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Observations on the stria vascularis of the guinea pig cochlea and the changes resulting from the administration of the diuretic furosemide.

An electron microscope examination of the stria vascularis of guinea pigs and the structural changes occurring after administration of furosemide has been made. The use of ruthenium-red, which stains cell coats, has shown that the atria is impermeable to the passive diffusion of material from both the endolymph and the spiral ligament. The first changes after administration of furosemide are observed 4 hours after injection; small spaces develop between the cells. After 5--6 hours the spaces have enlarged but the tight junctions between marginal cells remain intact. The intermediate cells show signs of atrophy. In severely affected animals the intermediate cells have so shrunk that the spaces are very large and the marginal cells clearly resolved. These latter cells show no structural damage. In this severely affected tissue it is seen that the blood vessels are entirely surrounded by marginal cell extensions. It is suggested that the intermediate cells may have a regulatory function. The damage appears to be recoverable, since 6 hours after injection the spaces have decreased in size although some signs of intermediate cell atrophy are still apparent.

Animals↗

The effects of calcium buffering and cyclic AMP on mechano-electrical transduction in turtle auditory hair cells.

1. The effects of intracellular Ca2+ buffering on hair cell mechanotransduction were studied in an intact cochlear epithelium where the endolymphatic and perilymphatic surfaces could be separately perfused with different Ca2+ solutions. 2. The speed and extent of transducer adaptation increased as the concentration in the patch electrode of the Ca2+ buffer BAPTA was lowered. In 0.1 mM BAPTA or less, the transducer adapted almost completely, with a mean time constant of 0.8 ms. 3. For a fixed internal BAPTA concentration, the transducer conductance varied with hair cell location, increasing towards the high-frequency end of the cochlea, and the time constant of adaptation decreased proportionally. At a given cochlear location, hair cells with larger transducer conductances displayed faster adaptation. We suggest that transducer adaptation accounts for a variable high-pass filter observed in the acoustic tuning curve. 4. The effects of perfusion of 50 microM Ca2+ endolymph depended on the BAPTA concentration of the electrode: with 3 mM BAPTA, adaptation was abolished, but in most recordings with 0.01 or 0.1 mM BAPTA, rapid adaptation was retained. The current-displacement curve was also shifted less the lower the intracellular BAPTA concentration. Cells in the high-frequency half of the papilla retained adaptation at a higher BAPTA concentration. 5. Treatment with the cAMP agonist, 8-bromo-cAMP, or with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, caused a rightward shift in the current-displacement curve which was independent of the internal BAPTA concentration. 6. We conclude that the free Ca2+ and cyclic nucleotide concentrations of the hair bundle modulate the position of the activation curve of the transducer. The factors which may be important for the correct functioning of adaptation in vivo are discussed.

8-Bromo Cyclic Adenosine Monophosphate↗

KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential.

Stria vascularis of the cochlea generates the endocochlear potential and secretes K(+). K(+) is the main charge carrier and the endocochlear potential the main driving force for the sensory transduction that leads to hearing. Stria vascularis consists of two barriers, marginal cells that secrete potassium and basal cells that are coupled via gap junctions to intermediate cells. Mice lacking the KCNJ10 (Kir4.1) K(+) channel in strial intermediate cells did not generate an endocochlear potential. Endolymph volume and K(+) concentration ([K(+)]) were reduced. These studies establish that the KCNJ10 K(+) channel provides the molecular mechanism for generation of the endocochlear potential in concert with other transport pathways that establish the [K(+)] difference across the channel. KCNJ10 is also a limiting pathway for K(+) secretion.

Animals↗

Computational model of vectorial potassium transport by cochlear marginal cells and vestibular dark cells.

