Comparative ototoxicity of ribostamycin, dactimicin, dibekacin, kanamycin, amikacin, tobramycin, gentamicin, sisomicin and netilmicin in the inner ear of guinea pigs.

Nine aminoglycoside antibiotics, ribostamycin (RSM), dactimicin (DAC), dibekacin (DKB), kanamycin (KM), amikacin (AMK), netilmicin (NTL), tobramycin (TOB), gentamicin (GM) and sisomicin (SISO) were administered intramuscularly to guinea pigs for 4 weeks, and ototoxicity and drug concentration in the inner ear fluid were determined. RSM and DAC showed the weakest ototoxicity against the cochlea and vestibular organs. AMK and KM were more toxic to cochlea than vestibular organs. DKB, TOM, GM and SISO were equally toxic to vestibular organs and cochlea. NTL was more toxic to vestibular organs than cochlea. As judged from the pinna reflex response and hair cell damage in the cochlea, the order of auditory toxicity was the following: SISO greater than GM greater than TOB greater than AMK greater than DKB greater than KM greater than NTL, DAC RSM, whereas the vestibular toxicity was in the following order: SISO greater than GM greater than DKB greater than TOB greater than NTL greater than AMK greater than KM greater than DAC, RSM. RSM, causing the weakest ototoxicity, showed a low drug concentration in the inner ear fluid, while GM, causing severe ototoxicity, showed the highest drug level under the same conditions.

A map of cochlear perilymph protein based on high-resolution two-dimensional electrophoresis.

For systematic characterization of cochlear perilymphatic (PL) fluid proteins and to compare the complex protein mixtures of PL fluid, cerebrospinal fluid (CSF) and blood plasma, we subjected eight postmortem PL fluid samples to two-dimensional (2D) electrophoresis. To avoid contamination of perilymph by blood and blood plasma, the samples were taken immediately after death and analyzed by keeping to certain selection criteria. When compared with CSF, PL fluid in the scala vestibule was found to have an albumin content of approximately 1-2g/l, or 10 times the level of CSF albumin. Due to the small volume of the sample obtained, it was not possible to concentrate the PL fluid. As a result, protein spots in the 2D-gel could only be detected with a highly sensitive silver stain. Visual inspection of the resulting 2D-electrophoretograms showed that most of the "CSF-specific" protein clusters were present in the PL fluid pattern.

Round window membrane and perilymph in experimental otitis media with effusion.

To investigate the influence of middle ear effusion (MEE) on perilymph (PL), an experimental otitis media with effusion (OME) was manufactured in chinchillas by injecting the tympanic cavity with immune complexes. The presence of MEE lasted for up to 9 days after the injection of immune complexes. Perilymph was aspirated on the fourth, tenth, and 21st days after the inoculation. The mean concentrations of albumin, immunoglobulin G, histamine, and prostaglandin E2 (PGE2) were significantly greater in PL from ears with induced OME than in that from normal control ears. The 3H-PGE2 placed on the round window membrane of pathologically affected ears passed into PL in significantly greater amounts than in normal control ears. The findings indicate that the immune complexes placed in the middle ear cavity affect the biochemical milieu of PL, and that MEE is a result of immune complexes.

Endotoxin permeability through the round window.

The permeability of the round window membrane to Salmonella typhimurium derived endotoxin was examined using a total of 17 chinchillas. One mg of endotoxin was instilled into the tympanic cavity via the superior bulla. Endotoxin activity in middle ear effusions (MEEs), perilymph (both inoculated and non-inoculated side), and sera was determined by Limulus lysate assay after 12, 24, 48, 72, and 120 h following endotoxin instillation. Endotoxin was detected in perilymph on the inoculated side by 12 h after endotoxin instillation and persisted for 5 days during the present measurement period. Endotoxin level peaked at 24-48 h post-instillation, and steadily declined afterwards. This result suggests that the maximum penetration occurred during the active inflammatory stage. Histologic investigation revealed marked pathological changes in the inner ear, including bleeding and inflammatory cell recruitment, mostly in the perilymphatic spaces (e.g. scalae tympani and vestibuli, spiral ligament), strial swelling, and sensory cell degeneration. These results suggest that endotoxin, when introduced into the middle ear, can permeate through the round window membrane and can cause inner ear tissue damage in this animal model.

