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The Journal of Neuroscience, February 1, 2003, 23(3):876
Early Exposure to Common Anesthetic Agents Causes Widespread
Neurodegeneration in the Developing Rat Brain and Persistent Learning
Deficits
Vesna
Jevtovic-Todorovic1,
Richard E.
Hartman2,
Yukitoshi
Izumi3,
Nicholas
D.
Benshoff3,
Krikor
Dikranian3,
Charles F.
Zorumski3,
John W.
Olney3, and
David F.
Wozniak3
1 Department of Anesthesiology, University of Virginia
Health System, Charlottesville, Virginia 22908, and Departments of
2 Neurology and 3 Psychiatry, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Recently it was demonstrated that exposure of the developing brain
during the period of synaptogenesis to drugs that block NMDA glutamate
receptors or drugs that potentiate GABAA receptors can
trigger widespread apoptotic neurodegeneration. All currently used
general anesthetic agents have either NMDA receptor-blocking or
GABAA receptor-enhancing properties. To induce or maintain a surgical plane of anesthesia, it is common practice in pediatric or
obstetrical medicine to use agents from these two classes in combination. Therefore, the question arises whether this practice entails significant risk of inducing apoptotic neurodegeneration in the
developing human brain. To begin to address this problem, we have
administered to 7-d-old infant rats a combination of drugs commonly
used in pediatric anesthesia (midazolam, nitrous oxide, and isoflurane)
in doses sufficient to maintain a surgical plane of anesthesia for 6 hr, and have observed that this causes widespread apoptotic
neurodegeneration in the developing brain, deficits in hippocampal
synaptic function, and persistent memory/learning impairments.
Key words:
NMDA antagonists; GABA agonists; isoflurane; midazolam; nitrous oxide; apoptosis
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Introduction |
Advances in pediatric and obstetric
surgery have resulted in an increased complexity, duration, and number
of anesthesia procedures. To minimize risks, it is necessary to
understand the effects of anesthetic drugs on the developing nervous
system. Presently used anesthetics act by two principal mechanisms: (1)
an increase in inhibition via GABAA receptors
(e.g., benzodiazepines, barbiturates, propofol, etomidate, isoflurane,
enflurane, and halothane) (Franks and Lieb, 1994 ), and (2) a decrease
in excitation through NMDA receptors [e.g., ketamine, nitrous oxide
(N2O), and xenon] (Lodge and Anis, 1982; Franks
et al., 1998; Jevtovic-Todorovic et al., 1998; Mennerick et al., 1998).
Recent findings indicate that drugs that act by either of these
mechanisms induce widespread neuronal apoptosis in immature rat brain
when administered during synaptogenesis (Ikonomidou et al., 1999, 2000;
Ishimaru et al., 1999). In addition, ethanol, an agent with both NMDA
antagonist and GABAmimetic properties, triggers a widespread pattern of
apoptotic neurodegeneration in the developing rat brain, which is a
composite of the patterns induced by NMDA antagonist and GABAmimetic
drugs (Ikonomidou et al., 1999). These findings raise questions
regarding the potential risk posed by currently used anesthesia
protocols. To address this issue, we studied the histopathological,
electrophysiological, and behavioral effects of exposure of 7-d-old
rats to N2O, isoflurane, and midazolam, three
agents commonly used in obstetric and pediatric anesthesia.
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Materials and Methods |
Animals. Seven-day-old male and female Sprague Dawley
rats were used for all experiments. At postnatal day 7 (P7),
experimental rats were exposed to 6 hr of anesthesia, and controls were
exposed to 6 hr of mock anesthesia. They were then allowed to recover and were divided into three randomly selected groups. One group was
used for histopathology studies at several acute postanesthesia intervals. The second group was used for behavioral studies, which involved evaluating the rats on several tests over a 160 d period. The third group was used to study long-term potentiation (LTP) in
hippocampal slices at P29-P33. Daily inspection and weighing was
performed on the latter two groups at P6-P21 to evaluate general health and development.
