A new three-dimensional model of the organization of proteoglycans and collagen fibrils in the human corneal stroma

The purpose of the present study was to re-evaluate the three-dimensional organization of colla-en fibrils and proteoglycans (PGs) in the human corneal stroma using an improved ultrastructural approach. After a short aldehyde prefixation, one half of seven fresh corneal buttons was stained for PGs with Quinolinic Phtalocyanin (QP) or Cupromeronic Blue (CB). Strips of 1 mm width were cut, subsequently treated with aqueous phosphotungstic acid (PTA) and further processed for light and electron microscopy. The other half of the corneas served as control and was routinely processed with OsO4. Embedding was as such that ultrathin sections could be cut precisely parallel (frontal sections) or perpendicular (cross sections) to the corneal surface. The mutual connections between collagen fibrils and PGs were studied and the length of PGs and their mutual distance were measured manually at a calibrated final magnification of 70 000 X. Prefixed fresh corneal tissue treated with QP and CB shows no signs of swelling and exhibits well contrasted PGs. In cross sections PGs form a repeating network of ring-like structures (similar to45 nm) around the collagen fibrils. In frontal sections PGs are aligned orthogonal to the collagen fibrils, are equidistant (similar to42 nm) attached to the collagen fibrils along their full length and have a thickness of similar to11 nm and a length of similar to54 nm. The observed maximal length of the PGs and the occurrence of ring-like structures enwrapping the collagen fibrils urged us to revisit the prevailing model of maurice (1962) on the organization of the corneal stroma. In the new model hexagonal arranged collagen fibrils are interconnected at regular distances with their next-nearest neighbours by groups Of Six PGs, attached orthogonal to the circumference of the fibrils. In this way a regular meshwork of ring-like structures enwrapping the collagen fibrils is formed. It is discussed that this new model more convincingly explains corneal resistance to compression and stretching and further rationalizes corneal transparency because of the low refractive index difference between the regularly arranged collagen fibrils and their inter-space filled with PGs. (C) 2003 Elsevier Ltd. All rights reserved.