The endolymphatic sinus is a possible detector of endolymph volume status.
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Animals↗
Biomedical subjects
Alec N Salt
Publications and source records attributed to Alec N Salt.
Cochlear pharmacokinetics with local inner ear drug delivery using a three-dimensional finite-element computer model.
HYPOTHESIS: Cochlear fluid pharmacokinetics can be better represented by three-dimensional (3D) finite-element simulations of drug dispersal. BACKGROUND: Local drug deliveries to the round window membrane are increasingly being used to treat inner ear disorders. Crucial to the development of safe therapies is knowledge of drug distribution in the inner ear with different delivery methods. Computer simulations allow application protocols and drug delivery systems to be evaluated, and may permit animal studies to be extrapolated to the larger cochlea of the human. METHODS: A finite-element 3D model of the cochlea was constructed based on geometric dimensions of the guinea pig cochlea. Drug propagation along and between compartments was described by passive diffusion. To demonstrate the potential value of the model, methylprednisolone distribution in the cochlea was calculated for two clinically relevant application protocols using pharmacokinetic parameters derived from a prior one-dimensional (1D) model. In addition, a simplified geometry was used to compare results from 3D with 1D simulations. RESULTS: For the simplified geometry, calculated concentration profiles with distance were in excellent agreement between the 1D and the 3D models. Different drug delivery strategies produce very different concentration time courses, peak concentrations and basal-apical concentration gradients of drug. In addition, 3D computations demonstrate the existence of substantial gradients across the scalae in the basal turn. CONCLUSION: The 3D model clearly shows the presence of drug gradients across the basal scalae of guinea pigs, demonstrating the necessity of a 3D approach to predict drug movements across and between scalae with larger cross-sectional areas, such as the human, with accuracy. This is the first model to incorporate the volume of the spiral ligament and to calculate diffusion through this structure. Further development of the 3D model will have to incorporate a more accurate geometry of the entire inner ear and incorporate more of the specific processes that contribute to drug removal from the inner ear fluids. Appropriate computer models may assist in both drug and drug delivery system design and can thus accelerate the development of a rationale-based local drug delivery to the inner ear and its successful establishment in clinical practice.
Animals↗
Simulation of application strategies for local drug delivery to the inner ear.
Local, rather than systemic, drug delivery to the inner ear is becoming more widely used to treat inner ear disorders. While many substances are undergoing preclinical and clinical studies, it is equally important to develop appropriate drug delivery systems. Pharmacokinetic studies are technically demanding in animals and almost impossible in humans. Computer simulations have helped establish the basic principles of drug distribution in the inner ear. The distribution of methylprednisolone in the guinea pig cochlea has been simulated for different drug delivery systems based on kinetic parameters established in prior studies. Results were compared for different rates of drug clearance from the middle ear. Absolute and relative drug levels in the perilymph were highly dependent on how long the drug remained in the middle ear. For a brief (30 min) application, the basal to apical drug gradient was higher than for longer delivery times. These findings show that controlling middle ear drug clearance is of critical importance.
Animals↗
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: 10.1016/j.jneumeth.2005.10.008 ). 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-muL 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.
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Cochlear microdialysis for quantification of dexamethasone and fluorescein entry into scala tympani during round window administration.
