RESUMEN
PURPOSE: To develop an automated tool that provides reliable, consistent, and accurate results for counting cell nuclei in tissue sections. METHODS: We propose a novel method based on an image processing algorithm to analyze large sets of digital micrographs. The nucleus detector design is based on a Laplacian of Gaussian filter. We use the leave-one-out cross validation method for estimating the generalization error, which is then used to choose the model and parameters of the proposed nucleus detector with both fluorescent and dye stained images. We also evaluate the performance of a nucleus detector by comparing the results with manual counts. RESULTS: When our nucleus detector is applied to previously unanalyzed images of feline retina, it correctly counts nuclei within the outer nuclear layer (ONL) with an average error of 3.67% ranging from 0 to 6.07%, and nuclei within the inner nuclear layer (INL) with an average error of 8.55% ranging from 0 to 13.76%. Our approach accurately identifies the location of cell bodies. Even though we have a relatively large error in the INL due to the large intra-observer variation, both manual counting and nucleus detector result in the same conclusion. This is the first time that cell death in the INL in response to retinal detachment is analyzed quantitatively. We also test the proposed tool with various images and show that it is applicable to a wide range of image types with nuclei varying in size and staining intensity. CONCLUSIONS: The proposed method is simple and reliable. It also has widespread applicability to a variety of sample preparation and imaging methods. Our approach will be immediately useful in quantifying cell number in large sets of digital micrographs and from high-throughput imaging. The tool is available as a plug-in for Image J.
Asunto(s)
Algoritmos , Núcleo Celular/ultraestructura , Diagnóstico por Computador , Procesamiento de Imagen Asistido por Computador , Microscopía , Retina/ultraestructura , Animales , Automatización , Gatos , Procesamiento de Imagen Asistido por Computador/normas , Microscopía Confocal , Reproducibilidad de los Resultados , Desprendimiento de Retina/patologíaRESUMEN
Retinal detachment, the separation of the neural retina from the retinal pigmented epithelium, starts a cascade of events that results in cellular changes throughout the retina. While the degeneration of the light sensitive photoreceptor outer segments is clearly an important event, there are many other cellular changes that have the potential to significantly effect the return of vision after successful reattachment. Using animal models of detachment and reattachment we have identified many cellular changes that result in significant remodeling of the retinal tissue. These changes range from the retraction of axons by rod photoreceptors to the growth of neurites into the subretinal space and vitreous by horizontal and ganglion cells. Some neurite outgrowths, as in the case of rod bipolar cells, appear to be directed towards their normal presynaptic target. Horizontal cells may produce some directed neurites as well as extensive outgrowths that have no apparent target. A subset of reactive ganglion cells all fall into the latter category. Muller cells, the radial glia of the retina, undergo numerous changes ranging from proliferation to a wholesale structural reorganization as they grow into the subretinal space (after detachment) or vitreous after reattachment. In a few cases have we been able to identify molecular changes that correlate with the structural remodeling. Similar changes to those observed in the animal models have now been observed in human tissue samples, leading us to conclude that this research may help us understand the imperfect return of vision occurring after successful reattachment surgery. The mammalian retina clearly has a vast repertoire of cellular responses to injury, understanding these may help us improve upon current therapies or devise new therapies for blinding conditions.
Asunto(s)
Retina/patología , Retina/fisiopatología , Desprendimiento de Retina/patología , Desprendimiento de Retina/fisiopatología , Animales , Modelos Animales de Enfermedad , HumanosRESUMEN
PURPOSE: Rod cGMP-phosphodiesterase, a key enzyme in visual transduction, is important for retinal integrity and function. Mutations in the gene encoding the phosphodiesterase beta-subunit (PDEbeta) cause retinal degeneration in animals and humans. Here the authors tested the hypothesis that elements in the 3' untranslated region (3' UTR) of the PDEbeta gene are involved in the regulation of PDEbeta expression. METHODS: Involvement of the 3' UTR of PDEbeta mRNA in the regulation of PDEbeta expression was assessed by Y-79 retinoblastoma cells or the heads of Xenopus laevis tadpoles with constructs containing the SV40 or PDEbeta promoter, the luciferase cDNA, and either the SV40 or the PDEbeta 3' UTR (or fragments of its sequence). RESULTS: Compared with the SV40 3' UTR (used as control), the entire PDEbeta 3' UTR decreased reporter gene expression in Y-79 retinoblastoma cells as well as in SY5Y neuroblastoma and 293 human embryonic kidney cell lines. However, the authors observed that two 100-nucleotide fragments from the PDEbeta 3' UTR increased while its noncanonical poly-adenylation signal abolished reporter gene expression in Y-79 retinoblastoma cells and in ex vivo experiments using Xenopus tadpole heads. In particular, an 11-nucleotide element (EURE) in one of the 100-nucleotide fragments was responsible for the upregulation of luciferase expression. CONCLUSIONS: These studies indicate that the 3' UTR of the PDEbeta mRNA is involved in the complex regulation of this gene's expression in the retina. Moreover, the results show that the PDEbeta poly-A signal has a dominant inhibitory effect over two other regions in the 3' UTR that stimulate gene expression.
