Xylazine

Hindlimb ischemia (HLI) was induced by ligation of femoral artery in male mice at 10-12 weeks of age. Mice were anesthetized via intraperitoneal injection of a combination of 75 mg/kg ketamine and 10 mg/kg xylazine (Alfasan Co, Netherlands) before the unilateral ligation was performed. In this unilateral ischemia model, the contralateral limb was considered as a control. Mice were kept warm on a heatpad at 36 ± 1.0 °C during the procedure. Blood perfusion was measured by imaging of plantar regions of interests with Laser Doppler Imager (Moor Instruments) and the average lower leg blood flow was presented as the ratio of ischemic to non-ischemic side at days 0, 3, 7, 14, 21 and 28 following HLI. Vasculature imaging of the thigh was performed with the Laser Speckle Contrast Imaging System RFLSI III (RWD Life Science Co.).

After the elevated plus maze test, mice were subdivided into two groups: one group was used to measure baseline serum corticosterone levels 2 days after the elevated plus maze test. The other group was used to measure stress-induced plasma corticosterone levels and tested 20 min after the elevated plus maze test. The cardiac puncture was used to collect the blood of mice. Animals were anesthetized by an i.p. injection of Ketamine hydrochloride (50 mg/kg; Alfasan) plus Xylazine (4 mg/kg; Alfasan). The entire sampling procedure was completed within 8 min after removing the animal from its living cage. This is rapid enough to perform a reliable assessment of baseline and stress-induced serum corticosterone levels at the time of sampling, without any undesirable effect of disturbance or anesthesia^{44 (link)}. The blood was collected into sterile tubes and allowed to clot on ice for a minimum of 15–20 min before centrifugation at 3000 rpm for 10 min. Then, serum was collected into sterile vials and stored at −20 °C until assayed. The specific quantitative sandwich ELISA kit (Bio-Medical Assay Company, Sensitivity: <3 ng/ml) was used to meaure corticosterone according to the manufacturer’s instruction. All samples and standards were assayed in triplicate.

Rats were fasted around 12 hours prior to surgery and they were anesthetized with 80 mg/kg ketamine (Rotexmeica, Germany) plus 10 mg/kg xylazine (Alfasan, Netherlands). The sham-operated group underwent all surgical procedures except that they do not expose to hepatic vascular ligation. The method used to induce hepatic IR is previously described.^{21 (link)} The lobar ischemia was induced by ligating the portal triad (portal vein, hepatic artery, and bile ductile) for 60 minutes and the reperfusion was followed for 24 hours. In the third group, after exposing the liver to 60 minutes ischemia, the hepatic post-conditioning was induced by 4 cycles of 30-second brief reperfusion followed by 30-second reocclusion before the commencement of reperfusion.^{22 (link)} The tofacitinib (Biology fermentation Company) diluted into DMSO was injected via peritoneal to the fourth group with a dose of 15 mg/kg (total 1 mL volume) 15 minutes before commencement of reperfusion. In the fifth group, tofacitinib as a JAK/STAT inhibitor was used to probe the role of this signaling pathway in the protection induced by hepatic post-conditioning against IR injury.

The adult female rats were ovariectomized bilaterally under anesthesia by ketamine 10% (100 mg/kg, Alfasan, the Netherlands) and xylazine 2% (10 mg/kg, Alfasan, the Netherlands). Both ovaries were surgically removed, except for the control group, after joining the uterine horn through a midline longitudinal incision. Postoperatively, morphine injection of (5 mg/kg S.C) was used to reduce pain in rats, and oxytetracycline spray was used to prevent infection.

A total amount of 450 minutes was recorded for each animal (150min in the base before neuropathy, 150min on day 3 after neuropathy surgery, and 150min on day 6 after neuropathy surgery). All sleep recordings were performed between 09:00 a.m. to 11:30 a.m. For the electrode implantation, the animals were anesthetized with ketamine (65mg/kg) (10%, Rotexmed, Germany) and xylazine (15mg/kg) (2%, Alfasan, the Netherlands) and fixed on a stereotaxic apparatus. Three stainless steel screw electrodes were implanted in the skull for EEG recording and the EMG was recorded from dorsal neck muscles via two stainless-steel wire electrodes. The EEG and EMG data were collected by the electrophysiology recording system (Ruby Mind, NY, US) in the 3,000Hz sampling rate. The signals were amplified and filtered (EEG 0-40Hz, EMG 1-400Hz)^{20 ,25} .

Bladder challenge assay was performed on days 48 and 180 after the first vaccination to evaluate the efficacy and sustainability of developed immune responses. Five mice out of 15 immunized mice were used for the first challenge and the remaining 5 mice were kept until day 180 were used for the second challenge assay. In brief, E. coli strain CFT073 was introduced transurethrally by micro catheter (B&D, USA) into the bladder of anesthetized mice with a mixture of ketamine and xylazine (70 mg/kg + 5 mg/kg) (Alfasan, the Netherland). After one week, kidneys and bladders of the challenged mice were isolated, homogenized, and cultured in LB agar medium to count the appeared colonies.