Involvement of corneal nerves in the progression of keratoconus.

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.

Cellular incursion into Bowman's membrane in the peripheral cone of the keratoconic cornea.

Analysis of corneal tissue from normal and keratoconic donors has revealed differences which may represent early signs in the pathogenesis of keratoconus. Peripheral areas of keratoconic tissue obtained from transplant surgery were targeted to ascertain cellular disposition and morphological changes which may be masked within the extensive damage of the central keratoconic cone. Peripheral keratoconic corneae exhibited discrete incursion of fine cellular processes into Bowman's membrane. These processes originated from keratocytes and were often observed in conjunction with a defined indentation from the basal epithelium. Comparison of the lysosomal enzymes cathepsin B and G with constitutively expressed cytoplasmic esterase determined that both cathepsins were elevated within keratocytes of keratoconic tissue compared with normal tissue. Some clusters of keratoconic keratocytes had elevated levels of cathepsin exceeding all others. Cathepsin-rich keratocytes localized with morphologically compromised regions of Bowman's membrane. The presence of cell nests deeper within the stroma indicated that the catabolic changes, which are visible within the acellular Bowman's membrane, are probably also occurring deeper within the stroma, but are masked and not readily detectable.

The effect of age on the macromolecular permeability of human Bruch's membrane.

PURPOSE: To determine whether age-related changes in Bruch's membrane affect its permeability to macromolecules. Such changes have been postulated to underlie some pathologic manifestations of age-related macular degeneration. METHODS: Bruch's membrane preparations were isolated from the macular region of donated human eyes of differing age and mounted in a modified Ussing chamber. Permeability to macromolecules was assessed by simultaneously placing a physiological concentration of serum proteins adjacent to the choroidal margin of the membrane preparation and a saline solution adjacent to the retinal pigment epithelial basement membrane. After 24 hours, the protein content of the saline solution was measured by standard assay and permeability calculated as the quantity of protein traversing the membrane preparation per unit area. The spectrum of proteins able to cross the membrane was assessed by subjecting the diffusate proteins to electrophoretic separation and the resultant gel to scanning densitometry. RESULTS: The permeability of Bruch's membrane to serum proteins decreased 10-fold from the first to the ninth decade of life, and on regression analysis this decline exhibited a linear relationship with donor age (P < 0.0005). Membrane preparations from young donors were permeable to proteins with a molecular weight in excess of 200 kDa, but with increasing age, the membrane progressively impeded the passage of high-molecular-weight entities. Even so, elderly membranes were still permeable to macromolecules with molecular weights in excess of 100 kDa. Results from the oldest preparation studied suggest that by the ninth decade, the membrane may selectively impede the flux of specific proteins, based on a criterion other than molecular weight. CONCLUSIONS: The results imply that, with increasing age, the capacity of Bruch's membrane to facilitate macromolecular exchange between the choroidal and the retinal pigment epithelial compartments is reduced.

Keratocyte networks visualised in the living cornea using vital dyes.

Fluorescent viability probes have been used to visualise and investigate the viability, morphology and organisation of the keratocyte within the stroma of the intact living cornea. The live cell probe, calcien-AM, in combination with a dead cell probe, ethidium homodimer (Live/Dead Assay, Molecular Probes, U.S.A.) proved superior to earlier generation vital dyes such as fluorescein diacetate or 5,6-carboxyfluorescein diacetate, initially used in combination with ethidium bromide. The ubiquitous distribution of esterase enzymes that cleave calcien-AM within the keratocyte cytoplasm produced a high concentration of fluorescently active calcein throughout the cell, including fine cell processes. Epi-illuminated fluorescence microscopy on transparent corneal dissections subsequently revealed details of keratocyte microanatomy and three-dimensional network organisation in situ. Three morphologically discrete subpopulations of keratocytes were identified: two formed relatively small bands of cells, immediately subjacent to either Bowman's or Descemet's membranes, the third subpopulation constituting the majority of keratocytes typically located within the corneal stroma. The results indicate that calcein-AM is able to penetrate intact living cornea revealing cell viability, and it also has the capacity to 'trace' cellular elements and reveal fine structure within a dense connective tissue matrix.

