The majority of morbidity and mortality in sickle cell disease is caused by vaso-occlusion: circulatory obstruction leading to tissue ischemia and infarction. The consequences of vaso-occlusion are seen clinically throughout the vascular tree, from the relatively high-oxygen and high-velocity cerebral arteries to the relatively low-oxygen and low-velocity postcapillary venules. Prevailing models of vaso-occlusion propose mechanisms that are relevant only to regions of low oxygen and low velocity, leaving a wide gap in our understanding of the most important pathologic process in sickle cell disease. Progress toward understanding vaso-occlusion is further challenged by the complexity of the multiple processes thought to be involved, including, but not limited to 1) deoxygenation-dependent hemoglobin polymerization leading to impaired rheology, 2) endothelial and leukocyte activation, and 3) altered cellular adhesion. Here, we chose to focus exclusively on deoxygenation-dependent rheologic processes in an effort to quantify their contribution independent of the other processes that are likely involved in vivo. We take advantage of an experimental system that, to our knowledge, uniquely enables the study of pressure-driven blood flow in physiologic-sized tubes at physiologic hematocrit under controlled oxygenation conditions, while excluding the effects of endothelium, leukocyte activation, adhesion, inflammation, and coagulation. We find that deoxygenation-dependent rheologic processes are sufficient to increase apparent viscosity significantly, slowing blood flow velocity at arterial oxygen tension even without additional contributions from inflammation, adhesion, and endothelial and leukocyte activation. We quantify the changes in apparent viscosity and define a set of functional regimes of sickle cell blood flow personalized for each patient that may be important in further dissecting mechanisms of in vivo vaso-occlusion as well as in assessing risk of patient complications, response to transfusion, and the optimization of experimental therapies in development.
Abnormal sickle red blood cell (sRBC) biomechanics, including pathological deformability and adhesion, correlate with clinical severity in sickle cell disease (SCD). Clinical intravenous fluids (IVFs) of various tonicities are often used during treatment of vaso-occlusive pain episodes (VOE), the major cause of morbidity in SCD. However, evidence-based guidelines are lacking, and there is no consensus regarding which IVFs to use during VOE. Further, it is unknown how altering extracellular fluid tonicity with IVFs affects sRBC biomechanics in the microcirculation, where vaso-occlusion takes place. Here, we report how altering extracellular fluid tonicity with admixtures of clinical IVFs affects sRBC biomechanical properties by leveraging novel in vitro microfluidic models of the microcirculation, including 1 capable of deoxygenating the sRBC environment to monitor changes in microchannel occlusion risk and an "endothelialized" microvascular model that measures alterations in sRBC/endothelium adhesion under postcapillary venular conditions. Admixtures with higher tonicities (sodium = 141 mEq/L) affected sRBC biomechanics by decreasing sRBC deformability, increasing sRBC occlusion under normoxic and hypoxic conditions, and increasing sRBC adhesion in our microfluidic human microvasculature models. Admixtures with excessive hypotonicity (sodium = 103 mEq/L), in contrast, decreased sRBC adhesion, but overswelling prolonged sRBC transit times in capillary-sized microchannels. Admixtures with intermediate tonicities (sodium = 111-122 mEq/L) resulted in optimal changes in sRBC biomechanics, thereby reducing the risk for vaso-occlusion in our models. These results have significant translational implications for patients with SCD and warrant a large-scale prospective clinical study addressing optimal IVF management during VOE in SCD.
Although homozygous sickle cell disease is often clinically severe, the corresponding heterozygous state, sickle cell trait, is almost completely benign despite the fact that there is only a modest difference in sickle hemoglobin levels between the two conditions. In both conditions, hypoxia can lead to polymerization of sickle hemoglobin, changes in red cell mechanical properties, and impaired blood flow. Here, we test the hypothesis that differences in the oxygen-dependent rheological properties in the two conditions might help explain the difference in clinical phenotypes. We use a microfluidic platform that permits quantification of blood rheology under defined oxygen conditions in physiologically sized microchannels and under physiologic shear rates. We find that, even with its lower sickle hemoglobin concentration, sickle trait blood apparent viscosity increases with decreasing oxygen tension and may stop flowing under completely anoxic conditions, though far less readily than the homozygous condition. Sickle cell trait blood flow becomes impaired at significantly lower oxygen tension than sickle cell disease. We also demonstrate how sickle cell trait can serve as a benchmark for sickle cell disease therapies. We characterize the rheological effects of exchange transfusion therapy by mixing sickle blood with nonsickle blood and quantifying the transfusion targets for sickle hemoglobin composition below which the rheological response resembles sickle trait. These studies quantify the differences in blood flow phenotypes of sickle cell disease and sickle cell trait, and they provide a potentially powerful new benchmark for evaluating putative therapies in vitro.
Objective Our goal was to develop a model of the microvasculature that would allow us to quantify changes in the rheology of sickle blood as it traverses the varying vessel sizes and oxygen tensions in the microcirculation. Methods We designed and implemented a microfluidic model of the microcirculation that comprises a branching microvascular network and physiologic oxygen gradients. We used computational modeling to determine the parameters necessary to generate stable, linear gradients in our devices. Sickle blood from 6 unique patients was perfused through the microvascular network and subjected to varying oxygen gradients while we observed and quantified blood flow. Results We found that all sickle blood samples fully occluded the microvascular network when deoxygenated, and we observed that sickle blood could cause vaso-occlusions under physiologic oxygen gradients during the microvascular transit time. The number of occlusions observed under 5 unique oxygen gradients varied among the patient samples, but we generally observed that the number of occlusions decreased with increasing inlet oxygen tension. Conclusions The model system we have developed is a valuable tool to address fundamental questions about where in the circulation sickle cell vaso-occlusions are most likely to occur and to test new therapies.
Understanding changes in the properties of semiconductor materials after immobilization of biomolecules on the surface is essential for the fabrication of well-tuned and programmable devices. The work examines changes in the properties of gallium phosphide (GaP) after modification with an organic linker, a single stranded DNA, and its complementary strand. We investigated changes in surface potential with Kelvin probe force microscopy (KPFM). Analysis revealed that a more ordered adlayer of ssDNA was present when a lower concentration of linker molecule was used. KPFM data combined with coverage data obtained from X-ray photoelectron spectroscopy (XPS) further confirmed this result. Successful hybridization with the complementary strand was confirmed by both KPFM and Raman spectroscopy. The results indicate that one can control the amount of DNA on the surface by changing the initial concentration of the organic linker, and thus modulate the surface potential of the semiconductor material.
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