Effects of sevoflurane on neuronal cell damage after severe cerebral ischemia in rats.

Abstract

The aim of this study was to investigate the neuroprotective effects of sevoflurane after severe forebrain ischemic injury. We also examined the relationship between the duration of ischemia and neuronal cell death.
Male Sprague-Dawley rats (300-380 g) were subjected to 6 (each n = 6) or 10 min (each n = 10) of near-complete forebrain ischemia while anesthetized with either 50 mg/kg of zoletil given intraperitoneally or inhaled sevoflurane (2.3%). Ischemia was induced by bilateral common carotid artery occlusion plus hemorrhagic hypotension (26-30 mmHg). Histologic outcomes were measured 7 days after ischemia in CA1 pyramidal cells of the rat hippocampus.
The mean percentage of necrotic cells in the hippocampal CA1 area decreased in the sevoflurane group compared to the zoletil group (25% vs. 40% after 6 min ischemia, respectively: P = 0.004 and 44% vs. 54% after 10 min of ischemia, respectively P = 0.03). The percentage of apoptotic cells was similar in all groups. The percentage of necrotic cells in each anesthetic groups was significantly higher in the 10 min ischemia group compared to the 6 min ischemia group (P = 0.004 in the sevoflurane group, P = 0.03 in the zoletil group).
The present data show that sevoflurane has neuroprotective effects in rats subjected to near-complete cerebral ischemia. Longer duration of ischemia is associated with more neuronal injury when compared to ischemia of shorter duration.

Full text

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Korean J Anesthesiol 2011 October 61(4): 327-331
http://dx.doi.org/10.4097/kjae.2011.61.4.327
Experimental Research Article
Copyright ⓒ the Korean Society of Anesthesiologists, 2011
www.ekja.org
Background: The aim of this study was to investigate the neuroprotective effects of sevoflurane after severe forebrain
ischemic injury. We also examined the relationship between the duration of ischemia and neuronal cell death.
Methods: Male Sprague-Dawley rats (300-380 g) were subjected to 6 (each n = 6) or 10 min (each n = 10) of
near-complete forebrain ischemia while anesthetized with either 50 mg/kg of zoletil given intraperitoneally or
inhaled sevoflurane (2.3%). Ischemia was induced by bilateral common carotid artery occlusion plus hemorrhagic
hypotension (26-30 mmHg). Histologic outcomes were measured 7 days after ischemia in CA1 pyramidal cells of the
rat hippocampus.
Results: The mean percentage of necrotic cells in the hippocampal CA1 area decreased in the sevoflurane group
compared to the zoletil group (25% vs. 40% after 6 min ischemia, respectively: P = 0.004 and 44% vs. 54% after 10
min of ischemia, respectively P = 0.03). The percentage of apoptotic cells was similar in all groups. The percentage of
necrotic cells in each anesthetic groups was significantly higher in the 10 min ischemia group compared to the 6 min
ischemia group (P = 0.004 in the sevoflurane group, P = 0.03 in the zoletil group).
Conclusions: The present data show that sevoflurane has neuroprotective effects in rats subjected to near-complete
cerebral ischemia. Longer duration of ischemia is associated with more neuronal injury when compared to ischemia
of shorter duration. (Korean J Anesthesiol 2011; 61: 327-331)
Key Words: Brain ischemia, Hippocampus, Inhalation anesthetics, Neuron.
Effects of sevoflurane on neuronal cell damage after severe
cerebral ischemia in rats
Hee-Pyoung Park1, Eun-Ju Jeong1, Mi-Hyun Kim1, Jung-Won Hwang2, Young-Jin Lim1, Seong-Won
Min3, Chong-Soo Kim3, and Young-Tae Jeon2
Department of Anesthesiology and Pain Medicine, 1Seoul National University Hospital, Seoul, 2Seoul National University Bundang
Hospital, Seongnam, 3Seoul City Boramae Hospital, Seoul, Korea
Received: January 25, 2011. Revised: May 17, 2011. Accepted: May 21, 2011.
Corresponding author: Young-Tae Jeon, M.D. Ph.D., Department of Anesthesiology and Pain Medicine, Seoul National University Bundang
Hospital, 300, Gumi-dong, Bundang-gu, Seongnam 463-802, Korea. Tel: 82-31-787-7493, Fax: 82-31-787-4063, E-mail: ytjeon@snubh.org
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium,
provided the original work is properly cited.
CC

