Ketorolac

For cocaine self-administration and yoked saline controls, rats underwent catheter implantation 5 to 7 days before the start of self-administration as previously described (Zhou et al., 2012 (link)). Briefly, rats were anesthetized (IP) using a mixture of ketamine hydrochloride (66 mg/kg; Vedco Inc., St. Joseph, MO) and xylazine (1.33 mg/kg; Lloyd Laboratories, Shenandoah, IA), followed by Equithesin (0.5 mL/kg) and Ketorolac (2 mg/kg; Sigma Aldrich) as a preoperative analgesic. One end of a silastic catheter was implanted into the right jugular vein, and the other end was attached to an infusion harness (Instech Solomon, Plymouth Meeting, PA) for IV drug delivery.

Rats were anesthetized with IP injections of ketamine (66 mg/kg; Vedco Inc, St. Joseph, MO, USA), xylazine (1.3 mg/kg; Lloyd Laboratories, Shenandoah, IA, USA), and equithesin (0.5 ml/kg; sodium pentobarbital 4 mg/kg, chloral hydrate 17 mg/kg, 21.3 mg/kg magnesium sulfate heptahydrate dissolved in 44% propylene glycol, 10% ethanol solution). Ketorolac (2.0 mg/kg, IP; Sigma Chemical, St. Louis, MO, USA) and cefazolin (0.2 g/kg, Patterson Veterinary, Saint Paul, MN, USA) were given before surgery as an analgesic and antibiotic, respectively. Catheters (constructed with Silastic tubing, Dow Corning Corporation, Midland, MI, USA) were inserted 4 cm into the right jugular vein and secured with silk sutures. The opposite end of the tubing ran subcutaneously and exited through a small incision on the back below the shoulder blades where an external port was exposed.

High-throughput screening for inhibitors of human caspase-4 was performed using the 1280 compound Prestwick Chemical Library at the University of Colorado High-throughput Screening Core Facility. The screen was designed in a 384-well format (Greiner 781207), using 33 μM compound, 1 μM recombinant human caspase-4 in high citrate buffer (50 mM Tris-HCl, pH 7.5, 1 M sodium citrate, 10 mM DTT, 10% sucrose), and 20 μM Promega Caspase-Glo® 9 Assay substrate (O’Brien et al., 2005 (link); Roschitzki-Voser et al., 2012 (link)). While designed for use with caspase-9, the LEHD substrate is also recognized by caspase-4 (Roschitzki-Voser et al., 2012 (link); Talanian et al., 1997 (link)). The assay Z’ factor is 0.66, recommending this method for high-throughput experimentation (Figure S1) (Zhang et al., 1999 (link)). Plates were read with endpoint luminescent analysis 20 minutes and 60 minutes after substrate addition. Compounds were considered to be hits if caspase-4 activity was inhibited to less than 25% relative to the solvent control. Using these criteria, we identified 27 hits, which are summarized in Table 1. Fenbufen, indoprofen, ketoprofen, ketorolac, felbinac, naproxen, ibuprofen, and tiaprofenic were purchased from Sigma Aldrich. Aspirin was purchased from TCI America. z-VAD-FMK was obtained from InvivoGen.