The Stromal Ultrastructure of Normal and Pathologic Human Corneas

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

An X-ray diffraction investigation of corneal structure in lumican-deficient mice

PURPOSE. The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate–containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency. METHODS. X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array. RESULTS. The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters. CONCLUSIONS. The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency

Neonatal development of the corneal stroma in wild-type and lumican-null mice

PURPOSE: Between days 8 and 14 of neonatal development, the corneal stroma of the mouse undergoes critical changes in tissue thickness, cell density, and light scattering. The authors investigate the stromal matrix structure in wild-type and lumican-deficient corneas in this developmental phase. METHODS: Wild-type (n = 44) and lumican-deficient (n = 42) mouse corneas at neonatal days 8, 10, 12, and 14 were investigated by synchrotron x-ray diffraction to establish the average collagen fibril spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal matrix. RESULTS: Collagen interfibrillar spacing in the normal mouse cornea became more closely packed between days 8 and 14, though not significantly so. In lumican-null mice, interfibrillar spacing was significantly elevated at days 8, 10, and 12, but not day 14, compared with that in wild-type mice. At all stages investigated, collagen fibrils were, on average, marginally thinner than normal in lumican-null mutants, and the spatial distribution of the fibrils was less well organized. CONCLUSIONS: Transient thickening of the corneal stroma of the normal mouse at eye opening is probably not caused by widespread, homogeneous rearrangement of collagen fibrils but more likely by a temporary increase in cell or stromal "lake" volume. Lumican, structurally influential in adult mouse corneas, is also a key molecule in the neonatal development of the stromal matrix

Developmental changes in patterns of distribution of fibronectin and tenascin-C in the chicken cornea: evidence for distinct and independent functions during corneal development and morphogenesis

The cornea forms the tough and transparent anterior part of the eye and by accurate shaping forms the major refractive element for vision. Its largest component is the stroma, a dense collagenous connective tissue positioned between the epithelium and the endothelium. In chicken embryos, the stroma initially develops as the primary stroma secreted by the epithelium, which is then invaded by migratory neural crest cells. These cells secrete an organised multi-lamellar collagenous extracellular matrix (ECM), becoming keratocytes. Within individual lamellae, collagen fibrils are parallel and orientated approximately orthogonally in adjacent lamellae. In addition to collagens and associated small proteoglycans, the ECM contains the multifunctional adhesive glycoproteins fibronectin and tenascin-C. We show in embryonic chicken corneas that fibronectin is present but is essentially unstructured in the primary stroma before cell migration and develops as strands linking migrating cells as they enter, maintaining their relative positions as they populate the stroma. Fibronectin also becomes prominent in the epithelial basement membrane, from which fibronectin strings penetrate into the stromal lamellar ECM at right angles. These are present throughout embryonic development but are absent in adults. Stromal cells associate with the strings. Since the epithelial basement membrane is the anterior stromal boundary, strings may be used by stromal cells to determine their relative anterior–posterior positions. Tenascin-C is organised differently, initially as an amorphous layer above the endothelium and subsequently extending anteriorly and organising into a 3D mesh when the stromal cells arrive, enclosing them. It continues to shift anteriorly in development, disappearing posteriorly, and finally becoming prominent in Bowman’s layer beneath the epithelium. The similarity of tenascin-C and collagen organisation suggests that it may link cells to collagen, allowing cells to control and organise the developing ECM architecture. Fibronectin and tenascin-C have complementary roles in cell migration, with the former being adhesive and the latter being antiadhesive and able to displace cells from their adhesion to fibronectin. Thus, in addition to the potential for associations between cells and the ECM, the two could be involved in controlling migration and adhesion and subsequent keratocyte differentiation. Despite the similarities in structure and binding capabilities of the two glycoproteins and the fact that they occupy similar regions of the developing stroma, there is little colocalisation, demonstrating their distinctive roles

Recapitulation of normal collagen architecture in embryonic wounded corneas

Wound healing is characterized by cell and extracellular matrix changes mediating cell migration, fibrosis, remodeling and regeneration. We previously demonstrated that chick fetal wound healing shows a regenerative phenotype regarding the cellular and molecular organization of the cornea. However, the chick corneal stromal structure is remarkably complex in the collagen fiber/lamellar organization, involving branching and anastomosing of collagen bundles. It is unknown whether the chick fetal wound healing is capable of recapitulating this developmentally regulated organization pattern. The purpose of this study was to examine the three-dimensional collagen architecture of wounded embryonic corneas, whilst identifying temporal and spatial changes in collagen organization during wound healing. Linear corneal wounds that traversed the epithelial layer, Bowman´s layer, and anterior stroma were generated in chick corneas on embryonic day 7. Irregular thin collagen fibers are present in the wounded cornea during the early phases of wound healing. As wound healing progresses, the collagen organization dramatically changes, acquiring an orthogonal arrangement. Fourier transform analysis affirmed this observation and revealed that adjacent collagen lamellae display an angular displacement progressing from the epithelium layer towards the endothelium. These data indicate that the collagen organization of the wounded embryonic cornea recapitulate the native macrostructure

