9%) was infused for 5 min. Then the catheter was withdrawn and the common hepatic duct clamp was removed. The duodenal puncture closed by a purse-string suture (7-0 monofilament). The traction sutures were removed and abdomen was closed in two layers. Animals were allowed to wake up and given JQ1 structure free access to food and water. Sham-operated animals underwent the same procedure without any infusion into the pancreas. Vehicle or the Rho-kinase inhibitor, Y-27632 [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; Calbiochem, San Diego, USA], was given (0.5�C5 mg?kg?1) i.p. 30 min prior to bile duct cannulation. In separate experiments animals were treated with 5 mg?kg?1 Y-27632 2 h after taurocholate challenge. Animals were killed 24 h after the induction of pancreatitis.

One group of mice received 5 mg?kg?1 Y-27632 alone without bile duct cannulation. Blood was collected from the tail vein for systemic leucocyte differential counts and determination of blood amylase levels. Blood samples were also collected from the inferior vena cava for flow cytometric studies of neutrophils. Pancreatic tissue was removed and kept in two pieces; one piece was snap-frozen in liquid nitrogen for biochemical analysis of myeloperoxidase (MPO), trypsinogen activating peptide (TAP) and MIP-2 and the other piece was fixed in formalin for later histological analysis. Systemic leucocyte counts Tail vein blood was mixed with Turks solution (0.2 mg gentian violet in 1 mL glacial acetic acid, 6.25% v/v) in a 1:20 dilution. Leucocytes were identified as monomorphonuclear (MNLs) and polymorphonuclear (PMNLs) cells in a Burker chamber.

Blood amylase Amylase was quantified in blood with a commercially available assay (Reflotron?, Roche Diagnostics GmbH, Mannheim, Germany). MPO assay Frozen pancreatic tissue was preweighed and homogenized in 1 mL mixture (4:1) with phosphate-buffered saline and aprotinin 10 000 KIE mL?1 (Trasylol?, Bayer HealthCare AG, Leverkusen, Germany) for 1 min. The homogenate was centrifuged (15339��g, 10 min) and the supernatant was stored at ?20��C and the pellet was used for MPO assay as previously described (Laschke et al., 2007). In brief, the pellet was mixed with 1 mL of 0.5% hexadecyltrimethylammonium bromide. Next, the sample was frozen for 24 h and then thawed, sonicated for 90 s, put in a water bath set at 60��C for 2 h, after which the MPO activity of the supernatant was measured.

The enzyme activity was determined spectrophotometrically as the MPO-catalyzed change in absorbance in the redox reaction of H2O2 (450 nm, with a reference filter 540 nm, 25��C). Values are expressed as MPO units g?1 tissue. Dacomitinib Flow cytometry For analysis of Mac-1 and CXCR2 expression on circulating neutrophils, blood was collected into syringes prefilled with 1:10 acid citrate dextrose at 24 h post taurocholate challenge.