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Effect of pentobarbital anesthesia on amikacin concentrations in plasma and perilymph and evaluation of multiple sampling in perilymph of guinea pigs.

The purpose of this study was to determine whether a multiple-sampling procedure could be used in guinea pigs to study the kinetics of amikacin in perilymph. Amikacin was infused intravenously for 6 h into conscious anesthetized guinea pigs, and the concentrations of the drug in plasma and perilymph were measured. From each anesthetized guinea pig, five to six perilymph samples were collected from one ear, and one sample was collected from the other ear at 6 h. The concentrations of amikacin in perilymph were dose proportional and increased slowly during the 6-h infusion. However, after 6 h of intravenous infusion, the concentrations of amikacin in perilymph of the multiply sampled ears were significantly higher than those of the singly sampled ears, indicating that the multiple-sampling procedure should not be used as is to study the kinetics of amikacin in perilymph. Amikacin concentrations in perilymph were linearly related to amikacin concentrations in plasma in pentobarbital-anesthetized animals, as had previously been observed for conscious guinea pigs. However, the slope of the regression line was only 0.09 for anesthetized animals compared with 0.24 for conscious animals. Drug concentrations in plasma were found to be threefold higher in anesthetized animals, whereas drug levels in perilymph were the same in both groups at similar dosing rates. These results indicate that the amikacin concentration in perilymph is not solely dependent upon its concentration in plasma and that other factor(s) can affect the entry of amikacin into the inner ear.

Amikacin

Protein distribution in the human perilymph. A comparative study between perilymph (post mortem), CSF and blood serum.

The large differences in the protein content of perilymph and serum as well as the perilymph volume limit of 10-15 microliter cause serious problems when collecting samples of uncontaminated perilymph in order to analyse its protein composition. In 8 out of 24 extremely carefully taken samples of perilymph removed during autopsy, we were able, by keeping to certain selection criteria, to carry out electrophoretic analyses which were suitable for reproduction and comparison. Comparison of human perilymph, CSF and serum, using SDS-Page and Western blotting, gave the expected agreement which had been suggested by tests on guinea pigs. However, a closer examination of individual proteins pointed to some relatively large differences in quantity and quality.

Animals

Facilitated transfer of glucose from blood into perilymph in the rat cochlea.

The transport of glucose into cochlear endolymph, perilymph of scala vestibuli and perilymph of scala tympani, and cerebrospinal fluid (CSF) was studied after intravenous administration of tracers of D-glucose, L-glucose, and 3-O-methyl-D-glucose in anesthetized rats. The data showed that D-glucose concentrations in perilymph of scala vestibuli, perilymph of scala tympani, and CSF were approximately 50%, and in endolymph less than 10%, that in plasma; D-glucose concentration in perilymph of scala vestibuli, perilymph of scala tympani, and CSF increased as a linear function of that in plasma; D-glucose entry into perilymph of scala vestibuli, perilymph of scala tympani, and CSF was more rapid than that of L-glucose; after infusion of 3-O-methyl-D-glucose, but not after that of mannitol, both the D-glucose concentration ratio of perilymph over plasma and D-glucose transfer into perilymph were lowered. These results indicate that D-glucose enters into perilymph of scala vestibuli by a facilitated transport, possibly located at the blood-perilymph barrier.

3-O-Methylglucose

Rapid protein test for perilymph fistula.

The existence of a perilymph fistula may be difficult to prove preoperatively or at surgery, except in obvious cases in which perilymph can be seen coming out of the inner ear around the stapes footplate or round window niche. Some surgeons doubt the common occurrence of spontaneous perilymph fistula. Most surgeons believe that a perilymph fistula is rare and is produced by some type of trauma and pressure change to the inner ear fluids. Analysis of fluid collected from the oval window area and round window niche may be a great help in confirming or disproving the diagnosis of perilymph fistula. After a myringotomy or tympanotomy, fluid collected in micropipets from the oval window or round window area is analyzed for protein concentration, using rapid protein indicator paper. The original technique was developed in the 1960's to analyze the inner ear fluid as a diagnostic procedure (i.e., diagnostic labyrinthotomy) in acoustic neuroma suspects. Normal perilymph has a protein content of approximately 200 mg %, which turns the indicator paper light green, whereas serum or transudate has a protein content of approximately 7000 mg %, which turns the indicator paper dark green. The protein concentration is determined by comparing the color of the indicator paper with the color developed by known protein standards. A middle ear tap may help avoid negative middle ear exploration for perilymph fistula and helps document the presence or absence of perilymph after the exploration.

