Changes in posterior scleral collagen microstructure in canine eyes with an ADAMTS10 mutation

Purpose: We aimed to characterize alterations in the posterior scleral collagen microstructure before detectable disease onset in a canine model of open-angle glaucoma caused by an ADAMTS10 mutation. Methods: Collagen orientation, anisotropy degree (proportion of preferentially aligned collagen), and relative density were measured at 0.4 mm spatial resolution using synchrotron wide-angle X-ray scattering. For statistical evaluation of structure parameters, regional averages of the peripapillary and mid-posterior sclera were compared between ADAMTS10 mutant (affected) dogs (n = 3) and age-matched (carrier) controls (n = 3). Results: No marked differences in the general pattern of preferential collagen fibril orientation were noted between the control and affected dogs. The peripapillary sclera of all specimens featured strongly aligned circumferential collagen ringing the optic nerve head. Collagen anisotropy was significantly reduced in the mid-posterior sclera of the affected dogs (carrier: 0.27±0.11; affected: 0.24±0.10; p = 0.032) but was not statistically significantly different in the peripapillary sclera (carrier: 0.46±0.15; affected: 0.45±0.17; p = 0.68). Collagen density was statistically significantly reduced in the affected dogs for the mid-posterior sclera (carrier: 28.1±9.14; affected: 18.3±5.12; p<0.0001) and the peripapillary sclera (carrier: 34.6±9.34; affected: 21.1±6.97; p = 0.0002). Conclusions: Significant alterations in the posterior scleral collagen microstructure are present before the onset of clinical glaucoma in ADAMTS10 mutant dogs. A reduction in fibrous collagen density is likely an important contributory factor in the previously reported mechanical weakening of the sclera in this model. Baseline scleral abnormalities have the potential to interact with intraocular pressure (IOP) elevations in determining the course of glaucoma progression in animal models of the disease, and potentially in human glaucoma

Microwave treatment of the cornea leads to localised disruption of the extracellular matrix

Microwave keratoplasty is a thermo-refractive surgical procedure that can correct myopia (short-sightedness) and pathologic corneal steepening by using microwave energy to cause localised shrinkage around an annulus of the cornea leading to its flattening and vision correction. The effects on the corneal extracellular matrix, however, have not yet been evaluated, thus the current study to assess post-procedure ultrastructural changes in an in-vivo rabbit model. To achieve this a series of small-angle x-ray scattering (SAXS) experiments were carried out across whole transects of treated and untreated rabbit corneas at 0.25 mm intervals, which indicated no significant change in collagen intra-fibrillar parameters (i.e. collagen fibril diameter or axial D-period), whereas inter-fibrillar measures (i.e. fibril spacing and the degree of spatial order) were markedly altered in microwave-treated regions of the cornea. These structural matrix alterations in microwave-treated corneas have predicted implications for corneal biomechanical strength and tissue transparency, and, we contend, potentially render microwave-treated corneas resistant to surgical stabilization using corneal cross-linking procedures currently employed to combat refractive error caused by corneal steepening

The structural response of the cornea to changes in stromal hydration

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 and intermolecular spacing. Both species showed a strong positive linear correlation between hydration and interfibrillar spacing2 and a non-linear, 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 crosslinking and intra/interfibrillar water partitioning

3D immuno-confocal image reconstruction of fibroblast cytoskeleton and nucleus architecture

Computational models of cellular structures generally rely on simplifying approximations and assumptions that limit biological accuracy. This study presents a comprehensive image processing pipeline for creating unified three‐dimensional (3D) reconstructions of the cell cytoskeletal networks and nuclei. Confocal image stacks of these cellular structures were reconstructed to 3D isosurfaces (Imaris), then tessellations were simplified to reduce the number of elements in initial meshes by applying quadric edge collapse decimation with preserved topology boundaries (MeshLab). Geometries were remeshed to ensure uniformity (Instant Meshes) and the resulting 3D meshes exported (ABAQUS) for downstream application. The protocol has been applied successfully to fibroblast cytoskeletal reorganisation in the scleral connective tissue of the eye, under mechanical load that mimics internal eye pressure. While the method herein is specifically employed to reconstruct immunofluorescent confocal imaging data, it is also more widely applicable to other biological imaging modalities where accurate 3D cell structures are required

Improving management of type 1 diabetes in the UK: the Dose Adjustment For Normal Eating (DAFNE) programme as a research test-bed. A mixed-method analysis of the barriers to and facilitators of successful diabetes self-management, a health economic analysis, a cluster randomised controlled trial of different models of delivery of an educational intervention and the potential of insulin pumps and additional educator input to improve outcomes

Peer reviewe

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

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