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-mL 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.
Objectives-The distribution of gentamicin along the fluid spaces of the cochlea following local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected and histological studies indicate that hair cell damage is greater at the base than at the apex following local gentamicin application. In the present study, gradients of gentamicin along the cochlea were measured.Methods-A recently-developed method of sampling perilymph from the cochlear apex of guinea pigs was used, in which the samples represent fluid originating from different regions along scala tympani. Gentamicin concentration was determined in sequential apical samples which were taken following up to three hours of local application to the round window niche.Results-Substantial gradients of gentamicin along the length of scala tympani were demonstrated and quantified, averaging more than 4000 times greater concentration at the base compared to the apex at the time of sampling. Peak concentrations and gradients for gentamicin varied considerably between animals, likely resulting from variations in round window membrane permeability and rates of perilymph flow. Conclusions-The large gradients for gentamicin demonstrated here in guinea pigs account for how it is possible to suppress vestibular function in some patients with a local application of gentamicin without damaging auditory function. Variations in round window membrane permeability and in perilymph flow could account for why hearing losses are observed in some patients.
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.
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.
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.
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