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Sodium loading of inner ear fluids.

A slow intracarotid infusion of hypertonic sodium chloride solution, 350 mEq/L, and potassium chloride, 5 mEq/L, was used to increase serum sodium chloride over a 90-minute period in 85 cats. A slow, steady rise in serum sodium occurred, which reached almost 208 mEq/L at the end of 90 minutes. A concomitant rise in sodium occurred in the cerebrospinal fluid and perilymph. In contrast, the sodium showed only a transient slight increase in the cochlear and vestibular endolymph. Endolymph potassium appeared to rise in order to balance out the increase in sodium concentration of the surrounding and extracellular fluids. The ratio of sodium to potassium ions in both the endolymph and perilymph compartment remained relatively constant before and after the infusion with hypertonic NaCl. After infusion, the total concentration of ions in endolymph was similar to that of perilymph. These experiments indicate that the endolymph compartment has a built-in mechanism for maintaining a low-sodium concentration while keeping ionic balance with the surrounding perilymph and serum.

Animals↗

Elemental composition of the developing inner ear.

The elemental composition of the inner ear fluid-filled compartments has been analyzed using the x-ray energy dispersive technique (CBA mouse). Special attention has been focused on the maturation of endolymph. A few days before and after birth the relative peak intensity of potassium (RK) in the vestibular endolymphatic compartment was slightly surpassing that in the cochlear part of the labyrinth. From the fourth day after birth (DAB) a rapid increase occurred concerning the RK. The highest RK was found in the endolymphatic space in the basal part of the cochlea followed by that in the vestibular endolymph. The lowest RK was measured in endolymph of the apical part of the cochlea. These obvious differences were abandoned already on the sixth DAB. A mature composition of endolymph was reached on the eighth DAB. The present technique does not allow analyses of differences between cochlear and vestibular endolymph with regard to minimal shifts.

Animals↗

Effects of sodium bromate on ionic concentrations and osmolalities of the cochlear fluids in guinea pigs.

Effects of sodium bromate on cochlear potentials and electrolyte composition of the cochlear fluids in guinea pigs were investigated following administration of sodium bromate into the cochlea, using perilymphatic perfusion. Cochlear microphonics and the whole nerve action potential of the auditory nerve were markedly suppressed. The K+ and Cl- activities in the endolymph as well as the endocochlear dc potential (EP) decreased significantly and irreversibly, in proportion to the concentration of sodium bromate. A negative EP never developed during the monitoring of 120 min. Microsamples of the endolymph showed substantial decreases of K+ and Cl- concentrations and an increase in the concentration of Na+. Osmolality of the endolymph was much lower than that of the perilymph. The severe edema of the stria vascularis and collapse of Reissner's membrane were histologically evident. These events suggest a breakdown of the endolymph-perilymph barrier, coincident with an inhibition of the strial active transport, as a result of the ototoxic action of sodium bromate. The possible ion and water movement across the endolymph-perilymph barrier in the presence of sodium bromate is discussed.

Acoustic Stimulation↗

Presence of plasma membrane-bound Ca(2+)-ATPase in the secretory epithelia of the inner ear.

The Ca2+ concentration of the endolymph is low, around 0.023 mM. Yet, because of the positive endocohlear potential, Ca2+ must be actively transported into the endolymphatic space. The mechanisms responsible for the active Ca2+ transport into the endolymph are not known. In this study, the presence of plasma membrane-bound Ca(2+)-ATPase (PMCA ATPase) in the endolymph-producing, secretory epithelia of the inner ear from guinea pig was investigated with immunoblotting and immunohistochemistry. The antibody used was a monoclonal antibody which recognizes an epitope shared by all four known isoforms of PMCA ATPase. With immunoblotting, a band corresponding to PMCA ATPase was found in the stria vascularis, the ampullary tissue, the utricle and the endolymphatic sac in assays from at least three different batches of tissue. With immunohistochemistry, a strong positive staining reaction for PMCA ATPase could be seen in the stria vascularis and the dark cells of the ampullary tissue and the utricle. The epithelial cells in the endolymphatic sac showed a moderate positive staining reaction. Accordingly, in this study the presence of PMCA ATPase was shown in all the endolymph-producing, secretory epithelia of the inner ear. These results indicate that PMCA ATPase plays a role in the regulation of the Ca2+ concentration in the endolymph.

Animals↗

Spatial orientation of caloric nystagmus in semicircular canal-plugged monkeys.

