Decreased Thickness and Integrity of the Macular Elastic Layer of Bruch's Membrane Correspond to the Distribution of Lesions Associated with Age-Related Macular Degeneration
2005; Elsevier BV; Volume: 166; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)62248-1
ISSN1525-2191
AutoresVictor Chong, Jason Keonin, Philip J. Luthert, Christina Frennesson, David M. Weingeist, Rachel L. Wolf, Robert F. Mullins, Gregory S. Hageman,
Tópico(s)Retinal Imaging and Analysis
ResumoAge-related macular degeneration (AMD) is a leading cause of blindness in the elderly. In its severest form, choroidal neovessels breach the macular Bruch's membrane, an extracellular matrix compartment comprised of elastin and collagen laminae, and grow into the retina. We sought to determine whether structural properties of the elastic lamina (EL) correspond to the region of the macula that is predilected toward degeneration in AMD. Morphometric assessment of the macular and extramacular regions of 121 human donor eyes, with and without AMD, revealed a statistically significant difference in both the integrity (P < 0.0001) and thickness (P < 0.0001) of the EL between the macular and extramacular regions in donors of all ages. The EL was three to six times thinner and two to five times less abundant in the macula than in the periphery. The integrity of the macular EL was significantly lower in donors with early-stage AMD (P = 0.028), active choroidal neovascularization (P = 0.020), and disciform scars (P = 0.003), as compared to unaffected, age-matched controls. EL thickness was significantly lower only in individuals with disciform scars (P = 0.008). The largest gaps in macular EL integrity were significantly larger in all categories of AMD (each P < 0.0001), as compared to controls. EL integrity, thickness, and gap length in donors with geographic atrophy did not differ from those of controls. These structural properties of the macular EL correspond spatially to the distribution of macular lesions associated with AMD and may help to explain why the macula is more susceptible to degenerative events that occur in this disease. Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly. In its severest form, choroidal neovessels breach the macular Bruch's membrane, an extracellular matrix compartment comprised of elastin and collagen laminae, and grow into the retina. We sought to determine whether structural properties of the elastic lamina (EL) correspond to the region of the macula that is predilected toward degeneration in AMD. Morphometric assessment of the macular and extramacular regions of 121 human donor eyes, with and without AMD, revealed a statistically significant difference in both the integrity (P < 0.0001) and thickness (P < 0.0001) of the EL between the macular and extramacular regions in donors of all ages. The EL was three to six times thinner and two to five times less abundant in the macula than in the periphery. The integrity of the macular EL was significantly lower in donors with early-stage AMD (P = 0.028), active choroidal neovascularization (P = 0.020), and disciform scars (P = 0.003), as compared to unaffected, age-matched controls. EL thickness was significantly lower only in individuals with disciform scars (P = 0.008). The largest gaps in macular EL integrity were significantly larger in all categories of AMD (each P < 0.0001), as compared to controls. EL integrity, thickness, and gap length in donors with geographic atrophy did not differ from those of controls. These structural properties of the macular EL correspond spatially to the distribution of macular lesions associated with AMD and may help to explain why the macula is more susceptible to degenerative events that occur in this disease. Bruch's membrane is a stratified extracellular matrix complex that lies between the retinal pigment epithelium (RPE) and the choroidal capillary bed, or choriocapillaris. It is comprised of two collagen-rich layers, referred to as the inner and outer collagenous layers, that flank a central domain of elastin and elastin-associated proteins.1Marshall J Hussain A Starita C Moore D Patmore A Aging and Bruch's membrane.in: Marmor M Wolfensberger T The Retinal Pigment Epithelium. Oxford University Press, New York1988: 669-692Google Scholar, 2Guymer R Bird A Bruch's membrane, drusen, and age-related macular degeneration.in: Marmor M Wolfensberger T The Retinal Pigment Epithelium. Oxford University Press, New York1988: 693-705Google Scholar A number of age-related changes have been described in Bruch's membrane,3Hogan M Bruch's membrane and disease of the macula.Trans Ophthalmol Soc UK. 1967; 87: 113-161PubMed Google Scholar, 4Hogan M Alvarado J Studies on the human macula. 