Advances in our understanding of stem cell biology and neuroscience have opened up new avenues of research for detecting early life stress-induced neurotoxicity and developing potential protection/prevention strategies against toxicant-induced neuronal injuries (Kiss et al., 1994
; Wang et al., 1994
; Brokhman et al., 2008
; Trujillo et al., 2009
). The classic definition of a stem cell requires that it has the capacity to self-renew and that it possesses potency. Self-renewal is defined as the ability of the stem cell to go through multiple cycles of cell division while maintaining its undifferentiated state (i.e., to generate daughter cells that are identical to it). Potency refers to the ability of the stem cell to differentiate into specialized cell types (Wilmut et al., 1997
; Shamblott et al., 1998
; Thomson et al., 1998
). Stem cell biology, when exploited along with molecular signaling and biological approaches including calcium imaging, genomics, proteomics, and metabolomics, can be utilized to enhance our understanding of complex biological processes such as apoptosis, and can provide the fundamental concepts necessary for constructing models of the building blocks of biological systems during development. As these models evolve and become linked together as integrative modules, they provide the intermediate components necessary for use in a developmental toxicology approach/platform.
A neural stem cell is a subclass of precursor cells that has several specific characteristics: (1) self-renewing – capable of making additional copies of itself by division; (2) multipotent – capable of making daughter cells other than itself including committed progenitors, neurons, astrocytes, and oligodendrocytes; and (3) capable of generating all or part of neural tissue. Neural stem cells or neural progenitor cells are generally uncommitted and so can change their fate after exposure to salient environmental cues. Evidence shows that gene expression and the capacity for self-renewal and differentiation of neural stem cells are spatially and temporally specified. Thus, neural stem cells are defined as cells with the ability to proliferate, self-renew, and produce a large number of functional progeny that can differentiate into neurons, astrocytes, or oligodendrocytes and this characteristic, thought to be controlled by genomic, biochemical, and physical factors, is known as multipotency (Wang et al., 1996
; Park et al., 2005
; Brokhman et al., 2008
The stem cell-derived models, especially human embryonic neural stem cells with their capacity for proliferation and potential for differentiation, have a great advantage for detecting potential anesthetic-induced neurotoxicity. This system provides a reliable, simple in vitro model, within a short time frame, for evaluating potential adverse effects and investigating the cellular mechanisms which may be associated with anesthetic-induced brain damage. Thus, stem cell-derived models should be one of the best systems in evaluating adverse effects of pediatric anesthetic exposure, because of: (1) source (some embryonic neural stem cells are directly from human fetuses); (2) specific cell types (the simplified in vitro system allows for examining adverse effects of anesthetics directly on neural stem cells, neurons, astrocytes, or oligodendrocytes; (3) using minimal numbers of animals in a short time frame; and (4) providing the opportunity for assessing the brain’s own regenerative capacity after experiencing events related to overdoses or prolonged exposures to drugs including some general pediatric anesthetics or environmental chemicals.
This review presents an overview of representative general anesthetics – primarily ketamine – as examples of how stem cells can be valuable in identifying the doses and time-course over which individual drugs produce damage and/or protect neural stem cells and cells derived from them, change their proliferation rate, and alter their fate (differentiation into neurons, oligodendrocytes, and astrocytes) in vitro. Here, a strategy of the use of embryonic neural stem cell cultures for monitoring potential adverse effects of ketamine on the neural stem cell expansion, proliferation, differentiation, and receptor function has been defined.
