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Dendritic cells (DCs) are a subset of leukocytes highly specialized in antigen-presentation to T cells, thus promoting the immune response. DCs occur in the meninges and choroid plexus. Brain DCs and brain-derived antigens are drained by cerebrospinal fluid in the afferent lymphatic vessels of cervical lymph nodes (cLNs) for antigen presentation. Information on the role of DCs in intracerebral immune response is still limited. We recently demonstrated (Laperchia et al., 2013) that in thy1GFP-M transgenic mice, engineered for the expression of green fluorescent protein (GFP) in a proportion of neurons, also myeloid DCs are GFP-tagged. Our in vivo analysis by two-photon microscopy on young (3-6 months) thy1GFP-M mice showed DCs floating in the cerebrospinal fluid or static at the pia mater/parenchyma interface. We are using this animal model to study brain DCs trafficking by two-photon microscopy in two different inflammatory conditions. The first concerns chronic encephalitis caused by the extracellular parasite Trypanosoma brucei. During the first, hemolymphatic stage of this infection, direct interactions between DCs and parasites were seen within meningeal and cortical microvessels. In the second stage, determined by parasite neuroinvasion, DCs invaded the brain parenchyma, exhibiting a random motion for target antigen recognition. With disease progression, intraparenchymal brain DCs were instead mainly arranged in static clusters which incorporated parasites for efficient antigen capture, and extravasated cytotoxic CD8+ T cells established contact with parasites. Ex vivo analysis on cLNs shown that the subcapsular zone was invaded by migratory DCs, and both migratory and resident DCs preferentially contacted CD8+ T cells. The second condition concerns normal aging (18-20 month old mice), which is known to be associated with low-grade chronic inflammation level and with functional impairments of the immune system, also known as immunosenescence. In these mice we observed that DCs infiltrate the brain parenchyma by transmigration from blood vessels and exhibit a motile behavior suggesting a scanning motion. Moreover, some DCs showed a progressive reduction of their motility until convergence, in about 30 minutes, into clusters whose functional significant has yet to be elucidated. Taken together, our studies enlightens key events of the intracerebral immune response both in presence of pathogens and in physiological aging., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

A wealth of recent evidence indicates that microglia activation is a polarized process, leading to a potentially neurotoxic M1 “classical activation” or potentially neuroprotective M2 “alternative activation”. The regulation of this process is, however, still largely unknown. We here investigated the induction of molecules which characterize M1 and M2 microglia responses after systemic (ip) and central (intracerebroventricular, icv) exposure to lipopolysaccharide (LPS). These challenges elicit different inflammatory responses of the brain parenchyma; in particular, leukocyte infiltration in the brain occurs after icv but not after ip LPS exposure. Young adult mice were subjected to ip or icv LPS injection, sacrificed at different time points from 2h up to 10 days, and compared with matched saline-treated control mice. Analyses with the pan-T cell marker CD3 confirmed the occurrence of T cells in the brain parenchyma after icv but not after ip LPS injections. Immunohistochemical phenotyping of microglia was pursued to reveal major histocompatibility complex class II (MHCII) antigen, a key molecule in M1 activation, and chitinase 3-like 3 (YM1), part of the M2 molecular repertoire. The M1 type of microglial response, revealed by upregulation of MHCII immunosignal, was very intense and occurred during the first 24h, with a peak at 2h after icv LPS, and at 6h after ip LPS. The M2 type of response, revealed by YM1 immunoreactivity in some microglial cells, followed the M1 type response evolving in the days which followed the first 24h, with an earlier peak (at 5 days) and more marked persistence (up to 10 days) after icv LPS, i.e., in the presence of T cell infiltration. Altogether the findings point out dynamic processes of microglia activation and its polarization over time, and a microglia – T cell dialogue with potential neuroprotective effects of T cell recruitment. Further investigation on the profiling of the M1 and M2 types of microglia activation, and of the possible molecules implicated in the microglia – T cell dialogue will further enlighten these crucial polarization mechanisms., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Astrocytic networks and gap junctional communication mediated by connexins (Cxs) have been repeatedly implicated in seizures, epileptogenesis, and epilepsy. However, the effect of seizures on Cx expression is controversial. The present study focused on the response of Cxs to status epilepticus (SE), which is in turn an epileptogenic insult. The expression of neuronal Cx36 and astrocytic Cx30 and Cx43 mRNAs was investigated in the brain of rats in the first day after pilocarpine-induced SE. In situ hybridization revealed a progressive decrease in Cx43 and Cx30 mRNA levels, significantly marked 24 h after SE onset in neocortical areas and the hippocampus, and in most thalamic domains, whereas Cx36 mRNA did not exhibit obvious changes. Regional evaluation with quantitative real-time-RT-PCR confirmed Cx43 and Cx30 mRNA downregulation 24 h after SE, when ongoing neuronal cell death was found in the same brain regions. Immunolabeling showed at the same time point marked a decrease in Cx43, microglia activation, and interleukin-1β induction in some microglial cells. The data showed a transient downregulation of astroglial Cxs in the cortical and thalamic areas in which SE triggers neurodegenerative events in concomitance with microglia activation and cytokine expression. This could potentially represent a protective response of neuroglial networks to SE-induced acute damage.