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These age-related alterations in Bruch's membrane could lead to a loss of the normal function of Bruch's membrane and promote degenerative changes in the aging eye. Various lines of evidence suggest that Bruch's membrane functions as a physical barrier to the egress of cells and vessels from the choroid into the sub-RPE and subretinal spaces. Disruption of, or damage to, this barrier is associated with loss of vision in a variety of ocular diseases, often resulting from the growth of new blood vessels—termed choroidal neovascular membranes (CNVM)—from the choroid into the sub-RPE and/or subretinal spaces. CNVM formation occurs in a number of macular degenerative diseases, including age-related macular degeneration (AMD), the leading cause of irreversible blindness in the developed world.31Bhagat N Flaxel C Nonexudative macular degeneration.in: Lim J Age-Related Macular Degeneration. Marcel Dekker, New York2002: 67-82Google Scholar Moreover, CNVM formation can be induced experimentally by thermal damage to Bruch's membrane in normal monkey eyes after laser photocoagulation32Ryan S Subretinal neovascularization. Natural history of an experimental model.Arch Ophthalmol. 1982; 100: 1804-1809Crossref PubMed Scopus (178) Google Scholar and potentially, after laser treatment of human CNVM.33Malthieu D Turut P Francois P Subretinal neovessels caused by photocoagulation.Bull Soc Ophthalmol Fr. 1988; 88: 645-646PubMed Google Scholar Interestingly, extramacular treatment is relatively ineffective, suggesting that the macula is inherently susceptible to choroidal neovascularization (CNV).34Lee S Shen W Yeo I Lai C Mathur R Tan D Constable I Rakoczy P Predilection of the macular region to high incidence of choroidal neovascularization after intense laser photocoagulation in the monkey.Invest Ophthalmol Vis Sci. 2003; 44 (E-abstract 3945)Google Scholar AMD-associated CNVM formation occurs in ∼4% of the population older than the age of 7535Smith W Assink J Klein R Mitchell P Klaver C Klein B Hofman A Jensen S Wang J de Jong P Risk factors for age-related macular degeneration: pooled findings from three continents.Ophthalmology. 2001; 108: 697-704Abstract Full Text Full Text PDF PubMed Scopus (805) Google Scholar and is primarily restricted to the macula. It accounts for 90% of severe visual loss associated with AMD, even though only ∼10 to 15% of individuals with AMD develop CNV. The macula is a unique 6-mm-diameter region of the posterior pole that lies directly in the visual axis of human and nonhuman primates (Figure 1A). This region of the retina develops postnatally and subserves fine acuity vision. The reason for the increased susceptibility of the macula to degeneration and CNVM formation in primates has not been elucidated. We have speculated that one possible explanation for the tendency of new vessels to breach the macular Bruch's membrane, as opposed to the extramacular regions, might be that there are regional differences in the structure and/or composition of Bruch's membrane. It is likely that the integrity and nature of its collagen and elastin components primarily dictate the basic structural and functional properties of Bruch's membrane. Hence, one might predict that the disruption of one or more layers of Bruch's membrane might precede CNV in AMD. Accordingly, we recently observed what we interpreted to be robust differences in the integrity and thickness of the elastic lamina (EL) of Bruch's membrane between the macular and extramacular regions in a series of human eyes. To evaluate further the topographical variation in the thickness and continuity of the EL, we assessed these parameters in a larger series of normal, unaffected donor eyes and compared them to those obtained from donors with early and late stages of AMD. The data collected herein show that the macular EL is substantially thinner and more porous than that in the extramacular region. This information has lead to the development of a working hypothesis that the structural attributes of the macular EL of Bruch's membrane may be related to, at least in part, the predilection of this region to degeneration in AMD, other macular dystrophies, and other conditions characterized by CNVM formation. The 121 human eyes used in this study were obtained from MidAmerica Transplant Services (St. Louis, MO), the Iowa Lions Eye Bank (Iowa City, IA), the Heartland Eye Bank (Columbia, MO), the Central Florida Lions Eye and Tissue Bank (Tampa, FL), and the Virginia Eye Bank (Norfolk, VA) after informed consent. Institutional Review Board committee approval for the use of human donor tissues was obtained from Human Subjects Committees at St. Louis University and the University of Iowa. All eyes were processed within 4 hours of death. The gross pathological features, as well as the corresponding fundus photographs and angiograms when available, of all eyes in this repository were read and classified by retinal specialists. Fundi were classified according to a modified version of the International AMD grading system.36Bird A Bressler N Bressler S Chisholm I Coscas G Davis M de Jong P Klaver C Klein B Klein R Mitchell P Sarks J Sarks S Soubrane G Taylor H Vingerling J An international classification and grading system for age-related maculopathy and age-related macular degeneration. The International ARM Epidemiological Study Group.Surv Ophthalmol. 