The combination of ketamine and chlorpromazine can be used for anesthesia in rats (35 (link),36 (link)). In the present study, the rats (n=6/subgroup) were anesthetized with a combination of ketamine (100 mg/kg) and chlorpromazine (5 mg/kg) via intra-peritoneal injection, according to our previous studies (37 (link),38 (link)). Following deep anesthesia determined by respiratory, palpebral reflex, pedal withdrawal reflex, and cutaneous reflex (39 (link),40 (link)), the animals were transcardially perfused with 400 ml of saline followed by 4% paraformaldehyde solution (pH 7.2-7.4). Following perfusion, the brain was dissected from the skull, and the auditory cortex was separated and immersed overnight in the same fixative. The right side was prepared for TUNEL staining and the left side was prepared for immunofluorescence. The following processes were performed, as previously described (41 (link)). Apoptosis in the auditory cortex of rats was detected using a TUNEL assay (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's protocol. DAPI staining solution (1 ยตg/ml; Beyotime Institute of Biotechnology, Haimen, China) was used to counterstain the nuclei. The labeled cells were detected with a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan).