Neurological deficits score assay and infarct volume measurement

The neurological deficit scores were evaluated in a blinded fashion 24 h after MCAO and reperfusion based on Bederson’s score standard [19]. Specifically, grade 0 represents no observed deficit; grade 1 represents flexion to contralateral torso and forelimb; grade 2 represents a decreased resistance to lateral push but without circling; grade 3 represents leaning to affected side; and grade 4 represents no spontaneous locomotor activity. Dead animals were excluded in the scoring scale. After evaluation of neurological deficits, the infarct volume was measured by TTC staining, which has been widely used for accurately reflecting the extent of irreversible ischemic damage in cerebral tissues [20]. Briefly, the brain was removed and frozen at -20°C for 30 min and then sectioned into slices 1 mm thick. The slices were then incubated in a 1.5% TTC solution at 37°C for 30 min and fixed in paraformaldehyde. Samples were digitally photographed and the percentage of the infarct volume with respect to the total brain volume was quantified with Photoshop CS4.