RESUMEN
PURPOSE: To quantify the extent of cellular proliferation and immunohistochemically characterize the proliferating cell types in epiretinal membranes (ERMS) from four different conditions: proliferative vitreoretinopathy (PVR), proliferative diabetic retinopathy, post-retinal detachment, and idiopathic ERM. METHODS: Forty-six ERMs were removed from patients undergoing vitrectomy and immediately fixed in paraformaldehyde. The membranes were processed whole and immunolabeled with either anti-MIB-1 or anti-SP6 to detect the K(i)-67 protein in proliferating cells, in combination with anti-glial fibrillary acidic protein or anti-vimentin to identify glia, anti-ezrin to identify retinal pigment epithelial cells, Ricinus communis to identify immune cells, and Hoechst to label nuclei. Digital images were collected using a laser scanning confocal microscope. The cell types were identified, their combined proliferative indices were tabulated as the average number of anti-K(i)-67-positive cells/mm(2) of tissue, and the number of dividing cells was related to the specific ocular condition and estimated disease duration. RESULTS: ERMs of all four types were shown to be highly cellular and contained proliferating cells identified as glia, retinal pigment epithelium, and of immune origin. In general, membranes identified as PVR had many more K(i)-67-positive cells in comparison to those in the other three categories, with the average number of K(i)-67-positive cells identified per mm(2) of tissue being 20.9 for proliferative diabetic retinopathy, 138.3 for PVR, 12.2 for post-retinal detachment, and 19.3 for idiopathic ERM. While all membrane types had dividing cells, their number was a relatively small fraction of the total number of cells present. CONCLUSIONS: The four ERM types studied demonstrated different cell types actively dividing at the time of removal, confirming that proliferation is a common event and does continue over many months. The low number of dividing cells at the time of removal in comparison to the total number of cells present, however, is an indicator that proliferation alone may not be responsible for the problems observed with the ERMs. Treatment strategies may need to take into consideration the timing of drug administration, as well as the contractile and possibly the inflammatory characteristics of the membranes to prevent the ensuing effects on the retina.
Asunto(s)
Proliferación Celular , Retinopatía Diabética/patología , Membrana Epirretinal/patología , Neuroglía/patología , Retina/patología , Desprendimiento de Retina/patología , Epitelio Pigmentado de la Retina/patología , Vitreorretinopatía Proliferativa/patología , Cuerpo Vítreo/patología , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/biosíntesis , Retinopatía Diabética/metabolismo , Retinopatía Diabética/cirugía , Membrana Epirretinal/metabolismo , Membrana Epirretinal/cirugía , Femenino , Proteína Ácida Fibrilar de la Glía/análisis , Proteína Ácida Fibrilar de la Glía/biosíntesis , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Antígeno Ki-67/biosíntesis , Masculino , Microscopía Fluorescente , Neuroglía/metabolismo , Retina/metabolismo , Retina/cirugía , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/cirugía , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/cirugía , Factores de Tiempo , Vimentina/análisis , Vimentina/biosíntesis , Vitrectomía , Vitreorretinopatía Proliferativa/metabolismo , Vitreorretinopatía Proliferativa/cirugía , Cuerpo Vítreo/metabolismo , Cuerpo Vítreo/cirugíaRESUMEN
PURPOSE: To study the fate of Müller's glia following experimental retinal detachment, using a "pulse/chase" paradigm of bromodeoxyuridine (BrdU) labeling for the purpose of understanding the role of Müller cell division in subretinal scar formation. METHODS: Experimental retinal detachments were created in pigmented rabbit eyes, and 3 days later 10 microg of BrdU was injected intravitreally. The retinas were harvested 4 h after the BrdU was administered (i.e., day 3) or on days 4, 7, and 21 post detachment. The tissue was fixed, embedded in agarose, and sectioned at 100 microm. The sections were labeled with various combinations of probes, including anti-vimentin and anti-S100 (as markers for Müller cells), anti-BrdU, anti-phosphohistone H3 (to identify mitotic cells), and the isolectin B4 (to identify macrophages and microglia). Images were captured using an Olympus Fluoview 500 confocal microscope. To aid in our understanding of how Müller cell nuclei undergo cell division, two additional procedures were used: 1) electron microscopy of normal cat and rabbit retinas and 2) a new method using 5-fluorouracil and subsequent anti-BrdU labeling to detect all Müller cell nuclei, using confocal imaging. RESULTS: Three days after detachment, anti-vimentin labeled all Müller cells, some of which were also labeled with anti-BrdU. On day 4, many of the anti-BrdU-labeled Müller cell nuclei appeared in columns with one labeled nucleus in the inner nuclear layer and another directly sclerad to it in the outer nuclear layer. By day 7, most anti-BrdU-labeled nuclei were observed in subretinal scars. At 3 weeks, some anti-BrdU-labeled nuclei that remained within the retina did not express vimentin or S100. Anti-phosphohistone H3-labeled (i.e., mitotic) cells, some of which were also labeled with anti-BrdU, were only observed in the outer nuclear layer on day 4, and these nuclei were surrounded by an accumulation of vimentin filaments. Isolectin B4-labeled microglia and macrophages also incorporated BrdU and were observed throughout the retina and in subretinal scars during all times of detachment. Electron microscopy and immunofluorescence labeling of the 5-fluorouracil-injected eyes revealed the presence of a unique structural relationship between Müller cell nuclei and intermediate filament proteins. CONCLUSIONS: Following retinal detachment, many Müller cell nuclei initially migrate to the outer retina, undergo mitosis, and eventually reside in subretinal glial scars, suggesting a possible link between the early division of Müller cells and the process of subretinal gliosis. In addition, a subpopulation of anti-BrdU-labeled cells, presumably once Müller cells, appears to stop expressing well accepted Müller cell marker proteins, suggesting a potential dedifferentiation of some of these cells over time. Additionally, Müller cell nuclei may use intermediate filaments as a "track" for migration into the outer retina and later as an important component of cell division by the accumulation of vimentin filaments around the mitotic nuclei.