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328www.ekja.org
Vol. 61, No. 4, October 2011
Sevoflurane and ischemia
Introduction
Numerous studies have demonstrated some degree of
a neuroprotective effect with isoflurane [1,2], sevoflurane
[3,4] and desflurane [2,5] after cerebral ischemia. The
neuroprotective effects of anesthetic agents depend on the
severity of ischemic injury. During less severe ischemia,
anesthetic agents (fentanyl, ketamine, and isoflurane) resulted
in no differences in outcome. In contrast, in animals sustaining
a severe insult, those that were anesthetized with isoflurane had
less damage than did rats given either ketamine or fentanyl [6].
Zoletil is a combination of a dissociative anesthetic, tileta-
mine hydrochloride, and a benzodiazepine, zolazepam hypo-
chloride. Tiletamine is a noncompetitive antagonist at the
phencyclidine site of N-methyl-D-aspartate receptor.
Sevoflurane has been shown to provide neuroprotection
against focal [3] or incomplete global cerebral ischemia [4]
in rats. To our knowledge, there has been no effort to define
the effects of sevoflurane against severe forebrain global
ischemia. The purpose of the present study was to determine
the neuroprotective effects of sevoflurane on severe forebrain
ischemic injury. We also examined the relationship between
ischemic duration and neuronal death. Neuroprotection was
assessed by histopathological evaluation 7 days after ischemia.
Degrees of neuronal damage in ischemic hippocampal CA1
cells were assessed by counting necrotic cells after H&E staining
and detection of DNA fragmentation was performed by terminal
deoxynucleotidyl transferase-mediated uridine 5'-triphosphate-
biotin nick end labeling (TUNEL) staining.
Materials and Methods
The following study was approved by the Institutional
Animal Care and Use Committee. Male Sprague-Dawley
rats (300-380 g, 10-16 wks) were fasted 12-16 h before the
experiments but were allowed free access to water. Rats were
then randomly assigned to one of 4 conditions based on the
anesthetics and duration of ischemia. In the 6 (n = 6) or 10 min
(n = 10) sevoflurane group, the animals were anesthetized with
5% sevoflurane in oxygen and during surgery, a level of 2.3%
sevoflurane in 100% O2 was maintained under spontaneous
breathing. In the 6 (n = 6) or 10 min (n = 10) zoletil group, 50 mg/
kg of zoletil (Virbac, Nice, France) was given intraperitoneally.
The tail artery was catheterized with a PE-50 catheter to allow
continuous recording of arterial blood pressure and blood
sampling. The common carotid arteries were encircled with
suture. The right jugular vein was cannulated with a silicone
catheter for drug infusion and blood withdrawal. A 22-gauge
needle thermistor was percutaneously placed adjacent to
the skull beneath the temporalis muscle, and pericranial
temperature was servoregulated (model TCAT-2 Temperature
Controller; Harvard apparatus, Holliston, MA, USA) at 37.5 ±
0.1oC by surface heating or cooling. Heparin (50 U) was given
intravenously.
Anesthetic conditions were established 30 min before
ischemia, during which time rats in both groups were allowed to
stabilize physiologically. Arterial blood gases and hemoglobin
were measured 10 min before and after ischemia.
Transient global ischemia was induced by bilateral common
carotid artery occlusion and bleeding to lower the mean arterial
pressure (MABP) to 26-30 mmHg using the method originally
described by Smith et al. [7]. The carotids were then occluded
with aneurysm clips. After 6 or 10 min of ischemia, the shed
blood was reinfused. After regaining consciousness, the animals
were maintained in an air conditioned room at 20oC. For each
recovery interval, a set of sham rats was generated (n = 4). These
animals were exposed to all aspects of sevoflurane anesthesia
and surgical preparation for ischemia. The carotids were not
occluded, and systemic hypotension was not used.
After completion of the ischemic protocol, rats were allowed
to recover for 7 days. All rats were anesthetized with sevoflurane,
and the brains were fixed in situ by intraaortic infusion with
buffered 10% formalin. Paraffin-embedded brain sections were
serially cut (5 μm thick) and stained with hematoxylin and eosin.
With the investigator blinded to group assignment, injury to the
hippocampal CA1 sector was evaluated by microscopy. The total
number of neurons (viable plus nonviable) was counted for each
animal. The percentage of necrotic (eosinophilic) CA1 neurons
was calculated as necrotic neurons/total neurons ×100. In each
animal, five optical fields of the hippocampal CA1 region were
examined.
For the detection of DNA fragmentation, TUNEL staining
was performed with the ApoptagⓇ Peroxidase In Situ Apoptosis
Detection Kit S7100 (Millipore Corporation, Billerica, USA).
After deparaffinizing with xylene and graded concentrations
of alcohol, brain sections were exposed to Proteinase K for 15
min at room temperature. Endogenous peroxidase activity was
quenched with 3% hydrogen peroxide in phosphate buffered
saline (PBS) for 5 min at room temperature. Sections were then
incubated with terminal deoxynucleotidyl transferase (TdT)
in a humidified chamber at 37oC for 1h. After incubation with
anti-digoxigenin-conjugate for 30 min at room temperature,
peroxidase substrate (0.05% diaminobenzidine, DAB) was
applied to develop color. The specimens were then washed with
distilled water and were counterstained with 0.5% methyl green
for 10 min at room temperature. TUNEL-positive neurons that
contained apoptotic bodies were identified as being neurons
undergoing apoptosis. The number of apoptotic cells was
counted under high-power microscopic magnification (×400).
In each animal, five optical fields of the hippocampal CA1

