Neurotoxicity of Anesthetic Drugs in theDeveloping BrainGreg Stratmann, MD, PhD
Anesthesia kills neurons in the brain of infantile animals, including primates, and causespermanent and progressive neurocognitive decline. The anesthesia community and regulatoryauthorities alike are concerned that is also true in humans. In this review, I summarize whatwe currently know about the risks of pediatric anesthesia to long-term cognitive function. Ifanesthesia is discovered to cause cognitive decline in humans, we need to know how toprevent and treat it. Prevention requires knowledge of the mechanisms of anesthesia-inducedcognitive decline. This review gives an overview of some of the mechanisms that have beenproposed for anesthesia-induced cognitive decline and discusses possible treatment options. Ifanesthesia induces cognitive decline in humans, we need to know what type and duration ofanesthetic is safe, and which, if any, is not safe. This review discusses early results ofcomparative animal studies of anesthetic neurotoxicity. Until we know if and how pediatricanesthesia affects cognition in humans, a change in anesthetic practice would be premature,not guided by evidence of better alternatives, and therefore potentially dangerous. TheSmartTots initiative jointly supported by the International Anesthesia Research Society andthe Food and Drug Administration aims to fund research designed to shed light on theseissues that are of high priority to the anesthesia community and the public alike and thereforedeserves the full support of these interest groups. (Anesth Analg 2011;113:1170–9)
For �150 years of anesthetic practice, it was believedthat as a general anesthetic wears off, the brain wouldreturn to the same state as before the anesthetic. We
are now beginning to understand that this basic premise ofanesthetic pharmacology is false. In 2003, Jevtovic-Todorovic et al.1 presented their sentinel findings that acombined anesthetic (midazolam, nitrous oxide, and isoflu-rane) administered to 7-day-old rats for 6 hours killsneurons in the developing brain and causes long-termimpairment of brain function. They showed that long-termpotentiation (LTP) in the hippocampus was impaired inanesthetized rats.1 LTP is a form of synaptic plasticity, oftenconsidered the electrophysiologic correlate of learning andmemory, and the hippocampus is a brain structure impor-tant for learning and memory. More importantly, theseauthors demonstrated a progressive deficit in spatial rec-ognition tasks administered both 4 weeks and 4.5 monthsafter anesthesia.1 Immediately, concern mounted withinthe anesthesia community2–5 and also within regulatory
authorities6 about whether these phenomena might applyto humans. Subsequently, it became clear that the histologicdata were reproducible not only in rodents but also invirtually every species tested,7 including primates,8–10 fur-ther heightening the degree of concern about anesthesia inthe immature human brain. A Food and Drug Administra-tion advisory committee meeting in 2007 concluded that nochange in clinical practice is justified based on availabledata,6 and a follow-up meeting in March 2011 upheld thisrecommendation.
It is uncertain if it will ever be feasible to test whetheranesthesia kills neurons in the brain of children. However,this may not be entirely necessary. A focus on anesthesia-induced neurodegeneration seems appropriate only if someaspect of brain function in humans was changed perma-nently by anesthesia, and if a causal link betweenneurodegeneration and long-term brain function couldbe demonstrated in animals. Let us examine these 2premises in more detail.
ANESTHESIA AND BRAIN FUNCTION IN HUMANSUntil recently, speculation as to whether developmentalanesthetic neurotoxicity might exist in humans occurredmostly on the basis of studies that were not specificallydesigned to address this question.2–5,7,11 Since 2009, 7publications appeared that were designed to shed light onwhether anesthesia in humans might impair brain functionlong-term.12–18 Unfortunately, for a number of reasonsdiscussed below, the issue remains far from being resolved.
Wilder et al.12 studied whether anesthesia administeredat younger than 4 years was associated with learningdisabilities between ages 5 and 19 years. A cohort of 5357children born in Olmsted County, Minnesota, between 1976and 1982 was assessed for the presence, type, and durationof anesthesia administered before age 4 years. Anesthesiaadministered for both surgical and diagnostic procedures
From the Department of Anesthesia and Perioperative Care, University ofCalifornia San Francisco, San Francisco, California.
Accepted for publication August 1, 2011.
This manuscript was handled by Peter J. Davis, MD.
The author declares no conflicts of interest.
Portions of this article are adapted from the California Society of Anesthe-siologists online Continued Medical Education Pediatric Anesthesia series,Module 4: Neurotoxicity of Anesthetic Agents in the Developing Brain by GregStratmann, MD, PhD. These sections are reproduced with permission of theCalifornia Society of Anesthesiologists. Release date: June 14, 2011. Availableat: http://www.csahq.org/cme2/course.module.php?course�11&module�34&terms�show.
Reprints will not be available from the author.
