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Ashdin PublishingJournal of Drug and Alcohol ResearchVol. 4 (2015), Article ID 235905, 8 pagesdoi:10.4303/jdar/235905

ASHDINpublishing

Review Article

Anesthetic Drug-Induced Neurotoxicity and Compromised Neural StemCell Proliferation

Cheng Wang, Fang Liu, Tucker A. Patterson, Merle G. Paule, and William Slikker Jr.

Division of Neurotoxicology, National Center for Toxicological Research, United States Food and Drug Administration, Jefferson, AR 72079, USAAddress correspondence to Cheng Wang, [email protected]

Received 25 January 2015; Revised 30 March 2015; Accepted 1 April 2015

Copyright © 2015 Cheng Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Introduction. Because of obvious concerns, it is not possibleto thoroughly explore the adverse effects of anesthetics in children.However, the availability of neural stem cell derived models hasprovided an invaluable tool to examine the etiology of neurotoxicityassociated with developmental exposure to general anesthetics.Stem cells and neurotoxicity. To evaluate the degree of anesthetic-induced toxicity, cultured neural stem cells were examined, usingspecific biomarkers, to determine the effects of anesthetics on NSCproliferation/differentiation, the nature of anesthetic-induced toxicity,and possible mechanisms underlying anesthesia toxicity. Anestheticsand proliferation. Using recently developed methods, such as dynamicmolecular imaging approaches, the relationships between anesthetic-induced NSC damage and alterations in NSC proliferation havebeen addressed. Summary. This review focuses on how anestheticsdirect or signal NSCs to undergo apoptosis or necrosis, how neuronaltransmitter receptors affect neurotoxicity, and how NSC models mayserve as the most expeditious platform for decreasing the uncertaintyin extrapolating preclinical data to the human condition.

Keywords neural stem cells; development; proliferation; anesthetics;neural stem cell damage

Core tip Stem-cell-derived models with their capacity to proliferateand differentiate provide advantages for detecting potential anesthetic-induced neurotoxicity. These systems provide reliable and simple invitro models, that can within a short time frame provide data for eval-uating the potential adverse effects of developmental anesthetic expo-sures and associated cellular mechanisms.

1. Introduction

The ability of cells to maintain a high degree of order in achaotic universe depends upon the accurate duplication ofvast quantities of genetic information carried in chemicalform as DNA. This process, called DNA replication, mustoccur before a cell can produce two genetically identicaldaughter cells. Neural stem cells (NSCs) are stem cells inthe nervous system that can self-renew and give rise to dif-ferentiated progenitor cells to generate lineages of neuronsas well as glia, such as astrocytes, and oligodendrocytes,a characteristic known as multipotency. Therefore, duringdevelopment NSCs are the sources of neurons, astrocytes,and oligodendrocytes in the central nervous system (CNS),

owing to their multipotency/pluripotency and ability toself-renew. NSCs and neural progenitor cells are presentthroughout development and persist in the adult nervoussystem. The proportion of subventricular zone (SVZ) stemcells declines with development, and multipotent stemcells are likely to be present only in regions of ongoingneurogenesis (e.g., anterior SVZ and SVZ underlyinghippocampus) in the adult CNS. While the nervous systemis one of the earliest organ systems that differentiate fromthe blastula-stage embryo, and this differentiation can bemimicked in culture, it is important to determine whetherstem cells from adult tissues have the same capability andpotential as embryonic stem cells.

Various factors contribute to determining the fates ofNSCs and affect their transition from maintenance to pro-liferation to differentiation. Utilization of embryonic NSC-based models should allow for the recapitulation of manyaspects of CNS development in vitro, and provide opportu-nity to identify the roles of specific chemical/cellular factorsduring developmental processes. Such models should thenallow for the evaluation as to whether anesthetics can affectmaintenance, proliferation, and/or differentiation.

In the developing brain, the nuclear and mitochondrialgenomes incur DNA damage, primarily mediated by freeradicals [1,2,3,4]. During embryonic development, accel-erated DNA transactions, which support rapid cell prolifera-tion, may even further jeopardize the integrity of DNA [5,6].

