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Pediatric Neuroscience: Review Article

Neurotoxicity of Anesthetic Drugs in the Developing Brain

Stratmann, Greg MD, PhD

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doi: 10.1213/ANE.0b013e318232066c
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For >150 years of anesthetic practice, it was believed that as a general anesthetic wears off, the brain would return to the same state as before the anesthetic. We are now beginning to understand that this basic premise of anesthetic pharmacology is false. In 2003, Jevtovic-Todorovic et al.1 presented their sentinel findings that a combined anesthetic (midazolam, nitrous oxide, and isoflurane) administered to 7-day-old rats for 6 hours kills neurons in the developing brain and causes long-term impairment of brain function. They showed that long-term potentiation (LTP) in the hippocampus was impaired in anesthetized rats.1 LTP is a form of synaptic plasticity, often considered the electrophysiologic correlate of learning and memory, and the hippocampus is a brain structure important for learning and memory. More importantly, these authors demonstrated a progressive deficit in spatial recognition tasks administered both 4 weeks and 4.5 months after anesthesia.1 Immediately, concern mounted within the anesthesia community25 and also within regulatory authorities6 about whether these phenomena might apply to humans. Subsequently, it became clear that the histologic data were reproducible not only in rodents but also in virtually every species tested,7 including primates,810 further heightening the degree of concern about anesthesia in the immature human brain. A Food and Drug Administration advisory committee meeting in 2007 concluded that no change in clinical practice is justified based on available data,6 and a follow-up meeting in March 2011 upheld this recommendation.

It is uncertain if it will ever be feasible to test whether anesthesia kills neurons in the brain of children. However, this may not be entirely necessary. A focus on anesthesia-induced neurodegeneration seems appropriate only if some aspect of brain function in humans was changed permanently by anesthesia, and if a causal link between neurodegeneration and long-term brain function could be demonstrated in animals. Let us examine these 2 premises in more detail.

ANESTHESIA AND BRAIN FUNCTION IN HUMANS

Until recently, speculation as to whether developmental anesthetic neurotoxicity might exist in humans occurred mostly on the basis of studies that were not specifically designed to address this question.25,7,11 Since 2009, 7 publications appeared that were designed to shed light on whether anesthesia in humans might impair brain function long-term.1218 Unfortunately, for a number of reasons discussed below, the issue remains far from being resolved.

Wilder et al.12 studied whether anesthesia administered at younger than 4 years was associated with learning disabilities between ages 5 and 19 years. A cohort of 5357 children born in Olmsted County, Minnesota, between 1976 and 1982 was assessed for the presence, type, and duration of anesthesia administered before age 4 years. Anesthesia administered for both surgical and diagnostic procedures was included in the analysis. The school district in which the study was performed routinely administered reading, writing, and math aptitude tests as well as intelligence tests. In this study, learning disability was defined as a performance on standardized achievement tests below a certain predicted score based on the child's IQ. If any of 3 different definitions used by the school district to identify disabled learning applied, the primary outcome of this study, learning disability, was considered to be present and study follow-up ceased at this point. Eleven percent of children underwent at least 1 anesthetic before age 4 years, of whom 24% underwent >1 anesthetic. Learning disabilities were more common in those children who had >1 anesthetic, and cumulative anesthetic duration of >2 hours was a risk factor for learning disability. Learning disability was not more common if only 1 anesthetic exposure occurred before age 4 years. Because children requiring >1 anesthetic were sicker than those requiring only a single anesthetic, the authors performed a subgroup analysis of children requiring >1 anesthetic with ASA physical status I and II and excluding those with ASA physical status III and IV. Despite including only less sick children with multiple anesthetic exposures, the association between learning disabilities and anesthesia persisted. Methodologic advantages of this study are that studying a birth cohort does not bias surgical procedures and comorbidities in the same way recruitment of a cohort of patients from an academic center might. Furthermore, controlling for IQ seems like an elegant approach to controlling for one of the strongest confounders of a child's ability to learn. General methodologic drawbacks include retrospective analysis of a retrospective cohort, which forces studying an outcome variable that is available rather than one that is chosen prospectively. Learning disability is a very nonspecific outcome and many underlying pathologies may impair a child's ability to learn, for example, motivation, attention, intelligence, sensory neural problems, or other, more-specific functional abnormalities, all of which may have relevance to anesthetic developmental neurotoxicity. Other drawbacks include that the anesthetic almost uniformly administered to the study cohort was halothane/nitrous oxide, which is now an outdated anesthetic in most pediatric anesthetic practices. Reporting the cumulative incidence of learning disabilities requires that follow-up is stopped when learning disability is detected. In other words, once a child meets the criterion for learning disabilities, it is assumed that learning disabilities persist and never resolve. This makes it impossible to comment on the true prevalence of the outcome. It is possible that children with learning disabilities at some point may have a change in performance that places them back in the normal range, an event that cannot be captured by the current study design. However, it might be possible that anesthesia-associated learning disabilities progress, as has been suggested for anesthesia-induced neurocognitive dysfunction in animals.1,1921 The current study design would not be able to detect progression of cognitive disability. Likewise, this methodology would not capture the spuriousness of the outcome. For example, a low aptitude score in any one of the tested domains, for whatever reason, would trigger the diagnosis of learning disability, as would a spuriously high IQ score, resulting in a predicted aptitude score that might render an otherwise normal aptitude to be classified as meeting the learning disability criterion.

