General Anesthetics Inhibit Erythropoietin Induction
under Hypoxic Conditions in the Mouse Brain
Tomoharu Tanaka1*, Shinichi Kai1, Tomohiro Koyama1, Hiroki Daijo1, Takehiko Adachi2, Kazuhiko
Fukuda1, Kiichi Hirota1*
1Department of Anesthesia, Kyoto University Hospital, Kyoto, Japan, 2Department of Anesthesia, Tazuke Kofukai Medical Research Institute, Kitano Hospital, Osaka,
Japan
Abstract
Background: Erythropoietin (EPO), originally identified as a hematopoietic growth factor produced in the kidney and fetal
liver, is also endogenously expressed in the central nervous system (CNS). EPO in the CNS, mainly produced in astrocytes, is
induced under hypoxic conditions in a hypoxia-inducible factor (HIF)-dependent manner and plays a dominant role in
neuroprotection and neurogenesis. We investigated the effect of general anesthetics on EPO expression in the mouse brain
and primary cultured astrocytes.
Methodology/Principal Findings: BALB/c mice were exposed to 10% oxygen with isoflurane at various concentrations
(0.10–1.0%). Expression of EPO mRNA in the brain was studied, and the effects of sevoflurane, halothane, nitrous oxide,
pentobarbital, ketamine, and propofol were investigated. In addition, expression of HIF-2a protein was studied by
immunoblotting. Hypoxia-induced EPO mRNA expression in the brain was significantly suppressed by isoflurane in a
concentration-dependent manner. A similar effect was confirmed for all other general anesthetics. Hypoxia-inducible
expression of HIF-2a protein was also significantly suppressed with isoflurane. In the experiments using primary cultured
astrocytes, isoflurane, pentobarbital, and ketamine suppressed hypoxia-inducible expression of HIF-2a protein and EPO
mRNA.
Conclusions/Significance: Taken together, our results indicate that general anesthetics suppress activation of HIF-2 and
inhibit hypoxia-induced EPO upregulation in the mouse brain through a direct effect on astrocytes.
Citation: Tanaka T, Kai S, Koyama T, Daijo H, Adachi T, et al. (2011) General Anesthetics Inhibit Erythropoietin Induction under Hypoxic Conditions in the Mouse
Brain. PLoS ONE 6(12): e29378. doi:10.1371/journal.pone.0029378
Editor: Olivier Baud, Hoˆpital Robert Debre´, France
Received June 19, 2011; Accepted November 28, 2011; Published December 27, 2011
Copyright:  2011 Tanaka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: 665tana@kuhp.kyoto-u.ac.jp (TT); hif1@mac.com (KH)
Introduction
Ischemic and hypoxic insults to the brain during surgery and
anesthesia result in life-threatening complications including stroke.
These complications occur at the rate of 0.08–0.7% in general
surgery and 1.4–3.8% in cardiac surgery [1]. Pharmacologic
interventions, including calcium channel blockers, free radical
scavengers, and glutamate antagonists, have been introduced to
prevent and/or ameliorate stroke [2]. Erythropoietin (EPO) is also
recognized as a promising molecule to introduce neuroprotection,
and encouraging results have been obtained from clinical trials
involving stroke patients [3,4,5]. Originally, EPO was widely
known as a hematopoietic growth factor produced in the kidney
and fetal liver [5]. Further investigations expanded this review by
showing that EPO and EPO receptor (EPOR) are present in the
human brain and synthesized locally by astrocytes and neurons
[6,7,8,9]. It is well documented in both experimental and clinical
studies that EPO produced in the brain acts in a paracrine or
autocrine manner to provide neuroprotection [10,11]. Endoge-
nous EPO in the brain is produced in an oxygen tension-
dependent manner [12] and reduces brain damage by inhibiting
apoptosis [13], suppressing glutamate release [14], and reducing
the production of proinflammatory cytokines [15].
Hypoxia-induced EPO upregulation in the brain is regulated
mainly by hypoxia-inducible factor (HIF)-1 and HIF-2 [16]. HIF is
a transcriptional factor that acts as a key regulator in cells exposed
to low oxygen [17,18]. In fact, HIF-1 was originally cloned as a
transcription factor responsible for hypoxia-induced EPO expres-
sion [17]. HIF is a heterodimeric DNA-binding complex
composed of two basic helix-loop-helix proteins of the PER-
ARNT-SIM (PAS) family: the constitutive non-oxygen-responsive
subunit HIF-1b (also termed as the aryl hydrocarbon receptor
nuclear translocator: ARNT) and one of either of the hypoxia-
inducible a-subunits HIF-1a or HIF-2a [19,20]. HIF-a proteins
are rapidly degraded in normoxia but highly induced by hypoxia
[19,20,21]. HIF-1a and HIF-2a share significant sequence
homology and both are regulated post-translationally by protein
degradation [19,20]. HIF-2a, originally termed endothelial PAS
domain protein 1 (EPAS1) because of its expression in endothelial
cells, exhibits a more restricted expression pattern than HIF-1a
[17,22]. Although both HIF-a subunits are able to bind the
consensus hypoxia-responsive element (HRE) in promoters that
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contain the sequence NCGTG, they seem to regulate a different
set of target genes depending on the cellular context and oxygen
concentration [23,24]. The factors and molecular mechanisms
that potentially determine this isoform-specific target gene
selectivity remain poorly defined. Interestingly, although HIF-1a
was originally identified to bind to HRE in the 39-enhancer of the
EPO gene, there is now considerable evidence that HIF-2a is the
main HIF-a-subunit controlling EPO gene expression both in vitro
and in vivo [25].
