Fixation-induced shrinkage of Reissner's membrane and its potential influence on the assessment of endolymphatic volume
A.S. Brunschwig, A.N. Salt, J.E. DeMott, M.M. Henson and S.L. Gewalt
Abstract
The quantification of endolymph volume by histological techniques or by magnetic resonance (MR) microscopy could be influenced significantly by shrinkage of Reissner's membrane during fixation. Since Reissner's membrane is anchored to bone at its medial and lateral sides, shrinkage of a distended membrane would tend to pull it towards a straight position. The goal of this study was to determine the amount of shrinkage of Reissner's membrane induced by various fixation protocols and to establish a protocol suitable for MR microscopy which minimizes soft tissue shrinkage. Fresh guinea pig cochleae were dissected in an artificial perilymph, and fragments of Reissner's membrane were isolated. Each individual specimen was observed in a petri dish using an inverted microscope and its size documented. A selected fixative was then infused while the specimen was observed and recorded on video tape. Size changes of the specimen were quantified, usually over a 20 minute period. Heidenhain-Susa, a fixative widely used for histological study of cochleae, caused substantial shrinkage of Reissner's membrane during the first few minutes. The mean decrease in specimen length was 15.1 % ± 1.1% (SE; n=8). It can be shown that the distension of Reissner's membrane in mild to moderate hydrops would be nullified by a 15% shrinkage of the tissue. Other fixation protocols produced far less shrinkage. The protocol showing least shrinkage was 3.1% glutaraldehyde in Hanks' buffered salt solution, which produced a mean length decrease of 0.3% ± 1.5% (SE; n=4).For MR microscopy, mercuric chloride (HgCl2), a component of Heidenhain-Susa, greatly enhances the contrast of Reissner's membrane against the background of the cochlear fluid spaces, which appear white in the MR image. We have found that Reissner's membrane looks black in specimens fixed with HgCl2-containing solutions, whereas it appears white in formalin- fixed specimens. However, the inclusion of HgCl2 in fixation media results in greater shrinkage of Reissner's membrane, when used as either a primary or secondary fixative. Protocols for MR microscopy are presently being optimized to maximize contrast while minimizing Reissner's membrane shrinkage. In conclusion, the degree of Reissner's membrane shrinkage shown here to be caused by some fixatives could result in systematic errors in the quantification of endolymph volume.
This study was supported by NIH grants numbered DC01368 (AS), DC00114 (MH) and 1P41RR05959 (SG)
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