A clinical, psychophysical, and electroretinographic survey of patients with autosomal dominant retinitis pigmentosa.

We have surveyed 104 patients (44 families) with autosomal dominant retinitis pigmentosa. The range of the survey includes clinical history, ocular examination, documentation of genetic history, Goldmann kinetic perimetry with IV/4 and I/4 white targets, two-colour static perimetry, and scotopic and photopic electroretinography. Comparison of interfamilial and intrafamilial patterns in the static perimetry data strongly suggests there may be at least two genetic subgroups within the disease characterised by the pattern of loss of rod function: in subgroup D (13 patients, 4 families) this is diffuse and severe, while in subgroup R (28 patients, 13 families) it is regional. In both D and R loss of cone function is regional, and in R it coincides with loss of rod function. In D patients the rod electroretinogram is absent; in all but two R cases it is present and usually substantial. All D patients were aware of night blindness before the age of 10, but most R patients not until after the age of 20. Many of the patients could not be classified because their disease was so advanced. The effect of disease duration on visual acuity and visual field area is described for all patients.

Rod and cone activity in patients with dominantly inherited retinitis pigmentosa: comparisons between psychophysical and electroretinographic measurements.

Extended electroretinographic (ERG) testing has been carried out in a series of patients with retinitis pigmentosa, dominantly inherited. In 36 of 57 cases only cone b waves were evoked. In 20 of these, psychophysical tests showed only cones mediated vision (Massof class I), while in 16 statis scotopic perimetry demonstrated residual rod function (class II). In the remaining cases, where rod ERGs were seen, the light intensities required to evoke responses were not greatly elevated. A computer model was constructed to relate psychophysical threshold measurements to ERG data. This analysis of the results suggests that in one subgroup of patients the scotopic ERG is smaller than expected from the losses of visual field and that in another the psychophysical elevation of rod visual threshold is greater than expected from ERG measurements.

A modified ERG technique and the results obtained in X-linked retinitis pigmentosa.

An electroretinographic (ERG) technique is described in which the relationship between scotopic b wave amplitude and stimulus light intensity is determined. The relative amplitude of scotopic to photopic responses is assessed by means of red light and flicker. The method is applied to the detection of ERG abnormalities in heterozygotes for X-linked retinitis pigmentosa. These have been found in only a proportion of cases. The ERG results can be used to suggest the nature of the retinal abnormality.

Evidence for functional endothelial cell damage in early diabetic retinopathy.

Two aspects of endothelial cell function were examined in two matched groups of male insulin-dependent diabetics, six with background retinopathy and seven without retinopathy. Leakage of fluorescein from the retinal capillaries was estimated by vitreous fluorophotometry. In addition, factors VIII/von Willebrand (vWF) and VIII-related antigen (VIII-RAG), which are synthesized by the endothelial cells, were measured, together with VIII-antihaemophilic factor (VIII-AHF). The patients without retinopathy had normal leakage of fluorescein in the macula (mean +/- SEM: 1.10 +/- 0.10 g X 10(-8)/ml) and the posterior vitreous (0.45 +/- 0.11 g X 10(-8)/ml), and normal circulating levels of vWF (123% of a normal reference plasma +/- 18%), VIII-RAG (137 +/- 14%) and VIII-AHF (112 +/- 18%). In contrast, the patients with background retinopathy showed higher leakage of fluorescein in the macula (6.34 +/- 1.74 g X 10(-8)/ml; p less than 0.01), and the posterior vitreous (3.09 +/- 0.94 g X 10(-8)/ml; p less than 0.02), as well as increased levels of vWF (177 +/- 16%; p less than 0.05). There was a trend towards increased VIII-RAG (195 +/- 24%; p less than 0.1), but not VIII-AHF (126 +/- 13%). Alterations of endothelial cell function thus accompany the development of retinopathy. It cannot be said from the present study whether these alterations also precede the appearance of retinopathy.