Ultrasound assessment of inflammation and renal tissue injury with microbubbles targeted to P-selectin

Background - Routine methods capable of assessing tissue inflammation noninvasively are currently not available. We hypothesized that tissue retention of microbubbles targeted to the endothelial cell adhesion molecule P-selectin would provide a means to assess inflammation with ultrasound imaging. Methods and Results - Phospholipid microbubbles targeted to P-selectin (MB_p) were created by conjugating monoclonal antibodies against murine P-selectin to the lipid shell. The microvascular behaviors of MB_p and control microbubbles without antibody (MB) or with isotype control antibody (MB_iso) were assessed by intravital microscopy of cremasteric venules of control and tumor necrosis factor (TNF)-α-stimulated wild-type mice. Retention of all microbubbles increased (P<0.05) with TNF-α treatment because of increased attachment to activated leukocytes. Extensive attachment of MB_p directly to the venular endothelium or to adherent platelet-leukocyte aggregates was observed in TNF-α-stimulated mice, resulting in 4-fold greater (P<0.01) retention of MB_p than either MB_iso or MB. Enhanced retention of MB_p was completely abolished in TNF-α-stimulated P-selectin-deficient mice. The ultrasound signal from microbubbles retained in inflamed tissue was assessed by contrast-enhanced renal ultrasound imaging of the kidneys of mice undergoing ischemia-reperfusion injury. In wild-type mice, this signal was significantly higher (P<0.05) for MB_p (12±2 U) than either MB_iso (6±3 U) or MB (5±3 U). In P-selectin-deficient mice, the signal for MB_p was equivalent to that from control microbubbles. Conclusions - Microvascular retention of microbubbles targeted to P-selectin produces strong signal enhancement on ultrasound imaging of inflamed tissue. These results suggest that site-targeted microbubbles may be used to assess inflammation, tissue injury, and other endothelial responses noninvasively with ultrasound.