Quantitative anatomy of the round window and cochlear aqueduct in guinea pigs.

In order to analyze the entry of solutes through the round window membrane, a quantitative description of round window anatomy in relationship to scala tympani is required. High-resolution magnetic resonance microscopy was used to visualize the fluid spaces and tissues of the inner ear in three dimensions in isolated, fixed specimens from guinea pigs. Each specimen was represented as consecutive serial slices, with a voxel size of approximately 25 microm(3). The round window membrane, and its relationship to the terminal portion of scala tympani in the basal turn, was quantified in six specimens. In each image slice, the round window membrane and scala tympani were identified and segmented. The total surface area of the round window membrane averaged 1.18 mm(2) (S.D. 0.08, n=6). The length and variation of cross-sectional area as a function of distance for the cochlear aqueduct was determined in five specimens. The cochlear aqueduct was shown to enter scala tympani at the medial limit of the round window membrane, which corresponded to a distance of approximately 1 mm from the end of the scala when measured along its mid-point. These data are of value in simulating drug and other solute movements in the cochlear fluids and have been incorporated into a public-domain simulation program available at http://oto.wustl.edu/cochlea/.

Ionic and potential changes of the endolymphatic sac induced by endolymph volume changes.

The endolymphatic sac (ES) is believed to be the locus for endolymph volume regulation in the inner ear. It has recently been shown that induced endolymph volume changes in the cochlea result in anatomical changes in the ES, suggesting that function of the sac varies according to endolymph volume status. In the present study we have recorded luminal concentrations of K(+) and Na(+) from the ES and the endolymphatic sac potential (ESP) during cochlear endolymph volume changes. ES recordings were made by an extradural approach, thereby preserving normal cerebrospinal fluid resting pressure. Cochlear endolymph volume changes were generated by performing injections or withdrawals through a pipette inserted into endolymph by a round window approach. The pre-treatment concentrations of K(+) and Na(+) in the ES were found to be 8.4 mM (S.D. 3.3, n=8) and 128. 6 mM (S.D. 18.4, n=10) respectively, and the mean ESP was 14.4 mV (S. D. 5.2, n=18). Endolymphatic injections were found to produce a sustained increase in the K(+) content of the ES by an average of 19. 9 mM and to decrease Na(+) by 30.7 mM measured 50 min after the start of injection. The time for K(+) increase to occur was found to correlate with the injected volume, with larger injected volumes producing a more rapid increase. Endolymphatic withdrawals were found to induce a slow decline in endolymphatic K(+) by an average of 3.4 mM measured at 50 min after withdrawal, although no significant change of Na(+) was detected. Volume-induced ESP changes were highly variable. Injections produced a small increase in the mean ESP and withdrawals produced a small decrease but neither change was statistically significant and some animals showed potential changes in the opposite direction. These data show that a change in cochlear endolymph volume status results in a physiologic response of the ES which is sustained for a considerable period. If the ES plays a part in the restoration of normal endolymph volume, this process appears to proceed slowly, based on the prolonged time courses of ionic changes observed.

Morphological changes of the endolymphatic sac induced by microinjection of artificial endolymph into the cochlea.

Morphological changes of the endolymphatic sac were analyzed in guinea pigs following microinjection of artificial endolymph into the cochlea or withdrawal of a quantity of native endolymph. Injections were performed into the second turn of scala media with a micro-pump at a rate of 60-100 nl/min, lasting for a period of 4, 7. 5, 15 or 18 min. In withdrawal experiments, endolymph was aspirated from the second cochlear turn over a period of 8 min. For each procedure the contralateral (non-treated) ear served as a histological control. Following artificial endolymph injections of 7. 5 min or more there was an almost total absence of the normal intraluminal homogeneous substance (HS) on the injected side. Our observations suggest that the disappearance of the HS occurs by both enzymatic and macrophagic activity. After endolymphatic withdrawals the ES was found to contain increased amounts of HS. The results could suggest that the volume of fluid in the ES, and hence the volume of the entire membranous labyrinth, may be regulated by a dynamic relationship between active secretion and enzymatic degradation of a lumen-expanding substance that is intimately related to the intraluminal macrophages. The exact mechanism governing these regulatory systems, and their relationship to ion and water movements across the epithelium of the sac, remain to be elucidated.

