4.6. Evaluation of Free Radical Production

Free radical production by transmigrating neutrophils is an important early event in inflammatory arthritis, and we have previously shown that it correlates well with other (functional, histological) features of disease severity [9]. L-012 (Wako Pure Chemical Industries Ltd., Osaka, Japan) is a luminol analog chemiluminescent probe that has been shown to be very sensitive toward reactive oxygen and nitrogen species in vivo. L-012 was dissolved in sterile physiologic saline, and was injected in a dose of 25 mg/kg i.p. under as described previously [42] 3 h after K/BxN serum challenge, and on days 1 and 4 of the experiment. Mice were imaged in ketamine/xylazine anesthesia 10 min after the injection using the IVIS Lumina III system with the following settings: 120 s acquisition, Binning = 8, F/Stop = 1). Identical ROIs were applied around the ankle joints and chemiluminescence was expressed as total radiance (total photon flux/s).