Evaluation of procedures to reduce fluid flow in the fistulized guinea-pig cochlea.

The rate of longitudinal flow of fluid in scala tympani (ST) has been quantified under a number of experimental conditions. The method used to measure flow involved using a tracer ion (trimethylphenylammonium: TMPA) as a volume flow marker. Movement of marked perilymph was monitored by ion-selective microelectrodes which were capable of detecting exceedingly low concentrations of TMPA. Our results show that when the cochlea is perforated at the apex, flow rates of 400-500 nl/min are induced in ST, compared to the normal very slow rate of 2 nl/min when the cochlea is sealed. This artifactual flow of CSF through the perforated cochlea can be reduced to 6.9 nl/min by releasing the hydrostatic pressure of cerebrospinal fluid (CSF) or further reduced to 1.8 nl/min by surgically obstructing the cochlear aqueduct. In addition, we observed no basally-directed flow in ST when the round window (RW) was perforated, demonstrating that perilymph is not produced in volume as previously assumed. This study demonstrates the importance of separating artifactual flows, induced by the experimental procedures required to access the cochlear fluids, from the low flow rates which occur in normal, physiologic conditions.     

Demonstration of a Longitudinal Concentration Gradient Along Scala Tympani by Sequential Sampling of Perilymph from the Cochlear Apex

Local applications of drugs to the inner ear are increasingly being used to treat patients' inner ear disorders. Knowledge of the pharmacokinetics of drugs in the inner ear fluids is essential for a scientific basis for such treatments. When auditory function is of primary interest, the drug's kinetics in scala tympani (ST) must be established. Measurement of drug levels in ST is technically difficult because of the known contamination of perilymph samples taken from the basal cochlear turn with cerebrospinal fluid (CSF). Recently, we reported a technique in which perilymph was sampled from the cochlear apex to minimize the influence of CSF contamination (J. Neurosci. Methods, doi: ). This technique has now been extended by taking smaller fluid samples sequentially from the cochlear apex, which can be used to quantify drug gradients along ST. The sampling and analysis methods were evaluated using an ionic marker, trimethylphenylammonium (TMPA), that was applied to the round window membrane. After loading perilymph with TMPA, 10 1-μL samples were taken from the cochlear apex. The TMPA content of the samples was consistent with the first sample containing perilymph from apical regions and the fourth or fifth sample containing perilymph from the basal turn. TMPA concentration decreased in subsequent samples, as they increasingly contained CSF that had passed through ST. Sample concentration curves were interpreted quantitatively by simulation of the experiment with a finite element model and by an automated curve-fitting method by which the apical–basal gradient was estimated. The study demonstrates that sequential apical sampling provides drug gradient data for ST perilymph while avoiding the major distortions of sample composition associated with basal turn sampling. The method can be used for any substance for which a sensitive assay is available and is therefore of high relevance for the development of preclinical and clinical strategies for local drug delivery to the inner ear.     

Perilymph Pharmacokinetics of Markers and Dexamethasone Applied and Sampled at the Lateral Semi-Circular Canal

Perilymph pharmacokinetics was investigated by a novel approach, in which solutions containing drug or marker were injected from a pipette sealed into the perilymphatic space of the lateral semi-circular canal (LSCC). The cochlear aqueduct provides the outlet for fluid flow so this procedure allows almost the entire perilymph to be exchanged. After wait times of up to 4 h the injection pipette was removed and multiple, sequential samples of perilymph were collected from the LSCC. Fluid efflux at this site results from cerebrospinal fluid (CSF) entry into the basal turn of scala tympani (ST) so the samples allow drug levels from different locations in the ear to be defined. This method allows the rate of elimination of substances from the inner ear to be determined more reliably than with other delivery methods in which drug may only be applied to part of the ear. Results were compared for the markers trimethylphenylammonium (TMPA) and fluorescein and for the drug dexamethasone (Dex). For each substance, the concentration in fluid samples showed a progressive decrease as the delay time between injection and sampling was increased. This is consistent with the elimination of substance from the ear with time. The decline with time was slowest for fluorescein, was fastest for Dex, with TMPA at an intermediate rate. Simulations of the experiments showed that elimination occurred more rapidly from scala tympani (ST) than from scala vestibuli (SV). Calculated elimination half-times from ST averaged 54.1, 24.5 and 22.5 min for fluorescein, TMPA and Dex respectively and from SV 1730, 229 and 111 min respectively. The elimination of Dex from ST occurred considerably faster than previously appreciated. These pharmacokinetic parameters provide an important foundation for understanding of drug treatments of the inner ear.     

