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.     

Marker retention in the cochlea following injections through the round window membrane

Local delivery of drugs to the inner ear is increasingly being used in both clinical and experimental studies. Although direct injection of drugs into perilymph appears to be the most promising way of administering drugs quantitatively, no studies have yet demonstrated the pharmacokinetics in perilymph following direct injections. In this study, we have investigated the retention of substance in perilymph following a single injection into the basal turn of scala tympani (ST). The substance injected was a marker, trimethylphenylammonium (TMPA) that can be detected in low concentrations with ion-selective microelectrodes. Perilymph pharmacokinetics of TMPA was assessed using sequential apical sampling to obtain perilymph for analysis. The amount of TMPA retained in perilymph was compared for different injection and sampling protocols. TMPA concentrations measured in fluid samples were close to those predicted by simulations when the injection pipette was sealed into the bony wall of ST but were systematically lower when the injection pipette was inserted through the round window membrane (RWM). In the latter condition, it was estimated that over 60% of the injected TMPA was lost due to leakage of perilymph around the injection pipette at a rate estimated to be 0.09muL/min. The effects of leakage during and after injections through the RWM were dramatically reduced when the round window niche was filled with 1% sodium hyaluronate gel before penetrating the RWM with the injection pipette. The findings demonstrate that in order to perform quantitative drug injections into perilymph, even small rates of fluid leakage at the injection site must be controlled.     

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 Kinetics of FITC-Dextran Reveals Homeostasis Dominated by the Cochlear Aqueduct and Cerebrospinal Fluid

Understanding how drugs are distributed in perilymph following local applications is important as local drug therapies are increasingly used to treat disorders of the inner ear. The potential contribution of cerebrospinal fluid (CSF) entry to perilymph homeostasis has been controversial for over half a century, largely due to artifactual contamination of collected perilymph samples with CSF. Measures of perilymph flow and of drug distribution following round window niche applications have both suggested a slow, apically directed flow occurs along scala tympani (ST) in the normal, sealed cochlea. In the present study, we have used fluorescein isothiocyanate-dextran as a marker to study perilymph kinetics in guinea pigs. Dextran is lost from perilymph more slowly than other substances so far quantified. Dextran solutions were injected from pipettes sealed into the lateral semicircular canal (SCC), the cochlear apex, or the basal turn of ST. After varying delays, sequential perilymph samples were taken from the cochlear apex or lateral SCC, allowing dextran distribution along the perilymphatic spaces to be quantified. Variability was low and findings were consistent with the injection procedure driving volume flow towards the cochlear aqueduct, and with volume flow during perilymph sampling driven by CSF entry at the aqueduct. The decline of dextran with time in the period between injection and sampling was consistent with both a slow volume influx of CSF (∼30 nL/min) entering the basal turn of ST at the cochlear aqueduct and a CSF-perilymph exchange driven by pressure-driven fluid oscillation across the cochlear aqueduct. Sample data also allowed contributions of other processes, such as communications with adjacent compartments, to be quantified. The study demonstrates that drug kinetics in the basal turn of ST is complex and is influenced by a considerable number of interacting processes.     

Radial communication between the perilymphatic scalae of the cochlea. I: Estimation by tracer perfusion.

The degree to which radial exchange between scala tympani (ST) and scala vestibuli (SV) can occur has not previously been quantified. We have measured the amount of cross-communication in the third turn of the guinea pig cochlea using an ionic tracer, trimethylphenylammonium (TMPA). TMPA was perfused through one scala while TMPA concentrations were measured simultaneously in the perfused and non-perfused scalae. On the basis of the time course of TMPA increase recorded in the non-perfused scala, we were able to calculate rate constants for cross-leak and clearance. Cross-leak in the third turn occurred remarkably rapidly with a rate constant from ST to SV of 0.049 min-1 and from SV to ST of 0.031 min-1. These correspond to transfer half times of 14.2 and 22.4 min respectively. This result demonstrates that ST and SV in the third turn cannot be regarded as independent compartments.     