It is known that the most commonly used generalanesthetics have either NMDA-type glutamate receptorblocking or GABA receptor enhancing properties. Agrowing body of data indicates that commonly usedgeneral anesthetics may cause neuronal apoptotic damagein several major brain regions in animal models includingrodents and nonhuman primates, during certain periodsof development, particularly the brain growth spurt [7,8,9,10,11,12]. Regarding an association between surgery

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early in life and later cognitive problems, it is true thatthere are no clinical data in humans showing neurotoxicityafter pediatric anesthesia exposure (because we are nottaking brain biopsies from children undergoing anesthesia).Therefore, it is still controversial about the contributionof anesthetics-mediated neuronal apoptosis (destruction ofexisting neurons) and impaired neurogenesis (inhibition ofgeneration of new neurons) in the developing brains to thesubsequent memory and learning disability.

Recent advances in our understanding of stem cellbiology and neuroscience have opened up new avenuesof research for detecting anesthetic-induced neurotoxicity,dissecting underlying mechanisms, and developing potentialprotective/preventative strategies against anesthetic-inducedneuronal injury. The application of stem cell modelstowards understanding issues relevant to developmentalneurotoxicology has the potential to advance our under-standing of brain-related biological processes, includingneuronal plasticity and toxicity [13,14,15,16,17]. Thisreview discusses how stem cells can be used as tools fordissecting out mechanisms underlying anesthetic-inducedneural stem cell damage.

2. Stem-cell-derived models and anesthetic-induced tox-icity

The nervous system is sensitive to the toxic effects of manychemicals including drugs, environmental agents, and cer-tain naturally occurring substances. Neurotoxicity can resultin temporary or permanent damage to the brain or peripheralnervous system and is also a major cause of neurodegen-erative diseases. The application of neural stem cell mod-els, coupled with advanced research approaches, will pro-vide valuable platforms from which to study factors that willpositively or negatively affect the consequences of pediatricanesthetic exposure.

Many of the anesthetic protocols used in children weredeveloped from those used in adults. However, it is clearthat the dynamic developmental physiology characteristicof early life stages is different from those occurring duringlater developmental stages and children seem to be morevulnerable to the adverse effects of anesthetic exposure [18].Recently, the availability of stem cells, especially neuralstem cells of embryonic origin, with their pluripotency andcapacity for proliferation, has provided a valuable tool forexamining the developmental effects of anesthetic agentsin vitro [19,20]. In fact, neural stem cell models mayrepresent some of the best systems available for evaluatingthe potential adverse health impacts of pediatric anesthetics.This capability stems from several attributes of neuralstem cells including (1) source (neural stem cells can beobtained directly from humans), (2) specificity of cell types(this approach allows for examining the adverse effectsof anesthetics on neural stem cells themselves, as well as

neurons, astrocytes and oligodendrocytes and the processesinvolved with differentiation into those cell types), (3)reduced animal use and shortened time frames for studycompletion, (4) ability to assess regeneration capacity afterexposure to toxicants, (5) potential to significantly impactbest practices for pediatric anesthetics [21], and (6) theability to mimic or model particular developmental stagesin animals and humans. Embryonic neural stem cells, espe-cially those of human origin, provide great opportunities foridentifying potential neurotoxicity associated with exposureto anesthetics such as ketamine. Combining these modelswith calcium imaging and molecular biological approachescreates great possibilities for elucidating mechanismsunderlying the etiology of the neurotoxicity associated withdevelopmental exposures to general anesthetics and mayalso help identify ameliorative strategies.

