Resumo

OBJECTIVES: An enriched environment was previously described to increase natural killer (NK) cell activity, which recognize and kill virally infected cells. However in the context of viral encephalitis the effect of an enriched environment on behavioral changes in correlation with virus clearance and neuropathology have not been studied in detail. In the present report we induced viral encephalitis by intranasal inoculation of Piry virus infected brain homogenate in albino Swiss mice, housed in impoverished or enriched environments. Neuropathological features of CA3 were correlated with behavioral changes at different time windows to address the following questions: 1) Does an enriched environment affect the disease progression of sub-lethal encephalitis, neuroinvasion and virus clearance from brain parenchyma? 2) Is there any correlation between performances on hippocampal-dependent tasks and the total number of activated microglia, perineuronal nets, and pyramidal neurons in the CA3 of diseased animals? METHODS: Stereology-based estimations of activated microglia perineuronal nets and neurons in CA3 were correlated with behavioral changes in the Piry virus model of encephalitis in albino Swiss mouse. Two-week-old albino Swiss mice were kept in an impoverished environment (IE, n=31) or enriched environment (EE, n=28) for three months and then submitted to the following tests: open field, burrowing, dark/light box, and elevated plus maze. After these tests, an equal volume of Piry virus (IEPy, EEPy) infected or normal (IEcont, EEcont) brain homogenates were nasally instilled. Eight days post-instillation (dpi), when behavioral changes became apparent, brains were fixed and processed to detect viral antigens, T lymphocytes, activated microglia, and perineuronal nets by immuno- or histochemical reactions. At 20 or 40 dpi, the remaining subjects were behaviorally tested and processed for the same markers. RESULTS: In IEPy, burrowing activity decreased and recovered earlier (8 – 10 dpi) than open field (20 – 40 dpi) but remained unaltered in the EEPy group. Before viral infection, EE subjects spent more time in the light and IE in the dark in DLB test. At 20 dpi, preferences were inverted in both groups, and at 40 dpi they disappeared. EEPy presented higher T-cell infiltration, smaller neuroinvasion, faster virus clearance, less microgliosis and less damage to the extracellular matrix than IEPy, whereas the number of neurons remained unchanged during disease evolution. CONCLUSIONS: Our study demonstrated for the first time that nasal instillation of Piry-infected brain homogenate into adult albino Swiss mice induces (i) encephalitis with neuroinvasion, mainly of the olfactory pathways, septum, and ventral hippocampus; (ii) the infection leads to an increase in CA3 microglial number and reduction of the perineuronal nets, but no change in the number of neurons; (iii) Piry infection activates the microglia and reduces perineuronal nets to a higher degree at 8 dpi, when behavioral abnormalities first become apparent; and (iv) environmental enrichment is associated with less intense behavioral changes, lesser degree of microglia activation and neuroinvasion, smaller reduction in the number of perineuronal nets, higher degree of T-cells infiltration and faster virus clearance and resolution of the disease when compared to the subjects of impoverished housing.