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Anesthetic Neurotoxicity: New Findings and Future Directions

2016; Elsevier BV; Volume: 181; Linguagem: Inglês

10.1016/j.jpeds.2016.10.049

1097-6833

Michael C. Montana, Alex S. Evers,

Cardiac, Anesthesia and Surgical Outcomes

The development and refinement of practices for the safe administration of anesthesia to children is a major success story in modern medicine. During the past several decades, there have been significant improvements in safety standards, cardiopulmonary monitoring, delivery systems, and airway management specific to the pediatric patient undergoing anesthesia. Millions of children receive anesthesia each year for surgical, procedural, or diagnostic purposes, and the majority of these patients receive a general anesthetic.1Wanderer J.P. Rathmell J.P. Complex information for anesthesiologists presented quickly and clearly.Anesthesiology. 2014; 120: A23Crossref PubMed Scopus (1) Google Scholar Parents and care providers can be confident that the vast preponderance of these children will have a safe outcome with a low likelihood of major morbidity or mortality.2van der Griend B.F. Lister N.A. McKenzie I.M. Martin N. Ragg P.G. Sheppard S.J. et al.Postoperative mortality in children after 101,885 anesthetics at a tertiary pediatric hospital.Anesth Analg. 2011; 112: 1440-1447Crossref PubMed Scopus (167) Google Scholar The last several decades also have seen the discovery, and subsequent verification, that agents commonly used to induce and maintain general anesthesia in humans exhibit evidence of neurotoxicity in animal models.3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar This realization dates to the early 1980s, when exposure of pregnant rat dams to chronic, low-level halothane was found to result in abnormal synaptogenesis and behavior in their offspring.4Uemura E. Levin E.D. Bowman R.E. Effects of halothane on synaptogenesis and learning behavior in rats.Exp Neurol. 1985; 89: 520-529Crossref PubMed Scopus (71) Google Scholar Further concerns arose from the discovery in 1998 that the N-methyl-D-aspartate (NMDA) receptor antagonist nitrous oxide (also known as laughing gas) can be neurotoxic in rodents.5Jevtovic-Todorovic V. Todorovic S.M. Mennerick S. Powell S. Dikranian K. Benshoff N. et al.Nitrous oxide (laughing gas) is an NMDA antagonist, neuroprotectant and neurotoxin.Nat Med. 1998; 4: 460-463Crossref PubMed Scopus (518) Google Scholar It was shown subsequently that when administered to neonatal rats during a period of critical synaptogenesis, a commonly used cocktail of anesthetics (including nitrous oxide, midazolam, and isoflurane) induces immediate widespread neuronal apoptosis and impairments in learning and memory that persist into adulthood.6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar Preclinical models from Caenorhabditis elegans to nonhuman primates now suggest that multiple anesthetic agents may be neurotoxic. These include positive allosteric modulators of the GABAA receptor (benzodiazepines, propofol, and the anesthetics isoflurane, sevoflurane, and desflurane), and the NMDA receptor antagonists ketamine and nitrous oxide.3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar The discovery of anesthetic neurotoxicity in animal models raises the disconcerting possibility that administration of what appears to be a safe general anesthetic may have long-lasting deleterious neurocognitive effects. These discoveries and concerns, however, represent an inversion of the traditional use of preclinical models to study human diseases. In most circumstances, a human malady is recognized clinically and is sufficiently prevalent or severe that researchers develop animal models to study disease process and to refine diagnostic and therapeutic approaches. Anesthetic neurotoxicity was first discovered in animal models, with the possibility of detriment to human patients arising from that discovery. This atypical knowledge acquisition makes it unclear what neurocognitive or behavioral components comprise the clinical syndrome of anesthetic-induced developmental neurotoxicity. This uncertainty presents parents, clinicians, and researchers with a conundrum: given the millions of children that undergo general anesthesia for surgical, procedural, and diagnostic purposes each year, anesthetic