REVIEW ARTICLE

CME
Neurotoxicity of Anesthetic Drugs in the
Developing Brain
Greg Stratmann, MD, PhD

Anesthesia kills neurons in the brain of infantile animals, including primates, and causes
permanent and progressive neurocognitive decline. The anesthesia community and regulatory
authorities alike are concerned that is also true in humans. In this review, I summarize what
we currently know about the risks of pediatric anesthesia to long-term cognitive function. If
anesthesia is discovered to cause cognitive decline in humans, we need to know how to
prevent and treat it. Prevention requires knowledge of the mechanisms of anesthesia-induced
cognitive decline. This review gives an overview of some of the mechanisms that have been
proposed for anesthesia-induced cognitive decline and discusses possible treatment options. If
anesthesia induces cognitive decline in humans, we need to know what type and duration of
anesthetic is safe, and which, if any, is not safe. This review discusses early results of
comparative animal studies of anesthetic neurotoxicity. Until we know if and how pediatric
anesthesia affects cognition in humans, a change in anesthetic practice would be premature,
not guided by evidence of better alternatives, and therefore potentially dangerous. The
SmartTots initiative jointly supported by the International Anesthesia Research Society and
the Food and Drug Administration aims to fund research designed to shed light on these
issues that are of high priority to the anesthesia community and the public alike and therefore
deserves the full support of these interest groups. (Anesth Analg 2011;113:1170 –9)

F or ⬎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 isoflu-
rane) 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 impor-
tant for learning and memory. More importantly, these
authors demonstrated a progressive deficit in spatial rec-
ognition tasks administered both 4 weeks and 4.5 months
after anesthesia.1 Immediately, concern mounted within
the anesthesia community2–5 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,8 –10 fur-
ther heightening the degree of concern about anesthesia in
the immature human brain. A Food and Drug Administra-
tion 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 perma-
nently 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.

From the Department of Anesthesia and Perioperative Care, University of
California 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 Greg
Stratmann, MD, PhD. These sections are reproduced with permission of the
California Society of Anesthesiologists. Release date: June 14, 2011. Available
at: 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 of
Anesthesia and Perioperative Care, University of California San Francisco,
Box 0464, Room U286, 513 Parnassus Ave., San Francisco, CA 94143.
Address e-mail to stratman@anesthesia.ucsf.edu.
Copyright © 2011 International Anesthesia Research Society
DOI: 10.1213/ANE.0b013e318232066c
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.2–5,7,11 Since 2009, 7
publications appeared that were designed to shed light on
whether anesthesia in humans might impair brain function
long-term.12–18 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