1995; 39: 367-374Abstract Full Text PDF PubMed Scopus (1626) Google Scholar For this investigation, donors were placed into one of four categories: young unaffected (<62 years), age-matched unaffected (≥62 years), early-stage AMD (the youngest was 62 years old), and late-stage AMD. Late-stage AMD donors were subdivided into those with 1) geographic atrophy (GA), 2) active CNVMs, and 3) disciform scars preceded clinically by CNVM (CNV/DS). Note that some individuals refer to early-stage AMD as age-related maculopathy, or ARM; this term will not be used herein. Donors were classified as unaffected if they had no macroscopic or funduscopic signs of macular pathology or any ophthalmic history of AMD. Early AMD donors were defined based on an ophthalmic history of AMD and the presence of significant numbers of macular drusen, pigment disruption, and/or other clinical signs of early AMD. Polyclonal antisera directed against tropoelastin (PCAB 94, gift from Dr. Robert Mecham, Washington University, St. Louis, MO; PR398, Elastin Products, Owensville, MO), as well as monoclonal antibodies directed against bovine elastin (clone BA-4, Sigma, St. Louis, MO; MM436, Elastin Products) and human aortic α-elastin (PR533, Elastin Products) were used to examine the EL of Bruch's membrane. For experiments using Elastin Products’ antibodies, antigens were retrieved according to the manufacturer's instructions. Reactivity of antibodies with the EL of Bruch's membrane was analyzed in a series of young ( 60 years) donors. Posterior poles, or wedges of posterior poles spanning between the ora serrata and the macula, were fixed in 4% (para)formaldehyde in 100 mmol/L sodium cacodylate, pH 7.4, as described previously.29Hageman G Mullins R Russell S Johnson L Anderson D Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.FASEB J. 1999; 13: 477-484PubMed Google Scholar After 2 to 4 hours of fixation, eyes were transferred to 100 mmol/L sodium cacodylate and rinsed (3 × 10 minutes), infiltrated, and embedded in acrylamide. These tissues were subsequently embedded in OCT, snap-frozen in liquid nitrogen, and stored at −80°C. In addition, unfixed posterior poles, or wedges thereof, were embedded directly in OCT, without acrylamide infiltration or embedment. Both fixed and unfixed tissues were sectioned to a thickness of 6 to 8 μm on a cryostat. Immunolabeling was performed as described previously,29Hageman G Mullins R Russell S Johnson L Anderson D Vitronectin is a constituent of ocular drusen and the vitronectin gene is expressed in human retinal pigmented epithelial cells.FASEB J. 1999; 13: 477-484PubMed Google Scholar, 30Mullins R Anderson D Russell S Hageman G Ocular drusen contain proteins common to extracellular deposits associated with atherosclerosis, elastosis, amyloidosis, and dense deposit disease.FASEB J. 2000; 14: 835-846Crossref PubMed Scopus (729) Google Scholar using Alexa 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR). Adjacent sections were incubated with secondary antibody alone, to serve as negative controls. Some immunolabeled specimens were viewed by confocal laser-scanning microscopy.37Anderson D Mullins R Hageman G Johnson LV A role for local inflammation in the formation of drusen in the aging eye.Am J Ophthalmol. 2002; 134: 411-431Abstract Full Text Full Text PDF PubMed Scopus (893) Google Scholar These cellular nuclei in these sections were counterstained with TO-PRO-3 (Molecular Probes). Ocular tissues used for transmission electron microscopical studies were fixed by immersion fixation in one-half strength Karnovsky's fixative, within 4 hours of death, for a minimum of 24 hours. Trephine-punched specimens (see below) were fixed, transferred to 100 mmol/L sodium cacodylate buffer, pH 7.4, and subsequently dehydrated, embedded in epoxy resin, sectioned, and photographed, as described previously.38Hageman G Mullins R Molecular composition of drusen as related to substructural phenotype.Mol Vis. 1999; 5: 28PubMed Google Scholar, 39Russell S Mullins R Schneider B Hageman G Basal laminar drusen are indistinguishable in location, substructure, and composition from drusen associated with aging and age-related macular degeneration.Am J Ophthalmol. 