Recreational use of ketamine (KET) has been increasing worldwide. hundred milligrams. If the high dose of KET was accompanied with ALC, the toxicity will be significantly increased. There have been some reports of the lethality from mixed-drug intoxication including ALC and KET19, 20. However, in most cases, the interplay between ALC and KET has not been fully characterized. In this study, PC12 cells were used to explore the neurotoxicity changes after exposure to ALC and/or KET. Our results exhibited that ALC potentiated Rabbit Polyclonal to PTTG KET-induced KU-57788 neurotoxicity in PC12 cells. The cell viability was significantly decreased, ROS levels were significantly increased and the ultrastructural changes were more notable when KET was combined with ALC compared with ALC or KET treatment alone. Moreover, main cultured cortical neurons were used to further examine the effect of ALC on KET-induced neurotoxicity. Our results provided the first evidence that the cell viability of main cultured neurons was also significantly decreased when KET was combined with ALC compared with ALC or KET treatment alone, which was consistent with the results obtained with PC12 cells. Previous studies have reported that KET has pro-apoptotic properties and induces neuroapoptosis in the developing brain and cultured neurons study using young mice or rats would be necessary to further characterize the nature and degree of neurotoxicity in animals. Physique 10 Mechanism summary of the ALC?+?KET-induced neurotoxicity. Materials and Methods Reagents KET hydrochloride was obtained from Gutian Medical Inc. (Fujian, China). ALC was purchased from Beijing Red Star Co. (Beijing, China). CNQX and Fura-4-Was were KU-57788 purchased from Sigma (USA). Dulbeccos altered eagle medium (DMEM)/F12, W27 product and fetal bovine serum (FBS) were purchased from Gibco (NY, USA). MTT was from Ameresco (USA). -tubulin was from Sigma (USA). Cytosine–D-arabinoside (Ara-C), DCFH-DA and DAPI were from Beyotime (Nanjing, China). Hoechst 33258, propidium iodide (PI) and AO-EB were from Solarbio (Beijing, China). Trizol reagent and HiFi-script KU-57788 cDNA Kit were purchased from CWBIO (Beijing, China). UltraSYBR Combination (low ROX) was purchased from LEWEITECH (Shijiazhuang, China). Main antibodies against Akt, p-Akt, CREB, p-CREB, PKA, CaMK-IV, Bcl-2, cleaved caspase-3 and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit and goat-anti-mouse) were purchased from Bioss (Beijing, China). Rabbit anti-caspase-3 and BDNF IgG and mouse anti–actin IgG were purchased from ZSGB-BIO (Beijing, China). Main antibody against Bax was from Proteintech (USA). Enhanced chemiluminescence was obtained from Amersham Biosciences (England, UK). PC12 cell cultures PC12 cell collection was obtained from Shanghai cell lender of Chinese Academy of Sciences. The cells were cultured in DMEM made up of 10% FBS, 100 U/ml streptomycin and 100 U/ml penicillin at 37?C in humidified atmosphere with 5% CO2. The culture medium was replaced every 48?h, and cell cultures were passaged at a ratio of 1:5 every 4 days. Main culture of rat cortical neuronal cells Main cultured neurons were prepared as previously explained54. Animal procedures were conducted in accordance with the National Institutes of Health lead for the care and use of Laboratory animals (NIH Magazines No. 8023, revised 1978) and approved by Ethics Committee of Shenyang Pharmaceutical University or college. Briefly, KU-57788 cerebral cortex of neonatal Sprague-Dawley rats (postnatal day 1) was dissected and placed in ice-cold DMEM, then mechanically dissociated and digested with trypsin. The cells (106?cells/ml) were distributed and grown in DMEM/F12 with 15% FBS, 100 U/ml penicillin, 100 U/ml streptomycin sulfates at 37?C in a humidified 5% CO2/95% air flow atmosphere. Neuron culture medium was replaced by serum-free DMEM/F12?+?2% W27 product at 48?hours, subsequently half of the culture medium was replaced every 48?hours. On the 4th day, cultures were fed with medium made up of Ara-C (2.5?g/ml) for 48?hours to prevent glial cell growth. The neurons cultured for 6 days were ready for the experiments. Measurement of cell viability Cell viability was assessed by the MTT assay according to our previous statement54. Briefly, the medium was incubated with 10?t of 5?mg/ml MTT solution before the end of the experiment for 4?h at 37?C. Then the culture medium with MTT was removed and 200?l dimethyl sulfoxide was added to each.