The same group16 reported later that year that general anesthesia for cesarean delivery does not increase the cumulative incidence of learning disabilities in the same12 birth cohort of children. This is consistent with their earlier study12 because cesarean delivery required one single, rather short anesthetic. Surprisingly, children born by cesarean delivery under regional anesthesia had a lower cumulative incidence of learning disability than those born by vaginal delivery.16 The significance of this finding is unclear and requires further study. However, this study suggests that a brief general anesthetic during late fetal life is not associated with later cognitive problems. The retrospective nature of this study confers the same limitations to interpretation of the data that apply to their previous study.12

Kalkman et al.13 approached the problem from a different and interesting angle. They argued that anesthesia is mostly administered to tolerate a surgical procedure. Therefore, to draw conclusions about the effects of anesthesia versus surgery on cognitive outcome, an unanesthetized control group undergoing surgery would be required or anesthesia would have to be administered to children who do not need it, neither one of which is ethically feasible. The authors further assumed that there is a distinct period of vulnerability to the effects of anesthesia on neurodevelopment, as suggested by animal studies using histologic outcomes.8,20,2224 Based on this assumption, the authors hypothesized that children anesthetized during the period of vulnerability (earlier in life) should have a worse cognitive outcome than children anesthetized after the period of vulnerability. They defined the period of vulnerability in humans as younger than 2 years of age.13 This design circumvents the issue of requiring an unobtainable control group and allows children anesthetized later in life to serve as controls. The authors used scores from the Child Behavioral Checklist to identify behavioral abnormalities and found that children anesthetized at younger than 2 years of age tended to have a higher incidence of clinically deviant behavior than children anesthetized at older than 2 years, undergoing the same (urological) procedures. The difference was even more pronounced between children undergoing anesthesia at younger than 6 months of age compared with older than 2 years. However, neither effect was pronounced enough to reach statistical significance. A sample-size calculation revealed that >6000 children would have to be studied to show the difference, given the effect size comparing children younger than 2 years with those of more than 2 years at the time of anesthesia, and >2200 patients if the sample-size calculation was based on the effect size comparing children younger than 6 months and older than 2 years of age at the time of anesthesia.13 Although the authors chose an innovative and logical approach to a difficult ethical dilemma, the validity of the observation is based on the assumption that the period of vulnerability in humans is limited to 2 years of age. This assumption may or may not be correct. It has been perpetuated over the years that the period of vulnerability coincides with the peak of synaptogenesis, which is also known as the brain growth spurt. The publication frequently cited in support of this is a scholarly article by Dobbing and Sands,25 which does not mention synaptogenesis. Instead, it synthesizes knowledge from various brain weight studies and proposes a hypothesis of how to relate vulnerability to environmental and nutritional challenges among different animal species. Appropriately, there are several notes of caution regarding the limitations for interpreting their hypothesis, for example, that the term “brain growth spurt” is an oversimplification because different areas of the same brain develop at different paces.25 Indeed, the peak of synaptogenesis in many structures of the rodent brain, including the cortex and the dentate gyrus of the hippocampus, does not occur until postnatal days 11 to 16, and synaptogenesis seems to persist until at least postnatal day 32.24,2630 Even within a given cortical neuron, synaptogenesis is not a uniform phenomenon.28 The period of vulnerability to anesthesia-induced neuronal apoptosis occurs before postnatal days 10 to 14,8,22,23 and is thus not well aligned with the peak of synaptogenesis. Most importantly, the period of vulnerability to the long-term behavioral effects of anesthetic drugs extends to at least postnatal days 14 to 17 in the rat.20 Rats reach sexual maturity at postnatal day 50.31 It must be concluded that the period of vulnerability to the outcome of interest—the long-term cognitive effects of anesthesia—well extend way past 2 years of age in humans. Consequently, the estimate by Kalkman et al. of the anesthetic effect on behavior might, if anything, be an underestimate.

