It is known that microglial morphology and function are related, but few studies have explored the subtleties of microglial morphological changes in response to specific pathogens. In the present report we quantitated microglia morphological changes in a monkey model of dengue disease with virus CNS invasion. To mimic multiple infections that usually occur in endemic areas, where higher dengue infection incidence and abundant mosquito vectors carrying different serotypes coexist, subjects received once a week subcutaneous injections of DENV3 (genotype III)-infected culture supernatant followed 24 h later by an injection of anti-DENV2 antibody. Control animals received either weekly anti-DENV2 antibodies, or no injections. Brain sections were immunolabeled for DENV3 antigens and IBA-1. Random and systematic microglial samples were taken from the polymorphic layer of dentate gyrus for 3-D reconstructions, where we found intense immunostaining for TNFα and DENV3 virus antigens. We submitted all bi- or multimodal morphological parameters of microglia to hierarchical cluster analysis and found two major morphological phenotypes designated types I and II. Compared to type I (stage 1), type II microglia were more complex; displaying higher number of nodes, processes and trees and larger surface area and volumes (stage 2). Type II microglia were found only in infected monkeys, whereas type I microglia was found in both control and infected subjects. Hierarchical cluster analysis of morphological parameters of 3-D reconstructions of random and systematic selected samples in control and ADE dengue infected monkeys suggests that microglia morphological changes from stage 1 to stage 2 may not be continuous.

We previously demonstrated in young mice that in comparison with animals raised in an impoverished environment (IE), animals from an enriched environment (EE) show more severe dengue disease, associated with an increased expansion of memory T target cells. Because active older adults show less functional decline in T-cell adaptive immunity, we hypothesized that aged mice from EE would show higher mortality and T-lymphocyte expansion than mice from IE. To test this hypothesis, we administered serial i.p. injections of anti-DENV2 hyperimmune serum, followed 24 h later by DENV3 (genotype III)-infected brain homogenate. Control mice received equal volumes of serum but received uninfected brain homogenate. The presence of virus or viral antigens was indirectly detected by real-time quantitative RT-PCR and immunohistochemistry. Compared to infected IE animals, EE mice, independent of age, showed higher mortality and more intense clinical signs. Compared to young mice, the higher mortality of aged mice was associated with a higher degree of T lymphocytic hyperplasia in the spleen and infiltration in kidneys, liver, and lungs, but less viral antigen immunolabeling. We propose that a higher expansion of T cells and serotype cross-reactive antibodies are associated with disease severity in aged mice.