Fig. 4

Activation characteristics of primary microglia. *p < 0.05; **p < 0.01; ***p < 0.001 compared to different experimental groups as marked by a horizontal bar. Characterization of the activated pro-inflammatory microglia phenotype by the expression of the inducible nitric oxide (NO) synthetase (iNOS) and release of NO after blockade of HCN and KCNQ/Kv7 channels with ZD7288 and XE-991 (30 μM each), respectively. Treatment with LPS (10 ng/ml) served as a positive control. a. iNOS expression was measured on RNA level by RT-qPCR (H(3) = 11.608, p < 0.009) and on b protein level by immunocytochemistry (H(3) = 17.665, p = 0.001). c Release of NO was measured by Griess assay (μmol/l; H(3) = 15.784, p = 0.001). Characterization of the anti-inflammatory phenotype of activated microglia by expression of CD206, release of insulin-like growth factor 1 (IGF1), and change of phagocytic capacity, after pharmacological block of HCN and KCNQ/Kv7 channels by 30 μM ZD7288 and XE-991, respectively. Treatment with IL4 (25 ng/ml) served as a positive control. d Regulation of CD206 expression on RNA level was measured by RT-qPCR (F (3, 20) = 13.981, p < 0.0001, ω = 0.62). e IGF1 release was measured by ELISA (pg/ml, H(3) = 15.805, p = 0.001). f Zymosan engulfment showed phagocytic activity of microglia (H(3) = 11.653, p < 0.009). Data were blank-corrected and normalized to control. g Representative immunocytochemical stainings for the microglia marker Iba1 (red), co-stained for iNOS (green), and Hoechst as a nuclear counterstain (blue); scale bars = 50 μm