The advantages and disadvantages of various study designs had been discussed in an editorial by the third contributors to the current human literature on long-term cognitive effects of anesthesia.11 According to these authors, the power of studying a prospective cohort must be balanced against the lead time for data to become available. For example, if enrollment of a randomized, controlled trial of regional versus general anesthesia for pediatric surgical procedures were completed today, data of remote neurobehavioral outcomes would not be available for years or perhaps decades. Given the urgency with which data on developmental neurobehavioral end points after anesthesia administration in humans are sought, a long lag time is arguably unacceptable. Thus, the authors11 concluded that an ideal combination of lag time and design strength would be prospective analysis of a retrospective cohort. The same group14 later studied whether hernia repair at age 3 years or younger is associated with subsequent behavioral and/or developmental disorders. A set of 383 medicare records listing procedure codes related to hernia repair was compared with a control set of 5050 age- and sex-matched medicare records not listing these procedure codes. Children younger than age 3 years were included. The behavioral outcome was defined as a diagnostic code for unspecified delay or behavioral disorder, mental retardation, autism, and language and speech disorder. If the behavioral outcome preceded the surgery, the record was excluded. After controlling for age, sex, race, and the presence of confounding diagnoses at birth, procedure codes indicating hernia repair were more than twice as likely to be associated with the behavioral outcome codes as when procedure codes for hernia repair were absent. The study design did not allow for assessing the type, frequency, and duration of the anesthetic in either the hernia repair or the control group. It was not possible to exclude that children in the control cohort did not have an anesthetic for procedures other than hernia repairs. Perhaps the most interesting finding of this study is the delay with which the behavioral outcome presented; in this case, 3 to 4 years after the surgery. This is reminiscent of animal studies, suggesting a progressive nature of the deficit.1,1921

Recently, the academic performance of a national cohort of Danish 15- to 16-year-old adolescents (n = 2689) who had undergone inguinal hernia repair between 1986 and 1990 at the age of 1 year or younger was compared with a random sample of 14,575 age-matched controls.17 When important confounders such as gender, birth weight, and parental age and education were controlled for, there was no evidence that the relatively brief (presumed by the authors to be 30–60 minutes) general anesthetic had affected academic achievement scores. All of the above confounders more strongly affected academic achievement than surgery plus anesthesia.17 This is despite the fact that children were younger than 12 months at the time of surgery, and thus may be considered more sensitive to the effects of anesthesia than older children.13 The authors appropriately concluded that these reassuring results cannot exclude deficits in more particular cognitive domains. It is understood that the effects of longer anesthetic durations are likewise not detectable with this study design.

Another human trial was designed to test whether there is a causality between anesthesia administered at younger than 3 years and between 3 and 12 years and cognitive performance. The authors studied 1143 pairs of monozygotic twins hypothesizing that if anesthesia and not the underlying disease caused cognitive disabilities, then the exposed twin should have a higher incidence of underachievement than the unexposed twin. Most pairs of twins in this study consisted of twins who were either both exposed or both not exposed to anesthesia. However, 71 twin pairs (15%) were discordant (one twin exposed, the other not exposed to anesthesia). Anesthesia was administered mostly for surgical procedures. Exposed twins had similar achievement scores on a nationwide test at 12 years of age to unexposed twins, and a similar incidence of cognitive problems, as assessed by a teacher questionnaire. The authors concluded that the comorbidity but not the combination of anesthesia and surgery is the cause of the cognitive problems. If these results can be duplicated, they would make a convincing argument that neither anesthesia nor surgery is a problem for the cognitive development of children.

DiMaggio et al.18 subsequently came close to doing just that by identifying 10,450 twins of unknown zygocity (i.e., “siblings”), 306 of whom had been exposed to anesthesia during a surgical procedure before age 3 years and 10,146 of whom had not. Of the 138 discordant pairs in which only 1 of the 2 twins was exposed to anesthesia, neither sibling of 107 pairs had International Classification of Diseases, Ninth Revision (ICD-9) diagnostic codes that would suggest a problem with brain development, and both siblings of 11 pairs had such ICD-9 codes subsequent to the procedure of the exposed twin.

When only 1 twin of a pair discordant for anesthetic exposure had an ICD-9 code suggesting a problem with brain function (n = 20), there was an even split of these codes between exposed (n = 9) and unexposed twins (n = 11). This supports the findings by Bartels et al.15 that there is no causal relationship between anesthetic exposure and brain dysfunction as measured by the occurrence of ICD-9 codes subsequent to the surgical procedure. A similar conclusion can be drawn from another finding from that same study,18 namely that the hazard ratio of behavioral/ developmental diagnosis was 1.6 when anesthesia occurred before the first occurrence of the ICD-9 code, but 1.3 when the ICD-9 code appeared before the anesthetic exposure. In this latter case, anesthesia could not possibly have caused the behavioral/developmental diagnosis. The fact there is nonetheless an association between anesthetic exposure and behavioral/developmental diagnosis in this case highlights the existence of confounders, which is unavoidable given the study design. The authors also found an increasing likelihood of an ICD-9 code of a behavioral/ developmental diagnosis with multiple anesthetics. Whether this represents an anesthetic dose response or a greater burden of disease is unclear.

In summary, the human literature is controversial as to whether anesthesia in infancy causes cognitive problems later in life. Furthermore, it is unclear what the period of vulnerability to anesthetic neurotoxicity is. We do not know whether there is a safe anesthetic technique or duration. The specific cognitive deficit caused by anesthesia, if any, that may underlie such outcomes as learning disabilities, has not been identified. None of the studies, alone or in combination, form a basis for informing clinical practice.