2002; 129: 205-214Abstract Full Text Full Text PDF Scopus (132) Google Scholar To establish the baseline structural and topographical characteristics of the EL, irrespective of aging or AMD, three separate analyses were performed. In the first analysis, baseline topographic data were collected from a series of oriented, 2-mm-diameter, full-thickness punchesof RPE-choroid-sclera that were collected using a trephine punch. These punches were taken at 2-mm intervals from the fovea to the ora serrata in the temporal, nasal, inferior, and superior quadrants in an eye from a 14-year-old donor and subsequently prepared for electron microscopy (Figure 1B). The average thickness and integrity of the EL in each of these punches were measured. A second series of 2-mm-diameter punches derived from two additional donors aged 7 and 25 were measured (these data were similar to those of the 14-year-old and are not depicted in this article). In the second analysis, measurements of EL thickness and integrity were made at two defined locations, 1 to 2 mm and 12 to 13 mm, from the foveal center, in the infero-temporal quadrant, in a series of donors (Figure 1C). Fifty-six eyes from 56 unaffected (non-AMD) donors, ranging in age from 6 hours after birth to 96 years of age (mean age, 51.4 years), were used in this analysis. At least five donor eyes from each decade of life were included in this analysis. Oriented, 4-mm-diameter, full-thickness punches of RPE-choroid-sclera were taken using a trephine punch and prepared for electron microscopy, as described above. In the third analysis, similar measurements of macular EL thickness and integrity were made from punches taken from 64 eyes derived from 64 human AMD donors, ranging in age from 62 to 99 years of age (Figure 1C). These included 24 donors with macular drusen, pigment disruption, and other signs of early-stage AMD; 15 donors with GA; 9 donors with active, patent CNVMs; and 16 donors with disciform scars (DS) that were preceded by documented CNVMs or in which a CNVM was present in the second eye. The mean ages of donors in these four categories were 79.0, 82.8, 83.7, and 88.2 years, respectively. Four random photographic images were taken from each punched specimen using a JEOL JEM 1220 microscope (JEOL USA Inc., Peabody, MA), as described previously.38Hageman G Mullins R Molecular composition of drusen as related to substructural phenotype.Mol Vis. 1999; 5: 28PubMed Google Scholar Images were collected at ×5000 actual magnification for the first analysis (see above) and ×2500 actual magnification for the second and third analyses (see above). The thickness of the EL of Bruch's membrane was measured at 20 points (five equal-distance points from each of the four images per specimen) using a micrometer. The average value of the 20 points was used in the statistical analyses. The integrity of the EL was defined as the total length of visually detectable elastin divided by the overall length of visible Bruch's membrane within each of the four images. Thus, 100% integrity represented a fully intact (nonporous) EL and 0% integrity represented complete absence of elastin. The average integrity value derived from the four images of each punch was used for statistical analyses. The largest, or maximum, gap length (ie, discontinuity in the EL) present within the elastin lamina from the four micrographs used for each donor was identified and measured. The largest gap length from all donors in any given category was averaged (Figure 2F). Statistical analyses were performed using Microsoft Excel (Microsoft Inc., Redmond, WA) and S-Plus Statistical Packages (Insightful Corp., Seattle, WA). The Mann-Whitney U-test (Wilcoxon rank-sum test) was used to compare data series and linear regression analysis was used to explore age-related changes. To compare normal, unaffected donors to donors with AMD, statistical analyses were performed using only the normal, unaffected donors >61 years of age (23 unaffected, 23 early-stage AMD, and 40 late-stage AMD total). The mean age of normal donors >61 was 79.8 years; this mean age was not significantly different from that of each of the three groups of donors with AMD. Some data are represented graphically in the form of box and whiskers plots. These graphs show values for the median (bolded line within box), the first and third quartiles as a solid box, and the minimum and maximum observation range as whiskers. Any extreme values, defined as those further than 1.5× the interquartile range from the median, are represented as solitary, horizontal lines. The reactivity of anti-elastin antibodies with the macular EL of Bruch's membrane differed markedly from that of extramacular regions in all donor eyes examined (Figure 2, A and B). Immunoreactive elastin was attenuated and highly discontinuous in the maculas of most donors, as compared to more peripheral regions where it was continuous and