neurotoxicity, although unproven in human patients, may represent a significant public health problem. Two recently published human studies that suggest a lack of harm in otherwise-healthy children following a short duration anesthetic (approximately 1 hour) deserve early mention. The first of these trials is the General Anaesthesia compared to Spinal anaesthesia (GAS) Trial, which randomized infants undergoing inguinal hernia repair to either an awake-regional technique or a general anesthetic.7Davidson A.J. Disma N. de Graaff J.C. Withington D.E. Dorris L. Bell G. et al.Neurodevelopmental outcome at 2 years of age after general anaesthesia and awake-regional anaesthesia in infancy (GAS): an international multicentre, randomised controlled trial.Lancet. 2016; 387: 239-250Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar Secondary outcomes assessed at 2 years of age showed no increased risk of adverse neurodevelopment in children exposed to a general anesthetic. The Pediatric Anesthesia & Neurodevelopment Assessment (PANDA) study compared children who had undergone inguinal hernia repair with general anesthesia before 3 years of age with an unexposed sibling.8Sun L.S. Li G. Miller T.L. Salorio C. Byrne M.W. Bellinger D.C. et al.Association between a single general anesthesia exposure before age 36 months and neurocognitive outcomes in later childhood.JAMA. 2016; 315: 2312-2320Crossref PubMed Scopus (537) Google Scholar No difference in IQ was found between exposed and unexposed siblings. Further details regarding these studies are discussed herein. The results from these trials are encouraging and suggest that a short-duration anesthetic in otherwise-healthy children may have limited effects. Nevertheless, the concerns regarding anesthetic neurotoxicity are myriad and nuanced. This commentary is intended as a review for pediatricians, anesthesiologists, and surgeons of the animal studies that first raised these concerns, the historical context of these studies, and the human studies that are either completed or ongoing. Exposure of rodents to anesthetic agents between postnatal day 7 and 14 is associated with decreased neuronal density, decreased neuronal numbers, and increased neuronal apoptosis.3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar, 9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar Multiple anesthetics have been implicated, including ketamine, propofol, and halogenated anesthetic gases. A cocktail of nitrous oxide, midazolam, and sevoflurane sufficient to maintain a surgical plane of general anesthesia for 6 hours results inapoptosis in multiple brain regions,6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar and even subanesthetic doses of propofol can be apoptogenic.10Cattano D. Young C. Straiko M.M. Olney J.W. Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain.Anesth Analg. 2008; 106: 1712-1714Crossref PubMed Scopus (218) Google Scholar Nonhuman primates also are vulnerable to neuronal damage. When administered to postnatal day 5-6 macaques, a combination of inhaled nitrous oxide and isoflurane sufficient to maintain surgical anesthesia for 8 hours produces apoptotic and possibly necrotic neuronal damage in multiple brain regions.11Zou X. Liu F. Zhang X. Patterson T.A. Callicott R. Liu S. et al.Inhalation anesthetic- induced neuronal damage in the developing rhesus monkey.Neurotoxicol Teratol. 2011; 33: 592-597Crossref PubMed Scopus (151) Google Scholar Evidence for anesthetic neurotoxicity has been found throughout the nervous system, including the hippocampus, striatum, thalamus, amygdala, cerebellum, cerebral cortex, and spinal cord.9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar The damage is not limited to neurons; apoptosis also has been observed in oligodendrocytes and other glial cells. In addition to neuronal damage, early-life anesthetic exposure in rodents has been associated with long-term behavioral deficits in spatial memory3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar; however, not all studies that have assessed behavior have found a difference between exposed and control animals.9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar Most studies that include behavioral outcomes have been performed in rodents; however, macaques exposed to a single 24-hour ketamine anesthetic also have long-lasting impairments in learning and motivation as compared with unexposed controls.12Paule M.G. Li M. Allen R.R. Liu F. Zou X. Hotchkiss C. et al.Ketamine anesthesia during the first week of life can cause long-lasting cognitive deficits in rhesus monkeys.Neurotoxicol Teratol. 