Crystallin distribution in Bruch's membrane-choroid complex from AMD and age-matched donor eyes

Crystallins were consistently found in a recent proteomic analysis of drusen from age-related macular degeneration (AMD) donor eyes. Here we compare the distribution of several crystallins in drusen, Bruch's membrane and choroid from AMD and non-AMD age-matched control eyes. Immunohistochemistry and Western blots of tissue samples were performed using antibodies to αA- and αB-crystallins. Bruch's membrane, drusen and the subjacent choroidal connective tissue from AMD tissues showed greater immunoreactivity for αA- and αB-crystallins than were observed in normal age-matched control tissues. Western blots also demonstrated more intense αA- and αB-crystallin signals from AMD tissues than were present in age-matched controls. These data indicate that αA- and αB-crystallins accumulate in Bruch's membrane and choroidal connective tissues to a greater degree in AMD than in normal aging. These findings suggest that the accumulation of these small heat shock proteins at this critical interface below the RPE reflects a disease-related stress response manifested during the progression of AMD.     

Corneal autofluorescence in diabetic and normal eyes

Corneal autofluorescence has been lately studied as a predictor of retinopathy severity in diabetic patients. We measured corneal autofluorescence in 138 eyes of 69 diabetic patients and 64 eyes of 32 healthy controls. Diabetic patients were subdivided by the severity of retinopathy according to the Modified Airlie House Classification (stage 1: no or minimal retinopathy; stage 2: minimal background retinopathy; stage 3: background retinopathy; stage 4: (pre-) proliferative retinopathy. The fluorescence peak value and the area underlying the corneal autofluorescence curve were measured with a scanning fluorophotometer (Fluorotron Master, Coherent Radiation Palo Alto CA) Healthy controls' values of corneal autofluorescence (peak value: mean 11.03±3.77 ng. equivalent fluorescein/ml; area: mean 163.85±61.40 scan-point × ng. equivalent fluorescein/ml) resulted similar (peak value: p=0.83; area: p=0.61) to those of diabetic patients without retinopathy (peak value: mean 11.2±3.4 ng.eq/ml; area: 170.07±57.23 scan-pnt.ng.eq/ml). A statistically significant difference was found between diabetic patients without retinopathy and patients with stage 2, 3, 4 retinopathy. No statistically significant difference was found both for the peak value (p=0.50) and for the area (p=0.61) between stage 3 and stage 4 retinopathy. The sensitivity and specificity of corneal autofluorescence as a screening test for diabetic retinopathy were 82% and 62% for the peak value, 87% and 60% for the area; the positive predictive value for the presence of diabetic retinopathy was 65% for the peak and 63% for the area.     

Cigarette smoke-related oxidants and the development of sub-RPE deposits in an experimental animal model of dry AMD

PURPOSE: Oxidative injury to the retinal pigment epithelium (RPE) has been proposed to be an important injury stimulus relevant to the accumulation of subretinal deposits in age-related macular degeneration (AMD). Cigarette smoking is a major risk factor for AMD, and cigarette smoke-related tar contains high concentrations of a potent oxidant, hydroquinone (HQ). This study was an investigation of the effects of cigarette smoke (CS) and HQ in the development of sub-RPE deposits in an experimental mouse model. METHODS: Sixteen-month-old C57BL/6 female mice were fed a high-fat diet (HFD) for 4.5 months. Mice were divided into two major experimental groups, one to examine the effects of cigarette smoke and one to study the effects of a defined cigarette smoke component such as HQ. In the first group, mice eyes were exposed to blue-green light (positive controls) or to whole cigarette smoke. A third group with no intervention served as the negative control. In the second experimental group, animals received a purified diet with HQ (0.8%) with low or high fat content for 4.5 months. Mice in both groups were euthanatized at 4.5 months and eyes processed for transmission electron microscopy. RESULTS: As previously demonstrated by our laboratory and others, most mice fed an HFD without other oxidant exposure demonstrated normal morphology or, in a few cases, small nodular basal laminar deposits. Eyes of mice exposed to whole cigarette smoke or to HQ in the food demonstrated a variable degree of basal laminar deposits and diffusely thickened Bruch's membrane. The choriocapillaris endothelium was variably hypertrophic. CONCLUSIONS: Exposure to cigarette smoke or the smoke-related redox molecule, HQ, results in the formation of sub-RPE deposits, thickening of Bruch's membrane, and accumulation of deposits within Bruch's membrane. Smoke-related oxidants may be another oxidative injury stimulus to the choriocapillaris and RPE, and may explain the association between cigarette smoking and early AMD.     

