The rats were separated into four groups, each composed of 10 individual rats: (i) 10 mg/kg SLD-treated CLP group; (ii) 20 mg/kg SLD-treated CLP group; (iii) CLP group; and (iv) sham-operated control group. The groups were housed in separate cages. A CLP polymicrobial sepsis model

was applied to the rats, induced through caecal ligation and two-hole puncture. Anaesthesia was induced through the intraperitoneal administration of thiopental 25 mg/kg. The abdomen was shaved and the peritoneum was Pexidartinib chemical structure opened. Once the diaphragm exposed the abdominal organs, the caecum was isolated and ligated with a 3/0 silk ligature just distal to the ileocaecal valve. Two punctures were made with a 22-gauge needle through the caecum distal to the point of ligation, and the caecum was returned to the peritoneal cavity. The abdominal incision was then closed with a 4/0 sterile synthetic absorbable suture. The wound was bathed in 1% lidocaine solution to ensure analgesia. The sham-operated group received laparotomies,

and the rats’ caeca were manipulated but not ligated or perforated. All the animals were given 2 ml/100 g body weight of normal saline subcutaneously at the time of surgery and 6 h afterwards for fluid resuscitation. Immediately after the surgical procedure was completed, the rats in the sham-operated and the SLD-treated CLP groups received 10- or 20-mg/kg doses of SLD, which were administered with an oral gavage suspended in saline. There are many sildenafil CHIR-99021 ic50 doses for rats, varying from 0·4 mg/kg to 90 mg/kg, with different administration routes [28–33]. The reason we selected 10- and 20-mg/kg doses of oral sildenafil is that 10 mg/kg/day of sildenafil would result approximately

in the same see more plasma concentration as 50 mg in humans [34]. These doses are very common for rats, and we first aimed to determine if it is protective in CLP-induced organ damage, as well as how the dose affects protection. Therefore, we used 10- and 20-mg/kg oral doses of sildenafil, as have previous authors [35–37]. An equal volume of saline was administered to the sham-operated control group and the CLP group. The rats were deprived of food postoperatively but had free access to water for the next 16 h, until they were killed. The survival rate in CLP-induced sepsis models varies according to the size of the needle used [38]. Otero-Anton et al. reported that mortality after CLP in rats increased gradually with the size of the caecal puncture. They evaluated 0·5-cm blade incision; 13-gauge, 16-gauge and 18-gauge puncture; and four punctures with a 22-gauge needle. Mortality increased gradually with the puncture size, from 27% with a 22-gauge needle to 95% with the blade incision during a week of observation [38]. In addition, in our previous studies we observed mortality within 12–20 h after sepsis induction with a 12-gauge needle [39–42]. However, in studies performed with 21- and 22-gauge needles, mortality was not as common [38,43,44].