Ouabain and streptomycin: their different loci of action on saccular hair cells in goldfish.

Ouabain, when applied to the periotic spaces, that is, to the basal end of the saccular hair cells, suppressed microphonic potentials of the inner ear in goldfish. In contrast, streptomycin produced such an effect only when it was applied directly into the endolymph, that is, to the hair-bearing ends of the hair cells.

The mutual independence of the endolymphatic potential and the concentrations of sodium and potassium in endolymph.

The relationship between the endolymphatic potential (EP) and the sodium and potassium concentration gradients between endolymph and interstitial fluid was studied both by measuring the EP at varying concentrations of sodium and potassium in endolymph and by measuring the effect of a depressed EP on the concentrations of these cations. Ethacrynic acid was used in dogs to change the concentration of sodium and potassium (meq/liter) in endolymph from 5.8 and 148 to 134 and 24.3, respectively. No change in the EP accompanied these alterations. In a second series of experiments the EP was reduced from + 72 mV to + 31 mV for a mean duration of 20 min. No change in the concentration of sodium and potassium in endolymph was found during the period of reduced EP. These data suggest that there is little relationship between the EP and the sodium and potassium concentrations in endolymph.

Acute perilymphatic perfusion of the guinea pig cochlea.

A method for the continuous perfusion of the perilymphatic space of the inner ear in the guinea pig is described. Artificial perilymph is supplied to the cochlea and drained away through a tubing system while flow rates from 10 microliters/min to 0.3 ml/min are established by gravity syphon pressure. Techniques are also presented which allow control over the temperature of the perfusate and over the level of dissolved oxygen in the perfusate. Alone with these variables, the pH of the artificial perilymph can be manipulated and various drugs can be added to the perfusate to test their effect on the inner ear. The function of the inner ear is monitored by continuous recording of the sound evoked bioelectric potentials, the cochlear microphonic and the compound action potential. The cochlear perfusion technique has many applications in the study of cochlear physiology and metabolism, and in testing the sensitivity of the inner ear to ototoxic drugs.

Amino acid content of guinea pig perilymph collected under conditions of quiet or sound stimulation.

With the cochlear aqueducts blocked, guinea pig cochleas were perfused with artificial perilymph. The collected perfusates were treated with 5-(dimethylamino)-1-naphthalenesulfonyl chloride (dansyl-Cl). The derivatized constituents were analyzed by high- performance liquid chromatography using fluorometric detection. The concentration of none of the compounds measured, including the putative transmitters glutamate and aspartate, was found to increase in the perfusate in response to sound stimulation. These results do not support the suggestion that one of these compounds might be primary afferent transmitter of audition.

Changes in endolymph chloride concentration following furosemide injection.

Endocochlear potential (EP) and chloride concentration in endolymph were monitored with microelectrodes in the basal turn of the cochlea of the chinchilla. After intravenous injection of furosemide (25-100 mg/kg), the EP dropped precipitously and rapidly reached its minimum value, however, the chloride activity in endolymph decreased more gradually. Possible mechanisms for this phenomenon include a reduced electrostatic attraction of chloride ions to the scala media due to a decreased EP and a reduction of passive influx of chloride into endolymph, resulting from a reduction of active inward potassium transport by furosemide.

Biochemical differences between perilymph, cerebrospinal fluid and blood plasma in the guinea pig.

Seven selected biochemical substances were systematically analyzed in perilymph (PL), cerebrospinal fluid (CSF) and blood plasma or serum of anesthetized guinea pigs in order to compare the composition of these fluids with regard to the origin and biochemical nature of PL. Compositional differences between the fluids were expressed by PL/CSF and PL/plasma concentration ratios of the chemical components measured. Contrary to the prevailing notion, the results suggest that the specific biochemical composition of PL corresponds neither to that of CSF nor to that of a simple ultrafiltrate of blood plasma. Furthermore, except for the total protein concentration, no significant differences were found in the composition of PL between the scala tympani and scala vestibuli.

The Ca2+ activity of cochlear endolymph of the guinea pig and the effect of inhibitors.

The Ca2+ concentrations in cochlear perilymph and endolymph of the guinea pig were measured with double-barreled Ca2+-selective microelectrodes and showed 1.76 +/- 0.74 X 10(-3) M and 2.20 +/- 0.19 X 10(-5) M, respectively. The electrochemical potential gradient for Ca2+ between perilymph and endolymph was 23.2 mV and the existence of an active transport mechanism from the former to the latter was suggested. Vanadate given perilymphatically decreased the Ca2+ concentration in endolymph with a slight elevation of the endocochlear potential and was suspected of blocking the active transport. The Ca2+ concentration in endolymph was abruptly increased by anoxia or the intravenous administration of 60 mg/kg furosemide and was slightly increased by the intravenous administration of 30 mg/kg furosemide or 100 mg/kg acetazolamide. The endolymphatic pH measured with pH-microelectrodes under various conditions indicates that the mechanism of increase in the Ca2+ concentration is attributed not to the liberation of Ca2+ from the surrounding tissues caused by a fall in pH but to the increased influx of Ca2+ from perilymph due to the depression of the endocochlear potential.