Clathrin and adaptin accumulation in drusen, Bruch's membrane and choroid in AMD and non-AMD donor eyes

Clathrin was identified in a recent proteomic analysis of Bruch's membrane from age-related macular degeneration (AMD) donor eyes. The present study was conducted to determine the localization of clathrin in AMD tissues and to compare this distribution and relative content with that in non-AMD control tissues. The distribution of adaptin, which is functionally linked to clathrin, was also evaluated. Human eyes were from donors between 66 and 94 years of age; 13 eyes were from donors with AMD and 13 from non-AMD donors. Bruch's membrane and choroid from the macula of each donor eye were prepared for immunohistochemistry and Western blotting. Differences in immunoreactivity were quantitated. Drusen, Bruch's membrane and choroid from AMD tissues showed greater immunoreactivity for clathrin and adaptin than did non-AMD tissues. Western blots also showed more intense clathrin and adaptin immunoreactivity in AMD tissues than were present in non-AMD samples. This study suggests that accumulation of clathrin and adaptin in drusen, Bruch's membrane and choroid may reflect a higher rate of clathrin mediated endocytosis in AMD tissues. Alternatively, the accumulation of these proteins in these extracellular compartments may reflect a higher susceptibility to oxidative damage.     

Drusenoid maculopathy in rhesus monkeys: autofluorescence, lipofuscin and drusen pathogenesis

PURPOSE: To examine patterns of retinal pigment epithelial autofluorescence and lipofuscin accumulation in relation to drusen and to explore the pathogenesis of drusen in rhesus monkeys. METHODS: The macular areas of six rhesus monkeys, euthanized at 19 to 28 years of age, were studied by bright field and fluorescence light microscopy and transmission electron microscopy. RESULTS: There was strong autofluorescence in the retinal epithelium that tended to diminish over drusen. Electron microscopy revealed that all retinal epithelial cells had large concentrations of lipofuscin bodies. The epithelial cells overlying drusen, however, tended to have less lipofuscin than epithelial cells not associated with drusen. Electron microscopy revealed that the epithelial cells overlying drusen were losing segments of cytoplasm containing lipofuscin bodies. Macrophage-like cells were consistently present in Bruch's membrane microns away from this lipofuscin-containing cytoplasmic material. CONCLUSIONS: Retinal epithelial cells overlying drusen have less lipofuscin than neighboring epithelial cells. The loss of lipofuscin seems due to a loss of cytoplasm containing lipofuscin that contributes to drusen formation. Macrophages in Bruch's membrane may be responsible for removing this lipofuscin debris. The results support in vivo studies showing reduced autofluorescence over drusen and support the "budding" of epithelial cytoplasm as a source of drusen material.     

Characteristics of drusen and Bruch's membrane in post-mortem eyes with age-related macular degeneration

Background: Different types of drusen and changes in Bruch's membrane have been associated with age-related macular degeneration (AMD). Methods: We compared 51 eyes with different stages of AMD with 40 age-matched controls using light microscopy. The degree of calcification of Bruch's membrane, fragmentation of Bruch's membrane, number of different types of drusen, and basal laminar deposit (BLD) were assessed. Results: In the macular area, the presence of basal laminar deposit was most strongly associated with the presence of AMD. There was a statistically significant difference observed in the degree of calcification and fragmentation of Bruch's membrane in eyes with AMD as compared to controls. Eyes with AMD displayed significantly more soft, confluent, and large drusen as compared to controls. Conclusion: Calcification and fragmentation of Bruch's membrane, soft, confluent, and large drusen and BLD but not hard drusen correlated strongly with the histologic presence of AMD. Calcification and fragmentation of Bruch's membrane seem to facilitate ingrowth of choroidal neovascular membranes with consecutive development of exudative AMD.      

