Studies on NF-(kappa)B activation during reperfusion injury following reversible acute ischemic stroke

We undertook this study to investigate whether agents that could block the NF-kB activation in human brain microvascular endothelial cells (HBMEC) would be effective in reducing reperfusion injury following reversible ischemic stroke in rats.; Low passage HBMECs were subjected to conditions of hypoxia/reoxygenation (H/R) in an anaerobic chamber. Activated NF-kB was detected by electrophoretic mobility shift assays (EMSA) and relative levels of ICAM-1 gene expression were estimated by Northern Blot and Dot Blot analysis. In a rat model of ischemic stroke with reversible middle cerebral artery occlusion (MCAO), the activated NF-kB was detected by EMSA and cerebral infarct volume was measured by TTC staining in each hemisphere during reperfusion. Components of the activated NF-kB complex were identified by antibodies using supershift assays.; When HBMECs were subjected to hypoxia followed by reoxygenation but not hypoxia alone, a NF-kB complex composed of P65 and P50 Rel proteins was rapidly activated within 15-30 minutes of reoxygenation. Four hours later, expression of the ICAM-1 gene was significantly upregulated. The antioxidant NAC, PDTC and the proteasome inhibitor TPCK blocked both the activation of NF-kB and the upregulation of ICAM-1 gene. In the rat MCAO stroke model, EMSA detected a transient, significant increase of activated NF-kB, consisting of P65 and P50 Rel proteins in the left (ischemic) hemispheres at 15 and 30 minutes after the onset of reperfusion. By 60 minutes, activated NF-kB fell to basal level, and subsequent activation of NF-kB was not observed at 5 hours 24 hours, 48 hours, 3 or 5 days of reperfusion. When the antioxidants NAC (150mg/kg) or DDTC (150mg/kg) were administered intraperitoneally to rats prior to the initiation of reperfusion, the activated NF-kB at 15 minutes was not seen. Moreover, NAC pretreatment led to a significant reduction (from {dollar}35.5pm2.8{dollar}% to {dollar}18.1pm2.1{dollar}%; n = 10, p {dollar}<{dollar} 0.01) in the percentage infarct volume of the affected hemispheres. However, when NAC was administered after the time period of the expected burst of NF-kB activation, there was a lower but still significant reduction (from {dollar}34.8pm3.8{dollar}% to {dollar}24.6pm3.8{dollar}%, n = 10, p {dollar}<{dollar} 0.05) in the infarct volume. Treatment of DDTC prior to the initiation of reperfusion did not reduce cerebral infarct volume ({dollar}36.25pm3.4{dollar}% and {dollar}35.5pm1.2{dollar}%, respectively, n = 6).; The data that NF-kB was rapidly activated by reoxygenation following hypoxia in HBMEC, as well as by ischemia/reperfusion in the rat MCAO model suggest that NF-kB activation in HBMEC may play a crucial role in mediating reperfusion tissue injury after acute ischemic stroke. (Abstract shortened by UMI.)...