2.15. Complete Freund's adjuvant (CFA)-induced paw inflammation

C57BL/6 male mice (7–8 weeks) were housed in an air-conditioned room with 12 h light–dark cycle, and animal studies were conducted according to the guidelines and protocol approved by Institutional Animal Care and Use Committee at Chang Gung University, Taiwan (IACUC approval No. CGU14-150). Mice were injected subcutaneously with carprofen (5 mg/kg) as analgesia and then intraperitoneally with CYR5099 (10 or 25 mg/kg body weight) or an equal volume of 0.9% saline under anesthesia (Isoflurane). After 1 h, paw inflammation was induced by intraplantar injection of CFA (1.5 mg/kg body weight in right-rear foot) or 0.9% saline (left-rear foot; as control). The change in paw thickness (nm) was determined before (baseline) and after CFA/saline injection at the indicated times. After injection of CFA for 4 h, L-012 (25 mg/kg body weight) was intraperitoneally administered. ROS production was determined using a Xenogen IVIS100 imaging system (PerkinElmer, Waltham, MA, USA) under anesthesia (30 mg/kg Zoletil 50 and 6 mg/kg xylazine) [17,29]. CO₂ euthanasia was used to stop the experiments. The hind paws were removed, fixed using 10% neutral buffered formalin, and embedded in paraffin blocks before cutting into 5-μm-thick sections. The staining of hematoxylin and eosin (H&E) and immunohistochemistry (Ly6G and MPO) was performed as previously described [17].