Cochlear marginal cells and vestibular dark cells transport potassium into the inner ear endolymph, a potassium-rich fluid, the homeostasis of which is essential for hearing and balance. We have formulated an integrated mathematical model of ion transport across these epithelia that incorporates the biophysical properties of the major ion transporters and channels located in the apical and basolateral membranes of the constituent cells. The model is constructed for both open- and short-circuit situations to test the extremes of functional capacity of the epithelium and predicts the steady-state voltages, ion concentrations, and transepithelial currents as a function of various transporter and channel densities. We validate the model by establishing that the cells are capable of vectorial ion transport consistent with several experimental measurements. The model indicates that cochlear marginal cells do not make a significant direct contribution to the endocochlear potential and illustrates how changes to the activity of specific transport proteins lead to reduced K(+) flux across the marginal and dark cell layers. In particular, we investigate the mechanisms of loop diuretic ototoxicity and diseases with hearing loss in which K(+) and Cl(-) transport are compromised, such as Jervell and Lange-Nielsen syndrome and Bartter syndrome, type IV, respectively. Such simulations demonstrate the utility of compartmental modeling in investigating the role of ion homeostasis in inner ear physiology and pathology.

Bartter Syndrome↗

A cationic nonselective stretch-activated channel in the Reissner's membrane of the guinea pig cochlea.

The Reissner's membrane (RM) separates in the mammalian cochlea the K(+)-rich endolymph from the Na(+)-rich perilymph. The patch-clamp technique was used to investigate the transport mechanisms in epithelial cells of RM freshly dissected from the guinea pig cochlea. This study shows a stretch-activated nonselective cationic channel (SA channel) with a linear current-voltage relationship (23 pS) highly selective for cations over anions [K+ approximately Na+ (1) > Ba2+ (0.65) > Ca2+ (0.32) >> Cl- (0.14)] and activated by the intrapipette gradient pressure. The open probability-pressure relationship is best fitted by a Boltzmann distribution (half-maximal pressure = 37.8 mmHg, slope constant = 8.2 mmHg). SA channels exhibit a strong voltage dependency and are insensitive to internal Ca2+, ATP, and fenamates but are blocked by 1 microM GdCl3 in the pipette. They are reversibly activated by in situ superfusion of the cell with hyposmotic solutions. Kinetic studies show that depolarization and mechanical or osmotic stretch modify the closed and open time constants probably by a different mechanism. These channels could participate in pressure-induced modifications of ionic permeability of the RM.

Animals↗

Determinants of semicircular canal afferent response dynamics in the toadfish, Opsanus tau.

1. Present results determine the relative contributions of the biomechanical and the posttransduction-current (PTC) mechanisms to the sensory process carried out by the horizontal semicircular canal (HC) in the oyster toadfish, Opsanus tau. The role of each element was estimated using in vivo measurements of hair cell receptor potentials and afferent responses elicited by electrical stimuli and mechanical HC indentation. Individual afferent response dynamics are defined here using first-harmonic transfer functions presented in the form of response gain and phase for sinusoidal stimuli from approximately 0.02-30 Hz. Comparison of the response dynamics for the two types of stimuli distinguishes the mechanical and the PTC transfer functions leading to the neural response. The results show that both mechanisms contribute significantly to the overall signal processing performed by the semicircular canals. 2. Endolymphatic polarization and HC indentation. Modulation of the endolymphatic potential by current injection induces a differential voltage across the apical face of the hair cells that drives the transduction current directly via the Nernst-Planck potential. Results show that the electrical impedance of the apical tight junctions is much larger than the basal impedance to ground in O. tau, such that leakage current to the basolateral space is negligible and the voltage-sensitive basolateral currents remain fully functional during polarization of the endolymph (in the frequency range tested). Extracellular afferent responses to endolymphatic polarization were combined with responses to HC indentation to separate the relative contributions of the mechanical and the PTC mechanisms to the overall afferent response dynamics. Data show that more than one-half of the overall signal processing, as defined by the first-harmonic transfer function, persists even when canal mechanics is bypassed. 3. Hair-cell receptor potential modulation during HC indentation. Sharp microelectrodes were used to record the modulation of hair-cell receptor potentials (intracellular voltages) in vivo during physiological levels of sinusoidal HC indentation. Receptor potentials exhibit modulations dominated by the first harmonic and centered about the resting potential. The average gain of the receptor-potential modulation for HC indentation is approximately 0.88 mV/microns indent, corresponding to a value of 0.22 mV/deg/s head velocity, centered near zero phase over the range tested from 0.1-10 Hz. The present receptor potential data fall well short of spanning the full range of gain and phase present in the afferent population. Rather, intracellular hair-cell responses are consistent with the frequency-dependent mechanical activation of the transduction current as determined above. 4. Origins of individual afferent responses. The population of afferent responses forms a continuous distribution that is discussed here in terms of three groups as defined by Boyle and Highstein: velocity-sensitive low gain (LG) afferents, velocity/acceleration-sensitive high gain (HG) afferents, and acceleration-sensitive (A) afferents. The response dynamics of individual afferents were found to be determined by a mix of biomechanical and biophysical factors that vary systematically between these afferent groups. All afferents show low-frequency phase advancement and gain decrease during HC indentation associated with the mechanical lower-corner frequency and high-frequency phase and gain enhancements associated with the PTC processing. In highly phase-advanced afferents (A type), the mechanical response is additive with the PTC processing to achieve broad-band acceleration sensitive neural responses.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Contribution of endolymphatic fluid shift to caloric response in plugged semicircular canals.