ANIMAL STUDIES

As discussed above, it is not entirely clear whether long-term cognitive dysfunction, the most worrisome feature of developmental anesthetic neurotoxicity, occurs in humans. In the meantime, animal models of anesthesia are important in furthering our understanding of the phenomenology, pharmacology, and the mechanism of anesthesia-induced neurocognitive dysfunction. To that end, a recent study21 in monkeys demonstrating a persistent and progressive decline in cognitive domains after ketamine anesthesia is the latest in a series of alarming studies suggesting that anesthesia given to immature mammals impairs brain function later in life.

Understanding the mechanism by which anesthesia impairs brain function months after anesthesia in infancy would allow us to develop rational preventive strategies, and is thus a very important, yet currently elusive, milestone in this field. Implicit in the concept of a mechanism is the concept of causality. Although causality might be impossible to prove, it is usually accepted on the basis of (good) enough evidence for and insufficient or bad evidence against such a link.32 If anesthesia caused cognitive dysfunction, the mechanism by which anesthesia caused cognitive dysfunction would be causally linked to both anesthesia and cognitive dysfunction. The following discussion suggests that the mechanism of anesthesia-induced cognitive dysfunction or decline, as the case may be,1,1921,33 is much less clear than previously thought. Specifically, I discuss evidence for each of 3 cellular phenomena to qualify as a mediating mechanism of anesthesia-induced cognitive decline: neurodegeneration, synaptogenesis, and hippocampal neurogenesis.

NEURODEGENERATION

It is now accepted, on the basis of overwhelming experimental evidence,7 that anesthesia causes neurodegeneration in a variety of animal species, including primates.8,9 Few would dispute that the possibility of anesthesia causing neurodegeneration in humans is real, although it will be very difficult to prove this definitively. Furthermore, whether or not anesthesia-induced neurodegeneration happens in humans is not nearly as important as whether anesthesia impairs cognition in humans. What is important, however, is to define the role of anesthesia-induced neurodegeneration in causing anesthesia-induced cognitive decline. Unless anesthesia-induced neurodegeneration mediated the anesthesia-induced cognitive outcome, it would merely be an epiphenomenon with little significance to cognitive function. When anesthesia was first shown to cause both neurodegeneration and cognitive decline in rats,1 a causal link between the 2 outcomes must have seemed so plausible that it was not as rigorously scrutinized as other, less intuitive potential mechanisms. To address this question in more detail, we must consider the evidence for and against such a causal link.