Anesthesia treatment. N2O and oxygen
were delivered using a calibrated flowmeter. Midazolam was dissolved in
10% DMSO immediately before administration. For control experiments,
10% DMSO was used. To administer specific concentrations of
N2O, both normobaric [1 atmosphere (atm),
100 vol%] and hyperbaric conditions were required. In
experiments requiring hyperbaric conditions, the N2O/oxygen mixture was introduced at a pressure
of 2.0 atm (200 vol% = 150 vol% N2O and 50 vol% oxygen) and sustained for the duration of the experiment
(Mahmoudi et al., 1989; Gonsowski and Eger, 1994). A relief valve on
the hyperbaric chamber allowed continuous escape of gases to avoid
accumulation of carbon dioxide. Animals were kept normothermic
throughout experiments. For N2O concentrations of
<80 vol%, normobaric conditions were used. The N2O/oxygen mixture was delivered to the chamber
while a relief valve was kept open so that pressure inside the chamber
remained at 1 atm. For control experiments, air was substituted for the gas mixtures. For experiments with isoflurane, we used an
agent-specific vaporizer that delivers a set percentage of anesthetic
into the chamber. After initial equilibration of the
N2O/oxygen/isoflurane or air/isoflurane
atmosphere inside the chamber, the composition of the chamber gas was
analyzed by mass spectrometry for N2O or nitrogen, isoflurane, carbon dioxide, and oxygen concentration. All
experiments were approved by The Animal Use and Care Committees of the
University of Virginia and Washington University School of Medicine.
Arterial blood gas analysis. To determine adequacy of
ventilation, arterial blood was sampled at the end of anesthesia by obtaining a single sample (100 µl) from the left cardiac ventricle using a 32 gauge hypodermic needle. Bicarbonate concentration (millimoles per liter), oxygen saturation (%), pH,
paCO2 (partial pressure of carbon dioxide in
mmHg), and paO2 (partial pressure of oxygen in
mmHg) were measured immediately after blood collection, using a Nova
Biomedical blood gas apparatus. Control samples were obtained from
air/DMSO-treated pups.
Histopathological studies. All pups were deeply anesthetized
and perfused with aldehyde fixatives for histopathology studies of the
brains. We have found that activated caspase-3 [immunocytochemical (ICC)] is an excellent method for marking neurons that are in an early
stage of apoptosis (Olney et al., 2002a,b), and DeOlmos silver staining
is very useful for mapping patterns of cell death in the developing
brain (Ikonomidou et al., 1999, 2000). To confirm the apoptotic nature
of the cell-death process, electron microscopy is the most reliable
available means (Dikranian et al., 2001). Pups used for studying
caspase-3 activation or for silver staining were perfused with a
mixture of paraformaldehyde (4%) in Tris buffer, pH 7.4, either 2 hr
(caspase) or 18 hr (silver) after cessation of anesthesia, whereas
those used for electron microscopy studies were perfused with a mixture
of paraformaldehyde (4%) and glutaraldehyde (1.5%) in cacodylate
buffer, pH 7.4, at 10 hr after anesthesia.
For activated caspase-3 ICC, 50-µm-thick vibratome sections were
processed by procedures described recently (Olney et al., 2002b), using
a primary anti-active caspase-3 antiserum raised in rabbits (D175; Cell
Signaling Technology, Beverly, MA). For silver staining, the fixed
brains were cut by vibratome into 50 µm sections and stained by the
DeOlmos cupric silver method, as described previously (DeOlmos and
Ingram, 1971; Corso et al., 1997). For electron microscopy, ultrathin
sections were stained with uranyl acetate/lead citrate and viewed in a
100C Jeol (Peabody, MA) electron microscope.
To determine the degree of neurodegeneration in a given brain region,
we used the optical dissector and fractionator method (West, 1999). A
counting frame (0.05 × 0.05 mm; dissector height, 0.05 mm) and a
high numerical aperture objective lens were used to visualize neurons.