Address correspondence to Greg Stratmann, MD, PhD, Department ofAnesthesia and Perioperative Care, University of California San Francisco,Box 0464, Room U286, 513 Parnassus Ave., San Francisco, CA 94143.Address e-mail to [email protected].
was included in the analysis. The school district in whichthe study was performed routinely administered reading,writing, and math aptitude tests as well as intelligencetests. In this study, learning disability was defined as aperformance on standardized achievement tests below acertain predicted score based on the child’s IQ. If any of 3different definitions used by the school district to identifydisabled learning applied, the primary outcome of thisstudy, learning disability, was considered to be present andstudy follow-up ceased at this point. Eleven percent ofchildren underwent at least 1 anesthetic before age 4 years,of whom 24% underwent �1 anesthetic. Learning disabili-ties were more common in those children who had �1anesthetic, and cumulative anesthetic duration of �2 hourswas a risk factor for learning disability. Learning disabilitywas not more common if only 1 anesthetic exposureoccurred before age 4 years. Because children requiring �1anesthetic were sicker than those requiring only a singleanesthetic, the authors performed a subgroup analysis ofchildren requiring �1 anesthetic with ASA physical statusI and II and excluding those with ASA physical status IIIand IV. Despite including only less sick children withmultiple anesthetic exposures, the association betweenlearning disabilities and anesthesia persisted. Methodo-logic advantages of this study are that studying a birthcohort does not bias surgical procedures and comorbiditiesin the same way recruitment of a cohort of patients from anacademic center might. Furthermore, controlling for IQseems like an elegant approach to controlling for one of thestrongest confounders of a child’s ability to learn. Generalmethodologic drawbacks include retrospective analysis ofa retrospective cohort, which forces studying an outcomevariable that is available rather than one that is chosenprospectively. Learning disability is a very nonspecificoutcome and many underlying pathologies may impair achild’s ability to learn, for example, motivation, attention,intelligence, sensory neural problems, or other, more-specific functional abnormalities, all of which may haverelevance to anesthetic developmental neurotoxicity. Otherdrawbacks include that the anesthetic almost uniformlyadministered to the study cohort was halothane/nitrousoxide, which is now an outdated anesthetic in most pedi-atric anesthetic practices. Reporting the cumulative inci-dence of learning disabilities requires that follow-up isstopped when learning disability is detected. In otherwords, once a child meets the criterion for learning disabili-ties, it is assumed that learning disabilities persist andnever resolve. This makes it impossible to comment on thetrue prevalence of the outcome. It is possible that childrenwith learning disabilities at some point may have a changein performance that places them back in the normal range,an event that cannot be captured by the current studydesign. However, it might be possible that anesthesia-associated learning disabilities progress, as has been sug-gested for anesthesia-induced neurocognitive dysfunctionin animals.1,19–21 The current study design would not beable to detect progression of cognitive disability. Likewise,this methodology would not capture the spuriousness ofthe outcome. For example, a low aptitude score in any oneof the tested domains, for whatever reason, would triggerthe diagnosis of learning disability, as would a spuriously
high IQ score, resulting in a predicted aptitude score thatmight render an otherwise normal aptitude to be classifiedas meeting the learning disability criterion.
The same group16 reported later that year that generalanesthesia for cesarean delivery does not increase thecumulative incidence of learning disabilities in the same12
birth cohort of children. This is consistent with their earlierstudy12 because cesarean delivery required one single,rather short anesthetic. Surprisingly, children born bycesarean delivery under regional anesthesia had a lowercumulative incidence of learning disability than those bornby vaginal delivery.16 The significance of this finding isunclear and requires further study. However, this studysuggests that a brief general anesthetic during late fetal lifeis not associated with later cognitive problems. The retro-spective nature of this study confers the same limitations tointerpretation of the data that apply to their previousstudy.12
Kalkman et al.13 approached the problem from a differ-ent and interesting angle. They argued that anesthesia ismostly administered to tolerate a surgical procedure.Therefore, to draw conclusions about the effects of anes-thesia versus surgery on cognitive outcome, an unanesthe-tized control group undergoing surgery would be requiredor anesthesia would have to be administered to childrenwho do not need it, neither one of which is ethicallyfeasible. The authors further assumed that there is a distinctperiod of vulnerability to the effects of anesthesia onneurodevelopment, as suggested by animal studies usinghistologic outcomes.8,20,22–24 Based on this assumption, theauthors hypothesized that children anesthetized during theperiod of vulnerability (earlier in life) should have a worsecognitive outcome than children anesthetized after theperiod of vulnerability. They defined the period of vulner-ability in humans as younger than 2 years of age.13 Thisdesign circumvents the issue of requiring an unobtainablecontrol group and allows children anesthetized later in lifeto serve as controls. The authors used scores from the ChildBehavioral Checklist to identify behavioral abnormalitiesand found that children anesthetized at younger than 2years of age tended to have a higher incidence of clinicallydeviant behavior than children anesthetized at older than 2years, undergoing the same (urological) procedures. Thedifference was even more pronounced between childrenundergoing anesthesia at younger than 6 months of agecompared with older than 2 years. However, neither effectwas pronounced enough to reach statistical significance. Asample-size calculation revealed that �6000 childrenwould have to be studied to show the difference, given theeffect size comparing children younger than 2 years withthose of more than 2 years at the time of anesthesia, and�2200 patients if the sample-size calculation was based onthe effect size comparing children younger than 6 monthsand older than 2 years of age at the time of anesthesia.13
Although the authors chose an innovative and logicalapproach to a difficult ethical dilemma, the validity of theobservation is based on the assumption that the period ofvulnerability in humans is limited to 2 years of age. Thisassumption may or may not be correct. It has been perpetu-ated over the years that the period of vulnerability coin-cides with the peak of synaptogenesis, which is also known
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as the brain growth spurt. The publication frequently citedin support of this is a scholarly article by Dobbing andSands,25 which does not mention synaptogenesis. Instead,it synthesizes knowledge from various brain weight studiesand proposes a hypothesis of how to relate vulnerability toenvironmental and nutritional challenges among differentanimal species. Appropriately, there are several notes ofcaution regarding the limitations for interpreting theirhypothesis, for example, that the term “brain growth spurt”is an oversimplification because different areas of the samebrain develop at different paces.25 Indeed, the peak ofsynaptogenesis in many structures of the rodent brain,including the cortex and the dentate gyrus of the hip-pocampus, does not occur until postnatal days 11 to 16, andsynaptogenesis seems to persist until at least postnatalday 32.24,26–30 Even within a given cortical neuron, synap-togenesis is not a uniform phenomenon.28 The period ofvulnerability to anesthesia-induced neuronal apoptosis oc-curs before postnatal days 10 to 14,8,22,23 and is thus notwell aligned with the peak of synaptogenesis. Most impor-tantly, the period of vulnerability to the long-term behav-ioral effects of anesthetic drugs extends to at least postnataldays 14 to 17 in the rat.20 Rats reach sexual maturity atpostnatal day 50.31 It must be concluded that the period ofvulnerability to the outcome of interest—the long-termcognitive effects of anesthesia—well extend way past 2years of age in humans. Consequently, the estimate byKalkman et al. of the anesthetic effect on behavior might, ifanything, be an underestimate.
