Marie Teixeira

In Vivo Study of the Electrochemical Composition of Luminal Fluid in the Guinea Pig Endolymphatic Sac

The aim of this study was to investigate the ionic composition (sodium, potassium) of the luminal fluid in the endolymphatic sac and to correlate it with the transepithelial potential. Experiments were performed in guinea pigs using either an intradural posterior fossa approach or a translabyrinthine approach. The endolymphatic sac transepithelial potential (ESP) was measured and the luminal fluid was sampled. The sodium, potassium and protein concentrations were determined. The results were: i) the luminal fluid in the endolymphatic sac differs in composition from perilymph, on the one hand, and from both cochlear and vestibular endolymph, on the other hand, indicating that the endolymphatic sac maintains chemical (sodium, potassium) and electrical (ESP) gradients; ii) the calculated osmolarity (Na + K) x 2 was about 230 mosm/l; iii) no correlation was observed between sodium and potassium concentrations; iv) large interindividual variations exist from one animal to another, suggesting physiological variations in the functional status of the endolymphatic sac. In conclusion, the variation in composition of the endolymphatic sac luminal fluid reflected variations in ion transport by the epithelium and thus a possible adaptation of the ion transport to different physiopathological conditions.

Blood-Perilymph Barrier and Ototoxicity: An In Vivo Study in the Rat

Cisplatin and gentamicin are two ototoxicants that are supposed to be transported by the paracellar route, i.e. via cellular junctions, to the perilymphatic compartment. This study was initiated to test the hypothesis that susceptive variation of individuals to ototoxic drugs may be explained by variability in transport properties. The transport of radioactive mannitol through the blood-perilymph barrier was correlated in vivo with the acute effect of cisplatin and gentamicin on auditory function. Transport of radioactive mannitol across the blood-perilymph barrier was monitored by sampling of scala vestibuli perilymph at 60 and 120 min after an intravenous infusion of the tracer to nephrectomized Long Evans rats. Counting of hair cell loss was performed in the animals receiving 16 mg/kg body weight cisplatin. The transport of radioactive mannitol across the blood-perilymph barrier did not correlate with the ototoxic effect of cisplatin, evaluated as changes in the auditory-evoked brainstem response thresholds or loss of outer hair cells. The results provide evidence that the barrier function is not involved in the interindividual variability of the ototoxic effect of cisplatin. Furthermore, it can be postulated that neither cisplatin nor gentamicin induce a disruption of endothelial cell junction stability in the inner ear.Cisplatin and gentamicin are two ototoxicants that are supposed to be transported by the paracellar route, i.e. via cellular junctions, to the perilymphatic compartment. This study was initiated to test the hypothesis that susceptive variation of individuals to ototoxic drugs may be explained by variability in transport properties. The transport of radioactive mannitol through the blood-perilymph barrier was correlated in vivo with the acute effect of cisplatin and gentamicin on auditory function. Transport of radioactive mannitol across the blood-perilymph barrier was monitored by sampling of scala vestibuli perilymph at 60 and 120 min after an intravenous infusion of the tracer to nephrectomized Long Evans rats. Counting of hair cell loss was performed in the animals receiving 16 mg/kg body weight cisplatin. The transport of radioactive mannitol across the blood-perilymph barrier did not correlate with the ototoxic effect of cisplatin, evaluated as changes in the auditory-evoked brainstem response thresholds or loss of outer hair cells. The results provide evidence that the barrier function is not involved in the interindividual variability of the ototoxic effect of cisplatin. Furthermore, it can be postulated that neither cisplatin nor gentamicin induce a disruption of endothelial cell junction stability in the inner ear.

High Variability of Perilymphatic Entry of Neutral Molecules Through the Round Window

Objective—To improve the efficacy of intratympanic therapy using perilymphatic entry through the round window membrane. Material and Methods—The perilymphatic entry characteristics of 2 neutral molecules, mannitol (182.2 Da) and inulin (7,000 Da), were studied. A polyethylene catheter was placed in contact with the guinea pig round window membrane and sealed with cyanoacrylate glue. This catheter was linked to an osmotic minipump that delivered 100 μl portions of 3H-mannitol or 3H-inulin solutions over a 7-day period at a constant rate (0.5 μl/h). Perilymph in the scala vestibuli and scala tympani, cerebrospinal fluid (CSF) and plasma were sampled after 4–7 days of delivery. Results—Despite the constant rate of infusion, perilymphatic radioactivity varied widely from one animal to the other, probably as a function of the position of the microcatheter within the round window niche and/or the permeability of the round window. Even a large molecule such as inulin entered the perilymphatic space. Seven days after the beginning of 3H-mannitol administration, the radioactivity was higher in the perilymph of the scala tympani than in that of the scala vestibuli. The perilymphatic radioactivity on Day 7 was ≈50% lower than that measured on Day 4 (p=NS). Finally, round window membrane delivery did not preclude distant spread of the molecules into the blood and CSF. Conclusions—Using round window membrane delivery, the perilymphatic entry of mannitol and inulin depended on their molecular weight. Intratympanic delivery induced a high inter-individual heterogeneity of the drug concentration within the inner ear, with subsequent variability of the therapeutic effects.

