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Responses of the endolymphatic sac to perilymphatic injections and withdrawals: evidence for the presence of a one-way valve

Alec N. Salt



, Helge Rask-Andersen


Department of Otolaryngology, Box 8115, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA b Department of Otolaryngology, University Hospital (Akademiska Sjukhuset) of Uppsala, 751 85 Uppsala, Sweden Received 16 October 2003; accepted 30 December 2003

Abstract Although the endolymphatic sac (ES) is thought to be a primary site for endolymph volume regulation, we have limited knowledge of how it responds to volume and pressure changes. In a prior publication, we demonstrated changes of Kþ , Naþ and endolymphatic sac potential (ESP) resulting from volume injections into, and withdrawals from, scala media of the cochlea. In the present study, we compared the influence of injections into and withdrawals from scala tympani of the cochlea on the endolymphatic sac. It is assumed that similar pressure changes are induced in endolymph and perilymph of both the cochlear and vestibular compartments of the ear. Pressure changes induced by the perilymphatic injections and withdrawals did not induce similar Kþ changes in the ES. The majority of perilymph withdrawals caused Kþ and ESP reductions in the sac, but few injections caused any measurable changes in the sac. Pressure measurements from the ES demonstrated that transmission of labyrinthine pressures to the lumen was directionally sensitive, with negative pressure transmitted more effectively than positive. In other experiments, application of infrasonic stimulation to the ear canal resulted in Kþ increase in the ES. These physiological measurements suggest that the endolymphatic duct may be closed by sustained positive pressure in the vestibule but open during pressure fluctuations. Study of the anatomy where the endolymphatic duct enters the vestibule suggests that the membranous sinus of the endolymphatic duct could act as a mechanical valve, limiting the flow of endolymph from the saccule to the endolymphatic sac when pressure is applied. This structure could therefore play an important role in endolymph volume regulation. Ó 2004 Elsevier B.V. All rights reserved.

Keywords: Endolymphatic hydrops; Endolymphatic sinus; Saccule; MeniereÕs disease

1. Introduction The endolymphatic sac (ES) is widely regarded as the locus of endolymph volume regulation. The strongest evidence supporting this view is the finding that surgical ablation of the ES in many species, including guinea pigs, produces severe endolymphatic hydrops (Kimura and Schuknecht, 1965; Kimura, 1967). Anatomic and physiologic studies suggest the ES is an active structure involved in ion transport (Rask-Andersen et al., 1981; Erwall et al., 1988; Lim, 1999; Rask-Andersen et al., 2000).


Corresponding author. Tel.: +314-362-7560; fax: +1-314-362-7522. E-mail address: [email protected] (A.N. Salt).

Abbreviations: ES, Endolymphatic sac; ESP, Endolymphatic sac potential; CSF, Cerebrospinal fluid 0378-5955/$ - see front matter Ó 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2003.12.018

The transport activity of the ES has been modulated by acute experimental manipulations of systemic fluid status, such as by glycerol administration (Erwall, 1988; Erwall et al., 1988; Takumida et al., 1991; Jansson et al., 1992; Jansson and Rask-Andersen, 1993). It has also been reported that function is altered by manipulations of endolymph volume. The ES response is bidirectional, giving different anatomic and physiological responses to endolymph volume increases and decreases (RaskAndersen et al., 1999; Salt and DeMott, 2000). Based on these studies, it was proposed that endolymph was resorbed by the sac under conditions of volume excess and secreted by the sac under conditions of volume deficiency. The possibility that endolymph may move in either direction between the labyrinth and the ES under different situations led us to consider the factors


