Aim. To study the relationships between phenotypes of extracranial arteries' plaques (stable/unstable), their calcification and its causes, in particular, vascularization.Material and methods. The study included 88 patients: patients (n=44) with ischemic stroke and those (n=44) with chronic brain ischemia. In all subjects, the parameters of systemic mineral homeostasis were assessed (total and ionized calcium, phosphate, total protein, albumin, and calcification propensity). Atherosclerotic plaques have been obtained during carotid endarterectomy, fixed in formalin, postfixed in 1% osmium tetroxide, stained in 2% osmium tetroxide, dehydrated in ascending ethanol series and acetone, stained with 2% alcoholic uranyl acetate and embedded into epoxy resin with its further polymerization. Epoxy resin blocks were grinded, polished, counterstained with Reynolds' lead citrate and sputter coated with carbon. Sample visualization was performed employing backscattered scanning electron microscopy. Number and area of calcium deposits and neointimal vessels were quantified using ImageJ. Statistical analysis was carried out using Mann-Whitney U-test and Spearman's rank correlation coefficient.Results. It was found that area of neointimal calcification, but not number of calcium deposits, was associated with the stable plaque phenotype. The stabilizing effect of calcification was manifested in retarding stenosis associated with plaque rupture and stroke. Calcification extent directly correlated with total and local plaque vascularization, which have been associated with unstable and stable plaque phenotype, respectively. In addition, plaque calcification negatively correlated with total protein and albumin, thereby reflecting the impaired systemic mineral homeostasis.Conclusion. Atherosclerotic plaque calcification and active local vascularization reduce stenosis extent and stabilize plaque. In contrast, total plaque calcification contributes to the atherosclerosis progression and promotes major acute cardiovascular events.
Aim. Using an original method based on backscattered scanning electron microscopy, to study the structural features of the mitral valve leaflets in infective endocarditis.Material and methods. We examined 9 mitral valves extracted during surgical interventions due to structural malfunction in patients with infective endocarditis (IE). The samples were fixed in buffered paraformaldehyde with osmium tetraoxide postfixation. After dehydration by increasing alcohol concentration and acetone, the samples were placed in epoxy resin. After the resin has polymerized, the samples were ground and then polished to the desired depth. To increase the electronic contrast, the samples were treated with a uranyl acetate alcohol solution during dehydration and with Reynolds' lead citrate after polishing the epoxy blocks. The samples were visualized by backscattered scanning electron microscopy at an accelerating 15-kV voltage.Results. Structural leaflet injuries caused by IE were most pronounced in the central part and the base. Necrotic areas were extensive electron-dense formations located in the central leaflet layers, or displaced towards the ventricular surface. The electron-dense material in the necrotic area was poorly structured and contained individual cells and bacteria. Bacteria were also present outside the necrotic area. Necrotic areas were surrounded by a layer of a modified extracellular matrix, usually covered with a fibrin layer. Among the extracellular matrix fibers, the macrophages, smooth myocytes and fibroblasts was noted. The fibrin layer, in addition to these cells, contained a large number of blood vessels and was often covered with endothelium.Conclusion. Infection of the mitral valve leaflets causes a simultaneous inflammatory response and regeneration activation. Without adequate regulatory factors, the processes of inflammation and connective tissue creation lead to structural and functional leaflet failure. Specific causes may be overgrowth of necrotic and inflammatory areas, edema and fiber orientation disorder, as well as leaflet rupture.
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