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)-alpha-stimulated wild-type mice. Retention of all microbubbles increased (P<0.05) with TNF-alpha 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-alpha-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-alpha-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.