No cell death is detected in the motor cortex after three injections of ketamine–xylazine (KX). (A) Cell apoptosis in the motor cortex was assessed by the immunostaining of cleaved caspase-3 on the day when the mice received the last injection of KX (20/3 mg/kg). Nucleic acid was stained with 4′,6′-diamidino-2-phenylindole (DAPI). Saline injection was used as control group. A 6-h-long anesthesia maintained by a higher dose of KX (40/4 mg/kg) in P7 mice was used as a positive control group. Three injections of KX at either P14 to P18 or P21 to P25 had no significant effect on cell apoptosis. Scale bar, 100 μm. (B) Quantification of cleaved caspase-3 positive cell number under various conditions. Comparison of means of each group was performed using a one-way ANOVA followed by Neuman–Keuls multiple comparison post hoc test. Data are presented as mean ± SD.