Migratory behavior of dendritic cells in the mouse brain cortex in normal and pathological conditions by multiphoton microscopy

Dendritic cells (DCs) have an immune surveillance role in the central nervous system. It is well known that in normal conditions they maintain the immuno-tolerance by T-cell suppression and that their presence is limited to the meninges, choroid plexus and rarely to the brain parenchyma. During inflammation, DCs infiltrate the brain parenchyma, enhance their antigen processing capacity and promote the initiation of immune responses by T-cell activation. Information on DCs in vivo in the CNS is very limited. We are investigating the dynamic behavior of the DCs in the mouse brain cortex in vivo in normal conditions and in a model of chronic neuroinflammation represented by infection with the parasite Trypanosoma brucei (Tb), the causative agent of African trypanosomiasis. This disease evolves in humans and experimental animals in two stages: an early stage, in which Tb invade peripheral organs through the hemolymphatic system; and a late meningoencephalitic stage with severe consequences on nervous system functions. In order to visualize dynamic cell processes in healthy mice and in mice infected with the non-human pathogenic subspecies Tb brucei, a little craniotomy was performed leaving the dura mater unperturbed and the bone flap was replaced by a coverglass. This chronic implantation provides an excellent optical access for multiphoton acquisition in vivo, allowing high-resolution imaging over time. Blood vessels were visualized by iv injection of TRITC-conjugated dextran. Four-dimensional reconstruction (x,y,z,t) of transcranial images were analyzed by Imaris software to track the migratory route of DCs, their position with respect to the vasculature over time, and their movement features (crawling, rolling, etc.). The initial results of this study show that in the healthy brain DCs are mainly localized in the meninges, they are motionless and exhibit a round shape. In addition, with the progression of Tb brucei infection DCs have been observed to penetrate the brain parenchyma from the meninges, their number increases and their motility is enhanced. At an advanced phase of the infection, phagosomelike structures are also observed in the brain parenchyma. The data suggest an involvement of DCs in pathogenetic mechanisms and severity of Tb brain infection. In vivo experiments with fluorescent Tb brucei are in progress to visualize in the brain potential interactions between the parasite and host immune cells represented by DCs.