RESUMO
PURPOSE: Lipofuscin contained in the retinal pigment epithelium (RPE) is the main source of fundus autofluorescence (FAF), the target of an imaging method useful for estimating the progression of geographic atrophy (GA) in clinical trials. To establish a cellular basis for hyperfluorescent GA border zones, histologic autofluorescence (HAF) was measured at defined stages of RPE pathologic progression. DESIGN: Experimental study. PARTICIPANTS AND CONTROLS: Ten GA donor eyes (mean age ± standard deviation, 87.1 ± 4.0 years) and 3 age-matched control eyes (mean age ± standard deviation, 84.0 ± 7.2 years) without GA. METHODS: The 10-micrometer-thick sections were divided into zones of RPE morphologic features according to an 8-point scale. Any HAF excited by 488 nm light was imaged by laser confocal microscopy. The HAF intensity summed along vertical lines perpendicular to Bruch's membrane at 0.2-µm intervals served as a surrogate for FAF. Intensity profiles in 151 zones were normalized to grade 0 at a standard reference location in each eye. Cross-sectional area, mean, and sum autofluorescence for individual RPE cells were measured (cellular autofluorescence [CAF]). MAIN OUTCOME MEASURES: Statistically significant differences in intensity and localization of HAF and CAF at defined stages of RPE morphologic progression for GA and control eyes. RESULTS: The RPE morphologic features were most abnormal (cell rounding, sloughing, and layering; grade 2) and HAF intensity profiles were highest and most variable immediately adjacent to atrophic areas. Peaks in HAF intensity frequently were associated with vertically superimposed cells. The HAF value that optimally separated reactive RPE was 0.66 standard deviations more than the mean for uninvolved RPE and was associated with a sensitivity of 75.8% and a specificity of 76.3%. When variable cell area was accounted for, neither mean nor sum CAF differed significantly among the RPE pathologic grades. CONCLUSIONS: Areas with advanced RPE alterations are most likely to exhibit clinically recognizable patterns of elevated FAF around GA, but may not predict cells about to die, because of vertically superimposed cells and cellular fragments. These data do not support a role for lipofuscin-related cell death and call into question the rationale of treatments targeting lipofuscin.
Assuntos
Atrofia Geográfica/patologia , Epitélio Pigmentado da Retina/patologia , Idoso , Idoso de 80 Anos ou mais , Cadáver , Angiofluoresceinografia , Fundo de Olho , Humanos , Microscopia ConfocalRESUMO
In geographic atrophy (GA), the non-neovascular end stage of age-related macular degeneration (AMD), the macular retinal pigment epithelium (RPE) progressively degenerates. Membrane cofactor protein (MCP, CD46) is the only membrane-bound regulator of complement expressed on the human RPE basolateral surface. Based on evidence of the role of complement in AMD, we hypothesized that altered CD46 expression on the RPE would be associated with GA development and/or progression. Here we report the timeline of CD46 protein expression changes across the GA transition zone, relative to control eyes, and relative to events in other chorioretinal layers. Eleven donor eyes (mean age 87.0 ± 4.1 yr) with GA and 5 control eyes (mean age 84.0 ± 8.9 yr) without GA were evaluated. Macular cryosections were stained with PASH for basal deposits, von Kossa for calcium, and for CD46 immunoreactivity. Internal controls for protein expression were provided by an independent basolateral protein, monocarboxylate transporter 3 (MCT3) and an apical protein, ezrin. Within zones defined by 8 different semi-quantitative grades of RPE morphology, we determined the location and intensity of immunoreactivity, outer segment length, and Bruch's membrane calcification. Differences between GA and control eyes and between milder and more severe RPE stages in GA eyes were assessed statistically. Increasing grades of RPE degeneration were associated with progressive loss of polarity and loss of intensity of staining of CD46, beginning with the stages that are considered normal aging (grades 0-1). Those GA stages with affected CD46 immunoreactivity exhibited basal laminar deposit, still-normal photoreceptors, and concomitant changes in control protein expression. Activated or anteriorly migrated RPE (grades 2-3) exhibited greatly diminished CD46. Changes in RPE CD46 expression thus occur early in GA, before there is evidence of morphological RPE change. At later stages of degeneration, CD46 alterations occur within a context of altered RPE polarity. These changes precede degeneration of the overlying retina and suggest that therapeutic interventions be targeted to the RPE.