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.     

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.     

Solute movement across the round window membrane in comparison with that across the blood-labyrinth barrier]

The permeability of the normal round window membrane of the guinea pig to trimethylphenylammonium (TMPA) was assessed using ion-selective electrodes and compared with the rate of TMPA entry from the systemic blood circulation into the scala tympani (ST) of the cochlea across the so-called blood-labyrinth barrier. While the round window niche was irrigated with artificial perilymph containing 1 mM TMPA, the TMPA concentration in ST of the basal turn rose rapidly so as to reach 20-50% of the irrigating medium concentration in one hour. Following this procedure, the concentration declined significantly faster when the niche was subsequently irrigated with TMPA-free artificial perilymph than when the niche was left free of any fluid. This result shows that the membrane is fairly permeable to TMPA in both directions. Furthermore, TMPA entry from blood to STs of the basal and third turns was observed while the plasma TMPA concentration was maintained at about 0.5 mM by continuous intravenous infusion of isotonic 50 mM TMPA medium (1 part 150 mM TMPA + 2 parts lactated Ringer solution). TMPA appeared to distribute evenly from the blood to both turns at a much slower rate than across the round window membrane. In another experiment, the round window niche was irrigated with TMPA-free artificial perilymph during the intravenous infusion of 50 mM TMPA medium. The TMPA concentration increase in ST of the basal turn was greatly suppressed, whereas that of the third turn was not affected for at least an hour.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of glycerol on inner ear fluid electrolytes and osmolalities in guinea pigs

Endolymph of the scala media (SM) and perilymph of the scala vestibuli (SV) and scala tympani (ST) were collected from the basal turn of anesthetized guinea pigs before and after intravenous administration of glycerol (3 g/kg). Sound-evoked responses were recorded during the test periods. Blood, CSF, and perilymph of the ST were also collected continuously after the injection. The osmolalities and chloride concentrations of the collected samples were determined. In another experiment, the continuous changes of potassium and chloride concentrations in endolymph and perilymph of the ST before and after the injection were measured by ion-selective electrodes. The osmolalities in CSF and perilymph lagged behind the increase in serum osmolality. The osmolalities in endolymph and perilymph increased gradually after the injection, reached maximum values after 90 minutes, and then decreased. The changes in chloride and potassium concentrations in endolymph and perilymph had similar tendencies. But the increases in chloride concentrations in perilymph of the SV and ST were much less than that in endolymph. We propose that most of the osmolality increase in perilymph is due to glycerol or other osmotically active substances and that the osmolality increase in endolymph is due to water shift.     

Systemic Lipopolysaccharide Compromises the Blood-Labyrinth Barrier and Increases Entry of Serum Fluorescein into the Perilymph

The blood vessels that supply the inner ear form a barrier between the blood and the inner ear fluids to control the exchange of solutes, protein, and water. This barrier, called the blood-labyrinth barrier (BLB) is analogous to the blood-brain barrier (BBB), which plays a critical role in limiting the entry of inflammatory and infectious agents into the central nervous system. We have developed an in vivo method to assess the functional integrity of the BLB by injecting sodium fluorescein into the systemic circulation of mice and measuring the amount of fluorescein that enters perilymph in live animals. In these experiments, perilymph was collected from control and experimental mice in sequential samples taken from the posterior semicircular canal approximately 30 min after systemic fluorescein administration. Perilymph fluorescein concentrations in control mice were compared with perilymph fluorescein concentrations after lipopolysaccharide (LPS) treatment (1 mg/kg IP daily for 2 days). The concentration of perilymphatic fluorescein, normalized to serum fluorescein, was significantly higher in LPS-treated mice compared to controls. In order to assess the contributions of perilymph and endolymph in our inner ear fluid samples, sodium ion concentration of the inner ear fluid was measured using ion-selective electrodes. The sampled fluid from the posterior semicircular canal demonstrated an average sodium concentration of 145 mM, consistent with perilymph. These experiments establish a novel technique to assess the functional integrity of the BLB using quantitative methods and to provide a comparison of the BLB to the BBB.     

