Research Paper Volume 12, Issue 24 pp 26121—26139

Subanesthetic isoflurane abates ROS-activated MAPK/NF-κB signaling to repress ischemia-induced microglia inflammation and brain injury

Figure 7. Sub-anesthetic ISO post-conditioning represses ROS-mediated activation of p38 MAPK/NF-κB signaling in OGD-stimulated microglial cells in co-cultures. (A) At the end of 3 h-OGD or Ctrl treatment, co-cultures were exposed to RA with or without 0.7% ISO for 30 min. All the cells were continuously cultured under normal conditions for 24 h after OGD exposure. Then, microglial cells were harvested for further analyses. The images of DCFH-DA-stained microglial cells were taken and ROS levels were calculated. Data represent the relative DCF fluorescence. Scale bar: 5 μm. (BD) Co-cultures with or without NAC (5 mM) pretreatment for 30 min were subjected to 3 h-OGD or Ctrl treatment and continuously cultured under normal conditions for 6 or 24 h after OGD stimulation. Then, microglial cells were harvested for assays. (B) Representative western blots show total and phosphorylated p38 MAPK and NF-κB p65 levels at 6 h after OGD exposure. β-actin and lamin B were used as the internal controls. (C) Quantification of PGE2 levels by RIA at 24 h after OGD exposure. (D) Quantification of NO production by Griess reagent at 24 h after OGD stimulation. Representative data are from three independent experiments and expressed as mean ± SD. Statistical significance: *P < 0.05 vs. Ctrl groups; #P < 0.05 vs. OGD + RA or OGD group. Ctrl: control; ISO: isoflurane; OGD: oxygen and glucose deprivation; RA: room air; NAC: N-acetyl-L-cysteine.