Previous work based on mRNA levels showed thatNMDA receptor NR1 expression in ketamine-exposed ratpup brains was significantly higher than in controls [12,22,23,24], and subsequent work also showed altered expressionlevels of the NMDA receptor NR2 family, including NR2Aand NR2C [23], after repeated ketamine exposure. Thisevidence of NMDA receptor upregulation suggests thatupon removal of ketamine from the extracellular milieu, thenow upregulated NMDA receptor population (a probablecompensation for prolonged NMDA receptor blockade byketamine) will “over” respond to normal levels of extracel-lular glutamate, resulting in glutamatergic excitotoxicity.Also, utilizing a primary neuronal culture system it wasdemonstrated that ketamine exposure has a significantimpact on subsequent intracellular Ca2+ homoeostasis:the amplitude of calcium influx caused by activatingconcentrations of NMDA was significantly increased inneurons from ketamine-exposed cultures compared withneurons from control cultures [24]. The NMDA-elicitedincreases in intracellular Ca2+ were blocked by chelation ofextracellular Ca2+ with EGTA, which clearly demonstratesthat the NMDA-evoked increases in intracellular calciumoriginated from an extracellular source, rather than froma depletion or releases of calcium from the endoplasmicreticulum [24]. Since ketamine has well-defined effects onthe NMDA receptor at anesthetic concentrations [25], thesedata provide further support for the hypothesis that con-tinuous blockade of NMDA receptors by anesthetics (e.g.,ketamine) causes a compensatory upregulation of NMDAreceptors. This upregulation makes neural cells bearingthese receptors more vulnerable to the excitotoxic effectsof glutamate, because these upregulated NMDA receptorsallow for the accumulation of toxic levels of intracellularfree calcium [11,26]. The elevated levels of intracellular freecalcium that exceed the buffering capacity of mitochondriainterfere with electron transport in a manner that results inthe increased production of reactive oxygen species (ROS).

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In a mechanistic study [24], administration of the anestheticagent ketamine markedly elevated both nuclear andmitochondrial levels of 8-oxoguanine, a marker of oxidativestress. The concordance between elevated 8-oxoguanine lev-els, enhanced DNA fragmentation, and increases in the num-ber of cells with DNA strand breaks, following ketamineexposure, suggests key roles for calcium homeostasis andmitochondrial ROS production in inducing neuronal DNAdamage and disruption of cell division cycles. Commonlyused anesthetics (e.g., propofol, sevoflurane, desflurane,and isoflurane) take their effects by either inhibiting theexcitatory neurotransmission mediated by NMDA receptorsor enhancing the inhibitory neurotransmission via theγ-aminobutyric acid type A receptor (GABAAR) [20,24,27,28]. Therefore, the neurotransmissions conductedby NMDARs and GABAARs are critical to the normaldevelopment of the CNS, and disturbances in glutamatergicor GABAergic signaling would compromise the synaptoge-nesis, neurogenesis, and neural circuit formation [29,30].

It is becoming increasingly apparent that mitochondrialie at the center of the cell death regulation process. Reactiveoxygen species (ROS), such as superoxide, hydrogenperoxide, and hydroxyl radicals, are typically produced inmitochondria as electrons leak from the electron transportchain and react with oxygen to form superoxide. ROSgenerated by mitochondria are not just toxic by-products ofrespiration, they are also important for cell signaling [31,32]. In a recent in vitro mechanistic study [24], an increasein the generation of ROS was associated with the increasedCa2+ influx seen in ketamine-exposed neurons in culture.These ROS appear to originate in mitochondria. Recentevidence suggests that general anesthetics, administeredduring the peak of synaptogenesis, caused protractedinjury to mitochondria including significant enlargement,impairment of their structural integrity, and a decrease intheir regional distribution [33]. Along with morphologicalchanges, the general anesthetics also cause functionalimpairment of immature neuronal mitochondria [34].Injured mitochondria may be a significant source of reactiveoxygen species [24,34]. Meanwhile, the role of the Bax,proapoptotic gene, in anesthetic-induced apoptosis has beenextensively studied [11,24,26]. As previously reported,Bax may open pores on the outer mitochondrial membrane,allowing cytochrome c to exit [35]. Cytochrome c, a com-ponent of the mitochondrial electron transfer chain, initiatescaspase activation when released from mitochondria duringapoptosis [36]. Cytosolic cytochrome c could bind to Apaf-1, a cytosolic protein containing a caspase-recruitmentdomain (CARD). The binding of nucleotide to the Apaf-1/cytochrome c complex triggers its oligomerization toform the apoptosome [37]. The CARDs of Apaf-1 becomeexposed in the apoptosome, which subsequently recruitmultiple procaspase-9 molecules to the complex and

