflammatory monocytes to barrier function after systemic exposure to lipopolysaccharide (LPS). We found that systemic LPS induced a profound change in vascular permeability, which correlated with minimal change in ABR threshold and EP. Macrophage depletion using CX3CR1-DTR mice did not alter the baseline permeability of cochlear vessels and resulted in preservation of barrier function in LPS-treated animals. We conclude that cochlear macrophages are not required to maintain the barrier in normal mice and activated macrophages are a critical factor in breakdown of the barrier after LPS. CCR2 null mice demonstrated that LPS induction of barrier leakiness occurs in the absence of CCR2 expression. Thus, enhanced aminoglycoside ototoxicity after LPS can be linked to the expression of CCR2 in inflammatory monocytes, and not to preservation of the blood-perilymph barrier in CCR2 knockout mice.

Estimation of perilymph enhancement after intratympanic administration of Gd-DTPA by fast T1-mapping with a dual flip angle 3D spoiled gradient echo sequence.

Eleven patients with suspected Ménière's disease received intratympanic (IT) administration of gadolinium (gadopentetate dimeglumine; Gd) prior to acquisition of 3-dimensional (3D) fluid-attenuated inversion recovery (FLAIR), 3D real inversion recovery (IR), and fast T1 mapping by dual flip angle 3D gradient echo (FT1-map) imaging sequences to evaluate the degree of perilymph enhancement. Though 3-dimensional FLAIR could detect lower concentrations of gadolinium than 3D real IR, in 2 patients, poor enhancement still prevented visualization of the endolymphatic space using 3D FLAIR. We could predict poor contrast enhancement in these 2 patients using the FT1-map technique.

Gadolinium distribution in cochlear perilymph: differences between intratympanic and intravenous gadolinium injection.

INTRODUCTION: Three-dimensional fluid-attenuated inversion recovery (3D-FLAIR) imaging 24 h after intratympanic gadolinium injection (IT method) or 4 h after intravenous injection (IV method) has been used to visualize endolymphatic hydrops in Ménière's disease. The aims of this study were to evaluate the difference in gadolinium distribution in cochlear perilymph between the two methods by comparing the enhancement of the basal and apical turns and clarify the pharmacokinetics in cochlear perilymph. METHODS: A total of 24 ears of 22 patients who underwent the IT method (gadolinium-diethylene-triamine pentaacetic acid was diluted eightfold with saline) and 28 ears of 17 patients who underwent the IV method (double dose of gadoteridol (0.5 mmol/ml); 0.2 mmol/kg body weight in total amount) at 3 T was analyzed retrospectively. Regions of interest of the perilymph of the cochlear basal turn (B), of the apical turn (A), and the medulla oblongata (M) were determined on each patient. The signal intensity ratios between B and M (BMR), A and M (AMR), and A and B (ABR) were subsequently evaluated. RESULTS: The IT-BMR (2.63 ± 1.22) was higher than the IV-BMR (1.46 ± 0.45) (p < 0.001). There was no significant difference between the IT- (1.46 ± 0.76) and IV-AMRs (1.21 ± 0.48) (p = 0.15). The IT-ABR (0.58 ± 0.17) was lower than the IV-ABR (0.84 ± 0.22) (p < 0.001). CONCLUSION: Gadolinium was predominantly distributed in the basal turn compared with the apical turn in the IT method, whereas it was more uniformly distributed in the IV method. These characteristics might reflect the distribution of therapeutic medications administered either intratympanically or systemically.

Imaging of endolymphatic and perilymphatic fluid after intravenous administration of single-dose gadodiamide.

To visualize endolymph as bright signal after intravenous injection of single-dose gadodiamide, we shortened the inversion time of heavily T(2)-weighted 3-dimensional (3D) fluid-attenuated inversion recovery (FLAIR) to 2050 ms. In 14 patients with suspected Ménière's disease, we observed high signal of vestibular endolymph in all ears, including 6 ears without vestibular endolymphatic hydrops. We observed high signal of cochlear endolymph in 17 ears with cochlear endolymphatic hydrops but not 11 ears without cochlear endolymphatic hydrops.

Comparison of contrast effect on the cochlear perilymph after intratympanic and intravenous gadolinium injection.

BACKGROUND AND PURPOSE: 3D-FLAIR imaging 24 hours after intratympanic gadolinium injection (IT-method) or 4 hours after IV injection (IV-method) has been used to visualize the endolymphatic hydrops in Ménière disease. The purpose of this study was to compare the degree of perilymph enhancement with the 2 methods and the perilymph contrast-effect difference with the IV-method in both sides in patients with unilateral Ménière disease. MATERIALS AND METHODS: Sixty-one patients with Ménière disease or sudden SNHL were included in this study. Thirty-nine patients who underwent the unilateral IT-method (Gd-DTPA was diluted 8-fold with saline) and 22 patients who underwent the IV-method (a double-dose of Gd-HP-DO3A; 0.4 mL/kg body weight [ie, 0.2 mmol/kg body weight]) at 3T were analyzed retrospectively. Regions of interest of the cochlear perilymph and the medulla oblongata were determined on each image, and the signal-intensity ratio between the 2 (CM ratio) was subsequently evaluated. The differences in the CM ratio between the 2 methods (Student t test) and the IV-method CM ratio between the affected and unaffected sides in patients with unilateral Ménière disease (paired t test) were evaluated. RESULTS: The IT-method CM ratio (2.98 ± 1.15, n = 39) was higher than the IV-method CM ratio (1.61 ± 0.60, n = 44; P < .001). In patients with unilateral Ménière disease who underwent the IV-method (n = 9), the CM ratio of the affected side (1.86 ± 0.74) was higher than that of the unaffected side (1.29 ± 0.31, P < .05). CONCLUSIONS: In general, the IT-method provides higher perilymph enhancement than the IV-method. In the patients with unilateral Ménière disease who underwent the IV-method, the affected side had a higher contrast effect.