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determining fluid movements across the duct. We initially expected that based on simple physical principles, the primary factor driving volume flow would be the pressure differential between the two ends of the duct. Since endolymphatic boundaries are highly compliant, the pressures of endolymph and perilymph in normal ears are almost identical (Bohmer, 1993; Andrews et al., 1991). Identical pressure changes are also measured in both endolymph and perilymph during most treatments that change labyrinthine pressure (Takeuchi et al., 1990; Takeda et al., 1990; Takeuchi et al., 1991; Wit et al., 2000). These studies suggest that pressure is constrained by the bony walls of the otic capsule, but not by the compliant boundary membranes of the endolymphatic space. It would therefore be expected that manipulations of perilymph pressure should directly influence pressure of endolymph at the saccular end of the endolymphatic duct. The present study therefore investigated the effects of perilymphatic pressure manipulations on the ES in order to determine under what conditions fluid movement through the endolymphatic duct occurred. Measurements of Kþ in the lumen of the endolymphatic sac have been previously used as an indicator of fluid status in the ES. The normal Kþ concentration of endolymph in the ES varies from 8 to 18 mM (Mori et al., 1987; Mori et al., 1998; Couloigner et al., 1999; Salt and DeMott, 2000) which is far lower than the 150 mM Kþ present in the saccule (Morgenstern et al., 1982). Injections into the endolymphatic space showed that endolymph movements into the sac were accompanied by a marked increase of Kþ concentration in the sac lumen, while endolymph withdrawals produced small reductions in Kþ (Salt and DeMott, 2000). The measurement of induced Kþ changes in the ES with ion-selective electrodes therefore provides a sensitive indicator of functional change.

2. Methods 2.1. Physiologic studies This study used pigmented NIH strain guinea pigs, anesthetized with sodium thiobutabarbital (Inactin, Sigma, St. Louis, 100 mg/kg, intraperitoneally). The trachea was cannulated and the animal was ventilated mechanically, adjusting tidal volume to maintain the end-tidal CO2 level close to 5%. Body temperature was maintained at 38 °C with a thermistor-controlled heating pad. The left jugular vein was cannulated for the administration of anesthetic supplements and for giving pancuronium bromide (Pavulon, Baxter, Irvine, approximately 0.05 mg, to effect) to minimize muscle artifacts and to aid in ventilation. The study used methods to place microelectrodes within the lumen of the endolymphatic sac identical to

those described by Salt and DeMott (2000). The upper margin of the niche in which the ES lies was exposed by an extradural approach. Care was taken not to disturb the ES itself, the dura mater or the sigmoid sinus near the sac. An ion-selective or pressure-sensing recording electrode was inserted blindly towards the middle of the niche until it entered the lumen of the sac, as indicated by registration of a stable positive potential, the endolymphatic sac potential (ESP). Kþ and ESP measurements were made in 10 animals and hydrostatic pressure and ESP measurements were made in 14 animals. Potassium-selective microelectrodes were made and calibrated according to methods described previously (Salt and DeMott, 2000). In brief, double-barreled pipettes were pulled and one barrel was silanized by exposure for 60 s to dimethyldichlorosilane vapor. The tips were beveled with a Narishige EG-40 pipette grinder set to a 45° angle until the diameter was in the range of 2­4 lm. The non-silanized reference barrel, used to record potential, was filled with 500 mM NaCl. The silanized barrel was filled with 500 mM KCl and a small column of Fluka 60398 ion exchanger (Sigma­Aldrich, St. Louis) was drawn into the tip by suction. Electrical connections were made to the electrodes by Ag/AgCl wires. The wires were connected to custom-made high-impedance electrometer, incorporating a differential amplifier to generate an ion-dependent, voltage-independent signal. Electrodes were calibrated before and after use in standards containing 2, 10 and 20 mM KCl in a background of 150 mM NaCl. Standards were maintained at 39 °C in a custom-made chamber with an outer jacket through which warmed water was circulated. Hydrostatic pressure in the endolymphatic sac was measured using a WPI model 900, servo-nulling micro pressure system (World Precision Instruments, Sarasota, FL). The recording pipettes were single barrel, beveled to a diameter of 5­7 lm and filled with 3 M KCl. The instrument provided output signals representing both the hydrostatic pressure at the pipette tip, and the potential. Manipulation of intracochlear pressure during endolymphatic sac recordings required the cochlea to be exposed. This was performed by a post-auricular approach, with the lateral bulla opened sufficiently to visualize the basal turn of the cochlea. A hole was drilled into scala tympani of the cochlea with a sharpened needle and a perfusion pipette with a bead of nail polish near the tip was inserted into the scala. The pipette was filled with artificial perilymph and mounted on a WPI Ultrapump that permitted fluid injections or withdrawals to be performed. The pipette was sealed into the bony otic capsule with Histoacryl tissue adhesive (Braun, Germany). The composition of the artificial perilymph used for injection is given elsewhere (Salt and DeMott, 1998). When injections or withdrawals are performed in an otherwise sealed cochlea it has been


