Quantifying biomarkers of axonal degeneration in early glaucoma to find the disc at risk.

To evaluate regional axonal-related parameters as a function of disease stage in primary open angle glaucoma (POAG) and visual field (VF) sensitivity. Spectral domain optical coherence tomography was used to acquire 20° scans of POAG (n = 117) or healthy control (n = 52) human optic nerve heads (ONHs). Region specific and mean nerve fibre layer (NFL) thicknesses, border NFL and peripapillary NFL, minimum rim width (MRW)/ area (MRA) and prelamina thickness; and volume were compared across POAG disease stages and with visual field sensitivity. Differences identified between early glaucoma (EG), preperimetric glaucoma (PG) and control (C) ONHs included thinner PG prelamina regions than in controls (p < 0.05). Mean border NFL was thinner in EG (p < 0.001) and PG (p = 0.049) compared to control eyes; and EG mean, and inferior and ST, border NFL was thinner than in PG (p < 0.01). Mean, superior and inferior PG peripapillary NFL were thinner than in controls (p < 0.05), and EG ST peripapillary NFL was thinner than in PG (p = 0.023). MRW differences included: PG SN and inferior less than in controls (p < 0.05); thinner EG mean regional, inferior, nasal, and ST MRW versus PG MRW (p < 0.05). Regional border NFL, peripapillary NFL, MRW, MRA, prelamina thickness (except centre, p = 0.127) and prelamina volume (p < 0.05) were significantly associated with VF mean deviation (MD). Novel axon-derived indices hold potential as biomarkers to detect early glaucoma and identify ONHs at risk.

Quantitative analysis of three-dimensional fibrillar collagen microstructure within the normal, aged and glaucomatous human optic nerve head.

The aim of this study was to quantify connective tissue fibre orientation and alignment in young, old and glaucomatous human optic nerve heads (ONH) to understand ONH microstructure and predisposition to glaucomatous optic neuropathy. Transverse (seven healthy, three glaucomatous) and longitudinal (14 healthy) human ONH cryosections were imaged by both second harmonic generation microscopy and small angle light scattering (SALS) in order to quantify preferred fibre orientation (PFO) and degree of fibre alignment (DOFA). DOFA was highest within the peripapillary sclera (ppsclera), with relatively low values in the lamina cribrosa (LC). Elderly ppsclera DOFA was higher than that in young ppsclera (p < 0.00007), and generally higher than in glaucoma ppsclera. In all LCs, a majority of fibres had preferential orientation horizontally across the nasal-temporal axis. In all glaucomatous LCs, PFO was significantly different from controls in a minimum of seven out of 12 LC regions (p < 0.05). Additionally, higher fibre alignment was observed in the glaucomatous inferior-temporal LC (p < 0.017). The differences between young and elderly ONH fibre alignment within regions suggest that age-related microstructural changes occur within the structure. The additional differences in fibre alignment observed within the glaucomatous LC may reflect an inherent susceptibility to glaucomatous optic neuropathy, or may be a consequence of ONH remodelling and/or collapse.

Bacterial contamination of human organ-cultured corneas.

PURPOSE: The aim of this study was to detect and identify bacterial contaminants in human corneoscleral tissue after organ culture storage. METHODS: Seventy-two corneoscleral rims and corneal buttons trephined from organ cultured corneoscleral discs using aseptic technique and 45 organ-cultured donor corneoscleral rims postpenetrating keratoplasty) were subjected to a mechanical extraction technique using a Stomacher laboratory blender. As a control, 28 of the corneoscleral rims and buttons were halved; one half of each corneoscleral rim and button was decontaminated in formalin for 48 hours before thorough washing in balanced salt solution. Corneal specimens, culture medium, and transport (5% dextran) medium were cultured in brain-heart infusion broth at 37 degrees C for 5 days. Bacterial isolates were identified after culture of turbid enrichment broth. RESULTS: Bacterial contamination was demonstrated in 29% (21 of 72) of the corneoscleral rims and 15% (11 of 72) of the corneal buttons that were trephined aseptically from corneoscleral discs and in 29% (13 of 45) of postkeratoplasty corneoscleral rims. Bacterial contaminants were not isolated from controls. Isolated microorganisms included coagulase-negative Staphylococcus, Staphylococcus aureus, Streptococcus viridans, Pseudomonas sp, and Bacillus sp. A correlation was not demonstrated between contamination and cause of death, death to enucleation, death to culture time, or time in culture. Postkeratoplasty endophthalmitis was not evident in the patients who had received corneal buttons from those corneoscleral discs that had contaminated corneoscleral rims. CONCLUSIONS: Bacterial contamination exists in corneoscleral tissue after organ culture storage. The difference in distribution of bacteria and percentage of contamination between the peripheral and central corneas causes us to question the value of routine postpenetrating keratoplasty corneoscleral rim cultures.