We studied caloric nystagmus before and after plugging all six semicircular canals to determine whether velocity storage contributed to the spatial orientation of caloric nystagmus. Monkeys were stimulated unilaterally with cold ( approximately 20 degrees C) water while upright, supine, prone, right-side down, and left-side down. The decline in the slow phase velocity vector was determined over the last 37% of the nystagmus, at a time when the response was largely due to activation of velocity storage. Before plugging, yaw components varied with the convective flow of endolymph in the lateral canals in all head orientations. Plugging blocked endolymph flow, eliminating convection currents. Despite this, caloric nystagmus was readily elicited, but the horizontal component was always toward the stimulated (ipsilateral) side, regardless of head position relative to gravity. When upright, the slow phase velocity vector was close to the yaw and spatial vertical axes. Roll components became stronger in supine and prone positions, and vertical components were enhanced in side down positions. In each case, this brought the velocity vectors toward alignment with the spatial vertical. Consistent with principles governing the orientation of velocity storage, when the yaw component of the velocity vector was positive, the cross-coupled pitch or roll components brought the vector upward in space. Conversely, when yaw eye velocity vector was downward in the head coordinate frame, i.e., negative, pitch and roll were downward in space. The data could not be modeled simply by a reduction in activity in the ipsilateral vestibular nerve, which would direct the velocity vector along the roll direction. Since there is no cross coupling from roll to yaw, velocity storage alone could not rotate the vector to fit the data. We postulated, therefore, that cooling had caused contraction of the endolymph in the plugged canals. This contraction would deflect the cupula toward the plug, simulating ampullofugal flow of endolymph. Inhibition and excitation induced by such cupula deflection fit the data well in the upright position but not in lateral or prone/supine conditions. Data fits in these positions required the addition of a spatially orientated, velocity storage component. We conclude, therefore, that three factors produce cold caloric nystagmus after canal plugging: inhibition of activity in ampullary nerves, contraction of endolymph in the stimulated canals, and orientation of eye velocity to gravity through velocity storage. Although the response to convection currents dominates the normal response to caloric stimulation, velocity storage probably also contributes to the orientation of eye velocity.

Animals↗

Electrochemical composition of the cochlear fluids in the early experimental hydrops. Preliminary results.

The composition of endolymph and perilymph was studied in the guinea pig cochlea after 2 and 6 weeks of blockage of the vestibular aqueduct in an experimental model of hydrops. Compound action potential was monitored several times in the observation period. The endocochlear potential was measured and the endolymph was sampled at the first and third turns of the scala media. The Na, K, and Cl concentrations were determined in nanolitre aliquots of endolymph and of perilymph, the latter sampled from the basal scala vestibuli. After 2 weeks, no change in endolymphatic electrochemical composition was observed. After 6 weeks, endocochlear potential was decreased by 25% at both cochlear turns; K concentration was decreased in endolymph of the basal turn and Cl concentration was decreased in both turns; the calculated osmolality (Na + K + Cl) was decreased in both turns. These results indicate that the blockage of the vestibular aqueduct induced early auditory dysfunction whereas alterations of the electrochemical composition of endolymph occurred later after a time lag of more than 2 and less than 6 weeks.

Action Potentials↗

Changes in the electrochemical composition of cochlear fluids after intracisternal application of doxorubicin in the rat.

Since doxorubicin (Adriamycin) is known to bind on membranous negative surface charges, its effect on the electrochemical composition of the cochlear fluids was studied in rats. Doxorubicin was infused into the cerebrospinal fluid via the lateral cerebral ventricle. The endocochlear resting potential was recorded, and endolymph and perilymph of the scala vestibuli were collected from the basal cochlear turn before and 1, 2, and 4 h after the drug application. Na+, K+, and Cl- concentrations and osmolality of the endolymph and perilymph were measured in 1 nl aliquots. In perilymph, Cl- concentration increased 4 h after doxorubicin treatment to reach a concentration 8 mM higher than recorded initially. In endolymph, the endocochlear potential decreased by 5 mV/h while its K+ concentration and osmolality increased by about 3 mM/h and 4 mosmol/kg H2O per hour, respectively. These results suggest that the negative surface charges demonstrated on Reissner's membrane may play a role in the homeostasis of endolymph.

Animals↗

X-ray microanalysis of inner ear fluids in the embryonic and newborn guinea pig.