Before new drugs for the treatment of inner ear disorders can be studied in controlled clinical trials, it is important that their pharmacokinetics be established in inner ear fluids. Microdialysis allows drug levels to be measured in perilymph without the volume disturbances and potential cerebrospinal fluid contamination associated with fluid sampling. The aims of this study were to show: (i) that despite low recovery rates from miniature dialysis probes, significant amounts of drug are removed from small fluid compartments, (ii) that dialysis sampling artifacts can be accounted for using computer simulations and (iii) that microdialysis allows quantification of the entry rates through the round window membrane (RWM) into scala tympani (ST). Initial experiments used microdialysis probes in small compartments in vitro containing sodium fluorescein. Stable concentrations were observed in large compartments (1000 microl) but significant concentration declines were observed in smaller compartments (100, 10 and 5.6 microl) comparable to the size of the inner ear. Computer simulations of these experiments closely approximated the experimental data. In in vivo experiments, sodium fluorescein 10 mg/ml and dexamethasone-dihydrogen-phosphate disodium salt 8 mg/ml were simultaneously applied to the RWM of guinea pigs. Perilymph concentration in the basal turn of ST was monitored using microdialysis. The fluorescein concentration reached after 200 min application (585+/-527 microg/ml) was approximately twice that of dexamethasone phosphate (291+/-369 microg/ml). Substantial variation in concentrations was found between animals by approximately a factor of 34 for fluorescein and at least 41 for dexamethasone phosphate. This is, to a large extent, thought to be the result of the RWM permeability varying in different animals. It was not caused by substance analysis variations, because two different analytic methods were used and the concentration ratio between the two substances remained nearly constant across the experiments and because differences were apparent for the repeated samples obtained in each animal. Interpretation of the results using computer simulations allowed RWM permeability to be quantified. It also demonstrated, however, that cochlear clearance values could not be reliably obtained with microdialysis because of the significant contribution of dialysis to clearance. The observed interanimal variation, e.g., in RWM permeability, is likely to be clinically relevant to the local application of drugs in patients.
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Perilymph sampling from the cochlear apex: a reliable method to obtain higher purity perilymph samples from scala tympani.
Measurements of drug levels in the fluids of the inner ear are required to establish kinetic parameters and to determine the influence of specific local delivery protocols. For most substances, this requires cochlear fluids samples to be obtained for analysis. When auditory function is of primary interest, the drug level in the perilymph of scala tympani (ST) is most relevant, since drug in this scala has ready access to the auditory sensory cells. In many prior studies, ST perilymph samples have been obtained from the basal turn, either by aspiration through the round window membrane (RWM) or through an opening in the bony wall. A number of studies have demonstrated that such samples are likely to be contaminated with cerebrospinal fluid (CSF). CSF enters the basal turn of ST through the cochlear aqueduct when the bony capsule is perforated or when fluid is aspirated. The degree of sample contamination has, however, not been widely appreciated. Recent studies have shown that perilymph samples taken through the round window membrane are highly contaminated with CSF, with samples greater than 2microL in volume containing more CSF than perilymph. In spite of this knowledge, many groups continue to sample from the base of the cochlea, as it is a well-established method. We have developed an alternative, technically simple method to increase the proportion of ST perilymph in a fluid sample. The sample is taken from the apex of the cochlea, a site that is distant from the cochlear aqueduct. A previous problem with sampling through a perforation in the bone was that the native perilymph rapidly leaked out driven by CSF pressure and was lost to the middle ear space. We therefore developed a procedure to collect all the fluid that emerged from the perforated apex after perforation. We evaluated the method using a marker ion trimethylphenylammonium (TMPA). TMPA was applied to the perilymph of guinea pigs either by RW irrigation or by microinjection into the apical turn. The TMPA concentration of the fluid sample was compared with that measured in perilymph prior to taking the sample using a TMPA-selective microelectrode sealed into ST. Data were interpreted with a finite element model of the cochlear fluids that was used to simulate each aspect of the experiment. The correction of sample concentration back to the perilymph concentration prior to sampling can be performed based on the known ST volume (4.7microL in the guinea pig) and the sample volume. A more precise correction requires some knowledge of the profile of drug distribution along the cochlear prior to sampling. This method of sampling from the apex is technically simple and provides a larger sample volume with a greater proportion of perilymph compared to sampling through the RW.
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Local inner-ear drug delivery and pharmacokinetics.