Species differences in inner ear fluids

Summary Inner ear fluids of guinea pigs and cats were analyzed for sodium, potassium, chloride, glucose, and total protein to determine species differences in chemical compositions. In the scala vestibuli perilymph and scala tympani perilymph, sodium, potassium, and choride levels in the guinea pig were lower than in the cat. The protein levels in the scala vestibuli perilymph and scala tympani perilymph of the guinea pig were lower than those of the cat. The glucose levels in the guinea pig were higher in the scala vestibuli perilymph and scala tympani perilymph, as compared to findings in the cat. Regarding the utricular endolymph, there were significant differences between guinea pigs and cats in sodium and potassium concentrations; the concentration in the former being higher in sodium and lower in potassium. These findings are pertinent for the phylogenetic studies on inner ear fluid biochemistry.This work was supported by Research Grant No. ROI-NS1026801 from the National Institute of Neurological Diseases and Strokes, USA     

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.     

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.     

Perilymph composition in scala tympani of the cochlea: influence of cerebrospinal fluid

A commonly used technique to obtain cochlear perilymph for analysis has been the aspiration of samples through the round window membrane. The present study has investigated the influence of the volume withdrawn on sample composition in the guinea pig. Samples of less than 200 nl in volume taken through the round window showed relatively high glycine content, comparable to the level found in samples taken from scala vestibuli. If larger volumes are withdrawn, lower glycine levels are observed. This is consistent with cerebrospinal fluid (having a low glycine content) being drawn into scala tympani through the cochlear aqueduct and contaminating the sample. The existence of a concentration difference for glycine between scala tympani perilymph and cerebrospinal fluid suggests the physiologic communication across the cochlear aqueduct is relatively small in this species. The observation of considerable exchange between cerebrospinal fluid and perilymph, as reported in some studies, is more likely to be an artifact of the experimental procedures, rather than of physiologic significance. Alternative sampling procedures have been evaluated which allow larger volumes of uncontaminated scala tympani perilymph to be collected.     

Effect of peroral glycerol administration on inner ear fluid electrolytes of guinea pigs

After the oral administration of 50% glycerol (12 mL/kg), serum, CSF and inner ear fluids from scala tympani perilymph, scala vestibuli perilymph, and scala media endolymph were collected from normal guinea pigs under sodium pentobarbital anesthesia (25-35 mg/kg). The sodium and potassium concentrations were determined by microflame photometry. Increases in sodium concentrations were found in CSF, scala tympani perilymph, scala vestibuli perilymph, and cochlear endolymph. No significant change was observed in the serum. These sodium increases were considered to be due to the dehydration caused by the osmotic action of glycerol. Potassium concentration was increased only in scala tympani perilymph. Oral administration of glycerol was found to be more gradual and effective in dehydration compared to intravenous injection.     

Effects of glycerol on sodium and potassium concentrations in guinea pig perilymph

Summary Serum, cerebrospinal fluid (CSF), scala vestibuli perilymph, and scala tympani perilymph were collected from 85 normal guinea pigs both before and after i.v. administration of glycerol (1 ml/kg), and the sodium and potassium concentrations were assessed using a microflame photometer. Marked increases in sodium concentrations were observed in scala tympani perilymph and CSF, while there was a slight decrease in the serum and there was no significant change in scala vestibuli perilymph. These increases in sodium concentrations are considered to occur in the dehydration process in the body fluids mentioned above. On the other hand, increase in the potassium concentrations was found only in scala vestibuli perilymph and thus cannot be explained by simple dehydration process. This potassium elevation in scala vestibuli perilymph should be understood by further experiment on endolymph. It became evident that scala vestibuli perilymph differs from scala tympani perilymph and CSF in the dynamics of electrolytes after glycerol administration. In this regard, the nature of the scala vestibuli as fluid space should be studied in future. The above findings obtained in the present study may imply the significance in elucidating the glycerol effect on hearing of endolymphatic hydrops case.This study was funded in part by a grant from the Ministry of Education, Science, and Culture, Japan     

