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Expression of inducible nitric oxide synthase (iNOS) in microglia of the developing quail retina
Ana Sierra1, Julio Navascués1, Miguel A Cuadros1
1Departamento de Biología Celular, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
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.
Insights
Immature microglial cells in quail retinas express inducible nitric oxide synthase (iNOS) during normal development. Lipopolysaccharide (LPS) exposure further upregulates iNOS in these activated microglial cells.
Area of Science:
- Neuroscience
- Immunology
- Developmental Biology
Background:
- Inducible nitric oxide synthase (iNOS) produces nitric oxide (NO) and is typically induced in activated immune cells like microglia.
- While iNOS is known in activated microglia, its expression in developing, undifferentiated microglia is less understood.
Purpose of the Study:
- To investigate the expression patterns of iNOS in microglial cells during normal quail retina development.
- To examine how lipopolysaccharide (LPS) affects iNOS expression and nitric oxide (NO) production in microglial cells.
Main Methods:
- Utilized quail retina as a model for both in situ developmental studies and in vitro organotypic cultures.
- Analyzed iNOS expression via immunolabeling, western-blot, and RT-PCR.
- Measured NO production using the fluorescent indicator DAR-4M AM.
Main Results:
- Amoeboid microglia in developing quail retinas express iNOS, with higher levels in migrating cells.
- iNOS expression decreases but is maintained as microglia differentiate into ramified forms.
- LPS treatment in vitro induced microglial activation, increased lysosomal compartments, and upregulated iNOS expression with NO production.
Conclusions:
- Immature, amoeboid microglial cells express iNOS during normal development, suggesting a basal level of activation.
- LPS exposure leads to overactivation of these microglia, significantly increasing iNOS expression and NO synthesis.
