Microglia of Rat Ventral Midbrain Recovers Its Resting State Over Time In Vitro: Let Microglia Rest Before Work

Cortical or total brain cultures of microglia are commonly used as a model to study the inflammatory processes in Parkinson's disease. Here we characterize microglia cultures from rat ventral midbrain and evaluate their response to zymosan A. We used specific markers of microglia and evaluated the morphology, the phagocytic activity and reactive oxygen species (ROS) levels of the cells. During the first 10 days in vitro (DIV), cultures presented predominantly cells with a round morphology, expressing CD68 and with high phagocytic activity and ROS production. After 13 DIV, this tendency was reversed, with cultures showing higher number of ramified cells and fewer CD68(+) cells along with lower phagocytic and ROS production capability, suggesting that microglia must be kept in vitro for at least 13 days to recover its resting state. The exposure of cultures with less than 10 DIV to zymosan A significantly decreased cell viability. Exposure of cultures with 13 DIV to zymosan A (0.05, 0.5, or 5 mu g/ml) increased the total cell number, the percentage of CD68(+) cells, and the phagocytic activity. Concentrations of zymosan A higher than 5 mu g/ml were also effective in activating microglia but significantly decreased the number of viable cells. In summary, microglial cells remain in the activated state for several days after the isolation process and, thus, stimulation of microglia recently isolated can compromise interpretation of the results. However, upon 13 DIV, cells achieve properties of nonactivated microglia and present a characteristic response to a proinflammatory agent. (C) 2009 Wiley-Liss, Inc.