Asunto(s)
Regiones no Traducidas 3'/genética , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/genética , Regulación Enzimológica de la Expresión Génica/fisiología , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Embrión no Mamífero/metabolismo , Genes Reporteros , Humanos , Riñón/embriología , Riñón/metabolismo , Datos de Secuencia Molecular , Neuroblastoma/genética , Retina/metabolismo , Neoplasias de la Retina/genética , Retinoblastoma/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Células Tumorales Cultivadas/metabolismo , Regulación hacia Arriba , Xenopus laevis/genéticaRESUMEN
PURPOSE: To determine the cellular consequences of retinal detachment in retinoschisin knockout (Rs1-KO) mice, a model for retinoschisin in humans. METHODS: Experimental retinal detachments (RDs) were induced in the right eyes of both Rs1-KO and wild-type (wt) control mice. Immunocytochemistry was performed on retinal tissue at 1, 7, or 28 days after RD with antibodies to anti-GFAP, -neurofilament, and -rod opsin to examine cellular changes after detachment. Images of the immunostained tissue were captured by laser scanning confocal microscopy. Quantitative analysis was performed to measure the number of Hoechst-stained photoreceptor nuclei and their density, number, and size of inner retinal cavities, as well as the number of subretinal glial scars. RESULTS: Since detachments were created with balanced salt solution, by examination, all retinas had spontaneously reattached by 1 day. Cellular responses common to many photoreceptor degenerations occurred in the nondetached retinas of Rs1-KO mice, and, of importance, RD did not appear to significantly accentuate these responses. The number of schisis cavities was not changed after detachment, but their size was reduced. CONCLUSIONS: These data indicate that large short-term RD in Rs1-KO mice, followed by a period of reattachment may cause a slight increase in photoreceptor cell death, but detachments do not accentuate the gliosis and neurite sprouting already present and may in fact reduce the size of existing retinal cavities. This finding suggests that performing subretinal injections to deliver therapeutic agents may be a viable option in the treatment of patients with retinoschisis without causing significant cellular damage to the retina.
Asunto(s)
Proteínas del Ojo/fisiología , Neuroglía/fisiología , Neuronas/fisiología , Células Fotorreceptoras de Vertebrados/patología , Desprendimiento de Retina/fisiopatología , Retinosquisis/fisiopatología , Animales , Recuento de Células , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente Indirecta , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Proteínas de Neurofilamentos/metabolismo , Opsinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Desprendimiento de Retina/metabolismo , Retinosquisis/metabolismoRESUMEN
PURPOSE: To determine the roles of glial fibrillary acidic protein (GFAP) and vimentin in Müller cell reactivity. METHODS: Retinal detachments were created in mice deficient for GFAP and vimentin (GFAP(-/-)vim(-/-)) and age-matched wild-type (wt) mice. The reactivity of the retina was studied by immunofluorescence and electron microscopy. RESULTS: Müller cell morphology was different and glutamine synthetase immunoreactivity was reduced in the undisturbed GFAP(-/-)vim(-/-) retinas. After retinal detachment, Müller cells formed subretinal glial scars in the wt mice. In contrast, such scars were not observed in GFAP(-/-)vim(-/-) mice. Müller cells, which normally elongate and thicken in response to detachment, appeared compressed, thin, and "spikey" in the GFAP(-/-)vim(-/-) mice. The end foot region of Müller cells in the GFAP(-/-)vim(-/-) mice often sheared away from the rest of the retina during detachment, corroborating earlier results showing decreased resistance of this region in GFAP(-/-)vim(-/-) retinas to mechanical stress. In regions with end foot shearing, ganglion cells showed intense neurite sprouting, as revealed by anti-neurofilament labeling, a response rarely observed in wt mice. CONCLUSIONS: Müller cells are subtly different in the GFAP(-/-)vim(-/-) mouse retina before detachment. The end foot region of these cells may be structurally reinforced by the presence of the intermediate filament cytoskeleton, and our data suggest a critical role for these proteins in Müller cell reaction to retinal detachment and participation in subretinal gliosis.