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shown that the cochlear aqueduct provides an outlet, which limits the pressure change induced (Wit et al., 2003). Endolymphatic injections were performed according to methods identical to those in our prior study (Salt and DeMott, 2000). In some experiments, infrasonic stimulation (0.3 or 1 Hz) was delivered to the external ear canal. The transducer used to deliver pressures was the pressure source of the WPI model 900 pressure measurement system, modified so that it could be driven by an external signal. This driver was connected to external ear canal through a hollow ear-bar. The system included a pressure measurement transducer that permitted the ear canal pressure to be monitored directly during stimulation. Prior studies have shown that infrasonic stimulation results in longitudinal endolymphatic movements in the cochlea (Salt and DeMott, 1999). 2.2. Anatomic studies The anatomy of the endolymphatic duct was first investigated in archival specimens of the guinea pig inner ear, generated as part of a prior study performed in collaboration with Dr. R. Kimura (Salt et al., 1995). In that study, control and hydropic ears were fixed with Heidenhan-Susa, decalcified in 5% trichloroacetic acid, dehydrated and embedded in celloidin. Horizontal sections 20 lm in thickness were cut and every tenth section was mounted and stained with hematoxylin and eosin. Due to the 200 lm separation of sections, the endolymphatic duct was not well described in all of these specimens. We therefore examined another guinea pig specimen, provided from Dr. KimuraÕs personal archive, which was prepared similarly cut in horizontal 20 lm sections. For this specimen, every section through the temporal bone was stained and mounted. This permitted the detailed structure of the endolymphatic duct to be observed. One additional specimen of the guinea pig endolymphatic duct was prepared for sectioning in an orientation orthogonal to the longitudinal axis of the endolymphatic duct. The guinea pig specimen was fixed with 3% glutaraldehyde, post-fixed in 1% osmic acid, dehydrated and embedded in Epon. The specimen was oriented and serial sections of 1­2 lm thickness were cut. The sections were mounted on glass slides and stained with toluidine blue. The sections were viewed with a light microscope. The Animal Studies Committee of Washington University approved the experimental procedures used in this study under protocol number 19990029.

similar pressure changes in both perilymph and endolymph of the cochlea, as shown in Fig. 1. Injection caused immediate pressure change in both perilymph and endolymph. The variation in magnitude of the pressure changes during perilymph injections is accounted for by variations in the resistance to fluid outflow provided by the cochlear aqueduct (Wit et al., 2003). Due to the non-compliant bony otic capsule, we would expect pressure throughout the vestibule to have shown comparable changes during injections and withdrawals. The magnitudes of perilymph pressure changes during injection were comparable with our prior study in which the injection at a rate of 1.5 lL/min into the sealed cochlea gave a mean pressure increase of 3.2 mm Hg (Salt and DeMott, 1998). The low initial pressure readings result from the animal orientation used in the study, in which the torso was substantially lower than the measurement point.

3. Results Manipulations of perilymphatic volume, by fluid injections or withdrawals from scala tympani, produced

Fig. 1. Cochlear pressure changes measured during injection and withdrawal from the basal turn of scala tympani at 1.5 lL/min. Data were recorded in two separate experiments from scala tympani of the basal turn (upper panel) or from scala media of the basal turn (lower panel).