Apoptosis in the endothelium of human corneas for transplantation.

PURPOSE: To determine whether endothelial cell loss of human corneas stored in organ culture before transplantation is due to apoptosis. METHODS: The corneal endothelium of human corneas, stored in organ culture at 34 degrees C for varying periods of time, were analyzed for the presence of apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL) technique. Corneal endothelial cell apoptosis was confirmed by Hoechst staining and immunolabeling with anti-caspase 3 active antibody. RESULTS: Apoptotic cells were identified in the corneal endothelium of human organ cultured corneas: their number and distribution demonstrated a close correlation with corneal folding and overall quality of the corneal endothelium. TUNEL-positive labeling of cells was confirmed as apoptotic by the presence of morphologic nuclear alterations identified by Hoechst staining and the presence of immunostaining for caspase-3 activity. Corneal endothelial cell apoptosis was independent of cause of donor death, death to enucleation time, and death to culture times. CONCLUSIONS: Corneal endothelial cell apoptosis appears to determine the suitability of a cornea for transplantation.

Age related changes in the non-collagenous components of the extracellular matrix of the human lamina cribrosa.

AIMS: To investigate age related alterations in the non-collagenous components of the human lamina cribrosa. METHODS: Fibronectin, elastin, and glial fibrillary acidic protein (GFAP) staining were assessed in young and old laminae cribrosae. An age range (7 days to 96 years) of human laminae cribrosae were analysed for lipid content (n=9), cellularity (n=28), total sulphated glycosaminoglycans (n=28), elastin content (n=9), and water content (n=56), using chloroform-methanol extraction, fluorimetry, the dimethylmethylene blue assay, and ion exchange chromatography, respectively. RESULTS: Qualitatively, an increase in elastin and a decrease in fibronectin and GFAP were demonstrated when young tissue was compared with the elderly. Biochemical analysis of the ageing human lamina cribrosa demonstrated that elastin content increased from 8% to 28% dry tissue weight, total sulphated glycosaminoglycans decreased, and lipid content decreased from 45% to 25%. There were no significant changes in total cellularity or water content. CONCLUSION: These alterations in composition may be indicative of the metabolic state of the lamina cribrosa as it ages, and may contribute to changes in mechanical integrity. Such changes may be implicated in the susceptibility of the elderly lamina cribrosa and also its response to glaucomatous optic neuropathy.

Age related compliance of the lamina cribrosa in human eyes.

AIMS: To investigate changes in the mechanical compliance of ex vivo human lamina cribrosa with age. METHODS: A laser scanning confocal microscope was used to image the surface of the fluorescently labelled lamina cribrosa in cadaver eyes. A method was developed to determine changes in the volume and strain of the lamina cribrosa created by increases in pressure. The ability of the lamina cribrosa to reverse its deformation on removal of pressure was also measured. RESULTS: Volume and strain measurements both demonstrated that the lamina cribrosa increased in stiffness with age and the level of pressure applied. The ability of the lamina cribrosa to regain its original shape and size on removal of pressure appeared to decrease with age, demonstrating an age related decrease in resilience of the lamina cribrosa. CONCLUSIONS: The mechanical compliance of the human lamina cribrosa decreased with age. Misalignment of compliant cribriform plates in a young eye may exert a lesser stress on nerve axons, than that exerted by the rigid plates of an elderly lamina cribrosa. The resilience of the lamina cribrosa also decreased with age, suggesting an increased susceptibility to plastic flow and permanent deformation. Such changes may be of importance in the explanation of age related optic neuropathy in primary open angle glaucoma.

Proteoglycans on normal and migrating human corneal endothelium.

Proteoglycans are of fundamental importance to the normal functioning of the cornea. They consist of a core protein to which one or more glycosaminoglycan chains are attached. Cell surface proteoglycans are known to mediate many aspects of cell behaviour including cell adhesion, control of extracellular matrix deposition, cell proliferation, cell migration, leukocyte adhesion and modulation of growth factor activity. This paper describes the first investigation into the distribution and function of the three main classes of proteoglycans on human corneal endothelium. Immuno-gold labelling techniques were used at the light, scanning and transmission electron microscope level to localise heparan sulphate, chondroitin sulphate and keratan sulphate proteoglycans on human corneal endothelium. Human corneas were freeze-wounded and kept in organ culture for 3 days in order to study the distribution of proteoglycans on migrating corneal endothelium. An Optimas image analysis system was used to quantify the change in proteoglycan labelling during cell migration. Labelling for chondroitin sulphate and heparan sulphate was at very low levels on normal corneal endothelium while keratan sulphate labelling was at high levels. The wound healing experiments showed that migrating cells had increased labelling for heparan sulphate and chondroitin sulphate with greatly decreased labelling for keratan sulphate. Statistical analysis showed these changes were highly significant (P<0.001). Transmission electron microscopy revealed that chondroitin sulphate and keratan sulphate were present throughout Descemet's membrane while heparan sulphate was concentrated at the interface of Descemet's membrane and the migrating corneal endothelial cells. The pattern of occurrence of chondroitin sulphate, heparan sulphate and keratan sulphate on the human endothelium in normal and wounded cornea suggests that these proteoglycans are linked to the process of cell migration.