Unbiased sampling of each brain region was performed by randomly
selecting 10-12 viewing fields over which the counting frame was
positioned for counting at different focal levels. The numerical
density of degenerating neurons in any given region was determined by
counting argyrophilic profiles in 50-µm-thick sections stained by the
DeOlmos silver method. Differences between anesthetic-treated and
control values for a given brain region were evaluated statistically by
Student's t test. To obtain an estimate of the severity of
induced degeneration in the anesthetic-treated brains, the numerical
density values for the anesthetic-treated animals were divided by those
for the controls. The result was expressed as a "fold" increase
over baseline, baseline being the rate at which neuronal cell death was
occurring as a natural phenomenon in control brains. Counting was done
by an experienced histopathologist who was unaware of the treatment condition.
Electrophysiological studies. Rats exposed for 6 hr to
anesthetic agents at P7 were killed at P29-P33 for preparation of
hippocampal slices using standard methods (Zorumski et al., 1996). At
the time of study, slices were placed in a submersion recording chamber at 30°C. Extracellular recordings were obtained from the apical dendritic region for analysis of population EPSPs using 2 M NaCl electrodes. Evoked responses were elicited
with 0.2 msec constant-current pulses through a bipolar electrode in
the Schaffer collateral pathway (CA1 hippocampal field) every 30-60
sec at an intensity sufficient to elicit 50% maximal EPSPs. After
establishing a stable baseline, LTP was induced by applying a single
100 Hz × 1 sec stimulus train using the same amplitude current.
Behavioral studies. Rats exposed to the anesthetic
"triple cocktail" or DMSO vehicle on P7 were evaluated behaviorally
at subsequent ages using the following measures [similar to those described in greater detail by Wozniak et al. (1989, 1990, 1991), Ho et
al. (2000), and Hartman et al. (2001)]: (1) Ascent test at P10, P12,
and P14 (to assess the acute response to drug treatment); (2)
auditory/tactile startle and prepulse inhibition (PPI) of startle at
P20; (3) a 1 hr locomotor activity test in a home-cage environment at
P21; and (4) a sensorimotor battery of tests at P22, including walking
initiation, ledge walking, inclined plane performance, and elevated
platform performance. (5) Spatial reference memory was evaluated using
the Morris water navigation test which included cued (P28), place (P32
and P131), and probe trials. (6) Spatial working memory was assessed at
P53, using a win-shift spatial discrimination protocol in the radial
arm maze.
Water navigation testing was conducted as two separate studies, each
involving different groups of experimental and control rats
(n = 9-11 per group). Except as is explained below,
the same conditions were used for both studies, as follows: rats were
tested in a small pool (100 cm inner diameter) as juveniles and in a larger pool (180 cm inner diameter) as adults. In the cued trials, rats
were tested for their ability to swim to a visible platform that was
switched to a new location for each trial. The place condition involved
testing the rats' ability to learn the location of a platform
(submerged, not visible) which remained in the same location for all
trials. When subjected to place testing as juveniles (P32), the rats
were given two blocks of trials (two trials per block) each day for
5 d, with an intertrial interval of 30 sec. When the rats were
retested as adults (P131), using a new platform location, a protocol
involving one block of trials (two trials per block) each day for
5 d was used in an effort to increase task difficulty and improve
test sensitivity. Study 2 was conducted exactly like study 1, except
that after 5 d of place testing as adults, rats in study 2 were
tested for an additional 5 d to determine whether they could
progressively improve their performance to an asymptotic level, to
provide evidence of learning. Probe trials were conducted after the
last place trial of juvenile testing and after the last place trials of
blocks 5 and 10 during adult testing. Probe trials involved removing
the platform and evaluating the rats' search patterns for the missing
platform by quantifying time spent in the target quadrant and number of
platform crossings.
The rats in both study 1 and study 2 were tested in the radial arm maze
at P53 according to an identical win-shift spatial discrimination
(working memory) protocol in each study.
