Abstract 1645: Examination of Leukocyte Trafficking in Ischemic Brains of CD73 Null Mice
Vascular endothelial cells play an essential role in inflammation and leukocyte trafficking through their surface enzymes, that modulate pro- and anti-inflammatory responses in injured tissue. The 5′ecto-nucleotidase CD73 breaks down extracellular adenosine monophosphate (AMP) to adenosine, a potent anti-inflammatory mediator. It has been shown that CD73 attenuates vascular leakage as well as leukocyte accumulation following hypoxia. Based on these findings, we hypothesized that adenosine produced as a result of CD73 catalysis triggers an endogenous protective mechanism in the ischemic brain, at least in part by regulation of leukocyte entry into the injured brain. To test this hypothesis, mice were subjected to the photothrombotic middle cerebral artery (MCA) occlusion model of stroke, in which an argon laser pulse was applied to the MCA, following intravenous Rose Bengal infusion. The results from 8 week old mice lacking CD73 (CD73−/−) were compared to wild-type (WT) control animals. Infarct volumes were compared between CD73−/− and WT control animals at 48 hours after stroke, at which time leukocyte influx was measured by flow cytometry. The kinetics of macrophage and microglial activation were determined by measuring surface expression of CD45, CD11b, and two costimulatory markers, CD80 and CD86. Ly6G expression was also assessed in order to distinguish neutrophils from microglia or macrophages. CD73−/− stroked hemispheres showed a 29.3% increase in total infiltrating cells compared with WT control hemispheres (3.9 x 105 vs. 3 x 105). This was confirmed by a 46.9% increase in infarct volume, measured by 2,3,5-Triphenyl Tetrazolium Chloride staining (52.0 mm3 vs 35.4 mm3). Although the ratio of microglia, macrophages and neutrophils was similar in CD73−/− mice and WT controls, within the cells of macrophage lineage, there was a larger subpopulation of highly activated cells of increased phagocytic ability in CD73−/− stroked hemispheres, as shown by CD80 expression (WT 1.8 x 104 vs CD73−/− 3 x 104). Together, these data suggest a role for CD73 in leukocyte trafficking in the ischemic brain. They also highlight flow cytometry as a valuable tool for monitoring the effect of critical vascular regulatory genes on ischemia-driven cerebral inflammation.