We previously reported that the volatile anesthetic halothane
inhibits hypoxia-induced activation of HIF-1 by distinct molecular
mechanisms [26]. Recently, however, another volatile anesthetic,
isoflurane, has been reported to upregulate HIF-1 activity in
Hep3B cells [27], cultured rat hippocampal neurons [28], and rat
myocardium [29]. Isoflurane-induced activation of HIF is now
considered a possible mechanism of anesthetic preconditioning
[27,28,29,30,31]. However, in vivo experiments of the brain have
not been reported, and while HIF-2 rather than HIF-1 mainly
regulates EPO in the brain [32], the effect of general anesthetics
on HIF-2 has not been well investigated. Considering the pivotal
role of EPO in inducing neuroprotection, the influence of general
anesthetics on EPO, especially in the brain, may have a major
impact on perioperative clinical management. In the present
study, we investigated the effect of general anesthetics, including
isoflurane, on hypoxia-induced upregulation of EPO in the mouse
brain and primary cultured astrocytes.
Results
Isoflurane inhibits the induction of EPO expression under
hypoxic conditions in the brain, but does not affect EPO
induction in the kidney
To examine the effect of general anesthetics on EPO expression
under hypoxic conditions, we exposed 6-week-old BALB/c mice
to 10% O2 (hypoxia) for 3 h with isoflurane. Hypoxic exposure
significantly increased EPO mRNA expression and isoflurane
suppressed hypoxia-induced EPO mRNA expression in a
concentration-dependent manner in the mouse brain (Figure 1A)
and spinal cord (Figure 1B). In the brain, a decrease was observed
at 0.25% and maximal suppression was achieved at 0.5%
(Figure 1A). Next, we performed a time course study with 0.5%
isoflurane. Hypoxic exposure increased EPO mRNA time-
dependently (Figure 1C). Isoflurane inhalation for more than 1 h
significantly suppressed induction (Figure 1C). We re-exposed
mice to hypoxia 24 h after hypoxic treatment with (hypoxia+iso/
hypoxia) or without (hypoxia/hypoxia) 0.5% isoflurane to
determine the reversibility of the suppressive effect of isoflurane
on EPO mRNA in the brain. As shown in Figure 1D, hypoxia-
induced upregulation of EPO mRNA was almost of a similar
extent between the two groups (hypoxia/hypoxia and hypox-
ia+iso/hypoxia); therefore, the suppressive effect of isoflurane was
not identified 24 h later. Next, in order to confirm the effect of
isoflurane at the protein level, we measured EPO protein
concentration in mouse brains using ELISA. We exposed mice
to 10% O2 (hypoxia) for 5 h with or without 0.5% isoflurane.
Hypoxic exposure induced a significant increase in EPO protein
immediately after the treatment, and the effect of isoflurane was
not apparent (Figure 1E). However, 24 h after the hypoxic
exposure, EPO protein concentration decreased in the mice
exposed to hypoxia with isoflurane, although still elevated in the
mice without isoflurane (Figure 1E). Hypoxia has been reported to
induce EPO mRNA upregulation in the kidney and brain [30].
Therefore, we measured EPO mRNA levels in the kidney.
Hypoxic exposure significantly increased EPO mRNA in the
kidney, but isoflurane did not affect the induction of EPO mRNA
levels (Figure 1F).
Sevoflurane, halothane, nitrous oxide (N2O),
pentobarbital, ketamine,and propofol suppress hypoxia-
induced up-regulation of EPO mRNA in mouse brains
To examine whether the effect of isoflurane could be observed
with other general anesthetics, we exposed 6-week-old BALB/c
mice to 10% O2 with 0.5% sevoflurane or halothane. As in the
case of isoflurane, both anesthetics also suppressed hypoxia-
induced EPO upregulation in the brain (Figure 2A) but did not
affect the expression of EPO in the kidney (Figure 2B). Next, we
investigated the effect of nitrous oxide (N2O). We exposed mice to
10% O2 and 90% N2O (hypoxia-N2O group), compared with
10% O2 and 90% N2 (hypoxia-N2 group). EPO mRNA in the
brains of the hypoxia-N2O group was significantly suppressed
compared with that in the hypoxia-N2 group (Figure 2C). Finally,
we tested the non-inhalational anesthetics pentobarbital, ketamine,
and propofol. Fifty mg/kg pentobarbital (Figure 2D), 400 mg/kg
ketamine (Figure 2E), and 200 mg/kg propofol (Figure 2F) also
suppressed hypoxia-induced EPO mRNA upregulation in the
brain.
Isoflurane inhibits the induction of EPO expression under
hypoxic conditions in the brain of one-week and sixteen-
week old C57BL/6N CrSlc mice
Age is an important factor that influences the response to
hypoxia-ischemia in the brain [33]. After our first experiment, we
performed the same experiment in mice of other ages and species.
One-week and sixteen-week-old C57BL/6N CrSlc mice were
exposed to 10% O2 (hypoxia) for 3 h with 0.5% isoflurane. As with
6-week-old BALB/c mice, EPO mRNA induction was significant-
ly suppressed with isoflurane in one-week (Figure 3A) and sixteen-
week (Figure 3B) old mice.
Systemic hemodynamics
To exclude the possibility of secondary effects, including
hypotension, influencing the brain’s hypoxic responses, we
examined the systemic hemodynamics of mice. Hemodynamic
parameters including heart rate (HR), systolic (SAP), diastolic
(DAP), and mean (MAP) arterial pressures were measured in 6-
week-old BALB/c mice exposed to 10%O2 (hypoxia), 10%O2 with
0.5% isoflurane (hypoxia+iso), or 10% O2 with 0.5% sevoflurane
(hypoxia+sev) for 3 h, compared with controls (Table 1). Control
mice were exposed to air without anesthetics. SAP and MAP
decreased in the hypoxia, hypoxia+iso, and hypoxia+sev groups
compared to the control group. However, there were no significant
differences in all hemodynamic parameters among the hypoxia,
hypoxia+iso, and hypoxia+sev groups.
