John E. DeMott

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.

Longitudinal endolymph flow associated with acute volume increase in the guinea pig cochlea

Endolymph volume disturbances were induced by microinjections of artificial endolymph into the second turn of the guinea pig cochlea at rates less than 60 nl/min. Induced longitudinal movements and area changes of endolymph were quantified in the basal turn using an ionic flow marker technique. Tetramethylammonium (TMA) was used as a flow marker by iontophoresing it into endolymph in micromolar amounts. TMA movements in the apical and basal directions were monitored by ion-selective electrodes. Changes in endolymph flow and cross-sectional area of scala media were derived using a mathematical model to interpret the recorded tracer time courses. The model was validated by performing comparable volume injections and flow measurements in fine-diameter plastic tubes. The rate of flow of endolymph measured prior to injection was close to zero, in agreement with prior studies. Based on the injection of different volumes into endolymph over a 15 min period, we found that injection of up to 80 nl of artificial endolymph into the second turn would not induce flow in the basal turn. However, above this amount, flow towards the base increased at a rate which correlated with the injected volume, with endolymph moving basally by a distance of 0.0067 mm/nl of artificial endolymph injected. Flow rates measured in the third turn, on the apical side of the injection were far lower and showed characteristics consistent with there being no outlet at the apex. These results suggest that small volume disturbances are corrected locally in the cochlea, but larger disturbances produce a longitudinal flow of endolymph out of the cochlea which represents a significant mechanism contributing to homeostasis. It can be concluded that structures outside the cochlea, such as the endolymphatic sac, do play a role in the correction of endolymph volume disturbances. Although the maintenance of endolymph composition is dominated by local ion transport mechanisms, the capacity of these local mechanisms to maintain normal endolymph volume appears to be limited.

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 Reissners 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 Reissners 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.

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.

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/.

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.

Endolymph calcium increases with time after surgical induction of hydrops in guinea-pigs

The ionized Ca2+ concentration in cochlear endolymph is normally extremely low. Previous studies have shown that endolymph Ca2+ levels become elevated when measured at long intervals after endolymphatic hydrops is surgically induced. The present study was designed to investigate how rapidly endolymph Ca2+ increases following endolymphatic duct ablation. Hydropic animals were tested at either 4 days, 4 weeks, 8 weeks or 16 weeks after surgery. In each animal endolymph Ca2+ and endocochlear potentials were measured in all four cochlear turns using double-barreled Ca(2+)-sensitive electrodes. Cochlear sensitivity was assessed using compound action potential thresholds. Our results confirm that hydropic animals show an elevation of endolymph Ca2+ and a reduction of EP which is initially small, but becomes more pronounced at longer times after surgery. At 16 weeks endolymph Ca2+ was increased by an average factor of 20 in the basal turn and 7.5 in the fourth turn. These findings suggest that endolymph Ca2+ changes may not be the primary factor responsible for hydrops generation, but probably contribute to cochlear dysfunction in later phases of hydrops. For some experimental groups, the elevation of AP threshold was more closely correlated with endolymph Ca2+ level than it was with endolymph volume. Endolymph Ca2+ changes must therefore be considered in order to account for dysfunction in the hydropic cochlea.

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.

Time course of endolymph volume increase in experimental hydrops measured in vivo with an ionic volume marker

A new method has been developed to measure the cross-sectional area (CSA) of scala media in the living cochlea. The method has some advantages over histological methods, in which tissues may shrink or move during processing. In the present study, scala media CSA was measured in the second turn of guinea-pig cochleas in which endolymphatic hydrops was induced surgically. The area measurement method used an iontophoretic injection of a volume marker into scala media, during which the concentration of marker in endolymph was monitored with an ion-selective microelectrode. The measured marker concentration was inversely proportional to the CSA of endolymph. The marker we used was the anion arsenic hexafluoride (AsF6-), which was almost ideal for the purpose as it was retained well in endolymph. Area was measured in normal animals and in hydropic animals at times from 4 days to 16 weeks after endolymphatic duct obstruction. The results showed that hydrops develops within days of ablation of the endolymphatic duct. The degree of hydrops was compared with electrophysiological measures of function, including the endocochlear potential, action potential thresholds and the amplitudes of the cochlear microphonic, summating potential and action potentials. In the initial stages of hydrops development, electrophysiological changes were small. In contrast, there were marked functional changes between 8 and 16 weeks, when endolymph volume was no longer increasing. If the same is true for dysfunction in the ears of patients with Ménières Disease, then it may not be possible to restore normal function simply by alleviating the hydrops.(ABSTRACT TRUNCATED AT 250 WORDS)

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.