2011; 33: 220-230Crossref PubMed Scopus (438) Google Scholar The aforementioned studies represent a mere fraction of the nearly 1000 articles related to anesthetic neurotoxicity that have been published (for an excellent review, see Disma et al9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar). The general consensus from these studies is that anesthetic agents may be neurotoxic; however, there is significant heterogeneity in the anesthetic(s) used, the duration of exposure, the age of the animals studied, the histologic methods by which neuronal damage was assessed, and the behavioral tasks performed. In mammals, NMDA- and GABA-mediated neuronal activity are important for synaptogenesis during a critical period of brain maturation.13Loepke A.W. McGowan Jr, F.X. Soriano S.G. CON: the toxic effects of anesthetics in the developing brain: the clinical perspective.Anesth Analg. 2008; 106: 1664-1669Crossref PubMed Scopus (87) Google Scholar The sequence of events involved in brain maturation including neurogenesis, synaptogenesis, myelination, and increases in brain weight, along with the development of behavioral milestones, proceed in an orderly and similar fashion in all mammalian species studied. These species include mice, rats, guinea pigs, cats, sheep, nonhuman primates, and humans.14Workman A.D. Charvet C.J. Clancy B. Darlington R.B. Finlay B.L. Modeling transformations of neurodevelopmental sequences across mammalian species.J Neurosci. 2013; 33: 7368-7383Crossref PubMed Scopus (519) Google Scholar The postconceptual and postnatal age at which synaptogenesis begins and its duration, however, vary significantly between species. Even within the same species, different brain regions are maximally vulnerable to anesthetic neurotoxicity at different developmental time points.3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar This interspecies variation and differential vulnerability underscores an important challenge in conducting and interpreting experiments involving animal models of neurodevelopment, that of determining what postconceptual age in a human patient corresponds to a given postconceptual age in another species, and what brain region(s) may be most vulnerable at that time point. For example, peak vulnerability to anesthetic neurotoxicity in rodents occurs at postnatal day 7 and remains present (albeit in different brain regions) up to postnatal day 21.3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar Synapse formation and elimination is thought to occur approximately between postconceptional days 10 and 60 in rats and days 25 and 1000 in humans.14Workman A.D. Charvet C.J. Clancy B. Darlington R.B. Finlay B.L. Modeling transformations of neurodevelopmental sequences across mammalian species.J Neurosci. 2013; 33: 7368-7383Crossref PubMed Scopus (519) Google Scholar There are myriad potential confounds in correlating interspecies brain maturation to exact postconceptual dates, however extending this argument to its logical conclusion suggests that the period of maximal vulnerability in humans actually may be in the second trimester and then last until nearly 3 years of age. Importantly, our goal here is not to use this interspecies comparison to define vulnerable dates in humans but rather to illustrate the complexity in applying animal data to human patients. The likelihood of apoptosis is influenced by the duration of exposure and the use of multiple vs single anesthetic agents. For example, studies have found no evidence of apoptosis when either nitrous oxide or isoflurane was administered as the sole anesthetic, only when administered in combination.6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar, 11Zou X. Liu F. Zhang X. Patterson T.A. Callicott R. Liu S. et al.Inhalation anesthetic- induced neuronal damage in the developing rhesus monkey.Neurotoxicol Teratol. 2011; 33: 592-597Crossref PubMed Scopus (151) Google Scholar Additional studies that use isoflurane as the sole anesthetic agent, however, have shown evidence of apoptosis in neurons and oligodendrocytes.6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar, 15Creeley C.E. Dikranian K.T. Dissen G.A. Back S.A. Olney J.W. Brambrink A.M. Isoflurane- induced apoptosis of neurons and oligodendrocytes in the fetal rhesus macaque brain.Anesthesiology. 