In energy-dispersive histograms, changes in the relative peak intensities were followed, especially C1 and K, which indicate the maturation of endolymph. The maturation of endolymph in the guinea pig occurs prior to birth. In X-ray histograms, distinct peaks for C1 and K, but also for Na, were observed approximately 20 days before (DBB). The lesser relative peak intensities for C1 and K as compared with mature endolymph indicate an immature endolymph composition at this stage of development. The relative peak intensities of C1 and K increased at approximately the 10-DBB stage and showed similar values as at birth.

Animals↗

Aspects on endolymphatic sac morphology and function.

Morphological evidence indicate that the main function of the endolymphatic sac is to act as a reabsorptive and defensive mechanism for the inner ear. This activity is markedly enhanced in labyrinthine trauma, such as injection of foreign particles into the labyrinth, blocking of the endolymphatic duct, and cryosurgical destruction of vestibular sensory epithelia. Light and dark epithelial cells of the intermediate portion of the sac are capable of reabsorbing endolymph and digesting cellular debris respectively. The extensive capillary network surrounding the endolymphatic sac exhibits endothelial characteristics suggestive of active fluid transport. The "dynamic-flow theory" of endolymph circulation suggests that a radial-flow should be considered for energy metabolism and ion exchange around the sensory cell regions whereas a longitudinal-flow should be considered for reabsorption of endolymph and disposal of high molecular waist products and debris by the endolymphatic sac. The earlier concepts of endolymph circulation thus need not any longer be considered conflicting.

Ear, Inner↗

Chemical composition in various compartments of inner ear fluid.

Sodium, potassium, chloride, glucose and total protein were determined in samples of scala vestibuli perilymph, scala tympani perilymph, CSF, cochlear endolymph and utricular endolymph from normal cats. Small but significant differences were evident in the concentrations of sodium and potassium between the scala vestibuli and scala tympani perilymph. It was also apparent that each compartment of endolymph has different values for sodium and potassium concentrations. Compared with the endolymph, the perilymph was found to contain a higher concentration of glucose and total protein. These findings are discussed from the view point of biological significance.

Cerebrospinal Fluid↗

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse.

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker -1 and Shaker -2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique. Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlear during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker -1 and Shaker -2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.

Adenylyl Cyclases↗

The ionic and electric environment in the endolymphatic sac of the chinchilla: relevance to the longitudinal flow.

The ionic composition of the endolymph in the endolymphatic sac (ES) of the chinchilla was measured using double-barreled ion-selective micro-electrodes. The DC potential of the ES was 9.3 +/- 1.8 mV (N = 18). The K+, Na+, and Cl- concentrations of the ES were 13.3 +/- 4.7 mM (N = 6), 129.0 +/- 8.8 mM (N = 6), and 124.3 +/- 16.6 mM (N = 6), respectively. In light of the chemical potentials of the cochlear endolymph previously reported [Ikeda and Morizono (1989), Hear. Res., 39, 279-286] the pressure gradient of the endolymph between the cochlea and ES was calculated to be 71.5 mmHg at 38 degrees C. The contribution of the osmotic and hydrostatic pressure gradients of the endolymph to the longitudinal flow is discussed.

Animals↗

Evaluation of cochlear function in an acute endolymphatic hydrops model in the guinea pig by measuring low-level DPOAEs.

During and after microinjection of artificial endolymph into scala media of the guinea pig, the 2f1- f2 -DPOAE at 4.5 kHz generated by low-level primaries was recorded. Reproducible changes were measured when 1.1 microl of artificial endolymph was injected at a rate of 1.65 nl/s (1.53-1.83). This volume corresponds with an acute endolymphatic hydrops of 23%. After the onset of injection the inner ear pressure immediately increased to a mean higher level of 22 Pa, whereas the 2f1- f2 -amplitude and -phase did not change for about 1 min. Thereafter, the amplitude decreased 2.6 dB (+/- 0.7) on average and slowly regained almost its initial value, with recovery frequently starting within the period of injection. In an attempt to explain the observed changes in 2f1- f2 -amplitude the basilar membrane displacement towards scala tympani at the 2f1- f2 generation site is estimated to be 19 nm for a 1.1 microl increase of endolymph volume. A small deflection of the outer hair cell stereocilia and as a consequence a change in cell conductance may explain the 2f1- f2 -amplitude changes. However, the precise mechanism of cochlear function change caused by endolymph volume increase (hydrops) remains to be elucidated.

Acute Disease↗

Endocochlear potential and scala media oxygen tension during partial anoxia.