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An example of the changes of endolymph sac Kþ and ESP recorded during injections into and withdrawals from scala tympani at 1.5 lL/min are shown in Fig. 2. The measured changes during volume injection in this preparation were small or nonexistent, while withdrawals at the same rate produced substantial decreases of endolymph Kþ and decreases of the ESP. Recovery of endolymph sac Kþ content and ESP from the withdrawal occurred rapidly after the withdrawal ceased. A summary of the endolymphatic sac changes resulting from injections and withdrawals is given in Fig. 3. In each panel, the individual responses to the injection or withdrawal are overlaid. Injections produced no endolymphatic Kþ increases in 10 of the 12 procedures, with only two injections (17%), both in the same animal, producing large Kþ increases. Similarly, recorded ESP changes were minimal during injections except for those for the two injections in the animal where Kþ increases were observed. In this animal, injection-induced ESP

Fig. 2. Changes of endolymph K+ (upper panel) and potential (lower panel) in the endolymphatic sac during injections into and withdrawals from scala tympani at 1.5 lL/min. Injections had little influence on both parameters, while withdrawals caused decreases of both K+ and potential.

increases were observed. In contrast, withdrawals from scala tympani produced decreases in Kþ and ESP in 6 of the 9 (66%) experiments. In one experiment, we compared changes in the endolymphatic sac induced by perilymphatic and endolymphatic injections, as shown in Fig. 4. The initial injection into perilymph at 1.5 lL/min had little effect on ESP and endolymph Kþ in the sac, except for an increase of noise in the Kþ trace due to electrical interference from the injection pump. In this experiment, an endolymph injection pipette was then inserted through the round window and organ of Corti and an injection into the endolymphatic space of the cochlea was performed at 79 nL/min. This injection caused a substantial Kþ increase and a small ESP increase that is similar to those observed in our prior studies using endolymph injections (Salt and DeMott, 2000). This experiment confirms in an individual preparation that the ES is far more sensitive to endolymphatic injections at low rate than it is to perilymphatic injections at higher rate. In some experiments, we measured pressure in the endolymphatic sac lumen while perilymphatic manipulations were performed. Pressure in the endolymphatic sac is more noisy and unstable than it is in the cochlea, due to the proximity of the sigmoid sinus and dura. Averaging of repeated samples was used to reduce the influence of this background noise. An example experiment showing simultaneously measured pressure and ESP changes is shown in Fig. 5. Perilymphatic injections at 1.5 or 3 lL/min produced no consistent changes of pressure or potential, while withdrawals produced pressure decreases in the sac with an associated reduction in ESP. The magnitude of the pressure and ESP changes during withdrawals increased with the higher withdrawal rate. The effects of low frequency sound on endolymphatic sac composition and ESP are shown in Fig. 6. In the figure, open symbols show data collected with no sound stimulus delivered. Prior to the data collection for each filled symbol, a burst of 5 cycles at 0.3 Hz (total burst time 1.6 s) was delivered to the external ear canal at a peak pressure of 8.8 mmHg. This stimulus is sufficient to displace the tympanic membrane and stapes a comparable distance to that observed during the contraction of middle ear muscles, which has previously been shown to induce longitudinal endolymph movements (Salt and DeMott, 1999). When the low frequency stimulus was applied, there was an increase in the Kþ concentration of the sac, with a delay of approximately 1 min. The increase peaked and declined back towards baseline when the infrasonic stimuli were stopped. ESP changes during the stimulation were unremarkable. The insensitivity of the endolymphatic sac to positive pressures in the vestibule led us to consider how this was possible mechanically. Prior to these studies, we had assumed that the endolymphatic duct provided an open


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Fig. 3. Summary of changes in endolymphatic K+ (upper row) and potential (lower row) during injections into (left column) or withdrawals from (right column) scala tympani at 1.5 lL/min. Traces represent individual injections or withdrawals. Withdrawals produced K+ and potential changes in 6 of the 9 experiments, while injections produced changes in only 2 of 12 experiments.

