Lack of effect of carbonic anhydrase inhibition on direct measurements of endolymph bicarbonate.

In the past, sodium, potassium, and chloride have been measured in endolymph directly, but bicarbonate has been measured only indirectly. We sampled endolymph directly while monitoring endocochlear potentials in normal and methazolamide-treated guinea pigs. Bicarbonate was determined in samples by use of a method that depends on reduction of NADH to NAD linked to malate formation from oxaloacetate. In 11 normal animals, the bicarbonate in endolymph was 20.2 mM +/- 4,4 mM (mean +/- standard deviation); in six of these, plasma bicarbonate was 23.1 mM +/- 3.5 mM. Nine animals treated with methazolamide (carbonic anhydrase inhibitor) had an endolymph bicarbonate of 19.5 mM +/- 3.9 mM; plasma bicarbonate in five of these was 25 mM +/- 3.2 mM. Carbonic anhydrase inhibition did not significantly affect endolymphatic bicarbonate levels.

Lectin detection of carbohydrates in the endolymphatic sac.

The carbohydrate contents of the guinea pig endolymphatic sac were investigated by the use of lectins. The lumen of the endolymphatic sac was filled with stainable precipitate containing N-acetyl glucosamine, mannose, glucose, galactose and fucose. N-Acetyl galactosamine was also detected but in minute amounts. This composition corresponded to other areas in the inner ear, such as the cupula, the otolithic membrane and the tectorial membrane. The function of these carbohydrates may play an important role in preventing the lumen of the endolymphatic sac from collapsing as well as in regulating transepithelial fluid transport.

The low temperature vacuum embedding technique for X-ray microanalysis of the developing inner ear.

The method of low-temperature embedding in vacuo using methacrylate resins, was tested for X-ray microanalysis of the embryonic inner ear of the CBA/CBA mouse. Fetal inner ears were examined on gestational days 16 and 18. The technique was evaluated in comparison with earlier used preparation techniques for X-ray microanalysis. With plastic embedding, an improvement of the morphological resolution was achieved, which allows reliable structural identification on the subcellular level. Furthermore, the possibility of orientating of the specimen prior to sectioning provides a better controlled and less time-consuming sectioning.

Magnesium ion activity in the mammalian endolymph measured with ion-selective microelectrodes.

The free Mg++ concentration in endolymph was measured with Mg++-selective microelectrodes based on the neutral ligand ETH 1117. The property of Mg++ microelectrodes was obtained from calibration solutions, containing various Mg++ concentrations with the background electrolytes resembling endolymph. The range between 10 and 0.1 mM Mg++ concentrations changed the potentials of Mg++ microelectrodes by 14.4 +/- 3.0 mV. The endocochlear potential and the Mg++ concentration in the endolymph were 82.0 +/- 5.0 mV and 0.77 +/- 0.29 mM in the guinea pig, and 84.4 +/- 4.9 mV and 1.12 +/- 0.24 mM in the chinchilla, respectively. These results are discussed in the light of the dependence of Na+, K+-ATPase and its interaction with Ca++.

Adaptation of mechanoelectrical transduction in hair cells of the bullfrog's sacculus.

Adaptation in a vestibular organ, the bullfrog's sacculus, was studied in vivo and in vitro. In the in vivo experiments, the discharge of primary saccular neurons and the extracellular response of saccular hair cells were recorded during steps of linear acceleration. The saccular neurons responded at the onset of the acceleration steps, then adapted fully within 10-50 msec. The extracellular (microphonic) response of the hair cells adapted with a similar time course, indicating that the primary sources of the neural adaptation are peripheral to the afferent synapse--in the hair cell, its mechanical inputs, or both. Evidence for hair cell adaptation was provided by 2 in vitro preparations: after excising the sacculus and removing the accessory structures, we recorded either the extracellular hair cell response to displacement of the otolithic membrane or the intracellular hair cell response to hair bundle displacement. In both cases the response to a step stimulus adapted. The adaptation involved a shift in the displacement-response curve along the displacement axis, so that the cell's operating point was reset toward the static position of its hair bundle. This displacement shift occurred in response to both depolarizing and hyperpolarizing stimuli. Its time course varied among cells, from tens to hundreds of milliseconds, and also varied with the concentration of Ca2+ bathing the apical surfaces of the hair cells. Voltage-clamp experiments suggested that the displacement shift does not depend simply on ion entry through the hair cell's transduction channels and can occur at a fixed membrane potential. The possible role of the displacement-shift process in the function of the frog's sacculus as a very sensitive vibration detector is discussed.

