Evaluation of neurological deficits

For determining neurological deficits of mice, neurological assessment was performed using a 5-point scoring system 24 hr after ischemia-reperfusion surgery, as follows: 0, normal motor function; 1, flexion of torso and contralateral forelimb when mouse was lifted by the tail; 2, circling to the contralateral side when mouse held by the tail on a flat surface but normal posture at rest; 3, leaning to the contralateral side at rest; 4, no spontaneous motor activity³⁴. Mice that did not show behavioral deficits immediately after reperfusion (neurological score = 0) were excluded from the study.

The pole test was adapted from Matsuura et al. with minor medications³⁵. Mouse was placed head upward near the top of a vertical wood pole (50 cm in length and 8 mm in diameter) with rough surface. The time taken to turn completely downwards (T/turn) and the total time to reach the floor with all four paws (T/floor) were recorded. If the mouse was unable to turn completely, the time to reach the floor was attributed to T/turn. Each animal was tested for 5 trials and the average score was taken as the final pole test score.

The balance and grip strength of mice were assessed using the wire-hanging test, as described previously³⁶. The test apparatus consisted of a steel wire (1 mm in diameter) stretching between two posts 60 cm above a foam pillow. Each mouse was trained to cling to the wire with their forepaws for two days before the test. On the third day, latencies to fall was tested twice (maximum: 3 min), and the average latencies were recorded.