Three‐dimensional morphometric analysis of microglial changes in a mouse model of virus encephalitis: age and environmental influences

Abstract

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.

Virus Infections on Prion Diseased Mice Exacerbate Inflammatory Microglial Response

“…Neuronal and glial immunolabeled cells were found in the limbic system, including olfactory, septal, hippocampal formation, including CA3 and dentate gyrus, and in the trigeminal pathway. These findings are in line with previous description [12]. …”

“…The present report is the first to use a real arbovirus infection associated with prion disease. The arbovirus Piry infection showed longer changes in burrowing activity (up to 3 weeks) suggesting possible activation of both acute and adaptive immune responses [12]. …”

“…Specific antibodies against Piry virus species were produced by the Department of Arbovirus and Hemorrhagic Fevers at the Instituto Evandro Chagas, as described elsewhere [18], and anti-IBA1 antibody by Wako Pure Chemical Industries, Ltd (Osaka, Japan). Detailed immunohistochemical procedures were described elsewhere [12, 19]. In brief, free-floating sections were rinsed in 0.1 M phosphate buffer and placed in a solution of 0.2 M boric acid (pH 9.0) at 70°C for 1 h for antigen retrieval, then rinsed in 0.1 M PBS with 5% Triton X-100, and incubated in a solution of methanol and 0.3% hydrogen peroxide for 10 min.…”

“…Emerging virus infections of the CNS are mainly associated with RNA viruses, many of which cause neurologic disease [10]. The Vesiculovirus Piry infection generates human disease characterized by rapid onset, high fever, headache, chills, photophobia, myalgia, dizziness, and weakness [11] and, in adult mice, a nonlethal CNS infection and injury to the limbic system including the hippocampus [12], a target region of the degenerative process induced by prion disease in mice [13]. This particularity to infect humans and damage the hippocampus of adult mice makes Piry virus a particularly interesting arbovirus species to study the interaction between the hippocampus underlying prion disease neurodegeneration and viral infection.…”

“…Thus, in the present report, we associated Piry virus, which generates symptoms of infectious disease in both human [16] and mice [11] to a mouse model of prion disease, to assess the influence of a nonlethal arbovirus encephalitis [12] on the progression of the ongoing hippocampal chronic neurodegeneration. We quantitated microgliosis using stereological unbiased method and assessed behavioral changes to measure directly the influence of a RNA virus infection on hippocampal microglial response and associated sickness behavior.…”