14 and as described.10 In this classification, three liver injury indices, sinusoidal congestion (score: 0-4), hepatocyte necrosis (score: 0-4), and ballooning degeneration (score: 0-4), are graded for a total score of 0-12. We also

quantified percent liver necrotic area as well as degree of hepatocyte apoptosis after liver IR. Hepatic apoptosis was quantified by counting the number of apoptotic hepatocytes per high-power field (400×) in necrotic and in nonnecrotic (viable) zones. Total apoptosis check details score per liver section was estimated by multiplying the number of apoptotic cells in necrotic area by percent liver necrotic area. Intestine H&E sections were also blindly evaluated for intestinal epithelial cell necrosis, development of a necrotic pannus over the mucosal surface, villous endothelial cell apoptosis, and swelling and blunting of villi because of villous mononuclear cell mucosal inflammation and edema. Renal H&E sections were evaluated for the

severity (score: 0-3) of renal cortical vacuolization, peritubular/proximal tubule leukocyte infiltration, proximal tubule simplification, and proximal click here tubule hypereosinophilia. Twenty-four hours after liver reperfusion, plasma, small intestine, and isolated crypt IL-17A levels were measured with mouse specific IL-17A ELISA kit according to the manufacturer’s instructions (eBiosciences). Tissues were homogenized in ice-cold RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, and 1% Triton-X [pH 7.4]) and samples processed for mouse-specific ELISA kits. Small intestine tissues from SOX9 flox/flox Villin Cre+/− (Paneth cell-deficient) or SOX9 flox/flox Villin Cre−/− (wildtype control) mice were homogenized in ice-cold RIPA buffer and processed for cryptdin-1 immunoblotting with Anti-(6C/A)-Crp1 antibody as described.10, 15 Small Intestine, and Kidney Inflammation. Liver, kidney, and small intestine inflammation after hepatic ischemia was determined with detection of neutrophil infiltration by immunohistochemistry 24 hours after hepatic IR as described16

and by measuring messenger RNA (mRNA) encoding markers of of inflammation, including keratinocyte-derived cytokine (KC), intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractive protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) 5 hours after liver IR (Supporting Table 1). Semiquantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed as described.10 We used two additional independent assays to assess the degree of liver and intestine apoptosis 24 hours after sham surgery or liver IR: in situ transferase-mediated dUTP nick-end labeling (TUNEL) assay and the detection of DNA laddering. For the TUNEL assay, formalin-fixed sections were deparaffinized in xylene and rehydrated through graded ethanol to water.