No abstract

The cornea is the main refracting lens in the eye. As part of the outer tunic it has to be resilient, a property conferred by the organisation of the constituent collagen. It also has to be sufficiently elastic to regain its exact shape when deformed, in order not to distort the retinal image. The basis of this elasticity is not fully understood. The purpose of this study was to characterise in three dimensions the arrangement and distribution of elastic fibers in the human corneal stroma, using serial block face scanning electron microscopy. We have demonstrated that there exists a complex network of elastic fibers that appear to originate in the sclera or limbus. These appear as elastic sheets in the limbus and peripheral cornea immediately above the trabecular meshwork which itself appears to extend above Descemet's membrane in the peripheral stroma. From these sheets, elastic fibers extend into the cornea; moving centrally they bifurcate and trifurcate into narrower fibers and are concentrated in the posterior stroma immediately above Descemet's membrane. We contend that elastic sheets will play an important role in the biomechanical deformation and recovery of the peripheral cornea. The network may also have practical implications for understanding the structural basis behind a number of corneal surgeries.

Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.

The optical and biomechanical properties of the cornea are largely governed by the collagen-rich stroma, a layer that represents approximately 90% of the total thickness. Within the stroma, the specific arrangement of superimposed lamellae provides the tissue with tensile strength, whilst the spatial arrangement of individual collagen fibrils within the lamellae confers transparency. In keratoconus, this precise stromal arrangement is lost, resulting in ectasia and visual impairment. In the normal cornea, we previously characterised the three-dimensional arrangement of an elastic fiber network spanning the posterior stroma from limbus-to-limbus. In the peripheral cornea/limbus there are elastin-containing sheets or broad fibers, most of which become microfibril bundles (MBs) with little or no elastin component when reaching the central cornea. The purpose of the current study was to compare this network with the elastic fiber distribution in post-surgical keratoconic corneal buttons, using serial block face scanning electron microscopy and transmission electron microscopy.We have demonstrated that the MB distribution is very different in keratoconus. MBs are absent from a region of stroma anterior to Descemet's membrane, an area that is densely populated in normal cornea, whilst being concentrated below the epithelium, an area in which they are absent in normal cornea. We contend that these latter microfibrils are produced as a biomechanical response to provide additional strength to the anterior stroma in order to prevent tissue rupture at the apex of the cone. A lack of MBs anterior to Descemet's membrane in keratoconus would alter the biomechanical properties of the tissue, potentially contributing to the pathogenesis of the disease.

Fuchs endothelial corneal dystrophy (FECD) is a genetic and oxidative stress disorder of post-mitotic human corneal endothelial cells (HCEnCs), which normally exhibit hexagonal shape and form a compact monolayer compatible with normal corneal functioning and clear vision. FECD is associated with increased DNA damage, which in turn leads to HCEnC loss, resulting in the formation rosettes and aberrant extracellular matrix (ECM) deposition in the form of pro-fibrotic guttae. Since the mechanism of ECM deposition in FECD is currently unknown, we aimed to investigate the role of endothelial-mesenchymal transition (EMT) in FECD using a previously established cellular in vitro model that recapitulates the characteristic rosette formation, by employing menadione (MN)-induced oxidative stress. We demonstrate that MN treatment alone, or a combination of MN and TGF-β1 induces reactive oxygen species (ROS), cell death, and EMT in HCEnCs during rosette formation, resulting in upregulation of EMT- and FECD-associated markers such as Snail1, N-cadherin, ZEB1, and transforming growth factor-beta-induced (TGFβI), respectively. Additionally, FECD ex vivo specimens displayed a loss of organized junctional staining of plasma membrane-bound N-cadherin, with corresponding increase in fibronectin and Snail1 compared to ex vivo controls. Addition of N-acetylcysteine (NAC) downregulated all EMT markers and abolished rosette formation. Loss of NQO1, a metabolizing enzyme of MN, led to greater increase in intracellular ROS levels as well as a significant upregulation of Snail1, fibronectin, and N-cadherin compared to normal cells, indicating that NQO1 regulates Snail1-mediated EMT. This study provides first line evidence that MN-induced oxidative stress leads to EMT in corneal endothelial cells, and the effect of which is further potentiated when redox cycling activity of MN is enhanced by the absence of NQO1. Given that NAC inhibits Snail-mediated EMT, this may be a potential therapeutic intervention for FECD.

Graphical abstract

PurposeThe presence of fibrillin-rich elastic fibers in the cornea has been overlooked in recent years. The aim of the current study was to elucidate their functional role using a mouse model for Marfan syndrome, defective in fibrillin-1, the major structural component of the microfibril bundles that constitute most of the elastic fibers.MethodsMouse corneas were obtained from animals with a heterozygous fibrillin-1 mutation (Fbn1+/−) and compared to wild type controls. Corneal thickness and radius of curvature were calculated using optical coherence tomography microscopy. Elastic microfibril bundles were quantified and visualized in three-dimensions using serial block face scanning electron microscopy. Transmission electron microscopy was used to analyze stromal ultrastructure and proteoglycan distribution. Center-to-center average interfibrillar spacing was determined using x-ray scattering.ResultsFbn1+/− corneas were significantly thinner than wild types and displayed a higher radius of curvature. In the Fbn1+/− corneas, elastic microfibril bundles were significantly reduced in density and disorganized compared to wild-type controls, in addition to containing a higher average center-to-center collagen interfibrillar spacing in the center of the cornea. No other differences were detected in stromal ultrastructure or proteoglycan distribution between the two groups. Proteoglycan side chains appeared to colocalize with the microfibril bundles.ConclusionsElastic fibers have an important, multifunctional role in the cornea as highlighted by the differences observed between Fbn1+/− and wild type animals. We contend that the presence of normal quantities of structurally organized elastic fibers are required to maintain the correct geometry of the cornea, which is disrupted in Marfan syndrome.

The primary aim of this study was to quantify the relationship between corneal structure and hydration in humans and pigs. X-ray scattering data were collected from human and porcine corneas equilibrated with polyethylene glycol (PEG) to varying levels of hydration, to obtain measurements of collagen fibril diameter, interfibrillar spacing (IFS) and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and IFS2 and a nonlinear, bi-phasic relationship between hydration and fibril diameter, whereby fibril diameter increased up to approximately physiological hydration, H = 3.0, with little change thereafter. Above H = 3.0, porcine corneas exhibited a larger fibril diameter than human corneas (p < 0.001). Intermolecular spacing also varied with hydration in a bi-phasic manner but reached a maximum value at a lower hydration (H = 1.5) than fibril diameter. Human corneas displayed a higher intermolecular spacing than porcine corneas at all hydrations (p < 0.0001). Human and porcine corneas required a similar PEG concentration to reach physiological hydration, suggesting that the total fixed charge that gives rise to the swelling pressure is the same. The difference in their structural responses to hydration can be explained by variations in molecular cross-linking and intra/interfibrillar water partitioning.