The purpose of this study was to clarify the role of endolymphatic fluid shift in caloric response, using frog posterior semicircular canals (PSCs). PSCs were sutured using 10-0 nylon thread and were used as a model of canal plugging. Compound action potentials (CAPs) of the PSC nerve evoked by a cooling stimulus were recorded. The CAPs after suturing the PSCs were found to be greater than those before suturing. This indicates that the fluid shift effect increases after canal suturing. Additionally, we present a clinical case in which caloric nystagmus was observed after lateral canal plugging. In this case MRI revealed the fluid space from the plugged portion toward the ampulla to be intact. There was another case with lateral canal plugging that showed the same findings on MRI. The above findings support the hypothesis that fluid shift is responsible for the caloric response without the convective flow of endolymph in the plugged canal.

Action Potentials↗

Na,K-ATPase subunit isoform expression in the guinea pig endolymphatic sac.

Na,K-ATPase subunit isoform expression was studied by immunocytochemistry in the guinea pig endolymphatic sac, using subunit isoform-specific polyclonal antibodies. Epithelial cells of the guinea pig endolymphatic sac were observed to contain the alpha 1- and beta 2-subunit isoforms, and to a lesser extent the beta 1-subunit isoform, of Na,K-ATPase. The alpha 1- and beta 2-subunit isoforms of Na,K-ATPase have been observed previously in other ion and fluid transporting regions of the membranous labyrinth, e.g., stria vascularis and vestibular dark cells. Combined data indicate that the alpha 1-, beta 2-form of Na,K-ATPase plays a role in the microhomeostasis of endolymph. The alpha 1 beta 2 Na,K-ATPase subunit isoform combination is different from typical ion and fluid transporting tissues, e.g., kidney and colon, and may reflect distinctive characteristics of inner ear Na,K-ATPase.

Animals↗

Effect of 4 kHz tone exposure on the guinea pig inner ear: relation in the change of cochlear microphonics, action potential, electrochemical potential and K+ ion concentration induced by noise exposure.

Using 110 male albino guinea pigs, and applying electrophysiological methods, we investigated the effects of 4 kHz tone at moderate sound pressure level on the inner ear at different exposure times (6, 12, 24 and 48 h). Longer than 12 h exposure to 4 kHz tone had a considerable effect on the hearing, inducing a decrease in the maximum output voltage of cochlear microphonics and an increase in the K+ concentration in the endolymph.

Action Potentials↗

Kinetics of gentamicin uptake and release in the rat. Comparison of inner ear tissues and fluids with other organs.

