From these isolated observations, it is not clear whether what signaling steps are invoked by H2S to activate KATP channels or cause mobilization of AnxA1, or whether these two signaling elements are mechanistically linked in the same cell type or to endothelial P-selectin expression

From these isolated observations, it is not clear whether what signaling steps are invoked by H2S to activate KATP channels or cause mobilization of AnxA1, or whether these two signaling elements are mechanistically linked in the same cell type or to endothelial P-selectin expression. of neutrophils is usually pulled out into long nanotubes (microvilli) that form tethers at the rear of the rolling leukocyte. These tethers eventually detach as the cell continues to roll, but do not retract. Instead, they persist and are slung in front of the rolling leukocyte, to interact again with P-selectin. The membranous nanotube is now referred to as a sling, which the neutrophil rolls over to again be retarded in its movement down the vessel wall as the sling transitions to a tethering function. Step 4 4: Integrin-ICAM-1-dependent adhesion activates intracellular signaling pathways that induce cytoskeletal changes and polarization of the cell that lead to luminal motility. The crawling neutrophils move along interendothelial junctions in search of preferred routes for diapedesis and are often observed moving against the direction of flow in this exploration. Step 6: Once through the endothelium, the diapedesing neutrophils crawl abluminally along pericyte processes, interacting with basement membrane structures at the same time. Steps 8 and 9: Penetration of pericyte gaps and regions of low matrix protein expression in the basement membrane. Abluminally crawling neutrophils breach the pericyte layer where gaps between these cells exist, which coincide with regions of low matrix protein deposition (depicted as a lighter shade of green) in the basement membrane. These actions occur more or less simultaneously and use different adhesion molecules to propel the leukocyte through these barriers. Step 10: imaging (Jenne, Wong, Petri, & Kubes, 2011; Ley et al., 2008). Use of laser scanning confocal microscopy further enhances the imaging power of intravital microscopy by scanning the laser beam over every point of the focal plane, collecting a stack of sections, and using computer reconstruction algorithms to render a three-dimensional image that can be rotated in all planes. Laser scanning takes too long to apply and collect images and is thus not suitable for leukocyte trafficking studies. However, faster imaging is achieved by use of spinning disk confocal microscopy, which simultaneously detects fluorescence emitted from multiple points and utilizes a charge-coupled device instead of a photomultiplier tube, thereby increasing the rate at which images can be captured (Nakano, 2002). Imaging at greater tissue depths, thinner optical sections, and with decreased photobleaching or phototoxicity can be achieved by use of Hoechst 34580 multiphoton confocal Hoechst 34580 microscopy (Denk & Svoboda, 1997; Ley et al., 2008; Li et al., 2012). Other methods that have been developed to study leukocyte trafficking in deep tissues include single photon emission computed tomography, positron emission tomography, magnetic resonance imaging, Hdac11 and bioluminescence imaging. These methods can noninvasively track leukocytes over long periods of time, however, they lack the spatial and temporal resolution to visualize single-cell dynamics in situ (Mempel, Scimone, Mora, & von Andrian, 2004). 4. ASSESSING LEUKOCYTE ROLLING, ADHESION, AND EMIGRATION IN THE INTACT MICROCIRCULATION Leukocyte rolling describes the low affinity adhesive conversation between leukocytes and the vascular endothelium whereby the force of blood flow induces a rotational motion (i.e., rolling) of the leukocyte along the vascular wall. Rolling behavior is usually most often quantified as the number (or flux) of rolling leukocytes, which is usually assessed by counting the number of leukocytes flowing past a fixed point (line drawn perpendicular to the long vessel axis on TV monitor) in the vessel of interest that are moving slower than the stream of erythrocytes. Another way to characterize the behavior of rolling leukocytes is usually to quantify their velocity (leukocyte rolling velocity, = 8= diameter. Wall shear Hoechst 34580 stress (= 4= mean blood flow velocity, = blood viscosity, and =.