Three-dimensional Supramolecular Organization of the Extracellular Matrix in Human and Rabbit Corneal Stroma, as Revealed by Ultrarapid-freezing and Deep-etching Methods

Abstract

The present work was carried out to clarify the three-dimensional fine structure of extracellular matrix in the cornea, using ultrarapid-freezing and deep-etching methods for electron microscopy. Fresh and glutaraldehyde-fixed samples of human and rabbit posterior corneas were ultrarapidly-frozen onto a copper block cooled by liquid helium or liquid nitrogen, freeze-fractured, deeply etched for 8–10min and rotary replicated with platinum-carbon. Replicas were examined in a transmission electron microscope equipped with a tilting device. Only structures with repeatedly observed, similar architectural profiles free from ice crystal damage, were taken into account. The very recognizable major collagen fibrils revealed 8–10nm subfibrils running helically along the fibril long axis. The other extracellular matrix components consisted of: (1) 8–12nm interfibrillar bridging filaments, frequently ornamented with globular domains, joining neighbouring collagen fibrils like steps of a ladder; (2) 10–20nm filaments with relatively large globular domains, running on the surface of collagen fibrils along their long axes, and projecting finger-like structures into interfibrillar spaces sometimes attaching to adjacent collagen fibrils; (3) 10–15nm beaded filaments with a periodicity of 75–110nm, forming extended networks, especially at the interlamellar interfaces; and (4) 8–14nm straight or sinuous strands consisting of 4–6nm repeating subunits or modules, forming extended sheets by lateral association at the Descemet’s membrane/stroma interface. In the light of the information available from studies on the localization of extracellular matrix components in the cornea, and by reference to the structural models of extracellular matrix molecules and macromolecular assemblies, we have related the deep-etched extracellular matrix structures described above to: (1) proteoglycans; (2) fibril-associated collagens with interrupted triple helices or FACIT collagens; (3) type VI collagen; and (4) fibronectin, respectively.