The Ca2+ activity of cochlear endolymph of the guinea pig and the effect of inhibitors.

The Ca2+ concentrations in cochlear perilymph and endolymph of the guinea pig were measured with double-barreled Ca2+-selective microelectrodes and showed 1.76 +/- 0.74 X 10(-3) M and 2.20 +/- 0.19 X 10(-5) M, respectively. The electrochemical potential gradient for Ca2+ between perilymph and endolymph was 23.2 mV and the existence of an active transport mechanism from the former to the latter was suggested. Vanadate given perilymphatically decreased the Ca2+ concentration in endolymph with a slight elevation of the endocochlear potential and was suspected of blocking the active transport. The Ca2+ concentration in endolymph was abruptly increased by anoxia or the intravenous administration of 60 mg/kg furosemide and was slightly increased by the intravenous administration of 30 mg/kg furosemide or 100 mg/kg acetazolamide. The endolymphatic pH measured with pH-microelectrodes under various conditions indicates that the mechanism of increase in the Ca2+ concentration is attributed not to the liberation of Ca2+ from the surrounding tissues caused by a fall in pH but to the increased influx of Ca2+ from perilymph due to the depression of the endocochlear potential.

[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.

Pigmented epithelial cells of the membranous saccular wall of the chinchilla.

The posterior-superior portion of the membranous wall of the chinchilla saccule is lined by pigmented epithelial cells which contain golden brown granules having three components (lipid droplets, granular matrix, and dense globules). Histochemical and electronmicroscopic studies indicate that the dense component is a form of melanin. The pigmented cells show morphological features suggestive of fluid transport function; they are also involved in decalcification and phagocytosis of otoconial crystals. These findings provide evidence that the saccular wall epithelium plays an active role in regulating endolymph composition and in clearance of debris from the endolymphatic space.

Otoconial pathology in a strain of the waltzing guinea pig.

The waltzing guinea pig is born with a clinically apparent vestibular dysfunction. In the present study otoconia in the maculae utriculi were studied with scanning electron microscopy in animals aged from newborn to six months old. Malformed otoconia, dumbbell-shaped and asymmetrically shaped otoconia occurred together with otoconia showing various degrees of morphologic degeneration. It is suggested that the otoconial pathologic condition starts prenatally and that an abnormal composition of endolymph may further contribute to the vestibular dysfunction.

Actin filaments, stereocilia, and hair cells of the bird cochlea. III. The development and differentiation of hair cells and stereocilia.

The cochleae of chick embryos of 8 days of incubation until hatching (21 days) were examined by scanning electron microscopy. Unlike what one would expect from the literature, the total number of hair cells per cochlea (10,405 +/- 529) is already determined and visible in a 10-day embryo and the growth of the cochlea is a result of the growth in size and surface area of the hair cells. We also find that the hair cells differentiate simultaneously throughout the cochlea and have followed the differentiation of individual hair cells throughout development. During development we find that the total number, hexagonal packing, and orientation of the stereocilia in each hair cell is determined early and accurately (9- to 10-day embryos). The stereocilia then begin to elongate in all the cells of the cochlea at approximately 0.5 micron/day. By Day 12 the tallest stereocilia in each cell are 1.5-1.8 micron long, the mature length for cells at the proximal end of the cochlea. At this point all stereocilia cease elongating, but those along the inferior edge gradually increase in width from 0.11 micron to maximally 0.19 micron in 17-day embryos. When the stereocilia on the inferior edge reach their mature width, widening ceases and the elongation of stereocilia in the distal hair cells begins again. When these stereocilia have attained their mature lengths, they stop growing. Thus elongation and widening of stereocilia are separated in time. During this period, 11 to 13 days, the shape of the tufts at the proximal end of the cochlea changes. This occurs because stereocilia in the front of each tuft are absorbed while others at the sides appear de novo. This rearrangement converts a circular bundle of stereocilia to a rectangular bundle.