The advantages and disadvantages of various studydesigns had been discussed in an editorial by the thirdcontributors to the current human literature on long-termcognitive effects of anesthesia.11 According to these au-thors, the power of studying a prospective cohort must bebalanced against the lead time for data to become available.For example, if enrollment of a randomized, controlled trialof regional versus general anesthesia for pediatric surgicalprocedures were completed today, data of remote neurobe-havioral outcomes would not be available for years orperhaps decades. Given the urgency with which data ondevelopmental neurobehavioral end points after anesthesiaadministration in humans are sought, a long lag time isarguably unacceptable. Thus, the authors11 concluded thatan ideal combination of lag time and design strength wouldbe prospective analysis of a retrospective cohort. The samegroup14 later studied whether hernia repair at age 3 yearsor younger is associated with subsequent behavioraland/or developmental disorders. A set of 383 medicarerecords listing procedure codes related to hernia repair wascompared with a control set of 5050 age- and sex-matchedmedicare records not listing these procedure codes. Childrenyounger than age 3 years were included. The behavioraloutcome was defined as a diagnostic code for unspecifieddelay or behavioral disorder, mental retardation, autism,and language and speech disorder. If the behavioral out-come preceded the surgery, the record was excluded. Aftercontrolling for age, sex, race, and the presence of confound-ing diagnoses at birth, procedure codes indicating herniarepair were more than twice as likely to be associated withthe behavioral outcome codes as when procedure codes forhernia repair were absent. The study design did not allow
for assessing the type, frequency, and duration of theanesthetic in either the hernia repair or the control group. Itwas not possible to exclude that children in the controlcohort did not have an anesthetic for procedures other thanhernia repairs. Perhaps the most interesting finding of thisstudy is the delay with which the behavioral outcomepresented; in this case, 3 to 4 years after the surgery. This isreminiscent of animal studies, suggesting a progressivenature of the deficit.1,19–21
Recently, the academic performance of a national cohortof Danish 15- to 16-year-old adolescents (n � 2689) whohad undergone inguinal hernia repair between 1986 and1990 at the age of 1 year or younger was compared with arandom sample of 14,575 age-matched controls.17 Whenimportant confounders such as gender, birth weight, andparental age and education were controlled for, there wasno evidence that the relatively brief (presumed by theauthors to be 30–60 minutes) general anesthetic had af-fected academic achievement scores. All of the aboveconfounders more strongly affected academic achievementthan surgery plus anesthesia.17 This is despite the fact thatchildren were younger than 12 months at the time ofsurgery, and thus may be considered more sensitive to theeffects of anesthesia than older children.13 The authorsappropriately concluded that these reassuring results can-not exclude deficits in more particular cognitive domains. Itis understood that the effects of longer anesthetic durationsare likewise not detectable with this study design.
Another human trial was designed to test whether thereis a causality between anesthesia administered at youngerthan 3 years and between 3 and 12 years and cognitiveperformance. The authors studied 1143 pairs of monozy-gotic twins hypothesizing that if anesthesia and not theunderlying disease caused cognitive disabilities, then theexposed twin should have a higher incidence of under-achievement than the unexposed twin. Most pairs of twinsin this study consisted of twins who were either bothexposed or both not exposed to anesthesia. However, 71twin pairs (15%) were discordant (one twin exposed, theother not exposed to anesthesia). Anesthesia was adminis-tered mostly for surgical procedures. Exposed twins hadsimilar achievement scores on a nationwide test at 12 yearsof age to unexposed twins, and a similar incidence ofcognitive problems, as assessed by a teacher questionnaire.The authors concluded that the comorbidity but not thecombination of anesthesia and surgery is the cause of thecognitive problems. If these results can be duplicated, theywould make a convincing argument that neither anesthesianor surgery is a problem for the cognitive development ofchildren.
DiMaggio et al.18 subsequently came close to doing justthat by identifying 10,450 twins of unknown zygocity (i.e.,“siblings”), 306 of whom had been exposed to anesthesiaduring a surgical procedure before age 3 years and 10,146of whom had not. Of the 138 discordant pairs in which only1 of the 2 twins was exposed to anesthesia, neither siblingof 107 pairs had International Classification of Diseases, NinthRevision (ICD-9) diagnostic codes that would suggest aproblem with brain development, and both siblings of 11pairs had such ICD-9 codes subsequent to the procedure ofthe exposed twin.