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Korean J Anesthesiol
Park, et al.
region were examined. The percentage of TUNEL-positive cells
was defined as the percentage of the number of TUNEL-positive
cells to the total cell number.
The quantitative data were presented as the mean ± SD.
Physiologic parameters were analyzed by repeated measures
analysis of variance. Statistical significance between groups
was analyzed using a Mann-Whitney U test. Differences were
considered significant when P < 0.05.
Results
Physiologic parameters are summarized in Table 1. There
were no significant differences in mean arterial pressure, pH,
PaCO2, PaO2, and hemoglobin concentration between the
control and the sevoflurane treated groups at any time point.
Of the 20 animals receiving 10 min of ischemia, five rats
in the zoletil treated group died before the determination of
neurologic damage. A majority of the postoperative deaths
occurred within 3 days of ischemia. No animals died after 6
min of ischemia and no neuronal death was observed in the
sham-operated group. The mean percentage of necrotic cells
in the zoletil 6 min group was significantly higher than in the
sevoflurane 6 min group (Fig. 1: P = 0.004) and similar results
were seen in the zoletil 10 and sevoflurane 10 group (Fig. 2:
P = 0.03). The percentage of necrotic cells in each anesthetic
group was significantly higher in the 10 min ischemia group
compared to the 6 min ischemia group (Fig. 1, 2: P = 0.004 in the
sevoflurane group, P = 0.03 in the zoletil group).
Increases in the percentages of TUNEL-positive cells were
observed in all groups as compared to the sham-operated group
(P < 0.001). No significant differences in TUNEL-positive cells
in the CA1 region were observed among the anesthetic groups
(Fig. 1 and 2).
Discussion
In the present study, we investigated the neuroprotective
effect of sevoflurane on near-complete cerebral ischemia. This
study demonstrated that histologic outcome after 6 or 10 min
Fig. 1. The percentage of necrotic and apoptotic cells in the hippo-
campus 7 days after forebrain ischemia. The percentage of necrotic
cells was lower in the sevoflurane 6 min ischemia group (*P < 0.05).
There is no significant difference between groups with respect to
apoptotic cell number. All data are expressed as mean ± SD.
Fig. 2. The percentage of necrotic and apoptotic cells in the hippo-
campus 7 days after forebrain ischemia. The percentage of necrotic
cells was lower in the sevoflurane 10 min ischemia group (*P < 0.05).
The percentage of necrotic cells in each anesthetic group is higher in
the 10 min ischemia group compared with the 6 min ischemia group
(†P < 0.05). There are no significant differences between groups with
respect to apoptotic cell numbers. All data are expressed as mean ± SD.
Table 1. Physiological Parameters 10 min before and after Ischemia
pH
PaCO2,
mmHg
PaO2,
mmHg
Hemoglobin,
g/dl
MAP,
mmHg
Blood glucose
(mg/dl)
Zoletil 6
Sevoflurane 6
Zoletil 10
Sevoflurane 10
Before
After
Before
After
Before
After
Before
After
7.33 ± .04
7.33 ± .02
7.32 ± .05
7.33 ± .03
7.36 ± .03
7.33 ± .02
7.31 ± .03
7.34 ± .04
45 ± 6
40 ± 9
48 ± 10
40 ± 11
43 ± 3
39 ± 3
48 ± 4
48 ± 6
230 ± 38
195 ± 39
250 ± 72
179 ± 21
240 ± 39
220 ± 20
203 ± 46
198 ±37
12.9 ± 1.9
12.5 ± 1.4
12.8 ± 1.4
11.4 ± 1.4
13.8 ± 1.6
14.0 ± 1.2
14.0 ± 1.2
13.8 ± 0.6
96 ± 30
88 ± 9
92 ± 13
87 ± 8
105 ± 13
116 ± 14
84 ± 12
92 ± 11
91 ± 9
94 ± 10
96 ± 10
92 ± 10
Data are expressed as mean ± SD.

