We used a well-established mouse model of partial warm hepatic IRI.2, 22, 23 Separate Gefitinib research buy groups of WT and ASC KO mice were injected with heparin (100 U/kg), and an atraumatic clip was used to interrupt the artery/portal vein blood supply to the left/middle liver lobes. After 90 minutes of ischemia, the clip was removed, and the mice were sacrificed
6 hours after reperfusion. Some of the ASC KO recipients received a single injection of recombinant high mobility group box 1 (rHMGB1; 0.8 mg/kg intraperitoneally; a gift from Dr. A. Tsung, University of Pittsburgh, or Sigma-Aldrich Corp., St. Louis, MO) immediately at reperfusion. Additional WT cohorts were given an antimouse IL-1β monoclonal antibody (mAb; 10 mg/kg intraperitoneally; Novartis, Inc., Basel, Switzerland) or control immunoglobulin G (IgG) 1 day before ischemia. An equivalent of antihuman IL-1β (canakinumab), this mAb binds to mouse IL-1β and neutralizes its activity by blocking its interaction with IL-1 receptors. Sham-operated controls underwent the same procedure but without vascular occlusion. Serum alanine transaminase (sALT) levels, an indicator of hepatocellular injury, were measured in blood samples with an autoanalyzer (ANTECH Diagnostics, Los Angeles, CA). Liver paraffin sections (5 μm) were stained
with hematoxylin and eosin. The severity of liver IRI was graded Selleckchem MK-3475 blindly with Suzuki’s criteria on a scale from 0 to 4.24 No
necrosis or congestion/centrilobular ballooning was given a score of 0, whereas severe congestion and >60% lobular necrosis were given a value of 4. Liver-infiltrating macrophages and neutrophils were detected with a primary mAb against CD11b and lymphocyte antigen 6 complex locus G (Ly6G; BD Biosciences, San Jose, CA), respectively. HMGB1 was detected in hepatocytes and liver-infiltrating macrophages/monocytes with a rabbit anti-HMGB1 antibody (Ab; Cell Signaling Technology, Danvers, MA). The secondary, biotinylated goat antirat IgG or goat antirabbit IgG (Vector, Burlingame, CA) was incubated with immunoperoxidase (ABC kit, Sinomenine Vector). Positive cells were counted blindly in 10 high-power fields (HPFs) per section (×400). Neutrophil influx in the liver tissue was assessed with the enzymatic activity of MPO.25 One unit of MPO activity was defined as the quantity of the enzyme degrading 1 μmol of peroxide per minute at 25°C per gram of tissue. Total RNA was extracted from frozen livers with an RNase mini kit (Qiagen, Valencia, CA); the RNA concentration was determined with a spectrophotometer. RNA (5.0 μg) was reverse-transcribed into complementary DNA. Quantitative polymerase chain reaction was performed with the DNA Engine with the Chromo 4 detector (MJ Research, Waltham, MA).