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Anesthesia and the Pediatric Brain
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 disabili-
ties 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. Methodo-
logic 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 pedi-
atric anesthetic practices. Reporting the cumulative inci-
dence 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 disabili-
ties, 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 sug-
gested for anesthesia-induced neurocognitive dysfunction
in animals.1,19 –21 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
November 2011 • Volume 113 • Number 5
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 retro-
spective 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 differ-
ent and interesting angle. They argued that anesthesia is
mostly 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 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.8,20,22–24 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 vulner-
ability 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 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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 REVIEW ARTICLE
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 hip-
pocampus, does not occur until postnatal days 11 to 16, and
synaptogenesis seems to persist until at least postnatal
day 32.24,26 –30 Even within a given cortical neuron, synap-
togenesis is not a uniform phenomenon.28 The period of
vulnerability to anesthesia-induced neuronal apoptosis oc-
curs before postnatal days 10 to 14,8,22,23 and is thus not
well 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 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 au-
thors, 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 neurobe-
havioral 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 out-
come preceded the surgery, the record was excluded. After
controlling for age, sex, race, and the presence of confound-
ing 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
1172 www.anesthesia-analgesia.org
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.1,19 –21
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 af-
fected 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 can-
not 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 monozy-
gotic twins hypothesizing that if anesthesia and not the
underlying disease caused cognitive disabilities, then the
exposed twin should have a higher incidence of under-
achievement 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 adminis-
tered 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.
ANESTHESIA & ANALGESIA
Anesthesia and the Pediatric Brain
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 high-
lights 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 anesthe-
sia, 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 impor-
tant 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, mile-
stone 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 evi-
dence 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 dis-
cussion suggests that the mechanism of anesthesia-induced
cognitive dysfunction or decline, as the case may be,1,19 –21,33
is much less clear than previously thought. Specifically, I
November 2011 • Volume 113 • Number 5
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 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 will
be very difficult to prove this definitively. Furthermore,
whether or not anesthesia-induced neurodegeneration hap-
pens 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 neurode-
generation 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 scruti-
nized 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 anesthe-
tized 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 cog-
nitive 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% isoflu-
rane, 75% nitrous oxide, and 1 mg/kg midazolam for 4
hours, but not fentanyl 15 mg/kg/h, caused acute neuro-
degeneration 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 ex-
ceeded 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,
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 REVIEW ARTICLE
which is usually at least 50%.35,36 However, neurons dying
during development are immature,37–39 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 deter-
mine 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 indi-
rectly 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 ␥-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 ad-
justing for important confounders. The discrepant results
do not allow us to conclude that neurodegeneration is not
important for cognitive outcome, partly because the anes-
thetic durations differed dramatically between the 2 stud-
ies. It is not known whether a triple anesthetic cocktail
administered for the duration required to perform a cesar-
ean delivery (presumably an hour or less) causes neurode-
generation or permanent neuronal deletion in an animal
model. The most compelling evidence that acute neurode-
generation causes lasting neuronal deletion resulted from 2
rat studies by the same group.41,42 Rats that were anesthe-
tized 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 se-
verely affected by acute cell death in mice. Because the
volume of these structures was not assessed, it is impos-
sible 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
1174 www.anesthesia-analgesia.org
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 neurode-
generation. However, despite extensive neurodegenera-
tion, 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 isoflu-
rane 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 develop-
mental 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 devel-
oping brain. Collectively, these findings suggest that the
degree to which an intervention causes acute neurodegen-
eration 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 inter-
ventions 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 counter-
act.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 analge-
sic 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 neurodegen-
eration 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
ANESTHESIA & ANALGESIA
Anesthesia and the Pediatric Brain
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 adju-
vant 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 neuroapop-
tosis 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 ␣-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 be-
havioral 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 vari-
ability.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 iso-
flurane was not prevented by a GABAA-receptor antago-
nist, 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. Hypo-
thermia (30°C) protected against isoflurane-induced and
ketamine-induced neurodegeneration.50 This indicates
either 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 GABAergic
drugs, which cause neuronal excitation in immature neu-
rons, rather than neuronal inhibition, as is true in mature
neurons. The mechanism of neuronal excitation in imma-
ture 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
November 2011 • Volume 113 • Number 5
therefore be no surprise that hypothermia failed to repro-
duce the isoflurane-induced neurodegeneration. Sevoflu-
rane has been shown to cause neuronal excitation in the
immature brain, which was actually postulated to be the
mechanism underlying sevoflurane-induced neurodegen-
eration.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 plasmino-
gen activator, which is released from the presynaptic
terminal 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 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. How-
ever, 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 si-
lenced by isoflurane. The authors48 also found that isoflu-
rane 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 Impor-
tantly, these authors48 demonstrated that their intervention
is nontoxic and does not cause an unwanted suppression of
naturally 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 the
effect of isoflurane-induced p75 neurotrophin receptor
signaling on synaptogenesis and neurodegeneration is me-
diated 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 neurotro-
phin receptor was inhibited or when the cytoskeleton was
stabilized, isoflurane-induced loss of dendritic spines and
www.anesthesia-analgesia.org 1175
 REVIEW ARTICLE
expression of cleaved caspase 3 was attenuated. This sug-
gests 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 pre-
vent 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 synap-
tic 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 perma-
nently alter cortical synaptic spine densities. This work
confirms 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 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 neurocog-
nitive 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 consoli-
date with the switch in the GABAergic phenotype from
excitatory to inhibitory at exactly the time at which vulner-
ability 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,
1176 www.anesthesia-analgesia.org
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 neu-
ronal 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 synap-
togenesis also prevents the anesthetic effect on cognitive
function.
Another possible mechanism is an anesthetic effect on
postnatal hippocampal neurogenesis.19,20 Postnatal neuro-
genesis occurs in only 2 brain areas, one of which is the
hippocampus.61– 63 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 irradiation66 –71; 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.1,19 –21 Iso-
flurane 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 neuro-
genesis mediated anesthesia-induced neurocognitive de-
cline, interventions that restore neurogenesis should rescue
the behavioral phenotype. Such interventions include en-
vironmental enrichment, voluntary exercise, caloric restric-
tion, or antidepressant drugs.74 – 80 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 contro-
versy 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 neu-
rotoxicity has been clearly demonstrated, these studies can
be performed relatively easily, with the caveat that anes-
thetic equipotency is vitally important for interpretation of
results of comparative studies. If an anesthetic results in
both greater anesthetic depth and greater anesthetic toxic-
ity than another anesthetic, then interpretation of the data
ANESTHESIA & ANALGESIA
Anesthesia and the Pediatric Brain
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 Specifi-
cally, 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 com-
bined, 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 imma-
ture rodents is not a stable anesthetic concentration but
decreases steadily with increasing duration of anesthe-
sia.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.84 – 87 An example of a
good 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 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.85– 87
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 prepa-
ration. 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
November 2011 • Volume 113 • Number 5
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 anesthe-
sia 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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