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Pharmacokinetics of kanamycin in the bullfrog, Rana catesbeiana.

1. Kanamycin disposition was studied in bullfrogs (Rana catesbeiana) following single doses IP. Both plasma t1/2 and Vd of the drug increased with increasing time after drug indicating redistribution and tight binding of kanamycin to deep tissue compartments. 2. Kanamycin was eliminated unchanged with a t1/2 plasma = 27 hr; perilymph = 89 hr; endolymph = 183 hr; aqueous humor = 54 hr; and CSF = 58 hr. 3. Kanamycin was absorbed by frogs from environmental water. 4. Environmental conditions must be carefully specified and monitored, as well as the physiological state of the animals when studying the effects of drugs on Amphibia.

Animals↗

Mixing model systems: using zebrafish and mouse inner ear mutants and other organ systems to unravel the mystery of otoconial development.

Human vestibular dysfunction is an increasing clinical problem. Degeneration or displacement of otoconia is a significant etiology of age-related balance disorders and Benign Positional Vertigo (BPV). In addition, commonly used antibiotics, such as aminoglycoside antibiotics, can lead to disruption of otoconial structure and function. Despite such clinical significance, relatively little information has been compiled about the development and maintenance of otoconia in humans. Recent studies in model organisms and other mammalian organ systems have revealed some of the proteins and processes required for the normal biomineralization of otoconia and otoliths in the inner ear of vertebrates. Orchestration of extracellular biomineralization requires bringing together ionic and proteinaceous components in time and space. Coordination of these events requires the normal formation of the otocyst and sensory maculae, specific secretion and localization of extracellular matrix proteins, as well as tight regulation of the endolymph ionic environment. Disruption of any of these processes can lead to the formation of abnormally shaped, or ectopic, otoconia, or otoconial agenesis. We propose that normal generation of otoconia requires a complex temporal and spatial control of developmental and biochemical events. In this review, we suggest a new hypothetical model for normal otoconial and otolith formation based on matrix vesicle mineralization in bone which we believe to be supported by information from existing mutants, morphants, and biochemical studies.

Animals↗

Driven equilibrium (drive) MR imaging of the cranial nerves V-VIII: comparison with the T2-weighted 3D TSE sequence.

PURPOSE: The aim of this study is to evaluate the efficacy of the driven equilibrium radio frequency reset pulse (DRIVE) on image quality and nerve detection when used in adjunction with T2-weighted 3D turbo spin-echo (TSE) sequence. MATERIALS AND METHODS: Forty-five patients with cranial nerve symptoms referable to the cerebellopontine angle (CPA) were examined using a T2-weighted 3D TSE pulse sequence with and without DRIVE. MR imaging was performed on a 1.5-T MRI scanner. In addition to the axial resource images, reformatted oblique sagittal, oblique coronal and maximum intensity projection (MIP) images of the inner ear were evaluated. The nerve identification and image quality were graded for the cranial nerves V-VIII as well as inner ear structures. These structures were chosen because fluid-solid interfaces existed due to the CSF around (the cranial nerves V-VIII) or the endolymph within (the inner ear structures). Statistical analysis was performed using the Wilcoxon test. P < 0.05 was considered significant. RESULTS: The addition of the DRIVE pulse shortens the scan time by 25%. T2-weighted 3D TSE sequence with DRIVE performed slightly better than the T2-weighted 3D TSE sequence without DRIVE in identifying the individual nerves. The image quality was also slightly better with DRIVE. CONCLUSION: The addition of the DRIVE pulse to the T2-weighted 3D TSE sequence is preferable when imaging the cranial nerves surrounded by the CSF, or fluid-filled structures because of shorter scan time and better image quality due to reduced flow artifacts.

Abducens Nerve↗

Does BAPTA leave outer hair cell transduction channels closed?

The calcium chelator BAPTA was iontophoresed into the scala media of the second turn of the guinea pig cochlea. This produced a reduction in low frequency cochlear microphonic (CM) measured in scala media and an elevation of the cochlear action potential (CAP) threshold that lasted for the duration of the experiment. Using two pipettes, one filled with KCl and the other KCl and BAPTA (50, 20 and 5 mM) it was possible to observe the effect of passing current through one electrode while measuring the endolymphatic potential (EP) with the other. The results demonstrated that current passed via the BAPTA pipette caused a sustained increase in EP of 8.2, 12.9 and 7.8 mV in the three animals used. This increase coincided with the decrease in low frequency CM that indicated a causal connection between the two. In a second series of experiments, pipettes with larger tips were inserted into scala media in the first cochlear turn and BAPTA was allowed to diffuse from the pipette. The results confirmed the relationship between EP increase and the fall of scala media CM. One interpretation of these results is that lowering the Ca2+ concentration of endolymph with BAPTA inhibits mechano-electrical transduction in outer hair cells (OHCs) and leaves the hair cell transduction channels in a closed state, thus increasing the resistance across OHCs and increasing the EP. These findings are consistent with a model of hair cell transduction in which tension on stereo cilia opens the transduction channels.