Cochlear Diseases

[Protein concentration in the guinea-pig perilymph].

The protein concentration of the guinea pig perilymph was investigated systematically using a micro-modification of the method of Lowry et al. Perilymph of scala vestibuli and of scala tympani was obtained from living animals and immediately post mortem by various methods. In living animals it is especially difficult to obtain samples without blood contamination. Another problem in the obtaining of perilymph from living animals is the contamination of tympanic perilymph samples with cerebrospinal fluid. This contamination diminishes the protein concentration of perilymph to a high degree. When the subarachnoid space is opened suboccipitally before perilymph extraction, there is no significant difference between protein content in tympanic and vestibular perilymph. The mean protein concentration in both cochlea scales is about 150 mg/100 ml. When samples are extracted post mortem from animals perfused intra-arterially, mean values of protein are in the same range. Without perfusion of animals, the mean value of tympanic samples extracted post mortem is significantly higher. Causes of artefacts in perilymph investigations are discussed.

Animals

Identification of perilymph proteins by two-dimensional gel electrophoresis.

Perilymph has a total protein component that is quantitatively distinct from serum and cerebrospinal fluid (CSF). The goal of this research was to determine if perilymph contains any qualitatively unique protein constituents that will distinguish it from serum or CSF. To test this hypothesis, matched sets of perilymph, serum, and CSF were obtained from 18 guinea pigs and seven human subjects. The purity of each sample was assured by measurement of the protein concentration of each sample and comparison of this parameter to known normal values for perilymph, serum, and CSF. Each sample was then subjected to two-dimensional gel electrophoresis, separating proteins by isoelectric point in the horizontal dimension and by relative molecular weight in the vertical dimension. All gels were processed under precisely identical physical conditions by use of a diamine silver stain. A small number of perilymph proteins not found in plasma were identified in both the guinea pig and the human specimens. The finding of unique perilymph proteins may permit the development of a sensitive marker that will aid in the diagnosis of perilymph fistula.

Animals

Peptides of the otosclerotic perilymph examined by analytical isotachophoresis.

Perilymphs of normal and otosclerotic origin were separated chromatographically on a Sephadex G-25 microcolumn. Peptide composition of the perilymphs was compared by capillary analytical isotachophoresis in the molecular mass range 0.3-5 kD. Otosclerotic perilymph samples contain a heterogeneous, UV-absorbing peptide subfraction which is not detected in the normal perilymph. Normal and otosclerotic perilymph, furthermore, contain four common subfractions detected in twice the normal concentration in the otosclerotic perilymph. These ITP subfractions are degraded during acid hydrolysis (6 M HCI). On the contrary, otosclerosis is a deficient state compared with the normal, as the number of peptides or oligoglycopeptides is twice as high in normal as in otosclerotic perilymph, beside the otosclerosis specific peptides.

Electrophoresis

Effects of perilymph volume adjustments on cochlear blood flow in the guinea pig.

Previous research suggests a potential relationship between perilymphatic pressure (Pp) and cochlear blood flow (CBF); however, the alterations in Pp necessary to produce changes in CBF have not been adequately described or quantified. The effects of perilymph volume changes on systemic blood pressure (BP) and CBF were presently investigated in the guinea pig cochlea. Five microliters of perilymph were displaced in each of three conditions: viz. evacuation of 5 microliters from the cochlea; replacement of these 5 microliters; and finally the addition of 5 microliters of artificial perilymph into the cochlea. All perilymph volume adjustments were completed in 1-microliter increments during which changes in CBG and BP were recorded. Significant alterations in CBF were observed during 1-microliter perilymph volume adjustments in each condition with no significant changes in systemic BP. The results from this study support our hypothesis that an inverse relationship exists between CBF and Pp in that decreases in perilymph volume yielded elevations in CBF while increases in perilymph volume yielded reductions in CBF.

Animals

[Sisomycin pharmacokinetics in the perilymph and blood serum--an approach to predicting its ototoxic effect].

