Ketamine-Induced Toxicity in Neurons Differentiated from Neural Stem Cells
- 937 Downloads
Ketamine is used as a general anesthetic, and recent data suggest that anesthetics can cause neuronal damage when exposure occurs during development. The precise mechanisms are not completely understood. To evaluate the degree of ketamine-induced neuronal toxicity, neural stem cells were isolated from gestational day 16 rat fetuses. On the eighth day in culture, proliferating neural stem cells were exposed for 24 h to ketamine at 1, 10, 100, and 500 μM. To determine the effect of ketamine on differentiated stem cells, separate cultures of neural stem cells were maintained in transition medium (DIV 6) for 1 day and kept in differentiation medium for another 3 days. Differentiated neural cells were exposed for 24 h to 10 μM ketamine. Markers of cellular proliferation and differentiation, mitochondrial health, cell death/damage, and oxidative damage were monitored to determine: (1) the effects of ketamine on neural stem cell proliferation and neural stem cell differentiation; (2) the nature and degree of ketamine-induced toxicity in proliferating neural stem cells and differentiated neural cells; and (3) to provide information regarding receptor expression and possible mechanisms underlying ketamine toxicity. After ketamine exposure at a clinically relevant concentration (10 μM), neural stem cell proliferation was not significantly affected and oxidative DNA damage was not induced. No significant effect on mitochondrial viability (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay) in neural stem cell cultures (growth medium) was observed at ketamine concentrations up to 500 μM. However, quantitative analysis shows that the number of differentiated neurons was substantially reduced in 10 μM ketamine-exposed cultures in differentiation medium, compared with the controls. No significant changes in the number of GFAP-positive astrocytes and O4-positive oligodendrocytes (in differentiation medium) were detected from ketamine-exposed cultures. The discussion focuses on: (1) the doses and time-course over which ketamine is associated with damage of neural cells; (2) how ketamine directs or signals neural stem cells/neural cells to undergo apoptosis or necrosis; (3) how functional neuronal transmitter receptors affect neurotoxicity induced by ketamine; and (4) advantages of using neural stem cell models to study critical issues related to ketamine anesthesia.
KeywordsKetamine N-methyl-D-aspartate (NMDA) receptors Development Differentiation Neurons Neurodegeneration
Central nervous system
Dulbecco’s modified Eagle’s medium
Enzyme-linked immunosorbant assay
Terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling
No external funding and no competing interests declared.
This document has been reviewed in accordance with United States Food and Drug Administration (FDA) policy and approved for publication. Approval does not signify that the contents necessarily reflect the position or opinions of the FDA. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the FDA.
- 11.Scallet AC, Schmued LC, Slikker W Jr, Grunberg N, Faustino PJ, Davis H, Lester D, Pine PS et al (2004) Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons. Toxicol Sci : Off J Soc Toxicol 81(2):364–370. doi: 10.1093/toxsci/kfh224 CrossRefGoogle Scholar
- 15.Zou X, Patterson TA, Divine RL, Sadovova N, Zhang X, Hanig JP, Paule MG, Slikker W Jr et al (2009) Prolonged exposure to ketamine increases neurodegeneration in the developing monkey brain. Int J Dev Neurosci : Off J Int Soc Dev Neurosci 27(7):727–731. doi: 10.1016/j.ijdevneu.2009.06.010 CrossRefGoogle Scholar
- 18.Amy L, Inselman CW, Fang L, and Hansen DK (2014) Handbook of nanotoxicology, nanomedicine and stem cell use in toxicology. Stem cells in toxicity testing in part three: stem cell toxicology. Wiley. doi: 10.1002/9781118856017
- 22.Johnson KM, Phillips M, Wang C, Kevetter GA (1998) Chronic phencyclidine induces behavioral sensitization and apoptotic cell death in the olfactory and piriform cortex. J Neurosci Res 52(6):709–722. doi: 10.1002/(SICI)1097-4547(19980615)52:6<709::AID-JNR10>3.0.CO;2-U CrossRefPubMedGoogle Scholar
- 23.Barreto-Chang OL, Dolmetsch RE (2009) Calcium imaging of cortical neurons using Fura-2 AM. J Vis Exp (23). doi: 10.3791/1067
- 24.Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF (2003) Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci : Off J Soc Neurosci 23(3):876–882Google Scholar
- 25.Paule MG, Li M, Allen RR, Liu F, Zou X, Hotchkiss C, Hanig JP, Patterson TA et al (2011) Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys. Neurotoxicol Teratol 33(2):220–230. doi: 10.1016/j.ntt.2011.01.001 PubMedCentralCrossRefPubMedGoogle Scholar
- 28.Wang C, Sadovova N, Hotchkiss C, Fu X, Scallet AC, Patterson TA, Hanig J, Paule MG et al (2006) Blockade of N-methyl-D-aspartate receptors by ketamine produces loss of postnatal day 3 monkey frontal cortical neurons in culture. Toxicol Sci : Off J Soc Toxicol 91(1):192–201. doi: 10.1093/toxsci/kfj144 CrossRefGoogle Scholar