communication between the saccule and the endolymphatic sac. Studies of the location where the endolymphatic duct opens into the vestibule, however, reveal that the anatomy in this region is complex. The region is shown as a horizontal section in Fig. 7, demonstrating the major structures on the posteromedial wall of the vestibule. The endolymphatic duct, entering the picture at the lower right side, does not immediately widen into the saccule when it enters the vestibule. Instead, it forms a membranous bulb. The terminology for the anatomy of this region was reviewed by Bast and Anson (1949), who termed the structure the endolymphatic sinus. The sinus narrows into the saccular duct before widening out again to become the saccule. In the section, the wall of the endolymphatic sinus facing the vestibule shows filamentous connections to the wall of the utricle. The relationship of the structure to the utricle is more apparent in serial sections from ventral to dorsal, as shown in Fig. 8. In sections 113 and 115 it is apparent that the wall of the endolymphatic sinus is continuous with the luminal wall of the endolymphatic duct, which enters the figures at the lower right side of each panel. Subsequent sections show a thickening of the wall of the sinus, which represents a tangential section through the

utricular duct that connects to the lumen of the utricle through the valve of Bast, as indicated in section 119. The anatomy of the sinus, sectioned orthogonal to the axis of the endolymphatic duct, is shown in Fig. 9. As the endolymphatic duct opens into the vestibule, the tissues forming the duct separate into two boundaries with a fluid space between, as seen in sections D, E and F. The outermost boundary becomes discontinuous (sections G and H) demonstrating fluid communication between this space and the vestibule. The luminal boundary of the endolymphatic duct continues to form the thin wall of the sinus. It is also notable that in this region (sections E and F) there is considerable corrugation of the membranes at the duct margins, consistent with the possibility that displacements of the membrane may occur. It is likely that pressure applied to the vestibule would collapse this boundary membrane, providing partial or complete closure of the lumen. Subsequent sections of the figure show a duct connecting to the utricle via the valve of Bast (sections J and K), followed by the sinus narrowing (section L) before widening to form the saccule (sections O and P). Based on the sections shown, we estimate that the endolymphatic sinus has dimensions of approximately


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Fig. 4. Comparison of the endolymphatic sac response to a perilymphatic injection, followed by the response in the same animal to an endolymphatic injection. The sac was insensitive to the perilymphatic injection at 1.5 lL/min, but was extremely sensitive to an endolymphatic injection at a lower rate.

Fig. 5. Changes of pressure (upper panel) and potential (lower panel) during injections into and withdrawals from perilymph of scala tympani at the indicated rates (in lL/min). Pressure and potential changes during injections were unremarkable, while withdrawals produced pressure decrease that depended on the rate of withdrawal.

70 Â 8 Â 45 lm, and a luminal volume of approximately 13 pL. In the guinea pig, the volume of the endolymphatic sinus is therefore smaller than the ES by a factor of about 104 .

4. Discussion Measurements of Kþ concentration, pressure and potential in the endolymphatic sac suggest that the communication pathway between the saccule and the endolymphatic sac is not patent under some conditions. Specifically when perilymphatic pressure is increased by injection, most animals showed no indication of any influence on the endolymphatic sac. Careful examination of the anatomy in the region of the endolymphatic duct demonstrates a structure, the endolymphatic sinus, which could possibly act as a

barrier to fluid entering the endolymphatic duct. The postulated action of fluid manipulations on the structure is shown schematically in Fig. 10. Injections into endolymph could expand the sinus with an associated movement of endolymph towards the endolymphatic sac. In contrast, injections into perilymph that increase pressure in the vestibule could cause the structure to collapse against the bony wall, limiting the volume of endolymph that is driven into the sac. Decreases in pressure of the vestibule would permit endolymph to be drawn from the endolymphatic sac into the sinus. Endolymph movements through the duct would then depend not only on the pressure difference between the vestibule and the endolymphatic sac, but also on factors that could influence distension of the sinus walls, including the pressure difference between endolymph and perilymph and possibly the rate of volume flow through the sinus.