2014; 120: 626-638Crossref PubMed Scopus (159) Google Scholar, 16Brambrink A.M. Back S.A. Riddle A. Gong X. Moravec M.D. Dissen G.A. et al.Isoflurane-induced apoptosis of oligodendrocytes in the neonatal primate brain.Ann Neurol. 2012; 72: 525-535Crossref PubMed Scopus (213) Google Scholar, 17Brambrink A.M. Evers A.S. Avidan M.S. Farber N.B. Smith D.J. Zhang X. et al.Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain.Anesthesiology. 2010; 112: 834-841Crossref PubMed Scopus (449) Google Scholar The conflict regarding the apoptogenic nature of single-agent anesthetics is not unique to isoflurane. Single doses of ketamine also have been suggested either to cause or not to cause apoptosis.18Rudin M. Ben-Abraham R. Gazit V. Tendler Y. Tashlykov V. Katz Y. Single-dose ketamine administration induces apoptosis in neonatal mouse brain.J Basic Clin Physiol Pharmacol. 2005; 16: 231-243Crossref PubMed Scopus (69) Google Scholar, 19Scallet A.C. Schmued L.C. Slikker Jr, W. Grunberg N. Faustino P.J. Davis H. et al.Developmental neurotoxicity of ketamine: morphometric confirmation, exposure parameters, and multiple fluorescent labeling of apoptotic neurons.Toxicol Sci. 2004; 81: 364-370Crossref PubMed Scopus (263) Google Scholar Studies that show no evidence of neuronal injury are not necessarily reassuring, because the majority of studies performed thus far indicate that the likelihood of neuronal damage increases when repeated doses of a single agent are given, when multiple agents are administered together, and especially when anesthesia is maintained for durations longer than 2-3 hours. Another challenge in extrapolating data from animal studies to humans lies in the use of immunohistochemical markers of neuronal apoptosis to signify neurotoxicity. Apoptosis during brain maturation is a physiologic process that is necessary for the removal of excess neurons produced during normal development.20Dekkers M.P. Nikoletopoulou V. Barde Y.A. Cell biology in neuroscience: death of developing neurons: new insights and implications for connectivity.J Cell Biol. 2013; 203: 385-393Crossref PubMed Scopus (124) Google Scholar It can be argued that anesthetic exposure results in an exaggerated, pathologic increase in neuronal apoptosis of otherwise-healthy cells,3Jevtovic-Todorovic V. Absalom A.R. Blomgren K. Brambrink A. Crosby G. Culley D.J. et al.Anaesthetic neurotoxicity and neuroplasticity: an expert group report and statement based on the BJA Salzburg Seminar.Br J Anaesth. 2013; 111: 143-151Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar, 21Jevtovic-Todorovic V. Olney J.W. PRO: anesthesia-induced developmental neuroapoptosis: status of the evidence.Anesth Analg. 2008; 106: 1659-1663Crossref PubMed Scopus (79) Google Scholar although the possibility exists that the increased apoptosis seen following anesthetic exposure in animals merely represents an acceleration of the death of neurons that would eventually have undergone physiologic pruning.13Loepke A.W. McGowan Jr, F.X. Soriano S.G. CON: the toxic effects of anesthetics in the developing brain: the clinical perspective.Anesth Analg. 2008; 106: 1664-1669Crossref PubMed Scopus (87) Google Scholar Regardless of whether it occurs under physiologic or pathologic conditions, the final steps of the apoptotic cascade include the activation of proteolytic effector caspases.20Dekkers M.P. Nikoletopoulou V. Barde Y.A. Cell biology in neuroscience: death of developing neurons: new insights and implications for connectivity.J Cell Biol. 2013; 203: 385-393Crossref PubMed Scopus (124) Google Scholar Many animal studies of anesthetic neurotoxicity use staining for activated caspase-3 to mark neurons that have passed the point of no return and are committed to cell death; however, caspase-3 staining is only able to reveal neurons in the early stages of apoptosis (approximately 6-12 hours after cascade initiation), as eventual phagocytosis of the dead neurons leaves no substrate for immunoreactivity. Furthermore, neuronal apoptosis may actually be one of the least-sensitive ways to assess altered synaptogenesis. Short durations of anesthetic exposure that do not induce neuronal death can still affect the density of dendritic spines,22Briner A. De Roo M. Dayer A. Muller D. Habre W. Vutskits L. Volatile anesthetics rapidly increase dendritic spine density in the rat medial prefrontal cortex during synaptogenesis.Anesthesiology. 