Contamination of perilymph sampled from the basal cochlear turn with cerebrospinal fluid

Our understanding of the perilymph kinetics of drugs depends largely on data obtained by the analysis of perilymph samples. Although a number of studies have demonstrated qualitatively that perilymph samples may be contaminated by cerebrospinal fluid (CSF), and some investigations adopt specific methods to minimize CSF contamination of their samples, many other studies fail to consider the influence of this potential artifact on their measurements. In the present study we have attempted to quantify the degree of CSF contamination of perilymph samples taken from the basal turn of the guinea pig cochlea using the ionic marker trimethylphenylammonium (TMPA). TMPA solution was irrigated across the round window membrane while a TMPA-selective electrode sealed into the perilymphatic space continuously monitored perilymph TMPA concentration. After a period of TMPA loading, a perilymph sample was aspirated and its TMPA content determined. Differences between the sample concentration and the measured TMPA time course during perilymph loading and sampling were analyzed using a finite element computer model for simulation of solute movements in the inner ear fluids. The experimental results were consistent with the aspirated fluid sample from the cochlea being replaced by CSF drawn into the perilymphatic space through the cochlear aqueduct. The dependence of perilymph sample purity on the location of sampling and on the volume withdrawn was quantified. These relationships are of value in the design and interpretation of experiments that utilize perilymph sampling.     

Volume flow rate of perilymph in the guinea-pig cochlea

The rate of longitudinal flow of perilymph has been measured using an ionic tracer technique. Spread of the tracer trimethylphenylammonium (TMPA) along the perilymphatic scalae was monitored with ion-selective microelectrodes following injection of a minute bolus (approximately 50 nl) of 150 mM TMPAC1 one turn away. This amount of TMPA had virtually no toxic effect on cochlear function. The spread of tracer by longitudinal volume flow and passive diffusion were separated by comparing tracer movements in both apical and basal directions along the scalae in two groups of animals. Experimental findings were compared with a mathematical model which combined diffusion and volume flow. Our results demonstrated that when electrodes were completely sealed into the cochlea, the rate of longitudinal volume flow in scala tympani was extremely slow, approximately 1.6 nl/min in the apical direction. Longitudinal flow was not detectable in scala vestibuli. When the otic capsule was perforated, flow rates of over 1 microliter/min were recorded in scala tympani, probably as a result of cerebrospinal fluid entry through the cochlear aqueduct. When the cochlea was sealed (with recording electrodes in place) and cerebrospinal fluid pressure was released, there was no significant basally-directed flow of perilymph in scala tympani. These findings support the concept that perilymph composition is maintained by local, cochlear mechanisms which do not involve longitudinal volume flow. They provide strong evidence that perilymph is not secreted in one region and resorbed at a spatially distant site.     

Quantification of solute entry into cochlear perilymph through the round window membrane.

