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 The dependence of perilymph sample purity on the location of sampling and on the volume withdrawn was quantified.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.     

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.     

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)     

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.     

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.     

Blockage of cochlear aqueduct for examination of perilymph (guinea pig) (author's transl)

To prevent the perilymph (guinea pig) from contamination with CSF during the sampling the aqueductus cochleae (AC) was blocked by injection of tissue adhesive into the meningeal aperture. The control of an exact blockage of AC was carriedout by examination of perilymph-outflow after opening the cochlea (injection of fluorescein-Na into the CSF-space), analysis of perilymph-protein-concentration, macroscopic and microscopic examination of the temporal bones. In all cochleae we have found the same morphological structures, notwithstanding whether the AC was blocked (for a time from 30 min to 7 weeks) or not: The cochlear aqueduct is filled with a mesh of mesenchymal tissue, which grows more dense towards the cochlear aperture andcontinues into the round window membrane. From scala tympani the AC is always limited by one layer of cells forming a sort of membrane (under light microscope). It seems possible that CSF moves in the inner of the round window membrane between AC and subepithelian space of middle ear mucosa, whereas perilymph of scala tympani is not in direct contact with the flow of CSF. The scala tympanic side of the round window membrane may be a big area for diffusion and there also may be an exchange between CSF and perilymph. The outflow of CSF into the cochlea after experimental opening of the cochlea is an artifact, caused by damage of pressure equilibration between CSF-space and cochlea. 30 min and 5--7 weeks after blockage no morphologicaland electrophysiological alterations from those of the control ears were to be seen. The protein concentration, however, increased significantly 5--7 weeks after blockage from normally about 200 mg/100 ml toalmost the double especially in the scala tympani (see Table 1).     

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.     

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.     

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.     

Concentration gradient along the scala tympani after local application of gentamicin to the round window membrane

OBJECTIVES: The distribution of gentamicin along the fluid spaces of the cochlea after local applications has never previously been demonstrated. Computer simulations have predicted that significant basal-apical concentration gradients might be expected, and histologic studies indicate that hair cell damage is greater at the base than at the apex after 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 the scala tympani. Gentamicin concentration was determined in sequential apical samples that were taken after up to 3 hours of local application to the round window niche. RESULTS: Substantial gradients of gentamicin along the length of the scala tympani were demonstrated and quantified, averaging more than 4,000 times greater concentration at the base compared with 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.     

Permeability of the round window membrane for prednisolone-21-hydrogen succinate. Prednisolone content of the perilymph after local administration vs. systemic injection

BACKGROUND AND OBJECTIVE: Prednisolone is the drug of first choice for the treatment of cochleovestibular disorders, such as sudden hearing loss. Because of the known side effects, the efficient drug levels to be achieved within inner ear fluids are limited by intravenous administration. The aim of the study was to determine the concentration in the perilymph of prednisolone-21-hydrogen succinate applied into the round window niche in comparison to the concentration after intraperitoneal application. METHODS: Application of prednisolone-21-hydrogen succinate (5 mg in 0.1 ml) on the round window membrane was performed after sedation under microscopic view directly into the round window niche of the guinea pig. In order to compare the results, perilymph samples after systemic application of 60 mg/kg body weight prednisolone were used. The time between application and taking specimens of perilymph from the cochlea varied. Specimens of perilymph were obtained after 15, 20, 80, 180, 330, and 960 min (10 specimens in each group, n = 60) by dissecting the cochlea and opening the apex cochleae. Levels of prednisolone-21-hydrogen succinate in perilymph were measured by isocratic high-pressure liquid chromatography (HPLC). RESULTS: The highest levels of prednisolone-21-hydrogen succinate were found after 180 min: 952.3 mg/l (95% confidence interval: 382.7). After 960 min the level was 18.72 mg/l (95% confidence interval: 16.9). In the group with systemic application, the levels measured were below 14.71 mg/l (95% confidence interval: 7.05). CONCLUSION: The results demonstrate that high levels of prednisolone-21-hydrogen succinate in perilymph are achievable by local application of a single dose into the round window niche. After application of 5 mg, the levels of prednisolone are measurable up to 16 h.     

