Previously described models of
endotoxin-induced
uveitis quantify neutrophil influx into the eye using biochemical or direct cell count methods 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 biochemical assay of
myeloperoxidase due to endogenous ocular
reductants and cellular constituents containing free
thiol functional groups. This 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 subplantar injection of
endotoxin. Twenty-four hours later, eyes were enucleated, homogenized, fractionated, and
myeloperoxidase activity of neutrophils sedimenting with the membranous pellet was extracted. Previously published extraction procedures yielded only 40% of total assayable
myeloperoxidase activity. Optimal recovery of
myeloperoxidase activity (>twofold increase) was achieved only with two sequential extractions using 50 mM
phosphate buffer (pH 7.4) containing 10 mM
N-ethylmaleimide, and subsequent solubilization of
myeloperoxidase activity by extraction with 0.5%
hexadecyltrimethylammonium bromide in 50 mM
phosphate buffer (pH 6.0). This modified extraction procedure and optimized
myeloperoxidase assay conditions (300 microM
hydrogen peroxide and 1.5 mM
o-dianisidine) were then used to enhance the
uveitis model. Maximum ocular neutrophil accumulation was observed at
endotoxin doses of 100-200 microg. Total ocular neutrophil infiltrations ranged from 250,000 to 800,000 cells/globe. This leukocyte influx was inhibited dose-dependently by topical
ocular administration of
dexamethasone, with half-maximal inhibition observed at a concentration of 0.01%, w/v. Further validated by the correlation of biochemical results with histological evaluation, the refined methodology described in this report has application in assessing the ophthalmic therapeutic potential of
antiinflammatory agents.