Influence of middle ear effusion on perilymph.

To investigate the influence of middle ear effusion (MEE) on perilymph (PL), an experimental otitis media with effusion (OME) was provoked in chinchillas by injecting the tympanic cavity with immune complexes. MEE remained for up to 9 days after the injection of immune complexes. PL was aspirated on the 4th, 10th, and 21st day after the inoculation. The mean concentrations of albumin, IgG, and prostaglandin E2 (PGE2) were significantly greater in PL from OME induced ears than in that form normal control ears. 3H-PGE2 placed on the round window membrane of pathologically affected ears passed into PL in significantly greater amounts than in normal controls. The findings of the present study indicate that MEE affects the biochemical environment of PL. However, this influence was transient and reversible.

Relationship between antigen levels in middle ear and antigen passage through round window membrane in antigen-induced otitis media.

By using an antigen-induced (Arthus) otitis media (OM) model in chinchillas sensitized with human serum albumin (HSA), we investigated the passage of HSA through the round window membrane (RWM) by measuring HSA levels in both the middle ear fluid (MEF) and the perilymph (PL). The effect of corticosteroid treatment on HSA passage was also studied. Mean HSA levels in both the MEF and the PL were proportional to the HSA dose administered (0.5, 5, 50 mg/ml). The proportion of HSA-positive PL samples was significantly lower in the sensitized group than that of the control (p less than 0.01). HSA levels in the PL appear to depend on HSA levels in the MEF, which were significantly depressed in the sensitized animals. These results suggest that an immunological reaction may be involved in the reduction of antigen (HSA) passage through the RWM by reducing antigen concentrations in the MEF. Corticosteroid treatment reduces the passage of HSA into the PL in non-sensitized animals.

Prostaglandins in type II collagen-induced ear diseases.

This study examines the role of prostaglandins in type II collagen-induced inner ear disease. Hartley guinea pigs were immunized with type II collagen in complete Freund's adjuvant. Auditory brain stem response was measured initially and at 2, 3, 4, and 6 months after immunization. The presence of PGE2 and 6-keto-PGF1 alpha in the perilymph was determined by radioimmunoassay. Perilymph from immunized animals contained about five times more PGE2 and about three times more 6-keto-PGF1 alpha than the perilymph from control animals. However, the two groups showed no difference in the level of either PGE2 or 6-keto-PGF1 alpha in cerebrospinal fluid and serum. Immunized guinea pigs also showed evidence of hearing loss. The temporal bones showed the following changes: spiral ganglion degeneration; mild to moderate degeneration in the organ of Corti; and infrequent and very mild endolymphatic hydrops. Thus, prostaglandins might play an important role in the pathogenesis of type II collagen-induced inner ear disease.

Round window membrane permeability to human serum albumin in antigen-induced otitis media.

The round window membrane (RWM) permeability to human serum albumin (HSA) was investigated in both normal chinchillas and chinchillas sensitized with HSA. The effect of a corticosteroid agent (triamcinolone) on the RWM permeability was also analyzed. It was found that HSA could not be detected in either the perilymph or the cerebrospinal fluid of normal chinchillas within 1 hour after instillation into the middle ear bulla. Perilymph levels of HSA peaked 24 hours after instillation. In antigen-induced otitis media, the HSA level in the perilymph at 12 hours after challenge was significantly higher than that in normal chinchillas (P less than .01), but it did not increase with the development of otitis media. A significant difference of HSA level in the perilymph of animals with and without steroid treatment was noted (P less than .025 at 24 hours and P less than .05 at 48 hours). It is concluded that little HSA passes through the normal RWM in a short time and that the RWM permeability to HSA increases in the early stage (about 12 hours) of antigen-induced otitis media. Corticosteroids can affect the RWM permeability to HSA by reducing the level of antigen-induced otitis media.

