Neurofilament localization and phosphorylation in the developing inner ear of the rat.

Detailed understanding of neurofilament protein distribution in the inner ear can shed light on regulatory mechanisms involved in neuronal development of this tissue. We assessed the spatio-temporal changes in the distribution of neurofilaments in the developing rat inner ear between embryonic day 12 and 30 days after birth, using antibodies against phosphorylated as well as non-phosphorylated light (NFL), medium (NFM) and heavy (NFH) neurofilament subunits. Our results show that during development, the onset of neurofilament expression in the rat inner ear is on embryonic day 12, earlier than previously shown. We demonstrate that neurofilament subunits of different molecular weight emerge in a developmental stage-dependent order. In addition, we determined that neurofilaments of the vestibular nerve mature earlier than neurofilaments of the cochlear nerve. Cochlear neurofilament maturation progresses in a gradient from base to apex, and from inner to outer hair cells. The sequential pattern of neurofilament expression we describe may help understand the consequences of certain mutations, and contribute to develop therapeutic strategies.

Advances in understanding the pathogenesis of pneumococcal otitis media.

In this review, a state of the art on otitis media research is provided with emphasis on the role of Streptococcus pneumoniae in the pathogenesis of this disease. Articles have been selected by MEDLINE search supplemented with a manual crosscheck of bibliographies. Pathogenic mechanisms in middle ear and eustachian tube are described. Furthermore, pneumococcal characteristics and pneumococcus-host interactions are highlighted as well as the possible role of biofilms in persistence or recurrence of otitis media. Because of the availability of new techniques, an increasing number of pneumococcal features contributing in the pathogenesis of otitis media are identified and in-depth knowledge of pneumococcus-host interactions has been gained. The present advances in research on otitis media open up new perspectives for therapeutic or preventive strategies.

Involvement of glycosaminoglycans in the attachment of pneumococci to nasopharyngeal epithelial cells.

Streptococcus pneumoniae is a major bacterial pathogen involved in the development of otitis media. The pathogenic mechanisms of this middle ear disease, including the bacterial adherence mechanisms to the mucosal epithelial cells of the host, are poorly understood. In this study, the role of glycosaminoglycans in the adhesion of pneumococci to mucosal epithelial cells is examined. Both nasopharyngeal epithelium from rats and an oral epithelial cell line were used for pneumococcal adherence experiments. Preincubation of pneumococci with heparin, heparan sulfate (HS) and to a lesser extent, chondroitin 4-sulfate (C-4S), was found to inhibit attachment of S. pneumoniae to oral epithelial cells, while dermatan sulfate and hyaluronate did not interfere with pneumococcal binding. Enzymatic removal of HS moieties by heparinase III from nasopharyngeal epithelial cells abolished the attachment of pneumococci to nasopharyngeal epithelium. This study demonstrates that heparin, HS and C-4S are involved in pneumococcal binding to mucosal epithelial cells. This knowledge may contribute to the development of a new prophylactic strategy for otitis media.

Bacterial otitis media: a new non-invasive rat model.

This study describes the development of a physiological rat model for otitis media. The model is based on the assumption that bacteria, intranasally introduced into the nasopharynx, will be transferred into the middle ear cavity during swallowing provided that the ambient air pressure is higher than the middle ear pressure. This model demonstrates that small pressure changes, generated in a pressure cabin under controlled conditions, can be used as driving force for the transfer of bacteria into the middle ear cavity resulting in bilateral otitis media. Because invasive techniques or biochemical agents are not applied, this model is suited to investigate immunological aspects of otitis media, including the effects of vaccination.

Changes in ultrastructural characteristics of endolymphatic sac ribosome-rich cells of the rat during development.

It has recently been demonstrated that endolymphatic sac (ES) ribosome-rich (dark) cells respond to induced endolymph changes and are thus likely to be involved in endolymph homeostasis. Therefore, we studied the ultrastructural characteristics of rat ES ribosome-rich cells during development in order to determine the cellular distribution of organelles involved in protein metabolism, secretion and absorption, indicative for their contribution to endolymph homeostasis. During embryonal stages ribosome-rich cells contain a limited number and variety of organelles and are predominantly involved in the production of components for cell growth and differentiation. In the young adult stage (P60) three different states of ribosome-rich cells may be distinguished. State A resembles a cell with only limited metabolic activities whereas state B is characterized by numerous different intracellular organelles and is considered to be involved in production and secretion as well as absorption and degradation of complex proteins. A third cellular state, state C, is filled with phagolysosomes and contains very few other organelles. This is considered to be a final (pre)apoptotic state. Autoradiography data suggest that ES ribosome-rich cells are capable of synthesis and secretion of tyrosine-containing proteins and may thus be involved in regulation of the osmolarity of endolymph based on the capacity to bind cations as well as water molecules. In addition, ES ribosome-rich cells appear to synthesize and secrete fucosylated glycoproteins into the endolymph. In conclusion, the present data suggest that ES ribosome-rich cells are actively involved in endolymph homeostasis through secretion and absorption of complex proteins and it is hypothesized that they are able to adapt their function or activities in response to changes in endolymph composition.

