RESUMO
Intraocular pressure (IOP) elevation is a principal risk factor for glaucoma. Using a microbead injection technique to chronically raise IOP for 15 or 30 d in mice, we identified the early changes in visual response properties of different types of retinal ganglion cells (RGCs) and correlated these changes with neuronal morphology before cell death. Microbead-injected eyes showed reduced optokinetic tracking as well as cell death. In such eyes, multielectrode array recordings revealed that four RGC types show diverse alterations in their light responses upon IOP elevation. OFF-transient RGCs exhibited a more rapid decline in both structural and functional organizations compared with other RGCs. In contrast, although the light-evoked responses of OFF-sustained RGCs were perturbed, the dendritic arbor of this cell type remained intact. ON-transient and ON-sustained RGCs had normal functional receptive field sizes but their spontaneous and light-evoked firing rates were reduced. ON- and OFF-sustained RGCs lost excitatory synapses across an otherwise structurally normal dendritic arbor. Together, our observations indicate that there are changes in spontaneous activity and light-evoked responses in RGCs before detectable dendritic loss. However, when dendrites retract, we found corresponding changes in receptive field center size. Importantly, the effects of IOP elevation are not uniformly manifested in the structure and function of diverse RGC populations, nor are distinct RGC types perturbed within the same time-frame by such a challenge.
Assuntos
Modelos Animais de Doenças , Progressão da Doença , Glaucoma/patologia , Glaucoma/fisiopatologia , Células Ganglionares da Retina/patologia , Células Ganglionares da Retina/fisiologia , Potenciais de Ação/fisiologia , Animais , Morte Celular/fisiologia , Feminino , Pressão Intraocular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Luminosa/métodos , Células Fotorreceptoras de Invertebrados/patologia , Células Fotorreceptoras de Invertebrados/fisiologia , Distribuição AleatóriaRESUMO
PURPOSE: Uveal melanoma (UM) is the most common primary cancer of the eye, resulting not only in vision loss, but also in metastatic death. This study attempts to identify changes in the patterns of gene expression that lead to malignant transformation and proliferation of normal uveal melanocytes (UVM) using the Suppressive Subtractive Hybridization (SSH) technique. METHODS: The SSH technique was used to isolate genes that are differentially expressed in the TP31 cell line derived from a primary UM compared to UVM. The expression level of selected genes was further validated by microarray, semi-quantitative RT-PCR and western blot analyses. RESULTS: Analysis of the subtracted libraries revealed that 37 and 36 genes were, respectively, up- and downregulated in TP31 cells compared to UVM. Differential expression of the majority of these genes was confirmed by comparing UM cells with UVM by microarray. The expression pattern of selected genes was analyzed by semi-quantitative RT-PCR and western blot, and was found to be consistent with the SSH findings. CONCLUSIONS: We demonstrated that the SSH technique is efficient to detect differentially expressed genes in UM. The genes identified in this study represent valuable candidates for further functional analysis in UM and should be informative in studying the biology of this tumor.