Intracochlear microprobe analysis.

Energy dispersive x-ray analysis (EDXA) or "microprobe analysis" provides cochlear physiologists with a means of accurately assessing relative ionic concentrations in selected portions of the auditory mechanism. Rapid freezing followed by lyophilization allows the recovery of fluid samples in crystalline form not only from perilymphatic and endolymphatic spaces, but also from much smaller subregions of the cochlea. Because samples are examined in a solid state, there is no risk of diffusion into surrounding or juxtaposed fluids. Samples of cochlear tissues may also be evaluated without the danger of intercellular ionic diffusion. During direct visualization by scanning electron microscopy, determination of the biochemical makeup of the material being examined can be simultaneously, assuring the source of the data collected. Other potential advantages and disadvantages of EDXA are reviewed. Initial findings as they relate to endolymph, perilymph, stria vascularis, and the undersurface of the tectorial membrane are presented.

Amino acid profiles in inner ear fluids and cerebrospinal fluid.

The levels of 19 amino acids in utricular endolymph, vestibular and cochlear perilymph, and cerebrospinal fluid of guinea pigs were determined using gradient elution reverse phase high performance liquid chromatography of the o-phthaldialdehyde-ethanethiol adducts with fluorescence detection. Aspartate and glutamate were significantly higher in endolymph than in perilymph, in agreement with earlier results on cochlear fluids based on enzymatic fluorometric techniques. All other amino acids tested were significantly lower in the endolymph, in most cases by an order of magnitude. Vestibular perilymph and perilymph of scala vestibuli are virtually identical. Amino acid levels were all higher in perilymph of scala vestibuli than in cerebrospinal fluid; two by an order of magnitude. All differences were statistically significant, with the exception of aspartate. Amino acid levels in perilymph of scala tympani were highly variable dependent upon sampling technique, and no definite values are therefore presented. Comparisons with results from other laboratories, technical pitfalls, and possible implications and interpretations of the results are presented.

Steep gradients of amino acids between cochlear endolymph and perilymph.

Levels of 19 free amino acids in cochlear endolymph and perilymph of scala vestibuli of the guinea pig were determined using reverse phase high performance liquid chromatography (HPLC) of the o-phthaldialdehyde-ethanethiol derivatives with fluorescence detection. Aspartate and glutamate were found to be significantly higher in endolymph than in perilymph, confirming results from another study using a different analytical method. The remaining 17 amino acids were significantly lower in the endolymph, in many cases by an order of magnitude. The data are compared with results on utricular endolymph and vestibular perilymph obtained by HPLC in another study from our laboratory. Possible implications and interpretations of the results are discussed.

Differential uptake of salicylate in serum, cerebrospinal fluid, and perilymph.

After intraperitoneal administration of salicylate in anesthetized rats and guinea pigs, we found that salicylate levels in perilymph (PL) are closely related to both drug levels in cerebrospinal fluid (CSF) and in serum, with higher levels systematically observed in PL than in CSF. Further analysis suggests that salicylate is not passively transported into PL across CSF but, rather, is transported from blood directly to PL. The time course of salicylate uptake in rats reveals maximum levels at 1 1/2 hours (serum) and two to four hours (CSF and PL). On the other hand, salicylate uptake into serum and CSF of guinea pigs exhibits a longer time course, with maximum levels reached at four hours (serum) and five hours (CSF). These data, not previously available, are basic to our understanding of salicylate-related auditory effects.

Lack of effect of carbonic anhydrase inhibition on direct measurements of endolymph bicarbonate.

In the past, sodium, potassium, and chloride have been measured in endolymph directly, but bicarbonate has been measured only indirectly. We sampled endolymph directly while monitoring endocochlear potentials in normal and methazolamide-treated guinea pigs. Bicarbonate was determined in samples by use of a method that depends on reduction of NADH to NAD linked to malate formation from oxaloacetate. In 11 normal animals, the bicarbonate in endolymph was 20.2 mM +/- 4,4 mM (mean +/- standard deviation); in six of these, plasma bicarbonate was 23.1 mM +/- 3.5 mM. Nine animals treated with methazolamide (carbonic anhydrase inhibitor) had an endolymph bicarbonate of 19.5 mM +/- 3.9 mM; plasma bicarbonate in five of these was 25 mM +/- 3.2 mM. Carbonic anhydrase inhibition did not significantly affect endolymphatic bicarbonate levels.

Endolymphatic sac obstruction. Biochemical studies.