Cochlear fluid space dimensions for six species derived from reconstructions of three-dimensional magnetic resonance images.

OBJECTIVES: To establish the dimensions and volumes of the cochlear fluid spaces. STUDY DESIGN: Fluid space volumes, lengths, and cross-sectional areas were derived for the cochleas from six species: human, guinea pig, bat, rat, mouse, and gerbil. METHODS: Three-dimensional reconstructions of the fluid spaces were made from magnetic resonance microscopy (MRM) images. Consecutive serial slices composed of isotropic voxels (25 microm3) representing the entire volume of fixed, isolated cochleas were obtained. The boundaries delineating the fluid spaces, including Reissner's membrane, were resolved for all specimens, except for the human, in which Reissner's membrane was not consistently resolved. Three-dimensional reconstructions of the endolymphatic and perilymphatic fluid spaces were generated. Fluid space length and variation of cross-sectional area with distance were derived by an algorithm that followed the midpoint of the space along the length of the spiral. The total volume of each fluid space was derived from a voxel count for each specimen. RESULTS: Length, volume, and cross-sectional areas are provided for six species. In all cases, the length of the endolymphatic fluid space was consistently longer than that of either perilymphatic scala, primarily as a result of a greater radius of curvature. For guinea pig specimens, the measured volumes of the fluid spaces were considerably lower than those suggested by previous reports based on histological data. CONCLUSIONS: The quantification of cochlear fluid spaces provided by this study will enable the more accurate calculation of drug and other solute movements in fluids of the inner ear during experimental or clinical manipulations.

Longitudinal endolymph movements and endocochlear potential changes induced by stimulation at infrasonic frequencies.

The inner ear is continually exposed to pressure fluctuations in the infrasonic frequency range (< 20 Hz) from external and internal body sources. The cochlea is generally regarded to be insensitive to such stimulation. The effects of stimulation at infrasonic frequencies (0.1 to 10 Hz) on endocochlear potential (EP) and endolymph movements in the guinea pig cochlea were studied. Stimuli were applied directly to the perilymph of scala tympani or scala vestibuli of the cochlea via a fluid-filled pipette. Stimuli, especially those near 1 Hz, elicited large EP changes which under some conditions exceeded 20 mV in amplitude and were equivalent to a cochlear microphonic (CM) response. Accompanying the electrical responses was a cyclical, longitudinal displacement of the endolymph. The amplitude and phase of the CM varied according to which perilymphatic scala the stimuli were applied to and whether a perforation was made in the opposing perilymphatic scala. Spontaneously occurring middle ear muscle contractions were also found to induce EP deflections and longitudinal endolymph movements comparable to those generated by perilymphatic injections. These findings suggest that cochlear fluid movements induced by pressure fluctuations at infrasonic frequencies could play a role in fluid homeostasis in the normal state and in fluid disturbances in pathological states.

Longitudinal endolymph movements induced by perilymphatic injections.

Endolymph movements and endocochlear potential (EP) changes were measured during disturbances of perilymphatic pressure. induced by injecting artificial perilymph into scala tympani (ST) or scala vestibuli (SV) of the guinea pig cochlea. Injections were performed either with or without an outlet made in the opposite perilymphatic scala. Injections into ST without an outlet induced large pressure changes but virtually no endolymph movement or EP change. Injection at the same rate into ST with an outlet in SV produced smaller pressure changes which were accompanied by a basally-directed displacement of endolymph and significant EP changes. The magnitude of endolymph displacements and EP changes varied as a function of injection rate. Injections into SV, either with or without an outlet in ST, produced apically-directed endolymph displacement and EP changes. For the SV injections without an outlet, the cochlear aqueduct and round window are likely to provide an outlet and compliance, permitting flow along the perilymphatic scalae to occur even when no ST outlet was provided. We conclude that endolymph movements are not dependent on the absolute pressure of the perilymph, but instead occur when small, sustained pressure gradients are present across the cochlear partition, corresponding to times when perilymph flow is induced. This study demonstrates that in the normal. sealed cochlea, endolymph and EP are insensitive to fluid injections into ST, but are sensitive to fluid injections into SV. Endolymph movements are therefore unlikely to be generated by cerebrospinal fluid pressure fluctuations (such as those produced by respiration, posture changes, coughing, sneezing, etc) which are transmitted to ST by the cochlear aqueduct.