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.

Ionic environment of cochlear hair cells.

The scala media of the adult cochlea in mammals comprises a morphologically closed compartment sealed with tight junctions of the intermediate to tight types. The unique ionic composition of endolymph is maintained by the stria vascularis through active reabsorption of sodium and active secretion of potassium against ionic gradients. The subtectorial space is only a partially closed compartment which communicates with the endolymph via holes in the tectorial membrane at its outer insertion to the organ of Corti. Hardesty's membrane divides the subtectorial space into two compartments: one facing the surfaces of inner hair cells and one facing the surfaces of outer hair cells. In the study of comparative anatomy, hair cells, e.g. in the lizard, basilar papilla are of two types: those covered with a tectorial membrane and those being free-standing lacking the tectorial membrane. The ionic environment of the hair cell surface seems to be the same, independent of whether covered with a tectorial membrane or not. The tectorial membrane itself is semipermeable to ions in the endolymphatic space. Only the surface structures of the hair cell with the sensory hairs facing the subtectorial space are exposed to the high concentration of potassium, whereas the remaining parts of the hair cell are surrounded by a fluid having a more normal extracellular type of ionic composition (cortilymph/perilymph). During embryonic development the ionic composition of endolymph develops in parallel with the morphologic maturation of the stria vascularis. A completely mature composition of endolymph is reached before any electrophysiological potentials in the cochlea can be elicited. The sensory hair surface of hair cells has reached a mature morphology prior to the maturation of endolymph. In several species the tectorial membrane is morphologically only partially mature when the increase of the potassium concentration of endolymph starts. Drugs primarily affecting the stria vascularis causing a transient change of the ionic composition of endolymph result in a transient dysfunction of inner ear potentials. If the ionic changes persist for longer time, morphological changes can occur in both the stria vascularis and the hair cells of the organ of Corti. Whether such changes are primarily caused by the ototoxic drug itself or by changes in the ionic composition of endolymph has to be explored further.

Changes in endolymph chloride concentration following furosemide injection.

Endocochlear potential (EP) and chloride concentration in endolymph were monitored with microelectrodes in the basal turn of the cochlea of the chinchilla. After intravenous injection of furosemide (25-100 mg/kg), the EP dropped precipitously and rapidly reached its minimum value, however, the chloride activity in endolymph decreased more gradually. Possible mechanisms for this phenomenon include a reduced electrostatic attraction of chloride ions to the scala media due to a decreased EP and a reduction of passive influx of chloride into endolymph, resulting from a reduction of active inward potassium transport by furosemide.

Furosemide ototoxicity: clinical and experimental aspects.

Furosemide is an ototoxic diuretic. Furosemide injection is followed by a rapid, but reversible decrease of the endocochlear potential and eighth nerve action potential with a more gradual decrease of the endolymph potassium concentration. In contrast to the reversible effects of furosemide alone on the cochlea, the combination of kanamycin with furosemide resulted in irreversible changes in cochlear function which were associated with elevated levels of kanamycin in the blood and perilymph of the experimental animals. There was a striking similarity between the blood level measured by high pressure liquid chromatography at the time of recovery of auditory function in experimental animals and the ototoxic blood levels proposed by others in clinical literature. These findings help to provide a pharmacologic basis for the clinical observation of furosemide-induced hearing loss.

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.

Permeability to chloride ions of the cochlear partition in normal guinea pigs.

The endocochlear potential and Cl- activities in the endolymph and perilymph of guinea pigs were simultaneously measured with a pair of double-barreled Cl--sensitive liquid-membrane electrodes. Under normal conditions the mean Cl- activity was 96.6 mEg/l in the endolymph and 93.5 mEq/l in the perilymph. The mean Cl- concentration determined by potentiometric titration was 132.3 mM in the endolymph and 121.8 mM in the perilymph. The permeability to Cl- of the cochlear partition was determined after active transport was abolished by permanent anoxia. The modified permeability coefficient and conductance for Cl- of the cochlear partition were computed from the rate of decrease in the Cl- concentration of the endolymph and its electrochemical potential difference between the endolymph and perilymph. The mean modified Cl- conductance and permeability coefficient of the cochlear partition were (10.7 +/- 4.25) X 10(-3) omega -1 . cm-3 and (22.58 +/- 8.95) X 10(-6) s-1 respectively, when averaged from 10 to 30 min after onset of anoxia. Our results indicate that the cochlear partition is relatively permeable to Cl-, when compared to its permeability to K+. Possible coupling between Cl- and water movement across the cochlear partition is discussed.

