INFLAMMATION-INDUCED MICROGLIAL POLARIZATION IN MICE OF DIFFERENT AGES AND MODELS OF NEURODEGENERATION

The response of microglia to a variety of insults is known since the discovery of microglia as components of the brain parenchyma. Microglia represent the main cell type of the central nervous system (CNS) immune response which serve as the effector arm of the innate immune system in the CNS. Data on non-neuronal cells (astrocytes, microglia, infiltrated T-cells) after inflammatory challenges in the rodent aging brain have implicated immune response changes with advancing age. In recent years, special attention has been devoted to microglia also as key element of neuroinflammation associated with aging-related neurodegenerative diseases. Interestingly, a number of recent studies have indicated that microglia activation is a polarized process, leading to a potentially neurotoxic M1 “classical activation” or a potentially neuroprotective M2 “alternative activation”. The regulation of the process of microglia polarization and its aging-related variations are, however, still largely unknown. In the general context of the multifaceted activation of microglial cells and of the impact of aging, the present doctoral project has been articulated in two experimental series. The first experimental series focused on the search of polarized microglia activation using lipopolysaccharide (LPS) systemic (ip) versus central (intracerebroventricular, icv) administration to normal mice of three different age groups (3, 12, and 20 months), and its correlation with T-cell recruitment to the brain parenchyma which occurs after icv, but not ip, LPS exposure. Immunohistochemical analyses based on qualitative observations and quantitative evaluation (densitometry of immunosignal intensity and cell counts) showed an early, intense and persistent M1 type microglia activation, revealed by upregulation of major histocompatibility class II (MHCII) and cluster of differentiation (CD) 86 antigens, with a gradual age-related amplification that was especially enhanced in the oldest mice. The M2 type microglia activation, revealed by Ym1 and CD206 immunoreactivities, was detected in a relatively restricted number of microglial cells and with a latency of 5 days and further persistence. Double immunofluorescence experiments in 3 month-old mice showed that M1 and M2 antigens were expressed by different microglial cell subsets. The overt onset of M2 microglia activation preceded T-cell recruitment to the brain parenchyma. Interestingly, the findings also showed an aging-related decline in M2 microglia activation. The second experimental series focused on the analysis of microglia activation phenotypes, together with the study of astrocytes, in models of aging-related neurodegenerative diseases. The first of these was the model of Alzheimer’s disease represented by double mutant PDAPP transgenic mice. Adult (4-6 month-old) and aged (24 month-old) transgenic mice and matched wild-type littermates were examined; quantitative evaluation (cell counts) was pursued in the hippocampus and ventral midbrain tegmentum. A significant enhancement of M1 type microglia activation, as well of astrocyte activation, was found in 24 month-old PDAPP mice compared to wild-type ones. No M2 type microglia activation was detected with the used markers. In 24 month-old PDAPP mice, amyloid-β accumulation was observed in the hippocampus, and activated microglia and astrocytes were closely associated with amyloid-β aggregates. The second murine model here examined was represented by mice with targeted deletion of the NFkB/c-rel gene (c-rel-/-). Previous data have documented a significant loss of substantia nigra dopaminergic neurons in male c-rel-/- mice of 18 months of age, with significant microglial, but not astrocytic, activation, and other molecular and functional changes indicating that these mice provide a model of Parkinsonism. Female c-rel-/- mice of 5 and 18 months of age and matched wild-type counterparts have been here examined. No significant loss in the number of tyrosine hydroxylase-immunoreactive neurons, though with significant enhancement of microglia activation (M1 type), but not of astrocytes, was found in the ventral midbrain tegmentum of female c-rel-/- mice of 18 months of age. No expression of the analyzed M2 microglia antigens was found in c-rel-/- mice. The data point to sex-related differences in the aging-related dopaminergic cell loss in c-rel-/- mice, which may not occur, or be slowed down, in females. Further experiments are needed to verify this hypothesis. Overall, dynamic processes of microglia polarization have been here analyzed after exogenous inflammatory challenges, revealing aging-related variations. In the study of gene-manipulated mouse models, an aging-related enhancement of M1 microglial activation, as well of astrocytic activation, has been documented in PDAPP mice, and enhancement of M1 microglial activation has been documented in c-rel-/- female mice.