title: Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis.
abstract: 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.
authors.affiliation: Ophthalmic Products Research, Alcon Laboratories, Inc., Fort Worth, Texas 76134-2099, USA.
authors.forename: G
authors.initials: G
authors.lastname: Graff
authors.forename: D A
authors.initials: DA
authors.lastname: Gamache
authors.forename: M T
authors.initials: MT
authors.lastname: Brady
authors.forename: J M
authors.initials: JM
authors.lastname: Spellman
authors.forename: J M
authors.initials: JM
authors.lastname: Yanni
mesh_terms: D000287:Administration, Topical; D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D005260:Female; D006801:Humans; D007958:Leukocyte Count; D008070:Lipopolysaccharides / Q000633:toxicity; D009504:Neutrophils / Q000201:enzymology* / Q000473:pathology*; D009195:Peroxidase / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D015203:Reproducibility of Results; D014605:Uveitis / Q000188:drug therapy / Q000201:enzymology / Q000473:pathology*
chemical_list: D000893:Anti-Inflammatory Agents; D008070:Lipopolysaccharides; D009195:Peroxidase