Fixation-induced shrinkage of Reissner's membrane and its potential influence on the assessment of endolymph volume

The quantification of endolymph volume by histological techniques or by magnetic resonance (MR) microscopy requires the inner ear to be first treated with chemical fixatives. If the fixative induces soft-tissue shrinkage, it would tend to return a distended Reissner's membrane towards a straight position, since this membrane is anchored to bone at its medial and lateral edges. The goal of this study was to determine the degree of Reissner's membrane shrinkage induced by different fixation protocols to establish methods which minimize tissue shrinkage. Fragments of fresh Reissner's membrane were dissected from isolated cochleae in an artificial perilymph. Specimens were viewed with an inverted microscope during infusion of fixatives, and changes recorded on video tape. Size changes of the specimen were quantified, usually over a 20 min period. Heidenhain-Susa, a fixative which is widely used in histological studies of hydropic cochleae, caused substantial shrinkage of Reissner's membrane, decreasing the length of specimens by an average of 15.1%. Other fixation procedures induced far less shrinkage. The use of 3.1% glutaraldehyde in Hanks' balanced salt solution produced a mean length decrease of only 0.3%. The inclusion in the fixation medium of 4.5% mercuric chloride, corresponding to the concentration which is present in Heidenhain-Susa and which acts to increase the contrast of Reissner's membrane in MR microscopy, contributes significantly to specimen shrinkage. We can conclude that the degree of endolymphatic hydrops may be underestimated in specimens fixed with media containing high levels of mercuric chloride.