Asunto(s)
División Celular , Linaje de la Célula , Núcleo Celular/patología , Cicatriz/patología , Neuroglía/patología , Retina/patología , Desprendimiento de Retina/patología , Animales , Bromodesoxiuridina/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patología , Microscopía Confocal , Neuroglía/metabolismo , Conejos , Retina/metabolismo , Vimentina/metabolismoRESUMEN
During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.
Asunto(s)
Envejecimiento/fisiología , Proteínas del Sistema Complemento/fisiología , Degeneración Macular/metabolismo , Coroides/metabolismo , Expresión Génica , Humanos , Degeneración Macular/genética , Polimorfismo de Nucleótido Simple , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
PURPOSE: To explore the role of integrin alpha5beta1 in proliferative vitreoretinopathy (PVR) pathogenesis by evaluating the expression alpha5beta1 on ARPE-19 cells and patient proliferative membranes, quantifying the inhibitory effects of JSM6427 (a small molecule alpha5beta1 inhibitor) on ARPE-19 cell adhesion and migration, and assessing the therapeutic potential of JSM6427 in a rabbit retinal detachment model. METHODS: Expression of alpha5beta1 was evaluated on activated ARPE-19 cells by flow cytometry and on PVR membranes by immunohistochemistry. ARPE-19 cells were used in fibronectin-dependent adhesion and migration assays with various concentrations of JSM6427; IC(50) was calculated. In the rabbit model, eyes were intravitreally injected with vehicle or JSM6427 on day 0 or 1 after retinal detachment; BrdU was administered intravitreally on day 3, and retinal tissues were harvested on day 3 (4 hours later) or 7. Retinal scarring, cellular proliferation, and inflammatory responses were quantified, and retinal morphology was analyzed in retinal sections. RESULTS: Activated ARPE-19 cells and PVR membranes expressed high levels of alpha5beta1; expression was low in control eyes. JSM6427 provided a dose-dependent blockade of ARPE-19 cell adhesion to fibronectin (IC(50), 7.1 +/- 2.5 microM) and inhibition of migration (IC(50), 6.0 +/- 4.5 microM). In the rabbit model, intravitreal injection of JSM6427 provided significant inhibition of proliferation of retinal cells (Müller cells, microglia, and macrophages) on days 3 and 7 after detachment and inhibition of inflammatory response and retinal scarring on day 7 after detachment. CONCLUSIONS: JSM6427 is a promising treatment for PVR, with data suggesting that inhibition of alpha5beta1-fibronectin interactions addresses multiple pathways involving retinal pigment epithelial, glial, and inflammatory cells.
Asunto(s)
Modelos Animales de Enfermedad , Integrina alfa5beta1/antagonistas & inhibidores , Propionatos/farmacología , Piridinas/farmacología , Pirrolidinas/farmacología , Desprendimiento de Retina/prevención & control , Epitelio Pigmentado de la Retina/efectos de los fármacos , Vitreorretinopatía Proliferativa/prevención & control , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Membrana Epirretinal/metabolismo , Femenino , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Inmunohistoquímica , Integrina alfa5beta1/metabolismo , Masculino , Conejos , Desprendimiento de Retina/metabolismo , Desprendimiento de Retina/patología , Epitelio Pigmentado de la Retina/metabolismo , Vitreorretinopatía Proliferativa/etiología , Vitreorretinopatía Proliferativa/metabolismoRESUMEN
PURPOSE: To test the effect of Palomid 529, an inhibitor of the Akt/mTOR pathway, on Müller cell proliferation, subretinal glial scar formation, and photoreceptor survival after experimental retinal detachment (RD). METHODS: Palomid 529 (600 microg) in balanced salt solution or balanced salt solution alone was injected intravitreally immediately after RD into the right eyes of 12 rabbits. Ten micrograms of BrdU was injected intravitreally on day 3. Animals were killed on day 3 or 7, at which time retinal sections were labeled with anti-BrdU to detect dividing cells, with anti-vimentin to identify Müller cells, and with the isolectin B4 to identify microglia and macrophages. Outer nuclear layer (ONL) thickness was measured from fluorescence-labeled nuclear-stained sections. Labeling was imaged using confocal microscopy. Six additional animals received either drug or balanced salt solution injections into normal eyes, and paraffin sections were stained with hematoxylin and eosin. RESULTS: In the drug-treated eyes there was a significant decrease in the number of anti-BrdU-labeled Müller cells, the number and size of subretinal scars, and the number of isolectin B4-labeled cells. The ONL was also significantly thicker, and there was no evidence of toxic effects. CONCLUSIONS: Palomid 529 is an effective suppressor of Müller cell proliferation, glial scar formation, and photoreceptor cell death in a rabbit model of RD. This suggests that inhibiting the Akt/mTOR signal transduction pathway may be an effective strategy to decrease proliferation and photoreceptor cell death induced by detachment and perhaps represents a novel therapy for related human diseases such as proliferative vitreoretinopathy.