Pharmacologic PAI inhibition mobilizes neutrophils into the circulation and improves their tissue infiltration, a process dependent on endogenous tPA and MMP-9. (A) Wright-Giemsa staining of MACS-isolated infiltrating Gr-1⁺ and Gr-1⁻ cells derived from ischemic tissues of C57BL/6 mice on day 5 after HL induction. (B) Immunofluorescent staining of Gr-1 was performed on nonischemic muscle tissues or on HL-ischemic muscle tissues derived from vehicle- or PAI-1 inhibitor-treated HL-ischemic tPA^+/+ and tPA^−/− mice 14 days after the HL procedure. PAI-1 inhibitor was administered daily on days 0-6 after the procedure. The arrows indicate Gr-1⁺ cells (scale bars, 200 mm). Nuclei were counterstained with DAPI (blue). (C) Quantification of Gr-1⁺ cells in ischemic muscle tissues (n = 3/group). (D-K) The total number of WBCs (D,H) and the number of neutrophils (E,I), CD11b⁺Gr-1⁺ cells (F,J), and monocytes (G,K) were determined in the peripheral blood of PAI-1 inhibitor-treated or vehicle-treated tPA^+/+ and tPA^−/− mice (for B-G, n = 4) and in MMP-9^+/+ and MMP-9^−/− mice (for H-K, n = 6) by counting (D,E,G,H,I,K) or by FACS analysis (F,J). (L-M) The total number of WBCs (L) and neutrophils (M) were counted in in MMP-9^+/+ and MMP-9^−/− mice (n = 4). *P < .05; **P < .001 for recombinant tPA-treated versus vehicle-treated C57BL/6 mice. Values represent the means ± SEM. Data are expressed as the absolute number of each cell type per milliliter of blood. *P < .05.