When only 1 twin of a pair discordant for anestheticexposure had an ICD-9 code suggesting a problem withbrain function (n � 20), there was an even split of thesecodes between exposed (n � 9) and unexposed twins (n �11). This supports the findings by Bartels et al.15 that thereis no causal relationship between anesthetic exposure andbrain dysfunction as measured by the occurrence of ICD-9codes subsequent to the surgical procedure. A similarconclusion can be drawn from another finding from thatsame study,18 namely that the hazard ratio of behavioral/developmental diagnosis was 1.6 when anesthesia occurredbefore the first occurrence of the ICD-9 code, but 1.3 whenthe ICD-9 code appeared before the anesthetic exposure.In this latter case, anesthesia could not possibly have causedthe behavioral/developmental diagnosis. The fact there isnonetheless an association between anesthetic exposureand behavioral/developmental diagnosis in this case high-lights the existence of confounders, which is unavoidablegiven the study design. The authors also found anincreasing likelihood of an ICD-9 code of a behavioral/developmental diagnosis with multiple anesthetics.Whether this represents an anesthetic dose response or agreater burden of disease is unclear.
In summary, the human literature is controversial as towhether anesthesia in infancy causes cognitive problemslater in life. Furthermore, it is unclear what the period ofvulnerability to anesthetic neurotoxicity is. We do notknow whether there is a safe anesthetic technique orduration. The specific cognitive deficit caused by anesthe-sia, if any, that may underlie such outcomes as learningdisabilities, has not been identified. None of the studies,alone or in combination, form a basis for informing clinicalpractice.
ANIMAL STUDIESAs discussed above, it is not entirely clear whether long-term cognitive dysfunction, the most worrisome feature ofdevelopmental anesthetic neurotoxicity, occurs in humans.In the meantime, animal models of anesthesia are impor-tant in furthering our understanding of the phenomenology,pharmacology, and the mechanism of anesthesia-inducedneurocognitive dysfunction. To that end, a recent study21 inmonkeys demonstrating a persistent and progressive declinein cognitive domains after ketamine anesthesia is the latest ina series of alarming studies suggesting that anesthesia givento immature mammals impairs brain function later in life.
Understanding the mechanism by which anesthesiaimpairs brain function months after anesthesia in infancywould allow us to develop rational preventive strategies,and is thus a very important, yet currently elusive, mile-stone in this field. Implicit in the concept of a mechanism isthe concept of causality. Although causality might beimpossible to prove, it is usually accepted on the basis of(good) enough evidence for and insufficient or bad evi-dence against such a link.32 If anesthesia caused cognitivedysfunction, the mechanism by which anesthesia causedcognitive dysfunction would be causally linked to bothanesthesia and cognitive dysfunction. The following dis-cussion suggests that the mechanism of anesthesia-inducedcognitive dysfunction or decline, as the case may be,1,19–21,33
is much less clear than previously thought. Specifically, I
discuss evidence for each of 3 cellular phenomena toqualify as a mediating mechanism of anesthesia-inducedcognitive decline: neurodegeneration, synaptogenesis, andhippocampal neurogenesis.
NEURODEGENERATIONIt is now accepted, on the basis of overwhelming experi-mental evidence,7 that anesthesia causes neurodegenera-tion in a variety of animal species, including primates.8,9
Few would dispute that the possibility of anesthesia caus-ing neurodegeneration in humans is real, although it willbe very difficult to prove this definitively. Furthermore,whether or not anesthesia-induced neurodegeneration hap-pens in humans is not nearly as important as whetheranesthesia impairs cognition in humans. What is important,however, is to define the role of anesthesia-induced neurode-generation in causing anesthesia-induced cognitive decline.Unless anesthesia-induced neurodegeneration mediatedthe anesthesia-induced cognitive outcome, it wouldmerely be an epiphenomenon with little significance tocognitive function. When anesthesia was first shown tocause both neurodegeneration and cognitive decline inrats,1 a causal link between the 2 outcomes must haveseemed so plausible that it was not as rigorously scruti-nized as other, less intuitive potential mechanisms. Toaddress this question in more detail, we must considerthe evidence for and against such a causal link.