facilitate their autoactivation. These executioner caspasessubsequently cleave many important intracellular substrates,leading to characteristic morphological changes in apoptosissuch as chromatin condensation, nucleosomal DNA frag-mentation, nuclear membrane breakdown, externalization ofphosphatidylserine, and formation of apoptotic bodies [38].

Taken together, the accumulated data [7,8,11,26,39]indicate that anesthetic-induced neural damage depends onthe amount (dose) given, the duration of exposure, the routeof administration, the receptor subtype activated, and thestage of the development at the time of exposure. Undoubt-edly, it is the most important that the dosage, duration, androute of administration are well controlled/monitored inclinical practice, pediatric anesthesia, and specific case-studies.

3. Anesthetic-induced damage and neural stem cell pro-liferation

NSCs in the nervous system can self-renew and give rise todifferentiated progenitor cells to generate lineages of neu-rons as well as glia, such as astrocytes and oligodendrocytes.This characteristic is known as multi- or pluripotency. NSCsare present throughout development and persist into adult-hood. Multiple classes of NSCs have been identified thatdiffer from each other in their ability to differentiation, theirresponses to cytokines, and their surface antigen characteris-tics. NSCs are valuable resources because of their potentialto proliferate and differentiate, thus, providing opportunitiesfor applications in neuroscience, in general, and for clinicalapplications specifically such as in the treatment of neurode-generative diseases and neurological disorders.

Self-renewal or self-replication is defined as the abilityof the stem cell to go through multiple cycles of cell divisionwhile maintaining an undifferentiated state (i.e., to generatedaughter cells that are identical to the mother cell). The self-replication of NSCs is characterized by two different typesof cell division: symmetrical and asymmetrical. Symmet-rical cell division refers to the generation of two daugh-ter cells that have the same fate; while asymmetrical celldivision refers to the generation of two daughter cells, onlyone of which remains the same as the mother cell, whilethe other does not. Typically, symmetrical cell renewal pre-cedes asymmetrical cell renewal, generating enough NSCsto develop the bulk of the CNS. Asymmetrical cell divisionsthen give rise to more differentiated neural cells, such asprogenitor cells, neurons or glia [40].

Enhancement of neurogenesis by pharmaceutical agentswould be predicted to improve functional outcome afterneuronal damage. Accordingly, there is great interest inusing NSCs as screening tools in the early stages of drugdevelopment for many neurological disorders. Inhibition ofneurogenesis, on the other hand, could be a key pathway foranesthetic-induced neurotoxicity during development [20].


Previously, a variety of cultured cell lines and animalmodels have been utilized to investigate the neurotoxiceffects of anesthetics in studies which have primarilyfocused on the postnatal period during the brain growthspurt [24,26,41,42]. Since the developmental stage at thetime of a chemical/toxicant exposure can critically influencethe outcome of neurodevelopment, an embryonic neuralstem cell model was utilized in a previous project [20]to study the toxic effects of anesthetics during earlydevelopmental stages. This provided an opportunity toallow for the assessment of the effect of the anestheticpropofol on neural stem cell proliferation and differentiationand to monitor associated biochemical outcomes includingoxidative DNA damage and apoptotic cascades [20].

