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.

Inducible nitric oxide synthase‐derived nitric oxide reduces vagal satiety signalling in obese mice

“…iNOS is expressed in multiple cell types, such as macrophages (Lumeng et al 2007), plasma cells (Fritz et al 2012), epithelia cells (Kolios et al 1998) and microglia cells (Sierra et al 2014), and is probably signalling from multiple sources in the present study. A number of studies have revealed that a high-fat diet increases NO production or iNOS expression in many different sites, such as plasma (Stanimirovic et al 2016), liver (Feng et al 2016), kidney (Sherif, 2014), small intestine (Ou et al 2012) and adipose tissue (Perreault & Marette, 2001), although not all studies have identified an increase (Perreault & Marette, 2001;Eccleston et al 2011;Stanimirovic et al 2016).…”

“…) and microglia cells (Sierra et al . ), and is probably signalling from multiple sources in the present study. A number of studies have revealed that a high‐fat diet increases NO production or iNOS expression in many different sites, such as plasma (Stanimirovic et al .…”

Onset of microglial entry into developing quail retina coincides with increased expression of active caspase-3 and is mediated by extracellular ATP and UDP

“…They were blocked for 15 minutes in PBS-Tr containing 5% normal goat serum (NGS) at room temperature and incubated overnight at 4°C in a mix of monoclonal anti-BrdU (1:800) (Sigma) and polyclonal anti-iNOS (1:500) (Abcam, Cambridge, United Kingdom) antibodies, both diluted in BSA-PBS-Tr. Polyclonal anti-iNOS antibody was used as microglial marker, because amoeboid microglia express iNOS during the normal embryonic development of quail retina [ 20 ]. The explants were subsequently rinsed in PBS-Tr and incubated with the secondary antibodies Alexa Fluor 594-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated goat anti-rabbit IgGs for 2 hours at room temperature.…”

“…Subsequent radial migration in a vitreal-to-scleral direction allows amoeboid microglia to reach the plexiform layers, where they differentiate into ramified microglia. Other studies in our lab showed that in vitro cultures of quail embryo retina explants mimic the migration and differentiation of microglial precursors in the in situ developing retina [ 19 , 20 ]. Therefore, these organotypic cultures of retina explants represent an excellent experimental model system to investigate possible molecular factors involved in the entry of microglial precursors into the retina.…”

The putative role of oxidative stress and inflammation in the pathophysiology of sleep dysfunction across neuropsychiatric disorders: Focus on chronic fatigue syndrome, bipolar disorder and multiple sclerosis

“…species, prostaglandins, NO, cyclooxygenase-2, adenosine and a range of other inflammatory and signalling molecules [73,74]. Experimental evidence gleaned from healthy human volunteers involving intravenous administration of inflammatory mediators such as lipopolysaccharides (LPS) revealed a significant reduction in the duration of REM sleep and a significant increase in the duration NREM sleep [75,76].…”