Antigenic as well as nonantigenic stimuli induce similar middle ear responses in the rat.

OBJECTIVES/HYPOTHESIS: The observation that during otitis media many different types of micro-organisms have been cultured from effusions indicate that, once present in the middle ear cavity, most types of micro-organisms are able to trigger an inflammatory reaction leading to otitis media. The present study was designed to determine the middle ear response after injection of different substances into the middle ear cavity. STUDY DESIGN: To determine whether and to what extent an inflammatory response of the middle ear depends on the entering agent, the response in the tympanic cavity was studied by otomicroscopy and histological examination after inoculation of various substances. METHODS: Lewis rats were inoculated in transtympanic fashion either with live or heat-killed bacteria (pathogenic and nonpathogenic), Keyhole limpet hemocyanin, active charcoal, or saline. The mucosal response of the challenged middle ears was studied histologically. RESULTS: Irrespective of the inoculated substance, no essential differences in the mucosal response were found. The intensity of the inflammatory response was greater when live bacteria were inoculated. CONCLUSIONS: The present study demonstrates that any substance reaching the middle ear cavity is likely to induce otitis media. These observations emphasize the role of the eustachian tube as "porte d'entrée" in the pathogenesis of this disorder. Determination of specific aspects of the eustachian tube involved in protection or in facilitating bacterial translocation will be important for the understanding of the pathogenesis of otitis media and the subsequent development of new therapeutic strategies. In addition, elucidation of bacterial factors involved in the process of colonization and translocation will be of equal importance.

Effect of exogenous surfactant on ventilatory and clearance function of the rat's eustachian tube.

HYPOTHESIS AND BACKGROUND: The Eustachian tube has three important functions with respect to the middle ear: ventilation, clearance, and protection. Surfactants are assumed to be important to maintain these functions. The administration of exogenous surfactant may therefore be effective to improve the function of the Eustachian tube. This randomized, double-blind, placebo-controlled study was designed to investigate the effect of exogenous surfactant on the function of the Eustachian tube in rats. MATERIALS AND METHODS: Exogenous surfactant was administered into the middle ear of 10 otologically healthy rats, and 10 other rats received placebo. The effect on the opening and closing pressure (passive ventilatory function) and the dye clearance time (clearance function) of the rat's Eustachian tube was measured. RESULTS: A significant decrease in the opening pressure was seen after the administration of surfactant. Both surfactant and placebo caused an increase in the closing pressure. A serious disturbance of the dye clearance time was induced in 13 rats, and the test failed in 1 rat. In the remaining 6 rats, no significant differences in the dye clearance time were observed between the two groups. CONCLUSIONS: Exogenous surfactant decreased the closing forces of the Eustachian tube even in otologically healthy rats. No significant effect on the mucociliary clearance was observed, but this may have resulted from the small number of rats. Additional randomized, double-blind, placebo-controlled trials should be conducted to determine the clinical relevance of these changes and to further assess the effect of surfactant on the function of the Eustachian tube.

Differences in endolymphatic sac mitochondria-rich cells indicate specific functions.

OBJECTIVE/HYPOTHESIS: The purpose of the study was to examine the specific involvement of endolymphatic sac mitochondria-rich cells in endolymph homeostasis. STUDY DESIGN: Transmission electron microscopy and immunohistochemistry were performed on the endolymphatic sac of young adult rats, and two important developmental stages were also investigated. METHODS: Ultrastructural characteristics of endolymphatic sac mitochondria-rich cells were studied more concisely and compared with renal mitochondria-rich cells (i.e., the intercalated cells). In addition, expression of cytokeratins 7 and 19 was determined. RESULTS: Until birth, only one type of mitochondria-rich cell is observed in the rat endolymphatic sac. In young adult animals, distinct differences in mitochondria-rich cell ultrastructure in the endolymphatic sac enables classification into subtypes or configurations. Comparison of endolymphatic sac mitochondria-rich cells with renal intercalated cells reveals striking similarities and provides additional information on their specific function in endolymph homeostasis. Furthermore, differences in cytokeratin expression are determined in endolymphatic sac mitochondria-rich cells. CONCLUSIONS: Differences in morphology of endolymphatic sac mitochondria-rich cells develop after birth and may reflect a distinct functional or physiological state of the cell. In analogy to renal intercalated cells, the distribution patterns of H+-adenosine triphosphatase and Cl-/HCO3- exchanger may differ between subtypes. We propose that subtype A mitochondria-rich cells, from which protruding A mitochondria-rich cells are the activated state, are involved in proton secretion (apical H+-adenosine triphosphatase) and thus are potential candidates for hearing loss accompanying renal tubular acidosis. Subtype B mitochondria-rich cells are the most likely candidates to be affected in Pendred syndrome because of the assumed function of pendrin as apical Cl-/HCO3- exchanger.