Functional IsK/KvLQT1 potassium channel in a new corticosteroid-sensitive cell line derived from the inner ear

Endolymph, a high K+/low Na+ fluid, participates in mechanoelectrical transduction in inner ear. Molecular mechanisms controlling endolymph ion homeostasis remain elusive, hampered by the lack of appropriate cellular models. We established an inner ear cell line by targeted oncogenesis. The expression of SV40 T antigen was driven by the proximal promoter of the human mineralocorticoid receptor (MR) gene, a receptor expressed in the inner ear. The EC5v cell line, microdissected from the semicircular canal, grew as a monolayer of immortalized epithelial cells forming domes. EC5v cells exhibited on filters of high transepithelial resistance and promoted K+ secretion and Na+ absorption. Functional MR and the 11β-hydroxysteroid dehydrogenase type 2, a key enzyme responsible for MR selectivity were identified. Expression of the epithelial sodium channel and serum glucocorticoid-regulated kinase 1 was shown to be up-regulated by aldosterone, indicating that EC5v represents a novel corticosteroid-sensitive cell line. Ionic measurements and 86Rb transport assays revealed an apical secretion of K+ at least in part through the IsK/KvLQT1 potassium channel under standard culture conditions. However, when cells were exposed to high apically K+/low Na+ fluid, mimicking endolymph exposure, IsK/KvLQT1 actually functioned as a strict apical to basolateral K+ channel inhibited by clofilium. Quantitative reverse transcriptase-PCR further demonstrated that expression of KvLQT1 but not of IsK was down-regulated by high K+ concentration. This first vestibular cellular model thus constitutes a valuable system to further investigate the molecular mechanisms controlling ionic transports in the inner ear and the pathophysiological consequences of their dysfunctions in vertigo and hearing loss.

Effects of acute and chronic hypertension on the labyrinthine barriers in rat.

Hearing loss, vertigo, and tinnitus have been related to arterial hypertension. The aim of the present work was to study the permeability of the blood-perilymph and of the labyrinthine barrier, between endolymph and perilymph, to small molecules during chronic and acute hypertension. Experiments were performed in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Acute hypertension was induced by phenylephrine. Perilymph was sampled from the first turn of the scala vestibuli and the Na, K, urea, and radioactive concentrations ((14)C-urea and (3)H-mannitol) were measured. In another experimental set, the endocochlear potential was recorded from the basal turn of scala media, before and after phenylephrine injection. The composition of the perilymph and the kinetic constants for (14)C-urea and (3)H-mannitol were similar in WKY and SHR, and not modified after acute hypertension. In endolymph, the endocochlear potential in SHR (+80+/-2.7 mV, n=24) was lower (P<0.001) than in WKY (+98+/-1.5 mV, n=29). The endocochlear potential was decreased by 40 mV during acute hypertensive peak in seven out of 19 WKY but not in SHR rats (n=13). In conclusion, chronic or acute hypertension did not severely alter the permeability to small molecules of the blood-perilymph barrier. The relationship between the low endocochlear potential and hypertension in SHR remains to be evaluated. After acute hypertensive peak, the presence of vascular protective mechanisms in the cochlea could account for the stable endocochlear potential recorded in SHR and 60% of normotensive rats.

Evidence for apical K conductance and Na-K-2Cl cotransport in the endolymphatic sac of guinea pig

The transepithelial potential in the endolymphatic sac (ESP) was recorded up to 60 min after apical injection of ouabain, bumetanide, quinine, barium, tetraethylammonium, and 4-aminopyridine. After control injection, ESP decreased by 74% and completely recovered at 30 min. After ouabain, barium, or quinine injection, the ESP time course was similar to that in the control group. After bumetanide, tetraethylammonium, or 4-aminopyridine injection, complete recovery was only observed at 60 min. These results suggest that apical K+ conductance and Na-K-2Cl cotransporter could be involved in the genesis of ESP.

Comparative entry of carboplatin and sucrose in endolymph in the rat cochlea

The permeability of the perilymphatic-endolymphatic interface for carboplatin was determined after lateral cerebral ventricle infusion of radioactive carboplatin (cis-diamine[1,1-cyclobutane-1-14C-dicarboxylate]platinum) to rats. [14C]sucrose, a similar weight molecule was used for comparison of the kinetics in the inner ear fluids. 14C-radioactivity was measured in perilymph and endolymph. The rate of elimination of the tracers from perilymph was equivalent indicating no difference in transport across the blood-perilymph barrier. The transport from perilymph to endolymph was very restricted for both substances. The present study indicates that the ototoxic effect of carboplatin cannot be explained by a specific endolymphatic transport mechanism.