Assuntos
Atrofia Geográfica/metabolismo , Proteína Cofatora de Membrana/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Feminino , Atrofia Geográfica/patologia , Humanos , Técnicas Imunoenzimáticas , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Epitélio Pigmentado da Retina/patologia , Simportadores , Doadores de TecidosRESUMO
To determine if complement anaphylatoxin-mediated inflammation contributes to the development and progression of experimental autoimmune uveitis (EAU), we induced disease in wild type and complement anaphylatoxin receptor-deficient mice (C3a receptor(-/-), C5a receptor(-/-) and C3aR(-/-)/C5aR(-/-)) and evaluated the eyes three weeks post-induction. No differences in disease severity or in disease incidence were seen between wild type controls and anaphylatoxin receptor-deficient mice. Our data indicate that C3a and C5a-mediated functions are not critical to the development of EAU.
Assuntos
Febre Uveoparotídea/patologia , Animais , Modelos Animais de Doenças , Humanos , Lectinas Tipo C/imunologia , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor da Anafilatoxina C5a/genética , Receptores de Complemento/genética , Febre Uveoparotídea/induzido quimicamente , Febre Uveoparotídea/genética , Febre Uveoparotídea/imunologiaRESUMO
To characterize the distribution of membrane-bound components of the complement system in normal human retina, eyes from eight human donors with no history of ocular disease, ranging in age from 47 to 85 years were examined using immunohistochemistry to localize the C3a receptor (C3aR), C5a receptor (C5aR), CD46, CD55, and CD59 in cryosections prepared from donor posterior segments. The C3aR was identified in the nerve fiber layer in a sawtooth-patterned band. Vimentin, used as a Müller cell marker, produced a similar staining pattern. The C5aR was detected on specific rounded structures in the inner plexiform layer and occasionally in the nerve fiber layer. CD46 produced markedly specific staining of the basolateral surface of the retinal pigment epithelium. CD55 was localized to the nerve fiber layer. Staining for these proteins was consistent across all eyes studied. CD59 was expressed throughout the nerve fiber layer and labeled vessels that extended through the ganglion cell, inner plexiform, and inner nuclear layers, but this pattern was only confirmable in a single subject. Complement anaphylatoxin receptors and regulatory proteins are localized in different but internally consistent patterns in normal adult human retina, independent of the age of the donor. C3aR and C5aR localization only in the inner retina contrasts with previously reported findings in the central nervous system of wide spread diffuse staining. The complement regulators CD55 and CD59 were found primarily on the inner retina, while CD46 was present exclusively in a polarized fashion on the RPE.
Assuntos
Receptores de Complemento/metabolismo , Retina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Complemento C3/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Proteína Cofatora de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Epitélio Pigmentado Ocular/metabolismo , Receptor da Anafilatoxina C5aRESUMO
In this report, we describe the effect of complement deficiency and inhibition on experimental autoimmune uveoretinitis (EAU). C57BL/6 mice genetically deficient in C3 (C3-/-) or expressing a soluble complement activation inhibitor (soluble complement receptor related protein Y or sCrry) in a CNS-targeted fashion, (sCrry/GFAP) were induced for EAU via peripheral immunisation with a peptide of amino acids 1-20 of human interphotoreceptor retinoid binding protein in complete Freund's adjuvant with concurrent intraperitoneal pertussis toxin. The incidence and severity of EAU in the mutant mice was compared with that in simultaneously induced C57BL/6 wild type mice. The sCrry protein was detected in retinal extracts from transgenic but not wild type mice by western blot. C3-/- mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 44 versus 89%, respectively, p=0.0417) and a significant reduction in the severity of EAU (median disease score values 0 versus 1.3, respectively, p=0.0253). Similarly, sCrry mice had a significant reduction in the incidence of EAU compared with wild type mice (incidence 57 versus 100% respectively, p=0.0033) and a significant reduction in the severity of EAU (median disease score values 0.18 versus 1.85, respectively, p=0.0054). A genetic deficiency of C3 and production of a soluble complement inhibitor targeted to the CNS and eye are protective against EAU.