Figure 2.

PI tissues show elevated acute neutrophil recruitment. Chronic ischaemia was induced in the cremaster muscles of LysM‐GFP mice, which have GFP^pos neutrophils. After 7 days, the tissues were fluorescently labelled with antibodies against PECAM‐1 (i.s.) and stimulated with TNF (300 ng), IL‐1β (50 ng), LPS (300 ng) or LTB₄ (100 nm, i.s.) 4 h before imaging. (A) Examples of LPS‐stimulated sham and PI tissues. (B) Quantification of neutrophil extravasation. (C) Still images from supplementary material Videos S1 and S2, showing neutrophil extravasation in sham and PI tissues after stimulation with topical LTB₄ (100 nm). Examples of postcapillary venules (PCVs) (arrows) and capillaries (arrowheads) are indicated. (D) Quantification of luminal neutrophil adhesion in small‐diameter capillary‐like vessels (<10 µm) and larger PCVs (>10 µm) 2 h after stimulation with TNF, IL‐1β, or LPS (i.s.). The data are normalized to luminal surface area. (E) Endothelial ICAM‐1 labelling intensity in PCVs in sham or 7‐day PI tissues (see supplementary material, Figure S1G, for images). (F) At 7 days PI, Cx3cr1‐GFP tissues were acutely stimulated with IL‐1β (50 ng) or LPS (300 ng) (i.s., 4 h), collected, and fluorescently labelled with antibodies against PECAM‐1 and the neutrophil marker S100A9. Correlations of the spatial distributions of Cx3cr1‐GFP^pos cells and neutrophils in 5 × 10⁴‐µm² regions of IL‐1β‐stimulated (left panel) or LPS‐stimulated (right panel) tissues (see supplementary material, Figure S1H, for an example image) are shown. (G) Depletion of Cx3cr1‐GFP^pos and Cx3cr1‐GFP^neg/dextran‐TRITC^pos cells by local application (i.s.) of clodronate liposomes on days 6–8 PI. (H) LPS‐induced or IL‐1β‐induced neutrophil recruitment in control or monocyte/macrophage‐depleted tissues. n = 6–12 animals per group. Data are presented as mean ± SEM, and statistically significant (t‐test) differences between sham and PI groups or correlations are indicated by asterisks: *p < 0.05, **p < 0.01, and ***p < 0.001. Differences between saline and stimulated groups, vessel size groups or control and clodronate‐depleted groups are indicated by hash symbols: ^# p < 0.05, ^## p < 0.01 and ^### p < 0.001.