Cd1 male mice

CD1 male mice, 6–8 weeks old, 20–25 g weight, from Jackson Laboratory (Bar Harbor, ME), kept under standardized housing and feeding conditions were used. The experiments were done under anesthesia, using ketamine (60 mg/kg), acepromazine (2.5 mg/kg) and xylazine (2.5 mg/kg) in 0.1–0.2 ml PBS. A specific ITSN-1 siRNA sequence (100 µg siRNA/mouse) was delivered by using cationic liposomes, by retro-orbital injection, into mouse lungs as described previously (Bardita et al., 2013 (link); Predescu et al., 2012 (link)). The siRNA sense sequence – 5′-GAGAGAGCCAAGCCGGAAAUU-3′ – (Dharmacon, Lafayette, CO) was used for knocking down mouse ITSN-1s. Chronic inhibition of ITSN-1s was achieved by repeated retro-orbital delivery of the siRNA_ITSN–liposome complexes every 72 h for 24 days as described previously (Predescu et al., 2012 (link)). Mice were killed at day 3, day 10, day 15 and day 24; three to four mice per experimental condition [controls (wild-type mice, vehicle- and non-specific siRNA-treated mice) and siRNA_ITSN-treated mice] were used; all experiments were repeated at least three times. No mouse mortality was recorded during the 24 days of the study. All experiments were approved and performed in accordance with the guidelines of Rush University Institutional Animal Care and Use Committee.

Male Fos^2A-iCreER mice (TRAP2) (Jax 030323) and adult male and female C57/BL6 mice (16–24 weeks old) were purchased from The Jackson Laboratory. Mice were housed in a temperature-controlled vivarium on a 12-hour light/12-hour dark cycle (lights on at 0700 hours; lights off at 1900 hours) with ad libitum access to food and water. CD1 male mice (16–26 weeks old,) and male SD rats (10–14 weeks old) were also purchased from The Jackson Laboratory and Charles River Laboratories, respectively. For carbamazepine, oral gavage of carbamazepine (Novartis, packaged by Precision Dose) at 60 mg/kg was used. For ketorolac (Hospira), oral gavage of 10 mg/kg was used. For tamoxifen (MilliporeSigma, WXBD4583V), intraperitoneal injection of 150 mg/kg dissolved in corn oil was used.

CD1 male mice, 6–8 weeks old, 20–25 g weight, from Jackson Laboratory (Bar Harbor, ME), kept under standardized housing and feeding conditions were used. All mouse studies were approved and performed under the guidelines of RUMC Institutional Animal Care and Use Committee. The experiments were done under anesthesia [ketamine (60 mg/kg), acepromazine (2.5 mg/kg) and xylazine (2.5 mg/kg)] in 0.1–0.2 ml phosphate buffered saline (PBS)]. Three to five mice per experimental condition (wt-mice, LPS- and LPS ± EVs-treated mice) were used; all experiments were repeated at least three times. No mouse mortality occurred during the study.