The administration of drugs to the inner ear via the round window membrane is becoming more widely used for both clinical and experimental purposes. The actual drug levels achieved in different regions of the inner ear by this method have not been established. The present study has made use of simulations of solute movements in the cochlear fluids to describe the distribution of a marker solute in the guinea pig cochlear fluid spaces. Simulation parameters were derived from experimental measurements using a marker ion, trimethylphenylammonium (TMPA). The distribution of this ion in the cochlea was monitored without volume disturbance using TMPA-selective microelectrodes sealed into the first and second turns of scala tympani (ST). TMPA was applied to perilymph by irrigation of the intact round window membrane with 2 mM solution. At the end of a 90 min application period, TMPA in the first turn, 1.4 mm from the base of ST, reached an average concentration of 330 microM (standard deviation (S.D.) 147 microM, n = 8). TMPA in the second turn, 7.5 mm from the base of ST reached a concentration of 15 microM (S.D. 33 microM, n = 5). The measured time courses of TMPA concentration change were interpreted using the Washington University Cochlear Fluids Simulator (V 1.4), a public-domain program available on the internet at http ://oto.wustl.edu/cochlea/. Simulations with parameters producing concentration time courses comparable to those measured were: (1) round window permeability: 1.9 x 10(-80 cm/s; (2) ST clearance half-time: 60 min; (3) longitudinal perilymph flow rate: 4.4 nl/min, directed from base to apex. Solute concentrations in apical regions of the cochlea were found to be determined primarily by the rate at which the solute diffuses, balanced by the rate of clearance of the solute from perilymph. Longitudinal perilymph flow was not an important factor in solute distribution unless the bony otic capsule was perforated, which rapidly caused substantial changes to solute distribution. This study demonstrates the basic processes by which substances are distributed in the cochlea and provides a foundation to understand how other applied substances will be distributed in the ear.     

Cerebrospinal fluid gusher during stapedectomy

Cerebrospinal fluid (CSF) otorrhea of labyrinthine origin presents through a congenital defect in the stapes footplate and is associated with repeated attacks of meningitis or as a profuse CSF flow (stapes gusher) during routine stapedectomy. These CSF leaks are thought to be due to an abnormal communication between the subarachnoid and perilymphatic spaces, either through the cochlear aqueduct or the internal auditory canal. A computerized tomographic diagnosis of X-linked congenital deafness associated with fixation of the footplate and perilymph gushers include enlarged internal auditory canal, hypoplasia of the cochlear base, absent bony modiolus, abnormal vestibular aqueduct, and enlarged labyrinthine facial nerve canal. Because there have been few reports of a successful stapedectomy with a significant gain in hearing performed in ears in which a stapes gusher is encountered, attention should be given more to the control of the catastrophic flow of perilymph rather than to the completion of the operative procedure.     

Gradients of organic substances between fluid compartments of the cochlea

We have previously demonstrated the presence of steep gradients of amino acids between cochlear endolymph and perilymph of scala vestibuli. However, only recently have we succeeded in developing a sampling technique which is capable of providing amino acid levels in scala tympani which are representative of the in vivo situation under physiological circumstances. This is achieved by sampling perilymph in a closed system, which precludes unphysiological contamination by CSF via the cochlear aqueduct. It is also shown that amino acids which exhibit very steep gradients between CSF and perilymph, such as glycine and alanine, can be used as endogenous markers to assess the degree of artifactual admixture of CSF when perilymph is sampled by conventional techniques in the open system. Moreover, pilot experiments indicate that net production of perilymph is very low when the cochlear aqueduct is blocked.     

In vivo observation of dynamic perilymph formation using 4.7 T MRI with gadolinium as a tracer

OBJECTIVE: To investigate the pharmacokinetics of gadolinium in the perilymphatic fluid spaces of the cochlea in vivo using high-resolution MRI to obtain information concerning perilymph formation. MATERIAL AND METHODS: A Bruker Biospec Avance 47/40 experimental MRI system with a magnetic field strength of 4.7 T was used. Anesthetized pigmented guinea pigs were injected with the contrast agent Gd-diethylenetriaminepentaacetic acid-bismethylamide and placed in the magnet. The signal intensity of Gd in the tissues was used as a biomarker for dynamic changes in the perilymphatic fluid. RESULTS: The most rapid uptake of Gd in the perilymphatic fluid spaces occurred in the lower part of the modiolus, followed by the second turn of the scala tympani. Within the scala tympani, the distribution of Gd in the basal turn was significantly lower than that in the other turns. Destruction of the cochlear aqueduct was followed by an increase in Gd uptake in the perilymph instead of a reduction. CONCLUSIONS: These findings offer further evidence that the pervasive perilymphatic fluid derives from the cochlear blood supply via the cochlear glomeruli, which are in close proximity to the scala tympani within the modiolus, and the capillary in the spiral ligament. Cerebrospinal fluid communicates with perilymph via the cochlear aqueduct but is not the main source of perilymph. These findings are of relevance to the treatment of inner ear diseases, as well as to our understanding of the flow and source of perilymphatic fluid.     