Der Verschluß des Aquaeductus cochleae für Perilymphuntersuchungen am Meerschweinchen

Summary To prevent the perilymph (guinea pig) from contamination with CSF during the sampling the aqueductus cochleae (AC) was blocked by injection of tissue adhesive into the meningeal aperture. The control of an exact blockage of AC was carried out by examination of perilymph-outflow after opening the cochlea (injection of fluorescein-Na into the CSF-space), analysis of perilymphprotein-concentration, macroscopic and microscopic examination of the temporal bones.In all cochleae we have found the same morphological structures, notwith-standing whether the AC was blocked (for a time from 30 min to 7 weeks) or not: The cochlear aqueduct is filled with a mesh of mesenchymal tissue, which grows more dense towards the cochlear aperture and continues into the round window membrane. From scala tympani the AC is always limited by one layer of cells forming a sort of membrane (under light microscope).It seems possible that CSF moves in the inner of the round window membrane between AC and subepithelian space of middle ear mucosa, whereas perilymph of scala tympani is not in direct contact with the flow of CSF. The scala tympanic side of the round window membrane may be a big area for diffusion and there also may be an exchange between CSF and perilymph. The outflow of CSF into the cochlea after experimental opening of the cochlea is an artifact, caused by damage of pressure equilibration between CSF-space and cochlea. 30 min and 5–7 weeks after blockage no morphological and electrophysiological alterations from those of the control ears were to be seen.The protein concentration, however, increased significantly 5–7 weeks after blockage from normally about 200 mg/100 ml to almost the double especially in the scala tympani (see Table 1).

Herrn Dr. Ritter (HNO-Klinik) danken wir für die Anfertigung eines Teils der fotografischen Aufnahmen und Herrn Doz. Dr. Werner (Institut für Pathologie) für seine Beratung in morphologischen Fragen.     

Zur Ausbreitung löslicher Substanzen nach Applikation in Perilymphe und Liquor

Summary New labelling methods using fluorochromes reduce the amount of substances to 1 g. The volume which is temporarily taken out and then reinjected into the fluid spaces is 0.1 l. At different times after the application, the heads of the guinea pigs are frozen by liquid air, and cut on a cryotome. The mounted object is then fotographed with an operating microscope in UV-light.Substances applicated into the perilymph of the scala tympani reach the subarachnoid space via the cochlear aqueduct in about 10 min. After filling the whole bulla tympanica, the dyes diffude into the cochlea mainly through the membrane of the round window. They are reaching the inner ear also through the oval window and by labyrinth crossing vessels. Substances applied to the cerebello-medular cistern could not be detected in the cochlea. After intraperitoneal administration the fluorochromes can be found in the cochlear nerve, in the spiral ligament, in perilymph and endolymph. Substances which have reached the cochlear endolymph can be found in the endolymphatic sac after 20 min.This leads to the conclusion that perilymph and endolymph are produced in the cochlea. The blood vessels are the origin of the substances in the inner ear fluids, but a blood-inner ear barrier seems to be existent.     

Kinetic experiments with radionuclides concerning the perilymph-blood barrier in a guinea pig model

Since 1950 many animal radiotracer experiments have been performed to study inner ear kinetics. For the most part in these studies, radionuclides were applied systemically, following which a discontinuous probing of inner ear fluids or of inner ear tissues was done. Two techniques have been developed in the Section for Experimental Otorhinolaryngology of the University of Würzburg. These have been adapted to the direct and continuous measurements of inner ear efflux kinetics for several hour periods. For this purpose, only a tiny amount of radiotracer need be applied directly to the inner ear. Experiments were done on the anesthetized guinea pig as an animal model. In the first technique, a collimator-detector system is focused precisely on the cochlea, which had been quickly resealed after application of the radionuclide bolus via two small holes in the basal turn of the cochlea. The second technique makes use of a perilymph cycling system, whereby a small outer volume includes a microcuvette with a so-called artificial round window. By this latter cycling technique, perilymph clearance kinetics of all kinds of radiotracers--with the exception of tritium labelled ones--can be measured. Calculations from clearance kinetics show that quite small particles with particle weights up to 100, such as the chlorine anion and the potassium cation, as well as urea, glycerol, pyruvate, and lactate, exhibit perilymphatic half-lives varying from 45 to 60 min. These half-live data are plausible in regard to cochlear blood flow measured previously via an independent technique developed by Angelborg et al.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Experimental studies on ischemia of the cochlea. 2. Pathobiochemistry]

