Measurement of total infarct volumes and neurological deficit scores

The brains harvested 24 h after MCAO were immediately sliced into 1 mm coronal sections to obtain 10 sections per brain from the olfactory bulb to the area immediately before the cerebellum. Sections were incubated with 2% 2,3,5-triphenyl-tetrazolium chloride (TTC) solution for 17 min at 25 °C and then soaked in 10% neutral buffered formalin for more than 2 h. Total infarct volumes in these sections were calculated using infarct areas as previously described (Jung et al. 2017) with a digital camera (Canon 20 D, Canon Korea, Korea) and ImageJ software (NIH, MD, USA).

At 24, 48, and 72 h after MCAO, neurological deficit scores were determined as previously described (Jung et al. 2017) using the following five-point scale: 0, no neurological deficit; 1, incomplete extension of the right forepaw and reduced grip when the tail was pulled; 2, voluntary movement in all directions and turning to the right when the tail was pulled; 3, walking or circling to the right and sensitive to nociception when stimulated; and 4, no response to stimulation or stroke-related death.