In Vivo Observation of Dynamic Perilymph Formation Using 4.7 T MRI with Gadolinium as a Tracer

Objective—To investigate the pharmacokinetics of gadolinium in the perilymphatic fluid spaces of the cochlea in vivo using high-resolution MRI to obtain information concerning perilymph formation. Material and Methods—A Bruker Biospec Avance 47/40 experimental MRI system with a magnetic field strength of 4.7 T was used. Anesthetized pigmented guinea pigs were injected with the contrast agent Gd-diethylenetriaminepentaacetic acid-bismethylamide and placed in the magnet. The signal intensity of Gd in the tissues was used as a biomarker for dynamic changes in the perilymphatic fluid. Results—The most rapid uptake of Gd in the perilymphatic fluid spaces occurred in the lower part of the modiolus, followed by the second turn of the scala tympani. Within the scala tympani, the distribution of Gd in the basal turn was significantly lower than that in the other turns. Destruction of the cochlear aqueduct was followed by an increase in Gd uptake in the perilymph instead of a reduction. Conclusions—These findings offer further evidence that the pervasive perilymphatic fluid derives from the cochlear blood supply via the cochlear glomeruli, which are in close proximity to the scala tympani within the modiolus, and the capillary in the spiral ligament. Cerebrospinal fluid communicates with perilymph via the cochlear aqueduct but is not the main source of perilymph. These findings are of relevance to the treatment of inner ear diseases, as well as to our understanding of the flow and source of perilymphatic fluid.     

Relationships of labyrinthine fluid pressures and blood flow.

Control of labyrinthine blood flow analogous to the autoregulation of cerebral blood flow has been suggested but not experimentally demonstrated. This study concerns the influence of systemic arterial pressure changes on the perilymph and CSP pressures in cats with the cochlear aqueduct (CA) blocked. No direct correlation was found between changes in arterial and CSF pressure. This seemed to be due to the efficient autoregulation of the global cerebral blood flow — a main factor for CSF pressure regulation. The CSF and central venous pressures induced little and much delayed influence on the perilymph pressure when the CA was blocked. However, there was a direct correlation between changes in systemic arterial pressure and the perilymph pressure. This relationship seemed to be mediated via changes in local labyrinthine blood flow. The study indicated a lack of autoregulation of labyrinthine blood flow and a direct correlation between labyrinthine fluid pressure and blood flow when the CA was obstructed.     

Effects of hypobaric pressure on the labyrinth. Cochlear aqueduct patent

Cats with the cochlear aqueduct patent were placed in a pressure chamber and exposed for 10 min to hypobaric pressures of 5.1 and 6.8 kPa relative to atmospheric pressure. The experiments were designed according to a program used for treatment of Meniere's disease. The perilymph, middle ear, cerebrospinal fluid (CSF), venous, arterial and chamber pressures were recorded. The results demonstrated that hypobaric effects on the labyrinth were mediated via pressure changes in the middle ear and not via a systemic vascular or CSF influence. A reduction in chamber pressure induced a relative increase in middle ear pressure. It was the rate of the hypobaric change as well as the patency of the cochlear aqueduct and the Eustachian tube function that determined the magnitude of the initial perilymph peak pressure and the duration of this pressure increase. A rapid versus a slow rate induced an initial perilymph increase of 3.4 and 2.2 kPa, respectively. This relative pressure increase was eliminated within 1 min via the patent aqueduct. Thus, neither did a longstanding perilymph pressure increase occur during the hypobaric exposure, nor did a prolonged significant reduction in perilymph pressure occur after atmospheric pressure was restored.     