It is difficult to comprehend how a one-time anesthetic exposure, which increases neuroapoptosis, can have a neurobehavioral consequence without the specific defect (apoptosis) or the defect's sequelae (decreased cell number, decreased synaptic connectivity, altered cell migration, etc.) persisting from the time of exposure. In other words, if months after anesthesia the brain of a formerly anesthetized person or animal was indistinguishable from a brain that was not exposed to anesthesia, it would be hard to argue that anesthesia caused the brain to be dysfunctional. Applied to neurodegeneration, this means that several months after anesthesia a causality between anesthesia-induced neurodegeneration and anesthesia-induced cognitive dysfunction would be difficult to accept unless neurodegeneration had somehow altered the brain of anesthetized animals. If neurons destroyed by anesthesia left a detectable gap in the brain or if the neuronal number was different from unanesthetized animals, a reasonable argument could be made that neurodegeneration qualifies as a potential mediating mechanism for the cognitive outcome. Rizzi et al.34 used pregnant guinea pigs to show that a triple anesthetic cocktail consisting of 0.55% isoflurane, 75% nitrous oxide, and 1 mg/kg midazolam for 4 hours, but not fentanyl 15 mg/kg/h, caused acute neurodegeneration in the offspring. They also found that the neuronal density in the first postnatal week was reduced by 30% to 50% in the offspring that had been exposed to anesthesia.34 The authors concluded that the observed degree of anesthesia-induced neuronal deletion far exceeded the approximately 1% neuronal deletion observed in their prior studies and therefore suggests that neurons are permanently lost.34 It could be argued, however, that the observed anesthesia-induced neuronal deletion is well in line with the normal rate of developmental apoptosis, which is usually at least 50%.35,36 However, neurons dying during development are immature,3739 but the postmitotic age of neurons killed by anesthesia is not yet known. Nonetheless, the authors34 observed a significant difference between neuronal deletion after the triple anesthetic versus the fentanyl infusion or no anesthetic. The absence of a behavioral assessment with concomitant assessment of neuronal density does not allow us to conclusively determine whether the neuronal density that was abnormal in the first postnatal week34 would also have been abnormal at the time of neurocognitive testing. This study is also interesting in that guinea pigs, like humans and unlike rats, have a relatively mature brain at birth. Anesthetizing the pregnant mother with drugs that cross the placenta is elegant in that it allows for hemodynamic monitoring or even hemodynamic control of the mother and thus indirectly of the fetus, which is very difficult in neonatal or infantile rodents. Furthermore, temperature and nutritional status can be more easily controlled than in newborn rodents. However, if anesthesia in utero is administered too close to delivery, maternal oxytocin might change neuronal vulnerability to anesthesia by temporarily shifting the chloride reversal and causing immature neurons to be inhibited by γ-aminobutyric acid (GABA), which is usually characteristic of mature neurons.40 This peripartum model of anesthesia is reminiscent of a human trial discussed above16 in which no adverse cognitive sequelae could be demonstrated by general anesthesia for delivery after adjusting for important confounders. The discrepant results do not allow us to conclude that neurodegeneration is not important for cognitive outcome, partly because the anesthetic durations differed dramatically between the 2 studies. It is not known whether a triple anesthetic cocktail administered for the duration required to perform a cesarean delivery (presumably an hour or less) causes neurodegeneration or permanent neuronal deletion in an animal model. The most compelling evidence that acute neurodegeneration causes lasting neuronal deletion resulted from 2 rat studies by the same group.41,42 Rats that were anesthetized on postnatal day 7 had neuronal deletion at postnatal day 3041 and ultrastructural abnormalities suggestive of ongoing cell death on day 21.42 This is approaching the age at which the same group previously demonstrated learning and memory deficits in rats.1 These results would be strengthened if it could be demonstrated that the total number of neurons was decreased long-term. This requires assessment of the volume of the structure of interest, which did not occur in the above studies. The results would be even stronger if animals with a proven learning and memory deficit had neuronal deletion, and strongest if those animals with the worst brain function were those with the greatest degree of neuronal deletion. Another group43 did not find a long-term effect of isoflurane during infancy on neuronal density in 2 brain regions most severely affected by acute cell death in mice. Because the volume of these structures was not assessed, it is impossible to know what the total number of neurons in these structures was. Interestingly, learning and memory were not affected in this study, which would suggest either that isoflurane does not affect long-term neurocognitive function or that the degree of acute cell death does not determine long-term neurocognitive outcome.43 The latter possibility was suggested by another study,44 demonstrating that 2 hours of isoflurane but not 1 hour of isoflurane at 1 minimum alveolar concentration (MAC) causes neurodegeneration. However, despite extensive neurodegeneration, mainly in the thalamus and cortex, no long-term neurocognitive sequelae were demonstrated by 2-hour isoflurane. From a functional standpoint, 2-hour isoflurane seems to be a safe dose in rats. This conclusion is supported by the finding that in the same study,44 4 hours of isoflurane caused long-term learning and memory problems. Hence, the absence of neurocognitive deficits after 2-hour isoflurane was not attributable to a general inability to demonstrate neurocognitive dysfunction. Isoflurane at 1 MAC given to rats at the peak of vulnerability to developmental anesthetic neurotoxicity (postnatal day 7) causes respiratory depression and hypercarbia. Hypercarbia alone for 4 hours caused a similar degree and distribution of cell death in the brain as 4-hour isoflurane, but instead of impairing brain function long-term, rats exposed to 4-hour hypercarbia at postnatal day 7 outperformed all other groups, including the control group. Hypercarbia alone caused robust improvement in long-term neurocognitive function despite causing extensive cell death in the developing brain. Collectively, these findings suggest that the degree to which an intervention causes acute neurodegeneration does not always determine long-term cognitive outcome.

An important prediction required by the concept that anesthetic neurodegeneration is responsible for later cognitive dysfunction is that interventions preventing anesthesia-induced neurodegeneration also prevent anesthesia-induced long-term neurocognitive sequelae. Examples of such interventions include melatonin,45 lithium,46 dexmedetomidine,47 inhibitors of the p75 neurotrophin receptor (TAT-conjugated Pep5 or Fc-p75NTR),48,49 hypothermia,50 and bumetanide,51 all of which have been shown to prevent anesthesia-induced neurodegeneration.

The rationale for using melatonin to counteract the effect of anesthesia is the demonstration that anesthesia causes neuronal apoptosis via a mitochondria-dependent pathway among others, which is associated with biochemical changes that melatonin had previously shown to counteract.45 Furthermore, melatonin has several other nonspecific, protective effects in the brain.45 Melatonin was found, in a dose-dependent manner, to reduce anesthesia-induced neuronal apoptosis in rats.45 The authors suggested that its bioavailability, lipophilicity, ability to cross the blood-brain barrier, absence of toxicity, and sleep-inducing and analgesic effects make it an ideal adjuvant for anesthesia.45 Surprisingly, it is not known whether melatonin reverses the long-term behavioral effects of anesthesia.

Another group46 showed that lithium protects against anesthetic neurotoxicity in the developing brain. They argued that lithium is known to counteract extracellular signal–regulated protein kinase inhibition and neurodegeneration caused by alcohol. Alcohol acts via antagonism of N-methyl-d-aspartate (NMDA) receptors and by facilitating GABAergic receptor transmission. Hence, they hypothesized that a combination of an NMDA antagonist anesthetic, ketamine, and a GABAergic drug, propofol, should cause similar effects as alcohol on the developing brain, and that these effects should be preventable by coadministration of lithium. This hypothesis was largely confirmed and the authors concluded that lithium may be an effective adjuvant to anesthesia, provided that it can be demonstrated that the inhibition of naturally occurring apoptosis, which is also caused by lithium, has no ill effects.46 This may be an important caveat, because naturally occurring neuroapoptosis is critically important for brain development,36 and when this process is inhibited, learning and memory are impaired.52 It is not known whether lithium can prevent anesthesia-induced neurocognitive decline.