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Fig. 6. Effect of infrasonic stimulation of the cochlea on the endolymphatic sac. Prior to data collection for each of the filled symbols, 5 cycles of stimulation at 0.3 Hz were delivered to the external ear canal. The pressure fluctuations result in an increase of K+ in the sac lumen. The K+ concentration recovers when stimulation is stopped.

Fig. 7. Horizontal section through the vestibule of the guinea pig at the location where the endolymphatic duct enters. The duct opens into a bulb-like structure, the endolymphatic sinus. The calibration bar is 25 lm.

The sinus of the endolymphatic duct is therefore likely to play a key role in the regulation of endolymph volume. Previously it has been assumed that the endolymphatic sac itself detects and corrects abnormal endolymph volume states. The endolymphatic sac is poorly situated to detect abnormal endolymph volumes by mechanical means. Since the sac is intimately in contact with both the sigmoid sinus and the dura mater, it is exposed to both vascular pulsations and respirationinduced CSF pressure changes. The pressure measured in the endolymphatic sac was found to fluctuate more than that in the cochlea. This is because the influence of CSF pressure fluctuations on the cochlea is attenuated through the combination of the narrow cochlear aqueduct and compliant round window membrane (Gopen et al., 1997). Similarly, in the guinea pig, anatomic studies show looped structures, termed springcoils, in arterioles of the cochlea (Axelsson and Ryan, 2001), which are believed to attenuate vascular pulsations. Since the sac does not have either of these specializations, it is difficult to believe that it could detect the minuscule endolymph overpressure resulting from a slight ReissnerÕs membrane distension in the distant higher turns of the cochlea. Recordings from vestibular afferents show high sensitivity to extremely small dilational pressures (the pressure of endolymph with respect to perilymph) and a marked insensitivity to ambient (perilymphatic) pressure changes (Yamauchi et al., 2001). These findings suggest the magnitude of the endolymphatic overpressure during small endolymph volume disturbances is likely to be extremely small, and probably less than the 10À3 Pa (approx. 1 lm water) that was found to induce vestibular stimulation. The mechanism by which the ES detects abnormal volume states has not previously been established. Based on our findings, we propose that the ES does not itself detect endolymph volume change, but instead the endolymphatic sinus performs this function. This would be achieved if the distension of the sinus varied with changes of endolymph volume so that movement of endolymph towards the sac would be limited under conditions of endolymph volume insufficiency and allowed to occur when volume was too high. Understanding how the sinus is likely to function, however, requires consideration of both the sustained and fluctuating pressures that act on the system. The pressure that must be detected to regulate endolymph volume is presumed to be sustained, representing the minuscule endolymph overpressure or underpressure with respect to perilymph that corresponds to the endolymph volume disturbance. However, absolute pressure in the ear (relative to atmospheric pressure) is never static but is subject to changes arising from many sources due to the anatomic connections of the labyrinth to other compartments. CSF pressure fluctuations are transmitted to the ear via the cochlear aqueduct and endolymphatic


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Fig. 8. Serial 20 lm thick sections through the sinus of the endolymphatic duct. The numerals indicate the section number, with lower numbers representing more ventral sections. The figure shows the relationship to the endolymphatic duct (entering at the lower right of sections 113 and 115) and to the valve of Bast (section 119). Abbreviations are S: endolymphatic sinus, U: utricle. The calibration bar is 25 lm.

Fig. 9. Serial sections through the sinus of the endolymphatic duct made orthogonal to the duct axis. The numerals indicate the section number. Abbreviations are: ED: endolymphatic duct, V: vestibule, U: utricle, I: endolymphatic sinus, S: saccule. The sinus is seen as a narrow structure (sections F­H) that could be collapsed by positive pressure in the vestibule. The calibration bar is 20 lm.

