Ermination of your concentration of circulating leukocytesThe total quantity of circulating leukocytes and neutrophils was evaluated in blood samples obtained by means of a cannula within the femoral artery. Samples have been collected prior to ischaemia (time 0), 120 min after ischaemia and 30 and 120 min just after reperfusion. The amount of total circulating leukocytes was determined by counting leukocytes in a modified Neubauer chamber after staining with Turk’s remedy and differential counts by evaluating the percentage of every single leukocyte on blood films stained with Could runwald iemsa.Evaluation of adjustments in vascular permeabilityThe extravasation of Evans blue dye in to the tissue was applied as an index of enhanced vascular permeability (Souza et al., 2000a). Evans blue (20 mg kg) was administered i.v. (1 ml kg) via a femoral vein 2 min before reperfusion with the ischaemic artery. At 30 (inside the mild injury model) or 120 min (inside the serious injury model) soon after reperfusion, fragments of the duodenum (10 cm) have been reduce open and allowed to dry in a petri dish for 24 h at 371C. The dry weight from the tissue was determined and Evans blue extracted utilizing 3 ml of formamide (24 h at room temperature). The amount of Evans blue inside the tissue was obtained by comparing the optical density (OD) of the extract with that of a common Evans blue curve study at 620 nm in an ELISA plate reader. Benefits are presented as the amount of Evans blue in mg per 100 mg of tissue. The right ventricle was flushed with 20 ml of PAR2 custom synthesis phosphatebuffered saline to wash the intravascular Evans blue within the lungs. The left lung was then excised and made use of for Evans blue extraction. The appropriate lung was employed for the determination of myeloperoxidase (MPO) as described below.Measurement of haemoglobin levelsThe levels of haemoglobin in tissue were utilised as an index of tissue haemorrhage. Tissues were carefully washed with excess saline to eliminate blood attached towards the intestinal epithelia or serosa. No attempt was made to perfuse the vessels with saline as no obvious hyperaemia was present. Immediately after washing, a sample of roughly one hundred mg of duodenum was removed and homogenized in Drabkin’s colour reagent in line with the instructions of your manufacturer (Analisa, Belo Horizonte, Brazil). The suspension was centrifuged for 15 min at 3000 g and filtered utilizing 0.two mm filters. The resulting solution was study using an ELISA plate reader at 520 nm and compared against a regular curve of haemoglobin.Measurement of cytokine levels in serum, intestine and lungsTNF-a, IL-1b, IL-6, IL-10 and CINC levels were measured in serum and intestine of animals employing ELISA strategies previously described (Hagan et al., 1993; Rees et al., 1999a, b; Francischi et al., 2000). Serum was obtained from coagulated blood (15 min at 371C, then 30 min at 41C) and stored at 01C till further evaluation. Serum samples had been analysed at a 1 : three dilution in PBS. In all, one hundred mg of duodenum or lung of sham-operated and NPY Y5 receptor Compound reperfused animals were homogenized in 1 ml of PBS (0.4 M NaCl and ten mM NaPO4) containing antiproteinases (0.1 mM PMSF, 0.1 mM benzethonium chloride, ten mM EDTA and 20 KI aprotinin A) and 0.05 Tween 20. The samples have been then centrifuged for 10 min at 3000 g and the supernatant quickly applied for ELISA assays at a 1 : 5 dilution in PBS. ELISA plates (Nunc MaxiSorb) have been coatedMPO levelsThe extent of neutrophil accumulation inside the intestine and ideal lung tissue was measured by assaying MPO activity as previously described (Matos et.