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 5. Sub-anesthetic ISO post-conditioning inhibits OGD-led NF-κB p65 activation and PGE2 and NO production in microglial cells in co-cultures. (A, B) 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 6 or 12 h after OGD stimulation. Then, microglial cells were harvested for subsequent studies. (A) Representative western blots show total and phosphorylated IKKβ and NF-κB p65 levels at 6 h after OGD exposure. β-actin and lamin B were used as the internal controls. (B) NF-κB p65 DNA-binding activity was quantified using the TransAM NF-κB p65 transcription factor assay kit at 12 h after OGD insult. (C, D) Co-cultures with or without NAI (2 μM) pretreatment for 30 min were subjected to 3 h-OGD or Ctrl treatment and continuously cultured under normal conditions for 24 h after OGD stimulation. Then, microglial cells were collected for further analyses. (C) Quantification of PGE2 by RIA. (D) Quantification of NO production by Griess reagent. 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.