Ketamine Induces Toxicity in Human Neurons Differentiated from Embryonic Stem Cells via Mitochondrial Apoptosis Pathway

Abstract

Ketamine is widely used for anesthesia in pediatric patients. Growing evidence indicates that ketamine causes neurotoxicity in a variety of developing animal models. Our understanding of anesthesia neurotoxicity in humans is currently limited by difficulties in obtaining neurons and performing developmental toxicity studies in fetal and pediatric populations. It may be possible to overcome these challenges by obtaining neurons from human embryonic stem cells (hESCs) in vitro. hESCs are able to replicate indefinitely and differentiate into every cell type. In this study, we investigated the toxic effect of ketamine on neurons differentiated from hESCs. Two-week-old neurons were treated with different doses and durations of ketamine with or without the reactive oxygen species (ROS) scavenger, Trolox. Cell viability, ultrastructure, mitochondrial membrane potential (ΔΨ m ), cytochrome c distribution within cells, apoptosis, and ROS production were evaluated. Here we show that ketamine induced ultrastructural abnormalities and dose-and timedependently caused cell death. In addition, ketamine decreased ΔΨ m and increased cytochrome c release from mitochondria. Ketamine also increased ROS production and induced differential expression of oxidative stress-related genes. Specifically, abnormal ultrastructural and ΔΨ m changes occurred earlier than cell death in the ketamine-induced toxicity process. Furthermore, Trolox significantly decreased ROS generation and attenuated cell death caused by ketamine in a dose-dependent manner. In conclusion, this study illustrates that ketamine time-and dosedependently induces human neurotoxicity via ROS-mediated mitochondrial apoptosis pathway and that these side effects can be prevented by the antioxidant agent Trolox. Thus, hESC-derived neurons might provide a promising tool for studying anesthetic-induced developmental neurotoxicity and prevention strategies.

Long-term NMDA receptor inhibition affects NMDA receptor expression and alters glutamatergic activity in developing rat hippocampal neurons

“…It is hypothesized that the increase in the NR1 of the NMDA receptor is responsible for apoptosis as the increased expression of the NR1 subunit evokes a raise in Ca 2+ -entry (Slikker et al, 2007;Bosnjak et al, 2012;Liu et al, 2013;Bai et al, 2013). Although [Ca 2+ ] i is necessary for neuronal development, pathologically enhanced Ca 2+ -influx promotes an increase in the [Ca 2+ ] i concentration, results in a rise of reactive oxygen species, increases mitochondrial membrane permeability by members of the bcl-2 family (bcl-2 and BAX) and ultimately, releases cytochrome C (Bosnjak et al, 2012;Bai et al, 2013).…”

“…Although [Ca 2+ ] i is necessary for neuronal development, pathologically enhanced Ca 2+ -influx promotes an increase in the [Ca 2+ ] i concentration, results in a rise of reactive oxygen species, increases mitochondrial membrane permeability by members of the bcl-2 family (bcl-2 and BAX) and ultimately, releases cytochrome C (Bosnjak et al, 2012;Bai et al, 2013). BAX and bcl-2 are proteins involved in apoptosis, both are localized in the inner mitochondrial membrane.…”

Down-regulation of MicroRNA-21 Is Involved in the Propofol-induced Neurotoxicity Observed in Human Stem Cell–derived Neurons

“…(H1 cell line, WiCell Research Institute Inc., Madison, WI) were cultured on a feeder layer of mouse embryonic fibroblasts (MEFs) that were mitotically inactivated using mitomycin C (Sigma-Aldrich, St. Louis, MO) as previously described by our laboratory 29,30 . Briefly, culture dishes were coated with 0.1% gelatin overnight in a humidified incubator.…”

Anesthetic Neurotoxicity in Pediatric Patients

“…Three hours exposure to ketamine did not produce any signiicant histochemical change, whereas profound brain cell death was observed in the frontal cortex among subjects that were under the efect of ketamine for 9 or 24 hours. In cell culture study of Bosnjak et al [25], they demonstrated that ketamine decreases neuronal viability time and dose dependently, leads to neuronal ultrastructural abnormalities, causes depolarization of mitochondrial membrane potential, induces apoptotic pathway, causes cytochrome c release from mitochondria into cytosol, and induces free oxygen radical production.…”