Middle ear gas loss in inflammatory conditions: the role of mucosa thickness and blood flow.

Several middle ear (ME) pathologies are associated with ME gas deficit. These include in particular the chronic otitis media diseases that are associated with inflammation (hence, increased ME mucosal blood flow) and/or reduced Eustachian tube (ET) function. The present study models the trans-mucosal gas exchange in normal and inflamed middle ears of rats. The model evaluates the role of the gas diffusion path in the ME mucosa using mucosa thickness as its index and the role of the mucosal blood flow rate on ME gas economy in order to compare between normal and inflamed MEs. An experimental method employing ME gas volume changes at constant pressure due to trans-mucosal gas exchange, and blood gas values from the literature, was used in anaesthetized rats to corroborate the model. Mucosa thickness was measured as an index of the gas diffusion path between the ME space and the ME circulation. ME inner surface area was estimated from its measured gas volume. Inflammation was inflicted by applying lipopolysaccharide (LPS) into one ear. The contralateral ear served as control. ME gas volume decreased significantly faster with time (p=0.02) in inflamed ears (-0.107 microL min(-1) +/- 0.034 S.D., n=10) versus control ears (-0.067 microL min(-1) +/- 0.036 S.D., n=10). Mucosa thickness was significantly thicker in inflamed ears (48.4 microm +/- 11.0 S.D.) versus controls (20.5 microm +/- 10.1 S.D.). The mathematical model, the experimental results, and the blood gas values were used to estimate the relative effective mucosal blood flow rate. The model predicts that in spite of almost doubling mucosa thickness in LPS treated ears, the increased gas loss in inflamed ears may be explained by increased mucosal blood flow rate. We suggest that the ability to estimate ME blood flow as obtained by applying the model to the measurements, is relevant to medical management of inflamed ME.

The role of PKCzeta in amikacin-induced apoptosis in the cochlea: prevention by aspirin.

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-kappaB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCzeta, a key regulator of NF-kappaB activation, in response to aminoglycoside treatment. Amikacin induced PKCzeta cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-kappaB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCzeta cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCzeta acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.

In vivo demonstration of the absorptive function of the middle ear epithelium.

The present study investigated in vivo fluid and ion transport across the middle ear epithelium. The tympanic membrane of rats was punctured under general anesthesia. A capillary tube was fitted to the external auditory canal and the bulla filled with various solutions. Middle ear (ME) fluid volume variations were then measured at constant pressure. When saline was used, a linear decrease of fluid volume was apparent. Replacement of sodium with a non-permeable cation (N-methyl-D-glucamin) reduced the absorption rate from 0.065+/-0.008 to 0.019+/-0.003 microl/min (P<0.05, n=6). Similarly, amiloride (10(-3)M), a sodium channel antagonist, reduced the absorption rate to 0.027+/-0.006 microl/min (P<0.05, n=6). Net absorption was abolished when chloride was substituted with gluconate: -0.008+/-0.004 microl/min (P<0.02, n=6), which might have been related (i) to the role of chloride as a diffusible anion through the paracellular pathway, or (ii) to the secretion of chloride through apical channels. However in this condition, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, a chloride channel blocker, did not affect the rate of fluid exchange -0.008+/-0.007 microl/min (P=0.75, n=6). This model provides the first in vivo evidence for the absorptive function of the ME. Fluid introduced into the ME cavity disappears due to active transport through the mucosa. This process is sodium-dependent and can be hindered by high concentration of amiloride. The rate of absorption is high enough to allow total clearance of fluid from the cavity of the middle ear within 13 h. This process might play a role in the maintaining a fluid-free and gas-filled middle ear cavity.

Experimental model for investigating trans-mucosal gas exchanges in the middle ear of the rat.

OBJECTIVE: The total pressure in the middle ear depends on the air composition of this gas pocket, i.e. on gas exchanges occurring through either the Eustachian tube (ET) or mucosa. The aim of this study was to develop an experimental model to investigate the exclusive role of trans-mucosal gas exchanges in the middle ear (ME). MATERIAL AND METHODS: Both tympanic membranes of 20 Sprague-Dawley rats were punctured under general anesthesia. Rats were divided into two equal groups. Group 1 had no ET obstruction. In Group 2, the ET was blocked, after velar incision, by cauterization and application of cyanoacrylate glue into the lumen. One open transparent glass tube containing a droplet of colored water was placed horizontally and connected hermetically to each ear canal. The ME was then flushed with room air through the tube. Variations in ME gas volume were measured by reading the displacement of the liquid droplet in the horizontal tube. The kinetics of variations in gas volume between groups were displayed and statistically compared using a two-sided t-test. RESULTS: The pattern of variations in ME gas volume with time was similar in the two groups. Both were characterized by a decrease with three phases and an elimination rate of approximately 0.152 +/- 0.026 microl/min. There was no significant difference in the mean rate of ME volume changes between the two groups. CONCLUSION: This experimental model allows investigation of trans-mucosal gas exchanges. These exchanges exhibit an absorptive function resulting in a negative pressure that must be compensated, under physiological conditions, by air flow through the ET.

