Ketamine Induces Neuroapoptosis in Stem Cell–Derived Developing Human Neurons Possibly through Intracellular Calcium/Mitochondria/microRNA Signaling Pathway

Ketamine, one of the commonly used agents in pediatric anesthesia, has been linked to neurodegeneration and cognitive dysfunction in developing animal models. Previous studies on developing neurons derived from human embryonic stem cells (hESCs) indicate that ketamine induces neuroapoptosis and the mechanisms remain largely unknown. This study aims to investigate the effect of ketamine on intracellular calcium, mitochondrial signaling, and microRNA profiles in hESCs-derived 2-week-old neurons. The neurons were exposed to ketamine for 6 or 24 hours. Neuroapoptosis was assessed by TUNEL staining. Intracellular calcium level was analyzed using Fluo-4 AM staining. The mitochondria-related neuroapoptosis pathway including mitochondrial membrane potential, cytochrome c release from mitochondria to cytosol, and mitochondrial fission was also investigated. miScript miRNA arrays were used in microRNA target identifica- tion studies. The results showed that ketamine exposure induced neuroapoptosis and alterations in intracellular calcium levels. In addition, ketamine decreased mitochondrial membrane potential, resulted in cytochrome c release from mitochondria into cytosol, and increased mitochondrial fission. Among 88 microRNAs investigated, let-7a/e, miR- 21, miR-23b, miR-28-5p, and miR-423-5p were found downregulated, while miR-96 was upregulated in the neurons treated with ketamine. Collectively, our findings indicate that ketamine induces neuroapoptosis possibly through the dysregulated intracellular calcium, mitochondria, and microRNA pathway. embryoid bodies (EBs) were present 24 h after dispase diges-tion. Five days after digestion, EBs were moved to neural induction media containing DMEM/F12 supplemented with 1% N 2 (Invitrogen), 1% nonessential amino acids, 1 mg/ mL heparin (Sigma) and 5 ng/mL bFGF. On day 9, the EBs were plated to matrigel-coated 35-mm dishes. Rosette-like structures were present within 5 days of the EBs plating down. The rosettes were manually separated from the surrounding cells using a serological pipette approximately 2 days after the morphology was clearly visible. The rosette cells were then transferred to matrigel-coated culture dishes and cultured in neural expan-sion media containing DMEM/F12 supplemented with 2% B27 without vitamin A, 1% N 2 (Invitrogen), 1% nonessential amino acids, 20 ng/mL bFGF and 1 mg/mL heparin. The neural stem cells (NSCs) were passaged enzymatically every 5 days. NSCs were cultured in 60-mm matrigel-coated dishes (500,000 cells/dish) in neuron differentiation media containing neurobasal media (Gibco) supplemented with 2% B27, 100 ng/mL ascorbic acid (Sigma-Aldrich), 0.1 μM cyclic adenosine monophosphate, 10 ng/mL brain-derived neurotrophic factor, 10 ng/mL insulin-like growth factor 1 (PeproTech Inc., Rocky Hill, NJ) and 10 ng/mL glial cell-derived neurotrophic factor. The media was changed every other day. Following 2 weeks of culture, the cells displayed clear neuronal morphology and were used for the studies. enhances human stem cell proliferation induces apoptosis