Wojciech S S Karwatowski

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.

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.

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

AIMS/BACKGROUND--The morphological changes in Bruchs 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 Bruchs 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 Bruchs membrane samples the constituent amino acids and collagen crosslinks were measured. RESULTS--The presence of collagen types I, III, IV, and V in Bruchs 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 Bruchs 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 Bruchs membrane with age and interfere with the function of the retinal pigment epithelium, with subsequent consequences for the overlying photoreceptors.

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.

Clinical evaluation of scanning laser polarimetry: I Intraoperator reproducibility and design of a blood vessel removal algorithm

AIMS To evaluate the reproducibility of the retardation values (change in polarisation) obtained with the scanning laser polarimeter in a series of normal subjects and glaucoma patients. To improve the analysis of the raw data by devising and evaluating a blood vessel removal algorithm. METHODS Scanning laser polarimetry was performed on 10 normal subjects and 10 glaucoma patients. A series of six images was obtained from each eye. The normal subjects were re-imaged 3 months after their initial assessment. The retardation values obtained from each eye were analysed using the authors’ own methods, including the use of an algorithm to remove blood vessels from the polar profiles. The reproducibility of these measurements and the performance of the blood vessel removal algorithm were assessed. RESULTS The “individual point” coefficient of variation was approximately 12.5% for normal subjects and 17.0% for glaucoma patients. The “integral” coefficient of variation for these groups was approximately 5.5% and 9.5% respectively. The reproducibility of the measurements did not improve with an increased number of measurements. There was no difference in the reproducibility of the measurements in normal subjects over time. The blood vessel removal algorithm improved the reproducibility of the measurements when the shape of the profile was assessed. CONCLUSION The intraoperator reproducibility of retardation values obtained with the scanning laser polarimeter is satisfactory for its use as a clinical tool. The use of a blood vessel removal algorithm improves the reproducibility of the measurements and also assists the clinician in the interpretation of the polar profiles. Furthermore, it allows the construction of normal database polar profiles, thereby enabling the identification, location and quantification of retinal nerve fibre layer damage in an “at risk” individual’s polar profile.

Ocular Elasticity: Is Engineering Stiffness a More Useful Characterization Parameter than Ocular Rigidity?

PURPOSE The purpose of this study is to present an analysis of the pressure-volume relation of the eye in basic engineering terms, so as to characterize the deformability of the ocular shell based on its intrinsic stiffness (Young modulus) and morphology, as opposed to the empirical measure of ocular rigidity. METHODS Starting from the structural mechanics equations describing the stress of spherical thin-walled vessels, the differential equation governing the eye pressure-volume relation is derived. This analysis, which is more rigorous than previously published derivations, assumes that the ocular shell has a Poisson ratio of 0.5. This assumption is experimentally confirmed by ultrasonic measurements of changes in bovine corneal thickness with intraocular pressure. RESULTS Even with a number of simplifying assumptions, this basic analysis yields a complex result, showing that the Young modulus of the ocular shell material increases rapidly with distension of the eye, and is approximately proportional to the fourth power of the ocular shell radius. CONCLUSION Due to the complexity of the phenomenon, engineering analysis does not lead to a simple picture of pressure-volume relation of the eye. However, it does explicitly separate the material properties of the ocular shell from morphologic contributions to pressure-volume relation of the eye. This approach allows pathologic changes in the pressure-volume relation of the eye to be related more easily to the fundamental structural mechanisms governing the nonlinear mechanical properties of ocular shell materials.

Clinical evaluation of scanning laser polarimetry: II Polar profile shape analysis

AIMS To devise a method to describe and quantify the shape of polar profiles obtained with the scanning laser polarimeter and to compare this measurement with other polar profile measurements in a series of normal subjects and glaucoma patients. METHODS Scanning laser polarimetry was performed on 54 normal subjects and 74 glaucoma patients. The retardation values obtained from one randomly chosen eye of each subject were analysed using our own methods, including the use of an algorithm to remove blood vessels from the polar profiles, an algorithm to standardise the glaucoma profiles to a normal database, and a further algorithm to evaluate the profile shape. The measurements of profile shape were compared with measurements of the absolute and standardised retinal nerve fibre layer thickness obtained with the scanning laser polarimeter. RESULTS There was no significant difference between the mean retardation values for the normal and glaucomatous subjects in either hemiretina. However, standardisation of the glaucoma retardation values to a normal database produced significant differences at p <1 × 10−8 in the mean retardation values for these two groups in both hemiretinas. Profile shape measurement analysis produced similar significant differences between the mean retardation values for the normal and glaucomatous subjects in both hemiretinas, although the degree of separation was greater following standardisation of the retardation values. CONCLUSION The use of an algorithm to standardise an individual’s retardation values in conjunction with a blood vessel removal algorithm enables an improvement in the ability of the scanning laser polarimeter to discriminate between normal and glaucomatous patients. The polar profile shape algorithm is independent of standardisation and significantly improves the discrimination between normal and glaucomatous patients, as well as providing additional information regarding the retinal nerve fibre layer.