Perilymphatic calcium and VIII nerve action potentials during gentamicin bolus i.v. injections. A preliminary study.

Life-threatening human sepsis is often treated using bolus intravenous aminoglycoside injections with transient deafness as a reported side effect. Young adult cats were given various high dosages of gentamicin in bolus injections (1 ml in 30 s). Scala vestibuli calcium and gentamicin, blood gentamicin and compound action potentials of the VIII nerve were measured shortly after the injections and 45 min later. An obvious relationship could be demonstrated between levels of gentamicin, decreased calcium content and acoustic thresholds elevation. Even after a high dose of 175 mg/kg that abolished action potentials at t = 2 min, recovery invariably occurred. It is believed that the reported transient hearing loss in humans may be partly attributable to a temporary blockage of calcium by gentamicin.

Absence of ototoxicity of teichomycin A2 in guinea pigs.

Teichomycin A2 is a new antibiotic that is similar to vancomycin. Because vancomycin is reported to be ototoxic, teichomycin A2 was tested for ototoxicity. No evidence of ototoxicity was found. Furthermore, ethacrynic acid, a diuretic that augments the ototoxicity of many drugs, did not enhance ototoxicity with teichomycin A2.

[Sample distribution of inner ear fluids in the submicroliter range]. / Probenverteilung von Innenohrflüssigkeiten im Submikroliterbereich.

Biochemical analysis of inner ear fluids is fraught with numerous problems. After developing accurate techniques to measure inorganic and organic substances in fluid amounts of 0.1 microliter the problem of measuring and handling series of samples arose. It became necessary to develop methods to handle inner ear fluid taken without sophisticated techniques, i.e. to measure, to store and to dispatch inner ear fluid samples. The fluid volume is measured with a "nanocap" technique and is then transferred to cellulose acetate membranes. A 3 mm long glas capillary tube held by a special device is dipped into the inner ear fluid sample. The capillary tube is then placed on a membrane piece that adheres to the tube. The higher capillary forces of the membrane transfer the fluid out of the glass tube into the membrane which is put into a plastic reaction tube and stored. The reproducibility of the method was tested by flame photometric assays of standard sodium solutions and of perilymph from the scala tympani of guinea pigs. The variation coefficient is below 3% for a standard solution and below 5% for perilymph. The method developed to measure and handle inner ear fluids with the aid of "nanocap" and membrane pieces is easier and faster than the conventional techniques.

Differential uptake of salicylate in serum, cerebrospinal fluid, and perilymph.

After intraperitoneal administration of salicylate in anesthetized rats and guinea pigs, we found that salicylate levels in perilymph (PL) are closely related to both drug levels in cerebrospinal fluid (CSF) and in serum, with higher levels systematically observed in PL than in CSF. Further analysis suggests that salicylate is not passively transported into PL across CSF but, rather, is transported from blood directly to PL. The time course of salicylate uptake in rats reveals maximum levels at 1 1/2 hours (serum) and two to four hours (CSF and PL). On the other hand, salicylate uptake into serum and CSF of guinea pigs exhibits a longer time course, with maximum levels reached at four hours (serum) and five hours (CSF). These data, not previously available, are basic to our understanding of salicylate-related auditory effects.

Production of endolymph in the semicircular canal of the frog Rana esculenta.