Data analyses. The data from each behavioral test in both
study 1 and study 2 were subjected to an ANOVA involving treatment and
study as between-subjects variables. In no case was a treatment by
study interaction found; therefore, for additional analysis, the data
from studies 1 and 2 were combined (and the variable study was
deleted), except for data pertaining to the last 5 d of place
testing as adults, which were generated only in study 2. Data from the
preweaning tests were typically analyzed using ANOVA models that
involved treatment and litter as between-subjects variables and test
sessions as a within-subjects variable. Data from postweaning tests
were typically analyzed using ANOVA models with treatment and gender as
between-subjects variables and blocks of trials as a within-subjects
variable. Pairwise comparisons were conducted after significant effects
of treatment or significant interactions involving treatment and other
relevant variables, and p values exceeding Bonferroni
corrected levels were noted when appropriate by the symbol  . The
Huynh-Feldt statistic was used to adjust p values to help
protect against violations of compound symmetry when more than two
levels of a within-subjects variable were used in an ANOVA model.
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Results |
Neonatal anesthetic treatment does not induce metabolic or
respiratory distress
To assess the effects of selected anesthetics on the
developing brain, we exposed P7 rats to midazolam, isoflurane, and
N2O, either individually or in clinically
relevant combinations, for 6 hr. In animals exposed for 6 hr to these
drugs, either individually or in combination, including the triple
cocktail (isoflurane plus N2O plus midazolam)
(Table 1), there were no signs of
metabolic or respiratory distress. Oxygen saturation,
paO2, paCO2, and pH did not
differ significantly from control animals exposed to air plus vehicle
for 6 hr. A slight increase in pH in both groups is attributable to
transient hyperventilation caused by cardiac puncture.
Neonatal anesthetic treatment induces extensive
apoptotic neurodegeneration
In vehicle-treated animals (10% DMSO plus air), both cupric
silver and activated caspase-3 ICC staining revealed a sparsely scattered pattern of baseline physiological cell death (Fig.
1a,f,h,j). Animals treated
with N2O alone (50, 75, or 150 vol%) or
midazolam alone (3, 6, or 9 mg/kg, i.p.) showed no significant increase in apoptotic neurodegeneration compared with control animals
(data not shown). However, animals that were treated with isoflurane alone (0.75, 1.0, or 1.5 vol%) exhibited dose-dependent
neurodegeneration (p < 0.05). The most
vulnerable brain regions were the laterodorsal and anteroventral
thalamic nuclei, where even the lowest isoflurane concentration
(0.75%) caused a significant increase in neuronal degeneration
(16-fold and ninefold increase, respectively). The parietal cortex
(layer II) was also affected in an apparently dose-dependent manner,
although the damage was significantly greater than controls at only the
highest isoflurane concentration (1.5 vol%) (p < 0.05).
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Figure 1.
Triple anesthetic cocktail induces apoptotic
neurodegeneration. a-l are light micrographic scenes
from various brain regions of either a control rat (a,
f, h, j) or a rat
exposed to the triple anesthetic cocktail (b-e,
g, i, k,
l). Some sections are stained by the DeOlmos
silver method (a, b, d,
f, g, k), and the
remainder are immunocytochemically stained to reveal caspase-3
activation (c, e, h-j,
l). The regions illustrated are the posterior
cingulate/retrosplenial cortex (a-c), subiculum
(d, e), anterior thalamus
(f, g), rostral CA1 hippocampus
(h, i), and parietal cortex
(j-l). The individual nuclei shown in the
anterior thalamus (f, g) are
laterodorsal (LD), anterodorsal (AD),
anteroventral (AV), anteromedial
(AM), and nucleus reuniens (NR).
m and n are electron micrographic scenes
depicting the ultrastructural appearance of neurons undergoing
apoptosis. The cell in m displays a very early stage of
apoptosis in which dense spherical chromatin balls are forming in the
nucleus at a time when the nuclear membrane remains intact and very few
changes are evident in the cytoplasm. The cell in n
exhibits a much later stage of apoptosis in which the entire cell is
condensed, the nuclear membrane is absent, and there is intermixing of
nuclear and cytoplasmic constituents. These are hallmark
characteristics of neuronal apoptosis as it occurs in the in
vivo mammalian brain.
