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.
Highlights
Microglia are cells responsible for innate cell immunity in the central nervous system (CNS); they have a ramified morphology in the adult CNS and continuously survey the local environment by extending and retracting their highly motile cell processes [1,2]
QH1/ Inducible nitric oxide synthase (iNOS) double immunolabeling of E9 retinas revealed iNOSpositive amoeboid microglial cells in the vitreal part of the retina (Figure 1C, D), they were more abundant than in E8 retinas and showed a lower anti-iNOS fluorescence intensity (Figures 1 and 3).The iNOS immunolabeling of amoeboid microglial cells bore some resemblance to the QH1 immunostaining, but they were clearly different
Given the association between iNOS expression and microglial activation [13,34,37], these findings indicate a certain degree of activation (‘‘baseline activation’’) in amoeboid microglia during normal development. iNOS expression becomes downregulated as amoeboid microglia differentiate into ramified microglia, suggesting a decrease in this baseline activation during the microglial differentiation process
Summary
Microglia are cells responsible for innate cell immunity in the central nervous system (CNS); they have a ramified morphology in the adult CNS and continuously survey the local environment by extending and retracting their highly motile cell processes [1,2]. Under pathological conditions in the adult CNS, ramified microglia become activated, retracting their processes and acquiring a macrophage-like rounded morphology [9,10,11] reminiscent of amoeboid microglia during development. Activated microglia upregulate their migratory, proliferative and phagocytic capacities to perform their macrophage-like defensive functions, thereby becoming similar to amoeboid microglia. NO is a liposoluble radical gas that traverses freely across cell membranes and acts as a cellular signaling molecule that participates in various biological effects without the need for a specific membrane receptor. Simultaneous production of NO and superoxide by activated microglia, under proinflammatory conditions, gives rise to the formation of peroxynitrite, a powerful oxidant that induces neuronal death [18,27,28,33,34,35]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
