The Neuroprotective Effect Of Interrupted Reperfusion On Ischemic Brain And Its Underlying Mechanisms

Cerebrovascular diseases have become the second cause of death in China, and about 80% are ischemic vascular diseases. Re-establishing perfusion as soon as possible is the clinical goal to minimize ischemic injury, but it also leads to additional damage which is called ischemia/reperfusion (I/R) injury. Interrupted reperfusion intervention (IRIV), which consists of several cycles of brief ischemia and reperfusion followed by permanent reperfusion, is neuroprotective in animal models, but the underlying mechanisms are still unclear.Based on Doppler measurement of regional cerebral blood flow and the beam walking test, we established a stable global cerebral ischemia model by bilateral common carotid arterial occlusion in C57BL/6 mice.Then, we evaluated the neuroprotective effects of different IRIV protocols (3 cycles of 10 s/10 s,15 s/15 s,30 s/30s occlusion/reperfusion) and determined the therapeutic time window of the best protocol by behavioral evaluations (beam walking, pole test and Morris water maze). We found that 3 cycles of 15 s occlusion/15 s reperfusion was most protective, and its therapeutic time window was within 60 min. An IRIV of 15 s/15 s decreased the oxidative stress after I/R injury and increased the survival rates of neurons.NADPH oxidase plays a critical role in the superoxide anion generation which is triggered by the onset of reperfusion. To determine whether NADPH oxidase participates in the neural protection against global I/R injury following IRIV, mice were given IRIV and (or) the NOX inhibitor apocynin, and were evaluated for neurological functional deficits by beam walking, pole test and Morris water maze. Brain tissues were used to measure the expression of gp91phox and p47phox, the translocation of p47phox?the activity of NOX and the activation of Racl. We found that the neuroprotective effect of IRIV was mediated by decreasing the expression and translocation of p47phox, as well as the activation of Racl, and subsequently reduced the assembly and activation of NOX.The NMDA receptor participates in the activation of NOX. We treated mice with NMDA, MK-801 or vehicle combined with IRIV to determine whether the NMDA receptor participates in the suppression of NOX activity by IRIV. The Ca2+ concentration, the expression and activation of Racl, the activity of NOX and the expression of NR 2A and NR 2B were measured. The results showed that IRIV suppressed NOX activity and this was partly mediated by decreasing the expression of NR 2A and NR 2B, and the activation of the NMDA receptor.Furthermore, to assess the pathway between the NMDA receptor and NOX, we established an oxygen and glucose deprivation/reperfusion (OGD/RP) model with cultured hippocampal neurons. The neurons were treated with NMDA (activator of the NMDA receptor), MK-801 (inhibitor of the NMDA receptor), PMA (activator of cPKC), Go6983 (inhibitor of cPKC) or vehicle at the beginning of reperfusion, and the Ca2+ concentration, the expression of Racl, gp91phox and p47phox, and the activity of NOX were measured. We found that a novel NMDA receptor-Ca2+in-cPKC-Racl pathway was activated by OGD/RP injury.In conclusion, IRIV decreases the activation of NOX after global cerebral ischemia/reperfusion injury, which at least though the suppression of the NMDAR-Ca2+-cPKC-Racl pathway.