Bq123

BQ123 and/or BQ788 (both from Sigma, St. Louis, MO, USA) dissolved in sterile phosphate-buffered saline (PBS) were administered to corresponding SHAM and SCI mice (SHAM + BQ123, SHAM + BQ788, SHAM + BQ123 + BQ788, SCI + BQ123, SCI + BQ788 and SCI + BQ123 + BQ788) via intraperitoneal injection (10 mg/kg, respectively) (27 (link)–29 (link)) at 1 day before SCI and then further treated once a day for 6 weeks for behavioral testing or for the indicated time-points (2, 4, 6 and 24 h and 4 days) for other experiments post-injury. PBS for vehicle control (VEH) was administered in corresponding SHAM and SCI mice (SHAM + VEH and SCI + VEH). Significant side effects resulting from BQ123 and/or BQ788 treatment, such as changes in body weight or an increase in mortality, were not observed during our experiments.

50 μl of vehicle (0.9% saline, Fresenius Kabi) or endothelin-1 (20 pmol, Sigma-Aldrich) were injected intradermally in the back of the neck. Where the ET_AR antagonist BQ-123 was used, 10 pmol of endothelin-1 were injected, with or without 10 nmol of BQ-123 (Sigma-Aldrich) in one intradermal injection. Each mouse was then placed in a cage with wooden chips and recorded for 1 h using a digital video camera. Afterwards, AniTracker® v1.2 was used to score the videos for the duration and frequency of grooming and scratching behavior. A scratching episode was defined as a bout of scratching by either hind paw; from the time point it was lifted until placed back on the ground. The videos were scored by observers blinded to the genotype and treatment given. The results for each group are expressed as the mean frequency of scratching episodes/60 min, the mean duration of scratching episodes/60 min, and, where relevant, as the mean length of scratching episodes (total duration/total frequency) and as the mean frequency of scratching episodes per each 10 min interval. Results are presented as mean ± SEM.

HCMs were purchased from ScienCell Research Laboratories (Carlsbad, CA, USA) and grown in cardiac myocyte medium supplemented with 5% fetal bovine serum (FBS), 1% cardiac myocyte growth supplement, and 1% penicillin/streptomycin solution. Cells were cultured on a poly-L-lysine-coated dish in a humidified incubator with 5% CO₂ at 37°C. Cells between passages 3–5 were used in all experiments. The Safety Committee of Biological Experiments at Kaohsiung Medical University approved the use of HCMs for in vitro experimental use. The following drugs were used: fenofibrate (a PPARα agonist, Sigma), GW6471 (GW, PPARα antagonist, Santa Cruz), BQ-123 [BQ123, endothelin A receptor (ETA) antagonist, Sigma], BQ788 [BQ788, endothelin B receptor (ETB) antagonist, Sigma], PD98059 (ERK inhibitor, Cell Signaling), and SB203580 (p38 inhibitor, Cell Signaling). None of these drugs significantly influence cell viability (>90%).