*2*.*4*. *Neurological Evaluation*

Postischemic neurological deficits were evaluated after 24 h of reperfusion on a 5-point scale as follows: grade 0, no deficit; grade 1, failure to fully extend the right forepaw; grade 2, spontaneous circling or walking to a contralateral side; grade 3, walking only when stimulated; grade 4, unresponsive to stimulation and a depressed level of consciousness; and grade 5, death [25,27]. Before MCAO, the neurological score was zero in all the rats. The rats that did not exhibit

#### *2*.*5*. *Detection of O2 − Production in the Brain*

Detection of intracellular O2 − production in the ischemic penumbral region of the cortex after MCAO/Re was performed by histochemical staining of freshly frozen brain sections (8 μm thick) with the fluorescent probe dihydroethidium (DHE). The brain sections were immediately incubated with DHE (10 μmol/L, Sigma-Aldrich Japan, Tokyo, Japan) in phosphate-buffered saline for 30 min at 37 °C [28,29]. To determine the fluorescence intensity of oxidized DHE, 3 visual fields within the penumbral cortex regions of each hemisphere were photographed using a confocal laser scanning microscope (Fluoview FV1000, OLYMPUS, Tokyo, Japan) with excitation at 510 nm and emission at 580 nm. Fluorescence intensity of oxidized DHE was quantified using imaging software (FV10-ASW 1.7, OLYMPUS, Tokyo, Japan). Analyses of immunohistochemistry were performed by an investigator blinded to the treatment protocol.
