5��C and 37.5��C. After anesthesia the rats underwent transurethral catheterization with a PE50 tubing passing into the bladder in kinase inhibitor Navitoclax order to empty it. The catheter was secured with a hitch suture onto the lower abdomen, oriented in a manner which did not alter the normal urethral axis. CLP or sham surgery was then performed. After surgery, rats were returned to their home cages in the same environmental conditions. Observations were made at times 0 (baseline), 3 and 7 hours after surgical procedures.The CLP-induced sepsisCLP was used to induce sepsis [42]. Briefly, a 3-cm midline laparotomy was made first through the skin and then through the linea alba to expose the cecum with the adjoining intestine. The cecum was exteriorized and ligated at its base, below the ileocecal valve in a non-obstructing manner with a 3.

0 silk. Then the ligated cecum was punctured with a 16-gauge needle, allowing entrapped fecal material to leak into the normally sterile peritoneal cavity. The cecum was then repositioned in the peritoneal cavity and the abdomen was closed in two layers. Sham-operated animals received laparotomy only. Samples of peritoneal fluid were taken at two experimental times (3 and 7 hour) and cultured for the growth of Gram-positive/Gram-negative isolates. Samples were incubated on Mannitol Salt Agar for 24 hours at 37��C for Gram-positive strains and layered on MacConkey Agar III for 24 hours at 37��C for Gram-negative culture. Species identification was determined by the automated Vitek2 system (bioM��rieux, Marcy l’Etoile, France), using a panel Gram-Positive (GP) card.

To demonstrate the occurrence of the inflammatory response, TNF-�� levels in systemic blood were measured. Blood samples were drawn via cardiac puncture at the same times (0, 3, and 7 hours) in the two experimental groups (at least n = 5 animals per group each time). The blood was immediately centrifuged at 4,000 rpm for 15 minutes at 4��C, plasma was collected, divided into aliquots and stored at -80��C until assayed. The TNF-�� plasma level was measured using an enzyme-linked immunosorbent assay according to the manufacturer’s instructions (Bender MedSystems, Vienna, Austria). The sensitivity of the assay was 11 pg/mL.Assessment of renal and GFB damage during sepsisFunctional assessment of damageCreatinine serum levels and creatinine clearance (calculated as urinary creatinine/seric creatinine*urinary volume/minute and expressed as mL/min/100 g body weight) were assessed as an index of acute renal injury.

Albumin urinary content was measured as an index of GFB change in perm-selectivity and expressed as albumin/urinary creatinine ratio to normalize for urinary volume. Urine and blood were sampled at 0, 3, 7 hours following CLP or sham-operation (at least n = 4 rats per group each time). Serum and urinary creatinine levels Dacomitinib were measured by an enzymatic method on an ADVIA 2400 Chemistry System Analyzer (Siemens Healthcare, Milan, Italy).