Biochemical analyses of organic substances (protein, glucose, amino acids) as well as inorganic substances (potassium and sodium) in inner ear fluids were performed for the first time under local ischemic conditions. The perilymph of the thrombosed right ear was collected from various animals after different durations of ischemia. The normal left cochlea served as control. The concentrations of potassium and sodium and the total protein content remain unchanged in the perilymph of the guinea pig during the first two hours of ischemia. The decrease in the glucose concentration in the perilymph shortly after the thrombosis is a new finding. It points to the possible existence of a reservoir of energy carriers in the fluid of the inner ear that become available to cochlear tissues during ischemia. The increase in the amino acid concentration during ischemia suggests metabolic processes in the cochlea. The biochemical analysis of the perilymph during ischemia of the cochlea provides evidence of two possible mechanisms that secure the survival of the organ of Corti. The glucose reserves of the perilymph are utilized by the cochlear tissues and the concentration of certain amino acids secure the supply.     

Kinetic experiments with radionuclides concerning the perilymph-blood barrier in a guinea pig model

Summary Since 1950 many animal radiotracer experiments have been performed to study inner ear kinetics. For the most part in these studies, radionuclides were applied systemically, following which a discontinuous probing of inner ear fluids or of inner ear tissues was done. Two techniques have been developed in the Section for Experimental Otorhinolaryngology of the University of Würzburg. These have been adapted to the direct and continuous measurements of inner ear efflux kinetics for several hour periods. For this purpose, only a tiny amount of radiotracer need be applied directly to the inner ear. Experiments were done on the anesthetized guinea pig as an animal model. In the first technique, a collimator-detector system is focused precisely on the cochlea, which had been quickly resealed after application of the radionuclide bolus via two small holes in the basal turn of the cochlea. The second technique makes use of a perilymph cycling system, whereby a small outer volume includes a microcuvette with a socalled artificial round window. By this latter cycling technique, perilymph clearance kinetics of all kinds of radiotracers — with the exception of tritium labelled ones — can be measured. Calculations from clearance kinetics show that quite small particles with particle weights up to 100, such as the chlorine anion and the potassium cation, as well as urea, glycerol, pyruvate, and lactate, exhibit perilymphatic half-lives varying from 45 to 60 min. These half-live data are plausible in regard to cochlear blood flow measured previously via an independent technique developed by Angelborg et al. For particle weights distinctly beyond 100, half-lives increased gradually according to the operation of a perilymph-blood barrier. For a few tracers such as theophylline, nicotinamideadenine-dinucleotide, urografin, and biligrafin, individual effects are superimposed, giving rise to rather fast kinetics. In contrast, the ototoxic drugs ethacrynic acid and tobramycin exhibit a certain retardation in their clearance kinetics. Very small non-polar gaseous particles such as hydrogen and xenon show extremely short perilymphatic/cochlear half-lives. The half-life of hydrogen is about 4 min which accounts for a maximum clearance consistent with total cochlear blood flow.Dedicated to Professor Dr. Walter Kley, former head of the ENT Department, University of Würzburg     

Fluorescein Kinetics in Perilymph and Blood: A Fluorophotometric Study

A modified slit lamp fluorophotometer was used to determine fluorescein concentration changes in the perilymph, cerebrospinal fluid and blood of chinchillas after intravenous injection of 0.2 ml of fluorescein sodium. This new technique provides a means of determining quantitative changes of fluorescein concentration in the perilymph without the need to withdraw fluid samples through the round window membrane or cochlear wall. Fluorescein was observed to enter the perilymph between 1 and 2 minutes after injection, and it reached its peak concentration in a mean time of 23 minutes. The mean peak concentration was 4.20x10^?6 g/ml. Both increasing and decreasing fluorescein concentration changes in the perilymph followed an exponential time course. Although the observations of cerebrospinal fluid fluorescence were thought to represent a composite of the fluorescence of the cerebrospinal fluid itself and the underlying brain stem blood vessels, the peak fluorescence did not exceed that observed in the perilymph. These observations support the view that most of the perilymph is produced in the cochlea by ultrafiltration from the cochlear blood vessels. The slit lamp fluorophotometer appears to be a satisfactory means of recording fluorescein concentration changes in the perilymph without disturbing the cochlear physiology by penetrating the labyrinth to obtain fluid samples.     

Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,对照组（16只）导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异（P〉0．05）;鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异（P〉0．05）,8kHz较术前增高（P〈0．05）,16kHz、20kHz较术前明显增高（P〈0．01）,术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01）,但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。