Investigation of oxidative damage at cellular level in an experimental ischemic stroke model

The aim of this study is direct intravital monitorization of the pharmacological effects of two well-known neuroprotectant antioxidant molecules having different blood-brain barrier (BBB) permeability against ischemia/reperfusion at vascular and parenchymal cellular level to gain insight to their failure in clinical trails. For this purpose, 1-hr distal middle cerebral artery ischemia was induced in Swiss albino and pan-tissue-mt-cpYFP transgenic mice. Superoxide (O2•̄) formation during recanalization was monitored in vivo by conventional fluorescence (via dihydroethidium fluorescence) and confocal (via mt-cpYFP fluorescence) microscopy in vascular and parenchymal compartments separately. Radical scavengers (N-tert-butyl alpha phenyl nitrone- PBN- and S-PBN) were evaluated for suppression of O2•̄ signal, cerebral blood flow, histopathology, neurological deficit and microvascular flow velocities. PBN (100mg/kg, 15min before recanalization) suppressed both the vascular and parenchymal O2•̄ signal almost completely from the beginning of recanalization and reduced the ischemic area from 1557±313μm2 to 552±116μm2 (p=0.01). The vascular antioxidant effect of BBB impearmeable S-PBN (156mg/kg) started instantly, whereas its parenchymal effect was partial (64%) and delayed by 30-40 min. Similarly to PBN, S-PBN reduced the ischemic area from 1557±313μm2 to 807±71μm2 (p=0.03), improved post-ischemic blood flow, reduced capillary obstructions in brain sections (105±18 vs. 158±24 p=0.05) and increased capillary erythrocyte velocities (1±0.1 mm/s vs. 0.3±0.1 p<0.001). The parenchymal effect of S-PBN was thought to result from improved tissue perfusion. When S-PBN was administered at a dose corresponding to the clinically ineffective dose of NXY-059 (an S-PBN-like molecule), its parenchymal effect disappeared. In conclusion, intravital examination of neuroprotective drug effects at the cellular level may promote a more successful translation of preclinical data to the clinic. Genetically encoded biosensors can significantly advance antioxidant research.