The kinetics of entry and release of gentamicin was investigated in fluids and tissues of the inner ear of the rat, as well as in renal cortex, and in organs that do not share susceptibility to the toxic effects of aminoglycosides. Various modes of administration were used to achieve different patterns of drug plasma concentrations. Electrophysiological and histological examinations were performed to correlate pharmacokinetics and ototoxicity. Results show that: the uptake of the drug by the inner ear tissues is dose dependent and manifests a rapid saturation kinetics with a concentration plateau of about 1 micrograms/mg of protein. The low ratio of the perilymph and endolymph to plasma concentrations argues against the concept of an accumulation of the drug in the inner ear over drug levels in plasma, which has been considered as the basic mechanism of ototoxicity. In renal cortex, the kinetics appears similar to that of the inner ear but the concentrations achieved are 10-fold higher than in cochlear tissues. In other organs (liver, heart, lung, and spleen), no saturation could be demonstrated within the duration of the experiment. Ototoxicity seems to be related to the penetration of the drug into compartment(s) from which the half-life of disappearance is extremely slow. Rapid uptake, early saturation, and long exposure of the tissues to the drug may account for the development of toxicity in inner ear and kidney.

Animals↗

Rupture pressures of membranes in the ear.

The rupture pressures of the tympanic membrane, Reissner's membrane, the round window membrane, and the annular ligament have all been measured in cadaver ears from Norwegian cattle. For the tympanic membrane, a static overpressure was applied to the ear canal; for Reissner's membrane, to the endolymph; and for the round window membrane, to the perilymph. The rupture pressure of the annular ligament equals the rupture force to the footplate divided by the area of the oval window. The mean rupture pressures are 0.39 atm for the tympanic membrane, 0.047 atm for Reissner's membrane, greater than 2 atm for the round window membrane, and 29.4 atm for the annular ligament. This last pressure corresponds to 0.68 kilogram force applied to the footplate. The ruptures of the tympanic membrane appeared without exception as small tears in the pars flaccida. The rupture pressure of the tympanic membrane was also measured in a few ears from foxes.

Animals↗

Volumetric and dimensional analysis of the guinea pig inner ear.

The objective of this study was to provide accurate volumetric data on the fluid spaces and soft tissue in the guinea pig inner ear by measuring all histologic serial sections by means of Metamorph Imaging Software at 400x to 1,000x magnification. The total endolymph volume of the inner ear was 4.691 mm3, of which 1.501 mm3 was in the cochlea, 3.090 mm3 in the vestibular labyrinth, and 0.100 mm3 in the endolymphatic duct and sac. The total perilymph volume was 15.938 mm3, of which 8.867 mm3 was in the cochlea and 7.071 mm3 in the vestibular labyrinth. The volume of the organ of Corti per millimeter length increased toward the apex, but the volumes of the stria vascularis, spiral ligament, and spiral limbus decreased. The volume of the macula utriculi was larger than that of the macula sacculi. The measurement of the luminal surface area of the stria vascularis was 3.944 mm2, and that of the vestibular dark cells was 5.772 mm2.

Animals↗

Attachment of the tectorial membrane revealed by scanning electron microscope.

Our present-day descriptions of the causes of many forms of sensorineural deafness are based on what is known about the morphology of the organ of Corti. Unfortunately, this information is still woefully inadequate for a complete interpretation of normal events within this structure. Nevertheless there has been no shortage of theories based, in part at least, on assumptins of what the unknown morphology might be. One of the least understood structures is the tectorial membrane, for which the extent and position have had many descriptions. A review of these reveals that the observations vary with the techniques used, and different techniques have various effects on the sticky gel of the tectorial membrane which is reported to be 90% water. Here is described a technique whereby the bulk frozen-hydrated tectorial membrane is viewed by scanning microscopy. In its natural living state the tectorial membrane is sealed to the Hensen's cells, protecting the reticular lamina from endolymph.

Animals↗

Cellular changes in Reissner's membrane in endolymphatic hydrops.

A quantitative morphologic study of the cellular changes in human Reissner's membrane (RM) was performed under light microscopy on 30 normal temporal bones from 15 subjects, and on 20 temporal bones from 10 subjects with endolymphatic hydrops. The cellular density of the epithelial and mesothelial layers of normal RMs decreased with increasing age. In endolymphatic hydrops, cellular proliferation and decreased cellular density of a distended RM were observed, and these changes occurred more remarkably in epithelial cells. Decreased cellular density in RM was correlated to the degree of endolymphatic hydrops. These findings indicate an involvement of the epithelial cellular changes in the pathogenesis of endolymphatic hydrops. Our results suggest that cellular changes in RM may cause dysfunction of radial flow leading to ionic disturbance of endolymph, which provides a possible mechanism of hearing loss in endolymphatic hydrops.

