A. A. Hussain

Age-related alterations in the diffusional transport of amino acids across the human Bruch's-choroid complex.

Photoreceptor maintenance is dependent on effective delivery of nutrients from the choroidal circulation by way of the acellular Bruchs membrane and the retinal pigment epithelium. Aging of Bruchs membrane is associated with thickening, increased cross linking of fibers, and deposition of debris culminating in reduced porosity. The present study has investigated the effects of aging on the diffusional transport of eight amino acids across Bruchs membrane in 19 human donors. Diffusion studies were carried out in Ussing chambers, and the amount of time-dependent transfer of amino acids across the preparation was quantified by reverse-phase high-performance liquid chromatography. Diffusion rates for all amino acids showed a significant linear decline with aging of donor. The importance of this reduction in delivery of amino acids is discussed with reference to both normal physiology and age-related macular degeneration.

Macromolecular diffusion characteristics of ageing human Bruch's membrane: implications for age-related macular degeneration (AMD).

Macromolecular species such as retinal binding protein, transferrin, ceruloplasmin, etc., released by the fenestrated choroidal capillaries must diffuse across Bruchs membrane for interaction with the basal membranes of the retinal pigment epithelium (RPE) for delivery of essential metabolites to the neural retina. The patency of this pathway through ageing Bruchs was examined by quantifying the diffusional flux of a 21.2 kDa fluorescein-isothiocyanate labelled dextran. Dextran flux measurements across Bruchs membrane from the macular region of the human fundus showed a highly significant decrease (p < 0.001) with ageing of donor such that diffusional transport in the ninth decade was about 6.5% of that in the first decade of life. Peripheral regions also showed a highly significant decline (p < 0.001) but ageing changes were considerably slowed in comparison to the macula with diffusional rates in the ninth decade being about 44% of that in the first decade. Peripheral samples from AMD donors displayed diffusional rates that were lower than the control population. The age-related decline in macromolecular diffusion across Bruchs membrane suggests that in the elderly, the patency of the conducting pathways may be compromised and in the more advanced ageing of Bruchs associated with AMD, the metabolic trafficking of carrier proteins may be severely impaired.

An experimental study of the elastic properties of the human Bruch’s membrane-choroid complex: relevance to ageing

Aim: To investigate the mechanical properties (stress-strain relation, elasticity, hysteresis, response to stress spikes and drops) of isolated human Bruch’s membrane-choroid, as well as the effect of ageing and aged related macular degeneration (AMD). Methods: 13 Bruch’s membrane-choroid complexes were obtained from human donors (21–97 years). Two samples (aged 85 and 95) showed signs of AMD including large, soft drusen, choroidal neovascularisation, and/or disciform scars. Various hydrostatic pressures (stress) were applied to the choroidal surface of mid-peripheral samples mounted in a modified open Ussing chamber. Linear scans of the tissue were recorded by optical coherence tomography (OCT) and the pressure induced deformation (strain), elasticity, hysteresis, and response to pressure spikes and drops measured. Results: The elasticity of human Bruch’s membrane-choroid complex decreased linearly with ageing (p<0.001) after the age of 21 with an approximate reduction of 1% per year. The decrease was not exaggerated in AMD. The recoil capacity of Bruch’s membrane-choroid was not affected by ageing. The response to pressure spikes/drops was similar in age matched normal and AMD eyes. The results suggest that although the aged induced decrease in Bruch’s membrane elasticity may contribute to breaks in this membrane in AMD leading to neovascularisation this is not sufficient. The presence of other factors is required for its development. Conclusion: The elasticity of Bruch’s membrane-choroid complex decreases with age while recoil capacity does not. The decrease was not exaggerated in AMD.

Human Transscleral Albumin Permeability and the Effect of Topographical Location and Donor Age

PURPOSE To quantify the permeability coefficient of albumin across human sclera and to assess topographical and age-related variation. METHODS Equatorial superotemporal scleral tissue from 15 donor eyes (mean age 60 years; range 39-84) was mounted in a modified Ussing chamber. Additional tissue was taken from the anterior and posterior superotemporal regions of six eyes, and equatorial superonasal, and inferotemporal regions of a further six eyes. Fluorescein isothiocyanate (FITC)-labeled, 0.412 mM, bovine albumin was placed in one hemichamber facing the internal scleral surface, and the rate of transscleral flux was determined over 24 hours, at 25 degrees C, with a spectrophotometer. RESULTS Permeability coefficient for equatorial superotemporal scleral tissue at 25 degrees C (+/-SD) was 0.83 +/- 0.50 x 10(-6) cm . s(-1). The permeability coefficient adjusted for 37 degrees C (+/-SD) was 1.43 +/- 0.86 x 10(-6) cm . s(-1). The effect of donor age was assessed for the 15 equatorial superotemporal samples. Regression analysis showed a significant decline in scleral diffusion of albumin with increasing donor age (P = 0.0166). There was no significant difference in diffusion over the different topographical regions tested. The partition coefficient of permeability to albumin also showed a decline with increasing donor age (P = 0.001). CONCLUSIONS The permeability and partition coefficients of human sclera both significantly decline with increasing donor age. Permeability coefficient shows no significant variation over the different topographical regions tested. The decrease in albumin permeability with increasing donor age may have pharmacokinetic implications when considering transscleral diffusion of high-molecular-weight compounds.

