Figure 5. Clopidogrel treatment alter circulating neutrophil in septic P2Y₁₂ null mice.

Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and hematology studies performed. Graphs show counts of (A) white blood cells (WBC), (B) neutrophils (PMN), (C) lymphocytes (LY), and (D) platelets in clopidogrel-treated WT (black) and P2Y₁₂ null mice (white). Both sham and CLP samples were analyzed. Values are expressed as 1×10³ cells/μL, mean ± S.E.M., (n = 8; *p<0.05 WT CLP versus KO CLP mice and *p<0.05 treated-CLP WT versus treated-CLP P2Y₁₂ null mice). (E) Blood samples were collected by cardiac puncture in 3.8% sodium citrate (10:1), and p-selectin expression on platelet surface was analyzed through flow cytometry. Representative flow cytometry histograms are shown for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Isotype control is shown in gray and P-selectin stained samples in black. (F) The percentage of aggregates is reported for CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. Blood samples were labelled with antibodies against CD61 (platelet marker) and CD11b (leukocyte marker). Activated leukocytes were gated based on CD11b expression, and cell shape and data were analyzed as a percentage of aggregates expressing both CD41 and CD11b. Values are expressed as percentage of CD41+/CD11b+ cells, mean ± SEM (n = 6). (G) Photomicrographs of hematoxylin- and eosin-stained tissue sections obtained after CLP surgery. Representative images of lung tissue specimens are shown for sham and CLP samples (Magnification 20, n = 5) in CLP and sham control mice in WT (black) and P2Y₁₂ null (white) mice. (H) Acute lung injury (ALI) score, was assessed in Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. (I) MPO analysis was performed in lung samples of sham and CLP mice for Sham and CLP clopidogrel-treated WT (black) and P2Y₁₂ null (white) mice. Values are expressed as rfu/min/mg, mean ± SEM (n = 5).