Ultrastructural studies of corneal scar tissue

Abstract

This thesis is concerned with the study of the ultrastructure of corneal scar tissue. The comeal scar tissue studied was produced in rabbits from three different corneal wounds. These were a manual anterior keratectomy, an excimer laser ablation and a full-thickness 2mm button wound. In each case, a number of animals was used which were sacrificed at different time intervals. For the manual keratectomy and laser ablation, electron microscopy, incorporating cuprolinic blue staining, was the main technique used to study the scar tissue arising from these wounds. The full-thickness wounds were examined using synchrotron X-ray diffraction at Daresbury, UK. From low angle and high angle X-ray diffraction patterns, average values were calculated for centre-to-centre collagen fibril separation, the spread of fibril separation, the fibril diameter and the intermolecular spacing within the collagen fibrils. The electron microscope studies showed that the same pattern of healing was observed in both types of wounds. An inflated population of keratocytes was observed under the epithelium at one week of healing. Around these keratocytes were vacuoles in the tissue, which were associated with large numbers of abnormally sized proteoglycan filaments. Up to 2 weeks of healing the population of abnormal filaments increased, as did the vacuoles in the tissue, after which they decreased. However, after nine months of healing the scar tissue is still clearly structurally distinguishable from original stromal tissue. A measure of the collagen fibril density (number per unit area), demonstrated a significant difference between the scar tissue and control. However, it also showed that the number density of fibrils increased with time in the scar tissue. A measure of the range of the collagen fibril diameters in scar tissue up to nine months, indicated that there is a small increase in the range of fibril diameters but the average diameter remained unchanged. Radial distribution functions, from normal corneal stroma and from nine month scar tissue, showed that the short range ordering of the collagen fibrils in the scar tissue was reduced. The results from the X-ray diffraction showed that, the average spacing between collagen fibrils remains slightly above normal. There is a small increase in the average fibril diameter and the intermolecular spacing is slightly lower than normal. The greatest change was in the range of interfibrillar spacing. The ratio of height to width of the distribution gives a quantitative estimate of this spread. After 3 weeks, this ratio was 0.3, increasing to 1.9 after 21 months; this is still significantly lower than the normal controls (6.2). X-ray diffraction of the Morquio syndrome cornea showed that the interfibrillar spacing was reduced 10% compared to normal and that the intermolecular spacing was normal. In the TEM, two populations of fibril diameters were observed, one at 24-30nm (the normal range for human corneal collagen fibrils), and the other at 32-44nm. Cuprolinic blue staining revealed abnormally large proteoglycan filaments scattered throughout the stroma many of which were associated with lacunae in the collagen matrix.