Effect of Glycerol on Electrochemical Composition of Endolymph and Perilymph in the Rat

Glycerol (2 g/kg body weight), or 0.15 M NaCl for control animals, was administered to rats by i.v. injection. The dose was chosen in order to obtain an osmolarity increase in plasma of about 15 mosm/l 1 h after the glycerol administration, an increase which is similar to that observed in the human glycerol dehydration test. Endolymph and perilymph were sampled from the basal turn of the cochlea; cerebrospinal fluid (CSF) was sampled from cisterna magna. Plasma osmolarity, endocochlear potential, Na and K concentrations in endolymph, perilymph and CSF were determined 1 and 2 h after the glycerol injection. Compared with control animals, glycerol induced an increase in Na and K concentration in perilymph and endolymph, respectively, 1 and 2 h after the glycerol injection. No modification of the endocochlear potential was observed. These results are compatible with an increase in inner ear fluids osmolarity induced by glycerol.

The effects of perilymphatic tonicity on endolymph composition and synaptic activity at the frog semicircular canal

The effects of changes in perilymphatic tonicity on the semicircular canal were investigated by combining the measurements of transepithelial potential and endolymphatic ionic composition in the isolated frog posterior canal with the electrophysiological assessment of synaptic activity and sensory spike firing at the posterior canal in the isolated intact labyrinth. In the isolated posterior canal, the endolymph was replaced by an endolymph-like solution of known composition, in the presence of basolateral perilymph-like solutions of normal (230 mosmol/kg), reduced (105 mosmol/kg, low NaCl) or increased osmolality (550 mosmol/kg, Na-Gluconate added). Altered perilymphatic tonicity did not produce significant changes in endolymphatic ionic concentrations during up to 5 min. In the presence of hypotonic perilymph, decreased osmolality, K and Cl concentrations were observed at 10 min. In the presence of hypertonic perilymph, the endolymphatic osmolality began to increase at 5 min and by 10 min Na concentration had also significantly increased. On decreasing the tonicity of the external solution an immediate decline was observed in transepithelial potential, whereas hypertonicity produced the opposite effect. In the intact frog labyrinth, mEPSPs and spike potentials were recorded from single fibers of the posterior nerve in normal Ringers (240 mosmol/kg) as well as in solutions with modified tonicity. Hypotonic solutions consistently decreased and hypertonic solutions consistently increased mEPSP and spike frequencies, independent of the species whose concentration was altered. These effects ensued within 1-2 min after the start of perfusion with the test solutions. In particular, when the tonicity was changed by varying Na concentration the mean mEPSP rate was directly related to osmolality. Size histograms of synaptic potentials were well described by single log-normal distribution functions under all experimental conditions. Hypotonic solutions (105 mosmol/kg) markedly shifted the histograms to the left. Hypertonic solutions (380-550 mosmol/kg, NaCl or Na-Gluconate added) shifted the histograms to the right. Hypertonic solutions obtained by adding sucrose to normal Ringers solution (final osmolality 550 mosmol/kg) increased mEPSP and spike rates, but did not display appreciable effects on mEPSP size. All effects on spike discharge and on mEPSP rate and size were rapidly reversible. In Ca-free, 10 mM EGTA, Ringers solution, the sensory discharge was completely abolished and did not recover on making the solution hypertonic. These results indicate that perilymphatic solutions with altered tonicity produce small and slowly ensuing changes in the transepithelial parameters which may indirectly affect the sensory discharge rate, whereas relevant, early and reversible effects occur at the cytoneural junction. In particular, the modulation of mEPSP amplitude appears to be postsynaptic; the presynaptic effect on mEPSP rate of occurrence is presumably linked to local calcium levels, in agreement with previous results indicating that calcium inflow is required to sustain basal transmitter release in this preparation.

In vitro electrogenic K secretion in the frog semicircular canal: absence of effect of streptomycin.

In vitro, the frog semicircular canal secretes an endolymph-like fluid, i.e. a K-rich, positively polarized fluid. This electrogenic K secretion involved basolateral Na+, K(+)-ATPase and Na-K-Cl co-transporter and a luminal protein possessing sulfhydryl groups blocked by N-ethylmaleimide. Streptomycin, an ototoxic antibiotic, is known to block the non-specific mechano-dependent channels in the sensory cells of the ampulla of the semicircular canal. The aim of the present study was to investigate the possible effect of streptomycin on the K fluxes in the ampulla of the semicircular canal. The posterior frog semicircular canal was isolated and the lumen was filled with perilymph-like solution containing or not containing 0.5 mM streptomycin. The luminal K concentration and the transepithelial potential were measured and the unidirectional K fluxes calculated. The K influxes (into the lumen, pmoles/min/mm2) were 114 +/- 25.9 and 111 +/- 3.2 (mean +/- SE, n = 3) in the absence and presence of streptomycin, respectively. The transepithelial potential was not altered (4.0 +/- 1.08 mV versus 3.4 +/- 1.03 mV, n = 3). When ouabain (10(-3)M) was added to the basolateral solution together with luminal streptomycin, no further alteration occurred as compared with the effect of ouabain alone. These results suggest that in these conditions, the sensory organ does not have a major role in the endolymphatic K secretion in the ampulla of the frog semicircular canal.