Influence of auditory deprivation upon the tonopic organization in the inferior colliculus: a Fos immunocytochemical study in the rat.

The frequency organization in the inferior colliculus of neonatally-deafened rats was investigated using electrical stimulation of the cochlea and immunoreactivity for Fos as a marker of neuronal activity. An electrode implanted either at the base or at the apex of the right cochlea delivered a unique 45-min stimulation at two different level intensities and at two time points, i.e. either at 4 weeks or at 4 months. In 4-week-old rats stimulated at 5x threshold, a site-for-site organization was observed since basal or apical stimulation induced a strong labelling in the ventro-medial or in the dorsolateral part of the left inferior colliculus, respectively. In 4-month-old rats, stimulation of the base induced an extremely weak Fos labelling without any specific location in the left inferior colliculus while stimulation of the apex induced a diffuse labelling with two discrete bands being distinguishable in the left inferior colliculus. In 4-week-old rats stimulated at 15x threshold, basal stimulation elicited a diffuse Fos-like immunoreactivity in the left inferior colliculus while apical stimulation yielded a response restricted to the dorsal part of the left inferior colliculus. In 4-month-old rats, no response was detected in the left inferior colliculus after stimulation of the basal part of the cochlea. Stimulation of the apex could still induce a labelling in the dorsolateral left inferior colliculus. Thus, the inferior colliculus exhibits an adult-like tonotopic organization early on independently of any acoustic stimulation. Prolonged absence of auditory input dramatically alters this organization in the inferior colliculus, especially for high frequencies. From a clinical standpoint, these results could argue for early implantation in deaf children.

Steroidogenic enzyme expression in the rat cochlea.

OBJECTIVE: Steroid hormones, and particularly mineralocorticoids, are candidates for controlling the homeostasis of endolymph as steroid receptors are widely expressed in the cochlea. In contrast, experiments on adrenalectomized animals have shown that an absence of steroids had little effect on the ionic composition of endolymph and hearing ability. We thus hypothesized that local production of steroids in the inner ear may regulate cochlear fluid exchanges. RESULTS: Using reverse transcription-polymerase chain reaction techniques, we showed that transcripts encoding the P450 side-chain cleavage, the 3beta hydroxysteroid dehydrogenase (3beta HSD) and the 17alpha hydroxylase (P450 C17) were expressed in the lateral wall, organ of Corti and modiolus. The mRNA encoding aldosterone synthase was expressed in the modiolus and lateral wall while the P450 11beta1 hydroxylase was not detected at all in any of these tissues. In situ hybridization experiments on cochlear sections confirmed that the 3beta HSD transcripts were expressed in the spiral ligament and modiolus and possibly in the hair cells of the organ of Corti. However, it was not possible to detect P450 C17 transcripts. Immunohistochemistry performed with an antibody raised against the various 3beta HSD isoforms confirmed the localization found using in situ hybridization. CONCLUSIONS: These results suggest that enzymes of the steroid pathway leading to mineralocorticoids and sex steroid hormones, but not glucocorticoids, may be expressed in the rat cochlea. This work is in line with the recent description of local production of steroid hormones in the brain (neurosteroids), heart and skin. In the cochlea, the local production of mineralo and sex steroids could account for a paracrine role and could induce specific gene expression through steroid receptors or act via a non-genomic mechanism on membrane receptors or ionic exchangers. Experiments aimed at demonstrating enzymatic activities within cochlea tissues are in progress.

[Contribution to the study of endolymph homeostasis]. / Contribution à l'étude de l'homéostasie de l'endolymphe.

The secretory structures of the cochlea including stria vascularis and spiral ligament are responsible for the secretion of endolymph, a fluid characterized by a high potassium concentration [150-180 mM], a low sodium concentration [< 1 mM] and a positive potential [80-100 mV]. This intra-cellular-like fluid fills the endolymphatic compartment and is essential in the transduction process which takes place in the organ of Corti. Yet, the mechanisms which control the homeostasis of this fluid remain largely unknown. To approach this issue we investigated the possibility of a steroid synthesis by the rat cochlea which might modulate the secretion of endolymph. Results show that inner ear expresses mARN encoding some of the enzymes of the steroid pathways. We also investigated the presence of the gastric H+, K(+)-ATPase pump. Results from RT-PCR show that the gastric H+, K(+)-ATPase alpha and beta subunits are expressed in the rat inner ear-lateral wall, organ of Corti and spiral ganglion neurons. The presence of the alpha subunit in inner ear was confirmed by immunoblot. Immunohistochemistry localized this protein in the intermediate cells of the stria vascularis, in the spiral ligament and in spiral ganglion neurons. Along with the vacuolar H(+)-ATPase, the gastric H+, K(+)-ATPase could be involved in the maintenance of H+ equilibrium in endolymph. All these arguments suggest that among the various types of so-called "sensorineural" deafness, some entities including some forms of congenital hearing loss and diuretic-induced ototoxic deafness should be classified as endolymphatic deafness. Such identification seems necessary since these entities result from different pathogenetic mechanisms and might benefit from the development of new therapies.