To elucidate the possibility of predicting the level of aminoglycoside antibiotic penetration into the fluids of the internal ear by the antibiotic blood levels, the pharmacokinetics of sisomicin in the perilymph and blood serum was studied on guinea pigs. The antibiotic was administered to the animals subcutaneously in doses of 50, 100 and 200 mg/kg. On the basis of the comparison of the sisomicin concentrations in the perilymph normalized against the dose it was concluded that the pharmacokinetics of sisomicin in the perilymph and blood serum of the animals was linear. Comparison of the areas under the curves of the antibiotic concentration versus time in the perilymph (AUCp) and blood serum (AUCs) showed that the tissue availability of the antibiotic in this study characterized by its penetration into the perilymph and defined by the ratio of the AUCp to AUCs amounted to 55 per cent. In a two-compartment model it was not possible to predict the antibiotic levels in the perilymph by concentrations in the blood. However, by the antibiotic blood levels it was possible to characterize in a complex the pharmacokinetic behaviour of the antibiotic in the perilymph by predicting the areas under the respective curves of the antibiotic concentration versus time. The proportional relation between the values of the AUCp and AUCs suggested that the level of the antibiotic penetration into the internal ear and consequently the intensity of the potential ototoxic effect could be more reliably predicted not by separate values of the antibiotic concentration but by the areas under curves of aminoglycoside concentrations versus time.

Animals

A patient-oriented approach to perilymph fistula.

The subject of perilymph fistula is controversial in part because the preoperative diagnosis of perilymph fistula is difficult. Patients suffering auditory and vestibular symptoms secondary to perilymph fistula, therefore, present a dilemma to the practicing physician. In some instances, patients with auditory and vestibular symptoms in the absence of perilymph fistula will be subjected to middle ear exploration without benefit, while in other instances patients with auditory and vestibular symptoms secondary to perilymph fistula will be denied surgical treatment. Auditory and vestibular symptoms are a quality of life issue. Therefore, in an effort to provide care of the highest quality the patient must be actively involved in the decision process. Such involvement can be meaningful only after the patient is carefully counseled as to the pros and cons of surgery, as well as the alternatives. A prognostic paradigm based on the personal experience of one of us (W.L.M.) with perilymph fistula patients is described and is used in patient consultation in an effort to facilitate decision making.

Fistula

Protein profiles of perilymph and endolymph of the guinea pig.

Results of protein separation of guinea pig plasma, perilymph, and endolymph by means of high-resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented. Several proteins are present in perilymph at levels in basic accord with the total protein gradient with respect to plasma; however, others are present in perilymph at levels comparable to plasma levels, and one protein low molecular weight protein, PLS:33, is eight times higher. In addition, a high molecular weight protein is shown to be present at similar levels in the two compartments. These findings indicate that ultrafiltration cannot be the sole mechanism of perilymph production. Endolymph proteins are uniformly five to eight times lower than perilymph levels, essentially following the total protein concentration gradient between the two compartments. This supports the view that endolymph is derived from perilymph rather than directly from blood.

Animals

Evidence that amikacin ototoxicity is related to total perilymph area under the concentration-time curve regardless of concentration.

Previous studies have failed to fully establish whether ototoxicity is related in any way to the levels of an aminoglycoside antibiotic in the perilymph. To study this we exposed guinea pigs to continuously infused amikacin at four different dosing rates under conditions parallel to those used in our previous study which related ototoxicity to total plasma area under the concentration-time curve regardless of the level in plasma. It was found that at all dosing rates, levels in the perilymph and ratios of levels in perilymph/plasma remained constant as the dosing duration increased from nonototoxic to strongly ototoxic. Plasma and perilymph amikacin levels were found to be linear functions of the dosing rate even at ototoxic dosing exposures, and ratios of levels in perilymph/plasma did not differ between dosing rates. The total perilymph area under the concentration-time curve was not different between dosing rates either for a total dose associated with threshold ototoxicity or for one associated with severe ototoxicity. The results suggest that amikacin ototoxicity is related to the integral of the concentration in the perilymph over the total time of amikacin exposure regardless of the level in the perilymph.

Amikacin

Quantitative assessment of perilymph sources.

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

Animals

Elimination kinetics of furosemide in perilymph and serum of the chinchilla. Neuropharmacologic correlates.