Several drugs that are applied directly to the inner ear are in widespread clinical use for the treatment of inner-ear disorders. Many new substances and drug delivery systems specific to the inner ear are under development and in some cases are being evaluated in animal experiments and in clinical studies. However, the pharmacokinetics of drugs in the inner ear is not well defined and the field is plagued by technical problems in obtaining pure samples of the inner-ear fluids for analysis. Nevertheless, a basic understanding of the mechanisms of drug dispersal in the inner ear has emerged, which facilitates the design and interpretation of future pharmacokinetic studies.
Animals↗
ATP-gamma-S shifts the operating point of outer hair cell transduction towards scala tympani.
ATP receptor agonists and antagonists alter cochlear mechanics as measured by changes in distortion product otoacoustic emissions (DPOAE). Some of the effects on DPOAEs are consistent with the hypothesis that ATP affects mechano-electrical transduction and the operating point of the outer hair cells (OHCs). This hypothesis was tested by monitoring the effect of ATP-gamma-S on the operating point of the OHCs. Guinea pigs anesthetized with urethane and with sectioned middle ear muscles were used. The cochlear microphonic (CM) was recorded differentially (scala vestibuli referenced to scala tympani) across the basal turn before and after perfusion (20 min) of the perilymph compartment with artificial perilymph (AP) and ATP-gamma-S dissolved in AP. The operating point was derived from the cochlear microphonics (CM) recorded in response low frequency (200 Hz) tones at high level (106, 112 and 118 dB SPL). The analysis procedure used a Boltzmann function to simulate the CM waveform and the Boltzmann parameters were adjusted to best-fit the calculated waveform to the CM. Compared to the initial perfusion with AP, ATP-gamma-S (333 microM) enhanced peak clipping of the positive peak of the CM (that occurs during organ of Corti displacements towards scala tympani), which was in keeping with ATP-induced displacement of the transducer towards scala tympani. CM waveform analysis quantified the degree of displacement and showed that the changes were consistent with the stimulus being centered on a different region of the transducer curve. The change of operating point meant that the stimulus was applied to a region of the transducer curve where there was greater saturation of the output on excursions towards scala tympani and less saturation towards scala vestibuli. A significant degree of recovery of the operating point was observed after washing with AP. Dose response curves generated by perfusing ATP-gamma-S (333 microM) in a cumulative manner yielded an EC(50) of 19.8 microM. The ATP antagonist PPADS (0.1 mM) failed to block the effect of ATP-gamma-S on operating point, suggesting the response was due to activation of metabotropic and not ionotropic ATP receptors. Multiple perfusions of AP had no significant effect (118 and 112 dB) or moved the operating point slightly (106 dB) in the direction opposite of ATP-gamma-S. Results are consistent with an ATP-gamma-S induced transducer change comparable to a static movement of the organ of Corti or reticular lamina towards scala tympani.
Adenosine Triphosphate↗
Acute endolymphatic hydrops generated by exposure of the ear to nontraumatic low-frequency tones.
Low-frequency sounds presented at high nontraumatizing levels induce temporary hyperacusis in humans and animals. One explanation of this finding is that the basilar membrane operating point may be disturbed by an endolymph volume change. This possibility was investigated using volume and flow markers iontophoresed into the endolymphatic space of guinea pigs. Marker concentrations were measured with ion-selective microelectrodes placed apically and basally to the iontophoresis site during exposure of the ear to low-frequency tones. Concentration changes were interpreted quantitatively using a finite-element model of the endolymphatic space that allowed changes of endolymph cross-sectional area and flow to be derived. Stimulation with a 200 Hz tone at 115 dB SPL for 3 min produced marker concentration changes consistent with the induction of transient endolymphatic hydrops and a basally directed displacement of endolymph. Endocochlear potentials were greater than normal after the exposure when hydrops was present. During identical tone exposures of animals without marker, we found that action potential (AP) threshold changes and endolymph potassium changes associated with the hydropic state were small. Marker concentration changes were compared with changes in endocochlear potential and AP thresholds for a range of exposure frequencies and levels. AP hypersensitivity occurred with 200 Hz exposure levels below those inducing endolymph volume disturbances. Endolymph volume changes are thought to be the result of, rather than the cause of, changes in operating point of the cochlear transducer. The observations that auditory threshold and endolymph potassium changes are minimal under conditions where substantial endolymphatic hydrops is present is relevant to our understanding of the hearing loss in patients with Meniere's disease.