Scleral hydraulic conductivity and macromolecular diffusion in patients with uveal effusion syndrome

PURPOSE To determine whether uveal effusion syndrome (UES) is caused by altered scleral permeability to water and large molecules. METHODS Transscleral water movement was measured using surgically removed sclera clamped in a modified Ussing chamber and connected to a water column set at intraocular pressure. Sclera was also clamped between two hemichambers, and transscleral diffusion of FITC-dextrans (4.4-77 kDa) was measured with a spectrophotometer. Clinical data were prospectively collected using postal questionnaires. RESULTS Ten patients (mean age, 63 years; mean spherical equivalent, +4.7 D) had a median preoperative visual acuity of 0.20 that improved to 0.33 after surgery. Nine eyes showed visual improvement, three worsened, and two were unchanged. Histology showed disorganization of collagen fibrils, with amorphous deposits expanding the interfibrillary spaces. The mean thickness (+/-1 SD) of the excised scleral specimens was 585 +/- 309 microm, and the mean specific hydraulic conductivity was 23.9 +/- 27.5 x 10(-14) cm(2), compared with 5.8 +/- 3.9 x 10(-14) cm(2) in age-matched control specimens (P = 0.068). Three specimens had hydraulic conductivity above the 95% CI of the controls. Control eyes showed a significant reduction in diffusion coefficient (D) with age. Eyes had a mean D of 5.69 +/- 5.35 x 10(-8) cm(2) x s(-1), similar to control eyes (6.14 +/- 2.40 x 10(-8) cm(2) x s(-1), 20 kDa dextran). In one eye, the result was higher than the 95% CI of the control; in three, it was lower. CONCLUSIONS UES is not caused by reduced scleral hydraulic conductivity, which tends to be higher than expected. Reduced macromolecular diffusion may impede the normal transscleral egress of albumin with subsequent osmotic fluid retention in some, but not all eyes.

Increased sequestration of matrix metalloproteinases in ageing human Bruch's membrane: implications for ECM turnover.

PURPOSE The ageing of Bruchs membrane is associated with progressive reduction in the degradation of the capacity for ECM turnover mediated by the matrix metalloproteinase (MMP) system. In this study, the free and bound pools of all gelatinase species were quantified to aid in assessing the likelihood of reduced availability of pro-MMPs for activation in ageing Bruchs membrane. METHODS Bruchs membrane from macular locations (10 eyes; donor age range, 21-84 years) was mounted in Ussing chambers and eluted with phosphate-buffered saline to release the free pool of MMPs. Free and bound pools of MMPs were subjected to gelatin zymography, and individual gelatinase species were quantified by densitometric scans. RESULTS The zymograms displayed six gelatinase species: four corresponding to the pro- and active forms of MMP-2 and -9 and two high-molecular-weight polymeric forms designated HMW1 and -2, corresponding to approximate molecular masses of 195 and 391 kDa, respectively. The ageing of Bruchs membrane was associated with an exponential increase in the percentage of pro-MMPs bound to the membrane (pro-MMP-2: %age bound = 0.54 exp(0.04 x age), r = 0.87, P < 0.01; and pro-MMP-9: %age bound = 5.0 exp(0.03 x age), r = 0.8, P < 0.01). A similar exponential increase was seen in the percentage of bound HMW1 species (%bound = 11.7 exp(0.018 x age; P < 0.05). The HMW2 species was virtually all bound to the membrane, but some release was observed in the very elderly. CONCLUSIONS The ageing of Bruchs membrane was associated with progressive sequestration of MMPs reducing the free concentration and potential for activation. These changes may underlie the reduction in degradation that leads to the age-related increase in the thickness of the membrane.