Spectral sensitivity in eyes with macular holes and their fellow eyes

Background: This prospective study was undertaken to investigate whether spectral sensitivity can be useful in determining the prognosis of fellow eyes of eyes with macular holes. Methods: Spectral sensitivity measurements using a one degree test spot presented at a rate of 1 Hz and 25 Hz on a bright (1000 td) white background were carried out on 10 patients aged between 67 and 74 years (mean age 70.3 ± 2.6 years). Each patient had a full thickness macular hole in one eye and a normal contralateral fellow eye. The spectral sensitivity measurements were made with eccentric fixation in the eyes with macular holes and with central fixation in the normal fellow eye. A year later, the patient files were reviewed to look at the patient's ocular condition. Another 10 subjects between the ages of 50 and 80 years (mean age 69.5 ± 4.2 years) were also seen. These control group subjects had visual acuities of 6/9 or better with minimal ocular media changes and no ocular or systemic pathology that could affect colour vision. Results: The 1 Hz and 25 Hz spectral sensitivities of all patients were reduced for both eyes. Despite the good eye without a macular hole having a VA of 6/6, the spectral sensitivity was similar to that of the eye with the macular hole and markedly reduced visual acuity. Conclusion: The present investigation enabled us to examine the chromatic and achromatic mechanisms by testing spectral sensitivity at 1 Hz and 25 Hz, respectively. The data revealed that both chromatic and achromatic processing could be damaged in the eye with a macular hole. Surprisingly, the spectral sensitivities of both 1 Hz and 25 Hz are equally reduced in the good fellow eye with no macular hole. A one‐year follow‐up showed that two of the 10 patients (20 per cent) did eventually develop a macular hole in the normal fellow eye. This indicates that there is some subclinical foveal dysfunction in the normal fellow eye, the nature of which is unclear.     

Sensitivities in older eyes with good acuity: eyes whose fellow eye has exudative AMD

We compared several indices of foveal visual function between two groups of people aged 60 and older. One group was comprised of individuals who had good acuity in one eye, but had a history of exudative aging macular degeneration (AMD) in the other eye. We measured visual function in these individuals' good eyes only. The second group was a normative group; it was comprised of individuals who had good acuity in each eye. None of the eyes which we tested from either group had funduscopic evidence of macular pathology other than macular drusen and/or hypopigmentation. We found that eyes whose fellow eye had suffered from exudative AMD themselves suffered compromised foveal function, even when they retained 20/20 or better acuity. Losses of sensitivity mediated by blue-sensitive cones tended to be greater for 1 degree than for 3 degrees diameter test stimuli. Absolute sensitivity losses at long test wavelengths were probably due to several factors, including decreased effective cone photopigment density. Slow rates of recovery during dark adaptation were associated with the presence of many macular drusen and/or macular hypopigmentation. Eyes whose fellow eye had suffered from exudative AMD had more macular drusen and hypopigmentation than eyes whose fellow eye had not suffered from exudative AMD.     

Infrared and fundus autofluorescence imaging in eyes with optic disc pit maculopathy

Background: To characterize the infrared (IR) and fundus autofluorescence (FAF) images of eyes with optic disc pit maculopathy. Methods: A retrospective non‐comparative study of three patients with optic disc pit maculopathy who were followed by optical coherence tomography (OCT) and retinal angiography (Heidelberg retina angiograph 2) before and after vitreous surgery without laser photocoagulation. Results: The areas of serous retinal detachment and inner retinal schisis were dark in the IR and FAF images preoperatively; and they changed to brighter areas following reattachment of the retina. There was an increase in the granular hyperfluorescence in the FAF images accompanied by an increase in the number of subretinal precipitates. OCT showed a thickening of the photoreceptor outer segments. The outer retinal layer defect initially appeared bright in the IR images and hypofluorescent or hyperfluorescent in the FAF images. The outer retinal layer defect became larger with a reduction of the outer retinal schisis and increased retinal detachment, but then became smaller with the resolution of the retinal detachment. All cases had a resolution or reduction of the retinal detachment and retinal schisis after the vitrectomy. Conclusion: The IR and FAF images in eyes with optic disc pit maculopathy reflect the changes in the inner and outer retinal layers corresponding with the stage of recovery of the disease.     

Comparison of morphology of human macular and peripheral Bruch's membrane in older eyes

Deposits in macular human Bruch's membrane (BrM) increase with age and have been postulated to be associated with age-related maculopathy. We used two ultrastructural methods to compare these deposits by electron microscopy in macular and peripheral BrM of eight eyes from donors 63-86 years of age. Quick-freeze/deep-etch (QFDE) was used to prepare replicas that showed the ultrastructure of deposits, and osmium-tannic acid-paraphenylenediamine (OTAP) was used to preserve small extracellular lipid particles. We found that an accumulation of lipoprotein-like particles (LLPs) occurred in the peripheral BrM just as it does in the macular region, but with perhaps a somewhat slower time course. The "lipid wall," reported in macular BrM, was also found occasionally in the peripheral regions. The same processes that lead to age-related accumulation of LLPs in macular BrM appear to also occur in the peripheral regions.     

