Age-related increase in activated microglial populations. (A) High-magnification visualization of resting/surveilling, or non-activated, and activated microglia reveals differences in both immunoreactivity and morphology with aging. Non-activated microglia expressed Iba1 but not CD74, and had long, thin, branched processes. Microglia undergoing mild activation displayed increased Iba1 immunoreactivity, weak CD74 immunoreactivity, and enlarged somata, while moderately activated microglia demonstrated increased Iba1 immunoreactivity, intense CD74 immunoreactivity, enlarged somata, thickened proximal processes, and retracted distal processes. Notably, activated microglia maintained a ramified rather than ameboid morphology, indicating that microglial activation had not progressed to a reactive, phagocytic phenotype. (B) Quantitation of activated microglia (calculated as the ratio of Iba1+/CD74+ to total Iba1+ cells) revealed significant increases in all three hippocampal subregions studied. In adult rats, a small fraction of total microglia were mildly or moderately activated in CA3, with far fewer activated microglia evident in CA1 and DG. The percentage of microglia undergoing mild or moderate activation was significantly increased, by several fold, throughout the hippocampus of both aged cognitively intact and aged cognitively impaired rats compared to adults. No differences between the degree of microglial activation (i.e., mild versus moderate) were observed between aged intact and aged impaired rats in CA1, CA3 or DG. Increased numbers of activated microglia in aged rats were not due to proliferation/infiltration, as total populations of microglia (Iba1+ cells) were not different between groups. ***p < 0.001, one-way ANOVA with SNK post hoc testing.