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to Leukocyte Adhesion, Vascular Leakage, and Apoptotic Cell Death in Endotoxin-Induced Uveitis In Vivo
1From the Department of Vitreoretinal Surgery, Center for Ophthalmology, and Center for Molecular Medicine, University of Cologne, Cologne, Germany.
PURPOSE. To investigate the effect of TNF-
on leukocyte adhesion, vascular leakage, and apoptotic cell death in endotoxin-induced uveitis (EIU) in the rat.
METHODS. EIU was induced in Long-Evans rats by a single footpad injection of lipopolysaccharide (LPS; 350 µg/kg) from Salmonella typhimurium. A single injection of recombinant TNF receptor P75 (etanercept) was given subcutaneously 24 hours before the administration of LPS. Twenty-four hours after administration of LPS, leukocyte adhesion was evaluated in vivo with SLO-acridine orange angiography and ex vivo with concanavalin A lectin staining of retinal flatmounts. Neutrophil activation was quantified by a myeloperoxidase activity assay. Vascular leakage was assessed by Evans blue extravasation. Retinal cell death was assessed with TUNEL staining and quantified with a modified ELISA protocol. Involvement of caspase-3 and -8 was determined by M30 antibody staining, Western blot analysis, and a test for enzymatic activity.
RESULTS. Twenty-four hours after the LPS injection, significant increases in leukocyte rolling, adhesion, and activation were observed. In addition, increased levels of apoptosis in the vascular endothelium and the ganglion cell and inner nuclear layers and activation of caspase-8 and -3 were observed. After administration of the TNF- inhibitor, significant reduction in the leukocyte rolling, adhesion, activation, and apoptosis in all the affected layers was observed. The quantitative analysis of vascular leakage revealed a significant decrease after treatment with etanercept. Retinal cell death quantification showed a significant decrease after treatment with the TNF- inhibitor.
CONCLUSIONS. Anti-TNF- treatment reduces the LPS-induced increases in leukocyte rolling, adhesion, and vascular leakage in this rat model of inflammatory uveitis. These results suggest the involvement of TNF- in inflammatory uveitis and its potential use as a therapeutic agent in the reduction of ocular inflammation.
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