Resumo

Neospora caninum is a protozoa that causes neurologic disease in dogs and abortion in cattle. The parasite can be detected in many animal tissues and it forms cysts in central nervous system (CNS). When infected by N. caninum tachyzoites, primary cultures of astrocytes has changed their morphology and released high levels of IL-6 and IL-10. This Th2 pattern suggested that glial cells could be responsible by nervous tissue protection. In order to study glial response to injuries caused by N. caninum infection in cultures with or without neuron/glia interactions, this study has evaluated the effect of this parasite infection in mixed glia (astrocyte/microglia) culture and in neuron/glia co-cultures stimulated with 300 IU/mL of IFN-γ or 1.0 μg/mL of LPS 72h post infection. The number of tachyzoites reduced 38.5 and 64.6% (mixed glia culture) and 54.1 and 55.7% (neuron/glia co-cultures) when stimulated with IFN- γ and LPS, respectively. In glia cultures the IFN-y and LPS treatment do not influence cell viability, measured by lactate dehydrogenase activity in the culture medium. However, in infected culture, cell viability decreased 96.0 and 266.4% in not treated and IFN-y stimulated cultures. In neuron/glia infected cultures without treatments, cell viability decrease (61.4%), LPS treatment did not influence cell viability and IFN-γ has caused toxic effects in both infected and not-infected cells. Microscopic phase contrast analysis showed in neuron/glia co-cultures high sensibility to parasite cytopathic effect. The morphological analysis by immunocytochemistry confirmed, in both cultures, an accentuated astrogliosis, characterized by morphological alterations and increase of fibrilar glial acid protein expression. As this phenomenon was not restricted to infected cells, these data suggest a paracrine effect. In neuron/glia co-cultures, the immunodetection of tubulin βIII (specific neuron marker) has showed a basal neurite outgrowth in not infected cultures. However, in infected cultures, it was observed a drastic impairment of neurite, followed by neuritis fragmentation. Our results show a large difference in immune/inflammatory response by glia cells in two cultures models. In glia culture, these cells response can control parasite proliferation until under IFN-γ or LPS stimulus. In neuron/glia co-cultures there was a large parasite proliferation in non treated cells, following by an expressive cellular lost and neuron degeneration. These results probably suggest that glia has failed in neuron protection to parasite infection in this cell interaction model.