IMPROVED MYELOPEROXIDASE ASSAY FOR QUANTITATION OF NEUTROPHIL INFLUX IN A RAT MODEL OF ENDOTOXIN-INDUCED UVEITIS

Previously described models of endotoxin-induced uveitis quantify neut rophil influx into the eye using biochemical or direct cell count meth ods that result in an underestimation of ocular leukocyte accumulation following the inflammatory stimulus. We have optimized the rat model of endotoxin-induced uveitis by first overcoming interference in the b iochemical assay of myeloperoxidase due to endogenous ocular reductant s and cellular constituents containing free thiol functional groups. T his was accomplished by simultaneously 1) extensively diluting soluble , interfering substances and 2) blocking tissue sulfhydril functional groups during tissue homogenization. Uveitis was induced in rats by su bplantar injection of endotoxin. Twenty-four hours later, eyes were en ucleated, homogenized, fractionated, and myeloperoxidase activity of n eutrophils sedimenting with the membranous pellet was extracted. Previ ously published extraction procedures yielded only 40% of total assaya ble myeloperoxidase activity. Optimal recovery of myeloperoxidase acti vity (>twofold increase) was achieved only with two sequential extract ions using 50 mM phosphate buffer (pH 7.4) containing 10 mM N-ethylmal eimide, and subsequent solubilization of myeloperoxidase activity by e xtraction with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosph ate buffer (pH 6.0). This modified extraction procedure and optimized myeloperoxidase assay conditions (300 mu M hydrogen peroxide and 1.5 m M o-dianisidine) were then used to enhance the uveitis model. Maximum ocular neutrophil accumulation was observed at endotoxin doses of 100- 200 mu g. Total ocular neutrophil infiltrations ranged from 250,000 to 800,000 cells/globe. This leukocyte influx was inhibited dose-depende ntly by topical ocular administration of dexamethasone, with half-maxi mal inhibition observed at a concentration of 0.01%, w/v. Further vali dated by the correlation of biochemical results with histological eval uation, the refined methodology described in this report has applicati on in assessing the ophthalmic therapeutic potential of antiinflammato ry agents. (C) 1998 Elsevier Science Inc.