thick. This same pattern was observed with all elastin antibodies tested. The differences between these same structural attributes in the macular and extramacular regions are even more striking when viewed by transmission electron microscopy (Figure 2, C and D). In the center of the fovea, elastin was particularly sparse and thin (Figure 2E). These data provided the impetus for a more robust evaluation of these structural parameters in a larger set of samples derived from donors with and without a history of AMD. The mean integrity of the EL measured in the series of punches derived from a 14-year-old donor (Figure 1B) is depicted in Figure 3, A and B. The mean integrity was lowest (∼40%) in the central macula (fovea) and increased rapidly, approaching ∼80% at the main retinal vascular arcades (+8 mm and −8 mm) in the temporal, superior, and inferior regions. In the nasal quadrant, the integrity was lowest around the optic disk, especially on the nasal aspect, and rapidly increased in regions approaching 8 to 10 mm from the fovea, just outside the major vascular arcades. The mean thickness values exhibited a similar distribution, except there was not a significant fovea-associated dip (Figure 3, C and D). A calculated color-coded schematic map of the mean EL integrity of this eye is depicted in Figure 4A. In general, the area of the fundus with the thinnest and most porous EL corresponds spatially to the same region where the majority of lesions associated with AMD are manifest (Figure 4, B and C).Figure 4A: A derived, color-coded schematic of the integrity of the elastic layer (relative percent scale shown to right) of Bruch's membrane based on the data shown in Figure 3, A and B. The black circle represents the optic nerve head and the curved black lines emanating from it are the approximate positions of the major retinal vessels. Note that the integrity is lowest in the foveal region (arrow), central macula, and on the nasal aspect of the optic nerve head. These regions of decreased integrity correspond spatially to the distribution of the majority of macular lesions, such as GA (B; arrows depict the margin of macular atrophy) and CNVMs (C; arrow) that occur in individuals with AMD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Scatter plots for the mean integrity and thickness of the EL of Bruch's membrane in a series of normal, unaffected human donors are depicted in Figure 5. The mean integrity value of the EL in this series of normal human donors was 37.2% (SD = 7.9%) in the macula and 92.3% (SD = 4.7%) in the extramacula (Figure 5A). The mean thickness of the EL from the same series of donors was 134.2 nm (SD = 32.0 nm) in the macula and 391.7 nm (SD = 120.1 nm) in the extramacula (Figure 5B). This represents a statistically significant difference in both the integrity (P < 0.00001) and thickness (P < 0.00001) of the EL between the macular and extramacular regions. When the unaffected donors were split into young and age-matched groups, aging effects in both the macular and extramacular regions were observed. The thickness, but not the integrity, of the EL was significantly higher in the age-matched group, as compared to the young group, in both the macular and peripheral regions (P = 0.033 and P < 0.001, respectively). Scatter plots comparing the mean integrity and mean thickness values for each donor (Figure 6) suggest that a strong relationship exists between integrity and thickness of the EL in all regions in which these parameters were measured (exponential fit R2 = 0.8735).Figure 6Scatter plot showing the relationship between EL integrity and thickness for all donors examined in this investigation. Note that there is a strong relationship between these two parameters in all regions measured (exponential fit R2 = 0.8735).View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the macular area, there was an apparent thinning of EL thickness and a loss of EL integrity from elderly normal individuals, through early-stage AMD to late-stage AMD (Table 1; Figure 7, Figure 8, Figure 9, Figure 10). For macular integrity, significant differences were observed between unaffected, age-matched controls and the following: all AMD (P = 0.003), early-stage AMD (P = 0.028), late-stage AMD (P = 0.003), active CNVM (P = 0.02), and CNV/DS (P = 0.003). For macular thickness, significant differences were observed between age-matched controls and donors with CNV/DS (P = 0.008), the latter which accounted for the apparent significant differences observed in the AMD (P = 0.035) and late-stage AMD (P = 0.024) groups (Table 1). These changes in EL thickness were in the opposite direction to those seen in normal aging. In contrast to the macula, no significant differences were found in the extramacular region
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