Acute Disease↗
Responses of the endolymphatic sac to perilymphatic injections and withdrawals: evidence for the presence of a one-way valve.
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 K(+), Na(+) 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 K(+) changes in the ES. The majority of perilymph withdrawals caused K(+) 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 K(+) 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.
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The influence of transducer operating point on distortion generation in the cochlea.
Distortion generated by the cochlea can provide a valuable indicator of its functional state. In the present study, the dependence of distortion on the operating point of the cochlear transducer and its relevance to endolymph volume disturbances has been investigated. Calculations have suggested that as the operating point moves away from zero, second harmonic distortion would increase. Cochlear microphonic waveforms were analyzed to derive the cochlear transducer operating point and to quantify harmonic distortions. Changes in operating point and distortion were measured during endolymph manipulations that included 200-Hz tone exposures at 115-dB SPL, injections of artificial endolymph into scala media at 80, 200, or 400 nl/min, and treatment with furosemide given intravenously or locally into the cochlea. Results were compared with other functional changes that included action potential thresholds at 2.8 or 8 kHz, summating potential, endocochlear potential, and the 2 f1-f2 and f2-f1 acoustic emissions. The results demonstrated that volume disturbances caused changes in the operating point that resulted in predictable changes in distortion. Understanding the factors influencing operating point is important in the interpretation of distortion measurements and may lead to tests that can detect abnormal endolymph volume states.
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[1D-and 3D- computer simulation for experimental planning and interpretation of pharmacokinetic studies in the inner ear after local drug delivery].
The local delivery of drugs to the cochlea is a promising alternative to systemic treatment of inner ear disorders. Whilst new drugs are being developed for this purpose, it is important to determine the time course and total dose required for the various target regions within the inner ear. Due to the small fluid spaces of the inner ear and the resulting experimental and analytical difficulties, many animal studies have only obtained one sample per animal. This results in limited information about drug time courses at specific locations in the inner ear. We show here how computer models considering general pharmacokinetic principles and inner ear geometry are used for application of the 3R-principle in animal research while avoiding experimental sampling artefacts. This can be achieved by: (1) careful planning and interpretation of experiments to study pharmacokinetics in the inner ear, (2) optimising volume sampling techniques, (3) facilitating the use of advantageous, continuous sampling methods like microdialysis and (4) developing a 3D-model that will permit consideration of the complex geometry of the inner ear when transferring results from one species to another.
Animal Testing Alternatives↗
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.
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Quantitative interpretation of corticosteroid pharmacokinetics in inner fluids using computer simulations.
The delivery of drugs to the inner ear by applying them directly onto the round window membrane is a promising way to treat human inner ear disorders. To further develop this strategy, and to design controlled clinical trials, additional preclinical studies are necessary. It is especially important to derive the time course and total dose for the various target regions within the inner ear. Since direct pharmacokinetic measurements in the human cochlea are not possible, simulations provide a valuable tool for the interpretation and planning of animal studies, for evaluating changes of application protocols and drug delivery systems, and for extrapolating the results from animal studies to the human. The present study has analyzed two previously published data sets in which concentration time courses of corticosteroids in the cochlear fluids were reported. Drug movements were simulated with a finite element computer model of the inner ear fluids. The time course of corticosteroid pharmacokinetics could be approximated for each study by consideration of the specific experimental paradigm. Although the experimental studies reported considerably different drug levels in the fluid samples taken from the cochlea, these differences were largely explained by considering the experimental design of the respective studies. After correction for experimental differences, the calculated perilymph levels of drug were within a factor of two of each other. The simulations demonstrated that an important factor controlling the drug level achieved is the time the drug solution remains in the middle ear. It can be concluded that small differences in delivery protocols may cause large variations in the drug levels achieved in the inner ear fluids.
