In vivo visualization of dendritic cells in the mouse brain cortex by multiphoton microscopy in normal conditions and parasitic infection

We are investigating with real-time multiphoton microscopy cells in the brain of GFPM mice, in which about 10% of the neurons express green fluorescent protein (GFP). Through a chronic optical brain window, we have observed in these mice not only fluorescent neuronal subsets but also fluorescent cells which are negative to a variety of tested neuronal antigens, including those of neuronal progenitors, and which exhibit motility. These cells, which are frequently observed to penetrate the brain parenchyma from the meningeal surface, are likely to represent dendritic cells (DCs) on the basis of their immunophenotype (CD68+, CD11b+, F4/80-), the characterization of which is currently being finalized. DCs are professional antigen-presenting cells, which notably play a key role in immuno-tolerogenic and immune responses, and their role in the brain is still largely unexplored. We have investigated these cells in vivo in young adult GFPM mice, both in normal conditions and after infection with the parasite Trypanosoma brucei brucei (T.b.). Infection with T.b. subspecies causes human African trypanosomiasis (also called sleeping sickness), and the non-human pathogenic subspecies T.b. brucei is widely used in rodent models of this disease. In our study, in vivo investigations are complemented by immunolabelling on cryosectioned brains for the study of areas inaccessible in vivo. The findings we have hitherto obtained show that in control non-infected brains DCs are mainly localized in the meninges, they exhibit a round shape and are motionless, and occasionally they move and change their shape. T.b. brucei-infected GFPM mice have been investigated at two time points during the meningoencephalitic phase of the disease, i.e., in the initial phase of parasite invasion of the brain parenchyma and at a more advanced stage of parasite neuroinvasion. At the first of these time points, represented by day post-infection (dpi) 16, we have observed fluorescent DCs rapidly moving (crawling, rolling, etc) within the brain parenchyma, mostly close to blood vessel walls. At a more advanced stage of infection (dpi 22), DCs have been mainly detected within the brain parenchyma, and a large number of fluorescent cell debris (perhaps as a result of autophagy processes) as well as DCs of a relatively small size have also been observed. In vivo experiments with fluorescent T.b. brucei are currently in progress to visualize the eventual occurrence of dynamic interactions of DCs with the parasite.