Adolescent↗

Effect of ototoxic drug administration to the endolymphatic sac.

Aminoglycosides were applied to the endolymphatic sac of guinea pigs, and the inner ears were studied by light microscopy. In group 1 gentamicin sulfate application on the lateral surface of the endolymphatic sac produced sensory cell atrophy limited to the macula sacculi. In group 2 gentamicin injection into the sac by extradural and intradural approaches often showed lesions in the maculae sacculi and to the sac from a lateral opening resulted in a similar pattern of degeneration but with less severity in comparison to the injection method. Mild to severe endolymphatic hydrops was sometimes observed. The study shows diffusion of drugs taking place against the longitudinal flow of endolymph. This procedure may be applicable for the treatment of Meniere's disease.

Acoustic Maculae↗

Primary culture of strial marginal cells of guinea pig cochlea: growth, morphologic features, and characterization.

To further investigate the cellular mechanisms involved in the formation of endolymph, primary cultures of marginal cells of guinea pig were established. Minute explants obtained by mechanical dissociation of stria vascularis were plated on collagen type I precoated impermeable substrate in serum-free, hormone-supplemented medium. A confluent layer of epithelial-like cells was obtained within 2 weeks. The cultured cells formed domes, demonstrating that they retain some of their transepithelial properties. Polarization was also suggested by electron microscopic observation of apical microvilli and tight junctions. Immunohistochemical methods revealed that the cultured cells coexpressed cytokeratin and vimentin, demonstrating their epithelial origin, although some degree of dedifferentiation occurred. Thus, a primary culture of marginal cells can be established that may be a suitable model for an in-depth investigation of the function of the marginal cells.

Animals↗

Endolymphatic hydrops induced by immune response of the endolymphatic sac: relation to perilymph antibody levels.

This study investigated the effect of a secondary immune response in the endolymphatic sac on the development of endolymphatic hydrops in guinea pigs. Following secondary keyhole limpet hemocyanin (KLH) challenge directly to the sac in systemically presensitized animals, hydrops rapidly developed within 1 week, then gradually reduced by 4 weeks. After 5 weeks, hydrops gradually recurred and developed again. Mean perilymph anti-KLH antibody levels rose from day 1 to 4 weeks and then decreased. The elevation of perilymph anti-KLH antibody levels on day 2 was due to an increased vascular permeability and there was a local regeneration by the challenge of the sac after day 7. The development of hydrops correlated well with mean perilymph anti-KLH antibody levels in the period from day 1 to day 7. After 2 weeks, there was no correlation between them. These results suggest that an immune response of the sac may induce an excess secretion of endolymph in the initial stage and may further lead to irreversible dysfunction of the sac at a later stage.

Animals↗

Alterations of charge barrier in the inner ear following immune reactions.

We investigated electron microscopically the changes of anionic sites of a charge barrier in the capillary basement membrane of the stria vascularis and endolymphatic sac following inner ear immune reactions. Hartley guinea pigs were immunized with bovine type II collagen, keyhole limpet hemocyanin, or horseradish peroxidase, with boosted and challenged antigens through the stylomastoid foramen. Animals were killed painlessly from several days up to 56 days after the antigen challenge. Polyethylenimine was used as a cationic tracer in order to observe the localization of anionic sites of the charge. In the animals immunized with bovine type II collagen or horseradish peroxidase, a significant decrease of anionic charge in the stria and the sac was found in the early stage of immunization. However, the keyhold limpet hemocyanin immunization group did not show any remarkable changes in the charge because of its lesser transfer into the inner ear due to of its high molecular weight and negative surface charge. A decrease of the charge under immunologic conditions may induce a hyperpermeability of vessels and a malabsorption of endolymph, and thus may cause endolymphatic hydrops.

Animals↗