Isoflurane inhibits the induction of HIF-2a protein
expression under hypoxic conditions in the brain of mice
EPO is induced under hypoxic conditions, mainly through
activation of HIF-1 and HIF-2 [16,34]. Therefore, we investigated
the effect of isoflurane on the expression of HIF-1a and HIF-2a
proteins. We exposed 6-week-old BALB/c mice to 10% O2
(hypoxia) for 3 h with or without 0.5% isoflurane. Control mice
were exposed to air without isoflurane (normoxia). The protein
expression of HIF-1a, HIF-2a, and ARNT (HIF-1b) was
investigated with an immunoblot assay. As shown in Figure 4A,
HIF-1a protein was expressed under normoxic conditions and this
expression was not significantly altered in response to either
hypoxic exposure or isoflurane. In contrast, hypoxic exposure
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induced a marked accumulation of HIF-2a protein, which was
clearly suppressed by isoflurane (Figure 4A). ARNT protein
expression was almost stable under all conditions (Figure 4A). Next
we assayed the expression of HIF-1a and HIF-2a immunohisto-
chemically, and found positive immunostaining of HIF-1a was
observed globally in the frontal cortex under all conditions
(Figure 4B). HIF-2a was also admitted to a certain degree under
hypoxic condition, but barely observed under normoxia or
hypoxia with isoflurane exposure (Figure 4B).
Effect of isoflurane on the expression of HIF target genes
Despite significant similarities in their DNA binding and
dimerization domains, it has been demonstrated that HIF-1 and
HIF-2 have unique, as well as common, target genes [35]. As
shown in Figure 5A, HIF-1 specifically regulates glycolytic genes,
including lactate dehydrogenase A (LDHA), whereas HIF-2
exclusively regulates POU transcription factor Oct-4, cyclin D1,
and transforming growth factor a (TGF-a) [35,36]. Other
hypoxia-inducible genes, such as vascular endothelial growth
factor (VEGF), glucose transporter 1 (GLUT1), and EPO, are
regulated by both HIF-1 and HIF-2 in a cell-type-specific manner
[35]. Therefore, we investigated the influence of hypoxia or
isoflurane on HIF target genes in the mouse brain. VEGF mRNA,
as well as EPO, was significantly induced by hypoxic exposure
(10% O2, 3 h) and was significantly suppressed with isoflurane
(0.5%) (Figure 5B). In contrast, the expression of LDHA did not
change significantly with hypoxic exposure and isoflurane
inhalation (Figure 5B). Next, we studied the change of HIF target
genes in the kidney. Although hypoxic exposure significantly
induced EPO mRNA in the kidney (Figure 1F), other HIF target
genes, VEGF and LDHA, were not elevated but rather suppressed
(Figure 5C). The effect of isoflurane on VEGF and LDHA was not
significant (Figure 5C).
Recently, several reports have shown that isoflurane activates
HIF-1 and upregulates HIF target genes [27,28,29,31]. Most of
these studies were performed under normoxic conditions.
Therefore, we investigated the influence of isoflurane on the
expression of HIF target genes in mouse brains under normoxic
conditions. We exposed mice to air with 0.5% or 1.0% isoflurane
for 3 h and measured the mRNA levels of the HIF target genes,
including EPO, VEGF, LDHA, and GLUT1 (Figure 5D).
Isoflurane inhalation did not significantly change the expression
of the mRNA for any of these HIF target genes.
Isoflurane, pentobarbital, ketamine, and propofol
suppress, but morphine does not suppress hypoxia-
induced up-regulation of EPO mRNA in primary cultured
astrocytes
Astrocytes are now supposed to be the major source of EPO in
the CNS [6,7]. To investigate the direct effect of general
anesthetics on astrocytes, we performed in vitro experiments.
Primary cultured astrocytes were exposed to hypoxia (1% O2) with
or without various anesthetics for 4 h. Hypoxic exposure
significantly induced EPO mRNA, and isoflurane suppressed its
induction (Figure 6A). Not only isoflurane but also other anesthetics,
including pentobarbital, ketamine, and propofol, suppressed the
induction of EPO mRNA (Figures 6B–D). However, morphine, a
commonly used drug during the preoperative period, did not inhibit
the upregulation of EPO mRNA in astrocytes (Figure 6E). Next,
HIF-1a, HIF-2a, and ARNT protein accumulation were analyzed
in whole cell lysates from astrocytes. An immunoblot assay showed
distinct HIF-1a and HIF-2a protein accumulation under hypoxic
conditions, and the expression was significantly suppressed with
isoflurane, ketamine, and pentobarbital (Figure 7).
Effect of various anesthetics on oxygen consumption in
primary cultured astrocytes
To investigate the precise mechanism of how general anesthetics
suppress the induction of EPO under hypoxic conditions, we
finally examined the influence of general anesthetics on oxygen
consumption in astrocytes. General anesthetics, especially thio-
pental, are known to decrease metabolism and oxygen consump-
tion of the brain [37]. Therefore, the suppression of oxygen
consumption induced by general anesthetics may reduce the level
of hypoxia and consequently decrease HIF-a protein accumula-
tion and EPO induction. As indicated in Figure 8, 1 mM
pentobarbital as well as sodium azide, a cytochrome oxidase
inhibitor, reduced the oxygen consumption; however, 1 mM
ketamine and 100 mM propofol did not alter the oxygen
consumption significantly. These results suggest that, at least as
to ketamine and propofol, the decrease in oxygen consumption
was not the cause of the suppression of EPO induction.
