Retinal detachment is a serious cause of visual impairment. Gaining an understanding of its cellular effects can help improve management of the disease and contribute to our basic knowledge of the retina and its relationship to the pigment epithelium. We have recently identified specific cellular and biochemical changes in long-term detachments using an animal model. Gaining a further understanding of these changes using a combination of structural, biochemical, and molecular techniques is a major goal of this project. Specifically we will determine: 1) when the upregulation of intermediate filament proteins and the downregulation of enzymatic and vitamin A binding proteins occurs in Muller cells (by using immunocytochemistry and immunoblot analyses); 2) if the mRNA levels,for one protein in each of these classes, changes in concert with the changes in protein expression (by Northern and slot blot analyses; appropriate cellular expression of the mRNAs will be studied by in situ hybridization); 3) the onset and extent of opsin redistribution in photoreceptors (by immunocytochemistry); 4) if opsin synthesis and outer segment renewal continues (by immunoprecipitation and Lucifer yellow band displacement analyses); 5) if opsin mRNA levels change and if opsin mRNA distribution changes in cells that have redistributed opsin or whose morphology is severely disrupted (by Northern, slot blot and in situ hybridization studies); 6) the extent of the proliferative response and identification of the cell types involved (by continuous delivery of 3H-thymidine, LM and EM autoradiography); 7) if growth factors are involved (by immunocytochemistry) or if they can mimic the effects of detachment (by injection of growth factors into the eye or their addition to cultures of Muller cells); 8) if the changes in protein expression or redistribution or the proliferative response are arrested or modified by retinal reattachment. A greater understanding of the synaptic organization of the human retina will be gained by studying synaptic circuitry by serial-section electron microscopy of adult and developing human retinas and the analysis of Golgi- impregnated whole-mounts. In the latter, we will: 1) study the stained neurons in the 75 existing whole-mounts by standard Golgi descriptive techniques, and 2) analyze the synaptic input into the Golgi stained cells by electron microscopy (by obtaining new postmortem tissue from donor eyes and using a variety of fixation protocols to develop better structural preservation of the retina).
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