Developmental anesthetic neurotoxicity has largely been attributed to the combination of GABAergic and NMDA antagonist actions of anesthetic drugs. Dexmedetomidine is neither a GABAergic nor NMDA antagonist and has therefore been hypothesized to be free of developmental anesthetic toxicity.47 Furthermore, it has a number of antiapoptotic effects, and thus Sanders et al.47 hypothesized that it might protect against anesthesia-induced neuronal apoptosis. Dexmedetomidine reduced neuronal apoptosis caused by a subanesthetic dose of isoflurane for 6 hours in a dose-dependent manner, which was reversed by blocking the α-2 adrenoceptor, indicating that the protective effect is mediated by this receptor. Furthermore, dexmedetomidine prevented an isoflurane-induced impairment in trace fear conditioning at 40 days of age. This is the only study to date of an intervention that nonspecifically protected from anesthesia-induced cell death that also protected from anesthesia-induced neurocognitive dysfunction. The behavioral outcome was virtually devoid of interindividual variability, which is unusual for behavioral experiments when using rats from different litters.53 Even the rate of neuroapoptosis is usually subject to substantial litter variability.54,55 Either way, these results require confirmation in both animal and human studies before considering a change in practice. An important mechanistic finding of this study47 is that the neurodegeneration caused by isoflurane was not prevented by a GABAA-receptor antagonist, indicating that this receptor does not mediate the neurodegeneration caused by GABAergic drugs.47

Creeley and Olney50 advanced an interesting hypothesis on the basis of a 2-part assumption: anesthesia decreases neuronal activity in the developing brain with subsequent withdrawal of trophic support and neurodegeneration. They argued that another intervention known to decrease neuronal activity, hypothermia, should therefore cause neurodegeneration, and found the exact opposite. Hypothermia (30°C) protected against isoflurane-induced and ketamine-induced neurodegeneration.50 This indicates either that neuronal inactivity does not cause neurodegeneration or that anesthesia does not cause neuronal inactivity. This latter possibility is actually a given for GABAergic drugs, which cause neuronal excitation in immature neurons, rather than neuronal inhibition, as is true in mature neurons. The mechanism of neuronal excitation in immature neurons is immaturity of a chloride transporter before the second postnatal week in rats56 and the first 3 to 12 months in humans.57 Because isoflurane is a predominantly GABAergic anesthetic, it should cause neuronal excitation in immature brains or immature parts of the brain. It may therefore be no surprise that hypothermia failed to reproduce the isoflurane-induced neurodegeneration. Sevoflurane has been shown to cause neuronal excitation in the immature brain, which was actually postulated to be the mechanism underlying sevoflurane-induced neurodegeneration.51 It is not known whether hypothermia protects against anesthesia-induced neurocognitive dysfunction.

Substantial insight into what does mediate anesthesia-induced developmental neuroapoptosis is provided by 2 elegant studies.48,49 In the first study, Head et al.48 showed that inhibitors of the p75 neurotrophin receptor prevent isoflurane-induced cleaved caspase 3 expression in vitro and loss of dendritic spines and synapses in vivo. Brain-derived neurotrophic factor (BDNF) is excreted as pro-BDNF and cleaved by proteases, such as plasmin, to BDNF, which interacts with the TrkB receptor to signal survival. If pro-BDNF remains uncleaved, it interacts with the p75 neurotrophin receptor and acts as a cell death signal. Plasmin is cleaved from plasminogen by tissue plasminogen activator, which is released from the presynaptic terminal when neurons are firing. The authors48 interpreted their findings as confirmation that neuronal silencing caused a shift in the balance of BDNF signaling to preferentially occur via pro-BDNF as opposed to mature BDNF. The authors48 went to great lengths to elegantly exclude alternative interpretation of their findings. However, they did not show that isoflurane actually decreases neuronal firing in immature neurons. As stated above, isoflurane is not expected to decrease neuronal firing in immature neurons. In fact, the opposite is true in that isoflurane or any other GABAergic anesthetic should cause neuronal excitation.56 A possible explanation for these discrepancies comes from the observation that in a cell culture model, such as the one used by Head et al.,48 glucose is commonly used as an energy substrate, whereas the predominant energy substrate of the developing brain is ketone bodies.58,59 Glucose causes a shift in the chloride reversal potential of neurons in culture that makes them act like mature neurons.58,59 Mature neurons are indeed silenced by isoflurane. The authors48 also found that isoflurane decreases the number of immature dendritic spines in vitro and the number of synapses in 5- to 7-day-old mice. This reduction in synaptic density was attenuated by the p75 neurotrophin receptor blocker TAT-Pep5.48 Importantly, these authors48 demonstrated that their intervention is nontoxic and does not cause an unwanted suppression of naturally occurring neuronal apoptosis. This is an advantage over nonspecific modalities that ameliorate anesthesia-induced neurodegeneration, such as lithium, melatonin, dexmedetomidine, or hypothermia.