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When a nontoxic dose of midazolam (9 mg/kg, i.p.) was followed by 6 hr
of a low/minimally toxic concentration of isoflurane (0.75 vol%),
there was a significant increase in apoptotic neurodegeneration compared with this concentration of isoflurane alone. Under this condition (double GABAmimetic cocktail), the damage was evident primarily in the laterodorsal and anterodorsal thalamus and parietal cortex (layer II). However, when 6 hr of N2O at a
low/nontoxic concentration (75 vol%) was added to the above double
cocktail, this triple cocktail (midazolam plus 6 hr of
N2O plus isoflurane) resulted in a robust
neurodegenerative reaction entailing more severe damage in the thalamus
and parietal cortex than was caused by the double cocktail, and also
producing moderate to severe damage in many other brain regions. It
warrants emphasis that all triple cocktail-exposed pups invariably
sustained brain damage, and that the pattern of damage was identical in
each of these brains. We have compiled a comprehensive list of the
damaged brain regions (Table 2), and for
each region the severity of neurodegeneration in anesthesia-exposed
brains is rated in terms of how many fold greater it is compared with
the baseline rate in the same region of control brain (the baseline
rate being that attributable to physiological cell death that occurs
naturally in the developing brain). This list (Table 2) includes only
those brain regions, or subfields within specific regions, where the
rate of degeneration was at least 15-fold greater than the control
rate. In all of these regions, the numerical density of
degenerating neurons in the anesthetic cocktail rats was
significantly greater than the control value at a level of
p < 0.001.
The two histological methods used (activated caspase-3 ICC and silver
staining) to evaluate the neurodegenerative reaction in experimental
brains compared with controls were mutually confirmatory regarding the
pattern of neurodegeneration induced by the triple cocktail (Fig.
1a-l). Electron microscopic evaluation confirmed that the degenerating neurons displayed classical morphological changes
characteristic of apoptosis (Fig. 1m,n).
Triple anesthetic cocktail induces profound LTP suppression
To assess long-term electrophysiological effects of early
anesthetic exposure, we examined synaptic function and LTP in
hippocampal slices prepared at P29-P33 from rats treated at P7 with
10% DMSO (controls), N2O, isoflurane, midazolam,
or a combination of the three agents. Baseline EPSPs elicited by single
stimuli did not differ among the five groups (data not shown). LTP
induction was unchanged in DMSO-treated controls compared with naive
controls [changes in EPSP slope 60 min after tetanus in naive and DMSO animals were 144.1 ± 7.9% of baseline (n = 6)
and 139.4 ± 6.3% of baseline (n = 10),
respectively]. A less robust LTP response was observed in rats treated
with N2O (129.1 ± 5.9%; n = 8), isoflurane (125.1 ± 3.1%; n = 6), or
midazolam (120.9 ± 2.2%; n = 8), but only
midazolam-treated animals differed significantly from controls (p = 0.022 by t test). In contrast,
slices from rats treated with the triple anesthetic cocktail exhibited
profound suppression of LTP (105.0 ± 6.5% of baseline;
n = 10; p = 0.001), despite the
presence of robust short-term potentiation (Fig.
2).
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Figure 2.
Effects of anesthetic exposure on LTP in the CA1
region of rat hippocampal slices. a, The graph depicts
the time course of change in field EPSP slope (± SEM) in hippocampal
slices from control rats treated with DMSO ( ) and rats exposed to
the triple anesthetic combination (triple c.,  ). A
single 100 Hz × 1 sec tetanus was delivered at time 0 (arrow). b, Traces to the
right of the graph show examples of EPSPs before
(solid traces) and 60 min after (dashed
traces) the tetanus in slices from animals treated with the
various anesthetics. Hippocampal slices were prepared at P29-P33 from
rats treated at P7.
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Triple anesthetic cocktail induces long-term impairment in
spatial learning/memory
Because the anesthetic triple cocktail produced the most profound
neurodegenerative effects, we decided to determine whether developmental exposure to this treatment also had lasting effects on
behavior. To accomplish this, we assessed sensorimotor and cognitive
function from early stages of development into adulthood and found that
the anesthetic cocktail rats did not differ from controls in (1)
overall growth as indexed by daily body weight measurements, (2)
sensorimotor ability as measured by the ascent test (preweaning) and
sensorimotor battery (postweaning), (3) acoustic or tactile startle
reactivity or PPI as measured by startle/PPI testing, or (4) various
movement-related variables, as measured during a 1 hr locomotor
activity test.
