Fig. 4
The incubation with angiotensin receptor blockers (ARBs) of astrocytes affects the inflammatory phenotype of microglia. *p < 0.05, **p < 0.01, and ***p < 0.001 compared different experimental groups as marked by horizontal bar; graphs depict mean values ± standard error of the mean (SEM). A Experimental timeline. Three to five days after subculturing, astrocytes were left unstimulated (control), treated with 10 µM PD123319 or 10 µM telmisartan for 48 h. Next, fresh culture medium was added, and the cells were allowed to recover in regular culture medium over 24 h. This so-called conditioned medium (CM) of astrocytes was consecutively collected for further experiments. Following 24 h after seeding in subcultures, microglia were exposed to CM from resting astrocytes (CM Control), CM from astrocytes stimulated with 10 µM PD123319 (CM PD123319) or CM from astrocytes stimulated with 10 µM telmisartan (CM Telmisartan) over 24 h. Consecutively, RNA was isolated or microglia were fixed with 4% paraformaldehyde (PFA) for immunocytochemical staining. B Microglial viability was not compromised through the exposure to CM Control (98.4% survival rate), CM Telmisartan (99% survival rate) or CM PD123319 (99.1% survival rate) compared to untreated control cells (98.9% survival rate). C Expression of the pro-inflammatory microglia marker inducible nitric oxide-synthetase (iNOS) and the anti-inflammatory microglia marker CD206 between CM Control and CM Telmisartan. D Expression of the pro-inflammatory microglia marker IL-6 and the anti-inflammatory microglia marker IL-10 between CM Control and CM Telmisartan. E Expression of the pro-inflammatory microglia marker iNOS and the anti-inflammatory microglia marker CD206 between CM Control and CM PD123319. F Expression of the pro-inflammatory microglia marker IL-6 and the anti-inflammatory microglia marker IL-10 between CM Control and CM PD123319. G Representative immunocytochemical stainings of microglia with IbA1 + (red) and iNOS + (green) in unstimulated (control), CM control-preincubated (CM Control), CM telmisartan-preincubated (CM Telmisartan) or PD123319-preincubated (CM PD123319) conditions. Hoechst stained all cell nuclei blue; scale bars = 50 µm