Comparison of endolymph cross-sectional area measured histologically with that measured in vivo with an ionic volume marker.

In order to establish how endolymph volume is regulated, it is essential to be able to measure endolymph volume or cross-sectional area in vivo. We have developed methods to accomplish this by injecting the volume marker ion hexafluoroarsenate (AsF6) into endolymph by iontophoresis. For an injection at a constant rate, the endolymph concentration is inversely dependent on the cross-sectional area of the scala into which injection occurred. Marker concentrations were monitored by inserting ion-selective microelectrodes into endolymph near the injection site. In a previous study we quantified the degree of hydrops in animals following ablation of the endolymphatic sac. In the present study we validated the technique by comparing the endolymphatic cross-sectional area measured in vivo with AsF6 with that measured by established histologic procedures. The correlation between the two measures was good, with a coefficient of .903, although the area measured histologically was a little lower than that measured in vivo.

Endolymph volume changes during osmotic dehydration measured by two marker techniques.

The processes underlying endolymph volume regulation during osmotic disturbances were investigated in vivo using ionic volume markers. The markers utilized were tetramethylammonium (TMA+) or hexafluoroarsenate (AsF6-). Both ions were used in concentrations low enough not to be toxic, but readily detectable by ion-selective microelectrodes (typically < 1 mM). Two marker techniques were developed. In one, termed the 'perfused volume marker' (PVM) method, the marker was loaded into endolymph throughout the cochlea by perfusion of the perilymphatic space. Concentration changes of the marker were measured with a double-barreled ion-selective microelectrode. These recordings were insensitive to longitudinal movements of endolymph. The second technique, termed the 'iontophoresed volume marker' (IVM) method, utilized a localized, iontophoretic injection of marker into endolymph. In this method, marker changes were recorded from two ion-selective electrodes, one placed basal and one placed apical to the injection site. These data were used to compute changes in cross-sectional area and longitudinal movements of endolymph. Changes in endolymph volume were induced by perfusion of the perilymphatic space with hypertonic media. The endolymph potassium increase produced by osmotic dehydration was of similar magnitude and time course to that of a volume marker loaded by the PVM method. Using the IVM method, it was shown that these concentration increases arose by two distinct processes. One component was the area decrease of scala media. A second component was a small apically directed movement of endolymph during dehydration, thereby concentrating the available electrolytes within a smaller volume. This latter component was estimated to contribute approximately one third of the electrolyte increase during dehydration. Both the present and previous studies show that in the undisturbed state, longitudinal endolymph movements are extremely small and cannot make a significant contribution to ionic homeostasis. However, when endolymph volume is disturbed, longitudinal movements contribute to the electrolyte changes and are part of the compensation process. This study provides the first direct evidence supporting the long-standing hypotheses that local, radial homeostasis and longitudinal volume corrections both occur in the mammalian cochlea.

Detection and quantification of endolymphatic hydrops in the guinea pig cochlea by magnetic resonance microscopy.

