Sickle cell disease (SCD) is caused by a single point mutation in a hemoglobin gene. The defect results in red blood cells (RBC's) that are abnormally shaped and non-pliable causing anemia, hemolysis, sluggish blood flow and vaso-occlusion. The resultant vaso-occlusions cause ischemia in peripheral retina and possibly choroid. Neovascularization can eventually develop distant from the vaso-occlusion, followed by hemorrhage, fibrosis, and ultimately retinal detachment. The precise site and pathogenesis of the vaso-occlusions remain obscure. One goal of this proposal is to investigate, in transgenic mouse models of sickle cell disease and in human cadaver eyes, the role of four potential contributing factors in the vaso-occlusive process in retina and choroid: platelet adhesion and aggregation, sickled RBC's (sRBC's), altered fibrinolysis, and adhesion of leukocytes. Local factors like adenosine diphosphate (ADP) can stimulate in situ platelet aggregation. The concentration of ADP is controlled by endothelial ADPase, which we will visualize by enzyme histochemistry in whole retina from one eye of each subject by flat-embedding reacted retinas in glycol methacrylate. Retinal vascular ADPase activity will be analyzed en bloc by darkfield microscopy, will be quantified by image analysis, and areas of interest will be sectioned. The 2 mum sections permit high resolution morphometric analysis of the constituents (RBC's, fibrin, WBC's endothelium) in and adjacent to vaso-occlusions with special stains. The fellow eye from each subject will be cryopreserved and sectioned for immunohistochemical analysis of the fibrinolytic system: tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), fibrin, and von Willebrand;s factor (vWf, for intrinsic clotting cascade and the condition of endothelial cells). Leukocyte/endothelia cell adhesion molecules (ICAM-1, VCAM-1, E-selectin, and P-selectin) will also be analyzed immunohistochemically and neutrophils identified by enzyme histochemistry in serial sections. Immunohistochemical analysis of basic fibroblast growth factor (bFGF) and transforming growth factor-b (TGF-b) will also be performed because of their role in controlling the fibrinolytic system in endothelium, stimulating fibrosis, and potentially affecting the neovascular processes which follow ischemia. We will also investigate the adherence and obstruction of retinal vessels by fluorescently tagged human sRBC's in rat and transgenic mouse sRBC's in normal mouse. After incubating retinas for ADPase identified under darkfield illumination (ADPase activity in blood vessels). This study will determine which of 4 transgenic mouse models investigated has retinal and choroidal pathological changes similar to the human. Furthermore, this study should help elaborate the mechanism of retinal and choroidal vaso-occlusion in SCD and the primary determinant of eventual neovascularization, hemorrhage, fibrosis, retinal detachment, and blindness.
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