The Stromal Ultrastructure of Normal and Pathologic Human Corneas

Abstract

This thesis describes results and observations from an ultrastructural study of the stroma of various human corneo-scleral tissues. The major components of the stromal extracellular matrix are collagen fibrils and proteoglycan macromolecules. Their character and distribution in normal human cornea and sclera are first studied. The main thrust of the research then progressed to elucidating the ultrastructure in two pathologic conditions where proteoglycan anomalies were known to occur; macular corneal dystrophy and corneal oedema. Transmission electron microscopical studies, employing the proteoglycan-specific stain Cuprolinic blue, demonstrated that the arrangement of proteoglycans, with respect to the collagen fibrils, in normal human cornea differs from other mammals in that there is more 'b' band association. Meridional X-ray diffraction showed that the axial electron density of human scleral collagen was similar to rat tail tendon collagen. When used in conjunction with Cupromeronic blue-staining, it verified as non-artifactual the electron microscopical observation that proteoglycans associate with collagen near the 'd/e' staining bands in the gap zone. Transmission electron microscopy revealed that macular dystrophy corneal stromas contain numerous collagen-free lacunae. Cuprolinic blue-staining further revealed that some of these lacunae contain congregations of various sized proteoglycan filaments. Enzyme digests identified these filaments as belonging to the chondroitin/ dermatan sulphate population of proteoglycans. It was concluded that aggregation of chondroitin/dermatan sulphate proteoglycans often occurs in the macular dystrophy stroma. X-ray diffraction data supported the electron microscopical observation of normal collagen fibrils in the macular dystrophy cornea. However, meridional X -ray data, from Cuprolinic blue-stained specimens, pointed to an abnormal distribution of proteoglycans along the collagen fibrils. Equatorial X-ray diffraction results indicated heterogeneous close packing of normal diameter collagen fibrils throughout the macular dystrophy stroma, this effect was deemed responsible for the central corneal thinning in vivo; a clinical feature of macular dystrophy. By using fresh tissue in the X-ray experiments, it was shown that that cryostorage of excised corneal buttons had no effect on the fibril dimensions. A collaboration was set up to analyse serum and corneal tissue immunochemically from the macular dystrophy patients, to characterise the type of macular dystrophy under investigation. There were no specific ultrastructural differences between type I and type II macular dystrophy stromas; an overall structural heterogeneity exists which indicates that the classification system is not, as yet, complete. High-angle X-ray patterns from macular dystrophy corneas contained two “extra reflections” not obtained from other human corneas, normal or pathologic. The reflections arise from 4.61Å and 9.62Å periodic structures which are considered to be glycosaminoglycan in origin. Electron microscopy revealed the presence of “wavy” lamellae and various sized collagen free “lakes” in the stroma of the oedematous hum an cornea, with the posterior portion containing by far the largest “lakes”. The existence of stromal “lakes” was further supported by the equatorial. X-ray diffraction data. Cuprolinic blue-stained transmission electron micrographs demonstrated a D-periodic association of proteoglycans with collagen, which suggested a depletion of keratan sulphate in the oedematous stroma; this was backed-up by immunochemical evidence. Large proteoglycan filaments (possibly chondroitin/dermatan sulphate) were observed in some parts of the extracellular matrix. Scheie’s syndrome corneal stromas, which contain no α-L-iduronidase, also contained dense Cuprolinic blue-stained deposits. The possibility exists that aggregation of corneal chondroitin/dermatan sulphate is a common factor of several corneal pathologies