gms | German Medical Science

102. Jahrestagung der DOG

Deutsche Ophthalmologische Gesellschaft e. V.

23. bis 26.09.2004, Berlin

Ultrastructural preservation of epiretinal membranes after cryofixation and mild aldehyde fixation

Meeting Abstract

  • corresponding author R. Schumann - Department of Ophthalmology, Ludwig-Maximilians-Universität of Munich, Munich
  • A. Kampik - Department of Ophthalmology, Ludwig-Maximilians-Universität of Munich, Munich
  • J. Werner - Department of Ophthalmology, Ludwig-Maximilians-Universität of Munich, Munich
  • M. Rohleder - Department of Ophthalmology, Ludwig-Maximilians-Universität of Munich, Munich
  • A. Gandorfer - Department of Ophthalmology, Ludwig-Maximilians-Universität of Munich, Munich

Evidenzbasierte Medizin - Anspruch und Wirklichkeit. 102. Jahrestagung der Deutschen Ophthalmologischen Gesellschaft. Berlin, 23.-26.09.2004. Düsseldorf, Köln: German Medical Science; 2004. Doc04dogP 125

Die elektronische Version dieses Artikels ist vollständig und ist verfügbar unter: http://www.egms.de/de/meetings/dog2004/04dog616.shtml

Veröffentlicht: 22. September 2004

© 2004 Schumann et al.
Dieser Artikel ist ein Open Access-Artikel und steht unter den Creative Commons Lizenzbedingungen (http://creativecommons.org/licenses/by-nc-nd/3.0/deed.de). Er darf vervielf&aauml;ltigt, verbreitet und &oauml;ffentlich zug&aauml;nglich gemacht werden, vorausgesetzt dass Autor und Quelle genannt werden.


Gliederung

Text

Objective

Cryotechniques and mild chemical preparation procedures have been demonstrated to preserve antigenicity in immunocytochemistry. However, the potential to maintain the ultrastructure of epiretinal membranes (ERM) remains uncertain. We compared two fixation techniques to find the most reliable method in preserving the ultrastructure of ERM for immunocytochemical analyses.

Methods

Twenty-four specimens obtained from fifteen patients with macular pucker who underwent vitrectomy were processed by: (1) mild chemical fixation at +4°C with a mixture of 2% paraformaldehyde and 0,05% glutaraldehyde without osmiumtetroxide-postfixation or (2) cryofixation with liquid nitrogen by plunging or impact freezing without any chemical additives but 1-hexadecene and 40% saccharose as cryoprotectants. Filter paper served as supportive substance for cryotechniques. Dehydration and embedding followed using pure acetone and Unicryl (BBI) or Unicryl-like medium.

Results

Ultramicroscopic analysis revealed considerable morphologic destruction of cryofixed ERM. The structure of the inner limiting membrane (ILM) and of collagen fibrils was well preserved but cellular details could not be shown. In contrast, mild chemically fixed ERM demonstrated precise localization of the ILM and collagen fibrils as well as nucleus membranes, nucleoli, chromatin and cytoplasmic microfilaments.

Conclusions

Cryofixation methods are limited by ice crystal growth and by the requirement of supportive tools like filter paper. Regarding to the reliable preservation of ultrastructural features, the mild aldehyde fixation with 2% paraformaldehyde and 0,05% glutaraldehyde is recommended for routine research preparation of epiretinal tissue in immunocytochemistry. Further investigation is needed to elucidate the antigen retrieval of ERM processed by cryofixation techniques and mild chemical procedures with respect to dehydration and embedding methods.