Action Potentials↗

Pathogenesis of cytomegalovirus-associated labyrinthitis in a guinea pig model.

Cytomegalovirus infects fetuses through the placenta, resulting in various congenital disorders in newborns, including hearing loss. We developed a monoclonal antibody to guinea pig cytomegalovirus (GPCMV) that was available for immunohistochemistry, and investigated the expression of the GPCMV antigen in animal models of direct and congenital infections. Injection of GPCMV, directly to the inner ear, increased the sound pressure level and resulted in labyrinthitis with severe inflammation. Immunohistochemistry detected GPCMV-infected cells mainly in the scala tympani, scala vestibule and spinal ganglion, but rarely in the cochlear duct. Injection of GPCMV to 5-week pregnant guinea pigs resulted in severe labyrinthitis in fetuses. Immunohistochemistry detected GPCMV-infected cells in the perilymph area and spinal ganglion, but not in the endolymph area, including hair cells. These data suggest that the virus spreads via the perilymph and neural routes in the inner ear of both models of direct and congenital infections.

Animals↗

Experimental measurement of the stiffness of the cupula.

An experimental procedure is described which consists of cutting the canal duct, inserting a micropipette and administering known volumetric displacements to the cupula. The cupula is made visible by dying the endolymph. Known displacements are administered to the cupula, and the time constant of the return to its equilibrium position is measured. With this information, the stiffness of the cupula is calculated. The experiment was successfully carried out on five White King pigeons. The mean stiffness found in somewhat less than other results reported in the literature, and reasons for this discrepancy are noted.

Animals↗

Etiology, pathophysiology of symptoms, and pathogenesis of Meniere's disease.

Endolymphatic hydrops is the pathologic feature associated with Meniere's disease. The development of endolymphatic hydrops appears to arise from multifactorial inheritance with alteration of endolymphatic homeostasis. Various factors associated with the phenomenon of hydrops include functional or anatomic obstruction of endolymphatic flow, malabsorption of endolymph, genetic anomalies, vasodilation, allergy, viral infection, and autoimmunity.

Animals↗

Breakdown of stria vascularis blood-labyrinth barrier in C3H/lpr autoimmune disease mice.

Sensorineural hearing loss related to autoimmune disease is a well-recognized condition, although the exact pathophysiologic mechanisms remain unclear. One current theory postulates immune complex-induced interference with blood-labyrinth barrier integrity in the stria vascularis. The C3H/lpr autoimmune mouse was chosen to study the permeability of capillaries in the stria vascularis because this mouse model has demonstrated abnormalities of the stria vascularis and shifts in the auditory brain stem response threshold during active disease. C3H/lpr mice with active disease were compared with younger mice without disease, as well as age-matched C3H/HeJ control mice. The mice were injected with the tracer ferritin and examined by transmission electron microscopy to evaluate the integrity of the capillary tight junctions in the stria vascularis. Four of five mice with active disease were noted to have extensive leakage of ferritin into the perivascular tissues. Neither the young, disease-free autoimmune mice nor the nonautoimmune control mice demonstrated vessel leakage. Thickening of the basement membrane was also noted in the diseased animals. The results imply that active disease leads to a breakdown in the blood-endolymph barrier, which could underlie the hearing loss accompanying autoimmune and other immune diseases.

Age Factors↗

Maturation of junctional complexes during embryonic and early postnatal development of inner ear secretory epithelia.

The development and maturation of tight junctions in secretory epithelia of the inner ear, i.e., the stria vascularis in the cochlea and the dark cell epithelium around the vestibular organs, have been analyzed with the freeze-fracture technique. Inner ears were followed from the otocyst stage to the mature stage (mouse; gestational age 20 to 21 days). Before epithelial cells differentiate into specific tissues, tight junctions have a loose network of sealing elements with zero to four sealing strands. During and after cytodifferentiation, the numbers of strands increase both in the marginal cells of the stria vascularis and in the dark cells around the vestibular organs. Large intra- and intercellular variations in the numbers of sealing strands of the tight junction occur prior to birth in both types of secretory epithelia. In the dark cells the mature structure of the tight junction with five to eight sealing elements is reached at birth. Maturation of marginal cell junctions occurs during the first few days postnatally. Thus, the tight junctions are morphologically mature before the development of the high potassium concentration in the endolymph.

Animals↗

Ototoxicity of cis-dichlorodiammine platinum (II) in guinea pigs.