duct (Carlborg and Farmer, 1983; Bohmer, 1993). CSF pressure fluctuations occur with respiration, posture changes, coughing or sneezing. Movements of the stapes also induce pressure changes in the labyrinth. Although moderate sounds at acoustic frequencies may not be relevant, there are many sources of low frequency (infrasonic) stapes movements that can cause substantial pressure changes in the labyrinth. Stapes movements are

induced by pressure changes in the middle ear (such as from swallowing, sniffing or performing the valsalva maneuver), middle ear muscle contractions (induced by swallowing, chewing or loud sound), atmospheric pressure changes (such as driving up hills, flying or diving) or induced pressure changes in the ear canal (such by pushing a fingertip into the canal opening). Middle ear pressure changes caused by some of the above factors


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Fig. 10. Schematic showing the proposed influence of fluid manipulations on sinus of the endolymphatic duct. Injections into endolymph (upper right) would be expected to stretch the structure, increasing the sinus volume. Increase of perilymph pressure by injection (lower left) would collapse the structure, limiting the amount of endolymph driven into the sac. Decrease of perilymph pressure by withdrawal (lower right) would permit endolymph to move from the sac to the sinus, thereby causing pressure and composition changes in the sac.

can also transiently influence perilymph pressure by acting through the round window membrane. Vascular pulsations can influence each of the compartments (cochlea, vestibule, middle ear, cranium, etc). In summary, there are numerous sources contributing to the ongoing perilymph pressure fluctuations that occur in the inner ear. Our data suggest that during those periods when the labyrinth has positive pressure with respect to the endolymphatic sac, a small volume of endolymph will be pushed towards the sac before the duct closes. As pressure cycles negative, the sinus would be re-inflated. This would account for the lack of Kþ increase in the sac when a sustained positive pressure was applied, but a Kþ increase when cyclical, infrasonic stimulation was applied. An important factor in such a mechanism is that the volume pushed into the endolymphatic sac during these pressure fluctuations would depend on the degree of distension of the sinus. Under conditions of above normal endolymph volume, if the sinus was distended, then more endolymph would be pushed into the sac with each positive pressure cycle. Under conditions where endolymph volume was low, the sinus membrane would be closer to the bony wall and would tend to collapse more readily with less volume pushed towards the sac. We accept that the above explanation of our results contains a number of speculative elements. We still do not know whether pressure fluctuations in the labyrinth play a necessary role in endolymph volume regulation by inducing fluid movements. It remains possible that the induced fluid movements and resulting ion changes in the sac are phenomena unrelated to volume regulation. Nevertheless, the present study demonstrates both

a physiologic and anatomic complexity of the endolymphatic duct that must be incorporated into future hypotheses related to ES function. Some of the variation of findings in the present study could be related to variation in the effectiveness of the sinus acting as a valve. During injections into perilymph of one preparation, substantial Kþ increases were observed. For this individual it must be concluded that the duct remained open during the pressure application. While this may have resulted from endolymph enlargement or an abnormal endolymphatic sinus, we have no corroborating evidence to support such an explanation. Similarly, for those withdrawal experiments where no Kþ and ESP changes in the sac were observed, it is not certain whether this represents an insensitivity of the sac to the treatment or a closed endolymphatic duct or sinus. The variation we observed here contrasts with the endolymph injection study (Salt and DeMott, 2000) where Kþ changes were consistently observed. It suggests that there may be many factors influencing fluid movement through the endolymphatic duct, and that the perilymph pressure and the effectiveness of the endolymphatic sinus as a valve may be just some of the relevant variables. The comparison of ion and potential changes in the sac caused by endolymph or perilymph manipulations indicate a number of differences in response characteristics. The changes observed with perilymph manipulation were generally of similar direction to those in our previous study with endolymphatic manipulations. Injections produced Kþ and ESP increases while withdrawals caused Kþ and ESP decreases. The decrease of


