A wide-angle x-ray diffraction study of the developing embryonic chicken cornea

In terrestrial vertebrates the cornea is the main refractive component of the eye. Its remarkable mechanical toughness and almost 100% light-transparency are largely a consequence of the unique collagenous architecture of the corneal stroma. We have used WAXS methods to investigate stromal remodelling in the embryonic chicken cornea in the latter stages of development. Collagen organisation at day 13-15 of embryogenesis is dominated by a four-fold orthogonal arrangement of fibrils. Thereafter this preferential alignment recedes, seemingly because further collagen is deposited in a more isotropic manner, masking the initial orthogonal template. In contrast, the mean lateral spacing of fibril-forming collagen molecules remains unaltered over this developmental period. Our observations have important implications for the biomechanical strength and shape of the cornea

Delayed reorganisation of F-actin cytoskeleton and reversible chromatin condensation in scleral fibroblasts under simulated pathological strain

Mechanical loading regulates the functional capabilities of the ocular system, particularly in the sclera (‘white of the eye’) – the principal load-bearing tissue of the ocular globe. Resident fibroblasts of the scleral eye wall are continuously subjected to fluctuating mechanical strains arising from eye movements, cerebrospinal fluid pressure and, most influentially, intra-ocular pressure (IOP). Whilst fibroblasts are hypothesised to actively participate in scleral biomechanics, to date limited information has been reported on how the macroscopic stresses and strains are transmitted via their cytoskeletal networks. In this study, the effect of applying either a ‘physiological load’ (simulating healthy IOP) or a ‘pathological load’ (simulating an elevated glaucomatous IOP) to bovine scleral fibroblasts, as a model of human glaucoma, was conducted to characterise cytoskeletal organisation, chromatin condensation and cell dimensions using immunofluorescence confocal microscopy. Quantification of cell parameters and cytoskeletal element anisotropy were subsequently performed using FibrilTool, and chromatin condensation parameter assessment through a bespoke MATLAB script. The novel findings suggest that physiological load-induced F-actin rearrangement is transient, whereas pathological load, recapitulating in vivo glaucomatous IOP levels, had a reversible and inhibitory influence on remodelling of the cytoskeletal architecture and, further, induction of chromatin condensation. Ultimately, this could compromise cell behaviour. These findings could provide valuable insight into the mechanism(s) used by scleral fibroblasts to mechanically adapt to support biomechanical tissue integrity, and how it could be potentially modified for therapeutic avenues targeting mechanically mediated ocular pathologies such as glaucoma

Effects on collagen orientation in the cornea after trephine injury

Purpose: Structural changes are well known to occur in the cornea after injury. The aim of this study was to investigate collagen orientation changes in the cornea during a short-term wound healing process. Methods: Seven bovine corneas were injured using a penetrating 5 mm biopsy punch and were subsequently organ cultured for up to two weeks. Six uninjured corneas acted as controls. The trephine wounded samples were snap frozen in liquid nitrogen either immediately after injury (0 h) or after 1 or 2 weeks in culture. Control/uninjured samples were snap frozen on arrival (0 h) or after 1 or 2 weeks in culture. Wide angle X-ray diffraction data were collected from each cornea at the UK Synchrotron Radiation Source or at the European Synchrotron Radiation Facility. Data analysis revealed information about collagen orientation and distribution in the corneal stroma during wound healing. For histology, two trephine wounded corneas at 0 h and 1 week and one control/uninjured cornea at 0 h were fixed in 10% neutral buffered formalin and processed for wax embedding. Wax sections were subsequently counterstained with haematoxylin and eosin to observe tissue morphology and the time course of complete re-epithelialization. Results: Immediately after injury, collagen organization was altered in a small area inside the wound but remained similar to the control/uninjured sample in the remainder of the tissue. After one week, the trephine wounded corneas showed complete re-epithelialization and evidence of swelling while collagen adopted a radial arrangement inside and outside the wound. Conclusions: Remarkable changes in collagen fibril orientation were observed in trephine wounded corneas. Orientation changes immediately after wounding are likely to be due to the mechanical deformation of the tissue during the wounding process. However, tissue swelling and changes in collagen orientation at later stages probably reflect the processes of tissue repair. These differences will determine corneal stability and strength following trauma and possibly refractive surgery

Morphometric, hemodynamic, and biomechanical factors influencing blood flow and oxygen concentration in the human lamina cribrosa

Purpose: We developed a combined biomechanical and hemodynamic model of the human eye to estimate blood flow and oxygen concentration within the lamina cribrosa (LC) and rank the factors that influence LC oxygen concentration. Methods: We generated 5000 finite-element eye models with detailed microcapillary networks of the LC and computed the oxygen concentration of the lamina retinal ganglion cell axons. For each model, we varied the intraocular pressure (IOP) from 10 mm Hg to 55 mm Hg in 5-mm Hg increments, the cerebrospinal fluid pressure (13 ± 2 mm Hg), cup depth (0.2 ± 0.1 mm), scleral stiffness (±20% of the mean values), LC stiffness (0.41 ± 0.2 MPa), LC radius (1.2 ± 0.12 mm), average LC pore size (5400 ± 2400 µm2), the microcapillary arrangement (radial, isotropic, or circumferential), and perfusion pressure (50 ± 9 mm Hg). Blood flow was assumed to originate from the LC periphery and drain via the central retinal vein. Finally, we performed linear regressions to rank the influence of each factor on the LC tissue oxygen concentration. Results: LC radius and perfusion pressure were the most important factors in influencing the oxygen concentration of the LC. IOP was another important parameter, and eyes with higher IOP had higher compressive strain and slightly lower oxygen concentration. In general, superior–inferior regions of the LC had significantly lower oxygen concentration than the nasal–temporal regions, resulting in an hourglass pattern of oxygen deficiency. Conclusions: To the best of our knowledge, this study is the first to implement a comprehensive hemodynamical model of the eye that accounts for the biomechanical forces and morphological parameters of the LC. The results provide further insight into the possible relationship of biomechanical and vascular pathways leading to ischemia-induced optic neuropathy