Figure 3. Assessment of survival rates and inflammatory parameters in Plg^–/– mice and their WT littermates during nonsevere sepsis.

(A) Plg^+/+ and Plg^–/– mice were subjected to nonsevere (30G needle) CLP. The survival rates (n = 6 mice) were monitored for 6 days. In another set of experiments (n = 4–7), cells present in the peritoneal cavity were harvested 12 hours after CLP. (B–D) The number of total cells (B), mononuclear cells (C), and neutrophils (D) were evaluated by counting cytospin slides stained with May–Grünwald–Giemsa. (E) The number of M1 (F4/80^low GR1⁺ CD11b^med) macrophages was determined by flow cytometry. (F) The percentage of efferocytosis was determined by morphological counting of cytospin slides treated with May–Grünwald–Giemsa stain. (G) Expression of fibrinogen in the liver was determined by Western blotting with anti–β-actin used as the loading control. (H) Densitometric analysis from Western blotting gels is also represented. (I) The levels of TNF, IL-10, and IL-6 were quantified by ELISA in cell-free peritoneal lavages. (J and K) The peritoneal fluid (J) and blood (K) samples were plated in brain–heart infusion medium for the analysis of bacterial load. (L) ALT activity was measured from plasma samples. (M) Representative slides of H&E-stained lungs of Plg^+/+ and Plg^–/– mice are shown. Bottom row: Higher-magnification images (scale bar: 10 μm) of the micrographs in the Upper row (scale bar: 50 μm). (N) Histopathological score (maximum score = 5) evaluated focal hemorrhage (arrow), edema (arrowhead), vascular hyperemia (#), and inflammatory infiltrate (*). Results are shown as the mean ± SEM or median of 4–7 mice per group. The experiments were performed 2 times with similar results. ^#P < 0.05 and ^##P < 0.01 when comparing Plg^+/+ and Plg^–/– mice by log-rank test (survival curves), unpaired 2-tailed Student’s t test or Mann-Whitney U test. Outliers were removed from the graphs when detected.