Effect of EDTA on cytokeratin detection in the inner ear.

Immunohistochemical studies on the epithelium of the adult inner ear are difficult to perform without decalcification of the bony capsule. In this study, we examined the effect of decalcifying agents on the immunoreactivity of various cytokeratin antigens in the cochlear duct epithelium of 2-day-old rats, allowing the comparison of fresh and decalcified specimens. Decalcification of unfixed tissue in a solution containing EDTA or EGTA and polyvinylpyrrolidone, at pH 7.4 and 4 degrees C for a maximum period of 2 days, not only preserved the antigen epitopes but even enhanced the staining intensities in comparison with fresh specimens. This enhancement effect, caused by chelating agents and found to be blocked by prior fixation with acetone, is suggested to be caused by unmasking of the antigenic epitopes.

Effects of endolymphatic sac obliteration on the cochlear duct glycocalyx.

The ultrastructural effects of surgical obliteration of the endolymphatic duct and sac on the cochlear duct glycocalyx were investigated using osmium tetroxide-potassium rutheniumcyanide postfixation. After 2 and 3 months a less prominent glycocalyx contrast staining was observed, together with disappearance of interconnecting material between the stereocilia of the inner and outer hair cells. Possible factors involved in this phenomenon are discussed.

Recent advances in laser microprobe mass analysis (LAMMA) of inner ear tissue.

Maintenance of ionic gradients within the various fluids compartments of the inner ear requires transport active cellular systems at different locations. LAMMA analysis is ideally suited for detection of ions in microquantity on cellular levels overcoming many technical difficulties. The present paper summarizes the results of microprobe analysis obtained with laser induced mass spectrometry (LAMMA) supplemented by X-ray microprobe analysis of epithelial cell layers adjacent to the endolymphatic space in the cochlear duct, in the vestibular organ and in the endolymphatic sac. The possible role of inner ear as well as ocular melanin in the mechanisms of active ion transport is discussed.

Frozen-section autoradiography of the cochlea: evidence for differential distribution of amino acids.

A histological method is described for embedding the whole cochlea in an egg yolk medium that permits frozen sectioning in the mid-modiolar plane. Resulting tissues can be processed using standard autoradiographic techniques and stained with Nissl or silver impregnation methods. Following identical intracochlear injections of tritiated proline or leucine and frozen sectioning of embedded cochleae, differential uptake patterns within the cochlea were revealed for these amino acids. Frozen sectioning of the cochlea promises to provide an efficient means to examine specific labeling patterns within the cochlear duct for compounds suspected of contributing to cochlear function by permitting the application of multiple histological techniques including routine stains and anterograde and retrograde tracers.

Intracellular and extracellular ion content of the endolymphatic sac.

The Cl- activity in the endolymph of the endolymphatic sac and in the cochlear duct was measured with Cl- sensitive double-barreled microelectrodes. The Cl- activity in the endolymphatic sac fluid was lower than in the cochlear duct. A small, positive, DC potential was recorded in the endolymphatic sac. During anoxia, the DC potential decreased while the Cl- activity in the endolymphatic sac increased. The K/Na ratio in the epithelial cells and subepithelial tissue of endolymphatic sac was measured using the LAMMA technique. The K/Na ratio in the epithelial cells decreased after ethacrynic acid injection (60 mg/kg i.v.). These findings suggest that chloride in the endolymphatic sac is actively transported inward and outward.

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse.

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker -1 and Shaker -2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique. Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlear during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker -1 and Shaker -2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.

X-ray microanalysis of fluid spaces in the frozen cochlea.

The cochlea is quick-frozen and then opened while under liquid nitrogen to expose the scalae of all turns in the mid-modiolar section. Still under liquid nitrogen, the cochlea is transferred to the cold stage of a pre-vacuum chamber especially constructed for attachment to the scanning electron microscope. Under partial vacuum, it is moved to the cold stage in the high vacuum chamber of a field-emission scanning electron microscope. Energy dispersive X-ray analysis demonstrates the relative levels of sodium, potassium, and chlorine in the fluid spaces of the cochlea.