This study was done to determine the comparative elimination kinetics of furosemide from chinchilla perilymph and serum, and to correlate perilymph concentration with changes in endocochlear potential. The elimination kinetics of furosemide (FU) were determined in sera and perilymph obtained from chinchillas injected with 100 mg/kg i.v. of FU. Concentrations of FU exhibited a linear decay pattern in serum and perilymph over the initial 60 minutes. The rate of decline of furosemide levels in perilymph was about four times slower than the rate of fall in serum. Chronic treatment (25 mg/kg i.p. every 12 hours) did not appear to influence the level of drug at 60 minutes after a dose of FU (100 mg/kg IV). Chinchillas were also studied following doses of FU ranging from 25--200 mg/kg i.v. to see the effect on endocochlear potential (EP). A positive correlation was found between FU dosage, the maximum millivolt reduction of EP and the time to initiation of recovery of EP. The perilymph concentration of furosemide when the EP began to recover was 5 microgram/ml (1.5 x 10(-5) M). Knowledge of furosemide kinetics may ultimately be applied to prevent ototoxicity in patients.

Animals

[Transmission of changes in the external ear atmospheric pressure to the perilymph].

Using guinea pig, pressures in the external ear canal, in the middle ear and in the perilymph were registered simultaneously, while pressure was applied to the external ear canal using an impedance audiometer. In the first experiment, applied pressure was changed in the range from 200 mmH2O to -200 mmH2O with and without the opening of the otic bulla. The change in the perilymphatic pressure with the opening was smaller than that without the opening. The result indicates that the external ear pressure is transmitted to the perilymph not only via the ossicular chain but also via the middle ear cavity without the opening, while it is exclusively transmitted via the ossicular chain with the opening. Pressure transmission to the perilymph was significantly impaired either by disrupting the ossicular chain or by closing the round window niche, especially by the latter. Thus the middle ear cavity itself plays an important role in pressure transmission from the external ear canal to the perilymph mainly via the round window. In the second experiment, applied pressure to the external ear canal was changed in the range from 1000 mmH2O to -1000 mmH2O after the Eustachian tube being closed. Between 400 mmH2O and -200 mmH2O, the middle ear and perilymphatic pressures paralleled well with the applied pressure. Beyond these levels, the middle ear pressure increased or decreased in response to the applied pressure but the perilymphatic pressure reversed against the middle ear pressure. Communication between perilymph and cerebrospinal fluid via the cochlear aqueduct is thought to be a major factor causing this reversal which, in turn, aggravates pressure gradient between the middle ear and the perilymph.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Effect of exogenous arachidonic acid metabolites applied on round window membrane on hearing and their levels in the perilymph.

Our previous studies revealed that treatment with sodium salicylate or indomethacin caused hearing loss, a decrease in prostaglandin (PG) levels, and an increase in leukotriene (LT) levels of the arachidonic acid (AA) cascade in the perilymph. We suspected that decreased PG-levels and/or elevated LT-levels in the inner ear may be responsible for the salicylate ototoxicity. In order to test this hypothesis, effects of exogenous treatments with PGs, PG-analog, LTs, and other lipoxygenase products on hearing and levels of AA metabolites in the perilymph were studied in chinchillas. Cyclooxygenase products, PGI2, 6-keto-PGF1 alpha, Iloprost (PGI2 analog), PGE2, and LTB4, LTC4, and 15-hydroxyeicosatetraenoic acid (15-HETE) in the lipoxygenase products in the dose of 150 ng were applied on the round window membrane (RWM); cochlear function tested by auditory brainstem response (ABR) and samples of perilymph were collected at 0.5, 1, 2, and 4 hours after the application. Samples of perilymph were assayed for all spectra of AA metabolites by high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). PG-treated animals developed minimal or no hearing loss. LT-treated animals exhibited hearing loss of 20 to 40 dB, peaking at one hour after the treatment. Elevated levels of arachidonic acid metabolites were measured in the perilymph of the ears treated with respective AA metabolites, with peak levels at one hour from the application. The findings of this study indicate that hearing loss can be induced by altered levels of PGs or LTs in the perilymph. This is another strong evidence that salicylate induced ototoxicity can be mediated by abnormal arachidonic acid metabolism in the inner ear.

Animals

Organic acid transport into the cochlear perilymph.

The passage of exogenous organic and inorganic substances from blood into perilymph is likely to be controlled by the blood-perilymph barrier. This report reviews published data on the transfer of ten exogenous organic acids from the blood into the perilymph of experimental animal models. Although the range of the molecular weights of these acids is within half an order of magnitude, major differences exist in the perilymph concentration as a percentage of simultaneous serum concentration. Furthermore, these studies show that, contrary to previous suggestions, non-ototoxic compounds can achieve marked concentration gradients within the perilymph.

Acids