This project proposes to employ intravital video-microscopy and radionuclide imaging to analyze and quantitate the abnormal microvascular flow dynamics of genetic variants of sickle erythrocytes and their propensity to induce vaso-occlusion and microvascular derangement. A secondary objective is to analyze endothelial markers that reflect its injury consequent to the vaso-occlusion, using biochemical and immunological assays. Our hypothesis is that polymer fraction of deoxy HbS in erythrocytes of genetic variants of sickle cell disease (HbSS, HbSC and HbSS Thal) through the effect of MCHC, is the primary determinant of cellular flow dynamics and vaso-occlusion in the microcirculation. This hypothesis will be tested by four experimental approaches: (a) determination of the flow dynamics i.e., flow velocity, flux, transit times, of normal and sickle erythrocytes, and various density-separated and osmotically altered fractions of these cells, flowing through the microcirculation of rat mesentery and cremaster muscle under varying oxygen saturation; (b) determination of the pressure-flow-resistance relationships of a rat hindlimb vascular preparation perfused with normal or sickle erythrocytes, and biochemical and immunological analysis of endothelial markers in the venous effluents that indicate endothelial injury consequent to vaso-occlusion; (c) radionuclide imaging of the kinetics and sites of vaso-occlusion, and the cellular and rheological factors involved, using indium-111 oxine labeled normal or sickle erythrocytes and various density fractions of these cells. These data on the frequency of occlusion at microvascular bifurcations and residence times of the cells at these sites will be supplemented with fluorescent-labeled normal and sickle cells and intravital microscopy; (d) determination of the filterability of the normal and sickle erythrocytes mentioned above flowing through polycarbonate (Nucleopore) filters under varying conditions of oxygen saturation, and pore geometry. These data will be analyzed in terms of pore resistance of a red cell and its transit through the pore. The result of these studies will provide very significant insight into the mechanisms of vaso-occlusive events in sickle cell disease.
