Abstract
Abstract: :
Purpose: We have previously shown that peroxynitrite formed during experimental autoimmune uveoretinitis (EAU) causes the peroxidation of docosahexaenoic acid (22:6), a principal unsaturated fatty acid in the photoreceptor membrane to its hydroperoxide (22:6HP). In the present study, we examined the effects of 22:6HP formation on the activation, chemotactic activity, and intracellular signaling pathways of the rat retinal microglia.Methods: Retinal microglia was isolated and cultured from 7–day– old Lewis rats. Immunocytochemical studies were performed using primary antibodies specific for microglia (CD11b) and astrocytes (GFAP). Chemotactic activity of the microglia towards 22:6HP was assayed and compared with both 22:6 and with fractalkine used as a positive control. The effect of 22:6HP on the actin fiber organization of microglia was visualized using actin–specific stain (tetramethylrhodamine isothiocyanate–labelled phalloidin). The expression of a small GTPase Rac, microglia/macrophage–specific calcium binding protein Iba1, and actin in 22:6HP activated microglia was studied by confocal microscopy. The gene expression of Rac and Iba1 in activated microglia was analyzed by real–time PCR.Results: Immunocytochemical studies indicated over 95% of isolated microglia stained for CD11b and about 5% stained for GFAP. Chemotaxis assay showed 22:6HP as a chemoattractant for the microglia. The 22:6HP caused a redistribution and polymerization of f–actin in microglia cells, with intense staining within the lamellipodia. These cells also showed co–localization of Iba1 in the cell periphery and lamellipodia. Similarly, Rac was also localized in the lamellipodia and periphery of the cell, whereas in the 22:6 treated cells they were concentrated towards the nucleus. Iba1 and Rac mRNA were upregulated 2.0– and 1.5 fold respectively in these activated cells. Conclusions: The present study reveals that 22:6HP activates retinal microglia and induces chemotaxis. Microglia may be activated by a signaling pathway in which Rac is activated by the calcium binding protein Iba1. The involvement of Iba1 in the signaling pathway of Rac GTPase might be an important step that regulates actin cytoskeleton reorganization and lamellipodia formation in 22:6HP activated microglia, and in EAU microglial migration towards the photoreceptors could be mediated by 22:6HP.
Keywords: microglia • microscopy: light/fluorescence/immunohistochemistry • uveitis-clinical/animal model