It is difficult to comprehend how a one-time anestheticexposure, which increases neuroapoptosis, can have aneurobehavioral 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, ifmonths after anesthesia the brain of a formerly anesthe-tized person or animal was indistinguishable from a brainthat was not exposed to anesthesia, it would be hard toargue that anesthesia caused the brain to be dysfunctional.Applied to neurodegeneration, this means that severalmonths after anesthesia a causality between anesthesia-induced neurodegeneration and anesthesia-induced cog-nitive dysfunction would be difficult to accept unlessneurodegeneration had somehow altered the brain ofanesthetized animals. If neurons destroyed by anesthesialeft a detectable gap in the brain or if the neuronal numberwas different from unanesthetized animals, a reasonableargument could be made that neurodegeneration qualifiesas a potential mediating mechanism for the cognitiveoutcome. Rizzi et al.34 used pregnant guinea pigs to showthat a triple anesthetic cocktail consisting of 0.55% isoflu-rane, 75% nitrous oxide, and 1 mg/kg midazolam for 4hours, but not fentanyl 15 mg/kg/h, caused acute neuro-degeneration in the offspring. They also found that theneuronal density in the first postnatal week was reduced by30% to 50% in the offspring that had been exposed toanesthesia.34 The authors concluded that the observeddegree of anesthesia-induced neuronal deletion far ex-ceeded the approximately 1% neuronal deletion observedin their prior studies and therefore suggests that neuronsare permanently lost.34 It could be argued, however, thatthe observed anesthesia-induced neuronal deletion is wellin line with the normal rate of developmental apoptosis,
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which is usually at least 50%.35,36 However, neurons dyingduring development are immature,37–39 but the postmitoticage of neurons killed by anesthesia is not yet known.Nonetheless, the authors34 observed a significant differencebetween neuronal deletion after the triple anesthetic versusthe fentanyl infusion or no anesthetic. The absence of abehavioral assessment with concomitant assessment ofneuronal density does not allow us to conclusively deter-mine whether the neuronal density that was abnormal inthe first postnatal week34 would also have been abnormalat the time of neurocognitive testing. This study is alsointeresting in that guinea pigs, like humans and unlike rats,have a relatively mature brain at birth. Anesthetizing thepregnant mother with drugs that cross the placenta iselegant in that it allows for hemodynamic monitoring oreven hemodynamic control of the mother and thus indi-rectly of the fetus, which is very difficult in neonatal orinfantile rodents. Furthermore, temperature and nutritionalstatus can be more easily controlled than in newbornrodents. However, if anesthesia in utero is administered tooclose to delivery, maternal oxytocin might change neuronalvulnerability to anesthesia by temporarily shifting thechloride reversal and causing immature neurons to beinhibited by �-aminobutyric acid (GABA), which is usuallycharacteristic of mature neurons.40 This peripartum modelof anesthesia is reminiscent of a human trial discussedabove16 in which no adverse cognitive sequelae could bedemonstrated by general anesthesia for delivery after ad-justing for important confounders. The discrepant resultsdo not allow us to conclude that neurodegeneration is notimportant for cognitive outcome, partly because the anes-thetic durations differed dramatically between the 2 stud-ies. It is not known whether a triple anesthetic cocktailadministered for the duration required to perform a cesar-ean delivery (presumably an hour or less) causes neurode-generation or permanent neuronal deletion in an animalmodel. The most compelling evidence that acute neurode-generation causes lasting neuronal deletion resulted from 2rat studies by the same group.41,42 Rats that were anesthe-tized on postnatal day 7 had neuronal deletion at postnatalday 3041 and ultrastructural abnormalities suggestive ofongoing cell death on day 21.42 This is approaching the ageat which the same group previously demonstrated learningand memory deficits in rats.1 These results would bestrengthened if it could be demonstrated that the totalnumber of neurons was decreased long-term. This requiresassessment of the volume of the structure of interest, whichdid not occur in the above studies. The results would beeven stronger if animals with a proven learning andmemory deficit had neuronal deletion, and strongest ifthose animals with the worst brain function were thosewith the greatest degree of neuronal deletion. Anothergroup43 did not find a long-term effect of isoflurane duringinfancy on neuronal density in 2 brain regions most se-verely affected by acute cell death in mice. Because thevolume of these structures was not assessed, it is impos-sible to know what the total number of neurons in thesestructures was. Interestingly, learning and memory werenot affected in this study, which would suggest either thatisoflurane does not affect long-term neurocognitive functionor that the degree of acute cell death does not determine
long-term neurocognitive outcome.43 The latter possibilitywas suggested by another study,44 demonstrating that 2hours of isoflurane but not 1 hour of isoflurane at 1minimum alveolar concentration (MAC) causes neurode-generation. However, despite extensive neurodegenera-tion, mainly in the thalamus and cortex, no long-termneurocognitive sequelae were demonstrated by 2-hourisoflurane. From a functional standpoint, 2-hour isofluraneseems to be a safe dose in rats. This conclusion is supportedby the finding that in the same study,44 4 hours of isoflu-rane caused long-term learning and memory problems.Hence, the absence of neurocognitive deficits after 2-hourisoflurane was not attributable to a general inability todemonstrate neurocognitive dysfunction. Isoflurane at 1MAC given to rats at the peak of vulnerability to develop-mental anesthetic neurotoxicity (postnatal day 7) causesrespiratory depression and hypercarbia. Hypercarbia alonefor 4 hours caused a similar degree and distribution of celldeath in the brain as 4-hour isoflurane, but instead ofimpairing brain function long-term, rats exposed to 4-hourhypercarbia at postnatal day 7 outperformed all othergroups, including the control group. Hypercarbia alonecaused robust improvement in long-term neurocognitivefunction despite causing extensive cell death in the devel-oping brain. Collectively, these findings suggest that thedegree to which an intervention causes acute neurodegen-eration does not always determine long-term cognitiveoutcome.
An important prediction required by the concept thatanesthetic neurodegeneration is responsible for later cognitivedysfunction is that interventions preventing anesthesia-induced neurodegeneration also prevent anesthesia-inducedlong-term neurocognitive sequelae. Examples of such inter-ventions include melatonin,45 lithium,46 dexmedetomidine,47
inhibitors of the p75 neurotrophin receptor (TAT-conjugatedPep5 or Fc-p75NTR),48,49 hypothermia,50 and bumetanide,51 allof which have been shown to prevent anesthesia-inducedneurodegeneration.
The rationale for using melatonin to counteract the effectof anesthesia is the demonstration that anesthesia causesneuronal apoptosis via a mitochondria-dependent pathwayamong others, which is associated with biochemicalchanges that melatonin had previously shown to counter-act.45 Furthermore, melatonin has several other nonspecific,protective effects in the brain.45 Melatonin was found, in adose-dependent manner, to reduce anesthesia-inducedneuronal apoptosis in rats.45 The authors suggested that itsbioavailability, lipophilicity, ability to cross the blood-brainbarrier, absence of toxicity, and sleep-inducing and analge-sic effects make it an ideal adjuvant for anesthesia.45
Surprisingly, it is not known whether melatonin reversesthe long-term behavioral effects of anesthesia.
