Characterization of a motile population of cells in the cerebral cortex and meninges of thy1GFP-M transgenic mice by multiphoton microscopy

The combination of the most advanced brain imaging techniques and transgenic animals expressing fluorescent proteins has provided a powerful tool to visualize with high-resolution dynamic biological processes that occur in vivo at the cellular and subcellular levels. The present doctoral thesis project has been aimed at the characterization and investigation, with two-photon microscopy, of a motile population of cells observed at the brain surface in GFP-M transgenic mice. This mouse line has been engineered for the expression of green fluorescent protein (GFP) in about 10% of the neurons at different locations, but GFP-labeled non-neuronal cells had not been previously characterized. The first extensive experimental series presented in this thesis focuses on the characterization of these non-neuronal GFP-tagged cells in GFP-M mice. In two-photon microscopy, through a chronic optical brain window and in a thinned skull preparation, such fluorescent cells have been observed in the meninges, in the upper cortical layers close to meninges as well as in perivascular spaces, occasionally moving from the pial and subpial surface into the brain parenchyma with evident changes of shape. Confocal microscopy analyses on cryosectioned brain sections has shown that these cells are immunonegative to several neuronal antigens, including those of neuronal progenitors, while they express antigens most commonly expressed by dendritic cells (DCs), namely major histocompatibility complex class II (MHC-II), and CD11c. Together with other analyses, including quantitative evaluation, the first experimental series has allowed to identify GFP-labeled non-neuronal cells in GFP-M mice as a subset of DCs, accounting for the majority of these cells in the brain of GFP-M mice. In the second experimental series of the doctoral thesis project, GFP-labeled DCs have been examined in a model of chronic neuroinflammation represented by infection with Trypanosoma brucei (T.b.). This parasite is the causative agent of the severe disease human African trypanosomiasis (also called sleeping sickness) and causes infections also in animals. The experimental model has been set up by infecting GFP-M mice with the parasite subspecies T.b. brucei (which is not pathogenic for humans and therefore safe for laboratory work). In humans and in animals African trypanosome infection progresses in two stages: a first hemolymphatic stage of systemic invasion, which evolves in the second, meningoencephalitic stage when the parasites cross the blood-brain barrier and invade the brain parenchyma. Infected GFP-M mice have been analyzed by multiphoton microscopy at two time points during the meningoencephalitic stage of the disease: early after the initial parasite invasion of the brain parenchyma (at 16 days post-infection, dpi) and at a more advanced phase (22 dpi). The results of the present investigation have shown that in the healthy brain of control GFP-M mice sessile DCs are mainly localized in meninges, whereas in T.b. brucei-infected animals at 16 dpi DCs have been observed to accumulate within the brain parenchyma, where they exhibited high motility. Moreover, some DCs showed adhesion and crawling on vascular endothelia. Interestingly, at an advanced phase of the infection (22 dpi), DC clusters with very dynamic membrane extensions, indicating active phagocytic events, were observed in the brain parenchyma. Comparing parameters of the DCs motility (mean velocity, motility coefficient, meandering factor) at these two time points, DCs appeared initially in rapid probing motions, and exhibited later motility in a more restricted tissue volume and their velocity decreased. These features could be aimed at an efficient antigen presentation to T-cells. In conclusion, the doctoral thesis project has shown for the first time the presence of fluorescent DCs in GFP-M mice, which thus could be used as a novel animal model for the study of these key actors of immunity. This finding has allowed to investigate the in vivo behavior of DCs in both basal and pathological conditions. Moreover, the data here obtained suggest that DCs could play a key role in pathogenic mechanisms of brain infection during African trypanosomiasis.