Ketamine, a non-competitive NMDA receptor antagonist, is a widely used general pediatric anesthetic agent. Lines of evidence have demonstrated that ketamine causes neuronal cell death in several major brain areas of experimental animals at an early developmental stage, e.g., during the brain growth spurt (Ikonomidou et al., 1999
; Scallet et al., 2004
; Slikker et al., 2007
; Zou et al., 2009a
). Apoptosis is a common cause of ketamine-induced neuronal cell death in rodents (Zou et al., 2009b
). Previous works based on mRNA and protein levels have revealed that NMDA receptor NR1 expression in ketamine-treated rat pup brain was significantly higher than that in control rats (Slikker et al., 2007
; Zou et al., 2009b
; Shi et al., 2010
). This is indicative of a compensatory up-regulation of NMDA receptors on the neurons, along with continued or prolonged NMDA receptor blockade by NMDA antagonists (e.g., ketamine). Previous evidence suggested that upon removal of ketamine from the extracellular milieu, the now up-regulated NMDA receptor population (compensatory regulation as a consequence of continued or prolonged NMDA receptor blockade) will “over” respond to normal levels of extracellular glutamate, resulting in glutamatergic excitotoxicity. However, several important questions remain unanswered.
NMDA receptors constitute a sub-family of glutamate receptors identified by specific molecular composition and pharmacological and functional properties. NMDA receptors are densely localized on neurons of most major brain areas and are physically connected to proteins involved in cell-signaling cascades (Arundine and Tymianski, 2003
; Arundine et al., 2003
). Glutamate is the primary excitatory neurotransmitter in the CNS of mammals. In addition to ionotropic receptors responsible for fast excitatory neurotransmission in the CNS, glutamate also activates a number of metabotropic glutamate (mGlu) receptors, which belong to the G-protein coupled receptor family of receptors. Glutamate stimulates the opening of the channels that the ionotropic receptors regulate to enable the influx of various ions and excessive activation of NMDA-type glutamate receptors is implicated in the pathophysiology of several neurological conditions including hypoxia-ischemia and seizure-mediated excitotoxic damage, neuropathic pain, and opiate dependence. Exploring the mechanisms by which anesthetic agents might disturb NMDA receptor expression patterns should help identify avenues for protection or prevention of potential anesthetic-induced neuronal damage.
Since NMDA receptors are highly calcium permeable, the interactions between altered ionotropic receptors (e.g., compensatory up-regulation of NMDA receptor) and intracellular calcium signaling [Ca2+]i, as well as how enhanced Ca2+ flux associated with ketamine exposure influences reactive oxygen species (ROS) generation and subsequent neuronal apoptosis, could appropriately be clarified, by monitoring changes in intracellular calcium concentration, e.g., Fura-2 AM live cell calcium imaging. Thus, the relationship between anesthetic (ketamine)-induced NMDA receptor dysregulation and signal transduction, could systematically be analyzed. Also, whether enhanced Ca2+ flux associated with up-regulated NMDA receptors (as a consequence of ketamine exposure) could increase ROS generation and subsequent neuronal apoptosis could be demonstrated.
The cartoon (above) indicates a potential specific involvement of NMDA receptor-mediated excitation in ketamine-induced neurotoxicity. Continuous blockade of NMDA receptors by NMDA antagonists, such as ketamine, causes a compensatory up-regulation of the NMDA receptor. This regulation could make cells bearing the receptors more vulnerable, after ketamine washout, to glutamate, because this up-regulation allows for a toxic accumulation of intracellular calcium. Therefore, prolonged exposure of neural stem cells to ketamine results in intracellular Ca2+ overload that exceeds the buffering capacity of the mitochondria and interferes with electron transport in a manner that results in an elevated production of ROS. Studies in vitro and in intact cells have shown that caspase-3 specifically activates the endonuclease, CAD (Caspase-Activated Deoxyribonuclease). CAD then degrades chromosomal DNA within the nuclei and causes chromatin condensation. Also, ketamine may affect neural stem cell proliferation by slowing down, or even stopping the cell cycle, finally resulting in cell death.
Taken together, the use of neural stem cell models, especially those of human origin, when combined with calcium imaging and molecular biology approaches, holds promise for helping to elucidate relevant mechanisms underlying the etiology of the neurotoxicity associated with developmental exposures to the general anesthetics, and may also help identify avenues of protection or prevention. Data/observations related to NMDA receptor expression and function could provide further support to the idea, that in addition to NMDA-type glutamate receptor expression levels, the specific signal transduction (e.g., Ca2+ influx) plays a critical role in anesthetic (ketamine)-induced neurotoxicity.