title: Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina.
abstract: Inducible nitric oxide synthase (iNOS), which produce large amounts of nitric oxide (NO), is induced in macrophages and microglia in response to inflammatory mediators such as LPS and cytokines. Although iNOS is mainly expressed by microglia that become activated in different pathological and experimental situations, it was recently reported that undifferentiated amoeboid microglia can also express iNOS during normal development. The aim of this study was to investigate the pattern of iNOS expression in microglial cells during normal development and after their activation with LPS by using the quail retina as model. iNOS expression was analyzed by iNOS immunolabeling, western-blot, and RT-PCR. NO production was determined by using DAR-4M AM, a reliable fluorescent indicator of subcellular NO production by iNOS. Embryonic, postnatal, and adult in situ quail retinas were used to analyze the pattern of iNOS expression in microglial cells during normal development. iNOS expression and NO production in LPS-treated microglial cells were investigated by an in vitro approach based on organotypic cultures of E8 retinas, in which microglial cell behavior is similar to that of the in situ retina, as previously demonstrated in our laboratory. We show here that amoeboid microglia in the quail retina express iNOS during normal development. This expression is stronger in microglial cells migrating tangentially in the vitreal part of the retina and is downregulated, albeit maintained, when microglia differentiate and become ramified. LPS treatment of retina explants also induces changes in the morphology of amoeboid microglia compatible with their activation, increasing their lysosomal compartment and upregulating iNOS expression with a concomitant production of NO. Taken together, our findings demonstrate that immature microglial cells express iNOS during normal development, suggesting a certain degree of activation. Furthermore, LPS treatment induces overactivation of amoeboid microglia, resulting in a significant iNOS upregulation.
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Ana
authors.initials: A
authors.lastname: Sierra
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Julio
authors.initials: J
authors.lastname: Navascués
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Cuadros
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Ruth
authors.initials: R
authors.lastname: Calvente
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Martín-Oliva
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: Rosa M
authors.initials: RM
authors.lastname: Ferrer-Martín
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: María
authors.initials: M
authors.lastname: Martín-Estebané
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: María-Carmen
authors.initials: MC
authors.lastname: Carrasco
authors.affiliation: Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
authors.forename: José L
authors.initials: JL
authors.lastname: Marín-Teva
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D030161:Avian Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D003370:Coturnix; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D018613:Microscopy, Confocal; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D012160:Retina / Q000196:embryology / Q000201:enzymology* / Q000254:growth & development; D020133:Reverse Transcriptase Polymerase Chain Reaction; D046509:Tissue Culture Techniques
chemical_list: D030161:Avian Proteins; D008070:Lipopolysaccharides; D052247:Nitric Oxide Synthase Type II