Preparation of Bruch's membrane and analysis of the age-related changes in the structural collagens.

AIMS/BACKGROUND: The morphological changes in Bruch's membrane and its constituent collagen seen during aging have been studied extensively but the chemical nature of the collagen and any aging changes have not previously been evaluated. METHODS: A method for preparing purified Bruch's membrane from human cadaver eyes by dissection preceded by trypsin digestion was developed. Following pepsin digestion, the constituent collagens were analysed by SDS-PAGE and by immunoblotting. Cyanogen bromide digestion was used to ascertain the solubility of the collagen and the proportion of type I to type III collagen. After hydrolysis of Bruch's membrane samples the constituent amino acids and collagen crosslinks were measured. RESULTS: The presence of collagen types I, III, IV, and V in Bruch's membrane was confirmed. The proportion of type III collagen as a percentage of total fibrous collagens was calculated as being between 35% and 39%, with no significant difference between different macular and peripheral sites or with age. There was a highly significant decline in the solubility of Bruch's membrane collagen with age, from near 100% in the first decade of life to 40-50% in the ninth decade at both macular and peripheral sites. There was no significant change in the amount of enzymatically formed collagen crosslinks with age. Amino acid analysis indicated a significant increase in the amount of non-collagen protein with age in macular but not peripheral sites. CONCLUSION: Changes in the constituent collagens may contribute to the accumulation of debris in Bruch's membrane with age and interfere with the function of the retinal pigment epithelium, with subsequent consequences for the overlying photoreceptors.

The human optic nerve: fascicular organisation and connective tissue types along the extra-fascicular matrix.

Fibres in the mammalian optic nerve are arranged into fascicles between which there is an extra-fascicular matrix containing connective tissue, a feature similar to that found in association with fibres in peripheral nerves, but not otherwise found in the CNS. The relationship between these major features of the nerve architecture are not known. We have addressed this question by examining the fascicular organisation of the optic nerve and the distribution of connective tissue and specific collagen types in the human. We have also examined the spatial development of connective tissue in the human nerve to determine when and from where it originates. Fibres are grouped into fascicles at all locations along the nerve, except intracranially, close to the chiasm where this pattern is lost. Relatively large fascicular numbers are found directly behind the eye and in the region of the optic canal, but decline in the mid-orbital segment of the nerve. Connective tissue is present in the extra-fascicular matrix throughout the fasciculated segment, but in many cases it does not fully encircle fascicles. The proportion of matrix occupied by connective tissue is similar along the length of the nerve (approximately 60%). Within the matrix, collagen types I, III, IV, V and VI are present throughout fasciculated regions. Staining for types V and VI appeared relatively weak compared with that for the other types. Although the collagen types in the nerve are similar to those at the lamina cribrosa and in peripheral nerves, they did not appear to be differentially distributed as in regions of the PNS. Connective tissue enters the nerve at a number of wide-spread locations early in development, consistent with the notion that it enters the nerve with the blood supply. It is present within the matrix before it is established at the lamina cribrosa.

Changes in the collagenous matrix of the aging human lamina cribrosa.

AIMS: The age-related changes in the biochemical composition of the collagenous matrix of the human lamina cribrosa were investigated. METHODS: An age range (3 weeks to 92 years old) of human laminae cribrosae, dissected free of any surrounding structures which contained collagen, were analysed for collagen solubility (n = 58) total collagen content (n = 46), proportion of collagen types (n = 38), and collagen cross linking (n = 30), using hydroxyproline analysis, scanning densitometry of peptides after cyanogen bromide digestion, and high performance liquid chromatography, respectively. RESULTS: Age-related changes included an increase in total collagen and a decrease in the proportion of type III collagen within the lamina cribrosa. The collagen cross link pyridinoline was present at low levels, but demonstrated no trend with age. An age-related increase was found in pentosidine, an advanced glycation product. CONCLUSION: These changes in collagen composition imply that the mechanical properties of the lamina cribrosa are altered, resulting in a stiffer, less resilient structure with age. Such alterations in structure may contribute to the increased susceptibility of the elderly to axonal damage in chronic open angle glaucoma.