2010; 112: 546-556Crossref PubMed Scopus (193) Google Scholar which may have a persistently measurable phenotype in the absence of neuronal cell death. In future studies, magnetic resonance imaging and positron emission tomography may provide data on changes in brain structure, gray matter and axonal density, and surrogate markers for brain inflammation in both humans and animals23Backeljauw B. Holland S.K. Altaye M. Loepke A.W. Cognition and brain structure following early childhood surgery with anesthesia.Pediatrics. 2015; 136: e1-12Crossref PubMed Scopus (182) Google Scholar, 24Zhang X. Liu S. Newport G.D. Paule M.G. Callicott R. Thompson J. et al.In vivo monitoring of sevoflurane-induced adverse effects in neonatal nonhuman primates using small-animal positron emission tomography.Anesthesiology. 2016; 125: 133-146Crossref PubMed Scopus (47) Google Scholar; to date, a definitive phenotypic signature for late and persistent neurodegenerative changes has not been accepted universally. Although it is accepted generally that exposure to anesthetics during vulnerable periods results in neurodegenerative histopathologic changes, how much this affects the animal on an organismal level is less certain. As discussed, neurocognitive deficits have been found in adult animals exposed to anesthetics as neonates; however, there is some question as to how much the most commonly used rodent tasks, including locomotor activity, and assessments of spatial learning and memory, actually apply to human children.6Jevtovic-Todorovic V. Hartman R.E. Izumi Y. Benshoff N.D. Dikranian K. Zorumski C.F. et al.Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits.J Neurosci. 2003; 23: 876-882Crossref PubMed Google Scholar, 9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar, 25Loepke A.W. Istaphanous G.K. McAuliffe 3rd, J.J. Miles L. Hughes E.A. McCann J.C. et al.The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and memory.Anesth Analg. 2009; 108: 90-104Crossref PubMed Scopus (226) Google Scholar Studies of anesthetic neurotoxicity in nonhuman primates, which are more phylogenetically related to humans, may allow future preclinical models to be more directly applicable to our species. The National Center for Toxicological Research has used the Operant Test Battery to assess the effects of nonanesthetic drug exposure on monkeys for several decades, and the behavior of sufficiently trained rhesus monkeys can be comparable with that of children.26Paule M.G. Green L. Myerson J. Alvarado M. Bachevalier J. Schneider J.S. et al.Behavioral toxicology of cognition: extrapolation from experimental animal models to humans: behavioral toxicology symposium overview.Neurotoxicol Teratol. 2012; 34: 263-273Crossref PubMed Scopus (15) Google Scholar The use of this battery in preclinical models of anesthetic neurotoxicity may therefore allow for a closer comparison with human subjects. Studies on anesthetic neurotoxicity in many animal models are performed in a manner inconsistent with the modern operating suite and in the absence of surgical stimuli. The stressors of surgery themselves may be detrimental to neurodevelopment, and there is evidence that anesthesia and analgesia blunt these responses. For example, preterm infants administered fentanyl while undergoing patent ductus ligation have demonstrably improved postoperative outcomes compared with infants to whom opioid analgesia is not administered.27Anand K.J. Sippell W.G. Aynsley-Green A. Randomised trial of fentanyl anaesthesia in preterm babies undergoing surgery: effects on the stress response.Lancet. 