In a second study, the same group49 showed that the effect of isoflurane-induced p75 neurotrophin receptor signaling on synaptogenesis and neurodegeneration is mediated via activation of RhoA, a kinase causing actin depolymerization. This causes growth-cone collapse, loss of immature dendritic spines, and, presumably, the loss of synapses observed in their previous study.48 The authors also observed expression of cleaved caspase 3, a marker for apoptotic cell death. When signaling via the p75 neurotrophin receptor was inhibited or when the cytoskeleton was stabilized, isoflurane-induced loss of dendritic spines and expression of cleaved caspase 3 was attenuated. This suggests that anesthesia causes actin depolymerization via RhoA activation, which in turn causes loss of dendritic spines and apoptotic cell death. It is unknown whether p75NTR antagonism or cytoskeletal stabilization can prevent anesthesia-induced neurocognitive dysfunction.

In another elegant study, Briner et al.30 confirmed that propofol, either as a single shot of 40 mg/kg or given repeatedly over 6 hours at half that dose, decreased synaptic spine density in 5-day-old rodents and increased spine density in 15- to 25-day-old rodents. Amazingly, both the 6-hour duration as well as the single shot of propofol caused persistent changes into adulthood, indicating that a single, brief anesthetic to an anesthetic depth that would not permit a surgical procedure, is sufficient to permanently alter cortical synaptic spine densities. This work confirms results of decreased synaptogenesis at approximately postnatal day 5,48,49 and their own previous results24,60 of rapid increase in synaptogenesis after postnatal day 15 in the cortex and the hippocampus. Consistent with previous results,22 no neurodegeneration occurred in the cortex of 16-day-old rats,24 confirming that the period of vulnerability to anesthetic apoptotic cell death is limited to postnatal day 10. Importantly, it was recently shown that the period of vulnerability to anesthesia-induced neurocognitive decline extends to at least postnatal day 17 in rats,20 a time at which neurons are no longer sensitive to the apoptotic effects of anesthesia.20,22,24 If these results20 can be confirmed, the causal link between anesthesia-induced neurodegeneration and anesthesia-induced neurocognitive decline would be further weakened. Also, it would need to be explained how an anesthesia-induced decrease30,48,49 or increase in synaptogenesis24,30,60 could both be responsible for the same outcome (anesthesia-induced neurocognitive decline).

One interesting feature of the age-dependent switch in anesthetic effect on synaptogenesis24,30,48,49,60 is that it nearly parallels the age-dependent switch in the chloride reversal potential and thus the excitatory to inhibitory switch in GABA phenotype.56,57 However, mechanistically linking the developmental switch in GABA phenotype with the switch from an anesthesia-induced decrease to increase in dendritic spines, although possibly accounting for the ill effects of GABAergic drugs in the immature brain, would not readily account for cellular or behavioral phenomena caused by NMDA antagonists; for example, the progressive cognitive decline in monkeys treated with ketamine during early postnatal development.21

It has been assumed that anesthesia-induced neuronal silencing is responsible for anesthesia-induced effects on synaptogenesis and apoptosis, which is difficult to consolidate with the switch in the GABAergic phenotype from excitatory to inhibitory at exactly the time at which vulnerability to anesthesia-induced neuronal apoptosis ceases. This assumption was formally challenged in a recent study51 demonstrating that sevoflurane causes global brain excitation in rats, which is entirely compatible with a motionless animal. These nonconvulsive seizures were associated with neuronal cell death. Bumetanide, which inhibits the immature chloride transporter responsible for the excitatory action of GABA during early development, prevented both the sevoflurane-induced seizures and sevoflurane-induced neurodegeneration.51 Interestingly, bumetanide did not prevent the functional consequences of sevoflurane, namely, a reduction in hippocampal LTP, the electrophysiologic correlate to learning and memory.51 Anesthesia-induced neurodegeneration had previously been associated with reduced hippocampal LTP.1 The fact that prevention of anesthesia-induced neurodegeneration did not prevent the functional sequelae of anesthesia51 again draws into question the assumption that one causes the other.

If anesthesia-induced neurodegeneration does not cause anesthesia-induced neurocognitive decline, then what does? It is possible that the age-dependent anesthetic effects on synaptogenesis24,30,48,49,60 can have functional relevance independent of whether or not they cause neuronal apoptosis. One prerequisite to this claim—persistence of these effects until the time of neurocognitive testing—has been met.30 Now it must be demonstrated that an intervention that prevents the anesthetic effects on synaptogenesis also prevents the anesthetic effect on cognitive function.