The endocochlear potential and the scala media oxygen tension were measured from the basal coil of the guinea pig cochlea during levels of partial respiratory anoxia. This was accomplished by simultaneously positioning two electrodes (for endocochlear potential and PO2) into the endolymph by a transbasilar membrane approach, in order to minimize blood capillary damage. The intracochlear pO2 was stable until the respiratory oxygen concentration was reduced to 16 per cent on the average. Thereafter, a further reduction of oxygen in the respiratory mix reduced both the endolymph pO2 and the endocochlear potential. The coupling between the endocochlear potential and endolymph pO2 is proportional over a 70 to 80 per cent reduction in endolymph pO2, and at this point the endocochlear potential is reduced by 50 per cent. For partial levels of respiratory oxygen that are not too low (e.g., > 12 per cent oxygen) it is possible to maintain partial levels of intracochlear pO2 for at least six minutes. During this time period the endocochlear potential is also held at a stable but reduced level.

Animals↗

[The endolymphatic sac: its roles in the inner ear].

The endolymphatic sac is a non-sensory organ of the inner ear. It is connected to the endolymphatic compartment that is filled with endolymph, a potassium-rich fluid that bathes the apical side of inner ear sensory cells. The main functions ascribed to the endolymphatic sac are the regulation of the volume and pressure of endolymph, the immune response of the inner ear, and the elimination of endolymphatic waste products by phagocytosis. Functional alteration of these functions, leading to deficient endolymph homeostasis and/or altered inner ear immune response, may participate to the pathophysiology of Ménière's disease, an inner ear pathology that causes episodes of vertigo, sensorineural hearing loss and tinnitus, and is characterized by an increase in volume of the cochleo-vestibular endolymph (endolymphatic hydrops).

Endolymph↗

FXYD6 is a novel regulator of Na,K-ATPase expressed in the inner ear.

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Amino Acid Sequence↗

The influence of transducer operating point on distortion generation in the cochlea.

Distortion generated by the cochlea can provide a valuable indicator of its functional state. In the present study, the dependence of distortion on the operating point of the cochlear transducer and its relevance to endolymph volume disturbances has been investigated. Calculations have suggested that as the operating point moves away from zero, second harmonic distortion would increase. Cochlear microphonic waveforms were analyzed to derive the cochlear transducer operating point and to quantify harmonic distortions. Changes in operating point and distortion were measured during endolymph manipulations that included 200-Hz tone exposures at 115-dB SPL, injections of artificial endolymph into scala media at 80, 200, or 400 nl/min, and treatment with furosemide given intravenously or locally into the cochlea. Results were compared with other functional changes that included action potential thresholds at 2.8 or 8 kHz, summating potential, endocochlear potential, and the 2 f1-f2 and f2-f1 acoustic emissions. The results demonstrated that volume disturbances caused changes in the operating point that resulted in predictable changes in distortion. Understanding the factors influencing operating point is important in the interpretation of distortion measurements and may lead to tests that can detect abnormal endolymph volume states.

Animals↗

Functional importance of sodium and potassium in the guinea pig cochlea studied with amiloride and tetraethylammonium.

The effects of amiloride on the cochlear responses in the guinea pig were compared with those produced by tetraethylammonium (TEA). Amiloride has been reported to reduce membrane permeability to sodium in a wide variety of ion-transporting epithelia. TEA has been documented to suppress the active potassium permeability increase during the repolarization phase of the action potential in mammalian excitable cells, and to reduce the resting potassium conductance in mammalian smooth muscle cells. Perilymphatic perfusion of 10(-3) M amiloride or intravenous injection at a dose of 20 mg/kg suppressed the whole nerve action potential (AP) of the cochlea but did not significantly affect the cochlear microphonics (CM) or endocochlear potential (EP). Application of amiloride to endolymph by iontophoretic or perfusion techniques also produced no significant changes of CM and EP when compared with appropriate control procedures. Perilymphatic perfusion of 10(-2) M TEA did not suppress CM or EP but the AP was reduced. Iontophoretic application of TEA to the endolymph caused a marked suppression of CM while the EP was significantly increased. The effects of endolymphatic TEA application are consistent with the concept that the normal EP recorded from scala media is the algebraic sum of a positive electrogenic potential and a negative diffusion potential, the latter component being sensitive to potassium permeability changes of the endolymph-perilymph barrier. Maintenance of normal cochlear microphonics also appears dependent upon the maintenance of normal potassium permeability properties of the endolymph-perilymph barrier. The functional importance of normal sodium permeability properties appears less certain.

Action Potentials↗