Effects of intravenous glycerol injection on inner ear fluid electrolytes

Under sodium pentrobarbital anesthesia (20-30 mg/kg, i.p.), normal guinea pigs received an intravenous injection of glycerol (1.0 ml/kg). Serum, cerebrospinal fluids (CSF) and inner ear fluids were collected from the scala tympani perilymph, scala vestibuli perilymph and the scala media endolymph. The sodium and potassium concentrations were assessed using microflame photometry. Increases in sodium concentration were found in the CSF and the scala tympani perilymph; no significant changes were observed in the serum, scala vestibuli perilymph or the scala media endolymph. These sodium increases were considered to be a result of the dehydration process caused by the osmotic agent glycerol. Increases in potassium concentration were found only in the scala vestibuli perilymph.     

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.     

Symposium: new data for noise standards. II. Effects of insulin on glucose concentrations in inner ear fluids and cochlear microphonics

Fluids from various inner ear compartments were taken from 77 ears of 70 normal, healthy cats and were analyzed for glucose concentration by the microchemical technique employing the hexokinase method. Perilymph of the scala vestibuli, perilymph of the scala tympani and C.S.F. had almost identical concentrations of glucose with the values approximately 80 mg/100 ml. Glucose concentrations of each fluid showed the significant relationship with serum glucose level except that of utricular and cochlear endolymph. Utricular endolymph glucose level was found to be lower (41.9 mg/100 ml) than the above three fluids and higher than cochlear endolymph (11.2 mg/100 ml). The significant difference of glucose concentration found between cochlear and utricular endolymph in the present study is further support for the independent nature of each endolymphatic compartment. Sodium, potassium and glucose were analyzed in serum, C.S.F. and inner ear fluids up to five hours after intravenous regular insulin (30 units/kg). Sodium, potassium and glucose concentrations were unchanged in both cochlear and utricular endolymph during hypoglycemia. In contrast glucose concentrations in C.S.F., scala tympani perilymph, and scala vestibuli perilymph began to decrease at 90 minutes after regular insulin infusion and fell to their lowest levels in the period during 180 to 300 minutes after the infusion. A significant decrease of potassium concentration was found first in serum and later in the C.S.F., scala vestibuli, and scala tympani perilymph. For five hours after insulin infusion there is no significant change in cochlear microphonics during hypoglycemia (20 mg percent).     

Surgical techniques for cell transplantation into the mouse cochlea

This study investigated surgical procedures for cell transplantation into the mouse inner ear. Female C57BL/6 mice were used as recipient animals. Fetal mouse neural stem cells expressing green fluorescence were used as donor cells. Two methods, an injection of transplants from the lateral semicircular canal (LSCC) and from the cochlear lateral wall (CLW), were examined. Two weeks after transplantation, the distribution of transplant-derived cells in the cochlea was examined. Effects on auditory function were assessed by measurement of auditory brain stem responses (ABRs). Cochleae receiving cell transplantation from the LSCC exhibited robust survival of transplant-derived cells mainly in the scala vestibuli and scala tympani. Transplantation from the LSCC caused elevation of ABR thresholds by less than 10 dB SPL. However, transplantation from the CLW resulted in considerable hearing loss, even though transplant-derived cells settled in the scala media. These findings demonstrate that an approach from the LSCC can be utilized for cell transplantation into the perilymph without causing apparent auditory disorder, while an approach from the CLW delivers cells to the endolymph but appears to cause auditory dysfunction.