Discussion
EPO is a major hematopoietic growth factor that is mainly
produced in the kidney and fetal liver [5]. It is also known to
express in CNS tissue [5]. EPO mRNA is constitutively expressed
in the cortex and hippocampus of the brain [38]. Various studies
have focused on the function of EPO in CNS; for example, mice
lacking EPO or EPOR exhibited increased apoptosis in the brain
before they died from severe anemia in utero [39,40], and mice
lacking EPOR in the brain suffered from reduced neurogenesis or
impaired migration of neurons in a brain stroke model [41]. Thus,
EPO is considered to be a neuroprotective factor against hypoxic-
ischemic and traumatic injuries and essential for neuronal
development [5,12].
In the present study, we showed that the induction of EPO
expression under hypoxic conditions was suppressed by the
general anesthetic isoflurane in a concentration- and time-
dependent manner in the mouse brain. Other anesthetics,
including sevoflurane, halothane, N2O, pentobarbital, ketamine,
and propofol, showed a similar effect. As for the mechanism of this
suppression, we found that the accumulation of HIF-2a, but not
HIF-1a, protein under hypoxic conditions was suppressed with
isoflurane in the mouse brain. This finding is consistent with a
previous report indicating that EPO is a target gene for HIF-2a,
rather than HIF-1a, in CNS [32]. HIF-1a is expressed
ubiquitously, but the expression of HIF-2a is tissue-specific [32].
Figure 1. Effect of isoflurane on EPO expression in mouse CNS and kidney. (A, B, and F) Six-week-old BALB/c mice were exposed to 10% O2
(hypoxia) in the presence of various concentrations of isoflurane for 3 hours (n = 6–15), or (C) exposed to 10% O2 (hypoxia) with 0.5% isoflurane for
the indicated periods of time. (D) 24 hours after the hypoxic exposure with or without 0.5% isoflurane, 6-week-old BALB/c mice were re-exposed to
hypoxia (10% O2) for 3 hours. EPO mRNA in the brain (A, C, and D), spinal cord (B) and kidney (F) was assayed with real-time RT-PCR analysis. (E)
Immediately or 24 hours after the 5-hour hypoxic (10% O2) exposure with or without 0.5% isoflurane, EPO protein concentration (pg/ml) in the brain
was quantified with ELISA and divided by the total protein concentration (mg/ml) of each mouse brain. Number of animals per treatment conditions
is 6 (C–E). Data are presented as mean 6 SD. The expression levels of EPO were normalized to that of 18S and expressed relative to the mean of
control mice (A, B, C, D and F). *P,0.05, **P,0.01 versus control, N.S.; not significant (Mann-Whitney U-test).
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HIF-2a is expressed in astrocytes and endothelial cells in the CNS
[32]. Astrocytes are the major source of EPO in the CNS [6,7],
and hypoxia-induced EPO upregulation is dramatically reduced in
the astrocyte-specific HIF-2a knockout mouse [38]. In the present
study, various anesthetics, including isoflurane, pentobarbital, and
ketamine, suppressed the accumulation of HIF-2a protein under
hypoxic conditions in cultured astrocytes. Therefore, our results
indicated that the hypoxia-induced activation of HIF-2 in
astrocytes was inhibited by general anesthetics, which resulted in
a significant suppression of EPO production.
Recently, various studies on astrocytes have been performed,
and these cells are considered responsible for a wide variety of
functions in the CNS, including synaptic transmission and
information processing by neural circuit functions [42]. In the
present study, we showed that various anesthetics suppressed the
accumulation of HIF-2a protein and EPO upregulation under
hypoxic conditions in the mouse brain and cultured astrocytes.
Considering the fact that the accumulation of HIFa proteins is
induced by hypoxia, the suppression of oxygen consumption
induced by general anesthetics may reduce the level of hypoxia
and consequently decrease HIF-2a protein accumulation. Actu-
ally, most of all general anesthetics excluding ketamine and N2O
are known to decrease cerebral metabolic rate of oxygen [43]. But
the effect of anesthetics on metabolism of astrocytes is not well
investigated. In the current study, pentobarbital, well known for
suppressing the metabolism of the CNS [37], decreased oxygen
consumption of astrocytes. On the other hand, ketamine and
propofol did not change oxygen consumption. In addition, we
previously reported that hypoxic brain EPO induction was preserved
in hypothermic mice, although hypothermia is well known to reduce
cerebral oxygen consumption [44]. Therefore, these findings suggest
that the suppressive effect of various anesthetics against HIF-2a
protein accumulation and EPO induction cannot be explained only
by the decrease in oxygen consumption.
The main target of general anesthetics differs with various
anesthetics; for example, ketamine and N2O act via N-methyl-D-
aspartate (NMDA) receptors [45,46], whereas the volatile
anesthetics, propofol and barbiturates act via c-aminobenzoic
acid-A (GABA-A) receptors [47,48]. On the other hand, the effect
of general anesthetics on glial cells is not well understood, except
for the fact that volatile anesthetics inhibit the glutamate uptake of
astrocytes [49]. Our finding that various general anesthetics have
an EPO-suppressive effect in in vitro experiments suggests that
general anesthetics have some common direct effects on astrocytes.
