Microvascular blood flow and stasis in transgenic sickle mice: Utility of a dorsal skin fold chamber for intravital microscopy

Abstract

Vascular inflammation, secondary to ischemia-reperfusion injury, may play an essential role in vaso-occlusion in sickle cell disease (SCD). To investigate this hypothesis, dorsal skin fold chambers (DSFCs) were implanted on normal and transgenic sickle mice expressing human a and b s /b s-Antilles globin chains. Microvessels in the DSFC were visualized by intravital microscopy at baseline in ambient air and after exposure to hypoxia-reoxygenation. The mean venule diameter decreased 9% (P < 0.01) in sickle mice after hypoxia-reoxygenation but remained constant in normal mice. The mean RBC velocity and wall shear rate decreased 55% (P < 0.001) in sickle but not normal mice after hypoxia-reoxygenation. None of the venules in normal mice became static at any time during hypoxia-reoxygenation; however, after 1 hr of hypoxia and 1 hr of reoxygenation, 11.9% of the venules in sickle mice became static (P < 0.001). After 1 hr of hypoxia and 4 hr of reoxygenation, most of the stasis had resolved; only 3.6% of the subcutaneous venules in sickle mice remained static (P = 0.01). All of the venules were flowing again after 24 hr of reoxygenation. Vascular stasis could not be induced in the subcutaneous venules of sickle mice by tumor necrosis factor alpha (TNF-a). Leukocyte rolling flux and firm adhesion, manifestations of vascular inflammation, were significantly higher at baseline in sickle mice compared to normal (P < 0.01) and increased 3-fold in sickle (P < 0.01), but not in normal mice, after hypoxia-reoxygenation. Plugs of adherent leukocytes were seen at bifurcations at the beginning of static venules. Misshapen RBCs were also seen in subcutaneous venules. Am.

Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice

“…They can best be described as tools to understand human SCD (41). For our studies, the sickle mouse models mimic human SCD in several important ways: they have rbc sickling in response to hypoxia; hemolysis; organ pathology with rbc congestion and infarction as in the human disease; excessive ROS production; a vigorous and chronic inflammatory response with activated endothelium; and impaired blood flow, especially in response to hypoxia/reoxygenation (2,4,7,37,(42)(43)(44)(45). Most of the studies described in this paper focused on the less severe murine model of SCD, the S+S-Antilles mouse (46), and fewer on the more severe murine model, BERK mice (47).…”

“…Hematocrit levels and reticulocytes were measured in EDTA blood collected from the tail veins of mice as previously described (43). Plasma Hb was measured by Drabkin's reagent.…”

Intravital microscopy in a dorsal skinfold chamber: hemodynamics, tumor angiogenesis and inflammation

“…ImageJ, ImagePro). 21 The shearing image technique has been used intensively with a dorsal skinfold chamber because of its ease of use and ability to measure real time values. This technique uses a video signal combined with sync separation and adjustable delay to determine horizontal displacement.…”

“…Another technique used to measure red blood cell (RBC) velocity is an optical Doppler velocimeter. 21 Blood flow (Q) is then determined using the equation Q=V×π(D/2) 2 where V is the mean RBC velocity and D is the vessel diameter. Another recent study applied a polymeric micro-optical interface for flow monitoring in a hamster dorsal skinfold chamber model.…”

Original Research: Diametric effects of hypoxia on pathophysiology of sickle cell disease in a murine model

“…Thus, hypoxia/reoxygenation (H/R) is used to mimic I/R to dissect the pathogenesis of sickle cell disease (SCD) in transgenic sickle mice. H/R induces acute sickling, 1 stasis in dorsal microvessels, 2 endothelial activation, 3 abnormal leucocyte/endothelial interactions, including increased emigration across the endothelium, 4,5 conversion of xanthine dehydrogenase to xanthine oxidase, 1 activation of nuclear factor (NF)-kappa B, 5,6 and increased sensitivity to pain. 7 A role for H/R in acute hemolysis and clinical syndromes associated with hemolysis is unclear.…”