Transplantation of mouse embryonic stem cells into the cochlea of an auditory-neuropathy animal model: effects of timing after injury.

Application of ouabain to the round window membrane of the gerbil selectively induces the death of most spiral ganglion neurons and thus provides an excellent model for investigating the survival and differentiation of embryonic stem cells (ESCs) introduced into the inner ear. In this study, mouse ESCs were pretreated with a neural-induction protocol and transplanted into Rosenthal's canal (RC), perilymph, or endolymph of Mongolian gerbils either 1-3 days (early post-injury transplant group) or 7 days or longer (late post-injury transplant group) after ouabain injury. Overall, ESC survival in RC and perilymphatic spaces was significantly greater in the early post-injury microenvironment as compared to the later post-injury condition. Viable clusters of ESCs within RC and perilymphatic spaces appeared to be associated with neovascularization in the early post-injury group. A small number of ESCs transplanted within RC stained for mature neuronal or glial cell markers. ESCs introduced into perilymph survived in several locations, but most differentiated into glia-like cells. ESCs transplanted into endolymph survived poorly if at all. These experiments demonstrate that there is an optimal time window for engraftment and survival of ESCs that occurs in the early post-injury period.

Time-varying alterations in the f2-f1 DPOAE response to continuous primary stimulation. II. Influence of local calcium-dependent mechanisms.

The distortion product otoacoustic emission (DPOAE) corresponding to the frequency f2-f1 displays stereotyped, time-varying amplitude alterations during continuous primary tone stimulation. The origin of these alterations is unknown; however, evidence that efferent neurons contribute little to the changes has been presented (Kujawa et al., 1994a, 1995; Lowe and Robertson, 1995). The present investigation examines the hypothesis that these alterations in f2-f1 amplitude are a reflection of local, Ca(2+)-dependent mechanisms involving the outer hair cell (OHC) response to sustained stimulation. Experiments were performed using urethane-anesthetized guinea pigs with sectioned middle ear muscles. Intracochlear perfusion was employed to reversibly lower perilymph Ca2+ levels and to introduce antagonists and agonists of L-type Ca2+ channels. Manipulations that lowered available Ca2+ (zero Ca2+ artificial perilymph; zero Ca2+ with BAPTA) or that blocked its entry into the cell via L-type Ca2+ channels (nimodipine) reduced, prevented or reversed the perstimulatory changes in f2-f1 DPOAE amplitude. These perilymph manipulations also reduced the overall amplitude of this distortion component while perfusion of an L-type Ca2+ channel agonist (Bay K 8644) increased its amplitude. Mg2+ did not substitute for Ca2+, suggesting that these are not merely divalent cation effects. Results are consistent with the hypothesis that continuous stimulation-related changes in f2-f1 DPOAE amplitude are sensitive to perilymph Ca2+ levels and to the function of L-type Ca2+ channels. However, nimodipine also reduced the endocochlear potential (EP) and Bay K 8644 increased the EP. The sensitivity of both the perstimulatory changes in f2-f1 DPOAE amplitude and the EP to the latter drugs leaves their site(s) of action unresolved.

Electrochemical potentials and potassium concentration profiles recorded from perilymph, endolymph and associated inner ear tissues in adrenalectomized rats.

This study evaluated the electrochemical potentials and potassium concentration (Ck+) profiles in the perilymph, endolymph, marginal cells, and spiral ligament of adrenalectomized rats in which endogenous corticosteroids had been removed. Electrochemical potentials recorded at the four cochlear sites were not affected by adrenalectomy (ADX). Ck+ was greater in the endolymph of the ADX animals as compared to control animals. Additionally, there was an increase of Ck+ in the marginal cells, perilymph, and spiral ligament tissues of the ADX animals as compared to control animals, although the observed increases were not statistically significant. In a previous study (Ma et al., 1995a), it was found that potassium levels in the blood plasma of ADX animals were higher than those identified in normal rats; thus, ADX may have a systemic effect on Ck+ that is detectable in both tissues and fluids within the cochlea. Even though Ck+ was elevated within the cochlea in the ADX model, the functional response of the inner ear, as assessed electrophysiologically, was not altered.

Fluid pathways in temporal bones.

Five temporal bones were serially sectioned and studied concerning spread of erythrocytes and blood-derived precipitate in patients who died from subarachnoid hemorrhage. Erythrocytes followed the natural pathways--the cochlear aqueduct, the cochlear, vestibular, facial and glossopharyngeal nerves, and were demonstrable in the inner ear fluid spaces. The temporal bone marrow spaces were also filled with erythrocytes, particularly in the hypotympanal area. In the microimmunoelectrophoresis, no specific precipitation lines formed between the anti-CSF serum from rabbits and middle ear exudate from human ears with acute otitis media. Although, in animals, middle ear spaces have been shown to be connected to the middle ear space this does not seem to apply to human ears, and CSF fluid components are not involved in the formation of middle ear exudate.

[A biochemical and cytological explanation of cochlear otospongiosis]. / Une explication biochimique et cytologique de l'otospongiose cochleaire

In a study of 250 otosclerotic perilymphatic fluids, it was found statistically that in the presence of developing inner ear deafness, 90% of cases will demonstrate increased trypsin activity. Conversely, elevated antitrypsin levels are found in the absence of developing inner ear deafness.