Three-dimensional studies of the developing mammalian cornea.

This thesis aimed to understand the structural changes that occur during the development of the mammalian cornea. The imaging techniques used included novel three-dimensional serial block-face scanning electron microscopy, transmission electron microscopy, optical coherence tomography, X-ray diffraction and immunofluorescence. These techniques were utilised to investigate the human, mouse and the fibrillin-1 knockout mouse cornea. The mouse cornea had no collagenous primary stroma to direct mesenchymal cell migration. Stromal cell projections associated with adjacent corneal stromal cells and the corneal epithelium, and appeared to direct collagen alignment. The mouse stroma expressed types I, II and V collagen, and later type IX collagen in the epithelium. Proteoglycans were observed before collagen deposition in the mouse stroma, associated with stromal cells and collagen fibrils. A collagenous primary stroma was identified in the human embryonic cornea prior to mesenchymal cell migration. The corneal endothelium contained novel cell extensions that associated with the mesenchymal cells and the acellular collagenous matrix; these results suggested that the endothelium assists mesenchymal cell migration. The human adult cornea contained true elastic fibres in the peripheral posterior cornea with fibrillin-rich microfibrils in the central posterior cornea. The elastic fibres in the mouse contained only fibrillin-rich microfibrils. In the human, elastic fibres were detected from week 12 of development and had a distribution similar to the mature human cornea. This included elastic fibre sheets directly anterior to the endothelium and individual elastic fibres in the posterior peripheral stroma. The fibrillin-1 knockout mouse cornea had reduced stromal thickness and a disorganised extracellular matrix. It is thought that elevated transforming growth factor-beta disrupted the corneal architecture. This thesis has contributed novel findings of the events that develop the mammalian cornea. The results identified fundamental differences and similarities between the mouse and human models and have suggested new mechanisms in the developmental process

A comparative study of the mouse and human corneal elastic system [Abstract]

Purpose : To characterise and compare the elastic fibre system of mouse and human posterior cornea. Methods : 4 adult human corneas were obtained from a UK eye bank and 20 corneas taken from 3 month old C5BL/6 mice which had been euthanized. The samples were examined using Serial block face scanning electron microscopy (SBF-SEM) and transmission electron microscopy (TEM). The corneas were fixed and processed for SBF-SEM through a series of staining solutions prior to embedding and polymerization in epoxy resin blocks. The sample blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 1000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira 6.4 software. For electron microscopy ultrathin sections were prepared and examined in a JEOL 1010 TEM. The corneas were also examined using immunofluorescence labelling of elastin and fibrillin-1. Results : Immunofluorescence revealed an extensive fibrillin-1-rich microfibril system running throughout the mouse corneal stroma. Human corneas were also positive for fibrillin-1 within the posterior cornea, but in addition exhibited positive elastin staining confined to the posterior peripheral cornea. TEM confirmed the presence of true elastic fibres containing an amorphous elastin core in peripheral human corneas, which were absent within the mouse microfibrils. Clear structural differences at the convergence of the trabecular meshwork (TM) into the elastic fibre system were also observed between mouse and human cornea using SBF-SEM. In mouse cornea the TM merged directly with Descemet’s membrane whereas in human cornea the TM inserted into the elastic fibre system anterior to Descemet’s. Conclusions : Clear differences exist between the elastic fibre system and TM anatomy of mouse and human cornea. These differences suggest the fibre system of mouse and human have different biomechanical properties and function

A comparative study of the mouse and human corneal elastic system [Abstract]

Purpose : To characterise and compare the elastic fibre system of mouse and human posterior cornea. Methods : 4 adult human corneas were obtained from a UK eye bank and 20 corneas taken from 3 month old C5BL/6 mice which had been euthanized. The samples were examined using Serial block face scanning electron microscopy (SBF-SEM) and transmission electron microscopy (TEM). The corneas were fixed and processed for SBF-SEM through a series of staining solutions prior to embedding and polymerization in epoxy resin blocks. The sample blocks were trimmed and transferred to a Zeiss Sigma VP FEG scanning electron microscope equipped with a Gatan 3View system, where data sets of up to 1000 images were acquired of the block surface every 50nm through automated sectioning. Selected serial image sequences were extracted and 3D reconstructions were generated using Amira 6.4 software. For electron microscopy ultrathin sections were prepared and examined in a JEOL 1010 TEM. The corneas were also examined using immunofluorescence labelling of elastin and fibrillin-1. Results : Immunofluorescence revealed an extensive fibrillin-1-rich microfibril system running throughout the mouse corneal stroma. Human corneas were also positive for fibrillin-1 within the posterior cornea, but in addition exhibited positive elastin staining confined to the posterior peripheral cornea. TEM confirmed the presence of true elastic fibres containing an amorphous elastin core in peripheral human corneas, which were absent within the mouse microfibrils. Clear structural differences at the convergence of the trabecular meshwork (TM) into the elastic fibre system were also observed between mouse and human cornea using SBF-SEM. In mouse cornea the TM merged directly with Descemet’s membrane whereas in human cornea the TM inserted into the elastic fibre system anterior to Descemet’s. Conclusions : Clear differences exist between the elastic fibre system and TM anatomy of mouse and human cornea. These differences suggest the fibre system of mouse and human have different biomechanical properties and function