TRANSSCLERAL ALBUMIN DIFFUSION AND SUPRACHOROIDAL ALBUMIN CONCENTRATION IN UVEAL EFFUSION SYNDROME

Purpose: To test the hypothesis that uveal effusion syndrome is caused by reduced transscleral albumin permeability. Methods: Surgical scleral specimens were obtained from a 55-year-old patient with nanophthalmic uveal effusion syndrome. Specimens were clamped in a modified Ussing chamber, and the rate of transscleral diffusion of fluorescein isothiocyanate–albumin was measured over 12 hours, using a spectrophotometer and predetermined standard curves. The diffusion coefficient was determined at 20°C, and then adjusted to body temperature using Einsteins equation. Results in 3 scleral samples were compared with 10 age-matched controls. Albumin and total protein concentration were measured in choroidal fluid and serum. Results: Histologic staining with Alcian blue showed interfibrillary acid mucin deposits. Transmission electron microscopy showed deposits measuring 1 μm to 10 μm and collections of expanded, degenerate collagen fibrils. The mean (±SD) albumin diffusion coefficient was 12% of that in controls (1.22 ± 0.67 × 10−8 vs. 10.3 ± 7.0 × 10−8 cm2/second) and below the lower 95% confidence limit of the control group. The diffusion coefficient was calculated to increase 53% to 1.87 ± 1.03 × 10−8 cm2/second at 37°C. Choroidal albumin concentration was much higher than physiologic levels, measuring 200 g/L (total protein 321 g/L), 5 times the serum albumin concentration of 42 g/L (total protein 70 g/L). Conclusion: Nanophthalmic uveal effusion syndrome can be associated with reduced scleral permeability to albumin, and a very high concentration of retained suprachoroidal albumin. This will lead to an osmotic gradient that retains fluid and may partly explain the pathogenesis of uveal effusion syndrome in some patients.

High Molecular-Weight Gelatinase Species of Human Bruch's Membrane: Compositional Analyses and Age-Related Changes

PURPOSE The structural and functional demise of aging Bruchs membrane is associated with a reduction in the activity of the matrix metalloproteinase (MMP) degradation system. The gelatinase component of the MMP system consists of MMP2 and MMP9 and two high molecular-weight (HMW1, HMW2) species that are yet to be characterized and whose roles in the aging process are yet to be elucidated. The purpose of this study was to determine the age-related changes in levels of expression and subunit characterization of the HMW gelatinase species of Bruchs membrane. METHODS Gelatin zymography followed by densitometric scanning was used to quantify the level of the HMW species present. Gel-filtration chromatography allowed the fractionation of the gelatinases according to their molecular weight, and subsequent degradation of the HMW species with a mino-phenyl acetate activation, reduction, and alkylation produced subunit fragments for analysis. RESULTS Most of the HMW1 and HMW2 pool (80% and 87%, respectively) were tightly bound to the matrix. Aging was associated with significant increases in the levels of HMW1 and HMW2 (P < 0.005 and P < 0.05 respectively). On gel filtration, a single large macromolecular complex (LMMC) was observed containing HMW1, HMW2, MMP9, and some MMP2. Activation-mediated fragmentation of HMW1 and HMW2 showed them to be composed of heteropolymers of MMP2 and MMP9. CONCLUSIONS The age-related increase of HMW1 and HMW2, together with the formation of LMMC, resulted in the sequestration of MMP2 and MMP9, thereby reducing the free pool for activation. This is likely to contribute to reduced matrix degradation and turnover of Bruchs membrane in both normal aging and age-related macular degeneration.

No difference in serum sialic acid in type 2 diabetic patients from the United Arab Emirates with and without diabetic retinopathy.

Serum total sialic acid (TSA) has recently been shown to be related to diabetic retinopathy. However, there is some controversy as this may be true in European Type 2 diabetic patients but not South Asians. There are few data looking at serum TSA expression in Arab Type 2 diabetic patients from the United Arab Emirates (UAE) and we wished to test the hypothesis that there may be different serum TSA expression in Arab Type 2 diabetic patients as regard to retinopathy. Sixty-five Type 2 diabetic patients from the UAE were studied (19 male and 46 female, age 57.5+/-9.8 (45-74) years, duration of diabetes 9.4+/-5.7 (0-22) years. The serum TSA in 13 patients with diabetic retinopathy was 757+/-130 mg/l and 782+/-163 mg/l in those without retinopathy (NS). There was no significant correlation between serum TSA and patient age, serum fructosamine, diabetes duration, or blood pressure. As in South Asians serum TSA does not appear to be elevated in Type 2 diabetic patients from the United Arab Emirates with diabetic retinopathy.

Disturbed Matrix Metalloproteinase Pathway in Both Age-Related Macular Degeneration and Alzheimer's Disease

Purpose. Abnormal protein deposits including β-amyloid, found in ageing Bruchs membrane and brain, are susceptible to degradation by matrix metalloproteinases (MMPs). In ageing Bruchs membrane, these MMPs become less effective due to polymerisation and aggregation reactions (constituting the MMP Pathway), a situation much advanced in age-related macular degeneration (AMD). The likely presence of this MMP Pathway in brain with the potential to compromise the degradation of β-amyloid associated with Alzheimers disease (AD) has been investigated. Methods. Presence of high molecular weight MMP species (HMW1 and HMW2) together with the much larger aggregate termed LMMC was determined by standard zymographic techniques. Centrigugation and gel filtration techniques were used to separate and quantify the distribution between bound and free MMP species. Results. The MMP Pathway, initially identified in Bruchs membrane, was also present in brain tissue. The various MMP species displayed bound-free equilibrium and in AD samples, the amount of bound HMW1 and pro-MMP9 species was significantly reduced (p < 0.05). The abnormal operation of the MMP Pathway in AD served to reduce the degradation potential of the MMP system. Conclusion. The presence and abnormalities of the MMP Pathway in both brain and ocular tissues may therefore contribute to the anomalous deposits associated with AD and AMD.