Effects of cis-dichlorodiammine platinum (II), an agent that has potent antineoplastic activity, were studied in guinea pigs. With repeated administration of a daily dose of 1.5 mg/kg of cis-dichlorodiammine platinum, the cochlear microphonics were suppressed. Suppression of the cochlear microphonics was greater in the basal turn than in the third turn of the cochlea. There was a close correlation between loss of the cochlear hair cells and suppression of the cochlear microphonics. The endocochlear potential was decreased in the basal turn but remained unchanged in the upper turn of the cochlea. The sodium, potassium, and chloride concentrations in both endolymph and perilymph were not affected. Slight and moderate congestion and regeneration of tubular epithelium of kidney were observed in guinea pigs treated with cis-dichlorodiammine platinum.

Animals↗

Effects of hyperosmotic solutions on endolymphatic pressure.

Hydrostatic pressure changes in endolymph as well as perilymph and cerebrospinal fluid (CSF) following intravenous injection of glycerol, urea, and mannitol were studied in the guinea pig using a servo-controlled micropipet system. After the initial rise in pressure observed during and immediately after injection of each hyperosmotic agent, all pressures fell below baseline levels for about 20 minutes. The largest reductions in pressure with each of the hyperosmotics showed no significant difference between CSF and labyrinth. In the glycerol and urea studies, labyrinthine pressures stabilized at their minimum levels 20 to 40 minutes after injection, while CSF pressure recovered slowly. However, alteration of labyrinthine pressures induced by mannitol paralleled that of CSF. These results may suggest a possible osmotic dehydration effect in the labyrinth for glycerol and urea, but an ambiguous effect for mannitol.

Animals↗

Inwardly rectifying K+ currents in intermediate cells in the cochlea of gerbils: a possible contribution to the endocochlear potential.

The stria vascularis in the cochlea generates the endocochlear potential (EP) and secretes K+-rich endolymph; both are indispensable for normal sound transduction by hair cells. K+ conductance in the intermediate cell, one of the several types of cells constituting the stria vascularis, was investigated by the whole-cell patch-clamp technique. Inwardly-rectifying K+ (Kir) currents were the major currents observed. The currents were inhibited dose-dependently by Ba2+, quinine, verapamil and Cs+, but not by tetraethylammonium (20 mM), 4-aminopyridine (5 mM) or Cd2+ (1 mM). The similarity between the effect of inhibitors on Kir currents and on the EP (Takeuchi et al., Hearing Res., 101 (1996) 181-185) suggests a direct contribution of the Kir conductance to the generation of the EP.

4-Aminopyridine↗

Sodium- and potassium-activated ATPase in the mammalian vestibular system.

Autoradiographic and cytochemical procedures were employed to determine the cellular distribution of the Na,K-ATPase enzyme in the mammalian vestibular system. A light-microscope survey of vestibular tissues incubated with [(3)H]ouabain shows high densities of ouabain binding sites within the dark cell epithelium (DC) of the ampullae of the semi-circular canals, and to a lesser extent, the DC of the utricular macula. A moderate number of binding sites was found in nerve fibers penetrating the connective tissue beneath the sensory epithelium (SE) of the ampullae and the maculae. A small number of binding sites is distributed in the deep portion of the SE, both in the ampullae and in the maculae. These latter binding sites seem to be associated with nerve terminals and receptor cells. At the ultrastructural level, the vestibular dark cells exhibit extensive basolateral membrane infolding, a morphological hallmark of cells engaged in trans-epithelial ion transport. The cytochemical reaction product is K(+)-dependent, ouabain inhibitable, and is restricted to the basolateral membrane extensions, with little or no product on the luminal membrane. The extent of membrane infolding in dark cells of the utricle is less pronounced than that of the ampullar dark cells and the intensity of the cytochemical reaction appears to correlate with the extent of membrane infolding. The results support the widely held hypothesis that the vestibular dark cells play a role in endolymph production. They also suggest that the vestibular sensory epithelia may be a site of ion exchange.

Animals↗

Changes in cochlear function after double-membrane rupture in the guinea pig.

We measured the transiently evoked otoacoustic emissions (TEOAEs), compound action potentials (CAPs) and cochlear microphonics (CMs) in guinea pigs after rupture of the round window membrane alone (n = 5) or of the round window membrane with localized cochlear damage (n = 10). The localized cochlear damage entailed rupture of Reissner's membrane with damage to the stria vascularis. We determined the time course of changes in the total echo power (TEP) in TEOAEs and the minimal detectable levels of CAPs and CMs. The endocochlear potential (EP) was measured in the cochlea with localized damage. There were no changes in TEOAEs, CAPs or CMs in the guinea pigs subjected to round window membrane rupture alone, but the minimal detectable levels of CAPs and CMs were increased in all the guinea pigs in which TEOAEs were absent after rupture of the round window membrane with localized cochlear damage. Our results suggest that double-membrane rupture (rupture of the round window membrane with localized cochlear damage) produces acute sensorineural hearing loss. The hearing loss appeared to be related to damage to the cochlea, which may be induced by influx of potassium-rich endolymph into the perilymph, and by morphological damage to the scala media.