When tested in the Morris water maze on P28, anesthetic cocktail and
control rats performed similarly in terms of path length and latency
when learning to swim to a visible platform during cued trials (data
not shown). However, when we examined spatial reference memory
capabilities in the same animals during place trials (submerged
platform, fixed location) on P32, anesthetic cocktail rats showed
significant acquisition deficits (Fig.
3a). The deficit was evidenced
by the anesthetic cocktail rats showing slower acquisition rates during
the middle blocks of trials, although they improved and performed like
controls by the end of training and also exhibited control-like
performance levels during probe trials (data not shown). When retested
on the water maze as adults (P131), using a more difficult
place-learning protocol, anesthetic cocktail rats again demonstrated
impaired performance during place trials (Fig. 3b) but,
unlike the earlier findings, they also demonstrated impaired retention
performance during probe trials. Specifically, they spent significantly
less time searching the pool quadrant where the platform had been (time
in target quadrant) (Fig. 3c) and crossed over the former
platform location significantly fewer times than controls (data not
shown). Moreover, the control rats from study 2 that were subjected to
an additional 5 d of testing improved their performance to
asymptotic levels, suggesting that learning occurred, whereas the
anesthetic cocktail group from study 2 showed no such improvement (Fig.
3b). In addition, the control rats showed significantly
higher levels of retention during the probe trials in terms of target
quadrant time (Fig. 3c) and platform crossings (data not
shown). Swimming speeds were also analyzed during cued and place
trials, and no differences were observed, further suggesting that
swimming performance deficits were not responsible for the
place-learning impairments in the anesthetic cocktail rats (data not
shown).
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Figure 3.
Effects of neonatal triple anesthetic cocktail
treatment on spatial learning. a, Rats were tested at
P32 for their ability to learn the location of a submerged (not
visible) platform. An ANOVA of the escape path length data yielded a
significant main effect of treatment (p = 0.032) and a significant treatment by blocks of trials interaction
(p = 0.024), indicating that the performance
of the rats that received the anesthetic cocktail was significantly
inferior to that of control rats during place training. Subsequent
pairwise comparisons indicated that differences were greatest during
blocks 4, 5, and 6 (p = 0.003, 0.012, and
0.019, respectively). However, the rats receiving the anesthetic
cocktail improved their performance to control-like levels during the
last four blocks of trials. b, Rats were retested as
adults (P131) for their ability to learn a different location of the
submerged platform. The graph on the left represents the
path length data from the first five place trials when all rats were
tested. An ANOVA of these data yielded a significant main effect of
treatment (p = 0.013), indicating that the
control rats, in general, exhibited significantly shorter path lengths
in swimming to the platform compared with anesthetic cocktail rats.
Subsequent pairwise comparisons showed that differences were greatest
during block 4 (p = 0.001). The graph on the
right shows the data from study 2 rats that received 5 additional training days as adults. During these additional trials, the
control group improved their performance and appeared to reach
asymptotic levels, whereas the anesthetic cocktail rats showed no
improvement. An ANOVA of these data yielded a significant main effect
of treatment (p = 0.045) as well as a
significant treatment by blocks of trials interaction
(p = 0.001). Additional pairwise comparisons
showed that group differences were greatest during blocks 7, 8, and 10 (p = 0.032, 0.013, and 0.017, respectively).
c, Probe trial performance of anesthetic cocktail and
control rats during adult testing. Search behavior of the rats was
quantified when the submerged platform was removed from the pool after
the last place trials in blocks 5 and 10. The histogram on the
left presents data for rats of both studies 1 and 2 combined after five blocks of place trials were completed. The
histogram on the right presents data for rats of study 2 alone, after 10 blocks of place trials were completed. The
dotted line represents the amount of time that animals
would be expected to spend in the target quadrant based on chance
alone. Both histograms show that the control rats spent significantly
more time in the target quadrant than the anesthesia-exposed rats,
regardless of whether the probe tests were performed on both study
groups after five blocks or only on the study 2 rats after 10 trials.