This finding is quite surprising considering the diverse action
mechanism of general anesthetics. Anesthetics modulate functions
of macromolecules, which play an essential role in cellular signal
transduction. For example, protein kinase C (PKC) [50], mitogen-
activating protein kinases (MAPKs) [51], and reactive oxygen
species (ROS) [52] are modulated by anesthetics. PKC, MAPK,
and ROS are also identified to affect HIF activity by modulating
HIF-a protein translation rate, hydroxylation, and phosphoryla-
tion of HIF-a protein [53,54,55]. Therefore, general anesthetics
may affect astrocytes through modulation of such enzymes and
mediators. But most of the studies considering the effect of
anesthetics on HIF have focused on HIF-1 under normoxic
conditions, and the effect on HIF-2 under hypoxic conditions is
not well understood.
Another important finding of the present study is the difference
of behavior between HIF-1a and HIF-2a. Namely, HIF-1a was
Figure 2. Effect of various anesthetics on hypoxia-induced EPO upregulation in the brain. (A, B) 6-week-old BALB/c mice were exposed to
10% O2 (hypoxia) in the presence of 0.5% sevoflurane, halothane or isoflurane for 3 hours. (C) 6-week-old mice were exposed to 10% O2, 90% N2O
(hypoxia- N2O) for 3 hours and compared with 10% O2, 90% N2 (hypoxia- N2). (D–F) 6-week-old mice were exposed to 10% O2 with pentobarbital (D),
ketamine (E) or propofol (F). In the all experiments, control mice were exposed to air without anesthetics (normoxia). EPO mRNA in the brain (A, C–F)
and kidney (B) was assayed with real-time RT-PCR. Data are presented as mean 6 SD (n = 6). The expression levels of EPO were normalized to that of
18S and expressed relative to the mean of control mice. *P,0.05, **P,0.01 versus control, N.S.; not significant (Mann-Whitney U-test).
doi:10.1371/journal.pone.0029378.g002
Figure 3. Effect of age and species on hypoxic EPO induction in mice brains. (A) 1-week and (B) 16-week-old C57BL/6N CrSlc mice were
exposed to 10% O2 (hypoxia) in the presence of 0.5% isoflurane for 3 hours. Control mice were exposed to air without anesthetics (normoxia). EPO
mRNA in the brain was assayed with real-time RT-PCR. Data are presented as mean 6 SD (n = 3–5). The expression levels of EPO were normalized to
that of 18S and expressed relative to the mean of control mice. *P,0.05 versus control (Mann-Whitney U-test).
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expressed even under normoxic conditions and 3-h hypoxic
exposure did not affect HIF-1 protein accumulation distinctively in
mice brains. In contrast, HIF-2a was barely expressed under
normoxic conditions and clearly increased in response to hypoxic
exposure. In in vitro experiments, however, both HIF-1a and HIF-
2a protein accumulation were observed under a 1% O2 condition,
and various anesthetics significantly suppressed their induction.
Although both HIF-1a and HIF-2a are considered to accumulate
significantly under hypoxic conditions in in vivo experiments using
the mouse brain [56], some reports have shown that HIF-1a was
expressed even under normoxic conditions [57,58]. In most of
these previous reports, HIF-1a protein increased in response to
hypoxic exposure in the brain, but the extent varied [57,58,59]. A
possible explanation for the discrepancy is the difference of oxygen
concentration. Previous report showed that HIF-1a protein
accumulation was observed under 1% O2 condition but not 5%
O2 condition in the neuronal cell line SK-N-BE cells [23]. We
exposed mice to 10% O2 in our studies, but 10% O2 might not be
low enough to induce HIF-1a protein accumulation.
Figure 4. Mechanism of suppression by isoflurane against EPO upregulation under hypoxic conditions. (A) Expression analysis of
hypoxia-inducible factor (HIF)-1a, HIF-2a, and aryl hydrocarbon receptor nuclear translocator (ARNT, also termed as HIF-1b) in the whole brain by
immunoblotting. 6-week-old BALB/c mice were exposed to air (control), 10% O2 (hypoxia) or 10% O2 with 0.5% isoflurane (hypoxia+iso) for 3 hours.
Figures are representative of at least three independent experiments. (B) Immunohistochemical staining for HIF-1a and HIF-2a in the frontal cortex of
6-week-old BALB/c mice. Mice were exposed to air (control), 10% O2 (hypoxia) or 10% O2 with 0.5% isoflurane (hypoxia+iso) for 3 hours. Figures are
representative of 6 slices of 3 mice. Scale bars: 100 mm.
doi:10.1371/journal.pone.0029378.g004
Table 1. Systemic hemodynamics.
Mice n
HR,
beats/min
SAP,
mmHg
DAP,
mmHg
MAP,
mmHg
Control 7 450670 115613 62615 80612
Hypoxia 7 513649 10065.8* 50617 67612*
Hypoxia+Iso 6 463677 9368.8* 4267.3 5865.6*
Hypoxia+Sev 6 448634 8968.7* 54612 65610*
Six-week-old BALB/c mice were divided into 4 groups; control (exposed to air),
hypoxia (exposed to 10% O2 for 3 hours), hypoxia+iso (exposed to 10% O2 with
0.5% isoflurane for 3 hours) and hypoxia+sev (exposed to 10% O2 with 0.5%
sevoglurane for 3 hours). Immediately after the hypoxic exposure, heart rate
and blood pressure were measured.
There were no significant differences in any of the parameters among hypoxia,
hypoxia+iso and hypoxia+sev group mice. Values are shown as mean 6 SD.
* P,0.05 versus control.
HR = heart rate; SAP = systolic arterial pressure; DAP =diastolic arterial pressure;
MAP=mean arterial pressur.