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Vol. 61, No. 4, October 2011
Sevoflurane and ischemia
of severe forebrain ischemia were substantially better in rats
with sevoflurane versus zoletil anesthesia 7 days after ischemia.
Sevoflurane did not decrease ischemia-induced apoptotic cell
death 7 days after ischemia. Shorter duration of ischemia was
associated with less neuronal damage.
In rats subjected to incomplete cerebral ischemia, sevo-
flurane improved neurologic outcomes [4]. In the present study,
we focused on the severity of cerebral ischemia. Others have
shown that there was no effect of anesthetic agents on outcome
in less severe incomplete ischemia [6]. Cerebral blood flow
studies after the near-complete insult causing EEG isoelectricity
have consistently found flow to be severely reduced. In contrast,
examination of cerebral blood flow during less severe forebrain
injury found substantial scatter in the magnitude of cerebral
blood flow reduction [8]. The near-complete ischemia insult
was uniformly severe across all animals. In contrast, findings
in the incomplete model varied with respect to concordance
between cortical and hippocampal regions as well as between
the presence or absence of EEG activity [6]. In the current study,
sevoflurane showed better histologic outcomes in rats subjected
to severe cerebral ischemia. Six min of ischemia also induced
severe neuronal injury. In contrast to our study, a previous
study showed sevoflurane completely prevented the neuronal
eosinophilic damage (a valid indicator of necrosis) of the brain
in incomplete cerebral ischemia [9]. The neuroprotective effect
of anesthetic agents seems to be correlated with the severity
of neurologic injury. Anesthetic agents provide sustained
neuroprotection in the presence of less severe ischemic states
[9,10]. In contrast, in a rat focal ischemia model, isoflurane
did not lead to sustained neuroprotection after 14 days [11].
This suggests that the severity of cerebral ischemia is beyond
the neuroprotective potential of anesthetic agents to prevent
necrotic cell death.
The mechanisms by which volatile anesthetic agents
potentially protect the brain are still unclear. Cerebral
protection with sevoflurane has been explained by a reduction
in cerebral requirements, similar to barbiturates or isoflurane.
The neuroprotective effects of volatile anesthetic agents are
associated with decreased apoptotic cell death in the post-
ischemic period [12-15]. Anesthesia with sevoflurane induces
a sustained inhibition of neuronal injury by promoting anti-
apoptotic pathways [9,16]. It was demonstrated that sevoflurane
prevents the ischemia-induced increase of the pro-apoptotic
protein Bax 4 h after injury induced by incomplete cerebral
ischemia [16]. Sevoflurane increases the hippocampal con-
centration of the anti-apoptotic proteins Bcl-2 and Mdm-
2 in addition to inhibiting the upregulation of Bax [9]. In the
present study, sevoflurane did not decrease TUNEL positive
cells. Kawaguchi et al.[13] showed that volatile anesthetics
delayed but did not prevent neuronal apoptosis after focal
cerebral ischemia. We propose that any anti-apoptotic effect of
sevoflurane might disappear 7 days after ischemia.
In the present study, shorter durations of ischemia had
significantly less damage than longer durations (i.e. 6 vs 10 min).
Consistent with our study, the magnitude of ischemic injury is
dependent on the duration of ischemia [17]. Sevoflurane did not
prevent neuronal damage following ischemia of 6 min duration.
We speculate that even short durations of severe hypotension
can cause severe damage which cannot be prevented by
sevoflurane.
We used 2.3 vol% sevoflurane for maintenance of anesthesia.
One minimal alveolar concentration of sevoflurane is 2.3 vol%
in adult rats [18]. Therefore, the sevoflurane concentration used
in this study was relevant.
There are several limitations in this study. First, the study was
limited to 7 days after ischemia. Longer periods of time may
be necessary to assess the permanent neuroprotective effect of
anesthetics. However, maintaining rats for such a long period
of time after cerebral ischemia is not feasible in our laboratory.
Second, we did not confirm EEG isoelectricity. However, it
is well demonstrated that bilateral common carotid artery
occlusion plus hemorrhage (30 mmHg) induces near-complete
ischemia in the CA1 region of rats [6,19]. Third, we classified
necrosis and apoptosis. Attempts to classify CA1 neuronal
death as either necrosis or apoptosis may be misleading when
attempting to understand mechanisms of death of differentiated
neurons. These neurons might possess some of the phenotypic
characteristics of necrotic neurons but, in contrast, possess all
of the biochemical and molecular characteristics needed to die
via apoptosis.
In summary, this study shows that sevoflurane improves
neurological outcome after severe cerebral ischemia in rats
compared with animals anesthetized with zoletil. Longer
duration of cerebral ischemia is associated with more neuronal
damage than shorter durations of ischemia.
Acknowledgements
Supported by grant to 04-2007-002 from the SNUBH Research
Fund.
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