Another group46 showed that lithium protects againstanesthetic neurotoxicity in the developing brain. Theyargued that lithium is known to counteract extracellularsignal–regulated protein kinase inhibition and neurodegen-eration caused by alcohol. Alcohol acts via antagonism ofN-methyl-d-aspartate (NMDA) receptors and by facilitatingGABAergic receptor transmission. Hence, they hypothesizedthat a combination of an NMDA antagonist anesthetic,ketamine, and a GABAergic drug, propofol, should cause
similar effects as alcohol on the developing brain, and thatthese effects should be preventable by coadministration oflithium. This hypothesis was largely confirmed and theauthors concluded that lithium may be an effective adju-vant to anesthesia, provided that it can be demonstratedthat the inhibition of naturally occurring apoptosis, whichis also caused by lithium, has no ill effects.46 This may be animportant caveat, because naturally occurring neuroapop-tosis is critically important for brain development,36 andwhen this process is inhibited, learning and memory areimpaired.52 It is not known whether lithium can preventanesthesia-induced neurocognitive decline.
Developmental anesthetic neurotoxicity has largely beenattributed to the combination of GABAergic and NMDAantagonist actions of anesthetic drugs. Dexmedetomidineis neither a GABAergic nor NMDA antagonist and hastherefore been hypothesized to be free of developmentalanesthetic toxicity.47 Furthermore, it has a number ofantiapoptotic effects, and thus Sanders et al.47 hypothesizedthat it might protect against anesthesia-induced neuronalapoptosis. Dexmedetomidine reduced neuronal apoptosiscaused by a subanesthetic dose of isoflurane for 6 hours ina dose-dependent manner, which was reversed by blockingthe �-2 adrenoceptor, indicating that the protective effect ismediated by this receptor. Furthermore, dexmedetomidineprevented an isoflurane-induced impairment in trace fearconditioning at 40 days of age. This is the only study to dateof an intervention that nonspecifically protected fromanesthesia-induced cell death that also protected fromanesthesia-induced neurocognitive dysfunction. The be-havioral outcome was virtually devoid of interindividualvariability, which is unusual for behavioral experimentswhen using rats from different litters.53 Even the rate ofneuroapoptosis is usually subject to substantial litter vari-ability.54,55 Either way, these results require confirmation inboth animal and human studies before considering achange in practice. An important mechanistic finding ofthis study47 is that the neurodegeneration caused by iso-flurane was not prevented by a GABAA-receptor antago-nist, indicating that this receptor does not mediate theneurodegeneration caused by GABAergic drugs.47
Creeley and Olney50 advanced an interesting hypothesison the basis of a 2-part assumption: anesthesia decreasesneuronal activity in the developing brain with subsequentwithdrawal of trophic support and neurodegeneration.They argued that another intervention known to decreaseneuronal activity, hypothermia, should therefore causeneurodegeneration, and found the exact opposite. Hypo-thermia (30°C) protected against isoflurane-induced andketamine-induced neurodegeneration.50 This indicateseither that neuronal inactivity does not cause neurodegen-eration or that anesthesia does not cause neuronal inactiv-ity. This latter possibility is actually a given for GABAergicdrugs, which cause neuronal excitation in immature neu-rons, rather than neuronal inhibition, as is true in matureneurons. The mechanism of neuronal excitation in imma-ture neurons is immaturity of a chloride transporter beforethe second postnatal week in rats56 and the first 3 to 12months in humans.57 Because isoflurane is a predominantlyGABAergic anesthetic, it should cause neuronal excitationin immature brains or immature parts of the brain. It may
therefore be no surprise that hypothermia failed to repro-duce the isoflurane-induced neurodegeneration. Sevoflu-rane has been shown to cause neuronal excitation in theimmature brain, which was actually postulated to be themechanism underlying sevoflurane-induced neurodegen-eration.51 It is not known whether hypothermia protectsagainst anesthesia-induced neurocognitive dysfunction.
Substantial insight into what does mediate anesthesia-induced developmental neuroapoptosis is provided by 2elegant studies.48,49 In the first study, Head et al.48 showedthat inhibitors of the p75 neurotrophin receptor preventisoflurane-induced cleaved caspase 3 expression in vitroand 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. Ifpro-BDNF remains uncleaved, it interacts with the p75neurotrophin receptor and acts as a cell death signal.Plasmin is cleaved from plasminogen by tissue plasmino-gen activator, which is released from the presynapticterminal when neurons are firing. The authors48 inter-preted their findings as confirmation that neuronal silenc-ing caused a shift in the balance of BDNF signaling topreferentially occur via pro-BDNF as opposed to matureBDNF. The authors48 went to great lengths to elegantlyexclude alternative interpretation of their findings. How-ever, they did not show that isoflurane actually decreasesneuronal firing in immature neurons. As stated above,isoflurane is not expected to decrease neuronal firing inimmature neurons. In fact, the opposite is true in thatisoflurane or any other GABAergic anesthetic should causeneuronal excitation.56 A possible explanation for thesediscrepancies comes from the observation that in a cellculture model, such as the one used by Head et al.,48
glucose is commonly used as an energy substrate, whereasthe predominant energy substrate of the developing brainis ketone bodies.58,59 Glucose causes a shift in the chloridereversal potential of neurons in culture that makes them actlike mature neurons.58,59 Mature neurons are indeed si-lenced by isoflurane. The authors48 also found that isoflu-rane decreases the number of immature dendritic spines invitro and the number of synapses in 5- to 7-day-old mice.This reduction in synaptic density was attenuated by thep75 neurotrophin receptor blocker TAT-Pep5.48 Impor-tantly, these authors48 demonstrated that their interventionis nontoxic and does not cause an unwanted suppression ofnaturally occurring neuronal apoptosis. This is an advan-tage over nonspecific modalities that ameliorate anesthesia-induced neurodegeneration, such as lithium, melatonin,dexmedetomidine, or hypothermia.
