Figure 9: Overexpression of ADFm specifically in endothelial cells attenuates proinflammatory responses after ischaemic injury in vivo.

tFCI was induced in WT or Tg-ADFm mice followed by 24 h of reperfusion. (a) Representative images from the inner border of infarction in the cortex after tFCI or the corresponding region after sham operation, showing immunostaining for the following markers: MPO (green, neutrophil), F4/80 (green, macrophage), NeuN (red, neuron), Iba1 (red, microglia/macrophage) and CD206 (green, activated M2 microglia/macrophage). Square: the region enlarged in high-power images. Scale bar, 20 μm. (b) MPO⁺, F4/80⁺ and Iba1/CD206⁺ cells were counted in the areas described in a, and data were expressed as the number of cells per mm³. n=6 mice per group. (c,d) Flow cytometric quantification of GR1⁺, CD45^high (circle) and CD45^int (oval) cells among the CD11b⁺ cell populations in WT and Tg-ADFm brains after tFCI or sham operation. Each fluorescence-activated cell sorting (FACS) sample was from two pooled brains. Data represent four independent experiments. Both immunofluorescent staining and FACS analyses demonstrated that endothelial ADFm overexpression impeded the infiltration of blood neutrophils/macrophages into the stroke brain without altering the numbers of resident microglia. (e) A panel of inflammatory markers was examined using the quantitative inflammation array in microvessel extracts from the ischaemic hemisphere at 24 h after tFCI. Levels of inflammatory markers were expressed relative to WT sham. ADFm overexpression significantly reduced the expression of several inflammation markers, including CCL2, IL-17, CXCL1, IL-6, ICAM-1 and CCL5. n=4 mice per group. *P≤0.05, **P≤0.01, ***P≤0.001 versus WT.