Comprehensive Neurological Assessment After MCAO in Mice

Mice were assessed for neurological functions on day 1, 3, and 7 after the MCAO procedure, by corner turning test, the modified Neurological Severity Score (mNSS), foot fault test, and Rotarod test. All the outcome evaluation was conducted by independent investigators blinded to the group allocation to minimize bias. The mNSS scoring system quantified performance on motor, sensory, reflex, and balance functions, with higher scores indicating more severe neurological impairment. The corner turning test was conducted to assess behavior performance regarding sensorimotor and postural asymmetries. Mice were placed into a 30° corner joined by two connected board walls, where they could rear and turn either left or right to leave the corner. Ten trials were repeated for each mouse to calculate the percentage of left turns. The foot fault test was performed to assess sensorimotor function by calculating the percentage of foot faults out of total footsteps. Briefly, mice were placed in a grid and allowed to move freely for 5 min. A foot fault was recorded when the limb slipped through the grid hole or rested with the grid at wrist level. The Rotarod test was performed to evaluate balance and motor coordination. Mice were conditioned on a rotating rod that accelerated from 4 to 40 rpm within 300 s, which were repeated three times with 5-min intervals. Latency to fall off the rotating rod was automatically recorded up to 300 s. Data were calculated as mean values from three trials.
To minimize the experiment bias, all the neurobehavioral tests were performed on the same day, in the same mouse coding order of the same batch of animals, and also in the same experiment order, that is, mNSS score, corner turning test, foot fault test, and Rotarod test. Of note, each test was separated by at least 30-min intervals, which allowed for a sufficient rest for experimental animals.