The pulmonary microvasculature contains neutrophils in about 60 times the concentration observed in the large systemic vessels due to longer capillary transit times of neutrophils than red blood cells (RBC). These long transit times are attributed to the mechanical characteristics of capillaries and neutrophils, particularly the dimensions of capillary segments and neutrophils, the longer deformation times of neutrophils compared to RBC, and the unique multisegmented anatomy of the capillary bed that provides numerous parallel pathways for RBC to detour around segments temporarily obstructed by neutrophils. The proposed studies will investigate neutrophil transit though the pulmonary microvasculature by examining the mechanical properties of capillaries and neutrophils. Our central hypothesIs is that, in normal lungs, neutrophil transit through the pulmonary microvasculature is determined by the geometry and compliance of the capillary bed, the pressure gradient across a capillary segment, and the mechanical properties of neutrophils. The studies described in the specific aims will provide new information about each contributing factor and will begin to examine their roles.
Aim 1 will determine the compliance of capillary segments using confocal microscopy by measuring the cross-sectional area of capillary segments at several transmural pressures and lung volumes within ranges that occur during breathing.
Aim 2 will develop a computational model to estimate the pressure gradients, blood flow and distribution of blood transit times across individual capillary segments and simulated arteriolar-venular pathways during breathing, as well as determining the effect of a neutrophil temporarily occluding a segment on these parameters in adjacent segments.
Aim 3 will determine the site of neutrophils within the bed and shape changes in neutrophils and capillary segments using confocal microscopy.
Aim 4 will determine the mechanical properties of quiescent and activated neutrophils, both at their periphery and within their cytoplasm, using magnetic twisting cytometry, which probes facets of neutrophil mechanics not previously studied. The role of the cytoskeleton in determining the mechanical properties will also be examined.
Aim 5 will compare computed and observed distributions of neutrophil transit times to determine which mechanical model of neutrophil structure is most consistent with the distribution of neutrophil transit times observed in vivo. Whether changes in the mechanical properties of neutrophils induced by inflammatory stimuli can account for their sequestration within the capillary bed will also be evaluated. These studies will provide new information about the mechanical behavior of the individual capillary segments and the entire bed, as well as the mechanical properties of neutrophils and how they pass through the normal capillary bed, and will help to focus future studies of neutrophil structure and transit. These mechanical events are also critical during the inflammatory process, where activation of neutrophils causes alterations in their mechanical properties and edema formation alters compliance of capillaries.
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