Another possible mechanism is an anesthetic effect on postnatal hippocampal neurogenesis.19,20 Postnatal neurogenesis occurs in only 2 brain areas, one of which is the hippocampus.6163 Inhibition of dentate neurogenesis is sufficient to impair learning and memory in a manner similar to anesthesia.64,65 Of particular interest is the time course of the deficits. Neurogenesis is exquisitely sensitive to brain irradiation6671; children who underwent brain irradiation developed progressive cognitive decline over a number of years.72 The deficit caused by anesthesia is hippocampus dependent and seems to progress.1,1921 Isoflurane has been shown to impair neurogenesis,19,20 as does phenobarbital.73 These effects persist until the time of neurocognitive testing.20,73 If an anesthetic effect on neurogenesis mediated anesthesia-induced neurocognitive decline, interventions that restore neurogenesis should rescue the behavioral phenotype. Such interventions include environmental enrichment, voluntary exercise, caloric restriction, or antidepressant drugs.7480 We have shown that environmental enrichment reverses the behavioral effects of anesthesia, even when instituted with a 3-week delay after anesthetic exposure (unpublished observation). The treatment efficacy of environmental enrichment may or may not be attributable to its effects on neurogenesis.

WHICH ANESTHETIC IS THE SAFEST?

This question is slowly beginning to be addressed in comparative toxicity studies in animals. Human studies have not addressed this issue at all and given the controversy as to whether or not functional sequelae of anesthesia in infancy even exist in humans, the argument might be made that comparative studies are not quite yet indicated. In animal models, whereby anesthetic developmental neurotoxicity has been clearly demonstrated, these studies can be performed relatively easily, with the caveat that anesthetic equipotency is vitally important for interpretation of results of comparative studies. If an anesthetic results in both greater anesthetic depth and greater anesthetic toxicity than another anesthetic, then interpretation of the data becomes difficult. For example, when it was determined that a 3-drug anesthetic combination causes a greater degree of neurodegeneration than 2 or 1 anesthetic drug, the 3 drugs were simply added to one another, which would have resulted in a greater anesthetic depth than the 2-anesthetic combination or the single anesthetic.1 Specifically, the single GABAergic volatile drug isoflurane caused mild cell death at 0.75% atm, which was aggravated by an otherwise nontoxic dose of midazolam (9 g/kg), and made even worse by an otherwise nontoxic dose of nitrous oxide (75% atm).1 This has been interpreted as greater toxicity when GABAergic and NMDA antagonist drugs are combined, but it is unclear whether this does not also reflect an effect of anesthetic depth. In anesthetic practice as well as in research, MAC is used to express anesthetic potency and anesthetic depth.81 Unlike in adult rodents, MAC in immature rodents is not a stable anesthetic concentration but decreases steadily with increasing duration of anesthesia.82,83 The reason for this phenomenon is unclear as is whether or not this occurs in humans. This steady decrease in anesthetic requirements makes comparative studies of volatile anesthetic drugs a challenge.8487 An example of a good study comparing isoflurane, sevoflurane, and desflurane is in press as of the time of this writing.83 In this study, desflurane caused greater neurotoxicity in the immature mouse brain than near equipotent doses of isoflurane or sevoflurane.83 Sevoflurane had been reported to have a more favorable neurotoxicity profile than isoflurane.8587 Two recent studies dispute these findings.83,84 However, in both of these studies, less sevoflurane was used than isoflurane, despite attempts to achieve equipotency. This is primarily a result of our incomplete understanding and agreement about how equipotency should be achieved in immature rodents. The simplest way would be to give a fraction of a published, constantly decreasing MAC.82,83,88 Alternatively, a constant anesthetic concentration can be expressed as percentage MAC over time and the area under the curve of this plot can be calculated.88 If the areas under the percentage MAC over time curve are within a certain limit of agreement (e.g., within 10% of each other), the anesthetic drugs were used at equipotency. The situation becomes more difficult when an inhaled drug is to be compared with an IV drug. Whereas MAC determination for volatile anesthetics is possible in immature rodents, the same would be much more difficult for IV drugs, because a constant plasma and brain concentration would have to be achieved to do so. This would require insertion of an IV cannula and infusion of drug with subsequent sampling of blood and/or brain tissue, a difficult experimental preparation. Furthermore, because it is conceivable that MAC for IV drugs also decreases over time in immature rodents, the above would have to be done at various time points. Hence, comparative studies between inhaled and IV drugs are currently very difficult to interpret. For example, it has been shown that sevoflurane has a favorable neurotoxicity profile over propofol but it is entirely unclear what the anesthetic depth of these animals was.89

CONCLUSION

Knowledge of developmental anesthetic neurotoxicity is rapidly accumulating but clarity about the mechanisms or the significance of this phenomenon for human pediatric anesthesia is not emerging. A change in clinical anesthetic practice is unwarranted, based on the currently available human literature and should probably not be based on animal studies. Most importantly, a change in clinical practice requires a superior alternative to current practice, and no evidence guides us as to what this might be. More research is urgently needed to determine whether anesthesia impairs brain function in humans, what the specific deficit is, and how it can be prevented and/or treated. This will require both human trials and good translational animal models and mechanistic studies. The SmartTots initiative, a joint effort of the International Anesthesia Research Society and the Food and Drug Administration, through funding such research, may go a long way toward meeting this important goal.

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