Acoustic Stimulation↗

Ultrastructural localization of the Na-K-Cl cotransporter in the lateral wall of the rabbit cochlear duct.

Localization of the immunoreactivity in the lateral wall of the rabbit cochlear duct was examined using a post-embedding immunogold method with a polyclonal antiserum raised against the rabbit parotid Na-K-Cl cotransporter. In the stria vascularis, the labeling was significant on the basolateral membrane infolding of marginal cells, whereas no labeling was seen on the luminal membrane of these cells. Immunoreactivity was also detected on the cell membranes of various other cells. These include fibrocytes of the spiral ligament and the spiral prominence, and vascular endothelial cells in the stria vascularis and the spiral ligament. In contrast, virtually no gold particles were seen on the membrane of intermediate cells, basal cells of the stria vascularis, the epithelial cells of the spiral prominence, or Reissner's membrane. Our result on the localization of the Na-K-Cl cotransporter in marginal cells is consistent with electrophysiological studies (Wangemann et al. (1995) Hear. Res. 84, 19-29). Our result on fibrocytes is discussed in relation to K+ circulation into endolymph from perilymph (Schulte and Steel (1994) Hear. Res. 78, 65-76).

Animals↗

Paracellular transport properties of inner ear barriers do not account for cisplatin toxicity in the rat.

The interindividual variability for the ototoxic effect of the antineoplastic drug cisplatin has still to be explained. To examine if the variability can be related to differences in drug kinetics, the effect of cisplatin on the paracellular transport properties of the inner ear barriers was studied in vivo in cisplatin treated Long-Evans rats. The concentration of [3H]mannitol was followed in plasma, scala vestibuli perilymph, and endolymph after an intravenous infusion of the tracer. Cisplatin had no effect on paracellular transport of the inner ear barriers 3 days after administration of 8 mg/kg cisplatin. However, an interindividual variability for the transport of [3H]mannitol across the blood-perilymph barrier was evident, indicating a variability for the passive transport of solutes to the inner ear.

Animals↗

Two types of K+ currents in marginal cells cultured from rat stria vascularis.

Membrane currents in marginal cells cultured from rat stria vascularis were studied using the whole-cell patch-clamp technique. Two types of voltage-dependent whole-cell currents were observed in the voltage range from -150 mV to +50 mV: an outwardly rectifying current and an inwardly rectifying current. The outwardly rectifying current, which was activated by depolarizing pulses more positive than -30 mV, was sensitive to TEA (20 mM) and relatively not to Ba2+ (0.5 mM). Tail current analysis revealed that the outward currents were primarily K+-selective. The conductance of the current was half-maximal at 0.5 mV and a substantial portion of current was not inactivated by the depolarizing prepulses from -30 mV to +20 mV. The inwardly rectifying current with rapid exponential activation was observed with hyperpolarizing voltage pulses. The zero-current potential of this current was dependent on extracellular K+ concentration. In contrast to the outwardly rectifying current, this current was blocked by extracellular application of Ba2+, not by TEA. The conductance of this current increased with the increase in external K+ concentration. Our data suggest that marginal cells cultured from rat stria vascularis express at least two types of voltage-dependent K+ currents which may serve as K+ secretory pathways into endolymph.

Animals↗

Differentiation of cyst-forming stria vascularis tissues in vitro.

The marginal cells of the stria vascularis possess distinctive morphological characteristics associated with their role in endolymph production. Interestingly, when stria-derived epithelial cells are grown in association with the underlying mesenchyme, the final differentiation of these cell types does not occur. Beyond the rudimentary polarity that is established, similar to that shown in epithelial monolayers, cells in culture bear only a slight resemblance to their marginal cell counterparts in vivo. The ultrastructural features that typify these epithelia, extensive cytoplasmic invaginations, with an abundance of mitochondria, and darkly stained cytoplasm, are not evident under standard culture conditions. In order to determine whether fluid transport, a key function of the stria vascularis, has an effect on the ultrastructural morphology, we examined de novo stria vascularis tissues that formed a fluid-filled cyst in vitro. We found that only cells associated with the luminal structure demonstrated dark cytoplasmic staining and amplification of the basolateral membrane of the marginal cells. Additionally, other epithelial features, such as mitochondria-rich and microvilli-rich cells, were observed in cyst-forming tissues. The enhancement of the marginal cell specializations was not as robust as that observed in vivo; however, they were clearly more extensive when compared to cells in the same culture that were not associated with a fluid-filled lumen. Thus it appears that fluid transport may be necessary to maximize differentiation of stria vascularis tissues in vitro.

Animals↗