RESUMO
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.
RESUMO
Many RNA virus CNS infections cause neurological disease. Because Piry virus has a limited human pathogenicity and exercise reduces activation of microglia in aged mice, possible influences of environment and aging on microglial morphology and behavior in mice sublethal encephalitis were investigated. Female albino Swiss mice were raised either in standard (S) or in enriched (EE) cages from age 2 to 6 months (young - Y), or from 2 to 16 months (aged - A). After behavioral tests, mice nostrils were instilled with Piry-virus-infected or with normal brain homogenates. Brain sections were immunolabeled for virus antigens or microglia at 8 days post-infection (dpi), when behavioral changes became apparent, and at 20 and 40 dpi, after additional behavioral testing. Young infected mice from standard (SYPy) and enriched (EYPy) groups showed similar transient impairment in burrowing activity and olfactory discrimination, whereas aged infected mice from both environments (EAPy, SAPy) showed permanent reduction in both tasks. The beneficial effects of an enriched environment were smaller in aged than in young mice. Six-hundred and forty microglial cells, 80 from each group were reconstructed. An unbiased, stereological sampling approach and multivariate statistical analysis were used to search for microglial morphological families. This procedure allowed distinguishing between microglial morphology of infected and control subjects. More severe virus-associated microglial changes were observed in young than in aged mice, and EYPy seem to recover microglial homeostatic morphology earlier than SYPy . Because Piry-virus encephalitis outcomes were more severe in aged mice, it is suggested that the reduced inflammatory response in those individuals may aggravate encephalitis outcomes.