Three-dimensional magnetic resonance microscopy (MRM) was used to study normal and hydropic cochleae of the guinea pig. With this technique consecutive serial slices representing the entire volume of isolated, fixed cochleae were obtained. The voxels (volume elements) making up the contiguous slices were isotropic (25 microns 3) and in each slice the boundaries of scala media, including the position of Reissner's membrane, were clearly delineated. Three-dimensional reconstructions of the endolymphatic and perilymphatic scale were generated. Custom software was developed to quantify cross-sectional area (CSA) of all scalae. In the normal cochlea all 3 scalae, including scala media, showed a gradual decrease in CSA from base to apex. Marked differences existed between our findings and previously reported cochlear dimensions, especially for the perilymphatic scalae in the basal turn. In hydropic cochleae the scala media was enlarged to a varying extent in different turns and marked changes in the degree of distension of Reissner's membrane occurred along the cochlea. MRM and subsequent computer analysis of the isotropic data provide excellent methods for imaging and quantifying the fluid spaces of normal and hydropic cochleae.

Minimal concentrations of metabolic substrates capable of supporting cochlear potentials.

The objective of this study was to determine the capability of glucose analogues, as well as lactate and pyruvate, to maintain the endolymphatic potential and the cochlear microphonics. In addition, the minimum concentration at which different substrates (including D-glucose) were able to sustain the potentials ("critical' concentration) was determined. Synthetic blood containing various substrates at different concentrations was perfused via the anterior inferior cerebellar artery. The critical concentration for D-glucose was found to be 15 mg% (0.83 mM). L-Glucose, galactose and fructose were not able to support the potentials at concentrations as high as 200 mg%. On the other hand, mannose was capable of supporting the potentials; however, the critical concentration (50 mg% or 2.8 mM) was substantially higher than that of D-glucose. Both lactate and pyruvate could support the potentials, but the critical concentrations (8.5 mM and 6.5 mM, respectively) were markedly higher than in the case of glucose, even when the difference of carbon equivalents was taken into consideration. The data are discussed in the context of the intermediary metabolism and possible carrier systems of the stria vascularis.

Effect of substrate-free vascular perfusion upon cochlear potentials and glycogen of the stria vascularis.

The effect of vascular perfusion of the anterior inferior cerebellar artery with synthetic blood containing no metabolic substrates upon the endolymphatic potential (EP) and the cochlear microphonics (CM) was determined in the guinea pig. In substrate-free perfusion the potentials were maintained for an average of 84 min. Subsequently, the EP declined at an average rate of 1.4 mV/min until a new steady-state level was temporarily established when the potential had dropped to about 30 mV. The decline of the CM appeared to be accounted for largely by the decline of the EP. During substrate-free perfusion prior to the onset of the decline of the potentials, the level of strial glycogen remained unchanged; glycogen decreased significantly only after the potentials had started to decline. When substrate-free vascular perfusion was accompanied by simultaneous substrate-free perilymphatic perfusion, the potentials started to decline immediately. On the basis of these data, we conclude that strial glycogen plays no role in the prolonged maintenance of the EP during substrate-free perfusion; rather, the potential seems to be maintained by entry of glucose (and presumably other substrates) from perilymph into the stria vascularis.

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.

Specificity of action of vanadate to the organ of corti.

Although vanadate strongly inhibits Na/K-ATPase activity of the stria vascularis in vitro, it initially causes no depression of the ouabain-sensitive endocochlear potential (EP) when perfused perilymphatically or via the vasculature. However, when the perilymph of scala tympani is replaced with artificial media containing 0.1 to 1 mM vanadate, there is a large (about 17 mV) increase in the EP of the second cochlear turn. Further experiments showed that the cochlear microphonics declined during the time in which the EP increased, and that the response of these two potentials to vanadate is greater in the second turn than in the first. Injection of 50 n1 of 1 mM vanadate (in artificial endolymph) into the endolymphatic space of the second turn caused no increase in the EP. These results support the notion that the early effects of vanadate are on the contra-luminal membranes of cells of the organ of Corti rather than on the stria vascularis. By superimposing anoxia or furosemide (i.v.) upon vanadate intoxication, we determined that the initial increase of the compound EP due to vanadate alone was due to a reduction in magnitude of the negative component of the EP. It is argued that of the three prevalent theories concerning the generation of the negative EP, the data tend to support the hypothesis that the intracellular potential of the hair cells gives rise to the negative EP.