In a second study, the same group49 showed that theeffect of isoflurane-induced p75 neurotrophin receptorsignaling on synaptogenesis and neurodegeneration is me-diated via activation of RhoA, a kinase causing actindepolymerization. This causes growth-cone collapse, lossof immature dendritic spines, and, presumably, the loss ofsynapses observed in their previous study.48 The authorsalso observed expression of cleaved caspase 3, a marker forapoptotic cell death. When signaling via the p75 neurotro-phin receptor was inhibited or when the cytoskeleton wasstabilized, isoflurane-induced loss of dendritic spines and
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expression of cleaved caspase 3 was attenuated. This sug-gests that anesthesia causes actin depolymerization viaRhoA activation, which in turn causes loss of dendriticspines and apoptotic cell death. It is unknown whetherp75NTR antagonism or cytoskeletal stabilization can pre-vent anesthesia-induced neurocognitive dysfunction.
In another elegant study, Briner et al.30 confirmed thatpropofol, either as a single shot of 40 mg/kg or givenrepeatedly over 6 hours at half that dose, decreased synap-tic spine density in 5-day-old rodents and increased spinedensity in 15- to 25-day-old rodents. Amazingly, both the6-hour duration as well as the single shot of propofolcaused persistent changes into adulthood, indicating that asingle, brief anesthetic to an anesthetic depth that wouldnot permit a surgical procedure, is sufficient to perma-nently alter cortical synaptic spine densities. This workconfirms results of decreased synaptogenesis at approxi-mately postnatal day 5,48,49 and their own previous re-sults24,60 of rapid increase in synaptogenesis after postnatalday 15 in the cortex and the hippocampus. Consistent withprevious results,22 no neurodegeneration occurred in thecortex of 16-day-old rats,24 confirming that the period ofvulnerability to anesthetic apoptotic cell death is limited topostnatal day 10. Importantly, it was recently shown thatthe period of vulnerability to anesthesia-induced neurocog-nitive decline extends to at least postnatal day 17 in rats,20
a time at which neurons are no longer sensitive to theapoptotic effects of anesthesia.20,22,24 If these results20 canbe confirmed, the causal link between anesthesia-inducedneurodegeneration and anesthesia-induced neurocognitivedecline would be further weakened. Also, it would need tobe explained how an anesthesia-induced decrease30,48,49 orincrease in synaptogenesis24,30,60 could both be responsiblefor the same outcome (anesthesia-induced neurocognitivedecline).
One interesting feature of the age-dependent switch inanesthetic effect on synaptogenesis24,30,48,49,60 is that itnearly parallels the age-dependent switch in the chloridereversal potential and thus the excitatory to inhibitoryswitch in GABA phenotype.56,57 However, mechanisticallylinking the developmental switch in GABA phenotype withthe switch from an anesthesia-induced decrease to increasein dendritic spines, although possibly accounting for the illeffects of GABAergic drugs in the immature brain, wouldnot readily account for cellular or behavioral phenomenacaused by NMDA antagonists; for example, the progressivecognitive decline in monkeys treated with ketamine duringearly postnatal development.21
It has been assumed that anesthesia-induced neuronalsilencing is responsible for anesthesia-induced effects onsynaptogenesis and apoptosis, which is difficult to consoli-date with the switch in the GABAergic phenotype fromexcitatory to inhibitory at exactly the time at which vulner-ability to anesthesia-induced neuronal apoptosis ceases.This assumption was formally challenged in a recentstudy51 demonstrating that sevoflurane causes global brainexcitation in rats, which is entirely compatible with amotionless animal. These nonconvulsive seizures wereassociated with neuronal cell death. Bumetanide, whichinhibits the immature chloride transporter responsible forthe excitatory action of GABA during early development,
prevented both the sevoflurane-induced seizures andsevoflurane-induced neurodegeneration.51 Interestingly,bumetanide did not prevent the functional consequences ofsevoflurane, namely, a reduction in hippocampal LTP, theelectrophysiologic correlate to learning and memory.51
Anesthesia-induced neurodegeneration had previouslybeen associated with reduced hippocampal LTP.1 The factthat prevention of anesthesia-induced neurodegenerationdid not prevent the functional sequelae of anesthesia51
again draws into question the assumption that one causesthe other.
If anesthesia-induced neurodegeneration does not causeanesthesia-induced neurocognitive decline, then whatdoes? It is possible that the age-dependent anestheticeffects on synaptogenesis24,30,48,49,60 can have functionalrelevance independent of whether or not they cause neu-ronal apoptosis. One prerequisite to this claim—persistenceof these effects until the time of neurocognitive testing—has been met.30 Now it must be demonstrated that anintervention that prevents the anesthetic effects on synap-togenesis also prevents the anesthetic effect on cognitivefunction.