The mechanisms of secretion of endolymph were studied in vitro in the isolated inner ear of the frog. Prior to in vitro experiments, the composition of perilymph was evaluated in vivo and compared to that of plasma. Composition of perilymph resembled that of an extracellular fluid, although Na and Cl concentrations were higher and K concentration was lower in perilymph than in plasma water. No difference in Ca and Mg concentrations was observed between these two fluids. Osmolality averaged 227 mosmol/kg H2O in perilymph and 183 mosmol/kg H2O in plasma. Endolymph in frog inner ear corresponded in chemical pattern to mammalian endolymph. K and Na concentrations in endolymph collected from the ampulla of the posterior vertical semicircular canal averaged 121.1 mM and 2.5 mM, respectively. Osmolality of endolymph was 237 mosmol/kg H2O. K and Na concentrations were unaltered when inner ears were incubated for 24 h either at 15 degrees C or at 4 degrees C. Addition of ouabain (10(-4) M) to the perilymph-like bathing solution altered greatly Na and K composition of endolymph after incubation for 3 h at 15 degrees C. The Na and K concentration gradients between endolymph and the bath were abolished after incubation for 24 h. Ligatures of the posterior vertical semicircular canal were performed at different sites to isolate some parts of the canal, i.e. the ampulla and the non-ampullar duct. K concentration in the ampulla after incubation for 24 h remained as high as 20 times that in the bath. This K gradient was abolished in the presence of ouabain (10(-4) M). High K concentration could be maintained in the non-ampullar part of the semicircular canal only if the latter communicated with the ampulla. It is concluded that endolymph is actively secreted into the ampulla of the semicircular canal. Na+-K+-activated ATPase in the ampullar dark cells may energize the ouabain sensitive ionic transports that are involved in the production of endolymph. Endolymph secreted into the ampulla would spread intraluminally to account for the high K and low Na concentrations of the fluid which fills the non-secretory part of the semicircular canal.

Biochemical aspects of inner ear fluids and possible implications for pharmacological treatment.

Inner ear fluids are in dynamic equilibrium with surrounding fluids, namely blood and cerebrospinal fluid. It is known that substances injected into the blood stream or cerebrospinal fluid are transported into the inner ear fluids. The rate of transport from blood into perilymph is inversely related to the molecular weight or molecular size. There appears to exist a blood-labyrinth barrier. In general, the morphology of capillaries in the spiral ligament and stria vascularis is similar to that of brain capillaries which contribute to formation of the blood-brain barrier. Information on the volume of the inner ear fluids is necessary for estimating the toxicity of the drugs as well as the effective concentration of systemically injected substances. Recently, alteration of the levels of arachidonic acid metabolites, especially the prostaglandins, in perilymph under experimental conditions has been reported. The responses of prostaglandin levels in perilymph to these experimental conditions (aspirin injection, antidiuretic hormone or epinephrine infusion) suggest that prostaglandins may play an important role under physiological conditions. Possible mechanisms of auditory dysfunction due to abnormal prostaglandin metabolism in the auditory system are discussed.

Non-collagen proteins of stapedial bone matrix in perilymph of otosclerotic patients.

Non-collagen proteins extracted from the otosclerotic stapes footplate, superstructure and temporal cortical bone were compared with the protein patterns of normal and otosclerotic perilymph by analytical isotachophoresis (ITP). Normal and otosclerotic perilymph yielded basically identical isotachophoretic subfractions. Thirteen subfractions were detected moving as anions at pH 9.6 in the normal perilymph, versus 16 subfractions of similar character in the otosclerotic perilymph. Of these 16 protein subfractions, one (which was not detected in the normal perilymph) could be found in the ITP-gram of the stapes footplate non-collagen proteins, but not of the stapes superstructure or temporal cortical bone. Our results support the concept that protein can enter the inner ear fluid spaces from the otosclerotic bone. The biological significance of these proteins has not yet been clarified.

The developing electrolytes concentrations of inner ear fluids in guinea pigs.

Cochlear endolymph, scala tympani perilymph, and scala vestibuli perilymph were collected from fetal, neonatal, and mature guinea pigs. The concentrations of Na and K in these fluids were then assessed using a microflame photometer. The electrolyte composition in the endolymph was already characterized by a high concentration of K and a low concentration of Na by the 49th day of gestation.