Studies of Corneal Structure and Transparency Steven John Gardner B.Phys., M.Sc. Structural Biophysics Group, Department of Optometry and Vision Sciences, Cardiff University. Thesis submitted to Cardiff University for the degree of Doctor of Philosophy March 2015 i ii Acknowledgements I’d most of all like to thank my supervisor, Prof. Keith Meek, for his support, knowledge and patience. I could not imagine having completed this thesis without his supervision. I also acknowledge the invaluable contribution of a wide array of present and former members of the school of optometry. Including Dr Carlo Knupp, for advice and knowledge of modelling methodology, Mr Nick White for advice on cell imaging methodology and for supervising the microscopy sections of this thesis, Dr Christian Pinali and Dr Rob Young for their expertise and teaching in electron microscopy methods, Dr Tina Kamma-Lorger and Dr Julie Albon for their training in cell culture protocol, Dr Sally Hayes and Mr Nick Hawksworth for securing and providing corneal tissue for study, summer intern student Ms Maeva Vallet for some enlightening discussions about the physics of latex beads, Dr Justyn Regini for fulfilling his role as pastoral advisor and more widely the whole of the structural biophysics group for providing a healthy and sociable working environment. I also wish to thank my parents, for the many sacrifices they’ve made to provide me with a comprehensive education. Last but certainly not least, I'd like to thank my long-suffering partner Jane, who has provided so much moral support since the beginning of my studies, and my son Henry. I hope he’s proud of his daddy. iii Abstract This thesis presents the results and conclusions of experiments designed to extend the current models for the origin of corneal transparency. The cornea is the transparent window at the front of the eye, which is responsible not only for the majority of refraction of light that enters, but also the protection against damage, infection and mechanical stress. The property of transparency is only realised by corneal and lens tissue in the human body. In the cornea, it has long been suspected to be caused by the precise arrangement of the fibrils of collagen that are contained within the central layer, the stroma, regulated by the sulphated proteoglycans (PG) that keep fibril spacing within acceptable boundaries. These models are consistent and give a complete description of the reasons for the transparency of a stroma that is entirely acellular. However, it is well known that the stroma is not acellular, and that the short-range order that is critical for transparency would necessarily be disturbed by the cells of the stroma, the keratocytes, which are at least an order of magnitude thicker than the maximum allowed range. Originally, an acellular stroma was considered to be a reasonable approximation due to the perceived sparsity of the cells, but more recent measurements have cast doubt upon this, and explanations have begun to focus on the properties of the cells themselves. One such property would be their refractive index (RI). If the cells could match their own RI to that of their surroundings then they would not scatter and hence would not cause a loss in transparency. This research attempts to measure that RI and by comparison with previously calculated values for the RI of the extra-cellular matrix, attempts to quantify the scale of the scattering that any mismatch would cause, using theoretical models based on both Mie scattering and finite- difference time-domain methods. In addition to models of healthy corneas, this thesis also provides results and conclusions drawn from studies of pathological corneas and discussions of how the pathology, and the treatments, iv can cause initial losses in transparency. The first such study concerned a cornea afflicted with keratoconus, a disorder of as yet unknown origin that causes the weakening of the corneal tissue, leading to a characteristic cone-shaped cornea, which had been treated with a full penetrating keratoplasty (PK) transplant before being donated. The study was conducted using the techniques of electron microscopy and x-ray diffraction, to both qualitatively and quantitatively analyse the properties of the fibrils and their spacing. This was done on both the original sections of diseased tissue and the donated sections, in order to investigate the idea of keratoconus recurring in previously healthy donated tissue. Any such discovery could provide evidence that keratoconus is not an entirely inherited disorder. The structural properties of the observed scar, that was present as a direct result of the PK procedure that was carried out decades before, were also investigated using the same methods. This investigation was designed to provide insights into the priorities of wound healing in the cornea, and whether any appreciable change in fibril spacing could account for the observed loss of transparency. The final study presented here is a novel tomographic reconstruction of a feature of the disease macular corneal dystrophy (MCD). MCD is a genetic disorder that affects the sulphation of the PG keratan sulphate. MCD gives rise to a multitude of abnormalities but one that has not been fully investigated is the apparent presence of areas within the stroma entirely populated by PGs, with no collagen present. This study attempts to reconstruct three-dimensional views of these lakes, as well as stromal lamellae, in order to investigate the interactions between free PGs and between PGs and collagen in MCD. v Contents Acknowledgements .............................................................................................................. iii Abstract ................................................................................................................................. iv List of Figures ....................................................................................................................... ix List of Abbreviations .......................................................................................................... xiii 1 Introduction ........................................................................................................................ 1 1.1 The Eye ....................................................................................................................... 1 1.2 The Cornea .................................................................................................................. 3 1.2.1 Epithelium and Bowman’s layer .......................................................................... 4 1.2.2 Stroma and Keratocytes ....................................................................................... 5 1.2.3 Proposed pre-Descemet’s layer............................................................................ 7 1.2.4 Descemet’s membrane and Endothelium............................................................. 7 1.3 Collagen ...................................................................................................................... 8 1.3.1 Corneal Collagen Types and Functions ............................................................... 8 1.3.2 Stromal Collagen Fibrils .................................................................................... 11 1.3.3 Lamellae ............................................................................................................. 12 1.4 Proteoglycans ............................................................................................................ 13 1.5 Theory of Transparency in the Healthy Cornea ........................................................ 16 1.6 Corneal Wound Healing ............................................................................................ 19 1.7 Pathological Abnormalities of the Cornea ................................................................ 22 1.7.1 Keratoconus ....................................................................................................... 22 1.7.2 Macular Corneal Dystrophy ............................................................................... 25 1.8 Background to Electron Microscopy......................................................................... 26 1.9 Background to X-ray Diffraction .............................................................................. 30 1.10 Aims and Objectives ................................................................................................. 37 2 Corneal Stromal Cell Refractive Index ............................................................................ 39 2.1 Introduction ............................................................................................................... 39 2.1.1 Refractive Index ................................................................................................. 39 2.1.2 The Transport of Intensity Equation .................................................................. 40 2.1.3 Calculation of Phase Images .............................................................................. 43 2.2 Materials and Methods .............................................................................................. 43 2.2.1 QPI Validation ................................................................................................... 44 2.2.2 Cell Culture of Activated Bovine Keratocytes .................................................. 46 2.2.3 Cell Culture of Inactive Bovine Keratocytes ....................................................