[Technics for postmortem removal of inner ear fluid]. / Techniken zur postmortalen Entnahme von Innenohrflüssigkeit.

Two methods for sampling inner ear fluid (a mixture of endolymph and perilymph) from corpses are described and compared. Using the classical method, a part of the petrous bone is chiselled out and, subsequently, a needle, attached to a 1-ml-syringe, is inserted through the oval foramen into the region of the utriculus. The inner ear fluid can then be removed. When the method proposed by Trela (1975) is applied, thin layers of the petrous crest are chiselled out until the common crus of the superior and posterior semi-circular becomes apparent. With a needle, attached to a 1-ml-syringe, the inner ear fluid can then be collected. The experiments show Trela's method to be simpler than the classical method. Moreover, only small amounts of inner ear fluid can be obtained by the latter technique. Trela's method is recommended for further studies on this fluid, which may be of forensic interest.

Principles in cochlear toxicity.

The hair cells of the cochlea (neuroepithelium) represent the primary target in most drug-induced ototoxic adverse effects on hearing (e.g. aminoglycoside antibiotics). To what extent an exogenically-induced morphologic damage to hair cells is reversible is not known. In aging structurally altered hair cells can persist for years likewisely not any longer participating in sensory transduction as the hair cells degenerate, secondary changes occur in the spiral ganglion cells and the neuronal pathways. Following heavy metal poisoning an adverse effect is observed on both central and peripheral innervation of the cochlea and only minor primary changes occur in the receptor cells. The link between function and morphology in the cochlea is very obvious regarding the high and middle frequencies with a distinct tonotopic localisation whereas for low frequencies (below 1 khz) such a specific morphologic correlation is lacking. Ototoxic effects primarily affecting the source for the production of endolymph, i.e. the stria vascularis, become manifest at all frequencies and at a rather early stage. Independent of type of substance penetrating into the inner ear, the substance has a considerably slower elimination rate as compared with all other compartments in the body. The toxicity of the drugs seems to be more related to its tissue binding capacity and saturation of receptor sites than related to the concentration of the drug in endo-or perilymph.

Elemental composition of individual cells and tissues in the cochlea.

Localization of elements at the cellular and sub-cellular levels was performed with the energy dispersive X-ray microanalysis technique, using shock-frozen, freeze-dried and araldite-embedded mouse (CBA/CBA) cochleae sectioned dry. Anatomical identification occurred in the STEM (scanning transmission electron microscopy) mode. In inner hair cell stereocilia the K/Na ratio was 70:1 but only 20:1 in the cytoplasm. In outer hair cell cytoplasm the K/Na ratio was 11:1 while the ratio in stereocilia was similar to that in inner hair cells. Ca was identified in stereocilia and the upper part of the cytoplasm of both outer and inner hair cells. The elemental composition in the subtectorial space is endolymph-like and that in the inner tunnel of the organ of Corti is similar to extracellular fluid. Considerable regional differences in elemental composition occur in the tectorial membrane with regard to P, K and Ca. The highest concentration of Ca occurs in the basal part of the tectorial membrane towards the sensory hairs. The highest concentration of K occurs in the basal and outer parts whereas the middle part of the tectorial membrane contains low levels of both K and Ca. The elemental composition changes in two main directions: 1) from the limbal (growing) region to the tip of the tectorial membrane, and, 2) from upper to lower surfaces. The three cell types of the stria vascularis differ considerably in elemental composition. The highest concentration of K occurs in marginal cells. The basal cells contain more K than do the intermediate cells. A significantly higher concentration of Ca, Cl and Na occurs in marginal cell cytoplasm than in any other cell type in the stria vascularis.