Enzyme Selective Flim in Microglia: Studying the Role of NADPH Oxidase in Chronic Neuroinflammation

Understanding the pathological mechanisms in the brain is the decisive step in developing novel specific therapeutic strategies and in curing chronic neuroinflammatory or neurodegenerative diseases like multiple sclerosis (MS). For this purpose, techniques which confer high molecular specificity and spatial resolution, which are non-invasive and allow for dynamics visualization in vivo are necessary. We already showed that NAD(P)H-based fluorescence lifetime imaging (FLIM) is the appropriate technique to detect the activation of NADPH oxidase in murine polymorphonuclear cells and in cells of Nicotiana tabacum during phagocytosis. The fluorescence lifetime of NAD(P)H if bound to NADPH oxidase increases to approx. 3600 ps as compared to the case of NAD(P)H-dependent metabolic enzymes, i.e. approx. 2000 ps. A central yet still not fully understood role in neuronal injury is assigned to microglial NADPH oxidase: this might be a neurotoxic effect due to direct production of reactive oxygen species or, indirectly, due to the induction of NO synthase or a protective role if NADPH oxidase is involved in the phagocytosis of noxious supra-molecular structures like beta-amyloids. By means of NAD(P)H-based FLIM we investigate the activation of NADPH oxidase in murine and human peripheral macropahges as compared to microglia, i.e. macrophages of the central nervous system. The same response to specific chemical activation and to phagocytosis of Stafiloccocus aureus functionalized beads is demonstrated for macrophages and microglia of new-born mice. However, this response is different for microglia of adult, healthy mice as also reflected by their reduced phagocytotic capacity. In pathological context, we investigate the activation of NADPH oxidase in microglia of adult, healthy mice as compared to microglia of mice affected by a murine model of MS. Thus, we were able to identify a new mechanism in chronic neuroinflammation.