Given the importance of NMDA-type glutamateand GABA receptors to the expression of anesthesia-induced neurotoxicity in vivo, it is vital to provide directevidence that cultured embryonic neural stem cells expressfunctional neurotransmitter receptors of these types. Ina recent mechanistic study [20], it was demonstratedthat no GABA receptor immunoreactive staining wasdetected on undifferentiated cultured rat NSCs. Andour recent preliminary data showed that there was noreceptor-associated intracellular calcium influx detected.However, evidence for the presence of functional NMDAreceptors [11,20,24] and strong immunoreactive stainingfor GABAA receptors [20] was detected on neuronsdifferentiated from the same neural stem cells. These dataclearly indicate that during early development embryonicneural stem cells do not express functional neurotransmitterreceptors for GABA or NMDA and, thus, any anesthetic-induced neural stem cell damage that occurs in such cellsmust involve different mechanisms. These might include theeffects of anesthetics to increase ROS production resultingin oxidative DNA damage, interruption of neural stem cellproliferation, and associated neural stem cell damage.

To investigate whether enhanced cell damage could beassociated with interruptions in cell division cycles, neu-ral stem cell proliferation rates after anesthetic exposureswere determined using an EdU (5-ethynyl-2′-deoxyuridine)assay [20]. EdU, like 5-bromo-2-deoxyuridine (BrdU), is athymidine analog that is incorporated into cells only dur-ing the S-phase of cell division and is used as a markerof cellular proliferation. After a 24-hour exposure of neuralstem cells to the anesthetic propofol (50μM), the numberof dividing cells was substantially decreased compared withcontrols suggesting a decrease in the neural stem cell pro-liferation rate. Coadministration of acetyl-L-carnitine [20],an antioxidant agent, effectively attenuated the decrease inneural cell proliferation produced by propofol (Figure 1).

In living cells, DNA is inherently unstable and itsintegrity relies on continuous DNA monitoring and repairingactivities [43]. During embryonic development, accelerated

DNA transactions, which support rapid cell proliferation,may even further jeopardize the integrity of DNA [5,6,44]. It has been shown that caspase-3 specifically activatesthe endonuclease caspase-activated deoxyribonuclease(CAD). CAD then degrades chromosomal DNA withinthe nuclei and causes chromatin condensation. It seemsthat anesthetics can affect neural stem cell proliferationby slowing down or even stopping the cell division cycle,eventually resulting in cell death.

The ameliorating effect of acetyl-L-carnitine on theeffects of propofol to inhibit cellular proliferation [20]strongly suggests that propofol can initially cause oxidativemitochondrial damage and dysfunction, subsequentlyaffecting neural stem cell proliferation by slowing downor even stopping cell division and eventually damaging thecells [45,46,47].

A growing body of data indicates that molecular imagingwith appropriate isotope-labeled biomarkers (radio-tracers)can help to detect and monitor aspects neurotoxicity in ani-mal models [48,49]. The high-resolution positron emissiontomography scanner (microPET) can provide in vivo molec-ular imaging at a sufficient resolution to resolve both majorstructures and neuronal activities in the brain. Therefore,imaging technology has great potential for advancing theunderstanding of brain-related biological processes, includ-ing neuronal plasticity, neuronal degeneration/regeneration,and neurotoxicity [48,49].

In ongoing studies designed to monitor endogenousneural stem cell proliferation in vivo, 3′-deoxy-3′-[18F]-fluoro-L-thymidine ([18F]-FLT), one of the most widelyused radiotracers for imaging cell proliferation [50],was utilized after sevoflurane exposure. Sevoflurane, avolatile anesthetic commonly used to anesthetize and sedatepediatric patients, is thought to act as an agonist at GABAA

receptors at anesthesia-relevant concentrations [51]. FLT, athymidine analog, is taken up by cells and phosphorylatedby thymidine kinase (TK) 1, leading to its intracellulartrapping. The phosphorylated FLT trapped within cells doesnot get incorporated into the cellular DNA because it lacksa 3′-hydroxyl. TK 1 is a cytosolic isozyme of TK and itsactivity closely parallels that of cellular proliferation [52].Therefore, the retention of [18F]-FLT within the cells servesas an in vivo marker that can be used for the visualization ofcell proliferation. In comparison with air-exposed animals,preliminary data indicate that sevoflurane exposure ata clinically-relevant concentration inhibited the uptakeof [18F]-FLT in the hippocampus of the developing brain.Here, the inhibitory effect of sevoflurane exposure manifestsin an age-dependent manner. These data suggest that byutilization of [18F]-FLT in conjunction with PET imaging,the relationships between anesthetic-induced neurotoxicityand endogenous neural stem cell activity following exposureto general anesthetics can be monitored in vivo. In addition,


