Differential cytokine expression by brain microglia/macrophages in primary culture after oxygen glucose deprivation and their protective effects on astrocytes during anoxia

Rawan Barakat, Zoran Redzic

Activation of microglia/macrophages following cerebral ischemia may be beneficial or detrimental for the survival of brain cells, an ambiguity in effects that has been explained by findings that ischemia can induce transformation of resting monocytes/macrophages into two different inflammation-related phenotypes, termed M1 and M2. The extent to which this differentiation depends on paracrine signaling from other brain cells is not clear. This study explored if oxygen glucose deprivation (OGD) can trigger expression of phenotype-specific markers in rat microglia/macrophages in primary culture, in absence/low abundance of other brain cells. Time pattern of these changes was assessed and compared to time-pattern that has been revealed in vivo previously. Effects of phenotype-specific cytokines on viability of astrocytes in primary culture during anoxia were also explored. Primary cultures of rat microglia/macrophages were exposed to 2h OGD and then incubated further under normal conditions; this was considered as a recovery period. Expression of mRNA for specific markers and secretion of phenotype-specific cytokines were explored at different time points by real time PCR and ELISA, respectively. Effects of cytokines that were secreted by microglia in primary culture after OGD on viability of astrocytes were determined. Expression and secretion of M2 phenotype-specific markers and/or cytokines after OGD increased early after OGD and then decreased in the later stages of the recovery period. Expression and secretion of M1 phenotype-specific markers and cytokines did not show a common time pattern, but there was a tendency for an increase during the recovery period. All M1 phenotype-specific and two out of the three tested M2 phenotype-specific cytokines revealed protective effects on astrocytes during near-anoxia by a marked reduction of apoptosis. Time-pattern of expression/secretion of phenotype-specific markers suggested that polarization of the brain microglia/macrophages in vitro to M2 and M1 phenotypes were largely independent and likely dependent on signaling from other brain cells, respectively. Time-pattern of polarization to the M2 phenotype partially resembled time-pattern that has been seen in vivo. Effects of M1 phenotype-specific cytokines on primary culture of astrocytes were protective, thus largely opposite to effects that have been observed in vivo.