Correlation of fundus autofluorescence with photoreceptor morphology and functional changes in eyes with retinitis pigmentosa

Purpose: To assess and correlate fundus autofluorescence (FAF) characteristics with photoreceptor morphology and functional features in eyes with retinitis pigmentosa (RP). Methods: Thirty‐four eyes of 17 patients with RP were examined. We compared FAF images obtained by confocal scanning laser ophthalmoscopy with Spectral‐domain optical coherence tomography (SD‐OCT) and retinal function assessed by microperimetry. Results: Normal FAF surrounded by a ring of increased FAF at the macular area was detected in 32 (94%) eyes. The diameter of the normal FAF was correlated significantly with the preserved area of the photoreceptor inner segment and outer segment (IS/OS) junction on SD‐OCT (R = 0.939, p < 0.001). The area outside the ring was associated with loss of IS/OS junction and external limiting membrane (ELM). The ring of increased FAF demarcated the border between the central retina with preservation of the IS/OS junction and ELM, and the adjacent eccentric retina with loss of these bands. In two eyes of one patient, there was no preservation of normal FAF at the macula and the photoreceptor IS/OS junction was not detected on SD‐OCT. The mean retinal sensitivity derived from microperimetry was correlated significantly with the area of normal FAF (R = 0.929, p = 0.007) and the preserved area of the IS/OS junction (R = 0.851, p = 0.032). Ten eyes had progressive reduction in size of the normal FAF inside the ring accompanied by decreased area of preserved IS/OS during 3.1 years. Conclusion: FAF appears to reflect the integrity of the photoreceptor layer. It may serve as a secondary outcome measure for novel therapeutic strategies for RP.     

视网膜色素上皮、Bruch膜及脉络膜老年性改变的定量分析

目的 研究及定量分析视网膜色素上皮（retinal pigment epithelium,RPE)、Bruch膜及脉络膜的老年性改变。方法 应用改良Masson三色组织染色法及电脑图像分析软件对36例不同年龄(3～93岁)正常眼RPE数量和高度、Bruch膜厚度、脉络膜毛细血管的密度及脉络膜厚度的老年性改变进行定量分析。 结果 与年龄有显著相关性的老年性改变包括RPE细胞数量的减少(8个/ mm²·年)、RPE细胞高度的增加(0.01μm/年) 以及Bruch膜增厚(0.02μm/年);脉络膜毛细血管密度及脉络膜厚度的改变与年龄之间无显著的相关关系。 结论 RPE细胞数量减少、高度增加以及Bruch膜的增厚可能是黄斑区最早的老年性改变。(中华眼底病杂志,2000,16:236-239)     

Correlation between the area of increased autofluorescence surrounding geographic atrophy and disease progression in patients with AMD

PURPOSE: To test the hypothesis that the extension of areas with increased fundus autofluorescence (FAF) outside atrophic patches correlates with the rate of spread of geographic atrophy (GA) over time in eyes with age-related macular degeneration (AMD). METHODS: The database of the multicenter longitudinal natural history Fundus Autofluorescence in AMD (FAM) Study was reviewed for patients with GA recruited through the end of August 2003, with follow-up examinations within at least 1 year. Only eyes with sufficient image quality and with diffuse patterns of increased FAF surrounding atrophy were chosen. In standardized digital FAF images (excitation, 488 nm; emission, >500 nm), total size and spread of GA was measured. The convex hull (CH) of increased FAF as the minimum polygon encompassing the entire area of increased FAF surrounding the central atrophic patches was quantified at baseline. Statistical analysis was performed with the Spearman's rank correlation coefficient (rho). RESULTS: Thirty-nine eyes of 32 patients were included (median age, 75.0 years; interquartile range [IQR], 67.8-78.9); median follow-up, 1.87 years; IQR, 1.43-3.37). At baseline, the median total size of atrophy was 7.04 mm2 (IQR, 4.20-9.88). The median size of the CH was 21.47 mm2 (IQR, 15.19-28.26). The median rate of GA progression was 1.72 mm2 per year (IQR, 1.10-2.83). The area of increased FAF around the atrophy (difference between the CH and the total GA size at baseline) showed a positive correlation with GA enlargement over time (rho=0.60; P=0.0002). CONCLUSIONS: FAF characteristics that are not identified by fundus photography or fluorescein angiography may serve as a prognostic determinant in advanced atrophic AMD. As the FAF signal originates from lipofuscin (LF) in postmitotic RPE cells and since increased FAF indicates excessive LF accumulation, these findings would underscore the pathophysiological role of RPE-LF in AMD pathogenesis.