大前庭水管综合征术前的影像学表现与人工耳蜗手术所见

目的 探讨大前庭水管综合征患儿术前影像学评估对人工耳蜗植入术的指导作用.方法 2010年3月至2011年1月间对7例大前庭水管综合征患儿进行了人工耳蜗术前影像学评估及人工耳蜗植入术.结果 7例患儿术中发现开窗后2例井喷,4例搏动,1例既无井喷亦无波动.结论 不合并内耳其他畸形的单纯前庭导水管扩大的患儿术中并不会引起井喷,仅在颅压过大时出现脑脊液搏动,只有合并内耳道底骨性缺损的患儿才会出现井喷.     

Marker entry into vestibular perilymph via the stapes following applications to the round window niche of guinea pigs

It has been widely believed that drug entry from the middle ear into perilymph occurs primarily via the round window (RW) membrane. Entry into scala vestibuli (SV) was thought to be dominated by local, inter-scala communication between scala tympani (ST) and SV through permeable tissues such as the spiral ligament. In the present study, the distribution of the ionic marker trimethylphenylammonium (TMPA) was compared following intracochlear injections or applications to the RW niche, with or without occlusion of the RW membrane or stapes area. Perilymph TMPA concentrations were monitored either in real time with TMPA-selective microelectrodes sealed into ST and SV, or by the collection of sequential perilymph samples from the lateral semi-circular canal. Local inter-scala communication of TMPA was confirmed by measuring SV and ST concentrations following direct injections into perilymph of ST. Application of TMPA to the RW niche also showed a predominant entry into ST, with distribution to SV presumed to occur secondarily. When the RW membrane was occluded by a silicone plug, RW niche irrigation produced higher concentrations in SV compared to ST, confirming direct TMPA entry into the vestibule in the region of the stapes. The proportion of TMPA entering by the two routes was quantified by perilymph sampling from the lateral semi-circular canal. The TMPA levels of initial samples (originating from the vestibule) were markedly lower when the stapes area was occluded with silicone. These data were interpreted using a simulation program that incorporates all the major fluid and tissue compartments of the cochlea and vestibular systems. From this analysis it was estimated that 65% of total TMPA entered through the RW membrane and 35% entered the vestibule directly in the vicinity of the stapes. Direct entry of drugs into the vestibule is relevant to inner ear fluid pharmacokinetics and to the growing field of intratympanic drug delivery.     

Quantitative assessment of perilymph sources.

The problem of the perilymph origin--influx of cerebrospinal fluid (CSF) versus ultrafiltration within the cochlea--cannot be solved by mere qualitative proofs of tracer passage through the cochlear aqueduct. In order to gain quantitative data on the possible perilymph sources, an experimental study was designed to follow the time course of dye concentrations in the cisternal CSF and in the perilymph after tracer injection into the CSF at the vertex. By comparing the resulting concentration peaks in both fluids, the mean peak of the perilymph tracer concentrations was found to reach 36% of the maximum CSF concentration only. It is concluded that the local perilymph production within the cochlea exceeds the influx of CSF by a ratio of about 2:1. A working hypothesis of the double perilymph origin is discussed.     

Potassium circulation in the perilymph of guinea pig cochlea

To determine whether any differences exist in potassium circulation between the scala vestibuli and scala tympani, we recorded the change in K⁺ activity in both scalae of the guinea pig cochlea at the basal and third turns, using a double-barrelled, K⁺-sensitive microelectrode after perfusion with artificial perilymph containing 20 mM KCl and 130 mM NaCl. K⁺ activity increased immediately after the start of perfusion and decreased after its completion. The rates of decrease of K⁺ activities were approximately 1.0 mEq/l per min in the scala vestibuli of the basal and third turns, also 1.0 mEq/l per min in the scala tympani of the basal turn, and approximately 0.5 mEq/l per min in the scala tympani of the third turn. The rate of decrease of K⁺ activity in the scala tympani was significantly slower in the third turn than in the basal turn. Blockage of the cochlear aqueduct depressed the rate of decrease of K⁺ activity in the scala tympani more in the basal turn than in the third turn. These results suggest that there is a difference in potassium circulation between the scala vestibuli and scala tympani, and that the cochlear aqueduct plays an important role in potassium circulation in the perilymph of the scala tympani.     

