On the three-dimensional structure of the proteoglycans in the cornea and how it controls corneal transparency

The external case limiting the eye must be tough to prevent foreign objects and germs from penetrating inside. It has to be light so that the eye is not unnecessarily heavy and tiny muscles can move it with high precision when we want to look around. But above all, at least at the front, it has to be transparent, so that light can reach the retina and we can see around us. But what strategy has nature developed to build a structure which is tough, light and also transparent? As engineers know, it is important to use the right materials. The main molecular component of the external case of the eye, and in particular that of its front window (called the cornea) is collagen. Collagen is a chain-like protein that assembles together to form long molecular ropes called fibrils. Collagen is light and because it assembles into fibrils, it is very tough as well. If we look at the collagen in the cornea using an electron microscope, we can see that the collagen fibrils run side by side to form sheet-like structures. The distance between individual neighbouring fibrils in a sheet is very precise and about 50 nm. The precision of the distance between the collagen fibrils is the key to transparency. To understand why, it is useful to think first of what happens when we throw a stone on the flat surface of a pond. When the stone hits the pond surface, wave circles are produced that move outwards from a central point. If we throw two stones, the waves produced by one stone meet those produced by the other stone and an interference pattern is produced; where the crests of two waves meet, we will have a wave crest that is twice as high. Where the throughs meet we will have a through which is twice as low, and where a crest meets a through, the wave will disappear. If we managed to drop a row of regularly spaced stones, one quite close to another, we would find that the circular waves would disappear in all directions, except the forward direction. This is because the crest of any wave would meet the through of another wave everywhere except in the forward direction. Light is itself a wave, and when it hits a collagen fibril, it produces waves not dissimilar from those produced by a stone on a pond surface. Because the collagen fibres are equally spaced, light cancels out in all directions except the forward direction and the light can cross the cornea to reach the retina. One crucial thing which is not known though, is how the collagen in the cornea is maintained equally spaced. We know that around the collagen fibrils there are other proteins called proteoglycans. Proteoglycans are seen in the electron microscope to reach out for adjacent collagen fibrils, building in this way a network that may help to keep the collagen fibrils equally spaced. In this project, we propose to find out the exact location and 3-dimensional spatial arrangement of the proteoglycans around and between the collagen fibrils; thus, we want to determine the exact mechanisms that keep the collagen fibrils in place. We also propose to measure corneal transparency in various areas of the cornea by shining light of known intensity through it and by measuring the intensity of the light coming out. Finally we want to correlate the measurement of the corneal transparency in different areas with the measurements taken in the microscope about the collagen fibril spacing, fibril diameters and proteoglycan location and 3-dimensional structure. In this way we will have an idea of how the 3-dimensional order is maintained in the cornea by the proteoglycans, what effects proteoglycans have on collagen fibril diameter and interfibrillar distance, and how important these parameters are for corneal transparency. This information will be very useful to doctors studying the illnesses of the eye in which the cornea loses its transparency, and also for bioengineers who want to build artificial corneas for people that have lost their corneal functionality.

The accepted explanation for corneal transparency is based on the high degree of positional order of the collagen fibrils within the cornea. In fact, incoming light scattered by all fibrils interferes destructively everywhere except from the forward direction, and this is possible only if the collagen fibrils are at precise positions with respect to each other. This precise positioning is achieved through proteoglycans that interconnect adjacent collagen fibrils together. The detailed modes of interaction between collagen fibrils and proteoglycans are not known. In addition, biochemical and X-ray data suggest variations in the proteoglycan composition between the centre and the periphery of the cornea. This may influence the collagen fibril arrangement and corneal transparency. In this project, we aim to carry out a series of three-dimensional reconstructions of the proteoglycan-collagen system in the cornea. This will allow us to uncover the structural roles of proteoglycans and their optimal three-dimensional organisation within corneas. Spectrophotometry experiments will also be carried out to determine the degree of transparency variation in different locations in the cornea. The spectrophotometry measurements will be correlated to fibrillar diameter and spacing, proteoglycan location and three-dimensional arrangement data obtained with the three-dimensional reconstructions. This project will establish the structural role of the proteoglycans in determining corneal transparency, it will generate a list of corneal structural parameters and find their optimal values for transparency, and will create a three dimensional description of a biological structure that can be used as a reference for the creation of synthetic transparent biomaterials.