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Figure 5. Effect of isoflurne on mRNA expression of HIF target genes. (A) HIF-1 and HIF-2 have unique, as well as common, target genes. HIF-
1 specifically regulates glycolytic genes, including lactate dehydrogenase A (LDHA), phosphoglycerate kinase (PGK), as well as carbonic hydrase-9 (CA
IX) whereas HIF-2 exclusively regulates POU transcription factor Oct-4, cyclin D1, and transforming growth factor a (TGF-a). Other hypoxia-inducible
genes, such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), and EPO are regulated by both HIF-1 and HIF-2. (B, C) 6-
week-old BALB/c mice were exposed to 10% O2 (hypoxia) for 3 hours with or without 0.5% isoflurane and compared with controls. Control mice were
exposed to air without isoflurane (normoxia). (D) 6-week-old BALB/c mice were exposed to 0.5% or 1.0% isoflurane in air for 3 hours. Data are
presented as mean6 SD (n = 6). The expression levels of EPO, VEGF, LDHA and GLUT1 were assayed using real-time RT-PCR and normalized to that of
18S and expressed relative to the mean of mice exposed to air without isoflurane (normoxia).
doi:10.1371/journal.pone.0029378.g005
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Anesthetics Inhibit Erythropoietin Induction
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EPO has now been considered to be one of the promising agents
for neuroprotection [3,4,5]. Actually, in the clinical trials,
erythropoietin showed neuroprotective effect against acute stroke
[60], hypoxic-ischemic encephalopathy in newborns [61] and
delayed ischemic deficits following aneurysmal subarachnoid
hemorrhage [62]. In the current study, we showed the induction
of EPO mRNA expression under hypoxic conditions was
suppressed with isoflurane in a concentration- and time-dependent
manner. Other anesthetics including sevoflurane, halothane, N2O,
pentobarbital, propofol and ketamine showed the same effect.
Most of all anesthetics suppressed EPO mRNA induction with
concentrations no more than clinically used, for example,
isoflurane, sevoflurane and halothane showed this effect with
0.5%. Therefore, considering the neuroprotective effect of EPO,
exposure to anesthetics beyond necessity should be avoided
especially in cases hypoxia in brain may happen at greater risk
like cardiovascular surgery.
According to the recent reports, anesthetic exposure in neonatal
animals leads to neuronal death in certain circumstances [63,64].
Such neurotoxicity has now been demonstrated for many
anesthetics, including isoflurane, ketamine, midazolam, pentobar-
bital, N2O, and propofol, and a positive correlation may exist
between increased levels of anesthesia and increased severity of
neuroapoptosis [65,66]. The precise mechanisms by which injury
is invoked are not clear, although an imbalance between excitatory
and inhibitory input in the CNS during synaptogenesis may
contribute to such an effect [65]. On the other hand, EPOR is
highly expressed in the developing mouse brain [7], and mice
lacking EPO or EPOR experienced increased apoptosis in the
brain before they died of severe anemia in the uterus [39,40]. We
did not investigate the effect of general anesthetics on brain EPO
under normoxic conditions in neonatal animals. However,
considering the pivotal role of EPO in brain development, general
anesthetics may cause neuroapoptosis by suppressing EPO
production in the brain. Further studies using neonatal animals
should be performed.
In conclusion, we demonstrated that isoflurane inhibited
hypoxia-induced EPO upregulation in the mouse brain and
cultured astrocytes, most likely through suppression of HIF-2
activity. Other general anesthetics showed the same effect. Our
findings suggest that general anesthetics have some direct effect on
astrocytes and a major impact on the hypoxic response of the
CNS.
Methods
Animals
This study (ID: Med Kyo 09504) was approved by the Animal
Research Committee of Kyoto University (Kyoto, Japan), and all
experiments were conducted in accordance with the institutional
and NIH guidelines for the care and use of laboratory animals. All
procedures were performed on BALB/c or C57BL/6N CrSlc
mice purchased from Japan SLC Inc., Shizuoka, Japan. Food and
water were provided ad libitum, and the mice were maintained
under controlled environmental conditions (24uC, 12-h light/dark
cycles).
Drugs and chemicals
Isoflurane and pentobarbital were obtained from Dainippon
Sumitomo Pharma Co., Ltd., Osaka, Japan; sevoflurane from
Maruishi Pharmaceutical Co., Ltd., Osaka, Japan; halothane from
Takeda Pharmaceutical Co., Ltd., Osaka, Japan; and propofol
from Astra-Zeneca, London, UK. Morphine and ketamine were
purchased from Sankyo Co., Ltd., Tokyo, Japan. Nitrous oxide
(N2O) (Wakayama Sanso, Wakayama, Japan), oxygen (O2) (Taiyo
Nippon Sanso, Tokyo, Japan), and nitrogen (N2) (Taiyo Nippon
Sanso) were also used.
Hypoxic treatment
Mice were placed in a polypropylene chamber, and O2 and N2
mixed gas with or without volatile anesthetics, including
isoflurane, sevoflurane, and halothane, was delivered to the
chamber at a flow rate of 3 l/min using an anesthetic machine
(Custom50; Aika, Tokyo, Japan). In the experiment using N2O,
O2 and N2O-mixed gas was administered at the same flow rate.