1987; 1: 62-66Abstract PubMed Scopus (406) Google Scholar In addition, neonates who received deep intraoperative anesthesia have improved hormonal and metabolic responses to surgery and lower mortality than infants anesthetized with a lighter technique.28Anand K.J. Hickey P.R. Halothane-morphine compared with high-dose sufentanil for anesthesia and postoperative analgesia in neonatal cardiac surgery.N Engl J Med. 1992; 326: 1-9Crossref PubMed Scopus (703) Google Scholar It should be noted that opioids were used for analgesia in both of these human studies and that, despite popular belief, halogenated inhaled anesthetics do not provide any significant measure of analgesia. There is also concern that surgical stressors may actually worsen anesthetic neurotoxicity. Studies performed in rats in the absence of opioid analgesia suggest that the combination of surgical stressors and inhaled anesthesia may be more detrimental than either alone. Both chemically induced and surgically induced pain exacerbated the immediate apoptosis and subsequently delayed cognitive impairment of a combined nitrous oxide/isoflurane anesthetic in postnatal day 7 rats.29Shu Y. Zhou Z. Wan Y. Sanders R.D. Li M. Pac-Soo C.K. et al.Nociceptive stimuli enhance anesthetic-induced neuroapoptosis in the rat developing brain.Neurobiol Dis. 2012; 45: 743-750Crossref PubMed Scopus (93) Google Scholar Surgery without anesthesia is clearly detrimental, and it is entirely possible that surgical stressors without adequate analgesia may exacerbate anesthetic neurotoxicity. What specific role adequate analgesia may play in blunting these effects remains to be elucidated. Physiologic monitoring of human subjects undergoing anesthesia and surgery in the modern era also includes continuous monitoring of oxygenation, ventilation, temperature, and circulation. Human patients can be assessed rigorously for hypoglycemia and electrolyte imbalances preoperatively, intraoperatively, and postoperatively. This detailed level of physiologic monitoring generally is not performed in preclinical testing of anesthetic neurotoxicity, although there are notable exceptions.9Disma N. Mondardini M.C. Terrando N. Absalom A.R. Bilotta F. A systematic review of methodology applied during preclinical anesthetic neurotoxicity studies: important issues and lessons relevant to the design of future clinical research.Paediatr Anaesth. 2016; 26: 6-36Crossref PubMed Scopus (58) Google Scholar It is possible that unrecognized deviations from physiologic norms (eg, hypoxia, hypoglycemia, hypotension) may compound or confound evidence for, or against, anesthetic neurotoxicity. Recently conducted studies have moved toward full physiologic monitoring and improved maintenance of homeostatic variables, including endotracheal intubation and mechanical ventilation, between exposed and control groups.17Brambrink A.M. Evers A.S. Avidan M.S. Farber N.B. Smith D.J. Zhang X. et al.Isoflurane-induced neuroapoptosis in the neonatal rhesus macaque brain.Anesthesiology. 2010; 112: 834-841Crossref PubMed Scopus (449) Google Scholar, 30Whitaker E.E. Bissonnette B. Miller A.D. Koppert T.L. Tobias J.D. Pierson C.R. et al.A novel, clinically relevant use of a piglet model to study the effects of anesthetics on the developing brain.Clin Transl Med. 2016; 5: 2Crossref PubMed Google Scholar These efforts should be applauded and continued in future studies so as to obtain data more relevant to human subjects. When administered to neonatal animals, multiple anesthetic agents commonly used in clinical practice have been shown to cause both neuronal damage and long-term behavioral changes. These effects have been repeated by multiple research groups and in multiple species, including nonhuman primates. There is good evidence that long-duration exposures, simultaneous exposures to multiple anesthetic agents, and repeated anesthetic exposures cause histologic damage. The degree of histologic damage appears to correlate with observed behavioral changes; however, evidence for neuronal damage, and especially for long-term behavioral changes, following short-duration and single-agent exposures is less robust. The question of how applicable animal studies are to human subjects also remains, and before focusing on the risks that anesthetics may pose to human patients, one must remember that many surgeries and procedures performed on children each year are demonstrably beneficial, and we do not have viable alternatives to the anesthetics currently in clinical use that are necessary to perform these procedures. These caveats aside, we feel that the data from animal studies raise and reaffirm the concern that anesthetics, although necessary for the safe conduct of a multitude of surgeries, may in fact be neurotoxic, especially when given for long durations and at high doses.