Inner ear fluids were studied biochemically in guinea pigs from 1 to 16 weeks after producing endolymphatic hydrops by obstructing the endolymphatic duct. Fluid collected from beneath the footplate showed changes of 50% of the animals indicating distension of the saccule. There was an increase in potassium concentration and decrease in sodium concentration of the collected fluid, indicating a traumatic mixing of endolymph and perilymph produced by rupture of the saccule during collection of the fluid. The smallest changes occurred at one week, and the greatest changes were found four months after endolymphatic sac obstruction. Similar findings were observed in studying inner ear fluids obtained from patients with Ménière's disease. Cochlear endolymph showed biochemical alterations after endolymphatic duct blockage. There was a two- to three-fold increase in sodium concentration and a decrease in potassium concentration with the total ionic concentration remaining approximately the same. Some guinea pigs showed similar changes in vestibular endolymph. This study indicates that in the guinea pig, endolymph obtained in the distended endolymph compartment has a slightly different sodium-potassium ratio as compared to the normal ear. In patients with Ménière's disease there may also be an elevation of endolymph sodium concentration. The significance of this change and hearing loss observed in experimental endolymphatic hydrops and Ménière's disease is open to speculation at the present time.

Vertigo of delayed onset after sudden deafness.

An entity of episodic true vertigo of delayed onset following sudden and profound sensorineural hearing loss is described. Data on 12 patients and three case reports are presented. The latency between sudden deafness and the onset of the vertigo varied from 1 to 68 years. The vestibular symptoms are identical to the vestibular symptoms of Ménière's disease, and there is some evidence that endolymphatic hydrops in the previously deafened ear represents at least part of the labyrinthine pathology. Labyrinthectomy in the deaf ear was curative. Tentatively, this entity is best considered a variant of Ménière's disease.

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.

Effect of intracochlear aminooxyacetic acid on cochlear potentials and endolymph composition.

Summary--Aminooxyacetic acid (AOAA; 1 MM) when perfused through the scala tympani of guinea pigs produced a biphasic reduction in the endocochlear potential, together with a shift in the input-output functions of the cochlear microphonics and the compound action potential of the cochlear nerve. Since the ionic (Na+, K+) content of the scala media was not alerted, it appears that such changes did not underlie the reduction recorded in the endocochlear potential. The negative direct current potential recorded in the organ of Corti did not appear to be affected by the drug. Attempts to antagonize the effects of AOAA with pyridoxine failed.

Hyaluronan content in human inner ear fluids.

Histochemical analyses of the luminal contents of the endolymphatic sac in numerous mammalian species have indicated a presence of acid mucopolysaccharides or proteoglycans. In the present study, one of these substances, hyaluronan (hyaluronic acid; HA), in endolymphatic sac endolymph and vestibular perilymph in humans, was biochemically determined with a highly specific radioassay. A considerable variation in HA concentration was noted between the individual ES endolymph samples, with a mean value of 2.50 micrograms/g (range 0.00-10.86). In perilymph the HA concentrations were less variable and the mean concentration was 0.91 micrograms/g (range 0.56-1.51). The difference between endolymph and perilymph HA concentration was not statistically significant (p greater than 0.05). The ability of HA to interact with proteoglycans, thereby forming giant hydrophilic molecules, could be important for ES function. These molecules may create a swelling pressure which is resistant to compressive forces that under conditions of increased intracranial hydrostatic pressure otherwise could cause ES collapse and impaired function.

Effects of pure tone on endocochlear potential and potassium ion concentration in the guinea pig cochlea.

Preliminary results presented here indicate a simultaneous decrease in endocochlear potential (EP) and in endolymphatic potassium ion concentration (K+e) under noise exposure. Maximum changes were observed after 20 min and 30 min of sound stimulation, respectively for EP and Ke+. EP recovery was faster than that of K+e. Noise-induced K+e alterations varied with the pure-tone frequency, and the locus along the basilar membrane where the measurements were performed. This supports the hypothesis that the noise has a tonotopic effect correlate with the shape of the travelling wave. Results obtained with ethacrynic acid, administered at the end of the recovery period which followed the noise exposure, indicate that the sound stimulation parameters which were used did not irreversibly alter the ionic transport in the cochlea.

[Electrophoretic examination of protein distribution in guinea pig perilymph]. / Elektrophoretische Untersuchungen zur Proteinverteilung in der Meerschweinchen-Perilymphe

Perilymph and serum of guinea pigs were investigated using immunoelectrophoresis and polyacrylamide gel electrophoresis. The molecular weights of the perilymph proteins were estimated by electrophoresis in a linear polyacrylamide gel gradient (3-20%). The immunoelectropherograms and the polyacrylamide gel electropherograms of equivalent amounts of perilymph protein and serum protein are nearly the same. The immunological detection of the perilymph proteins indicates that they are serum proteins. The clear identification of the individual proteins fails because of missing monospecific antisera against the individual proteins of guinea pig. High-molecular proteins (range of the molecular weight estimation 67 000-290 000) are also detectable in the perilymph according to the molecular weight estimation.

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.