G. Graff et al., IMPROVED MYELOPEROXIDASE ASSAY FOR QUANTITATION OF NEUTROPHIL INFLUX IN A RAT MODEL OF ENDOTOXIN-INDUCED UVEITIS, Journal of pharmacological and toxicological methods, 39(3), 1998, pp. 169-178
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.