Figure 1: Representative photographs of an EdU incorporation assay. Neural stem cells were exposed to propofol for24 h. The number of dividing cells was markedly decreased (b), (d), and (f) compared to controls (a), (c), and (e).Quantitative analysis of the EdU staining shows that the proliferation rate was significantly reduced after propofol exposure.Coadministration of acetyl-L-carnitine effectively blocked the enhanced cell death induced by propofol (g). Each conditionwas assessed at least in triplicate and experiments were repeated three times, independently. Data are presented as the means±SDs. A probability of ∗P < .05 was considered significant (one-way ANOVA). ∗∗P < .01. (Cited from [20].)

critical data can be obtained noninvasively, repeatedly, andquantitatively in the same subjects [53,54,55].

There are yet many questions to answer before thefindings of pediatric drug-induced neurotoxicity observedin animals can be related to effects in humans. Currently,[18F]-FLT is being used in conjunction with PET imagingin rodents to monitor effects on cellular proliferation ofexposure to the anesthetic sevoflurane. If such experimentsare successful in developing nonhuman primates, thensimilar studies should be able to help define the pathway totoxicity and allow cross-species comparisons.

4. Summary

Neural stem or progenitor cells are generally uncommittedand so can change their fate after exposure to salientenvironmental cues. Evidence shows that gene expressionand the capacity for self-renewal and differentiation ofNSCs are spatially and temporally specified. Stem-cell-derived models with their capacity to proliferate anddifferentiate provide advantages for detecting potential

anesthetic-induced neurotoxicity and underlying mech-anisms (Figure 2). These systems provide reliable andsimple in vitro models, that can within a short time frameprovide data for evaluating the potential adverse effects ofdevelopmental anesthetic exposures and associated cellularmechanisms [56]. The use of neural stem cell models,especially those of human origin, holds promise for helpingto elucidate relevant mechanisms underlying the etiology ofthe neurotoxicity associated with developmental exposuresto the general anesthetics. Stem-cell-derived models, whencombined with advanced research approaches such ascalcium and molecular imaging, may also help identifyavenues of protection or prevention.

Although stem cells in vitro (e.g., hESC-relatedneurogenesis models) can mimic the in vivo neuronaldevelopment process and provide a simple and unlimitedcell source for addressing anesthesia-related issues [57], it isdifficult to make an exact comparison between developingbrain (3D components) vis-a-vis neural stem cells foranesthetic-induced neurotoxicity. The 2D neural stem


Figure 2: Hypothesized pathways of anesthetics-induced neural stem cell and/or differentiated neuronal cell damage.

cell cultures, including adult NSC or even iPS-derived NSCmodels, are lack of 3D structure that more closely resemblesactual in vivo microenvironments that include extracellularmatrices, adhesion junctions, and so on. Also, it is difficultto determine the percent occurrence of neural stem cells inpediatric versus adult brains.

However, organotypic (3D) culture system providesan invaluable platform for extrapolating preclinical data tohuman conditions. The main advantage of using organotypicculture model is that this preparation maintains importantanatomical relationship and synaptic connectivity, whileat the same time preserving the advantages of an in vitropreparation. Comparable and functional high-throughput3D model platforms and microfluidic cell culture chips havequickly captured the attention of neuroscientists. Studiesare continuously revealing new information and it is hopedthat therapeutic applications will be developed from bothembryonic and adult sources.

Disclaimer This document has been reviewed in accordance withUnited States Food and Drug Administration (FDA) policy andapproved for publication. Approval does not signify that the contentsnecessarily reflect the position or opinions of the FDA. The findingsand conclusions in this report are those of the author and do notnecessarily represent the views of the FDA.

Conflict of interest The authors declare that they have no conflict ofinterest.

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