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Kþ with perilymph withdrawals likely represents a locally-derived change in ion transport activity (Salt and DeMott, 2000). One area of difference between perilymph and endolymph manipulations was in the relative magnitudes of endolymph Kþ and ESP changes. Endolymph manipulations usually produced large changes in Kþ concentration, especially for injections (as in Fig. 4), but changes of ESP were typically small, especially for endolymph withdrawals. In contrast, with perilymphatic withdrawals, substantial changes of ESP were seen (Figs. 3 and 5) even when induced Kþ changes were small. This difference is likely the result of pressure differences between the two experiments. Pressure decreases induced by perilymphatic withdrawals at 1.5 lL/ min will have been substantially larger than those induced endolymph withdrawals at 100 nL/min. The sac response appears to depend on the degree of mechanical and chemical disturbances, which differ for endolymphatic and perilymphatic manipulations. The observations reported here suggest that regulation of endolymph movements in and out of the endolymphatic sac may be more complex than was previously believed. This may have relevance to the causes of endolymphatic hydrops and MeniereÕs disease and suggests that future studies of the pathophysiological changes of the endolymphatic sinus may be worthwhile. They also suggest that endolymph volume regulation may not occur in a continuous, steady state fashion, but may instead occur as a discontinuous process during pressure fluctuations induced by swallowing, middle ear muscle contractions or from other sources. Dysfunction in this system could result from many causes, including not only the sinus but also of the many origins of pressure fluctuations in the ear. Our observations are relevant to a number of clinical studies that have reported an influence of pressure manipulations on symptoms of patients with MeniereÕs disease. Treatment in a hypobaric chamber was found to reduce vertigo and improve hearing in some patients (Ingelstedt et al., 1976; Van Deelen et al., 1987), an effect that has been attributed to an overpressure of the middle ear with respect to the surrounding atmosphere (Konradsson et al., 1999). In an animal study, hydropic guinea pigs exposed to positive pressures in the middle ear in a pressure chamber showed no significant change in the magnitude of hydrops, while animals with positive pressure applied to the intact ear canal for 1 h, twice a day showed significantly less hydrops (Sakikawa and Kimura, 1997). The difficulty in interpreting these studies is that all the pressure treatments have involved both transient pressure changes and sustained pressure delivery. Other investigators have combined slowly alternating pressure changes (6­9 Hz) with sustained pressure, delivered in a closed system to the middle ear space via a tympanostomy tube. This treatment was reported to improve hearing sensitivity and decrease the SP/AP ratio in MeniereÕs patients (Densert, 1987; Den-

sert et al., 1997). These studies led to the development of a portable device with similar characteristics, the Meniett (Medtronic Xomed, Jacksonville, FL), which is currently being used to treat patients with MeniereÕs disease. Initial clinical trials with the device have been encouraging (Odkvist et al., 2000; Gates and Green, 2002), although some of the improvements may be related to the tympanostomy tube which alone appears to have beneficial effects (Montandon et al., 1989; Barbara et al., 2001). The underlying mechanisms by which the Meniett influences patientÕs symptoms have not been established. Our observations with infrasound (Fig. 6) support the possibility that alternating infrasonic pressure changes can induce endolymph movements into the endolymphatic sac. However, there are still many unknown factors involved in this dependence, specifically what is the role of the sustained positive pressure and why is the tympanostomy necessary for the Meniett to be effective? In our present study, the low frequency stimulation causing Kþ rise in the ES was applied to the external canal without a tympanostomy. By shunting pressure differences across the tympanic membrane, the tympanostomy would dramatically attenuate the infrasound induced movements of the middle ear ossicles but it would also permit to applied pressures to access the cochlea through the round window membrane. The intralabyrinthine influences of such methodological differences have not yet been established.

Acknowledgements We thank Dr. Robert Kimura, for loaning us the slides of one guinea pig inner ear specimen. We also appreciate the technical assistance of John DeMott in the collection of electrophysiological data, of Ingvor Forsberg for processing one of the histological specimens, and of Ruth Gill for photographing specimens and preparing illustrations. This work was supported by research grant RO1 DC01368 to Alec Salt from the National Institute on Deafness and Other Communication Disorders (NIDCD), National Institutes of Health and by grants to Helge Rask-Andersen from the Swedish Research Council (VR Proj. No. 3908) and Stiftelsen Tysta Skolan.


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