d, e, Data from the radial arm maze test performed on
P53 to evaluate spatial working memory capabilities are shown.
d, A histogram showing that the anesthetic cocktail rats
required significantly more days to reach a criterion demonstrating
learning (8 correct responses out of the first 9 responses for 4 consecutive days) compared with controls. e, Plotting
the days to criterion data as the cumulative percentage of rats
reaching criterion in each group as a function of blocks of training
days shows that the acquisition rate of the anesthetic cocktail rats
began to slow around the fourth block of trials and remained slower
throughout the rest of the experiment. Numbers in
parentheses in each graph indicate sample sizes.
*p < 0.05; Bonferroni corrected level:
p < 0.005 in a;
p < 0.01 in b.
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When spatial working memory capabilities were tested in the radial arm
maze at P53, anesthetic cocktail rats were significantly impaired
relative to controls (Fig. 3d) in terms of days required to
reach a criterion demonstrating learning. Graphing these data in terms
of the cumulative percentage of rats reaching criterion as a function
of blocks of trials (Fig. 3e) reveals that the acquisition rate of the anesthetic cocktail rats began to slow compared with controls by the fourth block of trials and remained substantially slower for the remainder of training. The anesthetic cocktail rats also
made a larger number of errors in reaching criterion, although this
difference was marginally nonsignificant (p = 0.065; data not shown).
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Discussion |
Our findings indicate that exposure of infant rats to an
anesthetic cocktail (midazolam, isoflurane, N2O)
that is commonly used in pediatric anesthesia triggers apoptosis in
several major brain regions, resulting in deletion of many neurons from
the developing brain and residual learning/memory deficits, coupled with dysfunction of hippocampal synaptic mechanisms putatively associated with memory.
These findings are consistent with other recent evidence that apoptotic
neurodegeneration can be induced in the developing rodent brain by (1)
drugs that block NMDA receptors, (2) drugs that hyperactivate
GABAA receptors, or (3) ethanol, which has both
NMDA antagonist and GABAmimetic properties. It appears that more
profound neurodegeneration is induced if both NMDA and
GABAA receptors are simultaneously altered, in
that a more robust and widespread neurodegenerative response is seen
after exposure to ethanol than to either an NMDA antagonist or
GABAmimetic drug by itself. This principle is further corroborated by
the present demonstration that combining a nontoxic concentration of
the NMDA antagonist N2O with GABAmimetic agents
induced a much more severe and widespread pattern of neurodegeneration
than was induced by either drug category by itself, even at
substantially higher doses.
Our data indicate that exposure of infant rats to a clinically relevant
anesthesia protocol for 6 hr during synaptogenesis causes not only
acute deletion of many neurons from the developing brain but also
learning/memory disabilities that persist into adolescence and
adulthood. Animals exposed to this anesthesia protocol displayed
deficits in spatial reference memory capabilities as manifested by
slower place learning acquisition as juveniles and by significant
impairments in both spatial reference and working memory as adults. The
lack of differences between groups during the cued trials or with
regard to swimming speeds as well as the absence of performance
differences on a wide array of behavioral tests suggest that the
learning/memory deficits in the anesthetic cocktail rats did not result
from nonassociative performance factors such as sensorimotor
disturbances, altered emotionality, or changes in motivation. Our
additional finding that the anesthesia-exposed rats had lasting
deficits in hippocampal synaptic function may help explain the learning
deficits, in that the hippocampus is well known to play an important
role in memory and learning. However, it would probably be simplistic
to attribute these deficits solely to hippocampal damage, in view of
substantial evidence (Aggleton and Brown, 1999; Mitchell et al., 2002)
that fundamental memory functions are not mediated by the hippocampus
alone, but rather by a distributed network that includes, in addition
to the hippocampus, anterior thalamic nuclei, mammillary bodies, and
retrosplenial cortex. Each of the latter three structures was damaged
in the anesthesia-exposed brains more severely than the hippocampus.