Assuntos
Hibridização Genômica Comparativa/métodos , Olho/patologia , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Melanócitos/metabolismo , Melanoma , Neoplasias Uveais , Adulto , Idoso , Western Blotting , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Regulação para Baixo , Olho/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima , Neoplasias Uveais/genética , Neoplasias Uveais/patologiaRESUMO
PURPOSE: Müller cells are the principal glial cells of the retina. They span the entire thickness of the neural retina, and they are in close contact with neurons. Müller cells grow very slowly, and they undergo senescence with increasing passages. Moreover, successful primary cultures of Müller cells can be obtained only with donors no older than 35 years. These limitations of primary cultures motivated the characterization of cell lines. The purpose of this study was thus to compare normal human Müller cells (NHMCs) with two spontaneously generated human Müller cell lines from donors with type 1 and 2 diabetes (HMCLs). METHODS: Both cell lines were investigated for the expression of known markers of Müller cells as well as epithelial and endothelial cells by immunofluorescence and Western blot analyses. RT-PCR was also performed with growth factors that are typical of human Müller cells. RESULTS: In contrast to the typical fibroblast-like morphology of Müller cells, HMCLs showed an epithelial shape. Immunofluorescence analyses and Western blot showed that both NHMCs and HMCLs express the known markers of Müller cells. In addition, HMCLs express cytokeratins K8 and K18 as well as typical growth factors for NHMCs. Finally, HMCLs have reached 30 passages until now without any change in their morphology or expression of markers, whereas NHMCs cannot typically be passed beyond small number of passages. HMCLs are the only human Müller cells lines that have a normal karyotype. CONCLUSIONS: HMCLs can be used as a model to improve the understanding of Müller cells in the context of chronic diabetes.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/patologia , Neuroglia/patologia , Retina/patologia , Adulto , Idoso , Biomarcadores/metabolismo , Western Blotting , Bromodesoxiuridina/metabolismo , Linhagem Celular , Retinopatia Diabética/etiologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Marcadores Genéticos , Humanos , Cariotipagem , Masculino , Microscopia de Contraste de Fase , Neuroglia/metabolismo , RNA Mensageiro/metabolismo , Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Doadores de TecidosRESUMO
PURPOSE: Müller cells are the principal glial cells in the retina. They play important functions in this tissue. Alterations in Müller cell behavior were observed in the retinal tissue of patients with proliferative diabetic retinopathy. The purpose of this study was to determine the gene expression profile of normal human Müller cells (NHMCs) and to compare it with that of two spontaneously generated human Müller cell lines (HMCLs) from type 1 and type 2 diabetic donors by using serial analysis of gene expression (SAGE). METHODS: Approximatively 50,000 tags were sequenced for each SAGE library. Identification of the transcripts was obtained by matching the 15-bp tags with the UniGene and GenBank databases. Classification of the genes was based on the updated information of the genome directory found at the National Center for Biotechnology Information (NCBI) Web site. RESULTS: SAGE was used to characterize the entire transcriptome of human Müller cells and to compare these data with those from Müller cell lines generated from type 1 and type 2 diabetic donors. The transcriptome of NHMCs and HMCLs demonstrated that "metabolism" and "protein synthesis" are the two main categories of genes expressed by human Müller cells. Only 106 genes are differentially expressed between NHMCs and HMCLs. CONCLUSIONS: The SAGE libraries reported in this article provide the gene expression profile of NHMCs and HMLCs. It thus represents a valuable source of information regarding the function of Müller cells as well as their role in the development of diabetic retinopathy.
Assuntos
Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Retinopatia Diabética/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/fisiologia , Neuroglia/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Biologia Computacional , Retinopatia Diabética/etiologia , Etiquetas de Sequências Expressas , Humanos , Neuroglia/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
PURPOSE: Müller cells are the predominant type of glial cells in the retina. They play a critical role in the retina. The purpose of this study was to generate a profile of the genes specifically expressed by human retinal Müller cells and to identify genes that may be responsible for retinal diseases. METHODS: Subtractive hybridization is a method process by which two populations of mRNA are compared in order to obtain clones of genes expressed in one population but not in the other. A cDNA subtraction library was constructed using RNA isolated from human Müller cells and human astrocytes. PCR-select differential screening was used to further verify the differentially expressed cDNA clones. Positive clones were sequenced and analyzed using the NCBI BLASTN program to identify sequence homologies. RESULTS: We identified 194 clones specifically expressed in human Müller cells. Among these clones, 102 corresponded to known human genes. Of the remaining 94 clones, 75 corresponded to expressed sequence tags or genomic clones and 19 transcripts did not match with any sequence in databases, and are possibly novel genes. CONCLUSIONS: The analysis of the subtraction library revealed genes that are specifically expressed by human Müller cells. Some of these genes are unidentified, novel genes that are specific to Müller cells as determined by RT-PCR and Northern blot analyses. These novel genes thus represent candidate genes for retinal diseases.