Biochemical parameters of guinea pig perilymph sampled according to scala and following sound presentation.

Guinea pigs were exposed to sound varying from 2 to 8 kHz in frequency and 80-100 dB (SPL) in intensity for periods of 1 hr. The biochemical parameters, glucose, sodium, total protein, and the glycolytic enzymes, aldolase, phosphohexose isomerase, and total LDH as well as isozymes of the latter were ascertained for blood serum, perilymph, and, in some instances, cerebrospinal fluid. The three enzymes occurred at lower levels in perilymph as compared to blood serum. Except for a small difference in serum total protein, sound presentation incurred no significant effect on any of the above parameters. Definite differences in several metabolites were discerned for perilymph sampled according to scala and which were independent of the respective acoustical treatments. Thus, as compared to the scale tympani, the scala vestibuli perilymph displayed a higher glucose content and a diminished total LDH level and of the latter isozymes, LDH(1) ranged lower and LDH(2), higher. As further evidence pointing to cerebrospinal fluid as the possible origin of perilymph, similarities in glucose contents and LDH isozyme patterns were noted for both fluids.

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.

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.

Permeability to chloride ions of the cochlear partition in normal guinea pigs.

The endocochlear potential and Cl- activities in the endolymph and perilymph of guinea pigs were simultaneously measured with a pair of double-barreled Cl--sensitive liquid-membrane electrodes. Under normal conditions the mean Cl- activity was 96.6 mEg/l in the endolymph and 93.5 mEq/l in the perilymph. The mean Cl- concentration determined by potentiometric titration was 132.3 mM in the endolymph and 121.8 mM in the perilymph. The permeability to Cl- of the cochlear partition was determined after active transport was abolished by permanent anoxia. The modified permeability coefficient and conductance for Cl- of the cochlear partition were computed from the rate of decrease in the Cl- concentration of the endolymph and its electrochemical potential difference between the endolymph and perilymph. The mean modified Cl- conductance and permeability coefficient of the cochlear partition were (10.7 +/- 4.25) X 10(-3) omega -1 . cm-3 and (22.58 +/- 8.95) X 10(-6) s-1 respectively, when averaged from 10 to 30 min after onset of anoxia. Our results indicate that the cochlear partition is relatively permeable to Cl-, when compared to its permeability to K+. Possible coupling between Cl- and water movement across the cochlear partition is discussed.

Somatostatin along the auditory pathway.

Several structures associated with the auditory pathway have been examined by radioimmunoassay for their content of somatostatin. Of these, the medial geniculate body had the highest content, followed by the cochlear nucleus, inferior colliculus, auditory cortex and cochlea. Cochlear perilymph had no detectable somatostatin-like immunoreactivity.

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.

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.

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.

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.

Round window membrane and perilymph in experimental otitis media with effusion.

To investigate the influence of middle ear effusion (MEE) on perilymph (PL), an experimental otitis media with effusion (OME) was manufactured in chinchillas by injecting the tympanic cavity with immune complexes. The presence of MEE lasted for up to 9 days after the injection of immune complexes. Perilymph was aspirated on the fourth, tenth, and 21st days after the inoculation. The mean concentrations of albumin, immunoglobulin G, histamine, and prostaglandin E2 (PGE2) were significantly greater in PL from ears with induced OME than in that from normal control ears. The 3H-PGE2 placed on the round window membrane of pathologically affected ears passed into PL in significantly greater amounts than in normal control ears. The findings indicate that the immune complexes placed in the middle ear cavity affect the biochemical milieu of PL, and that MEE is a result of immune complexes.

Permeability of the middle ear to staphylococcal pyrogenic exotoxin in otitis media.

Middle ear permeability after instillation of staphylococcal pyrogenic exotoxin was studied in each of 12 cats, 6 of them with otitis media induced by obstructing their eustachian tubes. This is the first report that there is passage of toxin to the perilymph, cerebrospinal fluid and blood not only in diseased ears, but also in normal controls, 25 min and 12 h after exposure of the middle ear cavity and round window membrane to toxin. The data suggest a pathophysiological explanation for the association of otitis media and sensorineural hearing loss and/or endolymphatic hydrops; potentially both entities can be caused by exotoxins. It also documents the extraordinary capabilities of movement of staphylococcal exotoxin.

[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.

Measurement of oxygen tension in human perilymph.

The oxygen tension of the human perilymph has been determined without complications by using a technique based upon the polarographic principle. Carbon dioxide produced the most marked increase in oxygen tension in the human perilymph. No change in the perilymphatic PO2 was observed after administration of eupaverine and low molecular weight dextran. 5% CO2-95%O2 has been found to be the gas mixture producing the largest increase in oxygen tension in the human perilymph, without side effects. In order to achieve maximal oxygenation of the perilymph, the therapeutic administration of 5% CO2-95% O2 should continue for at least 30 minutes. At this preliminary stage it is not possible to assess the accuracy of the presented technique in determining the presence of a vascular disorder of the inner ear in patients.

Induced vestibular dysfunction in squirrel monkeys during rapid decompression.

The symptoms of postural instability and dizziness associated with decompression sickness could be ascribed to either damage of the vestibular apparatus or to central nervous system damage. However, a histological study of monkeys exposed to decompression reveals that these symptoms primarily result from damage to the vestibular apparatus (unless there are accompanying central deficits). Furthermore, the damage is of a type that causes new bone growth to occlude the otic fluid spaces of the semicircular canals. In some instances, there is sufficient bone growth to render the cristae ampullares as non-functional end organs. Such diminished vestibular function would present a serious threat to the diver.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

The developing electrolytes concentrations of inner ear fluids in guinea pigs.

Cochlear endolymph, scala tympani perilymph, and scala vestibuli perilymph were collected from fetal, neonatal, and mature guinea pigs. The concentrations of Na and K in these fluids were then assessed using a microflame photometer. The electrolyte composition in the endolymph was already characterized by a high concentration of K and a low concentration of Na by the 49th day of gestation.

The importance of potassium in the function of frog semicircular canals.

The slow potentials and afferent discharge of impulses in frog semicircular canals have been studied at different endolymphatic and perilymphatic K+ concentrations. Results indicate that the presence of K ions in the bathing fluids is essential for maintaining the receptor function in crista ampullaris, although very low concentrations of this ion in the perilymph are sufficient to preserve the receptor responsiveness to mechanical stimuli. The hypothesis is put forward that K+ may be pumped from the exterior of the canal towards the intracupular structures, where it accumulates. A K-rich endolymphatic environment does, however, appear to be necessary to ensure the resting activity of ampullar receptors and their ability to be "disfacilitated" during inhibitory cupula deflections.