The triple anesthetic drug protocol used in these experiments is one
that is commonly used in pediatric anesthesia practice. We selected a
dose of midazolam that is considered sedating (sedative dose is between
1 and 10 mg/kg, i.p., in rats) (Kissin et al., 1990) and followed this
with only those concentrations of N2O and
isoflurane that are required to induce and maintain a surgical plane of
anesthesia. The minimum alveolar anesthetic concentration that prevents
purposeful movement to supramaximal noxious stimulation in 50% of
subjects (MAC) for either N2O or isoflurane in
humans is ~67% of the MAC in rats (Hornbein et al., 1982; Mahmoudi
et al., 1989; Stevens and Kingston, 1992; Orliaguet et al., 2001). Thus, the concentrations of N2O (75%) and
isoflurane (0.75%) used in our rat study would be comparable with
exposing a human to 50% N2O and 0.5%
isoflurane, concentrations that are well within clinically used ranges.
It has been shown in previous studies (Ikonomidou et al., 1999, 2000)
that peak vulnerability to the apoptogenic action of NMDA antagonists
and GABAmimetics is during the synaptogenesis period, also known as the
brain growth-spurt period. This is a readily recognized period during
which the brain grows at an accelerated rate because newly
differentiated neurons throughout the brain are rapidly expanding their
dendritic arbors to provide the required surface area to accommodate
new synaptic connections. The brain growth spurt occurs in different
mammalian species at different times relative to birth. In rats, it
begins a day or two before birth and ends ~2 weeks after birth,
whereas in humans it starts at the beginning of the third trimester and
ends several years after birth (Dobbing and Sands, 1979). Comparing the
brain growth curves for rats and humans, the period of peak brain
growth occurs in rats between the fourth and tenth postnatal days and
in humans between the last month of gestation and first 6 months after
birth. Although neurodevelopmental age equivalencies between rats and humans cannot be specified with precision, our decision to use 7-d-old
rats for the present study was based on the assumption that this
neurodevelopmental age in the rat is equivalent to the human age from 0 to 6 months after a term birth, or perhaps from 0 to  12 months after
a premature birth. It is quite common, often out of necessity, but
sometimes on an elective basis, for surgical procedures requiring
general anesthesia to be performed on human infants in this
neurodevelopmental age range.
Various anesthesia protocols have been used in pediatric medicine for
many decades without clear evidence linking anesthesia exposure
to subsequent neurobehavioral disturbances. Establishing such a link is
rendered difficult by many confounding variables. For example, human
infants who undergo general anesthesia often have a history of
prematurity and/or adverse peripartum events, including prolonged
exposure to sedatives or anticonvulsants in an intensive care unit.
Moreover, linking neurobehavioral disturbances to perinatal drug
exposure is difficult unless gross signs of dysmorphogenesis are
present. For example, researchers were first alerted to the fetal
alcohol syndrome by conspicuous dysmorphogenic effects (craniofacial
malformations), and this led to the subsequent recognition that fetal
exposure to ethanol can cause a wide range of neurobehavioral
disturbances in the absence of dysmorphogenic effects (Streissguth and
O'Malley, 2000). Our findings indicate that an anesthesia protocol
that does not alter somatic development or induce sensorimotor
impairments in rats does result in learning/memory deficits that are
subtle enough to be easily overlooked. Additional animal research will
be needed to address the potential developmental neurotoxicity of other
clinically used anesthetic protocols. In addition, well designed
clinical investigations are needed to assess the potential relevance of
the animal findings to obstetric or pediatric anesthesia.
| |
FOOTNOTES |
Received Aug. 20, 2002; revised Nov. 4, 2002; accepted Nov. 8, 2002.
This work was supported in part by National Institutes of Health Grants
AG 11355, DA 05072, HD 37100, AG 18434, MH 45493, and AA 12951 and
Career Development Award K08 DA 00406 (V.J.-T.). We thank Adam
Myenberg, Anna Pieper, and Anthony Nardi for technical assistance.
Correspondence should be addressed to Vesna Jevtovic-Todorovic,
Department of Anesthesiology, University of Virginia Health System,
P.O. Box 800710, Charlottesville, VA 22908. E-mail: vj3w{at}virginia.edu.
| |
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TOP
ABSTRACT
Introduction