Adrenal Cortex Hormones↗
Quantitative anatomy of the guinea pig endolymphatic sac.
The endolymphatic sac is believed to represent one of the primary loci for endolymph volume regulation in the inner ear. Quantitative analysis of physiologic measurements from the endolymphatic sac requires knowledge of the anatomy of the structure, specifically the luminal volume and the variation of cross-sectional area with distance along the sac. Recently techniques have become available to make these measurements. In the present study, fixed, isolated specimens of the guinea pig endolymphatic sac were imaged by high-resolution magnetic resonance microscopy (MRM) or by histological serial sections. Structures were reconstructed and quantified using image analysis software. In specimens imaged by MRM the endolymphatic sac volume, including tissue and lumen, was 359 nl for the intraosseous region and 106 nl for the extraosseous region, totaling 465 nl for the entire structure. The luminal volumes were 131 nl for the intraosseous region and 13 nl for the extraosseous region, totaling 144 nl. In histological specimens the volume, including tissue and lumen, was 414 nl for the intraosseous region and 121 nl for the extraosseous region, totaling 535 nl for the entire structure. The luminal volumes were 152 nl for the intraosseous region and 26 nl for the extraosseous region, totaling 179 nl. Differences in volume estimates obtained by the two methods were not statistically significant and variation was dominated by inter-specimen variation. Pooling the data, the total volume of the endolymphatic sac in the guinea pig including tissue and lumen was 506 nl (S.D. 100, n=17) and the volume of the lumen was 169 nl (S.D. 48, n=14).
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Analysis of gentamicin kinetics in fluids of the inner ear with round window administration.
HYPOTHESIS: That a theoretical basis for quantifying drug distribution in the inner ear with local applications can be established. BACKGROUND: As methods of local drug delivery to the inner ear gain wider clinical acceptance it becomes important to establish how drugs are distributed in the ear as a function of time and for different delivery methods. METHODS: The time course of gentamicin concentration in the inner ear fluids was simulated with a program that considered general pharmacokinetic principles and incorporated inner ear dimensions and drug dispersal processes, including diffusion, clearance, and intercompartmental exchange. RESULTS: Cochlear fluid space dimensions of the chinchilla were derived from three-dimensional magnetic resonance images and were incorporated into the simulator. The published time course of gentamicin in vestibular perilymph of chinchillas was closely approximated by the adjustment of parameters defining round window membrane permeability, clearance, and interscala exchange. To simulate the time course, it was necessary for drug entry into the vestibule to be dominated by interscala exchange rather than longitudinal spread through the helicotrema. The effects of different round window delivery methods were also calculated. Perilymph drug levels and spatial distribution in the ear were shown to be markedly influenced by the time the applied drug remained in the middle ear. CONCLUSION: The development of local inner ear drug application strategies requires consideration of inner ear pharmacokinetic characteristics, delivery methods, and therapeutic range of the drug.
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Simulation of methods for drug delivery to the cochlear fluids.
The inner ear fluids are remarkably 'unstirred' so that it cannot be assumed that applied drugs are dispersed throughout the fluid spaces. Calculation of the effective concentrations achieved when drugs are applied directly to the inner ear is made possible by simulations combining the physical processes involved in solute dispersal, which are diffusion, longitudinal fluid flow and clearances to other compartments. The approach has been validated in numerous experiments in which ion-selective electrodes were used to characterize the spread of marker substances in the cochlear fluids. The model incorporates the known size and geometry of cochlear fluid spaces for 6 species, including the guinea pig and the human. The simulator allows the dispersal of drugs or other substances to be approximated with knowledge of relatively few parameters. The simulation program is available on the Internet at http://oto.wustl.edu/cochlea/.
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