Concentrations of O2, carbon dioxide (CO2), N2O, and volatile
anesthetics, including isoflurane, sevoflurane, and halothane, were
monitored continuously using an infrared analyzer (Capnomac
Ultima; Datex-Ohmeda, Helsinki, Finland). Mice were allowed to
adjust to the hypoxic environment by gradually decreasing the O2
level from 21% to 10% over 1 h, and they were maintained at
10% O2 for the indicated durations. Treatment with the volatile
anesthetics was initiated immediately after the adaptation to
Figure 6. Effect of various anesthetics on EPO expression in primary cultured astrocytes. Primary cultured astrocytes were exposed to 1%
O2 (hypoxia) in the presence of indicated concentrations of isoflurane (A), pentobarbital (B), ketamine (C), propofol (D) or morphine (E) for 4 hours. In
the all experiments, control was exposed to 20% O2. EPO mRNA was assayed with real-time RT-PCR. Data are presented as mean 6 SD (n = 4). The
expression levels of EPO were normalized to that of 18S and expressed relative to the mean of control. *P,0.05, **P,0.01 versus control, N.S.; not
significant (Mann-Whitney U-test).
doi:10.1371/journal.pone.0029378.g006
Figure 7. Expression analysis of HIF-1a, HIF-2a, and ARNT
protein in primary cultured astrocytes by immunoblotting.
Primary cultured astrocytes were incubated under hypoxic (1% O2)
conditions with or without 1.5% isoflurane, 1 mM pentobarbital, or
1 mM ketamine for 4 hours. Whole cell lysates were analyzed for HIF-1a,
HIF-2a, ARNT, GFAP, NeuN and b-actin protein expression by
immunoblot assay. Figures are representative of at least three
independent experiments.
doi:10.1371/journal.pone.0029378.g007
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hypoxia. In the experiments using pentobarbital, ketamine, and
propofol, the drugs were administered intraperitoneally immedi-
ately after hypoxic adaptation. The rectal temperature was
monitored using an ATB-1100 (Nihon Kohden, Tokyo, Japan),
and a heat lamp was used to maintain the temperature at 3661uC.
Arterial blood pressure was measured non-invasively using the tail-
cuff method immediately after completion of the hypoxic exposure
using an MK-2000ST (Muromachi Kikai Co., Ltd., Tokyo,
Japan). At the end of the experiments, the mice were killed by
cervical dislocation. The brains, spinal cords, and kidneys were
Figure 8. Effect of anesthetics on oxygen consumption of primary cultured astrocytes. Oxygen consumption curves generated using a
Clark electrode for primary cultured astrocytes suspensions. Arrows indicate addition of 2 mM sodium azide, 1 mM pentobarbital, 1 mM ketamine or
100 mM propofol. The slope of the curve is a measure of the rate of O2 consumption. Data are presented as mean 6 SD (n = 3).
doi:10.1371/journal.pone.0029378.g008
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rapidly removed, frozen in liquid nitrogen, and stored at 280uC
for subsequent determinations.
Reverse transcription and real-time quantitative
polymerase chain reaction
RNA was isolated from the frontal lobe of the brain using the
FastPureTM RNA Kit (Takara Bio, Inc., Shiga, Japan). First-strand
synthesis and real-time RT-PCR were performed using the One
Step SYBRTM PrimeScriptTM RT-PCR Kit II (Takara Bio)
according to the manufacturer’s instructions. PCR was performed
using the Applied Biosystems 7300 Real-Time PCR System
(Applied Biosystems, Foster City, CA). All PCR primers (Catalog
numbers: 18S: QT01036875; EPO: QT00170331; VEGF:
QT00160769; GLUT1: QT01044953) except lactate dehydroge-
nase A (LDHA) were purchased from Qiagen (Valencia, CA). The
sequences of the LDHA primers (Takara Bio) are 59-GGAT-
GAGCTTGCCCTTGTTGA-39 (forward) and 59-GACCAG-
CTTGGAGTTCGCAGTTA-39 (reverse). The fold changes in
expression of each target mRNA were calculated relative to 18S
rRNA.
ELISA of EPO
Samples were prepared according to the method described
previously [67]. Briefly, the entire brain was homogenized in
phosphate-buffered saline (PBS), centrifuged for 10 min at 5,000 g
at 4uC, and immediately frozen at 220uC. After two freeze–thaw
cycles to break the cell membranes, the brain homogenates were
assayed by an ELISA kit (R&D Systems Europe, Abingdon, UK)
according to the manufacturer’s instructions. The results were
expressed as the ratio of the quantity of EPO (in pg) to the quantity
of total protein (in mg) in the brain. The total protein
concentration was determined by the modified Bradford assay
(Nakalai Tesque, Inc., Kyoto, Japan) using bovine serum albumin
(BSA) as a standard.
Immunoblot assay
Nuclear extracts were prepared from a whole mouse brain using
a nuclear extraction kit (Active Motif, Carlsbad, CA). The aliquots
(100 mg protein) were fractionated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS/PAGE) (7.5% gel) and
subjected to an immunoblot assay following a protocol described
previously [68]. Primary antibodies raised against HIF-1a (AB
1536; R&D Systems, Minneapolis, MN), HIF-2a (NB100-480;
Novus Biologicals, Inc., Littleton, CO), ARNT (HIF-1b)
(#611078; BD Biosciences, San Jose, CA), glial fibrillary acidic
protein (GFAP) (#3670; Cell Signaling, Stockholm, Sweden),
neuronal nuclei (NeuN) (MAP377; Millipore, Billerica, MA), and
b-actin (A5316; Sigma-Aldrich, St. Louis) were used at a 1:1000
dilution. Horseradish peroxidase (HRP)-conjugated sheep anti-
mouse IgG (GE Healthcare, Piscataway, NJ) or donkey anti-rabbit
IgG antibodies (GE Healthcare) were also used at a 1:1000
dilution. The signal was detected with enhanced chemilumines-
cence (ECL) reagent (GE Healthcare).