A study of corneal thickness, shape and collagen organisation in keratoconus using videokeratography and X-ray scattering techniques

In keratoconus, the cornea becomes progressively ectactic resulting in severe visual impairment. Here, we use a combination of videokeratography and synchrotron X-ray diffraction to investigate the relationship between corneal shape and thickness, and the distribution and predominant orientation of stromal fibrillar collagen in five keratoconus corneas. In all but the least advanced case, the thinning and ectasia measured in vivo using corneal videokeratography was accompanied by corresponding changes in the relative distribution and orientation of stromal collagen in the excised corneal buttons. Although the most severe case of keratoconus possessed the most pronounced stromal collagen alterations, and only a minor disruption to stromal collagen arrangement was seen in the least advanced case, a variability in the extent of stromal collagen alteration was seen between these clinical extremes. The observed abnormalities in collagen distribution and orientation are consistent with a mechanism of keratoconus progression that involves inter-fibrillar or inter-lamellar slippage causing a redistribution of tissue within the cornea

An x-ray diffraction study of corneal structure in mimecan-deficient mice

PURPOSE: Keratan sulfate proteoglycans (KSPGs) in the corneal stroma are believed to influence collagen fibrillar arrangement. This study was performed to investigate the fibrillar architecture of the corneal stroma in mice homozygous for a null mutation in the corneal KSPG, mimecan. METHODS: Wild-type (n = 9) and mimecan-deficient (n = 10) mouse corneas were investigated by low-angle synchrotron x-ray diffraction to establish the average collagen fibrillar spacing, average collagen fibril diameter, and level of fibrillar organization in the stromal array. RESULTS: The mean collagen fibril diameter in the corneas of mimecan-null mice, as an average throughout the whole thickness of the tissue, was not appreciably different from normal (35.6 +/- 1.1 nm vs. 35.9 +/- 1.0 nm). Average center-to-center collagen fibrillar spacing in the mutant corneas measured 52.6 +/- 2.6 nm, similar to the 53.3 +/- 4.0 nm found in wild-type mice. The degree of local order in the collagen fibrillar array, as indicated by the height-width (H:W) ratio of the background-subtracted interfibrillar x-ray reflection, was also not significantly changed in mimecan-null corneas (23.4 +/- 5.6), when compared with the corneas of wild-types (28.2 +/- 4.8). CONCLUSIONS: On average, throughout the whole depth of the corneal stroma, collagen fibrils in mimecan-null mice, unlike collagen fibrils in lumican-null mice and keratocan-null mice, are of a normal diameter and are normally spaced and arranged. This indicates that, compared with lumican and keratocan, mimecan has a lesser role in the control of stromal architecture in mouse cornea

Cell regulation of collagen fibril macrostructure during corneal morphogenesis

While tissue form and function is highly dependent upon tissue-specific collagen composition and organization, little is known of the mechanisms controlling the bundling of collagen fibrils into fibers and larger structural designs that lead to the formation of bones, tendons and other tissues. Using the cornea as a model system, our previous 3 dimensional mapping of collagen fiber organization has demonstrated that macrostructural organization of collagen fibers involving interweaving, branching and anastomosing plays a critical role in controlling mechanical stiffness, corneal shape and refractive power. In this work, the cellular and mechanical mechanisms regulating critical events in the assembly of collagen macrostructure are analysed in the developing chicken cornea. We elucidated the temporal events leading to adult corneal structure and determined the effects of intraocular pressure (IOP) on the organization of the collagen macrostructure. Our findings indicate that the complex adult collagen organization begins to appear on embryonic day 10 (E10) after deposition of the primary stroma and full invasion of keratocytes. Importantly, organizational changes in keratocytes appearing at E9 preceded and predicted later changes in collagen organization. Corneal collagen organization remained unaffected when the development of IOP was blocked at E4. These findings support a primary role for keratocytes in controlling stromal organization, mechanical stiffness and corneal shape that are not regulated by the IOP. Our findings also suggest that the avian cornea represents an excellent experimental model for elucidating key regulatory steps and mechanisms controlling the collagen fiber organization that is critical to determining tissue form and function

Observations on nascent matrix structures in embryonic cornea: Important in cell interactions, or merely vestiges of the lens surface?

Here we present some new observations on early stages in chick corneal development obtained by remining of datasets obtained via serial block face scanning electron microscopy. We focus on matrix cords, proteoglycan-rich structures of apparent ectodermal origin, emerging from the epithelial basal lamina, which extend proximally into the growing collagenous matrix destined to become the corneal stroma. Cords have no known function. In their earliest manifestation, we describe how they appear to run continuously from epithelium to the lens, in contact with both tissues and may therefore be simply vestigial structures, remaining from the earlier detachment of the lens from its parent ectoderm. However, neural crest cells migrating to form the corneal endothelial monolayer appear to form close associations with cords via elaborate pseudopodial extensions. Presumptive endothelium and keratocytes, in the subsequent wave of neural crest cell influx, may conceivably utilise cords, as well as utilising collagenous fibrils of the interstitial matrix, as substrate cues in cell guidance, attachment and migration. The possibility also exists that cords fulfil a functional role in corneal morphogenesis via mechanotransduction through cell matrix interactions