Radial communication between the perilymphatic scalae of the cochlea. II: Estimation by bolus injection of tracer into the sealed cochlea.

Radial communication between ST and SV was measured in the sealed cochlea by monitoring the dispersal of an ionic tracer, trimethylphenylammonium (TMPA) injected in the form of a minute bolus. Tracer movements were recorded by a pair of ion-selective electrodes sealed into the injected and non-injected scalae close to the injection site. Measurements were made in the basal or third turn of the guinea pig cochlea. In the third turn, radial communication occurred rapidly with a ST half time from ST to SV of 25 min and from SV to ST of 26 min. In the basal turn the communication was markedly slower, with a ST half time from ST to SV of 170 min and from SV to ST of 240 min. However, the difference between the basal and third turns can be shown to arise almost totally from differences in cross-sectional area of the perilymphatic scalae. When normalized with respect to scala cross-section, the process of tracer movement across the spiral ligament is similar in the basal and third turns. These results demonstrate that radial communication between scala tympani and scala vestibuli is an important route which must be considered in studies involving perilymph.     

Dexamethasone concentration gradients along scala tympani after application to the round window membrane.

HYPOTHESIS: Local application of dexamethasone-21-dihydrogen-phosphate (Dex-P) to the round window (RW) membrane of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph. BACKGROUND: In recent years, intratympanically applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after RW application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur after RW application, with lower concentrations expected in apical turns. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph. METHODS: Dexamethasone-21-dihydrogen-phosphate (10 mg/ml) was administered to the RW membrane of guinea pigs (n = 9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which 10 samples, each of approximately 1 mul, were taken sequentially from the cochlear apex into capillary tubes. Dexamethasone-21-dihydrogen-phosphate concentration of the samples was analyzed by high-performance liquid chromatography. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in ST to be quantified. RESULTS: The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along ST. After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17,000. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially. CONCLUSION: The existence of substantial basal-apical concentration gradients of Dex-P in ST perilymph were demonstrated experimentally. If the variability in peak concentration and gradient is also present under clinical conditions, this may contribute to the heterogeneity of outcome that is observed after intratympanic application of glucocorticoids for various inner ear diseases.     

Direct measurement flow resistance of cochlear aqueduct in guinea pigs

OBJECTIVE: The cochlear aqueduct connects the scala tympani to the subarachnoid space and is the main pressure equalization canal for the inner ear. Increases in inner ear volume and pressure are thought to cause clinical symptoms such as vertigo, tinnitus and fluctuating hearing loss. In this study the flow resistance of the cochlear aqueduct was determined and its relation with inner ear pressure was studied. MATERIAL AND METHODS: Inner ear pressure was measured in the scala tympani through the round window using a micropipette. Through a second micropipette, artificial perilymph was infused into, or withdrawn from, the scala tympani at various constant rates. From the infusion rate and the change in perilymphatic pressure during infusion the flow resistance of the cochlear aqueduct was calculated. RESULTS: The flow resistance was found not to be constant but to depend on the position of the round window membrane and possibly on the magnitude and direction of fluid flow through the aqueduct. Measured flow resistance values were in the range 11-45 Pa s/nl. For very small flow values the flow resistance averaged over 6 animals was 21 Pa s/nl. CONCLUSIONS: The flow resistance of the cochlear aqueduct is not a constant value. The cochlear aqueduct is a canal with dynamic properties and may play a role in the complicated process of inner ear pressure regulation.     

Effects of persistent perilymph fistula on the inner ear

To study the effects of a persistent perilymph fistula on the cochlea, a small cannula was inserted into the scala tympani of the basal turn of cochlea in guinea pigs. A month later, cochlear morphology and blood flow were studied using either histological evaluation or the microsphere surface preparation technique. Some animals showed no cochlear morphologic changes or no cochlear blood-flow reduction, even if tubal patency was maintained and perilymph leakage lasted for 1 month. This suggests that a prolonged perilymph fistula, per se, causes no permanent cochlear damage. However, in some animals, hair cell damage and cochlear blood-flow disorders were observed. These observations and the causes of hearing loss in clinical cases of perilymph fistula were studied.