Immunohistochemistry
Immunohistochemistry was performed according to the proce-
dure described by Toda et al [69]. Mouse brains were kept at 4uC
overnight in 4% paraformaldehyde in 0.1 M phosphate buffer.
The brains were then rinsed in PBS, transferred to 70% ethanol,
and embedded in paraffin. Ten-micrometer coronal sections were
cut and mounted on slides using albumin water. Sections were
deparaffinized and rehydrated, and antigen retrieval was per-
formed using autoclaving. Briefly, a Coplin jar containing glass
slides in citrate buffer was covered with a loose fitting cap and
heated in a stainless steel pressure cooker for 5 min at 121uC. The
pressure cooker was removed from the heat source and cooled by
running under cold water with the lid on. The glass slides were
rinsed in distilled water. The incubation and washing procedures
were carried out at room temperature. After deparaffinization and
antigen retrieval by the methods noted above, endogenous
peroxidase activity was blocked by 0.3% H2O2 in methyl alcohol
for 30 min. The glass slides were washed in PBS (6 times, 5 min
each) and mounted with 1% goat normal serum in PBS for
30 min. Subsequently, rabbit polyclonal anti-HIF-1a (AB 1536;
R&D Systems) diluted 1:200 and rabbit polyclonal anti-HIF-2a
(NB100-480; Novus Biologicals) diluted 1:400 were applied
overnight at 4uC. They were then incubated with biotinylated
goat anti-rabbit serum (second antibody) diluted 1:300 in PBS for
40 min, followed by washes in PBS (6 times, 5 min each). Avidin-
biotin-peroxidase complex (ABC) (ABC-Elite, Vector Laborato-
ries, Burlingame, CA) at a dilution of 1:100 in BSA was applied for
50 min. After washing in PBS (6 times, 5 min each), coloring
reaction was performed using diaminobenzidine, and the nuclei
were counterstained with hematoxylin.
Cell culture
Primary cultures of cerebral cortical astrocytes were prepared
from 1- or 2-day-old C57BL/6N CrSlc mice according to the
method previously described [70]. Brains of mice were removed
under sterile conditions, and the meninges were carefully removed.
The tissue was dissociated by passing it through a 320-mm nylon
mesh with the aid of a rubber policeman. After washing with
Hanks’ balanced salt solution containing DNaseI, the cells were
suspended and passed through a 100-mm nylon mesh. Next, they
were plated on a plastic culture flask (density of 2 brains per flask)
in 10-ml tissue culture medium. The tissue culture medium
consisted of Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin, and 0.1 mg/ml streptomycin. The cultures were
maintained in a humidified atmosphere of 5% CO2 in air at
37uC. The medium was changed after 3 days, then twice weekly.
At the first medium change, the flasks were vigorously shaken in
order to remove oligodendrocytes and their precursors. All
experiments were performed in cells at day 14 in vitro.
Isoflurane exposure
Isoflurane exposure was performed as described previously [71].
Briefly, cell dishes were kept in the airtight chamber housed within
a water jacket incubator maintained at 37uC. An in-line calibrated
anesthetic agent vaporizer was used to deliver isoflurane to the gas
phase of the culture wells. Hypoxic gas (1% O2–5% CO2–94%
N2) was administered at a flow rate of 3 l/min, until the
appropriate effluent concentration of the anesthetic was achieved.
Effluent isoflurane, O2, and CO2 concentrations were continu-
ously monitored via a sampling port connected to an anesthetic
agent analyzer (Capnomac Ultima; Datex-Ohmeda, Helsinki,
Finland).
Protein extraction
Whole cell lysates were prepared using ice-cold lysis buffer
[0.1% SDS, 1% Nonidet P40 (NP40), 5 mM EDTA, 150 mM
NaCl, 50 mM Tris-Cl (pH 8.0), 2 mM DTT, 1 mM sodium
orthovanadate, and complete protease inhibitor (Roche Diagnos-
tics)] following a protocol described previously [72]. A total of
100 mg of protein was loaded onto a 7.5% SDS/PAGE gel for
immunoblot assay.
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Measurement of total cellular O2 consumption
Cells were trypsinized and suspended at 16107 cells per ml in
DMEM with 10% FBS and 25 mM HEPES buffer. For each
experiment, equal numbers of cells suspended in 0.4 ml were
pipetted into the chamber of an OxythermTM electrode unit
(Hansatech Instruments, Norfolk, UK), which uses a Clark-type
electrode to monitor the dissolved O2 concentration in the sealed
chamber over time. The data were exported to a computerized
chart recorder (Oxygraph; Hansatech Instruments) that calculated
the rate of O2 consumption. The temperature was maintained at
37uC during measurement. The O2 concentration in 0.4 ml of
DMEM medium without cells was also measured over time to
provide background values. Oxygen consumption experiments
were repeated three times.
Statistical analysis
Data are presented as the mean 6 SD. Statistical significance
was assessed by Mann-Whitney U-test for two group comparisons
and by Kruscal Wallis H-test, followed by Mann-Whitney U-test
with Bonferroni Correction for multiple group comparisons.
Significance was defined as a value of P,0.05.
Acknowledgments
We wish to thank Dr. Youshi Fujita (Department of Neurology, Graduate
School of Medicine, Kyoto University, Kyoto, Japan) for his advice on
immunoblotting and immunohistochemistry.
Author Contributions
Conceived and designed the experiments: TT KF KH. Performed the
experiments: TT SK TK HD. Analyzed the data: TT KH. Contributed
reagents/materials/analysis tools: TA. Wrote the paper: TT KF KH.
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Anesthetics Inhibit Erythropoietin Induction
PLoS ONE | www.plosone.org 14 December 2011 | Volume 6 | Issue 12 | e29378