Global Cerebral Ischemia in the Rat: Online Monitoring of Oxygen Free Radical Production Using Chemiluminescence in vivo

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Abstract
Using online in vivo chemiluminescence (CL), we studied for the first time continuously the production of reactive oxygen species (ROS) after global cerebral ischemia and the relationship of ROS production to CBF. In anesthetized rats equipped with a closed cranial window, the CL enhancer, lucigenin (1 m M), was superfused onto the brain topically. CL was measured through the cranial window with a cooled photomultiplier, and CBF was measured simultaneously with laser–Doppler flowmetry. Reperfusion after 10 min (n = 8) of global cerebral ischemia led to a CL peak to 188 ± 77% (baseline = 100%) within 10 ± 4 min. After 2 h of reperfusion, CL had returned to 102 ± 28%. Reperfusion after 20 min (n = 8) of ischemia increased CL to 225 ± 48% within 12 ± 3 min. After 2 h, CL was still increased (150 ± 44%, p < 0.05 compared with 10 min of ischemia). CL after 10 min of ischemia was neither affected by brain topical free CuZn-superoxide dismutase (SOD) (100 U/ml, n = 3) nor by i.v. administration of free CuZn-SOD (104 U/kg, followed by 104 U/kg/h, n = 3). The CBF hyperperfusion peak on reperfusion preceded the CL peak in all experiments by several minutes. In additional in vitro experiments we investigated the source of CL: Intracellular loading of lucigenin was demonstrated in cultured CNS cells, and a very similar pattern of CL as in the in vivo preparation after ischemia developed in rat brain slices after 15 min of hypoxia, which was unaffected by free CuZn-SOD (100 U/ml) but strongly attenuated by liposome-entrapped CuZn-SOD. We conclude that lucigenin-enhanced CL is a promising tool to study ROS production continuously from the in vivo brain of experimental animals and brain slices, and that the CL signal most likely derives from the intracellular production of superoxide. The production of ROS is preceded by reperfusion, is burst-like, and is dependent on the duration of the ischemic interval.