Another possible mechanism is an anesthetic effect onpostnatal hippocampal neurogenesis.19,20 Postnatal neuro-genesis occurs in only 2 brain areas, one of which is thehippocampus.61–63 Inhibition of dentate neurogenesis issufficient to impair learning and memory in a mannersimilar to anesthesia.64,65 Of particular interest is the timecourse of the deficits. Neurogenesis is exquisitely sensitiveto brain irradiation66–71; children who underwent brainirradiation developed progressive cognitive decline over anumber of years.72 The deficit caused by anesthesia ishippocampus dependent and seems to progress.1,19–21 Iso-flurane has been shown to impair neurogenesis,19,20 as doesphenobarbital.73 These effects persist until the time ofneurocognitive testing.20,73 If an anesthetic effect on neuro-genesis mediated anesthesia-induced neurocognitive de-cline, interventions that restore neurogenesis should rescuethe behavioral phenotype. Such interventions include en-vironmental enrichment, voluntary exercise, caloric restric-tion, or antidepressant drugs.74–80 We have shown thatenvironmental enrichment reverses the behavioral effectsof anesthesia, even when instituted with a 3-week delayafter anesthetic exposure (unpublished observation). Thetreatment efficacy of environmental enrichment may ormay not be attributable to its effects on neurogenesis.
WHICH ANESTHETIC IS THE SAFEST?This question is slowly beginning to be addressed incomparative toxicity studies in animals. Human studieshave not addressed this issue at all and given the contro-versy as to whether or not functional sequelae of anesthesiain infancy even exist in humans, the argument might bemade that comparative studies are not quite yet indicated.In animal models, whereby anesthetic developmental neu-rotoxicity has been clearly demonstrated, these studies canbe performed relatively easily, with the caveat that anes-thetic equipotency is vitally important for interpretation ofresults of comparative studies. If an anesthetic results inboth greater anesthetic depth and greater anesthetic toxic-ity than another anesthetic, then interpretation of the data
becomes difficult. For example, when it was determinedthat a 3-drug anesthetic combination causes a greaterdegree of neurodegeneration than 2 or 1 anesthetic drug,the 3 drugs were simply added to one another, whichwould have resulted in a greater anesthetic depth than the2-anesthetic combination or the single anesthetic.1 Specifi-cally, the single GABAergic volatile drug isoflurane causedmild cell death at 0.75% atm, which was aggravated by anotherwise nontoxic dose of midazolam (9 g/kg), and madeeven worse by an otherwise nontoxic dose of nitrous oxide(75% atm).1 This has been interpreted as greater toxicitywhen GABAergic and NMDA antagonist drugs are com-bined, but it is unclear whether this does not also reflect aneffect of anesthetic depth. In anesthetic practice as well as inresearch, MAC is used to express anesthetic potency andanesthetic depth.81 Unlike in adult rodents, MAC in imma-ture rodents is not a stable anesthetic concentration butdecreases steadily with increasing duration of anesthe-sia.82,83 The reason for this phenomenon is unclear as iswhether or not this occurs in humans. This steady decreasein anesthetic requirements makes comparative studies ofvolatile anesthetic drugs a challenge.84–87 An example of agood study comparing isoflurane, sevoflurane, and desflu-rane is in press as of the time of this writing.83 In this study,desflurane caused greater neurotoxicity in the immaturemouse brain than near equipotent doses of isoflurane orsevoflurane.83 Sevoflurane had been reported to have amore favorable neurotoxicity profile than isoflurane.85–87
Two recent studies dispute these findings.83,84 However, inboth of these studies, less sevoflurane was used thanisoflurane, despite attempts to achieve equipotency. This isprimarily a result of our incomplete understanding andagreement about how equipotency should be achieved inimmature rodents. The simplest way would be to give afraction of a published, constantly decreasing MAC.82,83,88
Alternatively, a constant anesthetic concentration can beexpressed as percentage MAC over time and the area underthe curve of this plot can be calculated.88 If the areas underthe percentage MAC over time curve are within a certainlimit of agreement (e.g., within 10% of each other), theanesthetic drugs were used at equipotency. The situationbecomes more difficult when an inhaled drug is to becompared with an IV drug. Whereas MAC determinationfor volatile anesthetics is possible in immature rodents, thesame would be much more difficult for IV drugs, because aconstant plasma and brain concentration would have to beachieved to do so. This would require insertion of an IVcannula and infusion of drug with subsequent sampling ofblood and/or brain tissue, a difficult experimental prepa-ration. Furthermore, because it is conceivable that MAC forIV drugs also decreases over time in immature rodents, theabove would have to be done at various time points. Hence,comparative studies between inhaled and IV drugs arecurrently very difficult to interpret. For example, it hasbeen shown that sevoflurane has a favorable neurotoxicityprofile over propofol but it is entirely unclear what theanesthetic depth of these animals was.89
CONCLUSIONKnowledge of developmental anesthetic neurotoxicity israpidly accumulating but clarity about the mechanisms or
the significance of this phenomenon for human pediatricanesthesia is not emerging. A change in clinical anestheticpractice is unwarranted, based on the currently availablehuman literature and should probably not be based onanimal studies. Most importantly, a change in clinicalpractice requires a superior alternative to current practice,and no evidence guides us as to what this might be. Moreresearch is urgently needed to determine whether anesthe-sia impairs brain function in humans, what the specificdeficit is, and how it can be prevented and/or treated. Thiswill require both human trials and good translationalanimal models and mechanistic studies. The SmartTotsinitiative, a joint effort of the International AnesthesiaResearch Society and the Food and Drug Administration,through funding such research, may go a long way towardmeeting this important goal.
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